AU2003231185B2 - Dengue tetravalent vaccine containing a common 30 nucleotide deletion in the 3'-UTR of dengue types 1,2,3 and 4, or antigenic chimeric dengue viruses 1,2,3, and 4 - Google Patents

Description

DENGUE TETRAVALENT VACCINE CONTAINING A COMMON 30

NUCLEOTIDE DELETION IN THE 3'-UTR OF DENGUE TYPES 1, 2, 3, AND 4,

OR ANTIGENIC CHIMERIC DENGUE VIRUSES 1, 2, 3, AND 4

Field of the Invention

The invention relates to a dengue virus tetravalent vaccine containing a common 30 nucleotide deletion (Δ30) in the 3 '-untranslated region of the genome of dengue virus serotypes 1, 2, 3, and 4, or antigenic chimeric dengue viruses of serotypes 1, 2, 3, and 4.

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 tropical Asian countries (WHO 1997 Dengue Haemorrhagic Fever: Diagnosis, Treatment, Prevention, and Control 2nd Edition, Geneva). There are four serotypes of dengue virus (DEN1, DEN2, DEN3, and DEN4) that annually cause an estimated 50-100 million cases of dengue fever and 500,000 cases of the more severe form of dengue virus infection known as dengue hemorrhagic fever/dengue shock syndrome (DHF DSS) (Gubler, DJ. and Meltzer, M. 1999 Adv Virus Res 53:35-70). This latter disease is seen predominantly 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 38:172-180; Halstead, S.B. et al. 1969 Am J Trop Med Hyg 18:997-1021; Thein, S. et al. 1997 Am J Trop Med Hyg 56:566-575). A dengue vaccine is needed to lessen 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 simultaneously induce immunity to all four serotypes. Ihimunity is primarily mediated by neutralizing antibody directed against the envelope (E) glycoprotein, a virion structural protein. Infection with one serotype induces long-lived homotypic immunity and a short- lived heterotypic immunity (Sabin, A. 1955 Am J Trop Med Hyg 4:198-207). Therefore, the goal of immunization is to induce a long-lived neutralizing antibody response against DEN1, DEN2, DEN3, and DEN4, 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-borne flavivirus present in tropical and semitropical regions of the world (Monafh, T.P. and Heinz, F.X. 1996 in: Fields Virology, Fields, D.M et al. eds. Philadelphia: Lippincott- Raven Publishers, pp. 961-1034).

Several live attenuated dengue vaccine candidates have been developed and evaluated in humans and 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. and Hotta, S. 1944 Jpn J Bacteriol 1:96-99; Sabin, A.B. and Schlesinger, R.W. 1945 Science 101:640; Wisserman, C.L. et al. 1963 Am J Trop Med Hyg 12:620-623). Although these candidate vaccine viruses were immunogenic in humans, their poor growth in cell culture discouraged further development. Additional live attenuated DEN1, DEN2, DEN3, and DEN4 vaccine candidates have been developed by serial passage in non-human tissue culture (Angsubhakorn, S. et al. 199 A Southeast Asian J Trop Med Public Health 25:554- 559; Bancroft, W.H. et al. 1981 Infect Immun 31:698-703; Bhamarapravati, N. et al. 1987 Bull World Health Organ 65:189-195; Eckels, K.H. et al. 1984 Am J Trop Med Hyg 33:684-698; Hoke, CH. Jr. et al. 1990 Am J Trop Med Hyg 43:219-226; Kanesa-Thasan, N. et al. 2001 Vaccine 19:3179-3188) or by chemical mutagenesis (McKee, K.T. et al. 1987 Am J Trop Med Hyg 36:435-442). 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 19:3179-3188; Bhamarapravati, N. and Sutee, Y. 2000 Vaccine 18:44- 47).

Two major advances using 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 mutations that have been introduced into the cDNA clone by site-directed mutagenesis (Lai, C.J. et al. 1991 PNAS USA 88:5139-5143). 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. and Lai, CJ. 1991 PNAS USA 88:10342-10346; Chen, W. et al. 1995 J Virol 69:5186-5190; Huang, C.Y. et al. 2000 J Virol 74:3020-3028; Plernev, A.G. and Men, R. 1998 PNAS USA 95:1746-1751). These techniques have been used to construct intertypic chimeric dengue viruses that have been shown to be effective in protecting monkeys against homologous dengue virus challenge (Bray, M. et al. 1996 J Virol 70:4162-4166). A similar strategy is also being used to develop attenuated antigenic chimeric dengue virus vaccines based on the attenuation of the yellow fever vaccine virus or the attenuation of the cell-culture passaged dengue viruses (Monath, T.P. et al. 1999 Vaccine 17:1869-1882; Huang, C.Y. et al. 2000 J Virol 74:3020-3028).

Another study examined the level of attenuation for humans of a DEN4 mutant bearing a 30-nucleotide deletion (Δ30) 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 70:3930-3937). Additional studies were carried out to examine whether this Δ30 mutation present in the DEN4 vaccine candidate was the major determinant of its attenuation for monkeys. It was found that the Δ30 mutation was indeed the major determinant of attenuation for monkeys, and that it specified a satisfactory balance between attenuation and immunogenicity for humans (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13).

Summary of the Invention The previously identified Δ30 attenuating mutation, created in dengue virus type 4 (DEN4) by the removal of 30 nucleotides from the 3'-UTR, is also capable of attenuating a wild-type strain of dengue virus type 1 (DENl). Removal of 30 nucleotides from the DENl 3'-UTR in a highly conserved region homologous to the DEN4 region encompassing the Δ30 mutation yielded a recombinant virus attenuated in rhesus monkeys to a level similar to recombinant virus DEN4Δ30. This establishes the transportability of the Δ30 mutation and its attenuation phenotype to a dengue virus type other than DEN4. The effective transferability of the Δ30 mutation, described by this work, establishes the usefulness of the Δ30 mutation to attenuate and improve the safety of commercializable dengue virus vaccines of any serotype. We envision a tetravalent dengue virus vaccine containing dengue virus types 1, 2, 3, and 4 each attenuated by the Δ30 mutation. We also envision a tetravalent dengue virus vaccine containing recombinant antigenic chimeric viruses in which the structural genes of dengue virus types 1, 2, and 3 replace those of DEN4Δ30; 1, 2, and 4 replace those of DEN3Δ30; 1, 3, and 4 replace those of DEN2Δ30; and 2, 3, and 4 replace those of DEN1Δ30. In some instances, such chimeric dengue viruses are attenuated not only by the Δ30 mutation, but also by their chimeric nature. The presence of the Δ30 attenuating mutation in each virus component precludes the reversion to a wild-type virus by intertypic recombination. In addition, because of the inherent genetic stability of deletion mutations, the Δ30 mutation represents an excellent alternative for use as a common mutation shared among each component of a tetravalent vaccine.

Brief Description of the DrawinRS Figure 1. The live attenuated tetravalent dengue virus vaccine contains dengue viruses representing each of the 4 serotypes, with each serotype containing its full set of unaltered wild-type structural and non-structural proteins and a shared Δ30 attenuating mutation. The relative location of the Δ30 mutation in the 3' untranslated region (UTR) of each component is indicated by an arrow.

Figure 2. A. The Δ30 mutation removes 30 contiguous nucleotides (shaded) from the 3' UTR of DEN4. Nucleotides are numbered from the 3' terminus. B. Nucleotide sequence alignment of the TL2 region of DENl, DEN2, DEN3, and DEN4 and their Δ30 derivatives. Also shown is the corresponding region for each of the four DEN serotypes.

Upper case letters indicate sequence homology among all 4 serotypes, underlining indicates nucleotide pairing to form the stem structure. C. Predicted secondary stracture of the TL2 region of each DEN serotype. Nucleotides that are removed by the Δ30 mutation are boxed (DENl - between nts 10562 - 10591, DEN2 Tonga/74 - between nts 10541 - 10570,

DEN3 Sleman/78 - between nts 10535 - 10565, and DEN4 - between nts 10478 - 10507). Figure 3. Niremia levels in rhesus monkeys inoculated with rDEΝ4 vaccine candidates bearing 5-FU derived mutations. Groups of four or two (rDEN4 and rDEN4Δ30) monkeys were inoculated with 5.0 log₁₀PFU virus subcutaneously. Serum was collected daily and virus titers were determined by plaque assay in Vero cells. The limit of virus detection was 0.7 log₁₀PFU/ml. Mean virus titers are indicated for each group. Figure 4. Niremia levels in rhesus monkeys inoculated with rDEΝ4 vaccine candidates bearing pairs of charge-to-alanine mutations in NS5. Groups of four or two (rDEN4 and rDEN4Δ30) monkeys were inoculated with 5.0 log₁₀PFU viras subcutaneously. Serum was collected daily and virus titers were determined by plaque assay in Vero cells. The limit of virus detection was 1.0 log_ι0PFU/ml. Mean virus titers are indicated for each group. Niremia was not detected in any monkey after day 4.

Figure 5. The Δ30 mutation attenuates both DEΝ1 and DEΝ4 for rhesus monkeys. Groups of 4 monkeys were immunized subcutaneously with 5.0 log₁₀ PFU of the indicated virus. Serum was collected each day following immunization and virus titers were determined and are shown as mean log₁₀ PFU/ml.

Figure 6. A. Diagram of the p2 (Tonga/74) full-length cDNA plasmid. Regions subcloned are indicated above the plasmid. Numbering begins at the 5' end of the viral sequence. B. The Δ30 mutation removes the indicated 30 nucleotides from the 3' UTR sequence to create p2Δ30. Figure 7. Niremia levels in rhesus monkeys inoculated with DEΝ2 (Tonga/74), rDEN2, and rDEN2Δ30 vaccine candidate. Groups of four monkeys were inoculated with 5.0 log₁₀PFU virus subcutaneously. Serum was collected daily and virus titers were determined by plaque assay in Vero cells. The limit of virus detection was 0.7 log₁₀PFU/ml. Mean virus titers are indicated for each group. Niremia was not detected in any monkey after day 8.

Figure 8. A. Diagram of the p3 (Sleman/78) full-length cDΝA plasmid. Regions subcloned are indicated above the plasmid. Numbering begins at the 5' end of the viral sequence. The sequence and insertion location of the Spel linker is shown. B. The Δ30 mutation removes the indicated 31 nucleotides from the 3' UTR sequence to create p3Δ30. Figure 9. A. Recombinant chimeric dengue viruses were constructed by introducing either the CME or the ME regions of DEN2 (Tonga/74) into the DEN4 genetic background. The relative location of the Δ30 mutation in the 3 ' UTR is indicated by an arrow and intertypic junctions 1, 2, and 3 are indicated. B. Nucleotide and amino acid sequence of the intertypic junction regions. Restriction enzyme recognition sites used in assembly of each chimeric cDNA are indicated.

Figure 10. Growth kinetics in Vero cells of chimeric rDEN2/4Δ30 viruses encoding single or combined Vero cell adaptation mutations. Vero cells were infected with the indicated viruses at an MOI of 0.01. At the indicated time points post-infection, 1 ml samples of tissue culture medium were removed, clarified by centrifugation, and frozen at - 80°C. The level of virus replication was assayed by plaque titration in C6/36 cells. Lower limit of detection was 0.7 log₁₀PFU/ml. Replication levels on day 4 post-infection are indicated by the dashed line.

Figure 11. A. Recombinant chimeric dengue viruses were constructed by introducing either the CME or the ME regions of DEN3 (Sleman/78) into the DEN4 genetic background. The relative location of the Δ30 mutation in the 3' UTR is indicated by an arrow and intertypic junctions 1, 2, and 3 are indicated. Restriction enzyme recognition sites used in assembly of each chimeric cDNA are indicated. B. Nucleotide and amino acid sequence of the intertypic junction regions. Restriction enzyme recognition sites used in assembly of each chimeric cDNA are indicated.

Figure 12. A. Recombinant chimeric dengue viruses were constructed by introducing either the CME or the ME regions of DENl (Puerto Rico/94) into the DEN4 genetic background. The relative location of the Δ30 mutation in the 3' UTR is indicated by an arrow and intertypic junctions 1, 2, and 3 are indicated. Restriction enzyme recognition sites used in assembly of each chimeric cDNA are indicated. B. Nucleotide and amino acid sequence of the intertypic junction regions. Restriction enzyme recognition sites used in assembly of each chimeric cDNA are indicated. Brief Description of the Sequences

Brief Description of the SEQ ID NOs

Detailed Description of the Preferred Embodiment Introduction A molecular approach is herewith used to develop a genetically stable live attenuated tetravalent dengue viras vaccine. Each component of the tetravalent vaccine, namely, DENl, DEN2, DEN3, and DEN4, must be attenuated, genetically stable, and immunogenic. A tetravalent vaccine is needed to ensure simultaneous protection against each of the four dengue viruses, thereby precluding the possibility of developing the more serious illnesses dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), which occur in humans during secondary infection with a heterotypic wild-type dengue virus. Since dengue viruses can undergo genetic recombination in nature (Worobey, M. et al. 1999 PNAS USA 96:7352-7), the tetravalent vaccine should be genetically incapable of undergoing a recombination event between its four virus components that could lead to the generation of viruses lacking attenuating mutations. Previous approaches to develop a tetravalent dengue virus vaccine have been based on independently deriving each of the four virus components through separate mutagenic procedures, such as passage in tissue culture cells derived from a heterologous host. This strategy has yielded attenuated vaccine candidates (Bhamarapravati, N. and Sutee, Y. 2000 Vaccine 18:44-7). However, it is possible that gene exchanges among the four components of these independently derived tetravalent vaccines could occur in vaccinees, possibly creating a virulent recombinant viras. Virulent poliovirases derived from recombination have been generated in vaccinees following administration of a trivalent polio viras vaccine (Guillot, S. et al. 2000 J Virol 74:8434-43).

The present invention describes: (1) improvements to the previously described rDEN4Δ30 vaccine candidate, 2) attenuated rDENlΔ30, rDEN2Δ30, and rDEN3Δ30 recombinant viruses containing a 30 nucleotide deletion (Δ30) in a section of the 3' untranslated region (UTR) that is homologous to that in the rDEN4Δ30 recombinant virus, (3) a method to generate a tetravalent dengue virus vaccine composed of rDENlΔ30, rDEN2Δ30, rDEN3Δ30, and rDEN4Δ30, 4) attenuated antigenic chimeric viruses, rDENl/4Δ30, rDEN2/4Δ30, and rDEN3/4Δ30, for which the CME, ME, or E gene regions of rDEN4Δ30 have been replaced with those derived from DENl, DEN2, or DEN3; alternatively rDENl/3Δ30, rDEN2/3Δ30, and rDEN4/3Δ30 for which CME, ME, or E gene regions of rDEN3Δ30 have been replaced with those derived from DENl, 2, or 4; alternatively rDENl/2Δ30, rDEN3/2Δ30, and rDEN4/2Δ30 for which CME, ME, or E gene regions of rDEN2Δ30 have been replaced with those derived from DENl, 3, or, 4; and alternatively rDEN2/lΔ30, rDEN3/lΔ30, and rDEN4/lΔ30 for which CME, ME, or E gene regions of rDENlΔ30 have been replaced with those derived from DEN2, 3, or 4, and 5) a method to generate a tetravalent dengue virus vaccine composed of rDENl/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, and rDEN4Δ30, alternatively rDENl/3Δ30, rDEN2/3Δ30, rDEN4/3Δ30, and rDEN3Δ30, alternatively rDENl/2Δ30, rDEN3/2Δ30, rDEN4/2Δ30, and rDEN2Δ30, and alternatively rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/lΔ30, and rDENlΔ30. These tetravalent vaccines are unique since they contain a common shared attenuating mutation which eliminates the possibility of generating a virulent wild-type virus in a vaccinee since each component of the vaccine possesses the same Δ30 attenuating deletion mutation. In addition, the rDENlΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30 tetravalent vaccine is the first to combine the stability of the Δ30 mutation with broad antigenicity. Since the Δ30 deletion mutation is in the 3' UTR of each virus, all of the proteins of the four component viruses are available to induce a protective immune response. Thus, the method provides a mechanism of attenuation that maintains each of the proteins of DENl, DEN2, DEN3, and DEN4 virases in a state that preserves the full capability of each of the proteins of the four virases to induce humoral and cellular immune responses against all of the stractural and non-structural proteins present in each dengue virus serotype.

As previously described, the DEN4 recombinant virus, rDEN4Δ30 (previously referred to as 2AΔ30), was engineered to contain a 30 nucleotide deletion in the 3' UTR of the viral genome (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13; Men, R. et al. 1996 J Virol 70:3930-7). Evaluation in rhesus monkeys showed the viras to be significantly attenuated relative to wild-type parental viras, yet highly immunogenic and completely protective. Also, a phase I clinical trial with adult human volunteers showed the rDEN4Δ30 recombinant viras to be safe and satisfactorily immunogenic (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13). To develop a tetravalent vaccine bearing a shared attenuating mutation in a untranslated region, we selected the Δ30 mutation to attenuate wild-type dengue virases of serotypes 1, 2, and 3 since it attenuated wild-type DEN4 virus for rhesus monkeys and was safe in humans (Figure 1).

The Δ30 mutation was first described and characterized in the DEN4 viras (Men, R. et al. 1996 J Virol 70:3930-7). In DEN4, the mutation consists of the removal of 30 contiguous nucleotides comprising nucleotides 10478 - 10507 of the 3' UTR (Figure 2 A) which form a putative stem-loop stracture referred to as TL2 (Proutslci, V. et al. 1997 Nucleic Acids Res 25:1194-202). Among the flavivirases, large portions of the UTR form highly conserved secondary structures (Hahn, C.S. et al. 1987 J Mol Biol 198:33-41; Proutski, N. et al. 1997 Nucleic Acids Res 25:1194-202). Although the individual nucleotides are not necessarily conserved in these regions, appropriate base pairing preserves the stem-loop structure in each serotype, a fact that is not readily apparent when only considering the primary sequence (Figure 2B, C). Immunogenic Dengue Chimeras and Methods for Their Preparation

Immunogenic dengue chimeras and methods for preparing the dengue chimeras are provided herein. The irnmunogenic dengue chimeras are useful, alone or in combination, in a pharmaceutically acceptable carrier as immunogenic compositions to minimize, inhibit, or immunize individuals and animals against infection by dengue viras. Chimeras of the present invention comprise nucleotide sequences encoding the immunogenicity of a dengue virus of one serotype and further nucleotide sequences selected from the backbone of a dengue viras of a different serotype. These chimeras can be used to induce an immunogenic response against dengue virus.

In another embodiment, the preferred chimera is a nucleic acid chimera comprising a first nucleotide sequence encoding at least one structural protein from a dengue viras of a first serotype, and a second nucleotide sequence encoding nonstructural proteins from a dengue viras of a second serotype different from the first. In another embodiment the dengue virus of the second serotype is DEΝ4. In another embodiment, the structural protein can be the C protein of a dengue viras of the first serotype, the prM protein of a dengue virus of the first serotype, the E protein of a dengue virus of the first serotype, or any combination thereof.

The term "residue" is used herein to refer to an amino acid (D or L) or an amino acid mimetic that is incorporated into a peptide by an amide bond. As such, the amino acid may be a naturally occurring amino acid or, unless otherwise limited, may encompass known analogs of natural amino acids that function in a manner similar to the naturally occurring amino acids {i.e., amino acid mimetics). Moreover, an amide bond mimetic includes peptide backbone modifications well known to those skilled in the art.

Furthermore, one of skill in the art will recognize that individual substitutions, deletions or additions in the amino acid sequence, or in the nucleotide sequence encoding for the amino acids, which alter, add or delete a single amino acid or a small percentage of amino acids (typically less than 5%, more typically less than 1%) in an encoded sequence are conservatively modified variations, wherein the alterations result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Naline (N); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W). As used herein, the terms "viras chimera," "chimeric virus," "dengue chimera" and

"chimeric dengue viras" means an infectious construct of the invention comprising nucleotide sequences encoding the immunogenicity of a dengue virus of one serotype and further nucleotide sequences derived from the backbone of a dengue viras of a different serotype. As used herein, "infectious construct" indicates a virus, a viral construct, a viral chimera, a nucleic acid derived from a virus or any portion thereof, which may be used to infect a cell.

As used herein, "nucleic acid chimera" means a construct of the invention comprising nucleic acid comprising nucleotide sequences encoding the immunogenicity of a dengue virus of one serotype and further nucleotide sequences derived from the backbone of a dengue virus of a different serotype. Correspondingly, any chimeric virus or virus chimera of the invention is to be recognized as an example of a nucleic acid chimera.

The structural and nonstructural proteins of the invention are to be understood to include any protein comprising or any gene encoding the sequence of the complete protein, an epitope of the protein, or any fragment comprising, for example, three or more amino acid residues thereof. Dengue Chimeras

Dengue virus is a mosquito-borne flaviviras pathogen. The dengue virus genome contains a 5' untranslated region (5' UTR), followed by a capsid protein (C) encoding region, followed by a premembrane/membrane protein (prM) encoding region, followed by an envelope protein (E) encoding region, followed by the region encoding the nonstructural proteins (ΝS1-ΝS2A-ΝS2B-ΝS3-ΝS4A-ΝS4B-ΝS5) and finally a 3' untranslated region (3' UTR). The viral structural proteins are C, prM and E, and the nonstractural proteins are NS1-NS5. The structural and nonstructural proteins are translated as a single polyprotein and processed by cellular and viral proteases.

The dengue chimeras of the invention are constructs formed by fusing structural protein genes from a dengue viras of one serotype, e.g. DENl, DEN2, DEN3, or DEN4, with non-structural protein genes from a dengue virus of a different serotype, e.g., DENl, DEN2, DEN3, or DEN4.

The attenuated, immunogenic dengue chimeras provided herein contain one or more of the structural protein genes, or antigenic portions thereof, of the dengue virus of one serotype against which immunogenicity is to be conferred, and the nonstructural protein genes of a dengue virus of a different serotype.

The chimera of the invention contains a dengue viras genome of one serotype as the backbone, in which the structural protein gene(s) encoding C, prM, or E protein(s) of the dengue genome, or combinations thereof, are replaced with the corresponding structural protein gene(s) from a dengue virus of a different serotype that is to be protected against. The resulting viral chimera has the properties, by virtue of being chimerized with a dengue virus of another serotype, of attenuation and is therefore reduced in virulence, but expresses antigenic epitopes of the structural gene products and is therefore immunogenic.

The genome of any dengue viras can be used as the backbone in the attenuated chimeras described herein. The backbone can contain mutations that contribute to the attenuation phenotype of the dengue virus or that facilitate replication in the cell substrate used for manufacture, e.g., Vero cells. The mutations can be in the nucleotide sequence encoding nonstructural proteins, the 5' untranslated region or the 3' untranslated region. The backbone can also contain further mutations to maintain the stability of the attenuation phenotype and to reduce the possibility that the attenuated virus or chimera might revert back to the virulent wild-type virus. For example, a first mutation in the 3' untranslated region and a second mutation in the 5' untranslated region will provide additional attenuation phenotype stability, if desired. In particular, a mutation that is a deletion of 30 nts from the 3' untranslated region of the DEN4 genome between nts 10478-10507 results in attenuation of the DEN4 viras (Men et al. 1996 J Virology 70:3930-3933; Durbin et al. 2001 Am J Trop Med 65:405-413, 2001). Therefore, the genome of any dengue type 4 virus containing such a mutation at this locus can be used as the backbone in the attenuated chimeras described herein. Furthermore, other dengue viras genomes containing an analogous deletion mutation in the 3' untranslated region of the genomes of other dengue virus serotypes may also be used as the backbone stracture of this invention.

Such mutations may be achieved by site-directed mutagenesis using techniques known to those skilled in the art. It will be understood by those skilled in the art that the virulence screening assays, as described herein and as are well known in the art, can be used to distinguish between virulent and attenuated backbone structures. Construction of Dengue Chimeras

The dengue virus chimeras described herein can be produced by substituting at least one of the stractural protein genes of the dengue virus of one serotype against which immunity is desired into a dengue viras genome backbone of a different serotype, using recombinant engineering techniques well known to those skilled in the art, namely, removing a designated dengue viras gene of one serotype and replacing it with the desired corresponding gene of dengue viras of a different serotype. Alternatively, using the sequences provided in GenBank, the nucleic acid molecules encoding the dengue proteins may be synthesized using known nucleic acid synthesis techniques and inserted into an appropriate vector. Attenuated, immunogenic virus is therefore produced using recombinant engineering techniques known to those skilled in the art.

As mentioned above, the gene to be inserted into the backbone encodes a dengue stractural protein of one serotype. Preferably the dengue gene of a different serotype to be inserted is a gene encoding a C protein, a prM protein and/or an E protein. The sequence inserted into the dengue virus backbone can encode both the prM and E structural proteins of the other serotype. The sequence inserted into the dengue virus backbone can encode the C, prM and E structural proteins of the other serotype. The dengue virus backbone is the DENl, DEN2, DEN3, or DEN4 viras genome, or an attenuated dengue viras genome of any of these serotypes, and includes the substituted gene(s) that encode the C, prM and/or E stractural protein(s) of a dengue viras of a different serotype, or the substituted gene(s) that encode the prM and/or E stractural protein(s) of a dengue viras of a different serotype.

Suitable chimeric virases or nucleic acid chimeras containing nucleotide sequences encoding structural proteins of dengue viras of any of the serotypes can be evaluated for usefulness as vaccines by screening them for phenotypic markers of attenuation that indicate reduction in virulence with retention of immunogenicity. Antigenicity and immunogenicity can be evaluated using in vitro or in vivo reactivity with dengue antibodies or immunoreactive serum using routine screening procedures known to those skilled in the art.

Dengue Vaccines The preferred chimeric viruses and nucleic acid chimeras provide live, attenuated virases useful as immunogens or vaccines. In a preferred embodiment, the chimeras exhibit high immunogenicity while at the same time not producing dangerous pathogenic or lethal effects.

The chimeric virases or nucleic acid chimeras of this invention can comprise the structural genes of a dengue virus of one serotype in a wild-type or an attenuated dengue virus backbone of a different serotype. For example, the chimera may express the structural protein genes of a dengue virus of one serotype in either of a dengue viras or an attenuated dengue viras background of a different serotype.

The strategy described herein of using a genetic background that contains nonstructural regions of a dengue virus genome of one serotype, and, by chimerization, the properties of attenuation, to express the stractural protein genes of a dengue virus of a different serotype has lead to the development of live, attenuated dengue vaccine candidates that express stractural protein genes of desired immunogenicity. Thus, vaccine candidates for control of dengue pathogens can be designed. Viruses used in the chimeras described herein are typically grown using techniques known in the art. Virus plaque or focus forming unit (FFU) titrations are then performed and plaques or FFU are counted in order to assess the viability, titer and phenotypic characteristics of the virus grown in cell culture. Wild type virases are mutagenized to derive attenuated candidate starting materials. Chimeric infectious clones are constructed from various dengue serotypes. The cloning of virus-specific cDNA fragments can also be accomplished, if desired. The cDNA fragments containing the stractural protein or nonstructural protein genes are amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from dengue RNA with various primers. Amplified fragments are cloned into the cleavage sites of other intermediate clones. Intermediate, chimeric dengue clones are then sequenced to verify the sequence of the inserted dengue-specific cDNA. Full genome-length chimeric plasmids constructed by inserting the structural or nonstractural protein gene region of dengue viruses into vectors are obtainable using recombinant techniques well known to those skilled in the art. Methods of Administration The viral chimeras described herein are individually or jointly combined with a pharmaceutically acceptable carrier or vehicle for administration as an immunogen or vaccine to humans or animals. The terms "pharmaceutically acceptable carrier" or "pharmaceutically acceptable vehicle" are used herein to mean any composition or compound including, but not limited to, water or saline, a gel, salve, solvent, diluent, fluid ointment base, liposome, micelle, giant micelle, and the like, which is suitable for use in contact with living animal or human tissue without causing adverse physiological responses, and which does not interact with the other components of the composition in a deleterious manner.

The immunogenic or vaccine formulations may be conveniently presented in viral plaque forming unit (PFU) unit or focus forming unit (FFU) dosage form and prepared by using conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets commonly used by one of ordinary skill in the art.

Preferred unit dosage formulations are those containing a dose or unit, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients particularly mentioned above, the formulations of the present invention may include other agents commonly used by one of ordinary skill in the art. The immunogenic or vaccine composition may be admimstered through different routes, such as oral or parenteral, including, but not limited to, buccal and sublingual, rectal, aerosol, nasal, intramuscular, subcutaneous, intradermal, and topical. The composition may be administered in different forms, including, but not limited to, solutions, emulsions and suspensions, microspheres, particles, microparticles, nanoparticles and liposomes. It is expected that from about 1 to about 5 doses may be required per immunization schedule. Initial doses may range from about 100 to about 100,000 PFU or FFU, with a preferred dosage range of about 500 to about 20,000 PFU or FFU, a more preferred dosage range of from about 1000 to about 12,000 PFU or FFU and a most preferred dosage range of about 1000 to about 4000 PFU or FFU. Booster injections may range in dosage from about 100 to about 20,000 PFU or FFU, with a preferred dosage range of about 500 to about 15,000, a more preferred dosage range of about 500 to about 10,000 PFU or FFU, and a most preferred dosage range of about 1000 to about 5000 PFU or FFU. For example, the volume of administration will vary depending on the route of administration. Intramuscular injections may range in volume from about 0.1 ml to 1.0 ml.

The composition may be stored at temperatures of from about -100°C to about 4°C.

The composition may also be stored in a lyophilized state at different temperatures including room temperature. The composition may be sterilized through conventional means known to one of ordinary skill in the art. Such means include, but are not limited to, filtration. The composition may also be combined with bacteriostatic agents to inhibit bacterial growth. Administration Schedule

The immunogenic or vaccine composition described herein may be administered to humans, especially individuals travelling to regions where dengue viras infection is present, and also to inhabitants of those regions. The optimal time for administration of the composition is about one to three months before the initial exposure to the dengue virus. However, the composition may also be administered after initial infection to ameliorate disease progression, or after initial infection to treat the disease. Adjuvants A variety of adjuvants known to one of ordinary skill in the art may be administered in conjunction with the chimeric viras in the immunogen or vaccine composition of this invention. Such adjuvants include, but are not limited to, the following: polymers, co- polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co- polymers, polymer p 1005, Freund's complete adjuvant (for animals), Freund's incomplete adjuvant; sorbitan monooleate, squalene, CRL-8300 adjuvant, alum, QS 21, muramyl dipeptide, CpG oligonucleotide motifs and combinations of CpG oligonucleotide motifs, trehalose, bacterial extracts, including mycobacterial extracts, detoxified endotoxins, membrane lipids, or combinations thereof. Nucleic Acid Sequences

Nucleic acid sequences of dengue viras of one serotype and dengue virus of a different serotype are useful for designing nucleic acid probes and primers for the detection of dengue viras chimeras in a sample or specimen with high sensitivity and specificity. Probes or primers corresponding to dengue virus can be used to detect the presence of a vaccine virus. The nucleic acid and corresponding amino acid sequences are useful as laboratory tools to study the organisms and diseases and to develop therapies and treatments for the diseases. Nucleic acid probes and primers selectively hybridize with nucleic acid molecules encoding dengue viras or complementary sequences thereof. By "selective" or "selectively" is meant a sequence which does not hybridize with other nucleic acids to prevent adequate detection of the dengue virus sequence. Therefore, in the design of hybridizing nucleic acids, selectivity will depend upon the other components present in the sample. The hybridizing nucleic acid should have at least 70% complementarity with the segment of the nucleic acid to which it hybridizes. As used herein to describe nucleic acids, the term "selectively hybridizes" excludes the occasional randomly hybridizing nucleic acids, and thus has the same meaning as "specifically hybridizing." The selectively hybridizing nucleic acid probes and primers of this invention can have at least 70%, 80%, 85%, 90%o, 95%, 97%, 98% and 99% complementarity with the segment of the sequence to which it hybridizes, preferably 85% or more.

The present invention also contemplates sequences, probes and primers that selectively hybridize to the encoding nucleic acid or the complementary, or opposite, strand of the nucleic acid. Specific hybridization with nucleic acid can occur with minor modifications or substitutions in the nucleic acid, so long as functional species-species hybridization capability is maintained. By "probe" or "primer" is meant nucleic acid sequences that can be used as probes or primers for selective hybridization with complementary nucleic acid sequences for their detection or amplification, which probes or primers can vary in length from about 5 to 100 nucleotides, or preferably from about 10 to 50 nucleotides, or most preferably about 18-24 nucleotides. Isolated nucleic acids are provided herein that selectively hybridize with the species-specific nucleic acids under stringent conditions and should have at least five nucleotides complementary to the sequence of interest as described in Molecular Cloning: A Laboratory Manual, 2nd ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989.

If used as primers, the composition preferably includes at least two nucleic acid molecules which hybridize to different regions of the target molecule so as to amplify a desired region. Depending on the length of the probe or primer, the target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions. For example, for the purpose of detecting the presence of dengue virus, the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other organisms.

The nucleic acid sequences encoding dengue virus can be inserted into a vector, such as a plasmid, and recombinantly expressed in a living organism to produce recombinant dengue virus peptide and/or polypeptides. The nucleic acid sequences of the invention include a diagnostic probe that serves to report the detection of a cDNA amplicon amplified from the viral genomic RNA template by using a reverse-transcription/polymerase chain reaction (RT/PCR), as well as forward and reverse amplimers that are designed to amplify the cDNA amplicon. In certain instances, one of the amplimers is designed to contain a vaccine virus-specific mutation at the 3 '-terminal end of the amplimer, which effectively makes the test even more specific for the vaccine strain because extension of the primer at the target site, and consequently amplification, will occur only if the viral RNA template contains that specific mutation.

Automated PCR-based nucleic acid sequence detection systems have been recently developed. TaqMan assay (Applied Biosystems) is widely used. A more recently developed strategy for diagnostic genetic testing makes use of molecular beacons (Tyagi and Kramer, 1996 Nature Biotechnology 14:303-308). Molecular beacon assays employ quencher and reporter dyes that differ from those used in the TaqMan assay. These and other detection systems may used by one skilled in the art.

EXAMPLE 1 Improvement of Dengue Virus Vaccine Candidate rDEN4Δ30 The safety of recombinant live-attenuated dengue-4 vaccine candidate rDEN4Δ30 was evaluated in twenty human volunteers who received a dose of 5.0 log₁₀ plaque forming units (PFU) (Durbin A.P. et al. 2001 Am J Trop Med Hyg 65:405-413). The vaccine candidate was found to be safe, well-tolerated and immunogenic in all of the vaccinees. However, five of the vaccinees experienced a transient elevation in alanine aminotransferase levels, three experienced neutropenia and ten vaccinees . developed an asymptomatic macular rash, suggesting that it may be necessary to further attenuate this vaccine candidate.

Currently, a randomized, double-blind, placebo-controlled, dose de-escalation study is being conducted to determine the human infectious dose 50 (HID₅₀) of rDEN4Δ30. Each dose cohort consists of approximately twenty vaccinees and four placebo recipients. To date, complete data for doses of 3.0 log₁₀ PFU and 2.0 log₁₀ PFU has been collected. rDEN4Δ30 infected 100% of vaccinees when 3.0 log₁₀ PFU was administered and 95% of vaccinees when 2.0 log₁₀ PFU was administered (Table 1). The vaccine candidate caused no symptomatic illness at either dose (Table 1). One vaccinee who received 3.0 log,₀ PFU experienced a transient elevation in alanine aminotransferase levels and approximately one fourth of the vaccinees experienced neutropenia at both doses (Table 1). Neutropenia was transient and mild. More than half of the vaccinees developed a macular rash at both doses; the occurrence of rash was not correlated with vaccination dose or with viremia (Table 1 and Table 2). Neither peak titer nor onset of viremia differed between the 3.0 log₁₀ PFU and 2.0 log₁₀ PFU doses, though both measures of viremia were significantly lower for these doses than for a dose of 5.0 log₁₀ PFU (Table 3). The vaccine candidate was immunogenic in 95% of vaccinees at both doses and neutralizing antibody did not decline between days 28 and 42 post-vaccination (Table 4). Although the HID₅₀ has not been determined yet, it is clearly less than 2.0 log₁₀ PFU. Interestingly, decreases in the dose of vaccine have had no consistent effect on immunogenicity, viremia, benign neutropenia or the occurrence of rash. Thus it will not necessarily be possible to further attenuate rDEN4Δ30 by decreasing the dose of virus administered, and other approaches must be developed.

Table 1. rDEN4Δ30 clinical summary

Log₁₀ pfu

1-.

L Looggi_j0o ppffuu//mL ^c Neutropenia defined as ANC < 1500/dl ^d T Max in volunteer = 100.4°F ^e ALT day 0 = 78, ALT max = 91 (day 14)

Table 2. Pattern of rash in vaccinees

^alog₁₀ pfu ^b Means in each column with different letters are significantly different (α = 0.05)

Table 3. rDEN4Δ30 viremia summary

^alog₁₀ pfu

"Means in each column with different letters are significantly different (α = 0.05) Table 4. hnmunogenicity of rDEN4Δ30

Two approaches have been taken to further attenuate rDEN4Δ30. This first is the generation and characterization of attenuating point mutations in rDEN4 using 5' fluorouracil mutagenesis (Blaney, J.E. Jr. et al. 2002 Virology 300: 125-139; Blaney, J.E. Jr. et al. 2001 J. Virol. 75: 9731-9740). This approach has identified a panel of point mutations that confer a range of temperature sensitivity (ts) and small plaque (sp) phenotypes in Vero and HuH-7 cells and attenuation (att) phenotypes in suckling mouse brain and SCID mice engrafted with HuH-7 cells (SCID-HuH-7 mice). In this example, a subset of these mutations has been introduced to rDEN4Δ30 and the phenotypes of the resulting viruses evaluated.

A second approach was to create a series of paired charge-to-alanine mutations in contiguous pairs of charged amino acid residues in the rDEN4 NS5 gene. As demonstrated previously, mutation of 32 individual contiguous pairs of charged amino acid residues in rDEN4 NS5 conferred a range of ts phenotypes in Vero and HuH-7 cells and a range of att phenotypes in suckling mouse brain (Hanley, K.H. et al. 2002 J. Virol. 76 525-531). As demonstrated below, these mutations also confer an att phenotype in SCID-HuH-7 mice. These mutations have been introduced, either as single pairs or sets of two pairs, into rDEN4Δ30 to determine whether they are compatible with the Δ30 mutation and whether they enhance the att phenotypes of rDEN4Δ30. A panel of rDEN4 viruses bearing individual point mutations have been characterized which possess temperature sensitive and/or small plaque phenotypes in tissue culture and varying levels of attenuated replication in suckling mouse brain when compared to wild type rDEN4 virus (Blaney, J. E. et al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 JYirol. 75:9731-9740). Three mutations have been selected to combine with the Δ30 deletion mutation to evaluate their ability to further restrict replication of rDEN4Δ30 in rhesus monkeys. First, the missense mutation in NS3 at nucleotide 4995 (Ser > Pro) which confers temperature sensitivity in Vero and HuH-7 cells and restricted replication in suckling mouse brain was previously combined with the Δ30 mutation (Blaney, J. E. et al. 2001 JVirol. 75:9731-9740). The resulting virus, rDEN4Δ30-4995, was found to be more restricted (1,000-fold) in mouse brain replication than rDEN4Δ30 virus (<5-fold) when compared to wild type rDEN4 viras. Second, a missense mutation at nucleotide 8092 (Glu > Gly) which also confers temperature sensitivity in Vero and HuH-7 cells and 10,000-fold restricted replication in suckling mouse brain was combined with the Δ30 mutation here. Third, a substitution in the 3' UTR at nucleotide 10634 which confers temperature sensitivity in Vero and HuH-7 cells, small plaque size in HuH-7 cells, and approximately 1,000-fold restricted replication in suckling mouse brain and SCLD mice transplanted with HuH-7 cells was combined with the Δ30 mutation here (Blaney, J. E. et al. 2002 Virology 300:125-139). For the present investigation, subcloned fragments of p4 (Durbin, A.P. et al. 2001

Am J Trop Med Hyg 65:405-13) containing the above mutations were introduced into the p4Δ30 cDNA clone. For transcription and recovery of virus, cDNA was linearized with Acc65ϊ (isoschizomer of Kpήl which cleaves leaving only a single 3' nucleotide) and used as template for transcription by SP6 RNA polymerase as previously described (Blaney, J. E. et al. 2002 Virology 300:125-139). C6/36 mosquito cells were transfected using liposome-mediated transfection and cell culture supernatants were harvested between days five and seven. Recovered viras was terminally diluted twice in Vero cells and passaged two (rDEN4Δ30-4995) or three (rDEN4Δ30-8092 and rDEN4Δ30-10634) times in Vero cells. The complete genomic sequences of rDEN4Δ30-4995, rDEN4Δ30-8092, and rDEN4Δ30-10634 viruses were determined as previously described (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413). As expected, each rDEN4Δ30 virus derivative contained the Δ30 mutation. Unexpectedly, in rDEN4Δ30-4995 virus, the nucleotide changes in the codon containing nucleotide 4995, resulted in a Ser > Leu amino acid change rather than a Ser > Pro change since the p4Δ30-4995 cDNA was designed to introduce the Ser > Pro change (Table 5). The p4Δ30-4995 cDNA clone was indeed found to encode a Ser > Pro change at nucleotide 4995, so it is unclear how the viras population acquired the Ser > Leu mutation. Nevertheless, this viras was evaluated to assess the phenotype specified by this missense mutation. rDEN4Δ30-4995 virus was also found to contain an incidental mutation at nucleotides 4725-6 which resulted in a single amino acid change (Ser > Asp). The rDEN4Δ30-8092 and rDEN4Δ30-10634 viruses contained the appropriate nucleotide substitutions as well as additional incidental mutations in E, NS4B and NS4B, respectively (Table 5). Table 5. Missense and UTR mutations present in rDEN4Δ30 virus derivatives bearing introduced point mutations.

^a Amino acid position in DEN4 polyprotein beginning with the methionine residue of the C protein (nucleotides 102- 104) as position 1. ^b Mutation restricts replication in mouse models of DEN4 infection which were introduced by Kunkel mutagenesis.

Replication of the three modified rDEN4Δ30 viruses were compared to rDEN4Δ30 and wild type rDEN4 virus in the suckling mouse brain model and SCID mice transplanted with HuH-7 cells (SCLD-HuH-7 mice). Experiments were conducted as previously described (Blaney, J. E. et al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 JVirol. 75:9731-9740). Briefly, for infection of suckling mouse brain, groups of six seven-day-old mice were inoculated intracerebrally with 4.0 log₁₀ PFU of viras and the brain of each mouse was removed five days later. Clarified supernatants of 10% brain suspensions were then frozen at -70°C, and the virus titer was determined by plaque assay in Vero cells. For analysis of DEN4 virus replication in SCID-HuH-7 mice, four to six week-old SCLD mice were injected intraperitoneally with 10⁷ . HuH-7 cells. Five to six weeks after transplantation, mice were infected by direct inoculation into the tumor with 4.0 log_ι0 PFU of viras, and serum for virus titration was obtained by tail-nicking on day 7. The viras titer was determined by plaque assay in Vero cells.

Wild type rDEN4 viras replicated to 6.0 log₁₀PFU/g in suckling mouse brain, and rDEN4Δ30 was restricted in replication by 0.7 logι₀PFU/g, which is similar to previous observations (Table 6) (Blaney, J. E. et al. 2001 J Virol. 75:9731-9740). rDEN4Δ30-4995, rDEN4Δ30-8092, and rDEN4Δ30-10634 viruses were found to have restricted replication in suckling mouse brain when compared to rDEN4 virus of 3.3, 2.8, and 2.4 log₁₀PFU/g, respectively. These results indicate that the additional attenuating mutations serve to further restrict replication of the rDEN4Δ30 virus in mouse brain ranging from 50-fold (rDEN4Δ30-10634) to 400-fold (rDEN4Δ30-4995). In SCID-HuH-7 mice, virus titer of rDEN4Δ30 virus was 0.4 log_I0PFU/ml lower than rDEN4 virus, which is also similar to previous studies (Blaney, J. E. et al. 2002 Virology 300:125-139). Each modified rDEN4Δ30 virus was found to be further restricted in replication in SCID-HuH-7 mice (Table 6). rDEN4Δ30-4995, rDEN4Δ30-8092, and rDEN4Δ30-10634 viruses had restricted replication in SCID-HuH-7 mice when compared to rDEN4 viras of 2.9, 1.1, and 2.3 log₁₀PFU/g below the level of wild type rDEN4 virus, respectively. Two important observations were made: (1) The 4995, 8092 and 10634 mutations were compatible for viability with the Δ30 mutation, and (2) These three modified rDEN4Δ30 viruses had between a 10 and 1,000-fold reduction in replication in comparison to rDEN4 wild-type viras, which allows virases with a wide range of attenuation in this model to be further evaluated in monkeys or humans.

Table 6. Addition of point mutations in NS3, NS5, or the 3' UTR to rDEN4Δ30 viras further attenuates the virus for suckling mouse brain and SCLD-HuH-7 mice.

^a Groups of 6 suckling mice were inoculated i.e. with 10⁴ PFU of viras. Brains were removed 5 days later, homogenized, and titered in Vero cells. ^b Comparison of mean viras titers of mice inoculated with mutant viras and concurrent rDEN4 wt control.

⁰ Groups of HuH-7-SCID mice were inoculated directly into the tumor with 10⁴PFU virus. Serum was collected on day 6 and 7 and titered in Vero cells.

Based on the findings in the two mouse models of DEN4 viras infection, each of the rDEN4Δ30-4995, rDEN4Δ30-8092, and rDEN4Δ30-10634 viruses was evaluated in the rhesus macaque model of DEN4 infection which has been previously described (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413). Briefly, groups of four (rDEN4Δ30-4995, rDEN4Δ30-8092, and rDEN4Δ30-10634) or two (rDEN4, rDEN4Δ30, mock) monkeys were inoculated with 5.0 log₁₀PFU viras subcutaneously. Monkeys were observed daily and serum was collected on days 0 to 6, 8, 10, and 12, and viras titers were determined by plaque assay in Vero cells for measurement of viremia. On day 28, serum was drawn and the level of neutralizing antibodies was tested by plaque reduction assay in Vero cells as previously described (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413).

Viremia was detected beginning on day 1 post-infection and ended by day 4 in all monkeys (Table 7, Figure 3). Viremia was present in each monkey infected with rDEN4, rDEN4Δ30, or rDEN4Δ30-10634 viras, but only 2 out of 4 monkeys infected with rDEN4Δ30-4995 or rDEN4Δ30-8092 virus had detectable viremia. As expected, infection with rDEN4 virus resulted in the highest mean number of viremic days per monkey (3.0 days) as well as mean peak viras titer (2.2 log₁₀PFU/ml). Monkeys infected with rDEN4Δ30 viras had both a lower mean number of viremic days per monkey (2.0 days) and mean peak virus titer (1.1 log₁₀PFU/ml) compared to rDEN4 viras. Groups of monkeys infected with each of the modified rDEN4Δ30 viruses had a further restricted mean number of viremic days with those inoculated with rDEN4Δ30-8092 virus having the lowest value, 0.5 days, a 4-fold reduction compared to rDEN4Δ30 virus. The mean peak virus titer of monkeys infected with rDEN4Δ30-4995 (0.9 log₁₀PFU/ml) or rDEN4Δ30-8092 (0.7 log₁₀PFU/mι) was also lower than those infected with rDEN4Δ30 virus. However, the mean peak viras titer of monkeys infected with rDEN4Δ30-10634 (1.3 log₁₀PFU/ml) was slightly higher than those infected with rDEN4Δ30 particularly on day 2 (Figure 3).

Table 7. Addition of point mutations to rDEN4Δ30 further attenuates the virus for rhesus monkeys.

^a Groups of rhesus monkeys were inoculated subcutaneously with 10⁵ PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10, 12, and 28. Virus titer was determined by plaque assay in Vero cells. ^b Niremia was not detected in any monkey after day 4.

Serum collected on day 0 and 28 was tested for the level of neutralizing antibodies against rDEΝ4. No detectable neutralizing antibodies were found against DEN4 on day 0, as expected, since the monkeys were pre-screened to be negative for neutralizing antibodies against flavivirases (Table 7). On day 28, monkeys infected with rDEN4 had a mean serum neutralizing antibody titer (reciprocal dilution) of 398 which was approximately twofold higher than monkeys infected with rDEN4Δ30 virus (1:181). This result and the twofold higher level of viremia in rDEN4 virus-infected monkeys are similar to results obtained previously (Durbin et al. 2001 Am. J. Trop. Med. Hyg. 65:405-413). Monkeys infected with rDEN4Δ30-4995 (1:78), rDEN4Δ30-8092 (1:61), and rDEN4Δ30-10634 (1:107) viruses each had a reduced mean serum neutralizing antibody titer compared to monkeys infected with rDEN4Δ30 viras. The four monkeys which had no detectable viremia did have serum neutralizing antibody titers indicating that they were indeed infected. Despite the slight increase in mean peak virus titer of rDEN4Δ30-10634 virus compared with rDEN4Δ30 viras, rDEN4Δ30-10634 virus had a lower mean serum neutralizing antibody titer compared to monkeys infected with rDEN4Δ30 virus. This and the lower mean number of viremic days per monkey suggests that the 10634 mutation can attenuate the replication of rDEN4Δ30 virus in monkeys. On day 28 after inoculation, all monkeys were challenged with 5.0 log₁₀PFU wild type rDEN4 virus subcutaneously. Monkeys were observed daily and serum was collected on days 28 to 34, 36, and 38, and virus titers were determined by plaque assay in Vero cells for measurement of viremia after challenge. Twenty eight days after rDEN4 virus challenge, serum was drawn and the level of neutralizing antibodies was tested by plaque reduction assay in Vero cells. Mock-inoculated monkeys had a mean peak viras titer of 2.3 log₁₀PFU/ml after challenge with a mean number of viremic days of 3.5 (Table 8). However, monkeys inoculated with rDEN4, rDEN4Δ30, or each of the modified rDEN4Δ30 virases had no detectable viremia, indicating that despite the decreased replication and immunogenicity of rDEN4Δ30-4995, rDEN4Δ30-8092, and rDEN4Δ30- 10634 virases, each was sufficiently immunogenic to induce protection against wild type rDEN4. Increases in mean neutralizing antibody titer were minimal (< 3 -fold) following challenge in all inoculation groups except mock-infected providing further evidence that the monkeys were protected from the challenge.

Table 8. rDEN4Δ30 containing additional point mutations protects rhesus monkeys from wt DEN4 viras challenge

^a 28 days after primary inoculation with the indicated virases, rhesus monkeys were challenged subcutaneously with 10⁵ PFU rDEN4 virus in a 1 ml dose. Serum was collected on days 28 to 34, 36, 38, and 56. Virus titer was determined by plaque assay in Vero cells.

Taken together, these results indicate that the three point mutations, 4995, 8092, and 10634) described above do further attenuate the rDEN4Δ30 vaccine candidate in suckling mouse brain, SCID-HuH-7 mice, and rhesus monkeys. Because of additional incidental mutations (Table 4) present in each modified rDEN4Δ30 virus, the phenotypes cannot be directly attributed to the individual 4995, 8092, and 10634 point mutations. However, the presence of similar mouse-attenuation phenotypes in other rDEN4 virases bearing one of these three mutations supports the contention that the 4995, 8092, and 10634 point mutations are responsible for the att phenotypes of the modified rDEN4Δ30 viruses. Since rDEN4Δ30-4995, rDEN4Δ30-8092, and rDEN4Δ30-10634 virus demonstrated decreased replication in rhesus monkeys while retaining sufficient immunogenicity to confer protective immunity, these virases are contemplated as dengue vaccines for humans.

DEN4 viruses carrying both Δ30 and charge-to-alanine mutations were next generated. A subset of seven groups of charge-to-alanine mutations described above were identified that conferred between a 10-fold and 1,000-fold decrease in replication in SCID- HuH-7 mice and whose unmutated sequence was well-conserved across the four dengue serotypes. These mutations were introduced as single pairs and as two sets of pairs to rDEN4Δ30 using conventional cloning techniques. Transcription and recovery of virus and terminal dilution of virases were conducted as described above. Assay of the level of temperature sensitivity of the charge-cluster-to-alanine mutant virases in Vero and HuH-7 cells, level of replication in the brain of suckling mice and level of replication in SCID- HuH-7 mice was conducted as described above.

Introduction of one pair of charge-to-alanine mutations to rDEN4 produced recoverable virus in all cases (Table 9). Introduction of two pairs of charge-to-alanine mutations produced recoverable virus in two out of three cases (rDEN4Δ30-436-437-808- 809 was not recoverable). rDEN4Δ30 is not ts in Vero or HuH-7 cells. In contrast, seven of the seven sets of charge-to-alanine mutations used in this example conferred a ts phenotype in HuH-7 cells and five also conferred a ts phenotype in Vero cells. All six viruses carrying both Δ30 and charge-to-alanine mutations showed a ts phenotype in both Vero and HuH-7 cells (Table 9). rDEN4Δ30 is not attenuated in suckling mouse brain, whereas five of the seven sets of charge-to-alanine mutations conferred an att phenotype in suckling mouse brain (Table 10). Four of the viruses carrying both Δ30 and charge-to-alanine mutations were attenuated in suckling mouse brain (Table 10). In one case (rDEN4Δ30-23 -24-396-397) combination of two mutations that did not attenuate alone resulted in an attenuated virus. Generally, virases carrying both Δ30 and charge-to-alanine mutations showed levels of replication in the suckling mouse brain more similar to their charge-to-alanine mutant parent viras than to rDEN4Δ30. rDEN4Δ30 is attenuated in SCID-HuH-7 mice, as are six of the seven charge-to- alanine mutant virases used in this example. Virases carrying both Δ30 and charge-to- alanine mutations tended to show similar or slightly lower levels of replication in SCID- HuH-7 mice compared to their charge-to-alanine mutant parent virus (Table 10). In three cases, virases carrying both Δ30 and charge-to-alanine mutations showed at least a fivefold greater reduction in SCID-HuH-7 mice than rDEN4Δ30.

The complete genomic sequence of five viruses (rDEN4-200-201, rDEN4Δ30-200- 201, rDEN4-436-437 [clone 1], rDEN4Δ30-436-437, and rDEN4-23-24-200-201) that replicated to > 10⁵ PFU/ml in Vero cells at 35°C and that showed a hundredfold or greater reduction in replication in SCID-HuH-7 mice was determined (Table 11). Each of the five contained one or more incidental mutations. In one virus, rDEN4Δ30-436-437, the one additional mutation has been previously associated with Vero cell adaptation (Blaney, J.E. Jr. et al. 2002 Virology 300:125-139). Each of the remaining virases contained at least one incidental mutation whose phenotypic effect is unknown. Consequently, the phenotypes described cannot be directly attributed to the charge-to-alanine mutations. However, the fact that rDEN4 and rDEN4Δ30 viruses carrying the same charge-to-alanine mutations shared similar phenotypes provides strong support for the ability of charge-to-alanine mutations to enhance the attenuation of rDEN4Δ30. Because rDEN4-436-437 [clone 1] contained 4 incidental mutations, a second clone of this viras was prepared. rDEN4-436- 437 [clone2] contained only one incidental mutation (Table 11), and showed the same phenotypes as rDEN4-436-437 in cell culture and SCID-HuH-7 mice. rDEN4-436-437 [clone 2] was used in the rhesus monkey study described below.

Table 9. Addition of charge-to-alariine mutations to rDEN4Δ30 confers a ts phenotype in both Vero and HuH-7 cells.

^a Underlined values indicate a 2.5 or 3.5 log₁₀PFU/ml reduction in titer in Vero or HuH-7 cells, respectively, at the indicated temperatur when compared to the permissive temperature (35°C). Amino acid pair(s) changed to pair of Ala residues.

⁰ Reduction in titer (log₁₀pfu/ml) compared to the permissive temperature (35°C).

Table 10. Addition of charge-to-alariine mutations attenuates rDEN4Δ30 in suckling mouse brain and enhances attenuation in SCID-HuH-7 mice.

^a Groups of six suckling mice were inoculated i.e. with 10⁴ PFU viras in a 30 μl inoculum. The brain was removed 5 days later, homogenized, and viras was quantitated by titration in Vero cells. ^b Determined by comparing the mean viral titers in mice inoculated with sample viras and concurrent wt controls (n = 6). The attenuatio (att) phenotype is defined as a reduction of > 1.5 log₁₀PFU/g compared to wt virus; reductions of > 1.5 are listed in boldface. ^c Groups of SCLO-HuH-7 mice were inoculated directly into the tumor with 10⁴ PFU viras. ^d Determined by comparing mean viral titers in mice inoculated with sample virus and concurrent wt controls. The attenuation phenotype i defined as a reduction of >1.5 logι₀ PFU/g compared to wt virus; reductions of >1.5 are listed in boldface.

Table 11. Missense and UTR mutations present in rDEN4 virus derivatives bearing charge-to-alanine and the Δ30 mutation.

"Asterisk indicates previously identified Vero cell adaptation mutation. ^bBold values indicate mutations designed to occur in the designated virus.

⁰ Amino acid position in the protein product of the designated DEN4 gene; numbering starts with the amino terminus of the protein.

Based on the attenuation in the SCID-HuH7 mouse model, four of the charge-to- alanine mutant viruses (rDEN4-200-201, rDEN4Δ30-200-201, rDEN4-436-437 [clone 2], rDEN4Δ30-436-437) were evaluated in rhesus macaques as described above. As with the study of viruses carrying attenuating point mutations, viremia was detected on day 1 post- infection and ended by day 4 in all monkeys (Figure 4, Table 12). Viremia was detected in most of the monkeys infected; only one of the four monkeys infected with rDEN4Δ30-200- 201 and one of the four monkeys infected with rDEN4Δ30-436-437 showed no detectable viremia. Monkeys infected with rDEN4 showed the highest mean peak viras titer; and in each case viruses carrying the Δ30 mutation showed an approximately 0.5 log decrease in mean peak viras titer relative to their parental viruses and a 0.5 to 2 day decrease in mean number of viremic days per monkey. Monkeys infected with viruses carrying both the Δ30 and charge-to-alanine mutations showed a two-fold reduction in mean peak viremia relative to those infected with rDEN4Δ30. This suggests that addition of the charge-to-alanine mutations further attenuates rDEN4Δ30 for rhesus macaques. As expected, none of the monkeys in this study showed detectable levels of neutralizing antibody on day 0. On day 28, every monkey infected with a virus showed a detectable levels of neutralizing antibody, indicating that all of the monkeys, even those that showed no detectable viremia, had indeed been infected. As in the study of attenuating point mutations, monkeys infected with rDEN4 had a mean serum neutralizing antibody titer (reciprocal dilution) which was approximately twice that of monkeys that had been infected with rDEN4Δ30. Monkeys infected with rDEN4-200-201 and rDEN4-436-437 [clone 2] had similar mean neutralizing antibody titers to rDEN4, and those infected with rDEN4Δ30-200-201 and rDEN4Δ30-436-437 had similar mean neutralizing antibody titers to rDEN4. In each case the addition of the Δ30 mutation to a virus resulted in a two-fold decrease in neutralizing antibody. Thus, although the addition of charge-to-alanine mutations to rDEN4Δ30 decreased mean peak viremia below that of rDEN4Δ30 alone, it did not affect levels of neutralizing antibody. Table 12. Addition of paired charge-to-alanine mutations to rDEN4Δ30 further attenuates the viras for rhesus monkeys.

^a Groups of rhesus monkeys were inoculated subcutaneously with 10⁵ PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10 and 28. Viras titer was determined by plaque assay in Vero cells. ^b Viremia was not detected in any monkey after day 4.

After challenge with rDEN4 on day 28, mock-infected monkeys had a mean peak virus titer of 1.5 log₁₀PFU/ml and a mean number of viremic days of 3.0 (Table 13). However, none of the monkeys previously inoculated with rDEN4, rDEN4Δ30 or the charge-to-alanine mutant viruses showed detectable viremia. Additionally, none of the monkeys showed a greater than four-fold increase in serum neutralizing antibody titer. Together these data indicate that infection with any of the viruses, including those carrying both Δ30 and the charge-to-alanine mutations, protected rhesus macaques from challenge with rDENA

Table 13. rDEN4Δ30 containing charge-to-alanine mutations protects rhesus monkeys from wt DEN4 virus challenge

^a 28 days after primary inoculation with the indicated viruses, rhesus monkeys were challenged subcutaneously with 10⁵ PFU rDEN4 virus in a 1 ml dose. Serum was collected on days 28 to 34, 36, 10, and 56. Viras titer was determined by plaque assay in Vero cells.

Addition of charge-to-alanine mutations to rDEN4Δ30 can confer a range of ts phenotypes in both Vero and HuH-7 cells and att phenotypes in suckling mouse brain and can either enhance or leave unchanged attenuation in SCID-HuH-7 mice. Most importantly, addition of these mutations can decrease the viremia produced by rDEN4Δ30 in rhesus macaques without decreasing neutralizing antibody titer or protective efficacy. Thus addition of such mutations to rDEN4Δ30 is contemplated as enhancing attenuation in humans. Also, mutations are contemplated as being added that do not change the overall level of attenuation, but stabilize the attenuation phenotype because they themselves are independently attenuating even in the absence of the Δ30 mutation. Charge-to-alanine mutations are particularly useful because they occur outside of the stractural gene regions, and so can be used to attenuate stractural gene chimeric viruses. Moreover, they involve at least three nucleotide changes, making them unlikely to revert to wild type sequence.

A series of point mutations that enhance the replication of rDEN4 in Vero cells tissue culture have been identified; these are primarily located in the NS4B gene (Blaney, J. E. et. al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 J Virol 75:9731-9740). Vero cell adaptation mutations confer two desirable features upon a vaccine candidate. First, they enhance viras yield in Vero cells, the intended substrate for vaccine production, and thus render vaccine production more cost-effective. Second, although each of these Vero adaptation mutations are point mutations, they are likely to be extremely stable during vaccine manufacture, because they give a selective advantage in Vero cells. At least one Vero cell adaptation mutation, at position 7129, was also shown to decrease mosquito infectivity of rDEN4; poor mosquito infectivity is another desirable characteristic of a dengue vaccine candidate. To investigate the generality of this finding, we tested the effect of the remaining Vero cell adaptation mutations on the ability of rDEN4 to infect Aedes aegypti mosquitoes fed on an infectious bloodmeal. Table 14 shows the infectivity of each viras carrying a single Vero cell adaptation mutation at high titer. Of these, only one mutation, at position 7182, was associated with a large decrease in mosquito infectivity. Thus 7182 may be a particularly valuable mutation to include in an rDEN4 vaccine candidate, as it has opposite effects on replication in Vero cells and in mosquitoes.

Table 14. Effect of Vero cell adaptation mutations on rDEN4 mosquito infectivity

"Virus titer ingested, assuming a 2 μl bloodmeal.

"Percentage of mosquitoes with IFA detectable antigen in midgut or head tissue prepared 21 days after oral infection.

EXAMPLE 2 Generation and Characterization of a Recombinant DENl Virus Containing the Δ30 Mutation

We first sought to determine if the Δ30 mutation was able to satisfactorily attenuate a wild-type DEN virus other than the DEN4 serotype. To do this, the Δ30 mutation was introduced into the cDNA for DENl (Western Pacific). The pRS424DENlWP cDNA clone (Puri, B. et al. 2000 Virus Genes 20:57-63) was digested with BamHI and used as template in a PCR using Pfu polymerase with forward primer 30 (DENl nt 10515-10561 and 10592-10607) and the M13 reverse sequencing primer (101 nt beyond the 3' end of DENl genome sequence). The resulting PCR product was 292 bp and contained the Δ30 mutation. The pRS424DENl WP cDNA was partially digested with Apa I, then digested to completion with Sac II and the vector was gel isolated, mixed with PCR product, and used to transform yeast strain YPH857 to yield growth on plates lacking tryptophan (Polo, S. et al. 1997 J Virol 71:5366-74). Positive yeast colonies were confirmed by PCR and restriction enzyme analysis. DNA isolated from two independent yeast colonies was used to transform E. coli strain STBL2. Plasmid DNA suitable for generating RNA transcripts was prepared and the presence of the Δ30 mutation was verified by sequence analysis.

For transcription and generation of virus, cDNA (designated ρRS424DENlΔ30) that was linearized with Sac II was used as template in a transcription reaction using SP6 RNA polymerase as described (Polo, S. et al. 1997 J Virol 71:5366-74). Transcription reactions were electroporated into LLC-MK2 cells and infection was confirmed by observation of CPE and immunofluorescence and harvested on day 14. Virus stocks were amplified on C6/36 mosquito cells and titered on LLC-MK2 cells. The genome of the resulting virus, rDENlΔ30, was sequenced to confirm the presence of the Δ30 mutation. The Δ30 mutation removes nucleotides 10562-10591 of DENl (Figure 2B, C), which corresponds to the TL2 of DENl . The virus replicates efficiently in Vero cell culture to titers of 6.5 log₁₀ PFU/ml, indicating that the Δ30 mutation is compatible with efficient growth of DENl in cell culture, a property essential for manufacture of the vaccine. Using similar techniques, parent viras rDENl was generated. Incidental mutations arising from viras passage in tissue culture were identified in both rDENl and rDENlΔ30 using sequence analysis and are listed in Table 15. An additional rDENlΔ30 virus was derived by transfection and amplification in Vero cells. Although this virus was not evaluated in the studies described below, its sequence analysis is included in Table 15. The properties of rDENlΔ30 as a vaccine in vivo were next examined. Table 15. Missense mutations present among the recombinant DENl virases and correlation of NS4B region mutations with those found in DEN4

^a Same nucleotide as 7154 in rDEN4. ^bSame nucleotide as 7162 in rDEN4

* Nucleotide and amino acid comparison of selected NS4B region:

7 7 7 7

DEN4 1 1 1 1 base 4 5 6 7 Number : 890123456789012345678901234567890123456789012345678901234567

++ ++ + +++++ + + + + ++ + ++++++++ ++ ++ ++ ++

D4 7128 CC^CAACCUUGACAGCAUCCUUAGUCAUGCUyUUAGUCCATUAUGC-^UAAUAGGCCCA

P T T L T A S L V M L L V H T A I I G P

Dl 7139 CCGCUGACGCUGACAGCGGCGGUAUUUAUGCUAGUGGCUCAJJUAUGCCAUAAU GGACCC

P L T L T A A V P M V A H T A I I G P

D2 7135 CCUAUAACCCUCACAGCGGCUCUUCU UUAUUGGUAGCACAUUAUGCCAUCAUAGGACCG

P I T L T A A L L L V A H T A I I G P

D3 7130 CCACϋAACUCUCACAGCGGCAGUUCUCCUGCUAGUCACGCAUUAUGCUAUUAUAGGUCCA

P T L T A A V L L L V T H T A I I G P

+ + + + + + + + + + + + +

D4 = rDEN4

Dl-rDENl(WP)

D2 = rDEN2(Tonga/74)

D3=rDEN3(Sleman/78)

+Homology among all four serotypes Nucleotides are underlined in even multiples of 10.

Evaluation of the replication, immunogenicity, and protective efficacy of rDENlΔ30 and wild-type parental rDENl virus (derived from the ρRS424DENlWP cDNA) in juvenile rhesus monkeys was performed as follows. Dengue virus-seronegative monkeys were injected subcutaneously with 5.0 log₁₀ PFU of viras in a 1 ml dose divided between two injections in each side of the upper shoulder area. Monkeys were observed daily and blood was collected on days 0-10 and 28 and serum was stored at -70°C. Titer of viras in serum samples was determined by plaque assay in Vero cells as described previously (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13). Plaque reduction neutralization titers were determined for the day 28 serum samples as previously described (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13). All monkeys were challenged on day 28 with a single dose of 5.0 log₁₀ PFU of wild-type rDENl and blood was collected for 10 days. Viras titer in post-challenge sera was determined by plaque assay in Vero cells. Monkeys inoculated with full-length wild-type rDENl were viremic for 2 - 3 days with a mean peak titer of 2.1 log₁₀ PFU/ml (Table 16), and monkeys inoculated with rDENlΔ30 were viremic for less than 1 day with a mean peak titer of 0.8 log₁₀ PFU/ml, indicating that the Δ30 mutation is capable of attenuating DENl. As expected for an attenuated virus, the immune response, as measured by neutralizing antibody titer, was lower following inoculation with rDENlΔ30 compared to inoculation with wild-type rDENl (Table 16), yet sufficiently high to protect the animals against wild-type DENl virus challenge. Wild-type rDENl viras was not detected in any serum sample collected following viras challenge, indicating that monkeys were completely protected following immunization with either full-length wild-type rDENl or recombinant virus rDENlΔ30. The level of attenuation specified by the Δ30 mutation was comparable in both the DENl and DEN4 genetic backgrounds (Figure 5).

Table 16. The Δ30 mutation attenuates rDENl for rhesus monkeys

* Rhesus monkeys were inoculated subcuateously with 5.0 log₁₀ PFU of viras. Serum samples were collected daily for 10 days. Serum for neutralization assay was collected on day 28. All monkeys were challenged on day 28 with 5.0 log₁₀ PFU of rDENl.

As previously reported, rDEN4 viras replicated to greater than 6.0 log₁₀PFU/ml serum in SCιD-HuH-7 mice, while the replication of rDEN4 virus bearing the Δ30 mutation was reduced by about 10-fold (Blaney, J.E. Jr. et al. 2002 Virology 300:125-139).

The replication of rDENlΔ30 was compared to that of wt rDENl in SCID-HuH-7 mice

(Table 17). rDENlΔ30 replicated to a level approximately 100-fold less than its wt rDENl parent. This result further validates the use of the SCID-HuH-7 mouse model for the evaluation of attenuated strains of DEN virus, with results correlating closely with those observed in rhesus monkeys.

Table 17. The Δ30 mutation attenuates rDENl for HuH-7-SCID mice

⁵ Groups of HuH-7-SCID mice were inoculated directly into the tumor with 4.0 log₁₀pfu virus. Serum was collected on day 6 and 7, and virus titer was determined by plaque assay in Vero cells.

⁶ Significant difference was found between rDENl and rDENl Δ30 virases, Tukey- Kramer test (P < 0.005).

Finally, the infectivity of rDENl and rDENl Δ30 for mosquitoes was assessed, using the methods described in detail in Example 5. Previously, the Δ30 mutation was shown to decrease the ability of rDEN4 to cross the mosquito midgut barrier and establish a salivary gland infection (Troyer, J.M. et al. 2001 Am J Trop Med Hyg 65:414-419).

However neither rDENl nor rDENl Δ30 was able to infect the midgut of Aedes aegypti mosquitoes efficiently via an artificial bloodmeal (Table 18), so it was not possible to determine whether Δ30 might further block salivary gland infection. A previous study also showed that the Δ30 had no effect on the infectivity of rDEN4 for Toxorhynchites splendens mosquitoes infected via intrathoracic inoculation (Troyer, J.M. et al. 2001 Am J

Trop Med Hyg 65:414-419), and a similar pattern was seen for rDENl and rDENlΔ30

(Table 18). The genetic basis for the inability of rDENl to infect the mosquito midgut has not been defined at this time. However, this important property of restricted infectivity for the mosquito midgut is highly desirable in a vaccine candidate since it would serve to greatly restrict transmission of the vaccine virus from a vaccinee to a mosquito vector. Table 18. DENl and DEN1Δ30 virases are both highly infectious for Toxorhynchites splendens, but do not infect Aedes aegypti efficiently.

"Amount of viras present in 0.22 μl inoculum. ^b Percentage of mosquitoes with IFA detectable antigen in head tissue prepared 14 days after inoculation.

⁰ Virus titer ingested, assuming a 2 μl bloodmeal.

Percentage of mosquitoes with IFA detectable antigen in midgut or head tissue prepared 21 days after oral infection. When virus infection was detected, but did not exceed a frequency of 50% at the highest dose of virus ingested, the MIDso was estimated by assuming that a 10-fold more concentrated virus dose would infect 100%) of the mosquitoes.

Thus, the Δ30 mutation, first described in DEN4, was successfully transferred to rDENl. The resulting virus, rDENlΔ30, was shown to be attenuated in monkeys and SCID-HuH-7 mice to levels similar to recombinant virus rDEN4Δ30, thereby establishing the conservation of the attenuation phenotype specified by the Δ30 mutation in a different DEN viras background. Based on the favorable results of rDEN4Δ30 in recent clinical trials (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13), it is predicted that rDENlΔ30 will be suitably attenuated in humans. To complete the tetravalent vaccine, attenuated rDEN2 and rDEN3 recombinant viruses bearing the Δ30 mutation are contemplated as being prepared (See Examples 3 and 4 below). The demonstration that the Δ30 mutation specifies a phenotype that is transportable to another DEN serotype has important implications for development of the tetravalent vaccine. This indicates that the Δ30 mutation is expected to have a corresponding effect on DEN2 and DEN3 wild-type virases. EXAMPLE 3 Generation and Characterization of a Recombinant DEN2 Virus Containing the Δ30

Mutation

Evaluation of rDENlΔ30 showed that it was satisfactorily attenuated. Based on this result, we sought to extend our technology to the creation of a DEN2 vaccine candidate. To do this, the Δ30 mutation was introduced into the cDNA of DEN2. A DEN2 virus isolate from a 1974 dengue epidemic in the Kingdom of Tonga (Tonga/74) (Gubler, D. J. et al. 1978 Am J Trop Med Hyg 27:581-589) was chosen to represent wt DEN2. The genome of DEN2 (Tonga/74) was sequenced in its entirety and served as consensus sequence for the construction of a full-length cDNA clone (Appendix 1). cDNA fragments of DEN2 (Tonga/74) were generated by reverse-transcription of the genome as indicated in Figure 6A. Each fragment was subcloned into a plasmid vector and sequenced to verify that it matched the consensus sequence as determined for the viras. This yielded seven cloned cDNA fragments spanning the genome. Cloned fragments were modified as follows: Fragment X, representing the 5' end of the genome was abutted to the SP6 promoter; Fragment L was modified to contain a translationally-silent Spel restriction site at genomic nucleotide 2353; Fragment R was modified to contain a translationally-silent Spel restriction site also at genomic nucleotide 2353, and to stabilize the eventual full-length clone, two additional translationally-silent mutations at nucleotides 2362 - 2364 and 2397 were created to ensure that translation stop codons were present in all reading frames other than that used to synthesize the viras polyprotein; Fragment A was modified at nucleotide 3582 to ablate a naturally occurring Spel restriction site and at nucleotide 4497 to ablate a naturally occurring Kpnl restriction site; Fragment C was modified at nucleotide 9374 to ablate a naturally occurring Kpnl restriction site; and Fragment Y, representing the 3' end of the genome was abutted to a Kpnl restriction site. Each fragment was added incrementally between the Ascl and Kpnl restriction sites of DEN4 cDNA clone p4 (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13) to generate a full-length DEN2 cDNA clone (p2) with the same vector background successfully used to generate rDEN4 and rDEN4Δ30. cDNA clone p2 was sequenced to confirm that the virus genome region matched the DEN2 (Tonga/74) consensus sequence, with the exception of the translationally-silent modifications noted above. The Δ30 mutation was introduced into Fragment Y to generate Fragment YΔ30. To create p2Δ30, the Fragment Y region of p2 was replaced with Fragment YΔ30 (Figure 6 A, B).

For transcription and generation of infectious virus, cDNA (p2 and p2Δ30) was linearized with Acc65l (isoschizomer of Kpnl which cleaves leaving only a single 3' nucleotide) and used as template in a transcription reaction using SP6 RNA polymerase as previously described (Blaney, J. E. et. al. 2002 Virology 300:125-139). Transcripts were introduced into Vero cells or C6/36 mosquito cells using liposome-mediated transfection and cell culture supernatants were harvested on day 7. rDEN2 virus was recovered from the p2 cDNA in both Vero and C6/36 cells, while rDEN2Δ30 was recovered from the p2Δ30 cDNA clone in only C6/36 cells (Table 19). The level of infectious viras recovered in C6/36 cells was comparable for the p2 and p2Δ30 cDNA clones when assayed by plaque titration and immunostaining in Vero or C6/36 cells. As previously observed, the efficiency of transfection in C6/36 cells was higher than that in Vero cells. Two rDEN2Δ30 viruses were recovered from independent cDNA clones, #2 and #10.

Table 19. rDEN2 viras is recovered in Vero and C6/36 cells, but rDEN2Δ30 virus is recovered only in C6/36 cells.

To produce working stocks of rDEN2 and rDEN2Δ30 virases, transfection harvests were passaged and terminally diluted in Vero cells, and genomic sequences of the viruses were determined. The Vero cell transfection harvest of rDEN2 viras was terminally diluted once in Vero cells, and individual viras clones were passaged once in Vero cells. To assess whether any homologous Vero cell adaptation mutations identified in the rDEN4 NS4B 7100-7200 region were present in these virus clones, seven independent terminally diluted clones were sequenced over this region. Each of the seven rDEN2 virases contained a single nucleotide substitution in this region at nucleotide 7169 (U > C) resulting in a Val > Ala amino acid change. This nucleotide corresponds to the 7162 mutation identified in rDEN4 (Blaney, J. E. et. al. 2002 Virology 300:125-139), which has a known Vero cell adaptation phenotype suggesting that this mutation may confer a replication enhancement phenotype in rDEN2 viras. One rDEN2 viras clone was completely sequenced and in addition to the 7169 mutation, a missense mutation (Glu > Ala) was found in NS5 at residue 3051 (Table 20). Table 20. Missense mutations which accumulate in rDEN2 and rDEN2Δ30 virases after

_^ transfection or passage in Vero cells.

^a Amino acid position in DEN2 polyprotein beginning with the methionine residue of the C protein (nucleotides 97-99) as position 1. ^b Virus was recovered in Vero cells and terminally-diluted once in Vero cells. Virus stock was prepared in Vero cells.

⁰ Same nucleotide position as 7162 in rDEN4. ^d Virus was recovered in C6/36 cells and passaged three times in Vero cells. Virus was then terminally diluted and a stock was prepared in Vero cells.

Because both rDEN2 and rDEN2Δ30 viruses grown in Vero cells acquired the same mutation at nucleotide 7169, which corresponds to the Vero cell adaptation mutation previously identified in rDEN4 at nucleotide 7162, it was reasoned that this mutation is associated with growth adaptation of rDEN2 and rDEN2Δ30 in Vero cells. In anticipation that the 7169 mutation may allow rDEN2Δ30 to be recovered directly in Vero cells, the mutation was introduced into the rDEN2Δ30 cDNA plasmid to create p2Δ30-7169. Transcripts synthesized from p2Δ30-7169, as well as p2 and p2Δ30 were introduced into Vero cells or C6/36 mosquito cells using liposome-mediated transfection as described above. Viras rDEN2Δ30-7169 was recovered from the p2Δ30-7169 cDNA in both Vero and C6/36 cells, while rDEN2Δ30 was recovered from the p2Δ30 cDNA clone in only C6/36 cells (Table 21). The 7169 mutation is both necessary and sufficient for the recovery of rDEN2Δ30 in Vero cells. Table 21. rDEN2Δ30-7169 viras containing the 7169 Vero cell adaptation mutation is recovered in both Vero and C6/36 cells

a Nucleotide 7169 in rDEN2 corresponds to nucleotide 7162 in rDEN4 which has been shown to be associated with growth adaptation in Vero cells.

To initially assess the ability of the Δ30 mutation to attenuate rDEN2 virus in an animal model, the replication of DEN2 (Tonga/74), rDEN2, and rDEN2Δ30 viruses was evaluated in SCID-HuH-7 mice. Previously, attenuation of vaccine candidates in SCID- HuH-7 mice has been demonstrated to be predictive of attenuation in the rhesus monkey model of infection (Examples 1 and 2). The recombinant virases tested in this experiment were recovered in C6/36 cells. The DEN2 Tonga/74 viras isolate, rDEN2, and two independent rDEN2Δ30 viruses, (clones 20 A and 21 A) which were derived from two independent p2Δ30 cDNA clones, were terminally diluted twice in C6/36 cells prior to production of a working stock in C6/36 cells. These virases should not contain any Vero cell adaptation mutations. DEN2 Tonga/74 viras replicated to a mean virus titer of 6.2 log₁₀PFU/ml in the serum of SCID-HuH-7 mice, and rDEN2 virus replicated to a similar level, 5.6 log₁₀PFU/ml (Table 22). Both rDEN2Δ30 viruses were greater than 100-fold restricted in replication compared to rDEN2 viras. These results indicate that the Δ30 mutation has an attenuating effect on replication of rDEN2 virus similar to that observed for rDEN4 and rDENl viruses. Table 22. The Δ30 mutation restricts rDEN2 viras replication in SCID-HuH-7 mice.

^a Groups of SCID-HuH-7 mice were inoculated directly into the tumor with 10⁴PFU virus grown in C6/36 cells. Serum was collected on day 7 and titered in C6/36 cells. ^b Comparison of mean viras titers of mice inoculated with mutant virus and concurrent rDEN2 control.

DEN2 viras replication in SCID-HuH-7 mice was also determined using DEN2 (Tonga/74), rDEN2, and rDEN2Δ30 which were passaged in Vero cells (see Table 20, footnotes b and d). Both rDEN2 and rDEN2Δ30 had acquired a mutation in NS4B, nucleotide 7169, corresponding to the 7162 mutation identified in rDEN4 as Vero cell adaptation mutation. In the presence of the 7169 mutation, the Δ30 mutation reduced replication of rDEN2Δ30 by 1.0 log₁₀PFU/ml (Table 23). Previously, using viras grown in C6/36 cells and lacking the 7169 mutation, the Δ30 mutation reduced replication of rDEN2Δ30 by about 2.5 log₁₀PFU/ml (Table 22). These results indicate that Vero cell growth adaptation in DEN2 may also confer a slight growth advantage in HuH-7 liver cells. Nevertheless, the attenuation conferred by the Δ30 mutation is still discernible in these Vero cell growth adapted viruses.

Table 23. The Δ30 mutation restricts Vero cell adapted rDEN2 virus replication in SCID-

HuH-7 mice.

" Groups of SCID-HuH-7 mice were inoculated directly into the tumor with 10⁴ PFU viras. Serum was collected on day 7 and titered in C6/36 cells. ^b Comparison of mean viras titers of mice inoculated with rDEN2Δ30 and rDEN2 control. Evaluation of the replication, immunogenicity, and protective efficacy of rDEN2Δ30 and wild-type parental rDEN2 viras in juvenile rhesus monkeys was performed as follows. Dengue virus-seronegative monkeys were injected subcutaneously with 5.0 log₁₀ PFU of virus in a 1 ml dose divided between two injections in each side of the upper shoulder area. Monkeys were observed daily and blood was collected on days 0 - 10 and 28 and serum was stored at -70°C. Virases used in this experiment were passaged in Vero cells, and recombinant virases contained the mutations shown in Table 20 (See footnotes b and d). Titer of viras in serum samples was determined by plaque assay in Vero cells as described previously (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13). Plaque reduction neutralization titers were determined for the day 28 serum samples as previously described (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13). All monkeys were challenged on day 28 with a single dose of 5.0 log_ι0 PFU of wt DEN2 (Tonga/74) and blood was collected for 10 days. Viras titer in post-challenge sera was determined by plaque assay in Vero cells. Monkeys inoculated with wt DEN2 (Tonga/74) or rDEN2 were viremic for 4 - 5 days with a mean peak titer of 2.1 or 1.9 log₁₀ PFU/ml, respectively.

Monkeys inoculated with rDEN2Δ30 were viremic for 2 - 3 days with a mean peak titer of 1.7 log₁₀ PFU/ml (Table 24, Figure 7), indicating that the Δ30 mutation is capable of attenuating DEN2, although not to the same low level observed in rDENl Δ30 (Table 16). As expected for an attenuated viras, the immune response, as measured by neutralizing antibody titer, was lower following inoculation with rDEN2Δ30 compared to inoculation with wt DEN2 (Tonga/74) or rDEN2 (Table 24), yet sufficiently high to protect the animals against wt DEN2 virus challenge (Table 25). Thus, the decreased number of days of viremia for rDEN2Δ30, the decreased mean peak titer, and the decreased serum antibody response indicate that the Δ30 mutation attenuates rDEN2 for rhesus monkeys.

Table 24. rDEN2Δ30 is slightly more attenuated for rhesus monkeys than rDEN2

^a Groups of rhesus monkeys were inoculated subcutaneously with 10⁵ PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10, 12, and 28. Virus titer was determined by plaque assay in Vero cells. ^b Viremia was not detected in any monkey after day 8.

Table 25. rDEN2Δ30 protects rhesus monkeys from wt DEN2 virus challenge

" 28 days after inoculation with the indicated viruses, monkeys were challenged subcutaneously with 10⁵ PFU DEN2 (Tonga/74) in a 1 ml dose. Serum was collected on days 28 to 34, 36, 38, and 56. Viras titer was determined by plaque assay in Vero cells. The infectivity of DEN2 (Tonga/74), rDEN2 and rDEN2Δ30 for Aedes aegypti mosquitoes via an artificial bloodmeal was evaluated using the methods described in detail in Example 5. However at doses of 3.3 to 3.5 log₁₀ pfu ingested, none of these three virases infected any mosquitoes, indicating that DEN2 (Tonga/74) is poorly infectious for Aedes aegypti. As with rDENl, the genetic basis for this lack of infectivity remains to be defined. The important property of restricted infectivity for the mosquito midgut is highly desirable in a vaccine candidate because it would serve to greatly restrict transmission of the virus from a vaccinee to a mosquito vector. Several missense mutation identified in rDEN4 have been demonstrated to confer attenuated replication in suckling mouse brain and/or SCID-HuH-7 mice (Blaney, J. E. et al. 2002 Virology 300:125-139; Blaney, J. E. et al. 2001 J Virol 75:9731-97 '40). In addition, missense mutations that enhance replication of rDEN4 virus in Vero cells have been characterized. The significant sequence conservation among the DEN viras serotypes provides a strategy by which the mutations identified in rDEN4 virases are contemplated as being used to confer similar phenotypes upon rDEN2 virus. Six mutations identified in rDEN4 virus that are at a site conserved in rDEN2 viras are being introduced into the p2 and p2Δ30 cDNA clones (Table 26). Specifically, two rDEN4 mutations, NS3 4891 and 4995, which confer Vero cell adaptation phenotypes and decreased replication in mouse brain, one mutation, NS4B 7182, which confers a Vero cell adaptation phenotype, and three mutations, NS1 2650, NS3 5097, and 3' UTR 10634 which confer decreased replication in mouse brain and SCID-HuH-7 mice are being evaluated. These mutations have been introduced into sub-cloned fragments of the p2 and p2Δ30 cDNA clones, and have been used to generate mutant full-length cDNA clones (Table 26), from which viras has been recovered in C6/36 cells (Table 27). The evaluation of these mutant rDEN2 virases is contemplated as determining that such point mutations can be transported into a different DEN virus serotype and confer a similar useful phenotype, as has been demonstrated for the Δ30 deletion mutation.

Table 26. Introduction of conserved point mutations characterized in rDEN4 virases into rDEN2 Tonga/74 viras.

^a Presence of the indicated mutation increases plaque size in Vero cells two-fold or greater than rDEN4 virus.

" Presence of the indicated mutation restricts replication in 7-day-old mouse brain greater than 100-fold compared to rDEN4 virus.

⁰ Presence of the indicated mutation restricts replication in SCID-HuH-7 mice greater than 100-fold compared to rDEN4 viras. ^d Amino acid position in DEN4 or DEN2 polyprotein beginning with the methionine residue of the C protein (nucleotides 102-104 or 97- 99, respectively) as position 1. ^e Primers were engineered which introduced (underline) translationally-silent restriction enzyme (RE) 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 RE site. The change preserves the codon for the amino acid substitution. ^f Nucleotide substitution in the 3' UTR is U > C in DEN4 and DEN2 viras.

Table 27. rDEN2 viruses containing conserved 5-FU mutations are recovered in C6/36 cells.

" Transfection has not yet been attempted.

EXAMPLE 4 Generation and Characterization of a Recombinant DEN3 Virus Containing the Δ30

Mutation

Because rDENlΔ30 was satisfactorily attenuated, we sought to extend our technology to the creation of a DEN3 vaccine candidate. To do this, the Δ30 mutation was introduced into the cDNA of DEN3, similar to the method used to create rDEN2Δ30. A DEN3 virus isolate from a 1978 dengue epidemic in rural Sleman, Central Indonesia (Sleman/78) (Gubler, D. J. et al. 1981 Am J Trop Med Hyg 30:1094-1099) was chosen to represent wt DEN3. The genome of DEN3 (Sleman/78) was sequenced in its entirety and served as consensus sequence for the construction of a full-length cDNA clone (Appendix 2). cDNA fragments of DEN3 (Sleman/78) were generated by reverse-transcription of the genome as indicated in Figure 8 A. Each fragment was subcloned into a plasmid vector and sequenced to verify that it matched the consensus sequence as determined for the viras. This yielded six cloned cDNA fragments spanning the genome. Cloned fragments were modified as follows: Fragment 5, representing the 5' end of the genome was abutted to the SP6 promoter preceded by an -4_?cl restriction site; Fragment 1L was modified to contain a translationally-silent Spel restriction site at genomic nucleotide 2345; Fragment IR was modified to contain a translationally-silent Spel restriction site also at genomic nucleotide 2345, and to stabilize the eventual full-length clone, three additional translationally-silent mutations at nucleotides 2354 - 2356, 2360 - 2362, and 2399 were created to ensure that translation stop codons were present in all reading frames other than that used to synthesize the viras polyprotein; Fragment 3 was modified at nucleotide 9007 to ablate a naturally occurring Kpnl restriction site; and Fragment 4, representing the 3' end of the genome was abutted to a Kpnl restriction site. Each fragment was added incrementally between the Ascl and Kpnl restriction sites of DEN4 cDNA clone p4 (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13) to generate a full-length DEN3 cDNA clone with the same vector background successfully used to generate rDEN4 and rDEN2. However, a stable, full- length clone could not be recovered in E. coli when fragments 1L and IR were combined into the same cDNA molecule. To overcome this instability, a synthetic DNA linker (Figure 8A) containing redundant termination codons in each of the forward and reverse open reading frames was introduced into the Spel restriction site at the same time that fragment 1L was added to complete the full-length cDNA construct. The resulting p3 clone containing the linker sequence was stable in E. coli, indicating that the linker sequence was sufficient to interrupt whatever deleterious element exists in this region. cDNA clone p3 was sequenced and the viras genome was found to match the DEN3 (Sleman 78) consensus sequence, with the exception of the linker sequence and translationally-silent modifications noted above (Appendix 2 - shown with the linker sequence removed). The Δ30 mutation was introduced into Fragment 4 to generate Fragment 4Δ30. To create p3Δ30, the Fragment 4 region of p3 was replaced with Fragment 4Δ30 (Figure 8 A, B).

For transcription and generation of infectious viras, cDNA plasmids p3 and p3Δ30 were digested with Spel and re-ligated to remove the linker sequence, linearized with Acc65l (isoschizomer of Kpnl which cleaves leaving only a single 3' nucleotide), and used as templates in a transcription reaction using SP6 RNA polymerase as previously described (Blaney, J. E. et. al. 2002 Virology 300:125-139). Transcripts were introduced into Vero cells or C6/36 mosquito cells using liposome-mediated transfection and cell culture supernatants were harvested on day 14. rDEN3 virus was recovered from the p3 cDNA in both Vero and C6/36 cells, while rDEN3Δ30 was recovered from the p3Δ30 cDNA clone in only C6/36 cells (Table 28). The level of infectious virus recovered in C6/36 cells was comparable for the p3 and p3Δ30 cDNA clones when assayed by plaque titration in Vero or C6/36 cells. As previously observed, the efficiency of transfection in C6/36 cells was higher than that in Vero cells. Two rDEN3Δ30 viruses were recovered from independent cDNA clones, #22 and #41. Table 28. rDEN3 viras is recovered in Vero and C6/36 cells, but rDEN3Δ30 virus is recovered only in C6/36 cells.

To produce working stocks of virases, transfection harvests will be passaged and terminally diluted in Vero cells, and genomic sequences of the viruses will be determined. To improve virus yield in Vero cells, the Vero cell adaptation mutation previously identified in rDEN4 at nucleotide 7162 was introduced into the homologous NS4B region of p3 and p3Δ30 to create p3-7164 and p3Δ30-7164. This mutation creates a Val to Ala substitution at amino acid position 2357. As demonstrated for rDEN2Δ30, this mutation allowed for the direct recovery of virus in Vero cells (Table 27) and is anticipated to have the same effect for rDEN3Δ30.

To. initially assess the ability of the Δ30 mutation to attenuate rDEN3 virus in an animal model, the replication of DEN3 (Sleman/78), rDEN3, and rDEN3Δ30 viruses will be evaluated in SCID-HuH-7 mice and rhesus monkeys. Previously, attenuation of vaccine candidates in SCID-HuH-7 mice has been demonstrated to be predictive of attenuation in the rhesus monkey model of infection (Examples 1 and 2). The evaluation of these mutant rDEN3 virases is contemplated as determining that the Δ30 deletion mutations can be transported into the DEN3 viras serotype and confer a similar useful phenotype, as has been demonstrated for DENl, DEN2, and DEN4.

In summary, the strategy of introducing the Δ30 mutation into wild-type DEN viruses of each serotype to generate a suitably attenuated tetravalent vaccine formulation is a unique and attractive approach for several reasons. First, the mutation responsible for attenuation is a 30-nucleotide deletion in the 3' UTR, thus assuring that all of the stractural and non-structural proteins expressed by each of the four components of the tetravalent vaccine are authentic wild-type proteins. Such wild-type proteins should elicit an antibody response that is broad based, rather than based solely on the M and E proteins that are present in chimeric dengue viras vaccine candidates (Guirakhoo, F. et al. 2001 J Virol 75:7290-304; Huang, C.Y. et al. 2000 J Virol 74:3020-8). The uniqueness of this approach derives from the fact that other live attenuated dengue viras vaccines have mutations in their structural or non-structural proteins (Butrapet, S. et al. 2000 J Virol 74:3011-9; Puri, B. et al. 1997 J Gen Virol 78:2287-91), therefore the immune response induced by these virases will be to a mutant protein, rather than a wild-type protein. Second, deletion mutations are genetically more stable than point mutations, and reversion of the attenuation phenotype is unlikely. In humans, DEN4Δ30 present in serum of vaccinees retained its Δ30 mutation, confirming its genetic stability in vivo (Durbin, A.P. et al. 2001 Am J Trop Med Hyg 65:405-13). The attenuating mutations in other existing dengue live attenuated vaccine candidates are based on less stable point mutations (Butrapet, S. et al. 2000 J Virol 74:3011-9: Puri, B. et al. 1997 J Gen Virol 78:2287-91). Third, since the Δ30 mutation is common to each of the four virases of the tetravalent vaccine, recombination between any of the four vaccine serotypes would not lead to loss of the attenuating mutation or reversion to a wild-type phenotype. Recombination between components of the trivalent polio vaccine has been observed (Guillot, S. et al. 2000 J Virol 74:8434-43), and naturally occurring recombinant dengue viruses have been described (Worobey, M. et al. 1999 RA S USA 96:7352-7) indicating the ability of this flaviviras to exchange genetic elements between two different virases. Clearly, gene exchange is readily achieved between different DEN viras serotypes using recombinant cDNA techniques (Bray, M. and Lai, C.J. 1991 PNAS USA 88:10342-6). Fourth, viruses with wild-type structural proteins appear more infectious than viruses with altered stractural proteins (Huang, C.Y. et al. 2000 J Virol 74:3020-80). This permits the use of a low quantity of each of the four viras components in the final vaccine, contributing to the low cost of manufacture. Low-cost manufacture is an essential element in defining the ultimate utility of a dengue viras vaccine. EXAMPLE 5 Generation and Characterization of Intertypic Chimeric DEN2 Viruses Containing the Δ30 Mutation

The four serotypes of dengue viras are defined by antibody responses induced by the stractural proteins of the viras, primarily by a neutralizing antibody response to the envelope (E) protein. These stractural proteins include the E glycoprotein, a membrane protein (M), and a capsid (C) protein. The mature virus particle consists of a well- organized outer protein shell surrounding a lipid bilayer membrane and a less-well-defined inner nucleocapsid core (Kuhn, R.J. et al. 2002 Cell 108:717-25). The E glycoprotein is the major protective antigen and readily induces viras neutralizing antibodies that confer protection against dengue virus infection. An effective dengue vaccine must therefore minimally contain the E protein of all four serotypes, namely DENl, DEN2, DEN3, and DEN4, thereby inducing broad immunity and precluding the possibility of developing the more serious illnesses DHF/DSS, which occur in humans during secondary infection with a heterotypic wild-type dengue viras. Based on a previously reported strategy (Bray, M. and Lai, C.J. 1991 PNAS USA 88:10342-6), a recombinant cDNA technology is being used to develop a live attenuated tetravalent dengue virus vaccine composed of a set of intertypic chimeric dengue virases bearing the structural proteins of each serotype.

Following the identification of a suitably attenuated and immunogenic DEN4 recombinant virus, namely DEN4Δ30 (Durbin, A.P et al. 2001 Am J Trop Med Hyg 65:405-13), chimeric viruses based on the DEN4 cDNA have been generated in which the C-M-E (CME) or M-E (ME) genes have been replaced with the corresponding genes derived from the prototypic DEN2 New Guinea C (NGC) strain (Figure 9A). To create the CME chimeric virases, the BgHl I Xhol region of the cDNA for either rDEN4 or rDEN4Δ30 was replaced with a similar region derived from DEN2. Likewise, to create the ME chimeric virases, the Pstl I Xhol region of the cDNA for either rDEN4 or rDEN4Δ30 was replaced with a homologous region derived from DEN2. The nucleotide and amino acid sequences of the resulting junctions are shown in Figure 9B. The GenBank accession number for the nucleotide sequence of rDEN4Δ30 is AF326837. The GenBank accession number for DEN2 NGC is M29095, which represents the mouse neuroviralent strain of DEN2 NGC and differs from the prototypic strain used here as previously documented (Bray, M. et al. 1998 J Virol 72:1647-51). For transcription and generation of viras, chimeric cDNA clones were linearized and used as template in a transcription reaction using SP6 RNA polymerase as described

(Durbin, A.P et al. 2001 Am J Trop Med Hyg 65:405-13). Transcripts were introduced into

Vero cells using liposome-mediated transfection and recombinant dengue virus was harvested on day 7. The genomes of the resulting viruses were confirmed by sequence analysis of viral RNA isolated from recovered viras as previously described (Durbin, A.P et al. 2001 Am J Trop Med Hyg 65:405-13). Incidental mutations arising from virus passage in tissue culture were identified in all virases and are listed in Table 29. Notably, each viras contained a missense mutation in NS4B corresponding to a previously identified mutation from rDEN4 and associated with adaptation to replication in Vero cells (See Table

30 for correlation of nucleotide positions between rDEN4 and chimeric virases). All virases replicated in Vero cells to titers in excess of 6.0 log₁₀ PFU/ml, indicating that the chimeric viruses, even those containing the Δ30 mutation, replicate efficiently in cell culture, a property essential for manufacture of the vaccine.

Table 29. Missense mutations observed among the Vero cell-grown chimeric DEN2/4 virases

^aSame nucleotide position as 7163 in rDEN4. "Same nucleotide position as 7546 in rDEN4. Table 30. Nucleotide (nt) length differences for DEN chimeric viruses compared to rDEN4.

Results of a safety, immunogenicity, and efficacy study in monkeys are presented in Table 31. Monkeys inoculated with wild-type DEN2 were viremic for approximately 5 days with a mean peak titer of 2.1 log₁₀ PFU/ml, while monkeys inoculated with any of the chimeric DEN2 virases were viremic for 1.2 days or less and had a mean peak titer of less than 1.0 log₁₀ PFU/ml. This reduction in the magnitude and duration of viremia clearly indicates that the chimeric virases containing either the CME or ME proteins of DEN2 were more attenuated than the parental DEN2 NGC viras. Neither the animals receiving the wild-type DEN2 nor the DEN2/4 chimeric viruses were ill. The decreased replication of the attenuated viruses in monkeys is accompanied by a reduction in the immune response of inoculated monkeys. This is indicated in Table 31 by approximately a 5-fold reduction in the level of neutralizing antibody following inoculation with the chimeric virases in comparison to titers achieved in animals inoculated with wild-type virus. Addition of the Δ30 mutation to the CME chimeric virus further attenuated the viras, such that rDEN2/4Δ30(CME) did not replicate in monkeys to a detectable level and did not induce a detectable immune response. This viras appeared over-attenuated, and if similar results were seen in humans, this viras would not be suitable for use as a vaccine. However, addition of the Δ30 mutation to the ME chimeric viras did not further attenuate this chimeric virus and the resulting rDEN2/4Δ30(ME) viras appears satisfactorily attenuated and immunogenic for use as a vaccine. Table 31. Chimerization between dengue viras types 2 and 4 results in recombinant virases which are attenuated for rhesus monkeys.

* Rhesus monkeys were inoculated subcutaneously with 5.0 log₁₀ PFU of viras. Serum samples were collected daily for 10 days. Serum for neutralization assay was collected on day 28. Serum samples obtained before virus inoculation had a neutralizing antibody titer of <5.

As described in the previous examples, SCID mice transplanted with the HuH-7 cells are a sensitive model for the evaluation of dengue viras attenuation. Each chimeric DEN2/4 viras was inoculated into groups of SCID-HuH-7 mice and levels of virus in the serum were determined (Table 32). Chimeric virases replicated to levels between 20- and 150-fold lower than either of the parental virases (rDEN4 and DEN2-NGC). CME chimeric virases were slightly more attenuated than the comparable ME chimeric virases, with the Δ30 mutation providing a 0.5 log₁₀ reduction in replication. This level of attenuation exerted by the Δ30 mutation was similar to that observed previously for rDEN4Δ30.

Table 32. Chimerization between dengue viras types 2 and 4 results in recombinant virases which are attenuated for HuH-7-SCID mice.

"Groups of HuH-7-SCID mice were inoculated into the tumor with 4.0 log₁₀ PFU of the indicated viras. Serum was collected on day 7 and virus titer was determined in Vero cells.

"Mean peak titers were assigned to statistical groups using the Tukey post-hoc test (P < 0.05). Groups with the same letter designation are not significantly different.

To evaluate the replication levels of each DEN2/4 chimeric viras in mosquitoes, two different genera of mosquitoes were experimentally infected. Aedes aegypti were infected by ingesting a virus-containing blood meal. By evaluating the presence of viras antigen in both the midgut and head tissue, infectivity could be determined for the local tissues (midgut), and the ability of virus to disseminate and replicate in tissues beyond the midgut barrier (head) could also be measured. The presence of viras in the head is limited by the ability of the ingested viras to replicate in the midgut and then disseminate to the salivary glands in the head, as well as the innate ability of the virus to replicate in the salivary glands. Intrathoracic inoculation of viras into Toxorhynchites splendens bypasses the mosquito midgut barrier. Parental virases rDEN4 and DEN2-NGC readily infect Ae. aegypti and T. splendens (Table 33), with DEN2-NGC appearing to be much more infectious in T. splendens. Each of the rDEN2/4 chimeric viruses was also tested in both mosquito types. In many cases it was not possible to inoculate Ae. aegypti with an undiluted viras stock of sufficient titer to achieve a detectable infection due to the very low infectivity of several of the virases. Nevertheless, it is clear that the rDEN2/4 chimeric virases are less infectious for the midgut and head. Parental virases rDEN4 and DEN2- NGC, administered at a maximum dose of approximately 4.0 log₁₀PFU, were detectable in 74% and 94% of midgut preparations, and 32% and 71% of head preparations, respectively. Among the chimeric virases, the highest level of infectivity, as observed for rDEN2/4Δ30(CME), resulted in only 26% infected midgut samples and 6% head samples. In the more permissive T. splendens, the rDEN2/4 chimeric viruses were generally less infectious than either parental virus, with CME chimeric viruses being less infectious than ME viruses. It has previously been reported for DEN4 that the Δ30 mutation does not have a discernable effect on virus infectivity in T. splendens similar to that observed here for the rDEN2/4 chimeric virases (Troyer, J.M. et al. 2001 Am JTrop Med Hyg 65:414-419).

Table 33. Dengue 2/4 chimeric virases are less infectious compared to either parental virus strain in mosquitoes

"Amount of viras present in 0.22 μl inoculum.

" Percentage of mosquitoes with IFA detectable antigen in head tissue prepared 14 days after inoculation.

⁰ Viras titer ingested, assuming a 2 μl bloodmeal.

Percentage of mosquitoes with IFA detectable antigen in midgut or head tissue prepared 21 days after oral infection. When viras infection was detected, but did not exceed a frequency of 50% at the highest dose of viras ingested, the MID₅₀ was estimated by assuming that a 10-fold more concentrated viras dose would infect 100% of the mosquitoes. ^enc = not calculated, since viras antigen was not detected.

Chimerization of the DEN2 structural genes with rDEN4Δ30 virus resulted in a viras, rDEN2/4Δ30(CME), that had decreased replication in Vero cells compared to either parent virus. To evaluate Vero cell adaptation mutations (Blaney, J. E. et al. 2002 Virology 300:125-139) as a means of increasing the viras yield of a DEN vaccine candidate in Vero cells, selected mutations were introduced into this chimeric virus. Accordingly, rDEN2/4Δ30(CME) virases bearing adaptation mutations were recovered, terminally diluted, and propagated in C6/36 cells to determine if the virus yield in Vero cells could be increased. rDEN2/4Δ30(CME) virases bearing Vero cell adaptation mutations were generated as follows. DNA fragments were excised from rDEN4 cDNA constructs encompassing single or double DEN4 Vero cell adaptation mutations and introduced into the cDNA clone of rDEN2/4Δ30(CME). The presence of the Vero cell adaptation mutation was confirmed by sequence analysis, and RNA transcripts derived from the mutant cDNA clones were transfected, terminally diluted, and propagated in C6/36 cells.

For evaluation of growth kinetics, Vero cells were infected with the indicated virases at a multiplicity of infection (MOI) of 0.01. Confluent cell monolayers in duplicate 25-cm² tissue culture flasks were washed and overlaid with a 1 ml inoculum containing the indicated virus. After a two hour incubation at 37°C, cells were washed three times in MEM and 5 ml of MEM supplemented with 2% FBS was added. A 1 ml aliquot of tissue culture medium was removed, replaced with fresh medium, and designated the day 0 time- point. At the indicated time points post-infection, 1 ml samples of tissue culture medium were removed, clarified by centrifugation, and frozen at -80°C. The level of virus replication was assayed by plaque titration in C6/36 cells and visualized by immunoperoxidase staining. The limit of detection was < 0.7 logι₀PFU/ml.

The growth properties of rDEN2/4Δ30(CME) viruses bearing single Vero cell adaptation mutations at NS4B -7153, -7162, -7163, -7182, NS5 -7630 or three combinations of mutations were compared in Vero cells with rDEN2/4Δ30(CME) viras (Figure 10). Without an introduced Vero cell adaptation mutation, rDEN2/4Δ30(CME) viras yield peaked at 4.4 log₁₀PFU/ml. Each individual adaptation mutation and the combined mutations conferred a substantial increase in replication. Specifically, rDEN2/4Δ30(CME)-7182 grew to the highest titer of 7.1 log₁₀PFU/ml, which was a 500- fold increase in yield. rDEN2/4Δ30(CME)-7162 had the lowest yield but still was increased 125-fold over the level of replication by rDEN2/4Δ30(CME) viras. Introduction of two adaptation mutations into rDEN2/4Δ30(CME) viras did not significantly increase viras yield over that of viruses bearing single Vero cell adaptation mutations. The observed mcrease of up to 500-fold in viras yield by the introduction of a Vero cell adaptation mutation into this chimeric vaccine candidate demonstrates the value of identifying and characterizing specific replication-promoting sequences in DEN virases. These results have particular significance for the development of a live attenuated dengue virus vaccine. First, it is clear that chimerization leads to attenuation of the resulting viras, as indicated by studies in rhesus monkeys, HuH7-SCID mice and mosquitoes. Although this conclusion was not made in the previous study with DEN2/DEN4 or DEN1/DEN4 chimeric virases (Bray, M. et al. 1996 J Virol 70:4162-6), careful examination of the data would suggest that the chimeric viruses are more attenuated in monkeys compared to the wild-type parent virases. Second, the Δ30 mutation can further augment this attenuation in a chimeric-dependent manner. Specifically, in this example, chimeric viruses bearing the CME region of DEN2 were over-attenuated by the addition of Δ30, whereas the attenuation phenotype of chimeric virases bearing just the ME region of DEN2 was unaltered by the addition of the Δ30 mutation. This unexpected finding indicates that in a tetravalent vaccine comprised of individual component virases bearing a shared attenuating mutation, such as the Δ30 mutation, only ME chimeric virases can be utilized since CME chimeric virases bearing the Δ30 mutation can be over- attenuated in rhesus monkeys and might provide only limited immunogenicity in humans.

EXAMPLE 6 Generation and Characterization of Intertypic Chimeric DEN3 Viruses Containing the Δ30 Mutation

Chimeric virases based on the DEN4 cDNA have been generated in which the CME or ME genes have been replaced with the corresponding genes derived from DEN3

(Sleman/78), a viras isolate from the 1978 dengue outbreak in the Sleman region of

Indonesia (Gubler, D.j. et al. 1981 Am J Trop Med Hyg 30:1094-1099) (Appendix 2). As described in Example 5 for the DEN2 chimeric virases, CME chimeric virases for DEN3 were generated by replacing the Bglϊl / Xhol region of the cDNA for either rDEN4 or rDEN4Δ30 with a similar region derived from DEN3 (Sleman/78) (Figure 11 A). Likewise, to create the ME chimeric virases, the Pstl / Xhol region of the cDNA for either rDEN4 or rDEN4Δ30 was replaced with a similar region derived from DEN3 (Sleman/78). The nucleotide and amino acid sequences of the resulting junctions are shown in Figure 1 IB.

The genomes of the resulting virases were confirmed by sequence analysis of viral RNA isolated from recovered virus as previously described (Durbin, A.P et al. 2001 Am J Trop

Med Hyg 65:405-13). Incidental mutations arising from virus passage in tissue culture were identified in all viruses and are listed in Table 34. Notably, each virus contained a missense mutation in NS4B corresponding to a previously identified mutation from rDEN4 and associated with adaptation to growth in Vero cells (See Table 30 for correlation of nucleotide positions between rDEN4 and chimeric viruses). All viruses replicated in Vero cells to titers in excess of 5.7 log₁₀ PFU/ml, indicating that the chimeric viruses, even those containing the Δ30 mutation, replicate efficiently in cell culture, a property essential for manufacture of the vaccine. Table 34. Missense mutations observed among Vero cell-grown chimeric DEN3/4 viruses

"Same nucleotide position as 7162 in rDEN4. "Same nucleotide position as 7183 in rDEN4.

As described in the previous examples, SCID mice transplanted with HuH-7 cells are a sensitive model for the evaluation of dengue viras attenuation. Each chimeric DEN3/4 virus was inoculated into groups of SCID-HuH-7 mice and levels of viras in the serum were determined (Table 35). While chimeric viras rDEN3/4 (CME) was not attenuated, the remaining chimeric virases replicated to levels between 40- and 400-fold lower than either of the parental virases (rDEN4 and DEN3-Sleman/78). In the CME chimeric viras, the Δ30 mutation providing a remarkable 2.7 log₁₀ reduction in replication. This level of attenuation conferred by the Δ30 mutation in the CME chimeric virus was much greater than that observed previously for rDEN4Δ30. The rDEN3/4 (ME) viras was 100-fold reduced in replication compared to either parent virus indicating that the ME chimerization was attenuating per se. Addition of the Δ30 mutation to rDEN3/4 (ME) did not result in additional attenuation. Table 35. Chimerization between dengue viras types 3 and 4 results in recombinant virases which are attenuated for HuH-7-SCID mice.

"Groups of HuH-7-SCID mice were inoculated into the tumor with 4.0 log₁₀ PFU of the indicated virus. Serum was collected on day 7 and virus titer was determined in Vero cells.

"Mean peak titers were assigned to statistical groups using the Tukey post-hoc test (P < 0.05). Groups with the same letter designation are not significantly different.

Evaluation of the replication and immunogenicity of the DEN3 chimeric recombinant viruses and wild-type DEN3 viras in monkeys was performed as described in

Example 5. Results of this safety and immunogenicity study in monkeys are presented in

Table 36. Monkeys inoculated with rDEN3/4(CME) and wild-type DEN (Sleman/78) were viremic for approximately 2 days with a mean peak titer of between 1.6 and 1.8 log₁₀

PFU/ml, respectively, indicating that chimerization of the CME structural genes of DEN3 did not lead to attenuation of viras replication, a different pattern than that observed for

DEN2 chimerization (Table 31). However, chimerization of the ME stractural genes resulted in attenuated virases with undetectable viremia in monkeys, although all monkeys seroconverted with a greater than 10-fold increase in serum antibody levels. As expected for an attenuated virus, the immune response, as measured by neutralizing antibody titer, was lower following inoculation with any of the chimeric virases compared to inoculation with wt DEN3 (Sleman/78), yet sufficiently high to protect the animals against wt DEN3 viras challenge (Table 37). It is clear that addition of the Δ30 mutation to rDEN3/4(CME) was capable of further attenuating the resulting viras rDEN3/4Δ30(CME). Table 36. The Δ30 mutation further attenuates rDEN3/4(CME) for rhesus monkeys

" Groups of rhesus monkeys were inoculated subcutaneously with 10⁵ PFU of the indicated virus in a 1 ml dose. Serum was collected on days 0 to 6, 8, 10, 1-2, and 28. Viras titer was determined by plaque assay in Vero cells.

" Viremia was not detected in any monkey after day 4.

Table 37. rDEN3/4 chimeric virases protect rhesus monkeys from wt DEN3 viras challenge

" 28 days after primary inoculation with the indicated virases, rhesus monkeys were challenged subcutaneously with 10⁵ PFU DEN3 (Sleman/78) viras in a 1 ml dose. Serum was collected on days 28 to 34, 36, 38, and 56. Viras titer was determined by plaque assay in Vero cells.

To evaluate the replication levels of each DEN3/4 chimeric viras in mosquitoes,

Aedes aegypti were infected by ingesting a virus-containing blood meal (Table 38).

Parental virases rDEN4 and DEN3 (Sleman/78) readily infect Ae. aegypti. Each of the rDEN3/4 chimeric virases was also tested. In many cases it was not possible to infect Ae. aegypti with an undiluted virus stock of sufficient titer to achieve a detectable infection due to the very low infectivity of several of the viruses. At a dose of approximately 2.8 - 2.9 log₁₀PFU, rDEN4, DEN3 (Sleman/78), and rDEN3/4(CME) were equally infectious and disseminated to the head with equal efficiency. For the remaining chimeric virases, infection was not detectable even at a dose of 3.4 log₁₀PFU, indicating that replication of rDEN3/4(ME) and rDEN3/4Δ30(CME) is restricted in Ae. aegypti. By comparing infectivity of rDEN3/4(CME) and rDEN3/4Δ30(CME), it is clear that the Δ30 mutation is capable of further attenuating the chimeric viras for mosquitoes. Table 38. Ability of DEN3/4 chimeric virases to infect Aedes aegypti fed an infectious bloodmeal.

" Amount of viras ingested, assuming a 2μ bloodmeal. ^b Number (percentage) of mosquitoes with detectable dengue viras in midgut tissue; mosquitoes were assayed 21 days post feed, and dengue viras antigen was identified by IFA.

⁰ When infection was detected, but did not exceed a frequency of 50% at the highest dose of viras ingested, the MΓD₅₀ was estimated by assuming that a 10-fold more concentrated virus dose would infect 100%) of the mosquitoes. ^d When no infection was detected, the M1D₅₀ was assumed to be greater than a 10- fold higher dose of viras than the one used. ^e Number (percentage) of mosquitoes with detectable dengue viras antigen in both midgut and head tissue. EXAMPLE 7 Generation and Characterization of Intertypic Chimeric DENl Viruses Containing the Δ30 Mutation

Chimeric virases based on the DEN4 cDNA have been generated in which the CME or ME genes have been replaced with the corresponding genes derived from DENl (Puerto Rico/94), a viras isolate from a 1994 dengue outbreak in Puerto Rico (Appendices 3 and 4). As described in Example 4 for the DEN2 chimeric virases, CME chimeric viruses for DENl were generated by replacing the Bglll / Xhol region of the cDNA for either rDEN4 or rDEN4Δ30 with a similar region derived from DENl (Puerto Rico/94) (Figure 12A). Likewise, to create the ME chimeric virases, the Pstl I Xhol region of the cDNA for either rDEN4 or rDEN4Δ30 was replaced with a similar region derived from DENl (Puerto Rico/94). The nucleotide and amino acid sequences of the resulting junctions are shown in Figure 12B.

For transcription and generation of virus, chimeric cDNA clones were linearized and used as template in a transcription reaction using SP6 RNA polymerase as described. Transcripts were introduced into C6/36 mosquito cells using liposome-mediated transfection and recombinant dengue virus was harvested between day 7 and 14. Viruses were subsequently grown in Vero cells and biologically cloned by terminal dilution in Vero cells. All virases replicated in Vero cells to titers in excess of 6.0 log₁₀ PFU/ml, indicating that the chimeric virases, even those containing the Δ30 mutation, replicate efficiently in cell culture. Genomic sequence analysis is currently underway to identify incidental mutations arising from viras passage in tissue culture.

To evaluate the replication levels of DEN1/4(CME) and rDENl/4Δ30(CME) chimeric virus in mosquitoes, Aedes aegypti were infected by ingesting a virus-containing blood meal (Table 39). Parental virus rDEN4 infects Ae. aegypti with an MID50 of 4.0 log₁₀PFU. However, parental viras DENl (Puerto Rico/94), is unable to infect Ae. aegypti at a dose of up to 3.4 log₁₀PFU. Thus CME chimeric viruses DEN1/4 and rDENl/4Δ30 share this inability to infect Ae. aegypti. Therefore, it is unnecessary in Ae. aegypti to evaluate the effect of the Δ30 mutation on the infectivity of the DEN1/4 chimeric virases, in a manner similar to that used for the DEN2/4 and DEN3/4 chimeric virases. Table 39. Inability of DEN1/4 chimeric viruses to infect Aedes aegypti fed an infectious bloodmeal.

"Amount of viras ingested, assuming a 2μl bloodmeal.

"Number (percentage) of mosquitoes with detectable dengue viras in midgut tissue; mosquitoes were assayed 21 days post feed, and dengue virus antigen was identified by IFA. ^cWhen infection was detected, but did not exceed a frequency of 50% at the highest dose of virus ingested, the MID₅₀ was estimated by assuming that a 10-fold more concentrated viras dose would infect 100% of the mosquitoes. ^d When no infection was detected, the MID₅₀ was assumed to be greater than a 10- fold higher dose of viras than the one used.

TSTumber (percentage) of mosquitoes with detectable dengue viras antigen in both midgut and head tissue.

As described in the previous examples, SCID mice transplanted with the HuH-7 cells are a sensitive model for the evaluation of dengue virus attenuation. Each chimeric DEN1/4 virus was inoculated into groups of SCID-HuH-7 mice and levels of viras in the serum were determined (Table 40). Chimeric virases replicated to levels between 15- and 250-fold lower than either of the parental virases, rDEN4 and DENl (Puerto Rico/94). CME chimeric viruses were more attenuated than the comparable ME chimeric virases, with the Δ30 mutation providing a 0.8 log₁₀ reduction in replication. This level of attenuation exerted by the Δ30 mutation in the CME chimeric virases was similar to that observed previously for rDEN4Δ30. However, the attenuating effect of the Δ30 mutation in the ME chimeric virases is indiscernible. Table 40. Chimerization between dengue viras types 1 and 4 results in recombinant viruses which are attenuated for HuH-7-SCID mice.

"Groups of HuH-7-SCID mice were inoculated into the tumor with 4.0 log₁₀ PFU of the indicated virus. Serum was collected on day 7 and viras titer was determined in Vero cells.

"Mean peak titers were assigned to statistical groups using the Tukey post-hoc test (R < 0.05). Groups with the same letter designation are not significantly different.

APPENDIX 1

Nucleotide and amino acid sequence of DEN2 (Tonga/74) cDNA plasmid p2

Bases 1 to 10713: DEN2 viras genome cDNA Bases 97 to 10269: DEN2 polyprotein ORF

Bases 97 to 438 : C protein ORF

Bases 439 to 936: prM protein ORF

Bases 937 to 2421 : E protein ORF Bases 2422 to 3477: NS1 protein ORF

Bases 3478 to 4131 : NS2A protein ORF

Bases 4132 to 4521: NS2B protein ORF

Bases 4522 to 6375: NS3 protein ORF

Bases 6376 to 6756: NS4A protein ORF Bases 6757 to 6825: 2K protein ORF

Bases 6826 to 7569: NS4B protein ORF

Bases 7570 to 10269: NS5 protein ORF

10 20 30 40 50 60 70 80 90 100

AGTTGTTAGTCTACGTGGACCGACAAAGACAGATTCTTTGAGGGAGCTAAGCTCAACGTAGTTCTAACTGTTTTTTGATTAGAGAGCAGATCTCTGATGA

Met>

110 120 130 140 150 160 170 180 190 200 ATAACCAACGGAAAAAGGCGAGAAACACGCCTTTCAATATGCTGAAACGCGAGAGAAACCGCGTGTCAACTGTACAACAGTTGACAAAGAGATTCTCACT

AsnAsnGlnArgLys ysAlaArgAsnThrProP eAsnMetLeuLysArgGluArgAsnArgValSerThrValGlnGlnLeuThrLysArgPheSerLeu> 210 220 230 240 250 260 270 280 290 300

TGGAATGCTGCAGGGACGAGGACCACTAAAATTGTTCATGGCCCTGGTGGCATTCCTTCGTTTCCTAACAATCCCACCAACAGCAGGGATATTAAAAAGA GlyMet euGlnGlyArgGlyProLeuLysLeuPheMetAla euValAlaPheLeuArgP eLeuThrIleProProThrAlaGlyIleLeuLysArg>

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CAAAAAACAGCATATTGACGCTGGGAAAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAGGACGCCAGAAAATGGAATGGTGCTGTTGA

10710 10720 10730 10740 10750 10760 10770 10780 10790 10800 ATCAACAGGTTCTGGTACCGGTAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATG

10810 10820 10830 10840 10850 10860 10870 10880 10890 10900 ATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA 10910 10920 10930 10940 10950 10960 10970 10980 10990 11000

CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA

11010 11020 11030 11040 11050 11060 11070 11080 11090 11100 GTTGCTCTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTC

11110 11120 11130 11140 11150 11160 11170 11180 11190 11200 AAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCA

11210 11220 11230 11240 11250 11260 11270 11280 11290 11300 AAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATC

11310 11320 11330 11340 11350 11360 11370 11380 11390 11400 AGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGT 11410 11420 11430 11440 11450 11460 11470 11480 11490 11500

CTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCATGTTTGACAGCTTATCATCGA

11510 11520 11530 11540 11550 11560 11570 11580 11590 11600 TAAGCTTTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTC

11610 11620 11630 11640 11650 11660 11670 11680 11690 11700

ACCCTGGATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGC

11710 11720 11730 11740 11750 11760 11770 11780 11790 11800 TGCTGGCGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACT

11810 11820 11830 11840 11850 11860 11870 11880 11890 11900

TGGAGCCACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCG

11910 11920 11930 11940 11950 11960 11970 11980 11990 12000 GTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCC

12010 12020 12030 12040 12050 12060 12070 12080 12090 12100 CCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAAT

12110 12120 12130 12140 12150 12160 12170 12180 12190 12200 GCAGGAGTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCA

12210 12220 12230 12240 12250 12260 12270 12280 12290 12300 CTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGA

12310 12320 12330 12340 12350 12360 12370 12380 12390 12400 TCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTAT 12410 12420 12430 12440 12450 12460 12470 12480 12490 12500

CGCCGGCATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGC

12510 12520 12530 12540 12550 12560 12570 12580 12590 12600 ATCGGGATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTT

12610 12620 12630 12640 12650 12660 12670 12680 12690 12700 CGATCACTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTG

12710 12720 12730 12740 12750 12760 12770 12780 12790 12800 CCTCCCCGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAG

12810 12820 12830 12840 12850 12860 12870 12880 12890 12900

CCAATCAATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTC

12910 12920 12930 12940 12950 12960 12970 12980 12990 13000

GGGCAGCGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAA

13010 13020 13030 13040 13050 13060 13070 13080 13090 13100 TCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTC

13110 13120 13130 13140 13150 13160 13170 13180 13190 13200 TGGAAACGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAA 13210 13220 13230 13240 13250 13260 13270 13280 13290 13300

GCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCA

13310 13320 13330 13340 13350 13360 13370 13380 13390 13400 TCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGG

13410 13420 13430 13440 13450 13460 13470 13480 13490 13500 AAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTG

13510 13520 13530 13540 13550 13560 13570 13580 13590 13600 TGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACG

13610 13620 13630 13640 13650 13660 13670 13680 13690 13700 GTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCAC 13710 13720 13730 13740 13750 13760 13770 13780 13790 13800

GTAGCGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTA

13810 13820 13830 13840 13850 13860 13870 13880 13890 13900

AGGAGAAAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGG

13910 13920 13930 13940 13950 13960 13970 13980 13990 14000

CGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT

14010 14020 14030 14040 14050 14060 14070 14080 14090 14100 GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCG

14110 14120 14130 14140 14150 14160 14170 14180 14190 14200

TTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC 14210 14220 14230 14240 14250 14260 14270 14280 14290 14300

ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATC

14310 14320 14330 14340 14350 14360 14370 14380 14390 14400 CGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGT

14410 14420 14430 14440 14450 14460 14470 14480 14490 14500 GCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAG

14510 14520 14530 14540 14550 14560 14570 14580 14590 14600 TTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA

14610 14620 14630 14640 14650 14660 14670 14680 14690 14700 TCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC 14710 14720 14730 14740 14750 14760 14770 14780 14790 14800

CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAG

14810 14820 14830 14840 14850 14860 14870 14880 14890 14900 CGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGAT

14910 14920 14930 14940 14950 14960 14970 14980 14990 15000 ACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCC

15010 15020 15030 15040 15050 15060 15070 15080 15090 15100 ATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAAGATCTGGCTAGCGAT

15110 15120 15130 15140 15150

GACCCTGCTGATTGGTTCGCTGACCATTTCCGGGCGCGCCGATTTAGGTGACACTATAG

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vo © vo © CN CN CO 10610 10620 10630 10640 10650 10660 10670 10680 10690 10700 ACAGCATATTGACGCTGGGAGAGACCAGAGATCCTGCTGTCTCCTCAGCATCATTCCAGGCACAGAACGCCAGAAAATGGAATGGTGCTGTTGAATCAAC

10710 10720 10730 10740 10750 10760 10770 10780 10790 10800 AGGTTCTGGTACCGGTAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCC

10810 10820 10830 10840 10850 10860 10870 10880 10890 10900 CATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCAT 10910 10920 10930 10940 10950 10960 10970 10980 10990 11000

AATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT

11010 11020 11030 11040 11050 11060 11070 11080 11090 11100 CTTGCCCGGCGTCAACACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGAT

11110 11120 11130 11140 11150 11160 11170 11180 11190 11200 CTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACA

11210 11220 11230 11240 11250 11260 11270 11280 11290 11300 GGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT

11310 11320 11330 11340 11350 11360 11370 11380 11390 11400 ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGA 11410 11420 11430 11440 11450 11460 11470 11480 11490 11500

AACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTTCAAGAATTCTCATGTTTGACAGCTTATCATCGATAAGCT

11510 11520 11530 11540 11550 11560 11570 11580 11590 11600 TTAATGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGGCACCGTGTATGAAATCTAACAATGCGCTCATCGTCATCCTCGGCACCGTCACCCTG

11610 11620 11630 11640 11650 11660 11670 11680 11690 11700 GATGCTGTAGGCATAGGCTTGGTTATGCCGGTACTGCCGGGCCTCTTGCGGGATATCGTCCATTCCGACAGCATCGCCAGTCACTATGGCGTGCTGCTGG

11710 11720 11730 11740 11750 11760 11770 11780 11790 11800 CGCTATATGCGTTGATGCAATTTCTATGCGCACCCGTTCTCGGAGCACTGTCCGACCGCTTTGGCCGCCGCCCAGTCCTGCTCGCTTCGCTACTTGGAGC

11810 11820 11830 11840 11850 11860 11870 11880 11890 11900 CACTATCGACTACGCGATCATGGCGACCACACCCGTCCTGTGGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCT

11910 11920 11930 11940 11950 11960 11970 11980 11990 12000 GGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGG

12010 12020 12030 12040 12050 12060 12070 12080 12090 12100 CCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGA

12110 12120 12130 12140 12150 12160 12170 12180 12190 12200 GTCGCATAAGGGAGAGCGTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATG

12210 12220 12230 12240 12250 12260 12270 12280 12290 12300 ACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCC

12310 12320 12330 12340 12350 12360 12370 12380 12390 12400 TGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGG 12410 12420 12430 12440 12450 12460 12470 12480 12490 12500

CATGGCGGCCGACGCGCTGGGCTACGTCTTGCTGGCGTTCGCGACGCGAGGCTGGATGGCCTTCCCCATTATGATTCTTCTCGCTTCCGGCGGCATCGGG

12510 12520 12530 12540 12550 12560 12570 12580 12590 12600 ATGCCCGCGTTGCAGGCCATGCTGTCCAGGCAGGTAGATGACGACCATCAGGGACAGCTTCAAGGATCGCTCGCGGCTCTTACCAGCCTAACTTCGATCA

12610 12620 12630 12640 12650 12660 12670 12680 12690 12700 CTGGACCGCTGATCGTCACGGCGATTTATGCCGCCTCGGCGAGCACATGGAACGGGTTGGCATGGATTGTAGGCGCCGCCCTATACCTTGTCTGCCTCCC

12710 12720 12730 12740 12750 12760 12770 12780 12790 12800 CGCGTTGCGTCGCGGTGCATGGAGCCGGGCCACCTCGACCTGAATGGAAGCCGGCGGCACCTCGCTAACGGATTCACCACTCCAAGAATTGGAGCCAATC

12810 12820 12830 12840 12850 12860 12870 12880 12890 12900 AATTCTTGCGGAGAACTGTGAATGCGCAAACCAACCCTTGGCAGAACATATCCATCGCGTCCGCCATCTCCAGCAGCCGCACGCGGCGCATCTCGGGCAG

12910 12920 12930 12940 12950 12960 12970 12980 12990 13000

CGTTGGGTCCTGGCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCG

13010 13020 13030 13040 13050 13060 13070 13080 13090 13100 ATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAAACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAA

13110 13120 13130 13140 13150 13160 13170 13180 13190 13200

CGCGGAAGTCAGCGCCCTGCACCATTATGTTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTG 13210 13220 13230 13240 13250 13260 13270 13280 13290 13300

GCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTA

13310 13320 13330 13340 13350 13360 13370 13380 13390 13400 ACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCCTTACACGGAGGCATCAGTGACCAAACAGGAAAAAA

13410 13420 13430 13440 13450 13460 13470 13480 13490 13500 CCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGCGGATGAACAGGCAGACATCTGTGAATC

13510 13520 13530 13540 13550 13560 13570 13580 13590 13600 GCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACA

13610 13620 13630 13640 13650 13660 13670 13680 13690 13700 GCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCG 13710 13720 13730 13740 13750 13760 13770 13780 13790 13800

ATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGA

13810 13820 13830 13840 13850 13860 13870 13880 13890 13900 AAATACCGCATCAGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAA

13910 13920 13930 13940 13950 13960 13970 13980 13990 14000 TACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT

14010 14020 14030 14040 14050 14060 14070 14080 14090 14100 TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCC

14110 14120 14130 14140 14150 14160 14170 14180 14190 14200

CCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCT

14210 14220 14230 14240 14250 14260 14270 14280 14290 14300 CACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAA

14310 14320 14330 14340 14350 14360 14370 14380 14390 14400 CTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA

14410 14420 14430 14440 14450 14460 14470 14480 14490 14500 GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA

14510 14520 14530 14540 14550 14560 14570 14580 14590 14600 GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT

14610 14620 14630 14640 14650 14660 14670 14680 14690 14700 GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTA 14710 14720 14730 14740 14750 14760 14770 14780 14790 14800

AATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCT

14810 14820 14830 14840 14850 14860 14870 14880 14890 14900 GTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCG

14910 14920 14930 14940 14950 14960 14970 14980 14990 15000 AGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAG

15010 15020 15030 15040 15050 15060 15070 15080 15090 15100 TCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAAGATCTGGCTAGCGATGACCCT

15110 15120 15130 15140 15150

GCTGATTGGTTCGCTGACCATTTCCGGGCGCGCCGATTTAGGTGACACTATAG

APPENDIX 3

Nucleotide and amino acid sequence of DENl (Puerto Rico/94) CME chimeric region '

Bases 1 to 88 (Bg-I-Q- DEN4 Bases 89 (Bglll) to 2348 (Xhol): DENl

Bases 2349 (Xhol) to 2426: DEN4

Bases 102 to 443 : C protein ORF Bases 444 to 941 : prM protein ORF Bases 942 to 2426: E protein ORF

10 20 30 40 50 60 70 80 90 100 AGTTGTTAGTCTGTGTGGACCGACAAGGACAGTTCCAAATCGGAAGCTTGCTTAACACAGTTCTAACAGTTTGTTTGAATAGAGAGCAGATCTCTGGAAA

110 120 130 140 150 160 170 180 190 200

AATGAACAACCAACGGAAAAAGACGGGTCGACCGTCTTTCAATATGCTGAAACGCGCGAGAAACCGCGTGTCAACTGGTTCACAGTTGGCGAAGAGATTC MetAsιτAsnGlnArgLysLysT rGlyArgProSerPheAsnMetLeuLysArgAlaArgAsnArgValSerT rGlySerGlnLeuAlaLysArgP e> 210 220 230 240 250 260 270 280 290 300

TCAAAAGGATTGCTTTCAGGCCAAGGACCCATGAAATTGGTGATGGCTTTCATAGCATTTCTAAGATTTCTAGCCATACCCCCAACAGCAGGAATTTTGG SerLysGlyLeu euSerGlyGlnGlyProMetLysLeuValMetAlaPheIleAlaPheLeuArgPheLeuAlaIleProProThrAlaGlyIleLeu>

310 320 330 340 350 360 370 380 390 400 CTAGATGGAGCTCATTCAAGAAGAATGGAGCGATCAAAGTGTTACGGGGTTTCAAAAAAGAGATCTCAAGCATGTTGAACATTATGAACAGGAGGAAAAA

AlaArgTrpSerSerPheLysLysAsnGlyAlaIle ysValLeuArgGlyPheLys ysGluIleSerSerMet euAsnIleMetAsnArgArgLysLys >

410 420 430 440 450 460 470 480 490 500 ATCTGTGACCATGCTCCTCATGCTGCTGCCCACAGCCCTGGCGTTCCATTTGACCACACGAGGGGGAGAGCCACACATGATAGTTAGTAAGCAGGAAAGA SerValT rMetLeu euMetLeuLeuProThrAla euAlaP eHis euT rT rArgGlyGlyGluProHisMetIleValSerLysGlnGluArg>

■ c-s © '

l VO © lO © CN CN co co

VO VO © VO ©

CN CN co APPENDIX 4

Nucleotide and amino acid sequence of DENl (Puerto Rico/94) ME chimeric region

Bases 1 to 404 (Pstl): DEN4 Bases 405 (Pstl) to 2345 (Xhol): DENl Bases 2346 (Xhol) to 2423: DEN4 Bases 102 to 440: C protein ORF Bases 441 to 938: prM protein ORF Bases 939 to 2423 : E protein ORF

10 20 30 40 50 60 70 80 90 100

AGTTGTTAGTCTGTGTGGACCGACAAGGACAGTTCCAAATCGGAAGCTTGCTTAACACAGTTCTAACAGTTTGTTTGAATAGAGAGCAGATCTCTGGAAA 110 120 130 140 150 160 170 180 190 200

AATGAACCAACGAAAAAAGGTGGTTAGACCACCTTTCAATATGCTGAAACGCGAGAGAAACCGCGTATCAACCCCTCAAGGGTTGGTGAAGAGATTCTCA MetAsnGlιιΑrgLysLysValValArgProProPheAsn etLeuLysArgGluArgAsrιArgValSerTlιrProGlnGlyLeuValLysArgPheSer>

210 220 230 240 250 260 270 280 290 300 ACCGGACTTTTTTCTGGGAAAGGACCCTTACGGATGGTGCTAGCATTCATCACGTTTTTGCGAGTCCTTTCCATCCCACCAACAGCAGGGATTCTGAAGA

ThrGlyLeuPheSerGlyLysGlyProLeuArgMetValLeuAlaP eIleT rPheLeuArgValLeuSerIleProProThrAlaGlyIleLeuLys>

310 320 330 340 350 360 370 380 390 400 GATGGGGACAGTTGAAGAAAAATAAGGCCATCAAGATACTGATTGGATTCAGGAAGGAGATAGGCCGCATGCTGAACATCTTGAACGGGAGAAAAAGGTC ArgTrpGlyGlnLeuLysLysAsnLysAlaIleLysIleLeuIleGlyPheArgLysGluIleGlyArgMetLeuAsnIle euAsnGlyArgLysArgSer>

410 420 430 440 450 460 470 480 490 500 TGCAGCCATGCTCCTCATGCTGCTGCCCACAGCCCTGGCGTTCCATTTGACCACACGAGGGGGAGAGCCACACATGATAGTTAGTAAGCAGGAAAGAGGA

AlaAlaMetLeuLeuMetLeuLeuProThrAlaLeuAlaPheHisLeuThrThrArgGlyGlyGluProHisMetIleValSerLysGlnGluArgGly>

510 520 530 540 550 560 570 580 590 600

AAGTCACTGTTGTTTAAGACCTCTGCAGGCATCAATATGTGCACTCTCATTGCGATGGATTTGGGAGAGTTATGCGAGGACACAATGACCTACAAATGCC LysSerLeuLeuPheLysThrSerAlaGlyIleAsnMetCysT rLeuIleAlaMetAspLeuGlyGluLeuCysGluAspThrMetThrTyrLysCys>

lO «o o lO ©

CN CN co

VO >o © VO O lO CN CN CO CO

>o 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, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. An immunogenic composition being tetravalent and containing a common 30 nucleotide deletion in the 3' untranslated region of dengue types 1, 2, 3, and 4 comprising a nucleic acid comprising a first nucleotide sequence encoding at least one stractural protein from a first dengue viras and a second nucleotide sequence encoding nonstructural proteins from a second dengue virus, wherein the second dengue virus is attenuated by a deletion of about 30 nucleotides from the 3' untranslated region of the dengue genome corresponding to the TL2 stem-loop stracture.

2. The composition of Claim 1, wherein the nucleic acid further comprises a mutation generating a mutant having a phenotype wherein the phenotype is temperature sensitivity in Vero cells or the human liver cell line HuH-7, host-cell restriction in mosquito cells or the human liver cell line HuH-7, host-cell adaptation for improved replication in Vero cells, or attenuation in mice or monkeys.

3. The composition of Claim 1 or 2, wherein the serotype of the first dengue viras is the same as the serotype of the second dengue viras.

4. The composition of Claim 3, wherein the serotype is type 1.

5. The composition of Claim 4, wherein the deletion is a deletion of about 30 nucleotides from the 3' untranslated region of the dengue type 1 genome corresponding to the TL2 stem-loop structure between about nucleotides 10562-10591.

6. The composition of Claim 3, wherein the serotype is type 2.

7. The composition of Claim 6, wherein the deletion is a deletion of about 30 nucleotides from the 3' untranslated region of the dengue type 2 genome corresponding to the TL2 stem-loop stracture between about nucleotides 10541-10570.

8. The composition of Claim 3, wherein the serotype is type 3.

9. The composition of Claim 8, wherein the deletion is by a deletion of about

30 nucleotides from the 3' untranslated region of the dengue type 3 genome corresponding to the TL2 stem-loop stracture between about nucleotides 10535-10565.

10. The composition of Claim 3, wherein the serotype is type 4.

11. The composition of Claim 10, wherein the deletion is a deletion of about 30 nucleotides from the 3' untranslated region of the dengue type 4 genome corresponding to the TL2 stem-loop stracture between about nucleotides 10478-10507.

12. The composition of Claim 1 or 2, wherein the serotype of the first dengue virus is different from the serotype of the second dengue virus and wherein nucleic acid is a nucleic acid chimera.

13. The composition of Claim 12, wherein the serotype of the second dengue virus having the deletion is type 1.

14. The composition of Claim 13, wherein the serotype of the first dengue viras is type 2.

15. The composition of Claim 13, wherein the serotype of the first dengue viras is type 3.

16. The composition of Claim 13, wherein the serotype of the first dengue viras is type 4.

17. The composition of any of Claims 13-16, wherein the first nucleotide sequence encodes at least two stractural proteins of the first dengue viras.

18. The composition of Claim 17, wherein the stractural proteins are prM and E proteins.

19. The composition of any of Claims 13-18, wherein the deletion is a deletion of about 30 nucleotides from the 3' untranslated region of the dengue type 1 genome corresponding to the TL2 stem-loop stracture between about nucleotides 10562 and 10591.

20. The composition of Claim 12, wherein the serotype of the second dengue virus having the deletion is type 2.

21. The composition of Claim 20, wherein the serotype of the first dengue viras is type 1.

22. The composition of Claim 20, wherein the serotype of the first dengue viras is type 3.

23. The composition of Claim 20, wherein the serotype of the first dengue viras is type 4.

24. The composition of any of Claims 20-23, wherein the first nucleotide sequence encodes at least two structural proteins of the first dengue viras.

25. The composition of Claim 24, wherein the structural proteins are prM and E proteins.

26. The composition of any of Claims 20-25, wherein the deletion is a deletion of about 30 nucleotides from the 3' untranslated region of the dengue type 2 genome corresponding to the TL2 stem-loop stracture between about nucleotides 10541 and 10570.

27. The composition of Claim 12, wherein the serotype of the second dengue viras having the deletion is type 3.

28. The composition of Claim 27, wherein the serotype of the first dengue viras is type 1.

29. The composition of Claim 27, wherein the serotype of the first dengue virus is type 2.

30. The composition of Claim 27, wherein the serotype of the first dengue virus is type 4.

31. The composition of any of Claims 27-30, wherein the first nucleotide sequence encodes at least two stractural proteins of the first dengue viras.

32. The composition of Claim 31, wherein the stractural proteins are prM and E proteins.

33. The composition of any of Claims 27-32, wherein the deletion is a deletion of about 30 nucleotides from the 3' untranslated region of the dengue type 3 genome corresponding to the TL2 stem-loop structure between about nucleotides 10535 and 10565.

34. The composition of Claim 12, wherein the serotype of the second dengue viras having the deletion is type 4.

35. The composition of Claim 34, wherein the serotype of the first dengue viras is type 1.

36. The composition of Claim 34, wherein the serotype of the first dengue viras is type 2.

37. The composition of Claim 34, wherein the serotype of the first dengue viras is type 3.

38. The composition of any of Claims 34-37, wherein the first nucleotide sequence encodes at least two stractural proteins of the first dengue viras.

39. The composition of Claim 38, wherein the stractural proteins are prM and E proteins.

40. The composition of any of Claims 34-39, wherein the deletion is a deletion of about 30 nucleotides from the 3' untranslated region of the dengue type 4 genome corresponding to the TL2 stem-loop stracture between about nucleotides 10478 and 10507.

41. The composition of any of Claims 1-40 wherein the one or more nucleic acid or nucleic acid chimera comprises a viras or viras chimera.

42. The composition of any of Claims 1-41 for use in the induction of an immune response.

43. A method of inducing an immune response in a subject comprising administering an effective amount of the composition of any of Claims 1-41 to the subject.

44. The method of Claim 43 wherein the subj ect is a human.

45. A tetravalent vaccine comprising the composition of any of Claims 1-41.

46. The vaccine of Claim 45 for use in the prevention of disease caused by dengue virus.

47. A method of preventing disease caused by dengue virus in a subject comprising administering an effective amount of the vaccine of Claim 45 to the subject.

48. The method of Claim 47 wherein the subject is a human.

49. An isolated nucleic acid probe or primer that selectively hybridizes with and possesses at least five nucleotides complementary to the nucleic acid or the complementary strand of the nucleic acid encoding the cleavage site that separates the capsid protein and the premembrane protein of the nucleic acid chimera comprising the composition of Claim 18, 25, 32, or 39.

50. A composition, optionally comprising a mutation selected from the group consisting of temperature sensitivity in Vero cells or the human liver cell line HuH-7, host- cell restriction in mosquito cells or the human liver cell line HuH-7, host-cell adaptation for improved replication in Vero cells, or attenuation in mice or monkeys, the composition comprising a member selected from the group consisting of:

(1) rDENlΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(2) rDENlΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4/lΔ30,

(3) rDENlΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30, (4) rDENlΔ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(5) rDENlΔ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4Δ30,

(6) rDENlΔ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (7) rDENlΔ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(8) rDENlΔ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(9) rDENlΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(10) rDENlΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/lΔ30, (11) rDENlΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(12) rDENlΔ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(13) rDENlΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(14) rDENlΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(15) rDEMΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30, (16) rDENlΔ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(17) rDENlΔ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4Δ30,

(18) rDENlΔ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/lΔ30,

(19) rDENlΔ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/2Δ30,

(20) rDENlΔ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/3Δ30, (21) rDENlΔ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4Δ30,

(22) rDENlΔ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/lΔ30,

(23) rDENlΔ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/2Δ30,

(24) rDENlΔ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/3Δ30,

(25) rDENlΔ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4Δ30, (26) rDENlΔ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/lΔ30,

(27) rDEMΔ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(28) rDENlΔ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/3Δ30,

(29) rDENlΔ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4Δ30,

(30) rDENlΔ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/lΔ30, (31) rDENlΔ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/2Δ30,

(32) rDENlΔ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(33) rDENlΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(34) rDENlΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/lΔ30,

(35) rDENlΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30, (36) rDENlΔ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(37) rDENlΔ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4Δ30,

(38) rDENlΔ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (39) rDENlΔ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(40) rDENlΔ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(41) rDENlΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(42) rDENlΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(43) rDENlΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(44) rDENlΔ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(45) rDENlΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(46) rDENlΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(47) rDENlΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(48) rDENlΔ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(49) rDENlΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(50) rDENlΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/lΔ30,

(51) rDENlΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(52) rDENlΔ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30,

(53) rDENlΔ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4Δ30,

(54) rDENlΔ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/lΔ30,

(55) rDENlΔ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(56) rDENlΔ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(57) rDENlΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4Δ30,

(58) rDENlΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(59) rDENlΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(60) rDENlΔ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(61) rDENlΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(62) rDENlΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(63) rDENlΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(64) rDENlΔ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(65) rDENl/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(66) rDENl/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/lΔ30,

(67) rDENl/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30,

(68) rDENl/2Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(69) rDENl/2Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4Δ30,

(70) rDENl/2Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (71) rDENl/2Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(72) rDENl/2Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(73) rDENl/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(74) rDENl/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(75) rDENl/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(76) rDENl/2Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(77) rDENl/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(78) rDENl/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(79) rDENl/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30, i (80) rDENl/2Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(81) rDENl/2Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4Δ30,

(82) rDENl/2Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/lΔ30,

(83) rDENl/2Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/2Δ30,

(84) rDENl/2Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/3Δ30,

(85) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4Δ30,

(86) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/lΔ30,

(87) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/2Δ30,

(88) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/3Δ30,

(89) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4Δ30, ι (90) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/lΔ30,

(91) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(92) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/2Δ30₅ rDEN4/3Δ30,

(93) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4Δ30,

(94) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/lΔ30,

(95) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/2Δ30,

(96) rDENl/2Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(97) rDENl/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(98) rDENl/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/lΔ30,

(99) rDENl/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30, ι (100) rDENl/2Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(101) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4Δ30,

(102) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (103) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(104) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(105) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(106) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(107) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(108) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(109) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(110) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(111) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(112) rDENl/2Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(113) rDENl/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(114) rDENl/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/lΔ30,

(115) rDENl/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(116) rDENl/2Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30,

(117) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4Δ30,

(118) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/lΔ30,

(119) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(120) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(121) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30₅ rDEN4Δ30,

(122) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(123) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(124) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(125) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(126) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(127) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(128) rDENl/2Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(129) rDENl/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(130) rDENl/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/lΔ30,

(131) rDENl/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30,

(132) rDENl/3Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(133) rDENl/3Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4Δ30,

(134) rDENl/3Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (135) rDENl/3Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(136) rDENl/3Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(137) rDENl/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(138) rDENl/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(139) rDENl/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(140) rDENl/3Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(141) rDENl/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(142) rDENl/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(143) rDENl/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(144) rDENl/3Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(145) rDENl/3Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4Δ30,

(146) rDENl/3Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/lΔ30,

(147) rDENl/3Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/2Δ30,

(148) rDENl/3Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/3Δ30,

(149) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4Δ30,

(150) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/lΔ30,

(151) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/2Δ30,

(152) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/3Δ30,

(153) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4Δ30,

(154) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/lΔ30,

(155) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(156) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/3Δ30,

(157) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4Δ30,

(158) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/lΔ30,

(159) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/2Δ30,

(160) rDENl/3Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(161) rDENl/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(162) rDENl/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/lΔ30,

(163) rDENl/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30,

(164) rDENl/3Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(165) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4Δ30,

(166) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (167) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(168) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(169) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(170) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(171) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(172) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(173) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(174) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(175) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(176) rDENl/3Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(177) rDENl/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(178) rDENl/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/lΔ30,

(179) rDENl/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(180) rDENl/3Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30,

(181) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4Δ30,

(182) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/lΔ30,

(183) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(184) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(185) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4Δ30,

(186) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(187) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(188) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(189) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(190) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(191) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30,

(192) rDENl/3Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(193) rDENl/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4Δ30,

(194) rDENl/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/lΔ30,

(195) rDENl/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/2Δ30,

(196) rDENl/4Δ30, rDEN2Δ30, rDEN3Δ30, rDEN4/3Δ30,

(197) rDENl/4Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4Δ30,

(198) rDENl/4Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (199) rDENl/4Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(200) rDENl/4Δ30, rDEN2Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(201) rDENl/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4Δ30,

(202) rDENl/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/lΔ30, (203) rDENl/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(204) rDENl/4Δ30, rDEN2Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(205) rDENl/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4Δ30,

(206) rDENl/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(207) rDENl/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/2Δ30, (208) rDENl/4Δ30, rDEN2Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(209) rDENl/4Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4Δ30,

(210) rDENl/4Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/lΔ30,

(211) rDENl/4Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/2Δ30,

(212) rDENl/4Δ30, rDEN2/lΔ30, rDEN3Δ30, rDEN4/3Δ30, (213) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4Δ30,

(214) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/lΔ30,

(215) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/2Δ30,

(216) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/lΔ30, rDEN4/3Δ30,

(217) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4Δ30, (218) rDENl/4Δ30₅ rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/lΔ30,

(219) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/2Δ30,

(220) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/2Δ30, rDEN4/3Δ30,

(221) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4Δ30,

(222) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/lΔ30, (223) rDENl/4Δ30, rDEN2/lΔ30₅ rDEN3/4Δ30, rDEN4/2Δ30,

(224) rDENl/4Δ30, rDEN2/lΔ30, rDEN3/4Δ30, rDEN4/3Δ30,

(225) rDENl/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4Δ30,

(226) rDENl/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/lΔ30,

(227) rDENl/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/2Δ30, (228) rDENl/4Δ30, rDEN2/3Δ30, rDEN3Δ30, rDEN4/3Δ30,

(229) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4Δ30,

(230) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/lΔ30, (231) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(232) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(233) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4Δ30,

(234) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/lΔ30, (235) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(236) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(237) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4Δ30,

(238) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/lΔ30,

(239) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/2Δ30, (240) rDENl/4Δ30, rDEN2/3Δ30, rDEN3/4Δ30, rDEN4/3Δ30,

(241) rDENl/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4Δ30,

(242) rDENl/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/lΔ30,

(243) rDENl/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/2Δ30,

(244) rDENl/4Δ30, rDEN2/4Δ30, rDEN3Δ30, rDEN4/3Δ30, (245) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4Δ30,

(246) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/lΔ30,

(247) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/2Δ30,

(248) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/lΔ30, rDEN4/3Δ30,

(249) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4Δ30, (250) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/lΔ30,

(251) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/2Δ30,

(252) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/2Δ30, rDEN4/3Δ30,

(253) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4Δ30,

(254) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/lΔ30, (255) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/2Δ30, and (256) rDENl/4Δ30, rDEN2/4Δ30, rDEN3/4Δ30, rDEN4/3Δ30.