TECHNICAL FIELD
The present invention relates to a method of prophylactic or therapeutic immunization of vertebrates against infections caused by vertebrate viruses. The method comprises of administration of a vertebrate, a vaccine composition containing a minimum of two components, one of which is a deoxyribonucleotide (DNA) vaccine comprising of a DNA molecule that encodes a polypeptide of the virus and the other component consisting of inactivated form of the virus. The protective immune responses induced by the administration of these two components together is superior to that induced by the administration of individual components alone. Thus, this invention can be used to develop a combination vaccine consisting of DNA vaccine and inactivated virus. In another aspect, this invention can be used to develop low cost inactivated virus-based vaccines that contain much lower amount of the said virus than that present in similar vaccines known in the prior art.
BACKGROUND OF THE INVENTION
Hver since Edward Jenner first documented the successful vaccination strategy for small pox more than two hundred years ago. vaccine development has undergone dramatic changes. The first generation vaccines involved the use of attenuated, live or killed pathogens as vaccines and this mode of vaccination was primarily responsible for eradicating diseases such as polio, small pox etc. Rapid progress in animal cell culture technology led to development of cell culture-based vaccines, wherein the pathogenic organisms were cultured in large scale, purified, inactivated and used as vaccines. The use of killed or inactivated viruses as vaccines is widely practiced for diseases such as polio, rabies, measles etc. In this method of vaccination, the virus is presented to the immune system in a non-infective form so that the individual can mount an immune response against it. Killed or inactivated viruses provide protection by directly generating T-helper and humoral immune responses against the immunogens of the virus. However, in this type of vaccination, because the virus does not replicate or undergo an infective cycle, cell-mediated immune response mediated by cytotoxic T

lymphocytes (CTLs) is not generated. In the absence of an efficient CTL response, these vaccines often do not confer complete protection against pathogen. Another major problem associated with killed or inactivated virus-based vaccines is their high cost of production. Although several such tissue culture-based inactivated virus vaccines are available for diseases such as rabies, the high cost of production of the virus at high titres using tissue culture methods renders these vaccines uneconomical in many parts of the world especially in the developing countries, where the demand for inactivated virus-based vaccines far exceeds the supply. Thus, a major draw back of these types of vaccines is their high cost of production and difficulty of producing them in large quantities. Strategies which decrease the quantity of inactivated virus in the vaccine formulation without compromising on vaccine potency can lower the cost of production of inactivated virus-based vaccines. Thus, there is a need to develop novel vaccines, which can overcome all or some of the drawbacks associated with inactivated virus-based vaccines.
The use of plasmid DNA as a vaccine assumes great significance since it can be produced at a very low cost and can be stored at room temperature. In the seminal study by Wolff el al (Wolff JA, Malone RW, Williams P. Chona W, Acsadi G, Jani A, Feigner PL. 1990. Direct gene transfer into mouse muscle in vivo. Science 247:1465-68) of "plasmid or naked" DNA vaccination in vivo, it was shown that direct intramuscular inoculation of plasmid DNA encoding several different reporter genes could induce protein expression within the muscle cells. This study provided a strong basis for the notion that purified/ recombinant nucleic acids ('"naked DNA") can be delivered in vivo and can direct protein expression. These observations were further extended in a study by fang el al (Tang, DC, De Vit M, Johnson SA. 1992. Genetic immunization is a simple method for eliciting an immune response. Nature 356:152-54), who demonstrated that mice injected with plasmid DNA encoding hGH could elicit antigen-specific antibody responses. Subsequently, demonstrations by Ulmer el al (Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Feigner PL. Dwarki VJ. Gromkowski SH, Deck RR. De Witt DM. Friedman A. Hawe LA. Leander KR. Martinez D, Perry HC, Shiver JW,

Montgomery DC. Liu MA. 1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259: 1745-49) and Robinson el ai (Robinson HJLJ_._Hun_t._l,A!_.WebsterJRCL_..!993. Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 11: 957-60) that DNA vaccines could protect mice or chickens, respectively, from influenza infection provided a remarkable example of how DNA vaccination could mediate protective immunity. The mouse study further documented that both antibody and CD8' cytotoxic T-lymphocyte (CTL) responses were elicited (Ulmer JB. Donnelly JJ, Parker SE, Rhodes GH. Feigner PL. Dwarki VJ, Gromkowski SH, Deck RR. De Witt DM. Friedman A. Hawe LA. Leander K.R. Martinez D, Perry 1IC. Shiver ,IW. Montgomery DC. Liu MA. 1993. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 259: 1745-49. Robinson HL. Hunt LA. Webster RG. 1993. Protection against a lethal influenza virus challenge by immunization with a haemagglutinin-expressing plasmid DNA. Vaccine 11: 957-60) consistent with DNA vaccines stimulating both humoral and cellular immunity. This was followed by several reports which demonstrated the utility of DNA vaccines to induce protective immune response against several infectious diseases including cancer in experimental models. The art is rich in literature on DNA vaccines as evident from several books (DNA Vaccines: Methods and Protocols. Robert G. Whalen. Douglas B. Lowrie (E:ditor) / Hardcover / Humana Press / August 1999. Gene Vaccination: Theory and Practice. Hyal Raz (Editor) / Hardcover / Springer-Verlag New York, Incorporated / July 1998. DNA Vaccines: A New Era in Vaccinology. Margaret A. Liu, Reinhard Kurth (Editor), Maurice R. Hilleman (Editor)/Paperback/New York Academy of Sciences /October 1995, Current
Topics in Microbiology and Immunology (yo_L_.226)_ entitled: DNA
Vaccination/Genetic Vaccination edited by David Weiner and Hilary Koprowski, Lewis PJ, Cox GJ. van Drunen Littel-vanden Hurk S, Babiuk LA. 1997. Polynu¬cleotide vaccines in animals: enhancing and modulating responses. Vaccine) and review articles (Babiuk LA. Lewis J, Suradhat S, Baca-Lstrada M, Foldvari M, Babiuk S (1999) Polynucleotide vaccines : potential for inducing immunity in

vaccines Allergy, 54: 47-48, Stevenson FK (1999) PNA vaccines against cancer: from genes to therapy Ann Oncol, 10: 1413-1418, Stevenson FK, Link CJ, Jr., Traynor A. YuJH^£ojil9^j3|^A^vaj:cinalion_against multiple myeloma Semin Hematol, 36: 38-42, Tuteja R (1999) PNA vaccines: a ray of hope Crit Rev Biochem Mol Biol. 34: 1-24. Watts AM, Kennedy RC (1999) PNA vaccination strategies against infectious diseases Int. J Parasitol, 29: 1149-1163, Weiner PB, Kennedy RC (1999) Genetic vaccines Sci Am. 281 : 50-57. Whitton JL. Rodriguez F. Zhang J. Hassett PB (1999) PNA immunization: mechanistic studies Vaccine, 17: 1612-1619) which have been published on this subject. A web site devoted to PNA vaccines also provides valuable information on the progress made in this area (www, dnavaccine.com). Further, guidelines for the use of PNA vaccines in animal and human clinical trials are also available (U.S.
Food and Prug Administration. Points to Consider.
http://www.fda.gov/cber/points.htiTil). Other information that is helpful in
understanding and practice of the PNA vaccine technology can be found in the
patent Nos.US05580859. US05589466. US0562OS96. US05736524,
US05939400, US05989553 US0606338S. US06087341. US06090790.
US06I 10898, US06I56322. US05576196, US05707812. US5643578.
US0557619. US0570781, US05916879, US05958895, US05830S76,
US05817637, US6133244. US6020319. US5593972. WO00/24428.
W099/51745, W099/43841. WO99/388S80. WO99/02I32, W098/145S6. WO98/08947. WO98/04720. WOOO/77188. WO00/77043, WO00/77218. WO00771SS. WO00037649. WO00044406. WO09904009. WO00053019. WO00050044. WO00012I27, WO09852603, WO9748370, and WO097730587. The vast literature available on PNA vaccines indicates that PNA vaccines hold a great deal of promise in the prevention and treatment of several infectious diseases including cancer. The general applicability of PNA vaccines to induce protective immunity has been well established in several animal models and this has led to phase 1 human clinical trials. Studies carried out in the last couple of years indicate that for certain diseases. PNA vaccination alone cannot produce the desired effect, but if used in combination with other prophylactic or

therapeutic strategies can yield the desired results. One such strategy, referred to in the prior art as the •prime-boost strategy' involves administration of a DNA vaccine to 'prime" the immune system and this is followed by the administration of recombinant protein or live attenuated vaccines or inactivated pathogen-based vaccines to 'boost' the immune system (De Rose R. McKenna RV, Cobon G, Tennent I. Zakrzewski H. Gale K. Wood PR, Scheerlinck JP, Willadsen P (1999) Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep Vet Immunol Immunopathol, 71: 151-160, Qkuda K, Xin KO, Tsuji T, Bukawa H. Tanaka S, Koff WC, Tani K, Honma K. Kawamoto S, Hamajima K, Fukushima ,1. 1997. DNA vaccination followed by macromolecular multicomponent peptide vaccination against H1V-1 induces strong antigen-specific immunity. Vaccine 15: 1049-56, Barnett SW, Raiasekar S, Legg 11, Doe B, Fuller PH. llaynes JR, Walker CM, Steimer KS. 1997. Vaccination with H1V-1 gp!20 DNA induces immune responses that are boosted by a recom-binant t gp!20 protein subunit. Vaccine 15:869-73, Letvin NL, Montefiori DC, Yasutomi Y, Perry HC, Davies ME, Lekutis C, Alroy M, Freed DC, Lord CI, Handt EK, Eui MA, Shiver JW. 1997. Potent, protective anti-H1V immune responses generated by bimodal HIV envelope DNA plus protein vaccination. Proc. Natal. Acad Sci. USA 94: 9378-83, Fuller PH. Corb MM. Barnett S, Steimer K, Haynes JR. 1997. Enhancement of immunodeficiency virus-specific immune responses in DNA-immunized rhesus macaques. Vaccine 15: 924-26, Hanke T. Blanchard TJ, Schneider J, Hannan CM, Becker M, Gilbert SCLHil! AY, Smjth GL_McMichael A. 19?8,_ KnhanccmeDt_of_MjjC cjass J: restricted peptide- specific I cell induction by a DNA prime/MVA boost vaccination regime. Vaccine 16:439-45, lladdad D, Liljeqvist S, Stahl S, Hansson MA i^erflmann P. AJijborg N, Berzins K I1999] Characterization of antibody responses to a Plasmodium falciparum blood- stage antigen induced by a DNA prime/protein boost immunization protocol Scand J Immunol, 49 : 506-514, Fensterle J, Grode L, Hess J, Kaufmann SH (1999) Effective DNA vaccination against listeriosis by prime/boost inoculation with the gene gun J Immunol, 163: 4510-4518, Sedegah M, Jones TR. Kaur M, Hedstrom R. Hobart P, Tine J A,

Hoftman SL. 1998. Boosting with recombinant vaccinia increases immunogenicity and protective efficacy of malaria DNA vaccine. Proc. Natl. Acad. Sci. USA 95: 7648-53, Schneid^JJ_Gjl^_SCJ^lajiciM^IJJHMkgJL Robson KJ, Hannan CM. Becker M, Sinden R. Smith GL, Hill AV. 1998, Enhanced immunogenicity for CD8 T cell induction and complete protective efficacy of malaria DNA vaccination by boosting with modified vaccinia virus Ankara. Nat. Med. 4:397-402. Kent SJ. Zhao A, Best SJ. Chandler JD, Boyle DB, Ramshaw IA. 1998. Enhanced T- cell immunogenicity and protective efficacy of a human immunodeficiency virus type 1 vaccine regimen consisting of consecutive priming with DNA and boosting with recombinant fowl pox virus. J. Virol. 72:10180-88. Xiang ZQ. Pasquini S. Ertl HC (1999) Induction of genital immunity by DNA__p_rjming_ and intranasal booster immunization with a replication-defective adenoviral recombinant J Immunol. 162: 6716-6723. Robinson ML, Montefiori DC, Johnson RP, Manson K.H, Kalish ML, Lifson JD, Rizvi TA, Lu S, Hu SL, Mazzara GP. Panicali PL, Herndon JG, Glickman R, Candido MA. Lydv SL, Wyand MS, McClure HM. 1999. Neutralizing antibody-independent containment of immunodeficiency virus challenges by DNA priming and recombinant pox virus booster immunizations, Nat. Med. 5:526-34, Richmond JF, Mustafa F, Lu S, Santoro JC, Weng J, O'Connell M, Fenyo EM, Hurwitz JL,....Montefiori DC, Robinson HL. 1997, Screening of HIV-1 Env glycoproteins for the ability to raise neutralizing antibody using DNA immunization and recombinant vaccina virus boosting. Virology 230: 265-74. Richmond JF. Lu S, Santoro JC, Weng 1, Hu SL, Montefiori DC, Robinson HL. 998. Studies of the neutralizing activity and avidity of anti-human immunodeficiency virus type I Env antibody elicited by DNA priming and protem. b^osAmg^
clinical development of a multi-CTL epitope based DNA prime MVA boost vaccine for AIDS Immunol Lett. 66: 177-181, Hanke T. Samuel RV. Blanchard TJ, Neumann VC. Allen I'M. Boyson JE. Sharpe SA. Cook N. Smith GL. Watkins Dl, Cranage MP. McMichael AJ (1999) Effective induction of simian immunodeficiency virus-specific cytotoxic T lymphocytes in macaques by using

a multicpitope gene and DNA prime-modified vaccinia virus Ankara boost vaccination regimen J Virol. 73 : 7524-7532. Calarota SA, Leandersson AC. Bratt G, Hinkula J. Klinman DM, . Wejnhold. K-LSandstrom.E....Wahrcri._BJI999} Immune responses in asymptomatic HIV-1-infected patients after H1V-DNA immunization followed by highly active antiretroviral treatment J I Immunol.. Caver TE. Lockey TD. Srinivas RV. Webster RG. Hurwitz JL (999) A novel vaccine regimen utilizing PNA. vaccinia virus and protein immunizations for H1V-1 envelope presentation Vaccine. 17: 1567-1572). In addition to the prime-boost strategy, several other strategies are described in the prior art for improving the potency of DNA vaccines. These include:
1. co-inoculation of multiple plasmids expressing different antigens of a
pathogen or
plasmids expressing multiple epitopes of different antigens of a pathogen (Bot A. Bot S, Bona C. 1998. Enhanced protection against influenza virus of mice immunized as newborns with a mixture of plasmids expressing hemagglutinin and nucleoprotein. Vaccine 16:1675-82, Kim JJ, Ayyavoo V, Bagara/zi ML. Chattergoon M, Boyer JD, Wang B. Weiner DB. 1997. Development of a multicomponent candidate vaccine for HIV-1. Vaccine 15: 879-83, Whitton JL, Rodriguez I', Zhang J, Hassett DE. 1999. DNA immunization: mechanistic studies. Vaccine 17:1612-19, Ciernik IF, Berzofsky J A. Carbone DP. 1996. Induction of cytotoxic T-lymphocyte and antitumor immunity with DNA vaccines expressing single T-cellepitopes, J. Immunol. 156: 2369-75, Iwasaki A, Dela Cruz CS, Young AR, Barber BH. 1999. Epitope-specific cytotoxic T lymphocyte induction by mini-gene DNA immunization. Vaccine 17: 2081-88. Yu Z, Karem K.L, Kanangat S. Majiickan E, Rouse B f. J.998_, Proteciion by mini- gencs:a novel approach of DNA vaccines. Vaccine 16: 1660-67. Hanke T, Schneider J. Gilbert SC, Hill AV. McMichael A. 1998. DNA multi-CTL epitope vaccines for HIV and, Plasmodium falciparum: immunogenicity inmice. Vaccine 16: 426-35. Thomson SA. Sherritt MA. Medveczky J. Elliott SL. Moss DJ, Fernando JG, Brown LE, Suhrbier A. 1998. Delivery of multiple CD8 cytotoxic 1' cell

epitopes by DNA vaccination. J. Immunol. 160:1717-23. Suhrbier A. 1997. Multi-epitope DNA vaccines. Immunol. Cell Biol. 75:402-8, Wang R, Doolan PL, Charoenvit Y, Hedstrom RC, Gardner NJ, Hobajt P, Tine J, Sedegah M. Fallarme V. Sacci JB Jr. Kaur M, Klinman DM, Hoffman SL. Weiss WR. 1998. Simultaneous induction of multiple antigen-specific cytotoxic T lymphocytes in nonhuman primates by immunization with a mixture of four Plasmodium falciparum DNA plasmids. Infect. Immun. 66: 4193-202. Shi YP, Hasnain SE. Sacci JB, Holloway BP, Fujioka H. Kumar N,„ WohJhueter R, Hoffman SL, Collins WE, Lai AA. 1999. Immunogenicity and in vitro protective efficacy of a recombinant multistage Plasmodium falciparum candidate vaccine. Proc. Natl. Acad. Sci. USA 96:1615-20. Mossman SP, Pierce CC. Robertson MN, Watson AJ, Montefiori DC, Rabin M, Kuller L. Thompson J, Lynch JB, Morton WR, Benveniste RE, Munn R, llu SL, Greenberg P, Haigwood N. (1999) Immunization against SIVmne in macaques using multigenic DNA vaccines J Med Primatol. 28: 206-213.)
2. co-inoculation of plasmids encoding cytokines or co-stimulatory
molecules and
Bio-lechnol. 15: 641-46, Tsuji T, Hamajima K, Ishii N, Aoki I, Fukushima J, Xin KQ. Kawamoto S, Sasaki S, Matsunaga K, Ishigatsubo Y. Tani K, Qkubo T, Qkuda K. 1997. Immunomodulatory effects of a plasmid expressing B7-2 on human immunodeficiency virus-1-specific cell-mediated immunity induced by a plasmid encoding the viral antigen. Eur../. Immunol. 27:782-87. Xiang Z, Ertl HC. 1995. Manipulation of the immune response to a plasmid-encoded -viral antigen by coinoculation with plasmids expressing cytokines. Immunity 2:129- 35, Lee AH, Suh YS, Sung YC. 1999. DNA inoculations with HIV-1 recombinant genomes that express cytokine genes enhance HIV-1 specific immune responses. Vaccine 17:473-79, Weiss WR, Ishii KJ, Hedstrom RC, Sedegah M, Ichino M, Barnhart K, Klinman DM, Hoffman SL. 1998. A plasmid encoding murine granulocyte-macrophage colony- stimulating factor increases protection conferred by a

malaria DNA vaccine. J. Immunol. 161: 2325-32, Svanholm C, Lowenadler B, Wigzell H. 1997. Amplification of T-cell and anti-body responses in DNA-based immunization with HIV-1 Nef by co-injection with a GM- CSF expression vector. Scand. ./. immunol 46: 298-303, Tsuji T, Hamaiima K, Fukushima J, Xin KQ, Ishii N. Aoki 1. Ishigatsubo Y, Tani K, Kawamoto S, Nitta Y, Miyazaki J. Koff WC. Okubo T, Okuda K. 1997. Enhancement of cell-mediated immunity against HIV-1 induced by coinoculation of plasmid-encoded HIV-1 antigen with plasmid expressing 1L-12. J. Immunol. 158:4008-13, Kim JJ, Ayyavoo V, Bagarazzi ML. Chat-tergoon MA, Pang K, Wang B, Boyer JD, Weiner DB. 1997. In vivo engineering of a cellular immune response by coadministration of 1L-12 expression vector with a DNA immunogen. ./. Immu-nol. 158: 816-26, Larsen PL, Dybdahl-Sissoko N, Mc-Gregor MW, Prape R, Neumann V, Swain WF, Lunn DP, Olsen CW. 1998. Coadministration of DNA encoding interleukin-6 and hemagglutinin confers protection from influenza virus challenge in mice. J. Virol. 72: 1704-8, Kim JJ, Simbiri KA. Sin Jl, Pang K. Oh J, Dentchev T. Lee P, Nottingham LK, Chalian AA, McCallus P, Ciccarelli E, Agadjanyan MG, Weiner PB. 1999. Cytokine molecular adjuvants modulate immune responses induced by PNA vaccine constructs for HIV-1 and SIV, ./. Interferon Cytokine Res, 19: 77-84, Chow YH, Chiang BL, Lee YL, Chi WK, Lin WC, Chen YT, Tao Mil. 1998, Pevelopment of Thl and Th2 populations and the nature of immune responses to hepatitis B virus DNA vaccines can be modulated by codelivery of various cytokine genes. 1 Immunol. 160: 1320-29, Geissler M, Gesien A, Tokushige K, Wands JR. 1997. Enhancement of cellular and humoral immune responses to hepatitis C_.YJrys core protein using DNA-based vaccines augmented with cytokine-expressing plasmids. J. Immunol. 158: 1231-37, Chow YH, Huang WL, Chi WK. Chu YD, Tao MH. 1997. Improvement of hepatitis B virus DNA
vaccines by plasmids coexpressing hepatitis B surface antigen and
interleukin-2. J. Virol. 71: 169-78, Kim JJ, Trivedi NN, Nottingham LK, Morrison L, Tsai A, Hu Y, Mahalingam S, Dang K, Ahn L, Doyle NK,

Wilson DM, Chattergoon MA, Chalian AA, Boyer JD. Agadjanyan MG, Weiner DB. 1998. Modulation of amplitude and direction of in vivo immune responses by to-administration of cytokine gene expression cassettes with DNA immunogens, Eur. ,/. Immunol. 28: 1089-103, Gurunathan S, Irvine K.R. Wu CY. Cohen J], Thomas E, Prussin C, Restifo NP, Seder RA. 1998. CD40 ligan/trimer DNA enhances both humoral and cellular immune responses and induces protective immunity to infectious and tumor challenge. J. Immunol. 161:4563-71, Maecker HT, Umetsu DT. DeKru^ff _RH, Levy _S. 1997, DNA vaccination with cytokine fusion constructs biases the immune response to ovalbumin. Vaccine 15: 1687-96, Hakim I. Levy S, Levy R. 1996. A nine amino acid peptide from 11.-10 augments antitumor immune responses induced by protein and DNA vaccines. ,/. Immunol. 157: 5503-11, Ishii K.J, Weiss WR, Ichino D, Verthelyi D, Klinman DM. 1999. Activity and safety of DNA plasmids encoding 11.-4 and IFN-c. Gene Ther. 6: 237-44, Okada E, Sasaki S. Ishii N. Aoki I, Yasuda T, Nishioka K. 1'ukushima J, Miyazaki J. Wahren B, Okuda K. 1997. Intranasal immunization of a DNA vaccjne with IL-I2- and granulocyte-macrophage colony stimulating factor (GM-CSF) expressing plasmids in lipo-somes induces strong mucosal and cell-mediated immune responses against HIV-1 antigens.,/. Immunol. 159: 3638-47).
3. Inclusion of certain compounds such as saponin, alum, chitosan, liposomes, cationic lipids etc., in the DNA vaccine preparations (Iwasaki. A, Stiernholm BJ, Chan AK, Berinstein NL, Barber BH, 1997. Enhanced C'I'L responses mediated by plasmid DNA immunogens encoding costimulatory molecules and cytokines. ./. Immunol. 158: 4591-601, Operschall E, Schuh f, Heinzerling L. Pavlovic J, Moelling K. (1999) Enhanced protection against viral infection by co-administration of plasmid DNA coding for viral antigen and cyrokines in mice J Clin Virol, 13: 17-27, Ishii N, Tsuji T, Xin KQ. Mohri IJL Okuda _K, 1998. Adjuvant effect of Uben-imex on a DNA vaccine for HIV-1. Clin. Exp. Immunol. Ill: 30-35, Sasaki S. Sumino K, llamajima K. Fukushima J, Ishii N, Kawamoto S, Mohri H, Kensil CR.

Okuda K, 1998. Induction of systemic and mucosal immune responses to human immunodeficiency virus type 1 by a DNA vaccine formulated with QS-21 saponin adjuvant via intramuscular and intranasal routes. J. Virol. 72: 4931-39, Sasaki S, Hamajima K. Fukushima J, lhata A, Ishii N, Gorai I, Hirahara F, Mohri H, Okuda K. 1998. Comparison of intranasal and intramuscular immunization against human immunodeficiency virus type 1 with a DNA-monophosphoryl lipid A adjuvant vaccine, Infect. Immun. 66: 823-26. Roy K. Mao HO, Huang SK, Leong KW. 1999. Oral gene delivery wjth_chitosan- DNA nanoparticlcs generates immunologic protection in a murine model of peanut allergy. Nat. Med. 5: 387-91, Klavinskis LS, Gao L, Bamfield C. Lehner T. Parker S. 1997. Mucosal immunization with DNA-liposome complexes. Vaccine 15: 818-20, Haigwood NL, Pierce CC, Robertson MN, Watson AJ, Montefiori DC, Rabin M, Lynch JB, Kuller L, Thompson J, Morton WR, Benveniste RE, Hu SL, Greenberg P, Mossman SP (1999) Protection from pathogenic SIV challenge using multigenic DNA vaccines Immunol Lett, 66: 183-188, Herrmann JE, Chen SC, Jones DH, Tin-sley-Bown A, Fynan EF, Greenberg HB, Farrar GH, 1999. Immune responses and protection obtained by oral immunization with rotavirus VP4 and VP7 DNA vaccines encapsulated in microparticles. Virology 259: 148-53, Chen SC, Jones DH, Fynan EF, Farrar GH, Clegg JC, Greenberg HB, Herrmann JE. 1998. Protective immunity induced by oral immunization with a rotavirus DNA vaccine encapsulated in microparticles. J. Virol. 72: 5757-61. Jones DH. Corris S. McDonald S, Clegg JC. Farrar GH. 1997. Poly (DL-lactide- co-glycolide)-encapsulated plasmid DNA elicits systemic and mucosal antibody responses to encoded protein after oral administration. Vaccine 15: 814-17. Lodmell PL. Ray NB, Ulrich JT, Ewalt LC (2000) DNA vaccination of mice against rabies virus: effects of the route of vaccination and the adjuvant monophosphoryl lipid A (MPL) Vaccine, 18: 1059-1066).
4. Inclusion of specific sequences referred to as the CpG motifs in the plasmid DNA (Krieg AM (1999) CpG DNA: a novel immunomodulator Trends

Microbiol 7, Sato Y, Roman M, Tighe J, Lee P. Corr M. Nguyen MP, Silverman Gl, Lotz M, Carson PA, Raz E. 1996. lmmunostimulatory PNA sequences necessary for effective intradermal gene immunization. Science 273: 352-54, Krieg AM. Yi AK, Matson S. Wald-schmidt TJ. Bishop GA. Teasdale R, Koretzky GA. Klinman PM. 1995. CpG motifs in bacterial PNA trigger direct B-cell activation. Nature 374: 546-49, Klinman PM, Yamshckikov G. Ishigat-subo Y. 197. Contribution of CpG motifs to the immunogenicity of PNA vaccines. J. Immunol. 158: 3635-39, Brazobt Millan CL, Weeratna R, Krieg AM, Siegrist CA, Pavis HL. 1998. CpG PNA can induce strong Thl humoral and cell-mediated immune responses against hepatitis 13 surface antigen in young mice. Proc. Natl. Acad Sci. USA 95: 15553-58, Klinman PM. 1998. Therapeutic applications of CpG-containing oligodeoxv nucleotides. Anli.sen.se Nucl Ac id Drug Dev. 8: 181-84, Klinman PM, Bamhart KM, Conover J. 1999. CpG motifs as immune adjuvants. Vaccine 17: 19-25. Zimmermann S, Kgeter O. Hausmann S, Lipford GB, Rocken M, Wagner H. Heeg K. 1998. CpG oligodeoxynuctgotjdes trigger protective and curative Thl responses in lethal murine leishmaniasis. ./. Immunol. 160: 3627-30, Roman M, Martin-Qrozco H, Goodman JS. Nguyen MP, Sato Y, Ronaghy A, Kornbluth RS, Richman P_PJ___Carson__ PA, Raz JE, _ 199_7, lmmunostimulatory PNA sequences function as T helper-1 promoting adjuvants. Nat. Med 3: 849-54, Oxenius A, Martinic MM, Hengartner H, Klenerman P (1999) CpG-containing oligonucleotides are efiicient adjuvants for induction of protective antiviral immune responses with T-cell peptide vaccines J Virol, 73 : 4120-4126. Paillard F (1999) CpG: the double-edged sword [comment| JJum_GencIb.eL.10: 2089^2090).
Further, a variety of strategies on modification of plasmid vector and/or the gene encoding the antigen were shown to improve the potency of PNA vaccines (Bryder K, Sbai H, Nielsen HV, Corbet S, Nielsen C, Whalen RG, Pomsgaard A (1999) Improved immunogenicity of HIV-1 epitopes in FIBsAg chimeric PNA vaccine plasmids by structural mutations of HBsAg PNA Cell Biol., Norman JA, Hobart

P, Manthorpe M Feigner P, Wheeler C. 1997. Development of improved vectors for DNA-based immunization and other gene therapy applications. Vaccine 15: 801-3, Wild J. Gruner B, Mclzger K, Kuhrober A. Pudollek HP. Hauser H, Schirmbeek R, Reimann J. 1998. Polyvalent vaccination against hepatitis B surface and core antigen using a dicistronic expression plasmid. Vaccine 16: 353-60, Uchijima M, Yoshida A, Nagata T, Koide Y. 1998. Optimization of codon usage of plasmid DNA vaccine is required for the effective MHC class l-resicted T cell responses against an intracellular bacterium. J. Immunol. 161: 5594-99, Andre S, Seed B, liberie J, Schraut W, Bultmann A, Haas J. 1998. Increased immune response elicited by DNA vaccination with a synthetic gp!20 sequence with optimized codon usage. ■/. Virol. 72: 1497-503, Vinner L, Nielsen HV, Bryder K, Corbet S, Nissen C, Fotnsgaard A. 1999. Gene gun DNA vaccination with Rev-independent synthetic H1V-1 gpl 60 envelope gene using mammalian codons. Vaccine 17: 2166-75, Inchauspe G. Vitvitski L, Major ME, Jung G, Spengler U, Maisonnas M, Trepo C. 1997. Plasmid DNA expressing a secreted or a nonsecreted form of hepatitis C virus nucleocapsid: comparative studies of antibody and T-helper responses following genetic immunization. DNA Cell Biol. 16: 185-95, Rice J, King CA, Spellerberg MB, Pairweather N, Stevenson FK. 1999. Manipulation of pathogen-derived genes to influence antigen presentation via DNA vaccines. Vaccine 17: 3030-38, Wu Y, Kipps TJ. 1997. Deoxyribonucleic acid vaccines encoding antigens with rapid, proteasome-dependent degradation are highly efficient induces of cytolytic T lymphocytes. J. Immunol. 159: 6037-43, lobery TW, Siliciano RF. 1997. Targeting of HIV-1 antigens for rapid intracellular degradation enhances cytotoxic T lymphocyte (CTL) recognition and the induction of de novo CTL responses in vivo after immunization. ./. Exp. Med. 185: 909-20, Rodriguez F, Zhang J, Whitton JL. 1997. DNA immunization: ubiquitination of a viral protein enhances cytotoxic T-lymphocyte induction and antiviral protection but abrogates antibody induction. J. Virol. 71: 8497-503, Chaplin PJ, De Rose R, Boyle JS, McWaters. P, Kelly J, Tennent JM, Lew AM, Scheerlinck JP (1999) Targeting improves the efficacy of a DNA vaccine against Corynebacterium pseudotuberculosis in sheep Infect

Immun, 67: 6434-6438, Forns X, Emerson SU, Tobin GJ, Mushahwar IK, Purcell RH, Bukh J (1999) DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope H2 protein expressed intracellular^ and on the cell surface Vaccine, 17: 1992-2002, Lewis PJ, van Drunen Littel-van den H. Babiuk LA (1999) Altering the cellular location of an antigen expressed by a DNA-based vaccine modulates the immune response J Virol, 73: 10214-10223, Li Z, Howard A, Kellev C. Delogu. G, Collins F. Morris S (1999) Immunogenicity of DNA vaccines expressing tuberculosis proteins fused to tissue plasminogen activator signal sequences Infect lmmun, 67: 4780-4786, Lowrie DB. Tascon RE, Bonato VL, Lima VM, Faccioli LH, Stavropoulos E, Colston MJ, Hewinson RG, Moelling K, Silva CL (1999) Therapy of tuberculosis in mice by DNA vaccination Nature, 400: 269-271, Nagata T, Uchijima M, Yoshida A, Kawashima M, Koide Y (1999) Codon optimization effect on translational efficiency of DNA vaccine in mammalian cells: analysis of plasmid DNA encoding a CTL epitope derived from microorganisms Biochem Biophys Res Commun. 261: 445-451, Svanholm C, Bandholtz L, Lobell A. Wigzell H (1999) Enhancement of antibody responses by DNA immunization using expression vectors mediating efficient antigen secretion J Immunol Methods, 228: 121-130, Torres CA, Yang K, Mustafa F, Robinson HL (1999) DNA immunization: effect of secretion of DNA-expressed hemagglutinins on antibody responses Vaccine. IS: 805-814).Thus. there is scope for the development of novel compositions that can improve the potency of DNA vaccines.
SUMMARY OF INVENTION
This invention describes a novel method of enhancing the potency of DNA vaccines by the addition of small quantities of inactivated virus to the DNA vaccine preparation. In one embodiment, this invention can lead to the development of combination vaccines containing DNA vaccine and inactivated virus. In another embodiment, this invention can lead to the development of vaccines containing much lower quantities of inactivated virus than that present in conventional inactivated virus-based vaccines. Phis invention also describes a method of producing a novel

vaccine composition against rabies virus.
BRIEF DESCRIPTION OF THE FIGURES:
Figure 1 is the schematic representation of the rabies DNA vaccine plasmid (pCMVRab) encoding the rabies virus glycoprotein.
DETAILED DESCRIPTION OF THE INVENTION
The present invention describes a novel method of immunization of vertebrates against infectious diseases using a vaccine formulation comprising minimally of killed or inactivated form of a virus and plasmid DNA encoding one or more polypeptides present in the inactivated virus. The method described in this invention can be used to develop a combination vaccine consisting mainly of the inactivated virus and plasmid DNA encoding one or more polypeptides of the virus. The vaccine formulation consisting of inactivated virus and DNA vaccine has a much higher potency than that containing inactivated virus alone or DNA vaccine alone, when used at the quantities present in the combination vaccine. As is true for all embodiments of the present invention, the vaccine formulation does not contain live or attenuated viruses.
The utility of this invention in developing combination vaccines is explained using rabies virus as an example but the methods of this invention are applicable to other vertebrate viruses since the mechanisms involved in the induction of protective immunity by DNA vaccines or inactivated virus-based vaccines are similar for all vertebrate viruses.
Potency as described in this invention refers to the ability of the vaccine to induce virus neutralizing antibodies and/or to protect the immunized host against subsequent virus challenge. In one embodiment, the potency of rabies vaccine is evaluated by measuring the rabies virus neutralizing antibody titre in the sera of immunized animals by the rapid fluorescent focus inhibition test or RFFIT (Smith J S. Yager P A. Bear G M. in Laboratory techniques in rabies, by Meslin F-X. Kaplan M M & Koprowski H, (WHO. Geneva) 1996. 181). In another embodiment, the potency of the rabies vaccine is evaluated by the level of

protection conferred by the rabies vaccine in the immunized host against rabies virus challenge.
The inactivated virus-based vaccines described in this invention can be produced by cell/tissue culture methods using any of the vertebrate cell lines known in the prior art. For example, inactivated rabies virus can be produced using vertebrate cells in culture as described in US patents 3423505, 3585266. 4040904, 3769415, 4115195. 3397267, 4664912, 4726946. In one embodiment, a purified chick embryo cell (PCEC) rabies vaccine produced from chick embryo cells were used in combination with rabies DNA vaccine. In another embodiment, purified Vero cell rabies vaccine (PVRV) produced from Vero cells were used in combination with rabies DNA vaccine. In yet another embodiment, rabies virus produced from baby hamster kidney (BHK) cells were used as the source of inactivated rabies virus. Further, the methods of the present invention are considered appropriate for the immunization of vertebrate species such as murine, canine, ovine or humans. In one embodiment, the murine species is a mouse. In another embodiment the canine species is a dog. In yet another embodiment, the bovine species is cattle.
The quantity of inactivated virus in the combination vaccine can vary widely depending on the immunogenicity and potency of the inactivated virus-based formulations. In one embodiment, the quantity of inactivated rabies virus in the combination vaccine is six hundred times less than that present in the tissue culture rabies vaccines known in the prior art. The amount of inactivated rabies virus to be used in combination with rabies DNA vaccine was determined based on careful titration of different dilutions of the inactivated rabies virus vaccine. The dose which failed to induce appreciable levels of VNA in the immunized host and/or failed to confer 100% protection following rabies virus challenge was chosen as the dose to be used in combination with DNA vaccine to develop a combination vaccine with higher potency. It is well known to those skilled in the art of producing rabies vaccines that the final concentration of the inactivated rabies virus and plasmid DNA to be present in the vaccine formulation should be determined by protocols described in the prior art such as those mentioned in U.S.

or British pharmacopoea using the vaccine standards obtained from World Health Organization (WHO).
As used in this invention, the plasmid DNA refers to extrachromosomal. covalently closed circular DNA molecules capable of autonomous replication in bacterial cells and consist of transcription units (i.e., nucleotide sequences encoding a polypeptides) similar or identical to those present in the virus of interest which are operably linked to transcriptional and translational regulatory sequences necessary for expression of the proteins in the cells of vertebrates.
The transcription unit of the plasmid described in the present invention may contain any of the large number of known eukaryotic promoters such as those found in the genomes of animal viruses and animals including humans, Further, the transcription unit may contain DNA encoding polypeptides of vertebrate pathogens such as viruses, bacteria, parasites etc. In one preferred embodiment, the transcription unit consists of DNA encoding the surface glycoprotein of rabies virus,
The introduction of DNA encoding polypeptides of vertebrate pathogens into the plasmids can be carried out by any of the known protocols described in the prior art (Sambrook J, Fritsch EF, Maniatis T, (1989) Molecular cloning; A laboratory Manual. 2nd ed. (Cold Spring Harbour laboratory press. Cold Spring Harbour. NY). Further, methods of introducing such plasmids into bacteria, culturing in nutrient media and purification of the plasmids from bacterial cultures can be carried out using any of the processes known in the prior art (Sambrook J, Fritsch FF, Maniatis T.11989) Mo]ecula_r_ cJorungL A laboratory Manual. 2nd ed. (Cold Spring Harbour laboratory press. Cold Spring Harbour. NY. Prazeres DMF. Fetreira GNM. Monteiro GA, Cooney CL. Cabral JMS. (1999) large scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and botlenecks. TIBTECH 17: 169-174).
Examples illustrating the various aspects of this invention are provided below. These examples are only illustrative and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1: Rabies DNA vaccine preparation and purification.
The rabies DNA vaccine plasmid was constructed as follows. The cytomegalovirus immediate early promoter and intron was isolated from the pCMVintBL plasmid (Manthorpe M, Cornefert-Jensen F, Hartikka J, Feigner J, Rundell A. Margalth M, Dwarki VJ. (1993) Gene therapy by intramuscular injection of plasmid DNA: studies on Firefly luciferase gene expression in mice-Mum. Gene Ther. 4: 419-421) by digestion with the restriction enzymes Hindlll and PstI and cloned at the Hindlli and PstI sites of the pEGFPI plasmid (Clontech, CA, USA) to obtain pCMVEGFP construct. The cDNA encoding rabies virus surface glycoprotein was isolated as a Bglll restriction fragment from ptgl55 vector (Keiny el al) and cloned into the Bamlll site of pCMVEGFP. Finally, the cDNA encoding EGFP was excised out as a Xbal-NotI fragment and the vector was re-ligated to obtain pCMVRab construct. Standard recombinant DNA techniques described in the prior art (Keiny, MP, Lathe R. Drillen R, Spehner D. Skory S, Schmitt P. W'iktor I. Koprowski H. Lccocq JP. (1984) Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature 312: 163-166) were used in the construction of pCMVRab, The schematic diagram of pCMVRab is shown in Figure 1. Large scale isolation and purification of pCMVRab were carried out by the alkaline lysis procedure (Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular cloning; A laboratory Manual, 2nd ed. (Cold Spring Harbour laboratory press. Cold Spring Flarbour, NY, Prazeres DMF, Fetreira GNM, Monteiro GA, Cooney CL, Cabral JMS. (1999) large scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and botlenecks. TIBTECH 17: 169-174). The Final plasmid preparation was dissolved in saline (0.15M NaCI) and used as rabies DNA vaccine. It is well known to those skilled in the art of preparing eukaryotic expression plasmids that several variations of the above protocol can be used for the development of rabies DNA vaccine plasmid. For example, the cDNA encoding rabies glycoprotein can be derived from other rabies virus strains such as Challenge Virus Standard (CVS), Street-Alabama-Dufferin (SAD), Evelyn Rokitniki Abelseth (ERA) etc. Similarly, the cytomegalovirus promoter can be obtained from several eukaryotic
20

expression plasmids that are available commercially.
Example 2: Preparation of inactivated rabies virus vaccine from tissue culture of vertebrate cells.
The inactivated rabies virus vaccine can be prepared by any of the methods disclosed in the prior art such as those described in US patents 3423505, 3585266, 4040904, 3769415, 4115195, 3397267, 4664912, 4726946. Briefly, vertebrate cells such as the Vero cells, baby hamster kidney (BHK) cells etc., are infected with rabies virus. The virus is separated from the cellular debris by filtration and then inactivated by the addition of beta-propiolactone or bromoethyleneimine. The virus is then concentrated and a portion is tested for freedom from infectious virus by inoculation of sensitive monolayer cell cultures and by intracerebral inoculation into mice. The concentrated virus preparation is mixed with adjuvants such as aluminium hydroxide (3 milligrams per dose) and the liquid vaccine is used for inoculation of animals. Alternatively, the virus is purified further by zonal centrifugation followed by lyophilization in presence of compounds such as human serum albumin, maltose etc., and the lyophilized preparation is used as rabies vaccine. In one embodiment, the Purified Vero cell derived rabies vaccine (PVRV) produced by Indian Immunologicals Ltd., Hyderabad, India and known as AbhayrabR was used as the source of inactivated rabies virus vaccine. In another embodiment, the purified chick embryo cell (PCEC) cell derived rabies vaccine produced by Hoechst Marion Roussel, India and known as RabipurR was used as the source of inactivated rabies virus vaccine. In another embodiment, the veterinary rabies virus vaccine produced from baby Hamster Kidney (BHK) cells by Indian Immunologicals Ltd.. Hyderabad. India known as RaksharabR was used as the source of inactivated rabies virus vaccine.
Example 3: Preparation of combination vaccine consisting of inactivated rabies virus and rabies DNA vaccine.
The inactivated rabies virus vaccines such as AbhayrabR. RaksharabR or RabipurR were diluted 625 fold with saline and 0.5 ml of the diluted sample was mixed

with 100 micrograms of rabies DNA vaccine and the mixture was used as combination rabies vaccine for immunization of mice, dogs or cattle. When necessary, aluminium hydroxide was added to a final concentration of 3.0 milligrams per dose. The final vaccine preparation may also contain preservatives such as Thiomerso! (0.015%) and can be used either as a liquid vaccine or as a lyophilized preparation.
Example 4: Potency of rabies DNA vaccine, inactivated rabies virus vaccine and the combination vaccine consisting of inactivated rabies virus and DNA vaccine as evaluated in a murine rabies virus challenge model.
The potency of rabies DNA was evaluated in a murine peripheral rabies virus challenge model. A group often mice were inoculated with 100 micrograms of rabies DNA vaccine per mice twice at an interval of two weeks. Two weeks after the administration of the second dose, mice were inoculated in the foot pads with virulent rabies virus of the challenge virus standard (CVS) strain and observed for 14 days. The results presented in table 1 indicate that only 80% of the mice inoculated with rabies DNA vaccine are protected from rabies virus challenge. We therefore examined whether addition of small quantities of inactivated rabies virus to the rabies DNA vaccine preparation can lead to higher levels of protection. Before addition of inactivated rabies virus to the DNA vaccine, we examined the potency of AbhayrabR, an inactivated rabies virus vaccine produced from Vero cells in the murine rabies virus challenge model. Two hundred microlitres of saline was added to each vial of AbhayrabR (>2.5 IU) and each mouse in a group often mice was injected intramuscularly with 0.1 ml of the vaccine. Five fold dilutions of AbhayrabR (1:5. 1:25. 1:125, 1: 625) were also prepared and 0.1 ml of each diluted Abhayrab preparation was injected per mouse by intraperitoneal route. The immunizations were repeated 14 days later. The mice were inoculated in the foot pads with virulent rabies virus of the CVS strain two weeks after administration of the second dose and observed for 14 days. The results presented in Table I indicate that undiluted AbhayrabR vaccine as well as that diluted 5. 25 or 125 fold confers 100% protection against rabies

virus challenge. Thus, these dilutions were not used in the combination with DNA vaccine since the inactivated virus vaccine at these dilutions conferred 100% protection. However, when diluted to 625 fold, the potency of the AbhayrabR was reduced significantly and only 50% of the mice were protected from rabies virus challenge. This dose of AbhayrabR which confers suboptimal protection was used to study the effect of inactivated rabies virus on the potency of rabies DNA vaccine. The immunization experiments were repeated and mice were inoculated twice at two week interval with 0.1 ml of 625 fold diluted AbhayrabR alone or 100 micrograms of rabies DNA vaccine alone or a combination of both, Mice were challenged two weeks later and the results presented in Table 1 indicates that AbhayrabR and rabies DNA vaccine confer 50% and 80% protection respectively but a combination of these two vaccines results in 100% protection against rabies virus challenge. Thus, addition of suboptimal dose of inactivated rabies virus to rabies DNA vaccine results in the development of a novel combination rabies vaccine with higher potency.
To study whether inactivated rabies virus produced from chick embryo cells can also enhance the potency of rabies DNA vaccine, virus challenge experiments were carried out using RabipurR. an inactivated rabies virus vaccine produced from chick embryo cells. Two hundred microlitrcs of saline was added to each vial of RabipurR (>2.5 1U) and 100 microlitres was diluted up to 625 fold with saline. The diluted vaccine (100 micorlitres) was injected either alone or in combination with rabies DNA vaccine into each mouse twice at two week interval and the mice were challenged two weeks after the last immunization as described above. The results presented in Table 1 indicate that 625 fold diluted RabipurR confers 60% protection against rabies virus challenge but in combination with rabies DNA vaccine, the level of protection is increased to 100%.
Thus, inactivated rabies virus preparation produced from either Vero cells or chick embryo cells can enhance the potency of rabies DNA vaccine.
Example 5: Potency of rabies DNA vaccine, inactivated rabies virus vaccine

and the combination vaccine consisting of inactivated rabies virus and DNA vaccine as evaluated by RFFTT in a murine model
The potency of" rabies vaccine can also be determined by their ability to induce virus neutralizing antibodies (VNA) in the immunized host as evaluated by the RFFIT (Smith et at). Briefly, different dilutions of the serum are incubated with a standard dose of challenge virus preparation in the wells of tissue culture chamber slides and the slides are incubated at 35°C in a humidified carbon dioxide incubator for 60-90 minutes. BHK cells are then added (1 x 10" cells per well) and mixture is incubated as described above for a further 20 hours. The slides are washed in phosphate buffered saline (PBS) and fixed in cold acetone. After drying, fluorescein isothiocyanate-conjugated anti-rabies nucleo protein is added at appropriate dilution and the incubation continued for 20-30 minutes at 37°C.
finally the slides are rinsed in PBS and observed under fluorescent microscope. The virus neutralizing titres are calculated using known protocols. National or international reference serum diluted to a potency of 1.0 lU/ml is used in each test for titrating the titre of the test sera and the results are expressed in terms of UJ/ml. A rabies vaccine which induces a VNA titre of 0.5 UJ/ml and above is considered to be protective. Thus, the level of VNA in the immunized host is a measure of the rabies vaccine potency and vaccine with better potency induce higher VNA titres. We, therefore, examined whether administration of combination vaccine consisting of 625 fold diluted AbhayrabR and rabies DNA vaccine (100 micrograms) can result in the induction of higher VNA in the immunized animal. Groups often mice were inoculated with rabies DNA vaccine alone, different dilutions of AbhayrabR or a combination of 625 fold diluted AbhayrabR and rabies DNA vaccine as described above. Mice were bled by retroorbital puncture before the administration of second dose (day 14 post immunization) as well as two weeks after the administration of the second dose (day 28 post immunization). Sera samples of mice in each group were pooled and the level of rabies VNA was examined by RFFIT. The results presented in Table 2 indicates that 14 days after the administration of a single dose of rabies DNA

vaccine alone, 1/625 fold diluted AbhayrabR or both results in the a VNA titre (IU/ml) of 0.287. 0.095 and 1.42 respectively. When the sera were analysed two weeks after the administration of the second dose, the VNA titre (IU/ml) in mice immunized with rabies DNA vaccine alone. 1/625 fold diluted AbhayrabR or both has increased to 1.96. 0.55 and 4.07 respectively. These results clearly indicate that a combination of inactivated rabies virus vaccine and rabies DNA vaccine induces higher levels of VNA in mice than either of them alone.
Example 6: Potency of rabies DNA vaccine, inactivated rabies virus vaccine and the combination vaccine consisting of inactivated rabies virus and DNA vaccine as evaluated by RFFIT in dogs.
1 o examine whether the results obtained in mice could be reproduced in other vertebrate species, the immunization experiments were repeated in dogs. Dogs of 3-4 months of age, seronegative for rabies VNA were immunized via intramuscular route with 625 fold diluted AbhayrabR. 100 micrograms of rabies DNA vaccine or both, twice at two week interval. Hach group consisted one animal. The animals were bled on day 7. 14, 28 and 35 days postimmunization and the rabies VNA titre in the sera was analysed by RFFIT. The results presented in Fable 3 clearly indicates that the VNA titre in the animal inoculated with 625 fold diluted AbhayrabK and DNA vaccine is higher at all time points tested than that in animals inoculated with either DNA vaccine alone or 625 fold diluted AbhayrabK alone.
Thus, inoculation of a combination of inactivated rabies virus and rabies DNA vaccine induces higher levels of rabies VNA not only in mice but also in dogs.
Example 7: Potency of rabies DNA vaccine, inactivated rabies virus vaccine and the combination vaccine consisting of inactivated rabies virus and DNA vaccine as evaluated by RFFIT in Cattle,
Having demonstrated the potency of the combination vaccine consisting of inactivated rabies virus and rabies DNA vaccine in mice and dogs, we then evaluated its immunogenicity in a large animal model, the cattle. In this

experiment. RaksharabR, the inactivated veterinary rabies virus vaccine produced from BHK cells as well as AbhayrabR were used. Cross bred calves of 12-16 months of age, seronegative for antibodies against rabies virus, were vaccinated via intramuscular route with 625 fold diluted AbhayrabR or RaksharabR either alone or in combination with 100 micrograms of rabies DNA vaccine twice at two week interval and blood samples were collected on day 7. 14, 21. 28 and 35 days post immunization. Bach group consisted of three cattle and at each time point, the cattle sera in each group were pooled and then subjected to RF1TI analysis. The results presented in Table 4 clearly indicate that cattle inoculated with inactivated rabies virus (either AbhayrabR or RaksharabR) and rabies DNA vaccine rabies virus vaccine alone or rabies DNA vaccine alone.
In another independent experiment, the effect of adjuvants such as aluminium hydroxide on the potency of combination vaccine (inactivated rabies virus + DNA vaccine) was evaluated. Cattle were immunized twice at two week interval (day 0 and day 14) with DNA vaccine, 625 fold diluted RaksharabR or both in the presence or absence of aluminium hydroxide. Cattle were bled at regular intervals and the level of VNA was evaluated by RFFIT. The results presented in Table 5 indicate that addition of aluminium hydroxide further enhances the potency of combination rabies vaccine.
These examples describing the use of rabies DNA vaccine in combination with different inactivated rabies virus vaccines should only be regarded as illustrating and not limiting the invention, which is defined by the appended claims.
TABLE 1 Protection of mice inoculated with rabies DNA vaccine, inactivated rabies virus vaccine (AbhayrabR or Rabipur") or the combination vaccine from, rabies virus challenge

WE CLAIM:
1. A rabies vaccine formulation comprising a plasmid harbouring DNA sequences encoding the surface glycoprotein of rabies virus and inactivated rabies virus.
2. A rabies vaccine as claimed in claim 1. wherein the inactivated virus preparation is produced from vertebrate cells.
3. A rabies vaccine as claimed in claim 2, wherein the vertebrate cells are vero cells, baby hamster kidney cells or chick embryo cells.
4. A process of preparing a rabies vaccine formulation comprising a plasmid harbouring DNA sequences encoding the surface glycoprotein of rabies virus and inactivated rabies virus, which comprises the following steps:

a) Preparing a plasmid construct harbouring DNA-sequences encoding the surface glycoprotein of rabies virus;
b) Preparing an inactivated rabies virus preparation;
c) Mixing plasmid construct of step (a) at a dose ranging from 1 to 200 micrograms with inactivated rabies virus preparation of step (b) a buffer between pH 7.0 and 8.0, salts and stabilizers or preservatives;
d) Blending the preparation; and
e) Bottling the preparation.

5. The process of preparing a rabies vaccine formulation as claimed in claim 4 wherein the process further comprises the step of adding adjuvants.
6. The process of preparing a rabies vaccine formulation as claimed in claim 5, wherein the adjuvants comprise aluminium hydroxide.
7. The process oT preparing a rabies vaccine formulation as claimed in any one of claims 4 to 6 wherein the salt is sodium chloride.

8. The process of preparing a rabies vaccine formulation as claimed in any one of claims 4 to 7 wherein the stabilizers or preservatives comprise thiomersol. maltose and/or human serum albumin.
9. The process of preparing a rabies vaccine formulation as claimed in any one of claims 4 to 8 wherein the buffers comprise phosphate buffer and/or Tris buffer.
10. Rabies vaccine formulation consisting of plasmid harbouring DNA sequences encoding the surface glycoprotein of rabies virus and inactivated rabies virus for use in immunization of vertebrates against rabies.
11. The rabies vaccine formulation as claimed in claim 10 for use in immunization of cattle, dogs or humans.