AU624863B2

AU624863B2 - Recombinant vaccinia virus for prevention of disease caused by flavivirus

Info

Publication number: AU624863B2
Application number: AU42064/89A
Authority: AU
Inventors: Michael P. Bray; Robert M. Chanock; Ching-Juh Lai; Bangti Zhao
Original assignee: US Department of Health and Human Services; Government of the United States of America
Current assignee: United States Department of Commerce
Priority date: 1988-08-20
Filing date: 1989-08-21
Publication date: 1992-06-25
Anticipated expiration: 2009-08-21
Also published as: IL91304A0; EP0431064A4; EP0431064A1; WO1990001946A1; JPH03503364A; AU4206489A

Description

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OPI DATE 23/03/90 APPLN. ID 42064 89 pcr AOJP DATE 6/ /9 C U R PCT/US89/03589 INTERNATIONAL APPLICATION L D IiN T AT'i T COOPERATION TREATY (PCT) (51) International Patent Classification 4 (11) International Publication Number: WO 90/01946 A61K 39/12, C12N 15/00, 07/00 Al (43) International Publication Date: 8 March 1990 (08.03.90) (21) International Application Number: PCT/US89/03589 (74) Agents: OLIFF, James, A. et al.; Oliff Berridge, 277 S.

Washington Street, Alexandria, VA 22314 (US).

(22) International Filing Date: 21 August 1989 (21.08.89) (81) Designated States: AT (European patent), AU, BE (Euro- Priority data: pean patent), CH (European patent), DE (European pa- 239,205 20 August 1988 (20.08.88) US tent), FR (European patent), GB (European patent), IT (European patent), JP, LU (European patent), NL (European patent), SE (European patent).

(71) Applicant: THE UNITED STATES OF AMERICA, represented by THE SECRETARY, UNITED STATES DE- PARTMENT OF COMMERCE [US/US]; Washington, Published DC 20231 With international search report.

(72) Inventors: LAI, Ching-Juh 7353 Heatherhill Ct., Bethesda, MD 20817 BRAY, Michael, P. 5019 Acacia Avenue, Bethesda, MD 20814 ZHAO, Bangti 4932 Battery Lane, Bethesda, MD 20814 CHA- NOCK, Robert, M. 7001 Longwood Drive, Bethesda, MD 20817 (US).

(54) Title: RECOMBINANT VACCINIA VIRUS FOR PREVENTION OF DISEASE CAUSED BY FLAVIVIRUS (57) Abstract A recombinant vaccinia virus contains complete coding sequence for the expression of flavivirus antigenic proteins. A vaccine against flavivirus disease is also provided.

i S WO 90/01946 PCT/US89/03589 -1- RECOMBINANT VACCINIA VIRUS FOR PREVENTION OF DISEASE CAUSED BY FLAVIVIRUS Technical Field The present invention is related to the construction of recombinant vaccinia viruses. More particularly, the present invention is related to the construction of recombinant vaccinia viruses which are useful for the preparation of a vaccine for the prevention of diseases caused by flaviviruses, such as dengue virus, Japanese B encephalitis virus and tick-borne encephalitis virus.

Background of the Invention Certain diseases of public health concern, such as dengue disease and encephalitis cf certain types are known to be caused by viruses belonging to the flavivirus family. It has been estimated that up to 100 million illnesses occur every year in tropical areas of the world due to dengue virus alone. However, there is no effective and specific immunoprophylactic measure to control such flavivirus diseases.

SUMMARY OF THE INVENTION It is, therefore, an object of the present invention to provide a recombinant vaccinia virus which induces an immune response against a specific flavivirus disease in a host infected with said recombinant vaccinia virus.

Other objects and advantages of the present invention will become evident from the following detailed Sdescription of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: Figure 1 shows schematic construction of a dengue virus-vaccinia virus recombinant plasmid. The intermediate cloning vector pscll contains interrupted WO 90/01946 PCT/US89/03589 R L 2 thymidine kinase gene sequences (TKR amd TK and a bacterial P-galactosidase gene (LacZ) under the transcription control of the P11 vaccinia virus promoter. The vector DNA was partially digested with BamHI to open the BamHI site downstream of the P7.5 promoter for insertion of the 4.1 kilobase (Kb) fragment of dengue virus DNA located at the 5' end of the viral genone. This fragment of dengue cDNA contains the coding region for the three dengue structural proteins [capsid pre-membrane (pre-M), and envelope glycoprotein plus the first two downstream non-structural proteins NS1 and NS2a.

Recombinant plasmid containing the dengue virus DNA insert in the sense transcription orientation was selected and used for construction of the recombinant vaccinia virus.

Figures 2A and 2B demonstrate the identification of dengue virus proteins synthesized by Sthe recombinant vaccinia virus. (Figure 2A:) Immunoprecipitation of [3S]-methionine-labeled lysate from recombinant vaccinia virus-infected cells (multiplicity i| of infection, 10 PFU per cell) was carried out with one of the following specific antibodies: monoclonal antibody specific for the envelope glycoprotein rabbit antibodies prepared against dengue virus type 2 nonstructural protein 1(NS 1 monoclonal antibody 5C9 specific for the membrane glycoprotein precursor (preM); and polyvalent antibodies The labeled precipitates were analyzed on sodium dodecyl sulfate-12% Spolyacrylamide gels. Also shown are the labeled dengue N 30 virus protein markers obtained by immunoprecipitation with polyvalent antibodies of 35 S]-methionine-labeled lysate from dengue virus-infected CV-1 cells. (Figure 2B:) Labeled immunoprecipitates prepared as described above were analyzed by digestion with endoglycosidase H (endo H) and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Numbers at the left of the gels indicate protein sizes in kilodaltons.

s-1 DETAILED DESCRIPTION OF THE INVENTION The above and various other objects and advantages of the present invention are achieved by a recombinant vaccinia virus containing complete coding sequence for the expression of the major specific protective flavivirus antigens in cells infected with said recombinant vaccinia virus, said antigens inducing protective immunity against infection by said flavivirus in a susceptible host.

Accordingly, the present invention provides a recombinant vaccinia virus selected from the group consisting V(C-M-E-NS1-NS2a), V(RSV V(FLU HA-E), 0:9, and V(NS1-NS 2 whrein the viral proteins are obtained from a flavivirus.

.I The present invention further provides a recombinant >5 vaccinia virus consisting essentially of the coding sequence for C, M, E, NS1, and NS2 proteins, wherein the viral proteins are obtained from a flavivirus and 85 percent of the carboxyl terminal sequence of NS 2 A protein is deleted.

The present invention also provides a pharmaceutical composition, comprising an immunogenic amount of the above virus in a pharmaceuticallacceptable carrier.

e The present invention also provides a method for inducing protective immunity against a flavivirus disease, comprising innoculating a host acceptable to flavivirus with 2 5 an immunogenic amount of the above virus in at least a single dose.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference.

Unless mentioned otherwise, the techniques employed herein are standard methodologies well known to one or ordinary skill in the art.

Like other members of the flavivirus family, 3

PT/

L-.Y~P O* l ~-Il extracellular dengue virus has a relatively simple structure.

Virions contain only three virus-coded proteins, designated capsid protein, membrane protein, and envelope (E) glycoprotein. Intracellular virus, which is also infectious, lacks M but contains another glycoprotein, preM, from which M is derived by cleavage. Both C and M are internal proteins.

The surface envelope glycoprotein is the major site responsible for neutralization of infectivity by specific antibodies. The envelope glycoprotein also exhibits hemagglutinating activity and is responsible for adsorption to the cell surface. Several nonstructural proteins have also been identified in dengue virus-infected cells. Among them is nonstructural protein NS1, which is a glycoprotein and described as a soluble complement-fixing 1 35 i i i{ 3a vt <NT-i It 3../dtr i

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WO 90/01946 PCT/US89/03589 4 antigen. It is believed that NS1 plays an important role in mediating immunity, since the analogous NS1 glycoprotein of yellow fever virus has been shown to be a protective antigen in mice and in primates. Furthermore, the NS1 of dengue type 2 virus has been shown to be a protective antigen.

The dengue type 4 virus genome consists of a molecule of positive-stranded RNA 10,644 nucleotides in length. A full-length cDNA copy of the dengue type 4 virus has been prepared, and its complete nucleotide sequence determined. From such studies, it has been estimated that 96% of the dengue virus genome codes for a polyprotein which is cleaved by specific protease(s) to generate individual viral proteins. The three structural proteins, C-M-E, are located at the amino terminus, while nonstructural proteins NS1-NS2a-NS2b-NS3-NS4a-NS4b-NS5 are at the carboxy-terminus. It must be emphasized that because dengue virus gene expression involves proteolytic cleavage of the polyprotein, it cannot be assumed a priori that the expression of protective antigens such as E and NS1 from their cloned DNA sequences, would be useful in immunoprophylaxis. Such determination can be made only through actual testing. The construction of a vaccinia flavivirus recombinant is now illustrated by employing dengue virus genes as an example.

Vaccinia virus was used as a vector for construction of a live recombinant virus expressing dengue viral genes. The BglII DNA fragment (4,041 base pairs, nucleotides 88 to 4128) from the 5' terminus of dengue type 4 virus cDNA contains the coding region for the three structural proteins as well as nonstructural proteins NS1 and NS2a. This fragment was excised from the full-length dengue virus DNA copy and was inserted into the PSC11 vaccinia intermediate vector. The dengue virus DNA sequence was inserted in the BamHI site immediately downstream of the vaccinia P7.5 early-late promoter (Figure In this construct, the dengue virus coding L_ WO 90/01946 PCT/US89/03589 5 sequence was placed under the transcriptional control of the vaccinia virus P7.5 early-late promoter. The vector contained a bacterial P-galactosidase gene under the control of the vaccinia virus Pll late promoter, which provided a visual selectable marker. The chimeric genes were flanked by sequences of the vaccinia virus thymidine kinase gene, which directed homologous recombination of dengue virus sequences into the vaccinia virus genome following transfection of simian CV-1 cells previously infected with wild type vaccinia virus (WR strain).

Recombinant vaccinia virus harboring the dengue virus DNA insert was isolated and plaque purified two times on thymidine kinase minus (TK) cells in selective medium.

Other dengue cDNA fragments, vide infra, were inserted into a vaccinia recombinant virus by the method just described. Similarly, constructs of other flavivirusvaccinia recombinants are made by inserting flavivirus cDNA following the procedure described herein for dengue virus.

Dengue virus-specific proteins synthesized during infection with the recombinant virus were initially detected by indirect immunofluorescence. CV-1 cells infected with the recombinant virus exhibited fluorescent-stainable antigens in the cytoplasm when polyvalent dengue type 4 virus hyperimmune mouse antiserum was used, although the intensity of staining was less than that observed in dengue virus-infected cells. Similar immunofluorescence was observed in CV-1 cells infected with the recombinant virus when monoclonal antibody specific to the E glycoprotein was used.

To further identify this and other dengue virus proteins, recombinant virus-infected cells were radiolabeled with 3 ]methionine and the cell lysate was prepared for immunoprecipitation by specific antibodies.

Analysis of the labeled precipitate on sodium dodecyl sulfate-polyacrylamide gel (Figure 2A) showed that the polyvalent hyperimmune mouse antiserum precipitated three r "ya'd_ 'i 1. i i yli-"l WO 90/01946 PCr/US89/03589 6 1 i major bands with estimated molecular size 20, 40 to 46, and 55 to 60 kilodaltons (kDa), respectively. Minor bands of larger molecular size that were probably nonspecific were also observed. When an E glycoprotei.nspecific monoclonal antibody (1H10) was used, a 55 to kDa band consistent with the molecular size of the E glycoprotein was precipitated. Similarly, dengue type 2 virus NS1-specific antiserum (obtained from Dr. J.

Schlesinger) precipitated a 40- to 46-kDa band, which is the predicted size for the NS1 nonstructural glycoprotein. The third major band precipitated by dengue type 4 virus hyperimmune antiserum was approximately 20 kDa which is consistent with the size of the intracellular preM glycoprotein. A similar band was precipitated by preM-specific antibodies (monoclonal antibody 2H2 or 5C9, obtained from Dr. M. K. Gentry of WRAIR).

Thus, in recombinant virus-infected cells, the three glycoproteins encoded in the cloned DNA appeared to be cleaved and modified by glycosylation in a manner similar to that observed during dengue virus infection.

This suggests that the dengue viral structural proteins, as well as the NS1 nonstructural protein, were specifically processed by proteolytic cleavage of the polyprotein in the absence of dengue virus functions provided by the distal nonstructural proteins NS2b through However, as estimated by immunoprecipitation, the amount of dengue preM, E nad NS1 glycoproteins produced in recombinant virus infected cells was significantly less than in dengue type 4 virus-infected cells.

The pattern of glycosylation of the glycoproteins produced in recombinant virus-infected cells varied as indicated by their response to endoglycosidase H, which cleaves the mannose rich carbohydrate core. The preM protein band was completely sensitive to endoglycosidase H treatment, yielding a band of 17 kDa, a reduction of 3 kDA in molecular size. On the other hand, a significant portion of the carbohydrate of both the E

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WO 90/01946 PCT/US89/03589 7 and the NS1 glycoproteins appeared to be resistant to endoglycosidase H digestion.

Each of the dengue virus glycoproteins, i.e., preM, E and NS1, is preceded by a stretch of hydrophobic amino acids which can serve as a signal. This indicates that cellular signalase is probably responsible for proteolytic cleavage. Presumably, the cleavage mechanism which generates the three glycoproteins, also yields the capsid protein, which is located amino terminal to the preM glycoprotein.

As described herein above, initially a recombinant vaccinia virus containing a 4.0 Kb sequence from the 5' terminus of cDNA that codes for the three structural proteins [capsid membrane and envelope and nonstructural proteins NS1 and NS2a was constructed. Subsequently, another recombinant vaccinia virus expressing only the three structural proteins was constructed by deleting the coding sequence for nonstructural proteins. Protein analysis showed that cells irfected with the first recombinant virus v(C-M-E-NS1- NS2a) produced authentic pre-membrane (preM), envelope and NS1 glycoproteins as detected by radioimmunoprecipitation using specific antibodies. These dengue proteins showed a glycosylation pattern similar to that found for these proteins produced during dengue virus infection. The second recombinant virus produced dengue virus preM and E glycoproteins. As expected, NS1 was not synthesized by the second recombinant (Table 2).

The mouse model of dengue encephalitis was employed to evaluate protective immunity induced by these recombinants. Mice were inoculated by the intraperitoneal route with 107 plaque forming units (pfu) of recombinant vaccinia virus. Mice were reinoculated with the same dose of recombinant two weeks later. One week H 35 after the second inoculation themice were challenged with 100 LD 50 of denque virus intracerebrally. The animals i were then observed for signs of encephalitis and death.

t I 'I i WO 90/01946 PCT/US89/03589 8 Ninety-one percent of mice immunized with a control vaccinia recombinant virus expressing the envelope glycoprotein of HIV died after challenge (Tables 1 and In contrast, 96% of mice immunized with the vaccinia (C-M-E- NS1-NS2a) recombinant that expressed the three structural proteins and NS1, survived. Also, all 15 animals immunized with the vaccinia recombinant virus that expressed only the three structural proteins were completely protected. These results indicate that structural proteins alone are able to induce protective immunity. Since C and M are internal proteins, it is concluded that E is the major antigen responsible for protection conferred by the v(C-M-E) recombinant. This interpretation is supported by the complete resistance to intracerebral challenge induced by immunization with faccinia recombinants that expressed only the E glycoprotein. Data in Table 1 provide such evidence. A recombinant that expressed E including its N-terminal hydrophobic aignal sequence, or a respiratory syncytial virus (RSV) G glycoprotein (amino acids 1 through 70) E fusion protein or an influenza A virus hemagglutinin N terminal signal peptide E fusion protein induced complete protection against dengue virus challenge.

The seroresponse of animals immunized with these recombinants was tested by radioimmunoprecipitation of labeled antigens. Each of the animals immunized with the v(C-M-E-NS1-NS2a) recombinant that expressed the three structural proteins and NS1, developed NS1 antibodies, while the amount of antibody to E was low or not detectable. The low level of E antibody response was further confirmed by other serologic tests such as virus neutralization and ELISA. Also, mice infected with the vaccinia recombinant that expressed only the three structural proteins developed little or no detectable E antibody.

The nonstructural protein NS1 expressed by various vaccinia recombinant viruses was also evaluated V-i _1 WO 90/01946 PCT/US89/03589 9 for its antigenic properties and the most promising products were evaluated for their protective effect in mice. A total of 6 NS1 vaccinia recombinants were studied. These recombinants contained an insert in which: NS1 was preceded by the structural protein sequence (ii) NS1 was preceded only by its immediate upstream hydrophobic sequence or (iii) NS1 was not preceded by structural genes or its immediate upstream hydrophobic sequence. Each of these inserts terminated with the complete NS2a sequence or the N terminal 15% of the NS2a sequence. The v(C-M-E-NS1-NS2a) and v(NS1-NS2a) recombinants produced authentic NS1, whereas uther recombinants that lacked the NS1 N terminal hydrophobic signal or lacked the complete downstream NS2a sequence yielded a NS1 product of different molecular size and/or were not glycosylated as indicated by gel analysis. From these studies it was concluded that the N-terminal signal sequence of NS1 and the complete downstream NS2a sequence are required for proper processing and proteolytic cleavage of NS1.

The above mentioned NS1 recombinants were then evaluated for their prophylactic potential in mouse protection studies as described above. The results indicated that all but one of 28 mice inoculated with vaccinia (C-M-E-NS1-NS2a) recombinant expressing the structural proteins and NS1 survived dengue challenge (Table Significantly, the recombinant which expressed authentic NS1 in the absence of structural -4 proteins, v(NS1-NS2a), induced complete resistance to lethal challenge with dengue virus (Table Other vaccinia recombinants that expressed abnormal NS1 induced only partial protection. Thus, vaccination with a vaccinia recombinant that expressed an upstream Nterminal RSV G sequence (amino acids 1-70) fused to NS1 or (ii) NS1 plus 15% of NS2a, induced only partial protection.

These tests demonstrate that dengue envelope WO 90/01946 PCT/US89/03589 10 glycoprotein and nonstructural protein NS1 are separate protective antigens, each of which can induce complete resistance to encephalitis in the mouse model of dengue disease. These results now allow the development of vaccinia recombinants expressing E and/or NS1 glycoprotein as vaccines for protection of humans against dengue type 4 virus. Accordingly, a vaccine comprises an immunogenic amount of the recombinant vaccinia virus which induces protective immunity against a specific flavivirus in a host infected with said recombinant vaccinia virus. Conventional pharmaceutically acceptable carrier or vehicles such as non-toxic buffers, physiological saline and the like could be used together with adjuvants and booster inoculations, if necessary. Of course, the vaccine can be administered in a single or multiple dosages as indicated.

It is important to point out here that a vaccinia-dengue type 2 virus recombinant that contained the dengue sequences for C,M, and E controlled by a P11 vaccinia virus promoter was recently reported by Deubel et al, 1988 Gen. Virol. 69:1921-1929) and failed to protect monkeys against dengue. In contrast, the recombinant of the present invention was efficacious in protecting the test animals against viral infection.

Since the genome organization, replication strategy and gene expression of all flaviviruses are similar, recombinant vaccinia viruses having prophylactic property against other flaviviruses are made in a manner similar to the herein illustrated vaccinia-dengue construct.

A deposit of the recombinant virus has been made at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A., on August 19, 1988 under the accession number VR 2228.

The deposit shall be viably maintained, and replaced if it becomes non-viable, for a period of 30 years from the date of the deposit, or for 5 years from the last date of -1 PCr/US89/03589 WO 90/01946 11 request for a sample of the deposit, whichever is longer, and made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

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;i Ii- WO :90/01946 PCl'/US89/03589 12 TABLE NICE INFECED WITH VACCINIA RECOBIANTS EXPRESSING DENGUE ENVEMOPE GLYCOPROFEIN WITH OR WITHOUT NONSTRUCI'URAL NS1 GLYCOPRO'EIN DEVELOP RESISTANCE TO FATAL CH11AE1NE WITH DENGUE VIRUS NO. TtICH SURVIVED VACCINIA RECOM- EXPRESSION OF NO. OF AN 2NI'RACEREBRAL BINANT EXPRESSING MICE CHALLENE WITH 102 DENUE PROIEYIN(S) GLYC- AIHMEN- =CED ID 5 0 DUE TYPE SYLATED E TIC NS1 (10 PFU) 4 VIRUS V(C-M-E-NS1-NS2A) YES YES 28 27 96%) V(C-M-E-NS1-NS2AA)* YES NO 15 15 (100%) v(C-M-E) YES 19 19 (100%) v(E) YES 15 15 (100%) V(RSV YES 15 15 (100%) v(FLU H-E) YES 15 15 (100%) v(HIV GP160)tt- 46 4 OF C TERMI L SEQUENE OF NS 2 A DELETED.

SURVIVNG MI DEVELPED ML S S OF ENCEPHALITIS.

EXPRESSES FUSION PROI

T

EflN OF N TERMINUS OF RSV G (AM= ACIDS 1- PLUS E.

EXPRESSES FUSION PROTEIN OF INFLUENZA A VIRUS N TERMINAL SIGNAL PEPIIDE PLUS E.

COTROL VACCINIA RECOMBIANT EXPRESSN ENVELOPE GLYCOPROTEIN OF

HIV.

Sii r, I- WO 90014 PCT/US89/035 I I O 9/01 946 PCT/US89/03589 13 TABLE 2 MICE INFECED WITH VACC=2IA REXCXBINANT EXPRESSING& ONLY DENUE NONSTRUCTURAL, PROITEIN NS1 DEVELOP RESISTANCE TO FATAL CHALEMUE WITH DENGUE VIRUS NO. MUC SURVIVED AN VACCINIA REmDM- EXPRESSION OF NO. OF MICE 'INRAEREBRAL CHAL- BINANT EXPRESSIG AUMEMrIC NS1 INFED LENE WITH A L INDICATED (10 PFU) DENUE TYPE 4 VIRUS DE\7UE PROIEIN(S) v(NS1-NS2A) YES 15 15 (100%) V(NS1-NS2A NO 30 20 67%) v(RSV GNS1)+ NO 15 8 54%) v(HIVGP160) 46 4 OF C TERIINAL SEQUENCE OF NS 2 A DELETED.

EXPRESSES FUSION PROEI OF RSV G (ANO ACIDS 1-70) PLUS NS1.

CONTROL VACCINIA RECOMBINANT EXPRESS ING ENVELOPE GLYCOPROTEIN OF

HIV.

Claims

1. A recombinant vaccinia virus selected from the group consisting of V(C-A-'~SA/1-A52a)V(C-M-E), V(E), V(RSV V(FLU HA-E), and V(NS1-NS2A), wherein the viral proteins are obtained from a flavivirus.

2. A recombinant vacciniq virus consisting essentially of the coding sequence for C, M, E, NS1, and NS 2 A proteins, wherein the viral proteins are obtained from a flavivirus and 85 percent of the carboxyl terminal sequence of NS 2 A protein is deleted.

3. The virus of claim 1 wherein said flav- ivirus is a dengue type 4 virus.

4. The virus of claim 2 wherein said flav-a ivirus is a dengue type 4 virus.

The virus of claim 1, wherein said flav- ivirus is a Japanese B or tick-borne encephalitis virus.

6. The virus of claim 2, wherein said flavi- virus is a Japanese B or tick-borne encephalitis virus.

7. A pharmaceutical composition, comprising an immunogenic amount of the virus of claim 1 in a pharma- cic 'ca u acceptable carrier.

8. A pharmaceutical composition, comprising an immunogenic amount of the virus of claim 2 in a pharma- i ceutically acceptable carrier. S9. A method for inducing protective immunity against a flavivirus disease, comprising inoculating a host acceptable to flavivirus with an immunogenic amount of the virus of claim 1 in at least a single dose. A method for inducing protective immunity Sagainst a flavivirus disease, comprising inoculating a host acceptable to a flavivirus with an immunogenic amount of the virus of claim 2 in at least a single dose. SUBSTITUTE SHEET SUBSIUTE SHEET fiAZ>R IPEAUS i l^ W Tt. I 1 N O0