an ideal vaccine against anthrax should contain pa , lf , ef together , but at the same time it should be non - toxic and safe . purified recombinant proteins with defined composition may be used in the vaccine to minimize reactogenicity of the vaccine . further , these anthrax toxin proteins maybe rendered non - toxic by introducing mutations that affect the biological activity of the proteins without affecting their structure or immunogenicity . these non - toxic , mutant anthrax toxin proteins may be used together to create a safe , non - reactogenic and effective recombinant vaccine against anthrax . thus , the prime objective of this invention was to create a process for making a safe and effective , second - generation vaccine against anthrax comprising of non - toxic anthrax toxin proteins that have been produced by site - directed mutagenesis of the different functionally important domains of the toxin proteins . the inventors of this application have pcr amplified the genes for pa , lf and ef . they have cloned these genes in pqe30 expression vector ( gupta p ., et . al ., 1998 , infect . immun ., 66 , 862 - 865 ; gupta p ., et . al ., 1999 protein expr . purif . 16 , 369 - 376 ; kumar p ., et . al . 2001 , infect . immun ., 69 , 6532 - 6536 ). the vector contains t5 promoter and a 6x - histidine tag , which allows convenient purification of the recombinant proteins ( fig1 ). conditions for overexpression of the said genes using the above mentioned recombinant plasmids , from e . coli strains have been optimised by the inventors ( chauhan v ., et . al ., 2001 , biochem . biophys . res . commun ., 283 , 308 - 315 ). using the above mentioned recombinant plasmid , inventors of the present process , introduced mutations in the said genes to make the expressed recombinant proteins defective in their biological function , thereby rendering them non - toxic . the invention involves the expression and purification of the said mutant proteins from e . coli strains . it further involves full characterization of the purified mutant proteins to pinpoint the defect that renders them non - toxic . [ heading - 0068 ] mutations introduced in protective antigen as part of the invention 1 . mutations that make pa defective in binding to lf / ef . the inventors introduced series of mutations in the 1 st domain of pa . among the mutations introduced , the mutations at residues 202 , 203 , 205 , 207 and 205 + 226 + 236 were found to be defective in binding to lf . 2 . mutations that make pa defective in oligomerization . the authors of this invention introduced mutations in the 3 rd domain of pa . the mutation at the residues 552 , 574 , 552 + 554 , 562 + 574 566 + 574 , 552 + 554 + 562 + 566 + 574 resulted in mutant proteins that were defective in oligomerization . 3 . mutations that make pa translocation - defective . inventors have introduced mutations at residues 425 and 427 of loop 4 of domain 2 . these mutations impaired the translocation - ability of pa . 4 . mutations that make pa defective in insertion / translocation . authors have discovered that when mutations are introduced at the residues 346 , 352 and 346 + 350 + 352 in loop 3 of domain 2 , pa becomes biologically inactive . the mutant proteins were able to bind to the cell - surface receptors , get proteolytically activated to form oligomers and bind to lf . the biological inactivity of these mutant proteins may pertain to a defect in insertion / translocation . [ heading - 0073 ] mutations introduced in lethal factor as part of the invention mutations that make lf defective in binding to pa . the inventors of this process have introduced mutations in the 1 st domain of lf . they found that mutation at residues 148 , 149 , 151 , 153 , 187 , 190 and 187 + 188 + 189 + 190 impaired the binding of lf to pa . [ heading - 0075 ] mutations introduced in edema factor as part of the invention mutations that make ef defective in binding to pa . the inventors of this process have introduced series of mutations in the 1 st 250 residues of ef . it was found that mutation at residues 137 , 138 , 140 and 142 drastically impaired the binding of ef to pa . after the expression and purification of the mutant proteins the proteins were evaluated for their biological activity . inventors have found that the above - mentioned mutants of pa when added along with wild - type lf , were nontoxic to j774a . 1 cells . likewise mutants of lf when added along with wild - type pa were non - toxic to j774a . 1 cells . similarly , mutants of ef when added along with wild - type pa were unable to produce camp - toxicity in cho cells ( table 2 ). [ heading - 0079 ] the purified mutant protein was analyzed for their biological activity by assaying : ability of lethal toxin to kill macrophage cell lines like raw264 . 7 and j774a . 1 protective antigen , as the name suggests is a highly immunogenic protein . in fact it is a necessary component of the vaccine against anthrax . immunization with wild - type recombinant pa elicits high anti - pa titers and provides protection against anthrax lethal challenge in guinea pigs . it was further observed that mutant pa was as immunogenic as the wild - type pa and could easily substitute the wild - type pa in vaccine ( singh et . al . 1998 , infect . immun . 66 , 3447 - 3448 ). immunization studies also indicate a significant contribution of lf / ef to immunoprotection . on basis of these results the inventors have is developed a recombinant vaccine against anthrax , which comprises of mutants of all the three anthrax toxin components . the anthrax toxin based recombinant vaccine developed by the inventors has the following advantages : 1 . the process described here does not involve handling of b . anthracis cultures ( at any stage ). this process is therefore safe , cost - effective and does not require the sophisticated containment facilities . 2 . the vaccine developed by the inventors has well - defined composition and will therefore not have any batch to batch variation . 3 . the invention described here utilizes purified mutant anthrax toxin protein . as a result , this second - generation anthrax vaccine will not be reactogenic and will not cause any side - effects unlike the previous vaccine . 4 . additionally , this invention comprises of non - toxic mutant proteins , which when administered ( either alone or in combination ) do not cause any toxigenicity or pathogenicity as associated with the currently used vaccine . 5 . the invention described here is therefore safe and suitable for animal / human use . to introduce the desired mutations in the anthrax toxin proteins , complementary mutagenic primers were used ( refer table 1 ) to amplify the wild type anthrax toxin genes ( for pa or lf or ef ). high fidelity pfu dna polymerase was used for the pcr reaction . entire lengths of both the strands of the plasmid dna were amplified in a linear fashion during several rounds of thermal cycling , generating a mutant plasmid with staggered nicks on the opposite strands ( fig2 ). the amplification was checked by agarose gel electrophoresis of the pcr product . the product of the amplification was treated with dpni that specifically cleaves fully methylated g me6 atc sequences . the digestion reaction was carried out in 20 μl reaction volume with 10 ng of the amplified product , 2 μl of 10x dpni reaction buffer and 0 . 1 u of dpni . after dpni digestion , dpni resistant molecules that are rich in desired mutants were recovered by transformation of the dna into the appropriate e . coli strain . the mutations were confirmed by sequencing of the above constructs using perlcin elmer cycle dna sequencing kit . [ heading - 0098 ] expression and purification of the mutant anthrax toxin proteins the confined constructs were transformed into e . coli expression strains expressing t5 rna polymerase . transformed cells were grown in luria broth medium ( lb ) containing 100 μg / ml of ampicillin and 25 μg / ml of kanamycin , at 37 ° c ., to an od 600 of 0 . 8 . induction vas then done with 0 . 5 mm iptg and the incubation was continued at 37 ° c . for 3 to 4 hours . cells were then harvested by centrifugation at 6000 rpm for 10 minutes . the cells then lysed . the protein profile was analysed by sds - page and western blotting . the mutant pa proteins were purified using ni - nta metal - chelate affinity chromatography and other chromatographic techniques ( kumar p ., et . al . 2001 , infect . immun ., 69 , 6532 - 6536 ; gupta p ., et . al ., 1998 , infect . immun ., 66 , 862 - 865 ; gupta p ., et . al ., 1999 protein expr . purif . 16 , 369 - 376 ). the purified mutant proteins were analysed by sds - page and western blotting and were estimated using bradford &# 39 ; s method . for storage the purified proteins were dialysed against 50 mm hepes and stored as aliquotes at − 70 ° c . macrophage like cell line j774a . 1 was maintained in rpmi 1640 medium containing 10 % heat inactivated fcs , 25 mm hepes , 100u / ml penicillin and 200 μg / ml streptomycin in a humidified 5 % co 2 environment at 37 ° c . cho cells were maintained in emem medium containing 10 % heat inactivated fcs , 25 mm hepes , 100u / ml penicillin and 200 μg / ml streptomycin in a humidified 5 % co 2 environment at 37 ° c . to study the biological activity of the wild - type pa or its mutant proteins , varying concentrations of these proteins were added along with lf ( 1 μg / ml ) to j774a . 1 cells plated in 96 - wells plates . incubation was allowed for 3 hrs . at 37 ° c . and then cell viability ( bhatnagar et . al . 1989 , infect . immun ., 57 , 2107 - 2114 ) was determined using 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 2 , 5 - diphenyltetrazoliumbromide ( mtt ) dye ( bhatnagar r ., et . al ., 1999 , cell signal ., 11 , 111 - 116 ). mt - t dissolved in rpmi was added to each well at a final concentration of 0 . 5 mg / ml and incubated for another 45 min at 37 ° c . to allow uptake and oxidation of the dye by viable cells . the medium was replaced by 0 . 5 % ( w / v ) sodium dodecyl sulphate ( sds ), 25 mm hcl in 90 % isopropyl alcohol and the plate vortexed . the absorption was read at 540 nm using microplate reader ( biorad ). similarly , to study the biological activity of wild - type lf or its mutant proteins , varying concentrations of these proteins were added along with pa ( 1 μg / ml ) to j774a . 1 cells plated in 96 - wells plates . incubation was allowed for 3 hrs . at 37 ° c . and then cell viability as determined using mtt dye , as detailed above . to study the biological activity of wild - type ef or its mutant proteins , varying concentrations of these proteins were added along with pa ( 1 μg / ml ) to cho cells that were plated in 96 - wells plates . incubation was allowed for 3 hrs . at 37 ° c . and then the cells were microscopically examined for elongation . rise in intracellular camp levels of the cells upon toxin treatment was determined ( kumar p ., et . al ., 2001 , infect . immun ., 69 , 6532 - 6536 ) with camp eia kit of amersham pharmacia . further experiments were then done to understand how mutations were affecting the biological activity of the anthrax toxin mutant proteins . j774a . 1 cells were allowed to grow to confluence in 24 - well plates before incubating with 1 μg / ml of wild - type pa or its mutant protein at 4 ° c . for 2 hrs . the cells were then washed with cold rpmi , dissolved in sds lysis buffer and subjected to sds - page for electroblotting . the blot was developed with anti - pa antibodies to study the binding of wild - type pa or its mutant protein with the cell surface receptors [ heading - 0109 ] proteolytic cleavage of pa and mutant proteins in solution wild - type pa and its mutant proteins were tested for susceptibility to cleavage by trypsin . the proteins ( 1 . 0 mg / ml ) were incubated with 1 μg / ml of trypsin for 30 minutes at room temperature in 25 mm hepes , 1 mm cacl 2 , 0 . 5 mm edta ph 7 . 5 . the digestion reaction was stopped by adding pmsf to a concentration of 1 mm . for sds - page , the samples were boiled in sds sample buffer for 5 minutes and resolved on 12 % sds - page . j774a . 1 cells were washed twice with rpmi and then incubated with 1 μg / ml of wild - type pa or its mutant protein at 4 ° c . for 3 hrs . the cells were then washed with cold rpmi to remove unbound protein . the cells were further incubated with lf ( 1 . 0 μg / ml ) for 3 hours and then washed with cold rpmi to remove unbound lf . the cells were dissolved in sds lysis buffer and subjected to sds - page for electroblotting . the blot was developed with anti - lf antibodies to study the binding of the wild - type pa or its mutant protein to lf . pa upon proteolytic cleavage oligomerises to form heptamers . to study the ability of the wild - type pa and its mutant proteins to form oligomers , the proteins ( 1 mg / ml ) were digested with trypsin for 30 minutes at 25 ° c . the samples were brought to ph 5 . 0 by addition of 1m tris ph5 . 0 to a final concentration of 100 mm . and were boiled for 5 minutes in sds sample buffer ( 0 . 0625m tris - cl , 1 . 25 % sds , 2 . 5 %, β - mercaptoethanol and 5 % glycerol , ph6 . 8 ) before loading on a 3 - 12 % gradient gel . silver staining was done to detect the formation of oligomers . j774a . 1 cells were washed with cold rpmi and then incubated with 1 μg / ml of wild - type pa at 4 ° c . for 3 hrs . the cells were washed again with cold rpmi to remove unbound protein . wild - type lf / ef or the mutant proteins ( 1 . 0 μg / ml ) were then added and incubation was continued for 3 hours . the cells were then washed with cold rpmi to remove unbound lf / ef . later , the cells were dissolved in sds lysis buffer and subjected to sds - page for electroblotting . the blot was developed with anti - lf / ef antibodies to study the binding of lf / ef to cell - surface bound . residue change primers domain defect pa mutants : phe202 to alanine 5 ′ cttttcatgaatattagaaatccatgctgaaag i defective in binding to lethal factor leu203 to alanine cttttcatgaatattagaaatccatggtgaagcaaaagt i defective in binding to lethal factor pro205 to alanine cttttcatgaatattagaaatccatggtgaaagagcagttct i defective in binding to lethal factor ile207 to alanine tttggttaaccctttcttttcatgaatattagaaatccatggt i defective in gaaagaaaagttcttttatttttgacatcaaccgtatatcctt binding to lethal ctacctctaatgaatcagcgattcc factor pro205 + trp226 + to alanine cttttcatgaatattagaaatccatggtgaaagagcagttct and i defective in phe236 ggatttctaatattcatgaaaagaaaggattaaccaaatata binding to lethal aatcatctcctgaaaaagcgagcacggcttctgatccgtaca factor gtgatgccgaaaaggtt phe552 to alanine caagggaaagatatcaccgaatttgatgctaatttcgatc iii oligomerization defective ile574 to alanine gaattaaacgcgtctaacgcatatactg iii oligomerization defective phe552 + phe554 to alanine attttgagatgtttgttgatcggcattagcatcaaattc iii oligomerization defective ile562 + ile574 to alanine cagtatatgcgttagacgcgtttaattccgcttaactgattct iii oligomerization tggcattttgagatg defective leu566 + ile574 to alanine atcaggcagcggaattaaacgcgtctaacgcatatactg iii oligomerization defective phe552 + phe554 + to alanine cagtatatgcgttagacgcgtttaattccgctgcctgattctt iii oligomerization ile562 + leu566 + ggcattttgagatg and defective ile574 attttgagatgtttgttgatcggcattagcatcaaatt phe427 to alanine gtaattggagtagaactggcatcgtcttgtgc ii translocation defective asp425 residue gtaattggagtagaactgaaatcttgttcatttaatgcg ii translocation deleted defective phe427 residue gcacaagacgatagttctactccaattac ii translocation deleted defective trp346 to alanine cggtcgcaattgatcattcactatctctagcaggggaaagaa ii membrane ctgcggctgaaacaatg insertion / translocation defective leu352 to alanine cggtcgcaattgatcattcactatctctagcaggggaaagaa ii membrane cttgggctgaaacaatgggtgcaaataccgctgat insertion / translocation defective trp346 , met350 to alanine cggtcgcaattgatcattcactatctctagcaggggaaagaa ii membrane and leu352 ctgcggctgaaacagcgggtgcaaataccgctgat insertion / translocation defective lf mutants : tyr148 to alanine gtagaaggtaccgaaaaggcactgaacgttgcttat i defective in binding to protective antigen tyr149 to alanine gtagaaggtaccgaaaaggcactgaacgtttatgctgaa i defective in binding to protective antigen ile151 to alanine gtagaaggtaccgaaaaggcactgaacgtttatgaagcaggt i defective in binding to protective antigen lys153 to alanine gtagaaggtaccgaaaaggcactgaacgtttatgaaataggt i defective in gcaata binding to protective antigen asp187 to alanine tgtgggatgttccttaagctgattagtaaataaaagagcttgt i defective in tcatctga binding to protective antigen phe190 to alanine tgtgggatgttccttaagctgattagtagctaaaagatcttg i defective in binding to protective antigen asp187 , leu188 , to alanine tgtgggatgttccttaagctgattagtagctggagcagcttgt i defective in leu189 , phe190 tcatctga binding to protective antigen ef mutants : tyr137 to alanine ccttacttatgatatcaagagaaatcccc ttt cc aat ttc defective in binding agc ata tac ttc ttt act ttg ttc ac to protective antigen tyr138 to alanine ccttacttatgatatcaagagaaatcccc ttt cc aat ttc defective in binding ata agctac ttc ttt act ttg ttc ac to protective antigen ile140 to alanine ccttacttatgatatcaagagaaatcccc ttt ccagcttc defective in binding ata atatac ttc ttt act ttg ttc ac to protective antigen lys142 to alanine ccttacttatgatatcaagagaaatcccc gct cc aat ttc defective in binding ata atatac ttc ttt act ttg ttc ac to protective antigen
anthrax toxin , comprising of protective antigen , lethal factor and edema factor is a major virulent factor of b . anthracis . protective antigen , pa is the main component of all the vaccines against anthrax . the protective efficacy of pa is greatly increased if small quantities of lf of ef are incorporated into the vaccines . an ideal vaccine against anthrax should contain pa , lf and ef together , but this combination would be toxic . therefore , the biologically inactive mutant preparations of pa , lf and ef may be used together for better immunoprotection . the present invention describes the method for generation of recombinant vaccine against anthrax , comprising of non - toxic , mutant anthrax toxin proteins . the procedure involves site - directed mutagenesis of the native genes of the toxin proteins , the expression and purification of the mutant proteins and finally characterization of these proteins .