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 (102 CFU)

Boost(s):• Ag85 A-PPE44-RV2660 (PFP)• Volume: 100 μl formulated in IC-31 as adjuvant

Challenge: low-dose aerosol of M.tb (105 CFU)

groups BCG 1st 2nd dose

A √ placebo - IC31

B1 √ √ - 25 μg

B2 √ √ - 50 μg

C √ √ √ 25 μg

Experimental groups

0 9 12 16 19 29 w

BCG 1st 2nd challenge

0 9 13 16 26 w

BCG 1st challenge

• BCG at day 0• First boost 9 weeks later• Group C, received 2nd 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 BCG 1st dose

D1 √ placebo adjuvant

D2 √ √ 50 μg

Reproductive toxicity studies

0 1 8 10 w

BCG 1st

• 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 safetySurvivalchange in Feeding behaviorMeasuring 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

EfficacyCounting the bacterial load [CFU] in lung and spleen

( homogenizing the lungs and plating on nutrient agar H711, and calculating the number of colonies)

• Humoral immunity: IgG level

• T cell population in lung and spleen

• lymphocyte proliferation assay from spleen

• 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 (>105)