WO2010073257A1

WO2010073257A1 - An immunodominant b-cell epitope based protein vaccine against anthrax and method of preparation thereof

Info

Publication number: WO2010073257A1
Application number: PCT/IN2009/000230
Authority: WO
Inventors: Manpreet Kaur; Hema Chug; Rakesh Bhatnagar
Original assignee: Manpreet Kaur; Hema Chug; Rakesh Bhatnagar
Priority date: 2008-12-26
Filing date: 2009-04-08
Publication date: 2010-07-01

Abstract

The present invention relates to the development of immunodominant B-cell epitope based protein vaccine having SEQ ID No.: 1 eliciting immune response against anthrax. The invention also relates to a polynucleotide sequence having SEQ ID No.: 2 encoding epitope based protein vaccine. The invention also relates to the method of preparation and purification of epitope based protein vaccine. The present invention further relates to vaccine composition of B-cell epitope based protein vaccine against anthrax, its method of preparation and process of using the composition. The vaccine or the vaccine composition of the present invention generates high titers of antibodies having high affinity and avidity and are highly effective in in vitro neutralization of B. anthracis lethal toxin and conferring protection against in vivo lethal toxin challenge.

Description

AN IMMUNODOMINANT B-CELL EPITOPE BASED PROTEIN VACCINE AGAINST ANTHRAX AND METHOD OF PREPARATION THEREOF

FIELD OF THE INVENTION The present invention relates to the development of B-cell epitope based protein vaccine against anthrax. In particular, the present invention relates to immunodominant B-cell epitope based protein vaccine against anthrax that is customized to generate effective neutralizing humoral immune response which in turn is capable of conferring protection against subsequent lethal toxin challenge. The invention also relates to the method of preparation of this B-cell epitope based protein vaccine. The present invention further relates to vaccine composition of B-cell epitope based protein vaccine against anthrax, its method of preparation and process of using the composition. BACKGROUND OF INVENTION

Anthrax is an epizootic disease mainly affecting cattle and wild bovidae worldwide (Mock and Fouet, Ann. Rev. Microbiol, 55, 647-671, 2001). Anthrax is rare in human beings but can occasionally be caused through contact with contaminated farm animals and animal products (Leppla et. al., J. CHn. Investig., 110, 141-144, 2002). Of potential concern is the use of anthrax spores as bio-warfare agents. Although there have been several earlier hoaxes or failed attempts, 2001 saw the first effective anthrax terror attacks, infecting 11 and killing five people in the United States (Jernigan et. al., Emerg. Infect. Dis., 7, 933-944, 2001).

Pathogenesis of B. anthracis is mediated by two plasmids, pXOl and pXO2, which encode for primary virulence factors-toxins and capsule, respectively (Brey, Adv. Drug Deliv. Rev., 57, 1266-1292, 2005). pXOl encodes protective antigen (PA), lethal factor (LF); and edema factor (EF). These three proteins act in binary combinations (Stanley and Smith, J. Gen. Microbiol, 26, 49-53, 1961) to produce anthrax exotoxins; Lethal Toxin (LeTx) and Edema Toxin (EdTx) comprising of protective antigen (PA) with lethal factor (LF) and edema Factor (EF) respectively. Prior to exotoxin formation, PA binds to cellular receptors, undergoes proteolytic cleavage and forms heptameric oligomers. The heptamer competitively binds LF and EF, which are then translocated into the cytosol. LF is a zinc dependent protease that cleaves mitogen-activated protein kinases (MAPKKs) leading to toxic shock and death (Vitale et. al., Biochem. Biophys. Res. Comtnun., 248, 706-711, 1998; Duesbery et. al., Science, 280, 734-737, 1998). EF is an adenylate cyclase converting intracellular ATP into cAMP, therefore provoking a substantial increase in intracellular cAMP levels leading to edema (Leppla, Proc. Natl. Acad. ScL USA, 79, 3162-3166, 1982). pXO2 encoded capsule enhances virulence in vivo by inhibiting phagocytosis of the organism (Little and Ivins, Microbes and Infection, 2, 131-139, 1999).

Induction of neutralizing antibodies to PA is considered to be the key to protection against anthrax (Shlyakhov et. al., Vaccine, 15, 631-636, 1997; McBride et. al., Vaccine, 16, 810-818, 1998; Fowler et. al., /. Appl. Microbiol, 87, 305, 1999; Doling et. al., Infect Immun., 67, 3290-3296, 1999; Brossier et. al., Infect Immun., 68, 5371-5374, 2000; Crotty et. al., /. Immunol. Methods, 286, 111-112, 2004). Lethality of anthrax is primarily attributed to toxemia (Mock and Fouet., Ann. Rev. Microbiol, 55, 647-671, 2001) and PA is essential for host cell intoxication as PA contains the host cell receptor binding site (Escuyer and Collier, Infect. Immun., 59, 3381-3386, 1991), the cell binding component for both edema factor and lethal factor (Elliott et. al., Biochemistry, 39, 6706-6713, 2000) and facilitates the entry of the toxin complex into the host cell (Flick-Smith et. al., Infect. Immun., 70, 1653-1656, 2002). PA is also the dominant antigen in both natural and vaccine-induced immunity to anthrax infection (Flick-Smith et. al., Infect. Immun., 70, 1653-1656, 2002). However, the extent of immune response and protection conferred by PA based vaccines against lethal anthrax infection in different experimental models is variable (Brahmbhatt, et. al., Infect. Immun., 75, 5240-5247, 2007).

PA is sub-divided into 4 domains (Mock and Fouet, Ann. Rev. Microbiol, 55, 647-671, 2001); Domain 4 (aa 596 to 735) represents the 139 amino acids of the carboxy terminus of the PA polypeptide. It contains the host cell receptor binding region (Little et. al., Microbiology, 142, 707-715, 1996), which has been identified as being in and near a small loop located between amino acid residues 679 and 693 (Varughese et. al., Infect. Immun., 67, 1860-1865, 1999), and it is therefore essential for host cell intoxication, as previous studies have demonstrated that expressed forms of PA containing mutations (Varughese et. al., Infect. Immun., 67, 1860-1865, 1999) or deletions (Brossier et. al., Infect. Immun., 67, 964-967, 1999) in the region of domain 4 are nontoxic. Flick-Smith et. al. showed that domain 4 contains the dominant protective epitopes of PA (Flick- Smith et. al_v Infect Immun., 70, 1653-1656, 2002). Park et. al. demonstrated that DNA vaccine based on domain IV of PA linked to CRT generated potent humoral response and conferred significant protection against lethal toxin challenge (Park et. al., Infect. Immun., 76, 1952-1959, 2008). Thus, domain W of PA may be the key component for induction of protective immunity against anthrax.

Pertaining to anthrax therapy, only Anthrax Vaccine Adsorbed, AVA (BIOTHRAX™) is licensed in U.S. for human use. AVA is derived from aluminum hydroxide gel adsorbed - sterile culture filtrate of an avirulent non-encapsulated Sterne strain (V770-NPI-R). Even though, various studies have demonstrated its safety; still there is a lack of wider acceptance due to undefined nature of the vaccine and lengthy immunization schedule (six subcutaneous injections along with annual boosters). Therefore, the current scenario demands design and development of improved immunoprophylactic approaches.

A possible solution to this could be an epitope based protein vaccine. Epitope based vaccines provide several advantages-inclusion of protective epitopes along with exclusion of suppressive epitopes; avoidance of any infectious or autoimmune potential hazard, neither the possibility of virulence due to reversion and re- assortment associated with conventional vaccines nor any concern regarding genetic integration or recombination associated with DNA vaccines; this may result in safer vaccines with fewer side effects. They are chemically defined and undergo analytical preparations, thus, they are subjected to robust quality control procedures. They are relatively easy to construct and produce, are economical and highly stable. They can be stored in lyophilized form circumventing the requisite of "cold-chain" for storage and transport. This attribute is particularly important in countries with poor infrastructure.

Previous studies involving mapping of domain 4 have demonstrated that neutralizing epitopes mainly reside in the region PA671-721 (Little et. al., Microbiology, 142, 707-715, 1996). A particular loop (703-722) has been found to be more exposed than the other three domains (Petosa et. al., Nature, 385, 833-838, 1997). Such arrangements may make the epitopes in this region more prone to recognition by immune effector cells (Flick-Smith et. al., Infect. Immun., 70, 1653-1656, 2002). Also, an epitope corresponding to PA678-697 which resides in and near the solvent exposed loop (679-693) has been identified. Further, it is recognized by several neutralizing MAbs to PA (Little et. al., Microbiology, 142, 707-715, 1996). Some of these epitopes, in spite of their close proximity in domain 4, exhibit great disparity in their immunogenidty. This phenomenon might be a reflection of differential processing and presentation of these antigens. It has been shown for PA659-672 and PA717-730 that despite being in close proximity in domain 4 exhibit significant differences in their processing, thus, local context of an epitope may be crucial for MHC binding and presentation (Musson et. al., /. Biol Chem., 278, 52425-52431, 2003). Thus, there is immense scope of identification of novel B-cell epitopes and elucidation of their immunogenic potential and ability to confer protective immunity.

B-cell epitopes can be exploited for development of epitope-based marker vaccines and diagnostic tools for various diseases (Peng et. al., Virus Res., 35, 267-272, 2008). The ability to identify specific epitopes derived from infectious pathogens has significantly encouraged the development of epitope based vaccines. Advancements in technology, including rational epitope building approach, greater understanding of the molecular basis of antigen recognition and HLA binding molecules, expression library immunization, computer assisted prediction algorithms, application of transgenic mice for rapid screening and optimization of vaccine candidates has boosted work in this direction. Epitope based vaccines have been developed against a number of infectious agents, some of which have shown great promise both in animal models and clinical trials. These include human immunodeficiency virus (Pinto et. al., AIDS, 13, 2003-2012, 1999; Dorrell et. al., AIDS, 19, 1321-1323, 2005; Lorin et. al., Vaccine, 23, 4463-4472, 2005); hepatitis B virus (Li et. al., Int. Immunol, 17, 1293-1302, 2005; Engler et. al., MoI. Immunol, 38, 457-765, 2001); human papillomavirus (Reddy et. al., Immunology, 112, 321-327, 2004); and malaria (Kashala et. al., Vaccine, 20, 2263-2277, 2000; Nardin et. al., /. Infect. Dis., 182, 14864496, 2000). Phase I trial of a CD8⁺ T-cell peptide epitope-based vaccine for Infectious Mononucleosis has established that epitope based vaccination is mostly well tolerated and immunogenic in most individuals (Elliott et. al., /. Virol 82, 1448-1457, 2008). Contraceptive vaccines utilizing synthetic epitopes based on leurinizing hormone releasing hormone (LHRH) have been used to achieve effective contraception in mouse models (Zeng et. al., /. Immunol, 169, 4905-4912, 2002; Zeng et. al., MoI. Immunol, 44, 3724-3731, 2007). T cell epitope based vaccination has also been shown to protect mice against subsequent sensitization to the allergen (Marazuela et. aL, MoI. Immunol., 45, 438-445, 2008).

The present invention discloses the development of an immunodominant B-cell epitope protein vaccine derived from C-terminus of protective antigen of B. anthracis aiming at generation of potent neutralizing antibody/protective humoral immune response that would mediate protection against subsequent lethal toxin challenge. The invention also discloses a method of preparation of this B-cell epitope based protein vaccine. The invention further discloses a composition of B-cell epitope based protein vaccine against anthrax, method of preparation and process of using the composition.

OBJECTS OF THE INVENTION

An important object of the present invention is to provide an immunodominant B-cell epitope based protein vaccine having SEQ ID No.: 1 eliciting protective immune response against anthrax. Another object of the present invention is to provide a polynucleotide sequence having SEQ ID No.: 2 encoding B-cell epitope based protein vaccine.

Still another object of the invention is to prepare and purify B-cell epitope based protein vaccine.

Yet another object of present invention is to provide a vaccine composition comprising B-cell epitope based vaccine that generates high titers of neutralizing antibodies with high affinity and high avidity.

Still further object of present invention is to provide a vaccine composition comprising B-cell epitope based vaccine that confers protection against in vivo lethal toxin challenge. Yet another object of present invention is to provide a method of preparation and usage of vaccine composition.

SUMMARY OF INVENTION

The present invention discloses an immunodominant B-cell epitope based protein vaccine having SEQ ID No.: 1 against anthrax involving identification of novel B-cell epitope regions with immunodominant potential with the ultimate goal of enhancing neutralizing antibody-humoral response. In another embodiment of the present invention, a polynucleotide sequence having SEQ ID No.: 2 encoding B-cell epitope based protein vaccine is disclosed.

In still another embodiment of the present invention, a process for the preparation and purification of B-cell epitope based protein vaccine comprising the steps of cloning and transforming is disclosed.

In yet embodiment of the present invention, the composition comprising B-cell epitope based protein vaccine, method of preparation and usage of the composition is disclosed.

In another embodiment of the present invention, a vaccine or composition generating high titers of neutralizing antibodies with high affinity and high avidity is disclosed. The magnitude of protection against subsequent lethal toxin challenge correlated well with the neutralizing antibody response elicited and is at par with that generated by immunization with the whole protective antigen molecule.

In still another embodiment of the present invention, a vaccine or a composition that induces an efficient immune response via intraperitoneal route and may induce the same via various routes namely intradermal (direct injection, delivery on gold beads by gene gun), sub-cutaneous, nasal or oral is disclosed. This vaccine or a composition can immunize vertebrates such as fish; or amphibians such as frogs; or reptiles such as snakes or birds, or mammals such as mice, cats, dogs, goat, sheep, human beings or fowl such as ducks, turkeys, chicken and the like is disclosed.

In another embodiment of the present invention, a strategy that generates an immune response at par with the immune response generated by the whole molecule immunization is disclosed. BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

FIGURE 1: Electrophoretic analysis showing protein expression and purification of recombinant ID-II from E. coli BL 21 (DE3) cells. Proteins are resolved on 12% SDS- PAGE and stained with coomassie brilliant blue (A) or probed with anti-His antibody (B). Arrows point towards recombinant ID-II protein.

FIGURE 2: IgG response in immunized mice. Groups of 10 mice are immunized with antigen preparation at 0, 2 and 4 weeks. Serum samples collected 2 weeks after each immunization are analyzed in triplicates for antigen specific IgG antibodies by ELISA. PBS immunized control group did not show any significant IgG titer.

FIGURE 3: The isotype profile of antigen specific serum antibodies of mice immunized through intraperitoneal route. Experiments are done in triplicates for sera collected at 6 weeks and data are represented as mean absorbance ± S.D.

FIGURE 4: Direct binding assay of ID-II with PA antisera raised in mice. ID-II is coated on ELISA plate and incubated with increasing dilution of PA antisera. Percentage binding is calculated from ratio of absorbance (at 450nm) of PA antisera with ID-II to that of PA.

FIGURE 5a: Direct binding assay of PA with epitope antisera raised in mice. PA is coated on ELISA plate and incubated with increasing dilution of epitope antisera at 37⁰C for two hours. After washing, goat anti mouse IgG-HRP (1:10,000 dilution) is added and kept at 37⁰C for 1 h and subsequently, colour is developed.

FIGURE 5b: Immunoblotting of PA with peptide antisera.5 μg of protective antigen is resolved on 12% SDS- PAGE and electroblotted onto nitrocellulose membrane. The membrane is blocked in 5% skimmed milk powder at 4°C overnight. After three washings with PBST, membrane is incubated with mouse antisera at 1:1000 dilution of A: ID II, B: PA for 2 h at 37⁰C. After washing, bound antibodies are detected with goat anti- mouse IgG-AP (1:10000) and colour is developed using NBT and BCIP. FIGURE 6: Cytokine level (pg/ml) in culture supernatant of splenocytes primed with individual antigen and in vitro stimulated with PA. Results are expressed as mean concentration of cytokine (pg/ml) of triplicate wells.

FIGURE 7: For in vitro LeTx neutralization assay, sera are pooled from groups of immunized mice and incubated with recombinant PA for 1 hour. This mixture is then added to murine macrophage J774A.1 cells and incubated in the presence of LF for 4 h after which cell viability is measured. Percent neutralization is calculated using the following formula: [sample OD value-LeTx standard OD value]/[cells-only OD value-

LeTx standard OD value] X 100. Data are represented as the average % cell survival of two experiments.

FIGURE 8: Epitope immunization provides efficient protection against LeTx challenge. Immunized mice (n=6) are challenged with 2*LDso of LeTx mixture. Challenge is 100% fatal in control mice immunized with PBS. Average percent survival from two independent experiments is depicted by Kaplan-Meier curves.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses the development of immunodominant B-cell epitope based protein vaccine having SEQ ID No.: 1

[MGSSHHHHHHSSGLVPRGSHMASMTGGQQM

GRGSGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDKLA AALEHH HHHH] eliciting protective immune response against anthrax. This epitope based protein vaccine is encoded by polynucleotide sequence having SEQ ID No.: 2 [ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCG GCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGG ATTATTGTTAAATATTGATAAGGATATAAGAAAAATATTATCAGGTTATATTGT AGAAATTGAAGATACTGAAGGGCTTAAAGAAGTTATAAATGACAGATATGAT ATGTTGAATATTTCTAGTTTACGGCAAGATGGAAAAACATTTATAGATAAGCTT GCGGCCGCACTCGAGCACCACCACCACCACCACTGA].

The softwares used to select B-cell epitopes of PA include BCPred, Emboss, and BcePRED. The overlapping of these predicted epitopes results in the identification of a 51 amino acid long region for its use as target for development of anthrax vaccine.

This region is produced as the His tagged protein in pET28a/BL21(DE3) expression system. ID-II is PCR-amplified using pMWl as the template, using forward primer having SEQ. ID. No.: 3 [δ'GACCGGATCCGGATTATTGTTAAATATTGATAAG 3'] and reverse primer having SEQ. ID. No.: 04 ['5'

GCCCAAGCTTATCTATAAATGTTTTTCCATCTTG 3']. The primers are employed to incorporate BαraHI and HindϊLI restriction sites in the PCR amplified product, whose digestion is utilized for cloning the PCR amplified, BαmHI- HmdIII digested insert in pET28a vector. The pET.ID-II construct is transformed into E. coli BL 21 (DE3) cells. ID- II protein is purified under denaturing conditions using metal-chelate affinity chromatography.

Mice are injected intraperitoneally with ID-II or protective antigen protein suspended in buffer and emulsified with complete Freund's adjuvant. The humoral response is assessed with respect to the antibody titer and IgG subclass generated by ELISA in the sera. Secondary antibodies, anti-mouse IgG or its isotypes conjugated with horseradish peroxidase are incubated. Estimation of the enzymatic activity is carried out with TMB as the substrate. Significant levels of antigen specific antibodies are detected post priming with ID-II and PA; which continued to increase post second boost. For the B-cell epitope based vaccination, a predominant IgGl isotype is observed. The dominance in IgGl antibody isotype indicates that the selected epitope is capable of eliciting humoral immune response. On the other hand, a mixed IgGI/IgG2a/IgG2b response is seen against PA. Importantly, protection against anthrax toxin has been associated with the production of subclass IgGl antibodies or a Th2- type response. But here the antibody response generated against PA is characteristic of a mixed Thl-Th2 type immune response.

Direct binding assay is performed to assess the binding capacity of antigenic polypeptide to antisera raised against protective antigen. Binding of protective antigen with antisera raised against protective antigen is considered as the reference binding in both the assays. The results show that polyclonal sera raised against protective antigen represents the immunogenic repertoire of selected epitopes. Binding of PA by peptides antisera is also analyzed in a solid phase ELISA assay. Antisera raised against the antigenic polypeptide contain antibodies which bind to the native protein.

Reactivity of PA with polypeptide antisera is also observed by western blotting. The ability of polypeptide antisera to interact with denatured protein shows that it is raised against linear epitopes.

The affinity of antibodies raised against different peptides is measured by estimating the Kd. The data showed that epitope - ID-II generated antibodies with significantly high Ka value while PA produced higher affinity antibodies. The avidities of the specific antibodies to antigen are also compared by using graded concentrations of ammonium thiocynate. The binding of antibodies with less avidity to the antigen is disrupted at lower concentrations of ammonium thioacynate compared to antibodies with greater avidity to the antigen. The levels of type I (IL-2, IFN-γ) and type II (IL-4, IL-IO) cytokines are also quantified. PA immunized group displayed significantly high levels of IL-2 and IFN-γ. On the contrary, epitope vaccine immunized group produced low level of these type I cytokines. However, pronounced levels of IL-4 are generated.

To measure the cellular immune responses elicited by epitope based vaccination, PA-spedfic IFN-y and IL-4 responses upon re-stimulation with rPA in vitro by ELISPOT. The number of IFN-γ and IL-4-producing cells is determined by counting the number of spot-forming unit (SFU) in each well using AID Immunospot

(Cellular Technology Ltd.), and the results are expressed as numbers of SFU per 10⁶ cells.

An in vitro cell protection assay is carried out investigate the potential of antibodies generated in response to immunization with ID-II and PA for neutralization of LeTx, employing LeTx and murine J774A.1 macrophage cell line. Further, in vivo lethal toxin challenge is performed for evidence of morbidity or mortality and percent survival is determined.

The present invention is illustrated and supported by the following examples. These are merely representative examples and optimization details and are not intended to restrict the scope of the present invention in any way.

Abbreviations

Ab, antibody; AP, alkaline phosphatase; C terminus, carboxy terminus; ELISA, Enzyme Linked Imrnuno Sorbent Assay; FBS, fetal bovine serum; HEPES, N-(2- hydroxyethyl) piperazine-N'-(2-ethane-sulfonic acid) sodium salt; HRP, horse radish peroxidase; i.p., intraperitoneal; ID, immunodominant; IFNγ, interferon-γ; Ig, immunoglobulin; IL, interleukin; LD, lethal dose; LF, Lethal Factor; MHC, major histocompatibility complex; MTT, methylthiazolyl-diphenyl tetrazolium bromide; PA, protective antigen; PBS, phosphate buffered saline; PCR, polymerase chain reaction;

SDS, sodium dodecyl sulfate.

EXAMPLE 1

Epitope prediction and synthesis of candidate peptides

Three systems are used to select B-cell epitopes of PA. BCPred, based on string kernels (EL-Manzalawy et. al, 2008) predicted 1 epitope within domain IV of PA. Emboss, based on epitope antigenedty; predicted 2 epitopes (http://Bioinfo.bgu.ac.il/EMBOSS). The third system, namely, BcePRED, predicts B-cell epitopes based on physico-chemical properties that include hydrophilicity, flexibility/mobility, accessibility and antigenedty respectively; it predicted 2, 3 and 5 epitopes respectively (Saha and Raghava, In: G. Nicosia, V. Cutello, P.J. Bentley and J. Tunis (Eds.), ICARIS, LNCS, 2004). By overlapping these predicted epitopes, a 51 amino acid long region is identified (Table 1) and characterized it for its immunodominance and potential usage as target for development of anthrax vaccine. The identified region; ID-II is produced as the His tagged protein in pET28a/BL21(DE3) expression system. For the same, ID-II is PCR-amplified using pMWl (Chauhan et. al., Biochem. Biophys. Res. Commun., 283, 308-3015, 2001) as the template, using the following primers: Forward Primer: (SEQ ID No. 3)

5'G ACC GGA TCC GGA TTA TTG TTA AAT ATT GAT AAG 3'

Reverse Primer: (SEQ ID No. 4) 5' G CCC AAG CTT ATC TAT AAA TGT TTT TCC ATC TTG 3'

The primers are employed to incorporate BamHl and Hindis, restriction sites in the PCR amplified product, whose digestion is utilized for cloning the PCR amplified, BamHl- HindlU digested insert in pET28a vector (Novagen). The construct is sequenced to confirm the integrity of cloned region. This construct is designated as pET.ID-II is transformed into Escherichia coli BL 21 (DE3) cells and induced at λeoo ~ 0.8, by addition of 1 niM isopropyl β-D-thiogalactoside (IPTG) for 4 h. ID-II protein is purified under denaturing conditions using metal-chelate affinity (Ni-NTA) chromatography. Purified protein is analyzed on 15% SDS-PAGE and dialyzed against 10 mM HEPES buffer containing 10% glycerol (Fig. 1). The yield of ID-II protein is about ~ 5 mg/1.

Protective antigen is purified under denaturing conditions using metal-chelate affinity chromatography, as described elsewhere (Chauhan et. al., Biochem. Biophys. Res. Commun., 283, 308-3015, 2001). Lethal factor is purified under native conditions, as described previously (Gupta et. al., Infect Imtnun., 66, 66862-66865, 1998). Table 1: Sequence of selected immunodominant peptide and its corresponding position

Sequence Number Peptide sequence Designation

Corresponding to PA

626-676 GLLLNIDKDIRKILSGYIVEIEDTEG ID-II

LKEVINDRYDMLNISSLRQDGKTFI

EXAMPLE 2

Animal immunization and sera collection

Four to six week old female BALB/c mice are procured from NIN, Hyderabad and maintained in pathogen free environment in animal house facility. All experiments are performed in accordance with 'Indian Animal Ethics Committee's Regulations'. A group of 10 mice are injected intraperitoneally, with 50 μg of ID-II or protective antigen protein; suspended in 100 μl of PBS and emulsified with complete Freund's adjuvant on day 0. Same dose of booster is given on day 15 and 29 with incomplete Freund's adjuvant. Control mice are immunized with PBS. Blood is collected on day 0, 14, 28 and 42. The sera are separated and stored at - 8O⁰C till further use.

EXAMPLE 3

Serum antibody titers and estimation of IgG subclass Antigen-specific antibody (IgG total) and isotypes (IgGl, IgG2a, IgG2b) levels are determined by ELISA in the sera. Wells are coated with respective antigens; overnight at 4⁰C. After washing and blocking, serial dilutions of antisera (weeks 2, 4, 6) are added in triplicates and incubated for 2 h at 37⁰C. Secondary antibodies, anti- mouse IgG or its isotypes conjugated with horseradish peroxidase (Santa-Cruz) are incubated for 1 h at 37°C Estimation of the enzymatic activity is carried out with TMB as the substrate. The reaction is stopped with 50 μl of IM H3PO4 and the absorbance is measured at 450 nm using Microplate Reader (Bio Rad).

The antibody response is measured with respect to the titer and IgG subclass generated. Significant levels of antigen specific antibodies are detected post priming with ID-II and PA; which continued to increase post second boost. ID-II induced peak antibody titer at 6 weeks of 50,000 (p value < 0.001) (Fig. 2). PA immunized group produced high antibody titers which ranged from 5, 000 to 2,00,000 over a period of six weeks (p value < 0.001). Mice in the PBS immunized control group didn't have significant antibody level. To assess the induction of effective humoral response, IgG isotypes are assayed. For the B-cell epitope based vaccinations, a predominant IgGl isotype is observed. IgG2a and IgG2b responses, though shown are not robust (Fig 3). The dominance in IgGl antibody isotype indicates that the selected epitope is capable of eliciting humoral immune response. On the other hand, a mixed IgGI/IgG2a/IgG2b response is seen against PA. Importantly, protection against anthrax toxin has been associated with the production of subclass IgGl antibodies or a Th2-type response (Little et. al., Infect. Imtnun., 56, 1807-1813, 1988). But here the antibody response generated against PA is characteristic of a mixed Thl-Th2 type immune response. These results are consistent with previous studies (Gu et. al., Vaccine, 17, 340-344, 1999).

EXAMPLE 4 Direct binding assay

A direct binding assay is performed to assess the binding capacity of antigenic polypeptide to antisera raised against protective antigen. ELISA plates are coated with protective antigen (500 ng/well). After blocking and washings, ID-II anti- sera at 10 fold dilutions (from 100 to 100,000) are incubated at 37°C for 2 h. After three washings with PBS-T, goat anti-mouse IgG HRP conjugate (1:5000) is added and incubated for 1 h at 37 ^eC. Alternatively, reactivity of PA antisera with ID-II is also performed, wherein; ELISA plates are coated with ID-II (500 ng/well). Anti PA sera raised in mice is added at 10 fold dilutions (from 100 to 100,000) onto the ELISA plates and incubated at 37⁰C for 2 h. Colour is then developed as described above. Binding of protective antigen with antisera raised against protective antigen is considered as the reference binding in both the assays. As shown in Fig.4, at 100 times dilution of PA antisera, ID- II showed -91 % recognition. On further diluting the PA antisera, percent recognition decreased proportionately. In spite of this, at high dilution of PA antisera (1,00,000), ID-II showed significant recognition of 23%. This shows that polyclonal sera raised against protective antigen represents the immunogenic repertoire of selected epitopes. Binding of PA by peptides antisera is also analyzed in a solid phase ELISA assay. Antisera raised against ID-II showed 48.348% binding with PA (Fig. 5a). This indicates that antisera raised against the antigenic polypeptide contain antibodies which bind to the native protein.

EXAMPLE 5

Western blotting

Reactivity of PA with polypeptide antisera is also observed by western blotting (Fig. 5b). Five micrograms of PA is resolved on 12% SDS-PAGE and electro-blotted onto nitrocellulose membrane. The membranes are blocked in 5% skimmed milk powder in PBS at 4°C overnight. After three washings with PBST, membranes are incubated with ID-II and PA antisera (1:1,000) for 2 h at 37°C. After washing, bound antibodies are detected with alkaline phosphatase conjugated goat anti-mouse IgG (1:10,000, Sigma) and colour is developed using NBT-BCIP (Sigma). The ability of polypeptide antisera to interact with denatured protein shows that it is raised against linear epitopes.

EXAMPLE 6

Antigen binding characteristics: Affinity measurement The affinity of antibodies raised against different peptides is measured by estimating the Kd. In brief, mice antisera (1:100 dilution) is incubated with different concentrations of the protein (1- 100 nM) for 16 h at 25°C so as to attain antigen- antibody equilibrium. The antigen-antibody complexes are transferred onto the wells of the microtitre plates previously coated with the respective protein (500 ng/well) and blocked. The plates are incubated for 2 h at 37°C. After three washings with PBST, goat anti-mouse IgG HRP conjugate (1:5000) is added and incubated for 1 h at 37°C. Colour is developed as described above. Dissociation constants are then calculated using regression analysis and a simplification of the mathematical equation of Scatchard and Klotz (Friguet et. al., /. Immunol. Methods 77, 305-319, 1985). ^•

A_n 1+K,

A₀-A a₀

Wherein, Ao: the absorbance measured for the antibody in absence of peptide, A: the absorbance measured for the antibody in presence of peptide,

Kd: the disassociation constant, and flo: the total peptide concentration. The dissociation constant of antibodies with respective antigens is studied in the pooled sera of day 42 with peak antibody titers for respective groups. As shown in

Table 2, sera obtained from immunized groups showed that epitope - ID-II generated antibodies with significantly high Ka value of 12.945 nM while PA produced higher affinity antibodies (Kd 54.7640 nM) (p value < 0.005).

Table 2: Dissociation constant (Kd) of peptide antisera generated by intraperitoneal immunization in BALB/c mice.

Immunization Group Kd (nM) iϊ>ϊϊ 12.945 ± 0.3950

PA 54.764 ± 3.0584

Experiment is done in triplicates and data are represented as mean Ka ± S.D.

EXAMPLE 7 Antigen binding characteristics: Avidity measurement

Avidity describes the collective interactions between antibodies and a multivalent antigen. The avidities of the specific antibodies to antigen are compared by using graded concentrations of ammonium thioacynate (NHiSCN), a chaotropic agent, which disrupts antigen-antibody interaction (Pullen et. al., /. Immunol. Methods, 86, 83-87, 1986). For determining the same, proteins are coated (500 ng/well) onto the wells of ELISA plates. After washing and blocking, respective antiserum (1:100) is incubated for 2 h at 37°C. After washing the plate, gradient of ammonium thiocynate; 0.1-2.5 M in PBST is added to the wells and the plates are incubated for 15 min. to allow disruption of antigen-antibody interaction. After washing thrice with PBST, HRP conjugated goat anti-mouse IgG (dilution 1:5000) is added and assay continued as previously described for affinity measurement. The antibody concentration determined in the absence of ammonium thiocynate is assumed as total binding (100%) of specific antibodies. The relative avidity between different antisera is represented by the molarity of ammonium thiocynate required for 50% reduction in initial absorbance (corresponding to total binding). This is referred to as the avidity index of that particular serum.

The binding of antibodies with less avidity to the antigen is disrupted at lower concentrations of ammonium thiocynate compared to antibodies with greater avidity to the antigen. The effective concentrations of NHiSCN required to release 50% of immune complexes of ID-II is 0.875 M. PA required higher NHUSCN concentration (1.083 M) in comparison to ID-II for disruption of antigen-antibody complexes (Table 3). This is also verified by the affinity measurement as indicated by high disassociation constant.

Table 3: The avidity index represented by the molarity of ammonium thiocynate required for 50% reduction in initial absorbance (corresponding to total binding). Immunization Group Ammonium thiocynate concentration (M)

ID-II 0.875 PA 1.083

EXAMPLE 8

Evaluation of cytokine levels by ELISA

As cytokines play an important role in polarization of T-helper cell responses, the levels of type I (IL-2, IFN-γ) and type II (IL-4, IL-10) cytokines are quantified.

Mice are sacrificed two weeks post last immunization and splenic cells isolated are processed for analysis of cytokines produced. Splenic cells are prepared by grinding spleens between frosted slides. Erythrocytes are lysed with 0.1 M ammonium chloride. Remaining spleen cells are washed twice with DMEM medium and then are suspended in complete DMEM medium supplemented with 10% heat inactivated fetal bovine serum and lO"⁶ M 2-mercaptoet.hanol. Viability is determined by Trypan blue exclusion test. Splenocytes are cultured in triplicates (IXlO⁶ cells/well) in a 24-well culture plate (Costar), stimulated with/without 5 μg/ml PA. Concanavalin A (ConA, Sigma) is used as a positive control at 1 μg/ml concentration. Splenocytes are incubated at 37°C under 5% COi and 95% humidity and supernatants are harvested at 24, 48 and 72 h time intervals and the levels of cytokine are determined.

Levels of IL-2, IL-4, IL-10 and IFN-γ are determined using BD Opt EIA™ kits according to manufacturer's protocol (Pharmingen). Briefly, 96 well microliter ELISA plate is coated with capture antibody of the respective cytokines and incubated overnight at 4⁰C. Plate is aspirated and washed thrice and blocked with 200 μl of 2% BSA for 2 h at 37°C. After the incubation period, plate is aspirated and washed thrice and incubated with the harvested supernatants for 2 h at RT. The plate is then aspirated and washed five times; plate is incubated with the Detector (Anti-mouse IgG-HRP) for 1 h at RT. Following this, plate is aspirated and washed 7 times and incubated with 100 μl Substrate Solution for 30 min. in dark at RT. Reaction is stopped by adding 50 μl Stop Solution to each well. The absorbance is read at 450 run using a Microplate Reader (Bio Rad) within 30 min. of stopping the reaction. The concentrations of cytokines in the culture supernatants are calculated using a linear regression equation obtained from the absorbance values of the standards provided by the manufacturer.

PA immunized group displayed significantly high levels of IL-2 and IFN-γ, at 69.415 and 2272 pg/ml respectively as compared to all other groups (p<0.005). On the contrary, peptide primed and in vitro protective antigen pulsed groups produced low levels of these cytokines. However, the production of IL-4 is enhanced in peptide immunized mice which ranged from 171.92 to 210.25 pg/ml. Likewise; levels of IL-IO are also significantly high in peptides immunized mice with peak titers of 547.05 pg/ml. The results therefore, depict that the predicted epitope region had bias for Th2 type immune response. Contrarily, protective antigen produced mixed Thl/Th2 type of immune response. The cytokine profile is depicted in Fig. 6.

EXAMPLE 9

ELISPOT assay for IFN-γ and IL-4

To measure the cellular immune responses elicited by epitope based vaccination, splenocytes from immunized mice are isolated to determine PA-specific IFN-γ and IL-4 responses upon re-stimulation with rPA in vitro by ELISPOT. Briefly, multiscreen filtration plates (96-well, Millipore) are coated with 10 μg/ml of anti- mouse IFN-γ / IL-4 monoclonal antibody (BD Pharmingen) in 100 μl of PBS. After overnight incubation at 4⁰C, the wells are washed thrice with sterile MQ, and blocked with 150 μl per well of DMEM for 2 h at 37°C. Triplicate samples of splenocytes are plated at a final concentration of 1 x 10⁶ cells/ml in cell medium. Cells are stimulated with/without 5 μg/ml PA. Unstimulated and ConA (1 μg/ml) stimulated splenocytes respectively are taken as negative and positive controls for all vaccination groups.

Samples are incubated at 37⁰C under 5% COz for 36 h; after which, the cells are decanted and wells are washed 6 times with PBST and incubated for 2 h at 37⁰C, 5%

CO2, and 95% humidity with 2 μg/ml of biotinylated rat anti-IFN-γ / IL-4 antibody in

PBS/0.5% BSA. The plate is again washed 6 times with PBST and incubated with 100 μl of 1:1000 dilution of streptavidin-peroxidase for 45 min. at RT. The wells are then washed thrice with PBST followed by PBS. Spots are developed by incubating with substrate for 20 min. in dark. The number of IFN-γ and IL-4-producing cells is determined by counting the number of spot-forming unit (SFU) in each well using AID Immunospot (Cellular Technology Ltd.), and the results are expressed as numbers of SFU per 10* cells.

Table 4 shows that compared to the control group (PBS immunized), other groups developed distinct PA-spedfic IFN-γ and IL-4 responses (p<0.005). The magnitude of IFN-γ secreting splenocytes for epitope immunized groups ranged ~ 65 SFU/10⁶ splenocytes. However, PA immunized group mounted the maximum response, which is more than 2 times of that elicited by epitope based immunization. On the other hand, pattern of IL-4 secreting splenocytes is quite varied; wherein epitope group generated higher SFU as compared to PA. Therefore, epitope immunized group generated an overall higher count of IL-4 secreting cells as compared to IFN-γ secreting cells.

Table 4: Cytoldne-produdng cells measured by ELISPOT assay

Immunization Group IL-4 IFN-γ

SFC/IO* cells SFC/10⁶ cells

ID-II 210 + 10 65 + 3

PA 180 + 7 148 ± 8

PBS 11 + 1 3 ± 1

Splenocytes from immunized mice are isolated 2 weeks after the last immunization and are re-stimulated with PA (5 μg/ml) in vitro for 36 h.

Each value is representative of 2 mice tested per group and in triplicates. Results are expressed as mean + S.D.

EXAMPLElO In vitro neutralization assay

To investigate the potential of antibodies generated in response to immunization with ID-II and PA for neutralization of LeTx, an in vitro cell protection assay is carried out employing LeTx (665.27 ng/ml LF and 1000 ng/ml PA) and the murine J774A.1 macrophage cell line. Using a standard assay, serial dilutions of antibodies generated are incubated with PA and then LF, prior to addition of complex to J774A.1 cells, to assess toxicity leading to cell death or neutralization of toxicity leading to protection from cell death. J774A.1 macrophages are maintained in Dulbecco's modified Eagles' medium (DMEM) with 4.5 g/L glucose, 10% fetal bovine serum, and 2 mM L-glutamine and seeded in a flat bottomed 96-well microtiter plates at 4X 10⁴ cells/well one day prior to the experiment. The final concentration of LeTx used for the experiments is determined to result in 50% killing (1000 ng/ml of PA and 665.27 ng/ml of LF) of J774A.1 cells. Sera from the last boost are serially diluted and incubated with PA for 1 h to allow neutralization to occur.

This is followed by addition of LF and further incubation for 1 h. The antiserum±toxin mixture is added to the cells, followed 4 h later by MTT (3-(4, 5- dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, Sigma) at final concentration of 100 μg/well. After 1 h incubation to allow uptake and oxidation of the dye, the supernatant is aspirated and 100 μl of solubilization buffer (0.5% (w/v) SDS, 25 mM HCl in 90% isopropyl alcohol) is added to each well. The plate is read at 570 run to determine cell viability. Three wells containing only medium served as medium controls, and three wells containing PA and LF served as toxin controls.

Percent neutralization is calculated using the following formula: [sample optical density (O.D.) value-LeTx standard O.D. value]/[cells-only O.D. value-LeTx standard O.D. value] X 100. Data are represented as the average % cell survival of two experiments. 86.57% protection from LeTx associated cell death is observed when serum from mice immunized with ID-II is diluted out to 1:10 dilution (p<0.001) (Fig.7). No protection (cell survival) is observed when serum from PBS immunized mice is assessed for LeTx neutralizing activity. EXAMPLE 11

Protection against in vivo lethal toxin challenge

Challenge experiments are performed 2 weeks after the last immunization with 200 μg of PA and 200 μg of LF in 200 μl of lmg/ml BSA in PBS administered intravenously, equivalent to approximately 2xLDso. Following challenge, mice are monitored for evidence of morbidity or mortality for 10 days and percent survival is determined. Kaplan-Meier curves for survival of the peptide vaccinated mice against LeTx challenge are summarized in Fig. 8. Time-to-death analysis revealed that vaccination with ID-II construct is equally protective as the native PA encoding construct (p<0.005). Control mice (PBS immunized) died after receiving a lethal toxin injection within 2-5 days. There are no overt clinical signs of illness in surviving mice.

The experimental data are analyzed by Sigma Plot 10.1 and Microsoft Excel and are expressed as means ± standard deviations (S.D.). Comparisons between individual data points are made using a Student's t-test and levels of significance (p value) are determined. P value < 0.05 is considered statistically significant.

ADVANTAGES OF THE PRESENT INVENTION

• An immunodominant B-cell epitope based protein vaccine for inducing potent immune response against anthrax and method of preparing the same.

• A vaccine composition comprising epitope based protein vaccine with adjuvant that generates a potent humoral immune response that correlates well with the survival against anthrax challenge.

• Vaccine or its composition can induce an efficient immune response via various routes namely intraperitoneal, intradermal (direct injection, delivery on gold beads by gene gun), sub-cutaneous, nasal or oral and can immunize an animal which includes vertebrate or amphibian or reptile or mammal or fowl.

• Vaccine or composition generates an immune response that is at par with the immune response generated by the whole molecule immunization, surpassing the side effects of the latter.

• The present invention presents a strategy that can be designed for protection against pathogens such as viruses, prokaryotes and eukaryotes including unicellular and multicellular organisms.

Claims

We claim

1. An immunodominant B-cell epitope based protein vaccine having SEQ ID No.: 1 eliciting immune response against anthrax.

2. The epitope based protein vaccine as claimed in claim 1 wherein the said protein vaccine is encoded by a polynucleotide sequence having SEQ ID No.: 2.

3. The epitope based protein vaccine as claimed in claim 1 or 2 wherein the said protein vaccine has molecular weight of -11 kDa.

4. The epitope based protein vaccine as claimed in claims 1 to 3 wherein the said protein vaccine is derived from C-terminus of protective antigen.

5. The epitope based protein vaccine as claimed in claims 1 to 4 wherein the said protein vaccine is administered in a vertebrate or mammal or human being or fowl.

6. The epitope based protein vaccine as claimed in claims 1 to 5 wherein the said protein vaccine induces an efficient immune response when administered intraperitoneally.

7. The epitope based protein vaccine as claimed in claims 1 to 6 wherein the said protein vaccine potentiates an immune response by promoting processing of the said antigenic polypeptide via the MHC class II pathway.

8. The epitope based protein vaccine as claimed in claims 1 to 7 wherein the said protein vaccine generates high titers of antibodies having high affinity and high avidity.

9. The epitope based protein vaccine as claimed in claims 1 to 8 wherein the said protein vaccine neutralizes B. anthracis lethal toxin in an in vitro toxin neutralization assay using macrophage cells.

10. The epitope based protein vaccine as claimed in claim 9 wherein the said neutralization confers protection at par with that conferred by the whole protective antigen protein.

11. The epitope based protein vaccine as claimed in claims 9 or 10 wherein the said neutralization of B. anthracis lethal toxin challenge is 66%.

12. A process for preparing and purifying the epitope based protein vaccine as claimed in any preceding claims comprising the steps of - (i) identifying an epitope region by overlapping the predicted epitopes; (ii) amplifying the region obtained by step (i) with forward and reverse primers; (iii) digesting the amplified product obtained by step (ii) with restriction enzymes; (iv) cloning the digested product obtained by step (iii) in prokaryotic expression system; (v) transforming the cloned product obtained by step (iv) in an expression host;

(vii) purifying the protein obtained by step (vi) by metal chelate affinity chromatography.

13. The process as claimed in claim 5 wherein said predicted epitopes of step (i) include BCPred, Emboss and BcePRED.

14. The process as claimed in claim 5 wherein said forward and reverse primers of step (ii) have SEQ ID No.: 3 and SEQ ID No.: 4, respectively.

15. The process as claimed in claim 5 wherein said restriction enzymes of step (iii) are BfljnHI andHϊndlll.

16. The process as claimed in claim 5 wherein said prokaryotic expression system of step (iv) is pET28a vector under the control of T7 promoter.

17. The process as claimed in claim 5 wherein said transforming of step (v) is in expression host Escherichia colt BL 21 (DE3) cells.

18. The process as claimed in claim 5 wherein said purifying of step (vii) is with metal nickel.

19. The process as claimed in claims 11 to 17 wherein the yield of protein is about ~ 5 mg/1.

20. A vaccine composition against anthrax comprising 40-60 μg of an immunodominant B-cell epitope based protein vaccine as claimed in claims 1 to 10 with 40-60% v/v adjuvant.

21. The vaccine composition as claimed in claim 19 wherein said adjuvant is a pharmaceutically acceptable complete freund's adjuvant and incomplete freund's adjuvant.

22. The vaccine composition as claimed in claim 19 or 20 wherein said complete freund's adjuvant is composed of inactivated and dried mycobacteria, preferably M. tuberculosis and said incomplete freund's adjuvant is the complete freund's adjuvant without mycobacteria.