WO1988003032A1

WO1988003032A1 - Diagnosis of and vaccine for japanese encephalitis virus and related viruses

Info

Publication number: WO1988003032A1
Application number: PCT/US1987/002763
Authority: WO
Inventors: Maurielle J. Fournier; Thomas L. Mason; Phyllis C. Mcada; Peter W. Mason
Original assignee: Fournier Maurielle J; Mason Thomas L
Priority date: 1986-10-27
Filing date: 1987-10-21
Publication date: 1988-05-05
Also published as: JPH01501203A

Abstract

Nucleic acid of JEV is isolated and cloned. It is used to synthesize polypeptides corresponding to those of JEV and thence obtain antibodies to these polypeptides. Diagnostic nucleic acid probes for JEV are obtained, as are synthetic antigens and antibodies suitable for diagnosis of JEV, and for inducing immunity to JEV.

Description

DIAGNOSIS OF AND VACCINE FOR JAPANESE ENCEPHALITIS VIRUS AND RELATED VIRUSES This invention was made with government support including a grant from the U.S. Army Medical Research and Development Command, contract number DAMD

17-82-C-2237. The government has certain rights in the invention.

Background of the Invention This invention concerns nucleic acids, diagnostic tests, and vaccines related to Japanese encephalitis virus (JEV).

JEV is a flavivirus responsible for encephalitis in both humans and domesticated animals. JEV in this application includes all variants and strains of the virus, both virulent and non-virulent, which are present in humans and other animals. Examples of such strains are given in Banerjee, Indian J. Med. Res. 83:243, (1986). The virus occurs predominantly in the Far East and is most prevalent in the maritime regions of Siberia to eastern India, Sri Lanka, the north and central portions of Indonesia, Borneo and the Philippines. It is particularly widespread in China and, historically, was a health problem in Japan.

Vaccines intended to protect against JEV have been developed by the Japanese and Chinese. They consist of chemically attenuated whole virus preparations intended for use in both humans and domesticated animals, such as swine.

Currently, diagnosis of JEV infection entails the use of immunological methods, e.g., viral antibodies are detected with standard ELISA (enzyme linked immunosorbent assay) tests using whole virus preparations (Xiao et al., Virus Inf. Exch. Newsletter for S.E. Asia and the W. Pacific 2:7, 1984). Summary of the Invention The invention features polypeptide and nucleic acid products for use in the diagnosis of, and immunization against, JEV. Diagnosis of viral infection is based on the detection of specific viral nucleic acid, specific viral antigens or specific viral antibodies in biological samples from animals, such as humans or domesticated animals. Inoculation of animals with synthetic viral protein immunogens, or with vectors encoding such immunogens, elicits protective antiviral antibodies.

In one aspect the invention features substantially purified nucleic acid having a sequence of at least a 10 base pair sequence of DNA or RNA that corresponds identically to the nucleic acid sequence of the Japanese encephalitis virus, but which is not found in the nucleic acid sequence of Yellow fever virus (Rice et al., Science 229:726, 1985).

In preferred embodiments the 10 base pair sequence is chosen from a segment within, the sequence shown in Fig. 1, and is not found in the nucleic acid sequence of West Nile River virus (Castle et al., Virology 149 : 10, 1986; Wengler et al., Virology 147:264, 1985; Castle et al., Virology 145:227, 1985), or Murray Valley virus (Dalgarno et al., J. Mol. Biol. 187 :309, 1986), dengue virus or St. Louis encephalitis (SLE) virus (Porterfield In the Togaviruses, ed. Schlessinger, Academic Press, N.Y., p. 13-36, 1980).

The first aspect of the invention also features: substantially purified nucleic acid that hybridizes to nucleic acid of Japanese encephalitis virus but not to one or more of the above-listed related viruses under stringent conditions; substantially purified nucleic acid encoding a polypeptide having at least one antigenic determinant that is immunologically reactive with a Japanese encephalitis virus-encoded protein, but not to proteins encoded by related viruses; and substantially purified nucleic acid sequences encoding a polypeptide sequence encoded by Japanese encephalitis virus, but not by yellow fever virus. In preferred embodiments the nucleic acid sequence encodes a polypeptide sequence not encoded by West Nile River virus, Murray Valley virus, dengue virus or SLE virus; the encoded polypeptide raises immunological protection against Japanese encephalitis; the polypeptide is reactive with the major envelope protein (E) or the non-stuctural protein NS1 (NS1) of the Japanese encephalitis virus; the polypeptide is substantially similar to the major envelope protein (E) or protein NS1 of Japanese encephalitis virus; and the nucleic acid is present in a vector, chosen from a phage, plasmid, cosmid, or eukaryotic virus, such as baculovirus, vaccinia, rotavirus and adenovirus.

In a second aspect the invention features a substantially purified polypeptide synthesized by expression of the nucleic acids described above or from substantially purified nucleic acid substantially corresponding to a portion of the nucleic acid of JEV. Preferably the polypeptide is a protective immunogen in man, or domesticated animals.

In a third aspect the invention features a method of diagnosing Japanese encephalitis based on a biological sample, comprising providing the nucleic acids described above as probes and determining whether the probes hybridize to nucleic acid in the sample. In preferred embodiments the sample is obtained from infected cells or infected organisms; and the probe comprises nucleic acid encoding at least a part of the major envelope protein (E) or the non-structural protein (NS1) of Japanese encephalitis virus. A fourth aspect of the invention features a method of vaccinating an animal to raise protection against Japanese encephalitis, by inoculating the animal with a composition comprising the above-described polypeptides, or with the above-described nucleic acids. In preferred embodiments the inoculation is by injection, or by an insect vector; the vaccination induces immunity to yellow fever. West Nile River encephalitis, Murray Valley encephalitis, St. Louis encephalitis or dengue fever; and the animal is a human, a domesticated animal, or a bird.

A fifth aspect of the invention features a method of diagnosing Japanese encephalitis comprising detecting immunologically reactive polypeptides with the above-described antigenic polypeptides or with antibodies produced to these antigenic polypeptides; preferably the detection is by an ELISA test or western blot.

The isolation and cloning of the nucleic acid of JEV makes it possible to devise assays that are specific for JEV, and, if desired, differentiate JEV from the above-mentioned related viruses. Moreover, having cloned JEV, it is possible to use segments of the JEV genome as nucleic acid probes, or to express them in vectors to produce viral antigens, and thence antibodies which can be used in JEV assays (regardless of whether those assays are specific for JEV). These synthetic antigens are suitable for vaccines, reducing the risk of viral infection from the vaccine, as compared with chemically attenuated viruses. Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims. Description of the Preferred Embodiments The drawings will first briefly be described.

Drawings

Figure 1 is a partial nucleic acid sequence of the JEV genome;

Figure 2 is a schematic representation of the cloning of JEV cDNA;

Figure 3 A, B is a diagrammatic representation of the JEV genome, showing protein-encoding regions, restriction enzyme sites, and the regions present on clones; Figure 4 is a diagrammatic representation of the protein coding sequences of the JEV genome and the portions of cDNA inserts in λgtll recombinants;

Figures 5A and B are diagrammatic representations of the orientations of a JEV insert cDNA in pGEM-4;

Figure 6 is a photograph of a Coomassie blue-stained gel and a western blot analysis of λgtll recombinant-infected cell lysates probed with the monoclonal antibodies: anti-E. coli beta-galactosidase, anti-JEV-E protein, and anti-JEV-M protein; "std" refers to molecular weight standards;

Figure 7 is a photograph of a Coomassie blue-stained gel and a western blot analysis of λgtll recombinant-infected cell lysates, probed with monoclonal antibodies to either beta-galactosidase or JEV-E-protein;

Figure 8 is a graphical representation of the hydrophobicity of the JEV-E-protein showing the coding regions present in a series of J7-1 clones;

Figure 9 is a photograph of a western blot analysis of JEV virion proteins probed with antibodies affinity-purified from HMAF (murine ascites fluids); and Figure 10 is a photograph of a western blot analysis of lysates of JEV-infected mosquito cells probed with antibodies affinity purified from HMAF. Structure

Nucleic acid of JEV The preferred source of the nucleic acid of the invention is the JEV genome. A substantial part of the nucleic acid sequence of one JEV genome is shown in Fig. 1. The amino acid sequence of the proteins encoded by the sequence are given by a standard 1 letter code above the RNA sequence; the locations of genes are shown above this amino acid sequence.) Other suitable nucleic acid sequences are those which include at least 10 base pairs of the nucleic acid of JEV, and which are not found in the nucleic acid of the related yellow fever virus. Preferably the sequence also does not correspond to one in West Nile virus, Murray valley fever virus, dengue virus or St. Louis encephalitis virus . The nucleic acid may be obtained from the virus as described below, and inserted into a desired vector. Specific probes can be derived from this nucleic acid. By specific is meant that the probes contain regions which will hybridize under stringent conditions only to JEV viral nucleic acids, and not to viruses such as yellow fever virus. Preferably these conditions will also not allow hybridization of the nucleic acid of West Nile encephalitis virus, St. Louis encephalitis virus, Murray Valley encephalitis virus and dengue fever virus. By stringent conditions is meant conditions under which a probe nucleic acid sequence of about 15 base pairs will hybridize to another nucleic acid sequence having a sequence which corresponds to at least 80% of the probe sequence. Other nucleic acid sequences (e.g. artificially synthesized), which may include both protein coding and noncoding sequences specific to JEV and not to related viruses, are also suitable in the invention. The probes may be either cloned directly from the JEV genome or chemically synthesized, and may be present on vectors or maintained as linear nucleic acid molecules. Thus these probes are substantially pur if ied , that is , to the extent necessary for use as a probe or in an expression vector, they have been isolated from their native environment within JEV, and have been separated from nucleic acid which normally surrounds them.

Nucleic acid corresponding to segments of the JEV genome can be prepared as complementary DNA (cDNA). Collections or libraries of cDNA can be in a desired vector, such as a plasmid, cosmid, phage, or virus. The presence of the nucleic acid sequences useful in the invention can be detected by finding homology to the above-described regions of JEV, and by the absence of hybridization to DNA from cells uninfected with JEV. Alternatively expression vectors (such as λgtll) can be used. These vectors cause expression of viral antigenic proteins, from cells containing these vectors, which can be detected with antibodies reactive to that virus, or to individual viral proteins. Examples of two such libraries of the JEV genome and of screening procedures are given below. These examples are not limiting to the invention, and one skilled in the art will realize that there are many other techniques by which JEV nucleic acid can be isolated. Further, it is understood that these methods are suitable for the isolation of nucleic acid from all strains of JEV. In the following specific, non-limiting example, the JEV genome was cloned in a plasmid vector, using cDNA technology, and subsequently subcloned in a λ-expression vector. The λ-clones were utilized to prepare antigenic viral proteins, which in turn were used to prepare antiviral antibodies. Approximately 10 kb of the estimated 10.9 kb genome of JEV (Nakayama strain) was cloned as cDNA, the uncloned portion corresponds to about 430 bases of the 5'-terminus and 450 bases at the 3'-end.

Example 1: cDNA Library A genomic bank of viral cDNA was developed by reverse transcription with synthetic DNA primers. The cDNA products were cloned. Cloning was into the PstI site in the bla gene of pBR322, using poly-dG tailed vector. Two cycles of transcription were used, initiated from one primer complementary to the 3' terminus, and another complementary to an internal sequence that occurs about 2.5 kb frσrrfthe 5' end. Approximately 8.6 kb of unique cDNA was derived from the first sequence and an additional 1.35 kb from the second, Referring to Fig. 2, positive strand viral RNA was extracted from JEV grown on Vero cells, by the RNA extraction method described by Repik et al., J. Virol. 20:157, 1976 and Repik et al., Am. J. Trop. Med. Hyg. 32:577, 1983. cDNA cloning was initiated using synthetic DNA primers corresponding to the 3' end of the RNA genome (3'-TTGTGTCCTAGA-5') or a sequence approx. 2.5 kb from the 5' end of the RNA genome (3'-GACCTCGTGGTTTACACCCT-5', present in PM-6, described below). 10μg of JEV RNA was treated with 50mM methylmercury hydroxide, in a 5 μl volume, for 10 minutes at room temperature (20-25°C) in the presence of the 3' synthetic primer, and then quenched with 5-fold molar excess of β-mercaptoethanol. Reverse transcription was carried out at room temperature for 10 minutes and at 42°C for 1 hour in a 100 μl reaction containing 50 mM Tris/HCl (pH 8.3 at 42°C), 6 mM

MgCl₂, 100 mM KCl, 1 mM dCTP, dGTP, dTTP, 0.5 mM [³²P]-dATP (2 Ci/mmole), 200 μl/ml gelatin, 50 units

RNAsin, and 100 units reverse transcriptase. The reaction was terminated by adjusting to 20 mM EDTA, pH 8.0, and 0.2 M NaCl. The mixture was chloroform extracted, separated on a Sephadex-G100 column in 10 mM Tris/HCl pH 8.0, 1 mM EDTA, 0.2 M NaCl, and ethanol precipitated.

Following procedure (A) in Fig. 2, the templates for second strand synthesis were prepared by heating the RNA:cDNA hybrids (250 μg) to 100°C for 2 minutes in 5 mM NaCitrate, pH 7.6, 0.5 mM EDTA, followed by quick cooling in ice-water. Second strand synthesis was carried out at 16°C for 16 hours in 100 μl containing 100 mM Hepes-KOH pH6, 10 mM MgCl₂, 2.5 mM DTT, 70 mM KCl, 1 mM deoxynucleotide triphosphates, and 25 units Klenow Fragment (DNA polymerase). The reaction was terminated by adjusting to 20 mM EDTA, pH 8.0 and 0.2 M NaCl, followed by chloroform extraction, passage through a Sephadex G-100 column, and ethanol precipitation. The double-stranded DNA (ds DNA) was treated with 500 units/ml S-1 nuclease in 100 ul of 50 mM NaCitrate pH 4.5, 0.3 M NaCl, 1 mM ZnCl₂ and 0.5% glycerol at 37°C for 30 minutes. After adjusting the reaction to 20 mM EDTA, pH8, the reaction was chloroform extracted and the ds DNA was size fractionated by chromatography through a Sephacryl S-1000 column to eliminate fragments less than 500 bps in length, and ethanol precipitated in the presence of 10 μg oyster glycogen as a carrier.

The fractionated ds cDNA (8-10 ng) was oligo dC tailed at the 3' ends using terminal transferase. The reaction was carried out in 50 ul containing 140 mM cacodylic acid and 30 mM Tris base adjusted to pH 7 with KOH, 1 mM CoCl₂, 0.1 mM DTT, 100 ul/1 ml gelatin and 25 mM dCTP. The reaction was incubated at 25°C for 10 minutes and then started by the addition of 50 units terminal transferase. Aliquots of the reaction (10 ul) were removed at 2, 4, 6, 8, and 10 minutes and added to 10 mM EDTA on ice to terminate the reaction. The reaction was heated to 70°C for 5 minutes, chloroform extracted, ether extracted, and ethanol precipitated. Plasmid vector pBR322, dG tailed at the Pst-I site (New England Nuclear) was annealed to the dC tailed cDNA in equimolar amounts and used to transform E. coli

HQ1574 (a recA derivative of MC1061) by standard procedures. cDNA inserts in pBR322 should inactivate the bla gene to produce Amp^s Tet^R colonies. Referring to Fig. 2, procedure (B), the PM-7 insert was generated by primer extension from the 20-mer synthetic DNA primer (see above). The procedure was essentially as described above except the RNA:cDNA hybrids from first strand synthesis were not melted prior to second strand synthesis which was carried out with the addition of 0.5 units of E. coli RNaseH. The resulting ds cDNA was separated from free nucleotides on a Sephadex G-100 column and oligo dC tailed for transformation as described above. The Tet^R Amp^s transformed clones were characterized by cross-hybridization on dot blots and the size and overlapping regions between related clones were mapped by restriction analysis. The cDNA clones were classified initially by cross-hybridization and restriction enzyme screening assays. The analysis was carried out with several of the larger cDNA clones and yielded a partial physical map of the genome. The results from the hybridization screening suggested that up to 30% of the insert-containing clones in the initial collection were unrelated to the JEV genome. Evidence that the cDNAs of four members were indeed of viral origin came from northern hybridization assays with viral RNA. A first round of screening was by a dot-blot procedure with electrophoretically enriched full-length virion RNA. A second series was then carried out using total RNA from both virus-infected and uninfected Vero cells. Final proof that the genomic bank corresponds to JEV sequences came from DNA sequencing and identification of several proteins encoded.

The structures of 5 cDNA clones are shown in Figure 3. The cDNAs range in size from 2.2 to 3.3 kb and together account for 10 kilobases of unique information. Results from primer extension analyses revealed that short segments of approximately 430 and 450 bases present in the genome at the 5'-and 3'-termini are missing from the collection of cloned JEV cDNAs. Example 2: λgtll library λgtll is an expression vector used to express JEV clones. DNA can be inserted into a unique EcoRI site and thereby be placed under the control of the E. coli lac operon promoter. Expression is induced using the chemical IPTG (isopropylthiogalactoside). While we have used this specific expression vector to express the proteins encoded in the JEV genome, since it has proven useful for the immunological identification of many different types of proteins (Young et al., Science 222:778, 1983; Proc. Natl. Acad. Sci U.S.A. 80:1194, 1983), other expression vectors could be equally suitable.

To identify viral protein coding sequences, random fragments of JEV cDNA were prepared and cloned into the unique Eco RI site of the lacZ gene of λgtll and banks of recombinants were screened with anti-viral antibodies. The bacteriophage recombinants that contain cDNA fragments inserted in the appropriate orientation and reading frame should express the inserted sequences in the form of JEV-β-galactosidase fusion proteins.

Bacteriophage libraries containing fragments of IEV cD A fused to the EN coli lacZ gene were prepared in λgtll by a slight modification of the procedure of Nunberg et al. (Proc. Natl. Acad. Sci. U.S.A. 81:3675, 1984). Plasmids containing JEV cDNA were digested with DNAse I in the presence of Mn ⁺⁺ to yield random fragments ranging in size from 100 base pairs (bp) to

7.5 kb. These fragments were treated with EcoRI methylase (New England Biolabs) to protect naturally occurring EcoRI sites from subsequent digestion, treated with T4 polymerase (P.L. Biochemicals) to repair ends, ligated to oligonucleotide linkers (GGAATTCC) containing an EcoRI site, and cut with EcoRI restriction endonuclease. Fragments from 100 bp to 5 kb in length were then resolved on Sephacryl S-1000 to remove excess linkers and different size fragment pools were ligated into EcoRI cut, calf intestinal alkaline phosphatase (CIAP) treated, λgtll DNA (either prepared with Soehringer Mannheim CIAP, or purchased directly from Promega Biotech, Madison, WI).

Subclones of selected JEV-λgtll recombinants were generated by cutting the JEV-λgtll DNA at specific internal restriction sites and recloning the JEV cDNA. Following restriction endonuclease cutting, the mixture of JEV-λgtll DNA fragments was treated with T4 DNA polymerase, ligated to the above EcoRI linkers and cut with EcoRI (regenerating the EcoRI sites in the original clone). The mixture was then recloned into CIAP-treated λgtll.

In order to identify clones in the λgtll library that were able to express viral proteins, and thus contain viral nucleic acid, libraries of recombinant phage were grown on lawns of E. coli strain Y-1090 and immunologically screened using the procedures of Young et al. (supra), except that blocking (for 30 minutes at room temperature) and antibody incubations (for either 4 hours at room temperature or 12 to 16 hours at 4°C) were performed in 20 mM Tris, 150 mM NaCl, pH 8.1 (TBS) containing 3% BSA, and washing consisted of three 15 minute rinses at room temperature; one in TBS, a second in TBS + 0.1% NP-40 and a final rinse in TBS. Nitrocellulose filter replicas of phage plaques were blocked, incubated in monoclonal (mcAbs) or polyclonal antibodies from murine ascites fluids (HMAF, having antibodies to JEV proteins, Brandt et al., Amer. J.

Trop. Med. Hyg. 16:339, 1967), washed, and incubated with either ¹²⁵I-labeled rabbit anti-mouse IgG or goat anti-mouse IgG coupled to alkaline phosphatase (Cappel Inc.). The filters were washed and, depending on the probe used, either autoradiographed or incubated in a phosphatase substrate mixture containing 5-bromo-4-chloroindoxyl phosphate and nitro blue tetrazolium. Immunological screening of 7,500 recombinants, with inserts ranging from 0.5 to 5 kb in length, yielded 47 clones chat reacted with the polyclonal HMAF. Screening of 4,000 recombinants generated with 0.1 to 1.0 kb fragments yielded an additional 44 HMAF-reactive recombinants. Although some of the clones obtained from the library constructed with larger cDNA fragments contained inserts of up to 2.0 kb, most of the clones contained inserts less than 500 bps long. Preliminary analysis showed that two of these recombinants expressed E protein antigenic determinants, and that the majority of the 90 clones expressed antigenic determinants for the NS3 protein. In order to maximize the expression of the sequences thought to encode the structural proteins, λgtll libraries were constructed that targeted the 5' end of the genome. One of these libraries, generated with 0.5 to 2 kb fragments of plasmid PM7 (pPM7, Fig. 3) yielded 12 recombinants that reacted with monoclonal antibodies to either the E or M proteins of JEV. An additional 24 HMAF-reactive recombinants, several of which reacted with the E and M mcAbs, were obtained from a second library by cloning small fragments of cDNA generated from the 5' end of the genome. Some of these cloned sequences are shown in Fig. 4, labelled with the prefix J7- or J-. Use

Nucleic Acid Probes JEV nucleic acid is useful for the detection of viral nucleic acid in whole animals, birds and insects and also in cell cultures derived from those sources. That is it can be used as a diagnostic probe for detection of Japanese encephalitis virus. In general this is a standard procedure involving nucleic acid hybridization technology. In the case of JEV one diagnostic assay involves the detection of viral nucleic acid in human cerebrospinal fluid. For example, one procedure involves fixing the nucleic acid from the test material to a solid matrix, such as a membrane filter, incubating the filter with a radioactively-labeled DNA probe under conditions that allow hybridization of the viral nucleic acid and the probe, and then detecting any such hybridization by the presence of label on the membrane filter. Alternative labels include other biochemical, chemical or physical agents, such as fluorescent molecules and enzyme- or immunological- based detectors.

The following is an example of use of one diagnositc probe. The cDNA used is designated PM-1 and approximately corresponds to the PM-6 cDNA shown in Fig. 3. The assay is able to positively detect JEV nucleic acid in clinical specimens and to differentiate them from specimens that contain no JEV-related nucleic acid. Dengue virus was not detected, whereas nucleic acid of the Murray Valley virus and West Nile virus were. Purified cDNA fragments or whole plasmids were radiolabelled for use in hybridization experiments with

³² P-deoxycytidme 5' triphosphate (300 Ci per mmole) using New England Nuclear (Boston, MA) nick trans lat ion ki ts . The transcription vector pGEM-4, is a plasmid having promoter sites for the binding of SP6 and T7 RNA polymerases, and was obtained from Promega Biotec (Madison, WI). Prior to use as a cloning vector, the plasmid DNA was restricted with PstI and subsequently treated with one unit of alkaline phosphatase for one hour at 37°C, and re-purified using phenol: chloroform extraction followed by ammonium acetate-ethanol precipitation. Purified unlabelled JEV cDNA fragments were annealed, ligated, and transformed into Escherichia coli HB101. Insert positive bacterial clones were detected by colony hybridization using radiolabelled insert DNA, and inoculated into LB broth with ampicillin (50 ug/ml). After incubation for 16 hours, plasmid DNA was isolated and the orientation of the insert DNA was determined by restriction enzyme analysis. Representative bacterial cultures were twice selected by pure culturing methods. Milligram amounts of insert-positive, transcription plasmid DNA (template DNA) were obtained using rapid extraction methods. The template DNA was linea'rized with the Sal I restriction enzyme, and re-purified by phenol: chloroform extraction followed by ammonium acetate-ethanol precipitation. Transcription reactions were performed according to the Promega Biotec procedure, using 1 ug of template DNA and 15 units of T7 poiymerase in a reaction mixture containing 30 units of

RNAse in, 10 mM dithiothreitol, 50 uCi ³²P-uridine 5' triphosphate (800 Ci per mmole), 500 uM cold ribonucleotide triphosphates (ATP, CTP, and GTP), and 12 uM uridine 5' triphosphate in transcription buffer supplied by the manufacturer. After a one hour incubation at 37ºC, the DNA template was destroyed by adding 1 unit of RQl DNAse and incubating the reaction mixture for an additional 15 minutes. The radiolabelled RNA was purified by phenol: chloroform extraction and ammonium acetate-ethanol precipitation. In general RNA is more sensitive as a probe than the corresponding DNA from which it is transcribed. However, since the flaviviruses contain unsegmented RNA of a single orientation (plus strand, messenger RNA-like RNA), it is important to determine which transcribed RNA probes have the appropriate orientation for detecting virus, using restriction analysis. For example, the orientation of the PM-1 insert in new recombinants was determined using Sst I. The plasmids pEH1002 and pEH1005 are representative JEV cDNA recombinants with plus and minus orientations, respectively (Figure 5). RNA derived from the pEH1002 plasmid, not the pEH1005 plasmid, is able to detect JE virus. To obtain a suitable RNA probe, pEH1002 was transcribed using T7 polymerase in the presence of

³² P-uridine 5' triphosphate, and the relative specific activity of the probes was compared to nick-translated probes produced with the original double-stranded plasmids. In general, the transcription reactions produced nucleic acid probes of higher specific activity. Nick-translated DNA radiolabelled using ³² P-deoxycytidme 5' triphosphate resulted in the production of DNA with a mean specific activity of single-stranded RNA probes at 4.7 X 10⁸ dpm per ug. Virus preparations for sensitivity testing consisted of flavivirus-infected cell culture supernatants diluted in phosphate-buffered saline (PBS) to approximately 100-200 plaque-forming units per 100 ul, and then diluted two-fold serially. Specimens for specificity tests were diluted 1:2 before use. Sample aliquots of 50 or 100 ul were treated with three volumes of 6.15 M formaldehyde in 10X SSC, incubated at 60°C for 15 minutes, and applied to slots of a Schleicher & Schuell (Keene, N.H.) vacuum manifold apparatus containing BA45 nitrocellulose paper. The paper was dried under a heat lamp, and baked at 80°C in a vacuum oven.

Oven dried nitrocellulose papers were treated with prehybridization buffer, (90 mM citrate buffer, pH 7.4, 0.9 M NaCl, 0.1% Ficol, 0.1% polyvinylpyrridolidone, 0.1% bovine serum albumin, 0.1% sodium dodecyl sulfate CSDS), and 100 ug/ml of denatured salmon sperm DNA), and the solution was replaced with fresh prehybridization buffer supplemented with dextran sulfate (1 g per 10 ml) and 10⁵ cpm per ml of labelled cDNA probe, which was boiled for 5 minutes before use. The bags were re-sealed and incubated for 16 additional hours at the appropriate temperature. After the incubation period, the papers were washed in 2X SSPE (1X SSPE was 0.01 M phosphate buffer, pH 7.4, 0.15 M NaCl, and 1 mM EDTA) for two hours in heat-sealable bags at 70°C, 1.0% SDS, twice at room temperature and four times with 2X SSPE, 0.1% SDS at 65°C or with 2X SSPE, 0.1% SDS at room temperature. Bound radioactivity was detected using Kodak X-OMAT X-ray film, with a fluorescent screen incubated at -70°C until processing using the manufacturer's directions.

In each case, the test was sensitive to amounts as low as between 8 and 16 plague-forming units of JEV after a 24 hour film exposure. No reaction was detected with uninfected cell culture supernatant. RNA transcribed from the pEH1005 plasmid was more sensitive for JEV than corresponding nick-translated DNA. A strong reaction with JEV nucleic acid was observed after 6 hours of X-ray film exposure, and a significantly weaker reaction was detected with the closely related West Nile virus.

Figs. 3 and 4 show numerous other clones that contain JEV nucleic acid useful for diagnostic probes, e.g. the PM, J7 and J series of clones. Other probes may be derived as well from JEV nucleic acid (specifically from the PM series of clones). Of those, the probes from the E and NS1 regions are likely to yield probes specific for JEV, but not the above-listed related viruses. The specificity of a probe can be determined experimentally, by comparing its hybridization reaction with various viral nucleic acids; or by choosing a sequence of at least 10 base pairs which correspond with a JEV sequence but not with the nucleic acid sequence of any other related virus, such as any one of yellow fever virus, Murray Valley virus, West Nile River virus, dengue virus or St. Louis encephalitis virus.

Thus, radiolabelled nucleic acid probes are generally suitable for the rapid detection of the

Japanese encephalitis viruses. In general, it is now possible to produce probes of high specific activity from various regions of the JEV genome which can detect as little as 10 plaque-forming units of JEV. Transcription vectors allow the production of single-stranded RNA probes with far greater specific activity than that obtained using nick-translated DNA probes. RNA probes can be used at higher concentrations and produce more easily detectable signals than probes produced using the nick-translation method. DNA probes were generally more specific than RNA probes when tested against closely related viruses. Production of Immunological Polypeptides Polypeptides suitable for the invention include antigenic proteins produced from cloned JEV nucleic acid, or from nucleic acid substantially corresponding to the nucleic acid of JEV, and the antibodies raised to these proteins. By substantially corresponding is meant that the polypeptides produced have an amino acid sequence corresponding to at least one antigenic determinant of JEV, or a related virus. These proteins may be isolated from a desired host, such as recombinant transformed or transfected prokaryotic or eukaryotic cells or organisms. Preferred are cells transformed with expression vectors which include bacteriophage, plasmids, cosmids, and mammalian, plant; insect and bird viruses, including vaccinia, retro-, adeno-, and rota-viruses. The cells may be E. coli, Bacillus, fungal, plant or animal host cells. The proteins may be purely JEV-derived proteins or fusion proteins, i.e., associated with other non-viral protein. In general these antigenic proteins are produced by insertion of viral nucleic acid into an expression vector (such as λgtll, see above) and causing expression of the inserted nucleic acid. Antibodies can be produced by purifying these antigenic proteins and injecting them into suitable animals. The antibodies raised can then be purified and used as probes for the antigenic proteins.

An example of such clone construction is given above where the recombinant antigenic polypeptides are hybrid E. coli-JEV proteins synthesized in E. coli.

Another example involves the construction of trpE fusion proteins with JEV proteins. Fragments of viral cDNA were fused to the trpE gene in plasmid pATH (Dieckmann et al. J. Biol. Chem. 260: 1513, 1985) and the resulting vectors transformed into E. coli HB101. Fusion proteins were produced and could be readily purified by the method of Kleid et al. (Science 214:1125, 1980) with yields of 20-30 mg of fusion protein per liter of cell culture. Parts of the M and E structural proteins and the three major non-structural proteins (NS1, NS3, NS5) have been expressed in this way.

The above described fusion proteins are useful for the detection of antibodies to JEV virus in the body fluids of infected animals. These proteins can be used as probes in any standard format, such as ELISA, western blot or similar tests. In particular, both JEV-E- and NS1-trpE fusion proteins are effective antigens for detecting anti-JEV antibodies in human serum.

One suitable ELISA format consists of coating the walls of a plastic microtiter plate with recombinant antigen, binding the antibodies in the test serum, and detecting the bound antigen-antibody couples with an enzyme conjugated second antibody and a chromogenic enzyme substrate. The enzyme used is horseradish peroxidase, the substrate ortho-phenylenediamine. In order to demonstrate the efficacy of such probes the following examples are meant to be illustrative and not in any way limiting to the invention.

The trp-JEV fusion protein preparations described above are useful in ELISA tests (Engvall, Methods in Enzymology, 70:419, 1980). They can detect antibodies of the IgG class present in sera of mice immunized with JEV; and the E-protein construct can bind to different anti-E protein monoclonal antibodies. Some of these monoclonal antibodies cross react with the E-proteins of other flaviviruses, and several of them also neutralize JEV in vitro and in vivo. The E-protein construct can be used to detect antibodies of the IgG class in JEV-infected human sera obtained from Bangkok. Further, these fusion proteins, when injected (5-20 μg emulsified in complete Freund's adjuvant) into mice, induce production of antibodies that bind to proteins from virus-infected cells in ELISA assays; and to appropriate viral proteins in western blots.

Referring to Fig. 6, JEV proteins and JEV-β-galactosidase fusion proteins, from E. coli cells transformed with specific vectors as described above, were electrophoresed in polyacrylamide gels containing SDS (Laemmli, Nature 227:680, 1970). Proteins were transferred to nitrocellulose paper as described by Towbin et al. (Proc. Natl. Acad. Sci. U.S.A. 76:4350, 1979). The nitrocellulose transfers (western blots) were then blocked in incubation buffer (20 mM Tris pH 7.4, 0.9% NaCl, 1% BSA, 0.01% NaN₃), incubated in the appropriate antibody (monoclonals to β-galactosidase, JEV-E, or JEV-M proteins) diluted in incubation buffer, and washed 3 times in wash buffer (20 mM Tris pH 7.4,

0.9% NaCl, 0.1% BSA, 0.05% NP40, 0.01% NaN₃). After reaction with ¹²⁵I-rabbit anti-mouse IgG diluted in incubation buffer, the filters were washed 3 times in wash buffer, dried, and autoradiographed. All antibody incubations were for either 4 hours at room temperature or 12 to 16 hours at 4°C.

Examination of the levels of expression of

JEV-3-galactosidase fusion proteins produced by many different recombinants suggests that recombinants containing shorter JEV cDNA inserts produce larger amounts of fusion proteins and give stronger signals in plaque assays. These data suggest that either the JEV cDNA insert size or specific viral sequences spread throughout the genome may affect the level of fusion protein production by these recombinants.

Preliminary analysis of portions of the JEV-E protein coding sequences shows that specific viral sequences are detrimental to the expression of β-galactosidase fusion proteins in E. coli. A comparison of the amounts of fusion protein produced by recombinant J7-1 and the subclones J7-1S and J7-1A shows that the removal of C-terminal sequences by cleavage at specific restriction sites increases the levels of β-galactosidase fusion protein accumulation by 10 to 20 fold (Figure 7). The efficient expression of fusion protein by recombinant J7-1S-2, which carries an in-frame, tandem duplication of the insert from J7-1S, shows that the size of the viral cDNA insert alone is not the critical variable in determining the level of accumulation of fusion proteins. As shown in Figure 8, the increased expression of the fusion proteins encoded by recombinants J7-1S and J7-1A correlates with removal of the membrane anchor domain (hydrophobic domain) of the mature E protein. Furthermore, all of the clones that carry this hydrophobic sequence (J7-1, J7-2, J7-5, J7-6, J-101) only express low levels of JEV-β-galactosidase fusion protein. These results are consistent with reports that the expression of foreign hydrophobic sequences can be toxic to E. coli (Yelverton st al . , Sc ience 219 : 614 , 1983 ) .

The fusion proteins can also be used to isolate specific antibodies from immune serum. Standard techniques can be used for such a process. These isolated antibodies are useful for the detection of viral antigens. As an example specific antibodies to structural and non-structural viral proteins were isolated.

Antibodies that recognize specific JEV-β-galactosidase fusion proteins were affinity-purified from HMAF following binding to E. coli proteins immobilized on nitrocellulose filters, as follows. Nitrocellulose filter disks were saturated with 10 mM IPTG, dried and then overlayed on purified recombinant phage densely plated (1,500 to 3,000 pfu/90mm plate) on E. coli strain Y-1090. Following incubation for 4 to 16 hours, the filters were washed in TBS, blocked in TBS + 3% BSA and incubated in a 1:30 dilution of HMAF in 3% BSA + TBS for 4 hours at room temperature. The filters were then washed 4 times for 15 min. each, one in TBS, twice in TBS + 0.1% NP-40 and once again in TBS. The specifically bound antibodies were eluted from the filters during a 60 second incubation in 2 ml of buffer containing 50 mM glycine HCl pH 2.3, 500 mM NaCl, 0.5% Tween 20, 0.01% BSA and the eluate was rapidly neutralized with an equal volume of 100 mM Na₂HPO₄ (Smith et al., J. Cell Biol.

99:20, 1984). The elution and neutralization process was repeated and the eluates were combined, diluted with one-half volume of TBS + 3% BSA and stored at 4°C. These antibodies can be used to identify the immunoceactive viral polypeptides in ELISA tests or on western blots prepared from either purified virus or virus infected cells. Rather than using purified proteins, crude unfractionated E. coli proteins, adsorbed from lawns of λ lysed E. coli strain Y-1090, can also be used as immunoadsorbants.

The feasibility of the affinity purification of antibodies was shown by using JEV-λgtll recombinants that reacted with mcAbs to purify antibodies from HMAF. JEV-β-gaiactosidase fusion proteins were prepared from E. coli Y-1089 infected with recombinant phage at an m.o.i. of 2:1. The E. coli lysogens were then grown to an OD₆₀₀ of 0.5 at 30°C (1.5 to 2.0 hours) in the presence of 10 mM IPTG. The cultures were shifted to 42°C for 15 minutes and then incubated for an additional 2 hours at 37ºC. The cells were collected by centrifugation and resuspended in SDS sample buffer (Laemmli, supra) containing 1% SDS, 1% β-mercaptoethanol and 1 mM PMSF. Protein extracts were prepared for electrophoresis by heating, sonication, and clarification as described above. These proteins were used to prepare western blots and probed with antibodies affinity purified from HMAF with: lane A, λgtll;

B,J7-8; CJ7-3; D,J7-2; E,J7-15; F,J7-1, or probed with unfractionated HMAF (lane G), anti-E monoclonal antibody (lane H), or anti-M monoclonal antibody (lane I); lane J is a Coomassie blue-stained virion protein preparation. The reactivity of these affinity purified antibodies with western blots prepared from both virion proteins and virus infected cell lysates showed that all of the recombinant proteins that reacted with mcAbs were able to purify antibodies with similar specificity from the HMAF (Figures 9 and 10).

Referring to Fig. 9, the reactivity of these affinity purified antibodies with viral proteins confirms that clones J7-1 (lane F) and J7-2 (lane D) express portions of the E proteins and that J7-3 (lane C) expresses portions of the M protein coding sequence. Unlike the mcAbs, the affinity purified antibodies may react with multiple viral determinants expressed by the JEV-λgtll recombinants, thus supplying additional specific immunological reagents. In the case of clone J7-8 (lane B) which shows the same spectrum of mcAb reactivity as clone J7-3 (reactivity to M mcabs, but not E mcAbs), the affinity purified antibody data demonstrates that this clone produces a fusion protein containing portions of both M and E. This result shows that there are antigenic determinants on E in addition to those recognized by the 9 anti-E mcAbs evaluated. Physical mapping of clone J7-8 to the JEV genome demonstrates that it contains portions of M common to J7-3, and sequences of E separate from those that react with the 9 anti-E mcAbs. Thus these affinity purified antibodies provide specific and useful reagents for the identification of viral sequences expressed in JEV-λgtll clones and provide specific polyclonal reagents to the viral proteins.

Referring to Fig. 10, the use of the above method for both structural and non-structural virus proteins is demonstrated. Preparations of JEV structural proteins were obtained from gradient purified virions harvested from JEV-infected Vero cells (Castle et al., supra). Total protein extracts of C6/36 Aedes albopictus mosquito cells grown for 80 hours, following infection (m.o.i. of 0.01:1) with JEV, served as a source of non-structural proteins. Infected cells were scraped into 2% SDS, 50 πiM Tris pH 6.8, homogenized and frozen. Immediately before electrophoresis, the samples were diluted with SDS sample buffer (Laemmli, supra) containing 1% β-mercaptoethanol and 1 mM PMSF, heated to 70°C for 15 minutes, sonicated briefly and clarified by centrifugation for 5 minutes in a microcentrifuge. The proteins were then western blotted and probed with antibodies affinity purified from HMAF with proteins from: lane A, λgtll; B,J-2; CJ-15; D,J-56; E,J-49; F,J-9; G,J-101; H,J7-1; I,J7-6; J,J7-8; K,J7-3; lanes L-Q represent immunological reaction of non-structural proteins of JEV probed with: L,HMAF (1:3,000 dilution) O,HMAF (1:1,000 dilution); P, non-immune ascites fluid (1:3,000 dilution); Q, non-immune ascites fluid (1:000 dilution); lane R, Coomassie blue-stained proteins from a mosquito cell lysate.

Using a combination of analyses similar to those given above, the specificity of reactivity of affinity purified antibodies with viral proteins, and a rapid scanning of plagues from unidentified immunopositive clones with affinity purified antibodies of known reactivity, it is possible to identify viral protein sequences that are recognized by a large number of immunoreactive recombinants. The immunological identity of the sequences encoded by 54 of the 91 recombinants (selected with HMAF from libraries generated from cDNA plasmids comprising most of the viral genome (pPM1, pPM2, pPM4, and pPM5)) have been identified using these techniques. 2 of these recombinants express portions of the E protein; 4 recombinants express portions of NS1; 5 recombinants that express portions of NS1', a higher molecular weight form of NS1; and 43 recombinants express portions of NS3.

As mentioned above, specific antibodies to viral antigens can be readily isolated. It is also possible to generate antibodies by injection of the recombinant proteins into animals, such as mice, rabbits and goats. These antibodies are isolated by standard procedures and can be used to detect viral antigens using ELISA tests, western blots and similar tests. Protective Immunogens

Immunogens of the invention include any recombinant antigenic protein which can elicit an immune response when injected into animals, such as humans, birds, or domesticated animals. The proteins are produced as described above; an illustrative example is given below. The preferred recombinant proteins include the NS1 and E proteins of JEV. The following example demonstrates the ability of these. recombinant proteins to serve as protective immunogens.

The above described trpE-fusion proteins were emulsified in Freund's complete adjuvant and 5-20 μg injected into mice. Antibodies were produced and isolated and found to bind to appropriate viral proteins on western blots of infected cell proteins. The antibodies were shown to be functionally equivalent to monoclonal antibodies which are able to neutralize JEV infectious to humans. In this test virus neutralization was demonstrated as a loss of infectivity by a virus preparation following incubation with the test antibody. Thus, the antibodies provide protection against virus.

The recombinant trpE-JEV proteins induce antibodies to JEV E and NS1 protein segments. These antisera, in turn, include antibodies that share specificity with monoclonal antibodies which, when injected intraperitoneally into mice, provide passive protection against JEV infection, and are thus protective immunologically. The functional equivalency of these two sets of antibodies was demonstrated by ELISA and western blot methods. Other Embodiments Other embodiments are within the following claims.

Other expression systems for the production of protective immunogens include viral vectors such as vaccinia. DNA can be inserted readily into the viral genome and the genes expressed when the virus is inserted into its mammalian host. Thus live vaccinia virus, having specific JEV sequences, can be inoculated into an animal, caused to express the JEV proteins and thence induce immunity to JE, or related viruses. Inoculation may be by injection or by oral or nasal administration.

One method which can be used to reduce the level of JEV within a human or domestic animal population involves immunization of the reservoir host, e.g., birds. This could be done by infecting the birds with a specific bird virus encoding the immunogens of this invention, thereby inducing immunity in the bird population, and reducing infection of other animals from this source.

Chemical processes can be used to produce the specific nucleic acid sequences of JEV, or to produce the antigenic proteins. Such synthetic chemicals are also useful in the invention. Deposit

An E. coli strain, HB101, harboring the recombinant plasmid vector pPM-7, has been deposited with the American Type Culture Collection, and assigned the number 67192.

Applicants acknowledge their responsibility to replace these cultures should they die before the end of the term of a patent issued hereon, 5 years after the last request for a culture, or 30 years, whichever is the longer, and their responsibility to notify the depository of the issuance of such a patent, at which time the deposits will be made available to the public. Until that time the deposits will be made available to the Commissioner of Patents under the terms of 37 CFR Section 1-14 and 35 USC Section 112.

Claims

1. Substantially purified nucleic acid comprising at least a 10 base pair sequence of DNA corresponding to the nucleic acid sequence of the Japanese encephalitis virus, but not to the nucleic acid sequences of yellow fever virus.

2. The nucleic acid of claim 1 wherein said 10 base pair sequence does not correspond to the nucleic acid sequence of West Nile River virus or Murray Valley virus.

3. The nucleic acid of claim 1 wherein said 10 base pair sequence does not correspond to the nucleic acid sequence of St. Louis encephalitis virus or dengue fever virus.

4. The nucleic acid of claim 1 wherein said 10 base pair sequence is included within the sequence shown in Fig. 1.

5. Substantially purified nucleic acid that hybridizes to nucleic acid of Japanese encephalitis virus but not to yellow fever virus under stringent conditions.

6. The nucleic acid of claim 5 wherein said nucleic acid does not hybridize to the nucleic acid of West Nile River virus or Murray Valley virus under stringent conditions.

7. The nucleic acid of claim 5 wherein said nucleic acid does not hybridize to the nucleic ac id of St . Louis encepha l it i s v irus or dengue virus under stringent conditions.

8. Substantially purified nucleic acid encoding a polypeptide having at least one antigenic determinant that is immunologically cross-reactive with a Japanese encephalitis virus-encoded protein, but not to proteins encoded by yellow fever virus.

9. Substantially purified nucleic acid encoding a polypeptide having at least one antigenic determinant that is immunologically cross-reactive with a Japanese encephalitis virus-encoded protein, but not to proteins encoded by West Nile River virus or Murray Valley virus.

10. Substantially purified nucleic acid encoding a polypeptide having at least one antigenic determinant that is immunologically cross-reactive with a Japanese encephalitis virus-encoded protein, but not to proteins encoded by St. Louis encephalitis virus or dengue fever virus.

11. A substantially purified nucleic acid sequence encoding a polypeptide sequence encoded by Japanese encephalitis virus, but not by yellow fever virus.

12. A substantially purified nucleic acid sequence encoding a polypeptide sequence encoded by Japanese encephalitis virus, but not by West Nile River virus or Murray Valley virus.

13. A substantially purified nucleic acid sequence encoding a polypeptide sequence encoded by Japanese encephalitis virus, but not by St. Louis encephalitis virus, or dengue fever virus.

14. The nucleic acid of claim 8 wherein said polypeptide raises immunological protection against Japanese encephalitis.

15. The nucleic acid of claim 8 or 11 wherein said Japanese encephalitis virus-encoded protein is the major envelope protein E or the non-structural protein NS-1 of said Japanese encephalitis virus.

16. The nucleic acid of claim 8 or 11 wherein said polypeptide is substantially similar to the major envelope protein E or the non-structural protein NS-1 of Japanese encephalitis virus.

17. The nucleic acid of claim 1, 4, 5, 8, 11 or 14 present in a vector, wherein said vector is chosen from a phage, plasmid, cosmid or eukaryotic virus.

18. The vector of claim 17 wherein said vector is baculovirus, vaccinia, rotavirus or adenovirus.

19. A substantially pure polypeptide synthesized from substantially purified nucleic acid that substantially corresponds to the nucleic acid of JEV.

20. A substantially pure polypeptide synthesized from the nucleic acid of claims 1, 5, 8, or 11.

21. The polypeptide of claim 19 or 20 wherein said polypeptide is a protective imrauhogen in man, or animals,

22. A method of diagnosis of Japanese encephalitis in biological sample, comprising providing a nucleic acid probe comprising the nucleic acid of claim 1, 4 or 5 and determining whether said probe hybridizes to nucleic acid in said sample.

23. The method of claim 22 wherein said sample is obtained from infected cells or infected organisms.

24. The method of claim 23 wherein said probe comprises nucleic acid encoding at least a part of the maj or envelope prote in E or the non-structural protein NS-1 of Japanese encephalitis virus.

25. A method of vaccination of an animal to raise protection against Japanese encephalitis, comprising inoculating said animal with a polypeptide of claim 19 or 20.

26. A method of vaccination of an animal to raise protection against Japanese encephalitis, comprising inoculating said animal with the nucleic acid of claim 14.

27. The method of claim 25 wherein said inoculation is by injection .

28 . The method of claim 25 wherein said inoculation is by oral or nasal administration.

29. The method of claim 26 wherein said inoculation is by an insect vector.

30. The method of claim 25 wherein said vaccination induces immunity to the disease caused by another flavivirus.

31. The method of claim 25 wherein said animal is a human or a domesticated animal.

32. The method of claim 25 wherein said animal is a bird.

33. The method of claim 26 wherein said vaccination induces immunity to the disease caused by another flavivirus.

34. The method of claim 30 or 33 wherein said flavivirus is chosen from yellow fever virus, West Nile

River encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, or dengue fever virus.

35. The method of claim 26 wherein said animal is a human or a domesticated animal.

36. The method of claim 26 wherein said animal is a bird.

37. A method of diagnosis of Japanese encephalitis comprising detecting immunologically reactive polypeptides with an antigenic polypeptide of claim 19 or 20 or with antibodies produced to said antigenic polypeptides.

38. The method of claim 37 wherein said detecting is by an ELISA test or western blot.

39. Chemically synthesized polypeptides which are substantially similar to said polypeptides of claim 19.

40. Antibodies produced to a polypeptide of claim 18 or 19.

41. Antibodies produced to a polypeptide of claim 39.