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

RECOMBINANT RECOMBINANT VACCINEVACCINE

AGAINST ANTHRAX AGAINST ANTHRAX

ANTHRAXANTHRAX

Geographical Distribution of Anthrax

EpidemicEndemicSporadicProbably FreeFreeUnknown

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 anthracisBacillus anthracis as a as a

Biowarfare AgentBiowarfare 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 forProtective Antigen (PA), LethalFactor (LF) & Edema Factor (EF).

*pXO2: 96 kb. Codes for PolyD-Glutamic acid capsule.

VIRULENCEDETERMINANTS

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

ANTHRAX TOXIN COMPONENTS

Edema in Skin(Rabbits, Guinea Pigs)Increased Cyclic AMP

Lethality(Rats, Mice, etc.)

Macrophage Lysis

Lethal Factor(LF)

Mw 90 kDaZn Metallo-

protease

Edema Factor(EF)

Mw 89 kDaAdenylate

cyclase

Protective Antigen

(PA)Mw 83 kDaCell binding

moiety

MECHANISM OF TOXIN ENTRY

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

pXO1184kb

PCRpExp

PCR Amplified Gene

LOCALIZATION OF E.coli EXPRESSED PA

220 kDa

97 kDa

66 kDa

46 kDa

30 kDa

21.5 kDa14.3 kDa

On

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ells

Cel

ls w

ith

pQ

E30

Un

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d C

ells

wi t

h p

MW

Ind

uce

d C

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MW

Per

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n

Cyt

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ract

ion

Incl

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ody

frac

tion

Sta

nd

ard

PA

Mar

ker

PURIFICATON OF PA

220kDa116kDa 97kDa 66kDa 45kDa

E. c

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A

PA

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Pro

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Purification of PA from E.coli

Fractions Volume (ml)

Protein (mg/ml)

Activity (EC50)

a Purification

(fold)b

Cell lysatec 50 115.84 75.580 1

Affinity

purification

10 0.65 0.040 1890

FPLC 2 2.0 0.025 3023

EC50 is defined as the concentration of PA (μg/ml) along with LF

(1μg/ml) required killing 50% of the J774A.1 cells.

Purification fold was determined by dividing EC50 for cell lysate

with EC50 for fractions obtained from different columns.

Cell lysate prepared from 2 litres of culture.

BINDING OF PA TO CELL SURFACE RECEPTORS A

Protein CPM PA(ng) PA/cell protein b

(ng/mg)

nPA 82679 ±1169 6.89 7.25

rPA 79986 ±1388 7.34 7.72

a J774A.1 CELLS WERE INCUBATED WITH 1µg OF RADIOIODINATED PA (nPA AND rPA) FOR 3 HRS. AT 4C.

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 (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.

PA63LFPA20

PA

fro

m B

. an

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cis

Rec

omb

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A

LF

fro

m B

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B. a

nth

raci

s P

A+

LF

Rec

omb

inan

t P

A+

LF

BINDING OF LF TO RECEPTOR BOUND PA

J774A.1 CELLS WERE INCUBATED WITH 1µg OF RADIOIODINATED LF (nLF AND

rLF) ALONG WITH PA 1µg/ML FOR 12 HRS. AT 4 C. THE CELLS WERE WASHED

WITH HANK’S BUFFERED SALINE SOLUTION AND SOLUBLIZED IN 100 mM NaOH.

RADIOACTIVITY WAS COUNTED IN A GAMMA COUNTER.

PROTEIN CONTENT OF THE CELLS PER WELL WAS 1.1 ± 0.05 mg AS DETERMINED

BY LOWRY’S METHOD.

Protein PA / cell protein b

(ng/mg)

LF alone 0.180.02

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 (1g/ml) FOR 3HRS. AT 37C.

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

0.001 0.01 0.1 1

PA(g/ml)

%ag

e vi

abili

ty

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

5g 12/18 66 --

1g 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

1g 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 107 spores per ml.

EXPRESSION, OPTIMIZATION AND PURIFICATIONOF PA, LF and EF

Ni-NTA AFFINITYCHROMATOGRAPHY & GEL FILTRATION

GROWTH CONDITION OPTIMIZATIONFOLLOWED BY HIGH DENSITY FEDBATCH CULTURE

E. coli with expressionplasmid 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 : 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% purechromatography 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 (CH3)

M

M

M

M

Methylated Plasmid template

gene

Mutants confirmed by sequencing

PA STRUCTURE: FUNCTIONALLY IMPORTANT RESIDUES

DOMAIN 1: BINDINGTO LF/EF

DOMAIN 3: PAOLIGOMERIZATION

DOMAIN2: MEMBRANEINSERTION AND

TRANSLOCATION OFLF/EF

Leu 203Pro 205

Ile 207

Phe 202

DOMAIN 1b

RESIDUES OF PROTECTIVE ANTIGEN RESIDUES OF PROTECTIVE ANTIGEN INVOLVED IN BINDING TO LF/EFINVOLVED 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

Leu 352

Trp 346

Residues of PA Involved In Membrane Insertion And Residues of PA Involved In Membrane Insertion And Translocation of LF/EFTranslocation 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 RESIDUES OF PROTECTIVE ANTIGEN NEEDED FOR OLIGOMERIZATIONFOR OLIGOMERIZATION

Ile 562

Leu 566

Ile 574

Phe552 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 DOMAINSUBJECT: 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+ LSESbjct: 73 MKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSE 132

Query: 123 EEKNSMNSRGEKVPFASRFVFEKKRETPKLII-NIKDYAINSEQSKEVYYEIGKGISLDI 181 ++K + G+ +V+ K+ P L+I + +DY N+E++ +S DISbjct: 133 DKKKIKDIYGKDALLHEHYVYAKEGYEPVLVIQSSEDYVENTEKALNVYYEIGKILSRDI 192

Query: 182 ISKDKSLDPEFLNLIKXXXXXXXXXXXXFSQKFKEKLELNNKSIDINFIKENLTEFQHAF 241 +SK +FL+++ F +L+ + + F+++N E Q FSbjct: 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.

EF

LF

147Val-Tyr-Tyr-Glu-Ile-Gly-Lys 153 136Val-Tyr-Tyr-Glu-Ile-Gly-Lys 142 NH2 NH2

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 149Glu 150 Il e 151Lys 153Asp187Phe190

Grey residues: involved in binding

Yellow and green: dispensable ones

IPA binding

IIVIP2 like

IIIHelix bundle

MAPKK-2 IV Catalytic centre

Val 147

Tyr 148Tyr 149

Glu 150

Ile 151

Asp 187Gly 152Lys 153Leu 188

Leu 189

Phe190

IPA binding

PA BINDING DEFECTIVE MUTANTS OF EF

Domain 1

Domain 2Domain 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

MUTATEDRESIDUES BINDING/TOXICITY Val136 + + / + + Tyr137 –– –– / –– –– Tyr138 –– –– / –– –– Glu139 + + / + +

Ile140 –– –– / –– –– Gly141 + + / + + Lys142 –– –– / –– ––

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 1;50(2-3):235-42.

Additive

Molarity PA activity retained after 48 hrs of incubation at 37oC

None - ~5 Sorbitol 1.0 ~10 Xylitol 2.0 ~18 Trehalose 1.5 ~74 Sodium Citrate

1.0 ~67

MgSO4 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 37oC in comparison with native PA.

S.NO. MUTANT RESIDUAL ACTIVITY AFTER 48 HRS. OF

INCUBATION AT 37 OC 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 Identification of the transgene in genomic DNA by PCR amplificationgenomic 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 Immunoblot detection of protective antigen with antisera raised against purified recombinant PAantisera 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 PAFunctional assay of plant expressed PA

0

20

40

60

80

100

120

1 2 3 4 5 6 7 8 9 10

Number Of plant from which TSP was isolated

pe

rce

nta

ge

kil

lin

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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 Tomato Callus Differentiating On Selection MediumMedium

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.

Thank You