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  Pharmaceutical Patents  

 

Title:  Chimeric T helper-B cell peptide vaccine for Japanese encephalitis virus
United States Patent: 
7,425,335
Issued: 
September 16, 2008

Inventors:
 Gore; M. M. (Pune, IN), Kolaskar; Ashok (Pune, IN), Dewasthaly; S. S. (Pune, IN), Kulkarani-Kale; Urmila D. (Pune, IN), Sawant; Sangeeta (Pune, IN)
Assignee: 
The Secretary Department of Biotechnology (New Delhi, IN), National Institute of Virology (Pune, IN), University of Pune (Pune, IN)
Appl. No.: 
10/250,468
Filed:
 January 4, 2002
PCT Filed:
 January 04, 2002
PCT No.:
 PCT/IN02/00003
371(c)(1),(2),(4) Date: 
November 04, 2003
PCT Pub. No.:
 WO02/053182
PCT Pub. Date:
 July 11, 2002


 

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Abstract

A vaccine composition for humans and animals against Japanese encephalitis virus infection is a composition which comprises a chimeric synthetic peptide selected from envelope glycoprotein consisting of amino acids Egp 149-SENHGNYSAQVGASQ-163 (SEQ ID NO:1) AND Egp 429-SIGGVFNSIGKAVHQVFG-446 (SEQ ID NO:2) of Japanese encephalitis virus envelope glycoprotein, present in an amount sufficient to induce protective immunity against Japanese encephalitis virus infection.

Description of the Invention

FIELD OF INVENTION

This invention relates to chimeric T helper-B cell peptide as a vaccine for Japanese encephalitis virus.

BACKGROUND OF INVENTION

Amongst insect borne viral diseases, Japanese encephalitis and dengue have a notoriety of encompassing entire region of South East Asia. The envelope glycoprotein contains at least five determinants that seem to be correlated with the important biological properties of hemagglutination and neutralization. Envelope glycoprotein is responsible for the attachment of the virus and thus is associated with infectivity. The existing vaccine for Japanese encephalitis is purified, killed virus vaccine prepared from infant mouse brain that consists of mainly envelope glycoprotein of the virus. Three injections of mouse brain derived killed purified vaccine followed by a booster after 12 to 18 months, can give effective immunization as judged by induction of neutralizing antibodies. Mouse brain vaccine shows neutralizing antibodies against Indian strain also. The efficacy of the vaccine has been noted only if three doses are administered. U.S. Pat. No. 5,824,506 Chan, et al. Oct. 20, 1998 discloses Peptide antigens derived from the dengue virus type-2 glycoprotein NS1 are provided. The peptide antigens are specifically immunoreactive with sera from individuals infected with the dengue virus. The antigens are useful as diagnostic tools in determining whether an individual has been or is infected with dengue virus, and for discriminating between infection with dengue virus and infection with related flaviviruses. The antigens are also useful in vaccine compositions for immunizing individuals against infection with the dengue virus.

U.S. Pat. No. 5,494,671 Lai, et al. Discloses C-terminally truncated flavivirus envelope proteins 80-81% in size which are more immunogenic than their counterpart full-length proteins. The aforesaid patent further discloses recombinant viruses that encode the truncated protein and to host cells infected therewith. Host cells express the truncated protein on their outer membrane and secrete it into the medium. The patent discloses vaccines for use against flavivirus infection. The vaccines include either a recombinant vaccinia virus expressing the truncated envelope protein, and the truncated envelope protein produced by a recombinant baculovirus.

In China, attenuated Japanese encephalitis virus vaccine consisting of Japanese encephalitis virus strain SA 14-14-2 is being used. The attenuation has been carried out in hamster kidney cells and have unknown passage histories. Mice inoculated intracerebrally with the SA 14-14-2 vaccine strains survived without showing any signs of CNS involvement. The virus titers in brains persisted at low levels for several days and could not be detected after 10 days (Hase T, Dubois, Dr. Summers, P L, Downs, M B and Ussery, M A. (1993) Comparison of replication rates and pathogenicities between the SA14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons. Arch Virol 130 131-43. Dubois T., Dr. Summers, P L. Downs, M B and Ussery, M A (1993) Comparison of replication rates and pathogenicities between the SA14 parent and SA 14-14-2 vaccine strains of Japanese encephalitis virus in mouse brain neurons. Arch Virol 130 131-43). The safety and immunogenicity of this vaccine has been tested. 1,026 children between the ages of 5 and 12 years, were vaccinated with live-attenuated Japanese encephalitis virus vaccine. None of the group of 47 of the vaccinated children, has temperature >37.4.degree. C. Seroconversion rates in seronegative children were 100% (GMT 35.3) (Xin Y Y, Ming, Z G, Peng, G Y, Jain, A and Min, L H (1988) Safety of a live-attenuated Japanese encephalitis virus vaccine (SA14-14-2) for children. Am J Trop Med Hyg 39 214-7). Many attempts are being carried out to develop recombinant vaccine for Japanese encephalitis virus. These include expression of various proteins and then immunizing animals with the products. It was realized very early that expression and immunization with envelope glycoprotein alone was not very useful (Mason P W, McAda, P C, Dalrynple, J M, Fournier, M J and Mason, T L (1987) Expression of Japanese encephalitis virus antigens in Escherichia coli. Virology 158 361-72). Mice immunized with recombinant baculovirus infected cells containing envelope glycoprotein and NS-1 genes were challenged with Japanese encephalitis virus. Survival was increased from about 30% in unimmunized mice to 70% in envelope glycoprotein and polyprotein recipients but not in NS-1 recipients (McCown J, Cochran, M, Putnak, R, Feighny, R, Burrous, J, Henchal, E and Hoke, C (1990). Protection of mice against lethal Japanese encephalitis with a recombinant baculovirus vaccine. Am J Trop Med Hyg 42 491-9). Immunization of mice with purified extracellular subviral particles composed of prM and E proteins in recombinant vaccinia viruses could protect mice from 4.9.times.10.sup.5 LD50 of Japanese encephalitis virus. (Konishi E, Pincus, S, Paoletti, E, Shope, R E, Burrage, T and Mason, P W. (1992) Mice immunized with a subviral particle containing the Japanese encephalitis virus prM/M and E proteins are protected from lethal JEV infection. Virology 188 714-20). These particulate antigens were also shown to induce Japanese encephalitis virus specific CTL response in mice. (Konishi E, Win, K S, Kurane, I, Mason, P M, Shope, R E and Ennis, F A (1997) Particulate vaccine candidate for Japanese encephalitis induces long-lasting virus-specific memory T lymphocytes in mice. Vaccine 15 281-6).

Vaccinia recombinants that co-expressed the genes for premembrane and envelope glycoprotein elicited high levels of neutralizing and HI antibodies in mice and protected mice from a lethal challenge by Japanese encephalitis virus. Recombinants expressing only the gene for NS1 induced antibodies to NS1 but provided low levels of protection from a similar challenge dose of Japanese encephalitis virus. Immunization of mice with vaccinia recombinant viruses containing PrM gene along with NS-1 and envelope glycoprotein protected them from challenge with Japanese encephalitis virus. Pox virus (Canary pox and vaccinia) based Japanese encephalitis recombinant vaccines have been constructed and shown to produce Japanese encephalitis virus specific CTLs in mice. (Konishi, E, Kurane, I Mason, P W, Shope, R E and Ennis, F A (1997) Poxvirus-based Japanese encephalitis vaccine candidates induce J E virus specific CD8+ cytotoxic T lymphocytes in mice. Virology 227 353). Poxvirus-based recombinant J E vaccine candidates, NYVAC-JEV and ALVAC-JEV, encoding the Japanese encephalitis virus prM, E and NS1 proteins were examined for their ability to induce Japanese encephalitis virus-specific CTLs. The volunteers received subcutaneous inoculations with each of these candidates on days 0 and 28. Anti-E and anti-NS1 antibodies were elicited in a most vaccinees inoculated with NYVAC-JE virus and in some vaccinees inoculated with ALVAC-JEV, PBMCs obtained from approximately one half of vaccinees showed positive proliferation in response to stimulation with live Japanese encephalitis virus. Presence of the Japanese encephalitis virus-specific CDS+CD4- cytotoxic T cells in vitro-stimulated peripheral blood mononuclear cells obtained from two NYVAC-JEV and two ALVAC-JEV vaccinees was demonstrated. (Konishi E, Kurane, I, Mason, P M, Shope, R E, Kanesa-Thasan, N, Smucny, J J, Hoke, C H Jr and Ennis, F A (1998). Induction of Japanese encephalitis virus-specific cytotoxic T lymphocytes in humans by poxvirus-based J E vaccine candidates. Vaccine 16 842-9). A chimeric Yellow fever (YF)/JE virus (ChimeriVax-JE virus) was constructed by insertion of the prM and envelope glycoprotein genes of an attenuated human vaccine strain (SA14-14-2) of Japanese encephalitis virus between C and NS genes of a YF 17D infectious clone. Mice inoculated subcutaneously with one dose of >/=10.sup.3 pfu of ChimeriVax-JE virus were protected against IP challenge with a virulent Japanese encephalitis virus.

In recent years, it has been shown that fragments of proteins in the form of synthetic peptides can be used to induce T helper and antibody responses. Attempts to delineate B cell epitopes from Japanese encephalitis virus have resulted in delineation of Met 303 to Trp 396 as the shortest sequence capable of reacting with 10 MAbs. Disulfide bond between cys 304 and 335 was required for presentation of the binding site(s) for these MAbs. However, it was not an effective immunogen for inducing neutralizing or protective antibodies in mice (Mason P W, Dalrymple, J M, Gentry, M K, McCown, J M, Hoke, C H, Burke, D S, Fournier, M J and Mason, T L (1989) Molecular characterization of a neutralizing domain of the Japanese encephalitis virus structural glycoprotein. J Gen Virol 70 2037-49). The fragment carrying the coding sequence of amino acid 373-399 of envelope glycoprotein elicited the highest neutralizing antibody titer (1:75). HI antibodies were not induced by this fusion protein (Seif S A, Korita, K and Igarashi, A (1996) A 27 amino acid coding region of E virus protein expressed in E. coli as fusion protein with glutathione-S-transferase elicit neutralizing antibody in mice. Virus Res 43 91-6). Neutralizing antibody inducing epitopes have been detected on C terminal regions of envelope glycoprotein (Seif S A, Morita, K, Matsuo, S, Hasebe, F and Igarashi, A (1995) Finer mapping of neutralizing epitope(s) on the C-terminal of Japanese encephalitis virus E-protein expressed in recombinant Escherichia coli system. Vaccine 13 1515-21 and Jan L R, Yang, C S, Henchal, L S, Sumiyoshi, H, Summers, P L, Dubois D R and Lai, C J (1993) Increased immunogenicity and protective efficacy in outbred and inbred mice by strategic carboxyl-terminal truncation of Japanese encephalitis virus envelope glycoprotein. Am J Trop Med Hyg 48 412-23) Peptides from C protein have also been delineated for reactivity with sera from Japanese encephalitis and dengue patients. Pep91-105 and 8-22 belonged to group-specific epitopes that reacted with both Japanese encephalitis and dengue-1 patient sera. Pep 1-15 and 34-48 belonged to subcomplex-specific epitopes that reacted only with Japanese encephalitis but not with dengue-1 patient sera. (Huang J H, Wey, J J, Lee, H F, Tsou) T L, Wu, C S, Wu, J R, Chen, H M, Chin, C, Chien, L J, Chen, L K, Wu Y C, Pan, M J and Wang, T M (1996) Identification of immunodominant, group-specific and subcomplex-specific, continuous epitopes in the core regions of Japanese encephalitis virus using synthetic peptides. Virus Res 41 43-53).

DISADVANTAGES OF THE PRIOR ART

In case of mouse brain derived killed purified vaccine, three doses of the injectable vaccine are to be administered. In a trial with vaccine made in Japan was carried out in South Arcot district of Tamil Nadu, India. Of a total of 113 school children, 72% showed antibody response while the responders increased to 87.8% after booster dose of Biken Japanese encephalitis vaccine after one year. Only about 20 per cent of the children had persisting antibodies one year after the primary vaccination. (Mohan Rao C V R, Risbud, A R, Dandawate, C N, Umarani, U B, Ayachit, V M, Rodrigues, F M and Pavri, K M (1993) Serological response to Japanese encephalitis vaccine in a group of school children in South Arcot district of Tamil Nadu Indian J Med Res 97 53-59). The problems of strain variation and the protection offered by the inactivated vaccine based on Nakayama have always been noted.

In case of attenuated Japanese encephalitis vaccine used in China, although efficacy of the vaccine has been proven in many studies by now, there are some problems associated with the licensing of this vaccine all over the world. The passage history and the laboratory practices, which were used in generation of this vaccine, have not been known completely. Thus, attempts to re-invent the attenuated strain from the same SA14-14-2 strain have been carried out. In a Japanese study antibodies against both envelope glycoprotein and NS 1 were observed in mice infected with the attenuated Japanese encephalitis virus strain SA(A) derived from the live Japanese encephalitis vaccine strain SA14-14-2. (Lee T, Komiya, T, Watanabe, K, Aizawa, C and Hashimoto, H (1995) Immune response in mice infected with the attenuated Japanese encephalitis vaccine strain SA14-14-2. Acta Virol 39 161-4).

The problems associated with vaccine for Japanese encephalitis are, e.g. discrepancy in the age at which Japanese encephalitis vaccine should be administered. The cost of currently available vaccine is very high. The additional cost of administering three doses will also have to be taken into consideration. It is not known whether the effect of Japanese encephalitis vaccine will be long lasting, in absence of exposure to Japanese encephalitis after the third dose. Whether yearly boosters are required or not, until natural immunity due to natural Japanese encephalitis infection is not known. Thirdly, immune response to Japanese encephalitis virus is very low (Pavri K M (1984)) Problems of JE immunization in India. Proc. Of National Conference on Japanese encephalitis. Ind.J. Med. Res Suppl pp 81-84) In addition, the question of immunity to the local strains by Nakayama or Beijing strains of virus used in vaccine will have to be taken into consideration. As the vaccine is mouse brain derived there are allergic reactions to the vaccine and the frequencies of allergic mucocutaneous reactions varied from 1-17 per 10,000 vaccinees during 1983-1995 (Plesner A M and Ronne, T (1997) Allergic mucocutaneous reactions to Japanese encephalitis vaccine. Vaccine 15 1239-43).

13. How these drawbacks have been overcome by your invention. In recent years, it has been shown that fragments of proteins in the form of synthetic peptides can be used to induce T helper and antibody responses. (Ref) Thus, neutralizing antibody-inducing epitopes from envelope glycoprotein have been delineated. As these peptide sequences are not sufficient for inducing protective immunity, a chimeric peptide has been prepared incorporating T helper epitope along with the virus neutralizing antibody inducing B cell epitope. This will ensure that both T helper and B cell immunity is generated for the protection from Japanese encephalitis virus challenge. The said chimeric vaccine is designed and thus more than one chimeric peptide can be added to formulate the effective vaccine as per requirement in the future.

OBJECTS OF THE INVENTION

It is an object of the present invention to propose safe and effective vaccines against flaviviruses for humans and animals. Various other objects and advantages of the present will become apparent from the ensuing description.

DESCRIPTION OF THE INVENTION

According to this invention there is provided a vaccine composition for humans and animals against Japanese encephalitis virus infection, comprising a chimeric synthetic peptide, said chimeric peptide selected from envelope glycoprotein, consisting of: amino acids Egp 149-SENHGNYSAQVGASQ-163 (SEQ ID NO:1) and Egp 428-GSIGGVFNSIGKAVHQVFG-446 (SEQ ID NO:84) of Japanese encephalitis virus glycoprotein wherein chimeric peptide is in an amount sufficient to induce protective immunity against Japanese encephalitis virus infection.

In one embodiment, the present invention relates to vaccine composition for humans and animals against or Japanese encephalitis virus infection, comprising a chimeric synthetic peptide. Chimeric peptide was selected from envelope protein, consists of amino acids 149-SENHGNYSAQVGASQAAKF-167 (SEQ ID NO: 3) AND 427-GSIGGVFNSIGKAVHQVFG-445 (SEQ ID NO: 84).

In another embodiment, the present invention also relates to the vaccine composition wherein said peptide 149-SENHGHNYSAQVAGASQAAKF-167 (SEQ ID NO: 3) induces neutralizing antibodies against Japanese encephalitis virus.

In another embodiment, the present invention also relates to the vaccine composition wherein said peptide 149-SENHGHYSAQVGASQAAKF-167 induces neutralizing antibodies against Japanese encephalitis virus.

In a further embodiment, it also relates to a vaccine composition for humans and animals against Japanese Encephalitis Virus infection comprising a neutralizing antibody inducing peptide sequences from envelope glycoprotein of Japanese encephalitis virus. The said sequences are amino acids 39-PTLDVRMINIEA -50 (SEQ ID NO:4), 273-EYSSSVKLTSG-283 (SEQ ID NO:5).

In another embodiment, the present invention also relates to peptide sequences from Japanese encephalitis virus envelope glycoprotein, Capsid protein, Membrane protein, Non Structural protein-1 and Non Structural protein-3 capable of stimulating T helper cells from immunized animals.

In another embodiment, the present invention also relates to a combination of peptides mentioned above resulting in chimeric T helper B cell peptides capable of inducing protective immunity against Japanese encephalitis virus infection.

Either B or T lymphocytes through their receptors may define epitopes as the regions of part of proteins that are recognized. Based can the cell involved epitopes may be classified as B cell, T helper cell or CTL epitopes that stimulate B cells, T helper (CD4+) and CTLs respectively. MHC molecules present T cell epitopes to the TCR present on the T cells. B cell epitopes in contrast to the T cell epitopes have dependence on the three dimensional structure. B cell epitopes can be predicted using several different methods B cell determinants of envelope glycoprotein of Bakura (India) strain (733913) were identified. Briefly, antigenic propensity values are assigned to each of the twenty amino acid residues based an their frequency of occurrence in experimentally confirmed B cell antigenic determinants. These parameters along with appropriate cutoff values were used in a computer program developed. Table 1 (see Original Patent) shows the predicted B cell determinants of envelope glycoprotein of Japanese encephalitis virus.

The Table 1 depicts amino acid sequences predicted from envelope glycoprotein of Japanese encephalitis virus Amino acid sequence derived is from Japanese encephalitis virus strain Bankura (733913).

In order to arrive at the unique sequences that might be useful in vaccine development amino acid sequences from other related flaviviruses viz., WHV, MVEV, DENV and YFV were downloaded from protein data banks and multiple alignments were carried out using CLUSTAL program. Similarly in order to understand amino acid sequences from a few Japanese encephalitis virus strains were also subjected to multiple alignment.

The multiple alignments were carried cut CLUSTAL program.

The region in the envelope glycoprotein which were identified as being non-homologous with other flaviviruses were: 1. 149-SENHGNYSAQVGASQ-163 (SEQ ID NO. 1) 2. 40-TLDVRMINIEA50 (SEQ ID NO. 30) 3. 270-IVVEYSSSVKLTS-282 (SEQ ID NO. 31)

These sequences were conserved within different Japanese encephalitis virus strains (as shown in FIG. 2 (see Original Patent) of U.S. published patent application, 20040076634). This means that regions 40-50, 155-163 and 270-290 are unique to Japanese encephalitis virus. Antibodies induced against these peptides from envelope glycoprotein would thus have better chances of being neutralizing antibody. It should be noted here that there are no major differences between the amino acid sequence of Japanese encephalitis virus Nakayama strain and Bankura strain.
 

Claim 1 of 7 Claims

1. A synthetic polypeptide consisting of a B cell epitope linked to a T-cell epitope, wherein the epitopes are from the Japanese encephalitis virus envelope glycoprotein, and the B cell epitope has the sequence 149-SENHGNYSAQVGASQ-163 (SEQ ID NO:1) and the T cell epitope has the sequence 429-SIGGVFNSIGKAVHQVFG-446 (SEQ ID NO:2).
 

 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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