Transcript rBCG - stopenterics

Designing a new
prophylactic vaccine as a
replacement for BCG
B2
Sultan Gulce Iz
Neelam Taneja
Murtada Osman
Why do we need a better vaccine?
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BCG is the only vaccine currently
available
Effective against
 only disseminated and TB
meningitis
 not pulmonary TB in infants
and children
Does not prevent latent infection
Short immunity period no longer
significant than after 10 years
Aim: to design a prophylactic
vaccine to replace BCG
rBCG
 Restricted replication
 Have endosomal escape mechanism
 Over expressing some relevant antigens
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Recombinant BCG over expressing
which antigens?
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To find out the biomarkers,
microarray studies and
comparative transcriptome
analysis were done.
Blood samples and PBMCs
of naïve, latently infected,
having active pulmonary
disease, TB meningitis and
miliary tuberculosis were
screened.
http://www.microfab.com/technology/biomedic
al/MicroarraysPreSyn.html
Rationale Behind Our Design
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Replication restricted rBCG,
concerns of HIV+ (15 million people
suffering from coinfection with TB&HIV)
 Safety
Secreted and cell wall antigens which are
highly immunogenic
 Secretary antigens expressed at different
stages of infection
 Genes encoding dormancy antigens
 Endosome escape mechanism
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Why Recombiant BCG ? Not attenuated Mtb?
rBCG
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Used since 1920s
Excellent safety profile
Billions of doses have been
given to humans
Ability to generate rBCG
over expressing relevant
antigens related with
protection
Attenuated Mtb
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Not much known
Still in Phase I trials
Risk of reversion to
virulence, safety must be
proven with
immunocompromised SCID
and IFN-γ knock-out mouse
models
Selected antigens-I
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Ag85A, Ag85B
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Major target antigens
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Belonging to Ag85 complex (Ag85A, Ag85B, Ag85C; 3:2:1)
Secreted and cell wall antigens (Secreted by SecA1 mediated
secretory pathway, posses mycolyl transferase activity)
Induce strong T cell proliferation and IFN-γ production in healty
individuals latently infected with TB
Protective in small animals both in mice and guinea pigs
Present in all environmental mycobacteria
Doesn’t interfere with new diagnostic tests
Selected antigens-II
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PPE44; is secreted protein which is expressed by
Mtb and presented to the immune system
throughout the different stages of the infection
PE and PPE proteins
Restricted to pathogenic mycobacteria
 Located in Mtb cell wall and membrane
 Plays an important role in pathogenesis and persistance of
Mtb
 Share a number of characteristics with ESAT-6 and
CFP-10
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Selected antigens-III
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Genes encoding dormancy antigen DA1 (Dos-R regulated
genes were identified, Lin et al 2007)
 Many antigens encoded by DosR regulon elicit stronger
IFN-γ production by PBMC from latently infected individuals
compared to patients with TB disease
 DosR-regulon is expressed during natural infection and
suggests that immune responses against these antigens
may contribute to controlling latent Mtb infection.
Addition of late stage antigens induced in dormancy is
promising for multi-stage prophylactic vaccine candidates
Replication restricted rBCG
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∆ mbtB deletion mutant, generated by
allelic exchange
 Synthesis
of siderophore mycobactin is
disrupted
 Normal iron acquaisition prevented
Strain is mycobactin dependent
 Formulation of the vaccine must include
sufficient ferric mycobactin
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Endosomal escape mechanism
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Expresses perfringolysin A of Clostridium perfringens
 ∆UreC:pfoA, deletion of UreC and expressing of pH
independent Perfringolysin A
 accomplished by using an allelic exchange plasmid to
replace ureC with pfoA
Perforation allows
 improved antigen release into cytosol
 potent MHC I presentation, better CD8+ responses
Induces apoptosis of host cell which carries the
maycobacterial antigens as cargo
Uptake of this vehicles by dendritic cells may lead cross
priming of antigens to CD4+ and CD8+ cells
Also it can induce Th17 response
rBCG-PasteurB Construct
rBCG∆mbtB∆UreC:pfoA-85AB-PPE44-DA-1
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Derived from BCG Tice Strain (Organoni Shering-Plough)
∆ mbtB deletion mutant
∆UreC:pfoA, replace ureC with pfoA
Over-expresses Mtb antigens: Ag85A, Ag85B, PPE44, DA-1
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Construct is encoded on multicopy plasmid named pSNB
under the control of fbpA promoter in frame with Ag85A
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Electroporated into BCG mutant (rBCG∆mbtB∆UreC:pfoA)
fbpA promoter
Ag85A
Ag85B
PPE 44
DA-1
rBCG-PasteurB Formulation
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Avalibility : Freeze dried Powder and solvent for
injection
Dose : rBCG-PasteurB, live, attenuated 5x106 cfu for
infants < 3 months
Route: Intradermal
Delivery vehicle: Needle and Syringe
Reconstitution: Solvent (BCG formulation) with ferric
mycobactin (10 microgram /ml)
Adjuvant: No need for adjuvant
rBCG Pasteur B – Potential benefits
 Principle:
Effecter and memory T cells
 Pre-exposure: allow short term Mtb infection
and eradicate Mtb
 Benefit: Sterile Mtb eradication cancelling the
risk of TB reactivation
Safer for HIV patients, superior to native
BCG
 Better protection against Beijing strain
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Immune responses
& Antigens
Dormancy
antigens
Perforation
Memory T cells,
TNF, IFN-γ, IL-2
Our Vaccination Strategy
Pre exposure with
superior BCG
replacement
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Our vaccine will prevent
reactivation and delay TB in
adults as we have added
dormancy antigens but will
not prevent reinfection
The immunity is expected to
be limited in time-childhood
Therefore we need to boost
Ideal Strategy
Pre exposure with
superior rBCG
PasteurB
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Boost with subunit
vaccine having the same
antigens as the prime
vaccine and have an
strong Th1 adjuvant
(IC31)
Limitations
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Though restricted replication and safer for HIV still
needs to be tested in SCID mice
Complex formulation, mutants has to be stable (5o
generations ) and scale up
We do not know the exact concentrations of the
antigens (fold as compared to parent BCG by
immunoblot )
How enviromental mycobacteria will affect the
immunity produced by this vaccine ?
Th 17 response may also trigger the pathological
responses that occur during TB DISEASE
PROGRESSION
Thank you very much for your
attention!
Macrophage engulfing
rBCG Pasteur B vaccine
Back-up Slides
rBCG-PasteurB Formulation
rBCG∆mbtB∆UreC:pfoA-85AB-PPE44-DA-1
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Mycobactin mutant generated by allelic exchange and plasmid containing mutant
allele electroporated in BCG.
mbtB deletion mutant, an allelic exchange substrate was constructed by using a
PCR strategy in which a BCG mbtB locus with a 3.9-kb deletion was created and a
Kmr cassette from pUC19-Kmr was inserted at the site of the deletion.
The mutated allele was cloned into the allelic exchange vector pEX2, and the
plasmid was electroporated into BCG for allelic exchange.
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Integration of the PfoA(G137Q) gene into the rBCG (mbtB) mutant was
accomplished using an allelic exchange plasmid to replace ureC with pfoA(G137Q).
To generate BCG mutants over expressing the relavant antigens a DNA construct
was made in frame with the the endogenous promoter of Ag85A(≈ 500 bps upstream
of the fbpA) in frame with Ag85B, PP44,DA-1 in a plasmid named pNSB.
Each protein first introduced in a shuttle vector than cloned one by one into the final
vector, each protein has its own start and stop codons and relavant linkers which will
lead to a correct conformation of the proteins.
pNSB was electroporated into the rBCG (mbtB) and selection was done with
hygromycin.
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Some of the strategies to construct better
vaccines to replace BCG
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rBCGG30, over express Ag85B, (Tullius et al., 2008)
rBCGΔUreC:Hly (VPM1002) (Grode et al., 2005)
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Better CD4, CD8 responses even better Th17
Better protection not only against Mtb H37Rv also Beijing
genotypes
These two are further attenuated, safer than parent
BCG
Combination of Ag85A, Ag85B and TB10.4; safer but
not more immunogenic than parent BCG (Sun et al.,
2009, 2010)
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rBCG ΔureC:Hly (developed by Dr. Kaufman's group at the Max Planck Institute, Berlin,
Germany) was constructed to amplify the CD8+ T-cell response induced by BCG. It is a
recombinant BCG mutant that expresses a pore-forming protein from Listeria monocytogenes
(listeriolysin; Hly), which disrupts the phagosome membrane by a mechanism that requires an
acidic pH. The gene (ureC) encoding the urease enzyme that blocks the acidification of the
phagosome containing BCG was deleted as a means of providing the optimal pH for
listeriolysin function. This vaccine demonstrated improved safety in SCID mice and superior
potency than BCG control to reduce Mtb burden in vaccinated mice (e.g. mice vaccinated with
rBCG ΔureC:Hly showed a reduction in Mtb burden by ~1.0 log compared to mice immunized
with the BCG control).
rBCG ΔureC:Hly has entered phase I clinial trials in 2008. Yet, like rBCG30, it contains
antibiotic-resistance markers, which are forbidden by regulatory authorities.
Pore-forming molecules, such as listeriolysin from Listeria monocytogenes or perfringolysin
from Clostridium perfringens, have the potential to punch holes in the phagosomal membrane.
They are expected to increase the MHC-I presentation of BCG-derived antigens to CD8 T cells
by allowing some leakage of rBCG from the phagosome to the cytosol.
Why Recombiant BCG ? Not attenuated Mtb?
rBCG






Used since 1920s
Excellent safety profile
Millions of dozes have been
given to humans
Ability to generate rBCG
over expressing relavant
antigens related with
protection
Cannot be boosted with
repeated BCG vaccination
Is contraindicated for use in
HIV-positive infants
Attenuated Mtb
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Much not known
Still in Phase I trials
Risk of reversion to
virulence, safety must be
proven with
immunocompromised SCID
and IFN-γ knock-out mouse
models
Still contains over 120 genes
(lost in BCG) have potential
protective antigens