FIELD OF INVENTION
The present invention relates to anthrax recombinant fusion proteins, process of preparation of such proteins and process for their preparation and composition thereof Particularly the recombinant fusion proteins of the present invention comprise an Edema factor protein and Protective Antigen (PA) optionally with a linker The present invention further relates to anthrax recombinant fusion proteins comprising Edema factor protein, protective antigen and lethal factor protein optionally with a linker The present invention further relates to the immunogenic composition comprising said anthrax recombinant fusion proteins optionally with the pharmaceutically acceptable adjuvants or excipients or both Also provided are nucleic acids encoding the fusion proteins of the present invention Further provided are nucleic acids encoding the DNA construct of the present invention Further, the present invention relates to process of preparation of anthrax recombinant fusion proteins, compositions comprising said recombinant fusion proteins 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 pX02 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 pX02 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 exosponum 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 l) the translocation domain and the anthrax lethal factor (LF) binding domain of the native anthrax protective antigen (PA) protein, and u) a hgand 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 n) 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 coh 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 preexposure and post-exposure prophylactic 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-etfective 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 the anthrax recombinant fusion proteins and process for the preparation thereof
It is also an objective of the present invention to provide anthrax recombinant fusion proteins comprising Edema factor protein and protective antigen 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 a further objective of the present invention to provide anthrax recombinant fusion proteins comprising a native or mutated or truncated Edema factor protein and a native or mutated or mature Protective Antigen optionally with a linker
It is a further objective of the present invention to provide anthrax recombinant fusion proteins comprising a truncated Edema factor protein and a native or mutated or mature Protective Antigen optionally with a linker
It is a further objective of the present invention to provide anthrax recombinant fusion proteins comprising a native or mutated or truncated Edema factor protein, a native or mutated or truncated Lethal factor protein and a native or mutated or mature PA wherein the LFn acts as a linker optionally with one or more other linker(s)
It is a further objective of the present invention to provide anthrax recombinant fusion proteins comprising truncated Edema factor protein (SEQ ID No 2), and protective antigen optionally with a linker wherein linker is truncated lethal factor protein (SEQ ID No 1)
It is a further objective of the present invention to provide anthrax recombinant fusion proteins comprising EFn protein LFn protein and a native or mutated or mature PA wherein the LFn protein acts as a linker optionally with one or more other linker(s)
It is another objective of the present invention to provide anthrax recombinant fusion proteins comprising truncated Edema factor protein (SEQ ID No 2), lethal factor protein (SEQ ID No 1) and protective antigen optionally with a linker
It is another objective of the present invention to provide anthrax recombinant fusion proteins or a combination of such fusion proteins preferably with a truncated anthrax recombinant protein which can result in faster prophylactic and therapeutic immune responses immune responses, including both pre-exposure and post-exposure, by targeting all three toxin proteins namely PA, EF and LF
It is another objective of the present invention to provide process for preparation of anthrax recombinant fusion proteins
It is further an objective of the present invention to provide an immunogenic composition against an infection of B anthracis which comprises an anthrax recombinant fusion protein comprising a EFn protein and a native or mutated or mature PA optionally with a linker, and optionally with one or more suitable adjuvant(s)
It is further an objective of the present invention to provide an immunogenic composition against an infection of B anthracis which comprises a mixture of an anthrax recombinant fusion protein comprising a EFn protein and a native or mutated or mature PA optionally with a linker, and a truncated anthrax recombinant protein preferably LFn, optionally with one or more suitable adjuvant(s)
It is a further objective of the present invention to provide a composition comprising anthrax recombinant fusion protein along with EFn protein and/or LFn protein
It is a further objective of the present invention to provide a composition comprising anthrax recombinant fusion protein along with a recombinant fusion protein comprising EFn protein and LFn protein optionally with one or more other hnker(s)
It is a further objective of the present invention to provide an immunogenic composition comprising anthrax recombinant fusion proteins comprising truncated Edema factor protein (SEQ ID No 2), and protective antigen optionally with a linker wherein linker is truncated lethal factor protein (SEQ ID No 1)
It is a further objective of the present invention to provide an immunogenic composition comprising anthrax recombinant fusion proteins comprising truncated Edema factor protein (SEQ ID No 2), lethal factor protein (SEQ ID No 1) and protective antigen optionally with a linker
It is a still further objective of the present invention to provide an immunogenic composition comprising anthrax recombinant fusion protein selected from amino acid sequence set forth in
SEQ ID No 3 or SEQ ID No 4 or SEQ ID No 5 optionally with one or more pharmaceutically acceptable adjuvants
It is further an objective of the present invention to provide a pre-exposure and post-exposure prophylactic and/or therapeutic composition useful against an infection of B anthracis in a subject which comprises administration of an effective amount ot an anthrax recombinant fusion protein comprising a EFn protein and a native or mutated or mature PA optionally with a truncated anthrax recombinant protein preferably LFn, and optionally one or more adjuvant(s) to the said subject
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 an anthrax recombinant fusion protein comprising a EFn protein, a native or mutated or mature PA and a linker which is LFn protein and/or one or more amino acid residues 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 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 the DNA construct for the expression of the anthrax recombinant fusion protein
It is a further objective of a present invention to provide an immunogenic composition or vaccine composition which is potentially immunogenic and safe Particularly the compositions are useful as a prophylactic and/or a therapeutic vaccine against anthrax The present invention thus provides 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
BRIEF DESCRIPTION OF FIGURES
Figure-1 Schematic representation of expression system
Figure-2 Schematic representation of recombinant expression plasmid upPAK-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 coh 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 coh 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 (SFQ ID No 1)
Figure-8 SDS-PAGE analysis of rLFn expression
Figure-9 shows an EFn Recombinant Construct in pET28a wherein clone EFn has been
constructed for the expression of LFn in Ecoh 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 coh BL21 (DE3) cells for the expression of protein
Figure-10 shows a Nucleotide Sequence of truncated EF
Figure-11 shows a Protein Sequence of truncated EF (SEQ ID No 2)
Figure-12 SDS-PAGE analysis of rEFn expression
Figure-13 shows an EFn-+PA with LFn as linker Fusion Construct in pET28a, wherein recombinant fusion construct of EFn+PA fusion protein has been developed using LFn as the linker gene 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 Ecoh BL21 (DE3) cells for the expression of fusion protein
Figure-14 shows an EFn+PA with LFn as linker Fusion Construct (SEQ ID No 3)
Figure-15 shows the Primers for EFn, LFn and PA in Recombinant Construct EFn+PA with LFn as linker in pET28a
Figure-16 shows an EFn+PA with LFn and Glycine as linker Fusion Construct in pET28a, wherein recombinant fusion construct of EFn+PA fusion protein has been developed using LFn and poly-glycine gene as linkers The EFn, PA and LFn genes were amplified from the pXOl region of the B anthracis using specific primers Primers were designed to insert the polyglycine residues between each gene pET-28a expression vector with Kanamycin as selection marker was used for cloning of the iusion protein Transformation was done in E coh BL21 (DE3) cells for the expression of fusion protein
Figure-17 shows an EFn+PA with LFn and Glycine as linker Fusion Construct (SEQ ID No
4)
Figure-18 shows the Primers for EFn+ PA with LFn and Glycine as linker Fusion Construct
Figure-19 shows an EFn+PA with Glycine as linker Fusion Construct in pET28a, wherein recombinant fusion construct of EFn f-PA fusion protein has been developed The gene for EFn and PA was amplified from pXOl plasmid of B anthracis using specific primers and was inserted in pET-28a expression vector having Kanamycin as selection marker The
recombinant plasmid was then transformed into E coli BL21 (DL3) cells for the expression of
fusion protein
Figure-20 shows an EFn+PA with Glycine as linker Fusion Construct (SEQ ID No 5)
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 a native or mutated or truncated Edema factor protein preferably a truncated Edema factor protein and a native or mutated or mature Protective Antigen optionally with a linker
In an embodiment, the present invention provides an anthrax recombinant fusion protein comprising a native or mutated or truncated EF protein preferably a EFn, a native or mutated or truncated LF protein preferably a truncated Lethal factor protein and a native or mutated or mature PA wherein the LFn acts as a linker optionally with one or more other hnker(s) such as one or more amino acid residues In an embodiment, the anthrax recombinant fusion protein or a combination of such fusion protein preferably with a truncated anthrax recombinant protein can result 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 an immunogenic composition against an infection of B anthracis which comprises a mixture of an anthrax recombinant fusion protein comprising EFn protein and a native or mutated or mature PA optionally with a linker, and a truncated anthrax recombinant protein preferably LFn optionally with one or more suitable adjuvant(s) In another embodiment, the present invention provides a pre-exposure and postexposure prophylactic and/or a therapeutic composition useful against an infection of B anthracis in a subject which comprises administration of an effective amount of an anthrax recombinant fusion protein comprising EFn protein and a native or mutated or mature PA optionally with a truncated anthrax recombinant protein preferably LFn, and optionally one or more adjuvant(s) to the 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 protein such that the expression vector when incorporated into a suitable host allows expression of an anthrax recombinant fusion protein 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 preferred embodiment of the present invention, the anthrax recombinant fusion protein comprises EFn and a mature Protective Antigen optionally with a linker
A mature PA also referred to as PAs3 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 funn-mediated cleavage to form PA63, the binding of one of the other toxin components is possible Heptamenzation and pore formation after acidification leads to a structure similar to the staphylococcal alpha-hemolysin 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 protein is capable of producing at least one or more of the anti-PA and anti-EF antibodies that shall work as neutralizing molecules and therefore, shall interrupt binding activities of the anthrax toxin Furthermore, since the LF and the EF protein share high N-terminal amino acid homology, some of the antibodies raised against the N-terminal of EF will also react with LF Therefore, the present invention by using EFn and native or mutated or mature PA as antigens, may result in faster immune responses by targeting all three toxin proteins since the presence of different antibodies allows attack of different proteins at the same time Further, in post-exposure anthrax, EFn and LFn provides competitive binding to wild type EF and LF and hence provides more therapeutic protection
The action of the vaccine of the present invention will be more effective in the post exposure therapeutic cases, as the EFn protein given during the vaccination process will also compete with natural EF or LF for the binding with the site ot natural/wild type PA associated with the bacterial infection The wild type PA when bound to the EFn will not produce any toxic effect and thus help in providing a potent therapeutic product against the bioterronst attack or accidental spread of Anthrax spores
In an embodiment of the present invention, an anthrax recombinant fusion protein comprising EFn protein and a native or mutated or mature PA optionally with a linker is provided to target all three proteins 1 e, PA, LF, and EF Many of the antibodies produced against EFn or LFn may neutralize the toxic effect of the anthrax toxin by interrupting the binding of EF and/or LF with PA
In another embodiment, the present invention provides an anthrax recombinant fusion protein comprising a native or truncated EF protein, a native or truncated LF protein and a native or mutated or mature PA 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 selected from a group comprising a Lethal factor (LF), a region of LF and amino acids such as glycine, polyglycine or combinations thereof or amino acid residues Alternatively the linker is also herein referred to as a spacer peptide
In another embodiment, the present invention provides an anthrax recombinant fusion protein comprising truncated Edema factor protein (SEQ ID No 2) and protective antigen optionally with linker wherein linker is truncated lethal factor protein (SEQ ID No 1)
In another embodiment the present invention provides an anthrax recombinant fusion protein comprising truncated Edema factor protein (SEQ ID No 2), truncated lethal factor protein (SEQ ID No 1) and protective antigen 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 protective antigen 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 protective antigen optionally with linker wherein linker is truncated lethal factor protein (SEQ ID No 1)
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 comprising truncated Edema factor protein (SEQ ID No 2), truncated lethal factor protein (SEQ ID No 1) and protective antigen optionally with a linker
In another embodiment, the present invention provides an immunogenic composition comprising anthrax recombinant fusion protein (SEQ ID No 4)
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 protective antigen 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 (SEQ ID No 5)
In an embodiment, the present invention provides an anthrax recombinant fusion protein that eases the vaccine production by reducing the need for at least three separate production lines for fermentation and purification procedures for each protein 1 e PA, EFn and LFn individually By making a fusion protein, the same effect is achieved in a single production line Alternatively, by making a fusion protein comprising EFn protein and a native or mutated or mature PA and making a truncated anthrax recombinant protein preferably LFn requires two production lines for fermentation and purification procedures Both the EFn-PA fusion protein and the LFn protein can then be mixed in order to obtain the desired vaccine composition of present invention
In a preferred embodiment, the anthrax recombinant fusion proteins of the present invention are designed in a particular sequence so as to produce a high yield of the proteins The anthrax recombinant fusion proteins comprise of sequence comprising EFn linked with native or mutated or mature PA by the linker LFn, wherein the EFn and the LFn, and LFn and the native or mutated or mature PA are linked with glycine residue(s) The said particular sequence is surprisingly found to be highly expressive thus leading to a high yield Alternatively, the said particular sequence of the anthrax proteins improves the immunogenic and/or therapeutic properties and/or the safety profile of the vaccine made by using the novel fusion proteins of the present invention
In another embodiment of the present invention, at least two antigenic B anthracis proteins are expressed as a fusion protein in an E coh host cell such as the BL-21(DE3) (from Novagen) The 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 coh BL-21(DE3) expression system involves the development of expression vector (plasmids) to express the target recombinant fusion proteins in an E coh 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 subtihs 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), pBluescnpt SK, pBluescript KS (Stratagene), pi RC99a, 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 anthrax recombinant fusion proteins comprises the following steps
1 Development of a suitable DNA construct comprising a vector and DNA fragment encoding for the desired fusion protein,
2 Clone development for the production of recombinant fusion protein for example EFn-PA or EFn-(Linker)-PA or EFn-(Linker)-LFn-(Linker)-PA or the like,
3 Expression of the fusion protein in a suitable host, purification and characterization of recombinant fusion protein,
4 Optionally mixing a truncated anthrax recombinant protein such as LFn with the fusion protein obtained in step (3),
5 Optionally mixing these fusion proteins and adding one or more suitable adjuvant(s) to step (3) or step (4)
In another embodiment of the present invention, the process for preparation of an anthrax recombinant fusion protein comprises the following steps
1 Amplification of the PA and EFn and optionally LFn gene from the genomic DNA of Bacillus antharacis using specific primers optionally having glycine residues as linker,
2 Insertion of fusion protein of PA and EFn and optionally LFn protein in the expression vector,
3 Restriction digestion of the gene and vector,
4 Ligation and transformation into competent E coh DH5a cells,
5 Selecting the transformants on LB agar containing an antibiotic such as kanamycin,
6 Screening of the colonies on the agar plates by colony PCR for the presence of the desired gene,
7 Confirming the selected clone by digestion with restriction enzymes, and
8 Transforming the recombinant plasmid into host cells for the expression of the fusion protein
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 1PTG, 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, periplasms 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 an anthrax recombinant fusion protein shall work as neutralizing molecules and, therefore, will interrupt binding activities of the anthrax toxins with each other 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 rPA and EFn components has been found to enhance the anti-EFn antibody titer, in comparison to immunization with EFn alone LFn also plays a role in eliciting antibody response against anthrax The N-tcrminal 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 tor reducing the toxicity caused by wild type EF and LF
In another embodiment of the present invention is provided an immunogenic composition to prepare a vaccine against an infection of B anthraas in a subject and includes an effective immunizing amount of a recombinant fusion protein comprising EFn protein and a native PA optionally with a linker
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-lsoglutaminyl-L-alanine (l'-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 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 (in) 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 intranasal 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 of the present invention, the fusion proteins or compositions comprising such proteins may be delivered as topical/transdermal formulation e g skin delivery patches
In an embodiment is provided 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 an anthrax recombinant fusion protein comprising EFn protein, a native or mutated or mature PA and a linker which is LFn protein and/or one or more other amino acid residues, and optionally one or more adjuvant(s) to the said subject A subject, as used herein, is a mammal particularly domesticated livestock, animals including but not limited to dogs, cats, cows, bulls, steers, pigs, horses, sheep, goats, mules, donkeys, etc and humans 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 recombinant fusion proteins 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 coh Commercial vector pET 26b (+) were used to express PA gene After restriction digestion of the gene and vector, both were ligated and transformed into TOP10
(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
Example 2
Recombinant fusion construct EFn-LFn-PA fusion protein was developed using PA, EFn and LFn gene amplified from Bacillus anthracis 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 Composition

(Table Removed)
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 along with Tween 80 in the bottle and mix
• After mixing add required volume of D-mannitol solution and L- Arginine Hydrochloride solution and again mix it
• To this composition add required volume of 2-Phenoxyethanol and then add required volume of Sodium phosphate buffer solution to make up the volume, shake and store the final formulation.

We Claim :-
1 Anthrax recombinant fusion proteins comprising a native Edema factor protein (EF) or mutated Edema factor protein (EFm) or truncated Edema factor protein (EFn) and a native or mutated or mature Protective Antigen (PA) optionally with a linker
2 Anthrax recombinant fusion proteins according to claim 1 wherein the linker is selected from the group consisting of a native Lethal factor protein (LF), mutated Lethal factor protein (LFm), truncated Lethal factor protein (LFn), amino acid residues, glycine, polyglycine and combinations thereof
3 Anthrax recombinant fusion proteins according to claim 1 comprising truncated Edema factor protein (SEQ ID No 2) and a native or mutated or mature Protective Antigen optionally with a linker wherein the linker is truncated Lethal factor protein (SEQ ID No 1)
4 Anthrax recombinant fusion proteins according to claim 3 further comprising an amino acid linker wherein the amino acid is glycine
5 Anthrax recombinant fusion proteins according to claim 1, comprising EFn protein, LFn protein and a native or mutated or mature PA wherein the LFn protein acts as a linker optionally with one or more other hnker(s)
6 Anthrax recombinant fusion proteins according to claim 1 comprising truncated Edema factor protein (SEQ ID No 2), a mature Protective Antigen and truncated Lethal factor protein (SEQ ID No 1) optionally with a one or more other lmker(s)
7 Anthrax recombinant fusion proteins according to claim 6 comprising truncated anthrax recombinant proteins having faster, pre-exposure and post-exposure prophylactic and therapeutic immune responses targeting all three toxin proteins PA, EF and LF
8 Anthrax recombinant fusion proteins according to claim 1, wherein said fusion proteins are prepared by process comprising the steps of
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 for example EFn-PA or EFn-(Linker)-PA or EFn-(Linker)-LFn-(Linker)-PA or the like,
c Expression of the fusion protein in a suitable host, purification and characterization
of recombinant fusion protein d Optionally mixing a truncated anthrax recombinant protein such as LFn with the
fusion protein obtained in step (c)
9 Anthrax recombinant fusion proteins according to claim 1, wherein said fusion proteins are
prepared by process compnsing the steps of
a Amplification of the PA and FF and optionally LF gene from the genomic DNA of Bacillus antharacis using specific primers optionally with linker
b Restriction digestion of the gene and vector
c Ligation and transformation into competent E coli DH5α cells,
d Selecting the transformants on LB agar containing an antibiotic such as kanamycin,
e Screemng of the colonies on the agar plates by colony PCR for the presence of the
desired gene, f Confirming the selected clone by digestion with restriction enzymes, and
g Transforming the recombinant plasmid into host cells for the expression of the fusion protein
10 Immunogenic compositions against an infection of B anthracis comprising anthrax recombinant fusion proteins comprising a native Edema factor protein (EF) or mutated Edema factor protein (EFm) or truncated Edema factor protein (EFn) and a native or mutated or mature Protective Antigen (PA) optionally with a linker and optionally with one or more pharmaceutically acceptable adjuvants
11 Immunogenic compositions according to claim 10 comprising truncated Edema factor protein (SEQ ID No 2) and a native or mutated or mature Protective Antigen optionally with a linker wherein the linker is truncated Lethal factor protein (SEQ ID No 1)
12 Immunogenic compositions according to claim 11 comprising truncated Edema factor protein (SEQ ID No 2), mature Protective Antigen and truncated Lethal factor protein (SEQ ID No 1) optionally with a linker wherein the linker is selected from one or more amino acid residues
13 An immunogenic composition compnsing anthrax recombinant fusion proteins selected from amino acid sequence set forth in SEQ ID No 3 or SEQ ID No 4 or SEQ ID No 5 optionally with one or more pharmaceutically acceptable adjuvants
14 Immunogenic composition according to claim 10 wherein adjuvants can be selected from the group comprising aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-lsoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-aldnyl-D-isoglutamine (CGP 1 1637, referred to as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine (l'-2'-dipalmitoyl-sn-glycero hydroxylphosphoryloxy) ethylamine (CGP 19835A, referred to as MTP-PE), and RIBI, which contains three components extracted from bacteria,
dipalmitoyl-sn-glycero hydroxylphosphoryloxy) ethylamine (CGP 19835A referred to as MTP-PE), and R1BI, 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
15 Immunogenic composition according to claim 14 further comprises auxiliary substances or excipients
16 Immunogenic composition according to claim 15 wherein auxiliary substance is selected from the group comprising wetting or emulsifying agents, pH buffering agents
17 Immunogenic composition according to claim 16 wherein excipient is selected from the group comprising water, normal saline dextrose glycerol, ethanol and combinations thereof
18 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 10
19 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
20 The anthrax recombinant fusion proteins substantially as herein described and illustrated by the examples
21 The process for the preparation of anthrax recombinant fusion proteins substantially as herein described and illustrated by the examples.