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Designing a new prophylactic vaccine as a
replacement for BCG
B2Sultan Gulce IzNeelam TanejaMurtada Osman
Why do we need a better vaccine?
BCG is the only vaccine currently available
Effective againstonly 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
Recombinant BCG over expressing which antigens? 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/biomedical/MicroarraysPreSyn.html
Rationale Behind Our Design
Replication restricted rBCG, Safety concerns of HIV+ (15 million people
suffering from coinfection with TB&HIV) Secreted and cell wall antigens which are
highly immunogenic Secretary antigens expressed at different
stages of infection Genes encoding dormancy antigens Endosome escape mechanism
Why Recombiant BCG ? Not attenuated Mtb?
rBCG 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 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 Ag85A, Ag85B
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)
Major target antigens 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
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
Selected antigens-III 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
∆ 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
Endosomal escape mechanism 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 ConstructrBCG∆mbtB∆UreC:pfoA-85AB-PPE44-DA-1
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
Construct is encoded on multicopy plasmid named pSNB under the control of fbpA promoter in frame with Ag85A
Electroporated into BCG mutant (rBCG∆mbtB∆UreC:pfoA)
Ag85B PPE 44Ag85A DA-1
fbpA promoter
rBCG-PasteurB Formulation
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 benefitsPrinciple: Effecter and memory T cellsPre-exposure: allow short term Mtb infection
and eradicate MtbBenefit: Sterile Mtb eradication cancelling the
risk of TB reactivation Safer for HIV patients, superior to native
BCG Better protection against Beijing strain
Our Vaccination StrategyPre exposure with superior BCG replacement
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
Boost with subunit vaccine having the same antigens as the prime vaccine and have an strong Th1 adjuvant (IC31)
Pre exposure with superior rBCG PasteurB
Limitations 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
rBCG-PasteurB Formulation
rBCG∆mbtB∆UreC:pfoA-85AB-PPE44-DA-1
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.
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.
Some of the strategies to construct better vaccines to replace BCG
rBCGG30, over express Ag85B, (Tullius et al., 2008) rBCGΔUreC:Hly (VPM1002) (Grode et al., 2005)
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)
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 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