Transcript Document

Centre for Biotechnology
Jawaharlal Nehru University (JNU), New Delhi
RECOMBINANT
VACCINE
AGAINST ANTHRAX
ANTHRAX
Geographical Distribution of Anthrax
Epidemic
Endemic
Sporadic
Probably Free
Free
Unknown
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%
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.
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.
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
ANTHRAX : Primarily a disease of animals,
humans are accidental host.
Bacillus anthracis
pXO1
*pXO1:181 kb. Codes for
Protective Antigen (PA), Lethal
Factor (LF) & Edema Factor (EF).
pXO2
VIRULENCE
DETERMINANTS
*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
ANTHRAX TOXIN COMPONENTS
Edema Factor
(EF)
Mw 89 kDa
Adenylate
cyclase
Protective
Antigen
(PA)
Mw 83 kDa
Cell binding
moiety
Edema in Skin
(Rabbits, Guinea Pigs)
Increased Cyclic AMP
Lethal Factor
(LF)
Mw 90 kDa
Zn Metalloprotease
Lethality
(Rats, Mice, etc.)
Macrophage Lysis
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
:
CATALYTIC SITE
Zn
CONTAINING
CLONING, EXPRESSION AND PURIFICATION
OF PA, LF AND EF FROM E. coli:
CLONING IN
EXPRESSION VECTORS
PCR
pExp
pXO1
184kb
PCR Amplified Gene
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.
LOCALIZATION OF E.coli EXPRESSED PA
220 kDa
97 kDa
66 kDa
46 kDa
30 kDa
21.5 kDa
14.3 kDa
PURIFICATON OF PA
220kDa
116kDa
97kDa
66kDa
45kDa
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
(
1
μ
g
/
m
l
)
r
e
q
u
i
r
e
d
k
i
l
l
i
n
g
5
0
%
o
f
t
h
e
J
7
7
4
A
.
1
c
e
l
l
s
.
 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
J774A.1 CELLS WERE INCUBATED WITH 1µg OF RADIOIODINATED PA
(nPA AND rPA) FOR 3 HRS. AT 4C.
a
PROTEIN CONTENT OF THE CELLS PER WELL WAS 0.95 ± 0.05 mg AS
DETERMINED BY LOWRY’S METHOD.
b
BINDING OF RECOMBINANT PA TO LF
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 LF TO RECEPTOR BOUND PA
Protein
(ng/mg)
PA / cell protein b
LF alone
0.180.02
nPA+LF
3.70 0.12
rPA+LF
3.55 0.15
 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.
MACROPHAGE LYSIS ASSAY
120
NATIVE PA
ALONE
100
RECOMBINANT
PA ALONE
%age viability
80
60
NATIVE PA +LF
40
RECOMBINANT PA + LF
0.01
0.1
PA(  g/ml)
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.
20
0
0.001
BIOLOGICAL ACTIVITY OF PA
PURIFIED FROM Bacillus anthracis
AND E. coli DH5 CELLS.
1
Protective efficacy of the rPA against B. anthracis
S.
No.
Group
Conc.
used
1.
Unvaccinated control
(challenged)
Vaccinated control
(unchallenged)
Protective Antigen
en
from B. anthracis
PBS
00/18
0
--
50 g
18/18
100
--
5g
12/18
66
--
1g
6/18
33
--
50 g
12/18
66
100
10 g
12/18
66
100
5 g
12/18
66
100
1g
6/18
33
50
2.
3.
4.

Recombinant
protective antigen
Survivors/
%
Relative
Total
survival efficacy
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 PURIFICATION
OF PA, LF and EF
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
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
chromatography and Gel
: 90-95% pure
Filtration Yield
: 3-5 g/L equivalent to ~1million
shots compared to currently
available vaccines.
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.

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
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.
immunogenecity
and
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
gene
M
M
pExp
M
M
Long PCR with Pfu turbo using
adjacent, partially overlapping
oligonucleotides encoding the
desired mutation at the 5’ end
of the primer.
gene
PCR
amplified
nicked
plasmid
M
M
M
Methylated
Plasmid
template
M
: Mutation point
M : Methyl group (CH3)
DpnI Digetsion
(degrades methylated
template DNA while
spares unmethylated
PCR amplified product)
Transformation into competent
E. coli cells.
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
RESIDUES OF PROTECTIVE ANTIGEN
INVOLVED IN BINDING TO LF/EF
Phe 202
Ile 207
Leu 203
Pro 205
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
Residues of PA Involved In Membrane Insertion And
Translocation of LF/EF
Trp 346
Leu 352
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
Leu 566
Ile 562
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
Score = 123 bits (309), Expect = 9e-27
Identities = 77/225 (34%), Positives = 123/225 (54%), Gaps = 3/225 (1%)
Query: 63
Sbjct: 73
INNLVKTEFTNETLDKIQQTQDLLKKIPKDVLEIYSELGGEIYFTDIDLVEHKELQDLSE 122
+ ++VK E
E
K + + LL+K+P DVLE+Y +GG+IY D D+ +H L+ LSE
MKHIVKIEVKGEEAVKKEAAEKLLEKVPSDVLEMYKAIGGKIYIVDGDITKHISLEALSE 132
Query: 123 EEKNSMNSRGEKVPFASRFVFEKKRETPKLII-NIKDYAINSEQSKEVYYEIGKGISLDI 181
++K
+ G+
+V+ K+
P L+I + +DY N+E++VYYEIGK
+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
QUERY: THE SEQUENCE OF EF FIRST DOMAIN
SUBJECT: THE SEQUENCE OF LF FIRST DOMAIN
HOMOLOGOUS STRETCH OF LF/EF
NH2
147Val-Tyr-Tyr-Glu-Ile-Gly-Lys 153
LF
NH2
136Val-Tyr-Tyr-Glu-Ile-Gly-Lys 142
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.
LF Structure: Residues Needed For Binding To PA
Val 147
II
PA binding
binding
PA
Glu 150
Tyr 148
Ile 151
Tyr 149
Lys 153 Gly 152
Leu 189
II
VIP2 like
Leu 188
Asp 187
Phe190
III
Helix bundle
IV
Catalytic centre
MAPKK-2
Mutants of Domain 1 defective in binding to
PA:
Tyr 148
Grey residues: involved in binding
Tyr 149
Glu 150
Yellow and green: dispensable ones
Il e 151
Lys 153
Asp187
Phe190
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.
PA BINDING DEFECTIVE MUTANTS OF EF
Domain 3
Domain 2
Linker
Ca 2+
Calmodulin
Domain 1
PA binding
domain
MUTATED
RESIDUES
Val136
Tyr137
Tyr138
Glu139
Ile140
Gly141
Lys142
BINDING/TOXICITY
+ + / + +
–– –– / –– ––
–– –– / –– ––
+ + /+ +
–– –– / –– ––
+ + / + +
–– –– / –– ––
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
THERMOSTABILIZATION OF PA
• COSOLVENT MEDIATED: MgSO4 and Trehalose are the best among
the studied cosolvents.
Additive
None
Sorbitol
Xylitol
Trehalose
Sodium
Citrate
MgSO4
Molarity
PA activity retained after 48
hrs of incubation at 37oC
1.0
2.0
1.5
1.0
~5
~10
~18
~74
~67
3.0
~83
Ref: Radha C, Salotra P, Bhat R, Bhatnagar R. Thermostabilization of protective antigenthe binding component of anthrax lethal toxin. J.Biotechnol. 1996, Oct 1;50(2-3):235-42.
Gln277Ala and Phe554Ala increase
thermal stability.
Activity of PA mutants retained after 48 hrs of incubation
at 37oC in comparison with native PA.
S.NO. MUTANT
1.
Gln277Ala
RESIDUAL ACTIVITY
AFTER 48 HRS. OF
INCUBATION AT 37 OC
~45%
2.
Phe554Ala
~90%
3.
Native PA
0%
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
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
209 kDa
1
2
3
4
5
120 kDa
78 kDa
47.7 kDa
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
percentage killing of macrophage cells
120
100
80
60
40
20
0
1
2
3
4
5
6
7
8
9
10
Number Of plant from which TSP was
isolated
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
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.

biologically

PA gene was expressed in Tobacco & Tomatoes.

Clinical trials are being conducted.
&
Thank You