WO2008152654A1

WO2008152654A1 - Anthrax fusion proteins, compositions and uses thereof

Info

Publication number: WO2008152654A1
Application number: PCT/IN2008/000368
Authority: WO
Inventors: Rajesh Jain; Anil Kumar Chawla
Original assignee: Panacea Biotec Limited
Priority date: 2007-06-11
Filing date: 2008-06-11
Publication date: 2008-12-18

Abstract

The present invention relates to anthrax recombinant fusion proteins, process of preparation of such proteins and compositions thereof. Particularly the recombinant fusion proteins of the present invention comprise the Edema factor protein (EF) preferably a native Edema factor protein (EF) or mutated Edema factor protein (EFm) or truncated Edema factor protein (EFn) more preferably a truncated Edema factor protein (EFn) and the Lethal factor protein (LF) preferably a native Lethal factor protein (LF) or mutated Lethal factor protein (LFm) or truncated Lethal factor protein (LFn) more preferably a truncated Lethal factor protein (LFn) optionally with a linker. Also provided are nucleic acids encoding the fusion proteins of the present invention. Further, the present invention provides a composition against an infection of B. anthracis comprising the anthrax recombinant fusion protein mixed with a native or mature or mutated PA, and optionally with one or more suitable adjuvant(s) which may be useful as a pre-exposure and post-exposure prophylactic and/or therapeutic vaccine against anthrax.

Description

ANTHRAX FUSION PROTEINS, COMPOSITIONS AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to anthrax recombinant fusion proteins, process of preparation of such proteins and compositions thereof. Particularly the recombinant fusion proteins of the present invention comprise the Edema factor protein and the Lethal factor protein (LF) optionally with a linker. More particularly the recombinant fusion proteins of the present invention comprise truncated Edema factor protein and truncated Lethal factor protein (LF) optionally with a linker. Also provided are nucleic acids encoding the fusion proteins of the present invention. Further, the present invention provides a composition against an infection of B. anthracis comprising the anthrax recombinant fusion protein mixed with a native or mature or mutated PA, and optionally with one or more suitable adjuvant(s) or excipients or both. Furthermore, the present invention relates to process of preparation of anthrax recombinant fusion proteins, native or mature or mutated PA, compositions comprising said recombinant fusion proteins optionally mixed with native or mature or mutated PA and methods of using them. The compositions of the present invention may be useful as a pre-exposure and post-exposure prophylactic and/or therapeutic vaccine against anthrax.

BACKGROUND OF THE INVENTION

Anthrax is an infectious bacterial disease caused by Bacillus anthracis. It occurs most commonly in wild and domestic herbivores (sheep, goats, camels, antelope, cattle, etc.) but may also occur in humans. Infection can occur by cutaneous exposure, by ingestion (gastrointestinal anthrax), or by inhalation (pulmonary anthrax). 95% of anthrax infections in humans occur by cutaneous infection, either from contact with unvaccinated, infected animals in an agricultural setting, or by handling contaminated animal products (meat, leather, hides, hair, wool, etc.) in an industrial setting. Cutaneous anthrax is fatal in about 20% of cases if untreated, but it can usually be overcome with appropriate antimicrobial therapy. Inhalation or gastrointestinal anthrax infection is much more serious and much more difficult to treat. Inhalation anthrax results in respiratory shock and is fatal in 90%- 100% of cases; gastrointestinal anthrax results in severe fever, nausea and vomiting, resulting in death in 25%-75% of cases. An effective vaccine against pre-exposure prophylactic to anthrax was developed in United States in the 1950s and 1960s, and a vaccine was approved by FDA in 1970.

In recent years the threat of air borne transmission of anthrax has been thought to increase as B. anthracis was identified as a possible agent for biological warfare. This threat has now been realized in the past year in the form of mailed anthrax spores, resulting in several deaths in the US. Whereas historically only individuals at high risk, such as veterinarians, livestock handlers, wool shearers, abattoir workers, etc. were needed to consider being vaccinated, the threat to military personnel of the possibility of biological weapons deployment caused the United States military to adopt a sweeping anthrax vaccination program in 1997, under which it was intended to administer the anthrax vaccine to 2.4 million military personnel in all branches of service. The most effective known method for preventing anthrax is vaccination. The only mass produced anthrax vaccine, Anthrax Vaccine Adsorbed (or AVA, commercial name BioThrax"), is a noninfectious sterile filtrate of an attenuated V770-NP1-R strain of B. anthracis, adsorbed to aluminum hydroxide (alum) adjuvant, with <0.02% formaldehyde and 0.0025% benzethonium chloride added. The ability of the vaccine to elicit an immune response in humans is well- documented. The course of vaccination consists of six subcutaneous injections of 0.5 mL doses of vaccine over eighteen months, with annual boosters to maintain immunity. This vaccination is believed to provide immunity that is 90%- 100% effective against aerosol anthrax challenge, based on animal studies and incidental human data.

Anthrax, a potentially fatal disease, is caused by Bacillus anthracis. The virulence of this pathogen is mediated by a capsule of a poly-D-[small gamma, Greek] -glutamic acid and an exotoxin composed of three proteins. The three protein components are the protective antigen (PA, 83 KDa), lethal factor (LF, 90.2 KDa) and edema factor (EF, 88.8 KDa) encoded by genes pag, lef, cya respectively in pXOl plasmids. These proteins, non-toxic by themselves, form lethal toxins (LT) and edema toxins (ET) when combined with an activated PA. Plasmid pXOl and pXO2 encodes the three toxin components and enzymes for capsule biosynthesis, respectively. The genes coding for these three protein components and the capsule are found in the endogenous plasmids pXOl and pXO2, respectively. The capsule of Bacillus anthracis, composed of poly-D-glutamic acid, serves as one of the principal virulence factors during anthrax infection. By virtue of its negative charge, the capsule is purported to inhibit host defence through inhibition of phagocytosis of the vegetative cells by macrophages. In conjunction with lethal factor (LF) and edema factor (EF), whose target cells include macrophages and neutrophils, respectively, the capsule allows virulent anthrax bacilli to grow virtually unimpeded in the infected host. Spores germinating in the presence of serum and elevated CO[sub]2 release capsule through openings on the spore surface in the form of blebs which may coalesce before sloughing of exosporium and outgrowth of fully encapsulated vegetative cell.

US patent no. 5,591,631 discloses a nucleic acid encoding a fusion protein, comprising a nucleotide sequence encoding the protective antigen (PA) binding domain of the native lethal factor (LF) protein and a nucleotide sequence encoding a polypeptide, wherein said fusion protein lacks the catalytic domain of LF.

US patent no. 5,677,274 describes a method for targeting compounds having a desired biological activity not present on native anthrax lethal factor (LF) to a specific cell population, comprising: a) administering to the cell population a first compound comprising a first protein consisting essentially of: i) the translocation domain and the anthrax lethal factor (LF) binding domain of the native anthrax protective antigen (PA) protein, and ii) a ligand domain that specifically binds the first protein to a target on the surface of the cell population to bind the first compound to said surface; and b) administering to the resultant cell population a second compound comprising a fusion protein or conjugate consisting essentially of: i) the anthrax protective antigen (PA) binding domain of the native anthrax lethal factor (LF) protein, chemically attached to ii) a biological activity-inducing polypeptide to bind the second compound to the first compound on the surface of the cell population, internalize the second compound into the cell population, and effect the activity of the polypeptide therein.

US publication no. 20040028695 describes an immunogenic composition to prepare a vaccine against a lethal infection of B. anthracis in an animal comprising an effective immunizing amount of at least one recombinant B. anthracis PA (rPA) protein and at least one recombinant B. anthracis LF (rLF) protein.

PCT publication no. WO2003048390 describes a recombinant DNA Construct comprising an expression vector and a DNA fragment including genes for wild type Protective Antigen (PA) or wild type Lethal Factor (LF) or wild type Edema Factor (EF).

PCT publication no. WO2003040179 discloses a process for preparing anthrax protective antigen protein from E.coli using fed batch culture technique.

PCT publication no. WO200337370 discloses an antigenic pharmaceutical composition comprising Protective Antigen (PA) and Lethal Factor (LF), wherein said PA and/or LF lacks a functional binding site, thereby preventing said PA and LF from binding together via said binding site or thereby preventing said PA from binding to a native PA cell receptor via said binding site, and wherein said composition is substantially non-toxic to animal cells.

PCT publication no. WO2002100340 describes an immunogenic composition capable of raising an anti-5. anthracis antigen immune response in a mammal consisting essentially of recombinant B. anthracis Protective Antigen (rPA).

Most of the prior art vaccines either compromise on efficacy or on safety, for example the BioThrax® vaccine is known to possess high incidence of side effects, and other vaccines which comprise mutagenized PA are comparatively less immunogenic than the native or mature PA. Further, no vaccine is reported in the prior art which can be used both as a pre-exposure and post-exposure prophylactic and/or for the treatment of anthrax infection. Hence there still exists a need to develop new vaccine compositions against anthrax infection which are safe and effective and which preferably can be used as a pre-exposure and post-exposure prophylactic and/or therapeutic.

Further there still exists a need to develop two or more anthrax proteins selected from PA, EF and LF in such a manner so that the same can be obtained by a single or at the most two separate production lines for fermentation and purification procedures.

The present invention not only provides a cost-effective method to develop safe and effective vaccine against anthrax but also attempts to provide a single vaccine composition that may be useful both as a pre-exposure and post-exposure prophylactic and/or as a therapeutic product. SUMMARY OF THE INVENTION

It is an objective of the present invention to provide anthrax recombinant fusion proteins.

It is also an objective of the present invention to provide anthrax recombinant fusion proteins comprising Edema factor protein and lethal factor protein optionally with a linker.

It is also an objective of the present invention to provide nucleic acids encoding the anthrax recombinant fusion proteins.

It is further an objective of the present invention to provide anthrax recombinant fusion proteins comprising a native Edema factor protein or mutated Edema factor protein or truncated Edema factor protein, and a native Lethal factor protein or mutated Lethal factor protein or truncated Lethal factor protein optionally with a linker.

It is further an objective of the present invention to provide anthrax recombinant fusion proteins comprising a truncated Edema factor protein (EFn) and a truncated Lethal factor protein (LFn) optionally with a linker.

It is further an objective of the present invention to provide anthrax recombinant fusion proteins comprising a truncated Edema factor protein (SEQ. ID No. 2) and a truncated Lethal factor protein (SEQ. ID No. 1) optionally with a linker.

It is a further objective of the present invention to provide anthrax recombinant fusion proteins mixed with a native or mature or mutated PA.

It is a further objective of the present invention to provide anthrax recombinant fusion proteins comprising EFn and LFn optionally with a linker mixed with a native or mature or mutated PA.

It is still a further objective of the present invention to provide anthrax recombinant fusion proteins having amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 4.

It is another objective of the present invention to provide anthrax recombinant fusion proteins mixed with a native or mature or mutated PA that result in faster immune responses by targeting all three toxin proteins namely PA, EF and LF which can result in faster prophylactic and therapeutic immune responses.

It is another objective of the present invention to provide process for preparation- of anthrax recombinant fusion proteins.

It is another objective of the present invention to provide process for preparation of recombinant native or mature or mutated PA.

It is another objective of the present invention to provide process for preparation of anthrax recombinant fusion proteins comprising EFn and LFn optionally with a linker.

It is another objective of the present invention to provide an immunogenic composition comprising anthrax recombinant fusion proteins comprising edema factor protein and lethal factor protein optionally with a linker.

It is further an objective of the present invention to provide a composition against an infection of B. anthracis which comprises anthrax recombinant fusion proteins comprising EFn protein and LFn protein optionally with a linker, mixed with a native or mature or mutated PA, and optionally with one or more suitable adjuvant(s).

It is further an objective of the present invention to provide an immunogenic prophylactic or a therapeutic composition useful against an infection of B. anthracis in a subject which comprises administration of an effective amount of anthrax recombinant fusion proteins comprising EFn protein and LFn protein optionally with a linker, mixed with a native or mature or mutated PA and optionally one or more adjuvant(s) to the said subject.

It is another objective of the invention to provide an immunogenic composition comprising anthrax recombinant fusion protein comprising truncated edema factor protein (SEQ. ID. No. 2) and truncated lethal factor protein (SEQ. ID No. 1) optionally with a linker.

It is another objective of the invention to provide immunogenic compositions against an infection of B. anthracis comprising anthrax recombinant fusion proteins comprising truncated Edema factor protein (SEQ ID No. 2) and truncated Lethal factor protein (SEQ ID No. 1) mixed with a native or mutated or mature Protective Antigen (PA) optionally with a linker and one or more pharmaceutically acceptable adjuvant(s).

It is another objective of the invention to provide an immunogenic composition comprising anthrax recombinant fusion proteins selected from amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 4 optionally with one or more pharmaceutically acceptable adjuvant(s).

It is an objective of the present invention to provide a method of producing an immunogenic prophylactic or therapeutic response against an infection of B. anthracis in a mammal comprising the step of administering to a mammal the immunogenic composition of the present invention described herewith. ,

It is another objective of the present invention to provide a method to produce an immunogenic prophylactic or therapeutic response against an infection of B. Anthracis in a subject which comprises administration of an effective amount of anthrax recombinant fusion proteins comprising EFn protein and LFn protein optionally with a linker which is one or more amino acid residues, mixed with a native or mature or mutated PA, and optionally one or more adjuvant(s) to the said subject.

It is a further objective of a present invention to provide a DNA construct comprising an expression vector and a DNA fragment comprising nucleic acids encoding the anthrax recombinant fusion protein such that the expression vector when incorporated into a suitable host allows expression of an anthrax recombinant fusion protein.

It is a further objective of a present invention to provide a DNA construct comprising an expression vector and a DNA fragment comprising nucleic acids encoding the anthrax recombinant PA protein such that the expression vector when incorporated into a suitable host allows expression Of an anthrax recombinant protein.

It is further an objective of the present invention to provide nucleic acids encoding the DNA construct.

It is also an objective of the present invention to provide plasmids and primers and the nucleic acids encoding them which are useful in making a DNA construct for the expression of the anthrax recombinant fusion protein.

It is a further objective of a present invention to provide a vaccine composition which is potentially immunogenic but safe. Particularly the compositions are useful as a prophylactic and/or a therapeutic vaccine against anthrax.

BRIEF DESCRIPTION OF FIGURES

Figure- 1 Schematic representation of expression system

Figure-2 Schematic representation of recombinant expression plasmid "pPAK- NATPRO"

Figure-3 shows a codon optimized Sequence of PA.

Figure-4 shows a Protein Sequence of PA.

Figure-5 shows a LFn with Glycine as linker Recombinant Construct in pET28a, wherein clone LFn has been constructed for the expression of LFn in E.coli BL21(DE3). The genes were amplified from the pXOl region of the B. anthracis using specific primers and inserted in pET-28a expression vector having Kanamycin as selection marker. The recombinant plasmid was then transformed into E.coli BL21 (DE3) cells for the expression of protein. Figure-6 shows a Nucleotide Sequence of truncated LF. Figure 7 shows a Protein Sequence of truncated LF (SEQ. ID No. 1).

Figure-8 shows an EFn Recombinant Construct in pET28a, wherein clone EFn has been constructed for the expression of LFn in E.coli BL21(DE3). The genes were amplified from the pXOl region of the B. anthracis using specific primers and inserted in pET-28a expression vector having Kanamycin as selection marker. The recombinant plasmid was then transformed into E.coli BL21 (DE3) cells for the expression of protein

Figure-9 shows a Nucleotide Sequence of truncated EF.

Figure-10 shows a Protein Sequence of truncated EF (SEQ. ID No. 2).

Figure- 11 shows an EFn+LFn with glycine as linker Fusion Construct. Figure- 12 shows an EFn with LFn with Glycine as tail Fusion Construct pET28a (EFn+LFn) (SEQ. ID No. 3).

Figure- 13 shows the Primers for EFn with LFn as tail Fusion Construct.

Figure-14 SDS-PAGE analysis of rEFn expression

Figure- 15 SDS-PAGE analysis of rLFn expression Figure- 16 SDS-PAGE analysis of rEFn -rEFn expression

Figure- 17 shows EFn+LFn as linker Fusion Construct.

Figure-18 shows EFn+LFn as linker Fusion Construct pET28a (EFn+LFn) (SEQ. ID

No. 4).

Figure- 19 shows Primers for EFn with LFn as tail Fusion Construct.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides anthrax recombinant fusion proteins and nucleic acids encoding the anthrax recombinant fusion proteins. Particularly the present invention provides anthrax recombinant fusion proteins comprising Edema factor protein and Lethal factor protein optionally with a linker. Preferably the present invention provides anthrax recombinant fusion proteins comprising a truncated Edema factor protein (EFn) and a truncated Lethal factor protein (LFn) optionally with a linker. More preferably the present invention provides anthrax recombinant fusion proteins comprising EFn protein and LFn protein optionally with one or more other linker(s) such as one or more amino acid residues, mixed with a native or mature or mutated PA.

In an embodiment, the anthrax recombinant fusion proteins optionally mixed with a native or mature or mutated PA results in faster immune responses by targeting all three toxin proteins.

In an aspect of the present invention, the terms PA (Protective Antigen), LF (Lethal Factor) and EF (Edema Factor) shall refer to proteins which are wild types or which are recombinant types or both unless otherwise specifically stated.

In an embodiment, the present invention provides a composition against an infection of B. anthracis which comprises anthrax recombinant fusion proteins comprising EFn and LFn optionally with a linker, and optionally with one or more suitable adjuvant(s). In another embodiment is provided a pre-exposure and post exposure prophylactic and/or a therapeutic composition useful against an infection of B. anthracis in a subject which comprises administration of an effective amount of anthrax recombinant fusion proteins comprising EFn and LFn optionally with a linker, mixed with a native or mature or mutated PA and optionally one or more adjuvant(s) to said subject.

In another embodiment, the present invention also provides a DNA construct comprising an expression vector and a DNA fragment comprising nucleic acids encoding the anthrax recombinant fusion proteins such that the expression vector when incorporated into a suitable host allows expression of anthrax recombinant fusion proteins. In another embodiment is provided a vaccine composition which is potentially immunogenic and safe. Particularly the compositions are useful as a pre-exposure and post exposure prophylactic and/or a therapeutic vaccine against anthrax.

In a still another embodiment, the present invention provides nucleic acids encoding the DNA construct. In a further aspect of the present invention is provided plasmids and primers and the nucleic acids encoding them which are useful in making a DNA construct for the expression of the anthrax recombinant fusion proteins.

In a preferred embodiment of the present invention, the anthrax recombinant fusion proteins comprising EFn and LFn are formulated by mixing with a mature Protective Antigen (PA). A mature PA also referred to as PA₈₃ is one wherein there is a cleavage of 29 amino acid residues signal sequence from the native PA. In this state, the PA is inactive except for its binding affinity for a receptor. Only after the furin-mediated cleavage to form PA₆₃, the binding of one of the other toxin components is possible. Heptamerization and pore formation after acidification leads to a structure similar to the staphylococcal alpha-hemolysin.

In another embodiment of the present invention, the anthrax recombinant fusion proteins comprising EFn and LFn are formulated by mixing with a mutated Protective Antigen. Use of a mutated PA also prevents the binding of the Edema factor protein or Lethal factor protein and thus might avoid the formation of Edema Toxin or Lethal Toxin respectively leading to better product safety.

In an embodiment, the composition of the present invention comprising anthrax recombinant fusion proteins is capable of producing at least one or more of the anti-PA, anti-LF and anti-EF antibodies that shall work as neutralizing antibodies and, therefore, shall interrupt binding activities of the anthrax toxin. Therefore, the present invention, by using EFn, LFn and native or mature or mutated PA as antigens, may result in faster immune responses by targeting all three toxin proteins namely PA, EF and LF, since the presence of different antibodies allows attack of different proteins at the same time. In an embodiment of the present invention, anthrax recombinant fusion proteins comprising EFn and LFn optionally with a linker, mixed with a native or mature or mutated PA is provided to target all three proteins i.e., PA, LF, and EF. The linker molecule facilitates the independent folding of the EFn and LFn proteins of the fusion construct. This helps in imparting better immunogenic response of the EFn and LFn proteins. Many of the antibodies raised against EF or LF may neutralize the toxic effect of the anthrax toxin by interrupting the binding of EF and/or LF and PA.

In another embodiment, the present invention provides anthrax recombinant fusion proteins comprising a native or mutated or truncated EF protein and a native or mutated or truncated LF protein optionally with one or more linkers inserted at one or more sites of the fusion protein chain.

In another embodiment of the present invention, the linker is one or more amino acids such as glycine or amino acid residue. Alternatively the linker is also herein referred to as a spacer peptide. The linker is preferably selected from one or more amino acids, amino acid residues, glycine, polyglycine, and others thereof.

In another embodiment, the present invention provides an anthrax recombinant fusion protein comprising truncated Edema factor protein (SEQ. ID No. 2) and truncated lethal factor protein (SEQ. ID No. 1) optionally with a linker.

In another embodiment, the present invention provides an anthrax recombinant fusion protein comprising truncated Edema factor protein (SEQ. ID No. 2) and and truncated lethal factor protein (SEQ. ID No. 1) optionally with linker wherein linker is amino acid or amino acid residue such as glycine.

In another embodiment, the present invention provides an immunogenic composition comprising anthrax recombinant fusion protein comprising truncated Edema factor protein (SEQ. ID No. 2) and truncated lethal factor protein (SEQ. ID No. 1) optionally with a linker.

In another embodiment, the present invention provides an immunogenic composition comprising anthrax recombinant fusion protein comprising truncated Edema factor protein (SEQ. ID No. 2) and truncated lethal factor protein (SEQ. ID No. 1) optionally with a linker wherein linker is amino acid or amino acid residue such as glycine.

In another embodiment, the present invention provides an immunogenic composition comprising anthrax recombinant fusion protein comprising truncated Edema factor protein (SEQ. ID No. 2) and truncated lethal factor protein (SEQ. ID No. 1) optionally with a linker; and protective antigen.

In another embodiment, the present invention provides an immunogenic composition comprising anthrax recombinant fusion protein (SEQ. ID. No. 3).

In another embodiment, the present invention provides an immunogenic composition comprising anthrax recombinant fusion protein (SEQ. ID. No. 4).

In an embodiment, the present invention provides anthrax recombinant fusion proteins that ease the vaccine production by reducing the need for separate production lines for fermentation and purification procedures for each protein i.e. EFn and LFn individually. The said fusion protein can further be mixed with other antigens such as a native or mature or mutated PA.

In another embodiment of the present invention, at least two antigenic B. anthracis proteins are over-expressed as a fusion protein, in an E. coli host cell such as the BL- 21(DE3) (from Novagen). The over-expressed proteins are produced by an optimized fermentation method and purified by one or more techniques known to the art such as column chromatography. The purified proteins are useful in formulating an immunogenic composition, which is again useful as a vaccine against infections caused by B. anthracis. Development of an E. coli BL-21(DE3) over-expression system involves the development of expression vector (plasmids) to express the target recombinant fusion proteins in an E. coli BL-21(DE3). It might however be understood that alternative vectors/plasmids and/or expression host might be utilized for the production of the novel fusion proteins of the present invention such as the use of Bacillus subtilis as the host cell for the fusion protein expression.

In another embodiment of the present invention, the expression vector described herein is for use in a prokaryotic system; the vector also can contain elements required for replication in either a prokaryotic or eukaryotic host system or both, as desired. Such vectors, which include plasmid vectors and viral vectors such as bacteriophage, baculovirus, retrovirus, lentivirus, adenovirus, vaccinia virus, semliki forest virus and adeno-associated virus vectors, are well known and can be purchased from a commercial source (Promega, Madison Wis.; Stratagene, La Jolla Calif.; GIBCO/BRL, Gaithersburg Md.) or can be constructed by one skilled in the art. The following vectors are provided by way of example; Bacterial: pQE70, pQE30, pET28a (Qiagen), pBluescript SK, pBluescript KS (Stratagene); pTRC99a, pRlT2T (Pharmacia); Eukaryotic: pWLNEO, pXTI, pSG (Stratagene) pSVK3, pSVLSV40 (Pharmacia). Any other plasmid or vector may be used as long as they are replicable and viable in the host being used to express the fusion proteins of the present invention.

In another embodiment of the present invention is provided a process for preparation of anthrax recombinant fusion proteins. In a further embodiment of the present invention, the process for preparation of an anthrax recombinant fusion protein comprises the following steps: a. Development of a suitable DNA construct comprising a vector and DNA fragment encoding for the desired fusion protein, b. Clone development for the production of recombinant fusion protein EFn-LFn or EFn-(Linker)-LFn, c. Expression of the fusion protein in a suitable host, purification and characterization of recombinant fusion protein, d. Obtaining the native or mature or mutated PA, e. Mixing the fusion protein of step (3) with the native or mature or mutated PA, and f. Making a composition of fusion protein mixed with a native or mature or mutated PA as a vaccine optionally by adding one or more suitable adjuvant(s). In a further embodiment of the present invention, the process for preparation of a recombinant protective antigen (rPA) comprises the following steps: a. Development of a suitable DNA construct comprising a vector and DNA fragment encoding for the PA protein, b. Clone development for production of recombinant protective antigen (rPA), and c. Expression of the recombinant protective antigen (rPA) in a suitable host, purification and characterization of recombinant protective antigen (rPA),

In another embodiment of the present invention, the process for preparation of an anthrax recombinant fusion protein comprises the following steps: a. Amplification of the EFn and LFn gene from the genomic DNA of Bacillus anthracis using specific primers optionally having glycine residues as a linker, b. Insertion of fusion protein of EFn and LFn proteins in the expression vector, c. Restriction digestion of the gene and vector, d. Ligation and transformation into competent E. coli DH5α cells, e. Selecting the transformants on LB agar containing an antibiotic such as kanamycin, f. Screening of the colonies on the agar plates by colony PCR for the presence of the desired gene, g. Confirming the selected clone by digestion with restriction enzymes, and h. Transforming the recombinant plasmid into host cells for the expression of the fusion protein.

In another embodiment of the present invention, the process for preparation of an anthrax recombinant protective antigen comprises the following steps: a. Amplification of the PA gene from the genomic DNA of Bacillus anthracis using specific primers optionally having glycine residues as a linker, b. Insertion of PA proteins in the expression vector, c. Restriction digestion of the gene and vector, d. Ligation and transformation into competent E. coli DH5α cells, e. Selecting the transformants on LB agar containing an antibiotic such as kanamycin, f. Screening of the colonies on the agar plates by colony PCR for presence of the desired gene, g. Confirming the selected clone by digestion with restriction enzymes, and h. Transforming the recombinant plasmid into host cells for expression of PA.

In another embodiment of the present invention, the process for preparation of an anthrax recombinant fusion protein comprises the following steps: a. Amplification of EFn with LFn (as tail) gene from the genomic DNA of Bacillus anthracis using specific primers optionally having glycine residues as a linker, b. Insertion of EFn with LFn as tail protein in the expression vector, c. Restriction digestion of the gene and vector, d. Ligation and transformation into competent E. coli DH5α cells, e. Selecting the transformants on LB agar containing an antibiotic such as kanamycin, f. Screening of the colonies on the agar plates by colony PCR for the presence of the desired gene, g. Confirming the selected clone by digestion with restriction enzymes, h. Transforming the recombinant plasmid into host cells for the expression of the fusion protein, and i. Fermentation and purification of EFn+LFn protein.

The EFn+LFn protein thus obtained may be mixed with anthrax recombinant native or mature or mutated PA and made into a composition along with one or more suitable adjuvant(s). In an aspect of the present invention, the method used for the preparation of fusion proteins is selected from one of more methods known to the art.

In an embodiment of the present invention, the upstream process for an anthrax recombinant fusion protein production may be carried out by a batch process or preferably by using fed-batch culture technique to improve the yield of the fusion protein. The optimization of various fermentation parameters using fed-batch conditions shall improve protein yields and stability. Standardization of fermentation parameters shall require systematic design of experiments as follows: a) Different nitrogen source such as Soya peptone, yeast extract, yeast autolysate, corn steep liquor or the like or mixtures thereof in the fermentation media. b) Different carbon sources such as glucose, glycerol, galactose, maltose, fructose, lactose or the like or mixtures thereof in the fermentation media. c) Different concentration of inducers such as IPTG, amino acid, sugars or the like or mixtures thereof by auto induction method, and pH of induction process for the expression of recombinant fusion proteins. d) Different induction time such as Low OD, Mid and high OD based upon the growth profile and duration. e) Different induction duration, temperature, various stabilizers and other in-process control method(s) or combination thereof for the stability of the fusion protein. f) Different quantities of carbon and nitrogen sources at pre & post induction phases. g) Various feeding strategies on the basis of DO-Stat, pH-Stat and Gluco-Stat to achieve high cell density in fed batch fermentation process and to maximize yield of fusion protein.

In an embodiment, the fermentation media used in the present invention is devoid of any component obtained from an animal source.

In an embodiment of the present invention, the downstream purification process of the recombinant fusion proteins is based upon their localization in the cell as inclusion bodies, periplasmic fluid or extracellular secretions. Based on the nature of protein localization and its isoelectric point (PI), various chromatographic methods may be used such as anion exchange chromatography, hydrophobic interaction chromatography and size exclusion or gel filtration chromatography, difiltration or the like, or combinations thereof.

In an embodiment of the present invention, anthrax recombinant fusion proteins shall work as neutralizing antibodies and, therefore, will interrupt binding activities of the anthrax toxin. PA is a 735-amino acid polypeptide of about 83 kDa that binds to the surface of mammalian cells by cellular receptors. Once bound, PA is activated by proteolytic cleavage by cellular proteases to a 63-kDa and 20-kDa molecule. 20 kDa is released in the blood and 63-kDa molecule is capable of forming a ring-shaped heptamer in the plasma membrane of the targeted cell exposing PA binding sites for the N-terminal region of either LF or EF which are then internalized by endocytosis. The complex is then carried to an acidic compartment, where the low pH causes a conformational change in the PA63 pre-pore that forms a cation-specific channel and allows the EF and LF to enter into the cytosol. This process is known as endosomal acidification. Once in the cytosol, the EF and LF then carry out their respective damage -inducing processes. EF acts as a Ca2+ and calmodulin dependent adenylate cyclase that greatly increases the level of cAMP in the cell. This increase in cAMP upsets water homeostasis, severely throws the intracellular signaling pathways off balance, and impairs macrophage function, allowing the bacteria to further evade the immune system. LF also helps the bacteria evade the immune system through killing macrophages. Once in these cells, LF acts as a Zn²⁺-dependent endoprotease that snips off the N-terminus of mitogen-activated protein kinase kinases (MAPKK). This inhibits these kinases by not allowing them to efficiently bind to their substrates, which leads to altered signaling pathways and ultimately to apoptosis. Thus, the synergistic effect of these three proteins leads to cellular death through a cascade of events that allow the proteins to enter the cell and disrupt cellular function. PA is the major immunogen in the anthrax vaccines. Antibodies to PA neutralize anthrax toxin by blocking adherence of PA to host cells, binding of LF/EF to PA, or assembly of PA heptamer. LF represents both a prophylactic and therapeutic target because it has been reported to play a vital role at two different points in anthrax infection. It elicits humoral and cell-mediated immune response and memory. Combination of LFn and EFn components has been found to enhance the anti-EFn and anti-LFn antibody titer, in comparison to immunization with EFn or LFn alone. The N- terminal truncated LF and EF (about 1-255 amino acids) without the catalytic domain completely lack any toxic effect. The use of EFn and LFn serves as a neutralizing molecule for reducing the toxicity caused by wild type EF and LF.

The vaccine may be administered by conventional routes, e.g. intravenous, subcutaneous, intraperitoneal and mucosal routes. The said vaccine can be administered through jet injection with needle or without needle. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules. The active immunogenic ingredients are often mixed with excipients which are pharmaceutically acceptable and compatible with the active components. Suitable excipients are for example, water, saline, dextrose, glycerol, ethanol, sugars or the like and combinations or derivatives thereof. In addition, if desired, the vaccine may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the vaccine. Examples of adjuvants which may be effective include but are not limited to aluminum hydroxide, aluminium phosphate, calcium phosphate, immunostimulatory sequence (ISS), CpG, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),, N-acetyl-nor- muramyl-L-alanyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N- acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine ( 1 '-2'-dipalmitoyl-sn-glycero hydroxylphosphoryloxy) ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a squalene/Tween-80 emulsion, and other adjuvants known to the art or suitable mixtures thereof.

The vaccines of the present invention may be conventionally administered parenterally by injection, for example, either subcutaneously or intramuscularly. The said vaccine can be administered through jet injection with needle or without needle.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective. The quantity to be administered, which is generally in the range of 0.5 micrograms to 500 micrograms of protein per dose, depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered may depend on the judgment of the practitioner and may be particular to each subject. The vaccine may be given in a single dose schedule, or optionally in a multiple dose schedule. A multiple dose schedule is one in which, a primary course of vaccination may be with 1-12, preferably 1-6 separate doses, followed by other doses given at subsequent time intervals required to maintain and/or reinforce the immune response, for example, at 1- 4 months for a second dose, and if needed, a subsequent dose(s) after several months. The dosage regimen will also at least in part be determined by the need of the individual and be dependent upon the judgment of the practitioner. In addition, the vaccine containing the immunogenic antigen(s) may be administered in conjunction with other immunoregulatory agents, for example, immunoglobulins, as well as antibiotics.

Additional formulations which are suitable for other modes of administration include microcapsules, suppositories and, in some cases, oral formulations or formulations suitable for distribution as aerosols. For suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5 % to 10 %, preferably 1-2%.

Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10-95% of active ingredient, preferably 25-70%. In one embodiment the medicament may be administered intranasally (i.n.). An intranasal composition may be administered in droplet form having approximate diameters in the range of 10-5000 pm. Intranasal administration may be achieved by way of applying nasal droplets or via a nasal spray. It is possible that, following i.n. delivery of antibodies, their passage to the lungs may facilitated by a reverse flow of mucosal secretions.

In a different embodiment, the medicament may be delivered in an aerosol formulation. The aerosol formulation may take the form of a powder, suspension or solution. The size of aerosol particles is one factor relevant to the delivery capability of an aerosol. Thus, smaller particles may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol particles have a diameter distribution to facilitate delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution may be selected to target an articular section of the respiratory airway, for example the alveoli. The aerosol particles may be delivered by way of a nebulizer or nasal spray. In the case of aerosol delivery of the medicament, the particles may have diameters in the approximate range of 0-50 pm, preferably 1-5 pm. The aerosol formulation of the medicament of the present invention may optionally contain a propellant and/or surfactant. By controlling size of droplets which are to be administered to a subject to within the defined range of the present invention, it is possible to avoid/minimise inadvertent antigen delivery to the alveoli and thus avoid alveoli associated pathological problems such as inflammation and fibrotic scarring of the lungs. In another embodiment, the composition may be delivered as a transdermal formulation e.g. skin delivery patches.

In an embodiment is provided a method to produce an immunogenic pre-exposure and post exposure prophylactic or therapeutic response against an infection of B. Anthracis in a subject which comprises administration of an effective amount of anthrax recombinant fusion proteins comprising EFn protein, LFn and a linker which is a one or more amino acid residues, mixed with a native or mature or mutated PA and optionally one or more adjuvant(s) to the said subject. A subject, as used herein, is a mammal, particularly domesticated livestock and animals including but not limited to dogs, cats, cows, bulls, steers, pigs, horses, sheep, goats, mules, donkeys, etc. Preferably the subject is a human. A subject can be of any age at which the subject is able to respond to inoculation with the present vaccine by generating an immune response. The immune response so generated can be completely or partially protective against disease, cellular toxicity, debilitation or death caused by infection with B. anthracis. A subject is also one to which a composition comprising the fusion protein of the present invention can be administered prophylactically and/or therapeutically.

The following example is offered by way of illustration and is not intended to limit the scope of the invention in any manner whatsoever.

Example-1: Clone development and production of recombinant PA Protective antigen (PA) gene has been codon optimized and synthesized for protein product expression in E.coli. Commercial vector pET 26b (+) were used to express PA gene. After restriction digestion of the gene and vector, both were ligated and transformed into TOPlO {E.coli) competent cells. The transformants were plated on LB agar containing 50μg/ml kanamycin. The colonies on the plates were screened by colony PCR for the presence of the desired gene. The selected clone was confirmed by restriction mapping and sequencing. The recombinant construct was then transformed into E.coli BL21 (DE3) cells for the expression of protein. Below fig-1 illustrate the schematic representation of expression system. The expressed protein was confirmed for its size on SDS-PAGE gel and also by western blotting using Anti-PA monoclonal antibody available from US Biologicals.

The Primer Sequence of PA in pET26b is shown in Table- 1.

Table-l: Details of Primers Pl & P2

Pl 5'CAC TAT CAT ATG GAG GTG AAA CAG GAA AAC CGT C 3'

P2 5' CAC TAT GTC GAC TTA GCC AAT TTC GTA GCC C 3'

Nucleotide Sequence of PA and the Protein Sequence of PA are shown in figure-3 and figure-4 respectively.

Example 2:Clone development for production of recombinant fusion protein (EFn+LFn)

Recombinant fusion construct EFn-LFn was developed using EFn and LFn gene amplified from cya and lef gene of pXOl plasmid of Bacillus anthracis respectively, using specific primers having glycine residues as spacer peptide inserted in pET-28a expression vector which has Kanamycin as selection marker. The recombinant plasmid was then transformed into E.coli BL21 (DE3) cells for the expression of fusion protein.

Example 3 The recombinant PA and the EFn+LFn fusion protein are mixed together optionally with one or more suitable adjuvant(s).

Example 4

Preparation of cell bank E. coli -cell bank preparation was carried out in animal component free medium (ACFM). As outlined below, recombinant strain PB08 (BL21 (DE3)/pPAK) was plated on ACFM plates with kanamycin, and single colony was grown in liquid ACFM medium for pre-cell bank preparation. • To estimate the density of cells, the Optical density was observed at 600nm in spectrophotometer (OD_6Oo)- When the OD_60O reached to 0.8-1.5, the culture was harvested and centrifuged. The pelleted cells were resuspended in 1/10 volume of fresh ACFM broth containing 20 % glycerol and 50μg/ml kanamycin. • Aliquots were made in sterile cryovials and the aliquots were stored at minus 80 ⁰C.

Fermentation process:

The upstream process for an anthrax recombinant protective protein production was carried out by moderate cell density culture to improve the yield of the recombinant protein. The fermentation media used in the present invention is devoid of any component obtained from an animal source.

Harvesting:

The culture was harvested in a sterilize bottle & Centrifuged at 5000 rpm for 30 minutes at 5 ± 3 ⁰C ,the supernatant was discarded & the pellet was stored at minus 20 ⁰C in polypropylene bottles for future use.

Purification:

The purification of recombinant protective antigen comprises of the following strategy:

Inclusion body isolation:

The pellet obtained was resuspended in lysis buffer in a ratio (1 :10). The lysis was performed using High pressure Homogenizer at 20000-25000psi in two passages and carried centrifugation at 10,000g for 30 min. at 4 ⁰C. The pellet was washed for Inclusion bodies using washing buffer for 30 min. at 4 ⁰C and centrifuged at 10,000g for 30 min. at 4 ⁰C. The efficacy of this process was checked by running on SDS - PAGE & Western blot.

Urea Solubilization: The pellet obtained after inclusion body isolation procedure was resuspended in solubilization buffer (lgm/lOml) & was kept on magnetic stirrer for 1 hr maintaining temperature at 2-8 ⁰C for effective solubilization. It was then clarified by centrifugation at 10,000g at 4 ⁰C for 30mins. Supernatant containing protein of interest was filtered through 0.45μ filter and was preceded for chromatography step.

Purification by MacroprepQ:

The column packed with the resin (Macro prep High-Q from Bio-Rad) was firstly equilibrated with start buffer i.e. Buffer-A. After equilibration, 100ml of urea solubilized, clarified and 0.45 μm filtered sample was loaded. 2CV of Buffer A was passed through column to collect unbound sample. Refolding on column of sample protein was takes place by passing linear gradients of Buffer-A & B (from 100 % A to 100 % B ) extend further for 2 column volumes with Buffer- B followed by 0.5 CV of Buffer -C. The bound rPA was eluted with gradients of Buffer-D. The peak fractions thus collected was analysed for their concentration & purity with SDS-PAGE (Coomassie brilliant Blue + silver staining) & Western blot. The column was regenerated by 5 CV of Buffer E.

The purified protein collected after Macroprep Q was subjected to hydroxyapatitte chromatography. Mannitol ( @1% -10%) & Tween -80 ( @ 0.2% -2%) were added to the final purified protein obtained after hydroxyapatite chromatography. The final purified protein with additives were subjected to sterile filtration. The active raw material was tested by different analytical & bioanalytical tests (Reverse phase high- pressure liquid chromatography, Macrophage lysis assay, Host cell protein, host cell DNA, Endotoxin, Circular Dichromism etc.)

Example 5: Composition

The immunogenic composition of anthrax recombinant fusion proteins of the present invention can prepared by following process:

• Add aluminium phosphate gel in a sterile bottle.

• Adding required volume of the recombinant fusion proteins in the bottle and mix optionally with protective antigen. • After mixing add required volume of D-mannitol solution and again mix it.

• To this composition add required volume of 2-Phenoxyethanol and then add required volume of Sodium phosphate buffer solution (pH 7.6) to make up the volume; shake and store the final formulation.

Claims

CLAIM

1. Anthrax recombinant fusion proteins comprising the Edema factor protein and the Lethal factor protein optionally with a linker.

2. Anthrax recombinant fusion proteins according to claim 1, wherein Edema factor protein is selected from a group consisting of native Edema factor protein (EF), mutated Edema factor protein (EFm), truncated Edema factor protein (EFn) and combinations thereof and the Lethal factor protein is selected from a group consisting of native Lethal factor protein (LF), mutated Lethal factor protein (LFm), truncated Lethal factor protein (LFn) and combinations thereof optionally with a linker.

3. Anthrax recombinant fusion proteins according to claim 2 comprising truncated Edema factor protein (SEQ ID No. 2) and truncated Lethal factor protein (SEQ ID

No. 1) optionally with a linker.

4. Anthrax recombinant fusion proteins according to claim 1 and 3 mixed with a native or mature or mutated Protective Antigen (PA).

5. Anthrax recombinant fusion proteins according to claim 1 wherein the linker is selected from one or more amino acids, amino acid residues, glycine, polyglycine, and others thereof.

6. Anthrax recombinant fusion proteins having amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 4.

7. A process for preparation of anthrax recombinant fusion proteins according to claim 1 which comprises the following steps: a. Development of a suitable DNA construct comprising a vector and DNA fragment encoding for the desired fusion protein, b. Clone development for the production of recombinant fusion protein EFn- LFn or EFn-(Linker)-LFn, c. Expression of the fusion protein in a suitable host, purification and characterization of recombinant fusion protein, d. Obtaining the native or mature or mutated PA, e. Mixing the fusion protein of step (c) with the native or mature or mutated PA, and further making a composition of fusion protein mixed with a native or mature or mutated PA as a vaccine optionally by adding one or more suitable adjuvant(s).

8. A process for preparation of anthrax recombinant fusion proteins according to claim 1 which comprises the following steps: a. Amplification of the EFn and LFn gene from the genomic DNA of Bacillus anthracis using specific primers optionally having glycine residues as a linker, b. Insertion of fusion protein of EFn and LFn proteins in the expression vector, c. Restriction digestion of the gene and vector, d. Ligation and transformation into competent E.coli DH5α cells, e. Selecting the transformants on LB agar containing an antibiotic such as kanamycin, f. Screening of the colonies on the agar plates by colony PCR for the presence of the desired gene, g. Confirming the selected clone by digestion with restriction enzymes, and h. Transforming the recombinant plasmid into host cells for the expression of the fusion protein.

9. A process for preparation of anthrax recombinant fusion proteins according to claim 1 which comprises the following steps: a. Amplification of the PA gene from the genomic DNA of Bacillus anthracis using specific primers optionally having glycine residues as a linker, b. Insertion of PA proteins in the expression vector, c. Restriction digestion of the gene and vector, d. Ligation and transformation into competent E.coli DH5α cells, e. Selecting the transformants on LB agar containing an antibiotic such as kanamycin, f. Screening of the colonies on the agar plates by colony PCR for presence of the desired gene, g. Confirming the selected clone by digestion with restriction enzymes, and h. Transforming the recombinant plasmid into host cells for expression of PA.

10. A process for preparation of anthrax recombinant fusion proteins according to claim 1 which comprises the following steps: a. Amplification of EFn with LFn (as tail) gene from the genomic DNA of Bacillus anthracis using specific primers optionally having glycine residues as a linker, b. Insertion of EFn with LFn as tail protein in the expression vector, c. Restriction digestion of the gene and vector, d. Ligation and transformation into competent E.coli DH5α cells, e. Selecting the transformants on LB agar containing an antibiotic such as kanamycin, f. Screening of the colonies on the agar plates by colony PCR for the presence of the desired gene, g. Confirming the selected clone by digestion with restriction enzymes, h. Transforming the recombinant plasmid into host cells for the expression of the fusion protein, and i. Fermentation and purification of EFn+LFn protein.

11. A process for preparation of anthrax recombinant fusion proteins comprising recombinant protective antigen (rPA) according to claim 4 which comprises the following steps: a. Development of a suitable DNA construct comprising a vector and DNA . fragment encoding for the PA protein, b. Clone development for production of recombinant protective antigen (rPA), and c. Expression of the recombinant protective antigen (rPA) in a suitable host, purification and characterization of recombinant protective antigen (rPA)

12. Immunogenic compositions comprising anthrax recombinant fusion proteins according to claim 2, comprising EFn protein and LFn protein optionally with a linker, mixed with a native or mature or mutated PA and optionally one or more adjuvant(s).

13. Immunogenic compositions against an infection of B. anthracis comprising anthrax recombinant fusion proteins comprising truncated Edema factor protein

(SEQ ID No. 2) and truncated Lethal factor protein (SEQ ID No. 1) mixed with a native or mutated or mature Protective Antigen (PA) optionally with a linker and one or more pharmaceutically acceptable adjuvant(s).

14. An- immunogenic composition comprising anthrax recombinant fusion proteins selected from amino acid sequence set forth in SEQ ID No. 3 or SEQ ID No. 4 optionally with one or more pharmaceutically acceptable adjuvant(s).

15. Immunogenic composition according to claim 12 wherein adjuvant(s) can be selected from the group comprising aluminum hydroxide, N-acetyl-muramyl-L- threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D- isoglutamine (CGP 1 1637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl- D-isoglutaminyl-L-alanine (1 '-2'-dipalmitoyl-sn-glycero hydroxylphosphoryloxy) ethylamine (CGP 19835 A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria, monophosphoryl lipid A, trehalose dimycolate and cell wall skeleton (MPL + TDM + CWS) in a squalene/Tween-80 emulsion and mixtures thereof

16. Anthrax recombinant fusion proteins according to claim 1, wherein a DNA construct comprises an expression vector and a DNA fragment comprising nucleic acids encoding the anthrax recombinant fusion protein such that the expression vector when incorporated into a suitable host allows expression of an anthrax recombinant fusion protein.

17. A method of producing an immunogenic prophylactic or therapeutic response against an infection of B. anthracis in a mammal comprising the step of administering to a mammal the immunogenic composition of claim 12.

18. Anthrax recombinant fusion proteins according to claim 1 wherein a vaccine composition is potentially immunogenic and safe and useful as a prophylactic and/or a therapeutic vaccine against anthrax.

19. The anthrax recombinant fusion proteins substantially as herein described and illustrated by the examples.

20. The process for the preparation of anthrax recombinant fusion proteins substantially as herein described and illustrated by the examples.