Phase 2a Dose-Defining Safety and Immunogenicity Study of MTBVAC in South African Neonates Living in a High-Burden Tuberculosis-Endemic Region
Phase: PHASE2
Conditions: Tuberculosis
Sponsor: Biofabri, S.L

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A Phase 2a Randomised Controlled Dose-Defining Trial of the Safety and 
Immunogenicity of MTBVAC in Healthy, BCG Naïve, HIV Unexposed, South 
African Newborns  
 
Protocol Number:  202 
 
Study Phase: 2a 
 
Version:  V4.0 
Date:   
15 April 2021 
 
Trial Acronym:  MTBVAC-Newborns 
 
Sponsor:   
BIOFABRI, SL 
 
 
La Relva s/n 
36400 Porriño (Pontevedra), SPAIN 
Phone: +34 986 33 04 00 
 
Principal Investigator:  Dr. Michele Tameris 
 
Trial Center:   South African Tuberculosis Vaccine Initiative (SATVI) 
 Brewelskloof Hospital 
 Haarlem Street 
 Worcester 
 Western Cape 
 6850 
 South Africa 
 
Clinical Study Advisor:  Prof. Mark Hatherill 
 
 
 
 
 
 
 
 
 

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CONFIDENTIALITY STATEMENT 
The confidential information in this document is provided to you as an investigator, for review 
by you, your staff, and the applicable Institutional Review Board/Independent Ethics 
Committee. Your acceptance of this document constitutes an agreement that you will not 
disclose the Information herein to others without written authorization. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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STATEMENT OF COMPLIANCE 
 
The study will be carried out in accordance with this protocol, the guidelines and ethical 
principles set forth in the Declaration of Helsinki, Good Clinical Practice (ICH-GCP) and SA 
GCP. Any proposed changes to this protocol, or to informed consent or participant information 
documents, will be submitted to the Human Research Ethics Committee of the University of 
Cape Town for approval prior to implementation. Monitoring representatives of the South 
African Health Products Regulatory Authority (SAHPRA), or representatives of the Human 
Research Ethics Committee of the University of Cape Town, South Africa will have reasonable 
access to inspect facilities and study records at the study site. 
 
 
 
 

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1 
SIGNATURE SECTION 
The signature below constitutes the approval of this protocol and the attachments and 
provides the necessary assurances that this trial will be conducted according to all stipulations 
of the protocol, including all statements regarding confidentiality, and according to local legal 
and regulatory requirements and applicable ICH guidelines. 
 
PRINCIPAL INVESTIGATOR: ___________________________ 
Date: _________________ 
 
 
 
 
 
 
 
 
 
 
 

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TABLE OF CONTENTS 
 
..................................................................................................................................... Page 
Confidentiality Statement ............................................................................................................ 2 
Protocol Signature Sheet ............................................................................................................ 3 
Statement of Compliance ........................................................................................................... 4 
1 
SIGNATURE SECTION ..................................................................................................... 5 
Table of Contents ........................................................................................................................ 6 
List of Abbreviations.................................................................................................................... 9 
Protocol Summary .................................................................................................................... 11 
2 
Key Roles ......................................................................................................................... 13 
3 
Background Information and Scientific Rationale ........................................................... 14 
3.1 
Background Information ....................................................................................... 14 
3.2 
Rationale .............................................................................................................. 14 
3.3 
Potential Risks and Benefits ................................................................................ 16 
3.3.1 
Potential Risks ......................................................................................... 16 
4 
Objectives ........................................................................................................................ 19 
4.1 
Study Objectives .................................................................................................. 19 
4.2 
Study Outcome Measures ................................................................................... 19 
4.2.1 
Primary Outcome Measures .................................................................... 19 
4.2.2 
Secondary Outcome Measures ............................................................... 20 
5 
Study Design .................................................................................................................... 21 
6 
Study Enrollment and Withdrawal ................................................................................... 23 
6.1 
Participant Inclusion Criteria ................................................................................ 23 
6.2 
Participant Exclusion Criteria ............................................................................... 23 
6.3 
Treatment Assignment Procedures ..................................................................... 24 
6.3.1 
Randomization Procedures ..................................................................... 24 
6.3.2 
Masking Procedures ................................................................................ 24 
6.3.3 
Reasons for Withdrawal ........................................................................... 25 
6.3.4 
Handling of Withdrawals .......................................................................... 25 
6.3.5 
Termination of Study ................................................................................ 25 
7 
Study Intervention/Investigational Product ...................................................................... 26 
7.1 
Study Product Description ................................................................................... 26 
7.1.1 
Acquisition ................................................................................................ 26 
7.1.2 
Product Storage and Stability .................................................................. 26 
7.2 
Dosage, Preparation and Administration of Study Intervention/Investigational 
Product ................................................................................................................. 27 
7.3 
Accountability Procedures for the Study Intervention/Investigational Product(s)
.............................................................................................................................. 27 
7.4 
Assessment of Participant Compliance with Study Intervention/Investigational 
Product ................................................................................................................. 28 
7.5 
Concomitant Medications/Treatments ................................................................. 28 
8 
Study Schedule ................................................................................................................ 29 
8.1 
Screening ............................................................................................................. 29 
8.2 
Enrollment/Baseline ............................................................................................. 30 

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8.3 
Follow-up .............................................................................................................. 31 
8.4 
Final Study Visit ................................................................................................... 34 
8.5 
Early Termination Visit ......................................................................................... 34 
8.6 
Unscheduled Visit ................................................................................................ 35 
9 
Study Procedures/Evaluations ........................................................................................ 36 
9.1 
Clinical Evaluations .............................................................................................. 36 
9.1.1 
Screening for TB and referral for Isoniazid Preventive Therapy ............. 36 
9.2 
Laboratory Evaluations ........................................................................................ 37 
9.2.1 
Clinical Laboratory Evaluations ............................................................... 37 
9.2.2 
Specimen Preparation, Handling, and Shipping ..................................... 38 
10 
Assessment of Safety ...................................................................................................... 39 
10.1 
Specification of Safety Parameters ..................................................................... 40 
10.2 
Methods and Timing for Assessing, Recording, and Analyzing Safety 
Parameters ........................................................................................................... 41 
10.2.1 Adverse Events ........................................................................................ 41 
10.2.2 Reactogenicity (for Vaccine Studies and Some Therapeutic Trials) ...... 44 
10.2.3 Serious Adverse Events .......................................................................... 44 
10.2.4 Procedures to be Followed in the Event of Abnormal Laboratory Test 
Values or Abnormal Clinical Findings ...................................................... 45 
10.3 
Reporting Procedures .......................................................................................... 45 
10.3.1 Serious Adverse Events .......................................................................... 46 
10.3.2 Suspected and Confirmed Tuberculosis Events ..................................... 47 
10.3.3 Other Immediately Reportable Events .................................................... 47 
10.4 
Type and Duration of Follow-up of Participants after Adverse Events ............... 47 
10.5 
Pausing Rules ...................................................................................................... 48 
10.6 
Emergency unblinding ......................................................................................... 50 
10.7 
Safety Oversight (DSMB) .................................................................................... 50 
10.8 
Trial Steering Committee (TSC) .......................................................................... 51 
11 
Clinical Monitoring ............................................................................................................ 52 
11.1 
Site Monitoring Plan ............................................................................................. 52 
12 
Statistical Considerations ................................................................................................ 53 
12.1 
Study Hypotheses ................................................................................................ 53 
12.2 
Sample Size Considerations ............................................................................... 53 
12.3 
Planned Interim Analyses .................................................................................... 53 
12.3.1 Safety Review .......................................................................................... 53 
12.3.2 Immunogenicity or Efficacy Review ......................................................... 53 
12.4 
Final Analysis Plan............................................................................................... 54 
12.4.1 Participant Analysis Sets ......................................................................... 54 
12.4.2 Demographics and Protocol Compliance ................................................ 54 
12.4.3 Immunogenicity and Other Immunology Analyses .................................. 55 
12.4.4 Safety Analyses ....................................................................................... 55 
12.4.5 Computer Methods .................................................................................. 56 
13 
Source Documents and Access to Source Data/Documents ......................................... 57 
14 
Quality Control and Quality Assurance ........................................................................... 58 
15 
Ethics/Protection of Human Participants ......................................................................... 59 

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15.1 
Ethical Standards ................................................................................................. 59 
15.2 
Institutional Review Board ................................................................................... 59 
15.3 
Informed Consent Process .................................................................................. 60 
15.4 
Exclusion of Women, Minorities, and Children (Special Populations) ................ 62 
15.5 
Participant Confidentiality .................................................................................... 62 
15.6 
Study Discontinuation .......................................................................................... 63 
15.7 
Future Use of Stored Specimens ........................................................................ 63 
16 
Data Handling and Record Keeping ................................................................................ 64 
16.1 
Numbering and coding of participant folders ....................................................... 64 
16.2 
Study logs ............................................................................................................ 64 
16.3 
Data Management Responsibilities ..................................................................... 64 
16.4 
Data Capture Methods ......................................................................................... 65 
16.4.1 Source documentation ............................................................................. 65 
16.4.2 Case Report Forms (CRFs) ..................................................................... 65 
16.5 
Timing/Reports ..................................................................................................... 65 
16.6 
Types of Data ....................................................................................................... 66 
16.7 
Study Records Retention ..................................................................................... 66 
16.8 
Protocol Deviations .............................................................................................. 66 
17 
Publication Policy ............................................................................................................. 68 
18 
Changes to the Protocol .................................................................................................. 69 
18.1 
Changes from Version 3.0 to Version 4.0 ........................................................... 69 
19 
Literature References ...................................................................................................... 73 
Supplements/Appendices ......................................................................................................... 76 
Appendix A. Schedule of Events .............................................................................................. 77 
Appendix B. Table of toxicity reference ranges for grading of adverse events ....................... 78 
Appendix C. Table of toxicity reference ranges for grading SAFETY LABORATORY EVENTS
.......................................................................................................................................... 80 
Appendix D. Total Bilirubin Table for Term and Preterm Neonates ........................................ 82 
 
 

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LIST OF ABBREVIATIONS 
AE 
adverse event  
ALP 
alkaline phosphatase 
ALT 
alanine aminotransferase 
AST 
aspartate aminotransferase 
BCG 
Bacille Calmette-Guérin 
BUN 
blood urea nitrogen 
CFR 
Code of Federal Regulations 
CFU 
colony forming unit 
DAFF 
Department of Agriculture, Forestry and Fisheries 
DSMB 
Data Safety Monitoring Board 
eCRF 
electronic case report form 
EDC 
Electronic Data Capture 
EDCTP 
European and Developing Countries Clinical Trials Partnership 
ELISA 
enzyme linked immunosorbent assay 
GCP 
Good Clinical Practice 
GGT 
gamma glutamyl transferase 
HIV 
human immunodeficiency virus 
ICH 
International Council for Harmonisation of Technical Requirements for 
Pharmaceuticals for Human Use 
ICMJE 
International Committee of Medical Journal Editors 
ICS 
intracellular cytokine staining 
IEC 
Independent Ethics Committee 
IFN 
interferon 
Ig 
immunoglobulin 
IGRA 
interferon gamma release assay 
IRB 
Institutional Review Board 
IRE 
Immediately Reportable Event 
LFT 
liver function test 
LMM 
local medical monitor 
MedDRA 
Medical Dictionary for Regulatory Activities 
Mtb 
Mycobacterium tuberculosis  
PBMC 
peripheral blood mononuclear cell(s) 
PCR 
polymerase chain reaction 
PHA 
phytohemagglutinin 
PID 
participant identification number 
POI 
prevention of infection 
PT 
prothrombin time 
PTT 
partial thromboplastin time 
QFT 
QuantiFERON®-TB 
SAE 
serious adverse event 
SAER 
Supplemental SAE Report 
SAHPRA 
South African Health Products Regulatory Authority  
SATVI 
South African Tuberculosis Vaccine Initiative 

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SOP 
Standard Operating Procedure 
SUSAR 
suspected unexpected serious adverse reaction 
SSAR 
Standard Safety Analysis Report 
TB 
tuberculosis 
TBVI 
TuBerculosis Vaccine Initiative 
TCD 
Triclinium Clinical Development (Pty) Ltd 
TCD RA 
Triclinium Regulatory Department 
TNF 
tumor necrosis factor 
TSC 
Trial Steering Committee 
ULN 
upper limit of normal 
US 
United States 
WB 
whole blood 
WBC 
white blood cell 
WHO 
World Health Organization 

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PROTOCOL SUMMARY 
Title: 
A Phase 2a Randomised Controlled Dose-defining Trial of the 
Safety and Immunogenicity of MTBVAC in healthy, BCG naïve, 
HIV unexposed, South African newborns 
Phase: 
2a 
Population: 
Ninety-nine HIV unexposed, BCG naïve, newborns without 
known household exposure to M. tuberculosis  
Number of Sites: 
One site in South Africa: 
South African Tuberculosis Vaccine Initiative (SATVI) 
Brewelskloof Hospital 
Worcester 
South Africa 
Study Duration: 
The estimated study duration (first participant vaccinated to 
completion of data collection) is approximately 36 months. 
Study Participation 
Duration: 
365 days 
Description of Agent or 
Intervention: 
MTBVAC at three dose levels: 2.5 x 104 CFU, 2.5 x 105 CFU 
and 2.5 x 106 CFU. The active control is BCG vaccine.  
Participants will receive a single dose of MTBVAC or BCG 
administered intradermally on Study Day 0.   
Objectives: 
 
Primary: 
• 
To evaluate safety and reactogenicity of MTBVAC at 
escalating dose levels compared to BCG vaccine in healthy, 
BCG naïve, HIV unexposed, South African newborns 
• 
To evaluate the immunogenicity of MTBVAC at escalating 
dose levels compared to BCG vaccine in healthy, BCG naïve, 
HIV unexposed, South African newborns 
Secondary: 
• 
To evaluate QFT conversion rates in neonates receiving 
escalating dose levels of MTBVAC 
Future exploratory: 
Samples will be kept for future analyses:  
• 
To evaluate exploratory immune responses to escalating dose 
levels of MTBVAC and to BCG (including, but not necessarily 

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limited to, analysis of innate response cytokines, specific IgG 
antibody responses, donor-unrestricted T-cell responses). 
• 
To differentiate vaccine-induced immune responses from 
natural Mtb infection. 
 
Design: 
Double-blind, randomised, active BCG control, MTBVAC dose-
finding trial with 3 cohorts of healthy, BCG naïve, HIV 
unexposed, South African newborns (24 BCG; 75 MTBVAC) 
Estimated Time to 
Complete Enrollment: 
The estimated time to complete enrollment is approximately 9 
months. 
 
 
Schematic of Study Design:  
 
 
 
 

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2 
KEY ROLES 
Study Monitor 
TCD, Triclinium Clinical Development (Pty) Ltd. 
 
Design House 
 
121 Amkor Road 
 
Pretoria 
 
0157 
 
South Africa 
 
Study Manager 
Ingrid Murillo  
 
Biofabri. S.L. 
 
La Relva s/n 
 
36400 Porriño (Pontevedra), SPAIN 
 
Phone: +34 986 33 04 00 
 
e-mail:  ingrid.murillo@biofabri.es 
 
Scientific Advisor  
Prof. Carlos Martín  
 
Grupo de Genetica de Micobacterias 
 
CIBER de Enfermedades Respiratorias 
 
Departamento de Microbiologia 
 
Facultad de Medicina 
 
Universidad de Zaragoza 
 
C/ Domingo Miral sn 
 
50009 Zaragoza, SPAIN 
 
Tel.:  34   976   76 17 59 
 
Email: carlos@unizar.es 
 
Other Collaborators  
  
Mrs. Danielle Roordink, Stichting TuBerculosis Vaccine Initiative (TBVI), Netherlands 
 
Professor Mark Hatherill, University of Cape Town (UCT), South Africa  
 
Clinical and Regulatory  
TCD, Triclinium Clinical Development (Pty) Ltd. 
Affairs Monitor 
 Design House 
 
 121 Amkor Road 
 
 Pretoria 
 
 0157 
 
 South Africa 

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3 
BACKGROUND INFORMATION AND SCIENTIFIC 
RATIONALE 
3.1 
Background Information 
A new effective tuberculosis (TB) vaccine is essential to achieve World Health Organization 
(WHO) End TB goals and ultimately to eliminate TB by 2050 (1). Bacille Calmette Guerin 
(BCG), currently the only licensed vaccine against TB, protects against disseminated TB 
(miliary and meningitic disease) in children, but provides inconsistent protection against the 
predominant form of pulmonary TB in all age groups. A new improved TB vaccine is needed 
that is effective against all forms of TB disease in infants, children, and adults; and safe in 
immune-compromised persons, including those with HIV infection. One potential vaccination 
approach targets adults, the drivers of the TB epidemic, to prevent transmission within the 
community including to susceptible children. Modeling shows that a moderately effective adult 
TB vaccine would be the single best tool to achieve TB elimination by 2050 (2). However, the 
optimal strategy would be to combine serial mass campaigns in adults, and universal newborn 
vaccination to provide direct protection to susceptible children who experience a high 
incidence rate of TB infection. Implementation of this strategy will require demonstration of 
safety, immunogenicity, and efficacy of a new TB vaccine in both newborns and adults. 
The most robust evidence for vaccine-induced protection against TB disease in humans 
comes from children who received a live attenuated mycobacterial vaccine – BCG, a live-
attenuated Mycobacterium bovis originally isolated from cattle. Systematic review suggests 
that BCG confers variable protection against TB disease in infants and school-age children, 
provided that they have not already been infected with Mycobacterium tuberculosis (or 
potentially exposed to other non-tuberculous mycobacteria) (3). However, the majority of 
adults in countries with high TB endemicity are latently infected (4). Therefore, although a new 
effective adult vaccine would be highly desirable to prevent active TB disease and halt TB 
transmission, adults might be the most difficult population to protect. Newborn infants are the 
only human population without prior mycobacterial exposure in TB endemic countries. 
Therefore, parallel clinical developments of MTBVAC are crucial to provide safety, 
immunogenicity and efficacy data in M. tuberculosis naïve and M. tuberculosis exposed 
populations, in order to deliver an effective, long-term TB vaccine strategy for both adults and 
newborns. 
3.2 
Rationale 
BCG lacks >100 genes when compared to the human TB pathogen M. tuberculosis (5), that 
are thought to be pathogen-beneficial for the successful interaction of M. tuberculosis with the 
human immune system (6,7). Therefore, it is hypothesized that a successful vaccine against 
TB should incorporate several of these M. tuberculosis genes. Within European TB vaccine 
projects and TBVI, Prof. Carlos Martin (Unizar) and Prof. Brigitte Gicquel (Pasteur Institute, 
France) genetically engineered a uniquely attenuated strain, MTBVAC, based on a M. 
tuberculosis clinical isolate of the Euro-American lineage (8), the most widespread and 

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commonly transmitted lineage between humans by the aerosol route (9). The attenuation of 
MTBVAC is based on two independent, stable genetic deletions of two major virulence factors 
in M. tuberculosis: phoP coding for the transcription factor PhoP and fadD26 coding for the 
synthesis of PDIM (8,10,11). MTBVAC contains most of the genes deleted from BCG and 
presents a wider collection of mycobacterial antigens to the host immune system. Live-
attenuated strategies are also considered the best approach for conferring durable T-cell 
immune responses (12). It is our hypothesis that, as a live attenuated M. tuberculosis, 
MTBVAC provides a more durable T-cell response to a wider collection of mycobacterial 
antigens and confers improved protection compared to BCG. The Spanish vaccine 
manufacturer Biofabri has produced and characterized live-attenuated MTBVAC as a freeze-
dried preparation in compliance with GMP, WHO, EMA and FDA recommendations to assure 
the quality, safety and efficacy of BCG vaccines. The final freeze-dried MTBVAC product has 
shown a robust safety, biodistribution and persistence profile comparable to licensed BCG; 
fulfills regulatory requirements; and has shown promising immunogenicity and protective 
efficacy in relevant animal models (8). Recent studies in newborn mice show that MTBVAC is 
safe, does not affect growth and organ development and, notably, confers superior 
immunogenicity and efficacy against pulmonary forms of TB, compared to BCG (13). 
MTBVAC entered a first-in-human Phase 1a trial in 2012. Safety and immunogenicity of three 
escalating dose levels of MTBVAC (5 x 103, 5 x 104, 5 x 105 CFU) was evaluated in comparison 
with BCG SSI in healthy Swiss adults without history of BCG vaccination or exposure to TB or 
HIV (14). Vaccination with MTBVAC demonstrated an excellent safety profile, comparable to 
BCG. MTBVAC was at least as immunogenic as BCG; and at the same dose level as BCG, 
the MTBVAC group showed greater frequency of polyfunctional CD4+ central memory T-cells. 
A transient increase in CFP-10-specific ELISPOT positivity was observed at day 28 in three 
adult participants who received MTBVAC (14). 
Based on these promising first-in-human data, a Phase 1b evaluation of MTBVAC in healthy 
newborns started in 2015 at the SATVI site in South Africa (ClinicalTrials.gov NCT02729571). 
This trial evaluated safety and immunogenicity of three escalating dose levels of MTBVAC 
(2.5 x 103, 2.5 x 104, 2.5 x 105 CFU) for the first time in BCG naïve newborns in a TB endemic 
region. Twenty-eight infants received MTBVAC (9 in each of the first two dose groups, and 10 
in the third), preceded by a pilot safety group of adults, previously BCG-vaccinated at birth, 
who received MTBVAC (2.5 x 105 CFU). Vaccination of both adults and newborns was 
completed without any significant safety signal.  Immunogenicity results indicate that one dose 
of MTBVAC 2.5 x 104 or 2.5 x 105 CFU given within 96 hours of birth induced substantial 
cytokine-expressing (IFN-, IL-2, or TNF-α) antigen-specific CD4 T-cell responses that 
persisted for six months until the final study day. MTBVAC 2.5 x 105 CFU dose evoked the 
greatest magnitude response at day 70 post vaccination, whereas the lowest MTBVAC dose 
of 2.5 x 103 CFU was less immunogenic and did not induce a durable response at day 180. 
Vaccination with MTBVAC at 2.5 x 104 or 2.5 x 105 CFU is immunogenic in neonates from a 
TB endemic setting. 
Data from this Phase 1b infant trial indicates that MTBVAC vaccination may cause interferon 
gamma release assay (IGRA) conversion in infants, unrelated to natural M. tuberculosis 
infection. This phenomenon is analogous to the known effect of BCG vaccination on tuberculin 

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skin test (TST) conversion. Three of 8 (37,5%), six of 8 (75%) and 7 of 9 (77,8%) infants who 
received MTBVAC at 2.5 x 103, 2.5 x 104 and 2.5 x 105 dose respectively, demonstrated a 
positive QuantiFERON (QFT) test 180 days after MTBVAC administration, in the absence of 
known exposure to TB, compared to none of seven BCG recipients. At one year of age none 
of 8 (0%), two of 8 (25%) and 4 of 9 (44%) infants who received MTBVAC at 2.5 x 103, 2.5 x 
104 and 2.5 x 105 dose respectively, demonstrated a positive QFT result. This new data offers 
a strong indication of the immunogenicity of MTBVAC in newborns, which requires 
confirmation in a larger study. Further, since QFT conversion is used as a tool to diagnose M. 
tuberculosis infection in adults and older children, and as an endpoint in exploratory prevention 
of M. tuberculosis infection (POI) vaccine trials, it is critical that the mechanism of MTBVAC-
induced QFT conversion and reversion is fully explored. If MTBVAC is shown to be efficacious 
against TB disease, these data will be important for development of alternative interferon-
gamma release assays (IGRA) for M. tuberculosis infection that are not cross-reactive with 
MTBVAC. 
Biofabri, industrial manufacturer and exclusive licensee of MTBVAC, together with intellectual 
property owner Unizar, has set two independent clinical development pathways for MTBVAC. 
One pathway focuses on development of MTBVAC for prevention of TB disease in previously 
BCG vaccinated adults. This clinical trial is based on the clinical development pathway of 
MTBVAC as a preventive pediatric vaccine that could potentially replace newborn BCG 
vaccination. A Phase 2a dose-defining study is a pre-requisite for selecting optimal dose for 
future efficacy trials of MTBVAC in newborns in TB endemic countries. 
Reflecting the clinical strategy to move straight from the Phase IIa to Phase III study with a 
single selected dose, the design of the proposed Phase III incorporates three case-driven 
assessments for futility and efficacy. Such a pathway for the MTBVAC clinical development 
would allow for the neonatal vaccine to be made available sooner (estimated 5 years) with the 
potential to offer a greater magnitude of protection compared to current BCG vaccine. Such a 
saving in time would have a major public health benefit when considering that the BCG vaccine 
has been reported to have variable protective effect depending on the form of TB and the 
geographical location. The impact of TB in children is significant: in 2019, over one million (1 
190 000) children globally became ill with TB and 230 000 died from the illness and therefore 
it is prudent to develop a more effective vaccine as soon as possible, within safety and 
regulatory requirements.  
3.3 
Potential Risks and Benefits 
3.3.1 Potential Risks 
The safety profile of MTBVAC at the highest dose to be tested (2.5 x 106 CFU) is not yet known 
in infants. Therefore, vaccination of newborns will be staggered to allow gradual evaluation of 
safety and reactogenicity at each escalation of MTBVAC dose. Progression from the 2.5 x105 
to the 2.5 x106 CFU dose level, for which safety in infants has not yet been demonstrated, will 
require that all of the following criteria have been met: (1) no pre-defined pausing rules have 
been observed at lower MTBVAC dose levels in this trial; (2) favourable review of day 28 

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safety data of all lower MTBVAC dose groups by the Data Safety Monitoring Board (DSMB); 
(3) favourable review of safety data from the current pilot Phase 1b infant trial by the DSMB; 
and (4) favourable review of day 28 safety data for the 2.5 x 106 CFU dose level in QFT 
negative adults in the parallel adult trial by the DSMB. In the event that any one of these safety 
criteria are not met, Cohorts 1 and 2 will be expanded so that 33 additional neonates will 
randomly allocated to receive the 2.5 x 104 CFU dose (n=12), the 2.5 x 105 CFU dose (n=13) 
or BCG (n=8) by default. If there is a delay in adult data, dose escalation in infants will either 
be delayed, or Cohorts 1 and 2 be expanded as mentioned above. 
MTBVAC Data Safety Monitoring Board Charter has been created as a guidance. 
It is not yet known whether MTBVAC offers equivalent, better, or worse protection against TB 
compared to BCG. Therefore, measures to minimize risk of TB will be put in place throughout 
the study and, after completion of all Study D365 visits in each cohort and unblinding of the 
clinical team, ‘catch up’ BCG vaccination will be given to all MTBVAC vaccinated infants once 
the last participant of each cohort has performed the D365 visit, provided that the individual 
recipients have a negative screen for active TB disease and new household contacts, and do 
not have a new positive QFT result at D365. Risk of incident TB disease will be minimized by 
exclusion of infants with known TB exposure, including prior maternal or family history. 
Newborns born to HIV infected mothers, with increased risk of TB, will not be enrolled. All 
infants will be screened actively for TB symptoms at each study visit to diagnose TB disease 
early and pre-empt severe morbidity. Infants diagnosed with TB disease will remain in safety 
follow-up, but will be referred immediately for TB treatment, provided free of charge by health 
services. In the previous South African study we saw no confirmed TB cases in either adults 
or infants who received MTBVAC. One adult MTBVAC recipient with recent HIV 
seroconversion was diagnosed with aseptic meningitis and received 6 months treatment for 
unconfirmed TB meningitis. Two infant MTBVAC recipients (one QFT+; one QFT-) received 
TB treatment in the public health system for unconfirmed TB; a rate (2/28; 7%) that is similar 
to the rate of unconfirmed TB in a previous infant trial in this community (13% over 2 years)[29] 
The QFT assay is widely used in older children and adults as a tool to aid in the diagnosis of 
M. tuberculosis infection. Given the preliminary evidence of MTBVAC vaccine induced QFT 
conversion in the current Phase 1b infant study, it is postulated that infants who receive 
MTBVAC will demonstrate QFT conversion events two months after vaccination (D56), 
followed by a high rate of reversion, leading to declining rates of QFT positivity at D182 and 
D365. The same response kinetic was observed for BCG-specific T cell responses in a prior 
longitudinal study in newborns (15). The observed effect of MTBVAC on QFT conversion is 
analogous to the known effect of BCG on false positive TST conversion; MTBVAC might be 
expected to have a similar effect on TST conversion. 
Based on observed rates of QFT positivity 1 year after MTBVAC vaccination in infants, half or 
more of infants who receive MTBVAC may remain QFT positive at end of study and longer. 
However, it will not be possible to differentiate MTBVAC-induced QFT conversion from QFT 
conversion as a consequence of natural M. tuberculosis infection. Therefore, risk of natural 
M. tuberculosis infection will be minimized by exclusion of infants with known TB exposure, 
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MTBVAC administration are more likely to have converted due to vaccine effect than infants 
who demonstrate QFT conversion later during follow-up, who are more likely to have 
converted due to natural M. tuberculosis infection. Early (D56) post-vaccination QFT 
conversion in infants with low risk of natural M. tuberculosis exposure is assumed to be 
vaccine-induced and will not be treated with isoniazid preventive therapy (IPT). Infants who 
demonstrate early QFT conversion at D56 will undergo enhanced screening for potential new 
TB exposure and TB symptoms during all scheduled study visits, and in addition, during 
interval telephonic contacts at D120 and D270. QFT samples will be collected at D182 and 
D365 from infants who tested QFT positive at D56, and stored for later analysis, but since a 
positive QFT result would not be interpretable, these data will not be used for clinical decision-
making. All infants who received MTBVAC and tested QFT positive will receive a card at the 
end of study indicating that they have received an experimental vaccine that may interfere with 
interpretation of future IGRA and TST tests for TB infection.  
QFT assays will be repeated at D182 and D365 in infants who tested QFT negative at D56. 
These results will be made available for clinical decision-making. Infants with new QFT 
conversion events at D182 or D365, and any infant with a reported new household TB contact 
regardless of QFT status, will be referred to the public health services for IPT since these 
infants would potentially benefit from TB prophylaxis against M. tuberculosis infection as per 
SA national guidelines, provided that signs and symptoms of active TB disease are absent.  
Known Potential Benefits 
All infant participants will benefit from enhanced TB screening and facilitated referral and 
improved access to healthcare providers. 

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4 
OBJECTIVES 
4.1 
Study Objectives 
Primary Objectives 
The primary objectives of this study are: 
• 
To evaluate safety and reactogenicity of MTBVAC at escalating dose levels compared to 
BCG vaccine in healthy, BCG naïve, HIV unexposed, South African newborns. 
• 
To evaluate the immunogenicity of MTBVAC at escalating dose levels compared to BCG 
vaccine in healthy, BCG naïve, HIV unexposed, South African newborns. 
Secondary Objective 
The secondary objective of this study is: 
• 
To evaluate QFT conversion rates in neonates receiving escalating dose levels of 
MTBVAC. 
 
Future Exploratory Objective 
Samples will be kept for future analyses:  
• 
To evaluate exploratory immune responses to escalating dose levels of MTBVAC and to 
BCG (including, but not necessarily limited to, analysis of innate response cytokines, 
specific IgG antibody responses, donor-unrestricted T-cell responses). 
• 
To differentiate vaccine-induced immune responses from natural Mtb infection 
 
4.2 
Study Outcome Measures 
4.2.1 Primary Outcome Measures 
The primary outcome measures are: 
Safety and reactogenicity endpoints:  
• Solicited systemic adverse events:  fever, irritability, vomiting, diarrhea, drowsiness, poor 
feeding, skin rash. 
• Solicited injection site reaction adverse events: pain, redness, swelling, ulceration, 
drainage, and regional lymphadenopathy 
• Unsolicited adverse events and serious adverse events 

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Immunogenicity endpoints:  
• Frequencies of antigen-specific IFN-, TNF-, IL-2, IL-17 and/or IL-22-expressing CD4 and 
CD8 T cells measured by 12-hour whole blood (WB) intracellular cytokine staining (ICS) 
assay at Study Days 28, 56, 182, and 365. Stimulating agents at Study Days 28 and 56 will 
include MTBVAC, ESAT-6, CFP-10, EspC and a “megapool” of peptides containing 
immunodominant M. tuberculosis epitopes (16). Stimulating agents at Study Days 182 and 
365 will include MTBVAC, ESAT-6, CFP-10, EspC and a “megapool” of peptides containing 
immunodominant M. tuberculosis epitopes (16) as well as a modified version of the 
“megapool” but containing only those epitopes shared by both BCG and MTBVAC. 
4.2.2 Secondary Outcome Measures 
The secondary outcome measures are: 
• Quantitative and qualitative results of the QFT Gold Plus assay at Study Day 56, 182 and 
365. 
 
 

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5 
STUDY DESIGN 
This double-blind, randomized, active controlled, MTBVAC dose-finding trial will evaluate the 
safety, reactogenicity, immunogenicity, and potential for QFT conversion and reversion of the 
candidate TB vaccine MTBVAC in HIV uninfected BCG naïve newborns, administered in 3 
cohorts at a single intradermal dose of 2.5 x 104 CFU (Cohort 1, n=25), 2.5 x 105 CFU (Cohort 
2, n=25), and either 2.5 x 106 CFU (n=25) or 2.5 x 105 CFU (n=13) plus 2.5 x 104 CFU (n=12) 
(Cohort 3),  compared to BCG vaccine (24 BCG; 75 MTBVAC). 
 
Three dose levels of MTBVAC (2.5 x 103, 2.5 x 104, 2.5 x 105 CFU) have been tested in the 
current Phase 1b trial in healthy, BCG-naïve, South African newborns; and four dose levels (5 
x 103, 5 x 104, 5 x105, and 5 x 106 CFU) are being tested in a parallel trial among M. 
tuberculosis infected and uninfected South African adults. It is our hypothesis that the optimal 
safe and immunogenic MTBVAC dose for infants lies in the range of 2.5 x 104 (10-fold lower 
than BCG) to 2.5 x 106 CFU (10-fold higher than BCG). Therefore, this proposed infant trial 
aims to define the MTBVAC dose-response curve above and below the optimal MTBVAC 
dose, straddling a 3-log dose range. Final selection of the MTBVAC dose for use in the 3rd 
cohort will be data driven, based upon the most recent safety and immunogenicity data from 
all ongoing and completed trials of MTBVAC in infants and adults at that time. Infant safety 
data are not yet available for the highest (2.5 x 106 CFU) MTBVAC dose. Therefore, selection 
of the 2.5 x 106 CFU MTBVAC dose would be contingent upon demonstration of an acceptable 
safety profile (as defined in Section 3.3.1), both for all lower MTBVAC doses in infants, and 
for the 5 x 106 CFU MTBVAC dose in QFT negative adults (data expected end 2018/beginning 
2019). If these safety criteria for the highest MTBVAC dose are not fulfilled, the 2.5 x 105 CFU 
and 2.5 x 104 CFU MTBVAC doses would be selected for the last cohort, as described in 
Section 3.3.1. This approach will build upon data from the current infant pilot study and should 
allow detailed comparison of safety and immunogenicity of the postulated optimal MTBVAC 
dose within a 3-log dose range, in a larger infant study population. Refer to Figure 5-1. 
 
In addition, given the crucial importance of understanding the immunological basis for 
MTBVAC vaccine-induced QFT conversion, and defining the time course and magnitude (IFN-
g concentration) of MTBVAC vaccine-induced QFT conversion and reversion events, serial 
QFT assays will be performed in parallel with whole blood T cell assays. 
 
 
 

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Figure 5-1 
Study Flow Diagram 
 
 
 

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6 
STUDY ENROLLMENT AND WITHDRAWAL 
6.1 
Participant Inclusion Criteria  
Participants must meet all of the following criteria at the time of randomization: 
1. 
Male or female neonates within 96 hours of birth. 
2. 
Written informed maternal consent, including permission to access maternal antenatal, 
postnatal and infant medical records. 
3. 
Infant participants and their caregivers available for study follow-up and display the 
willingness and capacity to comply with study procedures. 
4. 
Neonates must be in good general health during pregnancy and delivery, as assessed by 
medical history and focused physical examination. 
5. 
Birth weight ≥ 2450 grams. 
6. 
Apgar score at 5 minutes ≥ 7. 
7. 
A maternal HIV test result (rapid test, ELISA or PCR) taken within 30 days of delivery 
must be available, documented and negative. 
8. 
Estimated gestational age ≥ 37 weeks. 
9. 
Mother has not participated in a clinical trial within 3 months prior to the infant’s birth, and 
if breast-feeding, mother should not participate in a clinical trial during the 12 months of 
study follow up.  
10. Mother has never participated in a TB vaccine trial before. 
11. Infant may not participate in any other clinical trial from birth, and for the 12 months of 
follow up. 
6.2 
Participant Exclusion Criteria  
Participants must have none of the following at the time of randomization: 
1. 
Participant must not have received BCG vaccination prior to enrolment. 
2. 
Significant antenatal or intrapartum complications that may affect the health of the 
neonate. 
3. 
Skin condition, bruising or birth mark at the intended injection site. 
4. 
Maternal HIV test (rapid test, ELISA or PCR) not performed within 30 days of delivery, 
HIV test results not available, or HIV test result known positive. 
5. 
History of close contact with a TB patient, antenatal or postnatal, whether maternal, other 
family member or another household member who has not yet completed TB treatment. 
6. 
Clinically suspected neonatal sepsis. 
7. 
Any severe congenital malformation. 
8. 
History or evidence of any systemic disease on examination, or any illness that in the 
opinion of the investigator may interfere with the evaluation of the safety and 
immunogenicity of the vaccine. Neonatal jaundice not considered clinically significant is 
not an exclusion. 

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6.3 
Treatment Assignment Procedures 
6.3.1 Randomization Procedures 
Participants will be randomized using a pre-prepared block randomization schedule, linked to 
study enrolment number. 
Ninety-nine (99) infant participants will be recruited, randomized and allocated into 3 groups: 
BCG (single dose level 2.5 x 105 CFU/0.05 mL, n=24); or MTBVAC at three different dose 
levels as a single intradermal dose of 2.5 x 104 CFU (Cohort 1, n=25), 2.5 x 105 CFU (Cohort 
2, n=25), and either 2.5 x 106 CFU (n=25) or 2.5 x 105 CFU (n=13) plus 2.5 x 104 CFU (n=12) 
(Cohort 3) (24 BCG; 75 MTBVAC).   
Vaccination of newborns will be staggered to allow gradual evaluation of safety and 
reactogenicity at each escalation of MTBVAC dose. Progression from the 2.5 x 105 to the 2.5 
x 106 CFU dose level, for which safety has not yet been demonstrated, will require that all of 
the following criteria have been met: (1) no pre-defined pausing rules have been observed at 
lower MTBVAC dose levels in this trial; (2) favourable review of day 28 safety data of all lower 
MTBVAC dose groups by the Data Safety Monitoring Board (DSMB); (3) favourable review of 
safety data from the current pilot Phase 1b infant trial by the DSMB; and (4) favourable review 
of day 28 safety data for the 2.5 x 106 CFU dose level in QFT negative adults in the parallel 
adult trial by the DSMB. In the event that any one of these safety criteria are not met, Cohorts 
1 and 2 will be expanded so that 33 additional neonates will randomly allocated to receive the 
2.5 x 104 CFU dose (n=12), the 2.5 x 105 CFU dose (n=13) or BCG (n=8) by default. If there 
is a delay in adult data, dose escalation in infants will either be delayed, or Cohorts 1 and 2 
be expanded as mentioned above. 
6.3.2 Masking Procedures 
MTBVAC and BCG vaccine will be prepared, and allocation concealed by the site pharmacist. 
All other site staff will be blinded to vaccine allocation, throughout the follow-up period. 
Individually-sealed code break envelopes will be held by the pharmacist in case emergency 
unblinding is necessary. 
Data pertaining to the MTBVAC vaccine and to BCG control will be collected in an observer-
blinded manner. By observer blinded, we mean that during the course of the study the vaccine 
recipient and those responsible for the evaluation of safety and reactogenicity study 
parameters, will all be unaware of which vaccine preparation was administered to a particular 
participant. To do so, the MTBVAC vaccine and BCG control will be prepared and blinded by 
the pharmacist, whereas, the vaccination will be administered by the Vaccination Nurse, a 
research nurse not involved in follow up and safety evaluations of participants. The principal 
investigator and the sub-investigators will be responsible for assessing safety and 
reactogenicity.  Blinding will be maintained throughout the vaccination and follow-up portions 
of the vaccine trial. It is not yet known whether MTBVAC offers equivalent or better protection 
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throughout the study and, after completion of all Study D365 visits in each cohort and 
unblinding of the clinical team, ‘catch up’ BCG vaccination will be given to all MTBVAC 
vaccinated infants once the last participant of each cohort performed the Post D365 visit.  
No set of individual codes will be held at Biofabri’s Headquarters. Biofabri’s Headquarters will 
be able to access the individual randomization code from the SATVI Pharmacy randomization 
register. The code will be broken by the SATVI Pharmacist (Study Contact for Emergency 
Code Break) only in the case of medical events that the investigator/physician in charge of the 
participant feels cannot be treated without knowing the identity of the study vaccine(s). 
Biofabri’s policy (incorporating ICH E2A guidance, EU Clinical Trial Directive) is to unblind any 
serious adverse event (SAE) report associated with the use of the investigational product, 
which is unexpected and attributable/suspected, prior to regulatory reporting. The SATVI 
Pharmacist is responsible for unblinding the treatment assignment in accordance with 
specified time frames for expedited reporting of SAEs (Refer to Section 10.9). 
6.3.3 Reasons for Withdrawal 
Participants are free to withdraw from participating in the study at any time. 
Rules governing the pausing and stopping of investigational product administration between 
individuals within a single cohort and between cohorts are presented in Section 10.5. 
6.3.4 Handling of Withdrawals 
Participants who withdraw from participation in the study will be asked to complete a final visit 
(see Schedule of Events in Appendix A for procedures to be conducted at next visit, which 
depend on timing of discontinuation from the study). Participants who discontinue the study 
will not be replaced. 
6.3.5 Termination of Study  
Rules for pausing or stopping the study are presented in Section 10.5. 

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7 
STUDY INTERVENTION/INVESTIGATIONAL PRODUCT 
7.1 
Study Product Description 
7.1.1 Acquisition 
MTBVAC (live attenuated derivative of Mtb) was developed at University of Zaragoza, Spain, 
by Prof. Carlos Martin, and licensed to Biofabri, SL. MTBVAC and excipient solution for dilution 
will be supplied by Biofabri. 
BCG vaccine (BCG SSI, a WHO pre-qualified BCG vaccine) 
Formulation, Packaging, and Labeling 
The composition of MTBVAC is shown in Table 7-1. Refer to the most recent version of the 
Vaccine Management Manual for further details on formulation and packaging for MTBVAC. 
 
Table 7-1 
Composition of MTBVAC Study Vaccine 
 
 
Vaccine Dosea 
Formulation 
(approximately per 
standard dose) 
 
 
Presentation 
 
 
Volume 
MTBVAC 
2.5 x 105 
1.5 – 8.5 x 105 CFU 
Sucrose  
Sodium glutamate  
Lyophilized pellet in 
vials 
(20 doses) 
0.05 mL/doseb 
MTBVAC 
2.5 x 106 
1.5 – 8.5 x 106 CFU 
Sucrose  
Sodium glutamate  
Lyophilized pellet in 
vials 
(20 doses) 
0.05 mL/doseb 
a MTBVAC 2.5 x 104 dose to be prepared by dilution with excipient solution of 2.5 x 105 
formulation. 
b After reconstitution with 1 mL/vial sterilized water for injection 
Refer to the package insert for formulation details of BCG. 
7.1.2 Product Storage and Stability 
Upon receipt of investigational product, the study vaccine manager must immediately inspect 
all vials for damage. Investigational product will be shipped with a continuous temperature-
monitoring device. Any damage or discrepancies from the packing list must be documented 
and promptly discussed with Biofabri and the study monitor to determine the appropriate 
action.  
MTBVAC must be stored at -15 to -30C in a secured location with no access for unauthorized 
personnel. MTBVAC excipient solution must be stored at 2-8C. 
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Refer to the most recent version of the Vaccine Management Manual for detailed instructions 
regarding investigational product storage. 
7.2 
Dosage, Preparation and Administration of Study 
Intervention/Investigational Product 
The administered volume is 0.05 mL/dose for all doses of investigational product and BCG. 
Refer to the most recent version of the Vaccine Management Manual for detailed instructions 
regarding investigational product preparation. Refer to Section 6.3.2 for masking procedures. 
Participants will be administered one dose of the MTBVAC vaccine or BCG active control on 
Study Day 0. The vaccine dose will be administered intradermally in the deltoid area. Opsite 
plaster (breathable) will be used to cover the site of injection immediately after vaccination; 
the plaster will be removed for the 60 minutes post-vaccination injection site examination and 
then replaced. The participant’s caregiver will be provided with additional plasters and will be 
instructed to change the plaster as needed. Participants will be instructed to protect the plaster 
and site of vaccination from water. Participants will be provided with a plastic sample bag and 
clear instructions on how to replace the plaster and will be asked to retain used plasters in the 
provided plastic bag and return it to clinic for destruction. 
Participants will be observed closely for at least 60 minutes following the administration of 
vaccines, with appropriate medical treatment readily available in case of a rare anaphylactic 
reaction following the administration of vaccines. 
7.3 
Accountability Procedures for the Study 
Intervention/Investigational Product(s) 
MTBVAC will be shipped from Biofabri to the clinical trial site. The frequency of product 
distribution and amount of product distributed will depend on the rate of enrollment of study 
participants. A WHO pre-qualified BCG vaccine will be supplied by the Sponsor from a 
registered local supplier or imported under the applicable South African Medicines Control 
Council regulations. 
The study vaccine manager is required to maintain accurate investigational product 
accountability records. Instructions and forms to be completed and kept for accountability will 
be provided to the study vaccine manager. If the study vaccine manager wishes to use clinical 
trial site-specific accountability forms, these must be reviewed and approved in advance by 
Biofabri. Upon completion of the study, all investigational product management records will be 
copied, and the copies returned to Biofabri or its designee. The originals must be maintained 
at the clinical trial site with the rest of the study records. 
Biofabri must provide authorization for the disposition of any unused investigational product 
or BCG. Unused investigational product will be destroyed according to the facility’s SOPs. Any 
disposal of investigational product conducted at the clinical trial site must be documented in 
the study file. 

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7.4 
Assessment 
of 
Participant 
Compliance 
with 
Study 
Intervention/Investigational Product  
The vaccines will be administered at the Vaccination clinic, SATVI, under the supervision of 
investigators or nurses trained in the management of anaphylactic reactions. 
7.5 
Concomitant Medications/Treatments 
The collection of information on concomitant medications used by participants following 
vaccination will coincide with the collection period of adverse events (see Section 10.1). In 
addition, information on all concomitant medications associated with the treatment of adverse 
events and serious adverse events will be collected throughout the study. 
Concomitant medication includes prescription and non-prescription drugs or other treatments, 
and any vaccines other than the investigational product. The name of the medication, 
treatment start and stop dates (or ‘ongoing’), and indication must be recorded on the 
Concomitant Medications electronic case report form (eCRF). The indication recorded on the 
Concomitant Medications eCRF must correspond to a medical term/diagnosis recorded on the 
Adverse Event (AE) eCRF, or to a pre-existing condition noted in the participant’s medical 
history, or be noted as prophylaxis, e.g., dietary supplement or routine vaccinations. 

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8 
STUDY SCHEDULE  
A Schedule of Events depicting all visit-specific procedures is provided in Appendix A, 
including the acceptable time windows. Detailed descriptions of procedures to be carried out 
at each visit are presented in Sections 0 through 8.6. 
All participants who are assigned a participant identification number and receive 
investigational product will be followed according to the protocol unless consent is withdrawn.  
Participants will be instructed to contact a study team member to report new signs and 
symptoms or new or worsening adverse events and will be referred for medical attention as 
applicable. 
During each clinic visit, participants will be reminded to notify a study team member of the 
following:  
• 
The occurrence of AEs and SAEs during the respective reporting periods.  
• 
Receipt of any concomitant medications during the applicable reporting period. 
• 
Plans to move or if contact information changes. 
• 
If participant has decided to withdraw from the study. 
• 
Change in general health status.  
• 
Any other change in status that may affect the participant’s participation (e.g. plan to 
participate in another investigational study). 
If the clinical trial site’s study team members are unable to establish contact with a participant 
who misses a scheduled study visit, the clinical trial site must make every possible effort to re-
establish contact and document such efforts. If contact is re-established, then the participant 
will resume participation in the study. If contact with the participant cannot be re-established 
by the participant’s calculated Study Day 365 visit date, then a determination of “lost to follow-
up” should be made on the date of the last attended study visit. 
Upon completion of the study, participants will be provided a letter and a card that briefly 
describe their participation in the study.  
8.1 
Screening  
Written informed consent must be obtained before any screening procedures are performed. 
 
Pregnant women will be approached during the third trimester of their pregnancy at the time 
of their antenatal visits to the state public healthcare clinics. Information on the trial will be 
presented to them, and if interested, they will be invited to undergo an informed consent 
session in their language of choice and asked to sign an Informed Consent Form (ICF). 
Informed consent sessions will be conducted in a private space conducive to confidential 
discussion, at the clinic, at the participant’s home, or at the SATVI site. Where feasible, at 
least 1 further antenatal contact will be made by the study team with the mother-to-be to 
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and her pregnancy. Clinic and antenatal clinic cards will be marked with an easily identifiable 
sticker or marking to enable the birthing unit staff to identify potential study participants.  
 
All women are offered HIV testing per standard of care at the antenatal clinics. After signing 
the ICF, study staff will request access to the HIV result as recorded in the clinic notes, as well 
as other relevant medical history from medical records. If the pregnant woman has declined 
to be tested or if the result is unavailable, or if the result is positive, her newborn will not be 
enrolled into the study. Counseling and treatment of HIV positive mothers will be provided per 
standard of care by the state healthcare system according to national guidelines. The ICF also 
includes as separate documents permission to photograph abnormal injection site reactions 
and to perform investigations for TB when considered necessary by an investigator. 
 
When women who have consented present in labour to the birthing unit, the study team will 
be notified by the mother-to-be on a dedicated SATVI telephone number provided to her 
antenatally, as well as by the birthing unit staff when they identify her newborn child as a study 
participant from information on her antenatal record. After delivery, a study research nurse will 
go to the birthing unit in person and will confirm with the woman that she still wishes to enroll 
her child into the trial and will apply the inclusion and exclusion criteria and ensure that routine 
BCG administration by the clinic staff is postponed. The mother will be given a date when she 
should attend the clinic for the study day 0 visit. If the mother’s discharge from the birthing unit 
is delayed beyond 96 hours the infant will not be eligible for the study. 
Screening procedures 
• 
Maternal: 
Written informed consent 
Assign study participant identification (PID) number  
Medical history 
Confirm HIV status (mother to be tested if result is not available or if the test was not 
done within 30 days prior to delivery) 
History of active TB or household TB contact 
 
• 
Infant: 
On Road to Health Card: confirm no BCG given at birthing unit, confirm birth weight, 
gestational age and Apgar score meet eligibility criteria. 
8.2 
Enrollment/Baseline 
Study Day 0 (within 96 hours of birth) 
Pre-vaccination: 
1. Verbally reconfirm maternal consent. 
2. Confirm absence of acute illness in infant. 
3. Medical history and update of concomitant medication. 
4. Screening for potential new TB exposure and TB symptoms.  
5. Vital signs: 

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− Heart rate, respiratory rate, weight, recumbent length. 
− Axillary temperature: must be <38°C. 
6. History and physical examination by investigator. 
7. Confirm eligibility for study entry and vaccination. 
8. Complete Vaccine Request Form, send to study vaccine manager (study pharmacist) 
who will randomise participant into a study arm according to the next available treatment 
number in the final randomisation schedule, and then prepare study vaccine. 
 
Vaccination (by Vaccination Nurse): 
1. Inspect syringe and vaccine volume, confirm PID number, date and time of dose 
preparation.  
2. Administer study vaccine intradermally into left deltoid area of the infant’s arm. Record 
date and time of vaccination, presence of a bleb. Apply Opsite plaster. 
 
Post-vaccination: 
1. Monitor participant for acute adverse events for at least 60 minutes after vaccination. 
2. Obtain 60±10 minutes post-immunization vital signs (pulse, axillary temperature). 
3. Examine site of injection 60±10 minutes post-immunization (includes measurement of 
redness, swelling, and ulceration); replace Opsite plaster. 
4. Record any adverse events, including systemic solicited adverse events, unsolicited 
adverse events, solicited and unsolicited injection site reactions, and serious adverse 
events (including concomitant medications). 
5. Distribute and review diary card and diary card instructions with participant; review 
injection site care instructions and distribute Opsite plasters and plastic Ziploc bags. 
8.3 
Follow-up  
Study Day 7 
Allowable window for clinic visit is 7±3 days from Study Day 0 visit date. 
1. Verbally reconfirm consent. 
2. Update medical history including use of medications.  
3. Screening for potential new TB exposure and TB symptoms. 
4. Record feeding practice.  
5. Vital signs (heart rate, respiratory rate, axillary temperature, weight and recumbent 
length). 
6. Focused physical examination. 
7. Collect and review solicited AE and medication diary card entries for completeness 
and accuracy. Redistribute AE and medication diary card. 
8. Record any adverse events, including systemic solicited adverse events, unsolicited 
adverse events, solicited and unsolicited injection site reactions, and serious adverse 
events (including concomitant medications).  
9. Examine site of injection (includes measurement of redness, swelling, and ulceration) 
and take a pus swab if discharge present. 

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10. Blood collection for: 
− 
Serum chemistry: includes AST, ALT, GGT, total bilirubin, creatinine.  
− 
Hematology: includes full blood count with differential, and platelet count.  
Study Day 28 
Allowable window for clinic visit is 28±3 days from Study Day 0 visit date. 
1. Verbally reconfirm consent. 
2. Update medical history including use of medications.  
3. Screening for potential new TB exposure and TB symptoms. 
4. Record feeding practice.  
5. Vital signs (heart rate, respiratory rate, axillary temperature, weight and recumbent 
length). 
6. Focused physical examination. 
7. Collect and review solicited AE and medication diary card entries for completeness 
and accuracy. 
8. Record any adverse events, including systemic solicited adverse events, unsolicited 
adverse events, solicited and unsolicited injection site reactions, and serious adverse 
events (including concomitant medications).  
9. Examine site of injection (includes measurement of redness, swelling, and ulceration) 
and take a pus swab if discharge present. 
10. Blood collection for: 
− 
Immunogenicity WB-ICS 
 
Study Day 56 
Allowable window for clinic visit is 56±7 days from Study Day 0 visit date. 
1. Verbally reconfirm consent. 
2. Update medical history including use of medications.  
3. Screening for potential new TB exposure and TB symptoms. 
4. Record feeding practice.  
5. Vital signs (heart rate, respiratory rate, axillary temperature, weight and recumbent 
length). 
6. Focused physical examination. 
7. Record any adverse events, including unsolicited adverse events, solicited and 
unsolicited injection site reactions, and serious adverse events (including concomitant 
medications).  
8. Examine site of injection (includes measurement of redness, swelling, and ulceration) 
and take a pus swab if discharge present. 
9. Blood collection for: 
− 
Immunogenicity WB-ICS 
− 
QuantiFERON®-TB Gold Plus test 

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Study Day 120  ±7 days and Study Day 270 ±7 days 
Home / telephonic visit if infant tested QFT positive at D56 to enquire about TB signs and 
symptoms and general well-being 
 
Study Day 182 
Allowable window for clinic visit is 182 ±14 days from Study Day 0 visit date. 
1. Verbally reconfirm consent. 
2. Update medical history including use of medications.  
3. Screening for potential new TB exposure and TB symptoms. 
4. Record feeding practice.  
5. Vital signs (heart rate, respiratory rate, axillary temperature, weight and recumbent 
length). 
6. Focused physical examination. 
7. Record any adverse events, including unsolicited adverse events and serious adverse 
events (including concomitant medications).  
8. Examine site of injection (includes measurement of redness, swelling, and ulceration) 
and take a pus swab if discharge present. 
9. Blood collection for: 
− 
Immunogenicity WB-ICS 
− 
QuantiFERON®-TB Gold Plus  
 
Partial Cohort Unblinding of Data Post Study Day 182 
 
Once the last Cohort 3 Study D182 visit has been completed, an independent biostatistician 
be unblinded to available study data. At this point Cohort 1 and Cohort 2 participants will have 
completed all study visits and these participants and clinical team will be unblinded post Study 
D365 visit. All available safety data for all cohorts up to the last Cohort 3 Study D182 visit 
completion; and immunogenicity data for all cohorts up to each cohort’s Study D182 visit, will 
be analysed by the independent, unblinded biostatistician and provided to DSMB for review to 
determine MTBVAC dose selection for the Phase III study.  The Sponsor will review blinded 
data only in order to keep the blind until post Study D365 visit. The DSMB will be provided 
with the same blinded data along with unblinding vaccine recipient lists. The DSMB review will 
allow for a blinded recommendation and input into the dose selection for the Phase III study. 
For all cohorts, unblinding of the clinical team and participants will take place post Study D365 
visit. 
 
Note: The immunology team will remain blinded until immunology database lock. 

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8.4 
Final Study Visit 
Study Day 365 
Allowable window for clinic visit is 365 ±14 days from Study Day 0 visit date. 
1. Verbally reconfirm consent. 
2. Update medical history including use of medications.  
3. Screening for potential new TB exposure and TB symptoms. 
4. Record feeding practice.  
5. Vital signs (heart rate, respiratory rate, axillary temperature, weight and recumbent 
length). 
6. Focused physical examination. 
7. Record any serious adverse events (including concomitant medications) and 
unsolicited adverse events.  
8. Examine site of injection (includes measurement of redness, swelling, and ulceration) 
and take a pus swab if discharge present. 
9. Blood collection for: 
− 
Immunogenicity WB-ICS 
− 
QuantiFERON®-TB Gold Plus  
 
Post Cohort Unblinding of Participants Post Study Day 365 
 
Participant, and subsequent clinical team unblinding will occur when all infants in a given 
cohort have attained their Study Day 365 visit.  
 
1. MTBVAC recipients who have tested QFT positive will be given a card stating that they 
have received an investigational vaccine that may interfere with interpretation of future 
IGRA and TST tests. 
2. BCG rescue dose will be offered to all MTBVAC recipients in this cohort, given that the 
individual recipients have a negative screen for active TB disease and new household 
contacts, and do not have a new positive QFT result at D365.  Any MTBVAC recipient 
receiving IPT will be offered the BCG rescue dose after completion of IPT.  
3. The site will contact the participant’s caregiver telephonically (or perform a home visit 
if no telephone available) 14 days post vaccination with the BCG rescue dose to 
enquire about the infant’s wellbeing and BCG injection site reaction, and respond 
appropriately to any adverse reaction.  
 
Note: The immunology team will remain blinded until immunology database lock. 
8.5 
Early Termination Visit 
Visit to be conducted if participant discontinues from study prior to Study Day 365. 
1. Update medical history including use of medications.  
2. Screening for potential new TB exposure and TB symptoms. 
3. Record feeding practice.  

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4. Vital signs (heart rate, respiratory rate, axillary temperature, weight and recumbent 
length). 
5. Focused physical examination. 
6. Record any adverse events, including unsolicited adverse events, and serious adverse 
events (including concomitant medications).  
7. Examine site of injection (includes measurement of redness, swelling, and ulceration) 
and take a pus swab if discharge present. 
8. Blood collection for: 
− 
QuantiFERON-PLUS TB  
− 
Confirm contact details for possible BCG rescue dose once all infants in cohort 
have completed D365 visit. 
 
8.6 
Unscheduled Visit  
For emergencies and other unscheduled visits to a medical facility other than the study clinic, 
medical records will, to the extent possible, be obtained by the investigator. 
Should visits to the clinical trial site occur outside of the scheduled study visits, these will be 
documented in the source notes as an unscheduled visit and captured in the Electronic Data 
Capture system as an unscheduled visit. Data will be reviewed as per all scheduled visits. 

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9 
STUDY PROCEDURES/EVALUATIONS 
9.1 
Clinical Evaluations 
Participants will have vital signs taken prior to study vaccination. Participants will remain in the 
clinic under close observation for at least 60 minutes after receiving investigational product. 
Vital signs will be repeated at 60±10 minutes post-vaccination before participants leave the 
clinic. Allergic reactions to vaccination are possible; therefore, appropriate paediatric drugs 
and medical equipment to treat acute paediatric anaphylactic reactions must be immediately 
available, and a medically qualified study team member trained to recognize and treat 
anaphylaxis must be present in the clinic during the entire vaccination procedure and post-
vaccination monitoring period. 
Clinical assessments to be performed at each visit are summarized in Appendix A and listed 
by visit in Section 8. 
Participants’ caregivers will receive, and be instructed in, the operation of a daily adverse 
event diary, ruler (for measurement of redness, swelling, and ulceration), and a digital 
thermometer to measure axillary temperature, to be used during the 7 days post-vaccination 
diary period after investigational product administration. The daily adverse event diary is a tool 
to help aid the principal investigator or designee to engage in a conversation with the 
participant’s caregiver about any AEs that may have occurred between visits. During 
scheduled visits through the specified diary period and after investigational product 
administration, the daily diary will be collected and reviewed by the principal investigator or 
designee at which time any clinical details required for complete understanding of the 
information recorded will be obtained. If possible, diaries not brought to the scheduled visit 
should be obtained before adverse event assessment can be performed and events discussed 
with the principal investigator or designee. If a diary is lost, the principal investigator or 
designee will discuss the occurrence of any solicited adverse events with the participant’s 
caregiver and document the discussion. Information on the diary card will not be directly 
recorded onto eCRFs. The diary card will be considered source documentation and adverse 
event information obtained from the diary card will be recorded and completely assessed on 
the adverse event eCRF. Axillary temperatures below 38C will not be considered fevers. 
Any change to an observation or event recorded by the participant’s caregiver on the diary 
card (e.g., the severity level of an event is changed after interviewing the participant’s 
caregiver), based on the investigator’s evaluation of the event, must be explained by notation 
in source documentation by the principal investigator or designee. 
9.1.1 Screening for TB and referral for Isoniazid Preventive Therapy 
All infants will undergo screening for potential new TB exposure and TB symptoms at each 
visit and caregivers will be encouraged to report such symptoms or any new household TB 
contact if they occur in between visits. If TB symptoms or potential new TB exposure are 

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detected these infants will be investigated for active TB disease and may be referred to the 
public health clinic for initiation of TB treatment if indicated or IPT if active TB is ruled out.  
Early (D56) post-vaccination QFT conversion in infants with low risk of natural M. tuberculosis 
exposure is assumed to be vaccine-induced and will not be treated with isoniazid preventive 
therapy (IPT). Infants who demonstrate early QFT conversion at D56 will undergo enhanced 
screening for potential new TB exposure and TB symptoms during all scheduled study visits, 
and in addition, during interval telephonic contacts at D120 and D270. QFT samples will be 
collected at D182 and D365 from infants who tested QFT positive at D56, and stored for later 
analysis, but since a positive QFT result would not be interpretable, these data will not be used 
for clinical decision-making. All infants who received MTBVAC and tested QFT positive will 
receive a card at the end of study indicating that they have received an experimental vaccine 
that may interfere with interpretation of future IGRA and TST tests for TB infection..  
QFT assays will be repeated at D182 and D365 in infants who tested QFT negative at D56. 
These results will be made available for clinical decision-making. Infants with new QFT 
conversion events at D182 or D365, and any infant with a reported new household TB contact 
regardless of QFT status, will be referred to the public health services for IPT since these 
infants would potentially benefit from TB prophylaxis against M. tuberculosis infection as per 
SA national guidelines, provided that signs and symptoms of active TB disease are absent.  
 
9.2 
Laboratory Evaluations 
9.2.1 Clinical Laboratory Evaluations  
Laboratory tests to be performed at each visit are summarized in Appendix A (including 
approximate phlebotomy volumes) and listed by visit in Section 8. 
9.2.1.1.1. Special Assays or Procedures 
A summary of immunologic assays to be performed on blood specimens is shown in Table 
9-1.  
 

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Table 9-1 
Summary of Immunology Laboratory Evaluations 
 
 
 
Sample Type 
 
 
 
Assay 
 
 
Immunology 
Endpoint 
Approximate 
Blood 
Volume 
(per Visit) 
 
 
 
Study Days 
Name and 
Location of 
Analysis 
Laboratory 
Whole blood 
12-hour WB ICSa 
for analysis of T cell 
responses 
Primary 
 
2 to 4 mLb 
28, 56, 182, 365 
SATVI, South 
Africa 
QFT Gold Plus QFT conversion by 
MTBVAC 
Secondary,c 
4 mL 
56, 182, 365 
SATVI, South 
Africa 
Supernatants 
from whole 
blood ICS 
assay 
ELISA/ Multiplex 
bead array for 
analysis of innate 
response cytokines 
Future Exploratory  
Not 
applicable 
28, 56, 182, 365 
SATVI, South 
Africa  
 
a. Stimulation conditions for days 28, 56, 182 and 365 include MTBVAC, CFP-10, ESAT-6, EspC and a 
“megapool” of peptides containing immunodominant M. tuberculosis epitopes, Nil and PHA. The higher 
blood volume at days 182 and 365 allows for an additional stimulation condition of a modified version of 
the “megapool” but containing only those epitopes shared by both BCG and MTBVAC to be added.  
b. Depending on age of infant as explained in footnote to Schedule of Events (Appendix A) 
c. If QFT positive at D56, D182 and D365 QFT samples will be stored for later analysis 
9.2.2 Specimen Preparation, Handling, and Shipping 
9.2.2.1 Instructions for Specimen Preparation, Handling, and Storage 
Staff at the clinical trial site will refer to the most current version of the Laboratory Manual 
(provided under separate cover) for further instructions and additional information on 
specimen collection and processing. 
9.2.2.2 Specimen Shipment 
Staff at the clinical trial site will refer to the most current version of the Laboratory Manual 
(provided under separate cover) for further instructions and additional information on 
specimen shipping. 

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10 
ASSESSMENT OF SAFETY 
Responsibilities for Ensuring the Safety of Trial Participants 
The national regulatory authority, the study sponsor (Biofabri), the institution through which 
the research is performed, and all members of the principal investigator’s clinical team, share 
responsibility for ensuring that participants in this trial are exposed to the least possible risk of 
adverse events that may result from participation in this protocol.  
Principal Investigator 
The principal investigator has a personal responsibility to closely monitor trial participants and 
an inherent authority to take whatever measures necessary to ensure their safety. The 
principal investigator has the authority to terminate, suspend or require changes to a clinical 
trial for safety concerns and may delay a participant’s investigational product administration 
or pause investigational product administration in the whole trial if the investigator has some 
suspicion that the investigational product might place a participant at significant risk. The 
principal investigator determines severity and causality with respect to the investigational 
product for each adverse event.  
Responsibilities of the principal investigator may be assigned to a designee who is a medically 
qualified team member (who must be a sub-investigator in the case of assessment of adverse 
events); however, the accountability for the specific task remains with the principal 
investigator. 
Study Sponsor 
The sponsor (Biofabri) also has an institutional responsibility to ensure participant safety. This 
responsibility is vested in the local medical monitor and a DSMB (see Section 10.7).  
Local Medical Monitor 
The local medical monitor is a credentialed physician or surgeon in their country of residence 
with the necessary expertise to act in such capacity. The local medical monitor reviews the 
safety of the product for protocols in a specific region and, in conjunction with the sponsor, 
determines expectedness of related serious adverse events. The local medical monitor, in 
consultation with the sponsor, may assess the causality for adverse events and may upgrade 
or downgrade the causality determined by the principal investigator. The local medical monitor, 
like the principal investigator, is blinded. 
Institutional Review Boards and Ethics Committees 
The IRB/IEC has institutional responsibility for the rights, safety, and welfare of participants in 
clinical trials. The IRB/IEC has the authority to terminate, suspend or require changes to a 
clinical trial.  

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National Regulatory Authority 
Since the national regulatory authority receives all expedited safety reports, it also has the 
authority to terminate, suspend or require changes to a clinical trial.  
10.1 Specification of Safety Parameters 
Safety parameters that constitute the primary outcome measures of the study are described 
in Section 4.2.1. The time periods for collection of adverse events are as follows. 
Unsolicited Adverse Events 
Unsolicited adverse events will be collected from informed consent until trial completion.  
Solicited Adverse Events 
Solicited systemic adverse events will be collected post-vaccination through the Study Day 28 
visit (with diary cards to be used for 7 days after vaccination).  
Solicited injection site reactions will be collected post-vaccination through the Study Day 56 
visit (with diary cards to be used for 28 days after vaccination). 
Solicited adverse events are events the participant is specifically asked about. These adverse 
events are commonly observed soon after receipt of vaccines. For this study, solicited adverse 
events to be collected include:  
• 
Solicited systemic adverse events: 
fever, irritability, vomiting, diarrhea, drowsiness, poor feeding, skin rash. 
• 
Solicited injection site reaction adverse events: 
pain, redness, swelling, ulceration, drainage, and regional lymphadenopathy.  
Solicited adverse events of injection site reactions will be considered causally related to 
investigational product. 
All participants’ caregivers will be provided a diary card to record axillary temperature and 
information regarding occurrences of these specific events for the first 7 days (for systemic 
adverse events) or 28 days (for injection site reaction adverse events) of the solicited adverse 
event reporting period.  
Serious Adverse Events 
Serious adverse events will be collected throughout the entire study period (i.e., 365 days 
post-vaccination). 

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Site of Injection Examination 
Site of injection examinations will be performed at the visits noted in the schedule of events in 
Appendix A. The following will be assessed at each examination:  
• 
Tenderness/pain, redness, swelling, warmth, ulceration, presence of infection/abscess, 
crusting, drainage, scarring, and regional lymphadenopathy. 
Any of the above-mentioned injection site reactions that meet adverse event criteria according 
to the Toxicity Table in Appendix C will be recorded as adverse events. 
 
10.2 Methods and Timing for Assessing, Recording, and Analyzing 
Safety Parameters 
10.2.1 Adverse Events 
Definition of an Adverse Event 
An adverse event (AE) means any untoward medical occurrence associated with the use of a 
drug in humans, whether or not considered drug related. An adverse event (also referred to 
as an adverse experience) can be any unfavorable and unintended sign (e.g., an abnormal 
laboratory finding), symptom, or disease temporally associated with the use of a drug and 
does not imply any judgment about causality. An adverse event can arise with any use of the 
drug (e.g., off-label use, use in combination with another drug) and with any route of 
administration, formulation, or dose, including an overdose. 
Medical conditions that exist prior to administration of the investigational product (pre-existing 
conditions) will be recorded in the participant’s medical history to establish baseline. Day-to-
day fluctuations in pre-existing conditions that do not represent a clinically significant change 
in the participant’s status will not necessarily be reported as adverse events. 
Any adverse change from the participant’s baseline condition (determined from screening 
evaluations conducted to confirm study eligibility) that occurs following the administration of 
the investigational product will be considered an adverse event. This includes the occurrence 
of a new adverse event or the worsening of a baseline condition, whether or not considered 
related to the investigational product. Intermittent conditions may be present on Study Day 0 
but may represent an adverse event if the intensity or duration of the event is worse than usual 
following receipt of investigational product. Adverse events include but are not limited to: 
adverse changes (including clinically significant laboratory and vital sign changes) from 
baseline that represent increases in toxicity grade according to the Toxicity Table (see protocol 
appendices), adverse changes in the general condition of the participant, signs and symptoms 
noted by the caregiver of the participant, concomitant disease with onset or increased severity 
after investigational product administration, and clinically significant abnormalities in 
laboratory safety parameters occurring after investigational product administration.  

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The reporting period for all adverse events is specified in Section 10.1. Adverse events will be 
reported using a recognized medical term or diagnosis that accurately reflects the event. 
Adverse event evaluations will be reviewed by the principal investigator or sub-investigator. 
AE information is to be completed by members of the study team designated in writing by the 
principal investigator. The onset and resolution dates of the event and action taken in response 
to the event will be documented.  
Assessing Severity 
The safety concepts of “severity” and “seriousness” are distinct concepts (see Section 10.2.3). 
“Severity” refers to a degree of clinical manifestation. “Seriousness” refers to defined 
outcomes from an adverse event. A severe adverse event is not always serious, and a serious 
adverse event is not always severe.  
For all adverse events, the investigator is responsible for assessing the severity of the event 
and the causal relationship of the event to the investigational product. 
The severity of all adverse events, including clinical findings and abnormal laboratory values, 
will be classified as one of the following grades:  
1. Mild  
2. Moderate  
3. Severe  
Toxicity Tables are provided in the protocol appendices for the assessment of severity of 
specified adverse events. The Toxicity Table Adverse Event Grades do not correlate directly 
with the classical severity grades of mild, moderate and severe. FOR THE PURPOSES OF 
RECORDING EVENTS ON THE eCRF, Toxicity Table Grade 1 events will be considered mild 
in severity, Toxicity Table Grade 2 events will be considered moderate in severity, and both 
Toxicity Table Grade 3 and 4 events will be considered as severe.  
For adverse events not listed in the Toxicity Table determination of severity requires some 
level of interpretation as outlined below. The degree of incapacity caused by the adverse event 
and the level of medical intervention required for treatment may be helpful in assessing the 
overall severity of the adverse event.  
For example: 
• 
“Mild” events are generally regarded as noticeable but have no impact on normal activities; 
they may or may not require over-the-counter treatment managed by the participant’s 
caregiver.  
• 
“Moderate” events generally have some impact on an individual’s normal activities and 
may require general symptomatic medical intervention by a healthcare professional or by 
the participant’s caregiver.  
• 
“Severe” adverse events may be incapacitating, leading to suspension of normal daily 
activities, and would generally require more immediate medical evaluation and intervention 
by a healthcare professional.  

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A change in severity of an adverse event will not be recorded as a new adverse event. Only 
the highest severity level that occurs during the entire period of the adverse event will be 
recorded on the eCRF with the onset and resolution dates encompassing the entire duration 
of the event.  
Assessing Causal Relationship (Relatedness) 
For all adverse events, the investigator and, for SAEs, the sponsor and local medical monitor, 
will determine a causal relationship to the investigational product without knowledge, for 
blinded studies, of whether active vaccine or active control was administered. A number of 
factors will be considered in making this assessment, including: 1) the temporal relationship 
of the event to the administration of the investigational product 2) whether an alternative 
etiology has been identified, and 3) biological plausibility.  
The investigator will use the following guidelines to assess the causal relationship of an 
adverse event to study vaccine:  
• 
Not Related to study vaccine (i.e., there is no evidence of a causal relationship; another 
etiology is known to have caused the adverse event. The alternative etiology should be 
documented in the participant’s study record). 
• 
Unlikely Related to study vaccine (i.e., there is less than a reasonable possibility that the 
adverse event was caused by study vaccine). 
• 
Possible relationship to study vaccine (i.e., there is a reasonable possibility that the 
adverse event was caused by study vaccine. There must be a plausible mechanism for 
the event to be related to study vaccine. The evidence is inadequate to accept or reject, 
or favours rejection of, a causal relationship; an association exists between the event and 
the study vaccine but there may also be an alternative etiology, such as characteristics of 
the participant’s clinical status or underlying condition).  
• 
Probable relationship to study vaccine (i.e., it is likely that the adverse event was caused 
by administration of the study vaccine. The evidence favours acceptance of a causal 
relationship; an association exists between the event and receipt of the study vaccine and 
there is a plausible mechanism for the event to be related to the study vaccine, and an 
alternative etiology is not apparent). 
• 
Definite relationship to study vaccine (i.e., the study vaccine is known to be the cause of 
the adverse event. The evidence establishes a causal relationship; an association exists 
between the event and receipt of the study vaccine and there is a plausible mechanism 
for the event to be related to the study vaccine and causes other than the study vaccine 
have been ruled out).  
It is expected that communication and consultation with the sponsor and local medical 
monitor/research monitor will occur in the assessment of the causality of serious adverse 
events. 
Every effort should be made by the investigator to determine the existence of any pre-existing 
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must be taken into consideration when assessing causal relationship of an adverse event. 
Pre-existing conditions should be recorded in the eCRF as baseline history and substantiated 
by appropriate source documentation. Intermittent conditions may not be present on Study 
Day 0 but may represent an adverse event if the intensity or duration of the event is worse 
than usual following investigational product. 
Definition of an Adverse Reaction 
An adverse reaction is an adverse event judged to be related to investigational product. 
Assessing Expectedness of Adverse Events 
Expected adverse events are adverse events consistent with the applicable product 
information provided by the sponsor (the investigator’s brochure for an investigational 
product). The sponsor, in conjunction with the local medical monitor/research monitor, 
determines expectedness of related serious adverse events. 
10.2.2 Reactogenicity (for Vaccine Studies and Some Therapeutic Trials) 
Reactogenicity events are referred to as solicited adverse events. Refer to Section 10.1 for a 
list of solicited adverse events (systemic and injection site reactions) and timing of collection. 
Diary cards will be provided to participants for recording of solicited systemic adverse events 
(for 7 days) and solicited injection site reactions (for 28 days; see Section 9.1 for further details 
regarding diary cards). 
10.2.3 Serious Adverse Events 
Definition of Serious Adverse Event 
Seriousness refers to the outcome of an adverse event. Seriousness is determined by both 
the investigator and the local medical monitor. If the investigator or local medical monitor 
determines an event to be serious, it will be classified as such. If any of the following outcomes 
are present, then the adverse event is serious: 
• 
It results in death (i.e., the AE caused or led to the fatality). Serious does not describe an 
event which hypothetically might have caused death if it were more severe. 
• 
It was immediately life-threatening (i.e., the AE placed the participant at immediate risk 
of dying. It does not refer to an event which hypothetically may have led to death if it were 
more severe).  
• 
It required inpatient hospitalization or prolonged hospitalization beyond the expected 
length of stay. Hospitalizations for scheduled treatments and elective medical/surgical 
procedures related to a pre-existing condition that did not increase in severity or frequency 
following receipt of investigational product, are not serious by this criterion. Hospitalization 
is defined as a hospital admission or an emergency room visit for a period greater than 24 
hours.  

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• 
It resulted in a persistent or significant disability/incapacity (i.e., substantial reduction of 
the participant’s ability to carry out activities of daily living). 
• 
Other medically important conditions that may not result in death, threaten life or require 
hospitalization (i.e., the AE does not meet any of the above serious criteria) may be 
considered a serious adverse event when, based on appropriate medical judgment, they 
may jeopardize the participant and require medical or surgical intervention to prevent one 
of the serious outcomes listed in these criteria (e.g., allergic bronchospasm requiring 
intensive treatment in an emergency room or at home; blood dyscrasias or convulsions 
that do not result in hospitalization). 
A serious adverse event is an adverse event meeting the outcome criteria for seriousness 
regardless of relationship to an administered medicinal product. Recording and follow up of 
serious adverse events is discussed in Section 10.4. 
Definition of Suspected Unexpected Serious Adverse Reaction (SUSAR) 
When an adverse event is judged to be related to an investigational product, is judged to be 
serious and unexpected, and is in a participant who received active vaccine, it is a SUSAR 
(suspected unexpected serious adverse reaction) and is subject to expedited reporting. 
10.2.4 Procedures to be Followed in the Event of Abnormal Laboratory 
Test Values or Abnormal Clinical Findings 
Results from clinical assessments and laboratory tests obtained on the study must be 
reviewed by the investigator, and the participant managed in accordance with the clinical trial 
site policies. The clinical significance of any change in a vital sign or laboratory parameter will 
be determined by the investigator and may be reported as an adverse event at the discretion 
of the investigator (using the toxicity tables in Appendix B, C and D to grade severity). 
Additional laboratory tests may be performed if the investigator deems them to be necessary 
to fully evaluate an adverse event. In the event that the investigator elects to order non-
protocol-specified laboratory tests, the investigator must record the rationale for the tests and 
a determination of clinical significance of the result in the source documents. The investigator 
must keep the medical monitor informed of vaccine-related adverse events of clinical 
significance.  
Abnormal results and findings will be discussed as applicable with the participant, and the 
participant will be referred for follow-up with their healthcare provider if necessary. 
10.3 Reporting Procedures 
The reporting period for all adverse events is specified in Section 10.1. Reporting procedures 
for adverse events are described in Section 10.2.1. 
 

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10.3.1 Serious Adverse Events 
Serious adverse events, which include SUSARs, will be reported by the PI to the sponsor, 
local medical monitor, UCT HREC and TCD in an expedited fashion. TCD RA in turn will notify  
SAHPRA according to their required timelines .  SUSARs are reported even after the trial is 
over, if the principal investigator/investigator or local medical monitor becomes aware of them. 
Serious adverse events will be assessed by the investigator and the local medical monitor 
according to their roles for severity, causal relationship to the study vaccine, and 
expectedness.  The onset and resolution dates of the event and the action taken in response 
to the event will be documented.  If the event has not resolved by the final study visit, it will be 
documented as “ongoing” on the eCRF. However, follow-up of the SAE must continue until 
resolved.  Information recorded on the eCRF must be substantiated in the source documents. 
SAEs are to be reported to the sponsor within 24 hours of the investigator site becoming aware 
of the event, even if all information concerning the event is not yet known at that time. SAHPRA 
stipulates that a SUSAR is to be reported to SAHPRA within 7 calendar days if it is fatal or life 
threatening, and within 15 calendar days if the SUSAR is not fatal or not life threatening. Other 
SAEs should be submitted as part of 6 monthly reports to SAHPRA in a line-listing format.  
The UCT HREC are to be notified as soon as possible but not later than 7 days of all SAE’s, 
SSAR’s and SUSAR’s. New information which may impact the conduct of a study should be 
reported to HREC within 3 days. 
Follow-up reports for the SAE are to be provided by the site as new information becomes 
available or when the status of the SAE changes. 
Fatal or life-threatening serious adverse events that the investigator suspects are related to 
the vaccination should be telephoned to the local medical monitor immediately upon the 
investigator’s awareness of the event.  If the local medical monitor or PI is required by the 
protocol or chooses to suspend enrollment s/he shall immediately create a written 
memorandum for record to the study file and immediately notify the sponsor, UCT HREC and 
SAHPRA of this act. 
Contact information for all safety personnel are contained in the Team Contact List which will 
be stored on site in the Site Regulatory Binder and maintained by the PI.  
The sponsor will notify the DSMB and the manufacturers of MTBVAC and BCG of all SUSARs 
within 3 working days of becoming aware of an event and will provide all follow-up information 
in a timely manner. 
Investigators must not wait to collect additional information to fully document the event before 
notifying the local medical monitor/research monitor of a serious adverse event. The initial 
notification should include the following (at minimum):  
• 
Protocol number and name and contact number of the investigator 
• 
Participant ID number (and initials and date of birth, if available) 

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• 
Date participant received investigational product 
• 
Serious adverse event(s) and date of event onset 
• 
Current status of participant  
10.3.2 Suspected and Confirmed Tuberculosis Events 
Any adverse event suspected to be or confirmed as tuberculosis disease will be assessed, 
documented and reported according to the same procedures as for serious adverse events 
(refer to section 10.3.1), even if the event does not meet any of the serious adverse event 
criteria as per section 10.2.3. Such suspected or confirmed tuberculosis disease events will 
be classified as Immediately Reportable Events (IREs). These events will be assessed by the 
investigator and the local medical monitor according to their roles for severity, causal 
relationship to the study vaccine, and expectedness.  The onset and resolution dates of the 
event and the action taken in response to the event will be documented.  If the event has not 
resolved by the final study visit, it will be documented as “ongoing” on the eCRF. However, 
follow-up of the IRE must continue until resolved.  Information recorded on the eCRF must be 
substantiated in the source documents. 
10.3.3 Other Immediately Reportable Events 
The investigator must report the following events to the sponsor within 24 hours of becoming 
aware of the event:  
• 
Withdrawal of consent during the study for safety reasons 
• 
Emergency unblinding (see Section 10.6) 
• 
Protocol violation affecting the safety of a participant or involving the vaccination process 
• 
Adverse event thought to be an allergic reaction to the investigational product  
• 
Any event that, in the opinion of the investigator, precludes further administration of the 
investigational product  
 
10.4 Type and Duration of Follow-up of Participants after Adverse 
Events 
The duration of follow up for AEs is described in Section 10.1. 
Treatment of any adverse events will be determined by the investigator using his/her best 
medical judgment and according to current clinical practice guidelines. All applied measures 
as well as follow-up will be recorded in the appropriate eCRF.  

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Adverse events will be considered resolved when the condition returns to normal or returns to 
the participant’s baseline status as established on Study Day 0, or when the condition has 
stabilized with the expectation that it will remain chronic. 
The investigator will continue follow-up on adverse events, including laboratory abnormalities 
and solicited adverse events, until the event has resolved, is otherwise satisfactorily explained, 
or the participant completes the study. The resolution date will be recorded on the eCRF as 
the last date on which the participant experienced the adverse event. If an adverse event 
resolution date is uncertain the investigator should estimate the completion date based on 
medical judgment and interview of the participant. Approximate dates of resolution from 
interviews may be taken as adverse event resolution dates. Some examples of estimation of 
adverse event resolution are: 1) an asymptomatic laboratory abnormality on one visit that has 
not been followed-up between visits but has resolved by the next visit may be assumed to 
have resolved by the midpoint of the inter-visit interval; 2) A resolved adverse event that was 
treated may be assumed to have been resolved by the end of treatment. Adverse events that 
are still present at the end of the trial should be recorded as ongoing. Information recorded on 
the eCRF must be substantiated in the source documents. If an adverse event evolves into a 
condition that becomes “serious,” it will be designated as serious on the AE eCRF and a 
Supplemental SAE Report (SAER) form will be completed. 
Follow-up for serious adverse events must continue until resolution and the outcome reported 
to the sponsor, even if this extends beyond the serious adverse event reporting period (i.e., 
after the final study visit). For analysis purposes, the outcome for serious adverse events will 
be determined on the final study visit. 
Outcome of all adverse events will be classified as one of the following: 
• 
Resolved  
• 
Resolved with sequelae 
• 
Ongoing 
• 
Death 
If at any time after completion of the serious adverse event reporting period (the final study 
visit) the investigator becomes aware of a serious adverse event that is suspected by the 
investigator to be related to the investigational product, the event must be reported to the 
sponsor. 
10.5 Pausing Rules 
These rules govern the pausing and stopping of investigational product administration at any 
time during the study such as between individuals within a single cohort, and between cohorts. 
Enrolment to study will be paused if certain pre-defined conditions are met. Follow-up of study 
participants and pre-enrolment recruitment may continue while a study pause is in effect. The 
trial can be paused independently by either the PI, or the local medical monitor. Written 
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UCT HREC, sponsor, DSMB and SAHPRA within 24 hours of its effect and a memorandum 
recorded in the study site file. 
If the principal investigator pauses study vaccine administration in a trial under the rules in this 
section and additional clinical information becomes available that reduces the principal 
investigator’s assessment of causality, severity or toxicity grade such that the adverse event’s 
causality, severity or toxicity grade no longer requires pausing then the principal investigator, 
with the agreement of the local medical monitor, may resume study vaccine administration 
with a memorandum to the study regulatory binder. In such a case a formal study pause need 
not be affected. 
In all cases where the study has been paused, the DSMB will be convened, except in the case 
where the PI revises his assessment of a study pause condition before the meeting is 
convened, as additional information becomes available to him, to review safety reports and 
any other pertinent data following the study pause. Additional information, results of 
investigations, or unblinded data may show that potential risks to participants are lower, or 
less generalized, than suspected. If, after meeting and deliberating on all relevant information, 
the DSMB determines that it is safe for the study to continue, the study pause may be lifted, 
and enrolment and vaccination may continue. Restrictions may be placed, if appropriate, on 
further study conduct, additional safety or monitoring measures may be put in place, or 
recruitment and vaccination may be stopped completely if participant safety cannot be 
guaranteed. The sponsor, PI, LMM, UCT HREC and SAHPRA will be notified in writing via fax 
or email of the decisions and recommendations, and a signed memorandum of the 
deliberations will be provided to the sponsor, UCT HREC, SAHPRA, and will also be kept in 
the site regulatory binder.  Any changes to the protocol required as a condition of study 
enrolment resumption must be approved by, the Institutional Review Board/Ethics Committee 
(UCT HREC) and SAHPRA. 
The following rules will trigger pausing by the principal investigator, local medical monitor, or 
the sponsor of further screening, enrollment, and investigational product administration, and 
DSMB review of safety data: 
 
1. Severe (e.g., painful, draining, infected [abscess]) or large (e.g., >10.00 cm induration and 
erythema, and >3.0 cm ulceration) injection site reaction, excluding local injection site 
reactions that decrease to <Grade 3 within 24 hours. 
OR 
2. Large (≥5 cm) local regional lymphadenopathy or suppurative lymphadenitis.  
OR 
3. One or more SAE(s) judged related to investigational product occurs. 
OR 
4. Anaphylaxis or bronchospasm within 4 hours of injection, indicative of an immediate 
hypersensitivity reaction to the study injection. 
OR 
5. Two or more participants in any given cohort experience a Grade 3 or higher event, 
excluding local injection site reactions, judged related to investigational product. 
OR 
6. An adverse event pattern of concern occurred. 

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If the principal investigator, local medical monitor or sponsor pauses the study, the decision 
will be recorded in a memorandum to the study file and will trigger DSMB review. If a 
recommendation to resume study enrollment and investigational product administration is 
made, the DSMB will record their judgment in a memorandum to the study file and notify the 
sponsor. The DSMB memorandum will be forwarded to the medical monitors and principal 
investigator. The DSMB may recommend resumption of enrollment with changes to the 
protocol if it judges that such changes will reduce safety risks. However, the final decision to 
resume study activities, amend the protocol, or terminate the study will be made by the 
sponsor. The clinical trial site will be allowed to resume activities only upon receipt of written 
notification from the sponsor. Decisions regarding pausing and resumption of the study will be 
communicated promptly to the IRB/IEC by the principal investigator and to the applicable 
national regulatory authority by the sponsor (or designee). 
In addition, the study may be discontinued at any time by the applicable national regulatory 
authority, the IRB/IEC, the sponsor, or other governmental agencies as part of their duties to 
ensure that research participants are protected. 
10.6 Emergency unblinding 
If the PI is of the opinion that unblinding of a particular participant’s randomization result is 
essential to treat an adverse event and that the participant’s safety would be compromised 
without this information, the PI may unblind the randomization result for a specific participant. 
In such cases, the PI should, if at all possible, consult the local medical monitor first for 
concurrence. If emergency unblinding has occurred, a memorandum will be filed in the site 
regulatory binder detailing the occurrence. 
The DSMB must, if possible, immediately be informed of any unblinding. 
10.7 Safety Oversight (DSMB) 
The DSMB, composed of a panel of independent experts, will operate according to its charter. 
If investigational product administration is paused for safety reasons by the local medical 
monitor or the principal investigator, the DSMB will be convened. Based on its review and the 
protocol pause rules (Section 10.5) the DSMB will make recommendations to the sponsor 
regarding further conduct of the study and further administration of investigational product. 
The DSMB may review an individual SAE or it may choose to review adverse events, serious 
adverse events, solicited adverse events, and laboratory and vital signs data. The DSMB may 
unblind any amount of safety information needed to conduct their assessment. All procedures 
associated with this review, including objectives, data handling, and elements to be included 
for review will be documented in DSMB minutes. The DSMB may recommend suspension or 
resumption of enrollment and investigational product administration after review of safety data. 
However, the sponsor will make the final decision to suspend or resume study activities. The 
recommendations of the DSMB, along with the sponsor’s decision, will be communicated to 
the investigators, IRB/IEC, and the national regulatory authorities. The sponsor or its designee 
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The DSMB will conduct three planned safety data reviews during dose-escalation, as 
described in Section 12.3.1. Two planned safety data reviews will solely focus on safety, with 
a third safety data review including the review of immunogenicity data together with safety 
data to confirm the safest and most immunogenic dose of MTBVAC, as described in Section 
12.3.1 and 12.3.2.  
10.8 Trial Steering Committee (TSC) 
 
The Sponsor will establish a Trial Steering Committee (TSC) before the start of the clinical 
trial. The TSC will, based on a TSC charter, supervise clinical trial conduct as well as consider 
the advice from the DSMB. An independent expert member will chair the TSC that will 
furthermore be composed of 2 additional independent expert members. A representative of 
EDCTP will participate as an observer. The TSC will monitor trial progress and conduct and 
provide advice on scientific credibility. The TSC will consider the recommendations of the 
DSMB and will provide additional advice to the Sponsor as to whether a trial needs to be 
stopped, suspended, or resumed on scientific grounds and/or efficacy. However, the sponsor 
will make the final decision to stop, suspend or resume study activities. The TSC will meet 
annually in person and via Skype on a regular basis, or ad hoc when required. The TSC will 
reach advice by consensus and will minute meetings.  
 
Any resolution leading to a protocol amendment, pausing rules or as per safety consideration, 
will be communicated to the regulatory authorities and Ethics Committee. This is in addition to 
the regular 6-monthly Study progress reports. 
 
A European Ethics Committee, “Comité Autonómico de Ética da Investigación de Galicia” 
(Sergas), will be involved in the study as ethical advisor. This Committee will be informed from 
the beginning and throughout the study on Study Progress Reports, any Protocol amendment 
as well as any Safety Event that may occur. 

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11 
CLINICAL MONITORING 
11.1 Site Monitoring Plan 
The study will be monitored regularly, according to the protocol-specific monitoring plan, by 
the sponsor or its designee throughout the study period. Monitoring will be a combination of 
on-site monitoring and central review of data in the electronic database. One hundred percent 
(100%) of data will be reviewed centrally and in-house review of data will be done on an 
ongoing basis with formal review meetings approximately monthly. On-site monitoring will 
occur approximately monthly during recruitment and vaccination periods and approximately 
every 2 months during follow up. On-site monitoring will include source data verification of 
approximately 80% of study data. 
 

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12 
STATISTICAL CONSIDERATIONS 
12.1 Study Hypotheses 
No formal hypothesis testing will be performed, since the trial is not powered to do any formal 
testing (see sample size section below). 
12.2 Sample Size Considerations 
The sample size for this study was selected as being adequate for a preliminary assessment 
of the safety profile of MTBVAC in neonates, rather than for statistical reasons. The sample 
sizes of Phase 1 and 2a clinical trials are conventionally not powered to detect rare or 
infrequent events, but rather common safety events, major safety signals, or important trends. 
The sample size of 99 infants was selected as adequate for such initial evaluation of the safety 
profile of MTBVAC, rather than for statistical reasons (17-25). 
12.3 Planned Interim Analyses 
12.3.1 Safety Review  
Pausing rules are described in Section 10.5. The DSMB (see Section 10.7) will conduct two 
safety data reviews during dose-escalation, at the following time points: 
• 
Participants in Cohort 2 have completed the Study Day 28 visit. 
• 
Participants in Cohort 3 have completed the Study Day 28 visit. 
Dose-escalation will proceed as described in Section 5, as long as no pausing rules are 
triggered.  
In addition to dose-escalation DSMB safety data reviews, an additional safety data review 
will take place at the following time point: 
• 
Participants in Cohort 3 have completed the Study Day 182 visit. 
Dose selection of MTBVAC for the Phase III study will proceed based on the results of the 
analysis of all of the safety data available for all cohorts up to the time that Cohort 3 Study 
D182 visit has been completed. 
12.3.2 Immunogenicity or Efficacy Review 
An immunogenicity review will be completed by the DSMB to determine the most immunogenic 
(and safe) MTBVAC dose for dose selection for the Phase III study. This analysis is planned 
to take place once the last Study Day 182 visit of Cohort 3 has been completed and all 
available immunology samples across Cohort 1, 2, and 3 at Study D28, D56 and D182 visits 
have been processed. No unblinding of the clinical and immunology team to the 

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immunogenicity data analysis is expected for this review. An independent, unblinded 
biostatistician will complete the analysis on the raw immunogenicity data which will be 
provided by the immunology team. The independent, unblinded biostatistician will analyse 
MTBVAC dose selection based on magnitude of MTBVAC-specific CD4 T cell response; 
Comparison of CD4 T cells responses across increasing doses of MTBVAC and BCG; as well 
as evaluation of selected MTBVAC dose superiority over BCG vaccination. The results of the 
analysed immunogenicity data will be provided by the independent, unblinded biostatistician 
directly to the DSMB in a blinded manner along with the vaccine recipient listing. Sponsor will 
be provided with the same blinded data for review, excluding the vaccine recipient listing to 
maintain blinding of Cohort 3. The  DSMB meeting will take place to assess the 
immunogenicity results, as described above, for all cohorts (Cohort 1, 2 and 3) up to Study 
D182 visit per cohort to determine the most relevant dose for the Phase III study. Unblinding 
of the clinical team will take place post Study Day 365 visit for all cohorts. In order to maintain 
data integrity and avoid compromising immunology analysis of samples collected at the last 
visit (Study Day 365 visit), the immunology team will remain blinded until immunology 
database lock.  
Efficacy is not being assessed in this trial. 
12.4 Final Analysis Plan 
The planned statistical analyses for this study are outlined below. A detailed statistical analysis 
plan will be created and finalized prior to database lock. Any deviations from the planned 
analyses will be described in the final study report.   
12.4.1 Participant Analysis Sets 
 
The full analysis set will include all screened participants. The safety analysis set will include 
all participants who received investigational product. The immunogenicity analysis set will 
include all participants who received a complete study vaccination and has at least one 
evaluable immunogenicity result.  Listings of participants who did not complete all trial 
procedures, such as missed visits or other protocol deviations that may affect the analyses, 
will be provided for the overall sample and by study arm. No imputation for missing data will 
be performed.  Data will be transformed as appropriate prior to analysis, but results calculated 
from transformed values will be reported in original units for clarity. This trial is not designed 
for treatment efficacy analysis, but immunogenicity analyses and analysis of clinical 
reactogenicity will be reported. 
12.4.2 Demographics and Protocol Compliance 
 
Baseline demographic (age, sex, and race) and other baseline characteristics will be 
summarized using descriptive statistics (at a minimum the following will be reported: means, 
medians, standard deviations, frequencies and proportions, as appropriate), for the overall 
sample (Safety Analysis Set) and by study group.  

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As participant enrollment to each cohort will be conducted based on timing of completion of 
study eligibility requirements, any imbalance in baseline characteristics between cohorts will 
also be examined.  
Listings of randomized participants with protocol deviations will be provided. 
12.4.3 Immunogenicity and Other Immunology Analyses 
Immunogenicity will be summarized for all time points as collected and as available. No 
imputation for missing data will be performed. Data will be transformed as appropriate prior to 
analysis. 
For each of the outputs from the relevant immunology parameters, descriptive statistics 
including means, standard deviations, medians, and interquartile range, will be determined. 
Between-group comparisons of means or medians at each time point, as well as changes 
between time points post revaccination, will be performed (informally) as outlined in the 
Statistical Analysis Plan.  
 
12.4.3.1 
Whole Blood ICS 
The primary variables of interest for the assessment of the immune response to the study 
vaccine will be the frequencies of MTBVAC-specific CD4+ and CD8+ T cells that produce any 
or a combination of relevant cytokines. Response will be measured by flow cytometry using a 
qualified whole blood intracellular cytokine staining (ICS) assay (24).  
T cell responses will be summarized by treatment group and time point. The difference in T 
cell response between treatment regimens across all post-immunization time points as 
measured by percentage CD4 and CD8 responses will also be modeled using linear mixed 
effects models or other regression models, as appropriate.  
12.4.3.2 
QFT Gold Plus 
Qualitative and quantitative results for the QFT Gold Plus test at each available time point will 
be summarized using participant count (percentage) summaries and descriptive statistics. The 
proportions of QFT positive participants at various time points may also be compared using 
chi-square tests or Fisher’s exact tests. Appropriate figures will be used to illustrate the 
observed responses over time. 
12.4.4 Safety Analyses 
12.4.4.1 
Adverse Events 
The primary endpoint is the frequency of adverse events (AEs) by MTBVAC dose compared 
to BCG.  In order to assess the primary endpoint, analysis of the data will be preceded by a 

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blinded data review meeting in order to address population determination and miscellaneous 
data queries pertinent to analysis of the primary endpoint. 
Safety and reactogenicity data will be evaluated by study arm and cohort in all participants in 
the safety analysis set. The primary variable for evaluation of safety will be the frequency of 
unsolicited, solicited adverse events and injection site reactions at all available post-
vaccination time points. The number and percentage of participants with adverse events will 
be summarized by MedDRA system organ class (SOC) and preferred term (PT), as well as 
by severity and relationship to study vaccine. Informal between-group comparisons will be 
performed using the Chi-square or Fisher Exact test.  The Cochran-Mantel-Haenzel test will 
be used to compare the effect of dose on reactogenicity (injection site adverse events). 
Separate listings will be provided for participants with serious adverse events. SAEs may also 
be summarized by MedDRA system organ class and preferred term. 
12.4.4.2 
Clinical Laboratory and Vital Sign Parameters 
For each clinical laboratory parameter and vital sign parameter pre-specified in the protocol, 
summary statistics for continuous parameters will be presented by treatment group for all pre- 
and post-vaccination assessments and for change from pre-vaccination to post-vaccination 
assessments.  
The number and percentage of participants with post-vaccination Grade 3 or higher clinical 
laboratory values or vital sign values (Appendices B, C and D) will be tabulated at each post-
vaccination time point and overall. Clinical laboratory and vital sign abnormalities will also be 
reported as adverse events as applicable and will be included in the summaries of adverse 
events 
12.4.5 Computer Methods 
Statistical analyses will be performed using SAS® (Version 9.4 or higher, SAS Institute Inc., 
Cary, NC, USA), under a Windows operating system. 

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13 
SOURCE DOCUMENTS AND ACCESS TO SOURCE 
DATA/DOCUMENTS 
For the purpose of monitoring and auditing the study, source documentation will consist of any 
existing medical records and study records developed and maintained by the investigator for 
this study. Monitor(s), auditor(s), IRB/IEC members, and the applicable regulatory authorities 
will be granted direct access to the participant’s original study medical records for verification 
of protocol-specified procedures and/or data. The above-mentioned agencies will only have 
access to a participant’s personal details if they come to the study site to monitor or audit the 
study. Any data sent to these agencies will be de-identified. 
 
 

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14 
QUALITY CONTROL AND QUALITY ASSURANCE 
The clinical trial site will be required to have a Quality Management Plan and an Investigator 
Oversight Plan in place for this trial. The Quality Management Plan will specify quality control 
procedures to be implemented by the trial site staff to ensure the protocol is being followed, 
data are entered accurately into the study database, investigational product is handled 
correctly, and all staff are appropriately trained. Additionally, the site will be required to have 
SOPs in place for management of the investigational product handling, adverse event and 
serious adverse events, and the informed consent process. 
Independent trial monitors will provide an additional layer of quality control through regular 
monitoring visits. Monitoring will include review of site compliance with the quality 
management plan, investigator oversight plan, SOPs, and the protocol. Further details on the 
site monitoring plan can be found in Section 11.1. 
An independent auditor will conduct a site audit during the trial to ensure that the trial is 
performed, and the data is generated, documented and reported in accordance with Good 
Clinical Practice and applicable regulatory requirements. 
 

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15 
ETHICS/PROTECTION OF HUMAN PARTICIPANTS 
15.1 Ethical Standards 
The study will be carried out in accordance with this protocol, the guidelines and ethical 
principles set forth in the Declaration of Helsinki (last updated 2013), Good Clinical Practice 
(ICH E6 (R2)) and Guidelines for Good Practice in the Conduct of Clinical Trials with Human 
Participants in South Africa (SA GCP), Second Edition, 2006 or latest version.  
Written maternal informed consent will be obtained from each participant prior to any protocol-
specified procedures being conducted.  
The protocol and informed consent form will be reviewed and approved by the IRB or IEC of 
the participating clinical trial site prior to any protocol-specified procedures being conducted. 
The investigator is responsible for ensuring that the protocol is reviewed by an IRB/IEC with 
the appropriate composition (per clinical trial site guidelines). The investigator will inform the 
IRB/IEC as to the progress of the study at least annually and as defined by IRB/IEC policy.  
HIV testing will be performed with appropriate pre- and post-test counseling. 
The protocol may not be modified without written approval from the sponsor. All changes to 
the protocol must be submitted to the IRB/IEC and must be approved by the IRB/IEC and 
applicable regulatory agencies prior to their implementation.  
The investigator or designated clinical trial site staff will notify the IRB/IEC and applicable 
regulatory agencies when the study has been completed (i.e., the clinical database has been 
locked). After the study has been unblinded, the participant should be informed by the 
investigator which treatment (MTBVAC or BCG) the participant received. 
15.2 Institutional Review Board 
All the documents the IRB/IEC may need to fulfill its responsibilities, such as the protocol, 
protocol amendments, informed consent form, information concerning participant recruitment, 
payment or compensation procedures, etc., will be submitted to the IRB/IEC by the 
investigator. The IRB’s/IEC’s written, unconditional approval of the appropriate version of the 
study protocol and the informed consent form will be in the possession of the 
investigator/clinical trial site staff prior to the conduct of any protocol-specified procedures.  
Modifications to the protocol may not be implemented without prior written IRB/IEC approval 
except when necessary to eliminate immediate hazards to the participants or when the 
modification involves only logistical or administrative aspects of the study. Such logistical or 
administrative modifications will be submitted to the IRB/IEC in writing by the investigator, and 
a copy of the correspondence to verify the submission will be maintained.  

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The investigator must inform the IRB/IEC of modifications to the informed consent form or any 
other documents previously submitted for review/approval and any new information that may 
adversely affect the safety of the participants or the conduct of the study, provide an annual 
update and/or request for re-approval, and advise the IRB/IEC when the study has been 
completed.  
Any clinical trial site-generated documents or forms to be provided to the participant (e.g., 
information cards, letters from the investigator), and all forms of study advertising (flyers, 
brochures, print advertisements, radio or television scripts, etc.) must be approved by Biofabri 
or its designee prior to the clinical trial site submitting them to the IRB/IEC. Approval from the 
IRB/IEC must be obtained prior to providing the documents or forms to the participant. 
15.3 Informed Consent Process 
For infant participants, consent will be obtained from the participant’s legal guardian. In most 
cases, this will be the participant’s mother who would be asked to give consent before the birth 
of her baby. In exceptional cases where re-consent needs to be signed after the infant’s birth 
e.g. amended Informed Consent Form (ICF), death of mother, parent no longer available, a 
legal guardian may sign consent on behalf of the participant. In cases where the mother is 
herself a minor (younger than 18 years of age), her legal guardian is required to sign informed 
consent on her behalf. 
Potential participants will be identified through approaching members of the community or at 
their mother’s routine antenatal clinic visits in liaison with the local state healthcare services. 
General information about the study will be given. Individual face-to-face informed consent 
sessions will then be held between potential participant’s legal guardian and the study team 
member prior to written consent and signing of the informed consent document. This 
discussion session will take place in the privacy of a dedicated room at the clinic or at the 
SATVI field site, or at the participant’s home. Adequate time will be allowed for discussion and 
questions, and all attempts will be made to create an unrushed, confidential, and safe 
atmosphere that is conducive to open discussion. Where possible, paternal consent will also 
be sought. 
The final approved English language informed consent documents will be translated and back 
translated by a reputable translation agency into required languages. The translated 
documents will then be submitted to the UCT HREC for acknowledgment. The information and 
informed consent document will be given to the potential participant’s legal guardian to read 
(or to be read to them) before the informed consent process begins. If possible, the document 
will be given to the participant’s legal guardian at least the day before the informed consent 
process.  
Written informed consent will be obtained from all potential participants’ legal guardians in 
their own language for study participation and for all study interventions, including access to 
medical records and the results of HIV testing, prior to enrolment in the study. If a participant’s 
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sessions will be conducted by study team members who have undergone training in the 
protocol and consent documents, who have been judged proficient in the informed consent 
process, and who have been delegated this task by the PI. The consenter may be a research 
nurse, field worker, or medical officer. Members of the research team will be drawn from similar 
communities as the participants and it is expected that within the study team there will be staff 
that are fluent in each of the local home languages (Afrikaans, English, and isiXhosa). A 
medically qualified member of the study team will be available to answer any questions that 
the consenter is unable to answer. The consenter will discuss with the potential participant’s 
legal guardian the study information and consent document that has been approved by the 
UCT HREC, and detail the reason for the study, the study procedures, risks, benefits, and the 
rights of participants, including the right not to take part, or to withdraw without prejudice. The 
aspects of the study that are research-specific and those that are standard of care will be 
explained.  
Since MTBVAC might interfere with interpretation of future IGRA and TST tests for M. 
tuberculosis infection after the end of the study, mothers will be informed of this phenomenon 
and the risk of receiving unnecessary IPT for a false positive test for M. tuberculosis infection. 
We will emphasise that all infants, especially those testing QFT positive at D56 will be carefully 
monitored for signs and symptoms of TB, with two extra telephonic contacts for infants QFT+ 
at D56; and that mothers of MTBVAC recipients who test QFT positive will receive a card at 
end of study stating that they have received an experimental vaccine that may interfere with 
future IGRA and TST tests for M. tuberculosis infection.  
Any questions the potential participant’s legal guardian may have will be answered. If all 
questions have been answered satisfactorily, the participant’s legal guardian has 
demonstrated adequate understanding, and the participant’s legal guardian indicates their 
wish to participate, they will be asked to give their informed consent by personally signing and 
dating the consent form in the presence of the staff member. The informed consent process, 
as well as specific questions or uncertainties that the participant’s legal guardian has 
expressed during the informed consent session, will be documented in the source 
documentation. A copy of the signed informed consent document will be given to the 
participant’s legal guardian for her records. 
Illiterate persons will indicate their consent using a thumbprint, in the presence of an impartial 
witness. The witness will not date the document for illiterate participants on behalf of the 
participant’s legal guardian. The date of an illiterate participant’s legal guardian’s informed 
consent will be determined by the date of the witness signature. In cases where the mother-
to-be of the potential participant is under the age of 18 years, and is therefore considered a 
minor, she must give her informed assent by co-signing the informed consent document, but 
written consent must be given by her guardian. The University of Cape Town REC and the 
national Department of Health require that informed consent must be obtained from a parent 
for participation in research by any minor under the age of 18 years. Parental consent will be 
obtained using the standard information and consent document. 
All participants will have the right to withdraw their consent at any time throughout the course 
of the study.  

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In addition to the main Inform Consent Form, parental / caregiver consent may be requested 
for TB investigations and for photographing the baby, with emphasis on the injection site, at 
vaccination or during follow up.  Photographs will be used for safety review and for study-
related presentation or publication purposes. 
Informed consent is an ongoing process. At every clinic visit it will be verbally reconfirmed that 
the participant is voluntarily consenting to the study and understands the significant aspects 
of the study (e.g., purpose, risks, duration). 
15.4 Exclusion of Women, Minorities, and Children (Special 
Populations) 
Only women will be asked to sign informed consent in this study due the nature of the intended 
study population. Male and female infant participants will be enrolled.  
Estimates of racial/ethnic group distribution of study participants are based upon demographic 
data from the regional health report and from the experience with enrolment in ongoing and 
completed SATVI studies. The expected racial/ethnic distribution of the population throughout 
the study region is: 99% Black (24% African and 75% so-called Colored ethnic groups); 0.1% 
Asian; and 1% White. It is expected that the racial/ethnic distribution of participants in this 
study will follow a similar pattern to study population in previous SATVI studies. However, the 
population of the local district from which participants will be enrolled is increasing due to in-
migration. It is thought that the majority of new residents are Black African, and the 
representation of this group may increase. No participants will be excluded from the study on 
the basis of race or ethnicity. 
Pregnant women who are approached for recruitment into the study will not be excluded on 
the basis of age alone. Under-age mothers-to-be who are not married are considered minors 
by law and will require written consent from their legal guardian. In such a case the mother-
to-be would also provide written assent. 
15.5 Participant Confidentiality 
To maintain confidentiality, participant identification numbers will be used to identify the 
participant’s laboratory specimens, source documents, eCRF, study reports, etc. All study 
records will be maintained in a secured location. Clinical information will not be released 
without written permission from the participant except as necessary for monitoring or auditing 
of the study by Biofabri or its designee or applicable regulatory authorities. If the participant 
gives their written informed consent to do so, participant contact details may be divulged to 
researchers at the University of Cape Town who might wish to contact the participant to take 
part in future research. 
Study data will be entered into a dedicated custom-designed electronic database that is secure 
and password restricted. Access to records will be restricted to study staff only, participant 
folders will be stored on site in lockable, fire proof cabinets, and access to computer records 

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will be password restricted. Participant and laboratory data with potential identifiers will be 
recorded on study data capture forms and the database using a unique and coded participant 
identification number only. Access to data with participant identifiers in study files, or 
elsewhere, will be restricted to the Principal Investigator, Co-Investigators, the clinical trial 
staff, and the SATVI Data Manager. The study monitor will have access to participant records 
only as necessary for performance of their study-specific functions.  
No participant data will be reported in such a way that participants might be recognised from 
any presentation or publication of the study findings. 
15.6 Study Discontinuation 
Rules governing the pausing and stopping of investigational product administration between 
individuals within a single cohort and between cohorts are presented in Section 10.5. There 
are no procedures for participants to continue therapy, as this is a single-dose study. 
15.7 Future Use of Stored Specimens 
Documentation will be kept per laboratory SOPs to reflect storage location of samples for later 
retrieval. Participant identifiers will not be displayed on samples or in laboratory 
documentation. Laboratory staff will remain blinded to study arm allocation and participant 
identity. Blood collected for storage may be stored for future use for additional TB-related tests 
after the completion of this study if consent was obtained for this. These samples will remain 
anonymous. Storage of blood samples will be discussed in the informed consent form (ICF) 
and the participant will be given the option to have the infant participant’s blood samples 
destroyed after study completion. A decision not to allow storage of blood samples will not 
influence participation in the main study and will not prejudice the participant in any way.  
 

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16 
DATA HANDLING AND RECORD KEEPING 
The investigator is responsible to ensure the accuracy, completeness, legibility, and timeliness 
of the data reported. All source documents should be completed in a neat, legible manner to 
ensure accurate interpretation of data. 
Data reported in the eCRF derived from source documents should be consistent with the 
source documents. 
16.1 Numbering and coding of participant folders 
All CRFs and electronic records will be identified by the study number only.  The study number 
will be the same as the assigned enrolment number at the time of maternal consent. No 
participant identifiers or initials will be used on CRFs or on database records. The use of some 
identifiers on source documentation may be unavoidable, but access to these records will be 
restricted to the core clinical study team and data entry staff only and will not be available to 
any other person. 
16.2 Study logs 
The following logs will be maintained by the study team: 
• 
Informed consent (IC) log: will contain particulars of pregnant women who were 
consented. This log will contain participant identifiers.  
• 
Screening and Enrolment log: will contain results of screening process This log will not 
contain participant identifiers and will not contain blinded data i.e. the outcome of 
randomization.  
• 
Randomization log: the results of randomization by enrolment number in the case of 
study stage 2. This log will be accessible to unblinded team members only. 
16.3 Data Management Responsibilities 
All source documents and laboratory reports must be reviewed by the clinical team and data 
entry staff, who will ensure that they are accurate and complete. Adverse events must be 
graded, assessed for severity and causality, and reviewed by the site investigator as described 
in Section 10.2.1. 
Data collection is the responsibility of the clinical trial staff at the site under the supervision of 
the site principal investigator. During the study, the investigator must maintain complete and 
accurate documentation for the study. 
Biofabri or its designee will be responsible for data management, quality review, analysis, and 
reporting of the study data. 

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16.4 Data Capture Methods 
Data quality control (QC) measures will be instituted in accordance with generic data QC 
procedures at the study site. All completed eCRFs will be checked for completeness, accuracy 
and legibility. Data reported on the eCRF that are derived from source documents or chart 
review, should be consistent with the source documents or the discrepancies should be 
explained.  
Data for this study will include safety (systemic solicited adverse events, injection site 
reactions, unsolicited adverse events, and serious adverse events) and laboratory 
(immunogenicity and QFT Gold Plus) outcome measures. 
16.4.1 Source documentation 
A folder for source documentation will be maintained by the site for each study participant. 
This folder will contain notes and records made by the study staff, including visits and 
procedures performed, results of observations, history taking and examinations, or any other 
notes relevant to the conduct of the study, protection and safety of the participant, or study 
data collected. Copies of relevant results or reports will be included in the source 
documentation folder. If copies of original documents are made, the staff member who makes 
the copy will certify that the copy is a true reflection of the original by following the SATVI site 
SOP for “certification of copies”. The source documentation participant folder will contain 
participant identifiers and will therefore only be accessible to the SATVI clinical team and 
monitors.  
16.4.2 Case Report Forms (CRFs) 
Relevant data will be transcribed by the clinical study team from source documentation to 
study eCRFs. eCRFs will not contain information that will identify the individual participating 
in the trial who will be identified only by the study number (screening number). eCRFs will not 
be completed for recruitment or failed screening procedures. 
The database will be password-restricted and regular back-ups will be made on secure data-
storage media. Electronic records on the database will contain relevant data to be used in the 
analysis of endpoints for the trial. The results of laboratory analysis may be kept on a separate 
secure database within the SATVI laboratory until laboratory analysis is complete but will be 
reconciled with the main study database before the database is locked. All data recorded on 
the study eCRFs will be captured in the database. 
16.5 Timing/Reports 
Line lists of all adverse events will be reported to the sponsor, UCT HREC, DSMB, SAHPRA 
and local medical monitor, on a 6-monthly basis or per the guidelines or SOPs of each of these 
bodies. Serious adverse events will be reported to the sponsor within 24 hours of detection, 
to UCT HREC and SAHPRA as per their respective guidelines. 

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Data will be reviewed on a regular basis throughout the trial. DSMB reviews of safety data will 
be conducted at specified time points during dose-escalation, and on an ad hoc basis if the 
study is paused for safety reasons (see Section 10.7 for further details of DSMB reviews).  A 
DSMB review on safety and immunogenicity data will be conducted after completion of the 
last Cohort 3 Study D182 visit. This safety and immunogenicity review will assist in dose 
selection for the Phase III study and will include all available safety data for all cohorts up to 
the last Cohort 3 Study D182 visit, as well as all immunogenicity data up to Study D182 visit 
per each cohort.  
Coding (medical history, concomitant medications, and adverse events) will occur on an 
ongoing basis throughout the study, including prior to DSMB reviews. A final coding review 
and approval will be carried out prior to database lock.  
The blind will be maintained throughout the trial, unless there is a need to unblind a participant 
for safety reasons (see Section 10.6) or unblinding is requested by the DSMB (see Section 
10.7). Unblinding of an independent biostatistician only is planned once the last Cohort 3 Study 
D182 visit has been completed in preparation for a safety and immunogenicity review (see 
Sections 8.3 and 12.3.2). Unblinding of the data for preparation of the final study report is 
planned when the database has been locked, which will occur after all data (with the exception 
of data relating to the immunology and exploratory objectives) have been entered, reviewed, 
and all queries related to the data have been addressed. 
16.6 Types of Data 
Data for this study will include safety (systemic solicited adverse events, injection site 
reactions, unsolicited adverse events, and serious adverse events) and laboratory 
(immunogenicity and QFT Gold Plus) outcome measures. 
16.7 Study Records Retention 
All study records (source documents, signed informed consent forms, IRB/IEC 
correspondence and approval letters, investigational product management records, and other 
essential documents) will be kept secured for at least a minimum of 15 years or until, at least, 
2 years after the last approval of a marketing application and until there are no pending or 
contemplated marketing applications or at least 15 years have elapsed since the formal 
discontinuation of clinical development of the investigational product. Additional storage will 
be according to the guidance of the applicable national regulatory authority or authorities. The 
investigator will ensure that study records are not disposed of or removed from the clinical trial 
site without prior notification and approval from Biofabri or its designee. 
16.8 Protocol Deviations 
All participant-level deviations from protocol procedures, evaluations, and/or visits must be 
documented. When possible, missed visits and procedures must be rescheduled and 

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performed at the nearest possible time point to the original schedule. Deviations will be 
reported to the IRB/IEC and applicable national regulatory authorities per their guidelines. 

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17 
PUBLICATION POLICY 
The final study report will be made available to the principal investigator for purposes of 
publications. The principal investigator and study staff must send all manuscripts, abstracts, 
and presentations using data from this study to Biofabri for review prior to their submission. 
Biofabri jointly and separately reserve the right to delete any part or parts of such materials 
deemed to be confidential or proprietary. 
The International Committee of Medical Journal Editors (ICMJE) member journals have 
adopted a trials-registration policy as a condition for publication. This policy requires that all 
clinical trials be registered in a public trials registry such as ClinicalTrials.gov, which is 
sponsored by the National Library of Medicine. It is the responsibility of the sponsor to register 
this trial in an acceptable registry prior to the start of participant enrollment. 
The ICMJE defines a clinical trial as any research project that prospectively assigns human 
subjects to intervention or comparison groups to study the cause-and-effect relationship 
between a medical intervention and a health outcome. Studies designed for other purposes, 
such as to study pharmacokinetics or major toxicity (e.g., Phase 1 trials), would be exempt 
from this policy. 
 
 

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18 
CHANGES TO THE PROTOCOL 
Changes between protocol versions are described below. 
18.1 Changes from Version 3.0 to Version 4.0 
 

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Section 
Number 
(Title) 
Change/Rationale 
Title page 
Protocol version and date updated. 
Section 2 
Key Roles  
TCD address updated (Drive vs Road). 
Section 3.2 
Rationale 
Grammar update. 
Rationale updated to include explanation for additional safety and immunogenicity 
analysis/review which will determine the optimal dose level for the Phase III study. Saving 
time to start Phase III will be a major public health benefit as indicated in the text. 
 
Included confirmation of when the clinical team would be unblinded, for clarity. 
Section 
6.3.2 
Masking 
Procedures  
Included confirmation of when the clinical team would be unblinded for clarity. Grammar 
update. Inclusion of “D” to confirm the Study D365, visit instead of just 365 visit, to clarify. 
Section 8.3 
Follow-up  
Updated naming of study visit to avoid ambiguity and for clarity of the addition of 7 (days). 

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Section 
Number 
(Title) 
Change/Rationale 
Section 8.3 
Follow-up 
(Study Day 
182) 
Unblinding of data for safety and immunogenicity to independent biostatistician in order to 
determine most optimal dose for Phase III study without compromising data integrity. 
Confirmation that the immune response to MTBVAC at D182 is predictive of the memory 
response at D365 is included below: 
The original study was designed to analyse the antigen-specific CD4 T cell response induced 
by escalating doses of MTBVAC at timepoints defined to represent an acceptable 
assessment of the immune response kinetics (at days 28, 56, 182 and 365 post vaccination). 
Based on previous studies (Soares et al, JID, 2013, Tameris et al, Lancet Resp. Med., 2019) 
vaccine-induced T-cell immune responses are expected to peak between 7 and 10 weeks 
post-vaccination (D28 and D56 in our trial), then to gradually wane over the subsequent 10 
months of follow up.  
In the context of an interim analysis necessary for the vaccine dose selection to inform the 
MTBVAC phase 3 trial, we determined if assessment of immune responses at D182 post-
MTBVAC vaccination is predictive of memory responses at D365. We reanalysed 
immunogenicity data from the phase IIa trial (Tameris et al, Lancet Resp. Med., 2019), and 
assessed the strength of correlation between MTBVAC-specific total CD4 T cell responses 
(IFN-g, IL-2, TNF, IL-17 and/or IL-22) at D180 and D360 following MTBVAC vaccination 
(Figure 1). At the vaccine dose analysed (2.5 x 103 CFU, 2,5 x 104 CFU and 2.5 x 105 CFU), 
the MTBVAC-specific CD4 T cell responses between D180 and D360 were strongly 
correlated, suggesting that D182 is predictive of vaccine induced memory response at 1 year 
post-vaccination. 
 
In addition, we will also determine if the cytokine co-expression patterns indicate evidence of 
excessive T cell differentiation, which can lead to poor long-term memory responses.  
 
 
Section 8.4 
Follow-up 
(Final Study 
Visit) 
 
Included confirmation of when the clinical team would be unblinded for clarity.  
Updated grammar. 

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Section 
Number 
(Title) 
Change/Rationale 
Section 10.7 
Safety 
Oversight 
(DSMB) 
Included when DSMB to review overall available safety data to confirm if any safety issues 
for any cohort doses. DSMB to confirm safest dose for Phase III study. 
Section 
12.3.1 
Planned 
Interim 
Analyses – 
Safety 
Review  
Included when DSMB to review overall available safety data to confirm if any safety issues 
for any cohort doses. DSMB to confirm safest dose for Phase III study. 
Section 
12.3.2 
Planned 
Interim 
Analyses – 
Immunogeni
city or 
Efficacy 
Review 
Included when DSMB to review overall immunogenicity data up to D182 for all cohorts to 
confirm which dose is most immunogenic and whether MTBVAC is superior to BCG 
immunogenically. This will assist in dose selection for the Phase III study. 
Section 
12.4.3 
Immunogeni
city and 
Other 
Immunology 
Analyses 
(Final 
Analysis) 
Clarification of supporting document involved. 
Section 16.4 
Data 
Capture 
Methods 
Grammar update. 
Inclusion of injection site reactions previously not included in previous versions of the 
protocol. 
Section 16.5 
Timing/Rep
orts 
To collate information mentioned in previous sections. Included when DSMB to review 
overall immunogenicity data up to D182 and all available safety data for all cohorts to 
confirm which dose is most safe and immunogenic. This will assist in dose selection for 
Phase III. 
Section 16.6 
Types of 
Data 
Inclusion of injection site reactions previously not included in previous versions of the 
protocol. 
Header 
Protocol version and date updated. 
 
 

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19 
LITERATURE REFERENCES 
1. 
WHO, Global Tuberculosis Report 2017.  
http://www.who.int/tb/publications/global_report/en/. 
 
2. 
Knight GM, Griffiths UK, Sumner T, et al. Impact and cost-effectiveness of new 
tuberculosis vaccines in low- and middle-income countries. Proc Natl Acad Sci U S A, 
111(43), 15520-15525 (2014). 
 
3. 
Mangtani P, Abubakar I, Ariti C, et al. Protection by BCG vaccine against tuberculosis: a 
systematic review of randomized controlled trials. Clin Infect Dis, 58(4), 470-480 (2014). 
 
4. 
Mahomed H, Hughes EJ, Hawkridge T, et al. Comparison of mantoux skin test with three 
generations of a whole blood IFN-gamma assay for tuberculosis infection. Int J Tuberc 
Lung Dis, 10(3), 310-316 (2006). 
 
5. Brosch R, Gordon SV, Garnier T, et al. Genome plasticity of BCG and impact on vaccine 
efficacy. Proc Natl Acad Sci U S A, 104(13), 5596-5601 (2007). 
 
6. 
Copin R, Wang X, Louie E, et al. Within Host Evolution Selects for a Dominant Genotype 
of Mycobacterium tuberculosis while T Cells Increase Pathogen Genetic Diversity. PLoS 
Pathog, 12(12), e1006111 (2016). 
 
7. 
Copin R, Coscolla M, Efstathiadis E, Gagneux S, Ernst JD. Impact of in vitro evolution on 
antigenic diversity of Mycobacterium bovis bacillus Calmette-Guerin (BCG). Vaccine, 
32(45), 5998-6004 (2014). 
 
8. 
Arbues A, Aguilo JI, Gonzalo-Asensio J, et al. Construction, characterization and 
preclinical evaluation of MTBVAC, the first live-attenuated M. tuberculosis-based vaccine 
to enter clinical trials. Vaccine, 31(42), 4867-4873 (2013). 
 
9. 
Stucki D, Brites D, Jeljeli L, et al. Mycobacterium tuberculosis lineage 4 comprises globally 
distributed and geographically restricted sublineages. Nat Genet, 48(12), 1535-1543 
(2016). 
 
10. Kamath AT, Fruth U, Brennan MJ, et al. New live mycobacterial vaccines: the Geneva 
consensus on essential steps towards clinical development. Vaccine, 23(29), 3753-3761 
(2005). 
 
11. Walker KB, Brennan MJ, Ho MM, et al. The second Geneva Consensus: 
Recommendations for novel live TB vaccines. Vaccine, 28(11), 2259-2270 (2010). 
 

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12. Scriba TJ, Kaufmann SH, Henri Lambert P, Sanicas M, Martin C, Neyrolles O. Vaccination 
Against Tuberculosis with Whole-Cell Mycobacterial Vaccines. J Infect Dis, 214(5), 659-
664 (2016). 
 
13. Aguilo N, Uranga S, Marinova D, Monzon M, Badiola J, Martin C. MTBVAC vaccine is 
safe, immunogenic and confers protective efficacy against Mycobacterium tuberculosis in 
newborn mice. Tuberculosis (Edinb), 96, 71-74 (2016). 
 
14. Spertini F, Audran R, Chakour R, et al. Safety of human immunisation with a live-
attenuated Mycobacterium tuberculosis vaccine: a randomised, double-blind, controlled 
phase I trial. Lancet Respir Med, 3(12), 953-962 (2015). 
 
15. Soares AP, Kwong Chung CK, Choice T, et al. Longitudinal changes in CD4(+) T-cell 
memory responses induced by BCG vaccination of newborns. J Infect Dis, 207(7), 1084-
1094 (2013). 
 
16. Lindestam Arlehamn CS, McKinney DM, Carpenter C, et al. A Quantitative Analysis of 
Complexity of Human Pathogen-Specific CD4 T Cell Responses in Healthy M. 
tuberculosis Infected South Africans. PLoS Pathog, 12(7), e1005760 (2016). 
 
17. Scriba TJ, Tameris M, Mansoor N, et al. Dose-finding study of the novel tuberculosis 
vaccine, MVA85A, in healthy BCG-vaccinated infants. J Infect Dis, 203(12), 1832-1843 
(2011). 
 
18. Luabeya AK, Kagina BM, Tameris MD, et al. First-in-human trial of the post-exposure 
tuberculosis vaccine H56:IC31 in Mycobacterium tuberculosis infected and non-infected 
healthy adults. Vaccine, 33(33), 4130-4140 (2015). 
 
19. Penn-Nicholson A, Geldenhuys H, Burny W, et al. Safety and immunogenicity of 
candidate vaccine M72/AS01E in adolescents in a TB endemic setting. Vaccine, 33(32), 
4025-4034 (2015). 
 
20. Tameris M, Hokey DA, Nduba V, et al. A double-blind, randomised, placebo-controlled, 
dose-finding trial of the novel tuberculosis vaccine AERAS-402, an adenovirus-vectored 
fusion protein, in healthy, BCG-vaccinated infants. Vaccine, 33(25), 2944-2954 (2015). 
 
21. Geldenhuys H, Mearns H, Miles DJ, et al. The tuberculosis vaccine H4:IC31 is safe and 
induces a persistent polyfunctional CD4 T cell response in South African adults: A 
randomized controlled trial. Vaccine, 33(30), 3592-3599 (2015). 
 
22. Scriba TJ, Tameris M, Smit E, et al. A phase IIa trial of the new tuberculosis vaccine, 
MVA85A, in HIV- and/or Mycobacterium tuberculosis-infected adults. Am J Respir Crit 
Care Med, 185(7), 769-778 (2012). 
 

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23. Kagina BM, Tameris MD, Geldenhuys H, et al. The novel tuberculosis vaccine, AERAS-
402, is safe in healthy infants previously vaccinated with BCG, and induces dose-
dependent CD4 and CD8T cell responses. Vaccine, 32(45), 5908-5917 (2014). 
 
24. Kagina BM, Mansoor N, Kpamegan EP, et al. Qualification of a whole blood intracellular 
cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity 
by flow cytometry. J Immunol Methods, 417, 22-33 (2015). 
 
25. Kagina BM, Abel B, Scriba TJ, et al. Specific T cell frequency and cytokine expression 
profile do not correlate with protection against tuberculosis after bacillus Calmette-Guerin 
vaccination of newborns. Am J Respir Crit Care Med, 182(8), 1073-1079 (2010). 
 
26. Andrews JR, Nemes E, Tameris M, et al. Serial QuantiFERON testing and tuberculosis 
disease risk among young children: an observational cohort study. Lancet Respir Med, 
(2017). 
 
27. Diel R, Vandeputte J, de Vries G, Stillo J, Wanlin M, Nienhaus A. Costs of tuberculosis 
disease in the European Union: a systematic analysis and cost calculation. Eur Respir J, 
43(2), 554-565 (2014). 
 
28. Zisowsky J, Krause A, and Dingemanse J. Drug Development for Pediatric Populations: 
Regulatory Aspects. Pharmaceutics 2, 364-388 (2010). 
 
29. Tameris, M.D., et al., Safety and efficacy of MVA85A, a new tuberculosis vaccine, in 
infants previously vaccinated with BCG: a randomised, placebo-controlled phase 2b trial. 
Lancet, 2013. 381(9871): p. 1021-8. 
 
 
 

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SUPPLEMENTS/APPENDICES 
 
 

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APPENDIX A. SCHEDULE OF EVENTS   
 
 
 
Evaluation 
 
 
Screen 
Study day 
Visit Window (hrs/days) 
D0 
D7 
D28 
D56 
D182 D365 
Post 
D365 
Early 
termination 
NA 
+96 
hrs 
±3d 
±3d 
±7d 
±14d 
±14d 
NA 
NA 
Informed consent process  
Ma 
 
 
 
 
 
 
 
 
Medical history  
M 
 
 
 
 
 
 
 
 
TB symptoms or household TB 
contact 
M 
x 
x 
x 
x 
x 
x 
x 
x 
Maternal HIV result available 
M 
 
 
 
 
 
 
 
 
Verbally reconfirm consent 
M 
M 
M 
M 
M 
M 
M 
M 
 
Verify eligibility criteria 
x 
x 
 
 
 
 
 
 
 
Vital signs, length, weight 
 
x 
x 
x 
x 
x 
x 
x 
x 
Record feeding practice 
 
x 
x 
x 
x 
x 
x 
x 
x 
Physical examination 
 
x 
x 
x 
x 
x 
x 
x 
x 
Recording of concomitant medication 
 
x 
x 
x 
x 
x 
x 
x 
x 
Randomization 
 
x 
 
 
 
 
 
 
 
MTBVAC/BCG administration 
 
x 
 
 
 
 
 
 
 
Safety blood sampling (mL)b 
 
 
2 
 
 
 
 
 
 
QuantiFERON-TB Gold Plus (mL)b 
 
 
 
 
4d 
4 
4 
 
4 
Whole blood (mL)b 
(12-hour WB-ICS) 
 
 
 
2 
2 
4 
4 
 
 
Distribute/review diary cards 
 
x 
x 
 
 
 
 
 
 
Solicited systemic adverse events 
(incl. concomitant medications) 
 
x 
x 
x 
 
 
 
 
 
Unsolicited adverse events (incl. 
concomitant medications) 
 
x 
x 
x 
x 
x 
x 
 
x 
Solicited and unsolicited injection site 
reaction adverse events (incl. 
concomitant medications) 
 
x 
x 
x 
x 
 
 
 
 
Serious adverse events (incl. 
concomitant medications) 
 
x 
x 
x 
x 
x 
x 
 
x 
Site of injection examination, swab if 
discharge  
 
x 
x 
x 
x 
x 
x 
 
x 
Rescue dose BCG post unblinding of 
cohortc 
 
 
 
 
 
 
 
x 
 
Approximate per visit phlebotomy 
volume (mL)a 
 
 
2 
2 
6 
8 
8 
 
4 
Cumulative phlebotomy volume (mL)b 
 
 
2 
4 
10 
18 
26 
 
NA 
 
a Maternal - the mother signs once, antenatally , and thereafter reconfirms consent orally at each visit 
b Acceptable infant blood sample volumes in clinical research are limited to <3% of the total blood volume (TBV), and 
no more than 3mL/kg over an 8 week period  
c Telephonic contact/ home visit approximately 14 days post BCG to confirm infant’s wellbeing incl. BCG injection site 
reaction 
d If QFT positive at D56 a home/telephonic visit will be done at D120±7d and 270±7d to monitor infant’s wellbeing, 
specifically enquiring about TB signs and symptoms 

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APPENDIX B. TABLE OF TOXICITY REFERENCE RANGES FOR GRADING OF ADVERSE 
EVENTS 
Local Site of Injection 
Symptoms  
Grade 1  
Grade 2  
Grade 3  
Grade 4  
Tenderness (pain when 
area is touched)  
Minimal or no limitation of 
use of limb  
Limitation of use of limb OR 
greater 
than 
minimal 
interference 
with 
usual 
activities  
Inability to perform usual 
activities  
N/A  
Erythema (Redness)*  
>2 - ≤20 mm maximum 
diameter 
>20 - ≤50 mm maximum 
diameter 
>50 mm maximum 
Local 
or 
extensive 
exfoliative dermatitis  
Induration * 
>2 - ≤20 mm maximum 
diameter 
>20 - ≤50 mm maximum 
diameter 
>50 mm maximum 
Local 
or 
extensive 
exfoliative dermatitis  
Ulceration * 
>2- ≤15 mm maximum 
diameter 
>15- ≤30 mm maximum 
diameter 
>30 mm maximum 
diameter 
Requires 
hospitalization 
or non-routine treatment  
Scar at Injection Site* 
>2- ≤15 mm maximum 
diameter 
>15- ≤30 mm maximum 
diameter 
>30 mm maximum 
diameter 
Keloid 
or 
extensive 
scarring  
Regional 
lymphadenopathy 
Non-tender, 
isolated, 
<3cm, 
mobile, 
non-
fluctuating 
Tender, 
multiple 
matted 
nodes, 
fixated, 
non-
fluctuating 
Supparative 
lymphadenitis 
Supparative 
lymphadenitis with fistula 
formation 
* In addition to grading, record maximum diameter in mm 
General symptoms 
Grade 1  
Grade 2  
Grade 3  
Grade 4  
Decreased oral intake  
Minimal decrease in oral 
intake  
Below 50% of normal oral 
intake in 24 hr  
No oral intake in 24hr  
N/A  
Vomiting  
1 episode in 24hr; no 
interference with activity  
2-3 episodes in 24 hr OR 
some interference with 
activity  
> 3 episodes in 24 hours 
OR prevents daily activity  
N/A  
Diarrhea  
Unformed stool OR 1-3 
more stools than baseline 
in 24 hr  
Partially liquid stools OR 4-6 
more stools than baseline in 
24hr  
Completely liquid stools 
OR >6 more stools than 
baseline in 24 hr  
N/A  
Irritability  
Easily consolable; 
minimal or no 
interference with activity  
Difficult to console; some 
interference with activity  
Inconsolable; prevents 
daily activity  
N/A  
Lethargy  
Minimal decrease in 
alertness; minimal 
interference with activity  
Some interference with 
activity  
Unable to achieve normal 
level of alertness; 
prevents daily activity  
N/A  
Illness or clinical adverse 
event 
Minimal or no 
interference with activity  
Some interference with 
activity not requiring medical 
intervention  
Prevents daily activity 
and requires medical 
intervention  
N/A  
Vital Signs  
Grade 1  
Grade 2  
Grade 3  
Grade 4  
Fever (Axillary)  
38.0 – 38.4°C  
100.4 – 101.1°F  
38.5 - 40°C  
101.2 - 104°F  
>40°C  
>104°F  
N/A  
Tachycardia – beats per 
minute (infants) 
 (adults) 
181-200  
 
201-220  
 
>220  
 
N/A  
101 – 115 
116 – 130 
>130 
ER visit or hospitalization 
for arrhythmia 
Bradycardia – beats per 
minute(infants)(adults) 
96-105  
91-95  
<91  
N/A  
50 – 54 
45 –  49 
<45 
ER visit or hospitalization 
for arrhythmia 
Tachypnea – breaths per 
minute (infants) 
(adults) 
61-65 
66-69 
>69  
N/A  
17 – 20 
21 – 25 
25 
Intubation 

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Hypertension (systolic) – 
mm Hg (adults only) 
141 – 150 
151 – 155 
>155 
ER visit or hospitalization 
for malignant 
hypertension 
Hypertension (diastolic) – 
mm Hg (adults only) 
91 – 95 
96 – 100 
>100 
ER visit or hospitalization 
for malignant 
hypertension 
Hypotension (systolic) – 
mm Hg (adults only) 
85 - 89 
80 – 84 
<80 
ER visit or hospitalization 
for hypotensive shock 
Respiratory Distress/ 
Hypoxia  
Wheezing, nasal flaring or 
retractions; minimal or no 
interference with activity  
Some 
interference 
with 
activity or pulse oximetry 
<95%  
Prevents normal activity 
or pulse oximetry <90%  
N/A  
All other AEs 
No 
interference 
with 
normal 
daily 
activity, 
minor 
severity. 
No 
treatment required. 
Minimal 
interference 
with 
normal 
daily 
activity, 
moderate severity, treatment 
may be required  
Significant 
interference 
with normal daily activity, 
treatment required 
Severe 
incapacitation, 
urgent 
treatment 
or 
hospital 
admission 
required 
 
 
 

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APPENDIX C. TABLE OF TOXICITY REFERENCE RANGES FOR GRADING SAFETY 
LABORATORY EVENTS 
Serum/Plasma Chemistry 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Sodium – hyponatremia 
mEq/L or mmol/L: 
 
132 – 134 
 
130 – 131 
 
<130 
 
N/A 
Sodium – hypernatremia 
mEq/L or mmol/L: 
 
145- 146 
147-148 
 
>148 
 
N/A 
Potassium – hyperkalemia 
mEq/L or mmol/L 
≤ 1 year: 
1 - 2years: 
 
 
5.3 – 5.4 
5.1 – 5.2 
 
 
5.5 – 5.6 
5.3 – 5.4 
 
 
>5.6 
>5.4 
N/A 
Potassium – hypokalemia 
mEq/L or mmol/L: 
 
3.5 – 3.6 
 
3.3 – 3.4 
 
3.1 – 3.2 
N/A 
Glucose – hypoglycemia 
mg/dL: 
mmol/L: 
 
55-59 
3.0 – 3.2 
 
50-54 
2.8 – 2.9 
 
<50 
<2.8 
N/A 
Glucose – hyperglycemia 
Fasting -  mg/dL: 
mmol/L: 
Random – mg/dL: 
mmol/L: 
 
101 – 110 
5.7 – 6.0 
110 - 125 
6.1 – 6.8 
 
111 – 125 
6.1 – 6.8 
126 - 200 
6.9 – 11.0 
 
>125 
6.8 
>200 
11.0 
N/A 
Blood urea nitrogen (BUN) – 
increased  
mg/dL: 
mmol/L: 
 
21 – 24 
7.5 – 8.9 
 
25 – 28 
9.0 – 10.0 
 
>28 
>10.0 
N/A 
Creatinine – increased 
mg/dL: 
umol/L: 
 
0.8 – 0.9 
66 - 82 
 
1.0 – 1.2 
83 – 100 
 
>1.2 
>100 
N/A 
Calcium – hypocalcemia 
mg/dL: 
mmol/L: 
 
8.0 – 8.4 
2.00 – 2.10 
 
7.5 – 7.9 
1.87 – 1.99 
 
<7.5 
<1.87 
N/A 
Calcium – hypercalcemia 
mg/dL: 
mmol/L: 
 
11.1-11.3 
2.78 – 2.84 
 
11.4 – 11.6 
2.85 – 2.92 
 
>11.6 
>2.92 
N/A 
Magnesium – hypomagnesemia 
mg/dL: 
mmol/L: 
 
1.3 – 1.5 
0.52 – 0.62 
 
1.1 – 1.2 
0.43 – 0.51 
 
<1.1 
<0.43 
N/A 
Phosphorus – hypophosphatemia 
mg/dL: 
mmol/L: 
 
2.3 – 2.5 
0.73 – 0.80 
 
2.0 – 2.2 
0.63 – 0.72 
 
<2.0 
<0.63 
N/A 
Albumin – hypoalbuminemia 
g/dL: 
g/L: 
 
2.5 – 2.7 
25 – 27 
 
2.2 – 2.4 
22 – 24 
 
<2.2 
22 
N/A 
Total protein – hypoproteinemia 
g/dL: 
g/L: 
 
4.4 – 4.6 
44 – 46 
 
4.1 – 4.3 
41 – 43 
 
<4.1 
41 
N/A 
Alkaline phosphatase (ALP) 
– increased 
1.1 – 2.0 x 
ULN** 
2.1 – 3.0 x ULN 
3.1 – 10 x ULN 
>10 x ULN 
Liver Function Tests (LFT) 
 AST, ALT, GGT – increased 
1.1 – 2.5 x ULN 
2.6 – 5.0 x ULN 
5.1 – 10 x ULN 
>10 x ULN 
Bilirubin (with any increase in LFT) 
- increased 
1.1 – 1.25 x 
ULN 
1.26 – 1.5 x 
ULN 
1.51 – 1.75 x 
ULN 
>1.75 x ULN 

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Serum/Plasma Chemistry 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Bilirubin (with normal LFT) 
- increased 
1.1 – 1.5 x ULN 
1.6 – 2.0 x ULN 
2.1 – 3.0 – x 
ULN 
>3.0 x ULN 
Cholesterol – increased 
mg/dL: 
mmol/L: 
 
171 - 185 
5.1 – 5.5 
 
186 – 199 
5.6 – 6.0 
 
>199 
6.0 
N/A 
Pancreatic enzymes  
amylase, lipase – increased 
1.1 – 1.5 x ULN 
1.6 – 2.0 x ULN 
2.1 – 5.0 x ULN 
>5.0 x ULN 
 
 
Hematology 
Grade 1 
Grade 2 
Grade 3 
Grade 4 
Hemoglobin g/dL 
< 21 days 
22 – 35 days 
36 days – 56 days 
≥ 57 days < 6 months 
6 mo - 2yr 
 
12.0 – 13.0 
9.5 – 10.5 
8.5 – 9.4 
9.0 – 9.4 
10.0 – 10.4 
 
10.0 – 11.9 
8.0 – 9.4 
7.0 –  8.4 
8.5 – 8.9 
9.5 – 9.9 
 
9.0 – 9.9 
7.0 – 7.9 
6.0 – 6.9 
<8.5 
<9.5 
 
<9.0 
<7.0 
<6.0 
N/A 
N/A 
WBC – increased 
cells/mm3 
18,700 – 22,000 
22,100 – 25,000 
>25,000 
N/A 
WBC – decreased 
 cells/mm3 
4,500 – 5,500 
3,500 – 4,400 
<3,500 
N/A 
Lymphocytes - decreased 
cells/mm3 
2,000-2,700 
1,500-1,900 
<1,500 
N/A 
Neutrophils - decreased  
Age (> 7 days – 3 mo)  
cells/mm3:  
(≥ 3 mo)  
cells/mm3: 
 
1,000 – 1,300 
 
750-990 
 
 
750 – 999 
 
500-740 
 
 
< 750 
 
<500 
 
 
N/A 
 
N/A 
Eosinophils – increased cells/mm3 
850 – 1,500 
1,501 – 5,000 
5,000 
N/A 
Platelets - decreased 
cells/mm3 
125,000 – 
140,000 
100,000 – 
124,000 
<100,000 
N/A 
Prothrombin Time 
(PT)/International normalized ratio 
(INR) 
– increased 
1.1 – 1.2 x ULN** 
1.3 – 1.4 x ULN 
>1.4 x ULN 
N/A 
Partial thromboplastin time (PTT) – 
increased  
1.1 – 1.2 x ULN 
1.3 – 1.4 x ULN 
>1.4 x ULN 
N/A 
Fibrinogen – increased 
mg/dL: 
g/L: 
 
400 – 500 
4.00 – 5.00 
 
501 – 600 
5.01 – 6.00 
 
>600 
6.00 
N/A 
Fibrinogen – decreased 
mg/dL: 
g/L: 
 
150 - 170 
1.50 – 1.70 
 
125 – 149 
1.25 – 1.49 
 
<125 
<1.25 
N/A 
 
 
 
 
 

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APPENDIX D. TOTAL BILIRUBIN TABLE FOR TERM AND PRETERM NEONATES 
PARAMETER 
GRADE 1 
MILD 
GRADE 2 
MODERATE 
GRADE 3 
SEVERE 
GRADE 4 
POTENTIALLY 
LIFE- 
THREATENING 
Total Bilirubina, High 
(mg/dL; µmol/L)
b 
Term Neonate
c 
< 24 hours of age 
4 to < 7 
68.4 to < 119.7 
7 to < 10 
119.7 to < 171 
10 to < 17 
171 to < 290.7 
≥ 17 
≥ 290.7 
24 to < 48 hours 
of age 
5 to < 8 
85.5 to < 136.8 
8 to < 12 
136.8 to < 205.2 
12 to < 19 
205.2 to < 324.9 
≥ 19 
≥ 324.9 
48 to < 72 hours 
of age 
8.5 to < 13 
145.35 to < 222.3 
13 to < 15 
222.3 to < 256.5 
15 to < 22 
256.5 to < 376.2 
≥ 22 
≥ 376.2 
72 hours to < 7 
days of age 
11 to < 16 
188.1 to < 273.6 
16 to < 18 
273.6 to < 307.8 
18 to < 24 
307.8 to < 410.4 
≥ 24 
≥ 410.4 
7 to 28 days of 
age (breast 
feeding) 
5 to < 10 
85.5 to < 171 
10 to < 20 
171 to < 342 
20 to < 25 
342 to < 427.5 
≥ 25 
≥ 427.5 
7 to 28 days of 
age (not breast 
feeding) 
1.1 to < 1.6 x ULN 
1.6 to < 2.6 x ULN 
2.6 to < 5.0 x ULN 
≥ 5.0 x ULN 
Preterm Neonate
c 
35 to < 37 
weeks’ 
gestational age 
Same as for Total 
Bilirubin, High, 
Term Neonate 
(based on days of 
age). 
Same as for Total 
Bilirubin, High, 
Term Neonate 
(based on days of 
age). 
Same as for Total 
Bilirubin, High, 
Term Neonate 
(based on days of 
age). 
Same as for Total 
Bilirubin, High, 
Term Neonate 
(based on days of 
age). 
32 to < 35 weeks’ 
gestational age 
and 
< 7 days of age 
NA 
NA 
10 to < 14 
171 to < 239.4 
≥ 14 
≥ 239.4 
28 to < 32 weeks’ 
gestational age 
and 
< 7 days of age 
NA 
NA 
6 to < 10 
102.6 to < 171 
≥ 10 
≥ 171 
< 28 weeks’ 
gestational age 
and 
< 7 days of age 
NA 
NA 
5 to < 8 
85.5 to < 136.8 
≥ 8 
≥ 136.8 
7 to 28 days of 
age (breast 
feeding) 
5 to < 10 
85.5 to < 171 
10 to < 20 
171 to < 342 
20 to < 25 
342 to < 427.5 
≥ 25 
≥ 427.5 
7 to 28 days of 
age (not breast  
feeding) 
1.1 to < 1.6 x ULN 
1.6 to < 2.6 x ULN 
2.6 to < 5.0 x ULN 
≥ 5.0 x ULN 
a Severity grading for total bilirubin in neonates is complex because of rapidly changing total bilirubin normal 
ranges in the first week of life followed by the benign phenomenon of breast milk jaundice after the first week 
of life.  Severity grading in this appendix corresponds approximately to cut-offs for indications for phototherapy 
at grade 3 and for exchange transfusion at grade 4. 
b A laboratory value of 1 mg/dL is equivalent to 17.1 µmol/L. 

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c Definitions: Term is defined as ≥ 37 weeks’ gestational age; near-term, as ≥ 35 weeks’ gestational age; preterm,   
   as < 35 weeks’ gestational age; and neonate, as 0 to 28 days of age.