Centre for Biotechnology Centre for Biotechnology Jawaharlal Nehru University (JNU), Jawaharlal Nehru University (JNU), New Delhi New Delhi
Feb 03, 2016
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
ly c
ells
Cel
ls w
ith
pQ
E30
Un
ind
uce
d C
ells
wi t
h p
MW
Ind
uce
d C
ell s
wit
h p
MW
Per
ipla
smic
fra
ctio
n
Cyt
osol
ic f
ract
ion
Incl
usi
on b
ody
frac
tion
Sta
nd
ard
PA
Mar
ker
PURIFICATON OF PA
220kDa116kDa 97kDa 66kDa 45kDa
E. c
oli
cel l
s ex
pre
ssin
g P
A
PA
in t
he
incl
us i
o n b
odie
s
Pro
tein
s af
ter
affi
ni t
y p
uri
fic a
tio n
PA
aft
er F
PL
C
PA
pu
rifi
e d f
rom
B. a
nth
raci
s
Mo l
e cu
lar
wei
ght
stan
dar
ds
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
thra
cis
Rec
omb
inan
t P
A
LF
fro
m B
. an
thra
cis
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
g o
f m
ac
rop
ha
ge
ce
lls
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