Most common infection (>95% ) Spores enter through abrasions in skin. Papule - vesicle - ulcer Up to 20% case fatality rate if untreated Mortality with treatment <1% Cutaneous Anthrax
Rare form of infection. Ingestion of insufficiently cooked, contaminated meat. Abdominal pain and fever. Fatal bacterium and toxemia then ensue. Mortality exceeds 50% if untreated. GASTROENTESTINAL ANTHRAX
Inhaled spores phagocytosed by macrophages transported To regional lymphnodes. Spores germination followed by toxin release. Extensive necrotic haemorrhage. Death from sepsis and shock. INHALATION ANTHRAX
Bacillus anthracis as a Biowarfare Agent Bacillus anthracis as a Biowarfare Agent Possible vehicle of mass death Weapon of mass destruction (WMD) Destructive capability of weaponized anthrax is equivalent to that of a nuclear bomb (Wein et.al. 2003) Poor Diagnosis [Webb et.al.]
VACCINES AGAINST ANTHRAX TILL DATE, VACCINE BASED ON LIVE STERNE’S STRAIN IS THE MOST POPULAR VETERINARY VACCINE AGAINST ANTHRAX WORLDWIDE. RUSSIA USES LIVE SPORE VACCINE FOR HUMANS IN UK CURRENTLY AVAILABLE HUMAN VACCINE CONSISTS OF ALUM PRECIPITATED CELL FREE FILTRATE OF STERNE STRAIN. IN US THE VACCINE IS ALUMINIUM HYDROXIDE ADSORBED CELL FREE FILTRATE OF A NON-CAPSULATING STRAIN OF B. anthracis. HOWEVER, CURRENTLY AVAILABLE VACCINES HAVE CERTAIN DEGREE (5-10%) OF RESIDUAL VIRULENCE AS THE BACTERIUM PRODUCES BOTH LF AND EF COMPONENTS.
Anthrax Vaccine Side Effects Soreness, redness at the site of shot given Headache Muscle ache Fatigue Nausea Chills and Fever Allergic reactions Need for development of improved anthrax vaccine devoid of side effects
pXO1 pXO2 Bacillus anthracis *pXO1 :181 kb. Codes for Protective Antigen (PA), Lethal Factor (LF) & Edema Factor (EF). *pXO2: 96 kb. Codes forPoly D-Glutamic acid capsule. VIRULENCE DETERMINANTS Bacillus anthracis (under microscope ) * Extra chromosomal genetic material : plasmid. Anthrax spores are highly stable under adverse conditions. ANTHRAX : Primarily a disease of animals, humans are accidental host. PA: MAIN IMMUNOGEN, PROVIDES PROTECTIVE IMMUNITY AGAINST ANTHRAX. MAIN COMPONENT OF ALL ANTHRAX VACCINES
THE PA 83 MONOMER DOMAIN 1: (RESIDUES 1-258) CONTAINS FURIN CLEAVAGE SITE WHICH DEFINES TWO SUB-DOMAINS: PA 20 FRAGMENT (RESIDUES 1-167) AND DOMAIN 1’ (RESIDUES 168-258). DOMAIN 2: (RESIDUES 259-487) PLAYS A ROLE IN MEMBRANE INSERTIONAND TRANSLOCATION. DOMAIN 3: (RESIDUES488-595) PLAYS A ROLE IN OLIGOMERISATION. DOMAIN 4: (RESIDUES 596-735) RECEPTOR BINDING DOMAIN.
THE PA63 HEPTAMER LOSS OF PA20 LEADS TO HEPTAMER FORMATION BY PA63. HEPTAMER IS WATER SOLUBLE AT NEUTRAL OR BASIC pH. HEPTAMER INSERTS INTO MEMBRANE AT ACIDIC pH FORMING CATION- SELECTIVE CHANNELS IN BOTH ARTIFICIAL LIPID BILAYERS AND CELLS.
LETHAL FACTOR DOMAIN I : INVOLVED IN PA BINDING DOMAIN II : RESEMBLES ADP RIBOSYLATING TOXIN OF B. cereus, AUGMENTS SUBSTRATE RECOGNITION DOMAIN III : ALONGWITH DOMAIN 2 AND 4 HELPS IN HOLDING THE 16 RESIDUE LONG N-TERMINAL TAIL OF MAPKK BEFORE CLEAVAGE. POSSIBLY INVOLVED IN MEMBRANE INSERTION. DOMAIN IV : Zn CONTAINING CATALYTIC SITE
CLONING, EXPRESSION AND PURIFICATION OF PA, LF AND EF FROM E. coli: References: 1.Gupta P, Waheed SM, Bhatnagar R. (1999) Expression and purification of the recombinant protective antigen of Bacillus anthracis. Protein Expr Purif. Aug;16 : 369-76. 2. Chauhan V, Singh A, Waheed SM, Singh S, Bhatnagar R. (2001) Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli. Biochem Biophys Res Commun. May 4;283 : 308-15 3. Gupta P, Batra S, Chopra AP, Singh Y, Bhatnagar R. (1998) Expression and purification of the recombinant lethal factor of Bacillus anthracis. Infect Immun. Feb;66 : 862-5. 4.Kumar P, Ahuja N, Bhatnagar R. (2001)Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen. Infect Immun. Oct; 69 : 6532-6. CLONING IN EXPRESSION VECTORS pXO1 184kb PCR pExp PCR Amplified Gene
LOCALIZATION OF E.coli EXPRESSED PA 220 kDa 97 kDa 66 kDa 46 kDa 30 kDa 21.5 kDa 14.3 kDa Only cells Cells with pQE30 Uninduced Cells with pMW Induced Cells with pMW Periplasmic fraction Cytosolic fraction Inclusion body fraction Standard PA Marker
PURIFICATON OF PA 220kDa 116kDa 97kDa 66kDa 45kDa E. coli cells expressing PA PA in the inclusion bodies Proteins after affinity purification PA after FPLC PA purified from B. anthracis Molecular weight standards
BINDING OF PA TO CELL SURFACE RECEPTORS A a J774A.1 CELLS WERE INCUBATED WITH 1µg OF RADIOIODINATED PA (nPA AND rPA) FOR 3 HRS. AT 4 C. b PROTEIN CONTENT OF THE CELLS PER WELL WAS 0.95 ± 0.05 mg AS DETERMINED BY LOWRY’S METHOD.
BINDING OF RECOMBINANT PA TO LF PA-LF complex LF (1 µg) was incubated with trypsin – nicked PA (1µg) and samples were analyzed on a 4.5% native PAGE. The gel was stained with Coomassie-blue. PA63 LF PA20 PA from B. anthracis Recombinant PA LF from B. anthracis B. anthracis PA+LF Recombinant PA+LF
BINDING OF LF TO RECEPTOR BOUND PA Protein PA / cell protein b (ng/mg) LF alone 0.18 0.02 nPA+LF 3.70 0.12 rPA+LF 3.55 0.15
MACROPHAGE LYSIS ASSAY BIOLOGICAL ACTIVITY OF PA PURIFIED FROM Bacillus anthracis AND E. coli DH5 CELLS. J774A.1 CELLS WERE INCUBATED WITH VARYING CONCENTRATIONS OF PA ALONE OR IN COMBINATION WITH LF (1 g/ml) FOR 3HRS. AT 37 C. PA FROM Bacillus anthracis, RECOMBINANT PA PA FROM Bacillus anthracis WITH LF, RECOMBINANT PA WITH LF. NATIVE PA ALONE RECOMBINANT PA ALONE NATIVE PA +LF RECOMBINANT PA + LF 0 20 40 60 80 100 120 0.0010.010.11 PA( g/ml) %age viability
Protective efficacy of the rPA against B. anthracis S. No. Group Conc. used Survivors/ Total % survival Relative efficacy 1. Unvaccinated control (challenged) PBS 00/18 0 -- 2. Vaccinated control (unchallenged) 50 g 18/18 100 -- 3. Protective Antigenen from B. anthracis 5 g 12/18 66 -- 1 g 6/18 33 -- 4. Recombinant protective antigen 50 g 12/18 66 100 10 g 12/18 66 100 5 g 12/18 66 100 1 g 6/18 33 50 Relative efficacy is defined as the percentage of rPA immunized animals survivingg after virulent spore challenge w.r.t., the Native PA from B. anthracis. Concentration of the anthrax spore vaccine used is 10 x 10 7 spores per ml.
EXPRESSION, OPTIMIZATION AND PURIFICATION OF PA, LF and EF Ni-NTA AFFINITY CHROMATOGRAPHY & GEL FILTRATION GROWTH CONDITION OPTIMIZATION FOLLOWED BY HIGH DENSITY FED BATCH CULTURE E.coli with expression plasmid construct PAG PURIFIED PA Same way LF and EF were purified
METHOD OF FEEDING: pH-DO-stat FEED: 25xcomplex media (LB + 25% w/v glycerol) INCREASE IN BIOMASS: OD 600 >100units WET CELL WEIGHT: 195 grams/litre DRY CELL WEIGHT : 52 grams/litre PA: 20-30% of total cell protein PURIFICATION: Ni-NTA affinity : 90-95% pure chromatography and Gel Filtration Yield: 3-5 g/L equivalent to ~1million shots compared to currently available vaccines. OVERPRODUCTION OF rPA
Technology transferred to Panacea Biotec Ltd. A Pharmaceutical Company already producing vaccines for Polio and Hepatitis B. Scientists from Panacea Biotech Ltd. have been given extensive training in JNU for making recombinant vaccine. JNU scientists have gone and helped Panacea Biotech Ltd to produce 5 batches of recombinant vaccine in GMP facility of Panacea Biotech Ltd. Technology transfer of PA production
Panacea Biotech Ltd., scientists have produced 5 batches of rPA for toxicity and efficacy studies under GMP. Toxicity studies on mice, and rats at Rallis India Ltd. Banglore have shown that recombinant anthrax vaccine (rPA) is not toxic. Pre–Exposure studies on immunogenecity and efficacy have been completed. Phase-I/II, open labeled, randomized, placebo controlled, ascending dose trial to evaluate the safety and immunogenecity of recombinant protective antigen (rPA) anthrax vaccine have been initiated in Oct. 2004 and likely to be completed by Dec. 2005.
Immunogenicity of Anthrax toxin components PA :Good Immunogen PA +LF+EF : Better Immunogen. LF and EF cannot be added in the vaccine due to associated toxicity. Mutants defective in any one of the steps of intoxication may be added in vaccine with PA without causing toxicity.
Generation of non toxic mutants of PA, LF, EF Long PCR with Pfu turbo using adjacent, partially overlapping oligonucleotides encoding the desired mutation at the 5’ end of the primer. M M M M pExp gene PCR amplified nicked plasmid DpnI Digetsion (degrades methylated template DNA while spares unmethylated PCR amplified product) Transformation into competent E. coli cells. : Mutation point M : Methyl group (CH 3 ) M M M M Methylated Plasmid template gene Mutants confirmed by sequencing
PA STRUCTURE: FUNCTIONALLY IMPORTANT RESIDUES DOMAIN 1: BINDING TO LF/EF DOMAIN 3: PA OLIGOMERIZATION DOMAIN2: MEMBRANE INSERTION AND TRANSLOCATION OF LF/EF
DOMAIN 1b RESIDUES OF PROTECTIVE ANTIGEN INVOLVED IN BINDING TO LF/EF Ref: Chauhan V, Bhatnagar R. Identification of amino acid residues of anthrax protective antigen involved in binding with lethal factor. Infect Immun. 2002 Aug;70(8):4477-84
Residues of PA Involved In Membrane Insertion And Translocation of LF/EF DOMAIN 2 Ref:Batra S, Gupta P, Chauhan V, Singh A, Bhatnagar R. (2001) Trp 346 and Leu 352 residues in protective antigen are required for the expression of anthrax lethal toxin activity. Biochem Biophys Res Commun. 281:186-92
RESIDUES OF PROTECTIVE ANTIGEN NEEDED FOR OLIGOMERIZATION DOMAIN 3 Ref: Ahuja N, Kumar P, Bhatnagar R.Hydrophobic residues Phe552, Phe554, Ile562,Leu566, and Ile574 are required for oligomerization of anthrax protective antigen.Biochem Biophys Res Commun. 2001 Sep 21;287(2):542-9.
SIMILARITY BETWEEN EF AND LF SEQUENCES QUERY: THE SEQUENCE OF EF FIRST DOMAIN SUBJECT: THE SEQUENCE OF LF FIRST DOMAIN Score = 123 bits (309), Expect = 9e-27 Identities = 77/225 (34%), Positives = 123/225 (54%), Gaps = 3/225 (1%) Query: 63 INNLVKTEFTNETLDKIQQTQDLLKKIPKDVLEIYSELGGEIYFTDIDLVEHKELQDLSE 122 + ++VK E E K + + LL+K+P DVLE+Y +GG+IY D D+ +H L+ LSE Sbjct: 73 MKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSE 132 Query: 123 EEKNSMNSRGEKVPFASRFVFEKKRETPKLII-NIKDYAINSEQSKEVYYEIGKGISLDI 181 ++K + G+ +V+ K+ P L+I + +DY N+E++ +S DI Sbjct: 133 DKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDI 192 Query: 182 ISKDKSLDPEFLNLIKXXXXXXXXXXXXFSQKFKEKLELNNKSIDINFIKENLTEFQHAF 241 +SK +FL+++ F +L+ + + F+++N E Q F Sbjct: 193 LSKINQPYQKFLDVLNTIKNASDSDGQDL--LFTNQLKEHPTDFSVEFLEQNSNEVQEVF 250 Query: 242 SLAFSYYFAPDHRTVLELYAPDMFEYMNKLEKGGFEKISESLKKE 286 + AF+YY P HR VL+LYAP+ F YM+K + E LK + Sbjct: 251 AKAFAYYIEPQHRDVLQLYAPEAFNYMDKFNEQEINLSLEELKDQ 295 VYYEIGK
The amino terminal region of LF and EF is required in binding to PA. Sequence analysis reveals that 1 to 300 amino acids have several homologous stretches. Maximum homology was observed at a stretch of seven residues (Val-Tyr-Tyr-Glu-Ile-Gly-Lys ). Therefore, in order to determine to the role of these residues each amino acid of this stretch was substituted with alanine. HOMOLOGOUS STRETCH OF LF/EF
LF Structure: Residues Needed For Binding To PA References: 1. Singh A, Chauhan V, Sodhi A, Bhatnagar R. Asp 187 and Phe 190 residues in lethal factor are required for the expression of anthrax lethal toxin activity. FEMS Microbiol Lett. 2002 Jul 2; 212(2):183-6. 2. Gupta P, Singh A, Chauhan V, Bhatnagar R. Involvement of residues 147VYYEIGK153 in binding of lethal factor to protective antigen of Bacillus anthracis. Biochem Biophys Res Commun. 2001 Jan 12;280(1):158-63. Mutants of Domain 1 defective in binding to PA: Tyr 148 Tyr 149 Glu 150 Il e 151 Lys 153 Asp187 Phe190 Grey residues: involved in binding Yellow and green: dispensable ones I PAbinding II VIP2 like III Helix bundle MAPKK-2 IV Catalytic centre Val 147 Tyr 148 Tyr 149 Glu 150 Ile 151 Asp 187 Gly 152Lys 153 Leu 188 Leu 189 Phe190 I PA binding
PA BINDING DEFECTIVE MUTANTS OF EF Domain 1 Domain 2 Domain 3 PA binding domain Linker Calmodulin Ca 2+ Ref: Kumar P, Ahuja N, Bhatnagar R. 2001. Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen. Infect Immun. Oct;69(10):6532-6 MUTATED RESIDUES BINDING/TOXICITY Val136 + + / + + Tyr137 –– –– / –– –– Tyr138 –– –– / –– –– Glu139 + + / + + Ile140 –– –– / –– –– Gly141 + + / + + Lys142 –– –– / –– ––
THERMOSTABILIZATION OF PA COSOLVENT MEDIATED: MgSO 4 and Trehalose are the best among the studied cosolvents. Ref : Radha C, Salotra P, Bhat R, Bhatnagar R. Thermostabilization of protective antigen- the binding component of anthrax lethal toxin. J.Biotechnol. 1996, Oct 1;50(2-3):235-42. Additive Molarity PA activity retained after 48 hrs of incubation at 37 o C None - ~5 Sorbitol 1.0 ~10 Xylitol 2.0 ~18 Trehalose 1.5 ~74 Sodium Citrate 1.0 ~67 MgSO 4 3.0 ~83
Gln277Ala and Phe554Ala increase thermal stability. Ref: Singh S, Ahuja N, Chauhan V, Rajasekaran E, Mohsin Waheed S, Bhat R, Bhatnagar R. Gln277 and Phe554 residues are involved in thermal inactivation of protective antigen of Bacillus anthracis. Biochem Biophys Res Commun. 2002 Sep 6;296(5):1058-2 Activity of PA mutants retained after 48 hrs of incubation at 37 o C in comparison with native PA. S.NO. MUTANT RESIDUAL ACTIVITY AFTER 48 HRS. OF INCUBATION AT 37 O C 1. Gln277Ala ~45% 2. Phe554Ala ~90% 3. Native PA 0%
Transgenic plants as a source of Edible vaccine against Anthrax Cloned and expressed in Tobacco plants. Ref: Aziz MA, Singh S, Anand Kumar P, Bhatnagar R. Expression of protective antigen in transgenic plants: a step towards edible vaccine against anthrax. Biochem Biophys Res Commun. 2002 Dec 6;299(3):345-51. Transgenic Tomatoes are in early stage of development.
Identification of the transgene in genomic DNA by PCR amplification 1 2 3 4 5 6 7 8 9 10 11 1.5kb Genomic DNA extracted from tobacco leaves was used as template. Primers flanking 1.5 Kb region within the PA gene were used to carry out the reaction.
Molecular analysis Protective antigen expression determined using immunoblot analysis. Functional efficacy established using cytotoxicity assay.
Immunoblot detection of protective antigen with antisera raised against purified recombinant PA 78 kDa 209 kDa 120 kDa 47.7 kDa 1 2 3 4 5 Lane 1-3: PA expressed in transgenic plants Lane 4 : rPA, Lane 5: Negative control i.e. Total soluble protein extracted from untransformed tobacco plants
Functional assay of plant expressed PA Total soluble protein from different plant samples was incubated along with 1ug/ml LF. The percentage killing of RAW264.7 cells ranged between 26% to 98% owing to different expression levels in different plants
Tomato Callus Differentiating On Selection Medium
Putative Transgenic Tomato Plants at Bottle Stage
Putative Tomato Transgenic Plants Transferred To Pots
CONCLUSIONS We have PCR amplified PA gene. Overexpressed in suitable Vectors. Bioprocess optimized upto near industrial production. Recombinant PA was found to be biologically & fundamentally identical to native antigen. Thermostabilization of PA has been achieved. Technology or producing recombinant vaccine transferred to M/s. Panacea Biotec Ltd. Non-toxic variant of PA, LF, & EF generated for next generation vaccine. PA gene was expressed in Tobacco & Tomatoes. Clinical trials are being conducted.