1. Clinical Phase I Trial Signe Sorup Alex Chaudhuri Sandrine Duron

2. Objectives Primary - To assess the safety and tolerability of one dose of rBCG-Pasteur B vaccine compared to BCG in healthy adults. The endpoints will be any local and systemic reactogenicity signs and symptoms, any derangement in laboratory measures or any report of adverse event during the first 4 weeks after the vaccination. Secondary - To evaluate the immunogenicity of one dose of rBCG Pasteur B vaccine compared to BCG. The endpoints will be measured by intracellular cytokine staining on whole blood

3. Design Randomised Double-blinded BCG controlled (Tice strain) Dose escalating Single center Setting - Denmark (Non-endemic + No routine BCG last 30 years)

4. Inclusion criteria Healthy adults, 20 - 30 years old on the day of trial enrollment Healthy by medical history + clinical examination Women - Non-pregnant, contraception during period of trial including total period of follow-up ( not to commence new OCP during trial) BMI 18-25 Written consent

5. Exclusion criteria Evidence / risk of latent TB o Positive Tuberculin Test or Positive Quantiferon (IGRA) GOLD o Prior contact with active pulmonary tuberculosis or in relevant health care setting o Abnormal chest X-ray Confounders of immunogenicity endpoints o BCG vaccination history or scar o Other vaccination within the 4 preceding weeks Significant language barrier

6. Exclusion criteria - 2 Safety concerns o HIV positive / other immunosuppression o High risk behaviour for acute HIV infection o Illicit drug abuse o Fever > 37.5 c o Current or recent (one month) acute illness o Chronic disease or medication o Abnormality on Full blood Count/Coagulation/Liver enzymes o Abnormal urinary analysis

7. Advertisement for volunteers Focus on students o Student magazines o Campuses o Community center

8. Method - schedule Day 8015*10 5 2 73 Time of entry Number of injections Controls (BCG TICE)rBCG Pasteur B* Day 12015*10 5 25*10 6 74 Day 4015*10 5 25*10 4 72 Day 015*10 5 25*10 3 71 Dose (CFU) Number of subjects Dose (CFU) Number of subjects arm * TICE BCG strain with overexpression of Ag85A, Ag85B, PPE44 and DA-1

9. Method - description of the product No obvious difference between the rBCG Pasteur B and BCG Reconstituted by the pharmacist o immediately prior to the injection o 0.1 ml of neutral solvent Route of administration: intra-dermal

10. Adverse events Any untoward medical event that occurs after administration of the injection regardless of whether it is directly attributable to the vaccine or not.

11. Reactogenicity o Common adverse events usually observed after immunization  Local reaction  Systemic reaction o Usually occur up to 2 - 3 days after immunization Adverse events

12. According to the FDA guidance toxicity grading scale o Mild/moderate/severe/potentially life-threatening AE Serious adverse events o Death o Life threatening adverse events o Hospitalisation or prolonged hospitalisation length o Significant disability or incapacity o Congenital abnormality, birth defect, unintentional fetal loss

13. Data collection Daily diary formated o Solicited and unsolicited adverse events o Filled by the volunteer Case report forms (CRF) o Filled by the physicians (diagnosis/syndroms) o Based on the volunteers diary Clinical examination o Physician o Pre vaccination o Day 0 (after injection)  Subjects observation for an hour after the injection  Vital signs monitoring at 30 and 60 minutes post-injection  Local reactions evaluation at 60 minutes o Day 7, 14, 28 Biological tests o Hematology/ liver enzymes/ renal function o Pre vaccination o Day 0, 7, 14, 28

14. Serious adverse events declaration Dedicated phone number Declaration as soon as possible o to the sponsor o to the institutional review board (IRB) Investigation Suspend further vaccine administration if possibly related to vaccine

15. Immunogenicity assessment Intra cellular cytokine staining (flow cytometry) o Total cytokine positive CD4 frequency o expression of IFN gamma, IL-2, TNF alpha singly or in combination (polyfunctional T cell) o Same with CD8 T cells Whole blood (frozen after stimulation) and PBMC for a subset of volunteers

16. Follow-up schedule

17. Endpoints Rate of all adverse event Rate of adverse events acording to severity Rate of AE’s possibly attributable to vaccine Immunological endpoints

18. Environmental and regulatory challenges Comply with local GMO guideline Biohazard facility Disposal of residual vaccine as medical waste Drug sensitivity of the new recombinant BCG Choose adults instead of infants o phase 1 trial : infants o issue of Informed consent