Designing a new prophylactic vaccine as a replacement for BCG

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

2. Why do we need a better vaccine? • 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

3. Aim: to design a prophylactic vaccine to replace BCG • rBCG • Restrictedreplication • Have endosomal escape mechanism • Over expressingsome relevant antigens

4. Recombinant BCG over expressing which antigens? http://www.microfab.com/technology/biomedical/MicroarraysPreSyn.html 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.

5. 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

6. Why Recombiant BCG ? Not attenuated Mtb? rBCG 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 • 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

7. 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

8. 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

9. 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

10. 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 sufficientferric mycobactin

11. 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

12. 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) fbpA promoter DA-1 Ag85B Ag85A PPE 44

13. 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

14. 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

15. Immune responses & Antigens Dormancy antigens Perforation Memory T cells, TNF, IFN-γ, IL-2

16. Our Vaccination Strategy Pre 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

17. Ideal Strategy Pre exposure with superior rBCG PasteurB Boost with subunit vaccine having the same antigens as the prime vaccine and have an strong Th1 adjuvant (IC31)

18. 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

19. Thank you very much for your attention! Macrophage engulfing rBCG Pasteur B vaccine

20. Back-up Slides

21. rBCG-PasteurB FormulationrBCG∆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.

22. 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)

23. 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.

24. Why Recombiant BCG ? Not attenuated Mtb? rBCG 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 • 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