AU2015203575B2 - Development of mutations useful for attenuating dengue viruses and chimeric dengue viruses - Google Patents

Abstract

A menu of mutations was developed that is useful in fine-tuning the attenuation and growth characteristics of dengue virus vaccinations.

Description

2015203575 26 Jun2015 AUSTRALIA Patents Act 1990

The Government of the United States of America, as represented by the Secretary, Department of Health and Human Services

COMPLETE SPECIFICATION STANDARD PATENT

Invention Title:

Development of mutations useful for attenuating dengue viruses and chimeric dengue viruses

The following statement is a full description of this invention including the best method of performing it known to us: -1-

DEVELOPMENT OF MUTATIONS USEFUL FOR ATTENUATING DENGUE VIRUSES AND CHIMERIC DENGUE VIRUSES 2015203575 26 Jun2015

Cross-Reference to Related Applications 5 This application is a divisional application under S. 79B of the Patents Act 1990 of

Australian Patent Application No. 2012200637 which is a divisional application of Australian Patent Application No. 2008203275 which is a divisional application of Australian Patent Registration No. 2002312011 filed May 22, 2002 which corresponds to International Application No. PCT/US02/16308 in the Australian national phase, and 10 claims priority to US 60/293049 filed May 22, 2001; the contents of each of the foregoing applications are hereby incorporated in its entirety by way of reference.

Field of the Invention A menu of mutations was developed that is useful in fine-tuning the attenuation and growth characteristics of dengue virus vaccines. 15 Background of the Invention

Dengue virus is a positive-sense RNA virus belonging to the Flavivirus genus of the family Flaviviridae. Dengue virus is widely distributed throughout the tropical and semitropical regions of the world and is transmitted to humans by mosquito vectors. Dengue virus is a leading cause of hospitalization and death in children in at least eight 20 tropical Asian countries (WHO, 1997. Dengue haemorrhagic fever: diagnosis, treatment prevention and control - 2nd ed. Geneva: WHO). There are four serotypes of dengue virus (DEN-I, DEN-2, DEN-3, and DEN-4) which annually cause an estimated 50 - 100 million cases of dengue fever and 500,000 cases of the more severe form of dengue virus infection, dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (Gubler, D.J. & Meltzer, 25 M. 1999 Adv Virus Res 53:35-70). DHF/DSS is seen predominately in children and adults experiencing a second dengue virus infection with a serotype different than that of their first dengue virus infection and in primary infection of infants who still have circulating dengue-specific maternal antibody (Burke, D.S. et al. 1988 Am J Trop Med Hyg 38:172-80; Halstead, S B. et al. 1969 Am J Trop Med Hyg 18:997-1021; Thein, S. et al. 1997 Am J 30 Trop Med Hyg 56:566-72). A vaccine is needed to lessen the disease burden caused by dengue virus, but none is licensed. Because of the association of more severe disease with secondary dengue virus infection, a successful vaccine must induce immunity to all four serotypes. Immunity is primarily mediated by neutralizing antibody directed against the envelope E glycoprotein, a virion structural protein. Infection with one serotype induces 35 long-lived homotypic immunity and a short-lived heterotypic immunity (Sabin, A. 1955 Amer J Trop Med Hyg 4:198-207). Therefore, the goal of immunization is to induce a long-lived neutralizing antibody response against DEN-I, DEN-2, DEN-3, and DEN-4, -1A- which can best be achieved economically using live attenuated virus vaccines. This is a reasonable goal since a live attenuated vaccine has already been developed for the related yellow fever virus, another mosquito-bome flavivirus present in tropical and semitropical regions of the world (Monath, T.P. & Heinz, F.X. 1996 in: Fields B.N. et al. eds. Fields 5 Virology Philadelphia: Lippincott-Ravan Publishers, 961-1034). 2015203575 26 Jun2015

Several live attenuated dengue vaccine candidates have been developed and evaluated in humans or non-human primates. The first live attenuated dengue vaccine candidates were host range mutants developed by serial passage of wild type dengue viruses in the brains of mice and selection of mutants attenuated for humans (Kimura, R. & 10 Hotta, S. 1944 Japanese J Bacteriology 1:96-99; Sabin, A.B. & Schlesinger, R.W. 1945 Science 101:640; Wisseman, C.L. Jr. et al. 1963 Am J Trop Med 12:620-623). Although these candidate vaccine viruses were immunogenic in humans, their poor growth in cell culture discouraged further development. Additional live attenuated DEN-I, DEN-2, DEN-3, and DEN-4 vaccine candidates have been developed by serial passage in tissue culture 15 (Angsubhakom, S. et al. 1994 Southeast Asian J Trop Med Public Health 25:554-9; Bancroft, W.H. et al. 1981 Infect Immun 31:698-703; Bhamarapravati, N. et al. 1987 Bull World Health Organ 65:189-95; Eckels, K.H. et al. 1984 Am J Trap Med Hyg 33:684-9; Hoke, C.H Jr. et al. 1990 Am J Trop Med Hyg 43:219-26; Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-88) or by chemical mutagenesis (McKee, K.T. Jr. et al. 1987 Am J Trap 20 Med Hyg 36:435-42). It has proven very difficult to achieve a satisfactory balance between attenuation and immunogenicity for each of the four serotypes of dengue virus using these approaches and to formulate a tetravalent vaccine that is safe and satisfactorily immunogenic against each of the four dengue viruses (Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-88; Bhamarapravati, N. & Sutee, Y. 2000 Vaccine 18 Suppl 2: 44-7). 25 Two major advances utilizing recombinant DNA technology have recently made it possible to develop additional promising live attenuated dengue virus vaccine candidates. First, methods have been developed to recover infectious dengue virus from cells transfected with RNA transcripts derived from a full-length cDNA clone of the dengue virus genome, thus making it possible to derive infectious viruses bearing attenuating 30 mutations which have been introduced into the cDNA clone by site-directed mutagenesis (Lai, C.J. et al. 1991 PNAS USA88:5139-43). Second, it is possible to produce antigenic chimeric viruses in which the structural protein coding region of the full-length cDNA clone of dengue virus is replaced by that of a different dengue virus serotype or from a more divergent flavivirus (Bray, M. & Lai, C.J. 1991 PNAS USA 88: 10342-6; Chen, W. et 35 al. 1995 J Virol 69:5186-90; Huang, C.Y. et al. 2000 J Virol 74:3020-8; Pletnev, A.G. & -IB-

Men, R. 1998 PNAS USA 95:1746-51). These techniques have been used to construct intertypic chimeric dengue viruses which have been shown to be effective in protecting monkeys against homologous dengue vims challenge (Bray, M. et al. 1996 J. Virol 70:4162-6). Despite these advances, there is a need to develop attenuated antigenic dengue vims vaccines that specify a satisfactory balance between attenuation and immunogenicity for humans. 2015203575 08 Sep 2017

Summary of the Invention

The invention provides mutations that confer temperature sensitivity in Vero cells or human liver cells, host-cell restriction in mosquito or human liver cells, host-cell adaptation for improved replication in Vero cells, or attenuation in mice, which mutations are useful in fine tuning the attenuation and growth characteristics of dengue vims vaccines.

This invention provides a flavivims having a phenotype in which the viral genome is modified by introduction of a mutation, singly or in combination, taken from the group consisting of the mutations of any one of tables 1 to 37 and preferably of Table 37.

This invention also provides a method of producing neutralizing antibodies against dengue vims comprising the administration of a therapeutically effective amount of a pharmaceutical composition comprising a pharmacologically acceptable vehicle and a flavivims according to any one of the embodiments as described herein.

This invention also provides a tetravalent vaccine comprising a pharmacologically acceptable vehicle and a flavivims according to any one of the embodiments as described herein.

This invention also provides a live attenuated vaccine comprising a pharmacologically accepted vehicle and a flavivims according to any one of the embodiments as described herein.

This invention also provides an inactivated vaccine comprising a pharmacologically accepted vehicle and a flavivims according to any one of the embodiments as described herein.

This invention also provides a cDNA molecule and/or an RNA molecule encoding a flavivims according to any one of the embodiments as described herein.

This invention also provides a method of preparing a flavivims comprising (a) synthesizing full length viral genomic RNA in vitro using a cDNA molecule that encodes a flavivims according to any one of the embodiments described herein; (b) transfecting cultured cells with the viral genomic RNA to produce vims; and (c) isolating the vims from the cultured cells. -2-

This invention also provides a pharmaceutical composition and/or a method of making a pharmaceutical composition comprising combining a pharmacologically acceptable vehicle and a flavivirus according to any one of the embodiments as described herein. 2015203575 08 Sep 2017

In one example, the invention provides a dengue virus comprising a mutation at a nucleotide position that corresponds to nucleotide 10634 of the sequence set forth in SEQ ID NO: 14.

In one example, the invention provides a nucleic acid molecule encoding a dengue virus comprising a mutation at a nucleotide position that corresponds to nucleotide 10634 of the sequence set forth in SEQ ID NO: 1 as described herein, wherein the nucleic acid molecule is a cDNA molecule or an RNA molecule.

In one example, the invention provides a method of preparing a dengue virus comprising: (a) synthesizing full-length viral genomic RNA in vitro using a cDNA molecule that encodes a dengue virus as described herein; (b) transfecting cultured cells with the viral genomic RNA and culturing the transfected cells to produce dengue virus; and (c) isolating the dengue virus from the cultured cells.

In one example, the invention provides a dengue virus produced by the method described herein.

In one example, the invention provides a pharmaceutical composition comprising a pharmacologically acceptable vehicle and a dengue virus described herein.

In one example, the invention provides a tetravalent vaccine comprising a pharmacologically acceptable vehicle and a dengue virus described herein.

In one example, the invention provides a live attenuated vaccine comprising a pharmacologically acceptable vehicle and a dengue virus described herein.

In one example, the invention provides an inactivated vaccine comprising a pharmacologically acceptable vehicle and a dengue virus described herein.

In one example, the invention provides a kit comprising: (i) a pharmaceutical composition or a tetravalent vaccine or a live attenuated virus or an inactivated vaccine described herein packaged in a container or dispenser device; and (ii) instructions for administration.

In one example, the invention provides a method of eliciting neutralizing antibodies against dengue virus in a subject comprising administering to the subject a pharmaceutical composition or a tetravalent vaccine or a live attenuated vaccine or an inactivated vaccine as described herein.

In one example, the invention provides for use of a dengue virus or a pharmaceutical composition or a tetravalent vaccine or a live attenuated vaccine or an inactivated vaccine as -3- described herein in the preparation of a medicament for treatment or prevention of infection by dengue virus. 2015203575 08 Sep 2017

In one example, the invention provides for use of a dengue virus or a pharmaceutical composition or a tetravalent vaccine or a live attenuated vaccine or an inactivated vaccine as described herein to elicit neutralizing antibodies against dengue virus in a subject.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

Brief Description of the Drawings

Figure 1 shows growth of wt DEN4 2A and vaccine candidate, 2ΑΔ30, in Vero and HuH-7 cells. Vero (A) or HuHO-7 (B) cells were infected with DEN4 2A or 2ΑΔ30 at a multiplicity of infection (MOI) of 10 or 0.01. Confluent cell monolayers in 25-mm tissue culture flasks ere washed and overlaid with a 1.5 ml inoculum containing the indicated virus. After a two hour incubation at 37°C, cells were washed three times in PBS and 7 ml of culture media supplemented with 2% FBS was added. A 1 ml aliquot of tissue culture medium was removed, replaced with fresh medium, and designated the 0 hour time-point.

At the indicated time points post-infection, samples of tissue culture media were removed and frozen at -70 °C. The level of viral replication was assayed by plaque titration in Vero cells. Briefly, serial ten-fold dilutions of cell culture media samples were inoculated onto confluent Vero cell monolayers in 24-well plates in duplicate and overlaid with Opti-MEM containing 0.8% methylcellulose. After five days, plaques were visualized by immunoperoxidase staining as described in Example 1.

Figure 2 shows generation of temperature-sensitive (ts) DEN4 viruses by 5-fluorouracil (5-FU) chemical mutagenesis. The wild-type DEN4 2A virus was derived from a cDNA clone of DEN4 strain 814669 (Dominica, 1981). Vero cells were infected with DEN4 2A and overlaid with culture media containing 1 mM 5-fluorouracil (5-FU) which resulted in a reduction of approximately 100-fold in viral replication when compared to untreated controls. Viral progeny from the 1 mM 5-FU-treated cultures were subjected -3A- to a single round of terminal dilutions generating 1,248 biologically cloned viruses which were screened for ts phenotypes by assessing virus replication at 35°C and 39°C in Vero and HuH-7 cells. Virus clones which demonstrated a 100-fold or greater reduction in titer at 39°C were terminally diluted an additional two times and amplified in Vero cells. 2015203575 26 Jun2015 5 Temperature-sensitive phenotypes of the 3x biologically cloned viruses were confirmed by evaluating efficiency of plaque formation (EOP) in the indicated cells as described in Example 1.

Figure 3 shows plaque size phenotypes of representative 5-FU mutant DEN4 viruses. Serial ten-fold dilutions of wild-type DEN4 2A-13 (A), 5-FU mutant, viruses #569 10 and #1189 (B), and 5-FU mutant viruses #1083 and #311 (C) were inoculated onto confluent Vero and HuH-7 cell monolayers in 24-well plates. After incubation at 35°C for two horns, monolayers were overlaid with 0.8% methylcellulose culture media. Following incubation at 35°C for five days, plaques were visualized by immunoperoxidase staining. Viruses which had a plaque size that was <1 mm (approximately <50% the size of wt 15 DEN4 2A-13) at the permissive temperature of 35°C were designated as having the small-plaque (sp) phenotype. Mutant viruses #569 and #1189 (B) were sp in both Vero and HuH-7 cells, and #311 and #1083 (C) were sp in only HuH-7 cells.

Figure 4 shows generation of recombinant DEN4 viruses. (A), The p4 cDNA clone is represented which was constructed from the 2A cDNA clone (derived from DEN4 20 814669) by site-directed mutagenesis. Restriction enzyme sites were introduced or removed to facilitate subsequent cloning of DEN4 recombinants bearing introduced attenuating mutations. Restriction enzyme sites are shown and define fragments of the genome that were sub-cloned into modified pUC-119 vectors for site-directed mutagenesis to introduce mutations identified in the 5-FU mutant viruses. (B), An outline of the 25 methods used to generate rDEN4 viruses is also represented and described in Example 1.

Figure 5 shows amino acid sequence of the rDEN4 NS5 gene (SEQ ID NO: 1). Eighty underlined amino acid pairs were mutagenized to alanine pairs; 32 pairs in boldface represent mutant viruses that could be recovered in either Vero or C6/36 cells; pairs in normal type represent mutant viruses that could not be recovered in either Vero or C6/36 30 cells. Boxed regions indicate putative functional domains, including an S-adenosylmethionine utilizing methyltransferase domain (SAM), an importin-β binding -4- domain adjacent to a nuclear localization sequence (importin-β - binding + NLS) and an RNA-dependent RNA polymerase domain (Polymerase). 2015203575 26 Jun2015

Figure 6 shows plaque size of mutant 5-1A1 in C6/36 cells. Note that 5-1A1 has a small plaque phenotype in C6/36 cells relative to that of the wild type virus. 5 Figure 7 shows growth of wild type rDEN4 and 5-1A1 in C6/36 cells. Cells were inoculated in triplicate with each virus at an MOI of 0.01, and the amount of virus present in the supernatants that were harvested on the indicated days was determined by plaque enumeration in Vero cells. The titers are expressed as log10PFU/ml ± standard error.

Figure 8 shows nucleotide alignment of the 3’ UTR of mosquito-borne and tick-10 borne flaviviruses. cDNA sequences are shown 5’ to 3’ and represent a portion of the UTR corresponding to DEN4 nucleotides 10417 to 10649 (3’ genome end). Nucleotide numbering represents the position in the alignment. Regions deleted or swapped are indicated using the nucleotide numbering of DEN4. GenBank accession numbers for mosquito-bome viruses: DEN4 (SEQ ID NO: 2): AF326825, DENI (SEQ ID NO: 3): 15 U88535, DEN2 (SEQ ID NO: 4): AF038403, DEN3 (SEQ ID NO: 5): M93130, West Nile virus (WN) (SEQ ID NO: 6): M12294, Japanese encephalitis virus (JE) (SEQ ID NO: 7): AF315119, Yellow fever virus (YF) (SEQ ID NO: 8): U17067; GenBank accession numbers for tick-borne viruses: Powassan virus (POW) (SEQ ID NO: 9): L06436, Louping 111 virus (LI) (SEQ ID NO: 10): Y07863, Tick-bome encephalitis virus (TBE) (SEQ ID 20 NO: 11): U27495, and Langat virus (LGT) (SEQ ID NO: 12): AF253419.

Figure 9 shows genetic map of plasmid p4. Dengue cDNA is shown as bold line, with the C-prM-E region exchanged during construction of chimeric dengue virus cDNAs indicated.

Figure 10 shows plaque size phenotypes of rDEN4 viruses encoding Vero 25 adaptation mutations. Serial three-fold dilutions of the indicated viruses were inoculated onto confluent Vero and C6/36 cell monolayers in 6-well plates. After incubation at 37°C (Vero) or 32°C (C6/36) for two hours, monolayers were overlaid with 0.8% methylcellulose culture media. Following incubation for five days, plaques were visualized by immunoperoxidase staining. Values below each well are the average plaque size in mm ± 30 standard error. For each of the virus-infected wells, 36 plaques were measured on the digital image of the 6-well plate on Adobe Photoshop at 300% view. -5-

Figure 11 shows growth curve in Vero cells of rDEN4 viruses encoding single Vero adaptation mutations. Vero cells were infected with the indicated viruses at an MOI of 0.01. Confluent cell monolayers in 25-cm2 tissue culture flasks were washed and overlaid with a 1.5 ml inoculum containing the indicated virus. After a two hour incubation 5 at 37°C, cells were washed three times in PBS and 5 ml of culture medium supplemented with 2% FBS was added. A 1 ml aliquot of tissue culture medium was removed, replaced with fresh medium, and designated the 0 hour time-point. At the indicated time points post-infection, samples of tissue culture medium were removed, clarified, and frozen at -70°C. The level of virus replication was assayed by plaque titration in Vero cells. Briefly, 10 serial ten-fold dilutions of cell culture media samples were inoculated onto confluent Vero 2015203575 26 Jun2015 cell monolayers in 24-well plates in duplicate and overlaid with Opti-MEM containing 0.8% methylcellulose. After five days, plaques were visualized by immunoperoxidase staining as described in Example 1. Limit of detection (L.O.D.) is > 0.7 log10PFU/ml.

Figure 12 shows growth curve in Vero cells of rDEN4 viruses encoding combined 15 Vero cell adaptation mutations. Vero cells were infected with the indicated viruses at an MOI of 0.01. Confluent cell monolayers in 25-cm2 tissue culture flasks were washed and overlaid with a 1.5 ml inoculum containing the indicated virus. After a two hour incubation at 37°C, cells were washed three times in PBS and 5 ml of culture medium supplemented with 2% FBS was added. A 1 ml aliquot of tissue culture medium was removed, replaced 20 with fresh medium, and designated the 0 hour time-point. At the indicated time points post-infection, samples of tissue culture medium were removed, clarified, and frozen at -70°C. The level of virus replication was assayed by plaque titration in Vero cells. Limit of detection (L.O.D.) is > 0.7 log10PFU/ml.

Brief Description of the Tables 25 Table 1. Susceptibility of mice to intracerebral DEN4 infection is age-dependent.

Table 2. Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypes of 5-FU mutant DEN4 viruses.

Table 3. Nucleotide and amino acid differences of the 5-FU mutant viruses which are ts in both Vero and HuH-7 cells. 30 Table 4. Nucleotide and amino acid differences of the 5-FU mutant viruses which are ts in only HuH-7 cells.

Table 5. Mutations which are represented in multiple 5-FU mutant DEN4 viruses. -6-

Table 6. Addition of ts mutation 4995 to rDEN4A30 confers a ts phenotype and further attenuates its replication in suckling mouse brain. 2015203575 26 Jun2015

Table 7. Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypes of 5-FU DEN4 mutant viruses which exhibit a small plaque (sp) phenotype. 5 Table 8. Viruses with both ts and sp phenotypes are more restricted in replication in mouse brain than those with only a ts phenotype.

Table 9. Nucleotide and amino acid differences of the 5-FU mutant DEN4 viruses which produce small plaques in both Vero and HuH-7 cells.

Table 10. Nucleotide and amino acid differences of the 5-FU mutant DEN4 viruses 10 which produce small plaques in only HuH-7 cells.

Table 11. Putative Vero cell adaptation mutations derived from the full set of 5-FU mutant viruses.

Table 12. Mutagenic oligonucleotides used to generate recombinant DEN4 viruses containing single 5-FU mutations. 15 Table 13. sp, ts and mouse attenuation phenotypes of rDEN4 mutant viruses encoding single mutations identified in six sp 5-FU mutant viruses.

Table 14. Phenotypes of rDEN4 mutant viruses encoding single mutations identified in 10 5-FU mutant viruses that are ts in both Vero and HuH-7 cells.

Table 15. sp, ts and mouse attenuation phenotypes of rDEN4 mutant viruses 20 encoding single mutations identified in 3 HuH-7 cell-specific ts 5-FU mutant viruses.

Table 16. Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypes of additional rDEN4 viruses encoding single 5-FU mutations.

Table 17. Growth of wt DEN-4 2A-13 in SCID mice transplanted with HuH-7 cells. 25 Table 18. Combination of ts mutations, NS3 4995 and NS5 7849, in rDEN4 results in an additive ts phenotype.

Table 19. The 5-FU mutations are compatible with the Δ30 mutation for replication in the brain of suckling mice.

Table 20. Temperature-sensitive and mouse brain attenuation phenotypes of viruses 30 bearing charge-cluster-to-alanine mutations in the NS5 gene of DEN4.

Table 21. SCID-HuH-7 attenuation phenotypes of viruses bearing charge-cluster-to-alanine mutations in the NS5 gene of DEN4. -7-

Table 22. Combination of paired charge-cluster-to-alanine mutations into doublepair mutant viruses. 2015203575 26 Jun2015

Table 23. Temperature-sensitive and mouse brain attenuation phenotypes of double charge-cluster-to-alanine mutants of the NS5 gene of rDEN4. 5 Table 24. SCID-HuH-7 attenuation phenotypes of double charge-cluster-to-alanine mutants of the NS5 gene of rDEN4.

Table 25. Phenotypes (temperature sensitivity, plaque size and replication in mouse brain and SCID-HuH-7 mice) of wt DEN4 and viruses containing the Δ30 and 7129 mutations. 10 Table 26. The 5-fluorouracil 5-1A1 small plaque mutant demonstrates a restriction of midgut infection following oral infection of Aedes aegypti mosquitoes.

Table 27. The 5-fluorouracil 5-1A1 small plaque mutant demonstrates a restriction of infection following intrathoracic inoculation of Toxorhynchites splendens mosquitoes.

Table 28. Mutagenesis primers for the deletion or swap of sequences in DEN4 15 showing conserved differences from tick-bome flaviviruses.

Table 29. Vims titer and plaque size of 3' UTR mutant vimses in Vero and C6/36 cells.

Table 30. Infectivity of wt DEN4 and 3' UTR mutants for Toxorhynchites splendens via intrathoracic inoculation. 20 Table 31. Infectivity of 3’ UTR swap mutant vimses for Aedes aegypti fed on an infectious bloodmeal.

Table 32. Putative Vero cell adaptation mutations derived from the set of 5-FU mutant viruses and other DEN4 vimses passaged in Vero cells.

Table 33. Sequence analysis of rDEN2/4A30 clone 27(p4)-2-2A2. 25 Table 34. Sequence analysis of rDEN2/4A30 clone 27(p3)-2-lAl.

Table 35. Recombinant vims rDEN2/4A30 bearing Vero adaptation mutations can be recovery and titered on Vero cells.

Table 36. Putative Vero cell adaptation mutations of dengue type 4 vims and the corresponding wildtype amino acid residue in other dengue vimses. 30 Table 37. Mutations known to attenuate dengue type 4 vims and the corresponding wildtype amino acid residue in other dengue vims. -8-

Brief Description of the Appendices 2015203575 26 Jun2015

Appendix 1. Sequence of recombinant dengue type 4 virus strain 2A (amino acid sequence SEQ ID NO: 13 and nucleotide sequence SEQ ID NO: 14).

Appendix 2. Sequence of recombinant dengue type 4 virus strain rDEN4 (amino 5 acid sequence SEQ ID NO: 15 and nucleotide sequence SEQ ID NO: 16).

Appendix 3. Sequence of recombinant dengue type 2 chimeric virus strain rDEN2/4A30 (amino acid sequence SEQ ID NO: 17 and nucleotide sequence SEQ ID NO: 18).

Appendix 4. Alignment of dengue virus polyproteins. DEN4 (SEQ ID NO: 19); 10 DEN1-WP (SEQ ID NO: 20); DEN2-NGC (SEQ ID NO: 21); DEN3-H87 (SEQ ID NO: 22).

Detailed Description of the Preferred Embodiment

To assemble a collection of useful mutations for incorporation in recombinant live dengue virus vaccines, site-directed and random mutagenesis techniques were used to 15 introduce mutations into the dengue virus genome. The resulting mutant viruses were screened for several valuable phenotypes, including temperature sensitivity in Vero cells or human liver cells, host cell restriction in mosquito cells or human liver cells, host-cell adaptation for improved replication in Vero cells, and attenuation in mice. The genetic basis for each observed phenotype was determined by direct sequence analysis of the virus 20 genome. Mutations identified through these sequencing efforts have been further evaluated by their re-introduction, singly, or in combination, into recombinant dengue virus and characterization of the resulting phenotypes. In this manner, a menu of mutations was developed that is useful in fine-tuning the attenuation and growth characteristics of dengue virus vaccines. 25 Example 1

Chemical Mutagenesis of Dengue Virus Type 4 Yields Temperature-Sensitive and

Attenuated Mutant Viruses A recombinant live attenuated dengue virus type 4 (DEN4) vaccine candidate, 2ΑΔ30, was found previously to be generally well-tolerated in humans, but a rash and an 30 elevation of liver enzymes in the serum occurred in some vaccinees. 2ΑΔ30, a nontemperature-sensitive (ts) virus, contains a 30 nucleotide deletion in the 3’ untranslated region (UTR) of the viral genome. In the present study, chemical mutagenesis of DEN4 -9- has been utilized to generate attenuating mutations which may be useful to further attenuate the incompletely attenuated 2ΑΔ30 candidate vaccine. Wild-type DEN4 2A virus was grown in Vero cells in the presence of 5-fluorouracil, and, from a panel of 1,248 clones that were isolated in Vero cells, twenty ts mutant viruses were identified which were ts in both 5 Vero and HuH-7 cells (n = 13) or in HuH-7 cells only (n = 7). Each of the twenty ts mutations possessed an attenuation (att) phenotype as indicated by restricted replication in the brains of seven day old mice. The complete nucleotide sequence of the 20 ts mutant viruses identified nucleotide substitutions in structural and non-structural genes as well as in the 5’ and 3’ UTR with more than one change occurring, in general, per mutant virus. A 10 ts mutation in the NS3 protein (nucleotide position 4,995) was introduced into a recombinant DEN4 virus possessing the Δ30 deletion creating the rDEN4A30-4995 recombinant virus which was found to be ts and to be more attenuated than rDEN4A30 in the brains of mice. A menu of attenuating mutations is being assembled that should be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in 15 increasing our understanding of the pathogenesis of dengue virus. 2015203575 26 Jun2015

The mosquito-bome dengue (DEN) viruses (serotypes 1 to 4) are members of the Flavivirus genus and contain a single-stranded positive-sense RNA genome of approximately 10,600 nucleotides (nt) (Monath, T.P. &amp; Heinz, F.X. 1996 in: Fields Virology B.N. Fields, et al. Eds. pp. 961-1034 Lippincott-Ravan Publishers, Philadelphia). 20 The genome organization of DEN viruses is 5’-UTR-C-prM-E-NSl-NS2A-NS2B-NS3-NS4A-NS4B-NS5-UTR-3’ (IJTR - untranslated region, C - capsid, PrM - pre-membrane, E — envelope, NS - non-structural) (Chang, G.-J. 1997 hi: Dengue and dengue hemorrhagic fever DJ. Gubler &amp; G. Kuno, eds. pp. 175-198 CAB International, New York; Rice, C.M. 1996 in: Fields Virology B.N. Fields et al. Eds. pp. 931-959 Lippincott-Raven Publishers, 25 Philadelphia). A single viral polypeptide is co-translationally processed by viral and cellular proteases generating three structural proteins (C, M, and E) and seven NS proteins. The disease burden associated with DEN virus infection has increased over the past several decades in tropical and semitropical countries. Annually, there are an estimated 50-100 million cases of dengue fever (DF) and 500,000 cases of the more severe and potentially 30 lethal dengue hemorrhagic fever / dengue shock syndrome (DHF/DSS) (Gubler, DJ. &amp; Meltzer, M. 1999 Adv Virus Res 53:35-70). -10-

The site of viral replication in DEN virus-infected humans and the pathogenesis of DF and DHF/DSS are still incompletely understood (Innis, B.L. 1995 in: Exotic viral infections J.S. Porterfield, ed. pp. 103-146 Chapman and Hall, London). In humans, DEN virus infects lymphocytes (Kurane, I. et al. 1990 Arch Virol 110:91-101; Theofilopoulos, A.N. et al. 1976 JImmunol 117:953-61), macrophages (Halstead, S.B. et al. 1977 JExp Med 146:218-29; Scott, R.M. et al. 1980 J Infect Dis 141:1-6), dendritic cells (Libraty, D.H. et al. 2001 J Virol 75:3501-8; Wu, SJ. et al. 2000 Nat Med 6:816-20), and hepatocytes (Lin, Y.L. et al. 2000 JMed Virol 60:425-31; Marianneau, P. et al. 1996 J Gen Virol 77:2547-54). The fiver is clearly involved in DEN virus infection of humans, as indicated by the occurrence of transient elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in the majority of dengue virus-infected patients and by the presence of hepatomegaly in some patients (Kalayanarooj, S. et al. 1997 J Infect Dis 176:313-21; Kuo, C.H. et al. 1992 Am J Trop Med Hyg 47:265-70; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S.F. et al. 2000 Southeast Asian J Trop Med Public Health 31:259-63). DEN virus antigen-positive hepatocytes are seen surrounding areas of necrosis in the fiver of fatal cases (Couvelard, A. et al. 1999 Hum Pathol 30:1106-10; Huerre, M.R. et al. 2001 Virchows Arch 438:107-15), and dengue virus sequences were identified in such cases using RT-PCR (Rosen, L. et al. 1999 Am J Trop Med Hyg 61:720- 4). Of potential importance to the etiology of severe dengue virus infection, three studies have demonstrated that the mean levels of serum ALT/AST were significantly increased in patients with DHF/DSS versus those with DF (Kalayanarooj, S. et al. 1997 J Infect Dis 176:313-21; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S.F. et al. 2000 Southeast Asian J Trop Med Public Health 31:259-63). A vaccine for DEN viruses is not presently licensed. Since previous infection with one dengue virus serotype can increase the risk for DHF/DSS following infection with a different serotype (Burke, D.S. et al. 1988 Am J Trop Med Hyg 38:172-80; Halstead, S.B. et al. 1969 Am J Trop Med Hyg 18:997-1021; Thein, S. et al. 1997 Am J Trop Med Hyg 56:566-72), it is clear that a dengue virus vaccine will need to protect against each of the four dengue virus serotypes, namely DENI, DEN2, DEN3, and DEN4. Several strategies are currently being actively pursued in the development of a five attenuated tetravalent DEN virus vaccine (Bancroft, W.H. et al. 1984 J Infect Dis 149:1005-10; Bhamarapravati, N. &amp; Sutee, Y. 2000 Vaccine 18:44-7; Guirakhoo, F. et al. 2000 J Virol 74:5477-85; -11-

Huang, C.Y. et al. 2000 J Virol 74:3020-8). Recently, we demonstrated that a live attenuated DEN4 vaccine candidate, 2ΑΔ30, was attenuated and immunogenic in a group of 20 human volunteers (see Example 8). This recombinant DEN4 virus contains a 30 nt deletion in the 3’ UTR which removes nucleotides 10,478-10,507 and was restricted in, 5 replication in rhesus monkeys. Levels of viremia in humans were low or undetectable, and virus recovered from the vaccinees retained the Δ30 mutation. An asymptomatic rash was reported in 50% of patients. The only laboratory abnormality observed was an asymptomatic, transient rise in the serum ALT level in 5 of 20 vaccinees. All vaccinees developed serum-neutralizing antibody against DEN4 . virus (mean titer: 1:580). 10 Importantly, 2ΑΔ30 was not transmitted to mosquitoes fed on vaccinees and has restricted growth properties in mosquitoes (Troyer, J.M. et al. 2001 Am J Trop Med Hyg 65:414-9). The presence of a rash and of the elevated ALT levels suggests that the 2ΑΔ30 vaccine candidate is slightly under-attenuated in humans. Because of the overall set of desirable properties conferred by the Δ30 mutation, chimeric vaccine candidates are being 15 constructed which contain the structural genes of dengue virus type 1, 2, and 3 and the DEN4 attenuated backbone bearing the genetically stable Δ30 mutation. 2015203575 26 Jun2015

Although the initial findings indicate the utility of the 2ΑΔ30 vaccine candidate, many previous attempts to develop live attenuated dengue virus vaccines have yielded vaccine candidates that were either over- or under-attenuated in humans (Eckels, K.H. et al. 20 1984 Am J Trop Med Hyg 33:684-9; Bhamarapravati, N. &amp; Yoksan, S. 1997 in: Dengue and dengue hemorrhagic fever D.J. Gubler &amp; G. Kuno eds. pp. 367-377 CAB International, New York; Imiis, B.L. et al. 1988 J Infect Dis 158:876-80; McKee, K.T., Jr. et al. 1987 Am J Trop Med Hyg 36:435-42). Therefore, we developed a menu of point mutations which confer temperature-sensitive (ts) and attenuation (att) phenotypes upon DEN4. These 25 mutations are envisioned as being useful to attenuate DEN4 viruses to different degrees and therefore as having purpose in fine-tuning the level of attenuation of vaccine candidates such as 2ΑΔ30. Addition of such mutations to 2ΑΔ30 or to other dengue virus vaccine candidates is envisioned as resulting in the generation of a vaccine candidate that exhibits a satisfactory balance between attenuation and immunogenicity for humans. 30 In the present example, chemical mutagenesis of DEN4 has been utilized to identify point mutations which confer the ts phenotype, since such viruses often are attenuated in humans. Additionally, because of the reported involvement of the liver in natural dengue -12- infection and the elevated ALT levels in a subset of 2ΑΔ30 vaccinees, mutagenized DEN4 viruses were also evaluated for ts phenotype in HuH-7 liver cells derived from a human hepatoma. Here, we describe the identification of 20 DEN4 ts mutant viruses each of which replicates efficiently in Vero cells, the proposed substrate for vaccine manufacture, 5 and each of which is attenuated in mice. Finally, the feasibility of modifying the attenuation phenotype of the 2ΑΔ30 vaccine candidate by introduction of a point mutation in NS 3 is demonstrated. 2015203575 26 Jun2015

Cells and viruses. WHO Vero cells (African green monkey kidney cells) were maintained in MEM (Life Technologies, Grand Island, NY) supplemented with 10% fetal 10 bovine serum (FBS) (Summit Biotechnologies, Fort Collins, CO), 2 mM L-glutamine (Life Technologies), and 0.05 mg/ml gentamicin (Life Technologies). HuH-7 cells (human hepatoma cells) (Nakabayashi, H. et al. 1982 Cancer Res 42:3858-63) were maintained in D-MEM/F-12 (Life Technologies) supplemented with 10% FBS, 1 mM L-glutamine and 0.05 mg/ml gentamicin. C6/36 cells (Aedes albopictus mosquito cells) were maintained in 15 complete MEM as described above supplemented with 2 mM non-essential amino acids (Life Technologies).

The wild type (wt) DEN4 2A virus was derived from a cDNA clone of DEN4 strain 814669 (Dominica, 1981) (Men, R. et al. 1996 J Virol 70:3930-7). Sequence of the cDNA of DEN 4 2A virus is presented in Appendix 1. The full-length 2A cDNA clone has 20 undergone several subsequent modifications to improve its ability to be genetically manipulated. As previously described, a translationally-silent Xhol restriction enzyme site was engineered near the end of the E region at nucleotide 2348 to create clone 2A-Xho\ (Bray, M. &amp; Lai, C.J. 1991 PNAS USA 88:10342-6). The viral coding sequence of the 2A-Xhol cDNA clone was further modified using site-directed mutagenesis to create clone p4: 25 a unique BbvCl restriction site was introduced near the C-prM junction (nucleotides 447 -452); an extra Xbal restriction site was ablated by mutation of nucleotide 7730; and a unique Sacll restriction site was created in the NS5 region (nucleotides 9318 - 9320). Each of these engineered mutations is translationally silent and does not change the amino acid sequence of the viral polypeptide. Also, several mutations were made in the vector region '30 of clone p4 to introduce or ablate additional restriction sites. The cDNA clone ρ4Δ30 was generated by introducing the Δ30 mutation into clone p4. This was accomplished by replacing the Mlul - Kpnl fragment of p4 (nucleotides 10403 - 10654) with that derived -13- from plasmid 2ΑΔ30 containing the 30 nucleotide deletion. The cDNA clones p4 and ρ4Δ30 were subsequently used to generate recombinant viruses rDEN4 (Appendix 2) and rDEN4A30, respectively. (The GenBank accession number for rDEN4 is AF326825 and the accession for rDEN4A30 is AF326827).

Chemical mutagenesis of DEN4. Confluent monolayers of Vero cells were infected with wt DEN4 2A at an multiplicity of infection (MOI) of 0.01 and incubated for 2 hours at 32°C. Infected cells were then overlaid with MEM supplemented with 2% FBS and 5-fluorouracil (5-FU) (Sigma, St. Louis, MO) at concentrations ranging from 10 mM to 10 nM. After incubation at 32°C for five days, cell culture medium was harvested, clarified by centrifugation, and frozen at -70°C. Clarified supernatants were then assayed for virus titer by plaque titration in Vero cells. Serial ten-fold dilutions of the clarified supernatant were prepared in Opti-MEM I (Life Technologies) and inoculated onto confluent Vero cell monolayers in 24-well plates. After incubation at 35°C for two hours, monolayers were overlaid with 0.8% methylcellulose (EM Science, Gibbstown, NJ) in Opti-MEM I supplemented with 2% FBS, gentamicin, and L-glutamine. Following incubation at 35°C for five days, plaques were visualized by immunoperoxidase staining. Vero cell monolayers were fixed in 80% methanol for 30 minutes and washed for 10 minutes with antibody buffer which consists of 3.5% (w/v) nonfat dry milk (Nestle, Solon, OH) in phosphate buffered saline (PBS). Cells were then incubated for one hour at 37°C with an anti-DEN4 rabbit polyclonal antibody preparation (PRNT50 of >1:2000) diluted 1:1,000 in antibody buffer. After one wash with antibody buffer, cells were incubated for one hour with peroxidase-labeled goat-anti-rabbit IgG (KPL, Gaithersburg, MD) diluted 1:500 in antibody buffer. Monolayers were washed with PBS, allowed to dry briefly, overlaid with peroxidase substrate (KPL), and plaques were counted.

Virus yields in cultures treated with 1 mM 5-FU were reduced 100-fold compared to untreated cultures, and the virus present in the supernatant from the 1 mM 5-FU-treated culture was terminally diluted to derive clones for phenotypic characterization. Briefly, 96 well plates of Vero cells were inoculated with the 5-FU-treated virus at an MOI that yielded 10 or fewer virus-positive wells per plate. After a five-day incubation at 35°C, cell culture media from the 96 well plates were temporarily transferred to 96 well plates lacking cells, and the positive cultures were identified by immunoperoxidase staining of the infected-cell monolayers. Virus from each positive well was transferred to confluent Vero cell -14- monolayers in 12 well plates for amplification. Cell culture medium was harvested from individual wells five or six days later, clarified by centrifugation, aliquoted to 96 deep-well polypropylene plates (Beckman, Fullerton, CA) and frozen at -70°C. A total of 1,248 virus clones were prepared from the 1 mM 5-FU-treated cultures. Two wt virus clones, 2A-1 and 5 2A-13, were generated in the same manner from the 5-FU untreated control cultures. 2015203575 26 Jun2015

Screening of clones for ts and att phenotypes. The 1,248 virus clones were screened for ts phenotype by assessing virus replication at 35°C and 39°C in Vero and HuH-7 cells. Cell monolayers in 96 well plates were inoculated with serial ten-fold dilutions of virus in L-15 media (Quality Biologicals, Gaithersburg, MD) supplemented 10 with 2% FBS, L-glutamine and gentamicin. Cells were incubated at the indicated temperatures for five days in temperature-controlled water baths, and presence of virus was determined by immunoperoxidase staining as described above. Vims clones which demonstrated a 100-fold or greater reduction in titer at 39°C were terminally diluted an additional two times and amplified in Vero cells. The efficiency of plaque formation 15 (EOP) at permissive and restrictive temperatures of each triply biologically cloned vims suspension was determined as follows. Plaque titration in Vero and HuH-7 cells was performed as described above except virus-infected monolayers were overlaid with 0.8% methylcellulose in L-15 medium supplemented with 5% FBS, gentamicin, and L-glutamine. After incubation of replicate plates for five days at 35, 37, 38, or 39°C in 20 temperature-controlled water baths, plaques were visualized by immunoperoxidase staining and counted.

The replication of DEN4 5-FU ts mutant viruses was evaluated in Swiss Webster suckling mice (Taconic Farms, Germantown, NY). Groups of six one-week-old mice were inoculated intracranially with 104 PFU of virus diluted in 30 μΐ Opti-MEM I. Five days 25 later, mice were sacrificed and brains were removed and individually homogenized in a 10% suspension of phosphate-buffered Hank’s balanced salt solution containing 7.5% sucrose, 5 mM sodium glutamate, 0.05 mg/ml ciprofloxacin, 0.06 mg/ml clindamycin, and 0.0025 mg/ml amphotericin B. Clarified supernatants were frozen at -70°C and subsequently vims titer was determined by titration in Vero cells, and plaques were stained 30 by the immunoperoxidase method described above.

Sequence analysis of viral genomes. The nucleotide sequence of the 5-FU-mutagenized DEN4 vimses was determined. Briefly, genomic viral RNA was isolated -15- from virus clones with the QIAamp viral RNA mini kit (Qiagen, Valencia, CA) and reverse transcription was performed using the Superscript First Strand Synthesis System for RT-PCR (Life Technologies) and random hexamer primers. Advantage cDNA polymerase (Clontech, Palo Alto, CA) was used to generate overlapping PCR fragments of 5 approximately 2,000 nt which were purified by HighPure PCR Product Purification System (Roche Diagnostics, Indianapolis, IN). DEN-specific primers were used in BigDye terminator cycle sequencing reactions (Applied Biosystems, Foster City, CA) and reactions were analyzed on a 3100 genetic analyzer (Applied Biosystems). Primers were designed to sequence both strands of the PCR product from which consensus sequences were 10 assembled. 2015203575 26 Jun2015

The nucleotide sequence of the 5’ and 3’ regions of the viral genome were determined as above after circularization of the RNA genome. The 5’ cap nucleoside of the viral RNA was excised using tobacco acid pyrophosphatase (Epicentre Technologies, Madison, WI) and the genome was circularized by RNA ligase (Epicentre Technologies). 15 A RT-PCR fragment was generated which overlapped the ligation junction (5’ and 3’ ends) and was sequenced as described above.

Generation of recombinant DEN4 viruses. The mutation at nt position 4,995 in NS3 was introduced into the p4 cDNA construct by site-directed mutagenesis (Kunkel,

T.A. 1985 PNAS USA 82:488-92). The StuI - BstBI (nt 3,619 - 5,072) fragment of p4 was 20 sub-cloned into a modified pUCl 19 vector. The U > C mutation at nt position 4,995 was engineered by site-directed mutagenesis into the p4 fragment, cloned back into the p4 cDNA construct, and the presence of the mutation was confirmed by sequence analysis. The Δ30 mutation was introduced into the 3’ UTR of the p4-4995 cDNA clone by replacing the Mini - Kpnl fragment with that derived from the p4A30 cDNA clone, and the presence 25 of the deletion was confirmed by sequence analysis. Full length RNA transcripts were prepared from the above cDNA clones by in vitro transcription. Briefly, transcription consisted of a 50 μΐ reaction mixture containing 1 pg linearized plasmid, 60 U SP6 polymerase (New England Biolabs (NEB), Beverly, ΜΑ), IX RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol), 0.5 mM 30 m7G(5 ')ppp(5 ')G cap analog (NEB), 1 mM each nucleotide triphosphate, 1 U pyrophosphatase (NEB), and 80 U RNAse inhibitor (Roche, Indianapolis, IN). This -16- reaction mixture was incubated at 40°C for 90 min and the resulting transcripts were purified using RNeasy mini kit (Qiagen, Valencia, CA). 2015203575 26 Jun2015

For transfection of C6/36 cells, RNA transcripts were combined with DOTAP liposomal transfection reagent (Roche) in HEPES-buffered saline (pH 7.6) and added to 5 cell monolayers in 6 well plates. After incubation at 32°C for 12-18 hours, the cell culture media were removed and replaced with MEM supplemented with 5% FBS, L-glutamine, gentamicin and non-essential amino acids. Cell monolayers were incubated for an additional 5 to 7 days and cell culture media were harvested, clarified by centrifugation, and assayed for the presence of virus by plaque titration in Vero cells. Recovered viruses 10 were terminally diluted twice as described above, and virus suspensions for further analysis were prepared in Vero cells.

In vitro (tissue culture) and in vivo replication of wt DEN4 and ΌΕΝ4Δ30. The level of replication of both wt DEN4 2A and the vaccine candidate, 2ΑΔ30, was evaluated in Vero (monkey kidney) and HuH-7 (human hepatoma) cells (Figure 1), the latter of which 15 has recently been found to efficiently support the replication of DEN2 vims (Lin, Y.L. et al. 2000 J Med Virol 60:425-31). The pattern of replication of wt DEN4 2A and 2ΑΔ30 was similar in both cell lines. Viral titers from cultures infected with 2ΑΔ30 at an MOI of 0.01 were slightly reduced compared to wt DEN4 2A at 72 hours, but at later time points their level of replication was equivalent. The efficient replication of both DEN4 viruses in 20 each cell line indicated that these continuous lines of cells would be useful for characterization of the ts phenotype of the 1248 potential mutant viruses.

The level of replication of DEN4 vims administered intracerebrally to Swiss Webster mice was first determined to assess whether mice could be used to efficiently evaluate and quantitate the attenuation phenotype of a large set of mutant viruses. Since the 25 susceptibility of mice to DEN infection is age dependent (Cole, G.A. &amp; Wisseman, C.L.Jr. 1969 Am J Epidemiol 89:669-80; Cole, G.A. et al. 1973 J Comp Pathol 83:243-52), mice aged 7 to 21 days were infected with 2A-13 (a clone of DEN4 wild type vims - see below), rDEN4 or rDEN4A30, and after five days the brain of each mouse was removed, and the level of viral replication was quantitated by plaque assay (Table 1). The results indicated 30 that the two wt DEN4 viruses and the rDEN4A30 vaccine candidate replicated to high titer (>6.0 log10PFU/g brain) in 7-day old mice and that the mean viral titers were similar among the three vimses. These results demonstrated the feasibility of using 7-day old mice to -17- screen a large set of mutant viruses, and the high level of replication of wild type and vaccine candidate permits one to quantitate the magnitude of the restriction of replication specified by an attenuating mutation over a 10,000-fold range.

Generation and in vitro characterization of DEN4 5-FU mutant viruses. A panel of 1,248 DEN4 virus clones was generated from a 5-FU-mutagenized suspension of wt DEN4 2A as described above (Figure 2). Each clone was tested in Vero and HuH-7 cells for the ts phenotype at 39°C, and putative ts mutant viruses were subjected to two additional rounds of biological cloning by terminal dilution, and the ts phenotype of each further cloned virus population was examined in more detail by determining their efficiency of plating (EOP) at permissive temperature (35°C) and at various restrictive temperatures (Table 2). One virus (clone 2A-13) without a ts phenotype, which was passaged in an identical fashion as the ts mutant viruses, served as the virus to which each of the ts mutant viruses was directly compared for both the ts and att phenotypes.

Thirteen 5-FU mutant viruses were identified which have a ts phenotype in both Vero and HuH-7 cells, and seven mutant viruses were ts only in HuH-7 cells (Table 2). Mutant viruses which were ts in Vero cells but not in HuH-7 cells were not identified. Temperature-sensitivity was defined as a >2.5 or >3.5 log10PFU/ml reduction in virus titer in Vero or HuH-7 cells, respectively, at an indicated temperature when compared to the permissive temperature of 35°C. Wild type DEN4 2A was found to have approximately a 0.5 and 1.5 log10PFU/ml reduction in virus titer in Vero or HuH-7 cells at 39°C, respectively. The Δ30 deletion did not confer a ts phenotype in Vero or HuH-7 cells and exhibited only a slight reduction in virus titer (2.2 log10PFU/ml) at 39°C in HuH-7 cells, which was less than 10-fold greater than the reduction of wt DEN4 2 A at that temperature. Several 5-FU mutant viruses had a greater than 10,000-fold reduction in virus titer at 39°C in both· Vero and HuH-7 cells. A complete shut-off in viral replication at 39°C in HuH-7 cells was observed in five virus clones (#571, 605, 631, 967, and 992) which were not ts in Vero cells. Mutations that selectively restrict replication in HuH-7 liver cells may be particularly useful in controlling the replication of dengue virus vaccine candidates in the liver of vaccinees.

Replication of DEN4 5-FU mutant viruses in suckling mice. The level of replication of each of the 20 ts DEN4 mutant viruses in mouse brain was determined (Table 2). The titers obtained were compared to that of the two wt viruses, 2A-13 and rDEN4, -18- which each replicated to a level of greater than 106 PFU/g of brain tissue, and to that of the 2ΑΔ30 mutant, which conferred only a limited 0.5 log10PFU/g reduction in mean virus titer compared to the wt controls. The observed reduction in the level of rDEN4A30 replication was consistent among 11 separate experiments. Interestingly, the rDEN4A30 virus, which was attenuated in both rhesus monkeys and humans (Example 8), was only slightly restricted in replication in mouse brain. Varying levels of restriction of replication were observed among the mutant viruses ranging from a 10-fold (#473) to over 6,000-fold (#686) reduction. Mutant viruses with ts phenotypes in both Vero and HuH-7 cells, as well as in HuH-7 cells alone, were found to have significant att phenotypes. Five of 13 5-FU mutant viruses with ts phenotypes in both Vero and HuH-7 cells and five of seven mutant viruses with ts phenotypes in HuH-7 cells alone had greater than a 100-fold reduction in virus replication. There appeared to be no direct correlation between the magnitude of the reduction in replication at restrictive temperature in tissue culture and the level of attenuation in vivo. The similar level of temperature sensitivity and replication of the rDEN4 wt and clone 2A-13 in mouse brain indicated that observed differences in replication between the ts mutant viruses and clone 2A-13 was not simply a function of passage in Vero cells, but reflects the sequence differences between these viruses.

Sequence analysis of DEN4 5-FU mutant viruses. To determine the genetic basis of the observed ts and att phenotypes, the complete nucleotide sequence of each ts mutant and of clone 2A-13 was determined and summarized in Table 3 (ts in Vero and HuH-7 cells) and Table 4 (ts in only HuH-7 cells).

The only type of mutation identified in the 20 mutant viruses sequenced was a nucleotide substitution (no deletions or insertions occurred), and these were present in each of the coding regions except C and NS4A. Three mutant viruses (#239, 489, and 773) contained only a single missense point mutation in NS3 at nt position 4,995 resulting in a Ser to Pro amino acid (a.a.) change at a.a. position 1,632. For #773, this was the sole mutation present (Table 3). The non-coding mutations in coding regions are not considered to be significant. The 17 additional mutant viruses had multiple mutations (two to five) in a coding region or in an UTR which could potentially confer the observed ts or att phenotypes. Five of the 17 mutant viruses with multiple mutations (#473, 718, 759, 816, and 938) also encoded the point mutation at nt position 4,995. The presence of the 4,995 -19- mutation was found in only DEN4 mutant viruses with ts phenotypes in both Vero and HuH-7 cells.

The sequence analysis indicated that 10 mutant viruses which were ts in Vero and HuH-7 cells and three mutant viruses which were ts in only HuH-7 cells contained mutations in only the 5’ and 3’ UTR and/or in a nonstructural protein. These mutations are especially suitable for inclusion in chimeric dengue virus vaccine candidates in which the structural genes derive from a DENI, DEN2, or DEN3 serotype and the remaining coding and non-coding regions come from an attenuated DEN4 vector. Mutations identified in 5-FU DEN4 mutant viruses which were ts in only HuH-7 cells (Table 4) may potentially be utilized in vaccine candidates, such as rDEN4A30, to selectively control the replication and pathogenesis of DEN4 in the liver. These combined results from the sequence analysis of 5-FU mutant viruses demonstrate the utility of chemical mutagenesis as a means of introducing attenuating mutations into the dengue virus genome.

The presence of a point mutation at nt position 4,995 in eight separate mutant viruses was described above. Five additional point mutations were also represented in multiple viruses including nt changes at position 1,455 in E, 7,162, 7,163 and 7,564 in NS4B, and 10,275 in the 3’ UTR (Table 5). The significance of the occurrence of these “sister” mutations in multiple viruses is discussed in Example 6. Interestingly, the wild-type, parallel-passaged virus, 2A-13, also contained a single mutation at the 7,163 nt position in NS4B.

Introduction of a ts mutation into rDEN4 and rDEN4A30. The presence of a single nucleotide substitution (U > C mutation at nt position 4,995 in NS3) in three separate mutant viruses (clones 239, 489, and 773) indicated that this mutation specified the ts and att phenotypes in each of the three mutant viruses. This mutation was cloned into cDNA construct of p4 and ρ4Δ30 and recombinant viruses were recovered and designated rDEN4-4995 and rDEN4A30-4995, respectively. These recombinant viruses were tested for ts and att phenotypes as described above (Table 6). As expected, introduction of mutation 4995 into rDEN4 wt resulted in a significant ts phenotype at 39°C in both Vero and HuH-7 cells. rDEN4-4995 grew to nearly wild-type levels at the permissive temperature, 35°C, in both cell types, but demonstrated a greater than 10,000-fold reduction at 39°C (shut-off temperature) in both Vero and HuH-7 cells. The addition of the 4995 mutation to -20- rDEN4A30 yields a recombinant virus, rDEN4A30-4995, that exhibits the same level of temperature sensitivity as rDEN4-4995 (Table 6).

The rDEN4 viruses encoding the 4995 mutation were next tested for replication in the brains of suckling mice (Table 6). The 4995 mutation conferred an att phenotype upon both rDEN4 and rDEN4A30. There was an approximately 1,000-fold reduction in virus replication compared to that of wt virus. The combination of point mutation 4995 and the Δ30 deletion did not appear to result in an additive reduction of virus replication. These results confirmed that the 4995 point mutation indeed specifies the ts and att phenotypes. Importantly, the utility of modifying tissue culture and in vivo phenotypes of the rDEN4A30 vaccine candidate by introduction of additional mutations was also demonstrated.

Discussion. Herein we teach how to prepare a tetravalent, five-attenuated dengue virus vaccine using rDEN4A30 as the DEN4 component and three antigenic chimeric viruses expressing the structural proteins (C, prM, and E) of DENI, DEN2, and DEN3 from the attenuated rDEN4A30 vector (Example 8). DEN4 virus rDEN4A30 containing the Δ30 deletion mutation in the 3’ UTR manifests restricted replication in humans while retaining immunogenicity. Since rDEN4A30 retains a low level of residual virulence for humans despite this restricted replication, the present study was initiated to generate additional attenuating mutations that are envisioned as being useful to further attenuate rDEN4A30 or other dengue viruses and that are envisioned as being incorporated into any of the three antigenic chimeric viruses or other dengue viruses as needed. Temperature-sensitive mutants of dengue viruses (Bhamarapravati, N. &amp; Yoksan, S. 1997 in: Dengue and Dengue Hemorrhagic Fever D.J. Gubler &amp; G. Kuno eds. pp. 367-377 CAB International, New York; Eckels, K.H. et al. 1980 Infect Immun 27:175-80) as well of other vimses (Skiadopoulos, M.H. et al. 1998 J Virol 72:1762-8; Whitehead, S.S. et al. 1999 J Virol 73:871-7) manifest restricted replication in vivo. We have generated a panel of 20 ts DEN4 mutant viruses, determined their genomic sequence, and assessed their in vivo attenuation phenotypes. The 20 ts DEN4 mutant vimses were generated by growth in the presence of 5-FU and were first selected for viability in Vero cells, the substrate planned for use in the manufacture of these vaccines, to ensure that the mutant vimses can be grown efficiently in a suitable substrate. -21-

Two classes of mutant viruses were obtained; those ts in both Vero and HuH-7 cells (n = 13) or those ts in only HuH-7 cells (n - 7). The viruses exhibited a range in their level of temperature sensitivity from a 100- to 1,000,000-fold reduction in replication at the restrictive temperature of 39°C. Since our DEN4 vaccine candidate retains a low level of virulence for the liver and other findings support the ability of dengue vimses to infect hepatocytes (Lin, Y.L. et al. 2000 J Med Virol 60:425-31; Marianneau, P. et al. 1997 J Virol 71:3244-9) and cause liver pathology (Couvelard, A. et al. 1999 Hum Pathol 30:1106-10; Huerre, M.R. et al. 2001 Virchows Arch 438:107-15), we sought to develop mutations that would selectively restrict replication of dengue 4 virus in liver cells. Toward this end, we identified seven mutant viruses which have a HuH-7 cell-specific ts phenotype. The mutations present in these viruses are the first reported in DEN viruses that confer restricted replication in liver cells and are envisioned as being useful in limiting virus replication and pathogenesis in the liver of vaccine recipients. The contribution of individual mutations identified in the HuH-7 cell-specific ts viruses to the observed phenotypes is envisioned as being assessed by introduction of the individual mutations into recombinant DEN4 viruses.

Recent evidence has indicated that the magnitude of the viremia in DEN-infected patients positively correlates with disease severity, i.e., the higher the titer of viremia the more severe the disease (Murgue, B. et al. 2000 JMed Virol 60:432-8; Vaughn, D.W. et al. 2000 J Infect Dis 181:2-9). This indicates that mutations that significantly restrict replication of vaccine candidates in vivo are the foundation of a safe and attenuated vaccine. Evaluation of DEN virus vaccine candidates for in vivo attenuation is complicated by the lack of a suitable animal model which accurately mimics the disease caused by dengue viruses in humans. In the absence of such a model, the replication of the panel of 5-FU mutant viruses in the brains of Swiss Webster suckling mice was assessed as a means to identify an in vivo attenuation phenotype since this animal model is well-suited for the evaluation of a large set of mutant viruses. Each of the 20 ts mutant viruses exhibited an att phenotype manifesting a 10- to 6,000-fold reduction in replication in the brain of mice as compared to wt DEN4 virus (Table 2). This indicates that there is a correlation between the presence of the ts phenotype in tissue culture and attenuation of the mutant in vivo confirming the utility of selecting viruses with this marker as vaccine candidates. However, there was no correlation between the level of temperature sensitivity and the -22- level of restriction in vivo. Furthermore, Sabin observed a dissociation between mouse neurovirulence and attenuation in humans by generating an effective live attenuated virus vaccine against DEN by passage of virus in mouse brain. This research actually resulted in a highly mouse-neurotropic DEN virus which, paradoxically, was significantly attenuated in humans (Sabin, A.B. 1952 Am J Trop Med Hyg 1:30-50). Despite this, attenuation for the suckling mouse brain has been reported for other live-attenuated DEN virus vaccine candidates including the DEN2 PDK-53 vaccine strain which is non-lethal in mice and DEN-2 PR-159/S-1 vaccine strain which was significantly attenuated compared to its parental wild-type virus (Bhamarapravati, N. &amp; Yoksan, S. 1997 in: Dengue and Dengue Hemorrhagic Fever DJ. Gubler &amp; G. Kuno eds. pp. 367-377 CAB International, New York; Butrapet, S. et al. 2000 J Virol 74:3011-9; Eckels, K.H. et al. 1980 Infect Immun 27:175-80; Innis, B.L. et al. 1988 J Infect Dis 158:876-80). Replication in rhesus monkeys has been reported to be predictive of attenuation for humans (Innis, B.L. et al. 1988 J Infect Dis 158:876-80). Recently, murine models of DEN virus infection have been developed using SCID mice transplanted with human macrophage (Lin, Y.L. et al. 1998 J Virol 72:9729-37) or liver cell lines (An, J. et al. 1999 Virology 263:70-7), but these mice have not as yet been used to assess att phenotypes of candidate vaccine viruses. Mutant viruses or recombinant viruses bearing one or more of these mutations described herein are envisioned as being tested for replication in rhesus monkeys (or other suitable animal model) as predictive for attenuation in humans.

The chemical mutagenesis of DEN4 virus and sequence analysis of resulting viruses described here has resulted in the identification of a large number of point mutations resulting in amino acid substitutions in all genes except C and NS4A as well as point mutations in the 5’ and 3’ UTR (Tables 3 and 4). This approach of whole-genome mutagenesis has the benefit of identifying mutations dispersed throughout the entire genome which are pre-selected for viability in the Vero cell substrate. Ten 5-FU mutant viruses which were ts in Vero and HuH-7 cells and three viruses which were selectively ts in HuH-7 cells contained only mutations outside of the genes encoding the structural proteins, i.e., in the 5’ and 3’ UTR or NS genes. These mutations along with the Δ30 deletion in the 3’ UTR are particularly suited for inclusion in antigenic, chimeric vaccines which consist of an attenuated DEN4 vector bearing the wild-type structural genes (C, prM, E) of the other DEN virus serotypes. Use of this strategy has several advantages. Each -23- antigenic chimeric virus that possesses structural proteins from a wild-type virus along with attenuating mutations in their UTRs or NS genes should maintain its infectivity for humans, which is mediated largely by the E protein, and, therefore, each vaccine component should be immunogenic (Huang, C.Y. et al. 2000 J Virol 74:3020-8). The replicative machinery of the tetravalent vaccine strains would share the same attenuating mutations in the NS genes or in the UTR which should attenuate each vaccine component to a similar degree and thereby minimize interference or complementation among the four vaccine viruses. In addition, wild-type E protein would be expected to most efficiently induce neutralizing antibodies against each individual DEN virus.

Sequence analysis of dengue viruses (Blok, J. et al. 1992 Virology 187:573-90; Lee, E. et al. 1997 Virology 232:281-90; Puri, B. et al. 1997 J Gen Virol 78:2287-91) and yellow fever viruses (Dunster, L.M. et al. 1999 Virology 261:309-18; Holbrook, M.R. et al. 2000 Virus Res 69:31-9) previously generated by serial passage in tissue culture have mutations throughout much of the genome, a pattern we have observed in the present study. Recent analysis of the DEN2 PDK-53 vaccine strain has identified the important mutations involved in attenuation which were located in non-structural regions including the 5’ UTR, NS1 and NS3 (Butrapet, S. et al. 2000 J Virol 74:3011-9). This DEN2 vaccine strain has been used to generate a chimeric virus with DENI C-prM-E genes (Huang, C.Y. et al. 2000 J Virol 74:3020-8). In separate studies, the sequence of the DENI vaccine strain 45AZ5 PDK-27 was determined and compared to parental viruses, but the mutations responsible for attenuation have not yet been identified (Puri, B. et al. 1997 J Gen Virol 78:2287-91).

Several amino acid substitutions were identified in more than one ts 5-FU mutant virus (Table 5). Lee et al. have previously reported finding repeated mutations in separate DEN3 virus clones after serial passage in Vero cells (Lee, E. et al. 1997 Virology 232:281-90). A mutation (K > N) identified in E at a.a. position 202 in a single DEN3 passage series was also found in our 5-FU mutant virus #1012 (K > E). Mutations observed in the 5-FU sister mutant viruses are envisioned as representing adaptive changes that confer an increased efficiency of DEN4 replication in Vero cells. Such mutations are envisioned as being beneficial for inclusion in a live-attenuated DEN virus vaccine by increasing the yield of vaccine virus during manufacture. Interestingly, three distinct amino acid substitutions were found in NS4B of the 5-FU sister mutant viruses. The exact function of this gene is unknown, but previous studies of live-attenuated yellow fever vaccines -24- (Jennings, A.D. et al. 1994 J Infect Dis 169:512-8; Wang, E. et al. 1995 J Gen Virol 76:2749-55) and Japanese encephalitis vaccines (Ni, H. et al. 1995 J Gen Virol 76:409-13) have identified mutations in NS4B associated with attenuation phenotypes.

The mutation at nt position 4995 of NS3 (S1632P) was present as the only significant mutation identified in three 5-FU mutant viruses (#239, #489, and #773). This mutation was introduced into a recombinant DEN4 virus and found to confer a ts and att phenotype (Table 6). These observations clearly identify the 4995 mutation as an attenuating mutation. Analysis of a sequence alignment (Chang, G.-J. 1997 in: Dengue and Dengue Hemorrhagic Fever DJ. Gubler &amp; G. Kuno, eds. pp. 175-198 CAB International, New York) of the four dengue viruses indicated that the Ser at a.a. position 1632 is conserved in DENI and DEN2, while DEN3 contains an Asn at this position indicating that the mutation is predicted to be useful in modifying the phenotypes of the other DEN virus serotypes. The NS3 protein is 618 a.a. in length and contains both serine protease and helicase activities (Bazan, J.F. &amp; Fletterick, R.J. 1989 Virology 171:637-9; Brinkworth, R.I. et al. 1999 J Gen Virol 80:1167-77; Valle, R.P. &amp; Falgout, B. 1998 J Virol 72:624-32). The 4995 mutation results in a change at a.a. position 158 in NS3 which is located in the N-terminal region containing the protease domain. Amino acid position 158 is located two a.a. residues away from an NS3 conserved region designated homology box four. This domain has been identified in members of the flavivirus family and is believed to be a critical determinant of the NS3 protease substrate specificity (Bazan, J.F. &amp; Fletterick, R.J. 1989 Virology 171:637-9; Brinkworth, R.I. et al. 1999 J Gen Virol 80:1167-77). However, the exact mechanism which results in the phenotype associated with the 4995 mutation has not yet been identified. The identification of the 4995 mutation as an attenuating mutation permits a prediction of its usefulness for the further attenuation of rDEN4A30.

We have determined the contribution of individual 5-FU mutations to the observed phenotypes by introduction of the mutations into recombinant DEN4 viruses as was demonstrated herein for the 4995 mutation (see Example 3). In addition, combination of individual mutations with each other or with the Δ30 mutation is useful to further modify the attenuation phenotype of DEN4 virus candidate vaccines. The introduction of the 4995 mutation into rDEN4A30 described herein rendered the rDEN4A30-4995 double mutant ts and 1000-fold more attenuated for the mouse brain than rDEN4A30. This observation has demonstrated the feasibility of modifying both tissue culture and in vivo phenotypes of this -25- and other dengue virus vaccine candidates. Once the mutations responsible for the HuH-7 cell-specific ts phenotype are identified as described above and introduced into the rDEN4A30 vaccine candidate, we envision confirming that these mutations attenuate rDEN4A30 vaccine virus for the liver of humans. A menu of attenuating mutations is envisioned as being assembled that is predicted to be useful in generating satisfactorily attenuated recombinant dengue vaccine viruses and in increasing our understanding of the pathogenesis of dengue virus (see Example 7).

Example 2

Chemical Mutagenesis of DEN4 Virus Results in Small-Plaque Mutant Viruses with Temperature-Sensitive and Attenuation Phenotypes

Mutations that restrict replication of dengue virus have been sought for the generation of recombinant live-attenuated dengue virus vaccines. Dengue virus type 4 (DEN4) was previously grown in Vero cells in the presence of 5-fluorouracil, and the characterization of 1,248 mutagenized, Vero cell-passaged clones identified 20 temperature-sensitive (ts) mutant viruses that were attenuated (att) in suckling mouse brain (Example 1). The present investigation has extended these studies by identifying an additional 22 DEN4 mutant viruses which have a small-plaque size (sp) phenotype in Vero cells and/or the liver cell line, HuH-7. Five mutant viruses have a sp phenotype in both Vero and HuH-7 cells, three of which are also ts. Seventeen mutant viruses have a sp phenotype in only HuH-7 cells, thirteen of which are also ts. Each of the sp viruses was growth restricted in the suckling mouse brain, exhibiting a wide range of reduction in replication (9- to 100,000-fold). Complete nucleotide sequence was determined for the 22 DEN4 sp mutant viruses, and nucleotide substitutions were found in the 3’ untranslated region (UTR) as well as in all coding regions except NS4A. Identical mutations have been identified in multiple virus clones indicating that they are involved in the adaptation of DEN4 virus to efficient growth in Vero cells.

The DEN viruses cause more disease and death of humans than any other arbovirus, and more than 2.5 billion people live in regions with endemic dengue infection (Gubler, D.J. 1998 Clin Microbiol Rev 11:480-96). Annually, there are an estimated 50-100 million cases of dengue fever (DF) and 500,000 cases of the more severe and potentially lethal dengue hemorrhagic fever / dengue shock syndrome (DHF/DSS) (Gubler, D.J. &amp; Meltzer, M. 1999 Adv Virus Res 53:35-70). Dengue fever is an acute infection characterized by -26- fever, retro-orbital headache, myalgia, and rash. At the time of defervescence during DF, a more severe complication of DEN virus infection, DHF/DSS, may occur which is characterized by a second febrile period, hemorrhagic manifestations, hepatomegaly, thrombocytopenia, and hemoconcentration, which may lead to potentially life-threatening shock (Gubler, DJ. 1998 Clin Microbiol Rev 11:480-96).

The sites of DEN virus replication in humans and their importance and relationship to the pathogenesis of DF and DHF/DSS are still incompletely understood (Innis, B.L. 1995 in: Exotic Viral Infections J.S. Porterfield, ed. pp. 103-146 Chapman and Hall, London). In addition to replication in lymphoid cells, it has become evident that the liver is involved in DEN infection of humans. Transient elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels are observed in the majority of DEN virus-infected patients and hepatomegaly is observed in some patients (Kalayanarooj, S. et al. 1997 J Infect Dis 176:313-21; Kuo, C.H. et al. 1992 Am J Trop Med Hyg 47:265-70; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S.F. et al. 2000 Southeast Asian J Trop Med Public Health 31:259-63). DEN virus antigen-positive hepatocytes are seen surrounding areas of necrosis in the liver of fatal cases (Couvelard, A. et al. 1999 Hum Pathol 30:1106-10; Huerre, M.R. et al. 2001 Virchows Arch 438:107-15), from which dengue virus sequences were identified using RT-PCR (Rosen, L. et al. 1999 Am J Trop Med Hyg 61:720-4). Of potential importance to the etiology of severe dengue virus infection, three studies have demonstrated that the mean levels of serum ALT and AST were significantly increased in patients with DHF/DSS compared to those with DF (Kalayanarooj, S. et al. 1997 J Infect Dis 176:313-21; Mohan, B. et al. 2000 J Trop Pediatr 46:40-3; Wahid, S.F. et al. 2000 Southeast Asian J Trop Med Public Health 31:259-63). As expected, elevation of serum fiver enzymes has previously been observed in clinical trials of DEN virus vaccine candidates (Example 8; Eckels, K.H. et al. 1984 Am J Trop Med Hyg 33:684-9; Edelman, R. etal. 1994 J Infect Dis 170:1448-55; Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188; Vaughn, D.W. et al. 1996 Vaccine 14:329-36).

Based on the increasing disease burden associated with DEN virus infection over the past several decades, a vaccine which confers protection against the four dengue virus serotypes is needed, but none is presently licensed. Because of the increased risk for severe DHF/DSS associated with secondary infection with a heterologous DEN virus serotype (Burke, D.S. et al. 1988 Am J Trop Med Hyg 38:172-80; Halstead, S.B. et al. 1977 J Exp -27-

Med 146:218-29; Thein, S. et al. 1997 Am J Trop Med Hyg 56:566-72), an effective vaccine must confer simultaneous protection against each of the four DEN virus serotypes. Several approaches are presently being pursued to develop a tetravalent vaccine against the dengue viruses (Bancroft, W.H. et al. 1984 J Infect Dis 149:1005-10; Bhamarapravati, N. &amp; Sutee, Y. 2000 Vaccine 18:44-7; Butrapet, S. et al. 2000 J Virol 74:3011-9; Guirakhoo, F. et al. 2000 J Virol 74:5477-85; Huang, C.Y. et al. 2000 J Virol 74:3020-8; Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188). One such approach, a live-attenuated DEN4 vaccine candidate, termed 2ΑΔ30, was both attenuated and immunogenic in a cohort of 20 volunteers (Example 8). The recombinant 2ΑΔ30 virus contains a 30 nt deletion in the 3’ UTR which removes nucleotides 10,478-10,507 and was found to produce a low or undetectable level of viremia in vaccinees at a dose of 105 PFU/vaccinee. An asymptomatic rash was reported in 50% of volunteers, and the only laboratory abnormality observed was an asymptomatic, transient rise in the serum ALT level in 5 of the 20 vaccinees. All 2ΑΔ30 vaccinees developed serum neutralizing antibodies against DEN4 virus (mean titer: 1:580), and 2ΑΔ30 was not transmitted to mosquitoes that fed experimentally on vaccinees (Troyer, J.M. et al. 2001 Am J Trop Med Hyg 65:414-9). Because of the desirable properties conferred by the Δ30 mutation, chimeric vaccine candidates are being constructed which contain the structural genes of DEN virus type 1,2, and 3, in the attenuated DEN4 background bearing the genetically stable Δ30 mutation. Attenuating mutations outside of the structural genes are particularly attractive for inclusion in antigenic chimeric vaccine candidates because they will not affect the infectivity or immunogenicity conferred by the major mediator of humoral immunity to DEN viruses, the envelope (E) protein.

The presence of rash and elevated ALT levels suggests that the 2ΑΔ30 vaccine candidate may be slightly under-attenuated in humans. Similarly, many previous attempts to develop live attenuated dengue virus vaccines have yielded vaccine candidates that were either over- or under-attenuated in humans, some of which also induced elevation of serum ALT and AST levels (Bhamarapravati, N. &amp; Yoksan, S. 1997 in: Dengue and Dengue Hemorrhagic Fever D J. Gubler &amp; G. Kuno eds. pp. 367-377 CAB International, New York; Eckels, K.H. et al. 1984 Am J Trop Med Hyg 33:684-9; Innis, B.L. et al. 1988 J Infect Dis 158:876-80; Kanesa-thasan, N. et al. 2001 Vaccine 19:3179-3188; McKee, K.T., Jr. et al. 1987 Am J Trop Med Hyg 36:435-42). Therefore, we have developed a menu of -28- point mutations conferring temperature-sensitive (ts), small-plaque (sp), and attenuation (att) phenotypes capable of attenuating DEN4 viruses to a varying degree (Example 1). We have previously described 20 mutant viruses that exhibit a ts, but not sp, phenotype in Vero cells or HuH-7 liver cells and that show attenuated replication in mouse brain (Example 1). 2015203575 26 Jun2015 5 Addition of such mutations to 2ΑΔ30 or to other dengue virus vaccine candidates is envisioned as yielding vaccine candidates that exhibit a more satisfactory balance between attenuation and immunogenicity.

In the present Example, we have extended our analysis of the panel of 1,248 DEN4 virus clones previously generated by mutagenesis with 5-fluorouracil (5-FU) (Example 1), 10 by identifying a set of 22 sp mutant viruses, some of which also have a ts phenotype. Small plaque mutant viruses were sought since such viruses are often attenuated in humans (Bhamarapravati, N. &amp; Yoksan, S. 1997 in: Dengue and Dengue Hemorrhagic Fever D J. Gubler &amp; G. Kuno eds. pp. 367-377 CAB International, New York; Butrapet, S. et al. 2000 J Virol 74:3011-9; Crowe, J.E.Jr. etal. 1994 Vaccine 12:783-790; Crowe, J.EJr. etal. 1994 15 Vaccine 12:691-699; Eckels, K.H. et al. 1980 Infect Immun 27:175-80; Innis, B.L. et al. 1988 J Infect Dis 158:876-80; Murphy, B.R. &amp; Chanock, R.M. 2001 in: Fields Virology D.M. Knipe, et al. Eds. Vol. 1, pp. 435-468 Lippincott Williams &amp; Wilkins, Philadelphia; Talcemoto, K.K. 1966 Prog Med Virol 8:314-48). Because natural infection with dengue viruses and vaccination with 2ΑΔ30 may be associated with liver toxicity in humans, we 20 identified mutant viruses with restricted replication in human liver cells. Accordingly, viruses were screened for plaque size and temperature-sensitivity in the human hepatoma cell line, HuH-7, as well as in Vero cells. Here we describe the ts phenotype, nucleotide sequence, and growth properties in suckling mice of 22 sp DEN4 mutant virus clones.

Cells and viruses. WHO Vero cells (African green monkey kidney cells) and 25 HuH-7 cells (human hepatoma cells) (Nakabayashi, H. et al. 1982 Cancer Res 42:3858-63) were maintained as described in Example 1. DEN4 2A virus is a wild type virus derived from a cDNA clone of DEN4 strain 814669 (Dominica, 1981) (Lai, C.J. et al. 1991 PNAS USA 88:5139-43; Mackow, E. et al. 1987 Virology 159:217-28). The nucleotide sequence of DEN4 2A, the parent of the 5-FU mutant viruses, was previously assigned GenBank 30 accession number AF375822 (Example 1). The DEN4 vaccine candidate, 2ΑΔ30, (Example 8) contains a 30 nt deletion in the 3’ untranslated region (UTR) which removes nucleotides 10,478-10,507 (Men, R. et al. 1996 J Virol 70:3930-7). The cDNA clones p4, -29- a modified derivative of the DEN4 2A cDNA clone, and ρ4Δ30 were used to generate recombinant wild type and attenuated viruses, rDEN4 and rDEN4A30, respectively (Example 8). GenBank accession numbers were previously assigned as follows (virus: accession number): DEN4 strain 814669: AF326573; 2ΑΔ30: AF326826; rDEN4: AF326825; rDEN4A30: AF326827.

Generation and biological cloning of mutant viruses with a sp phenotype. The generation of 1,248 vims clones from a pool of 5-fluorouracil-mutagenized DEN4 2A has been previously described (Example 1). Briefly, monolayers of Vero cells were infected with DEN4 2A at a multiplicity of infection (MOI) of 0.01 and overlaid with MEM supplemented with 2% FBS and 1 mM 5-fluorouracil (5-FU) (Sigma, St. Louis, MO), which reduced replication of DEN4 2A 100-fold. Vero cells in 96-well plates were inoculated with the 5-FU treated vims suspension, and vims clones were harvested from plates receiving terminally-diluted vims. A total of 1,248 vims clones were generated from the cultures treated with 1 mM 5-FU. Two vims clones, 2A-1 and 2A-13, were generated in the same manner from control cultures not treated with 5-FU and served as parallel-passaged control vimses with a wild type phenotype.

Evaluation of in vitro plaque size and temperature sensitivity. The 1,248 5-FU-mutagenized vims clones were screened for temperature sensitivity by assessing vims replication at 35°C (permissive temperature) and 39°C (restrictive temperature) in Vero and HuH-7 cells. Cell monolayers in 96-well plates were inoculated with serial ten-fold dilutions of virus and replicate plates were incubated at 35°C and 39°C for five days in temperature-controlled water baths. Vims replication was determined by immunoperoxidase staining as previously described (Example 1). A collection of 193 5-FU vims clones demonstrated a 100-fold or greater reduction in titer at 39°C in either cell line, and these presumptive ts vimses were further characterized. The efficiency of plaque formation (EOP) at permissive and restrictive temperatures and the plaque size of each of the 193 vims clones were determined as follows. Serial ten-fold dilutions of vims suspension were inoculated onto confluent Vero cell and HuH-7 cell monolayers in replicate 24-well plates. After incubation at 35°C for two hours, monolayers were overlaid with 0.8% methylcellulose (EM Science, Gibbstown, NJ) in L-15 medium (Quality Biologicals, Gaithersburg, MD) supplemented with 2% FBS, gentamicin, and L-glutamine. After incubation of replicate plates for five days at 35, 37, 38, or 39°C in temperature- -30- controlled water baths, plaques were visualized by immunoperoxidase staining and counted as previously described. Plaque size of each of the 193 viruses was evaluated at the permissive temperature (35°C) and compared to that of DEN4 2A-13 parallel-passaged control virus with a wild type plaque size. Mutant viruses incubated at the permissive 5 temperature of 35°C which had a plaque size <1 mm or <0.4 mm (approximately <50% the size of wild type DEN4 2A-13) in Vero or HuH-7 cells, respectively, were designated as having a sp phenotype. The level of temperature sensitivity and plaque size of each virus was confirmed in at least two separate experiments. Seventy-five viruses which were confirmed to have a putative ts and/or sp phenotype were biologically cloned an additional 10 two times and phenotypes were re-assessed. Twenty-two of the 75 terminally diluted viruses were found to have a sp phenotype. Sixteen of the 22 sp mutant viruses were also found to have a ts phenotype as defined by a 2.5 or 3.5 log10PFU/ml reduction in virus titer in Vero or HuH-7 cells, respectively, at restrictive temperature compared to the permissive temperature of 35°C as previously described (Example 1). Twenty of the 75 terminally-15 diluted viruses were found to have a ts phenotype without a sp phenotype and were previously described (Example 1). The remainder of the 75 viruses did not meet either criteria for a ts or sp mutant virus. 2015203575 26 Jun2015

Evaluation of sp mutant viruses for restricted replication in suckling mice.

Animal experiments were carried out in accordance with the regulations and guidelines of 20 the National Institutes of Health, Bethesda, MD. Growth of DEN4 5-FU mutant viruses was detennined in Swiss Webster suckling mice (Taconic Farms, Germantown, NY). Groups of six seven-day-old mice were inoculated intracerebrally with 104 PFU of virus in 30 μ1· Opti-MEM I (Invitrogen) and the brain of each mouse was removed five days later and individually analyzed as previously described (Example 1). Clarified supernatants of 25 10% suspensions of mouse brain were frozen at -70°C, and the virus titer was determined by plaque assay in Vero cells.

Determination of the complete genomic sequence of the sp mutant viruses. The nucleotide sequence of the 5-FU-mutagenized DEN4 viruses was determined as described in Example 8. Briefly, genomic RNA was isolated from virus clones and cDNA was 30 prepared by reverse transcription and served as template for the generation of overlapping PCR fragments. A panel of primers was designed to sequence both strands of the PCR product from which consensus sequences were assembled and analyzed. The nucleotide -31- sequence of the 5’ and 3’ regions of the virus genome was determined after circularization of the RNA genome as described in Example 8.

Identification of DEN4 5-fluorouracil mutant viruses with a sp phenotype. The generation of a panel of 1,248 virus clones from a wild type DEN4 2A vims suspension mutagenized by 5-FU has been described previously (Example 1). In the present study twenty-two mutant viruses with a sp phenotype were identified. The plaque size of representative mutant viruses is illustrated in Figure 3. The plaque size of DEN4 2A-13 vims (a parallel-passaged vims with a wild type phenotype derived from control cultures not treated with 5-FU) was consistently smaller in HuH-7 cells than that observed in Vero cells (Figure 3 A). Mutant vimses #569 and #1189 (Figure 3B) were sp in both Vero and HuH-7 cells. In contrast, 5-FU mutant virus clones #311 and #1083 (Figure 3C) were sp in only HuH-7 cells, suggesting a liver cell-specific defect in replication within this phenotypic group. As indicated in Table 7, five mutant vimses were found to have a sp phenotype in both Vero and HuH-7 cells while 17 vimses had a sp phenotype in only HuH-7 cells. Each 5-FU mutant vims clone was compared for a sp or ts phenotype with three control vimses, 2A-13, wild type rDEN4, and rDEN4A30. The recombinant vimses, rDEN4 and rDEN4A30, each had a plaque size in Vero and HuH-7 cells similar to that of DEN4 2A-13 indicating that the Δ30 mutation does not confer a sp phenotype (Table 7).

Most of the sp 5-FU mutant viruses also had a ts phenotype in Vero and/or HuH-7 cells (Table 7) since mutant viruses were initially screened for temperature sensitivity. Temperature-sensitivity was defined as a 2.5 or 3.5 log,0PFU/ml reduction in vims titer in Vero or HuH-7 cells, respectively, at restrictive temperature compared to the permissive temperature of 35°C as previously defined (Example 1). Three mutant vimses (#574, #1269 and #1189) were sp and ts in both Vero and HuH-7 cells, while nine mutant vimses (#506-326 in Table 7) were found to be ts in both cell types but sp only in HuH-7 cells. Four vimses (#1104, 952, 738, and 1083) were found to have a wild type phenotype in Vero cells but were both sp and ts in HuH-7 cells. These four mutant vimses each had a 6,000- to 600,000-fold reduction in vims titer at 39°C in HuH-7 cells with only a 6- to 40-fold reduction at 39°C in Vero cells. Finally, sp mutant vimses were identified which did not have a ts phenotype in either cell line; two of these vimses (#569 and #761) were sp in both Vero and HuH-7 cells and four viruses (#1096-1012) were sp in only HuH-7 cells -32- (Table 7). As described previously, the Δ30 mutation did not confer temperature-sensitivity in either cell line (Example 1).

The sp 5-FU mutant viruses have restricted replication in suckling mouse brain. The 22 sp DEN4 5-FU mutant viruses were evaluated for their ability to replicate in the brain of one-week-old suckling mice. As a marker for in vivo attenuation, their level of replication was compared with that of the parallel-passaged control virus with a wild type phenotype, 2A-13 (Table 7). Nineteen of 22 sp mutant viruses had a greater than 100-fold reduction in virus replication in the brain of suckling mice compared to 2A-13 and nine viruses had a reduction of greater than 10,000-fold.

The five mutant viruses which were sp in both Vero and HuH-7 cells were 5,000-fold to 100,000-fold restricted in replication compared to 2A-13. Two of these mutant viruses, #569 and #761, were not ts in either cell line but had a reduction in virus titer of greater than 10,000-fold in mouse brain, indicating that the sp phenotype in both Vero and HuH-7 cells is an important surrogate marker for attenuated replication in suckling mouse brain. 5-FU mutant viruses which were sp in only HuH-7 cells had a more variable range of replication in mouse brain. Three viruses had a mean reduction in virus titer of less than 10-fold when compared to 2A-13 virus. However, 8 of 13 viruses which were ts in Vero and/or HuH-7 cells but sp in only HuH-7 cells had a greater than 5,000-fold reduction in virus replication. The results of the in vivo replication analysis of the previously described 20 ts 5-FU mutant viruses (Example 1) and the 22 sp mutant viruses are summarized in Table 8. Mutant viruses with both a sp and ts phenotype were found to have a significantly greater level of attenuation in the brain of suckling mice when compared to viruses with only a ts phenotype.

Sequence analysis of the sp 5-FU mutant viruses. To initiate an analysis of the genetic basis of the ts, sp, or att phenotype of the 22 sp mutant viruses, the complete nucleotide sequence of each virus genome was determined and is summarized in Table 9 (sp in Vero and HuH-7 cells) and Table 10 (sp in only HuH-7 cells). All identified mutations were nucleotide substitutions, as deletions or insertions were not observed. Point mutations were distributed throughout the genome, including the 3’ UTR as well as in all coding regions. Because all 5-FU mutant viruses were found to have at least two mutations (two to six), the observed phenotypes cannot be directly attributed to a specific mutation. The majority of sp vimses also contained translationally silent point mutations (none to -33- four) in the structural or non-structural coding regions. However, these silent mutations are not expected to contribute to the observed phenotypes. Six of the 22 sp mutant viruses (Tables 9 and 10) were found to have mutations in only the NS genes and/or the 3’ UTR, indicating that the sp phenotype can be conferred by mutations outside of the structural genes.

Presence of identical mutations in multiple 5-FU mutant viruses. Analysis of the complete nucleotide sequence data for the 5-FU mutant viruses identified several repeated mutations which were present in two or more viruses. Such mutations were also identified previously during our analysis of twenty 5-FU mutant viruses with a ts but not sp phenotype (Example 1). Because these mutations occurred in viruses together with additional mutations, the contribution of the repeated mutations to the observed sp, ts, and att phenotypes remains empirical. Table 11 fists the repeated mutations found among the 20 ts (not sp) mutant viruses described previously (Example 1) and the 22 sp mutant viruses described here. Repeated mutations were identified in the following genes: two in E, two in NS 3, five in NS4B, one in NS 5, and two in the 3’ UTR. Interestingly, within a thirty nucleotide region of NS4B (nt 7153-7182), there were five different nucleotide substitutions which were found in sixteen viruses. Also at nt 7,546 in NS4B, an amino acid substitution (Ala —> Val) was found in 10 different 5-FU mutant viruses. Hie significance of these repeated mutations in NS4B as well as in other DEN4 genomic regions remains empirical, but a reasonable explanation for this phenomenon is that these mutations are involved in adaptation of DEN4 virus for efficient growth in Vero cells, as further discussed in Example 6.

Discussion. As part of a molecular genetic vaccine strategy, we have developed attenuating mutations that are envisioned as being useful in the development of a live attenuated tetravalent dengue virus vaccine. Specifically, mutations which restrict replication of the vaccine virus in human fiver cells were generated since there was some residual virulence of the rDEN4A30 vaccine candidate for the fiver of humans. Mutant vimses with a sp phenotype were sought in both Vero cells and HuH-7 human fiver cells, in order to identify host-range mutant viruses that were specifically restricted in replication in HuH-7 cells (sp in HuH-7 but not in Vero). Such mutations are envisioned as being useful in limiting replication of a candidate vaccine in the fiver of vaccinees while preserving both efficient replication in Vero cells and immunogenicity in vivo. -34-

Several observations from the present study indicate that sp mutations confer an att phenotype in vivo. This is not surprising since attenuation in suckling mouse brain has been reported for live DEN virus vaccine candidates possessing sp phenotypes, including the DEN2 PDK-53 and DEN2 PR-159/S-1 vaccine strains (Bhamarapravati, N. &amp; Yoksan, S. 1997 in: Dengue and Dengue Hemorrhagic Fever D.J. Gubler &amp; G. Kuno eds. pp. 367-377 CAB International, New York; Butrapet, S. et al. 2000 J Virol 74:3011-9; Eckels, K.H. et al. 1980 Infect Immun 27:175-80; Innis, B.L. et al. 1988 J Infect Dis 158:876-80). Each of 22 DEN4 5-FU mutant viruses with a sp phenotype (some of which were also ts) in. either Vero or HuH-7 cells manifested restricted replication in the brains of mice. Six 5-FU mutant viruses with a sp phenotype in the absence of a ts phenotype were more attenuated in the brains of suckling mice than mutant viruses with solely a ts phenotype (Example 1), indicating that the sp phenotype specifies a greater level of attenuation for mouse brain than does the ts phenotype. Mutant viruses with both a ts and sp phenotype had an even greater reduction in replication, further indicating that the attenuation conferred by the ts and sp phenotypes can be additive. Importantly, seventeen of the 22 sp mutant viruses were host-range sp mutant viruses, being sp only in HuH-7 cells. Since such mutations are envisioned as being useful in restricting the replication of a DEN4 virus in human fiver cells, we used nucleotide sequence analysis to determine the genetic basis of the sp phenotype.

Analysis of the complete genomic sequence of the 22 sp DEN4 viruses revealed substitutions in the 3’ UTR as well as coding mutations in all genes except NS4A. It was first noted that several specific mutations were present in two or more of the 22 sp DEN4 mutant viruses and that many of these same mutations were also previously identified among the set of 20 ts DEN4 mutant viruses (Example 1). Since flaviviruses can rapidly accumulate mutations during passage in tissue culture (Dunster, L.M. et al. 1999 Virology 261:309-18; Mandl, C.W. et al. 2001 J Virol 75:5627-37), many of these over-represented mutations, previously referred to as putative Vero cell adaptation mutations (Example 1), likely promote efficient replication in Vero cells and were selected unintentionally during the biological cloning of the mutant viruses. The effect of these mutations on DEN virus replication in Vero cells, the proposed substrate for vaccine manufacture, is discussed in Example 6.

The sp mutations identified among the 5-FU mutant viruses are envisioned as being useful in several different approaches for the development of DEN virus vaccine strains. -35-

As described above for the generation of antigenic chimeric viruses, one or more sp attenuating mutations are envisioned as being added to the attenuated DEN4A30 genetic background to supplement the att phenotype of the Δ30 mutation. A second approach is to introduce a sp attenuating mutation, with or without Δ30, into infectious cDNA clones of the other three DEN serotypes. The ability to transfer mutations among genetically-related viruses and maintain similar att phenotypes has been previously demonstrated (Skiadopoulos, M.H. et al. 1999 Virology 260:125-35). These distinct strategies are envisioned as being useful as separate or complementary approaches to the construction of a tetravalent DEN virus vaccine, underlining the importance of the identification of a large panel of att mutations within the DEN viruses.

Example 3

Recombinant DEN4 Viruses Containing Mutations Identified in 5-FU Mutant Viruses show Restricted Replication in Suckling Mouse Brain and in SCID Mice Transplanted with Human Liver Cells

Data was presented in Examples 1 and 2 that summarizes the generation, characterization and sequence analysis of 42 attenuated mutant DEN4 viruses. For three of the mutant viruses (#239, 489, and 773) with a single missense mutation at nt position 4995 in NS3, it was clear that the identified mutation specified the ts and att phenotypes. This conclusion was confirmed in Example 1 by tissue culture and in vivo characterization of rDEN4-4995, a recombinant virus into which the 4995 mutation had been introduced by site-directed mutagenesis. In this analysis, rDEN4-4995 exhibited the same level of temperature sensitivity and attenuation as 5-FU mutant viruses #239, 489, and 773. The individual mutation(s) in the remaining 5-FU mutant viruses that specify the observed phenotypes remains to be identified, since most of these viruses possess more than one nucleotide substitution. We have conducted an analysis to identify the mutations in a subset of the other 39 mutant viruses that specify the ts, sp, and att phenotypes by introduction of each mutation into the wt DEN4 cDNA (p4) and evaluation of the phenotypes of the resulting recombinant DEN4 viruses bearing the individual mutations. Previous studies of a DEN2 virus vaccine candidate (Butrapet, S. et al. 2000 J Virol 74:3011-9) as well as other virus vaccines (Whitehead, S.S. et al. 1999 J Virol 73:871-7) have demonstrated the utility of this approach for the identification of the genetic basis of attenuation. -36-

As described in Examples 1 and 2, 19 5-FU mutant viruses were identified which were found to contain coding mutations in only the NS genes and/or nucleotide substitutions in the 5’ or 3’ UTR which would facilitate the generation of antigenic chimeric viruses. In the present example, the genetic basis of the observed sp, ts, and 5 mouse brain att phenotypes was identified for these 19 viruses using reverse genetics to generate recombinant DEN4 (rDEN4) viruses containing individual mutations identified in the panel of DEN4 mutant viruses. In addition, the 19 5-FU mutant viruses were evaluated for replication in a novel small animal model for DEN4 virus replication, SCID mice transplanted with HuH-7 cells (SCID-HuH-7), and the genetic basis of the att viruses was 10 identified using mutant rDEN4 viruses. Also presented are findings describing the generation and characterization of a recombinant virus containing two of the identified attenuating mutations as well as combination of select 5-FU mutations with the Δ30 mutation. 2015203575 26 Jun2015

Generation of rDEN4 viruses containing 5-FU mutations. The methods used for 15 the generation of rDEN4 viruses are outlined in Figure 4 and are similar to those described in Example 1. Briefly, the p4 cDNA was digested with the appropriate restriction enzymes and the resulting fragments were subcloned into a modified pUC119 vector. For Kunkel mutagenesis, single-stranded DNA preparations of the pUC-NS vectors were made, and primers were designed to individually introduce mutations that were present in the 5-FU 20 mutant viruses. The sequences of the 41 mutagenic oligonucleotides used to generate the single-mutation recombinant viruses are presented in Table 12. Primers were designed to co-introduce or co-ablate a translationally-silent restriction enzyme site in the cDNA, which greatly facilitates the screening and identification of cDNA clones possessing the mutant sequence. Fragments containing the introduced mutations were cloned back into p4, and 25 nucleotide sequence analysis confirmed the presence of the nucleotide changes. A total of 33 rDEN4 viruses was generated which contained each of the individual mutations present in the 19 5-FU mutant viruses containing only coding mutations in the NS genes and/or nucleotide substitutions in the 5’ or 3’ UTR. An additional 8 rDEN4 viruses were generated from mutations identified in the remaining panel of 42 5-FU mutant viruses. 30 A cDNA clone was also generated which combined the mutations identified at nt position 4995 in NS3 and 7849 in NS5. The 7849 mutation was introduced into the p4-4995 cDNA clone by replacing the Xmal - Pstl fragment with that derived from the p4- -37- 7849 cDNA clone. The presence of both mutations was confirmed by sequence analysis. The Δ30 mutation was introduced into the 3’ UTR of the individual mutant cDNA clones by replacing the Mlul - Kpnl fragment with that derived from the p4A30 cDNA clone, and the presence of the deletion was confirmed by sequence analysis. 2015203575 26 Jun2015 5 Recombinant viruses were recovered by transfection of Vero or C6/36 cells with RNA transcripts derived from the mutant cDNA clones as described in Example 1. Recovered viruses were terminally diluted twice and working stocks of viruses were prepared in Vero cells. Each of the mutant cDNA clones was recovered after transfection as expected since the 5-FU mutant viruses containing these mutations were viable. 10 Characterization of ts and att phenotypes of the rDEN4 viruses containing introduced mutations. Of the 19 5-FU mutant viruses with mutations in only NS genes and/or the 5’ or 3’ UTR, six had an sp phenotype (Table 13), ten had a ts phenotype in Vero and HuH-7 cells (Table 14), and three had a ts phenotype in only HuH-7 cells (Table 15). For the six sp 5-FU mutant viruses, #738, 922, 1081, 1083, 1136, and 1189, seventeen 15 mutations identified by sequence analysis resulted in a coding change or a nucleotide change in the UTR and each was engineered into an individual DEN4 cDNA clone. Vims containing each defined mutation was successfully recovered and propagated and was tested for efficiency of plaque formation in Vero and HuH-7 cells at various temperatures, plaque size phenotype, and growth properties in suckling mice using methods previously 20 described in Examples 1 and 2.

Table 13 lists the phenotypes of the six sp 5-FU mutant parent viruses and those of the 17 rDEN4 viruses encoding single mutations present in the parent vims. For example, 5-FU mutant #1189 (parent), which was ts and sp in both cell lines and had an almost 10,000-fold reduction in replication in suckling mouse brain, contained 4 coding mutations 25 at nt position 3303 in NS1, 4812 and 5097 in NS3, and 7182 in NS4B. Analysis of the four rDEN4 viruses containing each of these mutations indicated that rDEN4-5097 had a ts, sp, and att phenotype while rDEN4-3303, rDEN4-4812, and rDEN4-7182 had no discernible phenotypes, indicating that the mutation at nt 5097 was responsible for the phenotype observed in the 5-FU parent, #1189. Thus, analysis of the relative contributions of the four 30 mutations present in the 5-FU mutant #1189 to its attenuation phenotype provides the framework for a similar analysis of the remaining 5-FU mutant vimses. This analysis specifically demonstrates the methods used to identify mutations contributing to the -38- observed phenotype. The ts, sp, and att phenotypes of 5-FU parent viruses #738, 922, 1081, and 1083, were similarly attributed to single mutations 3540, 4306, 2650, and 10634, respectively. However, two separate mutations (3771 and 4891) contributed to the phenotypes of 5-FU mutant virus #1136. 2015203575 26 Jun2015 5 Table 14 lists the genetic basis of the ts and mouse brain attenuation for the ten 5- FU mutant viruses with ts phenotypes in both Vero and HuH-7 cells. As described in Example 1, the 4995 mutation which is the only mutation present in three 5-FU mutant viruses, #239, #489, and #773, was found to confer a ts and att phenotype, confirming the genetic basis for the phenotypes exhibited by these viruses. In three separate experiments, 10 the rDEN4-4995 virus was found to have an approximately 1,000-fold decrease in replication in the brains of suckling mice when compared to that of wild-type virus (Table 6 and 14). The 4995 mutation is also present in 5-FU mutant viruses #473, #759, and #816, each of which has. additional mutations. The ts and att phenotypes observed in these viruses can be attributed to the 4995 mutation since the additional mutations did not show 15 discernible phenotypes. Interestingly, 5-FU mutant virus #938 has the 4995 mutation and an additional mutation at nt 3442 in NS1 with both mutations independently conferring restricted replication in mouse brain. The remaining three 5-FU parent viruses in Table 14, #173, #509, and #1033, were found to each contain a single mutation responsible for the att phenotype: 7849, 8092, and 4907, respectively. 20 Three 5-FU mutant viruses, #686, #992, and #1175 with HuH-7 cell-specific ts phenotypes are listed in Table 15. Mutations in NS3 (5695) and NS5 (10186) were found to confer the phenotypes observed for parent virus #992 and #1175. Interestingly, two mutations in NS2A, 3575 and 4062, were found to result in a synergistic increase in the level of attenuation. Both individual mutations had an approximately 100-fold decrease in 25 virus replication in the brain while the parent virus with both mutations had an almost 10,000-fold reduction. Table 16 lists two additional mutations with an att phenotype, 4896 and 6259 inNS3.

Replication of DEN4 viruses in SCID mice transplanted with HuH-7 cells.

Since DEN viruses replicate poorly in the liver of mice and corresponding studies are 30 impractical to conduct in non-human primates, an animal model that evaluates the in vivo level of replication of DEN virus in liver cells was developed based on a recent report examining the replication of DEN virus in SCID mice transplanted with a continuous cell -39- line of human liver tumor cells (An, J. et al. 1999 Virology 263:70-7). SCID mice transplanted with human continuous cell lines, primary cells, or organized tissues have similarly been used to study the replication of other viruses which lack a suitable small animal model (Mosier, D.E. 2000 Virology 271:215-9). In our study, SCID mice were 5 transplanted with HuH-7 cells since DEN4 virus replicated efficiently in these cells in tissue culture and since these were the cells used to define the host-range phenotype. These studies are envisioned as addressing the utility of examining DEN virus infection in SCID mouse-xenograft models for vaccine development (An, J. et al. 1999 Virology 263:70-7; Lin, Y.L. et al. 1998 J Virol 72:9729-37). 2015203575 26 Jun2015 10 To further examine the in vivo growth properties of the 19 5-FU mutant DEN4 viruses with mutations in only the NS genes and/or the 3’ UTR and selected corresponding rDEN4 mutant viruses, replication was assessed in SCID mice transplanted with HuH-7 cells (SCID-HuH-7). For analysis of DEN4 virus replication in SCID-HuH-7 mice, four to six week-old SCID mice (Tac:Icr:Ha(ICR)-PrMcs'cz^) (Taconic Farms) were injected 15 intraperitoneally with 107 HuH-7 cells suspended in 200 μΐ phosphate-buffered saline (PBS). In preparation for transplantation, HuH-7 cells were propagated in cell culture as described above and harvested by trypsinization at approximately 80% confluence. Cells were washed twice in PBS, counted, resuspended in an appropriate volume of PBS, and injected into the peritoneum of mice. Tumors were detected in the peritoneum five to six 20 weeks after transplantation, and only mice with apparent tumors were used for inoculation. Mice were infected by direct inoculation into the tumor with 104 PFU of virus in 50 μΐ Opti-MEM I. Mice were monitored daily for seven days and serum for virus titration was obtained by tail-nicking on day 6 and 7. Approximately 400 μΐ blood was collected in a serum separator tube (Sarstedt, Germany), centrifuged, and serum was aliquoted and stored 25 at -70°C. The virus titer was determined by plaque assay in Vero cells. Seven days after infection, most mice developed morbidity and all mice were sacrificed. Tumors were excised and weighed to confirm uniformity of the experimental groups.

Preliminary experiments indicated that SCID-HuH-7 mice inoculated with DEN4 2A-13 directly into the tumor developed viremia with maximum levels (up to 8.0 30 logI0PFU/ml serum) achieved on day 5 (Table 17). Virus could also be detected in brain, liver, and tumor homogenates. -40-

The level of viremia in SCID-HuH-7 mice infected with parental 5-FU or rDEN4 mutant viruses was compared with that of the parallel-passaged control virus, 2A-13, or rDEN4, respectively. Results of 4 separate experiments indicated that the vaccine candidate, rDEN4A30, had an almost 10-fold reduction in virus replication compared to 5 wild type rDEN4 (Table 13) which reflects the apparent attenuation of the rDEN4A30 vaccine candidate in humans (Example 8). Results in Tables 13 to 15 indicate that three 5-FU mutant viruses had a greater than 100-fold reduction in viremia in the SCID-HuH-7 mice compared to wild type 2A-13 virus: #1081, #1083, and #1189. The common phenotype among these viruses was a sp phenotype in HuH-7 cells. Analysis of the genetic 10 basis of the att phenotype in these parent 5-FU mutant viruses identified three individual mutations in NS1, NS3, and the 3’ UTR which conferred at least a 100-fold reduction in viremia. Specifically, rDEN4-2650 (NS1), rDEN4-5097 (NS3), and rDEN4-10634 (3’ UTR) manifested a 2.2, 3.6, and 4.3 log10PFU/ml reduction in peak titer of viremia compared to rDEN4, respectively. These mutations also conferred the att phenotype in 15 suckling mouse brain. 5-FU mutant virus #738 and #509 had a reduction in viremia in the SCID-HuH-7 mice compared to wild type 2A-13 of 1.9 and 1.5 log10PFU/ml, respectively, and the genetic basis for these phenotypes is envisioned as being assessed on an empirical basis. 2015203575 26 Jun2015

This analysis of the genetic basis of the phenotypes specified by the mutations in 20 the 5-FU mutant viruses that manifested restricted replication in SCID-HuH-7 mice indicated that (1) three separate mutations conferred the att phenotype; (2) these mutations were located in two proteins, NS1 and NS3, and in the 3’ UTR; (3) these three mutations were fully responsible for each of the cell culture (ts or sp) and in vivo (attenuation in mouse brain and SCID-HuH-7 mice) phenotypes of the parent viruses; and (4) two of the 25 three mutations specify the host-range sp phenotype (sp on HuH-7 only) and therefore are envisioned as being useful in a vaccine virus. Although the relevance of such SCID-transplant models to virus replication and disease in humans is unknown, the identification of three novel mutations which restrict DEN4 virus replication in SCID-HuH-7 mice is envisioned as facilitating an examination of the correlation between the att phenotype in 30 SCID-HuH-7 mice with that in rhesus monkeys or humans. Such mutations, specifically the host-range sp mutations, are envisioned as being useful in conjunction with the Δ30 or other mutation to decrease the residual virulence of rDEN4A30 or other dengue virus for -41- the human liver, and studies are envisioned as being conducted to construct such rDEN4 viruses and evaluate them in monkeys and humans (Example 8). 2015203575 26 Jun2015

Combination of two 5-FU mutations results in an additive is phenotype. The ability to combine individual mutations in rDEN4 virus as a means to modulate the 5 phenotype of the resulting double mutant virus is a major advantage of using recombinant cDNA technology to generate or modify dengue virus vaccine candidates. Addition of multiple ts and att mutations to recombinant vaccine viruses is envisioned as improving the phenotypic stability of the double recombinant due to the decreased possibility of coreversion of the two mutations to wild-type virulence (Crowe, J.EJr. et al. 1994a Vaccine 10 12:783-790; Skiadopoulos, M.H. et al. 1998 J Virol 72:1762-8; Subbarao, E.K. et al 1995 J Virol 69:5969-5977; Whitehead, S.S. et al. 1999 J Virol 73:871-7). The mutations identified at nt position 4995 in NS3 and 7849 in NS5 were combined in a single p4 cDNA clone and a recombinant virus, designated rDEN4-4995-7849, was recovered and evaluated for its ts and att phenotypes (Table 18). rDEN4-4995-7849 was more ts than either 15 recombinant virus containing the individual mutations (Table 18), indicating the additive effect of the two ts mutations. The rDEN4-4995-7849 virus had a greater than 10,000-fold reduction in replication in the brains of suckling mice. The reduction in replication of the double mutant virus was only slightly increased over that of rDEN4-7849, however, a difference in the level of replication between rDEN4-4995-7849 and rDEN4-7849 would be 20 difficult to detect since the level of replication of both viruses was close to the lower limit of detection (2.0 log10PFU/g brain).

Combination of selected 5-FU mutations with the Δ30 mutation confers increased attenuation of rDEN4A30 for the brains of suckling mice. To define the effect of adding individual mutations to the attenuated rDEN4A30 background, five 25 combinations have been constructed: rDEN4A30-2650, rDEN4A30-4995, rDEN4A30-5097, rDEN4A30-8092, and rDEN4A30-10634. Addition of such missense mutations with various ts, sp, and att phenotypes is envisioned as serving to decrease the reactogenicity of rDEN4A30 while maintaining sufficient immunogenicity.

The Δ30 mutation was introduced into the 3’ UTR of the individual mutant cDNA 30 clones by replacing the Mlul - Kpnl fragment with that derived from the ρ4Δ30 cDNA clone, and the presence of the deletion was confirmed by sequence analysis. Recombinant viruses were recovered by transfection in C6/36 cells for each rDEN4 virus. However, -42- upon terminal dilution and passage, the rDEN4A30-5097 virus was found to not grow to a sufficient titer in Vero cells and was not pursued further. This is an example of a cDNA in which the 5-FU mutation and the Δ30 mutation are not compatible for efficient replication in cell culture. To begin the process of evaluating the in vivo phenotypes of the other four 5 viruses which replicated efficiently in cell culture, rDEN4 viruses containing the individual mutations and the corresponding rDEN4A30 combinations were tested together for levels of replication in suckling mouse brain. The results in Table 19 indicate that addition of , each of the mutations confers an increased level of attenuation in growth upon the rDEN4A30 virus, similar to the level conferred by the individual 5-FU mutation. No 10 synergistic effect in attenuation was observed between the missense mutations and Δ30. These results indicate that the missense mutations at nucleotides 2650, 4995, 8092, and 10634 are compatible with Δ30 for growth in cell culture and in vivo and can further attenuate the rDEN4A30 virus in mouse brain. Further studies in SCID-HuH-7 mice, rhesus monkeys, and humans are envisioned as establishing the effect of the combination of 15 individual mutations and Δ30 upon attenuation and immunogenicity (Example 8). 2015203575 26 Jun2015

By identifying the specific mutations in the 5-FU mutant viruses which confer the observed phenotypes, a menu of defined is, sp, and att mutations is envisioned as being assembled (see Example 7). Numerous combinations of two or more of these mutations are envisioned as being selected with or without the Δ30 mutation. Such mutations and their 20 combinations are envisioned as being useful for the construction of recombinant viruses with various levels of in vivo attenuation, thus facilitating the generation of candidate vaccines with acceptable levels of attenuation, immunogenicity, and genetic stability.

Example 4

Generation of DEN4 Mutant Viruses with Temperature-Sensitive and Mouse 25 Attenuation Phenotypes Through Charge-Cluster-to-Alanine Mutagenesis

The previous Examples described the creation of a panel of DEN4 mutant viruses with ts, sp, and att phenotypes obtained through 5-FU mutagenesis. As indicated in these Examples, the attenuating mutations identified in the 5-FU mutant viruses are envisioned as having several uses including (1) fine tuning the level of attenuation of existing dengue 30 virus vaccine candidates and (2) generation of new vaccine candidates by combination of two or more of these attenuating mutations. In the current example, we created a second panel of mutant viruses through charge-cluster-to-alanine mutagenesis of the NS5 gene of -43- DEN4 and examined the resulting mutant viruses for the ts, sp, and att phenotypes as described in Examples 1 and 2. The charge-cluster-to-alanine mutant viruses recovered demonstrated a range of phenotypes including ts in Vero cells alone, ts in HuH-7 cells alone, ts in both cell types, att in suckling mouse brains, and att in SCID-HuH-7 mice. 2015203575 26 Jun2015 5 The usefulness of mutant viruses expressing these phenotypes has already been described, however charge-cluster-to-alanine mutant viruses possess some additional desirable characteristics. First, the relevant mutations are envisioned as being designed for use in the genes encoding the non-structural proteins of DEN4, and therefore are envisioned as being useful to attenuate DENI, DEN2, and DEN3 antigenic chimeric recombinants 10 possessing a DEN4 vector background. Second, the phenotype is usually specified by three or more nucleotide changes, rendering the likelihood of reversion of the mutant sequence to that of the wild type sequence less than for a single point mutation, such as mutations identified in the panel of 5-FU mutant viruses. Finally, charge-cluster-to-alanine attenuating mutations are envisioned as being easily combinable among themselves or with 15 other attenuating mutations to modify the attenuation phenotype of DEN4 vaccine candidates or of DENI, DEN2, and DEN3 antigenic chimeric recombinant viruses possessing a DEN4 vector background.

Charge-Cluster-to-Alanine-Mutagenesis. The cDNA p4, from which recombinant wild type and mutant viruses were generated, has been described in Examples 20 1,2, and 3 and in Figure 4. Charge-cluster-to-alanine mutagenesis (Muylaert, I.R. et al. 1997 J Virol 71:291-8), in which pairs of charged amino acids are replaced with alanine residues, was used to individually mutagenize the coding sequence for 80 pairs of contiguous charged amino acids in the DEN4 NS5 gene. Subclones suitable for mutagenesis were derived from the full length DEN4 plasmid (p4) by digestion with 25 XmaUPstl (pNS5A), PstVSacU (pNS5B) or SaclVMlul (pNS5C) at the nucleotide positions indicated in Figure 4. These fragments were then subcloned and Kunkel mutagenesis was conducted as described in Examples 1 and 3. To create each mutation, oligonucleotides were designed to change the sequence of individual pairs of codons to GCAGCX (SEQ ID NO: 69), thereby replacing them with two alanine codons (GCX) and also creating a BbvI 30 restriction site (GCAGC) (SEQ ID NO: 70). The Bbvl site was added to facilitate screening of cDNAs and recombinant viruses for the presence of the mutant sequence. Restriction -44- enzyme fragments bearing the alanine mutations were cloned back into the full-length p4 plasmid as described in Examples 1 and 3. 2015203575 26 Jun2015

Initial evaluation of the phenotype of the 32 charge-cluster-to-alanine mutant viruses revealed a range in restriction of replication in suckling mouse brain and SCID-5 HuH-7 mice. To determine whether attenuation could be enhanced by combining mutations, double mutant viruses carrying two pairs of charge-cluster-to-alanine mutations were created by swapping appropriate fragments carrying one pair of mutations into a previously-mutagenized p4 cDNA carrying a second pair of mutations in a different fragment using conventional cloning techniques. 10 Transcription and Transfection. 5’-capped transcripts were synthesized in vitro from mutagenized cDNA templates using AmpliCap SP6 RNA polymerase (Epicentre, Madison, WI). Transfection mixtures, consisting of 1 pg of transcript in 60 μΐ of HEPES/saline plus 12 μΐ of dioleoyl trimethylammonium propane (DOTAP) (Roche Diagnostics Corp., Indianapolis, IN), were added, along with 1 ml Virus production-serum 15 free medium (VP-SFM) to subconfluent monolayers of Vero cells in 6-well plates. Transfected monolayers were incubated at 35°C for approximately 18 hr, cell culture medium was removed and replaced with 2 ml VP-SFM, and cell monolayers were incubated at 35 °C. After 5 to 6 days, cell culture medium was collected, and the presence of virus was determined by titration in Vero cells followed by immunoperoxidase staining 20 as previously described. Recovered virus was amplified by an additional passage in Vero cells, and virus suspensions were combined with SPG (sucrose—phosphate—glutamate) stabilizer (final concentration: 218 mM sucrose, 6 mM L-glutamic acid, 3.8 mM potassium phosphate, monobasic, and 7.2 mM potassium phosphate, dibasic, pH 7.2), aliquoted, frozen on dry ice, and stored at -70°C. 25 cDNA constructs not yielding virus after transfection of Vero cells were used to transfect C6/36 cells as follows. Transfection mixtures, as described above, were added, along with 1 ml of MEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 2 mM non-essential amino acids, and 0.05 mg/ml gentamicin, to monolayers of C6/36 cells. Transfected cell monolayers were incubated at 32°C for 18 hr, cell culture medium was 30 removed and replaced with 2 ml fresh medium, and cell monolayers were incubated at 32°C. After 5 to 6 days, cell culture media were then used to infect Vero cells and -45- incubated for 5-6 days, at which time cell culture media were collected, frozen and titered as described above. 2015203575 26 Jun2015

Recovered viruses were biologically cloned by two rounds of terminal dilution in Vero cells followed by an additional amplification in Vero cells. Briefly, virus was initially 5 diluted to a concentration of approximately 20 PFU/ml in VP-SFM and then subjected to a series of two-fold dilutions across a 96-well plate. Vims dilutions were used to infect Vero cell monolayers in a 96-well plate and incubated for 5 to 6 days at 35°C. Following incubation, cell culture media were removed and temporarily stored at 4°C, and the viruspositive cell monolayers were identified by immunoperoxidase staining. Terminal dilution 10 was achieved when < 25% of cell monolayers were positive for vims. Cell culture medium from a positive monolayer at the terminal dilution was subjected to an additional round of terminal dilution. Following the second terminal dilution, vims was amplified in Vero cells (75 cm2 flask), collected and frozen as previously described.

Assays for temperature-sensitivity and mouse attenuation. Assay of the level of 15 temperature sensitivity of the charge-cluster-to-alanine mutant viruses in Vero and HuH-7 cells and their level of replication hi the brain of suckling mice were conducted as described in Example 1 and assay of the level of replication in SCID-HuH-7 mice was conducted as described in Example 3.

Charge-cluster-to-alanine mutant viruses are viable and show temperature- 20 sensitive and mouse attenuation phenotypes. Of 80 full-length DEN4 cDNA constructs containing a single pair of charge-to-alanine mutations, vims was recovered from 32 in either Vero or C6/36 cells (Figure 5). The level of temperature sensitivity of wt rDEN4, rDEN4A30, and the 32 mutant viruses is summarized in Table 20. One mutant vims (645-646) was ts in Vero but not HuH-7 cells and 7 mutant viruses were ts in HuH-7 but not 25 Vero cells. Such mutants whose temperature sensitivity is host-cell dependent are referred to as temperature-sensitive, host-range (tshr) mutants. Thirteen mutant vimses were ts in both cell types, and 11 mutant vimses were not ts on either cell type. Thus a total of 21 mutant vimses were ts with 8 mutant vimses exhibiting an tshr specificity. None of the mutant vimses showed a small plaque phenotype at permissive temperature. Mutant 30 vimses showed a wide range (0 to 10,000-fold) of restricted replication in suckling mouse brain (Table 20). Fourteen mutant vimses were attenuated in suckling mouse brain, arbitrarily defined as a > 1.5 log10-unit reduction in vims titer. There was no correlation -46- between attenuation in mouse brain and temperature sensitivity in either Vero cells (Kendall Rank correlation: P = 0.77) or HuH-7 cells (Kendall Rank correlation: P = 0.06).

Thirteen mutant viruses that either showed an att phenotype in suckling mouse brain or whose unmutated charged amino acid pair was highly conserved among the four DEN serotypes (see Example 7) were assayed for att in SCID-HuH-7 mice (Table 21). Three of these mutant viruses showed >100-fold decrease in replication relative to wild type DEN4. Overall, mean log reduction from wild type in suckling mice did not show significant correlation with mean log reduction in SCID-HuH-7 mice (Spearman rank correlation, N = 13, P = 0.06). However, mutant virus 200-201 was unusual in that it showed a high level of restriction in SCID-HuH-7 mice but little restriction in suckling mouse brain. When virus 200-201 was removed from the analysis, restriction of replication in suckling and SCID-HuH-7 mice showed a significant correlation (Spearman rank correlation, N = 12, P = 0.02).

Combining charge-cluster-to-alanine mutations present in two viruses into one virus can enhance its ts and att phenotypes. Six paired mutations were combined into fourteen double-pair mutant viruses, ofwhich six could be recovered in Vero or C6/36 cells (Table 22). All of the individual paired mutations used in double-pair mutant viruses were ts on HuH-7 cells, none was ts in Vero cells, and for all combinations at least one mutation pair conferred an att phenotype in suckling mouse brain. Evaluation of four of the doublepair mutant viruses (Table 23) revealed that combining charge-cluster-to-alanine mutation pairs invariably resulted in the acquisition of a ts phenotype in Vero cells (4 out of 4 viruses) and often resulted in a lowered shutoff temperature in HuH-7 cells (3 out of 4 viruses). In half of the viruses assayed, combination of charge-cluster-to-alanine mutation pairs resulted in enhanced restriction of replication (10-fold greater than either component mutation) in suckling mouse brain (Table 23) and in SCID-HuH-7 mice (Table 24).

Summary. The major usefulness of the charge-cluster-to-alanine mutations stems from their design: they are located in the DEN4 non-structural gene region and therefore are envisioned as being useful to attenuate DEN4 itself as well as antigenic chimeric viruses possessing the DEN4 NS gene region. Furthermore, they are predicted to be phenotypically more stable than the single-nucleotide substitution mutant viruses such as the 5-FU mutant viruses. Finally, combinations of mutations are envisioned as being created in order to fine-tune attenuation and to further stabilize attenuation phenotypes. -47-

Example 5

Identification and Characterization of DEN4 Mutant Viruses Restricted in

Replication in Mosquitoes SECTION 1. Identification of viruses showing restriction of replication in mosquitoes.

In Examples 1 and 4, DEN4 mutant viruses were generated through 5-FU mutagenesis and charge-cluster-to-alanine mutagenesis, respectively, in order to identify mutations that confer ts, sp and att phenotypes. Another highly desirable phenotype of a dengue vims vaccine is restricted growth in the mosquito host. A dengue vims vaccine candidate should not be transmissible from humans to mosquitoes in order to prevent both the introduction of a dengue vims into an environment in which it is currently not endemic and to prevent the possible loss of the attenuation phenotype during prolonged replication in an individual mosquito host. Loss of the attenuation phenotype could also occur following sustained transmission between humans and mosquitoes. Recently, loss of attenuation of a live attenuated poliovirus vaccine was seen following sustained transmission among humans (CDC 2000 MMWR 49:1094).

In the present example, a panel of 1248 DEN4 mutant vimses generated through 5-FU mutagenesis and 32 DEN4 mutant vimses generated through charge-cluster-to-alanine mutagenesis were assayed for restricted growth in mosquito cells. This is a useful preliminary assay for restriction in vivo, since restriction in cultured mosquito cells is often, though not always, associated with poor infectivity for mosquitoes (Huang, C.Y. et al. 2000 J Virol 74:3020-8). Mutant vimses that showed restriction in mosquito cells and robust growth in Vero cells (the substrate for vaccine development, as discussed in Example 6) were targeted for further characterization.

Generation and characterization of the 5-1A1 mutant. The generation and isolation of the panel of 1248 5-FU mutant vimses and the panel of 32 charge-cluster-to-alanine mutant vimses have been described in Examples 1, 2, and 4. Vero and C6/36 cells were maintained as described in Example 1.

Each of the 1248 5-FU mutant vimses and 32 charge-cluster-to-alanine mutant vimses was titered in C6/36 cell monolayers in 24-well plates at 32°C and 5% C02. After 5 days, plaques were immunostained with anti-DEN4 rabbit polyclonal antibody and counted as described in the preceding Examples. Mutant vimses were assayed for one of two phenotypes indicating restricted growth in mosquito cells: either sp in C6/36 cells,relative -48- to Vero cells or a > 3.5 log10PFU/ml decrease in titer between Vero and C6/36 cells at the permissive temperature for each cell type. Two mutant viruses, one generated by 5-FU mutagenesis (#5) and one generated by charge-cluster-to-alanine mutagenesis (rDEN4-356,357), showed reduced plaque size in C6/36 cells. After three terminal dilutions, the 5-FU mutant #5, designated 5-1 Al, maintained the reduced plaque size phenotype. Additionally, recombinant virus rDEN4-7546, tested for Vero cell adaptation (discussed in detail in Example 6) also showed reduced plaque size in C6/36 (Figure 10).

The multicycle growth kinetics of both 5-1A1 and the recombinant wild type rDEN4 in C6/36 cells were determined as described in Example 1. Briefly, cells were infected in triplicate at a multiplicity of infection of 0.01 and samples were harvested at 24-hr intervals. Samples were flash frozen and titered in a single assay in Vero cell monolayers.

Oral infection of mosquitoes. Aedes aegypti is one of the primary vectors of dengue virus (Gubler, D.J. 1998 Clin Microbiol Rev 11:480-96). This species was reared at 26°C and 80% relative humidity (RH) with a 16 hr daylight cycle. Adults were allowed continuous access to a cotton pad soaked in a 10% sucrose solution. Five to ten day old female Ae. aegypti which had been deprived of a sugar source for 48 hr were fed a bloodmeal consisting of equal volumes of washed human red blood cells, 10% sucrose solution, and dengue virus suspension. The infected blood meal was prepared immediately prior to feeding and offered to mosquitoes in a water-jacketed feeder covered in stretched parafilm and preheated to 38°C (Rutledge, L.C. et al. 1964 Mosquito News 24:407-419). Mosquitoes that took a full bloodmeal within 45 min were transferred to a new container by aspirator and maintained as described above. After 21 days, mosquitoes were stored at -20°C until dissection.

Intrathoracic inoculation of mosquitoes. The large, non-haematophagous mosquito Toxorhynchites splendens is a sensitive host for determining the infectivity of dengue virus. This species was reared at 24°C and 75% RH with a 12 hr daylight cycle. Larvae and pupae were fed on appropriately sized Aedes larvae; adults were allowed continuous access to a cotton pad soaked in a 10% sucrose solution. Groups of one to ten day old adult T. splendens of both sexes were immobilized by immersion of their container in an icewater bath and inoculated intrathoracically with undiluted virus and serial tenfold dilutions of virus in IX PBS. Virus was inoculated in a 0.22 μΐ dose using a Harvard -49-

Apparatus microinjector (Medical Systems Corp, Greenvale NY) and a calibrated glass needle (technique is a modification of the method described in Rosen and Gubler, 1974). 2015203575 26 Jun2015

Detection of viral antigen in body and head tissues by immunofluorescence assay (IFA). Head and midgut preparations of Aedes aegypti and head preparations of 5 Toxorhynchites splendens were made on glass slides as described in Sumanochitrapon et al. (Sumanochitrapon, W. et al. 1998 Am J Trop Med Hyg 58:283-6). Slides were fixed in acetone for 20 min, and placed at 4°C until processed by IFA. The primary antibody, hyperimmune mouse ascites fluid specific for DEN-4 (HMAF), was diluted 1/100 in PBS-Tween 20 (0.05%). Slides were incubated at 37°C in a humid chamber for 30 min, and 10 subsequently rinsed in PBS-Tween 20. The secondary antibody, FITC conjugated goat anti-mouse IgG (KPL, Gaithersburg, MD), was diluted 1/200 in PBS-Tween 20 with 0.002% Evan’s Blue. Slides were viewed on an Olympus BX60 microscope. The infectious dose required to infect 50% of mosquitoes (Π)50) was determined by the method of Reed and Muench (Reed, LJ. &amp; Muench, H. 1938 Am J Hyg 27:493-497). For Aedes 15 aegypti infections, two OIDS0 (oral infectious dose 50) values were calculated for each virus: the OID50 required to produce an infection in the midgut, with or without dissemination to the head, and the OID50 required to produce disseminated infection. For Tx. splendens one MID50 (mosquito infectious dose 50) value was calculated.

Statistical Analysis. The percentage of mosquitoes infected by different viruses 20 were compared using logistic regression analysis (Statview, Abacus Inc.).

Mutations restricting growth of DEN4 in mosquito cells but not Vero cells are rare. Out of 1280 mutant viruses initially assayed, only two, #5 and rDEN4-356,357, showed reduced plaque size in C6/36 cells and normal plaque size in Vero cells. One additional virus, rDEN4-7546 (described in Example 6), with reduced plaque size in C6/36 25 was detected in subsequent assays. Mutant virus #5 was cloned by three successive terminal dilutions and designated 5-1 Al; rDEN4-7546 and rDEN4-356,357 had already been twice-terminally diluted when they were tested in C6/36 cells. Virus 5-1A1 has been extensively characterized and its phenotypes are described in detail in the following section. rDEN4-356,357 and rDEN4-7546 are envisioned as being characterized in a 30 similar fashion.

Plaque size and growth kinetics of 5-1A1. 5-1A1 replicated to 6.7 log10PFU/ml in Vero cells with normal plaque size and replicated to 7.6 log10PFU/ml in C6/36 cells with -50- small plaque size (Figure 6, Table 25). In comparison, wild type DEN4 used as a concurrent control replicated to 7.3 log10PFU/ml in Vero cells, 8.3 log10PFU/ml in C6/36 cells, and showed normal plaque size in both cell types (Figure 6, Table 25). The growth kinetics of 5-1A1 was compared to that of wild type DEN4 by infecting C6/36 cells at an MOI of 0.01 and monitoring the production of infectious virus. The kinetics and magnitude of replication of 5-1A1 in C6/36 cells was comparable to that of wild type DEN4 (Figure 7). 5-1A1 is restricted in its ability to infect mosquitoes. 5-1A1 was evaluated for its ability to infect Aedes aegypti mosquitoes through an artificial bloodmeal (Table 26). In this assay the ability to infect the midgut of the mosquito and the ability for a midgut infection to disseminate to the head are measured separately. The oral infectious dose 50 (OID50) of wild type DEN4 for the midgut was 3.3 log10 PFU; the OID50 of wild type DEN4 for a disseminated infection was 3.9 log10 PFU. In contrast, 5-1 Al never infected 50% of mosquitoes at the doses used. In order to calculate the OID50 for midgut infections by 5-1A1, it was assumed that at a 10-fold higher dose, 100% of 25 mosquitoes would have become infected. Using this assumption, the conservative estimate of the OID50 for midgut infections by 5-1 Al was >3.9 log10PFU. Because 5-1 Al produced only 3 disseminated infections, we did not attempt to calculate an OID50 for this category. 5-1A1 was significantly restricted in its ability to infect the midgut relative to wild type DEN4 (logistic regression, N = 150, P < 0.001). Additionally, 5-1A1 produced very few disseminated infections, but because of low numbers this result was not amenable to statistical analysis. 5-1A1 was also significantly restricted in its ability to infect Tx. splendens mosquitoes following intrathoracic inoculation (Table 27). The MID50 of wild type DEN4 was 2.3 log10 PFU whereas the MID50 of 5-1A1 was estimated to be > 3.0 log10 PFU (logistic regression, N = 36, P < 0.01). 5-1A1 does not show a ts or an att phenotype. 5-1A1 was tested for temperature sensitivity in Vero and HuH-7 cells and for attenuation in suckling mouse brains as described in Example 1. The mutant virus was not temperature sensitive, as defined in Example 1, and was not attenuated in suckling mouse brain (Table 25).

Identification and confirmation of the mutation responsible for the phenotype of 5-1A1. The nucleotide sequence of the entire genome of 5-1A1 was determined as described in Example 1. Sequencing of 5-1 Al revealed three changes from the wild type -51- sequence: two translationally-silent point mutations at positions 7359 and 9047, and one coding point mutation (C to U) at position 7129 in the NS4B gene which resulted in a proline to leucine substitution. 2015203575 26 Jun2015

To formally confirm the effect of the C7129U mutation, the mutation was inserted 5 into the cDNA p4, which has been described in Examples 1, 2, and 3 and in Figure 4, using Kunkel mutagenesis as described in Examples 1 and 3. The mutagenized cDNA was transcribed and transfected as described in Example 3, and the resulting virus, after two terminal dilutions, was designated rDEN4-7l29-lA. Like 5-1A1, rDEN4-7129-lA showed normal plaque size and titer in Vero cells and reduced plaque size and normal titer in C6/36 10 cells (Table 25). rDEN4-7129-l A was not ts on either Vero or HuH-7 cells and was not att in suckling mouse brain. Additionally, rDEN4-7129-lA did not show the SCID-HuH-7 att phenotype described in Example 3 (Table 25). The ability of rDEN4-7129-lA to infect mosquitoes is envisioned as being tested in both Ae. aegypti and Tx. splendens.

To test the compatibility of the C7129U mutation and the Δ30 deletion, the C7129U 15 mutation was inserted into rDEN4A30 using previously described techniques. The resulting virus, designated rDEN4A30-7129, is envisioned as being tested for the phenotypes listed in Table 25.

In summary, three mutant viruses, 5-1A1, rDEN4-356,357 and rDEN4-7546, showed a particular combination of phenotypes characterized by normal plaque size and 20 replication to high titers in Vero cells and small plaque size but unrestricted growth in mosquito cells. 5-1A1 was further characterized and lacked temperature sensitivity in either Vero or HuH-7 cells and showed normal levels of replication in mouse brain and in SCID-HuH-7 mice and restricted infectivity for both Ae. aegypti and Tx. splendens mosquitoes. In comparison to wild type rDEN4, the 5-1A1 mutant had one coding 25 mutation: a point mutation (C to U) at nucleotide 7129 in NS4B resulting in a replacement of Pro with Leu. Because 5-1A1 contains only a single missense mutation, the phenotype of this mutant virus can be attributed to the effect of the mutation at position 7129. These results indicate that the 7129 mutation is responsible for the phenotype of decreased infectivity for mosquitoes and is predicted to be useful to restrict replication of vaccine 30 candidates in mosquitoes. To formally confirm this, we have inserted the 7129 mutation into a recombinant DEN4 virus. The resulting virus, designated rDEN4-7129-lA, shows -52- an absence of ts and att phenotypes similar to 5-1 Al. It is envisioned as being tested for mosquito infectivity. 2015203575 26 Jun2015

The 7129 mutation is a valuable point mutation to include in a DEN4 vaccine candidate and into each of the dengue virus antigenic chimeric vaccine candidates since its 5 biological activity is host specific, i.e., it is restricted in replication in mosquitoes but not in mammals. Moreover, as discussed in Example 6, the 7129 mutation has also been shown to enhance replication in Vero cells. Thus, its insertion into a vaccine candidate is envisioned as enhancing vaccine production in tissue culture without affecting the biological properties specified by other attenuating mutations. It is also envisioned as 10 providing a useful safeguard against mosquito transmission of a dengue virus vaccine. SECTION II. Design of mutations to restrict replication in mosquitoes

In Section 1 of Example 5, we screened a large panel of mutant viruses carrying both random mutations (generated with 5-fluorouracil) and specific mutations (generated through charge-cluster-to-alanine mutagenesis) for restricted growth in C6/36 cells, a proxy 15 measure for restriction in mosquitoes. However, in neither case were mutations designed for the specific purpose of restricting replication in mosquitoes. In this section, we identified nucleotide sequences in the 3’ UTR that show conserved differences between the mosquito-transmitted and tick-transmitted flaviviruses. We then altered those sequences in the DEN4 cDNA p4 by either deleting them altogether or exchanging them with the 20 homologous sequence of the tick-transmitted Langat vims. The resulting viruses were assayed for reduced plaque size and titer in both Vero and C6/36 cells and for infectivity for Ae. aegypti and Tx. splendens.

Identification and modification of particular 3’ UTR sequences showing conserved differences between vectors. Several studies (Olsthoom, R.C. &amp; Bol, J.F. 25 2001 RNA 7:1370-7; Proutski, V. et al. 1997 Nucleic Acids Res 25:1194-202) have identified conserved differences in the nucleotide sequences of the 3’ UTR of mosquito-transmitted and tick-transmitted flaviviruses. Such differences are concentrated in the 3 ’ terminal core region, the approximately 400 3’ terminal nucleotides. It has been suggested that these sequences may have a vector-specific function (Proutski, V. et al. 1997 Nucleic 30 Acids Res 25:1194-202). While such a function has not been identified, it may nonetheless be possible to disrupt vector infectivity by deleting or otherwise altering these nucleotides. -53-

To identify target sequences for this type of alteration, we constructed an alignment of the 35 UTR nucleotide sequences of seven mosquito-transmitted flaviviruses and four tick-transmitted flaviviruses (Figure 8). From this alignment, we identified several sequences that showed conserved differences between the mosquito-transmitted 5 flaviviruses and tick-transmitted flaviviruses. We then designed primers to alter these sequences in the wt DEN4 cDNA p4 (Figure 4) in one of two ways: 1) deletion of the nucleotides (Δ) or 2) replacement of the nucleotides with the homologous sequence from the tick-transmitted flavivirus Langat (swap). Langat was chosen as the template for swapped nucleotides because it is naturally attenuated (Pletnev, A.G. 2001 Virology 10 282:288-300), and therefore unlikely to enhance die virulence of rDEN4 vims derived from 2015203575 26 Jun2015 the modified cDNA. The DEN4 sequences altered and the mutagenesis primers used to do so are listed in Table 28. Nucleotides 10508-10530 correspond to the CS2 region identified in previous studies (Proutski, V. et al. 1997 Nucleic Acids Res 25:1194-202).

Mutagenesis of p4, transcription and transfection were conducted as previously 15 described in Section I of this Example. All five of the engineered viruses were recovered, and all were subjected to two rounds of terminal dilution as previously described.

Evaluation of phenotypes: cell culture. Viruses were titered in Vero and C6/36 cells as previously described, and the results are listed in Table 29. All of the viruses replicated to >5.0 log10 PFU/ml; one of them (rDEN4A10508-10530) replicated to > 8.0 20 logi0 PFU/ml. Only one of the viruses (rDEN4Al 0535-10544) was small plaque in C6/36 cells; this vims showed wild-type plaque size in Vero cells. Interestingly, another vims (rDEN4swapl0508-10539) showed wild type plaque size in C6/36 cells but was sp in Vero cells.

Evaluation of phenotypes: mosquito infectivity. To date one of the five vimses 25 has been tested for infectivity via intrathoracic inoculation in Tx. splendens, using previously described methods. Vims rDEN4A10508-10530 was dramatically restricted in infectivity relative to the wild type (Table 30). So few mosquitoes were infected that it was not possible to calculate an MIDJ0 for this vims.

One of the five vimses has been tested for infectivity of Ae. aegypti fed on an 30 infectious bloodmeal using previously described methods. rDEN4swap 10535-10544 (Table 31) caused significantly fewer midgut infections than wild type rDEN4, but the percentage of disseminated infections did not differ between rDEN4swapl0535-10544 and wild type -54- rDEN4. All of the viruses are envisioned as being tested for mosquito infectivity using both methods. 2015203575 26 Jun2015

Summary. In this example we have outlined two different strategies for preventing mosquito transmission of a dengue vaccine. First, several small substitution mutations, 5 including two point mutations and one paired charge-to-alanine substitution, have been shown to restrict the replication of DEN4 in mosquito C6/36 cells in cell culture, and one of these mutations (C7129U) has been shown to restrict the ability of DEN4 virus to infect mosquitoes. Second, we have created a variety of deletion and substitution mutations in regions of the DEN4 3’ UTR that show conserved differences between mosquito-10 transmitted and tick-transmitted flaviviruses. One of these viruses is sp in C6/36 cells and at least two of these viruses show some degree of restriction of mosquito infectivity. By design, the nucleotide sequences in which these mutations were made are highly conserved among the four dengue serotypes and among mosquito-transmitted flaviviruses in general, indicating that they are portable to other vaccine candidates for mosquito-borne 15 flaviviruses. All of the mutations discussed in this Example lie outside the structural genes and so are envisioned as being useful in constructing antigenic-chimeric vaccine candidates.

Example 6

Adaptation Mutations Which Enhance the Replication of DEN4 and DEN4 Chimeric 20 Viruses in Vero Cells.

Vero cells are a highly characterized substrate that should be suitable for the manufacture of live attenuated flavivirus vaccines, such as dengue virus and tick-bome encephalitis virus. In addition, Vero cells can also be used to grow flaviviruses to high titer for the preparation of an inactivated virus vaccine. Optimal sequences for the efficient 25 growth of dengue viruses in Vero cells have not been identified, but it is well known that flaviviruses accumulate mutations during passage in various cell cultures (Dunster, L.M. et al. 1999 Virology 261:309-18; Theiler, M. &amp; Smith, Ή.Η. 1937 J Exp Med 65:787-800). Inclusion of specific sequences in live attenuated viruses that enhance their replication in Vero cells and increase the number of doses of vaccine produced per unit substrate would 30 greatly facilitate their manufacture. Similarly, inclusion of Vero cell growth-promoting sequences in wild type viruses used for the preparation of an inactivated vims vaccine would also greatly facilitate the manufacture of the vaccine. The present example identifies -55- mutations that occur following passage of DEN4 virus and DEN2/4 chimeric viruses in Vero cells. Data derived from five sources provided information for this analysis making it possible to generate a list of Vero cell growth-promoting sequences.

Presence of identical mutations in multiple 5-FU mutant viruses. First, as described in Examples 1 and 2, the genomes of 42 dengue virus clones isolated from a 5-FU mutagenized stock of virus were completely sequenced. If mutations that enhance replication occurred during the passage of these 42 mutant viruses in Vero cells, then such mutations should reveal themselves by representation in more than one clone. Analysis of the 42 sequences revealed the occurrence of specific missense mutations in coding regions or nucleotide substitutions in UTRs in multiple clones that are not present in the 2A parental virus genome (Tables 11 and 32). These mutations, many of which occur within a 400 nucleotide section of the NS4B coding region, represent Vero cell-adaptation mutations. One mutation, such as the 4995 mutation, present in eight viruses was found to specify both ts and ait phenotypes (Examples 1 and 3). In contrast, the 7163 mutation, present in six viruses, does not specify a ts or att phenotype (Table 13) and thus is an example of a specific Vero cell growth-promoting mutation.

Presence of Vero cell adaptation mutations in other DEN4 viruses and DEN2/4 antigenic chimeric viruses. Second, the 2A-13 dengue virus that was used as a parallel passaged wild type control during the 5-FTJ experiments described in Example 1 was grown and cloned in Vero cells in the absence of 5-FU in a manner identical to that of the 5-FU treated viruses. Sequence analysis of this 5-FU untreated virus, designated 2A-13-1A1, revealed that the virus genome contained a mutation at nucleotide 7163 (Example 1 and Table 32), identical to the missense mutation previously identified in 6 of the 5-FU mutant viruses (Tables 11 and 32). This indicates that growth and passage of DEN4 virus in Vero cells is sufficient to acquire this specific mutation, i.e. mutagenesis with 5-FU is not required. Thus, information from two separate sources indicates that the 7163 mutation appeared in separate Vero cell passaged viruses, thereby strengthening the interpretation that this mutation is growth promoting.

Third, following passage of the 2ΑΔ30 and rDEN4A30 in Vero cells, sequence analysis revealed the presence of a mutation at nucleotides 7153 and 7163, respectively. These two mutations were also previously identified among the 5-FU treated viruses (Table 32). Again, identical mutations appeared following independent passage of virus in Vero -56- cells, corroborating the hypothesis that these mutations confer a growth advantage in Vero cells. 2015203575 26 Jun2015

Fourth, an antigenic chimeric dengue virus vaccine candidate was generated that expressed the structural proteins C, prM, and E from DEN2 on a DEN4 wild type genetic 5 background or an attenuated Δ30 genetic background. To construct this virus, the C, prM and E region of wild type cDNA plasmid p4 was replaced with a similar region from DEN2 virus strain NGC (Figure 10). Specifically, nucleotides between restriction sites Bglil (nt 88) and Xhol (nt 2345) of p4 were replaced with those derived from dengue type 2 virus. RNA transcripts synthesized from the resulting p4-D2 plasmid were transfected into Vero 10 cells and rDEN2/4 virus was recovered. A further attenuated version of this chimeric virus containing the Δ30 mutation, rDEN2/4A30, was recovered in C6/36 mosquito cells following transfection of cells with RNA transcripts derived p4A30-D2. However, rDEN2/4A30 could not be recovered directly in Vero cells. The rDEN2/4A30 mutant virus recovered in C6/36 cells replicated to very low levels in Vero cells (<1.0 log10PFU/ml) but 15 grew to high titer in C6/36 cells (>6.0 log10PFU/ml). Genomic sequence of the C6/36-derived virus matched the predicted cDNA sequence and is shown in Appendix 3. Nevertheless, when C6/36-derived rDEN2/4A30 was serially passaged 3 to 4 times in Vero cells, a virus population adapted for growth in Vero cells emerged. Vims from this Vero cell-adapted preparation was cloned and amplified in Vero cells to a titer >6.0 log10PFU/ml. 20 The genomic sequence was determined for 2 independent vims clones and compared to the predicted cDNA sequence (Table 33 and 34). Each cloned vims contains a mutation in a non-stmctural gene which coincides closely in location or sequence with a mutation previously identified among the panel of 5-FU mutagenized viruses. The other mutations in these two clones also might confer a growth advantage in Vero cells. Importantly, the 25 mutations identified in Tables 33 and 34 are absolutely required for replication in Vero cells, and it would not be possible to produce the rDEN2/4A30 vaccine candidate in Vero cells without the growth-promoting mutations identified in Tables 33 and 34.

Fifth, sequence analysis of the dengue 4 wild-type vims strain 814669 (GenBank accession no. AF326573) following passage in Vero cells identified a mutation in the NS5 30 region at nucleotide 7630 which had previously been identified among the panel of 5-FU mutagenized vimses (Table 32). This mutation at nucleotide 7630 was introduced into recombinant vims rDEN4 by site-directed mutagenesis as described in Table 16. The -57- resulting virus, rDEN4-7630, was not temperature sensitive when tested at 39°C, indicating that mutation 7630 does not contribute to temperature sensitivity.

Characterization of rDEN2/4A30 chimeric viruses containing single and multiple Vero cell adaptation mutations. The generation of chimeric virus rDEN2/4A30 provided a unique opportunity for evaluating the capacity of individual mutations to promote increased growth in Vero cells. Because rDEN2/4A30 replicates to very low titer in Vero cells, yet can be efficiently generated in C6/36 mosquito cells, recombinant virus bearing putative Vero-cell adapting mutations were first generated in C6/36 cells and then virus titers were detennined in both C6/36 and Vero cells. As shown in Table 35, addition of a single mutation to rDEN2/4A30 resulted in a greater than 1000-fold increase in titer in Vero cells, confirming the Vero cell adaptation phenotype conferred by these mutations. However, the combination of two separate mutations into a single virus did not increase the titer in Vero cells beyond the level observed for viruses bearing a single adaptation mutation. Inclusion of either the 7182 or 7630 mutation in the cDNA of rDEN2/4A30 allowed the virus to be recovered directly in Vero cells, circumventing the need to recover the virus in C6/36 cells.

Characterization of the growth properties of rDEN4 viruses containing single and multiple defined Vero cell adaptation mutations. To confirm the ability of Vero cell adaptation mutations to enhance growth of DEN4 viruses, site-directed mutagenesis was used to generate rDEN4 viruses encoding selected individual mutations as described in Examples 1 and 3. Five mutations in NS4B (7153, 7162, 7163, 7182, and 7546) from the list of repeated mutations in the 5-FU mutant viruses (Table 32) were introduced singly into the p4 cDNA clone. In addition, the mosquito-restricted, rDEN4-7129 virus was evaluated for enhanced growth in Vero cells since the location of this mutation is in the same region of NS4B. Each virus, including wild-type rDEN4, was recovered, terminally diluted, and propagated in C6/36 cells to prevent introduction of additional Vero cell adaptation mutations, however, because of its restricted growth in C6/36 cells, rDEN4-7129 was propagated only in Vero cells.

Plaque size was evaluated for each mutant rDEN4 virus in Vero cells and C6/36 cells and compared to wild-type rDEN4. Six-well plates of each cell were inoculated with dilutions of virus and plaques were visualized five days later. Representative plaques are illustrated in Figure 10 and demonstrate that the presence of a Vero cell adaptation -58- mutation does indeed confer increased virus cell to cell spread and growth specifically in Vero cells. In C6/36 cells, average plaque size was approximately 0.50 mm for both wild-type rDEN4 and each mutant virus (except for rDEN4-7546 and rDEN4-7129 which were smaller than wild-type; see Example 5). However, rDEN4 viruses expressing mutation 7162, 7163, 7182, and 7129 had a greater than two-fold increase in plaque size in Vero cells compared to wild-type rDEN4 virus. A smaller but consistent increase in plaque size was observed for rDEN4-7153 and rDEN4-7546.

Growth kinetics and virus yield in Vero cells was assessed for the same panel of rDEN4 viruses. Vero cells were infected at an MOI of 0.01 and samples were removed daily for 10 days, titered on Vero cells, and plaques were visualized. The results in Figure 11 indicate that the presence of a Vero cell adaptation mutation increased the kinetics of f virus growth, but had only a marginal effect on the peak virus yield. At day four postinfection, wild-type rDEN4 grew to 5.2 log10PFU/ml while the level of replication in rDEN4-7129-infected cells was 100-fold higher. The rest of the mutant rDEN4 viruses had an increased yield at day four ranging from 0.9 (rDEN4-7153) to 1.6 (rDEN4-7162 and -7163) log10PFU/ml. Interestingly, enhanced kinetics of virus growth correlated with increased plaque size in Vero cells. The peak virus yield was reached by day 6 postinfection for rDEN4-7129, -7162, -7163, and -7182 while wild-type rDEN4 did not reach peak titer until day 10. However, the peak virus yield was only slightly higher in rDEN4 viruses expressing Vero cell adaptation mutations.

In an effort to further enhance rDEN4 replication, especially the peak virus yield, combinations of selected Vero cell adaptation mutations were introduced into the rDEN4 background. Three viruses with dual mutations were generated: rDEN4-7153-7163, rDEN4-7153-7182, and rDEN4-7546-7630 and tested in a Vero cell time course infection as described above along with rDEN4 and rDEN4-7162 as a positive control (Figure 12). The viruses expressing combined mutations grew in a nearly identical manner to rDEN4-7162 indicating that these selected combinations did not enhance the kinetics or peak virus yield. Additional combinations of these and other Vero cell adaptation mutations are envisioned as increasing peak virus yield.

Discussion. Some of the growth promoting mutations listed in Table 32 are also found in homologous regions of DENI, DEN2, and DEN3 and are envisioned as serving to promote the replication of these viruses in Vero cells. Specifically, the growth promoting -59- mutations indicated in Table 32 that are present in a DEN4 virus are envisioned as being useful for importation into homologous regions of other flaviviruses, such as DENI, DEN2 and DEN3. Examples of such conserved regions are shown in Appendix 4 and are listed in Table 36. The nucleotides for both mutation 7129 and 7182 are conserved in all four dengue virus serotypes. It is also interesting to note that mutation 7129 not only increases growth in Vero cells (Figure 10), but it also forms small plaques in mosquito cells (Figure 6, Table 25). Lee et al. previously passaged DEN3 virus in Vero cells and performed limited sequence analysis of only the structural gene regions of the resulting viruses (Lee, E. et al. 1997 Virology 232:281-90). From this analysis a menu of Vero adaptation mutations was assembled. Although none of these mutations correspond to the Vero adaptation mutations identified in this Example, a single mutation at amino acid position 202 in DEN3 corresponds to mutation 1542 identified in 5-FU mutant virus #1012. The current Example emphasizes the importance in this type of study of determining the sequence of the entire viral genome.

Vero cell growth optimized viruses are envisioned as having usefulness in the following areas. First, the yield of a live attenuated vaccine virus in Vero cells is predicted to be augmented. The live attenuated vaccine candidate is conveniently a DEN4 or other dengue virus or a DEN1/4, DEN2/4, or DEN3/4 antigenic chimeric virus, or a chimeric virus of another flavivirus based on the DEN4 background. The increased yield of vaccine virus is envisioned as decreasing the cost of vaccine manufacture. Second, Vero cell adaptation mutations that are attenuating mutations, such as the 4995 mutation, are envisioned as being stable during the multiple passage and amplification of virus in Vero cell cultures that is required for production of a large number of vaccine doses. Third, Vero cell adaptation mutations are actually required for the growth of the rDEN2/4A30 vaccine candidate in Vero cells. Fourth, the increase in yield of a DEN wild type or an attenuated virus is envisioned as making it economically feasible to manufacture an inactivated virus vaccine. Fifth, the presence of the Vero cell growth promoting mutations in the DEN4 vector of the rDENl/4, rDEN2/4, and rDEN3/4 antigenic chimeric viruses or other flavivirus chimeric viruses based on DEN4 is envisioned as permitting the viruses to grow to a high titer and as thereby being useful in the manufacture of a inactivated virus vaccine. Sixth, the insertion of Vero cell growth promoting mutations into cDNAs such as rDEN2/4A30 is envisioned as permitting recovery of virus directly in Vero cells, for which -60- there are qualified master cell banks for manufacture, rather than in C6/36 cells for which qualified cell banks are not available. And seventh, insertion of the 7129 and 7182 mutations into DENI, DEN2, or DEN3 wt viruses is envisioned as increasing their ability to replicate efficiently and be recovered from cDNA in Vero cells. 2015203575 26 Jun2015 5 Example 7

Assembly of a List of Attenuating Mutations The data presented in these examples permits the assembly of a list of attenuating mutations that is summarized in Table 37. This list contains individual mutations identified in Tables 13 - 16, 20, and 21 that are known to independently specify an attenuation 10 phenotype. Mutation 7129 is also included since it is derived from virus 5-1A1 shown to be attenuated in mosquitoes. We envision using various combinations of mutations from this list to generate viruses with sets of desirable properties such as restricted growth in the liver or in the brain as taught in Example 3 (Table 18) and Example 4 (Tables 23 and 24). These mutations are also combinable with other previously described attenuating mutations 15 such as the Δ30 mutation, as taught in Example 1 (Table 6 ) and Example 3 (Table 19) to produce recombinant viruses that are satisfactorily attenuated and immunogenic. Mutations listed in Table 37 are also envisioned as being combined with other previously described attenuating mutations such as other deletion mutations or other point mutations (Blok, J. et al. 1992 Virology 187:573-90; Butrapet, S. et al. 2000 J Virol 74:3011-9; Men, R. et al. 20 1996 J Virol 70:3930-7; Puri, B. et al. 1997 J Gen Virol 78:2287-91).

The possibility of importing an attenuating mutation present in one paramyxovirus into a homologous region of a second paramyxovirus has recently been described (Durbin, A.P. et al. 1999 Virology 261:319-30; Skiadopoulos, M.H. et al. 1999 Virology 260:125-35). Such an importation confers an att phenotype to the second virus or, alternatively, 25 further attenuates the virus for growth in vivo. Similarly we envision importing an attenuating mutation present in one flavivirus to a homologous region of a second flavivirus which would confer an att phenotype to the second flavivirus or, alternatively, would further attenuate the virus for growth in vivo. Specifically, the attenuating mutations indicated in Table 37 are envisioned as being useful for importation into homologous 30 regions of other flaviviruses. Examples of such homologous regions are indicated in Appendix 4 for the mutations listed in Table 37. -61-

Example 8 2015203575 26 Jun2015

Evaluation of Dengue Virus Vaccine In Humans And Rhesus Monkeys

The present example evaluates the attenuation for humans and rhesus monkeys (as an animal model) of a DEN-4 mutant bearing a 30 nucleotide deletion (Δ30) that was 5 introduced into its 3' untranslated region by site-directed mutagenesis and that was found previously to be attenuated for rhesus monkeys (Men, R. et al. 1996 J Virol 70:3930-7), as representative of the evaluation of any dengue virus vaccine for attenuation in humans and rhesus monkeys (as an animal model).

Viruses and cells. The wild type (wt) DEN-4 virus strain 814669 (Dominica, 10 1981), originally isolated in Aedes pseudoscutellaris (AP61) cells, was previously plaque- purified in LLC-MK2 cells and amplified in C6/36 cells as described (Mackow, E. et al. 1987 Virology 159:217-28). For further amplification, the C6/36 suspension was passaged 2 times in Vero (WHO) cells maintained in MEM-E (Life Technologies, Grand Island, NY) supplemented with 10% FBS. Viruses derived from RNA transfection or used for clinical 15 lot development were grown in Vero (WHO) cells maintained in serum-free media, VP-SFM (Life Technologies).

Construction of DEN-4 deletion mutants. A 30 nucleotide (nt) deletion was previously introduced into the 3' untranslated region of the 2A cDNA clone of wt DEN-4 strain 814669 as described (Men, R. et al. 1996 J Virol 70:3930-7). This deletion removes 20 nucleotides 10478 - 10507, and was originally designated 3'd 172-143, signifying the location of the deletion relative to the 3' end of the viral genome. In the current example, this deletion is referred to as Δ30. The full-length 2A cDNA clone has undergone several subsequent modifications to improve its ability to be genetically manipulated. As previously described, a translationally-silent Xliol restriction enzyme site was engineered 25 near the end of the E region at nucleotide 2348 to create clone 2A-Xhol (Bray, M. &amp; Lai, CJ. 1991 PNAS USA 88:10342-6). In this example, the viral coding sequence of the 2A-Xltol cDNA clone was further modified using site-directed mutagenesis to create clone p4: a unique BbvCl restriction site was introduced near the C-prM junction (nucleotides 447 -452); an extra Xbal restriction site was ablated by mutation of nucleotide 7730; and a 30 unique Sacll restriction site was created in the NS5 region (nucleotides 9318 - 9320). Each of these engineered mutations is translationally silent and does not change the amino acid sequence of the viral polypeptide. Also, several mutations were made in the vector region -62- of clone p4 to introduce or ablate additional restriction sites. The cDNA clone ρ4Δ30 was generated by introducing the Δ30 mutation into clone p4. This was accomplished by replacing the Mini - Kpnl fragment of p4 (nucleotides 10403 - 10654) with that derived from plasmid 2ΑΔ30 containing the 30 nucleotide deletion. The cDNA clones p4 and 5 ρ4Δ30 were subsequently used to generate recombinant viruses rDEN4 and rDEN4A30, 2015203575 26 Jun2015 respectively.

Generation of viruses. Full-length RNA transcripts were synthesized from cDNA clones 2A and 2ΑΔ30 using SP6 RNA polymerase as previously described (Lai, CJ. et al. 1991 PNAS USA 88:5139-43; Men, R. et al. 1996 J Virol 70:3930-7). The reaction to 10 generate full-length RNA transcripts from cDNA clones p4 and ρ4Δ30 was modified and consisted of a 50 μΐ reaction mixture containing 1 pg linearized plasmid, 60 U SP6 polymerase (New England Biolabs (NEB), Beverly, ΜΑ), IX RNA polymerase buffer (40 mM Tris-HCl, pH 7.9, 6 mM MgCl2, 2 mM spermidine, 10 mM dithiothreitol), 0.5 mM m7G(5')ppp(5')G cap analog (NEB), 1 mM each nucleotide triphosphate, 1 U 15 pyrophosphatase (NEB), and 80 U RNAse inhibitor (Roche, Indianapolis, IN). This reaction mixture was incubated at 40°C for 90 min and the resulting transcripts were purified using RNeasy mini kit (Qiagen, Valencia, CA). For transfection of Vero cells, purified transcripts (1 pg) were mixed with 12 pi DOTAP liposome reagent (Roche) in saline containing 20 mM HEPES buffer (pH 7.6) and added to cell monolayer cultures in a 20 6-well plate. After 5-17 days, tissue culture medium was harvested, clarified by centrifugation, and virus was amplified in Vero cells. The presence of virus was confirmed by plaque titration. It should be noted that during the course of transfection and amplification of 2ΑΔ30 to create the vaccine lot, the virus underwent a total of 6 passages entirely in Vero cells. The remaining viruses, rDEN4 and τϋΕΝ4Δ30 were passaged 5 25 times in Vero cells to generate the virus suspension used for sequence analysis and studies in rhesus monkeys.

Vaccine Production. An aliquot of clarified tissue culture fluid containing vaccine candidate 2ΑΔ30 was submitted to DynCorp (Rockville, MD) for amplification of virus in Vero cells and production of a vaccine lot. For vaccine production, 2ΑΔ30 infected tissue 30 culture supernatant was harvested, SPG buffer added (final concentration: 218 mM sucrose, 6 mM L-glutamic acid, 3.8 mM potassium phosphate, monobasic, and 7.2 mM potassium phosphate, dibasic, pH 7.2), and the virus suspension was clarified by low speed -63- centrifugation. To degrade residual Vero cell DNA, the vaccine suspension was treated with Benzonase endonuclease (American International Chemical, Natick, MA), 100 U/ml and incubated for 1 hr at 37°C, followed by high-speed centrifugation (17,000 x g, 16 hr). The resulting virus pellet was gently rinsed with MEM-E, resuspended in MEM-E· 5 containing SPG, sonicated, distributed into heat-sealed ampoules, and stored frozen at -70°C. Final container safety testing confirmed microbial sterility, tissue culture purity, and animal safety. The 2ΑΔ30 vaccine lot (designated DEN4-9) has a titer of 7.48 loglOPFU/ml, with a single dose of 5.0 loglO PFU/ml containing <1 pg/ml Vero cell DNA and <0.001 U/ml Benzonase endonuclease. 2015203575 26 Jun2015 10 Sequence of cDNA clones and viral genomes. The nucleotide sequence of the viral genome region of cDNA plasmids 2A and p4 was determined on a 310 genetic analyzer (Applied Biosystems, Foster City, CA) using vector-specific and DEN-4-specific primers in BigDye terminator cycle sequencing reactions (Applied Biosystems). The nucleotide sequence of the genomes of the parental wt DEN-4 strain 814669 and of 15 recombinant viruses 2A wt, 2ΑΔ30 (vaccine lot), rDEN4, and rDEN4A30 was also determined. Viral RNA was extracted from virus preparations and serum samples using the QIAamp Viral RNA mini kit (Qiagen). Reverse transcription (RT) was perfonned using random hexamers and the Superscript First-Strand Synthesis System for RT-PCR (Life Technologies). Overlapping PCR fragments of approximately 2000 base pairs were 20 generated using optimized DEN-4 specific primers and Advantage cDNA polymerase (ClonTech, Palo Alto, CA). Both strands of purified PCR fragments were sequenced directly using dye-terminator reactions as described above and results were assembled into a consensus sequence. To determine the nucleotide sequence of the viral RNA 5' and 3' regions, the 5' cap nucleoside of the viral RNA was removed with tobacco acid 25 pyrophosphatase (Epicentre, Madison, WI) followed by circularization of the RNA using RNA ligase (Epicentre). RT-PCR was performed as described and a cDNA fragment spanning the ligation junction was sequenced using DEN-4 specific primers. GenBanlc accession numbers have been assigned as follows (virus: accession number): 814669: AF326573, 2ΑΔ30: AF326826, rDEN4: AF326825, andrDEN4A30: AF326827. 30 Human Vaccine Recipients. 20 normal healthy adult volunteers were recmited by the Johns Hopkins School of Hygiene and Public Health Center for Immunization Research (CIR) located in Baltimore, Maryland. The clinical protocol was reviewed and approved -64- by the Joint Committee for Clinical Investigation of the Johns Hopkins University School of Medicine and informed consent was obtained from each volunteer. Volunteers were enrolled in the study if they met the following inclusion criteria: 18-45 years of age; no history of chronic illness; a normal physical examination; human immunodeficiency virus 5 antibody negative, hepatitis B surface antigen negative, and hepatitis C antibody negative; 2015203575 26 Jun2015 . no stool occult blood; and normal values for complete blood cell count (CBC) with differential, hematocrit, platelet count, serum creatinine, serum aspartate amino transferase (AST), alanine amino transferase (ALT), alkaline phosphatase, bilirubin, prothrombin time (PT), partial thromboplastin time (PTT), and urinalysis. Female volunteers were required 10 to have a negative urine pregnancy test prior to vaccination and on the day of vaccination and to agree to use contraception or abstain from sexual intercourse for the duration of the study. Volunteers also lacked serological evidence of prior flavivirus infection as defined by hemagglutination-inhibition antibody titer < 1:10 to DEN-1, DEN-2, DEN-3, DEN-4, St. Louis encephalitis virus, Japanese encephalitis virus, or yellow fever virus and a plaque-15 reduction neutralization titer < 1:10 to DEN-4 and yellow fever virus.

Studies in Humans. Volunteers were immunized in three successive cohorts of four, six, and ten volunteers to assess the safety of the vaccine. In this study, an illness was defined as the following: dengue virus infection associated with a platelet count of < 90,000/mm3; serum ALT > 4 times normal; oral temperature >38°C for > 2 successive 20 days; or headache and/or myalgia lasting > 2 successive days. Systemic illness was defined as the occurrence of fever >38°C for > 2 consecutive days, or any 2 of the following for at least two consecutive study days: headache, malaise, anorexia, and myalgia/arthralgia. The trials were conducted between October and April, a time of low mosquito prevalence, to reduce the risk of transmission of vaccine virus from the volunteers to the community. 25 On the day of vaccination, vaccine candidate 2ΑΔ30 was diluted to 5.3 log10PFU/ml in sterile saline for injection, USP, and each volunteer was injected subcutaneously with a 0.5 ml containing 5.0 log10PFU of vaccine into the left deltoid region. Volunteers were given a home diary card on which they were to record their temperature twice daily for days 0-5 post-vaccination. The volunteers returned to the clinic each day for examination 30 by a physician and their diary cards were reviewed. The injection site was evaluated for erythema, induration, and tenderness. Clinical signs and symptoms such as headache, rash, petechiae, lymphadenopathy, hepatomegaly, abdominal tenderness, anorexia, nausea, -65- fatigue, myalgia, arthralgia, eye pain, and photophobia were assessed daily. Symptoms were graded as mild (no need for treatment or a change in activity), moderate (treatment needed or change in activity noted, yet still able to continue daily activity) or severe (confined to bed). Blood was drawn for CBC with differential and for virus quantitation on days 0, 2 and 4. Volunteers were admitted to the inpatient unit at the CIR on the sixth day after immunization. The study physician evaluated all volunteers each day by physical examination and interview. The volunteers had their blood pressure, pulse, and temperature recorded four times a day. Blood was drawn each day for CBC with differential and for virus quantitation and every other day for ALT measurement. Volunteers were confined to the inpatient unit until discharge on study day 15. On study days 28 and 42, volunteers returned for physical examination and blood was drawn for virus quantitation (day 28) and for serum antibody measurement (day 28 and 42).

Virus quantitation and amplification. Serum was obtained for detection of viremia and titration of vims in positive specimens. For these purposes 8.5 ml of blood was collected in a serum separator tube and incubated at room temperature for less than 30 min. Serum was decanted into 0.5 ml aliquots, rapidly frozen in a dry ice/ethanol bath and stored at -70°C. Serum aliquots were thawed and serial 10-fold dilutions were inoculated onto Vero cell monolayer cultures in 24-well plates. After one hour incubation at room temperature, the monolayers were overlaid with 0.8% methylcellulose in OptiMEM (Life Technologies) supplemented with 5% fetal bovine serum (FBS). Following incubation at 37°C for four days, vims plaques were visualized by immunoperoxidase staining. Briefly, cell monolayers were fixed in 80% methanol for 30 min and rinsed with antibody buffer (5% nonfat milk in phosphate buffered saline). Rabbit polyclonal DEN-4 antibodies were diluted 1:1000 in antibody buffer and added to each well followed by a one hr incubation at 37°C. Primary antibody was removed and the cell monolayers were washed twice with antibody buffer. Peroxidase-labelled goat-anti-rabbit IgG (KPL, Gaithersburg, MD) was diluted 1:500 in antibody buffer and added to each well followed by a one hr incubation at 37°C. Secondary antibody was removed and the wells were washed twice with phosphate buffered saline. Peroxidase substrate (4 chloro-l-napthol in H202) was added to each well and visible plaques were counted.

For amplification of vims in semm samples, a 0.3 ml aliquot of serum was inoculated directly onto a single well of a 6-well plate of Vero cell monolayers and -66- incubated at 37°C for 7 days. Cell culture fluid was then assayed for virus by plaque assay as described above. 2015203575 26 Jun2015

Serology. Hemagglutination-inhibition (HAI) assays were performed as previously described (Clarke, D.H. &amp; Casals, J. 1958 Am J Trop Med Hyg 7:561-73). Plaque-5 reduction neutralization titers (PRNT) were determined by a modification of the technique described by Russell (Russell, P.K. et al. 1967 J Immunol 99:285-90). Briefly, test sera' were heat inactivated (56°C for 30 min) and serial 2-fold dilutions beginning at 1:10 were made in OptiMEM supplemented with 0.25% human serum albumin. rDEN4A30 virus, diluted to a final concentration of 1000 PFU/ml in the same diluent, was added to equal 10 volumes of the diluted serum and mixed well. The virus/serum mixture was incubated at 37°C for 30 min. Cell culture medium was removed from 90% confluent monolayer cultures of Vero cells on 24-well plates and 50 μΐ of virus/serum mixture was transferred onto duplicate cell monolayers. Cell monolayers were incubated for 60 min at 37°C and overlaid with 0.8% methylcellulose in OptiMEM supplemented with 2% FBS. Samples 15 were incubated at 37°C for 4 days after which plaques were visualized by immunoperoxidase staining as described above, and a 60% plaque-reduction neutralization titer was calculated.

Studies in rhesus monkeys. Evaluation of the replication and immunogenicity of wt virus 814669, and recombinant viruses 2A wt, 2ΑΔ30 (vaccine lot), rDEN4, and 20 rDEN4A30 in juvenile rhesus monkeys was performed as previously described (Men R. et al. 1996 J Virol 70:3930-7). Briefly, dengue virus seronegative monkeys were injected subcutaneously with 5.0 log10 PFU of virus diluted in L-15 medium (Quality Biological, Gaithersburg, MD) containing SPG buffer. A dose of 1 ml was divided between two injections in each side of the upper shoulder area. Monkeys were observed daily and blood 25 was collected on days 0-10 and 28, and processed for serum, which was stored frozen at -70°C. Titer of virus in serum samples was determined by plaque assay on Vero cells as described above. Neutralizing antibody titers were determined for the day 28 serum samples as described above. A group of monkeys inoculated with either 2ΑΔ30 (n = 4) or wt virus 814669 (n = 8) were challenged on day 42 with a single dose of 5.0 log10 PFU/ml 30 wt virus 814669 and blood was collected for 10 days. Husbandry and care of rhesus monkeys was in accordance with the National Institutes of Health guidelines for the humane use of laboratory animals. -67-

Construction and characterization of DEN-4 wild type and deletion mutant viruses. The nucleotide and deduced amino acid sequences of the previously described wt 814669 virus, the DEN-4 2A wt virus derived from it (designated 2A wt), and the 2ΑΔ30 vaccine candidate derived from 2A wt virus were first determined. Sequence analysis showed that the wt 814669 virus used in this study had apparently accumulated 2 missense mutations (nucleotides 5826 and 7630) and 3 silent mutations during its passage and amplification since these mutations were not described in previously published reports of the viral sequence (GenBank accession number M14931) and were not present in the 2A cDNA derived from the virus. Sequence comparison between viruses 2A wt and vaccine lot 2ΑΔ30 revealed that 2ΑΔ30 accumulated 2 missense mutations (nucleotides 7153 and 8308) and also confirmed the presence of the Δ30 mutation (nucleotides 10478 - 10507) as well as an additional deletion of nucleotide 10475, which occurred during the original construction of the Δ30 mutation (Men, R. et al. 1996 J Virol 70:3930-7). This sequence analysis revealed significant sequence divergence between the biologically-derived wt 814669 virus and its recombinant 2A wt derivative and between the 2A wt and 2ΑΔ30 virus. Since the 2A wt and 2ΑΔ30 viruses differed at nucleotides other than the deletion mutation, the attenuation phenotype previously reported for 2ΑΔ30 (Men, R. et al. 1996 J Virol 70:3930-7) could not be formally ascribed solely to the Δ30 mutation and may have been specified by the mutations at nucleotides 7153, 8308,10475, or the Δ30 deletion.

To determine whether the Δ30 mutation was responsible for the observed attenuation of 2ΑΔ30, a second pair of viruses, one with and one without the Δ30 mutation, were produced for evaluation in monkeys. A new DEN-4 cDNA vector construct, designated p4, was derived from the 2A-Xhol cDNA clone and translationally-silent mutations were introduced to add or ablate several restriction enzyme sites. These sites were added to facilitate the future genetic manipulation of this DEN-4 wt cDNA by the introduction of other attenuating mutations if needed. The sequence of the genomic region of the p4 cDNA plasmid was identical to that of the 2A wt virus except for the engineered restriction site changes and a point mutation at nucleotide 2440 which was introduced during the original mutagenesis of the 2A cDNA plasmid to create the Xhol site (Bray, M. &amp; Lai, CJ. 1991 PNAS USA 88:10342-6). The Δ30 mutation and the neighboring deletion at nucleotide 10475 were co-introduced into the p4 plasmid by replacing a short restriction fragment with one derived from the cDNA clone of 2ΑΔ30. RNA transcripts derived from -68- the p4 cDNA clone and from its Δ30 derivative each yielded virus (designated rDEN4 wt and rDEN4A30, respectively) following transfection of Vero cells. Sequence analysis of the rDEN4 virus revealed that during its passage and amplification in Vero cells it accumulated 2 missense mutations (nucleotides 4353 and 6195), a silent mutation 5 (nucleotide 10157), and a point mutation in the 3' untranslated region (nucleotide 10452). In addition to containing the Δ30 and the accompanying deletion at nucleotide 10475, rDEN4A30 had also accumulated a missense mutation (nucleotide 7163) and a silent mutation (nucleotide 7295). 2015203575 26 Jun2015

Parental wt 814669 vims and recombinant viruses 2A wt, 2ΑΔ30, rDEN4, and 10 rDEN4A30 each replicate in Vero cells to a titer exceeding 7.0 log10PFU/ml, and their replication is not temperature sensitive at 39°C.

Virus replication, immunogenicity, and efficacy in monkeys. Groups of rhesus monkeys were inoculated with wt DEN-4 814669, 2A wt, rDEN4, 2ΑΔ30 and rDEN4A30 to assess the level of restriction of replication specified by the Δ30 mutation. Serum 15 samples were collected daily and titer of vims present in the serum was determined by plaque enumeration on Vero cell monolayer cultures. Monkeys inoculated with wt 814669 vims or its recombinant counterparts, 2A wt or rDEN4, were viremic for 3 to 4 days with a mean peak vims titer of nearly 2 log10PFU/ml. Monkeys inoculated with vims 2ΑΔ30 or rDEN4A30 had a lower frequency of viremia (83% and 50%, respectively),, were viremic 20 for only about 1 day, and the mean peak titer was 10-fold lower. Monkeys inoculated with DEN-4 814669, 2A wt, or rDEN4 vimses developed high levels of neutralizing antibody, with mean titers between 442 and 532, consistent with their presumed wild type phenotype. Monkeys inoculated with 2ΑΔ30 or rDEN4A30 developed a lower level of neutralizing antibody, with mean titers of 198 and 223, respectively. The decrease in neutralizing 25 antibody titer in response to 2ΑΔ30 and rDEN4A30 is consistent with the attenuation phenotype of these vimses. Monkeys inoculated with either 2ΑΔ30 (n = 4) or wt 814669 vims (n = 8) were challenged after 42 days with wt vims 814669. Dengue vims was not detected in any serum sample collected for up to 10 days following vims challenge, indicating that these monkeys were completely protected following immunization with 30 either wt vims or vaccine candidate 2ΑΔ30.

Since DEN-4 814669, 2A wt, and rDEN4 each manifest the same level of replication and immunogenicity in rhesus monkeys, it is reasonable to conclude that the -69- identified sequence differences between these presumptive wild type viruses that arose during passage in tissue culture or during plasmid construction do not significantly affect their level of replication in vivo. Similarly, the comparable level of attenuation of 2ΑΔ30 and rDEN4A30 indicates that the mutations shared by these viruses, namely, the Δ30 5 mutation and its accompanying 10475 deletion mutation, are probably responsible for the attenuation of these viruses rather than their incidental sequence differences. 2015203575 26 Jun2015

Clinical Response to immunization with 2ΑΔ30. The 2ΑΔ30 vaccine candidate was administered subcutaneously at a dose of 10s PFU to 20 seronegative volunteers. Each of the vaccinees was infected and the virus was well tolerated by all vaccinees. Viremia 10 was detected in 70% of the vaccinees, was present only at low titer, and did not extend beyond day 11.

None of the 20 vaccinees reported soreness or swelling at the injection site. Mild erythema (1-3 mm) around the injection site was noted on examination of 8 volunteers 30 minutes post-vaccination which resolved by the next day in 7 of those volunteers and by the 15 third day in the remaining volunteer. Mild tenderness to pressure at the vaccination site was noted in 2 volunteers and lasted a maximum of 48 hours. During physical examination, ten volunteers (50%) were noted to a have a very mild dengue-like erythematous macular rash (truncal distribution) which occulted with greatest frequency on day 10. None of the volunteers noted the rash themselves, and it was asymptomatic in each 20 instance. Rash was seen only in vaccinees with detectable viremia. Volunteers did not develop systemic illness. Seven volunteers noted an occasional headache that was described as mild, lasting less than 2 hours, and was not present in any volunteer on two consecutive days. One volunteer reported fever of 38.6°C and 38.2°C without accompanying headache, chills, eye pain, photophobia, anorexia, myalgia, or arthralgia as 25 an outpatient the evening of day 3 and day 5, respectively. However, this volunteer was afebrile when evaluated by the study staff on the morning of days 3, 4, 5 and 6. All other temperature measurements recorded by the volunteer or study staff were normal. Although tourniquet tests were not performed, two volunteers were noted to have petechiae at the site of the blood pressure cuff after a blood pressure measurement was performed (one on day 30 6, the other on days 7 and 10). Both of these volunteers had normal platelet counts at that time and throughout the study. -70-

Significant hematological abnormalities were not seen in any vaccinee. Three vaccinees with presumed benign ethnic neutropenia manifested an absolute neutrophil count (ANC) below 1500/mm3. These three volunteers had baseline ANCs which were significantly lower than the remaining 17 volunteers and which did not decrease 5 disproportionately to the other volunteers. Two of the three volunteers who became neutropenic never had detectable viremia. A mild increase in ALT levels was noted in 4 volunteers, and a more significant increase in ALT level (up to 238 IU/L) was noted in one volunteer. These ALT elevations were transient, were not associated with hepatomegaly, and were completely asymptomatic in each of the 5 volunteers. Elevated ALT values 10 returned to normal by day 26 post-vaccination. The volunteer with the high ALT value was also noted to have an accompanying mild elevation in AST on day 14 (104 IU/L) which also returned to baseline by day 26 post-vaccination. This volunteer did not have an associated increase in LDH, bilirubin, or alkaline phosphatase levels. 2015203575 26 Jun2015

Serologic response of humans to immunization with 2ΑΔ30. Each of the twenty 15 vaccinees developed a significant rise in serum neutralizing antibody titer against DEN-4 by day 28. The level of serum neutralizing antibody was similar in viremic (1:662) and non-viremic vaccinees (1:426). The DEN-4 neutralizing antibody titers of both groups had not changed significantly by day 42.

Genetic stability of the Δ30 mutation. RT-PCR and sequence analysis of viral 20 RNA isolated from serum samples (n - 6) collected from volunteers 6 to 10 days postvaccination confirmed the presence of the Δ30 mutation and neighboring deletion at nucleotide 10475.

Example 9

Pharmaceutical Compositions 25 Live attenuated dengue virus vaccines, using replicated virus of the invention, are used for preventing or treating dengue virus infection. Additionally, inactivated dengue virus vaccines are provided by inactivating virus of the invention using known methods, such as, but not limited to, formalin or β-propiolactone treatment. Live attenuated or inactivated viruses containing the mutations described above form the basis of an imprnve.fl 30 vaccine for the prevention or treatment of dengue infection in humans.

Pharmaceutical compositions of the present invention comprise live attenuated or inactivated dengue viruses, optionally further comprising sterile aqueous or non-aqueous -71- solutions, suspensions, and emulsions. The composition can further comprise auxiliary agents or excipients, as known in the art. See, e.g., Berlcow et al. eds. 1987 The Merck Manual, 15th edition, Merck and Co., Rahway, N.J.; Goodman et al. eds. 1990 Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, 5 Inc., Elmsford, N.Y.; Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. 1987; Osol, A. ed. 1980 Remington’s Pharmaceutical Sciences Mack Publishing Co, Easton, Pa. pp. 1324-1341; Katzung, ed. 1992 Basic and Clinical Pharmacology Fifth Edition, Appleton and Lange, Norwalk, Conn. 2015203575 26 Jun2015 10 A virus vaccine composition of the present invention can comprise from about 102 - 109 plaque forming units (PFU)/ml, or any range or value therein, where the virus is attenuated. A vaccine composition comprising an inactivated virus can comprise an amount of virus corresponding to about 0.1 to 50 μg of E protein/ml, or any range or value therein. 15 The agents may be administered using techniques well known to those in the art.

Preferably, agents are formulated and administered systemically. Suitable routes may include oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intradermal, intranasal, or intraocular 20 injections, just to name a few. For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as saline, phosphate buffered saline, Tris buffered saline, Hank’s buffered saline, growth media such as Eagle’s Minimum Essential Medium (MEM), and the like.

When a vaccine composition of the present invention is used for administration to 25 an individual, it can further comprise salts, buffers, adjuvants, or other substances which are desirable for improving the efficacy of the composition. Adjuvants useful with the invention include, but are not limited to: (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; (2) oil-in-water emulsion formulations (with or without other specific immmiostimulating agents such as muramyl 30 peptides or bacterial cell wall components), such as for example (a) MF59 (International Publication No. WO 90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE, although not required) formulated -72- into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP either microfluidized into a submicron emulsion or vortexed to generate a larger particle size emulsion, and (c) Ribi™ adjuvant system (RAS), (Ribi Immunochem, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox™); (3) saponin adjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, MA) may be used or particle generated therefrom such as ISCOMs (immunostimulating complexes); (4) Complete Freunds Adjuvant (CFA) and Incomplete Freunds Adjuvant (IFA); (5) cytokines, such as interleukins (EL-1, IL-2, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) mucosal

C adjuvants such as those derived from cholera toxin (CT), pertussis toxin (PT), E. coli heat labile toxin (LT), and mutants thereof (see, e.g., International Publication Nos. WO 95/17211, WO 93/13202, and WO 97/02348); and (7) other substances that act as immunostimulating agents to enhance the effectiveness of the composition.

The pharmacologically active compounds of this invention can be processed in accordance with conventional methods of galenic pharmacy to produce medicinal agents for administration to patients, e.g., mammals including humans.

The compounds of this invention can be employed in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for parenteral, enteral (e.g., oral) or topical application, which do not deleteriously react with the active compounds. Suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions, alcohols, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, hydroxy methylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like which do not deleteriously react with the active compounds. They can also be combined where desired with other active agents, e.g., vitamins. -73-

For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, including suppositories. Ampoules are convenient unit dosages. 2015203575 26 Jun2015

For enteral application, particularly suitable are tablets, dragees, liquids, drops, 5 suppositories, or capsules. A syrup, elixir, or the like can be used wherein a sweetened vehicle is employed.

For topical application, there are employed as non-sprayable fonns, viscous to semi-solid or solid forms comprising a carrier compatible with topical application and having a dynamic viscosity preferably greater than water. Suitable formulations include but are not 10 limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, aerosols, etc., which are, if desired, sterilized or mixed with auxiliary agents, e.g., preservatives, stabilizers, wetting agents, buffers or salts for influencing osmotic pressure, etc. For topical application, also suitable are sprayable aerosol preparations wherein the active ingredient, preferably in combination with a solid or liquid inert carrier material, is 15 packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant, e.g., a freon.

The vaccine can also contain variable but small quantities of endotoxin, free formaldehyde, and preservative, which have been found safe and not contributing to the reactogenicity of Ihe vaccines for humans. 20 Example 10

Pharmaceutical Purposes

The administration of the vaccine composition may be for either a "prophylactic" or "therapeutic" purpose. When provided prophylactically, the compositions are provided before any symptom of dengue viral infection becomes manifest. The prophylactic 25 administration of the composition serves to prevent or attenuate any subsequent infection. When provided therapeutically, the live attenuated or inactivated viral vaccine is provided upon the detection of a symptom of actual infection. The therapeutic administration of the compound(s) serves to attenuate any actual infection. See, e.g., Berkow et al. eds. 1987 The Merck Manual, 15th edition, Merck and Co., Rahway, N.J.; Goodman et al. eds. 1990 30 Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y.; Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, -74-

Baltimore, Md. 1987; Katzung, ed. 1992 Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Conn. 2015203575 26 Jun2015 A live attenuated or inactivated vaccine composition of the present invention may thus be provided either before the onset of infection (so as to prevent or attenuate an 5 anticipated infection) or after the initiation of an actual infection.

The vaccines of the invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby live attenuated or inactivated viruses are combined in a mixture with a pharmaceutically acceptable vehicle. A composition is said to be a "pharmacologically acceptable vehicle" if its administration can 10 be tolerated by a recipient patient. Suitable vehicles are well known to those in the art, e.g., in Osol, A. ed. 1980 Remington's Pharmaceutical Sciences Mack Publishing Co, Easton, Pa. pp. 1324-1341.

For purposes of administration, a vaccine composition of the present invention is administered to a human recipient in a therapeutically effective amount. Such an agent is 15 said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant. A vaccine composition of the present invention is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient that generates a host immune response against at least one dengue serotype, stimulates the production of neutralizing antibodies, or leads to protection against 20 challenge.

The "protection" provided need not be absolute, i.e., the dengue infection need not be totally prevented or eradicated, if there is a statistically significant improvement compared with a control population or set of patients. Protection may be limited to mitigating the severity or rapidity of onset of symptoms of the dengue vims infection. 25 Example 11

Pharmaceutical Administration A vaccine of the present invention may confer resistance to one or more dengue serotypes by immunization. In immunization, an live attenuated or inactivated vaccine composition is administered prophylactically, according to a method of the present 30 invention. In another embodiment a live attenuated or inactivated vaccine composition is administered therapeutically, according to a different method of the present invention. -75-

The present invention thus includes methods for preventing or attenuating infection by at least one dengue serotype. As used herein, a vaccine is said to prevent or attenuate a disease if its administration results either in the total or partial attenuation (i.e., suppression) of a symptom or condition of the disease, or in the total or partial immunity of 5 the individual to the disease. 2015203575 26 Jun2015

At least one live attenuated or inactivated dengue virus, or composition thereof, of the present invention may be administered by any means that achieve the intended purpose, using a pharmaceutical composition as previously described.

For example, administration of such a composition may be by various parenteral 10 routes such as subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, intranasal, oral or transdermal routes. Parenteral administration can be by bolus injection or by gradual perfusion over time. A preferred mode of using a pharmaceutical composition of the present invention is by intramuscular, intradermal or subcutaneous application. See, e.g., Berlcow et al. eds. 1987 The Merck Manual 15th edition, Merck and 15 Co., Rahway, N.J.; Goodman et al. eds. 1990 Goodman and Gilman's The Pharmacological Basis of Therapeutics, 8th edition, Pergamon Press, Inc., Elmsford, N.Y.; Avery's Drug Treatment: Principles and Practice of Clinical Pharmacology and Therapeutics, 3rd edition, ADIS Press, LTD., Williams and Wilkins, Baltimore, Md. 1987; Osol, A. ed. 1980 Remington's Pharmaceutical Sciences, Mack Publishing Co, Easton, Pa. 20 pp. 1324-1341; Katzung, ed. 192 Basic and Clinical Pharmacology, Fifth Edition, Appleton and Lange, Norwalk, Comi. A typical regimen for preventing, suppressing, or treating a dengue virus related pathology, comprises administration of an effective amount of a vaccine composition as described herein, administered as a single treatment, or repeated as enhancing or booster 25 dosages, over a period up to and including between one week and about 24 months, or any range or value therein.

It will be appreciated that the actual preferred amounts of active compound in a specific case will vary according to the specific compound being utilized, the compositions formulated, the mode of application, and the particular situs and organism being treated. 30 Dosages for a given host can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the subject compounds and of a known agent, e.g., by means of an appropriate, conventional pharmacological protocol. -76-

The dosage of a live attenuated virus vaccine for a mammalian (e.g., human) subject can be from about 103 -107 plaque forming units (PFU)/kg, or any range or value therein. The dose of inactivated vaccine can range from about 0.1 to 50 pg of E protein. However, the dosage should be a safe and effective amount as determined by conventional methods, 5 using existing vaccines as a starting point. 2015203575 26 Jun2015

The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. -77-

Table 1. Susceptibility of mice to intracerebral DEN4 infection is age-dependenta

Virus Mean virus titer (log10PFU/g brain) ± SE following inoculation at indicated age (days) 7 14 21 2A-13 >6.0 4.0 ±0.2 3.1 ±0.2 rDEN4 >6.0 3.3 ± 0.4 3.3 ± 0.2 rDEN4A30 >6.0 3.6 ±0.2 2.8 ± 0.3 2015203575 26 Jun2015 a Groups of 4 or 5 Swiss Webster mice were inoculated intracerebrally with 10s PFU virus in a 30 μΐ inoculum. After 5 days, brains were removed, homogenized and titered in Vero cells. SE = Standard error. 2015203575 26Jun2015

Mean virus titer (logioPFU/ml) at indicated temp. (°C) Virus replication in suckling miceb

Phenotype Virus Vero cells HuH-7 cells Mean titer+ SE Mean log^o TABLE 2 Temperature-sensitive (to) and mouse brain attenuation (att) phenotypes of 5-FU mutant DEN4 viruses. 35 37 38 39 Δ&amp; 35 37 38 39 Δ n (logioPFU/g brain) reduction from wtd wt (not to) 2A-13 7.8 7.7 7.6 7.3 0.5 7.8 7.7 7.4 6.4 1.4 66 6.6 ±0.1° - rDEN4 6.5 6.4 6.4 6.0 0.5 74 6.7 6.0 5.5 1.6 66 6.1 ±0.1° rDEN4A30 6.3 6.1 6.1 5.7 0.6 6.9 6.3 5.9 4.7 2.2 64 5.6±0.1c 0.5 to in Vero and 695 6.2 6.0 5.2 24>e 3.6 6.5 5.5 3.8 <1.6 >4.9 6 3.0 ±0.2 3.2 HuH-7 cells 816 6.8 6.4 5.8 33 2.9 7.5 6.2 5.5 34 4.4 6 3.3 ±0.4 2.9 773 7.4 6.6 6.0 Μ 43 7.7 64 5.2 11 4.6 12 3.7 ±0.1 2.6 489 7.3 6.6 6.1 33 4.0 7.3 6.7 5.4 10 4.3 6 4.5 ±0.5 2.3 173 7.0 6.1 32 23 4.1 7.0 T2 10 24 4.9 18 4.7 ±0.4 2.2 509 6.2 5.8 5.5 34 2.8 6.5 6.1 4.5 <1.6 >4.9 6 4.9 ±0.3 1.9 938 7.1 6.5 5.6 li 4.0 7.2 6.4 5.6 34 4.1 6 5.1 ±0.2 1.7 1033 6.7 6.0 5.9 44 2.6 6.9 5.6 4.7 <1.6 >5.3 12 4.7 ±0.2 1.7 239 7.6 6.8 5.6 3J 4.3 7.6 6.7 4.7 15 5.1 12 4.7 ±0.3 1.5 793 6.5 5.8 5.3 40 2.5 7.2 6.8 5.6 <1.6 >5.6 6 5.4 ±0.3 1.4 759 7.2 6.9 6.4 47 2.5 7.5 6.8 6.3 !! 4.4 12 5.1 ±0.1 1.4 718 6.1 5.9 5.3 33 2.6 7.0 6.5 5.7 L7 5.3 12 5.0 ±0.3 1.4 473 6.7 6.3 5.4 23 4.7 7.2 6.7 17 13 5.3 12 5.1 ±0.3 1.2 to in only 686 7.0 6.7 6.7 6.4 0.6 7.3 6.8 6.4 2.2 5.1 12 2.7 ±0.2 3.8 HuH-7 cells 967 6.8 6.4 6.4 5.1 1.7 6.8 6.4 5.4 <1.6 >5.2 6 3.6 ±0.2 2.9 992 7.3 7.1 6.8 5.9 1.4 7.4 6.9 5.0 <1.6 >5.8 6 3.8 ±0.1 2.7 571 6.9 7.0 6.4 4.6 2.3 7.0 6.3 5.2 <1.6 >5.4 6 4.4 ±0.4 2.4 605 7.6 7.5 7.1 6.9 0.7 7.8 7.2 6.8 <1.6 >6.2 12 4.5 ±0.4 2.1 631 7.1 6.9 6.8 5.0 2.1 7.3 7.1 6.5 <1.6 >5.7 12 4.8 ±0.3 1.9 1175 7.4 7.1 6.9 5.3 2.1 7.6 6.5 4.7 13 4.3 12 4.7 ±0.2 1.7 2015203575 26 Jun2015

Reduction in titer (log10PFU/ml) at 39°C compared to titer at permissive temperature (35°C).

Groups of 6 suckling mice were inoculated i.c. with 104 PFU virus in a 30 μΐ inoculum. Brains were removed 5 days later, homogenized, and titered in Vero cells..

Average of 11 experiments with a total of 64 to 66 mice per group.

Determined by comparing mean viral titers of mice inoculated with mutant virus and the 2A-13 wt control in the same experiment (n= 6 or 12).

Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temp when compared to titer at permissive temp (35°C).

Table 3. Nucleotide and amino acid differences of the 5-FU mutant viruses which are ts in both Vero and HuH-7 cells. 2015203575 26 Jun2015

Mutations in UTR or coding region that result in Mutations in coding region that do an amino acid substitution not result in an amino acid

Virus substitution

Nucleotide position Gene/ region Nucleotide Amino Acid change change^3 Nucleotide position Gene Nucleotide change 173a 7163 NS4B A> C L2354F 10217 NS5 A > U 7849 NS5 A> U N2583I 8872 NS5 A>G K2924R 23 9a 4995 NS3 U>C S1632P 7511 NS4B G> A 10070 NS5 U>C 473 a 4480 NS2B u>c V1460A 7589 NS5 G> A 4995 NS3 u>c S1632P 10070 NS5 U>C 489a 4995 NS3 u>c S1632P 2232 E U>C 3737 NS2A C>U 509a 4266 NS2B A>G S1389G none 8092 NS5 a>g E2664G 695 40 5’UTR u>c n/a 1391 E A>G 1455 E g>u V452F 6106 NS3 A> G E2002G 7546 NS4B c>u A2482V 718 2280 E u>c F727L none 4059 NS2A A>G 11320V 4995 NS3 U>c S1632P 7630 NS5 A>G K2510R 8281 NS5 U>C L2727S 759a 4995 NS3 U>C S1632P none 8020 NS5 A>U N2640I 773a 4995 NS3 U>C S1632P none 793 1776 E G>A A559T 5771 NS3 U>C 2596 NS1 G> A R832K 7793 NS5 U> A 2677 NS1 A>G D859G 4387 NS2B C>U S1429F 816a 4995 NS3 U >C S1632P 6632 NS4A G> A 7174 NS4B c> u A2358V 6695 NS4A G> A 938a 3442 NS1 A>G E1114G 747 prM U>C 4995 NS3 U>c S1632P 4196 NS2b U>C -81-

10275 3'UTR A>U n/a 6155 NS3 G> A 1033a 4907 NS3 A>U L1602F 548 prM C>U

8730 NS5 A>C N2877H

9977 NS5 G> A M3292I a Viruses that contain mutation(s) resulting in an a.a. substitution in only a NS gene(s) and/or nucleotide substitutions in the UTRs are indicated; i.e. no a.a. substitutions are present in the structural proteins (C-prM-E). 2015203575 26 Jun2015 b Amino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nt 102-104) as residue #1. Wild-type amino acid on left of amino acid position; mutant amino acid, on right. -82- 2015203575 26 Jun2015

Table 4. Nucleotide and amino acid differences of the 5-FU mutant viruses which are ts in only HuH-7 cells. Mutations in UTR or coding region that result in an Mutations in coding region that do • amino acid substitution not result in an amino acid Virus substitution Nucleotide position Gene/ region Nucleotide change Amino acid changeb Nucleotide position Gene Nucleotide change 571 586 prM U>C VI62 A 6413 NS4A U>C 7163 NS4B A> U L2354F 7947 NS5 G> A G2616R 605 1455 E G> U V452F none 7546 NS4B C>U A2482V 631 595 prM A> G K165R 1175 E G> A 6259 NS3 U>C V2053A 5174 NS3 A>G 7546 NS4B c>u A2482V 686a 3575 NS2A G> A Ml 1581 4604 NS3 A>G 4062 NS2A A> G T1321A 7937 NS5 A> G 7163 NS4B A>U L2354F 967 2094 E G> C A665P 4616 NS3 C>U 2416 E u>c V772A 7162 NS4B u>c L2354S 7881 NS5 G> A G2594S 992a 5695 NS3 A> G D1865G 3542 NS2A A>G 7162 NS4B u>c L2354S 1175a 7153 NS4B u>c V2351A 6167 NS3 U>C 10186 NS5 u>c I3362T 10184 NS5 G> A 10275 3'UTR A > U n/a a Viruses that contain mutation(s) resulting in an a.a. substitution in only a NS gene(s) and/or nucleotide substitutions in the UTRs are indicated; i.e. no a.a. substitutions are present in the structural proteins. b Amino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nt 102-104) as residue #1. Wild-type amino acid on left of amino acid position; mutant amino acid on right. -83- 2015203575 26 Jun2015 TABLE 5. Mutations which are represented in multiple 5-FU mutant DEN4 viruses. Nucleotide position Gene/ region Nucleotide change Amino acid change Number of viruses with "sister" mutations 1455 E G>U val > phe 2 4995 NS3 U>C ser > pro 8 7162 NS4B U>C leu > ser 2 7163 NS4B A > U or C leu > phe 3 7546 NS4B C>U ala > val 3 10275 3'UTR A>U n/aa 2 a not applicable -84- 2015203575 26Jun2015

Mean virus titer (log10PFU/ml) at indicated temp (°C) Replication in suckling miceb

Table 6. Addition of ts mutation 4995 to rDEN4A30 confers a ts phenotype and further attenuates its replication in suckling mouse brain.

Virus Vero cells HuH-7 cells Mean virus titer ± SE Meanlog10 35 37 38 39 Δ2 35 37 38 39 Δ (l°g10PFU/g brain) reduction from wf 2A-13 7.1 7.1 6.9 6.8 0.3 7.4 7.3 6.7 6.4 1.0 6.5 ±0.1 - rDEN4 7.0 6.8 6.6 6.4 0.6 7.5 7.3 6.7 6.4 1.1 6.1 ±0.2 - rDEN4A30 7.0 6.7 6.2 6.2 0.8 7.5 7.0 6.5 5.1 2.4 5.9 ±0.1 0.2 rDEN4-4995 5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >4.8 3.2 ± 0.2 2.9 rDEN4A30- 5.9 4.9 3.9 <1.6d >4.3 6.4 5.6 4.4 <1.6 >4.8 3.0 ± 0.3 3.1 4995 a Reduction in titer (log10PFU/ml) at 39°C compared to titer at permissive temperature (35°C). b Groups of 6 suckling mice were inoculated i.c. with 104 PFU virus in a 30 μΐ inoculum. Brains were removed 5 days later, homogenized, and titered in Vero cells. The limit of detection is 2.0 logi0PFU/g brain. c Determined by comparing mean viral titers of mice inoculated with sample virus and rDEN4 control. d Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temperature when compared to permissive temperature. 2015203575 26 Jun2015

Table 7. Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypes of 5-FU DEN4 mutant viruses which exhibit a small plaque (sp) phenotype.

Phenotype Mean virus titer (logI0PFU/ml) at indicated temp (°C) Replication in suckling miceb sp ts Virus Vcro cells HuH-7 cells Mean virus titer + SE Mean logi0

Vero HuH-7 Vero HuH-7 35 37 38 39 Aa 35 37 38 39 Δ n (log10PFU/g brain) reduction from wr — — — — 2A-13 7.9 7.5 7.7 7.2 0.7 7.9 7.7 7.3 6.9 1.0 66 6.6±0.1c — — — — — rDEN4 7.9 7.6 7.7 7.3 0.6 8.1 7.6 7.5 6.7 1.4 66 6.1 ±0.1c — — — — — rDEN4A30 7.3 6.6 6.6 6.1 1.2 7.3 7.2 6.9 5.9 1.4 64 5.6 ±0.1c 0.5 + + + + 574 6.6* 5.5 M <1.6e >5.0 6.6X 4.9 5.0 <1.6 >5.0 6 2.1 ±0.1 5.1 + + + + 1,269 5.3X 4.8 3.9 <1.6 >3.7 4.0X 2.4 2.0 <1.6 >2.4 6 2.7 ± 0.2 4.1 + + + + 1,189 6.3X 5.2 4.5 3JS 2.5 5.5X 3.7 2.3 <1.6 >3.9 12 3.2 ± 0.4 3.7 + + _ _ 569 5.8X 5.6 5.6 3.7 2.1 6.2X 6.0 5.7 5.0 1.2 12 1.9 ±0.1 4.6 + + — — 761 5.0X 4.7 4.2 2.7 2.3 5.6X 5.3 4.5 2.6 3.0 12 2.0 ±0.1 4.2 _ + + + 506 7.0 6.8 5.6 2J> 4.4 6.T 4.3 <1.6 24) 4.7 6 2.2 ±0.1 4.7 — + + + 1,136 5.1 4.2 245 <1.6 >3.5 5.T 3.0 3.0 <1.6 >4.1 6 2.9 ± 0.3 4.5 — + + + 1,029 6.9 5.8 5.8 Z9 4.0 7.0X 5.8 5.2 11 4.5 6 2.2 ±0.1 4.2 — + + + 1,081 6.9 5.8 4.7 3J) 3.0 5.8X 4.1 3.3 12 3.9 12 2.6 ±0.2 3.9 — + + + 529 6.9 6.5 5.9 44) 2.9 7.1x 5.3 4.4 <1.6 >5.5 6 3.1 ±0.7 3.8 — + + + 1,114 6.7 6.4 6.2 23 4.2 5.T 3.0 2.9 12 3.8 6 2.7 ±0.3 3.7 — + + + 922 7.3 7.2 6.8 3J5 3.5 7.4X 5.3 4.1 34) 4.4 12 3.5 ±0.1 2.9 — + + + 311 6.9 5.9 43 L5 5.4 7.Γ 5.4 3^6 <1.6 >5.5 12 6.1 ±0.3 0.9 — + + + 326 6.6 5.7 4.5 3Λ 3.5 7.0X 5.5 4.1 11 5.0 6 6.0 ±0.1 0.9 _ + _ + 1,104 7.1 6.8 6.8 6.1 1.0 Ί.Τ 6.4 5.8 23 4.4 6 2.2 ± 0.1 4.7 — + — + 952 7.1 7.0 6.7 5.6 1.5 13x 6.3 5.6 34) 4.3 6 2.4 ±0.3 4.5 — + — + 738 6.5 6.0 5.9 5.7 0.8 6.9X 6.1 5.0 11 3.8 12 4.4 ±0.4 2.3 — + — + 1,083 7.4 7.3 7.4 5.8 1.6 7.4X 6.6 4.5 <1.6 >5.8 12 4.5 ±0.4 2.0 _ + _ _ 1,096 7.5 7.1 6.9 5.5 2.0 7.5X 6.6 5.6 4.8 2.7 6 2.9 ±0.2 3.5 — + — — 1,021 7.0 6.9 6.6 6.3 0.7 6.9X 5.7 4.4 4.0 2.9 6 3.9 ±0.6 2.6 2015203575 26Jun2015 — + — — 1,023 6.6 6.4 6.0 5.8 0.8 6.P 5.6 4.7 3.3 2.8 12 4.2 ±0.3 2.3 — + — — 1,012_7.5 7.1 7,0 5.7 1.8 7.4* 6,8 6.8 5.6 1.8 6_6.1 ±0.1_0.8 a Reduction in mean virus titer (log10PFU/ml) at 39°C compared to permissive temperature (35"C). b Groups of 6 suckling mice were inoculated i.c. with 104 PFU virus. Brains were removed 5 days later, homogenized, and titered in Vero cells. 0 Average of 11 experiments with a total of 64 to 66 mice per group. d Determined by comparing mean viral titers of mice inoculated with mutant virus and concurrent 2A-13 wild type (wi) virus control (n = 6 or 12).

Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temperature when compared to permissive temperature (35°C). x Small plaque size at 35°C; small plaques have a diameter of <1.0 mm compared to wild type plaque diameter of 1.5 - 2.0 mm in Vero cells, or a diameter of <0.4 mm compared to wild type plaque diameter of 0.75 to 1.0 mm in HuH-7 cells.

Table 8. Viruses with both ts and sp phenotypes are more restricted in replication in mouse brain than those with only a is phenotype.

Cell culture Number Mean logJ0 reduction in virus titer phenotype of viruses from controlb’0 tsa 20 2.1 ±0.2 sp 6 3.0 ±0.6 ts / sp 16 3.5 ±0.3 2015203575 26 Jun2015 a 20 ts mutant viruses without an sp phenotype were previously described (Example 1). b Determined by comparing mean viral titers of groups of mice inoculated with mutant virus and concurrent 2A-13 parallel-passaged control virus. c Significant difference between is group and ts /sp group, Tukey-Kramer test (P < 0.05) -88- 2015203575 26 Jun2015

Table 9. Nucleotide and amino acid differences of the 5-FU mutant DEN4 viruses which produce small plaques in both Vero and HuH-7 cells. Virus Mutations in UTR or in coding regions that result in an amino acid substitution Mutations in coding regions that do not result in an amino acid substitution Nucleotide position Gene/ region Nucleotide change Amino acid change^ Nucleotide Gene Nucleotide position change 569 826 prM G> A R242K 1946 E C>U 832 prM C>U P244L 7546 NS4B C>U A2482V 10275 3'UTR A>U n/a 10279 3'UTR A>U n/a 574 1455 E G>U V452F 1349 E C>U 1963 E U>C V621A 3880 NS2A A>G K1260R 7546 NS4B C>U A2482V 7615 NS5 A> G N2505S 10413 3'UTR A>G n/a 761 424 C U>C I108T none 2280 E U>C F727L 7131 NS4B A> G T2344A 7486 NS4B A>G N2462S 1189a 3303 NS1 A>G R1068G 6719 NS4A U>C 4812 NS3 G> A V1571I 5097 NS3 G> A D1666N 7182 NS4B G> A G2361S 1269 2112 E U>C F671L 542 prM C>U 3256 NS1 G> A G1052E 3993 NS2A U>C F1298L 7183 NS4B G > U G2361V a Virus contains missense mutations in only the non-structural genes. b Amino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nt 102-104).

Wild type amino acid on left of amino acid position; mutant amino acid on right. -89- 2015203575 26 Jun2015

Table 10. Nucleotide and amino acid differences of the 5-FU mutant DEN4 viruses which produce small plaques in only HuH-7 cells. Vims Mutations in UTR or in coding regions that result in an amino acid substitution Mutations in coding regions that do not result in an amino acid substitution Nucleotide Gene/ position region Nucleotide change Amino acid change^ Nucleotide position Gene Nucleotide change 311 1519 E A> G N473S 6761 NS4A C>U 2305 E G> A R735K 10070 NS5 U>C 4896 NS3 G>U A1599S 326 1587 E C>U P496S 1523 E G> A 7546 NS4B C>U A2482V 6080 NS3 U>C 10070 NS5 U>C 506 1455 E G > U V452F 3887 NS2A A> G 1902 E G> A V601M 5789 NS3 G > C 7546 NS4B C>U A2482V 10275 3'UTR A>U n/a 529 777 prM U>C S226P none 4641 NS3 A>G 11514V 7153 NS4B U>C V2351A 8245 NS5 U>C I2715T 10279 3'UTR A> C n/a 738a 3540 NS2A G> A E1147K none 7162 NS4B U>C L2354S 922a 4306 NS2B A> G N1402S 7736 NS5 G> A 5872 NS3 C>U T19241 7163 NS4B A>U L2354F 10279 3'UTR A> C n/a 952 1449 E G>U V450L none 1455 E G > U V452F 7546 NS4B C>U A2482V 7957 NS5 U>C V2619A 9543 NS5 A>G 13148V 1012 1542 E A> G K481E 953 E A> G 7162 NS4B U>C L2354S 1205 E G> A 10542 3'UTR A> G n/a 4425 NS2B U>C a Vimses that contain missense mutations in only the non-structural genes and/or mutations in the UTRs. b Amino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nt 102-104).

Wild type amino acid on left of amino acid position; mutant amino acid on right. -90- 2015203575 26 Jun2015 TABLE 10. Continued Mutations in UTR or in coding regions that Mutations in coding regions that do result in an amino acid substitution not result in an amino acid substitution Nucleotide position Gene/ region Nucleotide change Amino acid change^ Nucleotide position Gene Nucleotide change 1021 2314 E U>C I738T 665 prM C > A 3205 NS1 C>U A1035V 5750 NS3 C>U 4029 NS2A U>C C1310R 9959 NS5 C>U 7163 NS4B A>C L2354F 10275 3'UTR A>U n/a 10279 3'UTR A>U n/a 1023 2283 E G> A G728R 1001 E C>U 7182 NS4B G>A G2361S 1958 E A> G 3873 NS2a U>C 8486 NS5 C>U 1029 850 prM C>U A250V 3867 NS2a C>U 3087 NS1 A>G T996A 4891 NS3 U>C I1597T 1081a 2650 NS1 A>G N850S 6326 NS3 C>U 7163 NS4B A>U L2354F 9146 NS5 C>U 1083a 3702 NS2A G> A A1201T 3353 NS1 A> G 7153 NS4B U>C V2351A 6155 NS3 G> A 10634 3'UTR u>c n/a 1096 892 prM G> A R264Q 665 prM C>A 7163 NS4B A>C L2354F 4427 NS2b G> A 8659 NS5 C>U P2853L 1104 1692 E G> A V531M none 5779 NS3 C>U A1893V 7546 NS4B C>U A2482V 1114 709 prM A> G K203R 1076 E U>C 3693 NS2A A> G 11198V 1182 E c>u 4614 NS3 U>C F1505L 5690 NS3’ c>u 7546 NS4B C>U A2482V 9942 NS5 A>G T3281A 1136a 3771 NS2A A> G R1224G 5621 NS3 A> G 4891 NS3 U>C I1597T 10275 3’UTR A > U n/a a Viruses that contain missense mutations in only the non-structural genes and/or mutations in the UTRs. b Amino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nt 102-104). Wildtype amino acid on left of amino acid position; mutant amino acid on right. -91- 2015203575 26 Jun2015

Table 11. Putative Vero cell adaptation mutations derived from the full set of 5-FU mutant viruses. Nucleotide position Gene / region -(a. a. #)b 5-FU mutant viruses Nucleotide change Amino acid change No. of viruses with the mutation 1455 E (452) G>U Val>Phe 5 2280 E (727) U>C Phe > Leu 2 4891 NS3 (1597) U>C lie > Thr 2 4995 NS3 (1599) U>C Ser > Pro 8 7153 NS4B (2351) U>C Val> Ala 3 7162 NS4B (2354) U>C Leu > Ser 4 7163 NS4B (2354) A > U or C Leu > Phe 7 7182 NS4B (2361) G> A Gly > Ser 2 7546 NS4B (2482) C>U Ala> Val 10 7630 NS5 (2510) A> G Lys > Arg 1 10275 3'UTR A > U n/aa 6 10279 3'UTR A > C n/a 4 a not applicable b Amino acid position in DEN4 polyprotein beginning with tire methionine residue of the C protein (nt 102-104) as residue #1. -92- 2015203575 26 Jun2015

Tabie 12. Mutagenic oligonucleotides used to generate recombinant DEN4 viruses containing single 5-FU mutations.

SEQID NO. Recombinant virus (rDEN4-) Nucleotide change Amino acid change Gene pUC clone RE sitea Oligonucleotide^ 23 40 U>C n/a 5'UTR pUC-Mel BsaWl CAGTTCCAAAcCGGAAGCTTG 24 2650 A> G Asn > Ser NS1 pUC-NSl BsiWl CCAACGAGCTAtcsTAcGTTCTCTGGG 25 3303 A> G Arg > Gly NS1 pUC-NSl StyI GATTGTGACCATeGcGGCCCATCTTTG 26 3442 A> G Glu > Gly NS1 pUC-NSl Blpl GGAGATTAGGCCscTGAGcGgtAAAGAAGAG 27 3540 G> A Glu > Lys NS2A. pUC-NSl Bsml GTTTGTGGAAaAATGtcTGAGGAGAA 28 3575 G> A Met > lie NS2A pUC-NSl Sspl CTAGGAAACACAT aATATTAGTTGTGG 29 3702 G> A Ala>Thr NS2A pUC-NS2A BglL CAGATCCACCTAaCCAT aATGGCAGTG 30 3771 A> G Arg > Gly NS2A pUC-NS2A Aval GGAAACTCACcTCssGAGAGACAGC 31 4059 A> G lie > Val NS2A pUC-NS2A BstEll TTGGGTAGAeeTcACcGCACTCATCC 32 4062 A> G Thr> Ala NS2A pUC-NS2A BsrBI GTAGAAATAeCcGCtCTCATCCTAG 33 4266 A> G Ser > Gly NS2B pUC-NS2A SnaBl GGCGGCTTACGTaATGgGaGGTAGCTCAGC 34 4306 A> G Asn > Ser NS2B pUC-NS2A AlwNl CTAGAGAAGGCaGCttctGTGCAGTGG 35 4480 U>C Val > Ala NS2B pUC-NS2A Mscl CCTTGGCcATTCCAGcaACAATGAC 36 4812 G> A Val > lie NS3 PUC-NS2A Apol GACGTTCAaaTttTaGCCATAGAACC 37 4891 U>C Ile>Thr NS3 pUC-NS2A Kasl CTGGAGAAAceGGcGCcGTAACATTAG 38 4896 G>U Ala > Ser NS3 pUC-NS2A BstEll GAAATTGGAtCsGTAACcTTAGATTTC 39 4907 A>U Leu > Phe NS3 PUC-NS2A AcR GGAGCAGTAACeTTtGATTTCAAACCC 40 4995 U>C Ser > Pro NS3 pUC-NS2A BsaJl GTTACCAAAcCtGGsGATTACGTC 41 5097 G> A Asp > Asn NS3 pUC-NS3 BspEl GATTAACTATcATGaACTTACACCC 42 5695 A>G Asp > Gly NS3 pUC-NS3 Baril GGAAAACCTTTGecACcGAGTATCC 43 5872 C>U Thr>ne NS3 pUC-NS3 BsrFl TCCAGTGAtaCCeGCtAGCGCTGCTC 44 6106 A> G Glu > Gly NS3 pUC-NS3 Mscl GCCTCAGAGGtGecCAAAGGAAG 45 6259 U>C Val > Ala NS3 pUC-NS3 BglΠ AC ATGGAGGcaGAsAT cTGGACT AGA 46 7153 U>C Val > Ala NS4B pUC-NS4A Mscl AAAGCATGeCcAAGGATGCTGTC 2015203575 26Jun2015

47 7162 u>c Leu > Ser NS4B pUC-NS4A Blpl GCATAATGGACectAAGCATGACTAAGG 48 h 7163 A>C Leu > Phe NS4B pUC-NS4A ApaLl TTATTGCATAeTGcACffAAAAGCATG 49 7174 C>U Ala > Val NS4B pUC-NS4A BsaAl GGGCCTATTATTaCeTAATGGAC 50 7182 G > A Gly > Ser NS4B pUC-NS4A n/a CTGCAATCCTGGtgaTATTATTGC 51 7546 C>U Ala > Val NS4B pUC-NS5A AcB. CTCATAAAGAAcGttCAAACCCT 52 7630 A>G Lys > Arg NS5 pUC-NS5A Hgal C ATTAGAC AGAcucG AGTTT GAAG 53 7849 A>U Asn > lie NS5 pUC-NS5A Hgal TGGCGACeCTCAAGAtaGTGACTGAAG 54 8020 A > U Asn>He NS5 pUC-NS5A Clal GAGTCATCaTCeAtaCCAACAATAG 55 r 8092 A> G Glu > Gly NS5 pUC-NS5A EcoBA CTTCAAAACCTGgcTTCTGCATCAAAG 56 8281 U>C Leu > Ser NS5 pUC-NS5B Xmril CAAAGATGTTGagcAACAGGTTCACAAC 57 8730 A> C Asn > His NS5 pUC-NS5B Aval GGAAAGAAGAAAcAcCCeAGACTGTGC 58 8872 A> G Lys > Arg NS5 pUC-NS5B Pvul GGGAACTGGTcGAtcsAGAAAGGGC 59 9977 G> A Met > lie NS5 pUC-NS5C Sfcl CCAGTGGATtACtACaGAAGATATGCTC 60 10186 U>C He > Thr NS5 pUC-NS5C AgeI CAGGAACCTGAcCGGtAAAGAGGAATACG 61 10275 A >U n/a 3'UTR pUC-NS5C n/a CT GTAATTACC AACAtCAAACACCAAAG 62 10279 A > C n/a 3'UTR pUC-NS5C n/a CC AACAAC AAcC ACCAAAGGCTATTG 63 10634 U>C n/a 3'UTR pUC-3TJTR n/a GGATTGGTGTTGTcGATCCAACAGG

Primers were engineered which introduced (underline) or ablated (hatched line) translationally-silent restriction enzyme sites.

Lowercase letters indicate nt changes and bold letters indicate the site of the 5-FU mutation, which in some oligonucleotides differs from the original nucleotide substitution change in order to create a unique restriction enzyme site. The change preserves the codon for the amino acid substitution. 2015203575 26Jun2015

TableE 13. sp, ts and mouse attenuation phenotypes of rDEN4 mutant viruses encoding single mutations identified in six sp 5-FU mutant viruses. 5-FU mutant virus

Virus

Gene/ region containing mutation

Mean virus titer (logjoPFU/ml) at _indicated temp (°C)_

Replication in suckling mice^

Replication in HuH-7-SCID micc^

Vero cells 35 39 Aa

Mean virus Mean logio-umt Mean peak virus Meanlogio-unit HuH-7 cells n titer ± SE reduction from n titer ± SE reduction from (log io PFU/ml value for wtc 35 39 (logioPFU /g brain) value for wt° serum) 2A-13 rDEN4 rDEN4A30 7.6 7.6 7.6 7.1 6.8 6.9 0.5 0.8 0.7 7.8 8.0 7.7 6.6 6.7 5.6 1.2 1.3 2.1 30 54 30 6.5 ±0.1 5.8 ±0.1 5.6 ±0.1 0.2 29 32 18 6.8 ±0.2 6.3 ± 0.2 5.4 ±0.2 0.9 738 parent 6.5 5.7 0.8 x6.9 3Te 3.8 12 4.4 ±0.4 2.3 9 5.4 ± 0.7 1.9 rDEN4-3540 NS2A 6.9 5.1 1.8 7.4 12 3.7 12 4.1 ±0.3 1.7 5 6.1 ±0.3 (+)0-1 rDEN4-7162 NS4B 7.2 6.8 0.4 7.4 6.6 0.8 8 5.6 ±0.3 0.3 5 6.8 ±0.6 0.3 922 parent 7.3 M 3.5 x7.4 34) 4.4 12 3.5 ±0.1 2.9 6 6.2 ± 0.2 0.4 rDEN4-4306 NS2B x5.0 22 2.8 x5.6 <1.6 >4.0 12 1.7 ±0.1 4.1 5 5.2 ±0.6 1.1 rDEN4-5872 NS3 5.7 22 3.2 x6.5 <1.6 >4.9 12 4.5 ±0.3 1.3 5 6.2 ±0.5 0.1 rDEN4-7163 NS4B 7.8 7.2 0.6 8.0 7.4 0.6 6 6.2 ±0.2 (+)0.1 6 5.8 ±0.6 (+)0.2 rDEN4- 10279 3'UTR 6.9 5.7 1.2 7.7 5.7 2.0 6 4.8 ±0.2 0.7 4 6.7 ±0.2 0.4 1081 parent 6.9 33 3.0 x5.8 M 3.9 12 2.6 ±0.2 3.9 4 4.2 ±0.5 2.4 rDEN4-2650 NS1 5.1 3.0 2.1 x5.5 2.8 2.7 12 3.0 ±0.3 2.8 6 4.7 ±0.5 2.2 rDEN4-7163 NS4B 7.8 7.2 0.6 8.0 7.4 0.6 6 6.2 ± 0.2 (+)0-1 6 5.8 ±0.6 (+)0.2 1083. parent 7.4 5.8 1.6 x7.4 <1.6 >5.8 12 4.5 ±0.4 2.0 9 4.4 ±0.3 2.9 rDEN4-3702 NS2A 6.8 5.6 1.2 7.6 4.7 2.9 18 4.9 ±0.3 0.9 7 6.3 ±0.3 0.2 rDEN4-7153 NS4B 7.7 7.2 0.5 8.0 6.9 1.1 6 5.7 ± 0.1 0.2 4 5.9 ±0.7 0.1 rDEN4- 10634 3'UTR 4.9 JL6 3.3 x5.7 <1.6 >4.1 12 2.4 ±0.3 3.4 7 3.3 ±0.4 3.6 2015203575 26Jun2015 parent 5.1 <1.6 >3.5 x5.7 <1.6 >4.1 6 2.9 ±0.3 4.5 7 4.5 ±0.4 1.2 rDEN4-3771 NS2A 7.0 4.6 2.4 x7.6 3J 3.9 12 2.6 ±0.4 3.2 4 6.4 ±0.2 (+)0.1 rDEN4-4891 NS3 7.1 <1.6 >5.5 x7.4 <1.6 >5.8 12 2.5 ±0.3 3.5 6 6.0±0.5 0.3 rDEN4- 10275 3'UTR 6.9 5.8 1.1 7.1 5.2 1.9 6 5.0 ±0.3 0.5 4 6.7 ± 0.3 0.4 parent x6.3 M 2.5 x5.5 <1.6 >3.9 12 3.2 ±0.4 3.7 13 2.3 ±0.3 3.8 rDEN4-3303 NS1 6.1 4.8 1.3 6.6 3.9 2.7 8 5.7 ±0.4 0.2 4 6.3 ±0.3 0.8 rDEN4-4812 NS3 7.0 6.3 0.7 7.1 6.3 0.8 12 4.8 ±0.2 1.0 5 6.1 ±0.5 (+)0.5 rDEN4-5097 NS3 x5.0 <1.6 >3.4 M.6 <1.6 >3.0 12 1.8 ±0.1 4.0 8 1.9 ±0.1 4.3 rDEN4-7182 NS4B 7.7 6.9 0.8 7.8 6.8 1.0 6 6.2 ±0.1 (+)0.1 6 6.3 ±0.3 (+)0.7 a Reduction in mean vims titer (Iogi0PFU/ml) at 39°C compared to permissive temperature (35°C). b Groups of 6 suckling mice were inoculated i.c. with 104 PFU of vims. Brains were removed 5 days later, homogenized, and titered in Vero cells. c Comparison of mean vims titers of mice inoculated with mutant virus and concurrent DEN4 control. Bold denotes >50- or >100-fold decrease in replication in suckling or SCID-HuH-7 mice, respectively. d Groups of HuH-7-SCID mice were inoculated directly into the tumor with 104 PFU vims. Serum was collected on day 6 and 7 and titered in Vero cells. e Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temp when compared to permissive temp (35°C). x Small plaque size at 35°C; small plaques have a diameter of <1.0 mm compared to wild type plaque diameter of 1.5 - 2.0 mm in Vero cells, or a diameter of <0.4 mm compared to wild type plaque diameter of 0.75 to 1.0 mm in HuH-7 cells. 2015203575 26 Jun2015

Table 14. Phenotypes of rDEN4 mutant viruses encoding single mutations identified in 10 5-FU mutant viruses that are ts in both Vero and HuH-7 cells.

Mean virus titer (log,0PFU/ml) at indicated temp (°C) Replication in 7-day miceb Replication m HuH-7-SCID 5-FU mutant viruses rDEN4-Mutation (nt position) Gene/ - Vero cells HuH-7 cells n Mean log10 reduction from (log10PFU/g brain) n Mean logI0 reduction from wf (log10PFU/ml serum) 35 37 39 39 Δ* 35 37 38 39 Δ 239,489 parent 7.6 6.8 5.6 33e 4.3 7.6 6.7 4.7 25 5.1 30 2.1 6 0.3 773 4995f NS3 5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >4.8 6 2.9 473 parent 6.7 6.3 5.4 2ti 4.7 7.2 6.7 3J L9 5.3 12 1.2 8 (+)0.3 4480 NS2B 6.7 6.3 6.0 5.7 1.0 7.6 7.2 6.0 5.2 2.4 6 0.7 4995f NS3 5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >4.8 6 2.9 759 parent 7.2 6.9 6.4 42 2.5 7.5 6.8 6.3 3d 4.4 12 1.4 5 (+)0.4 4995f NS3 5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >4.8 6 2.9 8020 NS5 7.1 6.6 6.7 5.9 1.2 7.4 7.1 6.1 5.4 2.0 6 0.5 816 parent 6.8 6.4 5.8 3$ 2.9 7.5 6.2 5.5 3JL 4.4 6 2.9 6 0.4 4995f NS3 5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >4.8 6 2.9 7174 NS4B 6.9 7.1 6.9 6.1 0.8 7.5 7.2 7.1 5.6 1.9 6 0.6 938 parent 7.1 6.5 5.6 3d 4.0 7.2 6.4 5.6 3d 4.1 6 1.7 6 0.5 3442 NS1 5.1 3.6 4.3 2d 3.0 5.9 4.9 3.9 <1.6 4.3 6 4.1 4995f NS3 5.7 4.9 3.6 <1.6 >4.1 6.4 5.7 4.0 <1.6 >4.8 6 2.9 10275 3' 6.9 6.4 6.4 5.8 1.1 7.1 6.8 7.1 5.2 1.9 6 0.5 UTR 173 parent 7.0 6.1 32 2J? 4.1 7.0 32 10 2d 4.9 18 2.2 6 1.1 7163 NS4B 7.8 7.7 7.6 7.2 0.6 8.0 7.7 7.5 7.4 0.6 6 (+)0.1 7849 NS5 7.0 6.7 3.7 2.1 4.9 7.7 5.5 3.6 2.4 5.3 6 3.1 8872 NS5 7.0 6.3 6.4 4Λ 2.6 7.4 6.4 5.1 29 4.5 6 0.1 2015203575 26 Jun2015 509 1033 parent 6.2 5.8 5.5 M 2.8 6.5 6.1 4.5 <1.6 >4.9 6 1.9 4266 NS2B 5.9 6.1 6.1 5.2 0.7 6.7 6.1 5.7 5.3 1.4 6 1.0 8092 NS5 5.0X 4.6 4.6 <1.6 >3.4 5.6X 4.8 4.4 <1.6 >4.0 12 4.0 parent 6.7 6.0 5.9 41 2.6 6.9 5.6 4.7 <1.6 >5.3 12 1.7 4907 NS3 6.7 6.0 5.8 40 2.7 7.1 6.1 6.8 23 4.8 12 1.8 8730 NS5 7.0 6.7 6.6 6.7 0.3 7.6 7.0 7.2 6.6 1.0 12 0.6 9977 NS5 5.6 5.5 4.6 4.1 1.5 6.4 6.1 6.2 4.6 1.8 6 0.7 1.5 0.7 3 Reduction in mean virus titer (log10PFU/ml) at 39°C compared to permissive temperature (35°C). b Groups of 6 suckling mice were inoculated i.c. with 104 PFU of virus. Brains were removed 5 days later, homogenized, and titered in Vero cells. c Comparison of mean virus titers of mice inoculated with mutant virus Mid concurrent DEN4 control. Bold denotes >50- or > 100-fold decrease in replication in suckling or SCED-HuH-7 mice, respectively. d Groups of HuH-7-SCID mice were inoculated directly into the tumor with 104 PFU virus. Serum was collected on day 6 and 7 and titered in Vero cells. e Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temp when compared to permissive temp (35°C). f Data represents the results from a single rDEN4-4995 virus. x Small plaque size at 35°C; small plaques have a diameter of <1.0 mm compared to wild type plaque diameter of 1.5 - 2.0 mm in Vero cells, or a diameter of <0.4 mm compared to wild type plaque diameter of 0.75 to 1.0 mm in HuH-7 cells. 2015203575 26 Jun2015

Table 15. sp, ts and mouse attenuation phenotypes of rDEN4 mutant viruses encoding single mutations identified in 3 HuH-7 cell-specific ts 5-FU mutant viruses. 5-FU rDEN4- Gene/ Mean virus titer (logioPFU/ml) at indicated temp (°C) Replication in 7-day miceb Replication in HuH-7-SCID miceb mutant viruses Mutation (nt position) region Vero cells HuH-7 cells n Mean logio reduction from wfi (logi nPFU/g n Mean logio reduction from wfi 35 37 39 39 Δ* 35 37 38 39 Δ brain) (logioPFU/ml serum) 686 parent 7.0 6.7 6.7 6.4 0.6 7.3 6.8 6.4 2^2 5.1 12 3.8 6 1.2 3575 NS2A 6.9 6.9 7.1 7.0 0.1 7.9 6.8 6.9 4.9 3.0 12 2.3 nde 4062 NS2A 6.8 6.6 6.3 4.7 2.1 6.9 6.8 7.0 <1.6 >5.3 12 2.2 nd 7163 NS4B 7.8 7.7 7.6 7.2 0.6 8.0 7.7 7.5 7.4 0.6 6 (+)0.1 nd 992 parent 7.3 7.1 6.8 5.9 1.4 7.4 6.9 5.0 <1.6 >5.8 6 2.7 7 1.3 5695 NS3 5.6 4.7 4.7 3.8 1.8 6.3 5.1 3.7 <1.6 >4.7 6 2.8 nd 7162 NS4B 7.2 7.3 6.6 6.8 0.4 7.4 7.3 7.3 6.6 0.8 8 0.3 nd 1175 parent 7.4 7.1 6.9 5.3 2.1 7.6 6.5 4.7 M 4.3 12 1.7 5 1.0 7153 NS4B 7.7 7.7 7.6 7.2 0.5 8.0 7.8 7.5 6.9 1.1 6 0.2 nd 10186 NS5 4.3 3.7 2.4 <1.6 >2.7 5.1 <1.6 <1.6 <1.6 >3.5 6 3.4 nd 10275 3'UTR 6.9 6.4 6.4 5.8 1.1 7.1 6.8 7.1 5.2 1.9 6 0.5 nd a Reduction in titer (log10PFU/ml) at 39°C compared to permissive temperature (35°C). b Groups of 6 suckling mice were inoculated i.c. with 104 PFU virus. Brains were removed 5 days later, homogenized, and titered in Vero cells. c Determined by comparing mean viral titers of mice inoculated with mutant virus and concurrent 2A-13 or rDEN4 wt control. d Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temp when compared to permissive temp (35°C). 100 2015203575 26Jun2015

Table 16. Temperature-sensitive (ts) and mouse brain attenuation (att) phenotypes of additional rDEN4 viruses encoding single 5-FU mutations. 5-FU mutant virus Virus Gene/ region containing mutation Mean virus titer (logioPFU/ml) at indicated temp (°C) Replication in suckling mice^ 35 Vero cells 37 38 39 Δ» 35 HuH-7 cells 37 38 39 Δ n Mean virus titer ±SE (logi()PFU/g brain) Mean logjo -unit reduction from value for wtc 695 rDEN4-40 5’UTR 7.4 7.2 6.7 6.2 1.2 7.6 7.5 7.1 5.8 1.8 nd* nd 718 rDEN4-4059 NS2A 7.0 6.7 6.4 6.2 0.8 7.7 7.1 7.0 6.6 1.1 nd nd 311 rDEN4-4896 NS3 7.0 6.1 5.9 42 2.8 6.9X 6.0 5.6 33 3.6 6 4.1 ±0.4 2.0** 695 rDEN4-6106 NS3 6.8 6.3 5.9 T9 2.9 7.1 6.0 5.2 3A 3.7 nd nd 631 rDEN4-6259 NS3 7.0 6.1 5.8 5.0 2.0 7.5 6.6 5.7 4.2 3.3 6 2.2 ±0.2 3.9** 695e rDEN4-7546 NS4B 7.5 7.6 7.4 6.6 0.9 7.7 7.6 7.3 5.7 2.0 nd nd 718 rDEN4-7630 NS5 7.0 6.9 6.9 6.4 0.6 7.4 7.4 7.2 6.8 0.6 6 5.0 ±0.3 0.5 718 rDEN4-8281 NS5 6.4 6.6 6.7 5.4 1.0 7.6 7.6 7.0 5.1 2.5 6 5.0 ±0.5 1.1 a Reduction in titer (logioPFU/ml) at 39°C compared to titer at permissive temperature (35°C). b 6 mice were inoculated i.c. with 10^ PFU virus in 30μ1 inoculum. Brains were removed 5 days later, homogenized, and titered on Vero cells. Limit of detection is 2.0 logioPFU/g. c Determined by comparing mean viral titers of mice inoculated with sample virus and wt rDEN4 control. d Underlined values indicate a 2.5 or 3.5 logioPFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temperature when compared to permissive temperature (35 °C). e The 7546 mutation is also present in nine other 5-FU mutant viruses. x Small plaque size at 35°C; small plaques have a diameter of <0.4 mm compared to wt plaque diameter of 0.75 to 1.0 mm in HuH-7 cells, f not determined ** The att phenotype is defined as a reduction of >1.5 logi()PFU/g compared to wt virus. 2015203575 26Jun2015

Table 17. Growth of wt DEN-4 2A-13 in SCID mice transplanted with HuH-7 cells.a Virus titer Dose (logioPFU/ml) Mouse # logjoPFU/ml serum logi()PFU/g tissue day 3 day 5 Brain Liver Tumor 4 87 2.7 5.9 2.0 6.9 8.0 88 2.0 5.9 3.8 3.3 8.0 89 <1.7 6.2 2.7 3.6 8.0 90 1.7 3.5 3.2 3.0 7.0 5 84 <1.7 7.2 3.2 4.0 7.0 85 1.7 6.6 3.6 6.3 5.8 6 91 4.4 8.3 6.0 7.3 8.0 92 4.2 7.7 3.3 6.9 7.3 93 4.0 6.6 3.3 5.7 8.4 94 4.3 8.1 5.8 7.8 7.5 a SCID mice were injected i.p. with 107 HuH-7 human hepatoma cells. Approximately 8 weeks later, groups of tumor-bearing SClD-HuH-7 mice were inoculated with virus directly into the tumor. Serum and tissues were collected on day 5, processed, and titered in Vero cells. -101- 2015203575 26Jun2015

Table 18. Combination of ts mutations, NS3 4995 and NS5 7849, in rDEN4 results in an additive ts phenotype.

Virus Mean virus titer (logioPFU/ml) at indicated temp (°C) Replication in suckling mice15 Vero cells HuH-7 cells Mean virus titer ± SE (logioPFU/g brain) Mean logio reduction from wtc 35 37 38 39 Δ&amp; 35 37 38 39 Δ 2A-13 wt 7.1 7.1 6.9 6.8 0.3 7.4 7.3 6.7 6.4 1.0 6.9 ±0.09 - rDEN4 wt 7.0 6.8 6.6 6.4 0.6 7.5 7.3 6.7 6.4 1.1 6.5 ±0.11 - rDEN4A30 7.0 6.7 6.2 6.2 0.8 7.5 7.0 6.5 5.1 2.4 5.9 ±0.21 0.6 rDEN4-4995 5.7 4.9 3.6 <1.6d >4.1 6.4 5.7 4.0 <1.6 >4.8 3.4 ±0.10 3.1 rDEN4-7849 7.0 6.7 3J_ 2Λ 4.9 7.7 5.5 M M 5.3 2.6 ±0.29 3.9 rDEN4-4995-7849 5.9 M <1.6 <1.6 >4.3 5.6 2.4 <1.6 <1.6 >4.0 2.3 ±0.20 4.2 a Reduction in titer (log10PFU/ml) at 39°C compared to titer at permissive temperature (35°C). 102 1 Groups of 6 suckling mice were inoculated i.c. with 104 PFU virus. Brains were removed 5 days later, homogenized, and titered in Vero cells. The limit of detection is 2.0 log10PFU/g. c Determined by comparing mean viral titers of mice inoculated with sample virus and rDEN4 wt control. d Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero cells or HuH-7 cells, respectively, at indicated temperature when compared to permissive temperature. 2015203575 26Jun2015

Table 19. The 5-FU mutations are compatible with the Δ30 mutation for replication in the brain of suckling mice. Virus No. of mice/ group Mean virus titer ± SE (log10PFU/g brain)a Mean logi0-unit reduction from wtb rDEN4 12 6.0 ±0.1 — rDEN4A30 12 5.3 ±0.1 0.7 rDEN4-2650c 12 3.7 ±0.2 2.3 rDEN4A3 0-2650 12 3.9 ± 0.1 2.1 rDEN4-4995d 6 3.5 ±0.2 2.5 rDEN4A3 0-4995 6 2.7 ±0.4 3.3 rDEN4-8092d 12 2.0 ±0.1 4.0 rDEN4A30-8092 6 3.2 ±0.2 2.8 rDEN4-10634° 12 3.8 ±0.1 2.2 rDEN4A30-10634 12 3.6 ± 0.1 2.4 a Groups of 6 suckling mice were inoculated i.c. with 104 PFU of virus. Brains were removed 5 days later, homogenized, and titered in Vero cells. b Comparison of mean virus titers of mice inoculated with mutant virus and rDEN4 control. 0 Mutation restricts growth in both mouse brain and HuH-7-SCID mice. d Mutation restricts growth in mouse brain only. The 8092 mutation has not been tested in SCID-HuH7 mice. -103- 2015203575 26 Jun2015 104

Table 20. Temperature-sensitive and mouse brain attenuation phenotypes of viruses bearing charge-cluster-to-alanine mutations in the NS5 gene ofDEN4. Mutation8 Changed #nt Mean virus titer (logi0 PFU/ml at indicated temperature (°C)b_ _Replication in suckling miced AA Pair changed 35 37 Vero Cells 38 39 Ac 35 HuH-7 Cells 37 38 39 Δ n Mean titer ± SE (logioPFU/g brain) Mean log reduction from wt' wt(rDEN4) n/a 0 8.1 8.1 7.9 7.6 0.5 8.3 5.0 7.5 7.5 0.8 48 6.0 ±0.16 - deletion (rDEN4A30) n/a 30 6.3 6.1 6.1 5.7 0.6 6.9 63 5.9 4.7 2.2 42 5.4 ±0.22 0.6 21-22 DR 4 7.2 6.8 6.7 6.1 1.1 7.6 7.1 7.0 4.7 2.9 6 5.0 ±0.50 0.6 22-23 RK 4 7.0 7.8 6.9 3.7 3.3 7.6 7.6 6.5 <1.7 >5.9 6 2.6 ±0.19 2.9 23-24 KE 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3 5.6 <1.7 >5.4 18 4.7 ± 0.09 1.5 26-27 EE 3 7.8 7.6 6.8 43 3.8 8.4 8.2 7.3 A9 3.5 6 5.7 ±0.30 +0.1 46-47 KD 3 7.4 7.4 7.3 7.0 0.4 7.8 7.8 7.3 6.8 1.0 6 5.4 ± 0.42 0.5 157-158 EE 3 6.5 7.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 6 2.8 ±0.31 2.7 200-201 KH 4 5.3 4.6 5.3 4.1 1.2 5.6 4.9 3.7 <1.7 >3.9 12 5.5 ± 0.45 0.8 246-247 RH 5 6.9 5.8 5.7 5.4 1.5 6.4 6.1 6.1 5.5 0.9 6 6.1 ±0.17 +0.5 253-254 EK 4 7.1 6.9 6.8 7.0 0.1 7.9 7.5 7.6 6.8 1.1 6 6.2 ±0.13 +0.6 356-357 KE 3 7.7 7.6 7.0 7.0 0.7 8.0 7.3 6.4 <1.7 >6.3 6 3.5 ± 0.58 2.0 387-388 KK 5 7.7 6.1 7.0 <1.7 >6.0 7.0 6.3 7.0 <1.7 >5.3 6 3.1 ±0.33 2.4 388-389 KK 5 5.1 4.5 <1.7 <1.7 >3.4 6.1 5.0 <1.7 <1.7 >4.4 6 5.0 ±0.23 1.4 396-397 RE 4 7.0 7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 18 5.4 ±0.35 1.1 397-398 EE 2 7.0 7.1 7.0 3d) 4.0 8.0 7.6 7.0 <1.7 >6.3 6 6.0 ± 0.22 0.8 436-437 DK 4 4.5 3.3 3.0 Z0 2.5 5.7 4.5 <1.7 <1.7 >4.0 12 2.3 ±0.14 3.9 500-501 RE 3 6.6 6.3 5.7 23 4.3 7.1- 6.5 <1.7 <1.7 >5.4 6 6.9 ± 0.49 +0.7 520-521 EE 3 5.6 4.7 4.3 <1.7 >3.9 6.7 5.7 <1.7 <1.7 >5.0 6 5.2 ± 0.48 0.2 523-524 DK 4 6.6 6.3 6.3 5.8 0.8 7.1 6.6 <1,7 <1.7 >5.4 6 4.2 ± 0.47 1.3 524-525 KK 5 7.1 6.9 6.9 6.6 0.5 7.8 7.4 7.0 53 2.5 6 3.4 ±0.54 2.1 525-526 KD 4 7.8 7.1 7.6 6.8 1.0 7.9 7.7 8.0 6.9 1.0 6 3.7 ± 0.64 1.8 596-597 KD 3 4.6 4.0 2.6 <1.7 >2.9 '5.7 4.9 4.0 <1.7 >4.0 6 5.9 ±0.14 0.5 641-642 KE 4 7.3 6.9 6.9 5.2 2.1 7.8 7.5 7.2 6.9 0.9 6 4.7 ± 0.45 1.2 642-643 ER 3 6.8 6.1 4.0 33 3.5 7.5 7.1 6.6 zo 4.5 12 2.6 ±0.15 3.6 645-646 EK 4 6.3 5.3 5.9 3J. 3.2 6.4 5.8 5.5 4.5 1.9 6 5.4 ±0.51 0.2 649-650 KE 3 6.9 6.8 6.9 6.3 0.6 7.1 7.3 7.5 7.0 0.1 12 6.4 ± 0.20 +0.2 654-655 DR 4 6.3 5.7 <1,7 <1,7 >4.6 7.0 7.1 4.6 <1.7 >5.3 12 1.8 ±0.10 4.0 2015203575 26Jun2015 750-751 RE 3 7.1 7.1 6.9 5.7 1.4 7.8 6.9 6.5 5.6 2.2 6 6.0 ±0.18 0.7 808-809 ED 3 4.6 4.1 <1,7 <1.7 >2.9 5.2 <1.7 <1.7 <1.7 >3.5 6 1.8 ±0.05 3.1 820-821 ED 2 6.3 6.3 5.6 <1.7 >4.6 6.9 6.0 5.7 <1.7 >5.2 6 5n5 ±0.33 1.2 827-828 DK 4 6.9 6.3 6.3 5.9 1.0 7.5 6.9 5.0 <1.7 >5.8 6 3.6 ±0.76 23 877-878 KE 3 7.6 7.3 7.0 7.0 0.6 7.9 7.9 7.3 5.8 2.1 12 4.4 ±0.65 1.8 878-879 EE 3 7.6 7.3 7.3 7.1 0.5 8.1 8.1 7.9 6.6 1.5 12 2.4 ±0.10 3.8 a Positions of the amino acid pair mutated to an alanine pair; numbering starts at the amino terminus of the NS5 protein. b Underlined values indicate a 2.5 or 3.5 loglO PFU/ml reduction in titer in Vero or HuH-7 cells, respectively, at the indicated temperatures when compared to permissive temperature (35°C). 0 Reduction in titer (log 10 PFU/ml) at 39°C compared to permissive temperature (35°C). d Groups of six mice were inoculated i.c. with 4.0 loglO PFU virus in a 30 μΐ inoculum. The brain was removed 5 days later, homogenized, and titered in Yero cells. 105 <; Determined by comparing mean viral titers in mice inoculated with sample vims and concurrent wt controls (n = 6). The attenuation phenotype is defined as a reduction of >1.5 loglO PFU/g compared to wt vims; reductions of >1.5 are listed in boldface. 2015203575 26 Jun2015

Table 21. SCID-HuH-7 attenuation phenotypes of viruses bearing charge-cluster-to-alanine mutations in the NS5 gene of DEN4. Mutation2 Replication in SCID-HuH-7 miceb AA changed n Mean peak virus titer ± SE (log10PFU/ml serum) Mean log reduction from wt° wt na 21 5.4 ±0.4 - A30 na 4 3.7 ±0.6 2.5 23-24 KE 19 4.7 ±0.5 1.3 157-158 EE 6 4.6 ±0.6 1.3 200-201 KH 12 3.7 ±0.2 2.6 356-357 KE 10 6.3 ± 0.7 (-) u 396-397 RE 12 4.4 ±1.3 1.2 397-398 EE 6 6.0 ±0.5 (-)0.1 436-437 DK 6 3.6 ±0.2 2.6 500-501 RE 8 . 5.1 ±0.4 1.1 523-524 DK 5 5.3 ± 0.7 0.6 750-751 RE 8 5.1 ±0.4 1.1 808-809 ED 8 3.2 ±0.4 3.0 827-828 DK 5 2.9 ± 0.2 1.6 878-879 EE 5 4.4 ±0.7 1.5 a Positions of the amino acid pair changed to a pair of alanines; numbering starts at the amino terminus of the NS5 protein. b Groups of SCID-HuH-7 mice were inoculated directly into the tumor with 104 PFU virus.

Serum was collected on days 6 and 7 and titered in Vero cells. c Comparison of mean virus titers of mice inoculated with mutant virus and concurrent DEN4 control. Bold denotes a > 100-fold decrease in replication. A (-) sign indicates an increase in replication relative to wt. -106- 2015203575 26 Jun2015

Table 22. Combination of paired charge-cluster-to-alanine mutations into double-pair mutant viruses. Mutation Pair 1 Mutation Pair 2 Recovered 23-24 200-201 Yes 23-24 356-357 Yes 23-24 396-397 Yes 23-24 523-524 Yes 23-24 827-828 No 157-158 200-201 No 157-158 356-357 No 157-158 396-397 No 157-158 523-524 Yes 157-158 827-828 No 827-828 200-201 No 827-828 356-357 No 827-828 396-397 •Yes 827-828 523-524 No -107- 2015203575 26 Jun2015

Table 23. Temperature-sensitive and mouse brain attenuation phenotypes of double charge-cluster-to-alanine mutants of the NS5 gene of rDEN4. 108

Mutationa Charged #nt Mean virus titer (loglO PFU/ml) at indicated temperature (°C)b Replication in suckling ; miced n Mean virus Mean log AAPair changed Vero Cells HuH-7 cells titer ± SE reduction 35 37 38 39 Δ° 35 37 38 39 Δ (logioPFU/g brain) from wte wt n/a 0 8.1 8.1 7.9 7.6 0.5 8.3 8.0 7.5 7.5 0.8 48 6.0 ±0.16 - A30 n/a 30 6.3 6.1 6.7 5.7 0.6 6.9 6.3 5.9 4.7 2.2 42 5.4 ±0.22 0.6 23-24 KE 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3 5.6 <1.7 >5.4 18 4.7 ± 0.09 1.5 200-201 KH 4 5.3 4.6 5.3 4.1 1.2 5.6 4.9 3.7 <1.7 >3.9 12 5.5 ±0.45 0.8 23-24; 200-201 KE, KH 7 7.1 6.5 6.6 <1.7 >5.4 7.8 7.3 <1.7 <1.7 >6.1 6 5.8 ±0.16 0.6 23-24 KE 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3 5.6 <1.7 >5.4 18 4.7 ± 0.09 1.5 356-357 KE 3 7.7 7.6 7.0 7.0 0.7 8.0 7.3 6.4 <1.7 >6.3 6 3.5 ±0.58 2.0 23-24; 356-357 KE, KE 6 23-24 KE 3 6.7 6.6 6.0 6.5 0.2 7.1 7.3 5.6 <1.7 >5.4 18 4.7 ± 0.09 1.5 396-397 RE 4 7.0 7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 18 5.4 ± 0.35 1.1 23-24; 396-397 KE, RE 7 6.3 4.9 <1.7 <1.7 >4.6 7.1 6.0 5.6 <1.7 >5.4 6 3.7 ± 0.44 2.7 157-158 EE 3 6.5 7.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 6 2.8 ±0.31 2.7 396-397 RE 4 7.0 7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 18 5.4 ±0.35 1.1 157-158; 396-397 EE, RE 7 6 2.0 ±0.12 4.8 157-158 EE 3 6.5 7.2 5.1 5.1 1.4 7.6 7.4 5.9 <1.7 >5.9 6 2.8 ±0.31 2.7 523-524 DK 4 6.6 6.3 6.3 5.8 0.8 7.1 6.6 <1.7 <1.7 >5.4 6 4.2 ± 0.47 1.3 157-158; 523-524 EE, DK 7 5.6 3.9 <1.7 <1.7 >3.9 6.3 4.1 <1.7 <1.7 >4.6 396-397 RE 4 7.0 7.3 6.5 5.5 1.5 7.5 7.6 7.5 <1.7 >5.8 6 4.8 ± 0.54 1.6 827-828 DK 4 6.9 6.3 6.3 5.9 1.0 7.5 6.9 5.0 <1.7 >5.8 6 3.6 ± 0.76 2.3 2015203575 26Jun2015 396-397;827-828 RE, DK 8 7.0 6.5 6.0 <1.7 5.3 >6.7 5.7 <1.7 <1.7 >5.0 6_4,7 ±0.10 1.2_ a Positions of the amino acid pair mutated to an alanine pair; numbering starts at the amino terminus of the NS5 protein. b Underlined values indicate a 2.5 or 3.5 log10PFU/ml reduction in titer in Vero or HuH-7 cells respectively, at the indicated temperatures when compared to permissive temperature (35°C). c Reduction in titer (log10PFU/ml) at 39°C compared to permissive temperature (35°C). d Groups of six suckling mice were inoculated i.c. with 4.0 log10PFU virus in a 30 μΐ inoculum. Brains were removed 5 days later, homogenized, and titered in Vero cells. e Determined by comparing mean viral titers in mice inoculated with sample virus and concurrent wt controls (n = 6); reductions > 1.5 are listed in boldface. 109 2015203575 26 Jun2015

Table 24. SCID-HuH-7 attenuation phenotypes of double charge-cluster-to-alanine mutants of the NS5 gene of rDEN4. Mutation* Replication in SCED-HuH-7 miceb Charged AAPair n Mean peak virus titer ± SE (log10PFU/ml serum) Mean log reduction from wt° wt n/a 21 5.4+0.4 - A30 n/a 4 3.7 + 0.6 2.5 23-24 KE 19 4.7 ± 0.5 1.3 200-201 KH 12 3.7+0.2 2.6 23-24; 200-201 KE, KH 13 3.4 + 0.1 2.9 23-24 KE 19 4.7 + 0.5 1.3 356-357 KE 10 6.3 ± 0.7 (+) 1.1 23-24; 356-357 KE, KE 4 3.6 + 0.3 2.3 23-24 KE 19 4.7 + 0.5 1.3 396-397 RE 12 4.4+1.3 1.2 23-24; 396-397 KE, RE 10 3.4 + 0.5 3.3 157-158 EE 6 4.6 ± 0.6 1.3 396-397 RE 12 4.4+1.3 1.2 157-158;396-397 EE, RE 6 2.2 + 5.2 3.6 157-158 EE 6 4.6 + 0.6 1.3 523-524 DK 5 5.3 + 0.7 0.6 157-158; 523-524 E E, D K 3 5.1 + 0.6 0.8 396-397 RE 12 4.4+ 1.3 1.2 827-828 DK 5 2.9 + 0.2 1.6 396-397;827-828 R E, D K 4 4.1 + 0.7 0.4 a Positions of the amino acid pair mutated to an alanine pair; numbering starts at the amino terminus of the NS 5 protein. b Groups of SCID-HuH-7 mice were inoculated directly into the tumor with 104 PFU of virus. Serum was collected on days 6 and 7 and titered in Vero cells. c Comparison of mean virus titers of mice inoculated with mutant virus and concurrent DEN4 control. Bold denotes a > 100-fold decrease in replication. A (+) sign indicates an increase in replication relative to wt. -110-

Ill 2015203575 26 Jun2015

Table 25. Phenotypes (temperature sensitivity, plaque size and replication in mouse brain and SCED-HuH-7 mice) of wt DEN4 and viruses containing the Δ30 and 7129 mutations. Virus ED Mutation3· Mean virus titer (log^Q PFU/ml) at indicated Replication in suckling mouse Replication in SCID-HuH-7 micee temperature (°C) brain0 35 VERO 39 Ab 35 HUH7 39 Δ C6/36 32 n Mean virus titer ± SE (logioPFU/g brain) Mean log reduction fromwtd n Mean peak virus titer ± SE (logioPFU/ml serum)f Mean log reduction from wtd 1-TD-1A wt 7.3 6.8 0.5 8 6.8 1.2 .8.3 36 6.1 ±0.21 - 21 5.4 ±0.4 - p4A30 A30 6.6 6.5 0.1 7.4 6.4 1.0 42 5.4 ±0.22 0.6 4 3.7 ±0.6 2.5 5-1A1 C7129U 6.7 6.5 0.2 7.5 6 1.5 7.6* 6 6.2 ±0.30 0.0 rDEN4-7129-lA C7129U 7.3 7.0 0.3 7.6 6.3 1.3 7.5* 6 7.2 ±0.12 (-)0.4 4 5.4 ± 0.8 (-)0.8 τϋΕΝ4Δ30-7129 C7129U + Δ30 7.0 7.1* a Position and identity of the mutated nucleotides. bReduction in titer (log10 PFU/ml) at 39°C compared to permissive temperature (35°C). c Groups of six suckling mice were inoculated i.c. with 4.0 log10 PFU virus in a 30 μΐ inoculum. The brain was removed 5 days later, homogenized, and titered in Vero cells. d Determined by comparing mean viral titers in mice inoculated with sample virus and concurrent wt controls (n = 6). The attenuation phenotype is defined as a >50- or > 100-fold decrease in replication in suckling or SCID-HuH-7 mice, respectively. A (-) sign indicates an increase in replication relative to the wt control. e Groups of SCID-HuH-7 mice were inoculated directly into the tumor with 104 PFU virus. Serum was collected on days 6 and 7 and titered in Vero cells.

Small plaque size. 2015203575 26 Jun2015

Table 26. The 5-fluorouracil 5-1A1 small plaque mutant demonstrates a restriction of midgut infection following oral infection of Aedes aegytpi mosquitoes. Virus tested Dose ingested (log10PFU) a No. mosquitoes tested Midgut-only infectionb Disseminated infection0 Total no. infected d,e wtDEN4 4.5 19 1 (5%) 17 (89%) 18 (95%) (2A-13) 3.5 26 9 (35%) 7 (27%) 16 (62%) 2.5 28 1 (4%) 0 OIDS0 = 3.9 1 (4%) OID50 = 3.3 5-1A1 3.5 34 4 (12%) 2 (6%) 6 (18%) 2.5 9 0 1 (11%) 1 (11%) 1.5 23 0 0 0 OIDS0 > 3.9 a Amount of virus ingested, assuming a 2 μΐ bloodmeal. b Number (percentage) of mosquitoes with detectable dengue virus antigen in midgut tissue, but no detectable dengue virus antigen in head; mosquitoes were assayed 21 days postfeed, and dengue virus antigen was identified by IFA. c Number (percentage) of mosquitoes with detectable dengue virus antigen in both midgut and head tissue. d Total number (percentage) of mosquitoes with detectable dengue virus antigen. e The proportion of total infections caused by wild type DEN4 was significantly higher than the proportion caused by 5-1A1 (logistic regression, N = 426, P < 0.0001). There were too few disseminated infection caused by 5-1A1 to permit statistical analysis. -112-

Table 27. The 5-fluorouracil 5-1A1 small plaque mutant demonstrates a restriction of infection following intrathoracic inoculation of Toxorhynchites splendens mosquitoes.

Virus tested Dose ingested (log10PFU)a No. mosquitoes tested No (%) infectedc wtDEN4 4.0 5 5 (100) (2A-13) 3.0 4 4 (100) 2.0 4 1(25) MIDS0 = 2.3 Iog10PFU 5-1A1 3.0 9 0 2.0 7 1(14) 1.0 7 0

MIDS0 > 3.0 log10PFU 2015203575 26 Jun2015 a Amount of virus inoculated in a 0.22 μΐ inoculum. b Number (percentage) of mosquitoes with detectable dengue virus antigen in head tissue; mosquitoes were assayed 14 days post-inoculation, and dengue virus antigen was identified by IF A. 0 The proportion of infections caused by wild type DEN4 was significantly higher than the proportion caused by 5-1A1 (logistic regression, N = 36, P < 0.01). -113- 2015203575 26 Jun2015

Table 28. Mutagenesis primers for the deletion or swap of sequences in DEN4 showing conserved differences from tick-borne flaviviruses. DEN4 nucleotides1 Type of mutation2 Mutagenesis Primer3 SEQ ID NO 10508-10530 Δ CTGGTGGAAGCCCAACACAAAAAC 64 10508-10530 swap CTGGTGGAAGGAAGAGAGAAATTGGCAACTCCCCAACACAAAAAC 65 10535-10544 Δ AGACCCCCCCAAGCATATTGAC 66 10535-10544 swap AGACCCCCCCAATATTTCCTCCTCCTATAGCATATTGAC 67 10541-10544 Δ CCCAACACAAAGCATATTGAC 68 1 Nucleotides numbered 5' to 3', in the opposite direction from Figure 5.3 114 2 Δ: deletion of specified DEN4 nucleotides; swap: exhange of specified DEN4 nucleotides with homologous sequence from Langat 2 no swap mutation was made for nucleotides 10541-10544 2015203575 26 Jun2015

Table 29. Virus titer and plaque size of 3' UTR mutant viruses in Vero and C6/36 cells. Virus Vero C6/36 Titer (log10 PFU/ml) Plaque size1 Titer (log10 PFU/ml) Plaque size rDEN4A10508-10530 8.1 wt 7.5 wt rDEN4swapl0508-10530 5.4 sp 6.6 wt rDEN4A10535-10544 5.8 wt 7.0 sp rDEN4swapl0535-10544 7.0 wt 7.3 wt rDEN4A 10541-10544 6.4 wt >7.0 wt 1 Plaque size is designated as designated cell type. equivalent to wild type (wt) or <50% of wild type (sp) on the

Table 30. Infectivity of wt DEN4 and 3' UTR mutants for Toxorhynchites splendens via intrathoracic inoculation. Virus Dose No. mosquitoes % Infected13 mid50 (log10PFU)a tested (logio PFU) rDEN4 wt 3.3 6 83 2.3 2.3 7 57 1.3 6 0 0.3 6 0 rDEN4A10508-10530 4.4 8 0 3.4 9 11 2.4 4 0 a Amount of virus inoculated in a 0.22 μΐ inoculum. b Percentage of mosquitoes with detectable dengue virus antigen in head tissue; mosquitoes were assayed 14 days post-inoculation, and dengue virus antigen was identified by IFA -115- 2015203575 26 Jun2015

Table 31. Infectivity of 3’ UTR swap mutant viruses for Aedes aegypti fed on an infectious bloodmeal. Virus Tested Dose ingested (log10PFU) a No. Mosquitoes Tested Total No. Infected15’c Disseminated Infectionsc’d rDEN4 3.8 18 11 (61%) 4 (22%) 2.8 15 5 (33%) 1 (6%) 1.8 15 0 OIDS0 = 3.4 0 OIDS0 = > 4.2 rDEN4swap 3.8 25 5 (20%) 2 (8%) 10535-10544 2.8 25 0 0 1.8 20 0 OIDS0 = > 4.2 0 a Amount of virus ingested, assuming a 2 μΐ bloodmeal. b Number (%) of mosquitoes with detectable dengue virus antigen in the midgut tissue; mosquitoes were assayed either 14 d post-feed and dengue virus antigen was identified by IF A. c At a dose of 3.8 log10PFU, rDEN4swap10535-10544 infected significantly fewer mosquitoes at the midgut than wt rDEN4 (Fisher’s exact test, N = 43, P< 0.01), although disseminated infections were not significantly different (Fisher’s exact test, N = 43, P=0.38). d Number (%) of mosquitoes with detectable dengue virus antigen in the head tissue. -116- 2015203575 26 Jun2015

Table 32. Putative Vero cell adaptation mutations derived from the set of 5-FU mutant viruses and other DEN4 viruses passaged in Vero cells. 117

Nucleotide position Gene / region 5-FU mutant viruses Other DEN viruses passaged in Vero cells (a.a. #)b Nucleotide change Amino acid change No. of viruses with the mutation Virus Nucleotide change Amino acid change 1455 E(452) G>U val > phe 5 2280½3 E(727) U>C phe > leu 2 48912»3 NS3 (1597) U>C ile > thr 2 4995 NS3 (1599) U>C ser > pro 8 7153 NS4B (2351) U>C val > ala 3 2ΑΔ30 U>C val > ala 7162 NS4B (2354) U>C leu > ser 4 2A-1 U>C leu > ser 7163 NS4B (2354) A > U or C leu > phe 7 rDEN4A30 A>U leu > phe 2A-13-1A1 A>U leu > phe 71821,2=3 NS4B (2361) G> A gly > ser 2 7546 NS4B (2482) C>U ala > val 10 76303 NS5 (2510) A> G lys > arg 1 814669 A>G lys > arg 10275 3'UTR A>U n/ac 6 10279 3'UTR A> C n/a 4 a Conservation with DENI, DEN2, or DEN3 is designated by superscript. Lack of conservation is designated by no superscript. b Amino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nt 102-104) as residue #1. c not applicable 2015203575 26 Jun2015

Table 33. Sequence analysis of rDEN2/4A30 clone 27(p4)~2-2A2. Nucleotide Gene Mutation Nucleotide Amino acid 743 M anchor G> A Gly > Glu 1493 E C>U Ser > Phe 7544* NS4B C>U Ala > Val * Same as DEN4 nucleotide position 7546

Table 34. Sequence analysis of rDEN2/4A30 clone 27(p3)-2-lAl. Nucleotide Gene Mutation Nucleotide Amino acid 1345 E U>C Tyr > His 4885* NS3 G> A Glu > Lys 8297 NS5 G> A Arg > Lys *Codon adjacent to 5-FU mutation 4891 -118- 2015203575 26 Jun2015

Table 35. Recombinant virus rDEN2/4A30 bearing Vero adaptation mutations can be recovery and titered on Vero cells. Virus Virus titer in indicated cell line1 (log10PFU/ml) C6/36 Vero Virus titer following recovery in Vero cells (log10PFU/ml) rDEN2/4A30 wt 5.2 1.7 <0.7 rDEN2/4A30-7153 5.4 5.2 <0.7 rDEN2/4A3 0-7162 5.4 5.3 nd2 rDEN2/4A30-7182 4.7 4.9 2.3 rDEN2/4A30-7630 5.3 4.8 1.3 rDEN2/4A30-7153-7163 5.1 4.7 nd rDEN2/4A30-7153-7182 4.1 3.2 nd rDEN2/4A30-7546-7630 5.2 5.2 nd 1 Virus recovered following transfection of C6/36 mosquito cells was terminally diluted once in C6/36 cells and titered simultaneously in C6/36 cells and Vero cells. 2 not determined

Table 36. Putative Vero cell adaptation mutations of dengue type 4 virus and the corresponding wildtype amino acid residue in other dengue viruses.

Mutation Amino acid position3 Mutant . residue Amino acid in indicated wt dengue virusb DEN4 DENI DEN2 DEN3 1455 452 F V I A A 2280 727 L Fc F F F 4891 1597 T I V I I 4995 1632 P S s S N 7129 2343 L P P P P 7153 2351 A V F F L 7162 2354 S L V V V 7163 2354 F L V V V 7182 2361 S G G G G 7546 2482 V A L T V 7630 2510 R K S s K

Amino acid position is given for the polyprotein of DEN4 b DEN4 = rDEN4 (GenBank AF326825); DENI = Western pacific (GenBank DVU88535); DEN2 = New Guinea C (GenBank AF038403); DEN3 = H87 (GenBank M93130) c Underlined nucleotides are shared between DEN4 and one or more additional DEN types. -119- 2015203575 26 Jun2015

Table 37. Mutations known to attenuate dengue type 4 virus and the corresponding wildtype amino acid residue in other dengue virus. Mutation Amino acid Mutant . Amino acid in indicated wt dengue vimsb position3 residue DEN4 DENI DEN2 DEN3 2650 850 S Nd N N N 3442 1114 G E E E E 3540 1147 K E E E E 3575 1158 I M L A M 3771 1224 G R R K R 4062 1321 A 1 L A 1 4306 1402 S N E D D CO g 4891 1597 T I V I I • r*H 4896 1599 s A A A A 4907 1602 F L L L L a 4995 1632 P S S S N 1—' 5097 1666 N D D D D 5695 1865 G D D D D 6259 2053 A V V V V 7129° 2343 L P P P P 7849 2583 I N K N K 8092 2664 G E Q Q Q 10186 3362 T I I I I 10634 3’ UTR - - - - - 22, 23 2509, 2510 AA RK KS KS RK CO 23,24 2510,2511 AA KE SE SE KE a o 157,158 2644, 2645 AA EE EE EA EE 200,201 2687, 2688 AA KH KH KY KH 356, 357 2843, 2844 AA KE KE KE KE <0 a 387,388 2874, 2875 AA KK RN KK RN 436, 437 2923, 2924 AA DK HR DK DK T"· i cd 1 524, 525 3011,3012 AA KK KI KK KI o -w Λ 525,526 3012, 3013 AA KD IP KE IP <3 CO 642, 643 3129,3130 AA ER ER IA KK 3 1—( O 654, 655 3141,3142 AA DR ER ER ER l <o pS) 808, 809 3295, 3296 AA ED ED ED ED u 827,828 3314, 3315 AA DK DK DK DK U 877,878 3364, 3365 AA KE NE NE NE 878,879 3365, 3366 AA EE EN EE EE a Amino acid position is given for the polyprotein of DEN4 b DEN4 = rDEN4 (GenBank AF326825); DENI = Western pacific (GenBank U88535); DEN2 = New Guinea C (GenBank AF038403); DEN3 = H87 (GenBank M93130) 0 This mutation results in decreased replication of DEN4 in mosquitoes. d Underlined nucleotides are shared between DEN4 and one or more additional DEN types. -120- 2015203575 26 Jun2015

Appendix 1 Sequence of recombinant dengue type 4 virus strain 2A

LOCUS SEP-2001 DEFINITION ACCESSION VERSION KEYWORDS SOURCE ORGANISM

Flaviviridae; AF375822 10649 bp ss-RNA linear VRL 19- Dengue virus type 4 recombinant clone 2A, complete genome. AF375822 AF375822.1 GI:14269097 Dengue virus type 4. Dengue virus type 4 Viruses; ssRNA positive-strand viruses, no DNA stage;

Flavivirus; Dengue virus group. REFERENCE 1 (bases 1 to 10649) AUTHORS Blaney,J.E. Jr., Johnson,D.H., Firestone,C.Y., Hanson,C.T., Murphy,B.R. and Whitehead,S.S. TITLE Chemical Mutagenesis of Dengue Virus Type 4 Yields Mutant Viruses

Which Are Temperature Sensitive in Vero Cells or Human Liver

Cells and Attenuated in Mice

JOURNAL MEDLINE PUBMED REFERENCE AUTHORS TITLE JOURNAL J. Virol. 75 (20), 9731-9740 (2001) 21443968 11559806 2 (bases 1 to 10649) Blaney,J.E. Jr., Johnson,D.H., Firestone,C.Y., Hanson,C.T., Murphy,B.R. and Whitehead,S.S. Direct Submission Submitted (02-MAY-2001) LID, NIAID, 7 Center Drive, Bethesda, 20892,

FEATURES source mat peptide mat peptide

CDS USA Location/Qualifiers 1. .10649 /organism="Dengue virus type 4" /virion /db_xref="taxon:11070" 102.. 440 /note="anchC" /product="anchored capsid protein" 102.. 398 /note="virC" /product="virion capsid protein" 102.. 10265 /codon_start=l /products"polyprotein precursor" /protein_ids"AAK58017.1" /db xref="GI :1426909811

/1 rans 1 at ions "MNQRKKWRPPFNMLKRERNRVSTPQGLVKRFSTGLFSGKGPLR MVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKRSTI TLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCED TVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLET -121- RAET WMS S EGAWKHAQRVE S WILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAP SYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVAL LRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDWDRGWGNGCGLFGKGGV VTCAKFSCSGKITGNLVQIENLEYTVWTVHNGDTHAVGNDTSNHGVTAMITPRSPSV EVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEV HWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKV RMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTWKVKYEGAGAPCKVPIEIRDVNKE KWGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFE STYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGF LVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVQADMGCVVSWSGKELKCGSGIFVVDN VHTWTEQYKFQPES PARLAS AILNAHKDGVCGIRSTTRLENVMWKQITNELNYVLWEG GHDLTWAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTPEARNSTFLIDGPDT SECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSSEVCDHRLMSAAIKDQKAVHADM GYWIESSKNQTWQIEKASLIEVKTCLWPKTHTLWSNGVLESQMLIPKSYAGPFSQHNY RQGYATQTVGPWHLGKLEIDFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCR SCTMPPLRFLGEDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVE ECLRRRVTRKHMILVWITLCAIILGGLTWMDLLRALIMLGDTMSGRIGGQIHLAIMA VFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELIDGISLGLILLKIV TQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFWTLIPLCRTSCLQKQSHWVEI TALILGAQALPVYLMTLMKGASRRSWPLNEGIMAVGLVSLLGSALLKNDVPLAGPMVA GGLLIAAYVMSGSSADLSLEKAANVQWDEMADITGSSPIIEVKQDEDGSFSIRDVEET NMITLLVKLALI TVS GLYPLAIPVTMTLWYMWQVKTQRS GALWDVPS P AATKKAAL S E GVYRIMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRNDMIS YGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAVTLDFKPGTSG SPIINRKGKVIGLYGNGWTKSGDYVSAITQAERIGEPDYEVDEDIFRKKRLTIMDLH PGAGKTKRILPSIVREALKRRLRTLILAPTRWAAEMEEALRGLPIRYQTPAVKSEHT GREIVDLMCHATFTTRLLSSTRVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAA AIFMTATPPGATDPFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAG NDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFWTTDISEMGANFRAGRVIDPR RCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSGDPLKKDED HAHWTEAKMLLDMIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKTFVELMRRGDL PVWLSYKVASAGISYKDREWCFTGERNNQILEENMEVEIWTREGEKKKLRPRWLDARV YADPMALKDFKEFASGRKSITLDILTEIASLPTYLSSRAKLALDNIVMLHTTERGGRA YQHALNELPESLETLMLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVA EIQPQWIAASIILEFFLMVLLIPEPEKQRTPQDMQLIYVILTILTIIGLIAANEMGLI EKTKTDFGFYQVKTETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAA IANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVWPTTLTASLVMLLVHYAIIGPGL QAKATREAQKRTAAGIMKNPTVDGITVIDLEPISYDPKFEKQLGQVMLLVLCAGQLLL MRTT WAFCE VLTL ATGPILTL WEGNPGRFWNTTIAVS TAN IFRGS YLAGAGLAF S LIK NAQTPRRGTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKI KHAV SRGS S KIRWIVERGMVKPKGKWDLGCGRGGWS YYMATLKNVTE VKG YTKGGPG HEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKM VEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRNSTHEMYWVSGAS GNIVSSVNTTSKMLLNRFTTRHRKPTYEKDVDLGAGTRSVSTETEKPDMTIIGRRLQR LQEEHKETWHYDQENPYRTWAYHGSYEAPSTGSASSMVNGWKLLTKPWDVIPMVTQL AMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEF ISKVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGK REKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLHRL GYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKILAKAIFKLTY QNKWKVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFTNMEVQLIRQMEAEGVITQ DDMQNPKGLKERVEKWLKECGVDRLKRMAISGDDCWKPLDERFGTSLLFLNDMGKVR KDIPQWEPSKGWKNWQEVPFCSHHFHKIFMKDGRSLWPCRNQDELIGRARISQGAGW SLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWM TTEDMLKVWNRVWIEDMPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAKN IHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESEGVL " mat_peptide 441..938 2015203575 26 Jun2015 /note="prM" 122- mat peptide /product="membrane precursor protein 714..938 mat ^peptide /note="M" /products"membrane protein" 939..2423 mat_peptide /note="E" /products"envelope protein" 2424 . .3479 mat_peptide /products"NS1 protein" 3480..4133 mat_peptide /products"NS2A protein" 4134..4523 mat_peptide /products"NS2B protein" 4524..6377 mat_peptide /products"NS3 protein" 6378. .6758 mat_peptide /products"NS4A protein" 6759..6827 mat_peptide /product="2K protein" 6828..7562 mat_peptide /products"NS4B protein" 7563..10262 /products"NS5 protein" BASE COUNT 3302 a 2212 c 2800 g 2335 t 2015203575 26 Jun2015

ORIGIN 1 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg cttaacacag 61 ttctaacagt ttgtttgaat agagagcaga tctctggaaa aatgaaccaa cgaaaaaagg 121 tggttagacc acctttcaat atgctgaaac gcgagagaaa ccgcgtatca acccctcaag 181 ggttggtgaa gagattctca accggacttt tttctgggaa aggaccctta cggatggtgc 241 tagcattcat cacgtttttg cgagtccttt ccatcccacc aacagcaggg attctgaaga 301 gatggggaca gttgaagaaa aataaggcca tcaagatact gattggattc aggaaggaga 361 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc aacgataaca ttgctgtgct 421 tgattcccac cgtaatggcg ttttccttgt caacaagaga tggcgaaccc ctcatgatag 481 tggcaaaaca tgaaaggggg agacctctct tgtttaagac aacagagggg atcaacaaat 541 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga cactgtcacg tataaatgcc 601 ccctactggt caataccgaa cctgaagaca ttgattgctg gtgcaacctc acgtctacct 661 gggtcatgta tgggacatgc acccagagcg gagaacggag acgagagaag cgctcagtag 721 ctttaacacc acattcagga atgggattgg aaacaagagc tgagacatgg atgtcatcgg 781 aaggggcttg gaagcatgct cagagagtag agagctggat actcagaaac ccaggattcg 841 cgctcttggc aggatttatg gcttatatga ttgggcaaac aggaatccag cgaactgtct 901 tctttgtcct aatgatgctg gtcgccccat cctacggaat gcgatgcgta ggagtaggaa 961 acagagactt tgtggaagga gtctcaggtg gagcatgggt cgacctggtg ctagaacatg 1021 gaggatgcgt cacaaccatg gcccagggaa aaccaacctt ggattttgaa ctgactaaga 1081 caacagccaa ggaagtggct ctgttaagaa cctattgcat tgaagcctca atatcaaaca 1141 taactacggc aacaagatgt ccaacgcaag gagagcctta tctgaaagag gaacaggacc 1201 aacagtacat ttgccggaga gatgtggtag acagagggtg gggcaatggc tgtggcttgt 1261 ttggaaaagg aggagttgtg acatgtgcga agttttcatg ttcggggaag ataacaggca 1321 atttggtcca aattgagaac cttgaataca cagtggttgt aacagtccac aatggagaca 1381 cccatgcagt aggaaatgac acatccaatc atggagttac agccatgata actcccaggt 1441 caccatcggt ggaagtcaaa ttgccggact atggagaact aacactcgat tgtgaaccca 1501 ggtctggaat tgactttaat gagatgattc tgatgaaaat gaaaaagaaa acatggctcg 1561 tgcataagca atggtttttg gatctgcctc ttccatggac agcaggagca gacacatcag 1621 aggttcactg gaattacaaa gagagaatgg tgacatttaa ggttcctcat gccaagagac 1681 aggatgtgac agtgctggga tctcaggaag gagccatgca ttctgccctc gctggagcca 1741 cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg acatcttaag tgcaaagtcc 1801 gtatggagaa attgagaatc aagggaatgt catacacgat gtgttcagga aagttttcaa 1861 ttgacaaaga gatggcagaa acacagcatg ggacaacagt ggtgaaagtc aagtatgaag -123- 1921 gtgctggagc tccgtgtaaa gtccccatag agataagaga tgtaaacaag gaaaaagtgg 1981 ttgggcgtat catctcatcc acccctttgg ctgagaatac caacagtgta accaacatag 2041 aattagaacc cccctttggg gacagctaca tagtgatagg tgttggaaac agcgcattaa 2101 cactccattg gttcaggaaa gggagttcca ttggcaagat gtttgagtcc acatacagag 2161 gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga ttttggttcc gttggtggac 2221 tgttcacatc attgggaaag gctgtgcacc aggtttttgg aagtgtgtat acaaccatgt 2281 ttggaggagt ctcatggatg attagaatcc taattgggtt cttagtgttg tggattggca 2341 cgaactcaag gaacacttca atggctatga cgtgcatagc tgttggagga atcactctgt 2401 ttctgggctt cacagttcaa gcagacatgg gttgtgtggt gtcatggagt gggaaagaat 2461 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2521 aatttcaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg 2581 gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag caaataacca 2641 acgagctaaa ctatgttctc tgggaaggag gacatgacct cactgtagtg gctggggatg 2701 tgaagggggt gttgaccaaa ggcaagagag cactcacacc cccagtgagt gatctgaaat 2761 attcatggaa gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2821 ttttaataga cggaccagac acctctgaat gccccaatga acgaagagca tggaactctc 2881 ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg aaattccgag 2941 aaggaagttc agaagtgtgt gaccacaggt taatgtcagc tgcaattaaa gatcagaaag 3001 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatag 3061 agaaagcatc tcttattgaa gtgaaaacat gtctgtggcc caagacccac acactgtgga 3121 gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac 3181 agcacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac ttaggcaaat 3241 tagagataga ctttggagaa tgccccggaa caacagtcac aattcaggag gattgtgacc 3301 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgct 3361 gccgctcctg cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga 3421 tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtgacggccg 3481 gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg tttgtggaag 3541 aatgcttgag gagaagagtc actaggaaac acatgatatt agttgtggtg atcactcttt 3601 gtgctatcat cctgggaggc ctcacatgga tggacttact acgagccctc atcatgttgg 3661 gggacactat gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca 3721 agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3781 cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat gaccttatgg 3841 aactcattga tggaatatca ctgggactaa ttttgctaaa aatagtaaca cagtttgaca 3901 acacccaagt gggaacctta gctctttcct tgactttcat aagatcaaca atgccattgg 3961 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4021 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4081 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4141 ctcttaacga gggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4201 agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4261 tgatgagtgg tagctcagca gatctgtcac tagagaaggc cgccaacgtg cagtgggatg 4321 aaatggcaga cataacaggc tcaagcccaa tcatagaagt gaagcaggat gaagatggct 4381 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4441 tgataacagt gtcaggtctc taccccttgg caattccagt cacaatgacc ttatggtaca 4501 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccctca cccgctgcca 4561 ctaaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttattcggga 4621 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa 4681 caagaggatc agtgatctgc cacgagactg ggagattgga gccatcttgg gctgacgtca 4741 ggaatgacat gatatcatac ggtgggggat ggaggcttgg agacaaatgg gacaaagaag 4801 aagacgttca ggtcctcgcc atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4861 ctggcctttt caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg 4921 gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg 4981 gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa agaattggag 5041 agccagatta tgaagtggat gaggacattt ttcgaaagaa aagattaact ataatggact 5101 tacaccccgg agctggaaag acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5161 aaaggaggct acgaactttg attttagctc ccacgagagt ggtggcggcc gagatggaag 5221 aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag 5281 gaagagagat tgtagacctc atgtgtcatg caaccttcac aacaagactt ttgtcatcaa 2015203575 26 Jun2015 -124- 5341 ccagggttcc aaattacaac cttatagtga tggatgaagc acatttcacc gatccttcta 5401 gtgtcgcggc tagaggatac atctcgacca gggtggaaat gggagaggca gcagccatct 5461 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag 5521 aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag 5581 actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5641 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 5701 agtatccaaa aacgaaactc acggactggg actttgtggt cactacagac atatctgaaa 5761 tgggggccaa ttttagagcc gggagagtga tagaccctag aagatgcctc aagccagtta 5821 tcctaccaga tgggccagag agagtcattt tagcaggtcc tattccagtg actccagcaa 5881 gcgctgctca gagaagaggg cgaataggaa ggaacccagc acaagaagac gaccaatacg 5941 ttttctccgg agacccacta aaaaatgatg aagatcatgc ccactggaca gaagcaaaga 6001 tgctgcttga caatatctac accccagaag ggatcattcc aacattgttt ggtccggaaa 6061 gggaaaaaac ccaagccatt gatggagagt ttcgcctcag aggggaacaa aggaagactt 6121 ttgtggaatt aatgaggaga ggagaccttc cggtgtggct gagctataag gtagcttctg 6181 ctggcatttc ttacaaagat cgggaatggt gcttcacagg ggaaagaaat aaccaaattt 6241 tagaagaaaa catggaggtt gaaatttgga ctagagaggg agaaaagaaa aagctaaggc 6301 caagatggtt agatgcacgt gtatacgctg accccatggc tttgaaggat ttcaaggagt 6361 ttgccagtgg aaggaagagt ataactctcg acatcctaac agagattgcc agtttgccaa 6421 cttacctttc ctctagggcc aagctcgccc ttgataacat agtcatgctc cacacaacag 6481 aaagaggagg gagggcctat caacacgccc tgaacgaact tccggagtca ctggaaacac 6541 tcatgcttgt agctttacta ggtgctatga cagcaggcat cttcctgttt ttcatgcaag 6601 ggaaaggaat agggaaattg tcaatgggtt tgataaccat tgcggtggct agtggcttgc 6661 tctgggtagc agaaattcaa ccccagtgga tagcggcctc aatcatacta gagttttttc 6721 tcatggtact gttgataccg gaaccagaaa aacaaaggac cccacaagac aatcaattga 6781 tctacgtcat attgaccatt ctcaccatca ttggtctaat agcagccaac gagatggggc 6841 tgattgaaaa aacaaaaacg gattttgggt tttaccaggt aaaaacagaa accaccatcc 6901 tcgatgtgga cttgagacca gcttcagcat ggacgctcta tgcagtagcc accacaattc 6961 tgactcccat gctgagacac accatagaaa acacgtcggc caacctatct ctagcagcca 7021 ttgccaacca ggcagccgtc ctaatggggc ttggaaaagg atggccgctc cacagaatgg 7081 acctcggtgt gccgctgtta gcaatgggat gctattctca agtgaaccca acaaccttga 7141 cagcatcctt agtcatgctt ttagtccatt atgcaataat aggcccagga ttgcaggcaa 7201 aagccacaag agaggcccag aaaaggacag ctgctgggat catgaaaaat cccacagtgg 7261 acgggataac agtaatagat ctagaaccaa tatcctatga cccaaaattt gaaaagcaat 7321 tagggcaggt catgctacta gtcttgtgtg ctggacaact actcttgatg agaacaacat 7381 gggctttctg tgaagtcttg actttggcca caggaccaat cttgaccttg tgggagggca 7441 acccgggaag gttttggaac acgaccatag ccgtatccac cgccaacatt ttcaggggaa 7501 gttacttggc gggagctgga ctggcttttt cactcataaa gaatgcacaa acccctagga 7561 ggggaactgg gaccacagga gagacactgg gagagaagtg gaagagacag ctaaactcat 7621 tagacagaaa agagtttgaa gagtataaaa gaagtggaat actagaagtg gacaggactg 7681 aagccaagtc tgccctgaaa gatgggtcta aaatcaagca tgcagtatct agagggtcca 7741 gtaagatcag atggattgtt gagagaggga tggtaaagcc aaaagggaaa gttgtagatc 7801 ttggctgtgg gagaggagga tggtcttatt acatggcgac actcaagaac gtgactgaag 7861 tgaaagggta tacaaaagga ggtccaggac atgaagaacc gattcccatg gctacttatg 7921 gttggaattt ggtcaaactc cattcagggg ttgacgtgtt ctacaaaccc acagagcaag 7981 tggacaccct gctctgtgat attggggagt catcttctaa tccaacaata gaggaaggaa 8041 gaacattaag agttttgaag atggtggagc catggctctc ttcaaaacct gaattctgca 8101 tcaaagtcct taacccctac atgccaacag tcatagaaga gctggagaaa ctgcagagaa 8161 aacatggtgg gaaccttgtc agatgcccgc tgtccaggaa ctccacccat gagatgtatt 8221 gggtgtcagg agcgtcggga aacattgtga gctctgtgaa cacaacatca aagatgttgt 8281 tgaacaggtt cacaacaagg cataggaaac ccacttatga gaaggacgta gatcttgggg 8341 caggaacgag aagtgtctcc actgaaacag aaaaaccaga catgacaatc attgggagaa 8401 ggcttcagcg attgcaagaa gagcacaaag aaacctggca ttatgatcag gaaaacccat 8461 acagaacctg ggcgtatcat ggaagctatg aagctccttc gacaggctct gcatcctcca 8521 tggtgaacgg ggtggtaaaa ctgctaacaa aaccctggga tgtgattcca atggtgactc 8581 agttagccat gacagataca accccttttg ggcaacaaag agtgttcaaa gagaaggtgg 8641 ataccagaac accacaacca aaacccggta cacgaatggt tatgaccacg acagccaatt 8701 ggctgtgggc cctccttgga aagaagaaaa atcccagact gtgcacaagg gaagagttca 2015203575 26 Jun2015 -125- 8761 tctcaaaagt tagatcaaac gcagccatag gcgcagtctt tcaggaagaa cagggatgga 8821 catcagccag tgaagctgtg aatgacagcc ggttttggga actggttgac aaagaaaggg 8881 ccctacacca ggaagggaaa tgtgaatcgt gtgtctataa catgatggga aaacgtgaga 8941 aaaagttagg agagtttggc agagccaagg gaagccgagc aatctggtac atgtggctgg 9001 gagcgcggtt tctggaattt gaagccctgg gttttttgaa tgaagatcac tggtttggca 9061 gagaaaattc atggagtgga gtggaagggg aaggtctgca cagattggga tatatcctgg 9121 aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca 9181 caagaatcac tgaggatgac cttcaaaatg aggaactgat cacggaacag atggctcccc 9241 accacaagat cctagccaaa gccattttca aactaaccta tcaaaacaaa gtggtgaaag 9301 tcctcagacc cacaccgaga ggagcggtga tggatatcat atccaggaaa gaccaaagag 9361 gtagtggaca agttggaaca tatggtttga acacattcac caacatggaa gttcaactca 9421 tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt 9481 tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta aagaggatgg 9541 caatcagtgg agacgattgc gtggtgaagc ccctagatga gaggtttggc acttccctcc 9601 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9661 gatggaaaaa ctggcaagag gttccttttt gctcccacca ctttcacaag atctttatga 9721 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9781 gaatctcgca gggagctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9841 cccagatgtg gtcgcttatg tacttccaca gaagggatct gcgtttagcc tccatggcca 9901 tatgctcagc agttccaacg gaatggtttc caacaagcag aacaacatgg tcaatccacg 9961 ctcatcacca gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag 10021 aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc 10081 tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc agagccacct 10141 gggcgaagaa cattcacacg gccataaccc aggtcaggaa cctgatcgga aaagaggaat 10201 acgtggatta catgccagta atgaaaagat acagtgctcc ttcagagagt gaaggagttc 10261 tgtaattacc aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggttt 10321 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat ccccagggag 10381 gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga ggagacccct 10441 cccatcactg acaaaacgca gcaaaagggg gcccgaagcc aggaggaagc tgtactcctg 10501 gtggaaggac tagaggttag aggagacccc cccaacacaa aaacagcata ttgacgctgg 10561 gaaagaccag agatcctgct gtctctgcaa catcaatcca ggcacagagc gccgcaagat 10621 ggattggtgt tgttgatcca acaggttct 2015203575 26 Jun2015 -126

Appendix 2 2015203575 26 Jun2015

Sequence of recombinant dengue type 4 virus strain rDEN4 LOCUS AF326825 10549 bp RNA VRL 03-JAN-2001 DEFINITION Dengue virus type 4 recombinant clone rDEN4, complete sequence. ACCESSION AF326825 VERSION AF326825.1 GI:12018169

KEYWORDS SOURCE Dengue virus type 4. ORGANISM Dengue virus type 4

Viruses; ssRNA positive-strand viruses, no DNA stage; Flaviviridae;

Flavivirus; Dengue virus group. REFERENCE 1 (bases 1 to 10649) AUTHORS Durbin,A.P.( Karron,R.A., Sun,W., Vaughn,D.W., Reynolds,M.J.,

Perreault,J.R., Men,R.H., Lai,C.J., Elkins,W.R.,

Chanock,R.M.,

Murphy,B.R. and whitehead,S.S. TITLE A live attenuated dengue virus type 4 vaccine candidate with a 30

JOURNAL REFERENCE AUTHORS TITLE JOURNAL MD FEATURES source nucleotide deletion in the 3 ' untranslated region is highly attenuated and immunogenic in humans Unpublished 2 (bases 1 to 10649)

Whitehead,S.S.

Direct Submission

Submitted (08-DEC-2000) LID, NIAID, 7 Center Drive, Bethesda, 20892, USA

Location/Qualifiers 1. .10649 /organism="Dengue virus type 4" /db_xref="taxon:11070" /clone="rDEN4"

mat_peptide mat_peptide CDS 102. .440 /product="anchored capsid (anchC) protein" 102.. 398 /products"virion capsid (virC) protein" 102.. 10265 /codon_startsl /products "polyprotein precursor " /protein_id="AAG45435.1" /db xref="GI:12018170"

/ translations "MNQRKKWRPPFNMLKRERNRVSTPQGLVKRFSTGLFSGKGPLR MVLAFITFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKRSTI TLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTLIAMDLGEMCED TVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGERRREKRSVALTPHSGMGLET RAETWMSSEGAWKHAQRVESWILRNPGFALLAGFMAYMIGQTGIQRTVFFVLMMLVAP SYGMRCVGVGNRDFVEGVSGGAWVDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVAL LRTYCIEASISNITTATRCPTQGEPYLKEEQDQQYICRRDWDRGWGNGCGLFGKGGV VTCAKFSCSGKITGNLVQIENLEYTWVTVHNGDTHAVGNDTSNHGVTAMITPRSPSV -127-

EVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPWTAGADTSEV HWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEVDSGDGNHMFAGHLKCKV RMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTTWKVKYEGAGAPCKVPIEIRDVNKE KWGRIISSTPLAENTNSVTNIELEPPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFE STYRGAKRMAILGETAWDFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGF LVLWIGTNSRNTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFWDN VHTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNELNYVLWEG GHDLTWAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTPEARNSTFLIDGPDT SECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSSEVCDHRLMSAAIKDQKAVHADM GYWIESSKNQTWQIEKASLIEVKTCLWPKTHTLWSNGVLESQMLIPKSYAGPFSQHNY RQGYATQTVGPWHLGKLEIDFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCR SCTMPPLRFLGEDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVE ECLRRRVTRKHMILVWITLCAIILGGLTWMDLLRALIMLGDTiyiSGRIGGQIHLAIMA VFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELIDGISLGLIIiLKIV TQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFVVTLIPLCRTSCLQKQSHWVEI TALILGAQALPVYLMTLMKGASRRSWPLNEGIMAVGLVSLLGSALLKNDVPLAGPMVA GGLLLAAYVMSGSSADLSIjEKAANVQWDEMADITGSSPIVEVKQDEDGSFSIRDVEET NMITLLVKLALITVSGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSE 2015203575 26 Jun2015 gvyrimqrglfgktqvgvgihmegvfhtmwhvtrgsvichetgrlepswadvrndmis YGGGWRLGDKiroKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAVTLDFKPGTSG SPIINRKGKVIGLYGNGWTKSGDYVSAITQAERIGEPDYEVDEDIFRKKRLTIMDLH PGAGKTKRILPSIVREALKRRLRTLILAPTRWAAEMEEAIjRGLPIRYQTPAVKSEHT GREIVDLMCHATFTTRLLSSTRVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAA AIFMTATPPGATDPFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAG NDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFWTTDISEMGANFRAGRVIDPR RCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSGDPLKNDED imHWTEAKMIjLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKrFVELMRRGDL PWLSYKVASAGISYEDREWCFTGERNNQILEENMEVEIWTREGEKKKLRPRWLDARV YADPMALKDFKEFAS GRKSITLDILTE IAS LPTYLS SRAKLALDNIVMLHTTERGGRA YQHALNELPESLETLMLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVA EIQPQWIAASIILEFFLiWLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGLI EKTKTDFGFYQVKTETTIJ^VDLRPASAWTIjYAVATTILTPMLRHTIENTSANLSIiAA IANQAAVLMGLGKGWPLHRMDLG VPLLAMGC Y S QVNPTTLTAS LVMLLVHYAIIGPGL QAiamlEAQKRTAAGIMKNPTVDGITVIDLEPISYDPKFEKQIjGQVMLLVLCAGQLLL MITTWAFCEVIiTIiATGPILTLWEGNPGRFWIJTTXAVSTANTFRGSYIjAGAGLAFSLIK NAQTPRRGTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKI KHAVSRGSSKIRWIVERGMVKPKGKWDLGCGRGGWSYYMATLKNVTEVKGYTKGGPG HEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKM VEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRIISTHEMYWVSGAS GNIVSSWTTSKMLLNRFTTRHRKPTYEKDVDLGAGTRSVSTETEKPDMTIIGRRLQR LQEEHKETWHYDQENPYRTWAYHGSYEAPSTGSASSMVNGWKLLTKPWDVIPMVTQL· AMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWAIiIiGKKKIIPRLCTREEF ISKVRSNAAIGAVPQEEQGWTSASEAVNDSRFWELVDKERAIiHQEGKCESCVYNMMGK REKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLHRL GYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKILAKAI FKLTY QNKWEVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFTNMEVQLIRQMEAEGVITQ DDMQNPKGLKERVEKWLKECGVDRLIOlMAISGDDCWKPIiDERFGTSLLFLNDMGKVR KDIPQWEPSKGWKNWQEVPFCSHHFHKIFMKDGRSLWPCRNQDELIGRARISQGAGW S LRETACIiGKAYAQMWSLMYFHRRDIiRIiASMAI CSAVPTEWFPTSRTTWSIHAHHQWM TTEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDIiWCGSLIGLSSRATWAKN IHTAITQ VRNLIGKEE YVD YMP VMKRY SAP S ES EGVL " mat_jpeptide 441..938 /product="membrane precursor (prM) protein" mat peptide mat_peptide 714.. 938 /product="membrane (M) protein" 939.. 2423 /product="envelope (E) protein" -128- 2424.. 3479 2015203575 26 Jun2015 mat peptide mat_peptide mat_peptide mat_peptide mat peptide mat_peptide mat_peptide mat_peptide /product="NSl protein" 3480.. 4133 /products"NS2A protein" 4134.. 4523 /product="NS2B protein" 4524.. 6377 /products"NS3 protein" 6378.. 6758 /products "NS4A protein" 6759.. 6827 /products"2K protein" 6828.. 7562 /products"NS4B protein" 7563.. 10262 /products"NS5 protein" rDEN4 sequence cggaagcttg aatgaaccaa ccgcgtatca aggaccctta aacagcaggg gattggattc aacgataaca tggcgaaccc aacagagggg cactgtcacg gtgcaacctc acgagagaag tgagacatgg actcagaaac aggaatccag gcgatgcgta cgacctggtg ggattttgaa tgaagcctca tctgaaagag gggcaatggc ttcggggaag aacagtccac agccatgata aacactcgat gaaaaagaaa agcaggagca ggttcctcat ttctgccctc acatcttaag gtgttcagga ggtgaaagtc tgtaaacaag caacagtgta tgttggaaac gtttgagtcc ttttggttcc aagtgtgtat cttaacacag cgaaaaaagg acccctcaag cggatggtgc attctgaaga aggaaggaga ttgctgtgct ctcatgatag atcaacaaat tataaatgcc acgtctacct cgctcagtag atgtcatcgg ccaggattcg cgaactgtct ggagtaggaa ctagaacatg ctgactaaga atatcaaaca gaacaggacc tgtggcttgt ataacaggca aatggagaca actcccaggt tgtgaaccca acatggctcg gacacatcag gccaagagac gctggagcca tgcaaagtcc aagttttcaa aagtatgaag gaaaaagtgg accaacatag agcgcattaa acatacagag gttggtggac acaaccatgt 1 agttgttagt ctgtgtggac cgacaaggac agttccaaat 61 ttctaacagt ttgtttgaat agagagcaga tctctggaaa 121 tggttagacc acctttcaat atgctgaaac gcgagagaaa 181 ggttggtgaa gagattctca accggacttt tttctgggaa 241 tagcattcat cacgtttttg cgagtccttt ccatcccacc 301 gatggggaca gttgaagaaa aataaggcca tcaagatact 361 taggccgcat gctgaacatc ttgaacggga gaaaaaggtc 421 tgattcccac cgtaatggcg ttttccctca gcacaagaga 481 tggcaaaaca tgaaaggggg agacctctct tgtttaagac 541 gcactctcat tgccatggac ttgggtgaaa tgtgtgagga 601 ccctactggt caataccgaa cctgaagaca ttgattgctg 661 gggtcatgta tgggacatgc acccagagcg gagaacggag 721 ctttaacacc acattcagga atgggattgg aaacaagagc 781 aaggggcttg gaagcatgct cagagagtag agagctggat 841 cgctcttggc aggatttatg gcttatatga ttgggcaaac 901 tctttgtcct aatgatgctg gtcgccccat cctacggaat 961 acagagactt tgtggaagga gtctcaggtg gagcatgggt 1021 gaggatgcgt cacaaccatg gcccagggaa aaccaacctt 1081 caacagccaa ggaagtggct ctgttaagaa cctattgcat 1141 taactacggc aacaagatgt ccaacgcaag gagagcctta 1201 aacagtacat ttgccggaga gatgtggtag acagagggtg 1261 ttggaaaagg aggagttgtg acatgtgcga agttttcatg 1321 atttggtcca aattgagaac cttgaataca cagtggttgt 1381 cccatgcagt aggaaatgac acatccaatc atggagttac 1441 caccatcggt ggaagtcaaa ttgccggact atggagaact 1501 ggtctggaat tgactttaat gagatgattc tgatgaaaat 1561 tgcataagca atggtttttg gatctgcctc ttccatggac 1621 aggttcactg gaattacaaa gagagaatgg tgacatttaa 1681 aggatgtgac agtgctggga tctcaggaag gagccatgca 1741 cagaagtgga ctccggtgat ggaaatcaca tgtttgcagg 1801 gtatggagaa attgagaatc aagggaatgt catacacgat 1861 ttgacaaaga gatggcagaa acacagcatg ggacaacagt 1921 gtgctggagc tccgtgtaaa gtccccatag agataagaga 1981 ttgggcgtat catctcatcc acccctttgg ctgagaatac 2041 aattagaacc cccctttggg gacagctaca tagtgatagg 2101 cactccattg gttcaggaaa gggagttcca ttggcaagat 2161 gtgcaaaacg aatggccatt ctaggtgaaa cagcttggga 2221 tgttcacatc attgggaaag gctgtgcacc aggtttttgg -129· 2281 ttggaggagt ctcatggatg attagaatcc taattgggtt cttagtgttg tggattggca 2341 cgaactcgag gaacacttca atggctatga cgtgcatagc tgttggagga atcactctgt 2401 ttctgggctt cacagttcaa gcagacatgg gttgtgtggc gtcatggagt gggaaagaat 2461 tgaagtgtgg aagcggaatt tttgtggttg acaacgtgca cacttggaca gaacagtaca 2521 aatttcaacc agagtcccca gcgagactag cgtctgcaat attaaatgcc cacaaagatg 2581 gggtctgtgg aattagatca accacgaggc tggaaaatgt catgtggaag caaataacca 2641 acgagctaaa ctatgttctc tgggaaggag gacatgacct cactgtagtg gctggggatg 2701 tgaagggggt gttgaccaaa ggcaagagag cactcacacc cccagtgagt gatctgaaat 2761 attcatggaa gacatgggga aaagcaaaaa tcttcacccc agaagcaaga aatagcacat 2821 ttttaataga cggaccagac acctctgaat gccccaatga acgaagagca tggaactctc 2881 ttgaggtgga agactatgga tttggcatgt tcacgaccaa catatggatg aaattccgag 2941 aaggaagttc agaagtgtgt gaccacaggt taatgtcagc tgcaattaaa gatcagaaag 3001 ctgtgcatgc tgacatgggt tattggatag agagctcaaa aaaccagacc tggcagatag 3061 agaaagcatc tcttattgaa gtgaaaacat gtctgtggcc caagacccac acactgtgga 3121 gcaatggagt gctggaaagc cagatgctca ttccaaaatc atatgcgggc cctttttcac 3181 agcacaatta ccgccagggc tatgccacgc aaaccgtggg cccatggcac ttaggcaaat 3241 tagagataga ctttggagaa tgccccggaa caacagtcac aattcaggag gattgtgacc 3301 atagaggccc atctttgagg accaccactg catctggaaa actagtcacg caatggtgct 3361 gccgctcctg cacgatgcct cccttaaggt tcttgggaga agatgggtgc tggtatggga 3421 tggagattag gcccttgagt gaaaaagaag agaacatggt caaatcacag gtgacggccg 3481 gacagggcac atcagaaact ttttctatgg gtctgttgtg cctgaccttg tttgtggaag 3541 aatgcttgag gagaagagtc actaggaaac acatgatatt agttgtggtg atcactcttt 3601 gtgctatcat cctgggaggc ctcacatgga tggacttact acgagccctc atcatgttgg 3661 gggacactat gtctggtaga ataggaggac agatccacct agccatcatg gcagtgttca 3721 agatgtcacc aggatacgtg ctgggtgtgt ttttaaggaa actcacttca agagagacag 3781 cactaatggt aataggaatg gccatgacaa cggtgctttc aattccacat gaccttatgg 3841 aactcattga tggaatatca ctgggactaa ttttgctaaa aatagtaaca cagtttgaca 3901 acacccaagt gggaacctta gctctttcct tgactttcat aagatcaaca atgccattgg 3961 tcatggcttg gaggaccatt atggctgtgt tgtttgtggt cacactcatt cctttgtgca 4021 ggacaagctg tcttcaaaaa cagtctcatt gggtagaaat aacagcactc atcctaggag 4081 cccaagctct gccagtgtac ctaatgactc ttatgaaagg agcctcaaga agatcttggc 4141 ctcttaacga gggcataatg gctgtgggtt tggttagtct cttaggaagc gctcttttaa 4201 agaatgatgt ccctttagct ggcccaatgg tggcaggagg cttacttctg gcggcttacg 4261 tgatgagtgg tagctcagca gatctgtcac tagagaaggc cgccaacgtg cagtgggatg 4321 aaatggcaga cataacaggc tcaagcccaa tcgtagaagt gaagcaggat gaagatggct 4381 ctttctccat acgggacgtc gaggaaacca atatgataac ccttttggtg aaactggcac 4441 tgataacagt gtcaggtctc taccccttgg caattccagt cacaatgacc ttatggtaca 4501 tgtggcaagt gaaaacacaa agatcaggag ccctgtggga cgtcccctca cccgctgcca 4561 ctaaaaaagc cgcactgtct gaaggagtgt acaggatcat gcaaagaggg ttattcggga 4621 aaactcaggt tggagtaggg atacacatgg aaggtgtatt tcacacaatg tggcatgtaa 4681 caagaggatc agtgatctgc cacgagactg ggagattgga gccatcttgg gctgacgtca 4741 ggaatgacat gatatcatac ggtgggggat ggaggcttgg agacaaatgg gacaaagaag 4801 aagacgttca ggtcctcgcc atagaaccag gaaaaaatcc taaacatgtc caaacgaaac 4861 ctggcctttt caagacccta actggagaaa ttggagcagt aacattagat ttcaaacccg 4921 gaacgtctgg ttctcccatc atcaacagga aaggaaaagt catcggactc tatggaaatg 4981 gagtagttac caaatcaggt gattacgtca gtgccataac gcaagccgaa agaattggag 5041 agccagatta tgaagtggat gaggacattt ttcgaaagaa aagattaact ataatggact 5101 tacaccccgg agctggaaag acaaaaagaa ttcttccatc aatagtgaga gaagccttaa 5161 aaaggaggct acgaactttg attttagctc ccacgagagt ggtggcggcc gagatggaag 5221 aggccctacg tggactgcca atccgttatc agaccccagc tgtgaaatca gaacacacag 5281 gaagagagat tgtagacctc atgtgtcatg caaccttcac aacaagactt ttgtcatcaa 5341 ccagggttcc aaattacaac cttatagtga tggatgaagc acatttcacc gatccttcta 5401 gtgtcgcggc tagaggatac atctcgacca gggtggaaat gggagaggca gcagccatct 5461 tcatgaccgc aacccctccc ggagcgacag atccctttcc ccagagcaac agcccaatag 5521 aagacatcga gagggaaatt ccggaaaggt catggaacac agggttcgac tggataacag 5581 actaccaagg gaaaactgtg tggtttgttc ccagcataaa agctggaaat gacattgcaa 5641 attgtttgag aaagtcggga aagaaagtta tccagttgag taggaaaacc tttgatacag 2015203575 26 Jun2015 -130· 2015203575 26 Jun2015 5701 5761 5821 5881 5941 6001 6061 6121 6181 6241 6301 6361 6421 6481 6541 6601 6661 6721 6781 6841 6901 6961 7021 7081 7141 7201 7261 7321 7381 7441 7501 7561 7621 7681 7741 7801 7861 7921 7981 8041 8101 8161 8221 8281 8341 8401 8461 8521 8581 8641 8701 8761 8821 8881 8941 9001 9061 agtatccaaa tgggggccaa tcctaccaga gcgctgctca ttttctccgg tgctgcttga gggaaaaaac ttgtggaatt ctggcatttc tagaagaaaa caagatggtt ttgccagtgg cttacctttc aaagaggagg tcatgcttgt ggaaaggaat tctgggtagc tcatggtact tctacgtcat tgattgaaaa tcgatgtgga tgactcccat ttgccaacca acctcggtgt cagcatcctt aagccacaag acgggataac: tagggcaggt gggctttctg acccgggaag gttacttggc ggggaactgg tagacagaaa aagccaagtc gtaagatcag ttggctgtgg tgaaagggta gttggaattt tggacaccct gaacattaag tcaaagtcct aacatggtgg gggtgtcagg tgaacaggtt caggaacgag ggcttcagcg acagaacctg tggtgaacgg agttagccat ataccagaac ggctgtgggc tctcaaaagt catcagccag ccctacacca aaaagttagg gagcgcggtt gagaaaattc aacgaaactc ttttagagcc tgggccagag gagaagaggg agacccacta caatatctac ccaagccatt aatgaggaga ttacgaagat catggaggtt agatgcacgt aaggaagagt ctctagggcc gagggcctat agctttacta agggaaattg agaaattcaa gttgataccg attgaccatt aacaaaaacg cttgagacca gctgagacac ggcagccgtc gccgctgtta agtcatgctt agaggcccag agtaatagat catgctacta tgaagtcttg gttttggaac gggagctgga gaccacagga agagtttgaa tgccctgaaa atggafctgtt gagaggagga tacaaaagga ggtcaaactc gctctgtgat agttttgaag taacccctac gaaccttgtc agcgtcggga cacaacaagg aagtgtctcc attgcaagaa ggcgtatcat ggtggtaaaa gacagataca accacaacca cctccttgga tagatcaaac tgaagctgtg ggaagggaaa agagtttggc tctggaattt atggagtgga acggactggg gggagagtga agagtcattt cgaataggaa aaaaatgatg accccagaag gatggagagt ggagaccttc cgggaatggt gaaatttgga gtatacgctg ataactctcg aagctcgccc caacacgccc _ ggtgctatga tcaatgggtt ccccagtgga gaaccagaaa ctcaccatca gattttgggt gcttcagcat accatagaaa ctaatggggc gcaatgggat ttagtccatt aaaaggacag ctagaaccaa gtcttgtgtg actttggcca acgaccatag ctggcttttt gagacactgg gagtataaaa gatgggtcta gagagaggga tggtcttatt ggtccaggac cattcagggg attggggagt atggtggagc atgccaacag agatgcccgc aacattgtga cataggaaac actgaaacag gagcacaaag ggaagctatg ctgctaacaa accccttttg aaacccggta aagaagaaaa gcagccatag aatgacagcc tgtgaatcgt agagccaagg gaagccctgg gfcggaagggg actttgtggt tagaccctag tagcaggtcc ggaacccagc aagatcatgc ggatcattcc ttcgcctcag cggtgtggct gcttcacagg ctagagaggg accccatggc acatcctaac ttgataacat tgaacgaact cagcaggcat tgataaccat tagcggcctc aacaaaggac ttggtctaat tttaccaggt ggacgctcta acacgtcggc ttggaaaagg gctattctca atgcaataat ctgctgggat tatcctatga ctggacaact caggaccaat ccgtatccac cactcataaa gagagaagtg gaagtggaat aaatcaagca tggtaaagcc acatggcgac atgaagaacc ttgacgtgtt catcttctaa catggctctc tcatagaaga tgtccaggaa gctctgtgaa ccacttatga aaaaaccaga aaacctggca aagctccttc aaccctggga ggcaacaaag cacgaatggt atcccagact gcgcagtctt ggttttggga gtgtctataa gaagccgagc gttttttgaa aaggtctgca cactacagac aagatgcctc tattccagtg acaagaagac ccactggaca aacattgttt aggggaacaa gagctataag ggaaagaaat agaaaagaaa tttgaaggat agagattgcc agtcatgctc tccggagtca cttcctgttt tgcggtggct aatcatacta cccacaagac agcagccaac aaaaacagaa tgcagtagcc caacctatct atggccgctc agtgaaccca aggcccagga catgaaaaat cccaaaattt actcttgatg cttgaccttg cgccaacatt gaatgcacaa gaagagacag actagaagtg tgcagtatca aaaagggaaa actcaagaac gattcccatg ctacaaaccc tccaacaata ttcaaaacct gctggagaaa ctccacccat cacaacatca gaaggacgta catgacaatc ttatgatcag gacaggctct tgtgattcca agtgttcaaa tatgaccacg gtgcacaagg tcaggaagaa actggttgac catgatggga aatctggtac tgaagatcac cagattggga atatctgaaa aagccagtta actccagcaa gaccaatacg gaagcaaaga ggtccggaaa aggaagactt gtagcttctg aaccaaattt aagctaaggc ttcaaggagt agtttgccaa cacacaacag ctggaaacac ttcatgcaag agtggcttgc gagttttttc aatcaattga gagatggggc accaccatcc accacaattc ctagcagcca cacagaatgg acaaccttga ttgcaggcaa cccacagtgg gaaaagcaat agaacaacat tgggagggca ttcaggggaa acccctagga ctaaactcat gacaggactg agagggtcca gttgtagatc gtgactgaag gctacttatg acagagcaag gaggaaggaa gaattctgca ctgcagagaa gagatgtatt aagatgttgt gatcttgggg attgggagaa gaaaacccat gcatcctcca atggtgactc gagaaggtgg acagccaatt gaagagttca cagggatgga aaagaaaggg aaacgtgaga atgtggctgg tggtttggca tatatcctgg -131- 9121 aggagataga caagaaggat ggagacctaa tgtatgctga tgacacagca ggctgggaca 9181 caagaatcac tgaggatgac cttcaaaatg aggaactgat cacggaacag atggctcccc 9241 accacaagat cctagccaaa gccattttca aactaaccta tcaaaacaaa gtggtgaaag 9301 tcctcagacc cacaccgcgg ggagcggtga tggatatcat atccaggaaa gaccaaagag 9361 gtagtggaca agttggaaca tatggtttga acacattcac caacatggaa gttcaactca 9421 tccgccaaat ggaagctgaa ggagtcatca cacaagatga catgcagaac ccaaaagggt 9481 tgaaagaaag agttgagaaa tggctgaaag agtgtggtgt cgacaggtta aagaggatgg 9541 caatcagtgg agacgattgc gtggtgaagc ccctagatga gaggtttggc acttccctcc 9601 tcttcttgaa cgacatggga aaggtgagga aagacattcc gcagtgggaa ccatctaagg 9661 gatggaaaaa ctggcaagag gttccttttt gctcccacca ctttcacaag atctttatga 9721 aggatggccg ctcactagtt gttccatgta gaaaccagga tgaactgata gggagagcca 9781 gaatctcgca gggagctgga tggagcttaa gagaaacagc ctgcctgggc aaagcttacg 9841 cccagatgtg gtcgcttatg tacttccaca gaagggatct gcgtttagcc tccatggcca 9901 tatgctcagc agttccaacg gaatggtttc caacaagcag aacaacatgg tcaatccacg 9961 ctcatcacca gtggatgacc actgaagata tgctcaaagt gtggaacaga gtgtggatag 10021 aagacaaccc taatatgact gacaagactc cagtccattc gtgggaagat ataccttacc 10081 tagggaaaag agaggatttg tggtgtggat ccctgattgg actttcttcc agagccacct 10141 gggcgaagaa cattcatacg gccataaccc aggtcaggaa cctgatcgga aaagaggaat 10201 acgtggatta catgccagta atgaaaagat acagtgctcc ttcagagagt gaaggagttc 10261 tgtaattacc aacaacaaac accaaaggct attgaagtca ggccacttgt gccacggttt 10321 gagcaaaccg tgctgcctgt agctccgcca ataatgggag gcgtaataat ccccagggag 10381 gccatgcgcc acggaagctg tacgcgtggc atattggact agcggttaga ggagacccct 10441 cccatcactg ataaaacgca gcaaaagggg gcccgaagcc aggaggaagc tgtactcctg 10501 gtggaaggac tagaggttag aggagacccc cccaacacaa aaacagcata ttgacgctgg 10561 gaaagaccag agatcctgct gtctctgcaa catcaatcca ggcacagagc gccgcaagat 10621 ggattggtgt tgttgatcca acaggttct 2015203575 26 Jun2015 -132- 2015203575 26 Jun2015

Appendix 3 Sequence of recombinant dengue type 2 chimeric virus strain rDEN2/4A30

LOCUS DEFINITION sequence. ACCESSION VERSION KEYWORDS SOURCE ORGANISM

Submission pending Dengue virus type 2 recombinant clone rDEN2/4A30, complete Submission pending

Dengue virus type 2 NGC. Dengue virus type 2 Viruses; ssRNA positive-strand viruses, no DNA stage;

Flaviviridae ; Flavivirus; Dengue virus group. REFERENCE 1 (bases 1 to 10616) AUTHORS . TITLE JOURNAL Unpublished FEATURES Location/Qualitiers source 1..10616 /organism="Dengue virus type 2" /c1one="rDEN2/4Δ30" mat_peptide 97..438 /product="anchored capsid (anchC) protein" mat_peptide 97..396 /products"virion capsid (virC) protein" CDS 97..10263 /codon_start=l /products"polyprotein precursor"

/translate on=MNNQRKKARNTPFNMLKRERNRVS TVQQLTKRF S LGMLQGRGPLK LFMALVAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRRTAG MIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTLMAMDLGELCED TITYKCPLLRQNEPEDIDCWCNSTSTWVTYGTCTTTGEHRREKRSVALVPHVGMGLET RTETWMSSEGAWKHAQRIETWILRHPGFTIMAAILAYTIGTTHFQRALIFILLTAVAP SMTMRCIGISNRDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDFELIKTEAKQPAT LRKYCIEAKLTNTTTESRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGI VTCAMFTCKKNMEGKWQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSIT EAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPWLPGADTQGS NWIQKETLVTFKNPHAKKQDVWLGSQEGAMHTALTGATEIQMSSGNLLFTGHLKCRL RMDKLQLKGMSYSMCTGKFKWKEIAETQHGTIVIRVQYEGDGSPCKIPFEIMDLEKR HVLGRLITVNPIVTEKDSPVNIEAEPPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMFE TTMRGAKRMAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGV IITWIGMNSRNTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFWDN VHTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNELNYVLWEG GHDLTWAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFTPEARNSTFLIDGPDT SECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGSSEVCDHRLMSAAIKDQKAVHADM GYWIESSKNQTWQIEKASLIEVKTCLWPKTHTLWSNGVLESQMLIPKSYAGPFSQHNY RQGYATQTVGPWHLGKLEIDFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCR SCTMPPLRFLGEDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVE ECLRRRVTRKHMILVWITLCAIILGGLTWMDLLRALIMLGDTMSGRIGGQIHLAIMA -133- VFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMELIDGISLGLILLKIV TQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLFVVTLIPLCRTSCIjQKQSHWVEI TALILGAQALPVYLMTLMKGASRRSWPIjNEGIMAVGLVSLLGSALLKNDVPLAGPMVA GGLLLAAYVMSGSSADLSLEKAANVQWDEMADITGSSPIVEVKQDEDGSFSIRDVEET NMITLLVKLALIWSGLYPLAIPVTMTLWYMWQVKTQRSGALWDVPSPAATKKAALSE GVYRIMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRNDMIS YGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIGAVTLDFKPGTSG SPIINRKGKVIGLYGNGWTKSGDYVSAITQAERIGEPDYEVDEDIFRKKRLTIMDLH PGAGKTKRILPSIVREALKRRLRTLILAPTRVVAAEMEEALRGLPIRYQTPAVKSEHT GREIVDLMCHATFTTRLLS STRVPNYNLIVMDEAHFTDPS S VAARGYISTRVEMGEAA AIFMTATPPGATDPFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAG NDIANCLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFWTTDISEMGANFRAGRVIDPR RCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYVFSGDPLKNDED HAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEFRLRGEQRKTFVELMRRGDL PVWLSYKVASAGISYEDREWCFTGERNNQILEENMEVEIWTREGEKKKLRPRWLDARV YADPMALKDFKEFASGRKSITIiDIIjTEIASLPTYLSSRAKIiALDliriVMLHTTERGGRA YQHALNELPESLETLMLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVA EIQPQWIAASIILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGLI EKTKTDFGFYQVKTETTILDVDLRPASAWTLYAVATTILTPMLRHTIENTSANLSLAA IANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNPTTLTASLVMLLVHYAIIGPGL QAKATREAQKRTAAGIMKNPTVDGITVIDLEPISYDPKFEKQLGQVMLLVLCAGQLLL MRTTWAFCEVLTLATGPILTLWEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIK NAQTPRRGTGTTGETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKI KHAVSRGSSKIRWIVERGMVKPKGKWDLGCGRGGWSYYMATLKNVTEVKGYTKGGPG HEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTIEEGRTLRVLKM VEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLVRCPLSRNSTHEMYWVSGAS GNIVSSVWTTSKMLLNRFTTRHRKPTYEKDVDLGAGTRSVSTETEKPDMTIIGRRLQR LQEEHKETWHYDQENPYRTWAYHGSYEAPSTGSASSMVNGWKLLTKPWDVIPMVTQL AMTDTTPFGQQRVFKEKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEF ISKVRSNAAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMGK REKKLGE FGRAKGS RAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSGVEGEGLHRL GYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQMAPHHKILAKAIFKLTY QNKWKVLRPTPRGAVMDIISRKDQRGSGQVGTYGLNTFTNMEVQLIRQMEAEGVITQ DDMQNPKGLKERVEKWLKECGVDRLKRMAISGDDCWKPLDERFGTSLLFLNDMGKVR KDIPQWEPSKGWKNWQEVPFCSHHFHKIFMKDGRSLWPCRNQDELIGRARISQGAGW SLRETACLGKAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWM TTEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSSRATWAKN IHTAITQVRNLIGKEEYVDYMPVMKRYSAP SESEGVL" mat_peptide 2015203575 26 Jun2015 439.. 936 /product="membrane precursor (prM) protein" 712.. 936 /product="membrane (M) protein" 937.. 2421 /product="envelope (E) protein" 2422.. 3477 /product="NSl protein" 3478.. 4131 /product="NS2A protein" 4132.. 4521 /product="NS2B protein" 4522.. 6375 /product="NS3 protein" 6376.. 6756 /product="NS4A protein" 6757.. 6825 /product="2K protein" 6826.. 7560 mat_peptide mat_peptide mat_peptide mat_peptide mat_peptide mat_peptide mat_j?eptide mat_j?eptide mat_j?eptide -134- /product="NS4B protein" mat_peptide 7561..10260 2015203575 26 Jun2015 /product="NS5 protein" rDEN2/4A30 sequence 1 agttgttagt ctgtgtggac cgacaaggac agttccaaat cggaagcttg 51 cttaacacag ttctaacagt ttgtttgaat agagagcaga tctctgatga 101 ataaccaacg aaaaaaggcg agaaatacgc ctttcaatat gctgaaacgc 151 gagagaaacc gcgtgtcgac tgtacaacag ctgacaaaga gattctcact 201 tggaatgctg cagggacgag gaccattaaa actgttcatg gccctggtgg 251 cgttccttcg tttcctaaca atcccaccaa cagcagggat actgaagaga 301 tggggaacaa ttaaaaaatc aaaagccatt aatgttttga gagggttcag 351 gaaagagatt ggaaggatgc tgaacatctt gaacaggaga cgcagaactg 401 caggcatgat cattatgctg attccaacag tgatggcgtt ccatttaacc 451 acacgtaacg gagaaccaca catgatcgtc agtagacaag agaaagggaa 501 aagtcttctg tttaaaacag aggatggtgt gaacatgtgt accctcatgg 551 ccatggacct tggtgaattg tgtgaagata caatcacgta caagtgtcct 601 cttctcaggc agaatgaacc agaagacata gattgttggt gcaactctac 651 gtccacatgg gtaacttatg ggacgtgtac caccacagga gaacacagaa 701 gagaaaaaag atcagtggca ctcgttccac atgtgggaat gggactggag 751 acacgaactg aaacatggat gtcatcagaa ggggcctgga aacatgccca 801 gagaattgaa acttggatct tgagacatcc aggctttacc ataatggcag 851 caatcctggc atacaccata ggaacgacac atttccaaag agccctgatt 901 ttcatcttac tgacagctgt cgctccttca atgacaatgc gttgcatagg 951 aatatcaaat agagactttg tagaaggggt ttcaggagga agctgggttg 1001 acatagtctt agaacatgga agctgtgtga cgacgatggc aaaaaacaaa 1051 ccaacattgg attttgaact gataaaaaca gaagccaaac aacctgccac 1101 tctaaggaag tactgtatag aggcaaagct gaccaacaca acaacagaat 1151 ctcgctgccc aacacaagga gaacctagcc taaatgaaga gcaggacaaa 1201 aggttcgtct gcaaacactc catggtggac agaggatggg gaaatggatg 1251 tggattattt ggaaaaggag gcattgtgac ctgtgctatg ttcacatgca 1301 aaaagaacat ggaaggaaaa gtcgtgcaac cagaaaactt ggaatacacc 1351 attgtgataa cacctcactc aggggaagag catgcagtcg gaaatgacac 1401 aggaaaacat ggcaaggaaa tcaaaataac accacagagt tccatcacag 1451 aagcagagtt gacaggctat ggcactgtca cgatggagtg ctctccgaga 1501 acgggcctcg acttcaatga gatggtgttg ctgcaaatgg aaaataaagc 1551 ttggctggtg cacaggcaat ggttcctaga cctgccgttg ccatggctgc 1601 ccggagcgga cacacaagga tcaaattgga tacagaaaga gacattggtc 1651 actttcaaaa atccccatgc gaagaaacag gatgttgttg ttttgggatc 1701 ccaagaaggg gccatgcaca cagcactcac aggggccaca gaaatccaga 1751 tgtcatcagg aaacttactg ttcacaggac atctcaagtg caggctgagg 1801 atggacaaac tacagctcaa aggaatgtca tactctatgt gcacaggaaa 1851 gtttaaagtt gtgaaggaaa tagcagaaac acaacatgga acaatagtta 1901 tcagagtaca atatgaaggg gacggttctc catgtaagat cccttttgag 1951 ataatggatt tggaaaaaag acatgtttta ggtcgcctga ttacagtcaa 2001 cccaatcgta acagaaaaag atagcccagt caacatagaa gcagaacctc 2051 cattcggaga cagctacatc atcataggag tagagccggg acaattgaag 2101 ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac 2151 aatgagggga gcgaagagaa tggccatttt aggtgacaca gcttgggatt 2201 ttggatccct gggaggagtg tttacatcta taggaaaggc tctccaccaa 2251 gttttcggag caatctatgg ggctgccttc agtggggtct catggactat 2301 gaaaatcctc ataggagtca ttatcacatg gataggaatg aactcgagga 2351 acacttcaat ggctatgacg tgcatagctg ttggaggaat cactctgttt 2401 ctgggcttca cagttcaagc agacatgggt tgtgtggcgt catggagtgg 2451 gaaagaattg aagtgtggaa gcggaatttt tgtggttgac aacgtgcaca 2501 cttggacaga acagtacaaa tttcaaccag agtccccagc gagactagcg -135- 2551 tctgcaatat taaatgccca caaagatggg gtctgtggaa ttagatcaac 2601 cacgaggctg gaaaatgtca tgtggaagca aataaccaac gagctaaact 2651 atgttctctg ggaaggagga catgacctca ctgtagtggc tggggatgtg 2701 aagggggtgt tgaccaaagg caagagagca ctcacacccc cagtgagtga 2751 tctgaaatat tcatggaaga catggggaaa agcaaaaatc ttcaccccag 2801 aagcaagaaa tagcacattt ttaatagacg gaccagacac ctctgaatgc 2851 cccaatgaac gaagagcatg gaactctctt gaggtggaag actatggatt 2901 tggcatgttc acgaccaaca tatggatgaa attccgagaa ggaagttcag 2951 aagtgtgtga ccacaggtta atgtcagctg caattaaaga tcagaaagct 3001 gtgcatgctg acatgggtta ttggatagag agctcaaaaa accagacctg 3051 gcagatagag aaagcatctc ttattgaagt gaaaacatgt ctgtggccca 3101 agacccacac actgtggagc aatggagtgc tggaaagcca gatgctcatt 3151 ccaaaatcat atgcgggccc tttttcacag cacaattacc gccagggcta 3201 tgccacgcaa accgtgggcc catggcactt aggcaaatta gagatagact 3251 ttggagaatg ccccggaaca acagtcacaa ttcaggagga ttgtgaccat 3301 agaggcccat ctttgaggac caccactgca tctggaaaac tagtcacgca 3351 atggtgctgc cgctcctgca cgatgcctcc cttaaggttc ttgggagaag 3401 atgggtgctg gtatgggatg gagattaggc ccttgagtga aaaagaagag 3451 aacatggtca aatcacaggt gacggccgga cagggcacat cagaaacttt 3501 ttctatgggt ctgttgtgcc tgaccttgtt tgtggaagaa tgcttgagga 3551 gaagagtcac taggaaacac atgatattag ttgtggtgat cactctttgt 3601 gctatcatcc tgggaggcct cacatggatg gacttactac gagccctcat 3651 catgttgggg gacactatgt ctggtagaat aggaggacag atccacctag 3701 ccatcatggc agtgttcaag atgtcaccag gatacgtgct gggtgtgttt 3751 ttaaggaaac tcacttcaag agagacagca ctaatggtaa taggaatggc 3801 catgacaacg gtgctttcaa ttccacatga ccttatggaa ctcattgatg 3851 gaatatcact gggactaatt ttgctaaaaa tagtaacaca gtttgacaac 3901 acccaagtgg gaaccttagc tctttccttg actttcataa gatcaacaat 3951 gccattggtc atggcttgga ggaccattat ggctgtgttg tttgtggtca 4001 cactcattcc tttgtgcagg acaagctgtc ttcaaaaaca gtctcattgg 4051 gtagaaataa cagcactcat cctaggagcc caagctctgc cagtgtacct 4101 aatgactctt atgaaaggag cctcaagaag atcttggcct cttaacgagg 4151 gcataatggc tgtgggtttg gttagtctct taggaagcgc tcttttaaag 4201 aatgatgtcc ctttagctgg cccaatggtg gcaggaggct tacttctggc 4251 ggcttacgtg atgagtggta gctcagcaga tctgtcacta gagaaggccg 4301 ccaacgtgca gtgggatgaa atggcagaca taacaggctc aagcccaatc 4351 atagaagtga agcaggatga agatggctct ttctccatac gggacgtcga 4401 ggaaaccaat atgataaccc ttttggtgaa actggcactg ataacagtgt 4451 caggtctcta ccccttggca attccagtca caatgacctt atggtacatg 4501 tggcaagtga aaacacaaag atcaggagcc ctgtgggacg tcccctcacc 4551 cgctgccact aaaaaagccg cactgtctga aggagtgtac aggatcatgc 4601 aaagagggtt attcgggaaa actcaggttg gagtagggat acacatggaa 4651 ggtgtatttc acacaatgtg gcatgtaaca agaggatcag tgatctgcca 4701 cgagactggg agattggagc catcttgggc tgacgtcagg aatgacatga 4751 tatcatacgg tgggggatgg aggcttggag acaaatggga caaagaagaa 4801 gacgttcagg tcctcgccat agaaccagga aaaaatccta aacatgtcca 4851 aacgaaacct ggccttttca agaccctaac tggagaaatt ggagcagtaa 4901 cattagattt caaacccgga acgtctggtt ctcccatcat caacaggaaa 4951 ggaaaagtca tcggactcta tggaaatgga gtagttacca aatcaggtga 5001 ttacgtcagt gccataacgc aagccgaaag aattggagag ccagattatg 5051 aagtggatga ggacattttt cgaaagaaaa gattaactat aatggactta 5101 caccccggag ctggaaagac aaaaagaatt cttccatcaa tagtgagaga 5151 agccttaaaa aggaggctac gaactttgat tttagctccc acgagagtgg 5201 tggcggccga gatggaagag gccctacgtg gactgccaat ccgttatcag 5251 accccagctg tgaaatcaga acacacagga agagagattg tagacctcat 5301 gtgtcatgca accttcacaa caagactttt gtcatcaacc agggttccaa 5351 attacaacct tatagtgatg gatgaagcac atttcaccga tccttctagt 2015203575 26 Jun2015 -136- 5401 gtcgcggcta gaggatacat ctcgaccagg gtggaaatgg gagaggcagc 5451 agccatcttc atgaccgcaa cccctcccgg agcgacagat ccctttcccc 5501 agagcaacag cccaatagaa gacatcgaga gggaaattcc ggaaaggtca 5551 tggaacacag ggttcgactg gataacagac taccaaggga aaactgtgtg 5601 gtttgttccc agcataaaag ctggaaatga cattgcaaat tgtttgagaa 5651 agtcgggaaa gaaagttatc cagttgagta ggaaaacctt tgatacagag 5701 tatccaaaaa cgaaactcac ggactgggac tttgtggtca ctacagacat 5751 atctgaaatg ggggccaatt ttagagccgg gagagtgata gaccctagaa 5801 gatgcctcaa gccagttatc ctaccagatg ggccagagag agtcatttta 5851 gcaggtccta ttccagtgac tccagcaagc gctgctcaga gaagagggcg 5901 aataggaagg aacccagcac aagaagacga ccaatacgtt ttctccggag 5951 acccactaaa aaatgatgaa gatcatgccc actggacaga agcaaagatg 6001 ctgcttgaca atatctacac cccagaaggg atcattccaa cattgtttgg 6051 tccggaaagg gaaaaaaccc aagccattga tggagagttt cgcctcagag 6101 gggaacaaag gaagactttt gtggaattaa tgaggagagg agaccttccg 6151 gtgtggctga gctataaggt agcttctgct ggcatttctt acaaagatcg 6201 ggaatggtgc ttcacagggg aaagaaataa ccaaatttta gaagaaaaca 6251 tggaggttga aatttggact agagagggag aaaagaaaaa gctaaggcca 6301 agatggttag atgcacgtgt atacgctgac cccatggctt tgaaggattt 6351 caaggagttt gccagtggaa ggaagagtat aactctcgac atcctaacag 6401 agattgccag tttgccaact tacctttcct ctagggccaa gctcgccctt 6451 gataacatag tcatgctcca cacaacagaa agaggaggga gggcctatca 6501 acacgccctg aacgaacttc cggagtcact ggaaacactc atgcttgtag 6551 ctttactagg tgctatgaca gcaggcatct tcctgttttt catgcaaggg 6601 aaaggaatag ggaaattgtc aatgggtttg ataaccattg cggtggctag 6651 tggcttgctc tgggtagcag aaattcaacc ccagtggata gcggcctcaa 6701 tcatactaga gttttttctc atggtactgt tgataccgga accagaaaaa 6751 caaaggaccc cacaagacaa tcaattgatc tacgtcatat tgaccattct 6801 caccatcatt ggtctaatag cagccaacga gatggggctg attgaaaaaa 6851 caaaaacgga ttttgggttt taccaggtaa aaacagaaac caccatcctc 6901 gatgtggact tgagaccagc ttcagcatgg acgctctatg cagtagccac 6951 cacaattctg actcccatgc tgagacacac catagaaaac acgtcggcca 7001 acctatctct agcagccatt gccaaccagg cagccgtcct aatggggctt 7051 ggaaaaggat ggccgctcca cagaatggac ctcggtgtgc cgctgttagc 7101 aatgggatgc tattctcaag tgaacccaac aaccttgaca gcatccttag 7151 tcatgctttt agtccattat gcaataatag gcccaggatt gcaggcaaaa 7201 gccacaagag aggcccagaa aaggacagct gctgggatca tgaaaaatcc 7251 cacagtggac gggataacag taatagatct agaaccaata tcctatgacc 7301 caaaatttga aaagcaatta gggcaggtca tgctactagt cttgtgtgct 7351 ggacaactac tcttgatgag aacaacatgg gctttctgtg aagtcttgac 7401 tttggccaca ggaccaatct tgaccttgtg ggagggcaac ccgggaaggt 7451 tttggaacac gaccatagcc gtatccaccg ccaacatttt caggggaagt 7501 tacttggcgg gagctggact ggctttttca ctcataaaga atgcacaaac 7551 ccctaggagg ggaactggga ccacaggaga gacactggga gagaagtgga 7601 agagacagct aaactcatta gacagaaaag agtttgaaga gtataaaaga 7651 agtggaatac tagaagtgga caggactgaa gccaagtctg ccctgaaaga 7701 tgggtctaaa atcaagcatg cagtatcaag agggtccagt aagatcagat 7751 ggattgttga gagagggatg gtaaagccaa aagggaaagt tgtagatctt 7801 ggctgtggga gaggaggatg gtcttattac atggcgacac tcaagaacgt 7851 gactgaagtg aaagggtata caaaaggagg tccaggacat gaagaaccga 7901 ttcccatggc tacttatggt tggaatttgg tcaaactcca ttcaggggtt 7951 gacgtgttct acaaacccac agagcaagtg gacaccctgc tctgtgatat 8001 tggggagtca tcttctaatc caacaataga ggaaggaaga acattaagag 8051 ttttgaagat ggtggagcca tggctctctt caaaacctga attctgcatc 8101 aaagtcctta acccctacat gccaacagtc atagaagagc tggagaaact 8151 gcagagaaaa catggtggga accttgtcag atgcccgctg tccaggaact 8201 ccacccatga gatgtattgg gtgtcaggag cgtcgggaaa cattgtgagc 2015203575 26 Jun2015 -137- 8251 tctgtgaaca caacatcaaa gatgttgttg aacaggttca caacaaggca 8301 taggaaaccc acttatgaga aggacgtaga tcttggggca ggaacgagaa 8351 gtgtctccac tgaaacagaa aaaccagaca tgacaatcat tgggagaagg 8401 cttcagcgat tgcaagaaga gcacaaagaa acctggcatt atgatcagga 8451 aaacccatac agaacctggg cgtatcatgg aagctatgaa gctccttcga 8501 caggctctgc atcctccatg gtgaacgggg tggtaaaact gctaacaaaa 8551 ccctgggatg tgattccaat ggtgactcag ttagccatga cagatacaac 8601 cccttttggg caacaaagag tgttcaaaga gaaggtggat accagaacac 8651 cacaaccaaa acccggtaca cgaatggtta tgaccacgac agccaattgg 8701 ctgtgggccc tccttggaaa gaagaaaaat cccagactgt gcacaaggga 8751 agagttcatc tcaaaagtta gatcaaacgc agccataggc gcagtctttc 8801 aggaagaaca gggatggaca tcagccagtg aagctgtgaa tgacagccgg 8851 ttttgggaac tggttgacaa agaaagggcc ctacaccagg aagggaaatg 8901 tgaatcgtgt gtctataaca tgatgggaaa acgtgagaaa aagttaggag 8951 agtttggcag agccaaggga agccgagcaa tctggtacat gtggctggga 9001 gcgcggtttc tggaatttga agccctgggt tttttgaatg aagatcactg 9051 gtttggcaga gaaaattcat ggagtggagt ggaaggggaa ggtctgcaca 9101 gattgggata tatcctggag gagatagaca agaaggatgg agacctaatg 9151 tatgctgatg acacagcagg ctgggacaca agaatcactg aggatgacct 9201 tcaaaatgag gaactgatca cggaacagat ggctccccac cacaagatcc 9251 tagccaaagc cattttcaaa ctaacctatc aaaacaaagt ggtgaaagtc 9301 ctcagaccca caccgcgggg agcggtgatg gatatcatat ccaggaaaga 9351 ccaaagaggt agtggacaag ttggaacata tggtttgaac acattcacca 9401 acatggaagt tcaactcatc cgccaaatgg aagctgaagg agtcatcaca 9451 caagatgaca tgcagaaccc aaaagggttg aaagaaagag ttgagaaatg 9501 gctgaaagag tgtggtgtcg acaggttaaa gaggatggca atcagtggag 9551 acgattgcgt ggtgaagccc ctagatgaga ggtttggcac ttccctcctc 9601 ttcttgaacg acatgggaaa ggtgaggaaa gacattccgc agtgggaacc 9651 atctaaggga tggaaaaact ggcaagaggt tcctttttgc tcccaccact 9701 ttcacaagat ctttatgaag gatggccgct cactagttgt tccatgtaga 9751 aaccaggatg aactgatagg gagagccaga atctcgcagg gagctggatg 9801 gagcttaaga gaaacagcct gcctgggcaa agcttacgcc cagatgtggt 9851 cgcttatgta cttccacaga agggatctgc gtttagcctc catggccata 9901 tgctcagcag ttccaacgga atggtttcca acaagcagaa caacatggtc 9951 aatccacgct catcaccagt ggatgaccac tgaagatatg ctcaaagtgt 10001 ggaacagagt gtggatagaa gacaacccta atatgaqtga caagactcca 10051 gtccattcgt gggaagatat accttaccta gggaaaagag aggatttgtg 10101 gtgtggatcc ctgattggac tttcttccag agccacctgg gcgaagaaca 10151 ttcacacggc cataacccag gtcaggaacc tgatcggaaa agaggaatac 10201 gtggattaca tgccagtaat gaaaagatac agtgctcctt cagagagtga 10251 aggagttctg taattaccaa caacaaacac caaaggctat tgaagtcagg 10301 ccacttgtgc cacggtttga gcaaaccgtg ctgcctgtag ctccgccaat 10351 aatgggaggc gtaataatcc ccagggaggc catgcgccac ggaagctgta 10401 cgcgtggcat attggactag cggttagagg agacccctcc catcactgac 10451 aaaacgcagc aaaagggggc ccaagactag aggttagagg agaccccccc 10501 aacacaaaaa cagcatattg acgctgggaa agaccagaga tcctgctgtc 10551 tctgcaacat caatccaggc acagagcgcc gcaagatgga ttggtgttgt 10601 tgatccaaca ggttct 2015203575 26 Jun2015 * -138- 2015203575 26 Jun2015 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3

DEN4 DENI

Appendix 4

Alignment of dengue virus polyproteins 1 MNQRKKWRP PFNMLKRERNRVS TPQGLVKR FSTGLFS GKGPLRMVLAF 49 WP 1 MNNQRKKTGRPS FNMLKRARNRVS TVS QL· AKRF S KGLLS GQGPMKLVMAF 50 •NGC 1 MNN QR KKARNT P FNML KRERNRV S TV Q QL· T KR F S LGML QGRGP LKL FMAL 50 H87 1 MNNQRKKTGKPSINMLKRVRNRV S TG S QLAKRF S RGLLN GQGPMKLVMAF 50 ***** ***** ****** * * * * * *e *e**^^^ ^ * 50 ITFLRVLSIPPTAGILKRWGQLKKNKAIKILIGFRKEIGRMLNILNGRKR 99 WP 51 IAFLRFLAIPPTAGILARWGSFKKNGAIKVLRGFKKEISNMLNIMNRRKR 100 NGC 51 VAFLRFLTIPPTAGILKRWGTIKKSKAINVLRGFRKEIGRMLNILNRRRR 100 H87 51 IAFLRFLAIPPTAGVLARWGTFKKSGAIKVLKGFKKEISNMLSIINKRKK 100 *** * ****** * *** ** ** * ** *** ** * * * 10 0 STITLLCLIPTVMAFSLSTRDGEPLMIVAKHERGRPLLFKTTEGINKCTL 14 9 •WP 101 SVTMLLMLLPTALAFHLTTRGGEPHMIVSKQERGKSLLFKTSAGVNMCTL 15 0 •NGC 101 TAGMIIMLIPTVMAFHLTTRNGEPHMIVSRQEKGKSLLFKTEDGVNMCTL 15 0 H87 101 TSLCLMMMLPATLAFHLTSRDGEPRMIVGKNERGKSLLFKTASGINMCTL 150 ββ*# ^** *ee* *** *** ^ *e*e ***** *^* *** 150 IAMDLGEMCEDTVTYKCPLLVNTEPEDIDCWCNLTSTWVMYGTCTQSGER 199 WP 151 IAMDLGELCEDTMTYKCPRITETEPDDVDCWCNATETWVTYGTCSQTGEH 200 NGC 151 MAMDLGELCEDTITYKCPFLRQNEPEDIDCWCNSTSTWVTYGTCTTTGEH 2 00 H87 151 1AMDLGEMCDDTVTYKCPHITEVEPEDIDCWCNLTSTWVTYGTCNQAGEH 2 00 ****** * ** ***** ** * ***** * *** **** ** 2 00 RREKRSVALTPHSGMGLETRAETWMSSEGAWKHAQRVESWILRNPGFALL 249 WP 201 RRDKRSVALAPHVGLGLETRTETWMSSEGAWKQIQKVETWALRHPGFTVI 250 NGC 2 01 RREKRSVALVPHVGMGLETRTETWMSSEGAWKHAQRIETWILRHPGFTIM 25 0 H87 2 01 RRDKRSVALAPHVGMGLDTRTQTWMSAEGAWRQVEKVETWALRHPGFTIL 25 0 ** ****** ** * ** ** **** **** * * ** *** 250 AGFMAYMIGQTGIQRTVFFVLMMLVAPSYGMRCVGVGNRDFVEGVSGGAW 299 WP 251 ALFLAHAIGTSITQKGIIFILLMLVTPSMAMRCVGIGNRDFVEGLSGATW 300 NGC 251 AAILAYTIGTTHFQRALIFILLTAVAPSMTMRCIGISNRDFVEGVSGGSW 3 00 H87 251 ALFLAHYIGTSLTQKWIFILLMLVTPSMTMRCVGVGNRDFVEGLSGATW 3 00 * * ** * * * * ** *** * ******* ** * 3 00 VDLVLEHGGCVTTMAQGKPTLDFELTKTTAKEVALLRTYCIEASISNITT 349 WP 301 VDWLEHGSCVTTMAKDKPTLDIELLKTEVTNPAVLRKLCIEAKISNTTT 350 NGC 3 01 VDIVLEHGS CVTTMAKNKPTLDFELIKTEAKQPATLRKYCIEAKLTNTTT 350 Ή87 3 01 VDWLEHGGCVTTMAKNKPTLDIELQKTE ATQLATLRKLCIEGKITNITT 350 ** ****** ***** ** ** * ** *** * ** 3 50 ATRCPTQGEPYLKEEQDQQYICRRDWDRGWGNGCGLFGKGGWTCAKFS 3 99 WP 351 DSRCPTQGEATLVEEQDTNFVCRRTFVDRGWGNGCGLFGKGSLITCAKFK 40 0 •NGC 3 51 DSRCPTQGEPSLNEEQDKRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFT 4 00 H87 3 51 DSRCPTQGEAILPEEQDQNYVCKHTYVDRGWGNGCGLFGKGSLVTCAKFQ 400 ******* * **** * *************** *** * 400 CSGKITGNLVQIENLEYTVWTVHNGDTHAVGNDTSNHGVTAMITPRSPS 449 WP 401 CVTKLEGKIVQYENLKYSVIVTVHTGDQHQVGNETTEHGTTATITPQAPT 450 -139- 2015203575 26 Jun2015 DEN2 DEN3 NGC 4 01 CKKNMKGKWQPENLEYTIVITPHSGEEHAVGNDTGKHGKEIKITPQSSI 450 H87 401 CLESIEGKWQHENLKYTVIITVHTGDQHQVGNET- -QGVTAEITSQAST 44 8 ★ * ★★ ititit * * * ★ ★ ★★★ if if if if DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 WP NGC H87 450 VEVKLPDYGELTLDCEPRSGIDFNEMILMKMKKKTWLVHKQWFLDLPLPW 499 451 SEIQLTDYGALTLDCSPRTGLDFNEMVLLTMEKKSWLVHKQWFLDLPLPW 500 451 TEAELTGYGTVTMECSPRTGLDFNEMVLLQMENKAWLVHRQWFLDLPLPW 500 449 AEAILPEYGTLGLECSPRTGLDFNEMILLTMKNKAWMVHRQWFFDLPLPW 498 * * ** * ** * ***** * * * * ** *** ****** 500 TAGADTSEVHWNYKERMVTFKVPHAKRQDVTVLGSQEGAMHSALAGATEV 549 WP 501 TSGASTSQETWNRQDLLVTFKTAHAKKQEVWLGSQEGAMHTALTGATEI 550 NGC 501 LPGADTQGSNWIQKETLVTFKNPHAKKQDWVLGSQEGAMHTALTGATEI 550 H87 499 TSGATTKTPTWNRKELLVTFKNAHAKKQEWVLGSQEGAMHTALTGATEI 548 ** * * **** *** * * ********** ** **** 550 DSGDGNHMFAGHLKCKVRMEKLRIKGMSYTMCSGKFSIDKEMAETQHGTT 599 WP 551 QTSGTTTIFAGHLKCRLKMDKLTLKGMSYVMCTGSFKLEKEVAETQHGTV 600 NGC 551 QMSSGNLLFTGHLKCRLRMDKLQLKGMSYSMCTGKFKWKEIAETQHGTI 600 H87 549 QTSGGTSIFAGHLKCRLKMDKLKLKGMSYAMCLNTFVLKKEVSETQHGTI 598 #*>*****>>>*t** e ***** ** ^ _ **ee****** 600 WKVKYEGAGAPCKVPIEIRDVNKEKWGRIISSTPLAENTNSVTNIELE 64 9 WP 601 LVQVKYEGTDAPCKIPFSSQDEKGVTQNGRLITANPIVTDKEKPVNIEAE 650 NGC 601 VIRVQYEGDGSPCKIPFEIMDLEKRHVLGRLITVNPIVTEKDSPVNIEAE 650 H87 599 LIKVEYKGEDAPCKIPFSTEDGQGKAHNGRLITANPWTKKEEPVNIEAE 64 8 * * * *** * * ** * * *** * 650 PPFGDSYIVIGVGNSALTLHWFRKGSSIGKMFESTYRGAKRMAILGETAW 699 WP 651 PPFGESYIWGAGEKALKLSWFKKGSSIGKMFEATARGARRMAILGDTAW 700 NGC 651 PPFGDSYIIIGVEPGQLKLNWFKKGSSIGQMIETTMRGAKRMAILGDTAW 700 H87 649 PPFGESNIVIGIGDKALKINWYRKGSSIGKMFEATARGARRMAILGDTAW 698 if if if if ^ ie it λ φ it * β *φφ★*★***_* * φ * ***φ******φ*** 700 DFGSVGGLFTSLGKAVHQVFGSVYTTMFGGVSWMIRILIGFLVLWIGTNS 74 9 WP 701 DFGSIGGVFTSVGKLIHQIFGTAYGVLFSGVSWTMKIGIGILLTWLGLNS 750 NGC 701 DFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSWTMKILIGVIITWIGMNS 750 H87 699 DFGSVGGVLNSLGKMVHQIFGSAYTALFSGVSWIMKIGIGVLLTWIGLNS 748 ** * * # * * φ φ * < * * φ * * φ * * # * * * * * * * * .. * , * * * 750 RNTSMAMTCIAVGGITLFLGFTVQADMGCVASWSGKELKCGSGIFWDNV 799 WP 751 RSTSLSMTCIAVGMVTLYLGVMVQADSGCVINWKGRELKCGSGIFVTNEV 800 NGC 751 RSTSLSVSLVLVGWTLYLGVMVQADSGCWSWKNKELKCGSGIFITDNV 800 H87 749 KNTSMSFSCIAIGIITLYLGVWQADMGCVINWKGKELKCGSGIFVTNEV 798 **.....* ^ ** # ** * * * * *** * ##*********# * 800 HTWTEQYKFQPESPARLASAILNAHKDGVCGIRSTTRLENVMWKQITNEL 849 WP 801 HTWTEQYKFQADSPKRLSAAIGKAWEEGVCGIRSATRLENIMWKQISNEL 850 NGC 801 HTWTEQYKFQPES P S KLASAIQKAHEEGICGIRSVTRLENLMWKQITPEL 850 H87 799 HTWTEQYKFQADSPKRVATAIAGAWENGVCGIRSTTRMENLLWKQIANEL 848 ********** ** β β e** * *^***** **_**. ^****^ ** 85 0 NYVLWEGGHDLTWAGDVKGVLTKGKRALTPPVSDLKYSWKTWGKAKIFT 899 WP 851 NH1LLENDMKFTVWGDVSGILAQGKKMIRPQPMEHKYSWKSWGKAKIIG 900 NGC 851 NHILSENEVKLTIMTGDIKGIMQAGKRSLQPQPTELKYSWKTWGKAKMLS 900 H87 849 NYILWENDIKLTVWGDITGVLEQGKRTLTPQPMELKYSWKTWGLAKIVT 898 * * * * . *.. **. . * . *****.** ** -140- 2015203575 26 Jun2015 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 ' DEN4 DENI DEN2 DEN3

DEN4 DENI WP NGC H87 900 PEARNSTPLIDGPDTSECPNERRAWNSLEVEDYGFGMFTTNIWMKFREGS 901 ADVQNTTFIIDGPNTPECPDNQRAWNIWEVEDYGFGIFTTNIWLKLRDSY 901 TESHNQTFLIDGPETAECPNTNRAWNSLEVEDYGFGVFTTNIWLKLREKQ 899 AETQNSSFIIDGPSTPECPSASRAWNVWEVEDYGFGVFTTNIWLKLREVY * * **** * *** **** ******** ****** * * 95 0 SEVCDHRLMSAAIKDQKAVHADMGYWIES S KNQTWQIEKASLIEVKTCLW WP 951 TQVCDHRLMSAAIKDS KAVHADMGYWIESEKNETWKLARAS FIEVKTCIW NGC 951 D VF CD S KLMSAAI KDNRAVHADMGYWIE S ALND T WKIE KAS FIE VKS CH W H87 94 9 TQLCDHRLMSAAVKDERAVHADMGYWIESQKNGSWKLEKASLIEVKTCTW ** ***** ** ************ * * ** **** * * 10 0 0 PKTHTLWSNGVLESQMLIPKSYAGPFSQHNYRQGYATQTVGPWHLGKLEI WP 1001 PKSHTLWSNGVLESEMIIPKIYGGPISQHNYRPGYFTQTAGPWHLGKLEL NGC 1001 PKSHTLWSNGVLESEMIIPKNFAGPVSQHNYRPGYHTQTAGPWHLGKLEM H87 999 PKSHTLWSNGVLESDMIIPKSLAGPISQHNHRPGYHTQTAGPWHLGKLEL ** *********** * *** ** **** * ** *** ********* 1050 DFGECPGTTVTIQEDCDHRGPSLRTTTASGKLVTQWCCRSCTMPPLRFLG WP 1051 DFDLCEGTTVWDEHCGNRGPSLRTTTVTGKTIHEWCCRSCTLPPLRFKG NGC 10 51 DFDFCEGTTVWTEDCGNRGPSLRTTTASGKLITEWCCRSCTLPPLRYRG H87 104 9 DFNYCEGTTWISENCGTRGPSLRTTTVSGKLIHEWCCRSCTLPPLRYMG ** * **** * * ********* ** ******* **** * 1100 EDGCWYGMEIRPLSEKEENMVKSQVTAGQGTSETFSMGLLCLTLFVEECL WP 1101 EDGCWYGMEIRPVKEKEENLVKSMVSAGSGEVDS FSLGLLCISIMIEEVM NGC 1101 EDGCWYGMEIRPLKEKEENLVNSLVTAGHGQIDNFSLGVLGMALFLEEML H87 1099 EDGCWYGMEIRPINEKEENMVKSLASAGSGKVDNFTMGVLCLAILFEEVM ************_ *****_* * _** * . *>>*-* ___ ** 1150 RRRVTRKHMILVWITLCAII LGGLTWMDLLRAIj IMLGDTMSGRIG- GQI WP 1151 RSRWSRKMLMTGTLAVFLLLTMGQLTWNDLIRIjCIMVGANASDKMGMGTT NGC 1151 RTRVGTKHAILLVAVSFVTLITGNMSFRDLGRVMVMVGATMTDDIGMGVT H87 1149 RGKFGKKHMIAGVLFTFVLLLSGQITWRGMAHTLIMIGSNASDRMGMGVT *. * . . *.. .. .** 1199 HLAIMAVFKMSPGYVLGVFLRKLTSRETALMVIGMAMTTVLSIPHDLMEL WP 12 01 YLALMATFRMRPMFAVGLLFRRLTSREVLLLTVGLSLVASVELPNSLEEL NGC 12 01 YLALLAAFKVRPTFAAGLLLRKLTSKELMMTTIGIVLLSQSTIPETILEL H87 1199 YLALIATFKIQPFLALGFFLRKLTSRENLLLGVGLAMAATLRLPEDIEQM ** * * * * * *** * * * 1249 IDGISLGLILLKIVTQFDNTQVGTLALSLTFIRSTMPLVMAWRTIMAVLF WP 1251 GDGLAMGIMMLKLLTOFQSHQLWATLLSLTFVKTTFSLHYAWKTMAMIIiS NGC 1251 TDALALGMMVLKMVRKMEKYQLAVTIMAILCVPNAVILQNAWKVSCTILA H87 1249 ANGIALGLMALKLITQFETYQLWTALVSLTCSNTIFTIjTVAWRTATIjILA * ** * * ** * WP NGC H87

WP 949 950 950 948 999 1000 1000 998 1049 1050 1050 1048 1099 1100 1100 1098 1149 1150 1150 1148 1198 1200 1200 1198 1248 1250 1250 1248 1298 1300 1300 1298 1299 WTLIPLCRTSCLQKQSHWVEITALILGAQALPVYLMTLMKGASRRSWPL 1301 IVSLFPLCLSTTSQK-TTWLPVLLGSLGCKPLTMFLITENKIWGRKSWPL 1301 WSVSPLFLTSSQQK-ADWIPLALTIKGLNPTAIFLTTLSRTNKKRSWPL 1299 GISLLPVCQSSSMRK-TDWLPMTVAAMGVPPLPLFIFSLKDTLKRRSWPL * ** * ****

134 9 NEGIMAVGLVSLLGSALLKNDVPLAGPMVAGGLLLAAYVMSGSSADLSLE 13 5 0 NEGIMAVGIVSILLSSLLKNDVPLAGPLIAGGMLIACYVISGSSADLSliE -141- 1348 1349 1349 1347 1398 1399 2015203575 26 Jun2015 DEN2 DEN3 DEN4 DENI DEN2 DEN3 NGC 1350 NE AIMAV GMV SI LAS SLLKNDIPMTGPLVAGGLLTV C YVLTGRS ADLELE 1399 H87 1348 NEGVMAVGLVS I LAS S LLRNDVPMAGPLVAGGLLI AC YVITGTSADLTVE 1397 * * .******* *_**_***__**__***_* _**__* **** * 13 99 KAANVQWDEMADITGSSPIIEVKQDEDGSFSIRDVEETNMITLLVKLALI 1448 WP 1400 KAAEVSWEEEAEHSGASHNILVEVQDDGTMKIKDEERDDTLTILLKATLL 1449 NGC 1400 RAADVKWEDQAEISGSSPILSITISEDGSMSIKNEEEEQTLTILIRTGLL 1449 H87 13 98 KAADVTWEEEAEQTGVSHNLMITVDDDGTMRIKDDETENILTVLLKTAXjL 1447 ** * * * * * ** * * * * * DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 144 9 TVSGL·YPLAIPVTMTL·WYMWQVKTQRSGAL·WDVPSPAATKKAAL·SEGVYR 1498 •WP 1450 AISGVYPMSIPATLFVWYFWQKKKQRSGVLWDTPSPPEVERAVLDDGIYR 1499 NGC 1450 VISGLFPVSIPITAAAWYLWEVKKQRAGVLWDVPSPPPVGKAELEDGAYR 1499 H87 1448 IVSGIFPYSIPATMLVWHTWQKQTQRSGVLWDVPSPPETQKAELEEGVYR 1497 ^ e * * * * * * * 14 9 9 IMQRGLFGKTQVGVGIHMEGVFHTMWHVTRGSVICHETGRLEPSWADVRN 154 8 •WP 150 0 IL QRGLLGRS QVGVGVFQEGVFHTMWHVTRGAVLMY QGKRLEP S WAS VKK 1549 NGC 15 0 0 IKQKGILGY S QIGAGVYKEGTFHTMWHVTRGAVLMHKGKRIEP S WAD VKK 1549 H87 1498 IKQQGIFGKTQVGVGVQKEGVFHTMWHVTRGAVLTHNGKRLEPNWASVKK 1547 * * * * * * * ** ********** * * ** ** * DEN4 DENI DEN2 DEN3 WP NGC H87 1549 DMISYGGGWRLGDKWDKEEDVQVLAIEPGKNPKHVQTKPGLFKTLTGEIG 1598 1550 DLISYGGGWRFQGSWNAGEEVQVIAVEPGKNPKNVQTAPGTFKTPEGEVG 1599 1550 DLISYGGGWKLEGEWKEGEEVQVLALEPGKNPRAVQTKPGLFKTNAGTIG 1599 154 8 DLISYGGGWRLSAQWQKGEEVQVIAVEPGKNPKNFQTMPGIFQTTTGEIG 1597 * ******* * * *** * ****** ** ** * * * * DEN4 DENI DEN2 DEN3 1599 AVTLDFKPGTSGSPIINRKGKVIGLYGNGWTKSGDYVSAITQAERIGEP 1648 WP 1600 AIALDFKPGTSGSPIVNREGKIVGLYGNGWTTSGTYVSAIAQAKASQEG 1649 NGC 1600 AVSLDFSPGTSGSPIIDKKGKWGLYGNGWTRSGAYVSAIAQTEKSIED 1649 H87 1598 AIALDFKPGTSGSPIINREGKWGLYGNGWTKNGGYVSGIAQTNAEPDG 1647 * *** ******** ** ********* * *** * * DEN4 DENI DEN2 DEN3 1649 - D YEVDEDIFRKKRLTIMDLHPGAGKTKRIL P SIVRE ALKRRLRTLI LAP 1697 WP 1650 PLPEIEDEVFRKRNLTIMDLHPGSGKTRRYLPAIVREAIRRNVRTLVLAP 1699 NGC 1650 - NPEIEDDIFRKRKLTIMDLHPGAGKTKRYL P AI VRE AIKRGLRTLI LAP 1698 •H87 164 8 PTPELEEEMFKKRNLTIMDLHPGSGKTRKYLPAIVREAIKRRLRTLILAP 1697 * * * ********* *** ******* * ****** DEN4 DENI DEN2 DEN3 1698 TRWAAEMEEALRGLPIRYQTPAVKSEHTGREIVDLMCHATFTTRLLSST 1747 WP 1700 TRWASEMAEALKGMPIRYQTTAVKSEHTGKEIVDLMCHATFTMRLLSPV 1749 NGC 1699 TRWAAEMEEALRGLPIRYQTPAIRAEHTGREIVDLMCHATFTMRLLSPV 1748 H87 1698 TRWAAEMEEAMKGLPIRYQTTATKSEHTGREIVDLMCHATFTMRLLSPV 1747 ***** ** ** * ****** * **************** **** DEN4 DENI DEN2 DEN3 174 8 RVPNYNLIVMDEAHFTDPSSVAARGYISTRVEMGEAAAIFMTATPPGATD 1797 WP 1750 RVPNYNMIIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMTATPPGSVE 1799 NGC 1749 RVPNYNLIIMDEAHFTDPASIAARGYISTRVEMGEAAGIFMTATPPGSRD 1798 H87 1748 RVPNYNLIIMDEAHFTDPASIAARGYISTRVGMGEAAAIFMTATPPGTAD 1797 ****** * ********* * ********** ***** ********* DEN4 DENI DEN2 DEN3 1798 PFPQSNSPIEDIEREIPERSWNTGFDWITDYQGKTVWFVPSIKAGNDIAN 1847 WP 1800 AFPQSNAVIQDEERDIPERSWNSGYDWITDFPGKTVWFVPSIKSGNDIAN 1849 •NGC 1799 PFPQSNAPIMDEEREIPERSWSSGHEWVTDFKGKTVWFVPSIKAGNDIAA 1848 H87 1798 AFPQSNAPIQDEERDIPERSWNSGNEWITDFVGKTVWFVPSIKAGNVIAN 1847 *****_ * * ******** .* .*.**. ************* ** -142- 2015203575 26 Jun2015 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN 2 DEN 3 DEN4 DENI DEN2 DEN3 1848 CLRKSGKKVIQLSRKTFDTEYPKTKLTDWDFWTTDISEMGANFRAGR VI 1897 WP 1850 CLRKNGKRWQLSRKTFDTEYQKTKNNDWDYWTTDISEMGANFRADRVI 1899 NGC 1849 CLRKNGKKVIQLSRKTFDSEYVKTRTNDWDFWTTDISEMGANFKAERVI 1898 H87 1848 CLRKNGKKVIQLS RKTFDTE Y QKTKLNDWD F WTTD ISEMGANFIADR VI 1897 **** ** ^ ^ ******** ** *** φ ************ * * *** 1898 DPRRCLKPVILPDGPERVILAGPIPVTPASAAQRRGRIGRNPAQEDDQYV 1947 •WP 1900 DPRRCLKPVILKDGPERVILAGPMPVTVASAAQRRGRIGRNQNKEGDQYI 1949 •NGC 1899 DPRRCMKPVILTDGEERVILAGPMPVTHSSAAQRRGRIGRNPKNENDQYI 1948 H87 1898 DPRRCLKPVILTDGPERVILAGPMPVTVASAAQRRGRVGRNPQKENDQYI 1947 * * * * * t ie * * * ★ * * * * * * * * * * e * * * #*********** * * * * # 1948 FSGDPLKNDEDHAHWTEAKMLLDNIYTPEGIIPTLFGPEREKTQAIDGEF 1997 WP 1950 YMGQPLNNDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEY 1999 NGC 1949 YMGEPLENDEDCAHWKEAKMLLDNINTPEGIIPSMFEPEREKVDAIDGEY 1998 •H87 1948 FMGQPLNKDEDHAHWTEAKMLLDNINTPEGIIPALFEPEREKSAAIDGEY 1997 * ** *** * * * ********* *******> ie ***** *****e 1998 RLRGEQRKTFVELMRRGDLPVWLSYKVASAGISYKDREWCFTGERNNQIL 2047 WP 2000 RLRGEARKTFVELMRRGDLPVWLSYKVASEGFQYSDRRWCFDGERNNQVL 2049 •NGC 1999 RLRGEARKTFVDLMRRGDLPVWLAYRVAAEGINYADRRWCFDGIKNNQIL 2048 H87 1998 RLKGESRKTFVELMRRGDLPVWLAHKVASEGIKYTDRKWCFDGERNNQIL 2047 * * # * * *****^ *********** ^ .**. * * * * * * * * e***#* 2048 EENMEVEIWTREGEKKKLRPRWLDARVYADPMALKDFKEFASGRKSITLD 2097 •WP 2 050 EENMDVEIWTKEGERKKLRPRWLDARTYSDPLALREFKEFAAGRRSVSGD 2099 •NGC 2049 EENVEVEIWTKEGERKKLKPRWLDARIYSDPLTLKEFKEFAAGRKSLTLN 2098 •H87 2048 EENMDVEIWTKEGEKKKLRPRWLDARTYSDPLALKEFKDFAAGRKSIALD 2097 ***e ***** ***#***^******* *.**..*.,**,**.**,*.. 2 098 ILTEIASLPTYLSSRAKLALDNIVMIjHTTERGGRAYQHAIjNELPESLETIj 2147 WP 2100 LILEIGKLPQHLTQRAQNALDNLVMLHNSEQGGKAYRHAMEELPDTIETL 2149 NGC 2099 LITEMGRLPTFMTQKARDALDNLAVLHTAEAGGRAYNHALSELPETLETL 2148 H87 2098 LVTEIGRVPSHLAHRTRNALDNLVMIjHTSEHGGRAYRHAVEELPETMETL 2147 * * **** ** * ** ** ** *** *** DEN4 DENI DEN2 DEN3 2148 MLVALLGAMTAGIFLFFMQGKGIGKLSMGLITIAVASGLLWVAEIQPQWI 2197 WP 2150 MLL·AL·IAVL·TGGVTL·FFLSGRGLGKTSIGLLCVIASSALLWMASVEPHWI 2199 NGC 2149 LLLTLLATVTGGIFLFLMSGRGIGKMTLGMCCIITASILIiWYAQIQPHWI 2198 H87 2148 LLLGLMILLTGGAMLFLISGKGIGKTSIGLICVIASSGMLWMADVPLQWI 2197 * * * * ** * * ** * * ** * ** DEN4 DENI DEN2 DEN3 2198 AASIILEFFLMVLLIPEPEKQRTPQDNQLIYVILTILTIIGLIAANEMGL 2247 WP 2200 AASIILEFFLMVLLIPEPDRQRTPQDNQLAYWIGLLFMILTAAANEMGL 2249 NGC 2199 AASIILEFFLIVLLIPEPEKQRTPQDNQLTYWIAILTWAATMANEMGF 2248 H87 2198 ASAIVLEFFMMVLLIPEPEKQRTPQDNQLAYWIGILTLAAIVAANEMGL 2247 * * **** ******* ********* ** * ***** DEN4 DENI DEN2 DEN3 2248 IEKTKTDFGFY----QVKTETTILDVDLRPASAWTLYAVATTILTPMLRH 2293 WP 2250 LETTKKDLGIGHAAAENHHHAAMLDVDLHPAS AWTL YAVATT IITPMMRH 2299 NGC 2249 LEKTKKDLGLG-SITTQQPESNILDIDLRPASAWTLYAVATTFVTPMLRH 2297 H87 224 8 LETTKRDLGMS-KEPGWSPTSYLDVDLHPASAWTLYAVATTVITPMLRH 2296 * ** * * ** ** ************* .*** **

DEN4 DENI 2294 TIENTSANLSLAAIANQAAVLMGLGKGWPLHRMDLGVPLLAMGCYSQVNP 2343 WP 2300 TIENTTANISLTAIANQAAILMGLDKGWPISKMDIGVPLLALGCYSQVNP 2349 -143- DEN2-NGC 2298 SIENSSVNVSLTAIANQATVLMGLGKGWPLSKMDIGVPLLAIGCYSQVNP 2347 DEN3-H87 2297 TIENSTANVSLAAIANQAVVLMGLDKGWPISKMDLGVPLLALGCYSQVNP 2346 *** * ** ****** **** **** ** ****** ******** 2015203575 26 Jun2015 DEN4 DEN1-WP DEN2-NGC DEN3-H87 DEN4 DENl-WP DEN2-NGC DEN3-H87 DEN4 DENI-WP DEN2-NGC DEN3-H87 DEN4 DEN1-WP DEN2-NGC DEN3-H87 DEN4 DEN1-WP DEN2-NGC DEN3-H87 DEN4 DEN1-WP DEN2-NGC DEN3-H87 DEN4 DENl-WP DEN2-NGC DEN3-H87 DEN4 DENl-WP DEN2-NGC DEN3-H87 DEN4 DENl-WP DEN2-NGC DEN3-H87 2344 TTLTASLVMLLVHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGITVID 23 93 2350 LTLTAAVFMLVAHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIVAID 23 99 2348 ITLTAALFLLVAHYAIIGPGLQAKATREAQKRAAAGIMKNPTVDGITVID 2397 2347 LTLIAAVLLLVTHYAIIGPGLQAKATREAQKRTAAGIMKNPTVDGIMTID 2396 ** * * ******************** ************* ** 2394 LEPISYDPKFEKQLGQVMLLVLCAGQLLLMRTTWAFCEVLTLATGPILTL 2443 24 0 0 LDPWYDAKFEKQLGQIMLLILCTSQILLMRTTWALCESITLATGPLTTL 244 9 2398 LDPIPYDPKFEKQLGQVMLLVLCVTQVLMMRTTWALCEALTLATGPISTL 2447 2397 LDPVIYDSKFEKQLGQVMLLVLCAVQLLLMRTSWALCEVLTLATGPITTL 2446 * * ** ******** *** ** * * *** ** ** ****** ** 2444 WEGNPGRFWNTTIAVSTANIFRGSYLAGAGLAFSLIKNAQTPRRGTGTTG 24 93 2450 WEGSPGKFWNTTIAVSMANIFRGSYLAGAGLAFSLMKSLGGGRRGTGAQG 2499 2448 WEGNPGRFWNTTIAVSMANIFRGSYLAGAGLLFSIMKNTTNTRRGTGNIG 2497 2447 WEGSPGKFWNTTIAVSMANIFRGSYLAGAGLALSIMKSVGTGKRGTGSQG 2496 *** ** ********* ************** * * **** * 2494 ETLGEKWKRQLNSLDRKEFEEYKRSGILEVDRTEAKSALKDGSKIKHAVS 2543 2500 ETLGEKWKRQLNQLSKSEFNTYKRSGIIEVDRSEAKEGLKRGEPTKHAVS 2549 2498 ETLGEKWKSRLNALGKSEFQIYKKSGIQEVDRTLAKEGIKRGETDHHAVS 2547 2497 ETLGEKWKKKLNQLSRKEFDLYKKSGITEVDRTEAKEGLKRGEITHHAVS 2546 ******** ** * ** ** *** **** ** * * **** 2544 RGSSKIRWIVERGMVKPKGKWDLGCGRGGWSYYMATLKNVTEVKGYTKG 2593 2550 RGTAKLRWFVERNLVKPEGKVIDLGCGRGGWSYYCAGLKKVTEVKGYTKG 2599 254 8 RGSAKLRWFVERNMVTPEGKWDLGCGRGGWS YYCGGLKNVREVKGLTKG 2597 2547 RGSAKDQWFVERNMVIPEGRVIDLGCGRGGWSYYCAGLKKVTEVRGYTKG 2596 ** * * *** * * * * ************ ** * ** * *** 2594 GPGHEEPIPMATYGWNLVKLHSGVDVFYKPTEQVDTLLCDIGESSSNPTI 2643 2600 GPGHEEPIPMATYGWNLVKLYSGKDVFFTPPEKCDTLLCDIGESSPNPTI 2649 2598 GPGHEEPIPMSTYGWNLVRLQSGVDVFFTPPEKCDTLLCDIGESSPNPTV 2647 2597 GPGHEEPVPMSTYGWNIVKLMSGKDVFYLPPEKCDTLLCDIGESSPSPTV 2646 ******* ** ***** * * ** *** * * *********** ** 2644 EEGRTLRVLKMVEPWLSSKPEFCIKVLNPYMPTVIEELEKLQRKHGGNLV 2693 2 65 0 EEGRTLRVLKMVEPWLRGN-QFCIKILNPYMPSWETLEQMQRKHGGMLV 2 698 2648 EAGRTLRVLNLVENWLNNNTQFCIKVLNPYMPSVIEKMEALQRKYGGALV 2697 2647 EESRTIRVLKMVEPWLKNN-QFCIKVLNPYMPTVIEHLERLQRKHGGMLV 2695 * ** *** ** ** **** ****** * * * *** ** ** 2694 RCPLSRNSTHEMYWVSGASGNIVSSVNTTSKMLLNRFTTRHRKPTYEKDV 2743 2699 RNPLSRNSTHEMYWVSCGTGNIVSAVNMTSRMLLNRFTMAHRKPTYERDV 2748 2698 RNPLSRNSTHEMYWVSNASGNIVSSVNMISRMLINRFTMRHKKATYEPDV 2747 2696 RNPLSRNSTHEMYWISNGTGNIVSSVNMVSRLLLNRFTMTHRRPTIEKDV 2745 * ************ * ***** ** * * **** * * * ** 2744 DLGAGTRSVSTETEKPDMTIIGRRLQRLQEEHKETWHYDQENPYRTWAYH 2793 2749 DLGAGTRHVAVEPEVANLDIIGQRIENIKNGHKSTWHYDEDNPYKTWAYH 2798 2748 DLGSGTRNIGIESEIPNLDIIGKRIEKIKQEHETSWHYDQDHPYKTWAYH 2797 2746 DLGAGTRHVNAEPETPNMDVIGERIKRIKEEHSSTWHYDDENPYKTWAYH 2795 ***_*** * * _** *. __ * _**** ''**'***** -144- 2794 GSYEAPSTGSASSMVNGWKLLTKPWDVIPMVTQLAMTDTTPFGQQRVFK 2843 WP 2799 GSYEVKPSGSASSMVNGWRLLTKPWDVIPMVTQIAMTDTTPFGQQRVFK 2848 NGC 2798 GSYETKQTGSASSMVNGWRLLTKPWDWPMVTQMAMTDTTPFGQQRVFK 2847 H87 2796 GSYEVKATGSASSMINGWKLLTKPWDWPMVTQMAMTDTTPFGQQRVFK 2845 2015203575 26 Jun2015 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3 DEN4 DENI DEN2 DEN3

DEN4 DENI . **** ******t****t******** ^ *****m*************** 2844 EKVDTRTPQPKPGTRMVMTTTANWLWALLGKKKNPRLCTREEFISKVRSN 2893 WP 2849 EKVDTRTPKAKRGTAQIMEVTARWLWGFLSRNKKPRICTREEFTRKVRSN 2898 NGC 2848 EKVDTRTQEPKEGTKKLMKITAEWLWKELGKKKTPRMCTREEFTRKVRSN 2897 H87 2 846 EKVDTRTPRPMPGTRKVMEITAEWLWRTLGRNKRPRLCTREEFTKKVRTN 2895 ******* ** * ** *** * * ** ****** *** * 2894 AAIGAVFQEEQGWTSASEAVNDSRFWELVDKERALHQEGKCESCVYNMMG 2943 WP 2899 AAIGAVFVDENQWNSAKEAVEDERFWDLVHRERELHKQGKCATCVYNMMG 2948 NGC 2898 AALGAIFTDENKWKSAREAVEDSRFWELVDKERNLHLEGKCETCVYNMMG 2947 H87 2 896 AAMGAVFTEENQWDSARAAVEDEEFWKLVDRERELHKLGKCGSCVYNMMG 2945 ** ** * * * ** ** * ** ** ** ** *** ******* 2 944 KREKKLGEFGRAKGSRAIWYMWLGARFLEFEALGFLNEDHWFGRENSWSG 2 993 WP 2949 KREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFMNEDHWFSRENSLSG 2998 NGC 2948 KREKKLGEFGKAKGSRAIWYMWLGARFLEFEALGFLNEDHWFSRENSLSG 2997 H87 2946 KREKKLGEFGKAKGSRAIWYMWLGARYLEFEALGFLNEDHWFSRENSYSG 2995 **********_***************_********,****** **** ** 2-994 VEGEGLHRLGYILEEIDKKDGDLMYADDTAGWDTRITEDDLQNEELITEQ 3043 WP 2999 VEGEGLHKLGYILRDISKIPGGNMYADDTAGWDTRITEDDLQNEAKITDI 3048 NGC 2 998 VEGEGLHKLGYILRDVSKKEGGAMYADDTAGWDTRITLEDLKNEEMVTNH 3047 H87 2996 VEGEGLHKLGYILRDISKIPGGAMYADDTAGWDTRITEDDLHNEEKITQQ 3045 ******* ***** * * ************** ** ** * 3044 MAPHHKILAKAIFKLTYQNKWKVLRPTPRGAVMDIISRKDQRGSGQVGT 3093 WP 3049 MEPEHALLATSIFKIjTYQNKWRVQRPAKNGTVMDVISRRDQRGSGQVGT 3098 NGC 3 048 MEGEHKKLAEAIFKLTYQNKWRVQRPTPRGTVMDIISRRDQRGSGQVGT 3 097 H87 3046 MDPEHRQLANAIFKLTY QNKWKVQRPTPKGTVMD11SRKDQRGSGQVGT 3095 * * ** *********** * ** * *** *** ********** 3 094 YGLNTFTNMEVQLIRQMEAEGVITQDDMQNPKGLKERVEKWLKECGVDRL 3143 WP 3099 YGLNTFINMEAQLXRQMESEGIFSPSELETPN-IiAERVIiDWIiKKHGTERIi 3147 NGC 3098 Y GLNTFTNME AQLIRQMEGEGVFKSIQHLT VT - EE IAVQNWL ARVGRERL 3146 H87 3 096 Y GLNTFTNME AQL IRQMEGEGVL S KADLENPHPLEKKITQWLETKGVERL 3145 ********** ******* ** ** * ** 3144 KRMAISGDDCWKPLDERFGTSLLFLiNDMGKVRKDIPQWEPSKGWKNWQE 3193 WP 314 8 KRMAISGDDCWKPIDDRFATALTALNDMGKVRKDIPQWEPSKGWNDWQQ 3197 NGC 3147 SRMAISGDDCWKPLDDRFASALTALNDMGKVRKDIQQWEPSRGWNDWTQ 3196 H87 3146 KRMAISGDDCWKPIDDRFANALLALNDMGKVRKDIPQWQPSKGWHDWQQ 3195 ************* * ** * *********** ** ** ** * 3194 VPFCSHHFHKIFMKDGRSLWPCRNQDELIGRARISQGAGWSLRETACLG 3243 WP 3198 VPFCSHHFHQLIMKDGREIWPCRNQDELVGRARVSQGAGWSLRETACLG 3247 NGC 3197 VPFCSHHFHELIMKDGRVLWPCRNQDELIGRARISQGAGWSLRETACLG 3246 H87 3196 VPFCSHHFHELIMKDGRKLWPCRPQDELIGRARISQGAGWSLRETACLG 3245 ********* _ ***** ***** ****_****.*************** 3244 KAYAQMWSLMYFHRRDLRLASMAICSAVPTEWFPTSRTTWSIHAHHQWMT 32 93 WP 3248 KSYAQMWQLMYFHRRDLRLAANAICSAVPVDWVPTSRTTWSIHAHHQWMT 3297 -145- 2015203575 26 Jun2015 DEN2-NGC DEN3-H87 DEN4 DEN1-WP DEN2-NGC DEN3-H87 DEN4 DENl-WP DEN2-NGC DEN3-H87 3247 KSYAQMWSLMYFHRRDLRLAANAICSAVPSHWVPTSRTTWSIHAKHEWMT 3296 3246 KAYAQMWTLMYFHRRDLRLASNAICSAVPVHWVPTSRTTWSIHAHHQWMT 3295 *e***** ************ ******* * *********** ^ 9 *** 3294 TEDMLKVWNRVWIEDNPNMTDKTPVHSWEDIPYLGKREDLWCGSLIGLSS 3343 3298 TEDMLSVWNRVWIEENPWMEDKTHVSSWEDVPYLGKREDRWCGSLIGLTA 3347 3297 TEDMLTVWNRVWIQENPWMEDKTPVESWEEIPYLGKREDQWCGSLIGLTS 3346 3296 TEDMLTVWNRVWIEDNPWMEDKTPVTTWEDVPYLGKREDQWCGSLIGLTS 3345 ***** *******# e** * *** * .**, ,******** ********t 3344 RATWAKNIHTAITQVRNLIGKEEYVDYMPVMKRYSAPSESEGVL 3387 3 348 RATWATNIQVAINQVRRLIGNENYLDFMTSMKRFKNESDPEGALW 33 92 3347 RATWAKNIQTAINQVRSLIGNEEYTDYMPSMKRFRREEEEAGVLW 3391 3346 RATWAQNILTAIQQVRSLIGNEEFLDYMPSMKRFRKEEESEGAIW 3390 ***** ** ** *** *** * * * *** * * Residue . Residue identity- similarity -146-

While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, and appendices, as well as patents, applications, and publications, referred to above, are hereby incorporated by reference. 2015203575 26 Jun2015 -147-

Claims (19)

1. THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

   1. A dengue vims comprising a mutation at a nucleotide position that corresponds to nucleotide 10634 of the sequence set forth in SEQ ID NO: 14.
2. 2. The dengue vims of claim 1, further comprising a Δ30 mutation.
3. 3. The dengue vims of claim 1 or 2, wherein the dengue vims is a dengue vims type 1, a dengue vims type 2, a dengue vims type 3, or a dengue vims type 4.
4. 4. The dengue vims of any one of claims 1 to 3, wherein the dengue vims is a chimeric vims.
5. 5. The dengue vims of claim 4, wherein the chimeric vims has a dengue 1 backbone, a dengue 2 backbone, a dengue 3 backbone, or a dengue 4 backbone.
6. 6. A nucleic acid molecule encoding a dengue vims according to any one of claims 1 to 5, wherein the nucleic acid molecule is a cDNA molecule or an RNA molecule.
7. 7. A method of preparing a dengue vims comprising: (a) synthesizing full-length viral genomic RNA in vitro using a cDNA molecule that encodes a dengue vims according to any one of claims 1 to 5; (b) transfecting cultured cells with the viral genomic RNA and culturing the transfected cells to produce dengue vims; and (c) isolating the dengue vims from the cultured cells.
8. 8. A dengue vims prepared according to the method of claim 7.
9. 9. A pharmaceutical composition comprising a pharmacologically acceptable vehicle and a dengue vims according to any one of claims 1 to 5 or 8.
10. 10. A tetravalent vaccine comprising a pharmacologically acceptable vehicle and a dengue vims according to any one of claims 1 to 5 or 8.
11. 11. A live attenuated vaccine comprising a pharmacologically acceptable vehicle and a dengue vims according to any one of claims 1 to 5 or 8.
12. 12. The live attenuated vaccine of claim 11 in unit dosage form comprising from about 102 -109 plaque forming units (PFU)/ml of the dengue virus.
13. 13. An inactivated vaccine comprising a pharmacologically acceptable vehicle and a dengue virus according to any one of claims 1 to 5 or 8.
14. 14. The inactivated vaccine of claim 13 in unit dosage form comprising from about 0.1 to 50 pg of E protein/'ml from the dengue virus.
15. 15. A kit comprising: (i) a pharmaceutical composition according to claim 9 or a tetravalent vaccine according to claim 10, or a live attenuated virus of claim 11 or claim 12, or an inactivated vaccine of claim 13 or claim 14 packaged in a container or dispenser device; and (ii) instructions for administration.
16. 16. A method of eliciting neutralizing antibodies against dengue virus in a subject comprising administering to the subject a pharmaceutical composition according to claim 9, or a tetravalent vaccine of claim 10, or a live attenuated vaccine of claim 11 or claim 12, or an inactivated vaccine of claim 13 or claim 14.
17. 17. The method of claim 18, wherein administration is by subcutaneous, intradermal, or intramuscular injection.
18. 18. Use of a dengue virus according to any one of claims 1 to 5 or 8 or a pharmaceutical composition according to claim 9, or a tetravalent vaccine of claim 10, or a live attenuated vaccine of claim 11 or claim 12, or an inactivated vaccine of claim 13 or claim 14 to elicit neutralizing antibodies against dengue virus in a subject.
19. 19. Use of a dengue virus according to any one of claims 1 to 5 or 8 or a pharmaceutical composition according to claim 9, or a tetravalent vaccine of claim 10, or a live attenuated vaccine of claim 11 or claim 12, or an inactivated vaccine of claim 13 or claim 14 in the preparation of a medicament for treatment or prevention of infection by dengue virus.