Pre-clinical studies  The animal model selected to perform the preclinical studies.  The protocol of vaccination  Evaluating the safety, immunogenicity and efficacy of the PFP vaccine.  The expect results. Out line:

Guniea pig as Animal model? guinea pig model has been extensively used to test vaccine candidates This model is more susceptible to the disease than the mouse Has elements of pathology very similar to that seen in humans

 Toxicity study 4 group 12 Hartley guinea pig per group (male/female) ~ 300 gm weight  Reproductive toxicity study 2 groups 6 female Hartley guinea pig per group ~ 500 gm weight Toxcicity study design  Single dose toxicity  Repeated dose toxicity  Immunotoxicty  Reproductive toxicity studies  Safty

Vaccination and challenge Protocol Immunization route: intradermaly ( Same route as planned for clinical trial addministration) Prime immunization: One Dose BCG (10 2 CFU) Boost(s): Ag85 A-PPE44-RV2660 (PFP) Volume: 100 μl formulated in IC-31 as adjuvant Challenge: low-dose aerosol of M.tb (10 5 CFU)

groups BCG1st2nddose A√ placebo - IC31 B1√√-25 μg B2√√-50 μg C√√√25 μg Experimental groups w BCG 1 st 2 nd challenge w BCG 1 st challenge BCG at day 0 First boost 9 weeks later Group C, received 2 nd boost 3 week later Challenge 4 weeks after final boost Animals bled 1 day before challenge 4 animals sacrificed at 1 hr, 3 & 13 weeks after challenge

groups BCG1stdose D1√ placeboadjuvant D2√√50 μg Reproductive toxicity studies w BCG 1 st BCG at day 0 Pregnancy 1 week later Boost 8 week after BCG immunisation Expected to deliver 2 weeks after boost

Evaluating the safety and efficacy

Evaluating the safety Survival change in Feeding behavior Measuring change in body weight Maternal toxicity Food consumption, body weight, Clinical observations, fertility index and gestational index

Histopathology analysis Lung section Comparing the histopathology of the lungs of animals in diffrent groups

Efficacy Counting the bacterial load [CFU] in lung and spleen ( homogenizing the lungs and plating on nutrient agar H711, and calculating the number of colonies)

Immunogenecity Humoral immunity: IgG level T cell population in lung and spleen lymphocyte proliferation assay from spleen

Expected outcome No change in feeding behaviour No body weight loss Fewer and smaller lesion in lung comparing to animals immunized with BCG lower CFU in lung and spleen, comparing to control group immunized with BCG (>10 5 )