KR100430960B1 - Infectious Japanese encephalitis virus cDNA clone and its vaccine to produce highly attenuated recombinant Japanese encephalitis virus - Google Patents

Abstract

The present invention relates to a recombinant, attenuated Japanese encephalitis virus comprising a single amino acid variation in an envelope protein. Specifically, the present invention relates to an isolated DNA molecule encoding the genome of the Japanese encephalitis virus strain. The present invention also relates to a method for producing Japanese encephalitis virus strain from cultured animal cells. In addition, the present invention relates to a pharmaceutical composition for preventing toxic Japanese encephalitis virus.

Description

Infectious Japanese encephalitis virus cDNA clones and vaccines thereof which produce highly attenuated reconstruction Japanese encephalitis virus

This application is part of a pending US serial number 08 / 348,882 filed on November 28, 1994, filed on November 28, 1994, which is now abandoned and is filed on June 15, 1993, filed on June 15, 1993. to be.

The present invention relates to a recombinant, attenuated Japanese encephalitis virus comprising a single amino acid variation in an envelope protein. Specifically, the present invention relates to an isolated DNA molecule encoding the genome of said Japanese encephalitis virus mutation. The present invention also relates to a method for producing Japanese encephalitis virus mutations from cultured animal cells. Furthermore, the present invention relates to a pharmaceutical composition for preventing toxic Japanese encephalitis virus.

Japanese Encephalitis Virus (JEV) is a flavivirus, a mosquito-borne medium that causes signs of severe encephalitis and neurological diseases and causes numerous cases of fatal acute encephalitis in Asia each year. Typically, signs of JEV infection appear after a culture period of 4-20 days. The patient comes to the hospital with signs of severe central nervous system infections such as high fever, seizures and coma. However, no effective treatment is available. See Hoke et al., J. Infect. Dis. 165: 631 (1992). About one third of hospitalized patients die within 10 days, and the other 1/3 recover with severe and permanent neuropsychiatric defects. More than 10,000 people are officially announced to die from JEV infection each year, but in practice far exceed those figures [Burke et al., "Japaneseencephalitis," in THE ARBOVIRUSES: EPIDEMIOLOGY AND ECOLOGY AND Volumn 3, (Monath TP, ed. ), pages 63-92 (CRC Press 1988).

The JEV genome contains about 11 kb of single-stranded RNA molecules encoding three structural proteins, namely capsid, membrane and envelope proteins, as well as seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. [Sumiyosi et al., Virology. 161: 479 (1987). The genomes of the virulent JVE strains JaOArS982, Nakayama, Beijing and SA ₁₄ , as well as the attenuated SA ₁₄ -14-2, have been analyzed at the molecular level, revealing similar structural configurations. Id .: McAda et al., Virology. 158: 348 (1987); Hashimoto et al., Virus Genes 1: 305 (1990); Nitayaphan et al., Virology. 177: 541 (1990). The relationship between certain JEV proteins and diseases on toxicity is far from understood.

Currently, there are three types of JEV vaccines: (1) vaccines obtained from formalin-treated non-infected virus grown in mouse brain or tissue culture, and (2) viruses attenuated by a series of passages in animals. (3) vaccines containing vectors that express flavivirus proteins, such as recombinant vaccinia vectors, but despite the importance of public health for JEV, currently available vaccines are effective for several reasons. none.

Formalin-inactivated, mouse-brain-derived JEV vaccines were developed during World War II and have been used in Japan for more than 20 years. The vaccine had reduced both morbidity and mortality due to JEV in Japan. Hoke et al., N. Engl. J. Med. 319: 608 (1988). However, as the standard of living improved, the prevalence of JEV decreased somewhat.

There are many problems with inactivated vaccines due to the fact that the virus must be generated from live mice, recovered and purified by laborious procedures, and killed by formalin treatment. Since the formalin-treated virus cannot replicate in the receptor, successful immunization requires repeated administration of large amounts of viral antigens and vaccines. In addition, the effects of immunization using killed vaccines do not last as long as the immunity provided by live vaccines. Finally, the need to remove mouse antigens and the need for careful screening of inactivation treatments raises vaccine manufacturing costs. Therefore, inactivated vaccines are expensive to manufacture, making them unsuitable for most developing countries where JEV is causing serious problems.

Substantial efforts have been made to develop live, attenuated vaccines. These efforts are motivated by the hope of producing attenuated viruses in humans when a series of JEV passages occur in mice. The most potent vaccines described below may not be ideal because the virus contains a number of mutations and the effects of these mutations are unclear.

Live, attenuated JEV strain SA ₁₄ -14-2 has been empirically produced by the cells, a series of passages in culture and laboratory animals inoculated. Comparison of the nucleotide sequences of the attenuated virus SA ₁₄ -14-2 and its parent virus (SA ₁₄ ) revealed that the attenuated virus had 57 nucleotides substituted (as a result, amino acid 24 was substituted). Aihara et al., Virus Genes 5: 95 (1995). However, the parent virus's own genome is very different from that of other wild type JEVs. This observation amplified the question of whether any strain derived from the parental virus could protect individuals exposed to various JEV strains.

The SA ₁₄ -14-2 virus vaccine has been used in humans in China. Human seroconversion (neutralizing antibodies) is between 60 and 90% [Ao et al., Chinese J. Microbiol. and Immunol. 3: 245 (1981). However, multiple vaccine administrations are required to induce seroconversion, and each administration must be high titer (about 106 PFU). In the method, SA ₁₄ -14-2 vaccine is similar to the above-mentioned inactivated vaccine. In addition, the emergence of Japanese encephalitis has been reported to occur in individuals receiving the vaccine despite immunization with two high-titer vaccine doses fiddm.

SA ₁₄ -14-2 has been inoculated with passaged further in the primitive of kidney cells (PCK) [see: Eckels, etc., Vaccine, 6: 513 (1988 )] produced a virus, SA-14-14-2-PCK Was determined and its virulence markers were specified in cell culture and laboratory animals. Nitayaphan et al., Virology. 177: 541 (1990); Hase et al., Arch. Virol. 130: 131 (1993). In laboratory mice, immunization using various doses and dosing regimens of SA-14-14-2-PCK results in low seroconversion rates as can be measured by the presence of serum to neutralize antibodies. In addition, regardless of the immunizing vaccine dose, nearly all of the immunized laboratory mice were killed after the cerebral antigen administration of the parental virus.

In addition, SA-14-14-2-PCK virus was experimentally used as a vaccine in China in the 1980s. However, the attempt ended in failure because the subject receiving the vaccine failed to produce JEV antibodies. JEV SA ₁₄ -14-2 the cumulative result obtained by using the derivatives and PCK was suggested that similar behavior with which the attenuated virus inactivation than the active vaccine vaccine.

Several promising JEV vaccines have been produced in which JEV proteins can be delivered into a host by a carrier or recombinant vector. The most promising vaccine to date is using a recombinant virus derived from the vaccinia virus named NYVAC. Neutralizing antibodies inoculated with vaccinia-JEV recombinant virus encoding JEV envelope proteins were detected. Although mice inoculated in laboratory mice were protected against intraperitoneal antigen administration using toxic viruses, neutralizing antibody titers and seroconversion rates were lower than observed after immunization with a killed vaccine. In addition, protection against JEV was only partial (see Yasuda et al., J. Virology. 64: 2788 (1990); Msson et al., Virology 180: 294 (1991); Jan et al., Am. J. of Trop. Med. and Hyg. 48: 412 (1993). Therefore, multiple immunizations with recombinant vectors are required to protect laboratory mice from lethal antigen administration. In addition, immunizations using vectors expressing only JEV envelope proteins do not stimulate humoral and cellular immune systems that respond to all JEV proteins actively expressed in JEV infected cells.

In summary, attenuated live vaccines are often preferred for other types of vaccines, because attenuated vaccines may (1) have a longer lasting immunogenicity based on both humoral and cellular responses. Induction, (2) inexpensive to manufacture, and (3) usually requiring only a single dose for immunization. On the other hand, the risk of vaccine reversal to toxicity is a continuing problem of empirically derived attenuated active vaccines.

Thus, there is a need for attenuated JEV vaccines that do not replicate in the nervous system and elicit an immune response against various JEV proteins. In addition, the genome of the attenuated JEV must be fully characterized so that the presence of the molecular determinant of the attenuated phenotype can be identified and demonstrated in the final vaccine preparation.

It is therefore an object of the present invention to provide an attenuated Japanese encephalitis virus suitable for stimulation of an immune response to JEV in an animal.

It is a further object of the present invention to provide a DNA molecule having a known nucleotide sequence encoding said mutant virus.

These and other objects are isolated variants of Japan, distinguished from the corresponding wild type JEV by substituting an acidic amino acid for a basic amino acid at position 138 of the envelope protein, wherein the acidic amino acid is glutamic or aspartic acid and the basic amino acid is lysine or arginine. In accordance with one aspect of the present invention by providing encephalitis virus (JEV).

In a preferred embodiment, the wild type JEV is JEV strain JaOArS982 and the basic amino acid of the mutant JEV is lysine. In a more preferred embodiment, the wild type JEV is JEV strain JaOArS982 and the basic amino acid of the mutant JEV is arginine.

The present invention also provides an isolated DNA molecule encoding the genome of the mutant JEV, wherein the mutant JEV is distinguished from the corresponding wild type JEV by a mutation consisting of one or two nucleotide substitutions in the gene encoding the envelope protein. do. Specifically, the present invention provides the DNA molecule, wherein the mutation is an acidic amino acid (glutamic acid or aspartic acid) at position 138 of the envelope protein of wild type JEV and a basic amino acid at position 138 of the envelope protein of mutated JEV (lysine). Or arginine).

The invention further provides a method of preparing a mutant JEV from cultured animal cells, comprising the following steps:

(a) synthesizing full length viral genomic RNA in vitro using a DNA molecule encoding mutant JEV as a template;

(b) generating a virus by transfecting the animal cells cultured using the viral genomic RNA; And

(c) separating the virus from the cultured animal cells.

The present invention also provides a method of preparing a neutralizing antibody against JEV in an animal comprising administering a therapeutically effective amount of a pharmaceutical composition comprising (a) a mutant JVE and (b) a pharmaceutically acceptable carrier. In this case, the mutant JEV is distinguished from the corresponding wild type JEV since the acidic amino acid is replaced with a basic amino acid at position 138 of the envelope protein.

The present invention also provides a pharmaceutical composition comprising the mutant JEV and a pharmaceutically acceptable vehicle. The pharmaceutical composition may be administered to the mucosa or may be administered in an injection form selected from the group consisting of subcutaneous injection, intradermal injection and intramuscular injection.

The present invention contemplates a method for preparing a mutant JEV, comprising the following steps:

(a) synthesizing full length viral genomic RNA in vitro using a DNA template comprising a cDNA insert of pM343 (ATCC No. 75490);

(b) transfecting the monolayer culture of animal cells using the viral genomic RNA, wherein the transfection forms virus-containing plaques of various sizes in the monolayer culture; And

(c) separating the virus from smaller plaques in monolayer culture.

In addition, the isolated mutant flaviviruses of the present invention are distinguished from the corresponding wild-type flaviviruses in that the basic amino acids in the envelope proteins of the mutant flaviviruses have been replaced with acidic amino acids in the envelope proteins of the wild-type flaviviruses. The non-virus is selected from the group consisting of Murray Bali encephalitis virus, St. Louis encephalitis virus, true tick-borne encephalitis virus, and Central European encephalitis virus, wherein the acidic amino acid is glutamic acid or aspartic acid and the basic amino acid is lysine or arginine .

As mentioned above, there is a need for a safe, effective and economical active JEV vaccine for use in humans. Previous attempts to develop attenuated active vaccines have used empirical methods of passage of wild-type viruses using mice as hosts. In this approach, the only hope is to introduce a large number of mutations randomly, and then hope that the mutations show an attenuated phenotype.

To overcome this limitation, we developed a system for producing JEV from infectious cDNA clones. What was unexpected was that the method produced a very attenuated virus. The structure and function of these mutant viruses were characterized and additional attenuated viruses were prepared using in vitro variations. These studies revealed that the location of neurotoxicity against JEV is amino acid position 138 in the envelope protein.

The methods described herein allow the preparation of highly improved, attenuated active vaccines for JEV. In addition, the identification of neurotoxic sites makes them available for vaccine production against other neurotoxic flaviviruses.

1. Construction of Infectious JEV cDNA Clone

Methods for constructing infectious JEV cDNA clones are described by Sumiyoshi et al., J. Virol. 66: 5425 (1992). Briefly, oligonucleotide primers representing the 3 'and 5' ends of the genome of the JEV strain JaOArS982 ("parent JEV") were synthesized. Nucleotide sequences of the parent JEV are disclosed in Sumiyoshi et al., J. Virology 161: 497 (1987). First strand synthesis was performed using parental JEV genomic RNA as the 3 'end primer and reverse transcriptase template. Double stranded cDNA was performed using the 5 'end primer and the first strand of cDNA as a template. The cDNA molecule was then digested with BamHI and SalI and the fragments inserted into pBR322. The cDNA library was cloned into the Competent E. coli using recombinant pBR322. It was constructed by transforming Coli HB101 cells.

Full length DNA copies of the JEV genome were constructed by in vitro linking two cDNA molecules, each encoding about one half of the viral genome. As illustrated, the full length JEV cDNA, called “IC37,” was prepared using 5′-1 / 2 cDNA pM343 comprising JEV sequences from nucleotide 1 to nucleotide 5576 (see FIG. 2). CDNA molecules containing 3′-1 / 2 of the JEV genome were obtained by separating 5,400 bp fragments from cDNA clones; The fragment contains a portion of the JEV genome from nucleotide 5577 to the 3 'end. Finally, the 3 'and 5' end clones were joined to make a full length JEV cDNA clone, IC37.

Infectious JEV was obtained using IC37 as a template for RNA transcription as described in Example 1 and Sumiyosi et al. (1992). Transcription products were transfected into cultured mammalian cells, and infectious Japanese encephalitis virus was recovered from the transfected cells. As described below, viruses produced from IC37 were found to be functionally identical to the parent JEV. Thus, the infectious virus obtained from IC37 cDNA was called "recombinant wild type JEV".

2. Construction of cDNA Molecules Encoding Mutant JSV

The above-described approach can be used to obtain Japanese encephalitis virus mutated genome. The ultimate goal of in vitro mutagenesis is to prepare efficient attenuated vaccines. As an optional test, one particularly useful mutation was introduced into a cDNA molecule called pH756, which encodes the 3 'end of the JEV genome.

As described in Example 2, site-directed mutagenesis was used to introduce a silent mutation that eliminates XbaI restriction sites. Mutated cDNA, "pH756X" and pM343 were linked together, and the linked DNA was subjected to a T7 polymerase reaction to prepare RNA transcripts. Cultured cells were transfected with RNA and infectious JEV was recovered from the transfected cells. Viral genomic DNA molecules were prepared by reverse transcription-polymerase chain reaction. Subsequent analysis of the sequence revealed that specific asymptomatic mutations were present in the recovered genome of the virus. The results demonstrated that certain mutations can be introduced into the JEV genome using the methods described herein and that the mutations are stable.

3. Preparation of Attenuated Recombinant JEV

As illustrated in Figure 1, various infectious JEV cDNA clones were constructed by linking combinations of 5 'and 3' cDNA clones. In particular, the 5 'fragments obtained from pM343 and pM433 were linked with the 3' fragments obtained from pH756, pH313, pH173 and pH756X. The cDNA clones pM343, pM433 and pH756 were deposited on June 17, 1993 in the United States Model Culture Collection (ATCC), Rockville, Maryland, United States, and the plasmids were respectively identified by ATCC No. Accession numbers 75750, 75491 and 75492 have been assigned. Japanese encephalitis virus JaoArS982 strain IC47 was deposited on June 17, 1993, in the US Bacterial Culture Collection, ATCC No. VR2412 was granted.

When cultured in hamster kidney cells, all recombinant viruses appear to grow at similar rates. However, the virus comprising the 5 'cDNA, pM433, produced significantly smaller plaques than the plaques produced by the toxic parent JEV. The small plaque may mean that the virus has been attenuated. As described herein, we found that full-length JEV cDNA clones derived from pM433 were nontoxic, while JEV cDNA clones derived from pM433 were toxic (see Examples 1, 2 and 4).

Nucleotide sequence analysis revealed that pM433 contained a single nucleotide substitution compared to pM343. In the envelope protein of pM343, the 138th amino acid is glutamic acid encoded by GAA, whereas in the envelope protein of pM433, the 138th amino acid is lysine encoded by AAA. The results represent the first and only, clear definition of neurotoxic location within the genome of a neurotrophic flavivirus.

In addition, these studies suggest that additional attenuated JEV can be produced by mutating wild type codon GAA with AAG, which also encodes lysine. Mutations can be introduced into cDNA molecules encoding the 5 'end of the JEV genome using known techniques such as oligonucleotide-directed mutagenesis, linker-scanning mutagenesis, mutagenesis using polymerase chain reactions, and the like. See, eg, Examples 1 and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, pages 8.0.3-8.5.9 (John Wiley & Sons 1990). See also McPherson, DIRECTED MUTAGENESIS: A PRACTICAL APPROACH (IRL Press 1991). ].

More generally, the discovery of a neurotoxic position suggests that the acidic amino acid at position 138 of the envelope protein is associated with toxicity, while the basic amino acid at that position exhibits an attenuated phenotype. Therefore, by mutating ¹³⁸ Glu to ¹³⁸ Asp, it should be possible to maintain a toxic phenotype. On the contrary, attenuated JEV should be prepared by mutating the acidic amino acid to the basic amino acid arginine at position 138 of the envelope protein. The attenuated mutants can be obtained by mutating wild type codon GAA to CGT, CGC, CGA, CGG, AGA or AGG.

The study presented in Example 7 confirms this hypothesis. Briefly, we found that recombinant JEVs containing ¹³⁸ Asp in envelope proteins are lethal to mice, whereas recombinant JEVs carrying AGA (Arg) codons at position 138 show an attenuated phenotype. The discovery of neurotoxic sites at position 138 of the envelope protein is applicable to the production of attenuated vaccines for other neurotoxic flaviviruses such as the Murray Bali encephalitis virus, St. Louis encephalitis virus and the Central European encephalitis virus. The virus can be obtained from public deposits such as the United States Model Culture Culture Collection (Murley Bali Encephalitis Virus: ATCC No. VR-77; St. Louis Encephalitis Virus: ATCC No. VR-80 and VR-1265).

Alternatively, DNA molecules encoding the flavivirus genome can be obtained by chemical synthesis using known nucleotide sequences. For example, the DNA molecule can be obtained by synthesizing the gene using long oligonucleotides that prime each other (Ausubel pages 8.2.8-8.2.13 and Wosnick et al., Gene 60: 115 (1987)). In addition, these techniques can be extended by synthesizing genes of 1.8 kb in length using polymerase chain reactions. See, Adang et al., Plant Molec. Biol. 21: 1131 (1993); Bambot et al., PCR Methods and Applications 2: 266 (1993). Mandl et al., Virology 194: 173 (1993) disclose the nucleotide sequence of flavivirus poisan, which is a true tick mediator, whereas the nucleotide sequence of encephalitis virus whose western subtype true tick is a mediator is described in Mandl et al., Virology 166: 197 (1988) and Mandl et al., Virology 173: 291 (1989).

In summary, it is possible to construct an attenuated JEV by mutating an acidic amino acid residue to a basic amino acid residue at position 138 of the envelope protein. Example 7 shows that a nontoxic JEV bearing a single nucleotide substitution retains the attenuated phenotype even after 10 passages in murine. Nevertheless, the preferred attenuated Japanese encephalitis virus retains two nucleotide substitutions, ensuring that less reversal to wild type occurs. Thus, the preferred attenuated virus contains an arginine residue at position 138 of the envelope protein because two nucleotide substitutions are necessary to convert the codon encoding arginine to the codon encoding either glutamic or aspartic acid. .

4. How to use attenuated recombinant JEV as a vaccine

As described above, the mutant JEV SA ₁₄ -14-2 represents an attenuated phenotype for laboratory animals such as mice, humans vaccinated using said strain stimulates neutralizing antibodies [see: Eckels, etc., Vaccine 6: 513 (1988). Meanwhile, SA14-14-2-PCK virus shows low seroconversion rate in mice, which does not stimulate neutralizing antibodies in the human body. Thus, studies using attenuated JEV demonstrate a correlation between the ability of attenuated JEV strains to induce seroconversion in mouse and human vaccines. Thus, the results of the in vivo studies presented herein indicate that the mutant Japanese encephalitis virus of the present invention is particularly effective in stimulating immune responses in human vaccines.

Because viruses are intracellular obligate parasites that cannot grow in inanimate media, attenuated JEVs must be propagated in biological media such as cultured mammalian cells and chick embryo cell cultures. Techniques for purifying attenuated viruses from host cells are well known to those skilled in the art. Wang et al., Chin. J. Virol. 6: 38 (1990). The literature describes a method for producing the SA ₁₄ -14-2 vaccine from infected tissue culture cells. See also, generally, REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition (Mack Publishing Co. 1990).

Dosage forms of attenuated viral vaccines and suitable dosages of such vaccines are well known to those skilled in the art (REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition (Mack Publishing Co. 1990)). For example, the attenuated JEV vaccine can be administered to human receptors by intramuscular injection, subcutaneous injection, or intradermal injection. In general, the dosage of the vaccine will vary depending on factors such as the age, weight, height, sex, general medical condition and previous medical history of the receptor. Generally, 0.5-1 ml of the vaccine will be administered as a combination containing 10 ² -10 ⁶ PFU / ml. JEV through the use of a live attenuated vaccine experience, SA ₁₄ -14-2 may provide guidance on the proper doses of the attenuated recombinant JEV described herein [see: Yu, etc., Chin. J. Microbiol. Immunol. 1: 77 (1981), Ao et al., Chin. J. Microbiol. Immunol. 3: 245 (1983); Yu et al., Am. J. Trop. Med. Hyg. 39: 214 (1988), and Tsai et al., “Japanese Encephalitis Vaccines,” in VACCINES, 2nd edition, Plotkin et al., Pages 671-713 (WB Saunders Co. 1994).

The vaccines of the present invention can be combined according to known methods to prepare pharmaceutically useful compositions so that the attenuated virus can be mixed with a pharmaceutically acceptable vehicle in a mixture. Such compositions are referred to as "pharmaceutically acceptable vehicles" if the receptor can tolerate administration of the compositions. Sterile phosphate-buffered saline is an example of a pharmaceutically acceptable vehicle. Other suitable vehicles are known to those skilled in the art (REMINGTON'S PHARMACEUTICAL SCIENCES, 18th edition (Mack Publishing Co. 1990)).

For the purpose of treatment, attenuated JEV and pharmaceutically acceptable vehicle are administered to a human receptor in a therapeutically effective amount. If the dose is physiologically significant, it is stated that the combination of virus and pharmaceutically acceptable vehicle is administered in a "therapeutically effective amount". The physiological equivalent of any reagent means that the presence of the reagent causes a physiologically detectable change in the receptor. Physiologically prominent any reagent herein means that its presence stimulates the production of neutralizing antibodies against Japanese encephalitis virus.

The present invention described generally above is more easily embodied by the following examples, which are provided by way of example but are not intended to limit the present invention.

Example 1

Development of infectious cDNA clones encoding wild type JEV or mutant JEV

A. Composition of JEV cDNA Clone

Infectious wild-type JEV cDNA clones encoding strain JaOArS982 are described by reference in Sumiyoshi et al., J. Virol. 66: 5425 (1992). Briefly, two cDNA clones (pM343 or pM433) encoding the 5'-1 / 2 of the JEV genome and cDNA (pH756) corresponding to the 3'-1 / 2 of the genome were in vitro linked to a T7 polymer. Two full length templates were prepared for Laase (see FIG. 2). 50 μl of the RNA synthesis reaction mixture contains 1 μg of cDNA template, 40 mM Tris-HCl (pH 8.0), NaCl 25 mM, MgCl ₂ 8 mM, spermidine 2 mM, dithiothritol 5 mM, CTP, UTP and GTP 1 mM, ATP 0.1 mM, m ⁷ G (5 ') ppp (5') A 1 mM, RNase inhibitor 5U and 0.2 μl of T7 RNA polymerase.

RNA molecules transcribed from the cDNA template were transfected into BHK-21 cells and later virus was recovered (Chou et al., Adv. Enzym.Relat. Areas Mol. Biol. 47:45 (1978). In this study, BHK-21 cells were minimally supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 0.1 mM MEM non-essential amino acid, 0.3% sodium bicarbonate, 10 U / ml penicillin and 10 μg streptomycin. Growing in essential medium (MEM). BHK-21 cell monolayers in 35 mm dishes were washed with phosphate-buffered saline (PBS), RNA transcripts and 10% lipofectin® (Bedesda Research Laboratories, Maryland, United States). Incubated for 10 minutes at room temperature with 400 μl of PBS containing, obtained from Incorporated. The cells were then washed with PBS and top coated with 1% agarose containing 2% FCS in MEM. After incubation for 2-3 days, the cells were stained with neutral red to visualize viral plaques.

Viruses recovered from full-length cDNAs derived from 5'-1 / 2 pM343 and 3'-1 / 2 pH756 were named IC37. The characteristics of this recombinant virus could not be distinguished from those of the parent JEV. Viruses recovered from cDNA templates prepared from pM433 and pH756 were named IC47. Surprisingly, the characteristics of IC47 were distinguished from those of the parental virus and IC37.

B. Inducing Site-Specific Mutations in Wild-type JEV Clones

cDNA clone pH756 contained an XbaI restriction site unique to nucleotide position 9131. In vitro position-directed mutagenesis was used to introduce nucleotide substitutions (ie, asymptomatic mutations) that eliminate the XbaI position without altering the amino acid sequence. Studier et al., J. Mol. Biol. 189: 113 (1986). The mutated cDNA was used as a template for T7 RNA polymerase and recovered using this procedure.

Genomic RNA was purified from the recovered virus particles, which RNA was used as a template for cDNA synthesis and polymerase chain reaction (PCR) amplification. Restriction analysis of the resulting cDNA indicated that the clone lacked the XbaI position at nucleotide 9131. The results indicated that (1) the recovered virus was derived from mutated cDNA and is non-infectious, and (2) site-specific mutations can be introduced into Japanese encephalitis virus by this technique.

C. Structure and function analysis of JEV cDNA clones in vitro

Proliferation rates of IC37 and IC47 parental viruses were compared in BHK-21 cells infected with multiple 10 PFUs per cell (FIG. 3). The virus was indistinguishable based on proliferation rate, but the plaque size of IC47 was much smaller than that of the parental virus and IC37.

Original BHK-21 cells were isolated from kidney tissue. To investigate the growth of IC37 and IC47 in cells derived from neural tissues, N18 cells were incubated with other viruses at multiple 10 PFU per cell. As shown in Figure 4, IC37 grew faster than IC47. This observation may be related to the low rate of IC47 proliferation in murine brain tissue and the attenuated phenotype of IC47 as described below.

To compare the stability of parental and IC47 viruses, a solution of culture medium containing 10% FCS and parental or IC47 1000 PFU / ml was incubated in a 37 ° C. CO ₂ incubator. Viruses were measured for titer at each time point by plaque assay. As shown in FIG. 5, no obvious difference in virus stability was found.

Nucleotide sequence analysis of cDNA revealed that pM343 (ie, 5'-1 / 2 of IC37) and pM433 (ie 5'-1 / 2 of IC47) differed in single codon at position 138 of the envelope protein. In the envelope protein of pM343, the 138th amino acid is glutamic acid encoded by GAA, while the 138th amino acid of the envelope protein of pM433 is lysine encoded by AAA. Since 3'-1 / 2 of IC37 and IC47 are derived from the same cDNA clone, the results showed that a single amino acid change at position 138 in the envelope protein results in altered plaque size. In addition, the mutations were shown to cause serious changes in neurotoxicity as described below.

Example 2

Neurotoxicity of Wild-type JEV, IC37, and IC47 Viruses in Mice

Neurotoxicity was compared using Swiss ICR mice against recombinant virus, IC37 and IC47, and parental JEV. In preliminary experiments, three week old mice were inoculated with 10 to 10 ⁶ PFU using either dosing method in either cerebellar (ic) or intraperitoneal (ip) administration. All mice intraperitoneally inoculated with the parental virus or IC37 were killed, but the mortality of mice intraperitoneally inoculated with other viruses was about 50%. In contrast, all mice inoculated with IC47 intraperitoneally or in the brain survived. These studies revealed that IC37 and the parent virus are indistinguishable in their ability to kill mice, but IC47 is nontoxic.

In a second study, eight Swiss ICR xcross mice were challenged with parental virus, IC37 or IC47. Three and five week old mice were inoculated intracranially at 100 PFU, while mice two and three weeks old were intraperitoneally inoculated at 100 PFU. After intraperitoneal inoculation with the parental virus or IC37, the mortality rate of mice at 2 weeks of age was 100%, and the mortality rate of mice at 3 weeks of age was 50% (see Figures A and B in Figure 6). Intracerebellar inoculation with parental virus or IC37 resulted in 100% lethality in mice 3 and 5 weeks old (see Figures 6 C and D). All mice inoculated with IC47 survived without signs of disease.

Example 3

Neutralizing Antibody Production and Viremia in Mice Infected with Parent Virus or IC47 Virus

For this study, three week old Swiss ICR mice were inoculated intraperitoneally with 100 PFU of parental or IC47 virus. Serum samples were collected at various time points and neutralizing antibodies and viremia were measured as follows. To measure neutralizing antibody titers, 10-160 fold dilutions of serum samples were prepared and incubated overnight at 4 ° C. with 50 PFU of parental virus. Viral titers of the reactions were determined by plaque assay in monolayers of BHK-21 cells, where BHK-21 cell monolayers in 35 or 60 mm dishes were inoculated with virus, washed with PBS and containing 2% FCS in MEM. Top applied with% agarose. After 2-3 days, the cells were stained with neutral red or crystal violet to visualize the plaques. A 70% reduction in plaques indicated the presence of neutralizing antibody titers.

Neutralizing antibodies were detected 3-4 days after inoculation in both treatment groups (see Figure 7). Differences in neutralizing antibody production were relatively minor.

To determine the presence of viremia, serum samples were added to the BHK-21 monolayer and virus titers measured by plaque assay. Viremia was detected with a maximum titer of about 10 ³ PFU / ml at day 3 in mice inoculated with parental virus (FIG. 7). When antibody titers began to rise, detectable viremia began to decrease; No virus was detected on day 4. Viremia is undetectable in serum taken from mice inoculated with IC47, but the virus will probably replicate in these mice because the mice exhibit an antibody response similar to that of the parental virus.

Example 4

Virus multiplication in the brain

To examine virus proliferation in brain tissues, mice were inoculated with 100 PFU of parental JEV, recombinant wild-type JEV IC37 or IC47 using intracranial or intraperitoneal administration methods. Brains were harvested several hours after inoculation and tissue samples were homogenized in 50% FCS (4: 1; w / v) in MEM. Viral titers of brain homogenates were determined by the plaque assay described above.

When the parental virus was inoculated into the cerebellum route, virus titers in the brain immediately increased and reached levels of 10 ⁷ PFU / mg or more on day 5 (see Figure 8 A). On day 7, all mice died of infection. After intraperitoneal inoculation of the parent virus, the virus appeared in the brain on day 5. Mice with brain virus titers greater than 10 ⁷ PFU / g were paralyzed and killed (see A, ▲ in FIG. 8), while mice with brain virus titers less than 10 ⁶ PFU / g remained healthy (Δ). Virus was removed from the brains of surviving mice on day 14.

In mice inoculated with IC47 in the brain, the virus began to grow immediately in brain tissue, but IC47 accumulated more slowly than with the parental virus in the brain. IC47 had lower titers than those observed for the parental virus (see Figure 8B). Maximum brain titers were about 10 ^4.7 PFU / g on day 5. IC47 was removed from the brain on day 8. No virus was detected in the brain tissues of mice inoculated by intraperitoneal administration using IC47.

Example 5

Immunity-Antigen Dosing Study

To determine whether IC47 can be used to provide protective immunity against the parental virus, three groups of eight mice were inoculated with 100 PFU of IC47. Two weeks old mice were inoculated intraperitoneally, and three weeks old mice were inoculated by either intraperitoneal or cerebellar administration. Two weeks after inoculation with IC47, mice were challenged with 100 PFU of parental virus into the cerebellum.

All non-immunized mice died on day 7, but all mice treated with IC47 survived the antigen administration without any signs of disease. The maximum viral titers of the antigen-administered mice were below 10 ⁷ PFU / g brain tissue and were 10 times lower than those observed in the brains of non-immunized mice (see FIG. 9). Parental virus was removed from brain tissue on day 14.

Example 6

Computer Simulation of Envelope Protein Structure

Several simulation programs were used to predict protein structure based on amino acid sequence data. These programs were used to compare the expected envelope protein structures of recombinant attenuated IC47 and recombinant wild type IC37, which carry ¹³⁸ Lys mutations. Secondary structures, including alpha-helix, beta sheets and turns, were predicted using the Shoe-Passman program and the Garnier-Osgutorp-Robinson program. See Chou et al., Adv. Enz. Relat. Areas Mol. Biol. 47:45 (1978); Garnier et al., J. Mol. Biol. 120: 97 (1978). Other programs were used to predict additional variables such as hydrophilicity, surface likelihood, flexibility and antigenicity. See Hopp et al., Proc. Nat'l Acad Sci. USA 78: 3824 (1981); Emini et al., J. Virology 55: 836 (1985); Jameson BA et al., Computer Applications in the Biosciences 4: 181 (1988).

There were no significant differences in simulation results for hydrophilicity, surface likelihood, flexibility and antigenicity. However, both the Shu-Passman and Garnier-Osgutorp-Robinson programs expected differences in the secondary structure of the two viruses. According to the Shu-Passman program, there were differences in turns, alpha helix and beta sheets. Garnier-Osgutorff-Robinson predicted a difference in turn between the envelope proteins of the two viruses. Thus, it was possible for ¹³⁸ Lys mutations to change the structure of the envelope protein to induce an attenuated phenotype of IC47.

Example 7

Characterization of Recombinant Viruses Having ¹³⁸ Asp and ¹³⁸ Arg Mutations in Envelope Proteins

Position-directed mutagenesis was used to prepare recombinant viruses carrying the following mutations at position 138 of the envelope protein: Arg (AGA) [" ¹³⁸ Arg"], Arg (AGG) and Asp (GAC) [" ¹³⁸ Asp "]. DNA sequencing was used to demonstrate nucleotide substitutions.

The ability of recombinant mutant viruses to induce neurotoxicity was examined by inoculating 100 wk of recombinant wild type IC37, IC47, ¹³⁸ Arg or ¹³⁸ Asp into the brain at 3 weeks of age. All mice inoculated with IC37 or ¹³⁸ Asp were killed. In contrast, all mice inoculated with IC47 or ¹³⁸ Arg survived without signs of disease (see Figure 10).

In addition, neurotoxicity analysis was performed using a group consisting of 8 mice 5 days old. In this study, all mice were killed 6 days after inoculation with IC37 (see FIG. 11). In addition, IC47 and ¹³⁸ Arg were neurotoxic in these newborn mice.

This study did not induce neurotoxicity in IC47 and ¹³⁸ Arg in 5 day old mice, or in 3 week old mice. The susceptibility pattern was similar to that observed for established yellow fever 17D and attenuated vaccines were used to protect humans against yellow fever virus.

To investigate whether ¹³⁸ Arg induces neutralizing antibody production, ICR mice 3 weeks old were inoculated with 100 PFU mice. Neutralizing antibody titers were measured on blood samples obtained 7 and 14 days post inoculation. Titer 1:80 was observed in both serum samples. The results indicated that ¹³⁸ Arg was immunogenic like IC47.

In the antigen dosing study, three weeks old mice were inoculated with 100 PFU of ¹³⁸ Arg. Two weeks later, the mice were injected with 100 PFU of IC37 into the cerebellum. All non-immunized mice died on day 7, but all mice immunized with ¹³⁸ Arg survived lethal antigen administration without signs of disease. Thus, ¹³⁸ Arg and IC47 appear to provide protective immunity to a similar degree.

IC47 and ¹³⁸ Arg determined the genetic stability of mutations that were passaged in the mouse brain to give an attenuated phenotype. In this study, both viruses were administered intracellularly to mice 3 weeks old and the mice were killed 4 days after injection. Brain homogenates were prepared as above and named P1. A portion of the P1 homogenate was used to inoculate the second group of mice and the homogenates from these mice were named P2. The procedure was continued for 10 asymptomatic passages.

Neurotoxicity was measured by administering ¹³⁸ Arg virus or IC47 virus 100 PFU isolated from the P10 homogenate into the cerebellum three weeks old. The results of this study indicate that the attenuated phenotype is stable after 10 passages. DNA sequence analysis of the virus isolated from P10 homogenates confirmed the stability of the IC47 and ¹³⁸ Arg mutations. After 10 passages, no reverse was found.

In summary, the ¹³⁸ Arg virus was highly attenuated, very immunogenic, induced protective immunity, genetically stable, and contained duplicate mutations in the codons encoding the 138th amino acid of the envelope protein. ^The neurotoxic properties of ¹³⁸ Arg virus were similar to those of yellow fever vaccine 17D. The attenuated phenotype of both IC47 and ¹³⁸ Arg virus was stable after 10 asymptomatic passages through the mouse brain. ^{Since the 138} Arg virus carries a redundant mutation and the IC47 virus carries a single mutation, the ¹³⁸ Arg virus may prove to be genetically more stable than IC47 over a long period of time.

Although certain preferred embodiments are mentioned in the foregoing, it is to be understood that the invention is not limited thereto. Various modifications of the disclosed embodiments will be apparent to those skilled in the art without departing from the scope of the invention as defined by the appended claims.

All documents and patent applications in the present invention are exemplary to those skilled in the art to which the present invention belongs. All documents and patent applications are incorporated herein by reference in their entirety, as each individual document or patent application specifically and individually references the entire contents thereof.

1 shows a diagram of the JEV genome as well as the recombinant JEV virus structure.

2 is a diagram showing an experimental design for recovering a recombinant wild type JEV from cDNA. 5'-1 / 2 JEV cDNA (pM343) and 3'-1 / 2 cDNA (pH756) were purified from their respective cDNA clones and linked in vitro to full-length for T7 RNA polymerase. DNA templates were prepared. JEV RNA was transcribed from the cDNA template and the RNA was transfected into monolayer BHK-21 cells. After 2-5 days of incubation, JEV plaques were visualized by staining with neutral red or crystal violet.

3 shows the rate of proliferation of parental JEV, recombinant wild type (IC37) and recombinant mutant JEV (IC47) in BHK cells.

4 shows the proliferation rate of recombinant wild type (IC37) and recombinant mutant JEV (IC47) in N18 cells.

5 depicts the stability of parental JEV or mutant JEV (IC47) in media.

FIG. 6 shows survival rates of Swiss ICR xcross mice challenged with parental JEV, recombinant wild type (IC37) or recombinant mutant JEV (IC47). A: Two weeks old mice were inoculated intraperitoneally (ip). B: Mice 3 weeks old were inoculated intraperitoneally (ip). C: Mice 3 weeks old were inoculated intracranially (ic). D Figure: Five week old mice were inoculated into the brain with parental and IC47 viruses.

7 shows the presence of neutralizing antibodies and in 3 week old mice inoculated with either parental JEV or recombinant mutant JEV (IC47).

8 shows the titer of either parental JEV or recombinant mutant JEV (IC47) in the brain of ICR mice. A Figure: Virus titers of mice inoculated intraperitoneally with the parental virus were measured from vacant mice (▲, ip-1) and healthy mice (Δ, ip-2). B: Inoculated with recombinant mutant virus.

FIG. 9 shows viral titers in IC47-immunized mouse brains challenged with parental virus into the brain.

10 shows the induction of neurotoxicity by recombinant wild type IC37, IC47 virus or ¹³⁸ Arg virus in three week old mice.

FIG. 11 depicts the induction of neurotoxicity by recombinant wild type IC37, IC47 virus or ¹³⁸ Arg virus in 5-day-old mice.

Claims (21)

An isolated, mutant Japanese encephalitis virus (JEV) distinguished from the corresponding wild type JEV by replacing an acidic amino acid (glutamic acid or aspartic acid) at position 138 of the envelope protein with a basic amino acid (lysine or arginine). The method of claim 1, Wild type JEV is JEV strain JaOArS982 and mutant JEV wherein the acidic amino acid is glutamic acid. The method of claim 2, Mutant JEV wherein the basic amino acid is lysine. The method of claim 3, Mutant JEV wherein the mutant JEV is IC47 (ATCC No, VR 2412). The method of claim 2, Mutant JEV wherein the basic amino acid is arginine. Mutations consisting of one or two nucleotide substitutions in the gene encoding the envelope protein resulted in the acidic amino acid (glutamic acid or aspartic acid) at position 138 in the envelope protein of wild type JEV being basic at position 138 in the envelope protein of mutant JEV. An isolated DNA molecule encoding the genome of a mutant JEV, distinct from wild type JEV, substituted with an amino acid (lysine or arginine). The method of claim 6, An isolated DNA molecule wherein the basic amino acid is lysine. The method of claim 7, wherein An isolated DNA molecule comprising the cDNA insert of pM433 (ATCC No. 75491). The method of claim 8, An isolated DNA molecule consisting of a cDNA insert of pM433 and a cDNA insert of pH756 (ATCC No. 75492). (a) in vitro synthesis of full length viral genomic RNA using the DNA molecule of claim 6 as a template; (b) transfecting the animal cells cultured using the viral genomic RNA to produce a virus; And (c) separating the virus from the cultured animal cells. A method of making a neutralizing antibody against JEV in an animal, except human, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising (a) the mutant JEV of claim 1 and (b) a pharmaceutically acceptable vehicle. The method of claim 11, The mutation JEV is IC47 (ATCC No. VS 2412). The method of claim 11, The basic amino acid is arginine. The method of claim 11, Method of administering the pharmaceutical composition to the mucosa. The method of claim 11, The pharmaceutical composition is administered in an injection form selected from the group consisting of subcutaneous injection, intradermal injection and intramuscular injection. A pharmaceutical composition comprising (a) the mutant JEV of claim 1 and (b) a pharmaceutically acceptable vehicle, wherein said composition provides prophylaxis against toxic Japanese encephalitis virus. The method of claim 16, Pharmaceutical composition wherein the basic amino acid is lysine. The method of claim 17, Pharmaceutical composition wherein the mutant JEV is IC47 (ATCC No. VR 2412). The method of claim 16, A pharmaceutical composition wherein the basic amino acid is arginine. (a) in vitro synthesis of full length viral genomic RNA using a DNA template comprising a cDNA insert at pH343 (ATCC No. 75490); (b) transfecting a monolayer culture of animal cells excluding humans using the viral genomic RNA to form virus-containing plaques of various sizes in the monolayer culture; And (c) separating the virus from the smaller plaque in the monolayer culture. The acidic amino acid (glutamic acid or aspartic acid) at position 138 in the envelope protein of the wild-type flavivirus is replaced with the basic amino acid (lysine or arginine) at position 138 in the envelope protein of the mutant flaviviruses, thereby matching the corresponding wild-type flavivirus. And the flavivirus is isolated, mutant flavivirus selected from the group consisting of Murray Bali encephalitis virus, St. Louis encephalitis virus, true tick mediated encephalitis virus and Central European encephalitis virus.