WO1991002819A1

WO1991002819A1 - Recombinant japanese encephalitis molecules and methods of use therefor

Info

Publication number: WO1991002819A1
Application number: PCT/US1990/004661
Authority: WO
Inventors: Mark A. Cochran; Gale E. Smith; Jane A. Thanassi
Original assignee: Microgenesys, Inc.
Priority date: 1989-08-18
Filing date: 1990-08-17
Publication date: 1991-03-07
Also published as: CN1052506A

Abstract

This invention provides recombinant Japanese encephalitis DNA molecules, vectors and the recombinant proteins which are produced by them. This invention also provides methods for detecting immunizing and treating Japanese encephalitis infection in a subject which comprises using the recombinant Japanese encephalitis proteins under suitable conditions.

Description

RECOMBINANT JAPANESE ENCEPHALITIS MOLECULES AND METHODS

OF USE THEREFOR

The invention described herein was made in the course of work under Grant Nos. DAM D17-82-C-2237, DAM D17-86- C-6156, and DAM D17-C-6612, Department of Defense. The U.S. Government has certain rights in this invention.

Background of the Invention

A major cause of encephalitis in the orient is the result of infection by the Japanese encephalitis virus (JEV) , a flavivirus. The virus is spread by a mosquito vector which infests the local rice fields.

Until quite recently, the only widely accepted and effective flavivirus vaccine was the attenuated yellow fever virus vaccine (Thieler et al.). Attempts to make attenuated vaccines for other flavivirus have, for the most part, met with limited success (Bancroft et al.; Innis et al. ) .

Since 1965, highly purified, formalin inactivated, mouse brain derived JEV vaccines have been used, and have been shown to be safe and effective (Hoke et al.).

The requirement for a high degree of purity generally means a relatively high cost; therefore, a vaccine which is not derived from mouse brain would be desirable (Monath, Editorial, NEJM, 8 Sept, 1988). By using recombinant DNA technology, it is hoped that such vaccines can be made. It is an object of this invention to provide a recombinant Japanese encephalitis molecule which is useful for detecting JEV infection, for immunizing against JEV infection and for treating JEV infection. It also is an object of this invention to provide a method of detecting Japanese encephalitis infection in a subject.

It is a further object of this invention to provide a safe and effective vaccine which can be manufactured at a relatively low cost.

Summary of the Invention

This invention provides recombinant Japanese encephalitis DNA molecules, vectors and the recombinant proteins which are produced by them.

This invention also provides methods for detecting, immunizing and treating Japanese encephalitis infection in a subject which comprise using the recombinant Japanese enchephalitis proteins under suitable conditions.

Brief Description of Figures

Figure 1 shows the restriction enzyme map of JEV;

Figure 2 illustrates the cloning process for the recombinant JEV viral vectors;

Figure 3 shows that protein reactive with JEV antibodies were produced by the recombinants; Figure 4 shows the survival of mice which received the baculovirus expressed, JEV polyprotein or E glycoprotein;

Figure 5 shows the results of the plague assay wherein JEV specific plague reduction was seen in mice which received recombinant JEV E protein;

Figure 6 shows the results of the immunoprecipitation assay;

Figure 7 summarizes the construction of JEV plasmid recombinants;

Figure 8 shows the Western blot analysis of recombinant proteins. Vector 8283 expressed a protein as determined by reaction with JEV E-specific monoclonal antibody. This result shows that expression proceeded through pM and M genes. The Western blots also revealed three polypeptides of approximately the same intensity: two closely spared bands at about 45K and another at 65K. This suggests that the two bands at 45K represent E-gene product which resulted from cleavage of the pM/M/E precursor which has a predicted size of 66-72K. Recombinant vector 8302 showed no E specific antibody as determined by Western blot;

Figure 9 shows Western blot analysis of recombinant proteins reacted with anti-NS-1 monoclonal antibody;

Figure 10 shows Western blots of recombinants reacted with anti-M and anti-E specific antibodies;

Figure 11 shows that anti JE-E HMAF reacts only to JE-E expressing recombinants, and that DEN 1-E HMAF reacts specifically to DEN 1-E expressing recombinants;

Figure 12 shows that there is a measurable degree of cross-reactivity to NS-1 expressing recombinants; and

Figure 13 shows that some recombinant vectors produce full length NS-1 gene product.

Detailed Description of the Invention

This invention provides a recombinant DNA molecule which comprises a full length or partial JEV envelope protein coding region.

This invention also provides a recombinant DNA vector construct which comprises a full length or partial JEV envelope protein coding region and a viral vector. In one embodiment of the invention, the viral vector is a baculovirus vector. The baculovirus vector may contain the ATG initiation codon, a hydrophobic membrane insertion (signal) sequence and a TAA translation stop codon. Suitable vectors which are useful in the practice of this invention comprise, but are not limited to, a baculovirus vector from the group of vectors designated MGS3+1, PUC19, MGS12, MGS3 and MGS3+2.

Recombinant JEV viral vectors also are provided by this invention, for example, vectors designated 8302, 8437, 8501, 8929, 8469, 8468, 8650, 8590, and 8716, as well as the recombinant JEV RNA and envelope protein which they each express. This invention further provides an insect cell which contains a recombinant DNA molecule which comprises a full length or partial JEV envelope protein coding region.

This invention also provides a composition which comprises the proteins which are expressed by the recombinant vectors designated 8302, 8437, 8501, 8929, 8469, 8468, 8650, 8590, and 8716 and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water emulsion, and various types of wetting agents. These compositions also may contain a suitable adjuvant.

This invention further provides the above composition wherein a detectable moiety is linked to the recombinant JEV envelope protein, such as ^*-\ "_t radioisotopes, e.g. P and S, dyes and photochemical agents.

A method of detecting the presence of Japanese encephalitis virus in a subject, e.g. a human patient, is provided which comprises contacting a suitable sample from the subject with the recombinant JEV envelope protein described hereinabove, under conditions such that an antibody-antigen complex is formed, and detecting any complex so formed, and thereby detecting the presence of Japanese encephalitis. In one embodiment of this invention, the recombinant JEV envelope protein is labeled with a detectable moiety as described hereinabove. Suitable samples which are useful in the practice of this invention comprise, but are not limited to: sera; central nervous system tissue; and spinal fluid.

A method of treating and/or preventing Japanese encephalitis in a subject, e.g. a human patient, also is provided which comprises administering to the subject a therapeutically acceptable amount of said JEV envelope protein composition, effective to prevent and/or treat Japanese encephalitis. The composition may be administered by any suitable method, such as intravenous infusion or intramuscular injection.

Experimental Methods

cDNA cloning of JEV

Japanese encephalitis virus (Nakaya a strain) was cloned and sequenced as previously described (McAda et al.; Mason et al.). Clones were selected which contained the coding region for E (PM-7) , NS1 (PM-6) or the entire polyprotein of the virus.

Baculovirus cloning and expression

Figure 7 summarizes the construction of plasmid JEV recombinants.

The E glycoprotein coding region was derived from clone PM-7 (Yl) by restriction digestion with Hpa II and Eco Rl to generate a 1263 base pair (Bp)^' fragment from nucleotide 656 to 1919 (see Figure 1) . The NS1 glycoprotein coding region was derived from clone PM-6 by cutting with Aps I to generate a 1277 bp fragment from nucleotide 2063 to 3340. The coding region for JEV genes from preM through E, herein designated "polyprotein", was constructed by fusing clones Yl and PM-6 at a common Sac I restriction site at nucleotide 1620. The genes then were cloned into baculovirus recombination vectors (pAcMGS) . The E gene was cloned into a vector which supplies an initiator ATG codon, a hydrophobic membrane insertion (signal) sequence and a TAA translation stop codon. The NS1 and polyprotein genes were cloned into a vector where the ATG and stop codons were supplied by the vector, but the signal sequence was supplied by the insert. From the recombination vectors, genes were recombined into the polyhedrin (Pn) gene of baculovirus (Autographa californica nuclear polyhedrosis virus) by co- transfection of Spodoptera fruqiperda insect cells with virus and vector DNA (see Figure 2) . Polyhedrin negative (Pn~) recombinants were identified by plaque morphology. In order to express the JEV genes, insect cells in monolayer or suspension culture were infected with recombinant virus at a multiplicity of 10 plaque forming units per cell. At 72 hours after infection, cells were harvested and lysed in Laemmli sample buffer containing SDS and 2-mercaptoethanol. The lysates were electrophoresed on polyacrylamide-SDS gels and either stained with coo assie blue or transferred to nitrocellulose. Antigenic bands were detected by Western blotting with the appropriate antisera.

Mouse protection assay

A mouse challenge model was developed using 6 week old, female, C57 black mice. Immunizing preparations were made by mixing equal volumes of unpurified spodoptera

₇ cell lysate (approximately 3.3 X 10 cells/ml) and Freund's complete adjuvant for the first dose and incomplete adjuvant for the next 2 doses. 0.2 ml of the mixture was given intramuscularly in the leg and an additional 0.2 ml of cell lysate without adjuvant was given intraperitoneally. Mice were inoculated on days 0, 3 and 14 and bled on day 21, one day before they were challenged. The virus challenge was prepared from a 1/10 dilution of a 20 percent suspension of Japanese encephalitis virus (Nakayama strain) infected suckling mouse brain. The challenge preparation contained approximately 100 mouse LD50's of Japanese encephalitis virus. The challenge was administered intraperitoneally on day 22, and mice were observed daily thereafter. Dates of death were recorded.

Plaque reduction neutralization test

Sera were tested at 1/10 dilution for their ability to neutralize JEV by plaque reduction as previously described (Russell and Nisalak) . Approximately 50 plaque forming units of Nakayama strain of Japanese encephalitis virus were used. Virus and serum were mixed together and maintained at 36"C for 60 minutes. Twenty-five square centimeter flasks containing confluent LLCMK2 cells were innoculated with the virus- serum mixture. After a 2 hour incubation at room temperature, flasks were overlayed with 1% purified agar (Difco, Detroit, MI) in medium 199 (Gibco, Grand Island, N. Y.). After incubating for 7 days at 36"C, cells were stained with 0.02% neutral red in saline and plaques were counted. Each mouse serum was tested in duplicate and the number of plaques reported was the average of the two determinations. The percent of reduction of plaques was calculated by comparison of the results obtained with sera from .control mice.

Radioimmuno-dot blot assay

A radioimmuno-dot blot assay was performed using either purified, formalin inactivated JEV (Biken vaccine, Japan) , unpurified, lysed JEV infected feline kidney (CRFK) cells (Crandall et al.), or uninfected CRFK cells as the test antigens. Sera from mice immunized with recombinant baculovirus preparations diluted 1/500. JEV specific, hyperimmune mouse ascitic fluid (HMAF) , prepared as previously described (McCown and Brandt) , diluted 1/500 was used as the positive serum control and ascitic fluid from non-immune (unimmunized) mice was used as the negative control. Antigens were spotted on nitrocellulose papers, and the papers blocked with 5% nonfat dry milk, 0.001% sodium ayide for 20 minutes. Sera were added to the papers and incubated overnight at room temperature. Bound antibody was detected using I labeled, goat anti- mouse IgG. After extensive washing, papers were counted in an LKB gamma counter. Results were corrected by subtraction of background and expressed as cpm. Counts equal to or greater than 2.5 times the nagative control were considered indicative of specific antibody.

Immunoprecipitations

Immunoprecipitations were done with mouse sera, pooled from each immunization group and diluted 1/100. The test antigen was a lysate of JEV infected CRFK cells labeled iji vivo with [ 35S]-methionine. After incubating the antibody and antigen overnight at 4°C, immune complexes were absorbed onto protein A sepharose beads (Pharmacia, Piscataway, NJ) . After extensive washing, antigens were eluted by boiling in Laemmli sample buffer (Laemmli) and run on a 12.5% polyaery1amide gel with sodium dodecyl sulfate (SDS) .

Figure 6 shows the i unoprecipitation of JEV proteins by sera from recombinant-immunized mice. JEV antigen was prepared from SDS lysates of Crandell's feline kidney cells (CRFK, Crandell et al., 1976) infected with JEV (Nakayma strain) and labeled with [³⁵S]- methionine. After immunoprecipitations, proteins were analyzed on a 12.5% polyacrylamide-SDS gel. Lane 1 shows mice immunized with recombinant E protein; lane 2 shows recombinant NS1 protein; lane 3 shows recombinant polyprotein; lane 4 shows B-galactosidase; lane 5 shows purified, formalin inactivated JEV virus (Biken vaccine) ; and lanes 6 through 10 are as lanes 1 through 5 except that the immunoprecipitations were carried out with a lysate of uninfected CRFK cells.

Experimental Results

Expression of JEV genes in baculovirus

JEV genes, previously shown to encode the virion E glycoprotein and the NS1 glycoprotein (McAda et al.; Mason et al.) were selected for expression as vaccine candidates (Figure 1) . In addition, the entire polyprotein coding region, from the structural precursor preM through nonstructural protein NS3 was selected for expression. The general scheme used for obtaining baculovirus recombinants expressing these JEV genes is diagrammed in Figure 2. Recombinant baculovirus were used to infect Spodoptera frugiperda cells and cell lysates were analyzed for JEV protein by electrophoresis on polyacrylamide-SDS gels which were than stained with coomassie blue R 250 or subjected to Western blotting (Brunett et al.) with JEV specific antisera.

As demonstrated in Figure 3, proteins reactive with JEV antibodies were produced. The stained gels were compared with the Western blots; with some recombinants there were stained protein bands in the same position as immunoreactive bands. These bands were not present in cells infected with B-yol expression recombinant baculovirus and thus appeared to be JEV specific. By densitometric scanning of the stained gels, the estimated quantities of E and NS1 proteins produced were proteins produced were about 1O and 3_fJ ug, respectively, per 10 cells.

Although cells infected with the polyprotein recombinant did not produced enough protein to be seen clearly on stained gels, proteins were seen on Western blots indicating that the polyprotein had been properly processed. Western blots indicated that the quantity of JEV processed was about 1/20th the amount expressed by the E recombinant. (See Figures 8 and 9)

Figure 10 shows Western blots of the recombinants reacted with anti-M; and anti-E specific antibodies. The results suggest that there is production of a polyprotein precursor of approximately 66-72K which is subsequently cleaved to yield a variety of products. However, pulse-chase experiments failed to demonstrate a precursor/product relationship. In view of this then, it appears that the polyprotein may be cleaved during synthesis. Recombinant v8929, as shown in Figure 10, expresses as well as the shorter recombinant v8283, and produces a full-length E gene product. In addition, Figure 12 shows that it produces a full- length MSI gene product. These results suggest that there is accurate cleavage of JEV proteins in the baculovirus infected cells and that the proteins have been modified by glycosylation.

JEV: E Expression

Two constructs were designed to express only the JEV E gene: V8302 and V8501. Recombinant v8501 was designed to express the E gene using a baculovirus specific signal peptide. Expression levels of this recombinant are good with the production of abundant polypeptides in the range of 46-50K. This size suggests that the product is glycosylated. As shown in Figure 3 these proteins are visible in a coomassie stained gel and are highly antigenic on Western blots as shown in Figure 10.

JEV: NS-1 Expression

Recombinant v8524 was designed to express the NS-1 gene using the signal supplied by 34 amino acids from the C- terminus of the preceding E gene. Expression levels of this recombinant are good with the production of an abundant polypeptide in the range is about 50K. This size suggests that the product of glycosylated. As shown in Figure 3 this protein is visible in a coomassie stained gel and is highly antigenic on Western blots, as hown in Figure 10. DEN1: E Expression

Four recombinants were designed to express the DEN1 E- gene. Recombinant v8590 was designed to express a C- terminal truncated E-gene using a signal supplied by the vector, and v8716 was designed to express using authentic signal supplied by the 36 amino acids at the C-terminal end of the preceding M gene. Expression levels of recombinant V8590 were good with the production of an abundant polypeptide at 55-58K. (See Figure 11) . This size suggests that the product is glycosylated. However, recombinant V8716 expressed poorly. Recombinants V8468/69 and V8650 also expressed poorly. The only difference between recombinant V8468/69 and V8590 is a C-terminal truncation.

DEN1: NS1 Expression

Recombinant v8858 was designed to express a full-length NS-1 product using 55 amino acids from the preceding E- gene as a signal. Expression levels of this recombinant are good with the production of an abundant polypeptide in the range of about 50K. This size suggests that the product is glycosylated. See Figure 12.

Comparison between all E constructs and NS-1 constructs was made to determine if there is any cross-reactivity in serum as determined by Western blot analysis. Figure 11 shows that anti JE-E HMAF reacts only to JE-E expressing recombinants, and likewise, DEN1-E HMAF reacts specifically to DEN1-E expressing recombiants. However, Figure 12 shows that there is a measurable degree of cross-reactivity to NS-1 expressing recombinants. While JE NS-1 HMAF (to clone 32) only reacted to JE NS-1, anti DEN2 NS-1 HMAF cross-reacted to JE NS-1 as well as DENl NS-1. There was difficulty with JE NS-1 HMAF to clone 32 in Western reactions. If this sera was more active, it is expected that there would be cross-reactivity to the DEN NS-1.

That the proteins were glycosylated was verified by treating cells with the glycosylation inhibitor tunica ycin and by showing that recombinant proteins were labeled with [ 3H]-glucosamιne (data not shown).

Protection of mice against live JE virus with baculovirus recombinant JEV proteins

Survival of mice which received the baculovirus expressed, JEV polyprotein or E glycoprotein was significantly improved over recipients of the NSl glycoprotein or beta-galactosidase (Figure 4 and table 1).

Table I Survival and immune status of immunized mice

* p <.005 when compared to negative control recipients (B-galactosidase expressed in the same baculovirus system) Sixty-eight percent of the mice which received polyprotein and 75% of those which received E glycoprotein survived challenge with live JEV, as compared with 30% of B-galactosidase recipients and 32% of NSl recipients (p<.005 for both when compared to the negative control) . These results suggest that even in the mice which died, there was some prolongation of survival from administration of the recombinant E glycoprotein and polyprotein preparations but not B- galactosidase. Of the 14 B-galactosidase recipients which died, 13 died early, at about 6 days following live virus challenge; whereas, for the polyprotein recipients, only 3/6 died early (p=.06 by Fisher's exact test) and of the E recipients 2/5 died early (p=.04 compared with control).

In comparison with the recombinant antigens, the formalin inactivated JEV virion preparation protected 100% of mice to which it was administered. This was true even at a virus dilution of 1:10,000 (date not shown) .

Detection of JEV neutralizing antibody in recombinant E protein and polyprotein immunized mice

When tested at a dilution of 1/10, JEV specific plaque reduction was seen with sera from mice which received purified, formalin inactivated JEV (Biken vaccine preparation) , baculovirus recombinant polyprotein or E glycoprotein (Figure 5) . Sera from 15/19 polyprotein recipients and 18/20 E glycoprotein recipients, as compared with 0/20 beta galactosidase and 1/19 NSl recipients showed a 50% or greater reduction plaques at the dilution tested (p<l x 10 -7) . The mean reduction in plaques was 58% for recipients of the polyprotein and 62% for the recipients of the E glycoprotein (p<.005 when compared to the mean of 8% observed in the beta galactosidase control group) . If all the groups are considered together, there was a significant relationship between the presence of neutralizing antibody in mice and their survival.

Immunoqenicity of recombinant E, NSl and polyprotein antigens

The antibody response of recombinant immunized mice to virus specific antigens was determined. The antigens tested were purified, formalin inactivated JE virus (Biken vaccine) and an unpurified lysate of JEV infected CRFK cells. A radioimmunoassay was used to quantitate the immune response. Sera from mice immunized with baculovirus recombinant B-galactosidase and unimmunized control mice did not react significantly with any JEV specific antigen preparation. However, sera from mice immunized with purified, formalin inactivated virus or with JEV infected mouse brain (HMAF) reacted strongly against both antigen preparations. Recipients of baculovirus recombinant E glycoprotein also had antibody to both the purified virus and the crude cell lysate. Recipients of the baculovirus recombinant NSl glycoprotein had antibody to the JEV lysate only, reflecting the absence of NSl in the purified virus preparation.

Mice which received recombinant proteins appeared to make less antibody or lower avidity antibody than mice immunized with purified, formalin inactivated JEV or JEV infected mouse brain. In order to determine if this difference in immunogenicity was quantitative, a radioimmunoassay with JEV specific HMAF was performed on serially diluted recombinant antigens as well as formalin inactivated virus. This assay showed that the recombinant proteins were at least as reactive antigenically as virus, with the E glycoprotein recombinant being almost twice as reactive.

In order to determine whether mice immunized with recombinant proteins responded specifically to the appropriate, authentic JEV proteins, post-immunization sera were used to immunoprecipitate [ 35S]-methionine labeled proteins from JEV infected CRFK cells (Figure

6) . Pooled sera from mice immunized with baculovirus recombinant E glycoprotein immunoprecipitated the same proteins as sera from mice immunized with purified JE virus (Biken vaccine) , although less efficiently, including a protein of the size expected for E (about

50 Kd) and a 21 Kd protein which was possibly a degradation product. Sera from mice immunized with the baculovirus recombinant NSl glycoprotein immunoprecipitated a heterodisperse protein, consistent in size with NSl (around 40 Kd) . This heterodispersity may reflect differences in the glycosylation state of the NSl protein (Russell et al., 1980). Interestingly, mice immunized with protein from a baculovirus recombinant containing the polyprotein coding region for C, preM, E and NSl made detectable immunoprecipitating antibody to E but not to NSl. A negative control pool of sera from mice immunized with baculovirus recombinant B-galactosidase did not immunoprecipitate any JEV proteins. Experimental Discussion

A number of systems exist in which Japanese encephalitis virus genes can be expressed. Most extensively evaluated to date are genes expressed in E. coli. Although viral proteins are made, bioassay has demonstrated that they are not protective (Hoke and McCown, personal communication) perhaps due to lack of glycosylation, altered folding or degradation.

The baculovirus system has been used because of its relative ease of manipulation and presumed safety and because a large number of proteins have been expressed in native form (reviewed by Summers et al.). A number of viral antigens also have been expressed, including those of human immunodeficiency virus, rotavirus herpes simple virus, and hepatitis B virus.

Baculovirus expressed proteins of HIV-1 form the basis of a vaccine for this disease which is currently being tested in humans.

It was demonstrated that Japanese encephalitis virus E glycoprotein and polyprotein expressed in baculovirus infected Spodoptera fuqiperda cells protect animals against live JE virus.

Immunization of mice with baculovirus expressed, JEV polyprotein or E glycoprotein protected them against live virus challenge. The baculovirus expressed proteins that were not only antigenic, but also elicited neutralizing antibodies in mice. In contrast,

E. coli expressed proteins, although they contained neutralizing epitopes by immunoassay with monoclonal antibodies, did not elicit neutralizing antibody nor were they protective (Hoke and McCown, unpublished results) .

These results show that the presence of neutralizing antibody in immunized mice appeared to correlate with protection. Only those mice which received polyprotein or E glycoprotein, both of which stimulated a neutralizing antibody response, were protected against live virus challenge. Therefore, demonstration of neutralizing antibody in the sera of immunized animals may predict the ability of an immunogen to elicit protective immunity ij vivo.

It appears that protection mediated by the expressed polyprotein coding region of JEV was due to the presence in those preparations of processed E glycoprotein, since the other major constituent, NSl glycoprotein, was by itself not protective (see below) .

The degree of protection and the quantity or avidity of antibody elicited by the recombinant E glycoprotein was less than that elicited by purified, inactivated JE virion. This could reflect either quantitative or qualitative differences in the immunogens. However, it may be the result that the recombinant E glycoprotein lacked some conformational features present in the virion-associated form of the protein. In this regard, Wengler et al. have presented evidence that the virion associated E glycoprotein of another flavivirus, tick- borne encephalitis virus, exists as a trimer.

The lack of protection mediated by the recombinant NSl glycoprotein may have been due to its non-authentic expression in spodoptera cells. However, the fact that mice immunized with recombinant NSl made antibody which reacted with this protein from JEV infected cells argues against this possibility, though the antibody produced may not have been of the variety, i.e. , cytolytic, thought to be protective (Schlesinger) . Alternatively, it may reflect differences in the pathogenesis of JEV as compared with yellow fever or dengue virus infections, where antibodies to NSl are protective (Schlesinger et al; Henchal et al.). If, for example, immunity to NSl serves to eliminate virus infected cells before the maturation of progeny, then, viruses which replicate more rapidly may be less affected. It should also be remembered that JEV is highly neurotropic, whereas, yellow fever an dengue viruses are generally visceratropic.

In summary, the results demonstrate that JEV proteins expressed in spodoptera cells infected with recombinant baculovirus are immunogenic and protective. Stimulation of virus neutralizing antibody by expressed proteins may be a marker for protection in vivo.

References

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McAda PC, Mason PW, Schmaljohn CS, Dalrymple JM, Mason TL and Fournier MJ, (1987) . Partial nucleotide sequence of the Japanese encephalitis virus genome. Virology 158:348-360.

Mason PW, McAda PC, Dalrymple JM, Fournier MJ, and Mason TL, (1987) . Expression of Japanese encephalitis virus antigens in Ecsherichia coli. Virology 158:361- 372.

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Claims

What is claimed is:

1. A recombinant DNA molecule which comprises a full length or partial JEV envelope protein coding region.

2. A recombinant DNA vector construct which comprises the recombinant DNA molecule of claim 1 and a viral vector.

3. A recombinant DNA vector construct of claim 2, wherein the viral vector is a baculovirus vector.

4. The recombinant DNA vector construct of claim 3, wherein the baculovirus vector is the vector from the group consisting of the vectors designated MGS3+1, PUC19, MGS12, MGS3 and MGS3+2.

5. A recombinant viral vector designated 8302.

6. A recombinant viral vector designated 8437.

7. A recombinant viral vector designated 8501.

8. A recombinant viral vector designated 8929.

9. A recombinant viral vector designated 8469.

10. A recombinant viral vector designated 8468.

ii. A recombinant viral vector designated 8650.

12. A recombinant viral vector designated 8590.

13. A recombinant viral vector designated 8716.

14. An insect cell which contains the recombinant DNA molecule of claim 1.

15. Recombinant JEV envelope protein expressed by the recombinant viral vector of claims 5, 6, 7, 8, 9, 10, 11, 12 and 13.

16. A composition which comprises the protein of claim 15 and a pharmaceutically acceptable carrier.

17. A method of detecting the presence of Japanese encephalitis virus in a subject which comprises contacting a suitable sample from the subject with the recombinant JEV envelope protein of claim 15, under suitable conditions such that an antibody- antigen complex is formed, and detecting any complex so formed, and thereby detecting the presence of Japanese encephalitis.

18. The method of claim 16, wherein the subject is a human.

19. The method of claim 19, wherein the suitable sample is selected from the group which comprises sera, central nervous system tissue and spinal fluid.

20. A method of treating Japanese encephalitis in a subject which comprises administering to the subject a therapeutically effective amount of the composition of claim 16.

21. A method for preventing Japanese encephalitis is a subject which comprises administering to the subject a therapeutically effective amount of the composition of claim 16, effective to prevent Japanese encephalitis.

22. The method of claims 20 and 21, wherein the subject is a human.