Designing a new prophylactic vaccine as a replacement for BCG B2 Sultan Gulce Iz Neelam Taneja Murtada Osman.

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

Memory T cells, TNF, IFN-γ, IL-2

Perforation

Dormancy antigensImmune responses

& Antigens

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

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

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