WO2009063507A1 - Dna vaccine against anthrax - Google Patents

Abstract

The present invention relates to development of a DNA vaccine against anthrax using the protease-cleaved fragment of protective antigen. This fragment is operably linked to gene targeting cassette, either on N-terminal as a peptide leader or on C-terminal as a membrane anchoring tag or both. The gene targeting cassette will target the expressed antigen to various subcellular locations that will aid the processing and presentation of the antigen of interest through both the cellular antigen presentation pathways which would result in an enhanced immune response against protective antigen.

Description

DNA VACCINE AGAINST ANTBDRAX FIELD OF INVENTION

The present invention relates to the development of DNA vaccine against anthrax that is tailored to generate effective neutralizing antibody response as well as cellular immune response utilizing a set of signal sequences that target the encoded antigen to both the antigen processing pathways for generating an immune repertoire that is capable of providing protection against subsequent challenge. BACKGROUND AND PRIOR ART OF THE INVENTION

Cellular intoxication with anthrax toxin begins when PA (protective antigen, an 83 kDa protein), the receptor binding moiety of anthrax toxin binds to cell surface exposed anthrax toxin receptors on mammalian cells (Scobie, KM. et al. & Bradley, K.A. et al). Once bound, it is proteolytically processed either on the surface of the host cells or in the serum {Ezzell, J. et al & Panchal, R.G. et al.) to a 63 kDa fragment that is capable of forming a ring-shaped heptamer in the plasma membrane of the target cell (Lacy, D. B. et al). The PA heptamer then binds to LF (Lethal factor) and EF (Edema factor) (Mogridge J. et al). The resulting complex gets subsequently internalized into clathrin-coated pits via receptor-mediated endocytosis (Ahrami, L. et al 2003 & 2004). Inside the endosome this heptamer undergoes a conformational change from a soluble protein to integral membrane protein that forms a pore in the lysosomal membrane through which LF and EF translocate in the cytosol. LF, a zinc metalloprotease, proteolytically cleaves short N-terminal fragments from mitogen or extracellular signal- regulated protein kinase kinasel (MEKl)₅ MEK2 and MEK3, the upstream activators of ERKl, ERK2 and p38, respectively and thus, inhibits macrophages (Duesbery, N. S. et al, Vitale, G. et al. & Park JM. et al.) and suppresses T-lymphocyte activation by disrupting antigen receptor signaling (Paccani S,R, et al, ). EF, an adenylate cyclase, upon calmodulin binding undergoes structural rearrangements that leads to its activation and induces substantial increase in conversion of intracellular ATP to cAMP. Subsequently, water homeostasis and cellular signaling of host are disrupted, leading to edema during cutaneous anthrax infection (Leppla, S.H. 1982 & 1984). Additionally, EF inhibits the ability of neutrophils to phagocytose the bacilli and produce oxidative burst {Wright, G.G et al, Ahuja N. et al. & O'Brien, J. et al), co- operates with lethal factor to impair cytokine secretion during infection of dendritic cells and suppresses innate immune response (Cleret A. et al).

Studies in non-human primates support the correlation between vaccine-induced neutralizing antibodies against protective antigen (PA) and protection against subsequent challenge with pathogenic B. anthracis strain (Pitt M.L.M. et al, Fellows P. F. et al, McBride B. W. et al. & Turnbull P. CB. et al). Several vaccine strategies have been exploited to address this. In this context DNA vaccines seem to hold an enormous potential. Immunization with DNA vaccine encoding the antigen of interest (Ag) has been used to induce both cellular and humoral immunity (Justewicz D.Met al., Huygen K et al., Davis H.L et al, Gretchen S. et al & Brave A et al). To further enhance the humoral as well as cell-mediated responses elicited by DNA vaccines many strategies such as, cation-lipid formulation of DNA vaccines (Hermanson G. et al), liposome mediated delivery (Ishii N. et al), micro- particle delivery (Oster CG. et al), were tested with the goal of enhancing the antibody responses. Systemic vaccine strategies based on prime boost regimens that imply a DNA prime immunization followed by a booster with recombinant PA have been tested (Galloway D. et al, Hahn U.K. et al, Price BM. et al). Additionally, DNA vaccine encoding a fragment of LF has been shown to provide protection against lethal toxin challenge (Price BM. et al).

Given these considerations we reasoned that generation of high-avidity neutralizing antibodies that can mediate protection against subsequent challenge with anthrax will require the antigen to be efficiently targeted to various antigen processing and presentation pathways. Also, there is an increasing body of evidence to suggest that both CD8+ and CD4+ T cells are critical for the generation of an effective immune response against an intracellular pathogen. Although both CD4+ and CD8+ T cells recognize non-native forms of the antigen in association with major histocompatibility complex (MHC) molecules, the presentation of the antigen to these two types of T lymphocytes occurs through distinct pathways (Ljunggren KG. et al, Catherine Ret al, Castiglioni P. et al, Waldmann H. et al). For processing through the MHC II pathway the antigen needs to be targeted to the endosomal or lysosomal compartment (Harding C. V. et al, Nuchiern J.G. el al, Germain R.N. et al). The traditional pathway for the antigen targeting to lysosomal compartment involves phagocytosis or endocytosis of the exogenous antigen or specific targeting to endosomal pathway by attachment of lysosomal targeting signals. In addition to efficient antigen targeting and presentation of antigen via the MHC II pathway, effective CD8+ T cell responses are dependent upon efficient processing through MHC I pathway alongwith the help provided by CD4+ T cells (Rock KL. et al). Subsequently, recognition of CD8+ T cells should be reflected in the magnitude of Ag-specific CD8+ T cell-mediated immune responses (Castiglioni P et al).

Therefore, the invention reports the application of, trafficking of protease-cleaved fragment of protective antigen to various compartments of the cell with the ultimate goal of improving the neutralizing antibody response that would mediate protection against subsequent anthrax toxin challenge. OBJECTS OF THE INVENTION

The primary object of the invention is to develop a set of DNA chimeras that encode various forms (secreted, cytosolic, endosomal, secreted-endosomal) of protease-cleaved fragment of protective antigen. Mice, upon immunization generated high titers of neutralizing antibodies titers which were long-lasting. Also, anti-PA antibodies that were generated had high-avidity towards PA as indicated by avidity indices. The magnitude of protection against anthrax toxin challenge correlated with the levels of PA-specific antibody response and the generation of effective cellular immunity.

Another object of the present invention is to provide for a PA63-based DNA vaccine/composition for inducing a potent anti-PA humoral as well as cellular immune response.

Yet another object of the present invention is to provide a DNA vaccine that generated a high-quality immune response as compared to a recombinant protective antigen-based vaccine formulation (rPA83/rPA63 with alhydroxy gel) or any other PA-based DNA vaccine formulations lacking the targeting signals.

Yet another object of the present invention is to provide a DNA vaccine comprising protease-cleaved fragment of protective antigen of anthrax operably linked to an N-terminal human TPA leader for secretion of the encoded protective antigen. It is another object of the present invention to provide a DNA vaccine comprising protease-cleaved fragment of protective antigen of anthrax operably linked to an N-terminal mouse Ubiquitin A76 leader for proteasomal targeting.

Yet another object of the present invention is to provide a DNA vaccine comprising protease-cleaved fragment of protective antigen of anthrax operably linked to a C-terminal human LAMPl anchor for targeting of the protective antigen to lysosomal/endosomal membrane.

Still another object of the present invention is to provide^' a DNA vaccine comprising protease-cleaved fragment of protective antigen of anthrax operably linked to an N-terminal human TPA leader for secretion and a C-terminal human LAMPl anchor for targeting of the protective antigen to lysosomal/endosomal membrane. It is a further object of the invention to provide a vaccine ^"composition that generates a potent humoral and cellular immune response that correlates well with the survival against toxin challenge. SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a DNA vaccine comprising a gene targeting cassette comprising a gene construct comprising a nucleotide sequence encoding a protective antigen linked to signal sequence(s) present on one or both sides of the protective antigen for targeting the said antigen to various antigen processing pathways to enhance cellular and humoral immune response against the said protective antigen.

In accordance with the embodiments of the invention, the said protective antigen is PA63 gene from Bacillus anthracis.

In accordance with the embodiments of the invention, the said PA63 gene encodes a 63kDa protective antigen protein.

In accordance with the embodiments of the invention, one or more of the said signal sequences are present on the N-terminal of said protective antigen.

In a preferred embodiment of the invention, the said N terminal signal sequence is a human-TPA leader (pTPA). In yet another embodiment of the invention, the said N terminal signal sequence is a, ubiquitin leader (pUQ).

In yet another embodiment of the invention, one or more of the said signal sequences are present on the C-terminal of said protective antigen.

^■ In a further embodiment of the invention, the said C terminal signal sequence is LAMPl.

In another embodiment of the invention, the said construct pPA63 (Seq ID 1 ) encodes the native form of protease cleaved fragment of protective antigen (Seq ID 6).

In yet another embodiment of the invention, the said construct pTPA-PA63 (Seq ID 2) encodes secreted form of the protease cleaved fragment of the said protective antigen (Seq ID 7). In yet another embodiment of the invention, the said construct pUQ-PA63 (Seq ID 3) encodes the cytosolic form of the protease cleaved fragment of the said protective antigen (Seq ID 8).

In yet another embodiment of the invention, the said construct pPA63-LAMPl (Seq ED 4) encodes the endosomal form of the protease cleaved fragment of the said protective antigen (Seq ID 9).

In yet another embodiment of the invention, the said construct pTPA-PA63-LAMPl (Seq ID 5) encodes the secreted endosomal form of the protease cleaved fragment of the said protective antigen (Seq ID 10). In a further embodiment of the invention, the said protective antigen is presented by MHC I molecules/pathway.

In a still further embodiment of the invention, the said protective antigen is presented by MHC II molecules/pathway. The present invention also relates to a DNA vaccine wherein the said gene cassette is comprised of a vector comprising at least one protective antigen and signal sequence on one or both sides of the protective antigen operably linked to the transcriptional factors for targeting the said protective antigen to various antigen processing pathways characterized in that an enhanced cellular and humoral immune response is produced on expression of such antigen. In accordance with the embodiments of the invention, the said vector, is an expression vector selected from a prokaryote or eukaryote.

In accordance with the embodiments of the invention, the said gene cassette is present in a prokaryotic or eukaryotic host cell.

In accordance with the embodiments of the invention, the said signal sequences are meant for trafficking of said protective antigen to various sub-cellular locations.

In accordance with the embodiments of the invention, the immune response is generated against the protective antigen only.

In accordance with the embodiments, the DNA vaccine of the invention provides for an enhanced cellular and humoral immune response is generated in the absence of an immune adjuvant/stimulant.

In accordance with the embodiments, the DNA vaccine of the invention generates neutralizing antibodies of high avidity towards the said protective antigen when administered in a vertebrate.

In accordance with the embodiments, the DNA vaccine of the invention generates effective cytotoxic T-Iymphocytes against the said protective antigen when administered in a vertebrate.

In accordance with the embodiments, the DNA vaccine of the invention is effective against anthrax toxin.

In accordance with the embodiments, the said vertebrate is mouse. The present invention also relates to a pharmaceutical composition comprising the said DNA vaccine and a pharmaceutically acceptable additive.

In accordance with the embodiments of the invention, the said pharmaceutical composition comprising the said DNA vaccine comprises a gene cassette encoding differentially targeted, processed and presented protective antigen characterized in that an enhanced cellular and humoral immune response is generated in the absence of an immune adjuvant.

In accordance with the embodiments of the invention, the said pharmaceutical composition comprising the said DNA vaccine comprises a gene cassette encoding differentially targeted, processed and presented protective antigen characterized in that an enhanced cellular and humoral immune response ^" is generated when administered in a vertebrate.

In accordance with the embodiments of the invention, the said pharmaceutical composition comprising the said DNA vaccine comprises a gene cassette encoding differentially targeted, processed and presented protective antigen when administered in a vertebrate elicits an enhanced cellular and humoral immune response in comparison to the immune response elicited by two subcutaneous doses of recombinant full-length protective antigen protein vaccine administered as alhydroxy gel formulation. In accordance with the embodiments of the invention, the said vertebrate is mouse. ,

BRIEF DESCRIPTION OF THE DRAWINGS

FIG.l. shows cloning of protease-cleaved fragment of protective antigen in expression plasmid. Lane 1&2. pPA63-Native clone and vector, Lane 3&4. pUQ-PA63 clone and vector, Lane 5&6. pTPA-PA63 clone and vector, Lane 7 & 8. pPA63- LAMPl clone and vector, Lane 9 & 10. pTPA-PA63 -LAMPl clone and vector respectively, Lane M-DNA ladder.

FIG.2. shows expression of different forms of protease-cleaved fragment of protective antigen in J774.A1 mouse macrophage-like cells. Cells were transfected with DNA vaccine constructs- and cell lysates were prepared 48 hrs post-transfection. Subsequently, the samples were separated on 12% SDS-PAGE gel under reducing conditions and transferred onto a nitrocellulose membrane. Blot was probed with anti-PA polyclonal serum.

FIG.3.A, B, C, D. show IgGl, IgA, IgG2a and IgG2b antibody responses to PA63-based DNA vaccine constructs in mice, respectively. Balb/c mice were intramuscularly injected with plasmid DNA three times (lOOμg) or twice with DNA vaccine followed by subcutaneous vaccination with PA63 protein formulated with IFA at 4-wk intervals. PA-specific antibody titers were determined by ELISA at 4 week intervals. (P < 0.001, vs vector immunized control). FIG.4. shows neutralization of anthrax toxin by serum from immunized mice. Various dilutions of sera pooled from mice vaccinated with DNA vaccine constructs was pre-incubated with PA for 1 h. This mixture was added to J774.A1 cells in the presence of LF for 3 h, and cell survival measured. Results represent the average of two independent experiments.

FIG.5. shows avidity of PA-specific antibodies generated in the mice immunized i.m. with various DNA vaccine constructs. The bars show the percent binding after urea treatment + SD from replicate measurements. Data represented is an average of two experiments. (P < 0.01 vs control) FIG. 6.A & B. shows cell-mediated immune responses generated by PA63-based DNA vaccine constructs in mice. Murine splenocytes were isolated 4 wk after last immunization and were re-stimulated with rPA63 protein in vitro. The number of IFN-γ (A) and IL4 (B) secreting cells as determined by ELISPOT assay. The bars represent mean ± SEM for triplicate wells of pooled splenocytes from four mice. Data are representative of two experiments with comparable results. (P < 0.01 vs vector; P < 0.0001 vs PBS).

FIG.7. A & B. shows that PA63 -based DNA vaccines induces humoral immune responses. Mice received three doses of DNA vaccines separated by 4 wk intervals, as described in the immunization schemes. 4 weeks after the last immunization, ASC, IgA (A) & IgG (B) against PA63 were evaluated by ELISPOT in splenosytes. The bars represent mean + SEM for triplicate wells of pooled splenocytes from four mice. Data are representative of two experiments with comparable results. (P < 0.01 vs vector; P < 0.0001 vs PBS).

FIG.8. shows in vitro cell-proliferative responses to PA63-based DNA vaccine constructs in mice. Murine splenocytes were isolated 4 wk after last immunization and were re-stimulated with rPA63protein. Cell proliferation was analyzed by MTT assay. The bars represent mean ± SEM of pooled data from two to three experiments. (P < 0.0001 vs PBS; P < 0.05 vs vector,, P < 0.01 vs media). FIG.9. shows cytotoxic T-lymphocyte responses are generated by PA63 -based DNA vaccination. CTL activity of effector cells prepared from vaccinated mice after in vitro stimulation. Effector CTLs were assayed for their ability to lyse target cells. Cytotoxicity (% lysis) was evaluated by neutral red uptake method. The effector- to-target-cell ratios (E:T ratios) examined were 5:1, 10:1 and 15:1. Data are expressed as mean values ± SEM of triplicates. DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the application of protective antigen (PA63) trafficking to various compartments of the cell with the ultimate goal of enhancing the neutralizing antibody and specific cellular immune response. In addition to chimera bearing sequence of the native form of protease-cleaved fragment of Protective antigen (pPA63-

Native; SEQ ID NO: 1), the present invention- describes a set of chimeras that bear sequences that will either secrete PA63 (pTPA-PA63; SEQ ID NO: 2.) or target PA63 to cytosolic compartment (pUQ-PA63; SEQ ID NO: 3), endosomal compartment (pPA63-

LAMPl; SEQ ID NO: 4) and secretory endosomal compartment (pTPA-PA63 -LAMPl;

SEQ ID NO: 5) of the cell.

The different chimeras provided by the present invention are deposited in the MTCC gene bank under the accession numbers: SEQ ID NO: 1 GENE BANK ACCESSION NUMBER EU249810

SEQ ID NO: 2 GENE BANK ACCESSION NUMBER EU249808 SEQ ID NOc 3 GENE BANK ACCESSION NUMBER EU249809 SEQ ID NO: 4 GENE BANK ACCESSION NUMBER EU249806 SEQ ID NO: 5 GENE BANK ACCESSION NUMBER EU249807

The sequences of these chimeras are given below: SEQ ID NO : 1 LENGTH : 1695 NAME : pPA63-Native TYPE : DNA

ORGANISM: Bacillus anthracis

SEQUENCE : 1

1 atgcctacgg ttccagaccg tgacaatgat ggaatccctg attcattaga ggtagaagga

61 tatacggttg atgtcaaaaa. taaaagaact tttctttcac catggatttc taatattcat 121 gaaaagaaag gattaaccaa atataaatca tctcc.tgaaa aatggagc.ar. ggr.ftctgat 181 'ccgtacagtg atttcgaaaa ggttacagga cggattgata agaatgtatc accagaggca 241 agacaccccc ttgtggcagc ttatccgatt gtacatgtag atatggagaa tattattctc 301 tcaaaaaatg aggatcaatc cacacagaat actgatagtc aaacgagaac aataagtaaa 361 aatacttcta caagtaggac acatactagt gaagtacatg gaaatgcaga agtgcatgcg 421 tcgttctttg atattggtgg gagtgtatct gcaggattta gtaattcgaa ttcaagtacg 481 gtcgcaattg atcattcact atctctagca ggggaaagaa cttgggctga aacaatgggt 541 ttaaataccg ctgatacagc aagattaaat gccaatatta gatatgtaaa tactgggacg 601 gctccaatct acaacgtgtt accaacgact tcgttagtgt taggaaaaaa tcaaacactc 661 gcgacaatta aagctaagga aaaccaatta agtcaaatac ttgcacctaa taattattat 721 ccttctaaaa acttggcgcc aatcgcatta aatgcacaag acgatttcag ttc.tactcca 781 attacaatga attacaatca atttcttgag ttagaaaaaa cgaaacaatt aagattagat 841 acggatcaag tatatgggaa tatagcaaca tacaattttg aaaatggaag agtgagggtg 901 gatacaggct cgaactggag tgaagtgtta ccgcaaattc aagaaacaac tgcacgtatc 961 agttttaatg gaaaagattt aaatctggta gaaaggcgga tagcggcggt taatcctagt 1021 gatccattag aaacgactaa accgggtatg acattaaaag aagcccttaa aatagcattt 1081 ggatttaacg aaccgaatgg aaacttacaa tatcaaggga aagacataac cgaatttgat 1141 tttaatttcg atcaacaaac atctcaaaat atcaagaatc agttagcgga attaaacgca 1201 actaacatat atactgtatt agataaaatc aaattaaatg caaaaatgaa tattttaata 1261 agagataaac gttttcatta tgatagaaat aacatagcag ttggggcgga tgagtcagta 1321 gttaaggagg ctcatagaga agtaattaat tcgtcaacag agggattatt gttaaatatt 1381 gataaggata taagaaaaat attatcaggt tatattgtag aaattgaaga tactgaaggg 1441 cttaaagaag ttataaatga cagatatgat atgttgaata tttctagttt acggcaagat 1501 ggaaaaacat ttatagattt taaaaaatat aatgataaat taccgttata tataagtaat 1561 cccaattata aggtaaatgt atatgctgtt actaaagaaa acactattat taatcctagt 1621 gagaatgggg atactagtac caacgggatc aagaaaattt taatcttttc taaaaaaggc 1681 tatgagatag gataa

SBQ ID NO: 2 LENGTH: 1764

NRME: pTPA-PA63 ^.-r. --- ^•-__ .

TYPE : DNA

ORGZ-NISM: Bacillus anthracis

SEQUENCE : 2

1 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 61 tcgcccagca tgcctacggt tccagaccgt gacaatgatg gaatccctga ttcattagag 121 gtagaaggat atacggttga tgtcaaaaat aaaagaactt ttctttcacc atggatttct 181 aatattcatg aaaagaaagg attaaccaaa tataaatcat ctcctgaaaa atggagcacg 241 gcttctgatc cgtacagtga tttcgaaaag gttacaggac ggattgataa gaatgtatca 301 ccagaggcaa gacaccccct tgtggcagct tatccgattg tacatgtaga tatggagaat 361 attattctct caaaaaatga ggatcaatcc acacagaata ctgatagtca aacgagaaca 421 ataagtaaaa atacttctac aagtaggaca catactagtg aagtacatgg aaatgcagaa 481 gtgcatgcgt cgttctttga tattggtggg agtgtatctg caggatttag taattcgaat 541 tcaagtacgg tcgcaattga tcattcacta tctctagcag gggaaagaac ttgggctgaa 601 acaatgggtt taaataccgc tgatacagca agattaaatg ccaatattag atatgtaaat 661 actgggacgg ctccaatcta caacgtgtta ccaacgactt cgttagtgtt aggaaaaaat 721 caaacactcg cgacaattaa agctaaggaa aaccaattaa gtcaaatact tgcacctaat 781 aattattatc cttctaaaaa cttggcgcca atcgcattaa atgcacaaga cgatttcagt 841 tctactccaa ttacaatgaa ttacaatcaa tttcttgagt tagaaaaaac gaaacaatta 901 agattagata cggatcaagt atatgggaat atagcaacat acaattttga aaatggaaga 961 gtgagggtgg atacaggctc gaactggagt gaagtgttac cgcaaattca agaaacaact 1021 gcacgtatca tttttaatgg aaaagattta aatctggtag aaaggcggat agcggcggtt 1081 aatcctagtg atccattaga aacgactaaa ccggatatga cattaaaaga agcccttaaa 1141 atagcatttg gatttaacga accgaatgga aacttacaat atcaagggaa agacataacc 1201 gaatttgatt .ttaatttcga tcaac.aaac-a tαtoaaaata tcaagaatca gttagcggaa 1261 ttaaacgcaa ctaacatata tactgtatta gataaaatca aattaaatgc aaaaatgaat 1321 attttaataa gagataaacg ttttcattat gatagaaata acatagcagt tggggcggat^' 1381 gagtcagtag ttaaggaggc tcatagagaa gtaattaatt cgtcaacaga gggattattg 1441 ttaaatattg ataaggatat aagaaaaαtα ttatcaggtt atattgtaga aattgaagat 1501 actgaagggc ttaaagaagt tataaatgac agatatgata tgttgaatat ttctagttta 1561 cggcaagatg gaaaaacatt tatagatttt aaaaaatata atgataaatt accgttatat 1621 ataagtaatc ccaattataa ggtaaatgta tatgctgtta ctaaagaaaa cactattatt 1681 aatcctagtg agaatgggga tactagtacc aacgggatca agaaaatttt aatcttttct 1741 aaaaaaggct atgagatagg ataa

SEQ ID NO: 3 LENGTH: 1926

NAME: pUQ-PA63

TYPE : DNA

ORGANISM: Bacillus anthracis

SEQUENCE : 3

1 atgcagatct tcgtgaagac cctgacgggc aagaccacca ctct^'tggggt cgagcccagt 61 gacaccatcg agaatgtcaa ggccaagatc caagacaagg aaggcatccc acctgaccag 121 cagaggctga tattcgcggg caaacagctg gaggatggcc gcaccctgtc cgactacaac 181 atccagaaag agtccacctt gcacctggtg ctgcgtctgc gcggtgccgc tatgcctacg 241 gttccagacc gtgacaatga tggaatccct gattcattag aggtagaagg atatacggtt 301 gatgtcaaaa ataaaagaac ttttctttca ccatggattt ctaatattca tgaaaagaaa 361 ggattaacca aatataaatc atctcctgaa aaatggagca cggcttctga tccgtacagt

^■ 5 421 gatttcgaaa aggttacagg acggattgat aagaatgtat caccagaggc aagacacccc 481 cttgtggcag cttatccgat tgtacatgta gatatggaga atattattct ctcaaaaaat 541 gaggatcaat ccacacagaa tactgatagt caaacgagaa caataagtaa aaatacttct 601 acaagtagga cacatactag tgaagtacat ggaaatgcag aagtgcatgc gtcgttcttt 661 gatattggtg ggagtgtatc tgcaggattt agtaattcga attcaagtac ggtcgcaatt

10 721 gatcattcac tatctctagc aggggaaaga acttgggctg aaacaatggg tttaaatacc 781 gctgatacag caagattaaa tgccaatatt agatatgtaa atactgggac ggctccaatc 841 tacaacgtgt taccaacgac ttcgttagtg ttaggaaaaa atcaaacact cgcgacaatt 901 aaagctaagg aaaaccaatt aagtcaaata cttgcaccta ataattatta tccttctaaa 961 aacttggcgc caatcgcatt aaatgcacaa gacgatttca gttctactcc aattacaatg

15 1021 aattacaatc aatttcttga gttagaaaaa acgaaacaat taagattaga tacggatcaa 1081 gtatatggga atatagcaac atacaatttt gaaaatggaa gagtgagggt ggatacaggc 1141 tcgaactgga gtgaagtgtt accgcaaatt caagaaacaa ctgcacgtat catttttaat 1201 ggaaaagatt taaatctggt agaaaggcgg atagcggcgg ttaatcctag tgatccatta 1261 gaaacgacta aaccggatat gacattaaaa gaagccctta aaatagcatt tggatttaac

20 1321 gaaccgaatg gaaacttaca atatcaaggg aaagacataa ccgaatttga ttttaatttc 1381 gatcaacaaa catctcaaaa tatcaagaat cagttagcgg aattaaacgc aactaacata 1441 tatactgtat tagataaaat caaattaaat gcaaaaatga atattttaat aagagataaa 1501 cgttttcatt atgatagaaa taacatagca gttggggcgg atgagtcagt agttaaggag 1561 gctcatagag aagtaattaa ttcgtcaaca gagggattat tgttaaatat tgataaggat

25 1621 ataagaaaaa tat.tatcagg ttatattgta gaaattgaag atactgaagg gcttaaagaa 1681 gttataaatg acagatatga tatgttgaat atttctagtt tacggcaaga tggaaaaaca 1741 tttatagatt ttaaaaaata taatgataaa ttaccgttat atataagtaa tcccaattdt 1801 aaggtaaatg tatatgctgt tactaaagaa aacactatta ttaatcctag tgagaatggg 1861 gatactagta ccaacgggat caagaaaatt ttaatctttt ctaaaaaagg ctatgagata

30 1921 ggataa

SEQ ID NO: 4'

LENGTH: 1815 . '

NAME: pPA63-LAMPl

35 TYPE : DNA

ORGANISM: Bacillus anthracis SEQUENCE : 4

1 atgcctacgg ttccagaccg tgacaatgat ggaatccctg attcattaga ggtagaagga 61 tatacggttg atgtcaaaaa taaaagaact tttctttcac catggatttc taatattcat

40 121 gaaaagaaag gattaaccaa atataaatca tctcctgaaa aatggagcac ggcttctgat 181 ccgtacagtg atttcgaaaa ggttacagga cggattgata agaatgtatc accagaggca 241 agacaccccc ttgtggcagc ttatccgatt gtacatgtag atatggagaa tattattctc 301 tcaaaaaatg aggatcaatc cacacagaat actgatagtc aaacgagaac aataagtaaa 361 aatacttcta caagtaggac acatactagt gaagtacatg gaaatgcaga agtgcatgcg

45 421 tcgttctttg atattggtgg gagtgtatct gcaggattta gtaattcgaa ttcaagtacg 481 gtcgcaattg atcattcact atctctagca ggggaaagaa cttgggctga aacaatgggt 541 ttaaataccg ctgatacagc aagattaaat gccaatatta gatatgtaaa tactgggacg 601 gctccaatct acaacgtgtt accaacgact tcgttagtgt taggaaaaaa tcaaacactc 661 gcgacaatta aagctaagga aaaccaatta- agtcaaatac ttgcacctaa taattattat 0 721 ccttctaaaa aσttggcgcc aatcgcatta aatgcacaag acgatttcag ttctactcca 781 attacaatga attacaatca atttcttgag ttagaaaaaa cgaaacaatt aagattagat 841 acggatcaag tatatgggaa tatagcaaca tacaattttg aaaatggaag agtgagggtg 901 gatacaggct cgaactggag tgaagtgtta ccgcaaattc aagaaacaac tgcacgtatc 961 atttttaatg gaaaagattt aaatctggta gaaaggcgga tagcggcggt taatcctagt 5 1021 gatccattag aaacgactaa accggatatg acattaaaag aagcccttaa aatagcattt 1081 ggatttaacg aaccgaatgg aaacttacaa tatcaaggga aagacataac cgaatttgat 1141 tttaatttcg atcaacaaac atctcaaaat atcaagaatc agttagcgga attaaacgca 1201 actaacatat atactgtatt agataaaatc aaattaaatg caaaaatgaa tattttaata 1261 agagataaac gttttcatta tgatagaaat aacatagcag ttggggcgga tgagtcagta 0 1321 gttaaggagg ctcatagaga agtaattaat tcgtcaacag agggattatt gttaaatatt 1381 gataaggata taagaaaaat atcatcaggt tatattgtag aaattgaaga tactgaaggg 1441 cttaaagaag ttataaatga cagatatgat atgttgaata tttctagttt acggcaagat 1501 ggaaaaacat ttatagattt taaaaaatat aatgataaat taccgttata tataagtaat 1561 cccaattata aggtaaatgt atatgctgtt actaaagaaa acactattat taatcctagt 1621 gagaatgggg atactagtac caacgggatc aagaaaattt taatcttttc taaaaaaggc 1681 tatgagatag gaggccttaa caacatgttg atccccattg ctgtgggcgg tgccctggca 1741 gggctggtcc tcatcgtcct cattgcctac ctcattggca ggaagaggag tcacgccggc 1801 tatcagacca tctaa SEQ ID NO: 5

LENGTH: 1884 ^'

NAME: pTPA-PA63-LAMPl

TYPE: DNA

ORGANISM: Bacillus anthracis SEQUENCE: 5

1 atggatgcaa tgaagagagg gctctgctgt gtgctgctgc ^"tgϊgtggagc agtcttcgtt ^" 61 tcgcccagca tgcctaσggt tccagaccgt gacaatgatg gaatccctga ttcattagag 121 gtagaaggat atacggttga tgtcaaaaat aaaagaactt ttctttcacc atggatttct 181 aatattcatg aaaagaaagg attaaccaaa tataaatcat ctcctgaaaa atggagcacg 241 gcttctgatc cgtacagtga tttcgaaaag gttacaggac ggattgataa gaatgtatca 301 ccagaggcaa gacaccccct tgtggcagct tatccgattg tacatgtaga tatggagaat 361 attattctct caaaaaatga ggatcaatcc acacagaata ctgatagtca aacgagaaca 421 ataagtaaaa atacttctac aagtaggaca catactagtg aagtacatgg aaatgcagaa 481 gtgcatgcgt cgttctttga tattggtggg agtgtatctg caggatttag taattcgaat 541 tcaagtacgg tcgcaattga tcattcacta tctctagcag gggaaagaac ttgggctgaa 601 aeaatgggtt taaataccgc tgatacagca agattagatg ccaatattag atatgtaaat 661 actgggacgg ctccaatcta caacgtgtta ccaacgactt cgttagtgtt aggaaaaaat 721 caaacactcg cgacaattaa agctaaggaa aaccaattaa gtcaaatact tgcacctaat 781 aattattatc cttctaaaaa cttggcgcca atcgcattaa atgcacaaga cgatttcagt 841 tctactccaa ttacaatgaa ttacaatcaa tttcttgagt tagaaaaaac gaaacaatta 901 agattag'a.ta cggatcaagt atatgggaat atagcaacat acaattttga aaatggaaga 961 gtgagggtgg atacaggctc gaactggagt gaagtgttac cgcaaattca agaaacaact 1021 gcacgtatca tttttaatgg aaaagattta aatctggtag aaaggcggat agcggcggtt 1081 aatcctagtg atccattaga aacgactaaa ccggatatga cattaaaaga agcccttaaa 1141 atagcatttg gatttaacga accgaatgga aacttacaat atcaagggaa agacataacc 1201 gaatttgatt ttaatttcga tcaacaaaca tctcaaaata tcaagaatca gttagcggaa 1261 ttaaacgcaa ctaacatata tactgtatta gataaaatca aattaaatgc aaaaatgaat 1321 attttaataa gagataaacg ttttcattat gatagaaata acatagcagt tggggcggat 1381 gagtcagtag ttaaggaggc tcatagagaa gtaattaatt cgtcaacaga gggattattg 1441 ttaaatattg ataaggatat aagaaaaata ttatcaggtt atattgtaga aattgaagat 1501 actgaagggc ttaaagaagt tataaatgac agatatgata tgttgaatat ttctagttta 1561 cggcaagatg gaaaaacatt tatagatttt aaaaaatata atgataaatt accgttatat 1621 ataagtaatc ccaattataa ggtaaatgta tatgctgtta ctaaagaaaa cactattatt 1681 aatcctagtg agaatgggga tactagtacc aacgggatca agaaaatttt aatcttttct 1741 aaaaaaggct atgagatagg aggccttaac aacafgttga tccccattgc tgtgggcggt 1801 gccctggcag ggctggtcct catcgtcctc attgcctacc tcattggcag gaagaggagt 1861 cacgccggct atcagaccat ctaa

When the chimera will be transformed in an expression system, the transformed or transfected cells host cells can be used as a source of the protein or polypeptide specified as amino acid sequence in SEQ ID NO: 6 (PA63 -Native), SEQ ID NO: 7 (TPA-PA63), SEQ

ID NO: 8 (UQ-PA63), SEQ ID NO: 9 (PA63-LAMP1), and SEQ ID NO: 10 (TPA-PA63-

LAMPl).

The amino acid sequences of expressed chimeras are given below: SEQ ID NO: 6 LENGTH: 564 NAME: PA63-Native TYPE: Protein ORGANISM: Bacillus anthracis

SEQUENCE : 6

MPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKG

LTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSK

NEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSST VAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQ TLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQ LRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARISFNGKDLNLVERRI AAVNPSDPLETTKPGMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIK NQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESWKEAHREVIN SSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSERQDGKTFIDFK KYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG

SEQ ID NO: 7 LENGTH: 587 NAME: TPA- PA63 TYPE: Protein ORGANISM: Bacillus anthracis

SEQUENCE : 7

MDAMKRGLCCVLLLCGAVFVSPSMPTVPDRDNDGIPDSLEVEGY TVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPE ARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAE VHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRY VNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQ DDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLP QIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNL QYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHY DRNNIAVGADESWKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVI NDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENG DTSTNGIKKILIFSKKGYEIG

SEQ ID NO: 8

LENGTH: 641

NAME: UQ-PA63

TYPE: Protein ORGANISM: Bacillus anthracis

SEQUENCE : 8

MQIFVKTLTGKTTTLGVEPSDTIENVKAKIQDKEGIPPDQQRLI

FAGKQLEDGRTLSDYNIQKESTLHLVLRLRGAAMPTVPDRDNDGIPDSLEVEGYTVDV

KNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHP LVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHAS FFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTG TAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFS STPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQE TTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQG KDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNN IAVGADESWKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRY DMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTST NGIKKILIFSKKGYEIG SEQ ID NO: 9 LENGTH: 604

NAME: PA63-LAMP1

TYPE: Protein

ORGANISM: Bacillus anthracls SEQUENCE : 9

MPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKG LTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAΆYPIVHVDMENIILSK NEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSST VAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQ TLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQ LRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARLIFNGKDLNLVERRI AAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIK NQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESWKEAHREVIN SSTEGLLLNIDKDIRKISSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFK KYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIGGL - NNMLIPIAVGGALAGLVLIVLIAYLIGRKRSHAGYQTI

SEQ ID NO: 10 LENGTH: 627 NAME: TPA-PA63-LAMP1 TYPE: Protein

ORGANISM: Bacillus anthracis SEQUENCE: 10

MDAMKRGLCCVLLLCGAVFVSPSMPTVPDRDNDGIPDSLEVEGY TVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPE ARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAE VHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLDANIRY VNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQ DDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLP QIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNL QYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHY DRNNIAVGADESWKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVI NDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENG DTSTNGIKKILIFSKKGYEIGGLNNMLIPIAVGGALAGLVLIVLIAYLIGRKRSHAGY QTI

Therefore, it is one object of the present invention whereby the protease-cleaved form of protective antigen will be targeted to various sub-cellular locations on account of the presence of various targeting signals present on the N-terminus or C-terminus or both. In one embodiment the PA63 protein will be secreted with the help of N-teminal

TPA leader and the expressed antigen will be taken up as an exogenous antigen by phagocytosis or endocytosis by APC (antigen presenting cell) and directly targeted to endosomal pathway for presentation of the peptides in context with MHC-II molecules.

In another embodiment the PA63 protein will be targeted to proteasome complex on account of the presence of an N-terminal Ubiquitin leader. Ubiquitination should target the protein for rapid cytoplasmic degradation by the proteasome and increase the availability of the antigenic peptides for presentation through the MHC I pathway. In further embodiment of the invention the antigenic protein will be targeted to the lysosomal /endosomal membrane with the assistance of C-terminal LAMPl signal. LAMPl and MHC II are closely linked in their colocalisation in MIIC (for MHC II containing compartments) therefore, the antigenic peptides after processing in the lysosomal compartment will be displayed in context with MHC II molecules.

In one another embodiment the antigenic protein will be secreted and further targeted to lysosomal membrane on account of the presence of an N-terminal TPA leader and C-terminal

LAMPl sequence respectively. Secreted protein will be taken up and targeted to lysosomal membrane for generation of peptides inside the endosomal compartment. These peptides will be specifically displayed in context with MHC II molecule on the surface of APC.

According to the present invention the antigenic protein PA63 will be trafficked to various subcellular locations. On reaching the cellular target, the signal sequence will be cleaved as a part of normal processing event. The protein will access both the antigen processing pathways and peptides generated from PA63 will be displayed in context with MHC I and MHC II molecules on the surface of APC.

The present invention also describes a non-invasive method of immunization whereby the genetic material/DNA vaccine plasmid encodes PA63 protein along with the N- and C-terminal signal sequences. Only the antigen of interest will be responsible for the generation of immune response. Another embodiment of the present invention relates to host cells transiently transfected or transformed with the DNA vaccine plasmids. The host cell can be prokaryotic (for eg. Bacteria) or eukaryotic (for eg. Insect or mammalian cell or cell line).

In yet another embodiment the invention presents a method/protocol to enhance the immune response generation by means of efficient targeting and presentation of the protein antigen. The response can be evaluated by cytokine profiling, neutralizing antibody titers, avidity index of antibodies generated, cytotoxic responses which can be correlated well with survival against challenge.

The invention provides for a DNA vaccine or an immunological composition that comprises a vector bearing a set of gene targeting signals 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. The present invention may be used to immunize an animal/individual whereby the animal/ individual can be vertebrate e.g. a fish, amphibian, reptile, bird, or mammal such as a cow, a dog, a cat, a goat, a sheep, a horse, a pig or a fowl such as ducks, turkeys, chicken etc.

The present invention also presents a strategy for DNA vaccine that can be designed for protection against other pathogens such as viruses, prokaryote and pathogenic eukaryotic organisms such as unicellular organisms and multi-cellular parasites. The strategy of invention is particularly useful to immunize against intracellular pathogens.

Even further still the vaccine can be given in a single dose or multiple doses in which the course of vaccination may be separated in part as DNA regimen and in part as a prime-boost regimen. The term used herein "prime-boost regimen" refers to a DNA prime followed by a protein boost or vice versa whereby the protein dose can be administered by intraperitoneal, subcutaneous or intradermal route or topical application as a patch.

Also, the present invention describes a DNA vaccine formulation (without adjuvant) that is superior in quality as compared to recombinant protective antigen based vaccine (administered with adjuvant) as demonstrated by the enhanced humoral and cellular immune responses.

The present invention therefore, provides DNA vaccine composition, which can serve as the prophylactic vaccine against anthrax 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 protein vaccine formulations or other compositions lacking the targeting signals.

From the present work those skilled in the art will recognize that one of the utilities of the instant invention is to generate an enhanced immune response against an antigen.

The present invention is described with reference to the following examples in mouse, which are given by way of illustration and should not be construed to limit the scope of the present invention. EXAMPLES The following MATERIALS AND METHODS were used in the examples that follow.

DNA vaccine plasmids. Protease-cleaved fragment of the structural gene for protective antigen (PA63) was amplified by PCR from Bacillus anthracis (Sterne strain) pXOl plasmid using the following primers:

Forward primer: (SEQ ID NO.l 1)

(5'-GCGCAT GCTCTTCCATGCCTACGGTTCCAGACC G -3') Reverse ,primer 1 with TAA stop codon : (SEQ ID NO.12)

(5 '-GCAGAAGCTCTTCGTTATCCTATCTCATAGCCTTTTTTA-S ')

Reverse primer 2 with GCC alanine linker: (SEQ ID NO.13)

(5'- GCAGAAGCTCTTCGGCCTCCTATCTCATAGCCT TTTTTA G-3')

I I I

The primers were designed to generate 5'-ATG and 3'-TAA for address tags that correspond precisely to start and stop codons (N-terminal, TPA Leader and Ubiquitin leader) or 5'-ATG and 3'-GGC alanine linker for address tags that facilitate C-terminal extensions needed for trafficking (Endosomal LAMPl anchor). The amplified product was i cloned in a eukaryotic expression vector bearing the targeting signals.

PIasmid DNA preparation. Plasmid DNA was processed by using Endo-free Giga kits (Qiagen, Valencia, CA). Traαsfection, western blot analysis. J774.A1 mouse macrophage-like cells were seeded at a concentration of 2.5 x 10⁴ cells/well into 6-well tissue culture plates (BD Falcon) until the cells reached approximately 50-70% confluence. Plasmid DNA transfection was performed with LipofectAMINE 2000 (Invitrogen) reagent, as specified by the manufacturer. For Western blot analysis, the transfected cells were washed twice with PBS (pH 7.4) and lysed in lysis buffer (20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0'l% sodium dodecyl sulfate (SDS) at 48 h after transfection. The solubilized proteins were 'separated by SDS-polyacrylamide gel electrophoresis under denaturing conditions with 50 mM dithiothreitol (DTT). Proteins from the gel were transferred to a nitrocellulose membrane and probed with rabbit anti-PA polyclonal serum followed by alkaline phosphatase-conjugated donkey anti-rabbit IgG (Amersham biosciences).

Vaccination and lethal toxin challenge. Six- to eight- week-old female BALB/c mice (National Institute of Nutrition, Hyderabad, India) were immunized intramuscularly (i.m.) with 100 μg of DNA suspended in phosphate-buffered saline (PBS; 50 μl per hind leg) administered via a 26-gauge, 1-ml hypodermic needle. All mice received Lhe first DNA booster dose 28 days after their initial DNA prime and received either a second DNA booster (lOOμg, i.m.) or an i.p. injection of rPA63 (12.5 μg formulation with Incomplete Freunds Adjuvant) 28 days thereafter as a part of prime-boost regimen. Sera were obtained from blood samples collected from the retro-orbital plexus prior to each immunization and on 4-week intervals during immunization. For lethal toxin challenge, 50 μg of PA83 and 22μg of LF were injected intravenously via the tail vein, and the mice were closely monitored for 10 days. ELISA detection of anti-PA reactivity in mouse serum. The anti-PA reactivity of immunized animal sera was determined by Direct ELISA. Two fold Serum dilutions in PBS were allowed to bind the coated antigen (PA63) for 2 hours at 37°C and then were allowed to bind goat anti- mouse IgG, IgGl, IgG2a, IgG2b and IgA HRP conjugate (Santacruz Biotechnology) for 1 h at 37°C. Plates were developed using TMB substrate (Amersham biosciences) and were analyzed at 492 nm in an ELISA reader (Benchmark Plus Microplate spectrophotometer, BioRAD). Negative control consisted of sera from mice immunized with PBS and vector. Endpoint antibody titers were defined as the last reciprocal serial serum dilution at which the absorption at 450 nm was greater than two times the background signal detected.

Lethal toxin neutralization assay. Letx neutralizing antibody assay was measured by J774A.1 mouse macrophage cytotoxicity assay described previously (Price B. M. et al.). Similar protocol was followed to test the biological activity of rPA63 and rPA83 with slight modification. J774.A1 mouse macrophage cells were incubated with different concentrations of the recombinant proteins with LF (l μg/ml) and percentage survival was calculated to test the biological activity of the purified proteins.

Determination of avidity of PA-specific IgG antibodies. Antibody avidity was measured by ELISA using urea as chaotropic agent to dissociate low-affinity antigen-antibody complexes using a protocol described previously (D' Souza V.A. et al).

In vitro cytokine production. In vitro cytokine production by spleen cells were determined by Cytokine-specific sandwich ELISA using OptEIA kit for the specific cytokine (BD Pharmingen) according to manufacturer's protocols. Enumeration of single IFN-? and IL-4-producing cells by enzyme-linked immunospot assay. Multiscreen filtration plates (96 wells; Millipore, France) were coated overnight at 4°C with 4 μg of rat anti-mouse IFN-T antibody (clone R4-6A2; PharMingen, San Diego, CA) and rat anti-mouse IL-4 antibody (clone 11 B 11) per ml, and then the plates were washed and blocked with complete medium (RPMI 1640 medium with glutamine supplemented with 10% fetal calf serum, antibiotics, and 5 x 10^~5 M β-2-mercaptoethanol). Serial twofold dilutions of the spleen cell suspensions were added to the wells. The cells were incubated for 36 h at 37⁰C, 5% CO₂ with rPA63 (lOμg/ml). Negative (without rPA63) and positive controls (Con A) were also run along the assay. After extensive washes, the plates were revealed by incubation with 4 μg/ml of biotinylated rat anti-mouse IFN-T antibody (clone R4-6A2; PharMingen) and biotinylated rat anti-mouse IL-4 antibody (clone BVD6-24G2) followed by incubation with streptavidin-alkaline phosphatase (PharMingen). Finally, spots were revealed using 5-bromo-4- chloro-3-indolylphosphate/nitroblue tetrazolium (Sigma Aldrich) as the substrate. The number of IFN-T and IL-4-producing cells was determined by counting the number of spot-forming (SFU) in each well with the ELISPOT reader system (IMMUNOSPOT, CTL technologies), and the results are expressed as numbers of SFU per 10⁶ cells.

Evaluation of antibody-secreting cells (ASC) by the ELISPOT assay. The numbers of PA-specific ASCs (IgA and IgG) were determined following methods previously described (Kuklin N. et al.). , ____ Lymphocyte proliferation assay. Spleen cells (3 x 10⁵ cells) isolated from each group of mice were seeded into each well of tissue culture 96-well plates (BD falcon) and were incubated with rPA63 protein at 37°C incubator with a 5% CO₂ atmosphere for 72 h. Proliferation was measured based on MTT-dye based assay. The stimulation index (SI) was calculated as the ratio of the average O.D. value of wells containing antigen-stimulated cells to the average O.D. value of wells containing only cells with medium.

Cytotoxicity assays. The target cells were incubated with effector cells obtained from vaccinated groups . of mice at different Effector: Target ratios (5: 1, 10:1 , and 15:1) and incubated for 16 h. Neutral red uptake assay, a non-radioactive assay procedure (He Y. et al.) was followed to assay CTL activity.

TABLE 1 given below shows various DNA vaccine constructs and the targeting signal attached. TABLE. 1.

PT ASMTD NAME EXPRESSED PRODTTCT pTPA-PA63 N-terminal TPA signal, and 63 kDa mature protein. pPA63-Native 63 kDa mature protein. pLAMPl-PA63 C-terminal LAMPl membrane anchor and 63 kDa mature protein. pUQ-PA63 N-terminal Ubiquitin leader and 63 kDa mature protein. pTPA-PA63-LAMPl N-terminal TPA signal, C-terminal LAMPl membrane anchor and 63kDa mature protein. EXAMPLE 1

Cloning of PA 63 structural gene and its in vitro expression from DNA vaccine constructs following transfection and immunoblot analysis

PA 63 structural gene was cloned in the eukaryotic expression vector (Fig.l). In vitro expression and authenticity of the DNA chimeras was evaluated by transient transfection and immunoblot blot analysis. As shown in Fig.2, under reducing conditions, anti-PA rabbit polyclonal serum reacted with distinct the 63 kDa protein (PA63) in the native construct and the lysates of pTPA-PA63, pUQ-PA63, pPA63-LAMPl and pTPA-

PA63-LAMP1 transfected J774.A1 mouse macrophage-like cells, respectively. The bands were distinguishable due to slight differences in the molecular weight because of the presence of different targeting signal. These results indicate that PA63 protein was expressed in conjunction with its targeting signal in the transfected cells.

EXAMPLE 2 Delivery of different DNA vaccine combinations using a heterologous prime boost regimen induced a strong and long lasting systemic and mucosal response against protective antigen.

To address the issue whether the targeting signals could deliver the DNA encoded antigen to the immune system and induce a humoral response was assessed. BALB/c mice were immunized i.m. at days 0, 28, 56 with various DNA vaccine constructs or twice with DNA vaccine constructs followed by a subcutaneous vaccination with PA63 (12.5 μg) protein formulated with IFA. Mice immunized with corresponding vector and PBS served as negative controls whereas mice immunized twice subcutaneously with rPA83 (12.5μg) and rPA63 (12.5 μg) with aluminium hydroxide adjuvant served as positive controls. Mice were bled at 4 week intervals following the final boost for over a period of 22 weeks, and sera were analyzed for the presence of anti-PA IgGl, IgG2a, IgG2b, and IgA antibodies by ELISA. As shown in Fig.3a, significant IgGl titers were observed following immunization with construct encoding pTPA-PA63 -LAMPl both in case of DNA and protein boost (pO.OOl vs PBS, p<0.01 vs vector). Notably, pTPA-PA63 construct also generated significant titers following a protein boost (p<0.002 vs PBS, ρ<0.05 vs vector). Mice immunized with pUQ-PA63, pTPA-PA63-LAMPl+ pUQ-PA63, pLAMPl-PA63, pTPA-PA63+pUQ-PA63, native constructs generated moderate to high IgGl anti-PA titers. Overall, the anti-PA IgGl response for pTPA-PA63 and pTPA-PA63 -LAMPl was W

approximately 1.5 log higher than the other groups and was maintained for upto a period of 22 weeks demonstrating that the humoral response was persistent. Evaluation of the anti-PA IgA end point titers showed no significant differences between different groups but titers were maintained for over a period of 16 weeks (Fig.3b). Furthermore, IgG2a and IgG2b responses were evaluated. IgG2a titers were highest in the group immunized with pTPA-PA63 -LAMPl construct and pUQ-PA63 (p<0.01 vs vector, p<0.05 vs vector respectively) (Fig.3c). IgG2b titers showed no statistically significant differences for each time period testes between each group (Fig.3d). We also examined the IgG subclass distribution as an indicator of the T-helper-cell subsets (TH1/TH2) induced by the heterologous prime boost strategy. For all groups (except the group immunized with pUQ- PA63 and native construct) showed IgGl titers that were 1.5 to 2.0 log higher than the IgG2a responses which clearly indicates a TH2 biased immune response. These data therefore demonstrate that plasmid encoding PA63 with various signal sequences i.e. N- terminal TPA leader, C-terminal LAMPl achor or both, were efficiently targeted to multiple antigen processing pathways as shown by the superior immune responses in mice compared to plasmid alone and, that type of humoral immune response generated was persistent.

EXAMPLE 3 Induction of high avidity neutralizing antibody responses following DNA vaccination.

Lethal toxin neutralization activity was measured in the serum from all animals 4- weeks after the last immunization using the J774.A1 cytotoxicity assay. Neutralization activity was expressed as percentage survival of the J774.A1 cells following a lethal toxin shock in the presence of anti-PA antibodies from the serum of immunized mice. Neutralization curves for each group of animals arc shown in Fig.4. The anti-PA antibodies from the serum were able to neutralize the lethal toxin. Highest neutralization activity was seen with serum from mice immunized with pTPA-PA63 -LAMPl (DNA, 90% and Protein boost, 70%; at serum dilution log 10) followed closely by the group immunized with pTPA-63 Pro (60%). Importantly, the pTPA-PA63 -LAMPl DNA boost showed higher neutralizing activity (70%) at a dilution of loglO as compared to pTPA- PA63 protein boost (60%). Also, the neutralization activity decline^ sharply in case of pTPA-PA63 that demonstrates that pTPA-PA63 -LAMPl generated a better neutralizing antibody response and was more efficient as a targeting signal. Serum dilutions at which 50% neutralization was achieved were significantly higher in all groups immunized with PA63- bearing a TPA leader sequence and LAMPl anchor and highest in the one harboring both i.e. N-terminal TPA leader and a C-terminal LAMPl anchor (p<0.05 vs UQ-PA63, p<0.01 vs native). No significant differences were observed in the mean neutralization activity between other groups. Overall, these results indicate a high neutralizing antibody response in groups immunized with pTPA-PA63, pLAMPl-PA63 and pTPA-PA63-LAMPl as compared to those immunized with UQ-PA63 and native constructs.

We further examined the avidity of anti-PA antibodies to estimate the ability of the vaccine to generate B-cell memory response. Once a memory response is established, it is characterized by the generation of high-avidity (high affinity antibodies against the target

Ag) antibodies on subsequent encounter with the antigen. We performed a urea based

ELISA as mentioned in the materials and methods. The results corroborated with those for

' the neutralization assay (Fig.5). Mice immunized with pTPA-PA63 -LAMPl (both protein and DNA boost) generated anti-PA antibodies that had the highest avidity to PA, with the group receiving the protein boost showed significant high avidity antibodies to PA (p<0.05 between both groups). Antibodies with significantly higher avidity were also generated in the groups immunized with pTPA-PA63 and pPA63-LAMPl following a protein boost

(p<0.001 vs vector immunized). PA-specific avidities elicited in mice immunized in other groups, pUQ-PA63 and native, were significantly lower that those elicited by pTPA-PA63-

LAMPl, pTPA-PA63 and pPA63-LAMPl (pO.OOOl vs all groups) although the differences between them were not statistically significant. The results therefore, depict that high avidity neutralizing antibodies were generated as a result of DNA vaccination with constructs bearing TPA, LAMPl or both signals.

EXAMPLE 4

Differences in the T-helper cell responses after vaccination.

As cytokines play an important role in polarization of T-cell responses, we therefore, quantified the levels of type I (IL2, IL 12, IFNγ) and type II (ILlO, IL4) cytokines in cell culture supernatants after re-stimulation with PA. Table.2 as shown below, describes the levels of all cytokines after re-stimulation of splenocytes in vitro with PA (10μg/ml). TABLE 2.

Cytokine production by spleen cells from the mice immunized with DNA vaccine constructs.

CYTOKINE PROFILE* VACCINE IL2^#§ IL12*^§ IFN/^§ IL4^#§ ILlO TT

COMBINATION (pg/ml) (pg/ml) (pg/ml) (pg/ml) (pg/ml)

PTPA-PA63 Pro 52.7±5.5 1162.8 + 25.5 1079.7+104.1 6.62 + 1.2 470.5+120.4

pTPA-PA63 DNA 43.3+6.8 227.1 ±59.9 189.9 ± 122.1 3.41 ±0.91 165.5+12.3

pUQ-PA63 Pro 25.8 + 5.7 135.2 + 23.5 1600.2 ±207.6 3.08 + 0.87 98.35 + 45.7

pUQ-PA63 DNA 9.72 + 3.2 150.4 ±21.6 1296.2 ±89.7 3.41 ±0.78 74.44 ±12.9

pTPA-PA63-LAMP1 Pro 90.9 + 15.1 2770.9+169.8 1251.8±334.9 41.6+ 5.9 277.7±15.5

pTPA-PA63 LAMP1 DNA 27.8 ± 2.6 1970.9+150.8 1003.12 ± 45.9 40.9 ± 9.0 196.3 ±35.9

PTPA-PA63-LAMP1+ 90.8 + 11.8 840.95 + 276.9 1111.2 ±34.8 41.6 ±5.6 143.3 ±20.8 pUQ-PA63 Pro pTPA-PA63-LAMP1+ 4.54 ±0.98 110.0 + 25.1 208.98 + 22.5 . 39.2 ±9.9 134.1 ±18.5 pUQ-PA63 DNA pPA63-LAMP1 Pro 61.5 ± 11.4 680.0 ±109.8 177.08 ± 34.9 53.0 ± 12.5 108.3 + 27.9

pPA63-LAMP1 DNA 19.8 ±4.8 780.9 ±100.8 186.04 ±56.8 41.0 ±12.8 135.5 + 45.1

pPA63^NATIVE Pro 91.4+19.1 133.8 ± 18.8 294.79 ± 40.9 3.45 ±0.89 154.1 ± 22.7

pPA63-NATIVE DNA 18.4 + 3.9 109.04±25.8 263.54±39.8 3.17±0.99 96.38±17.7

pTPA-PA63+ 74.5 ± 14.2 102.34 ±33.8 887.7 ± 58.99 5.4 ±1.5 85.8 ± 22.9 pUQ-PA63 Pro pTPA-PA63+ 25.5 ±4.9 172.85 + 22.9 900.12 ±65.23 3.61+1.1 87.22 ±16.9 PUQ-PA63 DNA rPA63 55.7 + 10.7 135.17 ±35.9 94.79 ±31.9 17.8 ±3.8 88.88 + 22.9

rPA83 128±21.1 360 ±57.8 122.2 ±34,8 17.3 + 4.7 81.66 + 33.9

PBS 1.26 ±0.23 1.95 ±0.11 1.79 ±0.34 1.01+0.23 2.66 ±0.33

VECTOR 1.28 ±0.56 1.12 ±0.25 1.10 ±0.56 1.23 ±0.34 1.23 ±0.45 *Groups of 8-10 mice were immunized i.m. with different DNA vaccine constructs. After 4 wk mice were euthanized to take out the spleens and spleen cells from the mice were cultured in the presence 10μg/ml of rPA63 protein to determine the cytokine concentration in the cell-free supernatant.

# Cell-free supernatants were collected 48 h (IL2, IL4, ILlO, IL 12).

£ Cell-free supernatants were collected 72 h later (IFNγ) and analyzed in triplicate by sandwitch ELISA.

*[[ Indirectly measured as IFNγ: ILlO ratio.

§ The results are given as mean ± SEM of three pairs. of mice_per group (p< 0.01 by__. Student's t test compared with the vector immunized mice).

As shown, spleen cells from mice immunized with pTPA-PA63 -LAMPl (both Protein and DNA boost) produced high levels of both type I and type II cytokines. Anti-PA THl :TH2 ratio (indirectly measured as IFNγ: ILlO ratio) induced after immunization with constructs encoding pUQ-PA63 (both protein and DNA boost) was quite significant as compared to the control (pO.OOl vs vector control). By contrast the TH1 :TH2 ratio for the group immunized with pTPA-PA63 -LAMPl constructs the ratio was lower, 4.5 and 5.1, respectively for protein and DNA boost, as compared to pUQ-PA63 (Protein boost 16.3, DNA boost 17.5). Also, spleen cells from mice immunized with the construct pTPA-PA63 showed significant levels of all cytokines (pO.OOl vs vector control) with protein boost showing better levels as compared to DNA boost. Notably, when mice were immunized together with pTPA-63 and pUQ63 constructs we observed a higher TH1:TH2 ratio than when immunized with pTPA-PA63 alone (10.3 vs 6.35). Similar results were found when the mice were immunized together with pTPA- PA63-LAMP1 and pUQ-PA63 construct as opposed to pTPA-PA63-LAMPl alone (8.93 vs 4.52). This depicts that TPA and LAMPl signals cause a bias towards TH2 type whereas UQ signal causes a bias towards a THl type immune response.

EXAMPLE 5

Enhanced cellular and humoral immune responses after DNA vaccination.

To evaluate the frequency of PA-specific effector T cells after DNA vaccination, we measured the frequency of IFNγ and IL4 production by spleen cells by using ELISPOT

(Fig.6a & 6b). It can be shown that compared to the control group vaccinated with vector and PBS₅ all other groups developed discernible PA-specific IFNγ and IL4 responses. The magnitude of IFNγ secreting spleen cells for most of the groups was found to be similar (> 200 SFU per 10⁶ splenocytes). But, groups that received pPA63-LAMPl, pTPA-PA63, pTPA-PA63-LAMPl alongwith the ones receiving two subcutaneous protein injections of rPA63 and rPA83 generated the highest count of IFNγ secreting cells (Fig.όa). Importantly, the mounted response in all the groups was significantly higher as compared to the control mice that received vector or PBS (p< 0.01).

We also quantified the PA-specific IL4 secreting T cells after vaccination. As can be seen in the Fig. 6b, the number of IL4 secreting T cells was significantly high (~ 400 SFU per 10⁶ splenocytes, p< 0.001 vs vector and PBS) in the groups that received pTPA-PA63- LAMPl (both DNA and protein boost), pPA63 -LAMPl (Protein boost) _.and pTPA-PA63 (Protein boost). Following closely were the groups that received two subcutaneous injections of rPA83 and rPA63. The polyclonal anti-PA response was nearly 2 times higher in groups receiving PA attached with signals TPA-LAMPl together, LAMPl alone or TPA alone vs UQ alone or in combination, native protein encoding constructs.

We also evaluated the number of IgG and IgA secreting cells in the total splenocyte population (Fig.7a & 7b). The results obtained paralleled those obtained for cytokine secreting cells. pTP A-P A63 -LAMPl immunized mice elicited the highest number of IgG (> 400 ASC per 10⁶ splenocytes) and IgA (< 700 ASC per 10⁶ splenocytes) antibody secreting cells. These values were significantly higher than all other groups (p< 0.001). Following closely was the group that received pTPA-PA63 protein boost which produced, < 400 IgG ASC per 10⁶ splenocytes and < 600 IgA ASC per 10⁶ splenocytes. Groups that received subcutaneous injection of rPA83 and rPA63 also showed significant IgG and IgA antibody secreting cells as compared to the control mice (p< 0.01).

EXAMPLE 6 Induction of potent cellular proliferation responses on DNA vaccine delivery.

Spleen cells from the immunized mice were harvested at 4 week following the last immunization, were pooled and were incubated for standard MTT-based in vitro lymphocyte proliferation assay with rPA63 (lOμg/ml) for 3 days. As shown in Fig.8, pTPA- PA63-LAMP1 DNA construct mounted the highest proliferation response as depicted by the PI, proliferation index, as high as 16.0 (pO.OOOl vs vector, p< 0.001 vs other groups), followed closely by the group receiving pTPA-PA63 protein boost (PI -13). Other groups that targeted PA with a LAMPl signal alone or received subcutaneous protein injections of rPA63 and rPA83 also depicted strong proliferative responses with PI < 12.0 which was significantly higher than the control group (p<0.001). Groups receiving DNA vaccine constructs encoding PA with a UQ targeting signal alone or in combination were not able to mount proliferation responses as high as the ones that encoded PA in conjunction with pTPA-LAMPl together or LAMPlalone. However, the responses were significant (p<0.01 vs control) with PI < 9.0 except for the group that received pA63-LAMPl+pUQ-PA63 together followed by a DNA or a protein boost with rPA63 (PI- 12.0).

EXAMPLE 7

Generation of potent cytotoxic T-Iymphocyte responses following vaccination. DNA vaccination generates both CD4+ and CD8+ T cell responses by presentation of antigenic peptides via MHC II and MHC I presentation pathways respectively. We wanted to evaluate the effector T-lymhocyte response by measuring the cytotoxic potential of the T lymphocytes generated as a result of vaccination. To decipher which one of the pathways was mainly responsible for presentation of peptides and hence the generation of cytotoxic potential, we disrupted the endosomal / lysosomal pathway by using lysosomotropic agent, which disrupts acidification of the endosomal compartment, with chloroquine treatment. Results as depicted in Fig.9 indicate that percentage lysis of target cells was maximum in case of effector T lymphocytes isolated from group immunized with pTPA-PA63-LAMPl . Notably, the percentage lysis of target cells sharpely declined when the target cell population was given chloroquine treatment (80% vs 30% at E: T ratio 15:1). A similar effect was seen with all other constructs that targeted PA63 various cellular compartments utilizing a LAMPl anchor and TPA leader alone or in- combination, giving a strong indication that antigen presentation of the encoded protein through these constructs was mainly dependent on endosomal pathway. On the contrary, mice receiving pUQ-PA63 construct did not show a significant decline in the percentage lysis of cellular target (50% vs 40% at E:T ratio 15:1) clearly indicating the involvement of cytosolic pathway for antigen presentation. Importantly, all the groups generated significant cytotoxic T lymphocyte response (p<0.01 vs vector control).

EXAMPLE 8 DNA vaccination protected mice against toxin challenge.

Finally, we addressed the question whether the immune responses induced by DNA vaccination protected the mice against lethal toxin challenge. On day 112 after the last immunization, we challenged the mice with a lethal toxin mixture (50μg PA and 22 μg LF) in a total volume of 100 μl via tail vein injection. Toxin challenge results as obtained are summarized in table 3.

TABLE. 3. PA63-based DNA vaccination confers protection against lethal anthrax toxin challenge. VACCINE COMBINATION NUMBER OF SURVIVORS/ % SURVIVAL⁵

^' NUMBER CHALLENGED pTPA-PA63 Pro 6/8 75 pTPA-PA63 DNA 5/8 63 pUQ-PA63 Pro 3/8 38 pUQ-PA63 DNA 3/8 38 pTPA-PA63 -LAMPl Pro 7/8 . 88

PTPA-PA63 -LAMPl DNA 7/8 88 pTPA-PA63 -LAMP1+ UQ-PA63 Pro 4/8 50 pTPA-PA63 -LAMP1+ UQ-PA63 DNA 4/8 50 pPA63-LAMPl Pro 6/8 75 pPA63-LAMPl DNA 5/8 63 pPA63-NATIVE Pro 2/8 25 pPA63-NATIVE DNA 2/8 25 pTPA-PA63 +UQ-PA63 Pro 4/8 50 pTPA-PA63 +UQ-PA63 DNA 3/8 38 rPA63 6/8 - 75 rPA83 . 6/8 75

PBS 0/8 0 VECTOR 0/8 0 ^'

§ Percentage survival was calculated after challenge with a lethal toxin mixture of PA (50 μg/mouse) and LF (22 μg/mouse) injected i.v. in immunized and control mice.

Control mice (vector control and PBS immunized) died within 12-14 hrs of lethal toxin injection but 7 out of the 8 mice immunized with pTPA-PA63-LAMPl construct survived the toxin challenge thus, eliciting a survival percentage, of 88%. Also, the groups immunized with pTPA-PA63 Protein boost and pPA63-LAMPl Protein boost showed a high survival frequency of 75%. Importantly, the survival percentage of the groups receiving DNA vaccine combination pTPA-PA63-LAMPl (both Protein and DNA boost) was higher as compared to those that received subcutaneous rPA83/rPA63 protein vaccination (88% vs 75%), indicating that DNA vaccine was more efficient in protection against toxin challenge. Other groups that received pPA63-LAMPl DNA boost and pTPA-PA63-LAMPl DNA boost showed a survival percentage of 63%. Rest of the groups showed a survival percentage ranging from 38-50% with the native construct showing a survival rate of only 25%. These results suggest that mice immunized with DNA vaccine combinations developed an effective anti-PA immune response which could protect ^■ the mice against lethal toxin challenge.

Claims

We Claim:

1. A DNA vaccine comprising a gene targeting cassette comprising a gene construct comprising a nucleotide sequence encoding a protective antigen linked to signal sequence(s) present on one or both sides of the protective antigen for targeting the said antigen to various antigen processing pathways to enhance cellular and humoral immune response against the said protective antigen.

2. A DNA vaccine as claimed in claim 1 wherein the protective antigen is PA63 gene from Bacillus anlhracis.

3. A DNA vaccine as claimed in claim 1 wherein the PA63 gene encodes a 63kDa protective antigen protein.

4. A DNA vaccine as claimed in claim 1 wherein one or more the signal sequences are present on the N-terminal of said protective antigen.

5. A DNA vaccine as claimed in claims 1 and 5 wherein the N terminal signal sequence is a human-TPA leader (pTPA).

6. A DNA vaccine as claimed in claims 1 and 5 wherein the N terminal signal sequence is a ubiquitin leader (pUQ).

7. A DNA vaccine as claimed in claim 1 wherein one or more signal sequences are present on the C-terminal of said protective antigen.

8. A DNA vaccine as claimed in claims 1 and 8 wherein the C terminal signal sequence is LAMPl.

9. A DNA vaccine as claimed in claims 1 to 4 wherein the said construct pPA63 (Seq

ID 1) encodes the native form of protease cleaved fragment of protective antigen

(Seq ID 6).

10. A DNA vaccine as claimed in claim 1 to 6 wherein the said construct pTPA-PA63

(Seq ID 2) encodes secreted form of the protease cleaved fragment of the said protective antigen (Seq ID 7).

11. A DNA vaccine as claimed in claim 1 to 7 wherein the said construct pUQ-PA63 (Seq ID 3) encodes the cytosolic form of the protease cleaved fragment of the said protective antigen (Seq ID 8).

12. .A DNA vaccine as claimed in claims 1 to 9 wherein the said construct ρPA63- LAMPl (Seq ID 4) encodes the endosomal form of the protease cleaved fragment of the said protective antigen (Seq ID 9).

13. A DNA vaccine as claimed in any preceding claims wherein the said construct pTPA-PA63-LAMPl (Seq ID 5) encodes the secreted endosomal form of the protease cleaved fragment of the said protective antigen (Seq ID 10).

14. A DNA vaccine as claimed in any preceding claims wherein the said protective antigen is presented by MHC I molecules/pathway.

15. A DNA vaccine as claimed in any preceding claims wherein the said protective antigen is presented by MHC II molecules/pathway.

16. A DNA vaccine as claimed in any preceding claims wherein the said gene cassette is comprised of a vector comprising at least one protective antigen and signal sequence on one or both sides of the protective antigen operably linked to the transcriptional factors for targeting the said protective antigen to various antigen processing pathways characterized in that an enhanced cellular and humoral immune response^' is produced on expression of such antigen.

17. A DNA vaccine as claimed in any preceding claims wherein the vector is an expression vector selected from a prokaryote or eukaryote.

18. A DNA vaccine as claimed in any preceding claims wherein the gene cassette is present in aφrokaryotic or eukaryotic host cell.

19. A DNA vaccine as claimed in any preceding claims wherein the signal sequences are meant for trafficking of said protective antigen to various sub-cellular locations.

20. A DNA vaccine as claimed in any preceding claims wherein the immune response is

, generated against the protective antigen only.

21. A DNA vaccine as claimed in any preceding claims wherein an enhanced cellular and humoral immune response is generated in the absence of an immune adjuvant/stimulant.

22, A DNΛ vaccine as claimed in any preceding claims generates neutralizing antibodies of high avidity towards the said protective antigen when administered in a vertebrate.

23. A DNA vaccine as claimed in any preceding claims generates effective cytotoxic T- lymphocytes against the said protective antigen when administered in a vertebrate.

24. A DNA vaccine as claimed in any preceding claims is effective against anthrax toxin.

25. A DNA vaccine as claimed in any preceding claims wherein the said vertebrate is mouse.

26. A pharmaceutical composition comprising the DNA vaccine as claimed in any preceding claims and a pharmaceutically acceptable additive.

27. A pharmaceutical composition comprising a DNA vaccine as claimed in any preceding claims comprising a gene cassette encoding differentially targeted, processed and presented protective antigen characterized in that an enhanced cellular and humoral immune response is generated in the absence of an immune adjuvant.

28. A pharmaceutical composition comprising a DNA vaccine as claimed in any preceding claims comprising a gene cassette encoding differentially targeted, processed and presented protective antigen characterized in that an enhanced cellular and humoral immune response is generated when administered in a vertebrate.

29. A pharmaceutical composition comprising a DNA vaccine as claimed in any preceding claims comprising a gene cassette encoding differentially targeted,

. processed and presented protective antigen when administered in a vertebrate elicits an enhanced cellular and humoral immune response in comparison to the immune response elicited by two subcutaneous doses of recombinant full-length protective antigen protein vaccine administered as alhydroxy gel formulation.

30. A pharmaceutical composition as claimed in any preceding claim wherein said vertebrate is mouse.