WO2009153801A1

WO2009153801A1 - Dna vaccine based on glycoprotein gene of rabies virus conjugated to lamp-1 signal sequence at the c-terminus and an adjuvant; and method of its preparation thereof

Info

Publication number: WO2009153801A1
Application number: PCT/IN2008/000578
Authority: WO
Inventors: Manpreet Kaur; Anant Rai; Rakesh Bhatnagar
Original assignee: Manpreet Kaur; Anant Rai; Rakesh Bhatnagar
Priority date: 2008-06-17
Filing date: 2008-09-10
Publication date: 2009-12-23

Abstract

A pharmaceutical composition of vaccine against rabies, which comprises of DNA vaccine based on glycoprotein gene of rabies virus conjugated to LAMP-1 signal sequence at the C-terminus, and an adjuvant; and method of its preparation thereof.

Description

DNA VACCINE BASED ON GLYCOPROTEIN GENE OF RABIES VIRUS CONJUGATED TO LAMP l SIGNAL SEQUENCE AT THE C-TERMINUS AND AN ADJUVANT; AND METHOD OF ITS PREPARATION THEREOF.

FIELD OF INVENTION

A pharmaceutical composition of vaccine against rabies, which comprises of DNA vaccine based on glycoprotein gene of rabies virus conjugated to LAMP-I signal sequence at the C- terminus, and an adjuvant; and method of its preparation thereof.

B cell immune responses are enhanced by presentation of antigen to CD4⁺ T cells using a chimeric nucleic acid immunogen or vaccine that links DNA encoding an antigen with DNA encoding a polypeptide that targets or translocates the antigenic polypeptide to lysosome/endosome. The present invention relates to the development of DNA vaccine against rabies wherein the glycoprotein gene of rabies virus is conjugated to lysosomal associated membrane protein (LAMP-I) signal sequence for targeting it to lysosomal membrane. This vaccine, on supplementation with an adjuvant leads to generation of an immune repertoire that is capable of providing effective neutralizing antibody response and hundred percent protection against subsequent intracerebral rabies virus challenge in mouse model.

The invention additionally relates to vaccine composition containing novel targeting sequence and adjuvant, method of preparing and process of using the pharmaceutical composition of the invention.

BACKGROUND

Rabies is acute progressive, fatal viral encephalitis (Smith, J.S. 1996. Clinical Microbiology Reviews, 9, 2: 166-176). It is caused by neurotropic RNA virus of family Rhabdoviridae, genus Lyssavirus (Rupprecht, C, Hanlon, C.A., and Hemachuda, T. 2002. Lancet infectious disease, 2, 6: 327-343). Pathogenesis: Rabies virus multiplies in muscle or connective tissues at the site of inoculation and then enters peripheral nerves at neuromuscular junctions and spreads up the nerves to the central nervous system. The virus can also enter nervous system directly without local replication. It multiplies in the brain and may then spread through peripheral nerves to the salivary glands and other tissues. Rabies virus is also found in pancreas, kidney, heart, retina, and cornea. Susceptibility to infection and the incubation period may depend on the host's age, genetic background, immune status, the viral strain involved, the amount of inoculum, the severity of lacerations and the distance the virus has to travel from its point of entry to the central nervous system. Presence of eosinophilic, cytoplasmic inclusions- Negri bodies in infected nerve cells is pathognomonic of rabies but its absence doesn't rule out rabies as a diagnosis as they are not observed in at least 20% cases.

The Clinical spectrum of the disease is divided into three phases: a) A short prodromal phase, lasting 2-10 days, shows non-specific symptoms including malaise, anorexia, headache, photophobia, nausea and vomiting, sore throat and fever. Usually there is an abnormal sensation around the site of infection. b) An acute neurologic phase in which there is signs of nervous system dysfunction such as nervousness, apprehension and hallucinations. General sympathetic overactivity is observed. Most of patients exhibit hydrophobia. c) Coma or convulsive seizures usually 2-7 days after onset of neurologic phase, followed by death. The major cause of death is respiratory paralysis.

More than 99% of infections in infected organisms that develop symptoms end fatally. Recovery and survival is extremely rare. It is thus essential that infected organisms at high risk receive preventive immunization and they be given post-exposure prophylaxis after any case of exposure. Such a vaccine against should be initiated promptly ensuring symptoms have not been initiated.

PRIORART Currently used vaccines against rabies consist of inactivated virus adapted to growth in different cell lines. These vaccines suffer from problems like low immunogenicity thereby requiring multiple dosages. This further augments cost of immunization apart from their high cost of production. They have problems of safety, possible reversion to virulence, risk of contamination with adventitious agents and lack of stability. They may also cause anaphylactic, neuroparalytic or encephalitic side reactions. This emphasizes on the need of a cost effective, potent vaccine which could elicit long term protective responses.

A possible solution to these problems could be a DNA vaccine, which may overcome such problems and may also be superior to the inactivated vaccines. The DNA based immunization has been used in various pathologic conditions and in laboratory animal models, frequently with satisfactory results. The World Health Organization (WHO) and the US Food and Drug Administration have expressed favourable recommendations towards this technology, as long as the necessary safety rules are applied (FDA 1996. Docket No. 96N- 0400).

DNA vaccines against rabies virus using plasmid encoding glycoprotein (G) and driven by SV40 promoter/enhancer (Xiang, Z.Q., Spitalnik, S., Tran, M., Wunner, W.H., Cheng, J., and Ertl, KC 1995. Virology, 199, 1: 132-140) or CMV promoter (Bahloul, C, Jacob, Y, Tordo, N, and Perrin, P. 1998. Vaccine, 16, 4: 417-425) have been evaluated in laboratory animal models and were shown to induce potent immune response protecting against rabies virus challenge. In contrast, plasmid encoding the rabies virus nucleoprotein induced only a low level of specific antibodies, which did not confer protection against challenge (Ertl, KC, Verma, P., He, Z, andXiang, Z.Q. 1995. Annals of the New York Academy of Sciences 772, 1: 77-87).

DNA vaccine expressing target proteins fused to signal sequences like Tissue Plasminogen Activator (TPA), Ubiquitin (Ub), Lysosomal Associated Membrane Protein (LAMP-I) have been found to elicit substantial protective immunity. DNA vaccines expressing mycobacterial proteins fused to TPA induce significant humoral and cell mediated responses. It has found to consistently induce higher levels of IgG antibody levels (Li, Z., Howard, A., Kelley, C, Delogu, G., Collins, F, and Morris, S. 1999. Infection and immunity, 67, 9: 4780-4786). Ub conjugated proteins accelerate protein turnover by undergoing rapid intracellular degradation. They may enhance induction of cellular immune response and can elicit cytokine responses in the absence of specific antibody production. DNA vaccine expressing mycobacterial proteins fused to Ub have been shown to dramatically elevate IFN-γ response (Delogu, G., Li, A., Repique, C, Collins, F., and Morris, S. L. 2002. Infection and immunity, 70, 1: 292- 302; Delogu, G., Howard, A., Collins, F., and Morris, S. L. 2000. Infection and immunity, 68, 6: 3097-3102). LAMP-I is a type of transmembrane protein localized predominantly to lysosomes and late endosomes. Antigen trafficking of LAMP-I -fused antigens to the cellular site of MHC class II processing and presentation pathway could enhance its presentation to MHC class II restricted CD4⁺ T cells (Arruda, L.B., Chikhlikar, P.R., August, J, and Marquest, E.T.A. 2004. Immunology, 112, 1: 126-135) and thus augment the humoral response.

Different routes of administration have been tried for inducing protective immunity. A single intramuscular injection of DNA vaccine using pCI-neo^R encoding glycoprotein (G) under the control of immediate early promoter/enhancer CMV- induced an early, strong and long- lasting production of neutralizing antibodies as well as specific T helper, T cytotoxic and NK cells (Bahloul, C, Jacob, Y., Tordo, K, and Perrin, P. 1998. Vaccine, 16, 4: 417-425) resulting in full protection against an intracerebral challenge. In case of intradermal injection using gene gun, small quantities are sufficient to induce strong and protective immune response (Lodmell, D.L., and Ewalt, LC. 2000. Vaccine, 18, 22: 2394-2398; Lodmell, D.L., Parnell, MJ, Bailey, J.R., Ewalt, LC, and Hanlon, CA. 2002. Vaccine, 20, 17: 2221-2228; Lodmell, D.L., Ray, KB., and Ewalt, LC. 1998. Vaccine, 16, 2: 115-118; Lodmell, D.L, Ray, KB., Ulrich, T., and Ewalt, LC. 2000. Vaccine, 6, 18: 1059-1066; Rai, A., Gupta, P.K., and Rai, K 2002. Indian J. Comp. Microbiol. Immunol. Inf. Dis. 23, 2: 123-126). In cats, pGQH was administrated intranasally and generated high neutralizing antibody titers; > 14 IU/ml (Tesoro-Cruz, E., Calderon-Rodriguez, R., Hernandez-Gonzalez, R., Blanco-Favela, F., and Aguilar-Setien, A., 2008. Vet. Res. 39, 2. Ahead of print). In another study, repeated DNA vaccine injections could raise protective antibodies in dogs (Osorio, J.E., Tomlinson, CC, Frank, R.S., Haanes, EJ., Rushlow, K., Haynes, J.R., and Stinchcomb, D.T. 1999. Vaccine, 17, 9-10: 1109-1116; Perin, P., Jacob, A., Setien, A., Loza-Rubio, E, Jallet, C, Desmezieres, E., Aubert, M, Cliquet, F., and Tordo, K 2000. Vaccine, 18, 5-6: 479-486). In one report, protection against peripheral challenge was observed only if the challenge was administered a short time after the last DNA vaccination. DNA vaccine in association with a single dose of anti-rabies immune serum has been used for protection in mice against rabies in accordance to post-exposure vaccination method (Lodmell, D.L., and Ewali, L.C 2001. Vaccine, 19, 2468-2473).

Some of the cytokines which may play key roles in the induction of protective immune response against rabies virus have been studied. They include TNF-α, IFN-γ, IL-2, IL-4 and IL-12. IL-12 strengthens the non-specific immune response by activating NK cells to produce IFN-γ and in synergy with IFN-γ, drives the differentiation of CD4⁺ T cells into ThI cells (Lodmell, D.L., andEwalt, LC. 2001. Vaccine, 19, 2468-2473):

In order to investigate the potential of rabies DNA vaccine for immunization in humans, cynomolgus monkeys were vaccinated with plasmid encoding glycoprotein (G). On challenge, DNA vaccination elicited primary and anamnestic-neutralizing antibody responses {Lodmell, D.L, Parnell, M.J., Bailey, J.R., Ewalt, LC, and Hanlon, CA. 2001. Vaccine, 20, 5: 838-844; Lodmell, D.L, Ray, N.B., Parnell, M.J., Ewalt, LC, Hanlon, CA. and Shaddock, J.H. 1998. Nat Med, 4, 8: 949-952). This finding indicates that DNA vaccine has potential in immunization of humans against rabies (Bahloul, C, Ahmed, S., Imen B'chir, B., Kharmachi, K, Hayouni, E.A., andDellagi, K. 2003. Vaccine, 22, 2: 177-184).

None of the above DNA vaccines could provide a full spectrum of neutralizing and long- lasting immune response, which is the primary aim of any successful vaccine. Also, the potential of rabies DNA vaccine for immunization in humans and its long term effects still remain to be investigated.

OBJECTIVES

In view of this background, following are the major objectives of this invention:

♦ The prime objective of the present invention is to provide a glycoprotein-based DNA vaccine for inducing a potent anti- glycoprotein humoral immunity. ♦ Another object of the present invention is to provide a DNA vaccine comprising the glycoprotein G conjugated to a C-terminus human LAMP-I anchor for targeting of glycoprotein to lysosomal/endosomal membrane.

♦ Still another object of the invention is to provide a pharmaceutical composition for enhancing the immune response.

♦ Yet another object of the invention is to provide a novel adjuvant formulation having enhanced immunogenic activity.

♦ Further object of the present invention is to enhance the potency of DNA vaccine utilizing a pharmaceutically acceptable adjuvant- Emulsigen®-D.

♦ Still further object of present invention is to provide a vaccine composition that generates a potent humoral immune response that correlates well with the survival against viral challenge.

♦ Yet another object of present invention is to provide a vaccine formulation comprising DNA immunization and adjuvant usage; to enhance the immune response.

SUMMARY OF INVENTION

To achieve the said objectives the present invention relates to application of trafficking of glycoprotein to the endosomal compartment of the cell with the ultimate goal of enhancing neutralizing antibody-humoral response. Thereby, the antigenic protein will be targeted to the lysosomal/endosomal membrane with the assistance of C-terminus LAMP-I signal sequence. After processing in these compartments, epitopes will be displayed on the cell surface in context with MKC-II molecules. This is a non-invasive method of immunization whereby the DNA vaccine encodes glycoprotein along with C-terminus LAMP-I. Thus, only the specific antigenic moiety will be responsible for generation of an effective immune response. The invention presents a method to enhance the immune response generation by means of efficient targeting and presentation of the protein antigen. The response can be evaluated by total and neutralizing antibody titers, cytokine profiling, which' can be correlated well with survival against viral challenge. Thus, we could formulate a DNA vaccine or a pharmaceutical composition that comprises a vector bearing a signal sequence which may help induce an efficient immune response via various routes namely intradermal (direct injection, delivery on gold beads by gene gun), nasal or oral , with and without adjuvants.

Further, addition of a pharmaceutically acceptable adjuvant enhanced the immune response and conferred hundred percent protection on intracerebral rabies virus challenge. Thus, the invention also describes a DNA vaccine formulation (with adjuvant) that is superior in quality as compared to inactivated virus based vaccine as demonstrated by the enhanced humoral immune responses.

Such a DNA vaccine may be used to immunize an animal whereby the animal can be vertebrate e.g. a fish, amphibian, reptile, bird, or mammal such as a mouse, a dog, a cat, a goat, a sheep, a horse, a pig, a cow, a human being or a fowl such as ducks, turkeys, chicken and the like. This study also presents a strategy for DNA vaccine that can be designed for protection against other pathogens such as viruses, prokaryotes and pathogenic eukaryotic organisms such as unicellular organisms and multi-cellular parasites. The strategy of invention is particularly useful for immunization against intracellular pathogens.

Therefore, this study presents the strategy or DNA vaccine composition; whereby this DNA vaccine composition can serve as the prophylactic vaccine against rabies and the strategy hereto can be applied to other pathogens and parasites (both intracellular and extracellular) that can help generate an immune response that will be of higher quality as compared to other vaccine formulations.

The present invention further relates to a pharmaceutical composition to induce an immune response against Rhabdo virus in an animal susceptible to rabies disease comprising a DNA vaccine construct that encodes and expresses the glycoprotein gene of rabies virus. The Rhabdo virus is rabies virus (RV).

The vaccine against rabies comprises of:

- endotoxin free DNA vaccine construct based on glycoprotein gene fused to LAMP-I signal sequence at the C-terrfϊinus (50-200μg) and

- an adjuvant (15-20% v/v).

The said signal sequence is pharmaceutically acceptable.

The said LAMP-I signal sequence targets the glycoprotein gene to lysosomal membrane to potentiate an immune response by promoting processing of the linked antigenic polypeptide via the MHC class II pathway or targeting of a cellular compartment that increases said processing.

The adjuvant is a pharmaceutically acceptable vehicle.

The adjuvant is Emulsigen ®-D. The adjuvant is oil in water emulsion containing Dimethyl Diocta Decyl Ammonium

Bromide.

The oil is mineral oil.

The DNA vaccine construct is a eukaryotic expression vector.

The expression of glycoprotein gene is controlled by cytomegalo virus (CMV) promoter. The DNA vaccine construct codes for the complete mature glycoprotein.

The encoded fusion gene has the sequence of SEQ ID A.

The fusion gene when expressed in eukaryotic expression system produces the chimeric protein of the sequence SEQ ID B.

The vaccine construct; SEQ ID A encodes endosomal form of glycoprotein gene with the assistance of C-terminal LAMP-I signal for targeting it to lysosomal membrane.

The signal sequence is cleaved as part of normal cell cycle to generate immune response against the encoded glycoprotein.

The DNA Vaccine construct based on glycoprotein gene fused to LAMP-I signal sequence at the C-terminus (90-1 lOμg) and an adjuvant (20% v/v) to achieve 100% protection on intracerebral viral challenge. The present invention also relates to a process for preparing the pharmaceutical composition comprising: a. preparing the endotoxin free DNA vaccine construct based on glycoprotein gene fused to LAMP-I signal sequence at the C-termirius; using Endo free maxi kit, b. dissolving the vaccine of step (a.) in buffered saline, c. mixing the endotoxin free DNA Vaccine of step (b.) at 50-200 μg concentration with 15-20% v/v of adjuvant and making up the volume to 100-200μl.

This invention will now be described with reference to the accompanying drawings and examples, which are given for the purpose of illustration only and not intended to limit the scope of the present invention.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Fig. 1: Schematic representation of plasmid DNA construct encoding rabies virus glycoprotein (RV-G). The DNA encoding the full length of RV-G (1575 nt), was amplified from pTargeT Rabgp, using Sapϊ restriction site in both forward and reverse primer. rRV-G was cloned upstream LAMP-I signal sequence. (CMV: cytomegalovirus promoter/enhancer; LAMP-I : human lysosomal associated membrane protein-1 signal sequence).

Fig. 2: Cloning of glycoprotein in expression plasmid. Lane 1: vector; Lane 2: pgp.LAMP-1 clone and Lane 3: 1 Kb DNA ladder (Fermentas Lifesciences, Canada).

Fig. 3a: Reactivity in immunoblot of recombinant RV-G expressed by BHK-21 mammalian cells. BHK-21 cells transfected with pgp.LAMP-1 or vector were processed for Western blot analysis; for which resolved cell lysates were transferred onto nitrocellulose membrane and presence of rabies glycoprotein was detected using mouse polyclonal anti-rabies hyperimmune sera. Lanes represent; 1 : Prestained protein molecular weight marker; 2: BHK- 21 cells transfected with pgp.LAMP- 1 ; 3 : BHK-21 cells transfected with vector; and 4 : Mock transfected BHK-21 cells. Fig. 3b: Flow cytometric analysis of cells expressing rabies glycoprotein. BHK-21 cells transfected with pgp.LAMP-1 vaccine construct were stained with anti-rabies hyperimmune sera as the primary antibody. The number of cells showing fluorescence, after staining with Alexa Fluor 488 labeled secondary antibody were analyzed using FLl and displayed as histograms. Actin was used as positive control.

Fig. 3c: Expression of glycoprotein in BHK-21 cells transfected with pgp.LAMP-1 vaccine construct. After transfection, cultures were fixed, treated with anti-rabies hyperimmune sera; followed by visualization by staining with - IF: FITC-conjugated goat anti-mouse IgG or IP: HRP-conjugated goat anti-mouse IgG.

Immuno-fluorescence/ Immunoperoxidase analysis of tranfected BHK-21 cells: IF/IP-A: the DNA vaccine constructs; IF/IP-B: vector control; IF/IP-C: Mock transfected cells. For immunofluorescence analysis, 1 -microscopic view at 4OX; 2-microscopic view under fluorescent microscope. For immunoperoxidase analysis, 1 -microscopic view at 4OX.

Fig. 4: Humoral immune response in mice vaccinated with pgp.LAMP-1, pgp.LAMP-1 + Emulsigen®-D, vector (100 μg/mice) or PBS on days 0, 21 and 42. On days 20, 41 and 62, mice were bled for analysis of anti-RV-G antibodies by ELISA. Microtitration plates were coated with recombinant glycoprotein (expressed in bacterial system; 500 ng/well) and incubated with 1 :50 dilutions of immune sera samples. ELISA antibody titers are presented as the mean from all mice in each group.

Fig. 5a: The isotype profile of the RV-G-specific IgGl (A) and IgG2a (B) titers in mice immunized by different protocols. Each group of mice (n=10) was immunized respectively by DNA vaccine construct ± Emulsigen®-D, vector or PBS. Mice were bled at days 20, 41 and 62 and glycoprotein-specific IgGl and IgG2a titers were detected by ELISA. Optical density was measured at 450 nm. Data shown represents geometric mean titers and standard errors for each group of animals. Fig. 5b: The IgGl/IgG2a Ratio was calculated by dividing the mean optical density obtained with the IgGl isotyping reagent by that obtained with the IgG2a isotyping reagent.

Fig. 6: Concentrations of cytokines in cell-culture supernatants of BALB/c mouse splenocytes. Splenocytes (5 X10⁵cells/ml) were stimulated with 5 μg/ml of BPL-inactivated PV-I l virus. 24, 48 and 72 h later, culture supernatants were collected and analyzed by a capture ELISA for IL-2, IL-4, IL- 12 and IFN-γ. Splenocytes from two mice immunized with DNA vaccine constructs were included in each experiment. Data are expressed as mean values ± SD of triplicates.

Fig. 7: Rabies virus neutralizing antibody (RVNA) titers in mice (A) and in dogs (B) vaccinated with DNA vaccine construct were determined. The bars represent the geometric mean of the RVNA titers obtained with individual serum samples. Both the groups showed RVNA titer higher than 0.5 IU/ml, which is the minimum adequate titer against rabies as recommended by WHO. Data represents RVNA titers on day 62.

Fig. 8: Survival percentage of mice immunized with rabies DNA vaccine. All mice were challenged intracerebrally with 20 LD50 of CVS strain of Rabies virus on day 21 after the last immunization and observed for 18 days for rabies specific symptoms or death.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations:

Ab, antibody; C terminus, carboxy terminus; CVS, challenge virus standard; ELISA, Enzyme Linked Immunosorbent Assay; gp, glycoprotein; Lm., intramuscular(ly); IFNγ, interferon-γ; Ig, immunoglobulin; IL, interleukin ; IU, international units; LAMP-I, human lysosomal associated membrane protein-1 signal sequence; LD, lethal dose; MHC, major histocompatibility complex; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PV, Virus Pitman-Moore; WHO, world health organization. EXAMPLES

The following materials and methods were used in the examples that follow:

Materials And Methods:

DNA vaccine plasmid: Glycoprotein antigen was PCR amplified using pTargeT-RabG as the template, with the following primers:

Forward primer: (SEQ ID NO. C)

5'- GCGCATGCTCTTCCATGGTTCCTCAGGCTCTCCTG -3' Reverse primer: (SEQ ID NO. D)

5'- GCAGAAGCTCTTCGGCCTCACAGTCTGGTCTCACC -3'

The primers were designed to generate 5'-ATG and 3'-GGC alanine linker for address tags that facilitate C-terminus extensions needed for trafficking (Endosomal LAMP-I anchor). The amplified product was cloned in a eukaryotic expression vector bearing the targeting signal (LAMP-I signal sequence).

Plasmid DNA preparation: Plasmid DNA was processed using Endo-free Maxi kit (Qiagen, Valencia, CA).

In vitro expression of the DNA vaccine candidate: The ability of the DNA vaccine construct to express glycoprotein antigen was studied in vitro in a mammalian cell culture system. In brief, BHK-21 cells were cultured and seeded at a density of 10⁶cells/ml in a 24- well tissue culture plate, a day prior to transfection. BHK-21 cells were subsequently transfected with 800 ng DNA complexed with 2 μl of Lipofectin (Invitrogen) and 8 μl of Plus Buffer (Invitrogen). 40 h post-transfection, cells were solubilized using RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate, ImM PMSF) supplemented with protease inhibitor cocktail (Sigma). 100 μg of total lysate was resolved by 12% SDS-PAGE, blotted on to nitrocellulose membrane and probed with mouse anti-Rabies polyclonal sera, followed by incubation with goat anti-mouse immunoglobulins conjugated with alkaline phosphatase (Sigma) and visualized with NBT/BCIP substrate (Amersham).

For flow cytometric analysis, BHK-21 cells transfected with the construct were probed with mouse anti-Rabies polyclonal sera, followed by staining with secondary antibody - Alexa Fluor 488- Anti mouse IgG and analyzed for fluorescence using Cell Lab Quanta™ SC MPL flow cytometer (Beckman Coulter Inc.). Ten thousand cells per sample were analyzed using FLl filter (525 nm) and percent green fluorescent cells were recorded using Quanta SC MPL Analysis Software Version 1.0 (Beckman Coulter Inc.).

Alternatively, the expression of glycoprotein was detected in transfected cells by indirect immunofluorescent and immunoperoxidase test. In brief, transfected cells were washed with PBS, fixed with acetone and then blocked with PBS containing 1% BSA. Cells were incubated with mouse anti-Rabies polyclonal sera for 2 h at 37 ºC. This was followed by washing and incubation with FITC-labeled anti-mouse IgG (1:100 dilution, Sigma-aldrich) or HRP-labeled anti-mouse IgG (1:100 dilution, Santa-Cruz). Cells stained with FITC-labeled anti-mouse IgG were washed with PBS, covered with glycerol phosphate (glycerol: phosphate:: 1:1) and then visualized under fluorescent microscope. Cells stained with HRP- labeled anti-mouse IgG were developed with TMB and then visualized under microscope.

Immunization of mice: Four to six-week old female BALB/c mice were used to verify the immunogenicity of the constructs. Mice were purchased from NIN, Hyderabad; and maintained in pathogen free environment at the Animal House Facility of JNU. Each group comprised often mice. Mice were vaccinated intramuscularly (i.m.) with 50, 100, or 200 μg Endotoxin-free plasmid DNA in 100-200 μl PBS/animal in the individual vaccine groups, with or without adjuvant; thrice at three-week intervals. For adjuvant supplemented group, Emulsigen®-D was used at 15-20% v/v. For example, 100 μg of DNA was mixed with adjuvant at 20% and volume was then made up to 200 μl. Emulsigen®-D; oil in water emulsion containing Dimethyl Diocta Decyl Ammonium Bromide (DDA) was procured from MVP laboratories Inc., Omaha. Control mice were immunized with only PBS. Blood samples were collected at days, 20, 41 and 62. Mice were bled; sera were prepared and stored at -80 °C.

Immunization of dogs; The DNA vaccine construct was injected intramuscularly in to groups of four dogs each receiving 100 μg of vaccine plasmid DNA at one site in quadriceps muscle; thrice at three-week intervals. Two dogs were immunized with vector as negative control. Two unimmunized dogs were kept as control. Blood samples were collected at day 62 post first immunization. Sera from each dog were analyzed for rabies virus neutralizing antibody titers by rapid fluorescent focus inhibition test (RFFIT).

Determination of anti-glvcoprotein antibody and its isotvpes: Antigen-specific Antibody (IgG total) and isotypes (IgGl, IgG2a) levels were determined by ELISA. Recombinant glycoprotein (expressed in bacterial system, 500 ng/well) in 100 μl of 0.1 M PBS was coated overnight at 4 ºC. Plates were then blocked with 2% BSA in PBS for 2 h at 37 ºC followed by three washings with PBS-Tween 20 (0.05%). This was followed by incubation with sera samples for 2 h at 37 ºC and washing with PBST. Secondary antibodies, anti- mouse IgG or its isotypes conjugated with horseradish peroxidase; raised in sheep (Santa-Cruz) were incubated for 1 h at 37 ºC. Estimation of the enzymatic activity was carried out with TMB as the substrate. The reaction was stopped with 50 μl of IM H₃PO₄ and the absorbance was measured at 450 nm, with 630 nm as the reference filter using Microplate Reader (Bio Rad). The antibody response generated in a group of vaccinated mice was represented as the geometric mean of the absorbance; the reaction being carried out in triplicates.

Virus Neutralizing Antibodies (VNA) assay: Mouse and dog sera were tested in vitro for the presence of virus neutralizing antibodies with the rapid fluorescence inhibition test (RFFIT), as described previously (Smith, J.S., Yager, P.A., Baer, G.M., 1996. Laboratory techniques in rabies. Geneva, Switzerland: WHO: 181-92). Briefly, sera from mice and dogs (corresponding to day 62 post first immunization) and reference (Standard Rabies Immune Globulin) were heat inactivated at 56 °C for 30 min.100 μl of various sera dilutions were mixed with 100 μl of the CVS-Il strain of Rabies virus (containing 50 FFD50) in 96-well tissue culture plate and incubated at 37 °C, 5% CO₂ for 90 min. After the incubation period, BHK-21 cells (IxIO⁵) were added to each well and the plates were incubated for 40 h, following which they were fixed with chilled acetone and stained with FITC-conjugated anti- Rabies monoclonal antibody (VMRD, USA) for 45 min. The wells were washed thrice with PBS, mounted in glycerolrPBS (1:1), and visualized under fluorescence microscope (Nikon, Japan). Data were expressed as the neutralizing antibody titer that is the reciprocal of the serum dilution resulting in a 50% reduction in the number of the virus-infected cell foci in the presence of the test serum.

T-cell restimulation assays: Splenic cells were prepared by grinding spleens between frosted slides. Erythrocytes were lysed with 0.1 M ammonium chloride. Remaining spleen cells were washed twice with DMEM medium and were then suspended in complete DMEM medium supplemented with 10% heat inactivated fetal bovine serum and 10^'6 M β- mercaptoethanol. Viability was determined by Trypan blue exclusion test. Splenocytes were cultured in triplicate (IXlO⁶ cells/well) in a 24-well culture plate (Costar), stimulated without antigen or with 5 μg/ml of BPL inactivated PV-11 virus or concanavalin A (ConA) (1 μg/ml; Sigma), and incubated at 37 ºC under 5% CO₂ and 95% humidity. Supernatants were harvested after 24, 48 and 72 h and the levels of cytokine were determined.

Evaluation of cytokine levels by ELISA: Levels of IL-2, IL-4, IL- 12, and IFN-γ were determined using BD Opt EIA™ kits according to manufacturer's protocol (Pharmingen). Briefly, 96 well microtiter ELISA plate was coated with capture antibody of the respective cytokines and incubated overnight at 4 ºC. Plate was aspirated and washed thrice and blocked with 200 μl of 2% BSA for 2 h at 37 ºC. After the incubation period, plate was aspirated and washed thrice and incubated with the harvested supernatants for 2 h at RT. The plate was then aspirated and washed five times; plate was incubated with Detector (Anti-mouse IgG- HRP) for 1 h at RT. Following this, plate was aspirated and washed 7 times and incubated with 100 μl Substrate Solution for 30 min in dark at RT. Reaction was stopped by adding 50 μl Stop Solution to each well. The absorbance was read at 450 nm using a Microplate Reader (Bio Rad) within 30 min of stopping the reaction. The concentrations of cytokines in the culture supernatants were calculated using a linear regression equation obtained from the absorbance values of the standards provided by the manufacturer.

Protective efficacy against intracerebral Rabies virus challenge: For challenge, immunized mice were inoculated with 20 LD50 of Rabies virus CVS-Il strain intracerebrally 21 days post-immunization. The challenged mice were observed for eighteen days for symptoms indicative of Rabies virus infection. Mice that developed complete bilateral hind leg paralysis, a sign of the terminus stage of Rabies, were euthanized for humanitarian reasons. Upon challenge, PBS or vector vaccinated mice died within six to thirteen days. Surviving mice were kept and observed for an additional two to three weeks to ensure that they survived the infection. Survivorship rates obtained with different groups were compared.

RESULTS

Cloning of Glycoprotein structural gene and in vitro expression analysis of the DNA vaccine construct following transfection

Glycoprotein structural gene was cloned in the eukaryotic expression vector (Figs. 1, 2). The sequence of the above mentioned chimera is deposited in Genbank under the accession number: EU715588. In vitro expression and authenticity of the DNA chimera was evaluated by transient transfection and immunoblot blot and flow cytometric analysis. As shown in Fig. 3a, anti-GP mice polyclonal serum reacted with a distinct 67 kDa protein band observed in the lysate of transiently transfected BHK-21 cells. There was no such band in the vector or mock transfected cells. The expression of glycoprotein in pgp.LAMP-1 plasmid transfected BHK-21 cells was also analyzed by flow cytometer (Fig. 3b). The percentage of cells positive for fluorescence was recorded and compared with the mock transfected cells. The transfected cells were expressing the Rabies glycoprotein as indicated by fluorescence (74.85%) whereas the control cells revealed a low fluorescence signal (8.39%). As the majority of cells showed expression of the Rabies glycoprotein, it was thus, concluded that all the constructs were capable of expressing the protein efficiently in transfected cells. Expression was also observed by immunoflourescence and immunoperoxidase tests (Fig. 3c).

Delivery of DNA vaccine induced an enhanced immune response against glycoprotein

To address the issue whether the targeting signals could deliver the DNA encoded antigen to the immune system and induce a humoral response; antibody titers were assessed. Antibody titers increased on increasing the immunization dosage from 50 to 100 μg. However, there wasn't any significant increase on further increasing the dose to 200 μg. BALB/c mice were immunized i.m. at days 0, 21, 42 with pgp.LAMP-1; with or without the adjuvant. Mice immunized with empty vector and PBS served as negative controls Mice were bled at days 20, 41 and 62 and the sera was analyzed for the immune response generated (Fig. 4). Sera were analyzed for the presence of anti-GP IgGl and IgG2a antibodies by ELISA. As shown in Fig. 5a, significant IgGl titers were observed following immunization with pgp.LAMP-1. Higher titers were observed for DNA along with adjuvant formulation.

We also examined the IgG subclass distribution as an indicator of the T-helper-cell subsets (Thl/Th2) induced by the immunization. Both groups showed IgGl titers that were higher than the IgG2a responses which clearly indicate a Th2 biased immune response (Fig. 5b).

These data therefore demonstrate that plasmid encoding GP-LAMP-I efficiently targeted the antigen to MHC-II processing pathways, thereby inducing humoral immune response, which was superior in comparison to empty vector and PBS controls. Use of Emulsigen®-D adjuvant further improved the immune response generated.

Differences in the T-helper cell responses after vaccination

T helper cells (Thl/Th2) play an important role in eliciting both humoral and cellular responses via expansion of antigen-stimulated B cells and CD8⁺ T cells or CTLs, respectively. Apart from IL-2, a ThO cytokine, the levels of ThI cytokines (IL-12, and IFN-γ) and T h2 cytokines (.IL-4) were measured as parameters or poianzation of immune response (Fig. 6). The synthesis of cytokines by splenocytes from immunized mice; stimulated with BPL inactivated PV-Il virus and Con-A was assayed by ELISA at 24, 48 and 72 h. A substantial increase in the levels of IL-4 in adjuvant supplemented DNA vaccine (250 pg/ml; at 48 h) as compared to the control (unstimulated) splenocytes was observed. Only plasmid DNA also generated high level of IL-4 (125 pg/ml; at 48 h). IL-12 and IFN-γ were also produced in significant amounts. There was no significant increase in the cytokine levels of mice immunized with vector or PBS.

Rabies virus neutralizing antibody (RVNA) response

The ability of the anti-rRV-G antibodies generated to neutralize the Rabies virus in vitro was assessed by RFFIT. The VN titre against Rabies in both groups of immunized mice was >0.5 IU/ml indicating protective status against Rabies as recommended by WHO. The highest geometric mean RVNA titer was observed for pgp.LAMP-1 supplemented with Emulsigen®-D (32 I.U./ml), which was followed by only plasmid- pgp.LAMP-1 group (16 I.U./ml). In comparison, vector or PBS immunized group didn't induce significant neutralizing antibodies (Fig. 7A). In dogs, 8 I.U./ml of RVNA titer was generated, which was also significantly protective, in comparison to vector or PBS immunized dogs (Fig. 7B).

DNA vaccination protected mice against viral challenge

Finally, we addressed the question whether the immune responses induced by DNA vaccination protected the mice against lethal challenge. On day 21 after the last immunization, we challenged mice with 20 LD50 of Rabies virus CVS-I l strain intracerebrally.

The lethality of the challenge was confirmed by death of all the mice in the control and vector immunized group within six to thirteen days post-challenge. All the mice (8 out of the 8 mice) immunized with pgp.LAMP-1 + Emulsigen®-D survived the viral challenge thus, eliciting a survival percentage of 100%. The other group, namely pgp.LAMP-1 showed a survival percentage of 62.5% (5 out ot 8 immunized mice survived the chalenge (fig. 8) Surviving mice were kept and observed for an additional two to three weeks to ensure that they survived the infection.

SEQUENCE LISTING

The chimera (pgp.LAMP-1) has the following sequence:

SEQ ID NO : A

LENGTH : 1695

NAME : pgp.LAMP-1

TYPE : DNA

ORGANISM : Rabies virus

1 ATGGTTCCTC AGGCTCTCCT GTTTGTACCC CTTCTGGTTT TTCCATTGTG 51 TTTTGGGAAA TTCCCTATTT ACACGATCCC AGACAAGCTT GGTCCCTGGA 101 GCCCGATTGA CATACATCAC CTCAGCTGCC CAAACAATTT GGTAGTGGAG 151 GACGAAGGAT GCACCAACCT GTCAGGGTTC TCCTACATGG AACTTAAAGT 201 TGGATACATC TTAGCCATAA AAATGAACGG GTTCACTTGC ACAGGCGTTG 251 TGACGGAGGC TGAAACCTAC ACTAACTTCG TTGGTTATGT CACAACCACG 301 TTCAAAAGAA AGCATTTCCG CCCAACACCA GATGCATGTA GAGCCGCGTA 351 CAACTGGAAG ATGGCCGGTG ACCCCAGATA TGAAGAGTCT CTACACAATC 401 CGTACCCTGA CTACCGCTGG CTTCGAACTG TAAAAACCAC CAAGGAGTCT 451 CTCGTTATCA TATCTCCAAG TGTAGCAGAT TTGGACCCAT ATGACAGATC 501 CCTTCACTCG AGGGTCTTCC CTAGCGGGAA GTGCTCAGGA GTAGCGGTGT 551 CTTCTACCTA CTGCTCCACT AACCACGATT ACACCATTTG GATGCCCGAG 601 AATCCGAGAC TAGGGATGTC TTGTGACATT TTTACCAATA GTAGAGGGAA 651 GAGAGCATCC AAAGGGAGTG AGACTTGCGG CTTTGTAGAT GAAAGAGGCC 701 TATATAAGTC TTTAAAAGGA GCATGCAAAC TCAAGTTATG TGGAGTTCTA 751 GGACTTAGAC TTATGGATGG AACATGGGTC GCGATGCAAA CATCAAATGA 801 AACCAAATGG TGCCCTCCCG ATCAGTTGGT GAACCTGCAC GACTTTCGCT 851 CAGACGAAAT TGAGCACCTT GTTGTAGAGG AGTTGGTCAG GAAGAGAGAG 901 GAGTGTCTGG ATGCACTAGA GTCCATCATG ACAACCAAGT CAGTGAGTTT 951 CAGACGTCTC AGTCATTTAA GAAAACTTGT CCCTGGGTTT GGAAAAGCAT 1001 ATACCATATT CAACAAGACC TTGATGGAAG CCGATGCTCA CTACAAGTCA 1051 GTCAGAACTT GGAATGAGAT CCTCCCTTCA AAAGGGTGTT TAAGAGTTGG 1101 GGGGAGGTGT CATCCTCATG TGAACGGGGT GTTTTTCAAT GGTATAATAT 1151 TAGGACCTGA CGGCAATGTC TTAATCCCAG AGATGCAATC ATCCCTCCTC 1201 CAGCAACATA TGGAGTTGTT GGAATCCTCG GTTATCCCCC TTGTGCACCC 1251 CCTGGCAGAC CCGTCTACCG TTTTCAAGGA CGGTGACGAG GCTGAGGATT 1301 TTGTTGAAGT TCACCTTCCC GATGTGCACA ATCAGGTCTC AGGAGTTGAC 1351 TTGGGTCTCC CGAACTGGGG GAAGTATGTATTTCTGAGTG CAGGGGCCCT 1401 GACTGCCTTG ATGTTGATAA TTTTCCTGAT GACATGTTGT AGAAGAGTCA 1451 ATCGATCAGA ACCTACGCAA CACAATCTCA GAGGGACAGG GAGGGAGGTG 1501 TCAGTCACTT CCCAAAGCGG GAAGATCATA TCTTCATGGG AATCACACAA 1551 GAGTGGGGGT GAGACCAGAC TGGGCCTTAA CAACATGTTG ATCCCCATTG 1601 CTGTGGGCGG TGCCCTGGCA GGGCTGGTCC TCATCGTCCT CATTGCCTAC 1651 CTCATTGGCA GGAAGAGGAG TCACGCCGGC TATCAGACCA TCTAA .

The chimera when expressed in eukaryotic expression system produces the emmene protem (GP -LAMP-I) of the following amino acid sequence:

SEQIDNO :B LENGTH : 564

NAME : GP.LAMP-1

TYPE : Protein

ORGANISM : Rabies virus

MVPQALLFVPLLVFPLCFGKFPIYTIPDKLGPWSPIDIHHLSC PNNLVVEDEGCTNLSGFSYMELKVGYILAIKMNGFTCTGVV TEAETYTNFVGYVTTTFKRKHFRPTPDACRAAYNWKMAGD PRYEESLHNPYPDYRWLRTVKTTKESLVIISPSVADLDPYDR SLHSRVFPSGKCSGVAVSSTYCSTNHDYTIWMPENPRLGMS CDIFTNSRGKRASKGSETCGFVDERGLYKSLKGACKLKLCG VLGLRLMDGTWVAMQTSNETKWCPPDQLVNLHDFRSDEIE HLVVEELVRKREECLDALESIMTTKSVSFRRLSHLRKLVPGF GKAYTIFNKTLMEADAHYKSVRTWNEILPSKGCLRVGGRCH PHVNGVFFNGIILGPDGNVLIPEMQSSLLQQHMELLESSVIP LVHPLADPSTVFKDGDEAEDFVEVHLPDVHNQVSGVDLGLP NWGKYVLLSAGALTALMLIIFLMTCCRRVNRSEPTQHNLRG TGREVSVTSQSGKIISSWESHKSGGETRLGLNNMLIPIAVGG ALAGLVLIVLIAYLIGRKRSHAGYQTI

The following primers were used for cloning ot trie DNA vaccine construct:

SEQ ID NO : C LENGTH : 35 NAME : Forward primer

TYPE : DNA

ORGANISM : None; synthetically generated GCGCATGCTCTTCCATGGTTCCTCAGGCTCTCCTG

SEQ ID NO : D

LENGTH : 35

NAME : Reverse primer

TYPE : DNA

ORGANISM : None; synthetically generated

GCAGAAGCTCTTCGGCCTCACAGTCTGGTCTCACC

Claims

We Claim

1. A pharmaceutical composition to induce an immune response against Rhabdo virus in an animal susceptible to rabies disease comprising a DNA vaccine construct that encodes and expresses the glycoprotein gene of rabies virus.

2. The immunogenic composition of claim 1 wherein the Rhabdo virus is rabies virus

(RV).

3. A pharmaceutical composition for the vaccine against rabies as claimed in claim 1 comprising:

-endotoxin free DNA vaccine construct based on glycoprotein gene fused to

LAMP-I signal sequence at the C-terminus (50-200μg) and

-an adjuvant (15-20% v/v).

4. A pharmaceutical composition for the vaccine against rabies as claimed in claim 3, wherein the said signal sequence is pharmaceutically acceptable.

5. A pharmaceutical composition for the vaccine against rabies as claimed in claim 4, wherein said LAMP-I signal sequence targets the glycoprotein gene to lysosomal membrane to potentiate an immune response by promoting processing of the linked antigenic polypeptide via the MHC class II pathway or targeting of a cellular compartment that increases said processing.

6. A pharmaceutical composition for the vaccine against rabies as claimed in claim 3, wherein the adjuvant is a pharmaceutically acceptable vehicle.

7. A pharmaceutical composition for the vaccine against rabies as claimed in claim 6, wherein the adjuvant is Emulsigen ®-D.

8. A pharmaceutical composition for the vaccine against rabies as claimed in claim 7, wherein the adjuvant is oil in water emulsion containing Dimethyl Diocta Decyl Ammonium Bromide.

9. A pharmaceutical composition for the vaccine against rabies as claimed in claim 8, wherein the oil is mineral oil.

10. A pharmaceutical composition for the vaccine against rabies as claimed in claim 1, wherein the DNA vaccine construct is a eukaryotic expression vector.

11. A pharmaceutical composition for the vaccine against rabies as claimed in claim 10, wherein expression of glycoprotein gene is controlled by cytomegalo virus (CMV) promoter.

12. A pharmaceutical composition for the vaccine against rabies as claimed in claims 10 and 11, wherein the DNA vaccine construct codes for the complete mature glycoprotein.

13. A pharmaceutical composition for the vaccine against rabies as claimed in claims 3 and 10, wherein the encoded fusion gene has the sequence of SEQ ID A.

14. A pharmaceutical composition for the vaccine against rabies as claimed in claims 3 and 10, wherein the fusion gene when expressed in eukaryotic expression system produces the chimeric protein of the sequence SEQ ID B.

15. A pharmaceutical composition for the vaccine against rabies as claimed in claims 3- 14 wherein the vaccine construct; SEQ ID A encodes endosomal form of glycoprotein gene with the assistance of C-terminal LAMP-I signal for targeting it to lysosomal membrane.

16. A pharmaceutical composition for the vaccine against rabies as claimed in claim 15 wherein the encoded glycoprotein antigen will be taken up as an exogenous antigen for presentation of peptides in context with MHC-II molecules.

17. A pharmaceutical composition for the vaccine against rabies as claimed in claims 15 and 16 that will aid the generation of antigenic peptides in lysosomal/endosomal compartment that will be displayed in context with MHC II molecules on the surface of antigen presenting cell.

18. A pharmaceutical composition for the vaccine against rabies as claimed in claims 15-

17, wherein the signal sequence is cleaved as part of normal cell cycle to generate immune response against the encoded glycoprotein.

19. A pharmaceutical composition for the vaccine against rabies as claimed in claim 3, wherein DNA Vaccine construct based on glycoprotein gene fused to LAMP-I signal sequence at the C-terminus (90-1 lOμg) and an adjuvant (20% v/v) to achieve 100% protection on intracerebral viral challenge.

20. A process for preparing the pharmaceutical composition comprising: i. preparing the endotoxin free DNA vaccine construct based on glycoprotein gene fused to LAMP-I signal sequence at the C-terminus using Endo free maxi kit, ii. dissolving the vaccine of step (i.) in buffered saline, iii. mixing the endotoxin free DNA Vaccine of step (ii.) at 50-200 μg concentration with 15-20% v/v of adjuvant and making up the volume to 100-200μl.

21. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 20, wherein the said signal sequence is pharmaceutically acceptable.

22. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 21, wherein said LAMP-I signal sequence targets the glycoprotein gene to lysosomal membrane to potentiate an immune response by promoting processing of the linked antigenic polypeptide via the MHC class II pathway or targeting of a cellular compartment that increases said processing.

23. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 20, wherein the adjuvant is a pharmaceutically acceptable vehicle.

24. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 23, wherein the adjuvant is Emulsigen ®-D.

25. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 24, wherein the adjuvant is oil in water emulsion containing Dimethyl Diocta Decyl Ammonium Bromide.

26. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 25, wherein the oil is mineral oil.

27. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 20, wherein the DNA vaccine construct is a eukaryotic expression vector.

28. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 27, wherein expression of glycoprotein gene is controlled by cytomegalo virus (CMV) promoter.

29. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claims 27 and 28, wherein the DNA vaccine construct codes for the complete mature glycoprotein.

30. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claims 20 and 27, wherein the encoded fusion gene has the sequence of SEQ ID A.

31. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claims 20 and 27, wherein the fusion gene when expressed in eukaryotic expression system produces the chimeric protein of the sequence SEQ ID B.

32. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claims 20-31, wherein the vaccine construct; SEQ ID A encodes endosomal form of glycoprotein gene with the assistance of C-terminal LAMPl signal for targeting it to lysosomal membrane.

33. The process for preparing the DNA vaccine composition against rabies according to claim 22 wherein the encoded glycoprotein antigen will be taken up as an exogenous antigen for presentation of peptides in context with MHC-II molecules.

34. The process for preparing the DNA vaccine composition against rabies according to _ . claims 22, 32 and ,33 that will aid the generation of antigenic peptides in lysosomal/endosomal compartment that will be displayed in context with MHC II molecules on the surface of antigen presenting cell.

35. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claims 32-34, wherein the signal sequence is cleaved as part of normal cell cycle to generate immune response against the encoded glycoprotein.

36. The process for preparing the pharmaceutical composition for the vaccine against rabies as claimed in claim 20, wherein DNA Vaccine construct based on glycoprotein gene fused to LAMP-I signal sequence at the C-terminus (90-1 lOμg) and an adjuvant

(20% v/v) to achieve 100% protection on intracerebral viral challenge.