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Centre for Biotechnology Jawaharlal Nehru University (JNU), New Delhi

Centre for Biotechnology Jawaharlal Nehru University (JNU), New Delhi. RECOMBINANT VACCINE AGAINST ANTHRAX. Geographical Distribution of Anthrax. Epidemic Endemic Sporadic Probably Free Free Unknown. Cutaneous Anthrax. Most common infection (>95% ) Spores enter through

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Centre for Biotechnology Jawaharlal Nehru University (JNU), New Delhi

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  1. Centre for Biotechnology Jawaharlal Nehru University (JNU), New Delhi

  2. RECOMBINANT VACCINE AGAINST ANTHRAX

  3. Geographical Distribution of Anthrax Epidemic Endemic Sporadic Probably Free Free Unknown

  4. Cutaneous Anthrax • 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%

  5. GASTROENTESTINAL 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.

  6. INHALATION ANTHRAX • Inhaled spores phagocytosed by macrophages transported • To regional lymphnodes. • Spores germination followed by toxin release. • Extensive necrotic haemorrhage. • Death from sepsis and shock.

  7. 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.]

  8. 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.

  9. 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

  10. ANTHRAX : Primarily a disease of animals, humans are accidental host. * pXO1 :181 kb. Codes for Bacillus anthracis Protective Antigen (PA), Lethal VIRULENCE Factor (LF) & Edema Factor (EF). DETERMINANTS pXO2 pXO1 * pXO2: 96 kb. Codes for Poly D- Glutamic acid capsule. Bacillus anthracis (under microscope) * Extra chromosomal genetic material : plasmid Anthrax spores are highly stable under adverse conditions. . PA: MAIN IMMUNOGEN, PROVIDES PROTECTIVE IMMUNITY AGAINST ANTHRAX. MAIN COMPONENT OF ALL ANTHRAX VACCINES

  11. MECHANISM OF TOXIN ENTRY

  12. 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.

  13. 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.

  14. 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 CONTAININGCATALYTIC SITE

  15. CLONING, EXPRESSION AND PURIFICATION OF PA, LF AND EF FROM E. coli: CLONING IN EXPRESSION VECTORS PCR pExp pXO1 184kb PCR Amplified Gene • References: • 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.

  16. 220 kDa 97 kDa 66 kDa 46 kDa 30 kDa 21.5 kDa 14.3 kDa LOCALIZATION OF E.coli EXPRESSED PA Only cells Cells with pQE30 Uninduced Cells with pMW Induced Cells with pMW Periplasmic fraction Cytosolic fraction Inclusion body fraction Standard PA Marker

  17. 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 220kDa 116kDa 97kDa 66kDa 45kDa PURIFICATON OF PA

  18. BINDING OF PA TO CELL SURFACE RECEPTORSA aJ774A.1 CELLS WERE INCUBATED WITH 1µg OFRADIOIODINATED PA (nPA AND rPA) FOR 3 HRS. AT 4C. b PROTEIN CONTENTOF THE CELLS PER WELL WAS 0.95 ± 0.05 mg AS DETERMINED BY LOWRY’S METHOD.

  19. PA-LF complex PA63 LF PA20 LF (1µg) was incubated with trypsin – nicked PA (1µg) and samples were analyzed on a 4.5% native PAGE. The gel was stained with Coomassie-blue. BINDING OF RECOMBINANT PA TO LF PA from B. anthracis Recombinant PA LF from B. anthracis B. anthracis PA+LF Recombinant PA+LF

  20. 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 BINDING OF LF TO RECEPTOR BOUND PA

  21. 120 NATIVE PA ALONE 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. 100 RECOMBINANT PA ALONE 80 %age viability 60 NATIVE PA +LF 40 20 RECOMBINANT PA + LF 0 0.001 0.01 0.1 1 PA( g/ml) m MACROPHAGE LYSIS ASSAY

  22. Protective efficacy of the rPA against B. anthracis S. Group Conc. Survivors/ % Relative No. used Total survival efficacy 1. Unvaccinated control PBS 00/18 0 -- (challenged) m 2. Vaccinated control 18/18 100 -- 50 g (unchallenged) m 3. Protective Antigen en 12/18 66 -- 5 g from B. anthracis m 6/18 33 -- 1 g m 4. Recombinant 12/18 66 100 50 g protective antigen m 12/18 66 100 10 g m 12/18 66 100 5 g m 6/18 33 50 1 g Relative efficacy is defined as the percentage of rPA immunized animals Ø surviving g after virulent spore challenge w.r.t., the Native PA from . B. anthracis 7 Concentration of the anthrax spore vaccine used is 10 x 10 spores per ml. Ø

  23. PAG E. coli with expression plasmid construct GROWTH CONDITION OPTIMIZATION FOLLOWED BY HIGH DENSITY FED BATCH CULTURE Ni-NTA AFFINITY CHROMATOGRAPHY & GEL FILTRATION PURIFIED PA Same way LF and EF were purified EXPRESSION, OPTIMIZATION AND PURIFICATION OF PA, LF and EF

  24. OVERPRODUCTION OF rPA METHOD OF FEEDING : pH-DO-stat FEED : 25xcomplex media (LB + 25% w/v glycerol) INCREASE IN BIOMASS : OD600>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.

  25. Technology transfer of PA production • 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.

  26. 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.

  27. 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.

  28. gene M M pExp M M Long PCR with Pfu turbo using adjacent, partially overlapp ing oligonucleotides encoding the desired mutation at the 5’ end of the primer. gene M PCR M amplified Methylated nicked Plasmid plasmid template M M DpnI Digetsion (degrades methylated : Mutation point template DNA while spares unmethylated M : Methyl group (CH ) 3 PCR amplified product) Transformation into competent cells. E. coli Mutants confirmed by sequencing Generation of non toxic mutants of PA, LF, EF

  29. DOMAIN 1: BINDING TO LF/EF DOMAIN 3: PA OLIGOMERIZATION DOMAIN2: MEMBRANE INSERTION AND TRANSLOCATION OF LF/EF PA STRUCTURE: FUNCTIONALLY IMPORTANT RESIDUES

  30. RESIDUES OF PROTECTIVE ANTIGEN INVOLVED IN BINDING TO LF/EF DOMAIN 1b 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

  31. 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

  32. 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.

  33. 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 VYYEIGK ++K + G+ +V+ K+ P L+I + +DY N+E++ +S DI Sbjct: 133 DKKKIKDIYGKDALLHEH YVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDI 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 SIMILARITY BETWEEN EF AND LF SEQUENCES QUERY: THE SEQUENCE OF EF FIRST DOMAIN SUBJECT: THE SEQUENCE OF LF FIRST DOMAIN

  34. HOMOLOGOUS STRETCH OF LF/EF 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.

  35. I Val 147 I PA binding Glu 150 PA binding Tyr 148 Ile 151 Tyr 149 Leu 188 Lys 153 Gly 152 II Asp 187 VIP2 like Leu 189 Phe190 Mutants of Domain 1 defective in binding to PA: Tyr 148 Tyr 149 Glu 150 Il e 151 Lys 153 Asp187 Phe190 III Helix bundle Grey residues: involved in binding Yellow and green: dispensable ones IV MAPKK-2 Catalytic centre 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.

  36. Domain 3 Domain 2 Linker MUTATED RESIDUES BINDING/TOXICITY Val136 + + / + + Ca 2+ Tyr137 –– –– / –– –– Domain 1 Tyr138 –– –– / –– –– Calmodulin Glu139 + + / + + PA binding domain Ile140 –– –– / –– –– Gly141 + + / + + Lys142 –– –– / –– –– PA BINDING DEFECTIVE MUTANTS OF EF 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

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