1. Preclinical evaluation of safety, efficacy, immunogenicity of a recombinant rBCG Pasteur B vaccine Group B4 March 16, 2011 Adane Miheret, Tewodros Tariku

2. Pre-clinical study Objective To assess the safety (Reactogenecity and toxicity) and tolerability of rBCG Pasteur B vaccine compared to BCG To assess the immunogenicity of rBCG Pasteur B vaccine compared to BCG To assess the protective efficacy of rBCG Pasteur B vaccine compared to BCG

4. This rBCG, a substitute for BCG, is prophylactic vaccine is intended to be used at birth to prevent infection from Mycobacterium tuberculosis. Source of the vaccine: PASTEUR B LAB Vaccine composition: rBCG∆mbtB∆UreC:pfoA-85AB-PPE44-DA-1 Vaccine reconstitution: Vaccine dose: 5x 10 5, 5x10 6, 5x10 7 cfu Number of dose: 1 dose Route of immunization: Subcutaneous (mice and guinea pig) and Intradermal (NHP 1.Vaccine

5. MiceGuinea pigNon-human primates Weight250-300 g 4 kg Age8 weeks4 years TypeBALB/cHartley strain Cynomolgus macaques Route of vaccine administration Subcutaneous Intradermal Method of M.tb exposure Aerosol Mycobacterium tuberculosis strain Virulent strain (H37Rv) Number of M.tb dose100 cfu500 cfu100cfu Interval between vaccination and challenge 4 weeks 2. Animal studies

6. Vaccine (n=105/105/6) Dose Low HighMedium Safety (5/group) General health Body weight Immunogenicity (5/group) Luminex Flow cytometry Protection (5 /group) Survival time Pathology Weight change Bacterial burden BCG (n=105/105/6) Route of immunization TNF, IL-2, IFN-γ, IL-10, TGF-β, IL- 10, IL-4 Flow chart Subcutaneous (mice and Guinea pig) Intradermal (NHP) Mice, Guinea Pigs & NHP

7. 3. Safety and toxicity testing in Mice and Guinea pig Immunocompetent Mice (n=30) Immunocompromised (SCID) mice (n=30) 1d 2wk 8wk 12wk 16 wk BCG rBCG follow General health status Weight loss Hematology Clinical chemistry Histopathology Sacrificed for pathological examination and check for clearance of the vaccine (spleen, lung & liver) Guinea pig (n=30) rBCG BCG

8. 4. Immunogenicity studies in animal models (Mice and Guinea Pigs) Vaccine (n=15) 4 week Sacrifice mice and homogenate the spleen and collect cells Parameters to be measured IL-2, IFN-γ, TNF-α, IL-10, IL-4, IL-17 producing CD4 and CD8 T cells = Sacrifice time vaccination BCG (n=15)

9. Vaccine (n=60) 4 week (challenge) 4 week n=5 8 week n=5 12 week n=5 24 week n=5 4. Efficacy studies in animal models (Mice, Guinea Pigs and NHP) Parameters to be measured Bacterial burden (lung, spleen, liver) Histopathological examination of lung, spleen, and liver Body weight BCG (n=60) = Sacrifice time Mice &Guinea Pigs (n=120) 24 week n=3 6 week (challenge) vaccine NHP= 6 40 week n=3

10. 5. Recommendation/ Expected outcomes Safety profile of the new vaccine is excellent even in immunocompromised animals. The vaccine would be very immunogenic and elicit protective Cell Mediated Immunity (CMI) polarized to Th1 type of response. The new vaccine would enhance survival of Guinea pig infected with high dose of H37Rv with a marked reduction in bacterial load in different organs.

11. Thank you for your attention!