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Centre for Biotechnology Jawaharlal Nehru University JNU, New Delhi

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CONTAINS FURIN CLEAVAGE SITE WHICH DEFINES TWO SUB-DOMAINS: PA 20 FRAGMENT ... 3-5 g/L equivalent to ~1million shots compared to currently available vaccines. ... – PowerPoint PPT presentation

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Title: Centre for Biotechnology Jawaharlal Nehru University JNU, New Delhi


1

Centre for Biotechnology Jawaharlal Nehru
University (JNU), New Delhi
2
RECOMBINANT VACCINE AGAINST
ANTHRAX
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Cutaneous Anthrax
  • Most common infection
  • (gt95 )
  • Spores enter through
  • abrasions in skin.
  • Papule - vesicle - ulcer
  • Up to 20 case fatality
  • rate if untreated
  • Mortality with treatment
  • lt1

10
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.

11
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.

12
 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.

13
VACCINES AGAINST
ANTHRAX TILL DATE, VACCINE BASED ON LIVE
STERNES 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.
14
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
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MECHANISM OF TOXIN ENTRY
24
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.
25
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.

26
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
27
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. Aug16 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
    4283 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. Feb66 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.

28
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
29
PURIFICATON OF PA
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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 LOWRYS METHOD.
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BINDING OF RECOMBINANT PA TO LF
PA from B. anthracis Recombinant PA LF from B.
anthracis B. anthracis PALF Recombinant PALF
34
BINDING OF LF TO RECEPTOR BOUND PA
35
MACROPHAGE LYSIS ASSAY
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EXPRESSION, OPTIMIZATION AND PURIFICATION
OF PA, LF and EF
38
OVERPRODUCTION OF rPA
METHOD OF FEEDING pH-DO-stat FEED
25xcomplex media (LB 25 w/v
glycerol) INCREASE IN BIOMASS
OD600gt100units 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.
39
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.

40
  • 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.
  • PreExposure 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.

41
Immunogenicity of Anthrax toxin components
  • PA Good Immunogen
  • PALFEF 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.

42
Generation of non toxic mutants of PA, LF, EF
43
PA STRUCTURE FUNCTIONALLY IMPORTANT RESIDUES
44
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 Aug70(8)4477-84
45
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. 281186-92
46
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 21287(2)542-9.
47
SIMILARITY BETWEEN EF AND LF SEQUENCES
QUERY THE SEQUENCE OF EF FIRST DOMAIN SUBJECT
THE SEQUENCE OF LF FIRST DOMAIN
48
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.
49
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 12280(1)158-63.
50
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. Oct69(10)6532-6
51
THERMOSTABILIZATION OF PA
  • COSOLVENT MEDIATED MgSO4 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 150(2-3)235-42.
52
Gln277Ala and Phe554Ala increase thermal
stability.
Activity of PA mutants retained after 48 hrs of
incubation at 37oC in comparison with native PA.

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
6296(5)1058-2
53
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 6299(3)345-51.
  • Transgenic Tomatoes are in early stage of
    development.

54
Identification of the transgene in genomic DNA by
PCR amplification
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.
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  • Molecular analysis
  • Protective antigen expression determined using
    immunoblot analysis.
  • Functional efficacy established using
    cytotoxicity assay.

56
Immunoblot detection of protective antigen with
antisera raised against purified recombinant PA



78 kDa


57
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
58
Tomato Callus Differentiating On Selection Medium
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Putative Transgenic Tomato Plants at Bottle Stage
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Putative Tomato Transgenic Plants Transferred To
Pots
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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.

62
Thank You
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