A Phase I, Randomised, Double-blind, Placebo-controlled, Parallel Group Dose Escalation Study to Evaluate the Safety, Tolerability and Immunogenicity of a Potential Oral Enteric Fever Vaccine (ZH9 + ZH9PA) in Healthy Participants
Phase: PHASE1
Conditions: Enteric Fever
Sponsor: Prokarium Ltd

 
 
 
 
 
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STUDY PROTOCOL 
 
STUDY TITLE: 
A Phase I, randomised, double-blind, placebo-controlled, 
parallel group dose escalation study to evaluate the safety, 
tolerability and immunogenicity of three doses of a 
potential oral enteric fever vaccine (ZH9 + ZH9PA) in 
healthy participants 18 to 45 years of age inclusive. 
 
 
 
STUDY NUMBER: 
PRK-ENT-001 
RD760/34119 
EudraCT NUMBER:  
2019-002047-22 
IRAS ID: 
266061 
INVESTIGATIONAL 
MEDICINAL 
PRODUCT 
(IMP)(s): 
ZH9PA 
ZH9/ZH9PA 
Placebo 
 
PLANNED STUDY DOSES: 
3 doses of 1x109 CFU of ZH9PA 
3 doses of 1x1010 CFU of ZH9PA 
3 doses of 1x1010 CFU of ZH9PA and 1x1010 CFU of ZH9 
3 doses of placebo 
 
PRINCIPAL 
INVESTIGATOR: 
 
Simbec-Orion Clinical Pharmacology,  
Merthyr Tydfil Industrial Park, Pentrebach, 
Merthyr Tydfil, 
CF48 4DR, United Kingdom (UK) 
 
 
STUDY SPONSOR: 
Prokarium Ltd. 
London Bioscience Innovation Centre, 
2 Royal College St, London, 
NW1 0NH, UK 
 
 
STUDY SPONSOR’S 
RESPONSIBLE PHYSICIAN: 
 
 
 
 

 
 
 
 
 
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STUDY MONITOR:  
 
 
 
 
ADDITIONAL 
DEPARTMENTS: 
 
 
 
 
PROTOCOL FINALISATION STATEMENT 
 
This protocol is not considered final unless accompanied by a letter of favourable opinion 
from the Research Ethics Committees and Notice of Acceptance from the relevant Competent 
Authority. 
 
Protocol Prepared by: LL 
 
 

 
 
 
 
 
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PROTOCOL AMENDMENT/REVISION HISTORY 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
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SYNOPSIS 
NAME OF COMPANY: Prokarium Ltd. 
NAME OF INVESTIGATIONAL MEDICINAL PRODUCT: ZH9PA and ZH9/ZH9PA 
NAME OF ACTIVE INGREDIENT: ZH9 and ZH9PA 
TITLE OF STUDY: A Phase I, randomised, double-blind, placebo-controlled, parallel group dose escalation 
study to evaluate the safety, tolerability and immunogenicity of three doses of a potential oral enteric fever 
vaccine (ZH9 + ZH9PA) in healthy participants 18 to 45 years of age inclusive. 
PRINCIPAL INVESTIGATOR: 
 
STUDY CENTRE: 
Simbec-Orion Clinical Pharmacology, 
Merthyr Tydfil, CF48 4DR, UK 
CLINICAL PHASE: I 
OBJECTIVES:  
Primary Objective 
• 
To evaluate the safety and tolerability of three doses of two dose levels of ZH9PA and of three doses 
of a single dose level of a combination of ZH9 and ZH9PA over 12 weeks (up to Day 84) after the 
first dose of vaccine. 
 
Secondary Objectives 
• 
To evaluate serum immunoglobulin (Ig) A and IgG responses against the following antigens: - S. 
enterica Ser. Paratyphi A lipopolysaccharide (LPS) 0:2 and Flagella H:a and S. enterica ser. Typhi 
LPS O:9 and Flagella H:d, induced by three vaccine doses, of two dose levels (1x109 colony forming 
units (CFU) and 1x1010 CFU) of ZH9PA alone or in combination with 1x1010 CFU ZH9, up to Day 
84 after the first dose of vaccine (Day 0). 
 
• 
To evaluate the mucosal IgA immune responses to LPS 0:2, LPS O:9, Flagella H:a and Flagella H:d, 
at 7 days after each vaccination, as determined by antibodies in lymphocyte supernatant (ALS) assay. 
 
• 
To evaluate Serum IgA and IgG responses against the four antigens, induced by each of the first two 
vaccine doses, on Days 21, and 42, by blood samples taken prior to each participant receiving the 
next dose of vaccine or placebo. 
 
• 
To evaluate the safety profile of the ZH9PA and combination ZH9/ZH9PA vaccines, over the 6 
months following the third dose of vaccine.  
METHODOLOGY: 
This is a Phase I, randomised, double-blind, placebo-controlled, parallel group, single-centre study involving 
45 healthy participants. The aim is to evaluate a combination vaccine against enteric fever comprising the live 
attenuated Salmonella strain ZH9 (previously tested in a number of clinical trials as a typhoid vaccine) and its 
modified derivative, ZH9PA, in which the typhoid antigens LPS O:9 and Flagellin H:d have been substituted 
by the Paratyphi A antigens, LPS O:2 and Flagellin H:a. ZH9PA has not previously been tested in humans; 
therefore, the first two cohorts comprise a dose escalation of this component alone, followed by a final cohort 
in which the combination is tested at a single dose level. The study will remain fully blinded until Day 70, 
when a stool sample is taken to assess if participants are shedding the bacterial strains of the vaccine in their 
faeces.  The Investigators and the Monitor may be unblinded to the treatment allocation of participants who 
have a stool culture positive for any Salmonella strain, as it will be presumed to be one of the vaccine strains, 
and the participant will receive a course of antibiotics. 
NUMBER OF PARTICIPANTS:  45 healthy participants 18 to 45 years of age, inclusive. 
 
 

 
 
 
 
 
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INCLUSION CRITERIA: 
To be confirmed at Screening 
1. Healthy male and female participants 18 to 45 years of age, inclusive. 
2. Female participant of childbearing potential willing to use 2 effective methods of contraception, 
i.e., established method of contraception + condom, if applicable (unless of non-childbearing 
potential or where abstaining from sexual intercourse is in line with the preferred and usual lifestyle 
of the participant) from the first dose until 2 months after the last dose of IMP.  
3. Female participant of non-childbearing potential. For the purposes of this study, this is defined as 
the participant being amenorrhoeic for at least 12 consecutive months or at least 4 months post-
surgical sterilisation (including bilateral fallopian tube ligation or bilateral oophorectomy with 
or without hysterectomy).  
4. Female participant of childbearing potential or non-childbearing potential with a negative 
pregnancy test at Screening. 
5. Female participant of post-menopausal status confirmed by demonstrating at Screening that the 
serum level of the follicle stimulating hormone (FSH) falls within the respective pathology 
reference range.  In the event a participant's menopausal status has been clearly established (for 
example, the participant indicates she has been amenorrhoeic for 10 years, confirmed by medical 
history, etc), but serum FSH levels are not consistent with a postmenopausal status, determination 
of the participant’s eligibility to be included in the study will be at the Investigator’s discretion 
following consultation with the Sponsor. 
6. Male participant willing to use an effective method of contraception or 2 effective methods of 
contraception, i.e., established method of contraception + condom, if applicable (unless 
anatomically sterile or where abstaining from sexual intercourse is in line with the preferred and 
usual lifestyle of the participant) from first dose until a stool sample tested for presence of the 
vaccine strains is negative. 
7. Participant with a body mass index (BMI) of ≥ 19 or ≤34 kg/m2 (BMI = body weight (kg) / 
[height (m)]2). 
8. No clinically significant history of liver or active gall bladder disease. 
9. No clinically significant history of ongoing gastro-intestinal disease or abnormality. 
10. No clinically significant history of previous allergy / sensitivity to ZH9/ZH9PA or sodium 
bicarbonate. 
11. No clinically significant history of anaphylactic shock following vaccination. 
12. No clinically significant history of hypersensitivity (e.g., hives/rash/swollen lips/difficulty with 
breathing) to azithromycin, ampicillin, trimethoprim-sulfamethoxazole or ciprofloxacin. 
13. No clinically significant abnormal laboratory test results (in the opinion of the investigator) for 
serum biochemistry, haematology and/or urine analyses within 28 days before receiving the first 
dose administration of the IMP.   
14. Participant with a negative urinary drugs of abuse (DOA) screen (including alcohol and cotinine) 
test results, determined within 28 days before the first dose administration of the IMP unless there 
is a documented medical explanation for the positive result other than drugs of abuse (e.g., the 
participant has been prescribed opioids for pain). (N.B.: A positive test result may be repeated at 
the Investigator’s discretion). 
15. Participant with negative human immunodeficiency virus (HIV), hepatitis B surface antigen 
(HBsAg)) and hepatitis C virus antibody (HCV Ab) test results at Screening. 
16. No clinically significant abnormalities in 12-lead electrocardiogram (ECG) or vital signs 
determined within 28 days before first dose of IMP.  

 
 
 
 
 
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17. Participant must be available to complete the study (including all follow up visits). 
18. Participant must be willing to consent to have data entered into The Over Volunteering 
Prevention System (TOPS). 
19. Participant must provide written informed consent to participate in the study. 
To be re-confirmed on Day 0 / prior to each dosing visit 
1. Participant continues to meet all screening inclusion criteria. 
2. Participant with a negative urinary drugs of abuse screen (including alcohol and cotinine) prior to 
dosing unless there is a documented medical explanation for the positive result other than drugs of 
abuse (e.g., the participant has been prescribed opioids for pain). (N.B.: A positive test result may 
be repeated at the Investigator’s discretion). 
3. Female participant of childbearing potential or non-childbearing potential with a negative 
pregnancy test on admission. 
EXCLUSION CRITERIA:  
To be confirmed at Screening: 
1. Participant with any clinically significant medical (cardiovascular disease, pulmonary, hepatic, 
gallbladder or biliary tract, renal, haematological, gastrointestinal, endocrine, immunologic, 
dermatological, neurological, autoimmune disease or current infection) or psychiatric condition 
(see also exclusion criterion number 214) that, in the opinion of the Investigator, precludes 
participation in the study.  This will include any clinically significant abnormal serum biochemistry 
results and/or haematological results and/or urine analytical results. 
2. Participant with a history of heart disease or of rheumatic fever. 
3. Participant with a significant acute febrile illness (including fever of 38.0°C or greater within 14 
days) of each dose of IMP (Days 0, 21 and 42).  
4. Participant who has chronic diseases: Chronic diseases will include all autoimmune and 
immunocompromising conditions and any other chronic condition, which at the judgment of the 
Investigator, may put the participant at higher risk of side effects from the study vaccine. 
Conditions in the latter category might include unexplained anaemia, hepato-biliary disease, 
uncontrolled hypertension, participant with prosthetic joints or heart valves, etc. 
5. Participant with sickle cell anaemia. 
6. Participant who has undertaken a course of antibiotics/antibacterials within 28 days prior to each 
dose of IMP (Days 0, 21 and 42). 
7. Use of prescription or non-prescription drugs within 28 days or 5 half-lives (whichever is longer) 
prior to receiving the first dose of IMP, unless in the opinion of the Investigator and Sponsor's 
Responsible Physician the medication will not interfere with the study procedures or compromise 
participant safety. 
8. Participant who uses antacids, proton pump inhibitors or H2 blockers on a regular basis or has 
consumed proton pump inhibitors or H2 blockers within 24 hours prior to each dose of IMP. 
9. Participant who has received investigational or licensed vaccines in the 28 days prior to dosing or 
anticipates receiving a vaccine other than study medication up to Day 84 of the study. 
10. Participant with symptoms consistent with Typhoid fever concurrent with travel to countries where 
typhoid infection is endemic (most of the developing world) within 2 years prior to first dose of 
IMP.   
11. Vaccination against Typhoid within 3 years prior to first dose of IMP.  
12. Ingestion of Typhoid bacteria in a challenge study within 3 years prior to dosing. 

 
 
 
 
 
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13. Participant who works as a commercial food handler. 
14. Participant who is a health care worker in direct contact with patients. 
15. Participant who is a childcare worker. 
16. Participant who has household contact with immuno-compromised individuals, pregnant women, 
children < 2 years of age or individuals > 70 years of age. 
17. Participant who has person(s) living with him/her who, in the opinion of the Investigator, may be 
at risk of disease if exposed to the vaccine strain. 
18. Participant with a known impairment of immune function or receiving (or has received in the 6 
months prior to study entry) cytotoxic drugs or immunosuppressive therapy (including systemic 
corticosteroids). 
19. Participant who is a current smoker (cigarettes, tobacco and/or e-cigarettes) or has stopped 
smoking in the last 3 months prior to Screening.  
20. A clinically significant history of drug or alcohol abuse [defined as the consumption of more than 
14 units of alcohol a week] within the past two years prior to Screening.  
21. Inability to communicate well with the Investigators (i.e., language problem, poor mental 
development or impaired cerebral function). 
22. Participation in a New Chemical Entity (NCE) clinical study within the previous 3 months or a 
marketed drug clinical study within the 30 days before the first dose of IMP.  (Washout period 
between studies is defined as the period of time elapsed between receiving the last dose of the 
previous study and receiving the first dose of the next study). 
23. Donation of 450 millilitres (mL) or more blood within the 3 months before the first dose of IMP. 
24. Participant who, in the opinion of the Investigator, is unsuitable for participation in the study. 
To be re-confirmed at Day 0 / prior to each dosing visit: 
1. Development of any exclusion criteria since the Screening visit. 
2. Use of prescription or non-prescription drugs since Screening, unless in the opinion of the 
Investigator and Sponsor's Responsible Physician, the medication will not interfere with the study 
procedures or compromise participant safety. 
3. Participation in a clinical study since Screening. 
4. Donation of 450 mL or more blood since Screening. 
IMP ADMINISTRATION:  
Cohort 1 will consist of 9 participants, who will receive 3 doses of 1x109 CFU of ZH9PA (6 participants) or 
placebo (3 participants).   
Cohort 2 will consist of 18 participants, who will receive 3 doses of 1x1010 CFU of ZH9PA (12 participants) or 
placebo (6 participants).   
Cohort 3 will consist of 18 participants, who will receive 3 doses of 1x1010 CFU of ZH9PA and 1x1010 CFU 
of ZH9 (12 participants) or placebo (6 participants).   
CRITERIA FOR EVALUATION:  
Primary Endpoint: 
• 
Local and systemic reactogenicity up to Day 84 as assessed by adverse events, laboratory safety tests 
(biochemistry, haematology, urinalysis), vital signs and physical examination. 
 
Secondary Endpoints: 
• 
Local and systemic reactogenicity; from Day 85 to Day 224 as assessed by adverse events, laboratory 
safety tests (biochemistry, haematology, urinalysis) if undertaken for cause; vital signs if undertaken 
for cause. 

 
 
 
 
 
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• 
Concentrations of specific serum IgA and IgG antibodies to LPS O:2 and O:9, flagellin H:a and H:d. 
• 
Fold increase in specific serum IgA and IgG antibody concentrations, in individual participants, 
against the antigens LPS O:2 and O:9, flagellin H:a and H:d. 
• 
Concentrations of specific mucosal IgA ALS assays, in individual participants, against antigens LPS 
O:2 and O:9, flagellin H:a and H:d. 
• 
Seroconversion rate (proportion of participants with 4-fold increase above baseline at any time post 
vaccination) against each of the above antigens. 
• 
Fold increase in specific mucosal IgA ALS assays, in individual participants, against the antigens 
LPS O:2 and O:9, flagellin H:a and H:d. 
 
STATISTICAL METHODS:   All statistical analysis will be performed using SAS® (version 9.3 or higher). 
 
Descriptive statistics for quantitative parameters will be provided using number of observations (N), mean, 
standard deviation (SD), minimum, median and maximum. Descriptive statistics for qualitative parameters will 
be provided using absolute frequencies (n) and relative frequencies (%). For immunological endpoints (IgG, 
IgA, concentrations and fold increase) additionally geometric means with corresponding 95% confidence 
intervals (CI) will be presented. 
 
For parameters with evaluation before vaccination and in case of repeated value(s), only the last observation 
prior to dosing will be used in descriptive and inferential statistics and derivations of other parameter values.  
After vaccination, only values of scheduled assessments (planned in the protocol) will be used. 
 
The sample size chosen for this study is not based on a formal statistical estimation but is considered to be 
adequate to meet the objectives of the study. A sufficient number of participants will be initially screened for 
enrolment to ensure that the planned sample size is achieved. 
DURATION OF STUDY: 
Approximately 36 weeks for each individual (from Screening to Post-Study 
Follow-Up). 
 

 
 
 
 
 
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TABLE OF CONTENTS 
1 
SIGNATURE PAGE ................................................................................. 3 
2 
PROTOCOL AMENDMENT/REVISION HISTORY ............................. 5 
3 
SYNOPSIS ................................................................................................ 7 
4 
TABLE OF CONTENTS ........................................................................ 12 
5 
ABBREVIATIONS USED IN THE PROTOCOL TEXT ...................... 17 
6 
ETHICS ................................................................................................... 19 
6.1 
Research Ethics Committee or Institutional Review Board .................... 19 
6.2 
Ethical Conduct of the Study ................................................................... 19 
6.3 
Participant Information and Consent ....................................................... 20 
7 
INVESTIGATORS AND STUDY ADMINISTRATIVE STRUCTURE
 ................................................................................................................. 21 
7.1 
Study Personnel ....................................................................................... 21 
7.2 
Indemnity Arrangements ......................................................................... 21 
8 
INTRODUCTION ................................................................................... 22 
9 
STUDY OBJECTIVES ........................................................................... 25 
9.1 
Primary Study Objective .......................................................................... 25 
9.2 
Secondary Study Objective ...................................................................... 25 
10 
INVESTIGATIONAL PLAN .................................................................. 26 
10.1 
Overall Study Design and Plan ................................................................ 26 
10.2 
Dose Escalation Procedures and Stopping Criteria ................................. 28 
10.2.1 
Dose Escalation Procedures ..................................................................... 28 
10.2.2 
Stopping Criteria ...................................................................................... 28 
10.2.2.1 
General Stopping Criteria ........................................................................ 28 
10.2.2.2 
Dose Escalation Stopping Criteria ........................................................... 29 
10.3 
Discussion of Study Design, including the Choice of Control Groups ... 29 
10.4 
Selection of Study Population ................................................................. 30 
10.4.1 
Inclusion Criteria ..................................................................................... 30 
10.4.2 
Exclusion Criteria .................................................................................... 32 
10.5 
Additional Advice and Restrictions for Study Population ...................... 34 
10.5.1 
Contraception ........................................................................................... 34 
10.5.2 
Sperm Donation ....................................................................................... 34 

 
 
 
 
 
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10.5.3 
Diet and Fluid Restrictions ...................................................................... 34 
10.5.3.1 
Meal Times/Fasts ..................................................................................... 34 
10.5.3.2 
Fluid Intake .............................................................................................. 34 
10.5.3.3 
Alcohol Intake ......................................................................................... 34 
10.5.3.4 
Caffeine .................................................................................................... 35 
10.5.3.5 
Poppy and Sesame Seeds ......................................................................... 35 
10.5.4 
Other Life-Style Restrictions ................................................................... 35 
10.5.4.1 
Strenuous Exercise ................................................................................... 35 
10.5.4.2 
Blood Donation ........................................................................................ 35 
10.5.4.3 
Travel Restrictions ................................................................................... 35 
10.5.5 
Removal of Participants from Therapy or Assessment ........................... 35 
10.6 
Investigational Medicinal Product ........................................................... 36 
10.6.1 
Identity ..................................................................................................... 36 
10.6.2 
Receipt and Storage ................................................................................. 37 
10.6.3 
Assembly and Release ............................................................................. 37 
10.6.4 
Administration ......................................................................................... 38 
10.6.4.1 
Placebo Training ...................................................................................... 38 
10.6.4.2 
Treatment Period ..................................................................................... 38 
10.6.5 
Return/Destruction ................................................................................... 39 
10.6.6 
Method of Assigning Participants to Treatment Groups ......................... 39 
10.6.7 
Selection of Doses in the Study ............................................................... 39 
10.6.8 
Timing of Dose for Each Participant ....................................................... 40 
10.6.9 
Blinding ................................................................................................... 40 
10.6.10 
Prior and Concomitant Therapy ............................................................... 41 
10.6.11 
Treatment Compliance ............................................................................. 42 
10.7 
Safety Variables ....................................................................................... 42 
10.7.1 
Safety Measurements Assessed and Flow Chart ..................................... 42 
10.7.1.1 
Demographic and Background Assessments ........................................... 46 
10.7.1.1.1 Demographics .......................................................................................... 46 
10.7.1.1.2 Medical History and Concurrent Conditions ........................................... 46 
10.7.1.1.3 Virology Tests ......................................................................................... 46 
10.7.1.1.4 Drugs of Abuse (including Alcohol and Cotinine) .................................. 46 

 
 
 
 
 
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10.7.1.1.5 Pregnancy Test, Menstrual and Obstetric History ................................... 46 
10.7.1.2 
Compliance with Inclusion/Exclusion Criteria ........................................ 46 
10.7.1.3 
Safety Assessments .................................................................................. 47 
10.7.1.3.1 Adverse Events ........................................................................................ 47 
10.7.1.3.2 Laboratory Safety Assessments ............................................................... 50 
10.7.1.3.3 Other Safety Assessments ........................................................................ 51 
10.7.1.3.4 Stool Bacteriology ................................................................................... 51 
10.7.1.3.5 Blood Cultures ......................................................................................... 52 
10.7.1.3.6 Concomitant Medication ......................................................................... 52 
10.7.2 
Appropriateness of Measurements .......................................................... 52 
10.7.3 
Pharmacodynamic Assessments .............................................................. 54 
10.7.4 
Other Assessments ................................................................................... 54 
10.8 
Data Quality Assurance ........................................................................... 54 
10.9 
Statistical Methods and Determination of Sample Size .......................... 56 
10.9.1 
Statistical and Analytical Plan ................................................................. 56 
10.9.1.1 
Study Variables/Endpoints ...................................................................... 56 
10.9.1.2 
Analysis Sets ............................................................................................ 57 
10.9.1.3 
Description of Statistical Methods ........................................................... 57 
10.9.1.3.1 Demographic and Background Data ........................................................ 57 
10.9.1.3.2 Efficacy Data ........................................................................................... 57 
10.9.1.3.3 Safety Data ............................................................................................... 58 
10.9.1.3.4 PD Data .................................................................................................... 59 
10.9.1.3.5 Other Data ................................................................................................ 59 
10.9.2 
Sample Size Calculation .......................................................................... 59 
11 
PRACTICAL CONSIDERATIONS ....................................................... 60 
11.1 
Storage of Data ........................................................................................ 60 
11.2 
Protocol Amendments ............................................................................. 60 
11.3 
Confidentiality ......................................................................................... 60 
11.4 
Study Report and Publication Policy ....................................................... 60 
11.5 
General Data Protection Regulation ........................................................ 61 
12 
REFERENCES ........................................................................................ 62 
APPENDIX 1: DECLARATION OF HELSINKI (BRAZIL, 2013) .......................... 64 

 
 
 
 
 
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APPENDIX 2: NORMAL RANGES FOR VITAL SIGNS AND ECG 
PARAMETERS ....................................................................................... 65 
 

 
 
 
 
 
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TABLES 
 
Table 10.7.1 Study Flow Chart ................................................................................ 43 
Table 10.7.2 Summary of Blood Volume ................................................................ 53 
Table 10.8.1 Summary of Source Documentation Location .................................... 55 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
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ABBREVIATIONS USED IN THE PROTOCOL TEXT 
• ABPI 
Association of the British 
 
Pharmaceutical Industry 
• AE(s) 
adverse event(s) 
• ALS 
antibodies in lymphocyte 
 
supernatant 
• BMI 
body mass index  
• bpm 
beat(s) per min 
• CFU 
colony forming units 
• CI 
confidence interval 
• cm 
centimetre  
• CTA 
Clinical Trial Authorisation 
• DOA 
drugs of abuse 
• eCRF 
electronic case report form 
• ECG 
electrocardiogram  
• ELISA 
enzyme-linked 
 
immunosorbent assay 
• EU 
European Union 
• FSH 
follicle stimulating hormone 
• g 
gramme(s) 
• GALT 
gut-associated lymphoid 
 
tissue 
• GCP 
Good Clinical Practice 
• GDPR 
General Data Protection 
 
Regulation 
• GMP 
Good Manufacturing Practice 
• HBsAg 
hepatitis B surface antigen 
• HCV Ab 
hepatitis C virus antibody 
• HIV 
human immunodeficiency 
 
virus 
• IB 
Investigator’s Brochure 
• ICH 
International Council on 
 
Harmonisation 
• Ig 
immunoglobulin 
• IMP 
investigational medicinal 
 
product(s) 
• ISF 
investigator site file 
• kg 
kilogram 
• LMICs 
low- and middle-income
 
countries 
• LPS 
lipopolysaccharide 
• Ltd 
Limited 
• MedDRA 
Medical Dictionary for 
Regulatory Activities 
• MHRA 
Medicines and Healthcare 
products Regulatory Agency 
• mL 
millilitre 
• min(s) 
min(s) 
• NCE  
new chemical entity 
• NSAID 
non-steroidal anti-
 
inflammatory drug 
• PBMC 
peripheral blood mononuclear 
 
cells 
• PD 
pharmacodynamic 
• PR interval 
time the electrical impulse 
 
takes to travel from the sinus 
 
node through the AV node 
 
and entering the ventricles; 
 
measured from the beginning 
 
of the P wave to the beginning 
 
of the QRS complex 
• QA 
quality assurance 
• QP 
qualified person 
• QRS 
QRS complex represents 
 
ventricular depolarisation 
• QT interval 
the time for both ventricular 
 
depolarisation and 
 
repolarisation to occur, and 
 
therefore roughly estimates 
 
the duration of an average 
 
ventricular action potential. 
• QTc 
corrected QT interval 
• REC 
Research Ethics Committee 
• SAE 
serious adverse event 
• SAP 
statistical analysis plan 
• SAR 
serious adverse reaction 
• SAS 
statistical analysis software by 
 
SAS Institute Inc., USA 
• SD 
standard deviation 
• Ser. 
serovar 
• SHM 
sample handling manual 
 
• SmPC 
Summary of Product 
 
Characteristics  

 
 
 
 
 
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• SOC 
system organ class 
• SOP 
standard operating 
 
procedure(s) 
• SPI2 
salmonella pathogenicity 
 
island 2 
• SUSAR 
suspected unexpected serious 
 
adverse reaction 
• TEAE 
treatment emergent adverse 
 
event 
• TOPS  
The Over Volunteering 
 
Prevention System 
• UK 
United Kingdom 
• VMF 
volunteer master file 
• WBC 
white blood cell(s) 
• w/v 
weight/volume 
 

 
 
 
 
 
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6 
ETHICS 
6.1 
Research Ethics Committee or Institutional Review Board  
This study protocol will be submitted to the Research Ethics Committee (REC) for review to 
confirm provision of a favourable opinion.  The favourable opinion of the REC must be 
obtained before commencement of any study procedures. 
The favourable opinion is conditional upon the Sponsor registering the clinical trial in a 
publicly accessible database, within 6 weeks of the first participant recruited.   
All substantial protocol amendments must receive favourable opinion from the REC 
responsible for the study.  Non-substantial amendments will not require favourable opinion 
from the REC. 
If the study is stopped due to adverse events (AEs) it will not be recommenced without 
reference to the REC responsible for the study. 
The outcome of the study (e.g., completed) will be reported to the REC responsible for the 
study within 90 days of completion of the last participant’s final study procedures.  In the event 
of the study being prematurely terminated a report will be submitted to the REC responsible 
for the study within 15 days. 
A summary of the clinical study report will be submitted to the REC responsible for the study 
within 1 year of completion of the last participant’s final study procedures. 
The REC will be informed that Simbec-Orion is a commercial organisation and that the study 
is funded by Prokarium Ltd. The participants who take part in the clinical study will be paid 
for their inconvenience and have been informed that there will be no benefits gained by their 
participation. All potential conflicts of interest will be declared by the Investigators.  
6.2 
Ethical Conduct of the Study 
The Principal Investigator shall be responsible for ensuring that the clinical study is performed 
in accordance with the following: 
• Declaration of Helsinki (Brazil, 2013)[1] (Appendix 1). 
• Association of the British Pharmaceutical Industry (ABPI) Guidelines for Phase 1 
Trials (2018)[2]. 
• ICH (International Council on Harmonisation) Guideline for Good Clinical Practice 
(GCP) E6 (R2) (CPMP/ICH/135/95) 1996[3]. 
• The Medicines for Human Use (Clinical Trials) Regulations 2004 (Statutory 
Instrument 2004 No. 1031) and subsequent amendments[4]. 
This clinical study has been registered in the EudraCT database and a Clinical Trials 
Authorisation (CTA) will be obtained from the Medicines and Healthcare products Regulatory 
Agency (MHRA) prior to the start of the study in accordance with Part 3, Regulation 12 of the 
UK Statutory Instrument. 

 
 
 
 
 
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6.3 
Participant Information and Consent 
Potential participants who volunteer for participation in the study will be informed of the aims, 
methods, anticipated benefits and potential hazards of the study and any possible discomfort it 
may entail.  Information will be given in both oral and written form and in the manner deemed 
appropriate by the Clinical Unit standard operating procedures (SOPs). Each participant will 
also be informed of his/her right to withdraw from the study at any time, for any reason. 
A written explanation (participant information sheet) and informed consent form will be 
provided, and the participant will be allowed sufficient time to consider the study information. 
Prior to signing the informed consent form, the participant will be given an opportunity to 
discuss any issues concerning the study with an Investigator who has suitable knowledge of 
the study and will have all questions answered openly and honestly. 
If the participant is willing to participate in the study, the informed consent form will be signed 
and personally dated by the participant and the person taking consent. The participant will 
receive a copy of the informed consent form together with the participant information sheet 
and the original signed informed consent form will be retained with the study records at the 
Investigator site.  In addition, the actions and completion of the consenting process will be 
recorded in the participant’s medical record (i.e., source document). 
 

 
 
 
 
 
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7 
INVESTIGATORS AND STUDY ADMINISTRATIVE 
STRUCTURE 
The study will be performed at a single site, Simbec-Orion Clinical Pharmacology Unit. 
 
  
The Project Manager will act on behalf of the Principal Investigator to ensure the smooth and 
efficient running of all aspects of the study. 
7.1 
Study Personnel 
  
 
 
  
 
 
 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 
 
 
7.2 
Indemnity Arrangements 
 
 
 
 
 
 

 
 
 
 
 
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8 
INTRODUCTION 
Prokarium is developing a bivalent vaccine against Enteric Fever, caused by infection with 
typhoidal serovars, namely Salmonella enterica serovar (ser.) Typhi and Salmonella enterica 
ser. Paratyphi A.  These serovars are human-adapted bacterial pathogens that cause related, 
potentially fatal, systemic diseases, collectively called enteric fever or typhoid; the name being 
derived from the ancient Greek typhos, an ethereal smoke or cloud that was believed to cause 
disease and madness.  The infectious reservoir for the bacteria is patients with an acute infection 
that shed the bacteria in their stools and chronic carriers, patients who have a reservoir of 
bacteria, usually in their gall bladder, who periodically shed viable bacteria in their stools.  The 
classic presenting symptoms include fever, malaise, diffuse abdominal pain, and constipation 
although approximately one third of patients develop diarrhoea after the occurrence of the 
fever[6].  Left untreated, enteric fever can progress to delirium, intestinal haemorrhage and 
perforation of the bowel and ultimately death.  Salmonella enterica ser. Typhi and  Salmonella 
enterica ser. Paratyphi A have been eradicated from developed countries but thrive in areas of 
poor sanitation and remain a significant health problem in low- and middle-income countries 
(LMICs) of Southeast Asia, the Indian subcontinent, Africa, and, to a lesser extent, South 
America[7].  It has been estimated that the total number of typhoid fever episodes in 2010 was 
13.5 million (interquartile range 9.1-17.8 million). The adjusted estimate accounting for the 
low sensitivity of blood cultures for isolation of the bacteria was 26.9 million (interquartile 
range 18.3-35.7 million) episodes[8].   
The disease is spread through the ingestion of contaminated food and water.  S. enterica Typhi 
and S. enterica ser. Paratyphi A are highly invasive and adapted to avoid the innate immune 
system.  Once ingested, the bacteria pass through the intestinal mucosa, mainly of the distal 
ileum.  The bacteria have specialised fimbriae that adhere to the epithelium over the lymphoid 
tissue of the Peyer’s patches in the ileum. The bacteria are taken up by the macrophages and 
pass into the lymphatic system and then induce their host macrophages to attract more 
macrophages to the gut-associated lymphoid tissue (GALT).  The Salmonella reproduce within 
the macrophages, as they are carried through the mesenteric lymph nodes to the thoracic duct 
and the lymphatics, and then through to the reticuloendothelial tissues of the liver, spleen, bone 
marrow and lymph nodes. Once in the reticuloendothelial system, multiplication continues over 
a period of 8 to 14 days until apoptosis of the macrophages results in a bacteraemia and 
systemic spread.  The bacteria then infect the gallbladder via either bacteraemia or direct 
extension of infected bile.  The result is that the organism re-enters the gastrointestinal tract in 
the bile and re-infects Peyer patches. Bacteria that do not re-infect the host are typically shed 
in the stool and are then available to infect other hosts. 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
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 the widespread use of antibiotics in developing 
countries is leading to the development of resistance in typhoidal serovars.  Hence, there is a 
need for a vaccine to protect patients against antibiotic resistant strains of bacteria[7]. 
ZH9 has previously been administered to humans at a maximum dose of 1.7 x 1010 CFU and 
been shown to be well tolerated and immunogenic.  It is anticipated that a similar dose of 
ZH9PA will be required.  It is intended that the vaccine contains equal amounts of ZH9 and 
ZH9PA and therefore, this study will explore two dose levels of ZH9PA and a single dose level 
of ZH9 plus ZH9PA given in combination. Previous work with ZH9 has identified that a single 
dose does not give optimal protection against a typhoid challenge strain but it was demonstrated 
a single dose provided a significant reduction in the microbiological burden of infection and 
alteration of the clinical disease profiles[12].  Hence, the safety and immunogenicity of three 
doses, administered 3 weeks apart will be investigated in this study. 
Further details of the non-clinical studies and a summary of the known and potential risks and 
benefits to human participants of ZH9 and ZH9PA can be found in the IB[11].  

 
 
 
 
 
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The study will be conducted in compliance with the protocol, GCP and the applicable 
regulatory requirement(s), as indicated within Section 6.2.5 of the ICH GCP E6 (R2) 
guidelines[3]. 
 

 
 
 
 
 
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9 
STUDY OBJECTIVES 
9.1 
Primary Study Objective 
The primary objective is to evaluate the safety and tolerability of three doses of two dose levels 
of ZH9PA and of three doses of a single dose level of a combination of ZH9 and ZH9PA over 
12 weeks (up to Day 84) after the first dose of vaccine. 
9.2 
Secondary Study Objective 
• To evaluate serum IgA and IgG responses against the following antigens: - S. enterica 
Ser. Paratyphi A LPS 0:2 and Flagella H:a and S. enterica ser. Typhi LPS O:9 and 
Flagella H:d, induced by three vaccine doses, of two dose levels (1x109 CFU and 1x1010 
CFU) of ZH9PA alone or in combination with 1x1010 CFU ZH9, up to Day 84 after the 
first dose of vaccine (Day 0). 
 
• To evaluate the mucosal IgA immune responses to LPS 0:2, LPS O:9, Flagella H:a and 
Flagella H:d, at 7 days after each vaccination, as determined by ALS assay. 
 
• To evaluate Serum IgA and IgG responses against the four antigens, induced by each 
of the first two vaccine doses, on Days 21, and 42, by blood samples taken prior to each 
participant receiving the next dose of vaccine or placebo. 
 
• To evaluate the safety profile of the ZH9PA and combination ZH9/ZH9PA vaccines, 
over the 6 months following the third dose of vaccine.  
 
 

 
 
 
 
 
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10 
INVESTIGATIONAL PLAN 
10.1 
Overall Study Design and Plan 
This is a Phase I, randomised, double-blind, placebo-controlled, parallel group, single-centre 
study involving 45 healthy participants. The aim is to evaluate a combination vaccine against 
enteric fever comprising the live attenuated Salmonella strain ZH9 (previously tested in a 
number of clinical trials as a typhoid vaccine) and its modified derivative, ZH9PA, in which 
the typhoid antigens LPS O:9 and Flagellin H:d have been substituted by the Paratyphi A 
antigens, LPS O:2 and Flagellin H:a. ZH9PA has not previously been tested in humans; 
therefore, the first two cohorts comprise a dose escalation of this component alone, followed 
by a final cohort in which the combination is tested at a single dose level. 
 
There will be 3 cohorts:  
Cohort 1 will consist of 9 participants, who will receive 3 doses of 1x109 CFU of ZH9PA (6 
participants) or placebo (3 participants).   
Cohort 2 will consist of 18 participants, who will receive 3 doses of 1x1010 CFU of ZH9PA 
(12 participants) or placebo (6 participants).   
Cohort 2 will not be initiated until all 9 participants of Cohort 1 have received 1 dose of ZH9PA 
or placebo, and the safety data, up to Day 7 are available.  These safety data will be assessed 
by the safety review group, at which point the decision will be made as to whether proceeding 
with administration of the first dose of the higher dose level of ZH9PA (1x1010 CFU) to Cohort 
2 is appropriate.   
Cohort 3 will consist of 18 participants, who will receive 3 doses of 1x1010 CFU of ZH9PA 
and 1x1010 CFU of ZH9 (12 participants) or placebo (6 participants).   
Cohort 3 will not be initiated until a minimum of 14 participants of Cohort 2 have received 1 
dose of ZH9PA or placebo, and the safety data, up to Day 7 are available.  These safety data 
will be assessed by the safety review group, at which point the decision will be made as to 
whether proceeding with administration of the first dose of the combination of ZH9PA and 
ZH9 to Cohort 3 is appropriate.  In the event that fewer than 18 participants are dosed in Cohort 
2, the remaining participants to complete the required number will be dosed with Cohort 3 or 
as a straggler group.   
Each cohort will start with a sentinel group that will consist of 2 participants, 1 will receive 
placebo and 1 will receive the relevant vaccine for that cohort.  No further participants will be 
dosed until 24 hours after the second participant has received his/her dose.  The rest of the 
cohort will be dosed provided there are no safety issues identified by the Investigator.  This 
design allows maintenance of the “blind” until Day 70.  
The clinical phase is anticipated to take place in Q4 2019 and Q2 2020. The end of the study 
is defined as last participant last visit. 
The study will take place in the Clinical Pharmacology Unit of Simbec-Orion (Clinical Unit) 
under full medical and nursing supervision. 

 
 
 
 
 
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A schedule of all study assessments is provided in Table 10.7.1. 
Screening (Day -28 to Day -1) 
Screening assessments will be performed from Day -28 to Day -1 to ensure the eligibility of 
participants. Assessments will be performed as per Table 10.7.1. 
 
Treatment Period (Day 0 to Day 84) 
Participants will be admitted to the Clinical Unit on the morning of Day 0 with confirmation 
of eligibility and baseline assessments performed as per Table 10.7.1. Following a 90 minute 
(min) fast prior to dose administration, participants will receive the first dose on Day 0, and 
then continue to fast for a further 90 mins post dose administration; water will be allowed. 
Following the completion of the 4h post-dose assessments and at the discretion of the 
Investigator, the participants will be discharged from the Clinical Unit. Participants will return 
to the Clinical Unit on Day 3 for safety assessments as per Table 10.7.1. For the 7 days 
following the first dosing visit (Day 1 to Day 7) participants will be required to record their 
body temperature at home twice a day i.e. once in the morning and once in the evening (a 
thermometer and a diary will be provided to record body temperature). Participants will return 
to the Clinical Unit on Day 7 with the diary and to give a blood sample for the peripheral blood 
mononuclear cells (PBMC)/ALS assay; assessments will be performed as per Table 10.7.1. 
Participants will return to the Clinical Unit on Day 14 for safety assessments; assessments will 
be performed as per Table 10.7.1. 
Participants will be admitted to the Clinical Unit on the morning of Day 21, with assessments 
performed as per Table 10.7.1. Following a 90-min fast prior to dose administration, 
participants will receive the second dose, and then continue to fast for a further 90 mins post 
dose administration; water will be allowed. Following the completion of the 4h post-dose 
assessments and at the discretion of the Investigator, the participants will be discharged from 
the Clinical Unit. Participants will return to the Clinical Unit on Day 24 for safety assessments 
as per Table 10.7.1. For the 7 days following the second dosing visit (Day 22 to Day 28) 
participants will be required to record their body temperature at home twice a day, i.e. once in 
the morning and once in the evening (a thermometer and a diary will be provided to record 
body temperature). Participants will return to the Clinical Unit on Day 28 with the diary and to 
give a blood sample for the PBMC/ALS assay; assessments will be performed as per Table 
10.7.1.  
Participants will be admitted to the Clinical Unit on the morning of Day 42, with assessments 
performed as per Table 10.7.1. Following a 90-min fast prior to dose administration 
participants will receive the third dose, and then continue to fast for a further 90 mins post dose 
administration; water will be allowed. Participants will return on Day 45 for safety assessments 
as per Table 10.7.1. For the 7 days following the third dosing visit (Day 43 to Day 49) 
participants will be required to record their body temperature at home twice a day, i.e. once in 
the morning and once in the evening (a thermometer and a diary will be provided to record 
body temperature). Participants will return to the Clinical Unit on Day 49 with the diary and to 
give a blood sample for the PBMC/ALS assay; assessments will be performed as per Table 
10.7.1.  

 
 
 
 
 
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Participants will return to the Clinical Unit on Day 70 to provide a stool sample for 
bacteriology. If the stool sample is positive for any Salmonella strain at Day 70, it will be 
presumed to be one of the vaccine strains, and participants will be asked to return to the Clinical 
Unit to receive a course of antibiotic therapy. Participants who receive a course of antibiotics 
will be required to provide a repeat stool sample for bacteriology testing on Day 84, to confirm 
that shedding of the vaccine strains has ceased, as per Table 10.7.1.   
Post-Study Follow-Up (Day 85 to Day 224)  
Safety follow-up telephone calls will be performed on Days 154 and 224 following the first 
dose of study vaccine administration to check the general health of the participant and to review 
clinically relevant AEs.  Clinically relevant adverse events are adverse events that required a 
visit to a healthcare professional. Assessments will be performed as per Table 10.7.1. 
10.2 
Dose Escalation Procedures and Stopping Criteria 
10.2.1 
Dose Escalation Procedures  
For each cohort, a summary of all relevant safety (AEs, laboratory safety tests, ECG, vital 
signs, subject recorded body temperature, concomitant medications) data (up to 7 days post-
first dose) will be produced on behalf of the Principal Investigator.  Planned doses may be 
modified following a review of emerging data. Progression to the next dose level and dose 
selection will be based on the available safety data from all participants for Cohort 1 prior to 
dosing Cohort 2 and a minimum of 14 participants1 for Cohort 2 prior to dosing Cohort 3.  .  
Dose escalation will be dependent upon the accrual of acceptable safety data. If it is not 
appropriate to escalate the dose according to the proposed dose escalation schedule, then the 
same dose, an intermediate dose or a lower dose may be given following discussion between 
the Sponsor and the Principal Investigator (or deputy).   
There will be a Telephone Conference at a pre-appointed time to involve the Simbec-Orion 
Project Manager and Principal Investigator (or deputy) and the Sponsor’s representative(s), 
including the Sponsor's Responsible Physician.  After discussion of all the data, the decision 
will be made whether to dose escalate and a written document (dose escalation approval form) 
signed by the Principal Investigator (or deputy) and Sponsor will be produced ratifying that 
decision.  Full minutes, to be agreed by all parties, will be produced for each discussion 
regarding dose escalation and filed in the Investigator Site File (ISF).  A copy of the signed 
dose escalation approval form will be provided to the Simbec-Orion Pharmacist and this will 
allow the IMP to be assembled for the next dose level.  
Dose escalation stopping criteria are detailed in Section 10.2.2.2. 
10.2.2 
Stopping Criteria 
10.2.2.1 General Stopping Criteria 
The study will be discontinued if any unacceptable safety findings are identified.  This decision 
will be made jointly by the Principal Investigator (or deputy) and the Sponsor.  A written 
 
1 With the 12:6 ratio of active to placebo, if all 6 participants randomised to placebo are in the first 14 
participants who are dosed, this will ensure the minimum of 8 participants will have received active vaccine. 

 
 
 
 
 
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document signed by the Principal Investigator (or deputy) and Sponsor will be produced 
ratifying the decision. 
Individual participants may also be withdrawn for any of the reasons outlined in Section 10.5.5. 
10.2.2.2 Dose Escalation Stopping Criteria 
Dose escalation will be temporarily stopped pending evaluation of all available data if any of 
the following criteria are fulfilled: 
• 
If one of the sentinel group or more than 20% of the participants in a cohort experience a 
severe systemic reaction (including anaphylactoid reactions) related to the ZH9PA/ZH9 
vaccine (study agent). 
• 
If one of the sentinel group or more than 20% of the participants in a cohort experience a 
persistent febrile reaction (>38.5°C for ≥12hours) related to the study agent. 
• 
If one or more participants experience a serious adverse event (SAE) related to the study 
agent. 
• 
If two or more participants in the same cohort, experience 'severe' non-serious adverse 
reactions (i.e. severe non-serious adverse events, independent of system-organ-class, 
considered at least possibly related, to the study agent). 
Individual participants may also be withdrawn for any of the reasons outlined in Section 10.5.5. 
10.3 
Discussion of Study Design, including the Choice of Control 
Groups  
This is a Phase I, first in human, randomised, double-blind, placebo-controlled, parallel group, 
study. Randomised, double-blind studies are the gold standard methodology to assess the 
safety, tolerability and efficacy of products and therefore, this design has been used for this 
study.    In the case of this study, it will remain fully blinded until Day 70, when a stool sample 
is taken to assess if participants are shedding the bacterial strains of the vaccine in their faeces.  
The Investigators and the Monitor will be unblinded to the treatment allocation of participants 
who have a positive result for Salmonella and the participant will receive a course of antibiotics. 
No benefit to participants is expected, as efficacy has not been evaluated for the ZH9/ZH9PA 
combination or for ZH9PA on its own; therefore, use of placebo as the control is considered 
acceptable in the healthy participants participating in the study.  The use of the placebo control 
allows an assessment of the immune response and provides a background incidence of adverse 
events. 
Previous work with ZH9 has shown that the double mutation does appropriately attenuate the 
bacteria, however it is known that the bacterial strains that make up the vaccine can be shed in 
the stools of participants.  The vaccine strains are genetically modified organisms, and as such 
it is necessary to reduce environmental exposure and stool cultures are taken to ensure that 
participants are not shedding. 
There remains a theoretical risk of the vaccine strains causing a systemic infection and so body 
temperatures will be taken for 7 days post each dose of vaccine.  The vaccine strains are 

 
 
 
 
 
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sensitive to several antibiotics, which can be used to treat an infection in the unlikely event 
such an event occurs. 
Three doses are to be studied, as vaccines usually require more than one dose to induce a robust 
immune response.  
 
 
10.4 
Selection of Study Population 
45 participants will be required to complete the study.   
The study is to be conducted in healthy participants, which will be the ultimate target 
population for a vaccine.  While it is anticipated an immune response will be induced in 
participants receiving the vaccine candidate, it is not known whether the immune response is 
protective and; therefore, participants are not expected to derive any therapeutic benefit from 
taking part in the study.  A healthy participant population with carefully considered inclusion / 
exclusion criteria will avoid the potential for interaction of ZH9PA/ZH9 with any underlying 
disease state or concomitant medication that it may be necessary for patients to take, while 
ensuring that participants are fit and well enough for participation in the study.   
ZH9PA has not been previously administered to humans; therefore, its effects in humans are 
as yet unknown. 
The following eligibility criteria are designed to select participants for whom protocol 
treatment and procedures are considered appropriate.  All relevant medical conditions should 
be taken into consideration when deciding whether this protocol is suitable for a particular 
participant. 
10.4.1 
Inclusion Criteria 
To be confirmed at Screening 
1. Healthy male and female participants 18 to 45 years of age, inclusive. 
2. Female participant of childbearing potential willing to use 2 effective methods of 
contraception, i.e., established method of contraception + condom, if applicable 
(unless of non-childbearing potential or where abstaining from sexual intercourse 
is in line with the preferred and usual lifestyle of the participant) from the first dose 
until 2 months after the last dose of IMP. 
3. Female participant of non-childbearing potential. For the purposes of this study, 
this is defined as the participant being amenorrhoeic for at least 12 consecutive 
months or at least 4 months post-surgical sterilisation (including bilateral fallopian 
tube ligation or bilateral oophorectomy with or without hysterectomy).  
4. Female participant of childbearing potential or non-childbearing potential with a 
negative pregnancy test at Screening. 
5. Female participant of post-menopausal status confirmed by demonstrating at 
Screening that the serum level of the follicle stimulating hormone (FSH) falls within 
the respective pathology reference range.  In the event a participant's menopausal 

 
 
 
 
 
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status has been clearly established (for example, the participant indicates she has 
been amenorrhoeic for 10 years, confirmed by medical history, etc), but serum FSH 
levels are not consistent with a postmenopausal status, determination of the 
participant’s eligibility to be included in the study will be at the Investigator’s 
discretion following consultation with the Sponsor. 
6. Male participant willing to use an effective method of contraception or 2 effective 
methods of contraception, i.e., established method of contraception + condom, if 
applicable (unless anatomically sterile or where abstaining from sexual intercourse 
is in line with the preferred and usual lifestyle of the participant) from first dose 
until a stool sample tested for presence of the vaccine strains is negative . 
7. Participant with a BMI of ≥ 19 or ≤34 kg/m2 (BMI = body weight (kg) / [height 
(m)]2). 
8. No clinically significant history of liver or active gall bladder disease. 
9. No clinically significant history of ongoing gastro-intestinal disease or 
abnormality. 
10. No clinically significant history of previous allergy / sensitivity to ZH9/ZH9PA or 
sodium bicarbonate. 
11. No clinically significant history of anaphylactic shock following vaccination. 
12. No clinically significant history of hypersensitivity (e.g., hives/rash/swollen 
lips/difficulty with breathing) to azithromycin, ampicillin, trimethoprim-
sulfamethoxazole or ciprofloxacin. 
13. No clinically significant abnormal laboratory test results (in the opinion of the 
investigator) for serum biochemistry, haematology and/or urine analyses within 28 
days before receiving the first dose administration of the IMP.   
14. Participant with a negative urinary DOA screen (including alcohol and cotinine) 
test results, determined within 28 days before the first dose administration of the 
IMP unless there is a documented medical explanation for the positive result other 
than drugs of abuse (e.g., the participant has been prescribed opioids for pain). 
(N.B.: A positive test result may be repeated at the Investigator’s discretion). 
15. Participant with negative HIV, HBsAg and HCV Ab test results at Screening. 
16. No clinically significant abnormalities in 12-lead electrocardiogram (ECG) or vital 
signs determined within 28 days before first dose of IMP.  
17. Participant must be available to complete the study (including all follow up visits). 
18. Participant must be willing to consent to have data entered into TOPS. 
19. Participant must provide written informed consent to participate in the study. 
To be re-confirmed on Day 0 / prior to each dosing visit 
1. Participant continues to meet all screening inclusion criteria. 
2. Participant with a negative urinary drugs of abuse screen (including alcohol and 
cotinine) prior to dosing unless there is a documented medical explanation for the 

 
 
 
 
 
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positive result other than drugs of abuse (e.g., the participant has been prescribed 
opioids for pain). (N.B.: A positive test result may be repeated at the Investigator’s 
discretion). 
3. Female participant of childbearing potential or non-childbearing potential with a 
negative pregnancy test on admission. 
10.4.2 
Exclusion Criteria 
To be confirmed at Screening 
1. Participant with any clinically significant medical (cardiovascular disease, 
pulmonary, hepatic, gallbladder or biliary tract, renal, haematological, 
gastrointestinal, 
endocrine, 
immunologic, 
dermatological, 
neurological, 
autoimmune disease or current infection) or psychiatric condition (see also 
exclusion criterion number 21) that, in the opinion of the Investigator, precludes 
participation in the study.  This will include any clinically significant abnormal 
serum biochemistry results and/or haematological results and/or urine analytical 
results. 
2. Participant with a history of heart disease or of rheumatic fever. 
3. Participant with a significant acute febrile illness (including fever of 38.0°C or 
greater within 14 days) of each dose of IMP (Days 0, 21 and 42).  
4. Participant who has chronic diseases: Chronic diseases will include all autoimmune 
and   immunocompromising conditions and any other chronic condition, which at 
the judgment of the Investigator, may put the participant at higher risk of side effects 
from the study vaccine. Conditions in the latter category might include unexplained 
anaemia, hepato-biliary disease, uncontrolled hypertension, participant with 
prosthetic joints or heart valves, etc. 
5. Participant with sickle cell anaemia. 
6. Participant who has undertaken a course of antibiotics/antibacterials within 28 days 
prior to each dose of IMP (Days 0, 21 and 42). 
7. Use of prescription or non-prescription drugs within 28 days or 5 half-lives 
(whichever is longer) prior to receiving the first dose of IMP, unless in the opinion 
of the Investigator and Sponsor's Responsible Physician the medication will not 
interfere with the study procedures or compromise participant safety. 
8. Participant who uses antacids, proton pump inhibitors or H2 blockers on a regular 
basis or has consumed proton pump inhibitors or H2 blockers within 24 hours prior 
to each dose of IMP. 
9. Participant who has received investigational or licensed vaccines in the 28 days 
prior to dosing or anticipates receiving a vaccine other than study medication up to 
Day 84 of the study. 
10. Participant with symptoms consistent with Typhoid fever concurrent with travel to 
countries where typhoid infection is endemic (most of the developing world) within 
2 years prior to first dose of IMP.   

 
 
 
 
 
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11. Vaccination against Typhoid within 3 years prior to first dose of IMP.  
12. Ingestion of Typhoid bacteria in a challenge study within 3 years prior to dosing. 
13. Participant who works as a commercial food handler. 
14. Participant who is a health care worker in direct contact with patients. 
15. Participant who is a childcare worker. 
16. Participant who has household contact with immuno-compromised individuals, 
pregnant women, children < 2 years of age or individuals > 70 years of age. 
17. Participant who has person(s) living with him/her who, in the opinion of the 
Investigator, may be at risk of disease if exposed to the vaccine strain. 
18. Participant with a known impairment of immune function or receiving (or have 
received in the 6 months prior to study entry) cytotoxic drugs or 
immunosuppressive therapy (including systemic corticosteroids). 
19. Participant who is current smoker (cigarettes, tobacco and/or e-cigarettes) or have 
stopped smoking in the last 3 months prior to Screening.  
20. A clinically significant history of drug or alcohol abuse [defined as the consumption 
of more than 14 units of alcohol a week] within the past two years prior to 
Screening.  
21. Inability to communicate well with the Investigators (i.e., language problem, poor 
mental development or impaired cerebral function). 
22. Participation in a NCE clinical study within the previous 3 months or a marketed 
drug clinical study within the 30 days before the first dose of IMP.  (Washout period 
between studies is defined as the period of time elapsed between receiving the last 
dose of the previous study and receiving the first dose of the next study). 
23. Donation of 450 mL or more blood within the 3 months before the first dose of 
IMP. 
24. Participant who, in the opinion of the Investigator, is unsuitable for participation in 
the study. 
To be re-confirmed at Day 0 / prior to each dosing visit 
1. Development of any exclusion criteria since the Screening visit. 
2. Use of prescription or non-prescription drugs since Screening, unless in the opinion 
of the Investigator and Sponsor's Responsible Physician, the medication will not 
interfere with the study procedures or compromise participant safety. 
3. Participation in a clinical study since Screening. 
4. Donation of 450 mL or more blood since Screening. 
 

 
 
 
 
 
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10.5 
Additional Advice and Restrictions for Study Population 
10.5.1 
Contraception 
To prevent pregnancy, female participants of childbearing potential and male participants and 
their female partner(s) must use 2 reliable forms of contraception, i.e.,  
• Condom + Established use of oral, injected or implanted hormonal contraceptive.  
• Condom + Intrauterine device. 
• Condom + Diaphragm with spermicide.  
• True abstinence, when this is in line with the preferred and usual lifestyle of the 
participant. [Periodic abstinence (e.g., calendar, ovulation, symptothermal, post-
ovulation methods) and withdrawal are not acceptable methods of contraception]. 
To prevent exposure of any partner (male or female) during non-vaginal intercourse to the 
semen from a male participant who has been exposed to the IMP, the following contraception 
must be used: 
• Condom.  
The chosen contraception method(s) must be followed from receiving the first dose of IMP 
until a stool sample tested for presence of the vaccine strains is negative for male participants 
and at least 2 months after receiving the last dose of IMP for female participants. 
Male participants must not have unprotected sexual intercourse with a female who is pregnant 
or breastfeeding during the study. 
10.5.2 
Sperm Donation 
Male participants must not donate sperm from receiving the first dose of IMP until a stool 
sample tested for presence of the vaccine strains is negative. 
10.5.3 
Diet and Fluid Restrictions 
10.5.3.1 Meal Times/Fasts 
Participants will fast for 90 mins prior to dose administration and 90 mins following dose 
administration. Water is permitted. 
10.5.3.2 Fluid Intake 
Water is allowed ad libitum. 
10.5.3.3 Alcohol Intake 
The consumption of alcohol will be limited to a maximum of 2 units per day from 7 days prior 
to the first dose of IMP.  Alcohol will be avoided completely for a period of not less than 2 
days prior to screening and each of the 3 doses of IMP.  Any deviation outside this alcohol 
intake restriction will be assessed on a case-by-case basis at Investigator’s discretion (provided 
the participant’s alcohol intake will not impact in the safety aspects and objectives of the study 
and the participant has a negative alcohol screen prior to dosing). 

 
 
 
 
 
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10.5.3.4 Caffeine 
Food or drink containing caffeine, including coffee, tea, cola, energy drinks or chocolates will 
be avoided completely whilst the participants are in attendance at the Clinical Unit (during each 
dosing period). 
10.5.3.5 Poppy and Sesame Seeds 
Participants will be advised that they must not eat food containing poppy or sesame seeds for 
3 days before each visit to the Clinical Unit, as consumption of poppy seeds can lead to a 
positive opiate result in the drugs of abuse test. 
10.5.4 
Other Life-Style Restrictions 
10.5.4.1 Strenuous Exercise 
Strenuous exercise must be avoided completely from 3 days before receiving the first dose of 
IMP until completion of the Day 84 visit. 
10.5.4.2 Blood Donation 
Participants will be advised that they should not donate blood for at least 3 months after 
receiving the last dose of IMP (Day 42). 
10.5.4.3 Travel Restrictions 
Participants will be advised that they should not travel outside of the UK until a stool sample 
cultured for Salmonella produces a negative result to confirm that shedding of the presumed 
vaccine strain(s) has ceased.  
10.5.5 
Removal of Participants from Therapy or Assessment 
Each participant will be informed of his/her right to withdraw from the study at any time and 
for any reason. 
An Investigator will withdraw a participant from the study at any time for any of the following 
reasons: 
• If a participant experiences a serious or intolerable AE, that prevents him/her from 
continuing participation in the study. 
• If a participant incurs a significant protocol violation which impacts on his/her 
safety or the scientific integrity of the study (this will be discussed on a case-by-case 
basis with the Sponsor). 
• At the request of the Sponsor. 
• If it is considered that the participant’s health is compromised by remaining in the 
study or the participant is not sufficiently cooperative. 
• If a participant is lost to follow-up. 
Participants who meet one or more of the following stopping criteria will receive no further 
doses of the study vaccine but will be followed up for safety: 

 
 
 
 
 
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• Severe systemic reaction, e.g., anaphylaxis. 
• Persistent febrile reaction (≥38.5°C for ≥12 hours) at the Investigator’s discretion 
• SAE related to the study agent (serious adverse reaction (SAR) or suspected 
unexpected serious adverse reaction (SUSAR)) 
The medical safety of the participant is of paramount importance when discussing study 
continuation.   
The reasons for any participant withdrawal will be recorded on the study completion form of 
the electronic case report form (eCRF). 
If a participant is withdrawn or chooses to withdraw from the study for any reason every 
possible effort will be made to perform the evaluations described for the post-study follow-up 
(see Table 10.7.1).  The data collected from withdrawn participants will be included in the 
study report. 
In the event of any abnormalities considered to be clinically significant, participants will be 
followed up with appropriate medical management until values are considered to be clinically 
acceptable.  Referral or collaborative care will be organised if considered necessary. 
A total of 45 participants are required to complete the study.  Participants who withdraw from 
the study before receiving any IMP will be replaced.  Participants who are withdrawn from the 
study due to significant drug-related AEs will not be replaced.  Replacement of all other 
participants withdrawn from the study after receiving IMP will be decided on a case-by-case 
basis by the Principal Investigator (or deputy) and Sponsor. 
10.6 
Investigational Medicinal Product  
10.6.1 Identity 
 
 
   
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
 
 
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10.6.2 
Receipt and Storage 
The Sponsor must notify the Principal Investigator, or the Project Manager, prior to dispatch 
of IMP supplies, and of the anticipated date of their arrival.  IMP should arrive at the study site 
at least 7 days before the first dosing day.  The Sponsor shall address all supplies to: 
The Production Manager 
The Pharmacy 
Simbec-Orion Clinical Pharmacology Unit 
Merthyr Tydfil Industrial Park 
Merthyr Tydfil CF48 4DR, UK 
Upon receipt, supplies will be dealt with as per Simbec-Orion SOP SR-IMP 053. Temperature 
monitors included with shipments will be downloaded. The Sponsor will confirm that the 
transportation conditions are acceptable. 
The IMPs will be stored securely under quarantine in a segregated, study-specific location, at 
< -70oC. The shipping documentation and bulk product Qualified Person (QP) certification will 
be reviewed. The supplies will subsequently be removed from quarantine and approved for use. 
10.6.3 
Assembly and Release 
The IMP will be assembled into unit doses by suitably trained Simbec-Orion staff according to 
the Simbec-Orion SOP SR-IMP 015.  

 
 
 
 
 
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The IMP will be labelled as specified in Annex 13 (manufacture of IMPs) of the European 
Commission guide to Good Manufacturing Practice (GMP)[13]. 
 
 
 
 
 
10.6.4 
Administration 
10.6.4.1 Placebo Training 
Each participant will be administered with 150 mL of sodium bicarbonate solution 2% 
weight/volume (w/v) at the Screening Visit, in order to practice oral administration prior to 
enrolment on the study. 
 
10.6.4.2 Treatment Period 
The following IMPs will be administered: 
Cohort 1 (n=9) 
3 doses of 1x109 CFU of ZH9PA (6 participants) 
Placebo (3 participants)  
Safety Review Meeting (up to Day 7 safety data) 
Cohort 2 (n=18) 
3 doses of 1x1010 CFU of ZH9PA (12 participants) 
Placebo (6 participants)  
Safety Review Meeting (up to Day 7 safety data) 
Cohort 3 (n=18) 
3 doses of 1x1010 CFU of ZH9PA and 1x1010 CFU of ZH9 (12 
participants) 
Placebo (6 participants)  
The IMPs will be administered following 90 mins of fasting (water is permitted). 
A dose leader design will be implemented with 2 participants being dosed on the first dosing 
day of EACH cohort.  Of these 2 participants, 1 will be randomised to receive active drug and 
1 randomised to receive placebo.  The remainder of the cohort will be randomised and dosed 
at least 24 h later pending an acceptable safety profile in the dose-leader group and will contain 
at least 1 additional placebo participant. The decision to proceed will be confirmed by the 
Investigator or delegate and communicated with the Sponsor.  This design allows maintenance 
of the “blind”. 
IMP administration will be documented in the eCRF. 
There will be 21 days between doses. 

 
 
 
 
 
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10.6.5 
Return/Destruction 
All used IMP containers and unused IMP (with the exception of any prepared solution that is 
not used for dosing) will be held under quarantine pending return/destruction.  
The Sponsor must provide approval for return/destruction of all remaining IMP within 8 weeks 
of study completion.   
All returns will be arranged at the earliest available delivery date.  
For IMP destruction, the Sponsor will receive the Certificate of Destruction 4 to 6 weeks from 
the date of removal from site. 
10.6.6 
Method of Assigning Participants to Treatment Groups 
Participants will be allocated to treatment groups according to a randomisation code produced 
by Simbec-Orion using the PROC PLAN procedure of Statistical Analysis Software (SAS®) 
(version 9.3 or higher).  The randomisation code will include 2 dose-leaders (1 active:1 
placebo) in each cohort.  
Participants will be numbered sequentially from 001 (i.e. 001, 002 etc.).  Replacement 
participants will be assigned the same randomisation as the participant they are replacing, 
however, 100 will be added to the number (i.e. 101 would replace 001 etc.). 
10.6.7 
Selection of Doses in the Study 
ZH9 is a live attenuated, oral vaccine, against typhoid fever.  It was previously identified as 
MICRO-TY. ZH9 and consists of the vaccine strain, S. Typhi (Ty2 aroC- ssaV-) ZH9. 
 
 
 
 
   
Individual participant treatments will be dispensed by the pharmacist or designee and labelled 
in accordance with Annex 13 of “The Rules Governing Medical Products in European 
Community, volume 4 Good Manufacturing Practice for Medicinal Products”[4]. 
 
The following dosing strategy will be employed: 
Cohort 
Dose schedule 
Dose level  
1 
Day 0, 21, 42 
1x109 CFU ZH9PA or placebo 
2 
Day 0, 21, 42 
1x1010 CFU ZH9PA or placebo 
3 
Day 0, 21, 42 
1x1010 CFU ZH9PA and 1x1010 
CFU ZH9 or placebo 

 
 
 
 
 
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The dose levels chosen are based on the previous clinical experience with ZH9.  These studies 
have determined that a dose of 1x1010 CFU is well tolerated and induces immune responses to 
LPS 0:9. ZH9PA is derived from ZH9, being modified to carry heterologous antigens.  Other 
potential products, derived from ZH9, have been used to deliver heterologous antigens in 
clinical trials i.e. ETEC LT antigen and Hepatitis B core antigen, at doses of 1x109 CFU and 
1x1010 CFU respectively.  Based on these data it is considered appropriate that the starting dose 
for ZH9PA is 1x109 CFU.  To date, ZH9 has been administered at a maximum dose of 1.7x1010 
CFU with no significant adverse effects; therefore, administering a maximum bacterial load of 
2x1010 CFU, of ZH9PA with ZH9 is considered appropriate. 
10.6.8 
Timing of Dose for Each Participant 
Two (2) dose leaders (1 active:1 placebo) will be dosed first in each cohort.  The remainder of 
the cohort will be dosed at least 24 h later pending an acceptable safety profile in the dose-
leader group.  A telephone call will be made to the dose leaders on the morning of Day 1 to 
assess the safety profile.  
10.6.9 
Blinding 
A designated individual from the IMP Management Department at Simbec-Orion will generate 
the randomisation code under the guidance of a statistician.  An independent unblinded dosing 
team will be assigned at the site to supervise administration of the IMP or placebo. This dosing 
team will have no other involvement in the study. All other site and Sponsor personnel involved 
in the study will be blinded with regard to the IMP being administered.  The Pharmacist (or 
designee) responsible for the preparation of participant doses and emergency code break 
envelopes will not be blinded and a copy of the original randomisation code will be issued to 
the pharmacist (or designee) for this purpose. 
Participant doses: Once the randomisation code has been authorised as per Simbec-Orion 
SOPs, each participant dose will be packaged and labelled for individual participants by 
designated individuals from the IMP Management Department at Simbec-Orion on behalf of 
the Sponsor.   
Code break envelopes: Once the randomisation code has been authorised as per Simbec-Orion 
SOPs, the Pharmacist (or designee) will produce individual sealed code-break envelopes that 
contain the treatment allocation(s) for each participant. The envelopes will be stored in the 
restricted access pharmacy. A set of code break envelopes will also be provided to the person 
responsible for PV. 
Emergency unblinding: Where the site requires emergency access to an individual participant 
code Simbec-Orion will break the blind via the code break envelopes stored in the pharmacy 
without prior consultation with the Sponsor. In such an event, the Sponsor’s Responsible 
Physician will be notified as soon as possible via email. Please refer to Simbec-Orion SOP SR-
CPU 041.   
Non-emergency unblinding: If an Investigator believes that knowledge of the IMPs received 
by a participant is essential for appropriate treatment of an AE, the code will be broken via the 
code break envelopes stored in the pharmacy. Where practical, the Investigator should ideally 

 
 
 
 
 
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consult the Sponsor before breaking the code.  In any event, the Sponsor will be informed as 
soon as practical whenever the code has been broken for a participant.   
If the blind needs to be broken for an individual participant, the date and reason will be recorded 
in the participant’s eCRF. The Investigator will not reveal the unblinded treatment code to any 
other member of the clinical team involved in the study or to the Study Monitor. If the code is 
broken for any individual participant, the participant will be withdrawn from the study and the 
procedures accompanying withdrawal performed. If the code is broken without justification, 
this will be deemed a serious protocol deviation. 
10.6.10 
Prior and Concomitant Therapy 
Prior Medication:  Prescription or non-prescription drugs, should not be taken within 28 days 
(or 5 half-lives (whichever is longer)) prior to receiving the first dose of IMP, unless in the 
opinion of the Investigator and Sponsor's Responsible Physician the medication will not 
interfere with the study procedures or compromise participant safety.  Prescription or non-
prescription drugs taken during the 28 days before receiving the first dose of IMP, and the 
reason for taking them, will be noted in the participant’s eCRF.  
Inclusion of participants who have taken prior medication will be reviewed on a case-by-case 
basis in relation to the safety aspects and objectives of this study.   
Concomitant Medication: Prescription or non-prescription drugs should not be taken 
throughout the duration of the study, with the exception of paracetamol (which may be taken 
as an analgesic to a maximum of 2 grammes (g) in 24 h).  
If intake of ANY medication is necessary during the study, the daily dosage, duration and 
reasons for administration will be recorded in the concomitant medication section of the 
participant’s eCRF. 
Prohibited Medications:  
28 days prior to first dose (Day 0) to Day 49: 
• Prescription and Non-prescription drugs (exception of 2 g per 24 hours of paracetamol). 
 
28 days prior to first dose (Day 0) to 28 days following the third dose on Day 42, i.e. Day 70: 
• Antibiotics  
Participants requiring antibiotics to treat an infection from Day 0 to Day 49 will be followed 
for safety but will not receive further doses of IMP. 
 
28 days prior to first dose (Day 0) to Day 84: 
• Non-steroidal anti-inflammatory drugs (NSAIDs) (participants will be advised to avoid 
NSAIDs if possible) 
• Steroids 
• Immunosuppressants 

 
 
 
 
 
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• Vaccines 
10.6.11 
Treatment Compliance 
Each dose of IMP will be taken under supervision and compliance will be documented.  The 
exact time a participant starts to drink the IMP and the exact time a participant finishes drinking 
the IMP will be recorded on the participant’s eCRF.  
10.7 
Safety Variables 
10.7.1 
Safety Measurements Assessed and Flow Chart 
A schedule of study assessments is provided in Table 10.7.1.   
Simbec-Orion personnel who have been appropriately trained will carry out study procedures. 
Where more than 1 procedure is scheduled for the same time-point, the following order of 
priority will apply:  
1. Safety and pharmacodynamic (PD) blood sampling  
2. Vital signs and 12-lead ECG (a window of ± 10 min in relation to the nominal time-point 
is allowed). 
3. Urine collection for urinalysis, urine DOA and urine pregnancy (if applicable) 
All baseline assessments will be performed prior to dosing on each dosing day.  

 
 
 
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Table 10.7.1 Study Flow Chart 
 
Screening 
Treatment Period1 
Post-Study Follow-Up 
Telephone Call2 
Week 
 
0 
 
1 
2 
3 
 
4 
6 
 
7 
10 
12 
22 
32 
Day 
-28 to -1 
0 
3 
7 
14 
21 
24 
28 
42 
45 
49 
70 
84 
154 
224 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
Visit Window (days) 
N/A 
+/-0 
+/-1 
+/-1 
+/-1 
+/-0 
+/-1 
+/-1 
+/-0 
+/-1 
+/-1 
+/-1 
+/-7 
+/-7 
+/-7 
Informed Consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/Exclusion3 
X 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
Demographics 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Height / Weight / BMI  
X 
 
 
 
 
 
 
 
 
 
 
 
X(weight 
only) 
 
 
Medical History & 
Concurrent Conditions 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Virology Tests  
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine DOA (including 
Alcohol and Cotinine)3 
X 
X11 
 
 
 
X11 
 
 
X11 
 
 
 
 
 
 
Pregnancy3, 4 
X(serum) 
X(urine) 11 
 
 
 
X(urine) 
11 
 
 
X(urine) 
11 
 
 
 
X(serum) 
 
 
FSH5 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Biochemistry3 
X 
X11 
 
X 
 
X11 
 
X 
X11 
 
X 
 
X 
 
 
Haematology3 
X 
X11 
 
X 
 
X11 
 
X 
X11 
 
X 
 
X 
 
 
Urinalysis 
X 
 
X 
 
 
 
X 
 
 
X 
 
 
X 
 
 
Vital Signs6 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
 
X 
 
 
Physical Examination 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
General Health Check 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
 
12-Lead ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
X 
 
 
Placebo training 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccine administration7 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
 
 

 
 
 
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Screening 
Treatment Period1 
Post-Study Follow-Up 
Telephone Call2 
Week 
 
0 
 
1 
2 
3 
 
4 
6 
 
7 
10 
12 
22 
32 
Day 
-28 to -1 
0 
3 
7 
14 
21 
24 
28 
42 
45 
49 
70 
84 
154 
224 
Visit 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
Visit Window (days) 
N/A 
+/-0 
+/-1 
+/-1 
+/-1 
+/-0 
+/-1 
+/-1 
+/-0 
+/-1 
+/-1 
+/-1 
+/-7 
+/-7 
+/-7 
Twice-Daily 
Temperature Records8 
 
 
 
 
 
 
 
 
 
Blood culture9 
 
 
Stool bacteriology 
(vaccine strains)10 
 
 
 
 
 
 
 
 
 
 
 
X 
(X) 
 
 
Administration of 
antibiotics10 
 
 
 
 
 
 
 
 
 
 
 
(X) 
 
 
 
Blood sample for serum 
Ig responses11 
 
X 
 
 
 
X 
 
 
X 
 
 
 
X 
 
 
PBMC collection for 
ALS assay11 
 
X 
 
X 
 
 
 
X 
 
 
X 
 
 
 
 
Adverse Events 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Concomitant Medication 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 

 
 
 
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Study Flow Chart Footnotes: 
All procedures on dosing days (Days 0, 21, 42) are to be completed prior to dosing (except post dosing vital signs and health check for discharge) 
1 The treatment period will be approximately 85 days duration, from the first dosing day (Day 0) until Day 84 (inclusive).  
2 Post-study follow-up telephone call will be performed on Days 154 and 224 after receiving the first dose of IMP (Day 0). 
3 Eligibility will be confirmed prior to dose administration on Day 0, Day 21 and Day 42. 
4 For female participants only. 
5 For postmenopausal female participants only 
6At Day 0, Day 21, and Day 42, vital signs to be performed pre-dose and at 4 hours post-dose. 
7 IMP will be administered fasted (90 mins prior to dose administration). Participants will fast for a further 90 mins post each dose administration. Participants in sentinel cohorts will be 
contacted by telephone approximately 24 hours following the Day 0 dose to assess their safety profile. 
8 Participants to record body temperature twice daily at home for 7 days post administration of each dose of IMP. 
9 Blood cultures to be performed to identify vaccine strains only if a participant experiences defined symptoms (e.g., fever greater than 37.8°C, white blood cell (WBC) >20x109 cells/L, 
respiratory rate >25 breaths per min, pulse rate >130 beats per min (bpm)) or clinically indicated. 
10 Participants will attend the Clinical Unit on Day 70 to provide a stool sample. If the stool sample is positive for any Salmonella strain at Day 70, it will be presumed to be one of the vaccine 
strains, and participants will be asked to return to the Clinical Unit to receive a course of antibiotic therapy, and a repeat stool culture will be obtained on Day 84 to confirm shedding of 
Salmonella, presumed to be one of the vaccine strains, has ceased.  
11 Samples to be collected prior to each dosing. 
 
 

 
 
 
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10.7.1.1 Demographic and Background Assessments 
The following demographic and background assessments will be performed during the study 
at the time-points specified in Table 10.7.1.  
10.7.1.1.1 Demographics 
Demographic data: age, year of birth, gender, race, height, weight and BMI.  
Height in metres (m) (to the nearest centimetre (cm)) and weight in kilogram (kg) (to the nearest 
0.1 kg) in indoor clothing and without shoes will be measured.  BMI = body weight (kg) / 
[height (m)]2 will be calculated. 
10.7.1.1.2 Medical History and Concurrent Conditions 
Relevant medical history and current conditions will be recorded in the eCRF.   
10.7.1.1.3 Virology Tests 
Virology tests: HBsAg, HCV Ab and HIV (antibodies to HIV-1 and HIV-2).  
HBsAg, HCV Ab, and HIV (antibodies to HIV-1 and HIV-2) virology tests will be analysed 
from the same serum sample for biochemistry analyses at Screening by Simbec-Orion 
Laboratory Services , using the Roche cobas® c6000 analyser series comprising of the c501 
and e601 modules or any other appropriate analyser.   
10.7.1.1.4 Drugs of Abuse (including Alcohol and Cotinine) 
Urine DOA screen (including alcohol and cotinine): Alcohol, amphetamines, barbiturate, 
benzodiazepine, cannabinoids, cocaine, cotinine, methadone, and opiate. 
A mid-stream urine sample will be collected into a universal collection/storage container.  At 
protocol-defined time-points when both urinalysis and drugs of abuse / alcohol screening are 
required, all urine analyses will be performed from a single approximately 20 mL urine sample. 
Urine samples for drugs of abuse (including alcohol and cotinine) will be analysed by Simbec-
Orion Laboratory Services, using the Roche cobas® c6000 analyser series comprising of the 
c501 and e601 modules or any other appropriate analyser. Assessments of urine sample quality 
(i.e., urine sample verification) will be performed by measuring urine creatinine for DOA. 
10.7.1.1.5 Pregnancy Test, Menstrual and Obstetric History 
Pregnancy tests will be performed on all female participants.  Pregnancy tests will be performed 
by Simbec-Orion Laboratory Services.  Serum pregnancy tests will be performed at time-points 
specified in Table 10.7.1.  Urine pregnancy tests will be performed manually using appropriate 
urine pregnancy test kits at time-points specified in Table 10.7.1.  
Serum FSH to confirm post-menopausal status will be analysed from the same serum sample 
for biochemistry analyses at Screening for postmenopausal females only.  Serum FSH analysis 
will be performed by Simbec-Orion Laboratory Services using the Roche cobas® c6000 
analyser series comprising of the c501 and e601 modules or any other appropriate analyser. 
10.7.1.2 Compliance with Inclusion/Exclusion Criteria 
An Investigator will assess all participants against the study inclusion and exclusion criteria at 
Screening.  Compliance will be re-confirmed on prior to each dose administration. 

 
 
 
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10.7.1.3 Safety Assessments 
The following safety assessments will be performed at the time-points specified in 
Table 10.7.1. 
10.7.1.3.1 Adverse Events 
AEs and SAEs that occurred during the study along with their severity and relationship to study 
drug will be reported. 
An AE is defined as per Statutory Instrument 2004 No. 1031[4]: 
Any untoward medical occurrence1 in a participant to whom a medicinal product has been 
administered, including occurrences which are not necessarily caused by or related to that 
product.2 
Notes: 
1 Whether subjective complaint or objective finding. 
2 An AE can therefore be any unfavourable and unintended sign (including an abnormal 
laboratory finding), symptom, or disease temporarily associated with the use of an IMP, 
whether or not considered related to the IMP. 
An unexpected adverse reaction is defined as: 
An adverse reaction, the nature, or severity of which is not consistent with the applicable 
product information (e.g., Investigator’s brochure for an unapproved investigational product 
of summary of product characteristics (SmPC) for an authorised product). 
AEs will be monitored throughout the study from the signature of participant informed consent 
through to the post-study follow-up visit.  All AEs will be recorded, whether considered minor 
or serious, drug-related or not. 
All abnormal laboratory findings considered to be clinically significant will be recorded as 
AEs. 
Recording of Adverse Events 
All of the following details will be recorded in the participant’s eCRF for each AE: 
• Full description of AE. 
• Date and time of onset. 
• Date and time of resolution. 
• Severity of event to be assessed by an Investigator in accordance with the 
definitions below. 
• Relationship to IMP to be assessed by an Investigator in accordance with the 
definitions below. 
• Action taken (if any). 
• Outcome and details of any further follow-up. 

 
 
 
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Grades of Adverse Event Severity 
The following grades will be used by an Investigator to describe the severity of AEs. 
The following are the only grades, which will be used to describe AE severity.  Only 1 severity 
grade will be used for each AE (e.g., mild - moderate is not acceptable). 
SEVERITY OF THE AE 
DEFINITION 
Mild  
The AE does not interfere with the participant’s daily routines.  It causes no 
more than slight discomfort or mild objective change in any of the safety 
parameters assessed during the study as determined by the Investigator. 
Moderate  
The AE interferes with some aspects of the participant’s daily routines or 
moderate objective change in any of the safety parameters assessed during 
the study as determined by the Investigator. 
Severe  
The AE causes inability to carry out the participant’s daily routine or severe 
objective change in any of the safety parameters assessed during the study as 
determined by the Investigator. 
Definitions of Adverse Event Causality 
The following definitions will be used by an Investigator to describe the relationship between 
an AE and the IMP. 
The following are the only definitions which will be used to describe the relationship between 
AEs and the IMP.  Only 1 relationship definition will be used for each AE (e.g., possible-
probable is not acceptable). 
RELATIONSHIP TO IMP 
DEFINITION 
Reasonable possibility of 
being related 
 
 
• There is a reasonable possibility of the event being related to the IMP.  This 
might be temporal or due to a physiological or pharmacodynamic process 
No reasonable possibility of 
being related. 
 
• No reasonable possibility of the event being related to the IMP, the IMP 
may not have been administered, no temporal relationship, and no known 
or understood physiological or pharmacological mechanism for the event 
to be related. 
Adverse events of special interest 
For this study, adverse events of special interest are: 
• Bacteraemia proven to be caused by one of the vaccine strains  
• Development of autoimmune diseases. 
Should one of these events occur the PI (or delegate) will alert the Sponsor’s Responsible 
Physician within 24 hours of becoming aware. 
Serious Adverse Events 
SAEs are defined in Statutory Instrument 2004 No. 1031 as any AE that 
a. results in death, 

 
 
 
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Note: Death is an outcome (of an AE, of progressive disease, etc.) and not an AE in 
itself. 
b. is life-threatening, 
A life-threatening event places the patient at risk of death at the time of the event. It 
does not refer to an event which hypothetically might have caused death if it were more 
severe. 
c. requires hospitalisation or prolongation of existing inpatients’ hospitalisation, 
“In-patient hospitalisation” means that the patient has been formally admitted to a 
hospital for medical reasons, for any length of time. It does not include presentation to 
or care within an emergency department. 
Complications that occur during hospitalisations are AEs. If a complication prolongs 
hospitalization, it is an SAE. 
d. results in persistent or significant disability or incapacity, or 
e. consists of a congenital anomaly or birth defect. 
f. Is a medically important event or reaction. 
Some medical events may jeopardise the participant or may require an intervention to prevent 
one of the above characteristics/consequences. Such events (referred to as ‘important medical 
events’) should also be considered as ‘serious’ in accordance with the definition. 
SAEs must be reported within 24 h of knowledge of the event by submitting an initial SAE 
report via email or fax to Diamond Pharma Services Ltd. 
E mail: pvservices@diamondpharmaservices.com 
SAE Fax: +44 (0) 1279 418964 
Suspected Unexpected Serious Adverse Reactions 
AEs which meet all of the following criteria 
• Serious. 
• Unexpected (i.e. is not consistent with the applicable product information e.g., 
Investigator's brochure for an unapproved IMP or SmPC) for an authorised product. 
• There is at least a reasonable possibility that there is a causal relationship between 
the event and the medicinal product (possible, probable, almost definite).  
will be classified as SUSARs and will be reported to the REC and to the MHRA in accordance 
with applicable regulatory requirements for expedited reporting.  It is the Sponsor’s 
responsibility to report SUSARs to the REC and MHRA. 
To ensure no confusion or misunderstanding of the difference between the terms “serious” and 
“severe,” which are not synonymous, the following note of clarification is provided: 
The term “severe” is often used to describe the severity of a specific event (as in mild, 
moderate, or severe myocardial infarction); the event itself, however, may be of relatively 
minor medical significance (such as severe headache). This is not the same as “serious,” which 
is based on participant/event outcome or action criteria usually associated with events that pose 

 
 
 
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a threat to a participant’s life or functioning. Seriousness (not severity) serves as a guide for 
defining regulatory reporting obligations. 
Monitoring of Participants with Adverse Events 
In the event of any abnormalities considered to be clinically significant by the investigating 
physician, participants will be followed up with appropriate medical management until: 
• It has resolved/returned to normal or baseline. 
• The event has stabilised at a level acceptable to the Investigator and is not considered 
to be clinically significant. 
Pregnancy 
Pregnancies must be reported within 24 h of knowledge  and should be followed up to 
determine outcome, including spontaneous or voluntary termination, details of the birth, and 
the presence or absence of any birth defects, congenital abnormalities, or maternal and/or AEs 
that occur in the new born child(e.g., meconium ileus, respiratory distress).   
Pregnancy outcomes must be collected for females who took the IMP and the female partners 
of any males who took the IMP.  Consent to report information regarding these pregnancy 
outcomes should be obtained from the mother. 
10.7.1.3.2 Laboratory Safety Assessments 
Laboratory Safety Screen 
Laboratory safety screen samples will be analysed by Simbec-Orion Laboratory Services.  
Printed laboratory test result reports will include normal reference ranges.  A decision 
regarding whether the laboratory test result outside the normal reference range is of clinical 
significance or not shall be made by an Investigator/designee and the report will be annotated 
accordingly.  Clinically significant laboratory test result abnormalities will be recorded on the 
AE page. The normal reference ranges for laboratory test parameters will also be filed in the 
ISF.  
Biochemistry Tests: Alanine transaminase, albumin, alkaline phosphatase, aspartate 
transaminase, bicarbonate, total bilirubin, direct bilirubin, calcium, cholesterol, chloride, 
creatinine, creatine kinase, c-reactive protein, glucose (random), gamma glutamyl transferase, 
total globulin, lactate dehydrogenase, potassium, inorganic phosphorus (or phosphate), total 
protein, triglycerides, sodium, urea and uric acid. 
Blood samples for biochemistry analyses for each time-point will be collected into an 
appropriately sized serum collection tube with or without a separator, and analysed by Simbec-
Orion Laboratory Services using the Roche cobas® c6000 analyser series comprising of c501 
and e601 modules or any other appropriate analyser. Assessments of blood sample quality (i.e., 
for sample verification) will be performed by measuring 3 indices [namely, Lipaemic (for 
Lipaemia), Haemolytic (for Haemolysis) and Icteric (for Icterus)] in serum. 
Haematology Tests: Haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular 
haemoglobin, mean corpuscular haemoglobin concentration, platelets, red blood cell count, red 
blood cell distribution width, WBC count, and WBC differential count (neutrophils, 

 
 
 
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lymphocytes, monocytes, eosinophils, and basophils) (reported in absolute and percentage 
values).   
Blood samples for haematology analyses for each time-point will be collected into a collection 
tube containing ethylenediaminetetraacetic acid and analysed by Simbec-Orion Laboratory 
Services using the Siemens ADVIA® 2120 or Siemens ADVIA® or an any other appropriate 
analyser. 
Urinalysis Tests: Specific gravity, pH, protein, glucose, ketones (or ketone bodies), 
urobilinogen, bilirubin, blood, leukocytes, and nitrite.  
A mid-stream urine sample for each time-point will be collected into a universal 
collection/storage container and divided into aliquots. Urinalysis will be performed by Simbec-
Orion Laboratory Services, using the Roche cobas® U6500 or any other appropriate analyser. 
In the event that the urinalysis automated ‘dipstick’ test result is positive for nitrites and/or 2+ 
or more reported for protein, blood, and/or leucocytes, then urine microscopy will be 
performed. The following test parameters will be reported: bacteria, casts (non-pathogenic), 
casts (pathogenic), crystals, epithelial cells, red blood cells and white blood cells.  The urine 
microscopy will be performed by Simbec-Orion Laboratory Services, using the Roche cobas® 
U6500 701 module or any other appropriate analyser/method. Separate aliquot samples will be 
sent for culture, if microscopy visualises bacteria (please refer to the Sample Handling Manual 
for processing and shipping instructions). 
10.7.1.3.3 Other Safety Assessments 
Vital signs: Systolic/diastolic blood pressure, pulse rate, oral body temperature. Measurements 
will be recorded in the supine position.  Blood pressure, pulse rate and oral body temperature 
will be measured by the DINAMAP* Compact Vital Signs Monitor (Model TS) or equivalent.  
Normal ranges for vital signs are presented in Appendix 2. 
Physical Examination: A physical examination will be performed by an Investigator.  The 
examination will include ear/nose/throat, ophthalmological, dermatological, cardiovascular, 
respiratory, gastrointestinal, central nervous system, lymph nodes and musculoskeletal). An 
Investigator can examine other body systems if required, at their discretion. 
12-Lead ECG: Heart rate, PR interval, QRS width, QT interval and QT interval corrected 
using Bazett’s formula and Fridericia's formula.  
12-lead ECG recordings will be made using a MAC 5500 or equivalent.  Each ECG trace will 
be labelled with the study number, participant number, participant year of birth.  An 
Investigator will provide an interpretation of each tracing.  Clinically significant abnormalities 
will be recorded on the AE page. Normal ranges for 12-lead ECG parameters are presented in 
Appendix 2. 
10.7.1.3.4 Stool Bacteriology 
Stool samples of up to 10 g of stool will be collected for stool bacteriology (vaccine strains) 
in appropriate stool collection kits at the timepoints specified in Table 10.7.1. 
Detailed sample collection and processing instructions will be documented in the SHM. 

 
 
 
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10.7.1.3.5 Blood Cultures 
Blood samples will be collected (as specified in Table 10.7.1) for blood cultures to be 
performed to identify ZH9 and/or ZH9PA only if the participant experiences defined symptoms 
(e.g., fever greater than 37.8°C, WBC >20 x 109 cells/L, respiratory rate >25 breaths per min, 
pulse rate >130 bpm) or clinically indicated. 8-10 ml of blood will be collection into bottles 
enriched with Aro.  Detailed sample collection and processing instructions will be documented 
in the SHM. 
10.7.1.3.6 Concomitant Medication 
All prior and concomitant medications taken during the study will be recorded in the 
participant’s eCRF (see Section 10.6.10). 
10.7.2 
Appropriateness of Measurements 
All measurements performed in the study are standard measurements. 
The total volume of blood to be collected from each participant during the study (approximately 
388 mL) is considered acceptable (Table 10.7.2).  If repeat or additional samples are required, 
the volume of blood taken will not exceed 500 mL. 

 
 
 
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Table 10.7.2 Summary of Blood Volume 
Procedure 
Visit 
No. of Samples 
Blood Volume per Sample (mL) 
Biochemistry 
Screening1 
1 
3.5 
Day 0 
1 
3.5 
Day 7 
1 
3.5 
Day 21 
1 
3.5 
Day 28 
1 
3.5 
Day 42 
1 
3.5 
Day 49 
1 
3.5 
Day 842 
1 
3.5 
Haematology 
Screening 
1 
3.0 
Day 0 
1 
3.0 
Day 7 
1 
3.0 
Day 21 
1 
3.0 
Day 28 
1 
3.0 
Day 42 
1 
3.0 
Day 49 
1 
3.0 
Day 84 
1 
3.0 
PD Assessment – PBMC/ALS assay 
Day 0 
1 
64 
Day 7 
1 
64 
Day 28 
1 
64 
Day 49 
1 
64 
PD Assessment – Serum Ig responses 
Day 0 
1 
20 
Day 21 
1 
20 
Day 42 
1 
20 
Day 84 
1 
20 
Total Blood Volume3 
388 
1 From the biochemistry blood sample collected at the Screening visit, the serum pregnancy test (for all female participants 
only), serum FSH (for postmenopausal female participants only) and virology screen (HBsAg, HCV Ab, and HIV) will be 
analysed from the same serum sample. 

 
 
 
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2 From the biochemistry blood sample collected at Day 84, the serum pregnancy test (for all female participants only) will be 
analysed from the same serum sample.   
3 The exact volumes of each sample may change but the total volume of blood drawn for any participant will not exceed 500 
mL. This total blood volume does not include blood samples required to be collected for performing blood cultures as 
specified in Table 10.7.1, and any repeat/unscheduled/additional blood sample collections required at the discretion of the 
Investigator/deputy. 
 
 
 
10.7.3 
Pharmacodynamic Assessments 
The following PD blood samples will be collected at the time-points specified in Table 10.7.1.  
• Serum Ig Responses 
• PBMC for ALS assay 
Detailed sample collection and processing instructions will be documented in the SHM. 
The IgG and IgA responses to four antigens (LPS O:9, LPS O:2; H:d, H:a) will be determined 
by a validated enzyme-linked immunosorbent assay (ELISA) end point titre method. 
PBMC will be cultured for 72 hours (+/- 3 hours) and the concentration of IgA in the 
supernatant specific for the same four antigens will be determined by a validated ELISA end 
point titre method. 
10.7.4 
Other Assessments 
Not applicable. 
10.8 
Data Quality Assurance 
At the time the study is initiated, a representative of the Sponsor will review the final protocol 
and eCRFs with the Principal Investigator and site staff.  During the course of the study the 
Monitor will visit the Clinical Unit to check the completeness of the participants records 
(including the volunteer (participant) master files (VMF), laboratory and 12-lead ECG print-
outs), the accuracy of entries into the eCRFs, the adherence to the final protocol and to ICH 
GCP E6 (R2) guidelines [3], the progress of enrolment and also to ensure the storage, handling 
and accountability of the IMP.  The Principal Investigator and key study personnel will be 
available to assist the Monitor during these visits. 
The Principal Investigator will give the Monitor, Auditor(s), the REC, and the MHRA direct 
access to relevant clinical records to confirm their consistency with the eCRF entries.  No 
information in these records about the identity of the participants will leave Simbec-Orion.  The 
Sponsor will maintain the confidentiality of all participant records, in line with Section 6.10 of 
the ICH GCP E6 (R2) guidelines[3] and the General Data Protection Regulation (GDPR) 
European Union (EU) 2016_679[14]. 
Study data will be fully documented in the eCRFs, study log books and paper source files.  
Signatures (dated via audit trail) will be given to account for all interventions in the study by 
research staff. 
Source data is all information in original records and certified copies of original records of 
clinical findings, observations, or other activities in a clinical study necessary for the 

 
 
 
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reconstruction and evaluation of the study.  Source data are contained in source documents 
(original records or certified copies). 
For the purposes of this study the source data will be recorded as detailed in Table 10.8.1. 
Table 10.8.1 Summary of Source Documentation Location 
Data 
Source Document 
VMF 
eCRF4 
Paper Source 
File 
Log Book 
Evidence of healthy participant 
status for entry into clinical study 
X 
 
 
 
Demographic data1 
X 
X 
 
 
Medical history 
X 
 
 
 
Inclusion and exclusion criteria 
 
X 
 
 
Informed consents2 
X 
X 
 
 
Participant participation in the 
clinical study 
X 
X 
 
 
Participant number in the clinical 
study 
 
X 
X 
 
Adverse events 
 
X 
 
 
Previous and on-going therapy 
X 
 
 
 
Concomitant therapy 
 
X 
 
 
Results of study examinations (e.g., 
12-lead ECGs, vital signs and 
laboratory safety tests)3 
 
X 
X 
 
Study visit dates 
 
X 
 
 
Administration of IMP 
 
X 
X 
 
Serum PD sample collection times 
 
X 
 
 
Serum PD sample tracking 
 
 
 
X 
Evidence of handwashing training 
 
 
 
X 
Sentinel dosing 24-hour telephone 
call 
 
 
 
X 
1. 
Only year of birth will be captured in the eCRF 
2. 
Copies of the informed consent form should be present in the VMF.  The original informed consent forms will be maintained 
in the study officer file during the clinical phase and will then be transferred to the Project Manager for archiving with the 
Investigator site file at the end of the study. 
3. 
The 12-lead ECG trace and laboratory safety test print-out will be stored in the paper source file. 
4. 
In the event of eCRF technical issues source data will be located in the paper source file. 
The above table indicates where source data will be recorded but for completeness the 
following information will also be recorded in the VMF: 
• Clinical study code. 
• Study visit dates (pre-dose; post-dose). 
• IMP administration (date of last dose). 
• Results of any key safety and efficacy measures from the clinical study that, in the 
opinion of an Investigator, should be noted. 
• Any concomitant medications used to treat the participant during the study that 
should be noted. 
The data collected in the eCRFs during the study will be subject to quality control checking by 
clinical staff prior to sign off. 

 
 
 
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Designated investigator staff will enter the data required by the protocol into the eCRFs using 
fully validated software that conforms to 21 CFR Part 11 requirements. Staff will not be given 
access to the eCRF until they have been trained. Automatic validation programs check for data 
discrepancies and, by generating appropriate error messages, allow the data to be confirmed or 
corrected before transfer of the data to the Biometrics group. The Investigator must certify that 
the data entered into the eCRF are complete and accurate.  
The study will be subject to an independent audit by the Simbec-Orion Quality Assurance Unit 
as outlined in Simbec-Orion SOP BD/324/9/01. 
Independent clinical quality assurance audits may be performed at any time during or following 
completion of the study by the Sponsor, or its authorised agents, and Regulatory Authorities 
and/or the REC. 
10.9 
Statistical Methods and Determination of Sample Size 
10.9.1 
Statistical and Analytical Plan 
A statistical analysis plan (SAP) will be written by Simbec-Orion and agreed by Prokarium 
prior to the locking of the database and subsequent reporting of the study data. 
10.9.1.1 Study Variables/Endpoints 
Primary Endpoint 
• Local and systemic reactogenicity up to Day 84 as assessed by adverse events, 
laboratory safety tests (biochemistry, haematology, urinalysis), vital signs and physical 
examination. 
 
Secondary Endpoints 
• Local and systemic reactogenicity; from Day 85 to Day 224 as assessed by adverse 
events, laboratory safety tests (biochemistry, haematology, urinalysis) if undertaken for 
cause; vital signs if undertaken for cause; physical exam if undertaken for cause. 
• Concentrations of specific serum IgA and IgG antibodies to LPS O:2 and O:9, flagellin 
H:a and H:d. 
• Fold increase in specific serum IgA and IgG antibody concentrations, in individual 
participants, against the antigens LPS O:2 and O:9, flagellin H:a and H:d. 
• Concentrations of specific mucosal IgA antibodies in lymphocyte supernatant (ALS) 
assays, in individual participants, against antigens LPS O:2 and O:9, flagellin H:a and 
H:d. 
• Seroconversion rate (proportion of participants with 4-fold increase above baseline at 
any time post vaccination) against each of the above antigens. 
• Fold increase in specific mucosal IgA antibodies in lymphocyte supernatant (ALS) 
assays, in individual participants, against the antigens LPS O:2 and O:9, flagellin H:a 
and H:d. 
 

 
 
 
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10.9.1.2 Analysis Sets 
Safety Set: All randomised participants who receive at least 1 dose of IMP will be included in 
the Safety Set. 
PD Set: All randomised participants who receive at least 1 dose of IMP and have at least one 
post-baseline antibody concentration will be included in the PD Set. 
Per Protocol Set: The Per Protocol Set will be comprised of all participants in the PD Set who 
comply with the following criteria: 
• Do not have an occurrence of vomiting or diarrhoea which may impact the systemic 
exposure of IMP; 
• Do not use a concomitant medication which may impact the immunogenicity results; 
• Do not incur a major protocol deviation which may invalidate or bias the 
immunogenicity results.  
10.9.1.3 Description of Statistical Methods 
All statistical analysis will be performed using SAS® (version 9.3 or higher). 
Descriptive statistics for quantitative parameters will be provided using number of observations 
(N), mean, standard deviation (SD), minimum, median and maximum. Descriptive statistics 
for qualitative parameters will be provided using absolute frequencies (n) and relative 
frequencies (%). For immunological endpoints (IgG, IgA, concentrations and fold increase) 
additionally geometric means with corresponding 95% confidence intervals (CI) will be 
presented. 
For parameters with evaluation before vaccination and in case of repeated value(s), only the 
last observation prior to dosing will be used in descriptive and inferential statistics and 
derivations of other parameter values.  After vaccination, only values of scheduled assessments 
(planned in the protocol) will be used. 
10.9.1.3.1 Demographic and Background Data 
All demographic and background data will be listed, in addition: 
Disposition: Participant disposition will be listed with any withdrawals flagged.  Frequencies 
(number and %) of the total number of participants dosed, completed and prematurely 
discontinued (including reason for discontinuation) from the study will be summarised by 
treatment and overall (all treatments combined).  Additionally, the number and percentage of 
participants included within each analysis set will be summarised. 
Demographics: Demographic data will be listed.  Descriptive statistics (number of participants 
in the analysis set (N), number of participants with non-missing observations (n), mean, 
standard deviation (SD), minimum, median and maximum) will be tabulated by treatment and 
overall for the continuous variables age, height, weight and BMI and frequencies (number and 
%) for the categorical variables race and gender. 
10.9.1.3.2 Efficacy Data 
Not applicable. 

 
 
 
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10.9.1.3.3 Safety Data 
All safety data will be listed, in addition: 
AEs: All AEs will be coded using the Medical Dictionary for Regulatory Activities (MedDRA) 
dictionary (the most up to date version will be used and this will be documented in the DMP) 
All AEs, including those which occurred prior to receiving the first dose of IMP, will be listed.  
Only treatment emergent adverse events (TEAEs), i.e., existing conditions that worsen or 
events that occur during the course of the study after administration of IMP, will be included 
within the summary tables.  AE summaries will be presented by treatment, all active treatments 
combined and overall.   
An overall summary of AEs will be produced including the number of TEAEs; the number and 
% of participants reporting at least 1 TEAE, serious TEAE, TEAE leading to withdrawal from 
the study; the number and % of participants reporting TEAEs by severity and relationship to 
IMP.   
The number of TEAEs and the number and % of participants reporting at least 1 TEAE will be 
tabulated by system organ class (SOC) and preferred term.  A participant reporting multiple 
episodes of a particular AE will only contribute 1 count towards the corresponding SOC and 
preferred term. 
The number of TEAEs and the number and % of participants reporting at least 1 TEAE will be 
tabulated by preferred term and sorted by descending frequency on the total number of 
participants with that AE.  A participant reporting multiple episodes of a particular AE within 
a treatment period will only contribute 1 count towards the corresponding preferred term. 
In addition, the number and % of participants reporting TEAEs will be tabulated by maximum 
severity and strongest relationship to IMP.  For the summary of TEAEs by severity, if a 
participant has multiple events occurring within the same SOC or preferred term the event with 
the highest severity will be counted.  Similarly, for TEAEs by relationship to IMP, if a 
participant has multiple events occurring within the same SOC or preferred term, the event 
with the highest association to IMP will be counted. 
The incidence of TEAEs will be compared between treatments using Fisher’s Exact Test (or 
an alternative statistical test if appropriate). 
Laboratory Safety: Biochemistry, haematology, and urinalysis parameters will be listed with 
any out of normal range values flagged.  Laboratory test results which are out of normal range 
will also be presented separately along with normal reference ranges. Descriptive statistics (N, 
n, mean, SD, minimum, median and maximum) of absolute and change from baseline (Day 0) 
values at each protocol-defined time-point will be tabulated by treatment. 
Vital Signs: Vital signs parameters will be listed with any out of normal range values flagged.  
Descriptive statistics (N, n, mean, SD, minimum, median and maximum) of absolute and 
change from baseline (Day 0 pre-dose) values at each time-point will be tabulated by treatment.   
12-Lead ECG: 12-Lead ECG parameters will be listed with any out of normal range values 
flagged.   

 
 
 
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10.9.1.3.4 PD Data 
Descriptive statistics (N, n, arithmetic mean, standard deviation (SD), minimum, median, inter-
quartile range, maximum, geometric mean with corresponding 95% CI) of serum IgG and IgA, 
mucosal IgA concentrations and fold-increase will be summarised by visit for each treatment 
group.  
 
The immune response (log-transformed serum IgG and IgA, mucosal IgA concentrations) will 
be subjected to an ANOVA/ANCOVA to compare pre-vaccination and post-vaccination levels 
within a treatment group for each visit. Similarly, the immune response (log-transformed serum 
IgG and IgA, mucosal IgA concentrations) and the magnitude of the immune response (fold-
increase) will be compared between treatment groups at each visit.  
 
In addition, the number of participants seroconverting per visit (i.e. with 4-fold increased level 
above baseline) will be compared between treatment groups using Fisher’s Exact Test (or an 
alternative if appropriate). 
10.9.1.3.5 Other Data 
Not applicable. 
10.9.2 
Sample Size Calculation 
The sample size chosen for this study is not based on a formal statistical estimation but is 
considered to be adequate to meet the objectives of the study. A sufficient number of 
participants will be initially screened for enrolment to ensure that the planned sample size is 
achieved.  

 
 
 
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11 
PRACTICAL CONSIDERATIONS 
11.1 
Storage of Data 
The Investigator site file and associated study documentation will be archived for at least 25 
years after the end of the study (last participant last visit) as per European Medicine Agency 
Guideline INS/GCP/856758/2018[15].  The study documentation may be transferred to an 
offsite storage facility during this period but will remain under the control of Simbec-Orion.   
The Sponsor has delegated the set up and maintenance of the Sponsor trial master file (TMF) 
to Simbec-Orion.  The TMF will be returned to the Sponsor at the end of the study, who will 
archive it for at least 25 years after the end of the study.  
11.2 
Protocol Amendments 
Changes in the study protocol must take the form of written protocol amendments and shall 
require the approval of all persons responsible for the study (see Section 1).  
A protocol amendment is deemed to constitute a substantial protocol amendment if it is 
considered to be likely to affect to a significant degree either: 
a. The safety or physical or mental integrity of the participants of the study. 
b. The scientific value of the study. 
c. The conduct or management of the study. 
d. The quality or safety of any IMP used in the study. 
Such amendments must be submitted to the REC responsible for the study and the MHRA for 
approval prior to implementation. 
Protocol amendments required for urgent safety reasons may be implemented immediately.  
However, the REC and MHRA must be notified in writing within 3 days of the measures taken 
and the reasons for implementation. 
All other amendments shall be deemed to be non-substantial and as such do not need the prior 
approval of the REC and the MHRA. 
11.3 
Confidentiality 
The confidentiality of the study must be maintained at all times and the Principal Investigator 
must not reveal any information relating to the study without express permission from the study 
Sponsor. 
11.4 
Study Report and Publication Policy 
Simbec-Orion will investigate and analyse the data generated with all due speed. 
A draft study report will be sent to the Sponsor for review.  The Sponsor will forward any 
comments on the draft study report to the Project Manager within 30 days of receipt.  Upon 
receipt of these comments a final, Quality Assurance (QA) approved report will be issued with 
all due speed.  A copy of the report will be forwarded to the Sponsor. 

 
 
 
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The Principal Investigator will obtain the Sponsor’s written permission before any information 
concerning this study is submitted for publication. 
11.5 
General Data Protection Regulation 
Personal data of the participant shall be processed in a manner that ensures it has appropriate 
security.  This includes protection against unauthorised or unlawful processing and against 
accidental loss, destruction or damage and by using appropriate technical or organisational 
measures. One such measure is by the Investigator ensuring that the participants’ personally 
identifiable information should be replaced through the use of pseudonymisation. 
On the eCRFs or other documents submitted to Prokarium will NOT be identified by their 
names but by the assigned participant number (panel/screening/participant number) to ensure 
confidentiality of the participant’s information and that data minimisation principles are 
maintained. If participant names are included in error on copies of documents submitted to 
Prokarium, the names will be erased or securely destroyed and the assigned participant number 
added to the document. 
Documents not intended for submission to Prokarium/Simbec-Orion (e.g., signed consent 
forms) should be maintained by the Investigator in strict confidence and not disclosed to any 
parties outside of this approved agreement. Forms should be protected by use of strong 
encryption. 

 
 
 
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12 
REFERENCES  
 [1] 
World Health Association Declaration of Helsinki. Recommendations guiding 
physicians in biomedical research involving human participants. Adopted by the 
18th WMA General Assembly, Helsinki, Finland, June 1964, and amended by the 
29th WMA General Assembly, Tokyo, Japan, October 1975, 35th WMA General 
Assembly, Venice, Italy, October 1983, 41st WMA General Assembly, Hong Kong, 
September 1989, and 48th WMA General Assembly, Somerset West, Republic of 
South Africa, October 1996, 52nd WMA General Assembly, Edinburgh, Scotland, 
October 2000, 53rd WMA General Assembly, Washington 2002 (Note of 
Clarification on Paragraph 29 added), 55th WMA General Assembly, Tokyo 2004 
(Note for Clarification on Paragraph 30 added), 59th WMA General Assembly, 
Seoul, October 2008 and 64th WMA General Assembly, Fortaleza, Brazil, October 
2013. 
[2] 
ABPI (The Association of the British Pharmaceutical Industry). Guidelines for 
Phase 1 Clinical Trials. 2018 edition. 
[3] 
ICH (International Council on Harmonisation) Guideline for Good Clinical Practice 
(GCP) E6(R2) (CPMP/ICH/135/95) 1996 
[4] 
The Medicines for Human Use (Clinical Trials) Regulations 2004 (Statutory 
Instrument 2004 No. 1031) and subsequent amendments. 
[5] 
Insurance and compensation in the event of injury in Phase I clinical trials 2012. 
Guidance developed by the ABPI (The Association of the British Pharmaceutical 
Industry), the BioIndustry Association and the Clinical Contract Research 
Association in consultation with the Department of Health and the National 
Research Ethics Service. 
[6] 
Raffatellu M, Wilson RP, Winter SE, Bäumler AJ. Clinical pathogenesis of typhoid 
fever.  J Infect Dev Ctries. 2008 Aug 30;2(4):260-6 
[7] 
Holt KE, Thomson NR, Wain J, Phan MD, Nair S, Hasan R, Bhutta ZA, Quail MA, 
Norbertczak H, Walker D, Dougan G, Parkhill J.  Multidrug-resistant Salmonella 
enterica serovar paratyphi A harbors IncHI1 plasmids similar to those found in 
serovar typhi. J Bacteriol. 2007 Jun;189(11):4257-64. 
[8] 
Buckle GC, Walker CL, Black RE. Typhoid fever and paratyphoid fever: 
Systematic review to estimate global morbidity and mortality for 2010. J Glob 
Health. 2012 Jun;2(1) 
[9] 
Tacket CO, Hone DM, Curtiss R, Kelly SM, Losonsky G, Guers L, et al. 
Comparison of the safety and immunogenicity of delta aroC delta aroD and delta 
cya delta crp Salmonella typhi strains in adult volunteers. Infect Immun 1992; 60: 
536-41 

 
 
 
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[10] 
Waterman SR, Holden DW. Functions and effectors of the Salmonella 
pathogenicity island 2 type III secretion system. Cell Microbiol. 2003 
Aug;5(8):501-11 
[11] 
Investigator’s Brochure for ENTERVAX Enteric Fever Vaccine version 2.0 release 
date 04-Nov-2019 
[12] 
Darton TC, Jones C, Blohmke CJ, Waddington CS, Zhou L, Peters A, Haworth K, 
Sie R, Green CA, Jeppesen CA, Moore M, Thompson BA, John T, Kingsley RA, 
Yu LM, Voysey M, Hindle Z, Lockhart S, Sztein MB, Dougan G, Angus B, Levine 
MM, Pollard AJ.  Using a Human Challenge Model of Infection to Measure Vaccine 
Efficacy: A Randomised, Controlled Trial Comparing the Typhoid Vaccines 
M01ZH09 with Placebo and Ty21a.  PLoS Negl Trop Dis. 2016 Aug 17;10(8) 
[13] 
European Commission. EudraLex The rules governing medicinal products in the 
European Union – Volume 4: Good Manufacturing Practice Guidelines. 
[14] 
The General Data Protection Regulation (EU) 2016/679 (25 May 2018). 
[15] 
European Medicines Agency Guideline on the content, management and archiving 
of 
the 
clinical 
trial 
master 
file 
(paper 
and/or 
electronic) 
EMA/INS/GCP/856758/2018 (06 Dec2018). 
 
 
 

 
 
 
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APPENDIX 1: DECLARATION OF HELSINKI (BRAZIL, 2013) 
 
https://www.wma.net/policies-post/wma-declaration-of-helsinki-ethical-principles-for 
medical-research-involving-human-participants/ 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
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APPENDIX 2: NORMAL RANGES FOR VITAL SIGNS AND ECG 
PARAMETERS 
Vital Sign Parameters  
Pulse Rate 
40-100 
Systolic Blood pressure 
90-140 
Diastolic Blood pressure 
50-90 
Oral Temperature 
35.0-37.5  
 
 
ECG Parameters  
Heart Rate (HR) 
40-100 bpm 
PR Interval  
120-220 ms 
QRS Width 
70-120 ms 
QT Interval  
N/A 
QTc Interval - QTcB 
Male: 350-430ms 
Female: 350-450ms 
QTc Interval -  QTcF 
Male: 350-450ms 
Female: 350-450ms