A Phase 2 Immunological Bridging Study Assessing the Non-inferiority of a New Formulation of ETVAX®. A Prospective Double-blind, Randomized Study in Healthy Volunteers.
Phase: PHASE2
Conditions: Healty Volunteers, Preventable Disease, Vaccine
Sponsor: Scandinavian Biopharma AB

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Clinical Study Protocol 
 
EudraCT No. 
 
2021-001541-13 
 
Investigational Product 
ETVAX®;  
An oral inactivated vaccine against 
ETEC containing five antigens; four 
different inactivated E. coli strains 
overexpressing the most prevalent 
ETEC colonization factors (CFs), 
i.e. CFA/I, CS3, CS5 and CS6, 
recombinant protein LCTBA and 
the adjuvant double mutant LT 
(dmLT)  
 
Study code:  
OEV-125 
Protocol Version and date 
Final 5.0, 23JUNE2022 
 
STUDY TITLE 
A Phase 2 immunological bridging study assessing the non-inferiority of a new formulation of 
ETVAX®. A prospective double-blind, randomized study in healthy volunteers. 
 
 
 
Test product and dosage  
 
New partially dried formulation of ETVAX® 
 
 
Comparator product and dosage  
Wet formulation of ETVAX® 
 
 
Duration of treatment  
Two vaccinations, two weeks apart. 
 
 
Medical Advisor  
(Sponsor signatory) 
Per Arne Parment, MD, PhD 
Scandinavian Biopharma Holding AB, Solna, Sweden 
Phone: +46 (0)70 644 47 71 
Email: per-arne.parment@scandinavianbiopharma.se 
 
 
 
 
Principal Investigator  
Dan Curiac, MD  
Clinical Trial Center, Göteborg, Sweden 
Phone: +46 (0) 700-823351 
Email: dan.curiac@vgregion.se 
 
 
 
 
 

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The following amendments have been made to the Final Clinical Study Protocol version 2.0:  
Amendment No.
 
 
Date of Amendment 
 
 
Revised protocol version (if applicable) 
1.0 
16 Jan 2022 
Version 3.0, dated 16 Jan 2022 
2.0 
15 Mar 2022 
Version 4.0 dated 15 Mar 2022 
3.0 
23 June 2022 
Version 5.0 dated 23 June 2022 
 
 
 

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PROTOCOL SYNOPSIS 
Study Title:  
A Phase 2 immunological bridging study assessing the non-inferiority of a new formulation of 
ETVAX®. A prospective double-blind, randomized study in healthy volunteers. 
 
Study code: OEV-125 
EudraCT No: 2021-001541-13 
Study period 
Estimated date of first subject enrolled: Q4 2021 
Estimated date of last subject completed, main study: Q3 2022 
Estimated date of last subject completed, exploratory study:  
 Q4 2022 
 
 
Phase of development: 2 
 
 
 
 
Principal Investigator 
Dan Curiac, MD 
Clinical Trial Center (CTC) 
Gröna Stråket 12 
SE-413 46 Göteborg, Sweden 
 
Study design 
A prospective double-blind, randomized, parallel-group study with the aim to demonstrate non-
inferiority, in terms of immunogenicity, between the wet formulation and a newly developed partially 
dried formulation of selected components of ETVAX®. 
 
Objectives  
Primary objective(s) 
 
The aim of this study is to demonstrate non-inferiority, in terms of immunogenicity, between 
the wet formulation and the newly developed partially dried formulation of selected 
components of ETVAX®. 
Secondary objectives  
To evaluate the safety and tolerability of the new formulation of the vaccine. 
 
 
 
 

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Number of subjects planned 
A total number of 126 subjects will be included in each arm of the study, i.e. 252 subjects in total. 
Assuming a 10% dropout rate the target number of subjects to be recruited per study arm is therefore  
140, i.e. 280 subjects in total. 
 
Diagnosis and eligibility criteria 
Healthy volunteers fulfilling the following criteria will be eligible for enrolment into the study: 
Inclusion criteria 
 
• 
Male or female aged 18-50 years, inclusive at the time of signing the informed consent. 
• 
Healthy constitution as established by medical history and physical examination. 
• 
Willing and able to give written informed consent for participation in the study. 
• 
Able to comply with study activities, as judged by the Investigator. 
• 
Female Participants: Women of child-bearing potential (for definition see Section 9.3.6): 
- Have to agree to use an acceptable birth control method during participation in the 
investigation (see Section 9.3.6). 
- A negative pregnancy test (beta human chorionic gonadotropin dipstick test in urine) at 
Visit 2/Day 1 will be required. 
• 
Male Participants:  
- Have to agree to remain abstinent (refrain from heterosexual intercourse) or use a condom, 
and agreement to refrain from donating sperm, as defined in Section 9.3.6. 
Exclusion criteria 
Subjects must not enter the study if any of the following exclusion criteria are fulfilled: 
 
• 
An acute or chronic medical condition that, in the opinion of the investigator/physician, 
would render ingestion of the investigational products unsafe or would interfere with the 
evaluation of responses. This includes, but is not limited to gastrointestinal diseases, and 
autoimmune diseases. 
• 
Current malignancy or history of malignancy during the last five years, based on anamnesis.  
• 
Gastroenteritis within two weeks prior to vaccination. 
• 
Regular use of laxatives, antacids or other agents that lower stomach acidity. 
• 
Any planned major surgery during the duration of the study. 
• 
After 10 minutes supine rest, any vital signs outside the following ranges: 
-Systolic BP > 160 mm Hg 
-Diastolic BP > 100 mm Hg 
-Heart rate < 40 or >85 beats per minute 
• 
Antibiotic therapy within two weeks prior to the vaccination.  

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Known Hepatitis A, B, C, and/or HIV infection. 
• 
Concomitant intake of immunomodulating drugs during the study period or less than 3 
months prior to the first immunization, with the following exceptions: oral anti-histamines 
are not allowed during the study period or less than 3 weeks prior to the first immunization.  
Local anti-histamine treatment is allowed during the study period. 
• 
Any other significant medical conditions (e.g. poorly controlled psychiatric condition) judged 
by the Investigator to preclude entry. 
• 
Intends to receive any other vaccine during the study period, or within two weeks prior to 
trial vaccination. 
• 
Has previously received Dukoral or any type of enterotoxigenic Escherichia coli (ETEC) or 
cholera vaccines. 
• 
Brought up in ETEC-endemic areas (e.g., urban and rural areas of Central and South 
America, Caribbean, most countries in Asia, Africa, etc.). 
• 
Has travelled to ETEC-endemic areas within the last 3 years OR spent > two months in 
ETEC endemic areas during the last 10 years. 
• 
Intends to travel to ETEC endemic countries during the study period. 
• 
Known or suspected history of drug, chemical or alcohol abuse, as deemed by the 
investigator/physician.  
• 
History of severe allergy/hypersensitivity or ongoing allergy/hypersensitivity, as judged by 
the Investigator, or history of hypersensitivity to drugs with a similar chemical structure or 
class to ETVAX®. 
• 
Participation in any other clinical study that included drug treatment with the last 
administration within the past 3 months prior to administration of treatment in this study. 
Patients consented and screened but not dosed in previous clinical studies are not excluded 
• 
Concomitant participation in any other clinical study.  
• 
Females who are pregnant as determined by urine test at inclusion and prior to each 
vaccination. 
• 
Females who are nursing.  
• 
Unable to participate in all study visits. 
• 
Any condition or circumstance which would make the subject unsuitable for participation in 
the study in the opinion of the investigator/physician. 
Methodology 
Consenting subjects will be screened for eligibility, according to study-specific inclusion/exclusion 
criteria within 30 days prior to the first administration of Investigational Medicinal Product (IMP) 
(Visit 1; Screening visit). Eligible subjects will be randomized on Day 1 (Visit 2) to receive either of 
the two oral formulations of ETVAX® (1:1) and consecutively included the study. The treatment 
allocation (Wet formulation/Partially dried formulation) will be double-blinded, i.e. it will not be 
disclosed to the subjects, site staff, laboratory staff or Sponsor. 
 
Study subjects will receive two oral doses, two weeks apart (Day 1/Visit 2 and Day 15 (±2) /Visit 3).  

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If a subject is unable to come for their third study visit on day 15±2 due to e.g. illness or rules 
regarding quarantine due to the COVID-19 epidemic, the second vaccine dose can be administered up 
to 28 days after the first dose. 
  
Dosing will occur at the clinic. After each dose, subjects will remain at the clinic for observation for 
15 min before leaving. 
 
At each dosing visit (Day 1 and Day 15) subjects will receive adverse event and medication diaries to 
be completed for six days after each vaccination (i.e. day of vaccination and five subsequent days). 
A follow-up visit will be performed 7 days (acceptable range: 6-10 days) after the last (second) dose 
in all study subjects.  
Blood samples will be collected on visit 2 and 4. 
For the exploratory analyses, subgroups of subjects (n=20-40, evenly distributed between the two 
treatment arms) will participate in additional follow-up visits 5± 1, 30± 7 and 75-125 days after the 
second dose. Blood samples will be collected on all exploratory visits. The extra visits and analyses 
for exploratory analyses may continue after the main part of the study has been completed and the 
database locked. 
Investigational Medicinal Product, dosage and mode of administration 
 
ETVAX® is an oral inactivated vaccine against ETEC containing four different inactivated E. coli 
strains overexpressing the most prevalent ETEC colonization factors (CFs), i.e. CFA/I, CS3, CS5 and 
CS6, a recombinant protein LCTBA and the adjuvant double mutant LT (dmLT).  
The product formulation of ETVAX® used so far in clinical trials (wet formulation) is a liquid 
suspension of inactivated bacteria and LCTBA in one vial, freeze-dried dmLT adjuvant in a second 
vial, and effervescent buffer granules in a separate sachet. Prior to administration, the buffer is 
dissolved in 150 ml tap water, followed by the addition of the content of the vaccine vial (inactivated 
bacteria mixed with LCTBA) and reconstituted adjuvant dmLT from the second vial.  
As a part of manufacturing development process, a method to spray-dry dmLT together with LCTBA 
protein has been established. In the new, partially dried formulation, the dmLT and LCTBA are spray-
dried and mixed with the buffer granules and stabilizing excipients in a sachet. In preparation of the 
partially dried formulation, the content of the buffer sachet (buffer, dmLT, and LCTBA) is dissolved 
in 150 ml tap water, followed by the addition of the vaccine vial content (inactivated bacteria). The 
new product formulation is thus very simple to prepare and suitable for use in the field. 
 
Non-Investigational Medicinal Products, dosage and mode of administration (if applicable) 
Not applicable in this study. 
Duration of treatment 
Subjects will receive two oral doses of the vaccine, two weeks apart. 
Duration of subjects’ involvement in the study 
The duration of subject’s participation in the study will be between 3 and 9 weeks for the main study, 
including a 0-4 week screening period, 2-4 week treatment period and 1 week of follow-up.  

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For subjects who also participate in the exploratory sub-study with follow-up until 75-125 days after 
dose 2, the total duration will be between 13 and 26 weeks, including follow-up visits on Days 30± 7 
and 75-125 after the second (last) dose. 
 
Efficacy assessments  
Seroconversion rates of IgA and/or IgG anti-LTB antibodies in serum: 
IgA and IgG antibodies specific for LTB will be measured in serum samples collected on Day 1/Visit 
2 and Day 7 after the second dose/Visit 4 using an Enzyme Linked Immunosorbent (ELISA) assay. 
The analyses will be performed by technical laboratory personnel at the Department for Microbiology 
and Immunology, University of Gothenburg, according to standardized laboratory procedures. The 
seroconversion rate for IgA and IgG will be determined as the frequencies of subjects having a ≥2-
fold increase in LTB-specific antibody levels in post compared to pre-vaccination samples. 
Safety assessments  
Physical examination 
A complete physical examination will include assessments of the head, eyes, ears, nose, throat, skin, 
thyroid, neurological, lungs, cardiovascular, abdomen (liver and spleen), lymph nodes and 
extremities. 
Vital signs  
Systolic and diastolic blood pressure and heart rate will be measured in the supine position after 10 
minutes of rest. Body temperature will be measured in the ear using a digital thermometer. 
Subject Diary 
The study diaries will contain questions regarding adverse events and current medication. The diaries 
should be filled in by the subjects on the day of vaccination and five subsequent days. Information 
about adverse events obtained from the diaries will be verified by interviewing the subject and will 
then be registered in the CRFs by the study nurse/physician. 
The following solicited AEs will be included in the diary: fever, nausea, vomiting, abdominal pain 
and loose stools/diarrhea. 
Statistical methods 
Assuming a seroconversion rate of 85% in both the wet and the partially dried formulation, and 
aiming to prove non-inferiority with margin -15% (on an absolute scale), 126 subjects are needed in 
each treatment arm to achieve 90% power. This calculation was done with Farrington-Manning’s 
method, assuming that the study will be evaluated by a two-sided 95% confidence interval for the 
difference between the seroconversion rates of the two treatment groups.  
A 10% dropout rate is assumed.  The target number of subjects to be recruited per study arm is 
therefore 140. 
The primary endpoint will be summarized for the Full analysis set (FAS) and the Per protocol 
analysis set (PPAS). The primary endpoint analysis of PPAS will be considered the primary analysis 
of the study. 

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The primary endpoint will also be summarized with descriptive statistics for the PPAS and FAS. 
 
 

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TABLE OF CONTENTS 
2 
PROTOCOL SYNOPSIS ........................................................................................ 3 
3 
TABLE OF CONTENTS ......................................................................................... 9 
4 
LIST OF ABBREVIATIONS AND DEFINITION OF TERMS ....................... 14 
5 
ETHICS AND REGULATORY REQUIREMENTS ......................................... 17 
5.1 
Ethical conduct of the study .............................................................................. 17 
5.2 
Ethics and regulatory review ............................................................................. 17 
5.3 
Subject information and consent ...................................................................... 17 
5.4 
Subject Information Card ................................................................................. 18 
5.5 
Subject data protection ...................................................................................... 18 
6 
INVESTIGATOR(S) AND STUDY ADMINISTRATIVE STRUCTURE ....... 18 
7 
INTRODUCTION .................................................................................................. 20 
7.1 
Project background ............................................................................................ 20 
7.2 
Investigational Medicinal Product (IMP) ........................................................ 21 
7.2.1 
Vaccine development........................................................................................ 21 
7.2.2 
Mucosal Adjuvants ........................................................................................... 22 
7.2.3 
Development and testing of double mutant LT (dmLT) .................................. 23 
7.2.4 
Non-clinical safety ............................................................................................ 24 
 
Immunogenicity and safety in mice of ETEC vaccine with and without 
dmLT............................................................................................................. 24 
 
cGLP Toxicology and Immunology Evaluation of ETEC vaccine with and 
without dmLT ............................................................................................... 25 
7.2.5 
Clinical experience ........................................................................................... 25 
 
Vaccine safety ............................................................................................... 25 
 
Vaccine immunogenicity .............................................................................. 26 
7.3 
Risk/benefit assessment ...................................................................................... 27 
7.3.1 
Benefits ............................................................................................................. 27 
7.3.2 
Risks ................................................................................................................. 27 
 
Risk assessment for the OEV-125 trial in relation to the COVID-19 epidemic
....................................................................................................................... 28 
8 
STUDY OBJECTIVES AND ENDPOINTS ........................................................ 28 
8.1 
Primary objective ............................................................................................... 28 
8.1.1 
Primary endpoint(s) .......................................................................................... 29 

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8.2 
Secondary objectives .......................................................................................... 29 
8.2.1 
Secondary endpoints ......................................................................................... 29 
8.3 
Exploratory Objective(s) .................................................................................... 29 
8.3.1 
Exploratory Endpoint(s) ................................................................................... 29 
9 
INVESTIGATIONAL PLAN ................................................................................ 30 
9.1.1 
Dose levels ........................................................................................................ 33 
9.1.2 
Stopping rules/Discontinuation Criteria ........................................................... 33 
9.1.3 
Internal Safety Review Committee (iSRC) ...................................................... 33 
9.2 
Rationale for study design, doses and control group ...................................... 33 
9.3 
Selection of study population ............................................................................. 35 
9.3.1 
Recruitment....................................................................................................... 35 
9.3.2 
Number of subjects ........................................................................................... 35 
9.3.3 
Screening and enrolment log ............................................................................ 35 
9.3.4 
Inclusion criteria ............................................................................................... 35 
9.3.5 
Exclusion criteria .............................................................................................. 36 
9.3.6 
Restrictions ....................................................................................................... 37 
9.3.7 
Removal of subjects from therapy or assessment ............................................. 38 
 
Subject withdrawal criteria and replacement ................................................ 38 
9.4 
Treatments .......................................................................................................... 38 
9.4.1 
Identity of Investigational Medicinal Product (IMP) ....................................... 39 
 
Inactivated E. coli ETEX 21 .......................................................................... 39 
 
Inactivated E. coli ETEX 22 .......................................................................... 39 
 
Inactivated E. coli ETEX 23 .......................................................................... 39 
 
Inactivated E. coli ETEX 24 .......................................................................... 40 
 
LCTBA protein.............................................................................................. 40 
 
Oral dmLT adjuvant ...................................................................................... 40 
 
Sodium bicarbonate protective buffer powder (Recip) used in the Wet 
formulation .................................................................................................... 41 
 
dmLT LCTBA spray-dried preparation ........................................................ 41 
 
dmLT LCTBA spray-dried powder together with effervescent powder and 
excipients. ..................................................................................................... 41 
 Product(s) to be tested and supply (where applicable) ................................. 41 
9.4.1.10.1 Wet formulation .................................................................................... 41 

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9.4.1.10.2 Partially dried formulation .................................................................... 42 
 Investigational Product: ................................................................................ 42 
9.4.2 
Packaging, labelling and storage of IMP .......................................................... 44 
 
Packaging ...................................................................................................... 44 
 
Labelling ....................................................................................................... 44 
 
Storage .......................................................................................................... 44 
9.4.3 
Doses and treatment regimens .......................................................................... 45 
 
Preparation and administration of investigational products .......................... 45 
9.4.3.1.1 Preparation of Wet formulation .............................................................. 45 
9.4.3.1.2 Preparation of partially dry formulation ................................................. 46 
 
Vaccine oral solution .................................................................................... 46 
9.4.4 
Product accountability ...................................................................................... 46 
9.4.5 
Method of assigning subjects to treatment groups ........................................... 47 
9.4.6 
Blinding ............................................................................................................ 47 
9.4.7 
Emergency decoding of blinded treatment ....................................................... 47 
9.4.8 
Prior and concomitant therapy .......................................................................... 47 
9.4.9 
Continuation of treatment ................................................................................. 48 
9.4.10 
Treatment compliance ...................................................................................... 48 
9.5 
Study assessments ............................................................................................... 48 
9.5.1 
Demographics and other baseline characteristics ............................................. 48 
 
Informed consent .......................................................................................... 48 
 
Demographic information ............................................................................. 48 
 
Medical/surgical history................................................................................ 48 
 
Prior and concomitant medications ............................................................... 48 
9.5.2 
Clinical efficacy assessments ........................................................................... 49 
 
Seroconversion rates of IgA and/or IgG anti-LTB antibodies in serum. ...... 49 
9.5.3 
Safety laboratory assessments .......................................................................... 49 
9.5.4 
Clinical safety assessments ............................................................................... 49 
 
Physical examination .................................................................................... 49 
 
Vital signs ..................................................................................................... 49 
 
Subject Diary ................................................................................................ 49 
9.5.5 
Exploratory assessments ................................................................................... 50 

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9.5.6 
Adverse events .................................................................................................. 51 
 
Definitions..................................................................................................... 51 
 
Assessment of severity/intensity ................................................................... 52 
 
Assessment of causal relationship ................................................................ 52 
 
Assessment of Outcome ................................................................................ 53 
 
Collecting and recording of AEs................................................................... 53 
 
Exceptions from expedited reporting of SAEs ............................................. 54 
 
Procedures in case of pregnancy ................................................................... 55 
 
Coding of AEs............................................................................................... 55 
9.5.7 
Appropriateness of measurements .................................................................... 55 
9.6 
Data quality assurance ....................................................................................... 55 
9.6.1 
Monitoring ........................................................................................................ 56 
9.6.2 
Audits and inspections ...................................................................................... 56 
9.6.3 
Case Report Forms (CRFs) ............................................................................... 56 
9.6.4 
Source Data ....................................................................................................... 57 
9.6.5 
Training of study staff ...................................................................................... 57 
9.7 
Statistical methods and determination of sample size..................................... 57 
9.7.1 
Description of study population ....................................................................... 58 
 
Demographics and baseline characteristics .................................................. 58 
 
Medical/surgical history and concomitant medication ................................. 58 
 
Analysis of efficacy (Seroconversion rates of IgA and/or IgG anti-LTB 
antibodies in serum) ...................................................................................... 58 
9.7.2 
Analysis of safety ............................................................................................. 59 
 
Vital signs ..................................................................................................... 59 
 
Adverse Events (AEs) ................................................................................... 59 
9.7.3 
Analysis of exploratory assessment .................................................................. 59 
9.7.4 
Analysis data sets .............................................................................................. 60 
 
Full Analysis Set (FAS) ................................................................................ 60 
 
Per Protocol Analysis Set (PPAS) ................................................................ 60 
 
Determination of sample size ........................................................................ 60 
9.7.5 
Statistical/analytical issues ............................................................................... 60 
 
Adjustments for covariates ........................................................................... 60 

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Handling of dropouts or missing data ........................................................... 60 
 
Multi-centre studies ...................................................................................... 60 
 
Multiple comparison/multiplicity ................................................................. 60 
 
Active-control studies intended to show equivalence  Not applicable ......... 61 
 
Examination of subgroups  No examination of subgroups is planned ......... 61 
 
Interim analyses and data monitoring ........................................................... 61 
9.8 
Data Management .............................................................................................. 61 
9.8.1 
The web based e-CRF ....................................................................................... 61 
9.8.2 
Entering of data into the e-CRF ........................................................................ 61 
9.8.3 
The query process ............................................................................................. 62 
9.8.4 
Source documents ............................................................................................. 62 
9.8.5 
Audit trail .......................................................................................................... 63 
10 
EMERGENCY PROCEDURES ........................................................................... 63 
10.1 
Emergency contacts ............................................................................................ 63 
10.2 
Procedures in case of medical emergency ........................................................ 63 
10.3 
Procedures in case of overdose .......................................................................... 63 
11 
STUDY MANAGEMENT ..................................................................................... 64 
11.1 
Changes in the approved study protocol .......................................................... 64 
11.2 
Study timetable ................................................................................................... 64 
11.3 
Discontinuation of the study .............................................................................. 64 
11.4 
Reporting and publication of study results ...................................................... 64 
11.5 
Disclosure and confidentiality ........................................................................... 65 
11.6 
Archiving ............................................................................................................. 65 
11.7 
Insurance/indemnity........................................................................................... 65 
11.8 
Study agreements................................................................................................ 66 
12 
REFERENCES ....................................................................................................... 67 
12.1 
Signature pages ................................................................................................... 71 
12.2 
Declaration of Helsinki....................................................................................... 73 
12.3 
Appendix 3: Clinical Toxicity Grading ............................................................ 74 
 
 

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LIST OF ABBREVIATIONS AND DEFINITION OF TERMS 
Abbreviation or term 
Explanation 
AE 
Adverse event 
ATC 
Anatomic Therapeutic Chemical 
CF 
CI 
CT 
CTC 
CS 
Colonization Factor 
Confidence Interval 
Cholera Toxin 
Clinical Trial Center at Sahlgrenska University Hospital 
Coli surface antigen 
CRF 
CRO 
CSR 
CTA 
CV 
DMID 
DMP 
dmLT 
Case Report Form 
Clinical Research Organization 
Clinical Study Report 
Clinical Trial Agreement 
Curriculum Vitae 
Division of Microbiology and Infectious Diseases  
Data Management Plan 
Double mutated heat-labile toxin 
ECL 
ELISA 
EPM 
ETEC 
FAS 
Electrochemiluminescence 
Enzyme Linked Immunosorbent Assay 
Etikprövningsmyndigheten 
Enterotoxigenic Escherichia coli 
Full analysis set 
GCP 
GLP 
GMP 
Good clinical practice 
Good laboratory practice 
Good manufacturing practise 
IB 
ICF 
ICH 
Investigator’s Brochure 
Informed Consent Form 
International Council for Harmonization 
IEC 
Ig 
Independent Ethics Committee 
Immunoglobulin 
IMP 
Investigational medicinal product 

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Abbreviation or term 
Explanation 
ISF 
iSRC 
LCTBA 
 
 
LMIC 
LT 
LV 
MedDRA 
NIH 
PPAS 
PBMC 
PBS 
PT 
QP 
RN 
Investigator Site File 
internal Safety Review Committee 
Hybrid protein between the B-subunit of the E. coli heat-
labile enterotoxin (LTB) and the B-subunit of the cholera 
toxin (CTB) 
Low and middle income countries 
Heat-labile toxin 
Läkemedelsverket 
Medical Dictionary for Regulatory Activities 
National Institutes of Health 
Per protocol analysis set 
Peripheral Blood Mononuclear Cells 
Phosfate Buffer Solution 
Preferred term 
Qualified Person 
Registered Nurse 
SAR 
Serious Adverse Reaction 
SAE 
Serious Adverse Event 
SBH 
SCRO 
SDV 
SIgA 
SOC 
ST 
SUSAR 
TD 
WOCBP 
Scandinavian Biopharma Holding AB 
Scandinavian CRO 
Source Data Verification 
Secretory IgA 
System Organ Class 
Heat-stable toxin 
Suspected Unexpected Serious Adverse Reaction 
Travellers’ Diarrhea 
Women of childbearing potential 
Enrolled subject 
Subject who has signed the Informed Consent Form (ICF) 
Screen failure 
Enrolled subject not included  

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Abbreviation or term 
Explanation 
Included subject 
Subject randomized 
Withdrawn subject 
Subject randomized but not completed 
Completed subject 
Subject completed the study period, including follow-up 
 
 
 

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5 
ETHICS AND REGULATORY REQUIREMENTS 
5.1 
Ethical conduct of the study 
The study will be performed in accordance with ethical principles that have their origin in the 
Declaration of Helsinki and are consistent with ICH/Good Clinical Practice (GCP) E6(R2), 
European Union (EU) Clinical Trials Directive, and applicable local regulatory requirements. 
The Declaration of Helsinki is included as Appendix 12.2 to the Protocol. 
5.2 
Ethics and regulatory review 
The Principal Investigator is responsible for submission of the Clinical Study Protocol, the 
Subject Information and Informed Consent Form (ICF), any other written information to be 
provided to the subjects and any advertisements used for recruitment of study subjects to 
Etikprövningsmyndigheten (EPM), the applicable Independent Ethics Committee (IEC), for 
approval.  
The Sponsor, Scandinavian Biopharma Holding AB (SBH), is responsible for submission of 
study documents to Läkemedelverket (LV), the applicable Competent Authority in Sweden. 
Approval must be obtained in writing from both Competent Authority (LV) and IEC (EPM) 
before the first study subject can be enrolled. 
 
SBH will provide the Competent Authority (LV), IEC (EPM) and Principal Investigator with 
safety updates/reports according to local requirements. 
 
5.3 
Subject information and consent 
It is the responsibility of the Investigator to give each potential study subject adequate verbal 
and written information regarding the objectives and the procedures of the study as well as 
any risks or inconvenience involved before including the subject in the study. The subject 
must be informed about the right to withdraw from the study at any time. The subject should 
be allowed sufficient time for consideration of the proposal.  
Furthermore, it is the responsibility of the Investigator to obtain signed informed consent from 
all subjects before including them in the study. The ICF must be signed and personally dated 
by the subject and by the person who conducted the informed consent discussion before any 
investigation-specific procedures are performed, including screening procedures. A copy of 
the Subject Information and the signed ICF will be provided to the subject. 
Documentation of the discussion and the date of informed consent must be recorded in the 
source documentation and in the Case Report Form (CRF). The signed Subject Information 
sheet and ICF should be filed by the Investigator for possible future audits and/or inspections.  
The final version of the Subject Information sheet and ICF is submitted to the IEC (EPM) and 
must not be changed without permission from SBH and EPM. 

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5.4 
Subject Information Card  
The subject will be provided with a Subject Information Card. The card will at least include 
the following information:  
• That he/she is participating in a clinical study 
• Subject study ID  
• That he/she is treated with the Investigational Medicinal Product (IMP) 
• The name and phone number of the Investigator  
• Name and address of the Sponsor 
5.5 
Subject data protection 
The Investigator must file a Subject Identification List which includes sufficient information 
to link records, i.e. the CRF and clinical records. This list should be preserved for possible 
future inspections/audits but should not be made available to SBH or representatives except 
for monitoring, inspection or auditing purposes. 
The ICF includes information that data will be recorded, collected and processed. In 
accordance with the European General Data Protection Regulation (2016/679), the data will 
not identify any persons taking part in the study.  
The potential study subject must be informed that by signing the ICF he/she approves that 
authorized representatives from SBH, the IEC (EPM) and Competent Authority (LV) have 
direct access to his medical records for verification of clinical study procedures.  
The subject has the right to request access to his personal data and the right to request 
rectification of any data that is not correct and/or complete.  
6 
INVESTIGATOR(S) AND STUDY ADMINISTRATIVE STRUCTURE 
 
This clinical study is sponsored by the Swedish company Scandinavian Biopharma Holding 
AB. The study will be conducted at Clinical Trial Center’s (CTC’s) facilities at Sahlgrenska 
Hospital in Gothenburg, Sweden.   
Immunological analyses will be performed at the Department for Microbiology and 
Immunology at the University of Gothenburg. 
Clinical Monitoring, Data Management, biostatistics and SUSAR reporting will be provided 
by Scandinavian CRO. Key members of the Sponsor, CTC, University of Gothenburg and 
CRO project teams and sub-contractors are presented below. 
 
 

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Sponsor 
Sponsor’s Signatory 
Scandinavian Biopharma Holding AB 
Industrivägen 1 
SE-171 48 Solna, Sweden 
Per Arne Parment, Medical Advisor  
Phone: +46 (0)70 644 47 71 
 
E-mail: @scandinavianbiopharma.se 
 
 
 
 
 
 
 
 
 
Academic co-investigator: 
Dept. of Microbiology and 
Immunology 
Sahlgrenska Academy 
University of Gothenburg 
Box 435, 405 30 Gothenburg 
 
 
Anna Lundgren, associate professor, senior lecturer 
Phone: +46 (0)70-2264574 
E-mail: anna.lundgren@microbio.gu.se 
 
 
Clinical Trial Center (CTC) 
Gröna Stråket 12 
SE-413 46 Göteborg, Sweden 
 
 
Principal Investigator 
Dan Curiac, MD 
Phone: +46 (0)700-823351 
Email: dan.curiac@vgregion.se  
 
  
 
 
 
 
 
Clinical Monitoring, 
Pharmacovigilance/SUSAR 
reporting, Data Management  
and Statistics 
Scandinavian CRO 
Skolgatan 8 
SE-750 15 Uppsala, Sweden 

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Laboratory  
 
Human Immunology Laboratory  
Dept. of Microbiology and Immunology 
Sahlgrenska Academy 
University of Gothenburg 
Box 435, 405 30 Gothenburg, Sweden 
Contact: Anna Lundgren 
Tel: +46 (0)70-2264574 
e-mail: anna.lundgren@microbio.gu.se 
 
 
Signatures required should be provided in appendix 12.1. 
7 
INTRODUCTION 
7.1 
Project background 
Enterotoxigenic Escherichia coli (ETEC) bacteria cause considerable physical suffering and 
malnutrition due to repeated bouts of illness in children in low- and middle-income countries 
(LMIC). Diarrhoea is the second-leading cause of death among children <5 years of age 
worldwide. The actual mortality numbers are difficult to estimate; global annual incidences of 
nearly 1.7 billion cases of childhood diarrhoeal disease with approximately 500 000 – 700 000 
deaths in children <5 years of age have been presented [1, 2]. The relative importance of 
ETEC versus other diarrhoeal pathogens for the total burden of diarrhoeal disease is debated 
[2, 3]. However, the significant morbidity caused by ETEC in young children is undisputed, 
and it is also clear that repeated ETEC episodes have significant impact on growth and mental 
development [4-6]. WHO’s PDVAC  (Product Development for Vaccines committee) has 
2020 expressed that an ETEC vaccine is a high priority for LMIC  [4, 5]. A vaccine against 
ETEC would have a significant impact on travelers as well. Approx. 80 million travelers visit 
subtropical/tropical countries annually [7]. Traveling to LMIC involves a risk of travel-related 
diseases and at least 35 million travelers per year are affected by travelers’ diarrhea (TD) [8, 
9]. ETEC is the major cause of TD, responsible for approximately 15 million annual cases [8, 
9].   
ETEC spreads through the faecal-oral route. Infection occurs when a person ingests food or 
liquid contaminated with ETEC bacteria. Human wastes (e.g. faeces) are the ultimate source 
of ETEC contamination. ETEC can adhere to the mucosal epithelial cells lining the small 
intestine using colonisation factors (CF, also called coli surface antigens, CS) [6]. Once 
attached to the mucosal surface, the bacterium starts to produce a heat labile (LT) and/or a 

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heat stable toxin (ST) triggering diarrhoea. The LT toxin is highly homologous to the cholera 
toxin (CT) and cause diarrhea via a highly similar mechanisms [6]. 
ETVAX® is an oral inactivated vaccine against ETEC containing four different inactivated E. 
coli strains overexpressing the most prevalent ETEC CFs, i.e. CFA/I, CS3, CS5 and CS6, a 
recombinant protein LCTBA and the adjuvant double mutant LT (dmLT) [6, 10]. ETVAX® is 
currently in late phase development and has been studied in several clinical phase 1 and 2b 
studies. Successfully completed clinical studies have shown that the vaccine is safe and 
immunogenic, giving rise to mucosal IgA responses to all vaccine CFs and to LTB, in 
children living in endemic areas as well as in Western healthy adults [11-15]. Our previous 
results also demonstrate that the vaccine gives rise to memory B cell responses that can be 
detected at least until 2 years after vaccination and that such long-term immunity may be 
reflected by the circulation of activated follicular helper T cells early after immunization [13, 
16]. 
The product formulation of ETVAX® used so far in clinical trials is a liquid suspension of 
inactivated bacteria and LCTBA in one vial, freeze-dried dmLT adjuvant in a second vial, and 
effervescent buffer granules in a separate sachet. Prior to administration, the buffer is 
dissolved in 150 ml tap water, followed by the addition of the content of the vaccine vial 
(inactivated bacteria mixed with LCTBA) and reconstituted and diluted adjuvant dmLT from 
the second vial. As a part of manufacturing development process, a method to spray-dry 
dmLT together with LCTBA protein has been established. In the new, partially dried 
formulation, the spray-dried dmLT and LCTBA are mixed with the buffer granules and 
stabilizing excipients in a sachet. In preparation of the partially dried formulation the content 
of the buffer sachet (buffer, dmLT, and LCTBA) are dissolved in 150 ml tap water, followed 
by the addition of the vaccine vial content (inactivated bacteria). The new product formulation 
is thus very simple to prepare and suitable for use in the field. 
A two-armed, non-inferiority, immunogenicity and safety study comparing the two 
formulations will be conducted in 280 adult volunteers at Clinical Trial Center (CTC) located 
at the Sahlgrenska University Hospital in Gothenburg, Sweden.  
 
Due to difficulties to get a reliable measurement of immune responses to the CFs in serum of 
non-primed (i.e. previously not immunized nor infected) individuals, only the immune 
response rates induced by the LCTBA part of the vaccine to the binding subunit of LT (LTB) 
will be measured. For oral vaccines, the responses are evoked in the gut and only some 
immune responses, e.g. against LTB for ETVAX®, are detectable in serum of immunised 
individuals [6, 12].  
 
7.2 
Investigational Medicinal Product (IMP) 
7.2.1  Vaccine development  

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Protection against ETEC is most likely mainly provided by antibodies against the different CFs 
and LT produced locally in the gut. Studies in experimental animals and human subjects 
strongly support that the CFs are key protective antigens against ETEC expressing homologous 
CFs. Thus, studies in a birth cohort in Bangladesh have shown that re-infections with ETEC 
expressing homologous CFs are very rare, at variance with re-infections with ETEC expressing 
heterologous CFs [17], supporting the importance of CF immunity in an ETEC vaccine. The 
current ETVAX® formulation includes four of the most prevalent CFs, i.e. CFA/I, CS3, CS5, 
CS6. 
Clinical trials both in travellers and in endemic countries have also shown that cholera toxin B 
subunit (CTB), which is highly homologous to LTB, may provide significant protection against 
disease caused by LT expressing ETEC. However, it has been suggested that an LT-like toxoid 
may provide stronger protection against LT ETEC, at least in young, immunologically naïve 
children in comparison to CTB. Thus, it has been previously shown that human subjects 
challenged with LT producing ETEC as well as Bangladeshi patients convalescing from E. coli 
LT diarrhoea responded with significantly stronger local and serum antibody responses to LT 
than to CT [18, 19]. ETVAX® includes LCTBA, a hybrid between LTB and CTB [20], which 
has been shown to induce strong neutralizing LTB-specific antibody responses in both serum 
and mucosal samples in several preclinical and clinical studies [10-12, 14, 15]. 
 
Since ETEC infections are confined to the mucosal surfaces in the gut, immune protection is 
most likely predominantly provided by locally produced secretory IgA (SIgA) antibodies 
against major protective antigens, i.e., CF and LT antigens [6]. However, strong LTB- 
specific IgA and IgG responses can also be detected in serum after vaccination with ETVAX® 
including LCTBA, and these responses correlate significantly with LTB-specific IgA 
responses measured in samples reflecting mucosal immunity, such as secretions from 
antibody secreting cells migrating to the intestinal mucosa [11, 12, 14]. Serum from 
immunized subjects also have strong LT toxin neutralizing capacity, and the functional 
antibody activity correlate well with antibody titers measured by ELISA [12]. Levels of LTB-
specific antibodies measured in serum after ETVAX® vaccination thus reflect both mucosal 
immune responses and antibody function. 
7.2.2 Mucosal Adjuvants 
The LT toxin has been shown to have inherent mucosal adjuvant properties for co-
administered antigens and thus has potential as a mucosal adjuvant for different co-
administered vaccines [21]. Several animal and human studies have evaluated LT as a 
mucosal adjuvant for co-administration with inactivated whole cell or purified subunit 
antigens derived from a wide range of bacterial and viral pathogens [21]. Despite the 
significant potential of LT as a mucosal adjuvant, oral LT has considerable enterotoxicity for 
humans, thereby limiting its use as an adjuvant [21, 22]. Therefore, efforts have been made to 
construct LT molecules with decreased enterotoxicity, but with undiminished adjuvant 
properties. As described in more detail below, single mutant LT (mLT) was developed by Dr. 
John Clements and colleagues at Tulane University [23], but a high oral dose (100 µg) was 
still found to be reactogenic when given alone. Also, despite impressive adjuvant activity, 
mLT retained some level of gastrointestinal reactogenicity in Phase I trials when combined 

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with bacterial vaccine antigens [24].  Subsequently, double mutant LT (dmLT) was developed 
by the investigative team at Tulane as a 2nd generation mucosal adjuvant with further reduced 
enterotoxicity [21, 25]; dmLT has been proven to be safe as a stand-alone  vaccine in a phase 
I single dose escalation study (Protocol 13-0013; clinicaltrials.gov NCT02531685) [26], and 
together with an attenuated ETEC vaccine in a 25 µg/dose phase 2b study (clinicaltrials.gov,  
NCT01060748)[27]. 
7.2.3 Development and testing of double mutant LT (dmLT) 
E. coli JM83(pLC403) codes for production of the genetically modified form of E. coli  LT in 
which the arginine at amino acid position 192 has been replaced with glycine and the leucine 
at amino acid position 211 has been replaced with alanine in the A subunit of LT [25]. These 
two amino acid substitutions are essential in known and putative proteolytic cleavage sites in 
the LT protein that are considered to be critical for activation of the secreted toxin molecules. 
The protein coded by this strain was designated LT(R192G/L211A), i.e. dmLT, and has been 
extensively evaluated for residual enterotoxicity in vitro and in vivo studies, and in pre-clinical 
animal studies for its ability to function as an adjuvant for co-administered antigens [21, 25]. 
Two oral doses of dmLT in rats were also found to lack local and systemic toxicity in a GLP 
toxicology study conducted by PATH and Division of Microbiology and Infectious Diseases 
(DMID), National Institutes of Health (NIH) [28]. Figure 1 shown below highlights the lack of 
residual enterotoxicity in the dmLT protein compared to native LT (dmLT is at least 50-fold 
less toxic) and mLT (dmLT is at least 10-fold less toxic) in the patent mouse assay. 
 
 
 

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Figure 1: Comparative enterotoxicity of native LT, mLT, and dmLT (Lot No. 1575) in the 
Patent Mouse Model. 
 
Gut-to-carcass ratios of 0.9 or above are considered indicative of enterotoxic activity. 
7.2.4 Non-clinical safety 
 Immunogenicity and safety in mice of ETEC vaccine with and without dmLT 
ETEC vaccine given alone or together with the dmLT adjuvant was studied in mice after oral 
immunizations for its capacity to induce intestinal and serum antibody responses to the different 
vaccine components. Small intestine or fecal IgA responses showed 2 to 50-fold increase in 
specific antibodies to the different vaccine components following immunization [10]. Immune 
responses were further increased when the vaccine was given in combination with the oral 
adjuvant (Figure 2). 
 
A significant IgA immune response was also obtained in serum to each of the vaccine 
components and this was even further pronounced for IgG + IgM type antibodies. In most cases, 
the serum immune responses were further enhanced in the presence of the adjuvant [10]. In 
addition to demonstrating immune enhancement, this study also showed that when adding 
different dosages of dmLT when administered with vaccine, the adjuvant effect of dmLT was 
especially strong for lower vaccine dosages [10]. This suggests the potential for vaccine dose 
sparing when an effective adjuvant is added, which could be especially valuable for use with 
ETVAX® in younger age groups in which fractional doses of vaccine may be needed [29]. 
 
 
 

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Figure 2.  Intestinal IgA antibody responses in mice following oral immunization with ETEC 
vaccine (V) with and without dmLT adjuvant. 
 
 
 cGLP Toxicology and Immunology Evaluation of ETEC vaccine with and without 
dmLT 
A repeated dose cGLP toxicity study was conducted in C57BL/6J mice, in which the main 
objective was to investigate whether ETEC vaccine produced toxic reactions after repeated per-
oral administration in the animals. ETEC vaccine was administered in two different doses, 5 x 
the human clinical dose and 25 x the human clinical dose, and administered with or without the 
addition of the dmLT adjuvant (lot 1575). Phosphate buffer solution (PBS) was used as the 
control item. 
In comparison to PBS treated animals the vaccines in high doses affected the general health 
slightly but no major pathological, clinical chemistry or haematological observations were 
found. Acute gastritis was seen in some animals that were most probably the results of previous 
and passing injury on a microscopic level. The gastritis was deemed to be transient and did not 
raise any safety concerns by the Swedish Medical Product Agency. Furthermore, symptoms 
consistent with acute gastritis have not been observed in any of the Phase I/II trials in which 
dmLT was co-administered with ETEC vaccine or when combined with a live-attenuated ETEC 
vaccine in doses of up to 25 µg. A strong anti-LCTBA response in serum was detected across 
all dosing groups in the mouse cGLP study while the serum anti-CFA/I response was somewhat 
weaker and dependent on the dosing group.  
 
In conclusion, the vaccine was shown to be safe and immunogenic when tested in mice; 
adjuvantation with dmLT increased the immune response without altering the safety profile. 
 
7.2.5 Clinical experience 
 Vaccine safety   
ETVAX® has been shown to have an excellent safety profile. In the OEV-121 study (Double-
blind, Randomised, Placebo Controlled Phase I study to Evaluate the Safety and 
Immunogenicity of an Oral inactivated Tetravalent ETEC Vaccine Alone and in Combination 
with dmLT Adjuvant in Healthy Adult Volunteers, EudraCT number 2011-003228-11) in 
adult Swedish volunteers, administration of two oral doses of ETEC vaccine was shown to be 
safe irrespective of given alone or in combination with either 10 or 25 µg dmLT, respectively, 
with no differences in adverse events (AEs) in vaccinees and placebo recipients [12]. In the 
OEV-121A study, a single dose of ETEC vaccine alone, without adjuvant, was administered 
to 57 subjects that had received either two doses of ETEC vaccine alone or ETEC vaccine 
+10 µg dmLT 1-2 years previously or had never been vaccinated against ETEC before (A 
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Alone and in Combination with dmLT Adjuvant to Induce Immunological Memory to Toxin 
and Colonization Factor Antigens in the Vaccine, EudraCT number 2013-003693-28) [13]. 
Few AEs were recorded in the subjects participating in the study and there were no significant 
differences between subjects receiving a first or third vaccine dose. 
  
ETVAX® safety was also documented in a recent age-descending study in Bangladesh (A 
Randomised, Double- blind, Placebo-controlled, Dose-Escalation Study Evaluating the 
Safety, Tolerability, and Immunogenicity of an Oral Inactivated ETEC Vaccine Alone and 
Together With dmLT Adjuvant in age-descending groups in Bangladesh, ClinicalTrials.gov 
number NCT02531802) encompassing 495 subjects (OEV-122 study) [14, 15]. In this study, 
immunisations started with full vaccine dose with or without 10 µg dmLT in adults, followed 
by administration of different fractional vaccine and dmLT doses in children 2-5 years, 1-2 
years and finally infants aged 6-12 months. In all age groups, the highest safe dose was 
identified and then given with different doses of dmLT. There was no progression to younger 
age groups before safety was assessed in the older groups. The vaccine was safe in the adults 
with no significant differences in AEs between vaccinees and placebo recipients [14].  
  
In the children, no clear safety signals were identified although vomiting occurred in children 
when higher doses were tested [15].  However, vomiting was decreasingly observed with 
decreasing dose and increasing age, similar to what had been observed in studies with a first- 
generation whole cell inactivated ETEC vaccine [30]. Furthermore, dmLT did not appear to 
contribute to tolerability issues. When vomiting occurred, the reaction was transient, self-
limited and generally mild, and mitigated by selection of lower doses, as was the objective of 
the dose escalation phases (i.e. identification of highest tolerated dose) [15].  
 
A phase 2b study of Finnish adult travellers to Benin (A randomized, placebo-controlled 
phase IIb (OEV-123) study to evaluate safety, immunogenicity, diagnostic methodology, and 
estimate vaccine efficacy of an oral enterotoxigenic Escherichia coli (ETEC) Vaccine 
(ETVAX®) for prevention of clinically significant ETEC diarrhea in healthy adult travelers 
visiting West Africa, EudraCT No.: 2016-002690-35) encompassing 749 randomized subjects 
(ITT set) whereof 374 received vaccine and 375 received placebo was recently finalized. In 
this study the vaccine was shown to have an excellent safety profile and no statistical 
difference regarding safety was found between vaccine and placebo recipients. 
 Vaccine immunogenicity 
The immunogenicity of ETVAX® has previously been documented in Swedish adults in the 
OEV-121 study. The results showed that a full adult dose of ETEC vaccine administered 
together with 10 µg dmLT adjuvant resulted in 93% IgA and/or IgG seroconversion (≥2-fold 
increase) against LTB in samples collected 7 days after the second dose [12]. The vaccine 
also induced strong IgA responses against both LTB and CFs in fecal samples and antibodies 
in lymphocyte supernatants (ALS) specimens, reflecting mucosal immune responses. Serum 
from immunized subjects also had strong LT toxin neutralization activity [12]. Serum 

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antibody responses against LTB also correlated significantly with LTB-specific mucosal 
responses measured in ALS specimens. dmLT adjuvant significantly enhanced ALS IgA 
responses to CS6, which is the antigen present in the lowest amount in the ETVAX vaccine, 
supporting the dose-sparing effect of dmLT previously observed in mice [10, 12].  
 
In the OEV-122 study 100% of the adult Bangladeshi subjects receiving the vaccine alone or 
with 10 µg dmLT responded with an at last 2-fold response in anti-LTB IgA antibodies in 
both serum and ALS specimens [14].  ALS IgA responses to CFs were also observed in 100% 
of subjects. The high response is probably reflecting that in Bangladesh a high percentage of 
the population has experienced ETEC infections prior to vaccination.  
Mucosal (ALS) as well as plasma IgA responses in Bangladeshi children 2-5 years were 
comparable to those in adults both in magnitude and frequencies [15]. Responses in children 
1-2 years were also good with ALS IgA responses to all five antigens in a majority of 
vaccinees and somewhat lower frequencies in plasma. With regard to immune responses in 
the 6-11 months old infants immune responses to the CFs were lower than in the older age 
groups. Furthermore, response rates in the infant placebo recipients were considerably higher 
than in older children, probably due to very high frequencies of asymptomatic ETEC 
infections in this age group as previously shown in a birth cohort study in Bangladesh [17]. In 
spite of this, mucosal immune responses (ALS and/or fecal SIgA) were significantly higher 
against all vaccine antigens in the infant vaccinees than in the placebo recipients [15].  
 
In the OEV-123 study 81% of the Finnish vaccine recipients responded with at least a 2-fold 
increase in anti-LTB IgA serum antibodies after vaccination with ETEC vaccine + 10 µg 
dmLT.  
 
7.3 
Risk/benefit assessment 
7.3.1 Benefits 
There are no major benefits for subjects participating in the study. However, subjects will 
receive a free health check-up and immunization may provide protection against some of the 
most common types of ETEC and against diarrhoea caused by Vibrio cholerae bacteria, which 
can be beneficial during travel to ETEC and cholera endemic countries.  
7.3.2 Risks 
Based on experiences from previous studies with ETVAX®, immunization is expected to give 
rise to only a low frequency of mild and transient symptoms, including a few loose stools, 
mild nausea/vomiting, and abdominal pain or bloating. In the previous trial including 129 
Swedish adults there were no significant differences in either frequency or severity of AEs 
between the study groups receiving ETEC vaccine with or without dmLT and the placebo 
group receiving bicarbonate buffer alone [12]. The excellent safety profile of the vaccine was 
supported by very few recorded AEs in Bangladeshi adults vaccinated with ETEC vaccine 

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with or without 10 µg dmLT, with no significant differences between subjects in the vaccine 
(n=30) or placebo groups (n=15) [14].In the recent clinical trial in Finland (OEV-123), no 
significant differences in AEs were recorded between subjects receiving ETVAX® (n=374) 
and placebo recipients (n=375).  
Since the new partially dried vaccine formulation contains the same vaccine components as 
the previously tested wet vaccine formulation, the risk of inducing different vaccine 
associated AEs in subjects receiving the new formulation is deemed to be low.  
Blood sampling of study participants may cause some pain at the time of sample collection 
and may result in the development of a bruise but has no other risks.  
The risk for individuals participating in this study is therefore assessed to be very small.  
 
 Risk assessment for the OEV-125 trial in relation to the COVID-19 epidemic  
The risk that the COVID-19 epidemic will influence the possibility to perform the trial 
according to plan, or influence the results of the trial, is continuously assessed by the study 
team.  Since the majority of the subjects are planned to be enrolled in Q4 2021-Q2 2022, 
when a majority of the adult Swedish population is predicted to have been vaccinated against 
COVID-19, the risk that the epidemic will have major influence on the study is deemed to be 
relatively small.  
However, the epidemic situation and changes in the national and local COVID-19 vaccination 
schedule will be closely monitored.  
Some COVID-19 related mitigation measures have already been implemented into the Study 
Protocol, e.g allowing a longer span between the dosing visits under certain circumstances, 
see section 9 for details. Detailed information about COVID-19 infections and vaccinations is 
also collected from all participants, primarily to support evaluation of exploratory endpoints, 
which include comparisons of immune responses to ETEC and other infections and 
vaccinations, including COVID-19. In addition, a risk assessment, including mitigations, 
taking into account the local COVID-19 situation and predictions provided by the authorities 
will be performed and updated within 60 days of trial initiation. This assessment will cover, 
but should not be limited to, the following aspects; participant and site staff safety, IMP and 
laboratory material logistics as well as monitoring. A separate Monitoring plan will also be 
provided, in which monitoring details will be further described. 
The risk-assessment will be documented in the Trial Master File (TMF) and will be updated if 
the situation changes after study start. 
8 
STUDY OBJECTIVES AND ENDPOINTS 
8.1 
Primary objective 
 
The aim of this study is to demonstrate non-inferiority, in terms of immunogenicity, between 
the “wet formulation” and the newly developed partially dried formulation of selected 

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components of ETVAX®. 
 
8.1.1 Primary endpoint(s) 
The primary endpoint to be measured for each patient in the study is response (yes/no) to a 
vaccine. A vaccine responder will be defined by a ≥2-fold increase in IgA and/or IgG 
antibody levels against LTB in serum between post- compared to pre-immunization samples. 
The response rates (seroconversion rates) of IgA and/or IgG anti-LTB antibodies in serum 
will be derived and compared between the two treatment groups.  
8.2 
Secondary objectives  
To evaluate the safety and tolerability of the new formulation of the vaccine. 
8.2.1 Secondary endpoints 
 
Occurrence of solicited symptoms for six days after each vaccination (day of vaccination and 
five subsequent days).  
8.3 
Exploratory Objective(s) 
 
To evaluate if ETVAX® vaccination induces circulating antigen specific memory B- and/or T 
cells that can be assessed using recently established laboratory assays. 
 
8.3.1 Exploratory Endpoint(s) 
Levels of IgA and IgG antibodies specific for LTB and CFs in cultures of peripheral blood 
mononuclear cells (PBMCs) isolated before and after vaccination stimulated with substances 
that activate memory B cells and transform them into antibody secreting plasmablasts.  
Frequencies of CD4+ T helper cells expressing activation markers and/or cytokines in blood 
samples collected before and after vaccination. 
Levels of cytokines in culture supernatants of samples collected before and after vaccination. 
 
 

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9 
INVESTIGATIONAL PLAN 
 
This is a prospective double-blind, randomized, parallel-group study with the aim to 
demonstrate non-inferiority, in terms of immunogenicity, between the wet formulation and a 
newly developed partially dried formulation of selected components of ETVAX®. 
The study will be conducted at one site; CTC in Gothenburg, Sweden. 
 
A total of 280 healthy volunteers is planned to be randomized into the study. 
Assuming a 10% drop-out rate, we expect 252 healthy volunteers to complete the study. 
 
 
Overall description of study design  
Consenting subjects will be screened for eligibility, according to study-specific 
inclusion/exclusion criteria within 30 days prior to the first administration of IMP (Visit 1; 
Screening visit). Eligible subjects will be randomized on Day 1 (Visit 2) to receive either of 
the two oral formulations of ETVAX® (1:1) and consecutively included the study.  
 
Screening visit and Day 1 (Visit 2) may be performed on the same day, provided that the 
Study protocol is adhered to in all aspects, including but not limited to the Subject 
Information and Consent procedures outlined in section 5.3. 
 
The treatment allocation (Wet formulation/Partially dried formulation) will be double-
blinded, i.e. it will not be disclosed to the subjects, site staff, laboratory staff or Sponsor. 
 
Study subjects will receive two oral doses, two weeks apart (Day 1/Visit 2 and Day 15 /Visit 
3). Preferably, subjects should receive their second dose on Day 15 (±2). However, if a 
subject is unable to come for the visit on day 15±2 due to e.g. illness or rules regarding 
quarantine due to the COVID-19 epidemic, the second vaccine dose can be administered up to 
28 days after the first dose.  
Dosing will occur at the clinic. After each dose, subjects will remain at the clinic for 
observation for 15 min before leaving. 
At each dosing visit (Day 1 and Day 15) subjects will receive adverse event and medication 
diaries to be completed for six days after each vaccination (i.e. day of vaccination and five 
subsequent days). 
 
A follow-up visit will be performed 7 days (acceptable range: 6-10 days) after the last 
(second) dose in all study subjects (Visit 4).  
Blood samples will be collected on visit 2 and 4. 
 
For the exploratory analyses, subgroups of subjects in the main study (n=20-40, evenly 
distributed between the two treatment arms) will participate in extra follow-up visits 5± 1 
(Visit 3b, n=40), 30± 7 (Visit 4b, n=20) and 75-125 (Visit 4c, n=20) days after the second 

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dose in addition to the regular visits (Visit 1-4) in the main study. Blood samples will be 
collected on all exploratory visits. The extra visits and analyses for exploratory analyses may 
continue after the main part of the study has been completed and the database locked.  
For subjects participating in main study, the total volume of blood drawn will not exceed 40 
ml. For subjects also participating in the exploratory analyses, the total volume will not 
exceed 250 ml. 
 
The timing and frequency of study visits and assessments in each study part are presented in 
the schedules of events in Table 9.1 and 9.2. 
Table 9.1 Schedule of events – Main Study assessments 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
aScreening visit and Day 1 (Visit 2) may be performed on the same day. 
bIf a subject is unable to come for the visit on day 15±2 due to e.g. illness or rules regarding quarantine due to the COVID-19 
epidemic, the second vaccine dose can be administered up to 28 days after the first dose. 
 cUntil subject receives IMP    
 
Visit 1 
Screening 
Visit 2 
Dose 1 
Visit 3 
Dose 2 
Visit 4 
 Follow-up 
Day/assessments 
 
Day -30 
to Day 1a 
 
Day 1a 
 
Day 15 b 
(±2 days) 
7 days (6-10 
days) after  
Dose 2 
Informed consent 
X 
 
 
 
Demographics and relevant 
medical/surgical history 
X 
 
 
 
Inclusion/exclusion criteria 
X 
X 
X 
 
Physical examination 
X 
 
 
X 
Vital signs  
(Blood pressure and Heart rate) 
X 
 
 
X 
Vital signs (Body temperature) 
X 
X 
 X 
X 
Pregnancy Test (Urine) 
X 
X 
X 
 
Randomization 
 
X 
 
 
Dosing  
 
X 
X 
 
Blood sampling for immunology 
(primary endpoint) 
 
X 
 
X 
Subject diary distribution  
 
X 
X 
 
Subject diary collection  
 
 
X 
X 
Baseline events 
Xc 
Xc 
 
 
Adverse Event and  
Serious Adverse Event reporting 
 
X 
X 
X 
Prior and concomitant medications 
X 
X 
X 
X 

 
 
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Table 9.2  Schedule of events – Main and Exploratory Study assessments 
aScreening visit and Day 1 (Visit 2) may be performed on the same day. 
 
bIf a subject is unable to come for the visit on day 15±2 due to e.g. illness or rules regarding quarantine due to the COVID-19 
epidemic, the second vaccine dose can be administered up to 28 days after the first dose. 
 
cVisit 4 should be performed on a separate day compared to Visit 3b. 
 E.g. if Visit 3b is performed on Day 6 after the second dose, then Visit 4 should be performed on Day 7-10. 
dExploratory analyses at Visits 2, 3b and 4 will be performed in 40 subjects. 
eExploratory analyses at Visits 4b and 4c will be performed in 20 subjects. 
fUntil subject receives IMP    
 
Visit 1 
Screening 
Visit 2 
Dose 1 
Visit 3 
Dose 2 
Visit 3b 
Exploratory 
Visit 4c 
Follow-up 
Visit 4b 
Exploratory 
Visit 4c 
Exploratory 
Day/assessments 
 
Day -30 to 
Day 1a 
 
Day 1a 
 
Day 15b 
(±2 days) 
5 ±1 day 
after Dose 2 
7 day (6-10 
days) after 
Dose 2 
30 ±7 days 
after Dose 2 
75-125 days 
after Dose 2 
Informed consent 
X 
 
 
 
 
 
 
Demographics and 
relevant 
medical/surgical history 
X 
 
 
 
 
 
 
Inclusion/exclusion 
criteria 
X 
X 
X 
 
 
 
 
Physical examination 
X 
 
 
 
X 
 
 
Vital signs (Blood 
pressure and Heart rate) 
X 
 
 
 
X 
 
 
Vital signs (Body 
temperature) 
X 
X 
X 
 
X 
 
 
Pregnancy Test (Urine) 
X 
X 
X 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
Dosing  
 
X 
X 
 
 
 
 
Blood sampling for 
immunology (primary 
endpoint) 
 
X 
 
 
X 
 
 
Blood sampling for 
exploratory 
immunological analyses 
 
Xd 
 
Xd 
Xd 
Xe 
Xe 
Subject diary 
distribution  
 
X 
X 
 
 
 
 
Subject diary collection  
 
 
X 
 
X 
 
 
Baseline events 
Xf 
Xf 
 
 
 
 
 
Adverse Event and 
Serious Adverse Event 
reporting 
 
X 
X 
 
X 
 
 
Prior and concomitant 
medications 
X 
X 
X 
 
X 
 
 

 
 
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9.1.1  Dose levels 
There is only one dose level in this study; full dose (1/1) of ETVAX®; a tetravalent ETEC 
vaccine including LCTBA and 10 µg double mutant LT (dmLT) and effervescent powder for oral 
solution. 
 
9.1.2  Stopping rules/Discontinuation Criteria 
Scandinavian Biopharma AB has the right to close the study at any time, although this should 
occur only after consulting involved parties. The IEC (EPM) and the Competent Authority (LV) 
must be informed. Events that may trigger premature termination of the study include, but are 
not limited to: new toxicity finding, results of any interim analysis, completed accrual and 
follow-up of participants, non-compliance with the protocol, change in development plans for 
the study vaccine, slow recruitment and poor quality data.  
9.1.3 Internal Safety Review Committee (iSRC) 
There will be no iSRC or Data Monitoring Committee for this study 
9.2 
Rationale for study design, doses and control group 
The new partially dried vaccine formulation is very simple to prepare and suitable for use in the 
field. In contrast, the old wet vaccine formulation needs to be prepared in several different steps, 
by trained nurses or pharmacists, and cannot be used in large Phase 3 studies. To ensure that the 
new partially dried formulation, which includes LCTBA toxoid and dmLT in the buffer powder, 
instead of addition of diluted dmLT from a lyophilized preparation to the vaccine, is not inferior 
to the old wet formulation of the vaccine, a two-armed, non-inferiority, immunogenicity and 
safety study comparing the two formulations will be performed in healthy adult Swedish 
volunteers. This study is important in order to ensure that the new vaccine formulation has a 
comparable immunogenicity profile as the old vaccine, before evaluating the new vaccine 
formulation in large Phase 3 studies in ETEC endemic areas. 
 
The study will include healthy Swedish volunteers 18-50 years of age and the inclusion and 
exclusion criteria will be very similar to previous studies performed at the same study site [11, 
12, 31], in order to generate immunogenicity and safety results that are comparable previous 
studies. The travel history of subjects will be recorded, and subjects likely to have been exposed 
to ETEC infection during travel will be excluded based on the criteria used in previous studies, 
in order to minimize variability in in immune responses as a result of on natural immunological 
priming.  
  
Since the LCTBA toxoid is formulated in a new way in the new vaccine, it is particularly 
important to confirm the immunogenicity of this component. For oral vaccines, the responses are 

 
 
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evoked in the gut and only some immune responses, e.g. against LTB for ETVAX®, are 
detectable in serum of immunised individuals [6, 12]. Due to difficulties to get a reliable 
measurement of immune responses to the CF antigens in serum of non-primed (i.e. previously 
not immunized nor infected) individuals, only the immune response rates raised by the LCTBA 
part of the vaccine to LTB will be measured in this study. Previous studies have shown that 
serum antibodies induced by ETVAX® have strong LT toxin neutralization activity [12]. 
Furthermore, the serum responses correlate significantly with LT-specific IgA responses 
measured in samples reflecting mucosal immunity, such as secretions from antibody secreting 
cells migrating to the intestinal mucosa [12]. Thus, measurement of serum antibody responses to 
LTB is a good proxy measurement of toxin neutralizing mucosal antibody responses. LTB-
specific antibody responses will be evaluated using assays employed in previous ETEC vaccine 
trials at the same laboratory (OEV-120, OEV-121 and OEV-121A CSR [11-13]), using a 2-fold 
increase in antibody responses over baseline as primary endpoint, consistent with the general 
praxis in the ETEC and mucosal vaccine field. 
We have developed a mouse assay to show the adjuvanting capacity of dmLT for release and 
stability studies (K2038) as well as studied the vaccine with the addition of dmLT in a mouse 
assay [10]. However, since the spray-dried intermediate and, to an even greater extent, the final 
formulation contains excipients, buffer components and stabilizers, which makes both the 
intermediate and the product insoluble at the concentrations and volumes that are required for 
dosing mice by gastric gavage, a non-inferiority study of the new vaccine formulation in mice is 
not feasible. 
The vaccine will be administered orally twice to each subject at a target interval of 14 days. If 
the subject is unable to come for the third study visit on day 15 due to eg illness or rules 
regarding quarantine due to the COVID-19 epidemic, the second vaccine dose can be 
administered up to 28 days after the first dose. A prolonged interval between vaccine doses did 
not affect the serum responses to CTB component of the cholera vaccine Dukoral, which is 
highly homologous to LCTBA, when tested in Swedish volunteers [32]. Thus, serum CTB-
specific IgA and IgA responses were comparable in both magnitude and frequency in subjects 
receiving Dukoral 14 days or 28-48 days apart, motivating the extended interval between doses 
in this ETEC vaccine trial compared to previous trials [32]. 
  
Many non-inferiority studies of parenteral vaccines use a 10% non-inferiority margin, but larger 
margins are also relatively common [33]. The 15% non-inferiority margin used in this non-
inferiority study is motivated by the higher individual variability in vaccine responses and lower 
responder frequencies often observed with mucosal compared to parenteral vaccines. As an 
example, a comparison an oral inactivated Shigella flexneri 2 a vaccine gave a maximum serum 
response of 70% [34] as compared to a S. flexneri 2a conjugate given parentally, where 92-100% 
of the vaccinees responded in serum [35]. The response rate for the whole cell components of 
ETVAX® in serum is quite low (3-19% in a Western adult population respond after two doses of 
vaccine), whereas the mucosal immune responses are much stronger (71-97%) [12]. The 
response against the LCTBA protein is quite different resulting in a 79-90% seroconversion after 
two doses (OEV-123 CSR, [12]).  Still, this is substantially lower than what is seen with many 
parenteral vaccines. Since no formal correlate of protection exists for ETEC or most other 
mucosal vaccines, the protective efficacy of the new formulation of the ETVAX® vaccine can 
only be supported by data from efficacy studies. If the new vaccine is found to be non-inferior to 

 
 
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the old vaccine in this study, this will support that it is reasonable both from an ethical and 
economical perspective to proceed to test the efficacy of this vaccine formulation in large Phase 
3 trials in ETEC endemic areas. 
9.3 
Selection of study population  
9.3.1 Recruitment 
The clinical part of the recruitment will be conducted by Clinical Trial Center (CTC) located at 
the Sahlgrenska University Hospital in Gothenburg, Sweden. This centre has helped recruiting 
the same category of volunteers, as will be recruited for the non-inferiority bridging study, for all 
previous studies of ETVAX® in Swedish adult volunteers [11, 12, 31].  
Previous experience indicate that the number of recruited subjects should not be a problem, and 
the historical drop-out rate has been low (less than 5%) [12]. The compliance is also expected to 
be excellent, based on previous experience. However, due to the ongoing COVID-19 epidemic, a 
higher drop-out rate is expected (estimation 10%), since even mild symptoms may prevent study 
visits (See section 9.4.3) 
9.3.2 Number of subjects 
A total number of 126 subjects needs to be included in each arm of the study in order to achieve 
90% power to assess the primary objective. Assuming a 10% dropout rate the target number of 
subjects to be recruited per study arm is therefore 140. 
9.3.3 Screening and enrolment log 
The clinic will keep a log of all subjects screened and included. The reason for screen failure 
should be stated for all subjects screened but not included. The reason for withdrawal should be 
stated for all subjects included but not completed. 
 
Subjects who have signed the ICF will be assigned a screening number. Subjects included will 
be assigned a subject number. 
9.3.4 Inclusion criteria 
For inclusion in the study, subjects must fulfil all the following criteria:  
1. Male or female aged 18-50 years, inclusive at the time of signing the informed consent. 
2. Healthy constitution as established by medical history and physical examination. 
3. Willing and able to give written informed consent for participation in the study. 
4. Able to comply with study activities, as judged by the Investigator. 
5. Female Participants:  
• Women of child-bearing potential (for definition see Section 9.3.6): 
o Have to agree to use an acceptable birth control method during participation 
in the investigation (see Section 9.3.6). 

 
 
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o A negative pregnancy test (beta human chorionic gonadotropin dipstick test in 
urine) at Visit 2/Day 1 will be required.  
 
6. Male Participants:  
• Have to agree to remain abstinent (refrain from heterosexual intercourse) or use a 
condom, and agreement to refrain from donating sperm, as defined in Section 9.3.6 
 
In order to receive Dose 2, subjects must fulfil all the criteria above. 
9.3.5 Exclusion criteria 
Subjects must not enter the study if any of the following exclusion criteria are fulfilled: 
1. An acute or chronic medical condition that, in the opinion of the investigator/physician, 
would render ingestion of the investigational products unsafe or would interfere with the 
evaluation of responses. This includes, but is not limited to gastrointestinal diseases, and 
autoimmune diseases. 
2. Current malignancy or history of malignancy during the last five years, based on 
anamnesis.  
3. Gastroenteritis within two weeks prior to vaccination. 
4. Regular use of laxatives, antacids or other agents that lower stomach acidity. 
5. Any planned major surgery during the duration of the study. 
6. After 10 minutes supine rest, any vital signs outside the following ranges: 
- 
Systolic BP > 160 mm Hg 
- 
Diastolic BP > 100 mm Hg 
- 
Heart rate < 40 or >85 beats per minute 
7. Antibiotic therapy within two weeks prior to the vaccination.  
8. Known Hepatitis A, B, C, and/or HIV infection. 
9. Concomitant intake of immunomodulating drugs during the study period or less than 3 
months prior to the first immunization,with the following exceptions: oral anti-histamines 
are not allowed during the study period or less than 3 weeks prior to the first 
immunization. Local anti-histamine treatment is allowed during the study period. 
10. Any other significant medical conditions (e.g. poorly controlled psychiatric condition) 
judged by the Investigator to preclude entry. 
11. Intends to receive any other vaccine during the study period, or within two weeks prior to 
trial vaccination. 
12. Has previously received Dukoral or any type of ETEC or cholera vaccines. 
13. Brought up in ETEC-endemic areas (e.g., urban and rural areas of Central and South 
America, Caribbean, most countries in Asia, Africa, etc.). 
14. Has travelled to ETEC-endemic areas within the last 3 years OR spent > two months in 
ETEC endemic areas during the last 10 years. 
15. Intends to travel to ETEC endemic countries during the study period. 
16. Known or suspected history of drug, chemical or alcohol abuse, as deemed by the 
investigator/physician.  

 
 
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17. History of severe allergy/hypersensitivity or ongoing allergy/hypersensitivity, as judged by 
the Investigator, or history of hypersensitivity to drugs with a similar chemical structure or 
class to ETVAX®. 
18. Participation in any other clinical study that included drug treatment with the last 
administration within the past 3 months prior to administration of study treatment in this 
study. Patients consented and screened but not dosed in previous clinical studies are not 
excluded. 
19. Concomitant participation in any other clinical study.  
20. Females who are pregnant as determined by urine test at inclusion and prior to each 
vaccination. 
21. Females who are nursing.  
22. Unable to participate in all study visits. 
23. Any condition or circumstance which would make the subject unsuitable for participation 
in the study in the opinion of the investigator/physician. 
 
In order to receive Dose 2, subjects must not fulfil any of the above criteria. 
 
9.3.6 Restrictions 
The following restriction must be respected by the study subjects during participation in the 
study: 
• Contraception (Women): A woman is considered of childbearing potential 
(WOCBP), i.e. fertile, following menarche and until becoming post-menopausal 
unless permanently sterile. Permanent sterilisation methods include hysterectomy, 
bilateral salpingectomy and bilateral oophorectomy. A postmenopausal state is 
defined as no menses for 12 months without an alternative medical cause.  
 
A WOCBP can participate in the study if practising abstinence (only allowed when 
this is the preferred and usual lifestyle of the subject)  
 
OR 
is willing to use a highly effective method of contraception with a failure rate of < 
1% to prevent pregnancy (from at least 4 weeks prior to dose until completion of 
the main study, i.e. the follow-up visit 7 days after the last dose): 
o combined (oestrogen and progestogen containing) hormonal contraception 
associated with inhibition of ovulation (oral, intravaginal, transdermal) 
o progestogen-only hormonal contraception associated with inhibition of 
ovulation (oral, injectable, implantable), intrauterine device (IUD) 
o intrauterine hormone-releasing system (IUS)  
 
• 
Contraception (Men):  
 

 
 
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o With a female partner of childbearing potential or pregnant female partner, 
men must remain abstinent or use a condom during the treatment period and 7 
days after the final dose of IMP. Men must refrain from donating sperm 
during the same period. Periodic abstinence (e.g. calendar, ovulation, 
symptothermal or postovulation methods) and withdrawal are not adequate 
methods for preventing drug exposure. 
 
• Blood donation: The subjects participating in the main study must not donate blood or 
plasma during the study until 1 month after the last visit. The subjects participating in the 
exploratory study must not donate blood or plasma during the study until 3 months after 
the last visit. 
• Fasting: The subject should not eat or drink for at least one hour before or after ingestion 
of the study vaccine 
9.3.7 Removal of subjects from therapy or assessment 
 Subject withdrawal criteria and replacement 
A study subject should be withdrawn from the study treatment if, in the opinion of the 
Investigator, it is medically necessary, or if it is the expressed wish of the subject. 
Irrespective of the reason for withdrawal and whenever possible, the subject should be examined. 
Relevant laboratory test samples should be obtained, and all relevant assessments should be 
completed, preferably according to the scheme for the final assessment. The CRF should be 
completed as far as possible and checked/verified by the Monitor. 
Subjects may be discontinued from the investigation at any time at the discretion of the 
Investigator for any of the following reasons:  
 
• Severe non-compliance to the Study Protocol procedures, as judged by the Investigator 
and/or SBH 
• Subject is lost to follow-up  
• Significant AEs posing a risk for the subject, as judged by the Investigator and/or SBH 
A withdrawn subject is not allowed to re-enter into the trial. 
9.4 
Treatments 
 
The wet formulation consists of a liquid suspension of inactivated bacteria (ETEX 21-24) and 
LCTBA in one vial, freeze-dried dmLT adjuvant in a second vial, and effervescent buffer 
granules in a separate sachet. Prior to administration, the buffer is dissolved in 150 ml tap water, 
followed by the addition of the content of the vaccine vial (inactivated bacteria mixed with 
LCTBA) and reconstituted and diluted adjuvant dmLT from the second vial.  

 
 
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In the partially dried formulation, dmLT and LCTBA are spray-dried and mixed with the buffer 
granules and stabilizing excipients in a sachet. Prior to administration, the content of the buffer 
sachet (buffer, dmLT, and LCTBA) is dissolved in 150 ml tap water, followed by the addition of 
a liquid suspension of inactivated bacteria (ETEX 21-24).  
The identity of the different components of the two vaccine formulations are described below.  
9.4.1 Identity of Investigational Medicinal Product (IMP) 
 Inactivated E. coli ETEX 21  
The E. coli ETEX 21 strain was developed using a recombinant plasmid expressing the entire 
CFA/I operon under a tac promoter as described with the difference that the antibiotic selection 
marker (ampicillin) was replaced with the gene encoding thymidinylate synthetase, (thyA) from 
V. cholerae [36]. The bacterial host for this construct is an Escherichia coli, CFA/I, O78, K-. The 
strain was originally isolated from a patient in Dhaka, Bangladesh in 1985 suffering from 
diarrhea due to an ETEC infection. This strain was used as a component of the first generation 
ETEC vaccine and has been given to at least 2000 subjects. The plasmid(s) encoding the ST and 
CFA/I native genes were removed by natural selection. Further modification was done by 
knocking out the thyA gene on the chromosome by inserting a kanamycin resistance gene in the 
thyA gene. Furthermore, in a second round of chromosomal deletion the kanamycin gene was 
deleted in its first 200 nucleotides making it kanamycin sensitive. The combination of the thyA 
expressing plasmid and the thyA deficient host strain enables antibiotic-free selection of the 
recombinant ETEX 21 strain. Inactivation of the strain was performed by mild formalin 
treatment [37]. 
 Inactivated E. coli ETEX 22  
The E. coli ETEX 22 strain was developed using a recombinant plasmid expressing the entire 
CS3 operon under a rns promoter which in turn is under the lac operator. The selection system 
for this plasmid is also based on the thyA gene from V. cholerae. The bacterial host for this 
construct is the same E. coli, CFA/I, O78, K- strain, as described for ETEX21. The combination 
of the thyA expressing plasmid and the thyA deficient host strain enables antibiotic-free selection 
of the recombinant ETEX 22 strain. Inactivation  of the strain was performed by mild formalin 
treatment [37]. 
 
 Inactivated E. coli ETEX 23  
The E. coli ETEX 23 strain was developed using a recombinant plasmid expressing the entire 
CS5 operon under a under a tac promoter. The selection system for this plasmid is also based on 
the thyA gene from V. cholerae. The bacterial host for this construct is the same E. coli, CFA/I, 
O78, K- strain, as described for ETEX 21 and ETEX 22.  The combination of the thyA 
expressing plasmid and the thyA deficient host strain enables antibiotic-free selection of the 
recombinant ETEX 23 strain. Inactivation of the strain was performed by mild formalin 
treatment [37]. 
 

 
 
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 Inactivated E. coli ETEX 24  
The E. coli ETEX 24 strain was developed using a recombinant plasmid expressing the entire 
CS6 operon under a tac promoter. The selection system for this plasmid is also based on the thyA 
gene from V. cholerae. The bacterial host for this construct is an E. coli K12 strain C600, 
previously used as a placebo in numerous Dukoral and ETEC clinical trials. This strain was 
modified by knocking out the thyA gene on the chromosome by inserting a kanamycin resistance 
gene in the thyA gene. This strain was used as host for the SBL109 strain, studied in a previous 
clinical trial (EudraCT: 2009-015741-23, OEV-120) as a component of the "Prototype ETEC 
Vaccine No 2" [11]. In a second round of chromosomal deletion the kanamycin gene was deleted 
in its first 200 nucleotides making it kanamycin sensitive. The combination of the thyA 
expressing plasmid and the thyA deficient host strain enables antibiotic-free selection of the 
recombinant ETEX 24 strain. Inactivation of the strain was performed by mild phenol treatment 
[38]. 
 
 LCTBA protein 
LCTBA is a hybrid protein between the B-subunit of the E. coli heat-labile enterotoxin (LTB) 
and the B-subunit of the cholera toxin (CTB). Seven amino acids in the CTB molecule have been 
replaced by amino acids at the corresponding positions of the LTB molecule [20]. The LCTBA 
encoding DNA was cloned on a plasmid under a tac promoter. The plasmid has the thyA gene 
from E. coli and is expressed in a V. cholerae strain that is deleted in its thyA gene, enabling 
antibiotic free selection of this plasmid. The V. cholerae strain that hosts the plasmid is a further 
development of the strain holding the rCTB213 encoding plasmid JS1569. The change is the 
deletion of the thyA gene, thus the genetic characteristics are ctxA, thyA, kan [39]. The 
LCTBA protein was evaluated for safety and immunogenicity in 1 mg and 4 mg dosages together 
with inactivated CFA/I over-expressing SBL 109 bacteria in the OEV-120 clinical trial. This 
study revealed that the LCTBA protein is safe and strongly immunogenic at both dosage levels 
tested [11]. 
 
 Oral dmLT adjuvant 
LT(R192G/L211A), or dmLT is a derivative of wild-type enterotoxigenic E. coli  heat-labile 
enterotoxin that has been genetically modified by replacing the arginine at amino acid position 
192 with glycine and the leucine at amino acid position 211 with alanine [25]. These two amino 
acid substitutions take place in proteolytic cleavage sites which are critical for activation of the 
secreted toxin molecules. The protein has been designated LT(R192G/L211A) and has been 
extensively evaluated in pre-clinical animal studies for its ability to adjuvant the immune 
responses for co-administered antigens [21]. The double mutant toxoid is safe and tolerable in 
humans at 5, 25, 50, and 100 µg doses [26].  
 
The protein dmLT has been manufactured under cGMP at IDT Biologika, Dessau, Germany. In 
addition, the stability testing of this clinical lot of dmLT will continue over the course of the 
clinical phase of this Phase II trial.   
 

 
 
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 Sodium bicarbonate protective buffer powder (Recip) used in the Wet formulation 
Protective buffer is used to neutralize gastric acidity upon ingestion of vaccine. The dried 
powder is supplied in moisture-proof sachets (5.6 g/sachet). 
For use, the carbonate buffer is dissolved in 150 ml of potable water. The Sodium hydrogen 
carbonate buffer has been produced by Recip AB in Sweden. 
 
  dmLT LCTBA spray-dried preparation  
The dmLT spray-dried preparation was produced under GMP at GEA Niro, Sörborg, Denmark. 
 
 dmLT LCTBA spray-dried powder together with effervescent powder and excipients. 
The complete dmLT/LCTBA effervescent powder was produced under GMP at 
Upperton Pharma Solutions, Nottingham NG7 2TN, United Kingdom  
Protective buffer is used to neutralize gastric acidity upon ingestion of vaccine in particular to 
protect the dmLT against degradation. The dried powder is supplied in moisture-proof sachets 
(10.84 g/sachet). 
For use, the carbonate buffer is dissolved in 150 ml of potable water.  
 Product(s) to be tested and supply (where applicable)   
9.4.1.10.1 Wet formulation  
 
Product name and applicable 
formulation(s) 
Manufacturer  
Details of product 
(approved for use/under 
development), GMP 
guarantee, supply and 
availability – see details 
above)  
QP release 
Freeze-dried formulation of 
dmLT 
 
IDT Biologika 
GmbH, Am 
Pharmapark, 
06861 Dessau-
Rosslau, 
Germany 
GMP guarantee, 
contracted 
CSM Europe,SA Belgium 
Wet formulation of CFA/I, 
CS3, CS5, CS6 and LCTBA 
 
Cobra 
Biologics, 
SE-864 23 
Matfors, 
Sweden 
GMP guarantee, 
contracted 
Cobra Biologics, 
Matfors, Sweden 

 
 
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9.4.1.10.2  Partially dried formulation 
 
Product name and applicable 
formulation(s) 
Manufacturer  
Details of product 
(approved for use/under 
development), GMP 
guarantee, supply and 
availability – see details 
above)  
QP release 
Spray-dried formulation of 
LCTBA, dmLT mixed with 
buffer  
 
Upperton 
Pharma 
Solutions 
GMP guarantee, 
contracted  
Upperton Pharma Solutions, 
Nottingham, England 
Wet formulation of CFA/I, 
CS3, CS5, CS6 
 
Cobra 
Biologics, 
SE-864 23 
Matfors, 
Sweden 
GMP guarantee, 
contracted 
Cobra Biologics, 
Matfors, Sweden 
  
 Investigational Product:  
 
New partially dried formulation 
Wet formulation 
Contents  
Adult 
dose 
(mg) 
Contents 
Adult 
dose 
(mg) 
Part 1: 
dry 
powder  
Sodium bicarbonate 
effervescent 
granules/dmLT/LCTBA 
Part 1: 
dry 
powder 
Sodium bicarbonate 
effervescent granules 
Sodium hydrogen 
carbonate 
3600 
Sodium hydrogen 
carbonate 
3600 
Citric acid, 
anhydrous 
1450 
Citric acid, 
anhydrous 
1450 
Sodium carbonate 
400 
Sodium carbonate 
400 
Sucrose 
1010 
Sucrose 
NA 
PEG4000 
1010 
PEG4000 
NA 
Raspberry flavor 
70 
Raspberry flavor 
70 

 
 
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Sodium saccharin 
30 
Sodium saccharin 
30 
Sodium citrate 
6 
Sodium citrate 
6 
Di-Sodium 
hydrogen phosphate 
di-hydrate 
12.7 
Di-Sodium 
hydrogen 
phosphate di-
hydrate 
NA 
Potassium 
phosphate mono 
basic 
1.6 
Potassium 
phosphate mono 
basic 
NA 
Sodium chloride 
5.3 
Sodium chloride 
NA 
Lactose 
monohydrate 
55.6 
Lactose 
monohydrate 
NA 
Maltodextrin 
40 
Maltodextrin 
NA 
Tween 80 
0.5 
Tween 80 
NA 
L-arginin HCl 
3150 
L-arginin HCl 
NA 
Hybrid protein 
LCTBA 
1.0 
Hybrid protein 
LCTBA 
NA 
dmLT 
0.010 
dmLT 
NA 
Part 2: 
Liquid 
Tap water 
q.s. ad 
150 ml 
Part 2: 
Liquid 
Tap water 
q.s. ad 
150 ml 
Part 3:  
liquid 
Excipient: Buffer 
Part 3:  
liquid 
Excipient: Buffer 
Phosphate 
buffered saline 
(PBS) 
q.s. ad 
3.3 ml 
Phosphate 
buffered saline 
(PBS) 
q.s ad 
3.3 ml 
dmLT* 
0.01 
Inactivated bacteria 
Inactivated bacteria and 
LCTBA 
E.coli CFA/I, 
ETEX21 
0.98 
E.coli CFA/I, 
ETEX21 
0.98 
E.coli CS3, 
ETEX22 
3.13 
E.coli CS3, 
ETEX22 
3.13 
E.coli CS5, 
ETEX23 
0.63 
E.coli CS5, 
ETEX23 
0.63 
E.coli CS6, 
ETEX24 
0.16 
E.coli CS6, 
ETEX24 
0.145 

 
 
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Hybrid protein 
LCTBA 
1 
Total volume of ETVAX® 
153.3 ml Total volume of ETVAX® 
153.3 ml 
 
NA; not applicable  
*; In the wet formulation, dmLT is pipetted into the 150ml of reconstituted buffer containing the vaccine  
 
9.4.2 Packaging, labelling and storage of IMP 
 Packaging 
The vaccine vials for both formulations are packed in boxes of 10 vials/box.  
The effervescent powder for the wet formulation is packed in boxes of 20 sachets/box. 
The dmLT vials for the wet formulation are packed in cardboard boxes with 30-50 vials/box. 
The spray-dried formulation of LCTBA and dmLT mixed with buffer for the partially dried 
formulation is packed in boxes of 10-30 sachets/box. 
 Labelling 
All IMP will be shipped directly to the study sites or to a local distribution site, according to 
local requirements. 
Labels will comply with applicable Good Manufacturing Practice (GMP) requirements 
(EudraLex VOLUME 4, Good manufacturing practices, ANNEX 13, Manufacture of 
investigational medicinal, products, February 2010, section 26). 
 
 Storage 
All study medication must be stored in a safe place with limited access, at the temperature given 
on the labels. Temperature logs should be kept for the area where the IMP is stored at the clinic. 
The temperature should be noted on a daily basis (working days only unless automatic readings 
are available). 
 
• Vaccine vials shall be stored at 2-8C. 
• Buffer sachets for the wet formulation shall be stored at 2-25C. 
• Buffer sachets for new partly dry formulation shall be stored at 2-8C. 
• dmLT vials shall be stored at -205C. 
• Reconstituted diluted (200 µg/ml) dmLT shall be stored at 2-8C. Maximum storage time 
is 48 hours. 

 
 
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9.4.3 Doses and treatment regimens 
 
Both vaccine formulations should be administered only by the oral route. The vaccine will be 
administered orally twice to each subject at a target interval of 14 days. If the subject is unable to 
come for the second dose at the third study visit on day 15 due to e.g. illness or rules regarding 
quarantine due to the COVID-19 epidemic, the second vaccine dose can be administered up to 
28 days after the first dose. A prolonged interval between vaccine doses did not affect the serum 
responses to the highly homologous CTB component of the cholera vaccine Dukoral when tested 
in Swedish volunteers [32]. Thus, serum CTB-specific IgA and IgA responses were comparable 
in both magnitude and frequency in subjects receiving Dukoral 14 days or 28-48 days apart. 
 
 Preparation and administration of investigational products 
To maintain blinding of the study, the investigational products will be prepared by un-blinded 
trained nurses on the day of vaccination (Day 1 and day 15) at the clinic.  
 
NOTE: The vaccine will be given to the participants by a blinded study nurse. 
 
Detailed instructions of vaccine preparation for both formulations, including dmLT reconstitution 
and preparation of stock dmLT will be provided in a separate IMP manual. 
 
9.4.3.1.1 Preparation of Wet formulation 
9.4.3.1.1.1 dmLT preparation 
A dmLT vial is retrieved from the freezer and thawed at room temperature. 700 µl of water for 
injection is added to reconstitute the dmLT. After careful swirling of the ampoule 500 to 700 µl 
are removed from the vial to an Eppendorf tube labelled “Stock dmLT”. A 1/5 dilution of this 
stock is done in PBS, resulting in a 200 µg/ml solution. From this preparation 50 µl (10 µg) is 
pipetted into the effervescent buffer vaccine preparation before administration to volunteers. 
 
 
9.4.3.1.1.2 Preparation of complete Wet formulation 
 
The vaccine supplied as a liquid, is mixed with the 150 ml of sodium bicarbonate buffer solution 
on the day of preparation for use on dosing day. The time of investigational product preparation 
will be recorded on the appropriate Accountability Record. The vaccine dose in sodium 
bicarbonate buffer can be stored at room temperature for up to 90 minutes prior to dosing. The 
buffer is used to prevent degradation of LCTBA hybrid protein, dmLT and sensitive CF antigens 
by the gastric acid. 
 

 
 
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Just prior to administration 10 µg of dmLT is added by pipette (50 µl). After addition of dmLT, 
the vaccine should be used within 30 minutes.  
 
9.4.3.1.2 Preparation of partially dry formulation 
 
The partially dry formulation of vaccine is prepared by adding the effervescent powder 
containing the dmLT and LCTBA to 150 ml of water (the time is recorded), after mixing the 
content of the vaccine vial is added to the mixture and the vaccine is administered to the 
volunteer within 30 minutes after adding the buffer powder to the water. The buffer is used to 
prevent degradation of LCTBA hybrid protein, dmLT and sensitive CF antigens by the gastric 
acid. 
 
 Vaccine oral solution 
Full dose of ETVAX®; a tetravalent ETEC vaccine including LCTBA + 10 µg double mutant LT 
(dmLT) adjuvant and effervescent powder for oral solution 
 
9.4.4 Product accountability 
The IMP will be released to the study site(s) after approvals of the study protocol have been 
received from the IEC and the Competent Authority.  
 
In order to maintain blinding of the study, the IMP preparation procedure will be performed by 
an un-blinded member of the site staff, not otherwise involved in the conduct of the study. The 
person who will prepare the IMP needs to be a Registered Nurse (RN). The preparation will be 
observed by an additional un-blinded person. Both the nurse preparing the vaccine and the 
observer need to sign the vaccine preparation documentation log. 
 
The study site will maintain a Drug Dispensing Log detailing the dates and quantities of IMP 
received, prepared, administered/dispensed to each subject as well as study medication returned 
or destroyed at the end of the study. Any discrepancies between dispensed and returned products 
must be explained and documented. Products deliberately and/or accidentally destroyed by the 
Investigator or the subject must be accounted for. 
 
All drug accountability procedures, including cold chain monitoring will be documented and will 
be responsible of the unblinded study staff. 
 
The unblinded Monitor will verify IMP accountability and make sure that all unused IMP is 
adequately destroyed/returned and documented.  
 

 
 
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9.4.5 Method of assigning subjects to treatment groups 
 
The randomization list will be generated by the SCRO biostatistician and the original 
randomization list will be kept at Scandinavian CRO. Subjects will be randomized to treatment 
with the wet formulation or new partially dry formulation of the vaccine (1:1).  
 
9.4.6 Blinding 
 
This is a double-blind study and the allocation of treatments will not be disclosed until clean file 
has been declared and the database has been locked for primary and secondary endpoint 
analyses.  
 
Hence neither the personnel directly involved in taking care of the study subjects or evaluating 
the results, nor the subjects themselves, will be informed which of the two different formulations 
of vaccine that is given. The two formulations will have identical appearance and the same smell. 
 
However, the person who prepares the vaccines immediately before immunization and the 
observer will be unblinded. Theses persons will not be directly involved in evaluation of adverse 
events or laboratory results. 
 
The unblinded SCRO Monitor will visit the investigational site to review documents and records 
related to the preparation of the vaccine, i.e. information unblinded to treatment allocation.  
 
SCRO pharmacovigilance personnel will be un-blinded for SUSAR reporting purposes but are 
not involved in data analyses. 
9.4.7 Emergency decoding of blinded treatment 
The treatment code may only be broken by the study medical staff in case of emergency when 
knowledge of the treatment received is necessary for the proper medical management of the 
subject. The code breaking procedure should be carefully documented. 
Decoding of the blinded treatment must be reported to SBH. 
9.4.8 Prior and concomitant therapy 
Therapies considered necessary for the subject’s welfare may be given at the discretion of the 
Investigator.  
All concomitant therapy used during and within 3 months prior to the study period must be 
recorded in the CRF. No other drug under investigation may be used concomitantly with the 
study medication. 
The subjects must not participate concurrently in any other clinical study. 

 
 
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9.4.9 Continuation of treatment 
This is a bridging study comparing two formulations of the unregistered product ETVAX® and 
thus there will be no treatment with ETVAX® available after end of study participation. 
9.4.10 Treatment compliance 
IMP will be administered at the research clinic (CTC) under medical supervision to ensure 
compliance.  
9.5 
Study assessments 
The timing and frequency of study visits and assessments are presented in the schedule of events 
in Table 9.1 and 9.2.  
Each study assessment is described in the sections below. 
9.5.1 Demographics and other baseline characteristics 
 Informed consent 
Signed informed consent must be obtained before any screening procedures are initiated. The 
informed consent procedure is further described in Section 5.3. 
 
  Demographic information 
The following demographic data will be recorded: race, ethnic origin, gender and date of birth. 
 
 Medical/surgical history  
Relevant medical/surgical history will be obtained by interview in order to verify that the 
eligibility criteria are met.  
 
In addition, information regarding COVID-19 (infections and vaccinations) as well as detailed 
travel history to ETEC and cholera endemic countries will be collected, primarily to support 
evaluation of the exploratory data. This information will not be captured in the CRF, but in a 
separate document. 
   Prior and concomitant medications 
Medication history (prior medications) from 3 months prior to study start until administration of 
the first dose of IMP will be recorded in the CRF. Prescription medications, Over-the-counter 
(OTC) medications, and herbal products should be asked for. Any medication used from first 
administration of IMP until the last follow-up assessment (concomitant medication) will be 
recorded.  
Prior and concomitant medications will be coded according to the WHO Anatomic Therapeutic 
Chemical (ATC) classification system. 

 
 
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9.5.2 Clinical efficacy assessments  
 Seroconversion rates of IgA and/or IgG anti-LTB antibodies in serum.  
IgA and IgG antibodies specific for LTB will be measured in serum samples collected on visit 2 
and 4 using an Enzyme Linked Immunosorbent (ELISA) assay. The analyses will be performed 
by technical laboratory personnel at the Department for Microbiology and Immunology, 
University of Gothenburg, according to standardized laboratory procedures. The seroconversion 
rate for IgA and IgG will be determined as the frequencies of subjects having a ≥2 increase in 
LTB-specific antibody levels in post compared to pre-vaccination samples. Specifically, this will 
be determined by dividing the antibody titer measured in samples collected 7 days after the 
second vaccination (Visit 4) with the titer measured in samples collected before vaccination 
(Visit 2). Samples collected before and after immunization will always be analyzed on the same 
ELISA-plate. Fold rises in ELISA titers ≥2 will be considered as significant responses for both 
IgA and IgG.  
9.5.3 Safety laboratory assessments 
 
There will be no safety laboratory assessments performed in this study.  
9.5.4 Clinical safety assessments 
 Physical examination 
A complete physical examination will include assessments of the head, eyes, ears, nose, throat, 
skin, thyroid, neurological, lungs, cardiovascular, abdomen (liver and spleen), lymph nodes and 
extremities. 
  Vital signs  
Systolic and diastolic blood pressure and heart rate will be measured in the supine position after 
10 minutes of rest. Body temperature will be measured in the ear using a digital thermometer. 
 
 Subject Diary 
The study diaries will contain questions regarding adverse events and current medication. The 
adverse event and medication diaries should be filled in by the subjects on the day of vaccination 
and five subsequent days. Information about adverse events and medication obtained from the 
diaries will be verified by interviewing the subject at the following clinic visit and will then be 
registered in the CRFs by the study nurse/physician, i.e data from study diaries will not be 
transcribed word by word into the eCRF. 
The following solicited AEs will be included in the diary: fever, nausea, vomiting, abdominal 
pain and loose stools/diarrhea. 
 

 
 
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9.5.5 Exploratory assessments 
All serum samples will be analyzed for LTB specific antibodies using an 
electrochemiluminescence (ECL) assay based on the Meso Scale Discovery platform in parallel 
to the ELISA IgA assay [14, 15]. The ECL results will be reported as exploratory data. Serum 
samples will also be analyzed for antibodies against additional antigens expressed by ETEC 
bacteria, such as O78 LPS, by ELISA, ECL or similar methods.  
Memory cell responses will be evaluated in subsets of subjects using analysis tools recently 
established for assessment of responses to a number of different infections and vaccines at the 
Dept. of Microbiology and Immunology at UG and the collaborating Clinical Immunology 
Laboratory at SU. These exploratory analyses will be used to validate the new methods for 
analysis of long-term immunity to ETEC in order to provide additional analysis tools to be used 
in subsequent ETEC vaccine trials and basic research studies.  
Effector T- and B-cell (plasmablast) responses will be analyzed in 40 subjects (20 immunized 
with the wet and 20 with the partially dry vaccine formulation). Memory T- and B-cell analysis 
will be performed in 20 subjects (10 immunized with the wet and 10 with the new partially dry 
vaccine formulation). Memory B cell responses will be assessed on day 30±7 and 75-125 after 
the second dose by stimulating PBMCs with substances  that transform memory B cells into 
antibody secreting plasmablasts. The production of IgA and IgG antibodies specific for LTB and 
CFs will be measured in culture supernatants by ECL and/or ELISA. The antigen specificity and 
isotype distribution of antibodies produced by activated memory B cells will be compared to 
serum antibody responses at all analysis time points after the second vaccination (day 5±1, 6-10, 
30±7 and 75-125) and to antibodies secreted by plasmablasts induced to circulate shortly after 
the second immunization (day 5±1). 
Effector and memory T cell responses will be assessed by flow cytometric analysis of activation 
markers on T helper cell subsets before and after stimulation of cells with LTB and CFs. The 
production of cytokines characteristic for different T cell subsets will be evaluated by flow 
cytometry, ELISA, ECL or other highly sensitive immunoassays. T cell responses will be 
compared in samples collected before vaccination and after the second dose (day 5±1, 6-10, 
30±7 and 75-125). The relation between different types of effector T cell responses and 
induction of early plasmablast and serum antibody responses (day 5±1 and 6-10) and memory B 
and T cell responses (day 30±7 and 75-125) will be analyzed. 
To check the general immune status of the volunteers and their ability to form long lived 
memory cells in relation to the ETEC specific immune responses measured after vaccination, 
immune responses to other common infections and vaccinations will be measured, such as 
cytomegalovirus, measles, SARS-CoV-2 and tetanus toxoid. Responses will be measured in 
samples from both the main study and the explorative study with techniques similar to those used 
to analyze ETEC specific immune responses, such as ELISA, electrochemiluminescence and 
flow cytometry.   
 

 
 
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9.5.6 Adverse events 
 Definitions  
Adverse Event (AE) 
An AE is any untoward medical occurrence in a clinical study subject administered a 
pharmaceutical product and which does not necessarily have a causal relationship with this 
treatment. An AE can therefore be any unfavourable and unintended sign (including clinically 
significant abnormal values from relevant tests, such as ECGs, vital signs), symptom, or disease 
temporally associated with the use of an IMP, regardless of whether it is considered related to 
the IMP. 
A baseline event is any medical event in a clinical study subject that occurs after signing the ICF 
up until the (first) administration of IMP. 
Clarifications: 
Diagnostic and therapeutic non-invasive and invasive procedures, such as surgery, should not be 
reported as AEs. However, the medical condition for which the procedure was performed should 
be reported if it meets the definition of an AE. For example, an acute appendicitis that begins 
during the AE reporting period should be reported as “acute appendicitis” and the resulting 
appendectomy noticed under Comments. Pre-study conditions, which led to elective surgery 
during the time of the study, are not to be reported as AEs. 
If an abnormal vital sign is associated with corresponding clinical signs and symptoms, the 
sign/symptom should be reported as the AE and the associated laboratory result or vital sign 
should be considered additional information that is to be collected in the e-CRF. 
Serious Adverse Event (SAE) 
An SAE is any AE that: 
− results in death 
− is life-threatening (this refers to an event in which the subject was at risk of death at the 
time of the event; it does not refer to an event that hypothetically might have caused 
death had it been more severe) 
− requires inpatient hospitalisation or prolongation of existing hospitalisation 
− results in persistent or significant disability/incapacity 
− is a congenital anomaly/birth defect 
− is medically important (this refers to an event that may not be immediately life-
threatening or result in death or hospitalisation, but may jeopardise the subject or may 
require intervention to prevent any of the SAEs defined above) 
Examples of medically important events are intensive treatment in an emergency room for 
allergic bronchospasm or blood dyscrasias, convulsions that do not result in hospitalisation, 
development of drug dependency and drug abuse. 
Planned hospitalisations or surgical interventions for a condition that existed before the subject 
signed the ICF and that did not change in intensity are not SAEs. 

 
 
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If there is any doubt as to whether an AE meets the definition of an SAE, a conservative 
viewpoint must be taken, and the AE must be reported as an SAE. 
 
Serious Adverse Reaction (SAR) 
The term SAR is to be used whenever either the Investigator or SBH or designee assessed the 
SAE as possibly or probably related to the IMP. 
Suspected Unexpected Serious Adverse Reaction (SUSAR) 
A SUSAR is any SAR whose nature or intensity is not consistent with the current version of the 
Investigator’s Brochure (IB). 
 Assessment of severity/intensity 
The Investigator must assess the severity/intensity of an AE using the following definitions, and 
record it on the Adverse Event Form in the CRF: 
Mild 
 
The AE does not interfere in a significant manner with the subject’s 
normal functioning level. It may be an annoyance. 
Moderate 
 
The AE produces some impairment of function but not hazardous to 
health.  It is uncomfortable and/or an embarrassment. 
Severe 
 
The AE produces significant impairment of functioning or incapacitation 
and/or it is a hazard to the subject. 
If an AE changes in severity, the most severe intensity should be recorded for that AE; i.e. the 
AE should not be recorded twice in the eCRF.  
For severity grading of AEs, please also see Appendix 3, Clinical Toxicity Grading. 
 Assessment of causal relationship 
The Investigator must assess the causal relationship between an AE and the IMP using the 
definitions below and record it on the Adverse Event Form in the CRF as well as on the Serious 
Adverse Event Report Form, if applicable: 
 
 
Probably 
− Probably related – the AE has a strong temporal relationship to the IMP 
or recurs on re-challenge, and another aetiology is unlikely or 
significantly less likely. 
Possibly 
− Possibly related – the AE has a suggestive temporal relationship to the 
IMP, and an alternative aetiology is equally or less likely. 
Unlikely 
− Unlikely related – the AE has no temporal relationship to the IMP or is 
due to underlying/concurrent illness or effect of another drug (that is, a 
causal relationship between the IMP and the AE is unlikely). 

 
 
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An AE is considered causally related to the use of the IMP when the causality assessment is 
probably or possibly. 
For a baseline event, a causality assessment is not relevant. 
   Assessment of Outcome 
The Investigator must assess the outcome of an AE using the definitions below and record it 
on the Adverse Event Form in the CRF: 
− Unknown - not known, not observed, not recorded, or refused 
− Recovering/resolving - indicates that the event is improving.  
− Recovered/resolved with sequelae - the subject recuperated but retained pathological 
conditions resulting from the prior disease or injury.   
− Recovered/resolved - indicates that the event has improved or recuperated. 
− Not recovered/not resolved - indicates that the event has not improved or recuperated. 
− Fatal - the termination of life as a result of an AE. 
   Collecting and recording of AEs 
AEs and baseline events identified using any of the following methods will be recorded: 
− AEs spontaneously reported by the subject 
− AEs observed by the Investigator or medical personnel 
− AEs elicited based on non-leading questions from the Investigator or medical 
personnel 
− AEs obtained from Subject Diaries, as described in section 9.5.4.3 
Collection of baseline events starts after the subject signs the ICF and continues until the first 
intervention with the IMP. 
AE collection starts with the first intervention with the IMP and continues until the last follow-up 
assessment. Any AE with start date on the day of first IMP intervention must be recorded with 
start time. 
At the Follow-up Visit, information on new AEs or SAEs, if any, and stop dates for AEs 
recorded and on-going during the dosing period must be recorded. 
All AEs, serious and non-serious, should be recorded in the CRFs.  Reporting of SAEs 
No distinction should be made between the investigational product and the reference product 
regarding reporting of SAEs as long as the code is not broken.  
Starting from administration of first dose of IMP, all SAEs must be reported by the Investigator 
to SCRO Pharmacovigilance, the Monitor and SBH within 24 hours of knowledge of the event, 
regardless of the time that may have elapsed from the time the event occurred to when the 
Investigator first learns of it.  
 
All SAEs should be reported to SCRO Pharmacovigilance: 

 
 
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E-mail: pharmacovigilance@pharmassist.se 
 
 
 
 
The Initial Report should contain as a minimum the following information: 
• Subject identification 
• Treatment specification (to be expressed in a blinded fashion, e.g. “Treatment 
A/Treatment B”) 
• AE diagnosis 
• Time specification for the medical event 
• Name of the original reporter 
• Seriousness classification  
A Serious Adverse Event Report Form must also be completed, signed by the Investigator and 
submitted to SCRO Pharmacovigilance, the Monitor and Sponsor no later than three calendar 
days after the initial information was received. Apart from the information above, this Follow-up 
Report should also contain the following information: 
• assessment of severity 
• assessment of causality 
Only SAEs that are both unexpected and related to the investigational product(s), Suspected 
Unexpected Serious Adverse Reactions (SUSARs), are subject to expedited reporting.  SBH are 
responsible for informing all Investigators concerned of relevant information about SUSARs that 
could adversely affect the safety of subjects.  
Further details are provided in a separate Safety Management Plan. 
 
  Exceptions from expedited reporting of SAEs 
An event need not to be reported as an SAE if it represents only a relapse or an expected change, 
or progression of a pre-existing condition and is without any other symptoms or signs than those 
present before treatment. This event needs only to be reported as an AE and should be described 
in the Study Protocol. 
AEs must be followed up until resolution or the follow-up assessment, whichever comes first. At 
the Follow-up Visit, information on new AEs, if any, and stop dates for previously reported AEs 
must be recorded. 
It is the responsibility of the Investigator to follow up on all SAEs until the subject has 
recovered, stabilized, or recovered with sequelae, and to report to SBH all relevant new 
information using the same procedures and timelines as those for the initial report. Relevant 
information includes discharge summaries, autopsy reports, and medical consultation. 
SAEs spontaneously reported by a subject to the Investigator within 30 days after the last follow-
up assessment should be reported to SBH even after the clinical investigation has been finished, 

 
 
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if, in the judgment of the Investigator, there might be an association between the event and the 
previous use of the IMP or as a result of the investigation procedures. 
   Procedures in case of pregnancy 
In case of pregnancy or suspicion of possible pregnancy, the subject must be discontinued from 
further dosing in the study. Pregnancy itself is not regarded as an AE unless there is a suspicion 
that the IMP may have interfered with the effectiveness of the contraceptive medication. 
However, the outcome of all pregnancies (spontaneous miscarriage, elective termination, normal 
birth or congenital abnormality) must be followed up and documented even after the Subject was 
discontinued from the study. 
All events of congenital abnormalities/birth defects are SAEs. Spontaneous miscarriages should 
also be reported and handled as AEs. Any pregnancy revealed during the study must be reported 
to SBH and the Monitor on the pregnancy report form. All outcomes of pregnancy must be 
reported to SBH and the Monitor on the pregnancy outcomes report form. 
 Coding of AEs 
All AEs will be coded according to Medical Dictionary for Regulatory Activities (MedDRA) by 
Scandinavian CRO. 
9.5.7 Appropriateness of measurements 
Standard safety assessment methods are used in this study and are very similar to procedures 
performed to monitor vaccine safety in a previous Phase IIb study of adult Finnish travellers 
before they left Finland to go to Benin in West Africa  (OEV-123, EudraCT No.: 2016-002690-
35). 
Due to difficulties to get a reliable measurement of immune responses to ETEC CFs in serum of 
non-primed (i.e. previously not immunized nor infected) Swedish individuals, only the immune 
response rates to LTB will be measured. For oral vaccines, the responses are evoked in the gut 
and only some immune responses, e.g. against LTB for ETVAX®, are detectable in serum of 
immunised individuals [6, 12]. Serum IgA responses to LTB correlate significantly with IgA 
responses measured in mucosal samples [12]. LTB-specific serum antibody levels measured by 
ELISA also closely reflect the neutralization capacity of the serum antibodies [12]. ELISA 
assays to measure LTB-specific IgA and IgG antibodies are well-established and have previously 
been used to evaluate antibody responses induced by ETVAX® vaccination in Swedish 
volunteers and in Bangladeshi adults and children [11-15]. The ELISA method used to measure 
antibodies in this trial has been validated by comparisons with ECL assays in previous studies 
[14]. Exploratory analyses of serum antibodies will also be performed using the ECL method. A 
cut-off of 2 for seroconversion has been used in many previous studies of oral vaccines, 
including all previous trials of ETVAX® .  
9.6 
Data quality assurance 
This study will be conducted in compliance with the protocol, relevant Standard Operating 
Procedures (SOPs), Good Clinical Practice (GCP) and the applicable regulatory requirement(s). 

 
 
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The Principal Investigator will provide SBH with all data produced during the study from the 
scheduled study assessments. He/she ensures the accuracy, completeness, legibility, and 
timeliness of the data reported to SBH in the CRF and in all required reports. 
9.6.1 Monitoring  
The investigational site will be visited both by the blinded Monitor and/or the blinded 
Scandinavian CRO Project Manager overseeing monitoring activities, as well as the unblinded 
Monitor periodically at times agreed on by the Investigator. It is the function of the Monitor to 
ascertain that all aspects of the CSP are complied with and that the conduct of the investigation 
conforms to applicable regulatory requirements and established rules for GCP.  
At the time of each monitoring visit, the blinded Monitor will review the CRFs to ascertain that 
all items have been completed and that the data provided are accurate and obtained in the manner 
specified in the CSP.  
The blinded Monitor will also check that the data in the CRF are consistent with the clinical 
records (Source Data Verification) and that investigational results are recorded completely and 
correctly.  
 
The blinded Monitor will check on the reporting of SAEs and record keeping. For this purpose, 
the Monitor must be given direct access to clinical records, original laboratory data, etc., as far as 
these relate to the investigation and without jeopardizing subject integrity. CRFs for all included 
subjects must be made available to the blinded Monitor for review.  
The unblinded Monitor will visit the investigational site to review documents and records related 
to the preparation of the vaccine, i.e. information unblinded to treatment allocation.  
 
A risk-based approach will be developed for the monitoring visits. Further details are provided in 
a separate Monitoring Plan.  
 
9.6.2 Audits and inspections 
Authorized representatives of SBH or a Competent Authority (e.g LV), may perform audits or 
inspections at the research clinic, including Source Data Verification (SDV). The purpose of an 
audit or inspection is to systematically and independently examine all study-related activities and 
documents, to determine whether these activities were conducted, and data were recorded, 
analysed, and accurately reported according to the protocol, ICH-GCP guidelines and any 
applicable regulatory requirements. The Investigator will contact SBH immediately if contacted 
by a Competent Authority about an inspection at the centre. 
The Investigator is required to inform SBH immediately of an inspection requested by the 
Competent Authority (e.g. LV).  
9.6.3 Case Report Forms (CRFs) 
A CRF must be completed and signed by authorized personnel for each included subject, 
according to the CRF completion instructions.  

 
 
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All data must be entered in English. The CRFs should always reflect the latest observations 
made during the subject’s participating in the study. Therefore, the CRFs should be completed as 
soon as possible during or after the subject’s visit.  
The Investigator must verify that all data entries in the CRFs are accurate and correct by signing 
the completed CRF. 
The completed CRFs should be made available for checking of completeness and accuracy by 
the blinded Monitor.  
 
Please see section 9.8 for further details. 
9.6.4 Source Data  
A separate source data document will be generated for the site before start of enrolment, 
specifying the location of the source of derived information appearing in the CRF. This 
document must be signed by the Principal Investigator and the Monitor to confirm agreement 
before start of recruitment. 
The Investigator should guarantee access to source documents to the Monitor, Competent 
Authority (LV) and the IEC (EPM), if required. 
9.6.5 Training of study staff  
Before inclusion of the first investigational subject the Monitor (both blinded and unblinded) 
and/or Project Manager will perform an initiation visit at the investigational site. The 
requirements of the Clinical Study Protocol and related documents will be reviewed and 
discussed, and the investigational staff will be trained in any study specific procedures and 
system(s) utilised. 
It is the responsibility of the Principal Investigator to ensure that all personnel involved in the 
study are fully informed of all relevant aspects of the study, and have a detailed knowledge of 
and training in the procedures that are to be executed by them, including good documentation 
practice. Any new information of relevance to the performance of this study must be forwarded 
to the staff involved in a timely manner. 
The Investigator will keep a list of all personnel involved in the study together with their 
function and study related duties delegated. A Curriculum Vitae (CV) will be available for all 
staff delegated study-specific duties. 
9.7 
Statistical methods and determination of sample size 
General descriptive statistics for categorial variables will be number of observations (n) and 
proportion (%) within treatment group. General descriptive statistics for continuous variables 
will be number of observations (n), Mean, Quartile 1 (Q1), Median, Quartile 3 (Q3), Geometric 
Mean, Standard deviation (SD) and Range (Min, Max). 
 
A detailed statistical analysis plan (SAP) will be written and finalized well in advance of 
database lock. The plan will follow the outline of the statistical analyses presented below, but 
details necessary to complete the statistical analyses will be given. 

 
 
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9.7.1 Description of study population 
 Demographics and baseline characteristics 
Descriptive statistics of demographics and other baseline characteristics will be presented for 
each vaccination group. 
 Medical/surgical history and concomitant medication 
Medical/surgical history and prior/concomitant medications will be presented by descriptive 
statistics for each vaccination group.  
 Analysis of efficacy (Seroconversion rates of IgA and/or IgG anti-LTB antibodies in 
serum) 
The primary endpoint, response, will be defined as “yes” for patients with at least a 2-fold 
increase in IgA and/or IgG antibody levels against LTB in serum between pre- and post-
immunization samples (Visit 4 versus Visit 2).  
The response rates (seroconversion rates) within treatment groups, 𝑝𝑤𝑒𝑡 and 𝑝𝑝𝑎𝑟𝑡𝑖𝑎𝑙𝑙𝑦_𝑑𝑟𝑖𝑒𝑑,  will 
be estimated as the proportion of responders (response = “yes”) in each treatment group. 
The primary analysis of the study seeks to compare the seroconversion rates using the difference 
𝑑=  𝑝𝑝𝑎𝑟𝑡𝑖𝑎𝑙𝑙𝑦_𝑑𝑟𝑖𝑒𝑑− 𝑝𝑤𝑒𝑡. 
The following non-inferiority hypothesis will be tested, using a -15% non-inferiority limit (on an 
absolute scale): 
H0: The seroconversion rate of the partially dried group is lower than that of the wet group, 𝑑< −0.15 
HA: The seroconversion rate of the partially dried group is higher than or equal to that of the wet group, 𝑑≥−0.15 
 
A two-sided 95% confidence interval (CI) for 𝑑 will be calculated by normal approximation. The 
lower limit of the CI will be compared to the non-inferiority limit -0.15. If the lower CI limit is 
larger than or equal to the non-inferiority limit, the null hypothesis will be rejected, and the result 
will be taken as supporting non-inferiority of the partially dried formulation compared to the wet 
formulation. 
The normal approximation requires at least 5 patients in each cell of the Treatment by Vaccine 
response contingency table. If assumptions are not fulfilled, a non-parametric CI will be used 
instead. 
The primary endpoint will be summarized for the full analysis set (FAS) and the per protocol 
analysis set (PPAS). The primary endpoint analysis of PPAS will be considered the primary 
analysis of the study. 
The primary endpoint, i.e. the proportion of responders with at least a 2-fold increase in LTB-
specific IgA and/or IgG antibody levels, will be summarized with descriptive statistics for the 

 
 
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PPAS and FAS. In addition, the proportion of responders with at least a 4-fold increase in IgA 
and/or IgG antibody levels will be presented. The levels of serum antibodies at visit 2 and visit 4 
and the fold rise (magnitude of response) will also be presented for both IgA and IgG. 
9.7.2 Analysis of safety  
 Vital signs 
Change from baseline in vital signs (blood pressure, heart rate and body temperature) will be 
presented by individual time courses for each parameter and subject and summarized by dose 
group. 
 Adverse Events (AEs) 
AEs and SAEs will be recorded from the start of IMP administration until the Follow-up Visit. 
AEs that occur before first treatment with IMP will be reported separately as baseline event.  
All AEs will be described in terms of the treatment/dose at which they occurred. AEs will be 
coded using the Medical Dictionary for Regulatory Activities (MedDRA) and summarized by 
Preferred Term (PT) and System Organ Class (SOC).  The number of subjects reporting AEs, 
and the number of AEs reported will be presented by dose group. The events will be tabulated by 
SOC, PT and by severity and relationship to IMP. SAEs will also be presented in separate 
tabulations. The number of subjects experiencing an AE in connection to each treatment will be 
presented. 
 
9.7.3 Analysis of exploratory assessment 
 
The exploratory assessments will be reported separately from the Clinical Study Report (CSR). 
The exploratory assessments may continue after unblinding of the main study. 
 
Results from exploratory analyses will be summarized using descriptive statistics. The following 
parameters will be included: for categorical variables, the number and percent in each category; 
for continuous variables, median (with quartiles), range (minimum, maximum), and geometric 
means. Magnitudes (fold-rises) of serum antibodies (against LTB measured by ECL and against 
additional bacterial antigens), memory B cell, antibody secreting cell and T cell responses will 
be determined at each time point post vaccination compared to pre vaccination. Frequencies of 
subjects responding with ≥2-fold increased levels of antibodies and markers reflecting T cell 
activity post vaccination compared to pre vaccination will be determined. Assessment of the 
change from the baseline measurement to a follow-up measurement will be analyzed using the 
Wilcoxon signed-ranks test, with adjustment for multiple testing using the Holm´s method, when 
applicable. Correlations between different types of responses will be analyzed using the 
Spearman test. P values <0.05 will be considered to be statistically significant.  
 

 
 
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9.7.4 Analysis data sets  
 Full Analysis Set (FAS) 
Unless otherwise stated, statistical evaluations will be based on all subjects who have received 
randomized treatment with IMP and have available data. Evaluations will be done according to 
actual treatment received regardless of treatment assigned at randomization. A treatment group 
will consist of all subjects treated with a specific formulation. The FAS data set will be used for 
the safety and tolerability assessments as well as for the efficacy endpoints. 
 Per Protocol Analysis Set (PPAS)  
The PPAS comprises data from all subjects randomized and treated with evaluable efficacy data, 
and no major protocol deviation that significantly affects the evaluability of the subject in the 
study. The PPAS will be used for presentation of the primary endpoint. 
 Determination of sample size 
Assuming a seroconversion rate of 85% in both the wet and the new formulation, and aiming to 
prove non-inferiority with margin -15% (on an absolute scale), 126 subjects are needed in each 
treatment arm to achieve 90% power. This calculation was done with Farrington-Manning’s 
method, assuming that the study will be evaluated by a two-sided 95% confidence interval for 
the difference between the two treatment groups’ seroconversion rates.  
A 10% dropout rate is assumed.  The target number of patients to be recruited per study arm is 
therefore 140. 
9.7.5 Statistical/analytical issues 
 Adjustments for covariates  
No adjustment for covariates in statistical analyses are planned, as the study is randomized and 
double-blinded. 
  Handling of dropouts or missing data 
 
The handling of dropouts and missing data will be provided in the SAP. 
 
 Multi-centre studies  
The study will be conducted at one site. 
 Multiple comparison/multiplicity 
Only a single primary hypothesis test will be performed, giving no need for multiple testing 
adjustment. All other tests will be considered descriptive and should be interpreted with caution. 
Exploratory statistical tests will be adjusted for multiple testing by Holm’s method. 

 
 
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Active-control studies intended to show equivalence 
 
Not applicable 
 
 Examination of subgroups 
 
No examination of subgroups is planned 
  Interim analyses and data monitoring 
 
There will be no interim analysis, internal Safety Review Committee or Data Monitoring 
Committee for this study. 
9.8 
Data Management 
 
Data management based on GCP refers to the activities defined to achieve safe routines to 
efficiently enter subject information into a database, avoiding errors. 
The data management activities, such as procedures of validating data entered in the e-CRF, will 
be described in a separate data management plan. The e-CRF will be designed in accordance 
with the Clinical Study Protocol.  
9.8.1 The web based e-CRF 
 
Clinical data (including AEs and concomitant medications) will be entered into a 21 CFR Part 
11-compliant e-CRF (Medicase) provided by Scandinavian CRO. The e-CRF includes password 
protection with 2-Factor Authentication and internal quality checks, such as automatic range 
checks, to identify data that appear inconsistent, incomplete, or inaccurate. Clinical data will be 
entered directly from the source documents, which are to be defined at the site before inclusion 
of the first subject. 
Authorised study site personnel designated by the Investigator will complete data collection. 
Appropriate training will be completed with the Investigator and all authorised study site 
personnel prior to any data being entered into the system for any study subject. Each user will 
have a personal and unique account. E-CRF data entries will be attributable to the unique user. 
Captured data will be monitored electronically. Source data verification (SDV) and source data 
review will be performed at the site as described in the monitoring guidelines. 
 
9.8.2 Entering of data into the e-CRF 
 
All data must be entered in English. 

 
 
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The e-CRFs should always reflect the latest observations made during the subject’s participation 
in the study. The e-CRFs should be completed as soon as possible during or after the subject’s 
visit. 
If some assessments are not done, or if certain information is not available, not applicable or 
unknown, this will be indicated in the e-CRF. 
Entries triggering internal e-CRF checks will be presented as errors and warnings. Forms with 
incomplete entries or that contains triggered errors cannot be set to completed state. 
The Investigator must verify that all data entries in the CRFs are accurate and correct by signing 
the completed CRF. 
Once all data have been entered, verified, and validated, the database will be locked. 
After the e-CRF has been locked, the data will be sent to the Sponsor and a copy to the research 
clinic to be filed in the Investigator Study File (ISF) for archiving. 
 
9.8.3 The query process 
 
The Monitor will review the e-CRFs and source documents to evaluate them for completeness 
and consistency. The e-CRF will be compared with the respective source documents to ensure 
that there are no discrepancies between critical data (SDV), in accordance with the monitoring 
guidelines. 
Data Manager will perform data validation checks on data exported from the e-CRF, as defined 
in the Data Management Plan (DMP). 
Queries will be raised in the e-CRF by Monitor or Data Manager respectively if inconsistencies 
or suspected errors are found. Queries shall be resolved, and data updated if applicable, in a 
timely manner by authorised study site personnel. The Monitor or Data Manager cannot enter or 
update data in the e-CRF. 
9.8.4 Source documents 
Data entered into the subjects’ medical record at the clinic and original analysis results at the 
laboratory will be considered source data. The e-CRF is considered as source data when data is 
entered directly into the e-CRF. A Data Management Plan (DMP) will be written, detailing the 
collection of data into the study database. 
Source documents are all documents used by the Investigator or hospital that relate to the 
subject’s medical history, that verifies the existence of the subject, the inclusion and exclusion 
criteria, and all records covering the subject’s participation in the study. Source documents 
include laboratory results, memoranda, material dispensing records, subject files, etc.  
The Investigator is responsible for maintaining source documents. These will be made available 
for inspection by the Monitor at each monitoring visit. All supportive documentation submitted 
with the e-CRF, such as laboratory or hospital records, should be clearly identified with the study 
ID and Subject Number. Any personal information, including name, should be removed or 

 
 
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rendered illegible to preserve individual confidentiality. 
 
 
9.8.5 Audit trail 
 
All changes made to data entered into the e-CRF will be recorded in a protected audit trail 
(logging name of the person making the change, date and time, and if relevant the reason for 
change). 
 
 
10 
EMERGENCY PROCEDURES 
10.1 
Emergency contacts 
In the case of a medical emergency the Investigator must contact the Chief Medical Officer at 
SBH (see below). 
Name  
Function in the study  
Phone number and e-mail 
Per Arne Parment 
Medical Advisor 
Phone: +46 (0)70 644 47 71 
E-mail:xxx@scandinavianbiopharma.se  
10.2 
Procedures in case of medical emergency 
The Investigator is responsible for ensuring that there are procedures and expertise available to 
cope with medical emergencies during the study. 
10.3 
Procedures in case of overdose 
An overdose is a dose in excess of the dose specified for each cohort in this Clinical Study 
Protocol. 
Over-dosing is highly unlikely to occur in the study since one vaccine and buffer container will 
be used per person. The risk for over-dosing is most relevant for the dmLT adjuvant component 
given to subjects receiving the wet/reference vaccine, where dmLT has to be reconstituted and 
added manually by a pipette to the other vaccine components. Ten-fold higher oral doses dmLT 
(100 µg) compared to the dose intended in this trial (10 µg) have previously been found to be 
safe in healthy adults [26]. Furthermore, four-fold higher doses (4 mg) of the LCTBA component 
have previously been given to Swedish volunteers without causing significant AEs [11]. There 
are no data on overdosing of the bacterial component of ETVAX® in humans. Over-dosing of 

 
 
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any of the vaccine components is expected to give rise to transient gastro-intestinal symptoms 
such as vomiting, nausea and loose stools.  
There is no known antidote and an overdose in a study subject should be monitored closely and 
treated symptomatically. Any overdose should be recorded as follows:  
An overdose with associated AE is recorded as the AE diagnosis/symptoms on the relevant AE 
modules in the e-CRF. 
An overdose without associated symptoms is only reported in the subject´s medical records. 
 
11 STUDY MANAGEMENT 
11.1 Changes in the approved study protocol 
Any proposed change to the approved Final Study Protocol (including appendices) will be 
documented in a written and numbered protocol amendment. All amendments including 
substantial changes to the protocol must be submitted to appropriate IEC and Competent 
Authority for approval, according to applicable national regulations.  
11.2 Study timetable 
The end of the clinical part of the study is defined as the last visit of the last subject participating 
in the investigation. 
The study is expected to start in Quarter 3, 2021.   
The main part of the study is expected to be completed by Quarter 3, 2022 and the exploratory 
part by Quarter 4, 2022. 
11.3 Discontinuation of the study 
The SBH reserves the right to discontinue the investigation at any time, but intends only to 
exercise this right for valid scientific or administrative reasons. 
After such a decision, the Investigator must inform all participating subjects and perform 
relevant assessments, preferably according to the scheme for the final assessments. All delivered 
and unused IMP and other study materials must be returned and all CRFs completed as far as 
possible. 
11.4 Reporting and publication of study results 
A Clinical Study Report, in compliance with ICH E3; Structure and content of Clinical Study 
Reports, describing the conduct of the study, the statistical analysis performed and the results 
obtained for the primary and secondary endpoints of the study, will be prepared. 

 
 
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The study results will be reported in the EudraCT database per applicable regulations within 12 
months after completion of the study.  
Results for the exploratory endpoints will be reported separately from the Clinical Study Report. 
If the study duration exceeds one year, SBH must submit an annual safety report to the 
Competent Authority (LV) and to the IEC (EPM). The report will summarize all SAEs and 
contain an update of the risk-benefit evaluation if there has been any change since the approval 
of the clinical study. 
Formal presentation or publication of data collected in this study should be considered as a joint 
publication by the Academic Co-Investigator and a person appointed by SBH. Authorship will 
be determined by mutual agreement.  
Before any publication (oral or written) of the results SBH will be given 30 days for review and 
comment on the manuscript. If the Academic Co-Investigator has not submitted the results for 
publication within six months after completion of the final Clinical Study Report, the SBH has 
the right to publish. In this event, the Academic Co-Investigator will be given 30 days to review 
and comment on the manuscript before it is submitted to a journal. 
11.5 Disclosure and confidentiality 
All unpublished information concerning the test product and research carried out by the SBH, 
including patent applications, manufacturing processes, scientific data from analysis of the 
primary and secondary endpoints etc., is considered confidential and the sole property of SBH. 
Scientific data resulting from the exploratory analyses is also confidential, but is the property of 
both SBH and the Academic Co-investigator. Disclosure to third parties must be limited to those 
undertaking legitimate peer review of the scientific and ethical aspects of the study and to those 
participating, including the recipients of drugs, so that customary medical care and informed 
consent can be achieved. 
11.6  Archiving 
The Investigator must arrange for retention at the investigational site of a list of the subjects and 
their identifying code, subject files and other study documents. The archiving period must be 
adapted to regulations in force and should not be shorter than ten years after the termination of 
the study and the presentation of the final report.   
It is the responsibility of the SBH to inform the investigator/institution as to when these 
documents no longer need to be retained. 
11.7 Insurance/indemnity         
Subjects will be covered through the Swedish Pharmaceutical Insurance 
(Läkemedelsförsäkringen) and the Patient Insurance (Patientförsäkringen).  
 

 
 
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11.8 Study agreements 
The Principal Investigator must comply with all the terms, conditions, and obligations of the 
Clinical Trial Agreement (CTA) for this study.  
Agreements between SBH and the study site must be in place before any study-related 
procedures can take place, or subjects be enrolled. 
 

 
 
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12 REFERENCES 
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Collaborators GBDDD (2017) Estimates of global, regional, and national morbidity, 
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Lanata CF, Black RE (2018) Estimating the true burden of an enteric pathogen: 
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WHO (April 2020) Preferred product characteristics for vaccines against enterotoxigenic 
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tpp/PPC_ETEC_April_2020_Public_Consultationpdf?ua=1  
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Svennerholm AM, Lundgren A (2012) Recent progress toward an enterotoxigenic 
Escherichia coli vaccine. Expert Rev Vaccines 11 (4): 495-507 DOI 10.1586/erv.12.12 
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Steffen R (2017) Epidemiology of travellers' diarrhea. J Travel Med 24 (suppl_1): S2-S5 
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Holmgren J, Bourgeois L, Carlin N, Clements J, Gustafsson B, Lundgren A, Nygren E, 
Tobias J, Walker R, Svennerholm AM (2013) Development and preclinical evaluation of 
safety and immunogenicity of an oral ETEC vaccine containing inactivated E. coli 
bacteria overexpressing colonization factors CFA/I, CS3, CS5 and CS6 combined with a 
hybrid LT/CT B subunit antigen, administered alone and together with dmLT adjuvant. 
Vaccine 31 (20): 2457-2464 DOI 10.1016/j.vaccine.2013.03.027 
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Lundgren A, Leach S, Tobias J, Carlin N, Gustafsson B, Jertborn M, Bourgeois L, 
Walker R, Holmgren J, Svennerholm AM (2013) Clinical trial to evaluate safety and 
immunogenicity of an oral inactivated enterotoxigenic Escherichia coli prototype vaccine 
containing CFA/I overexpressing bacteria and recombinantly produced LTB/CTB hybrid 
protein. Vaccine 31 (8): 1163-1170 DOI 10.1016/j.vaccine.2012.12.063 
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12 
Lundgren A, Bourgeois L, Carlin N, Clements J, Gustafsson B, Hartford M, Holmgren J, 
Petzold M, Walker R, Svennerholm AM (2014) Safety and immunogenicity of an 
improved oral inactivated multivalent enterotoxigenic Escherichia coli (ETEC) vaccine 
administered alone and together with dmLT adjuvant in a double-blind, randomized, 
placebo-controlled Phase I study. Vaccine 32 (52): 7077-7084 DOI 
10.1016/j.vaccine.2014.10.069 
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Lundgren A, Jertborn M, Svennerholm AM (2016) Induction of long term mucosal 
immunological memory in humans by an oral inactivated multivalent enterotoxigenic 
Escherichia coli vaccine. Vaccine 34 (27): 3132-3140 DOI 
10.1016/j.vaccine.2016.04.055 
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Akhtar M, Chowdhury MI, Bhuiyan TR, Kaim J, Ahmed T, Rafique TA, Khan A, 
Rahman SIA, Khanam F, Begum YA, Sharif MZ, Islam LN, Carlin N, Maier N, Fix A, 
Wierzba TF, Walker RI, Bourgeois AL, Svennerholm AM, Qadri F, Lundgren A (2019) 
Evaluation of the safety and immunogenicity of the oral inactivated multivalent 
enterotoxigenic Escherichia coli vaccine ETVAX in Bangladeshi adults in a double-
blind, randomized, placebo-controlled Phase I trial using electrochemiluminescence and 
ELISA assays for immunogenicity analyses. Vaccine 37 (37): 5645-5656 DOI 
10.1016/j.vaccine.2018.11.040 
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Qadri F, Akhtar M, Bhuiyan TR, Chowdhury MI, Ahmed T, Rafique TA, Khan A, 
Rahman SIA, Khanam F, Lundgren A, Wiklund G, Kaim J, Lofstrand M, Carlin N, 
Bourgeois AL, Maier N, Fix A, Wierzba T, Walker RI, Svennerholm AM (2020) Safety 
and immunogenicity of the oral, inactivated, enterotoxigenic Escherichia coli vaccine 
ETVAX in Bangladeshi children and infants: a double-blind, randomised, placebo-
controlled phase 1/2 trial. Lancet Infect Dis 20 (2): 208-219 DOI 10.1016/S1473-
3099(19)30571-7 
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Cardeno A, Magnusson MK, Quiding-Jarbrink M, Lundgren A (2018) Activated T 
follicular helper-like cells are released into blood after oral vaccination and correlate with 
vaccine specific mucosal B-cell memory. Sci Rep 8 (1): 2729 DOI 10.1038/s41598-018-
20740-3 
17 
Qadri F, Saha A, Ahmed T, Al Tarique A, Begum YA, Svennerholm AM (2007) Disease 
burden due to enterotoxigenic Escherichia coli in the first 2 years of life in an urban 
community in Bangladesh. Infect Immun 75 (8): 3961-3968 DOI IAI.00459-07 [pii] 
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Svennerholm AM, Holmgren J, Black R, Levine M, Merson M (1983) Serologic 
differentiation between antitoxin responses to infection with Vibrio cholerae and 
enterotoxin-producing Escherichia coli. J Infect Dis 147 (3): 514-522 DOI 
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Levine MM, Young CR, Black RE, Takeda Y, Finkelstein RA (1985) Enzyme-linked 
immunosorbent assay to measure antibodies to purified heat-labile enterotoxins from 
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serodiagnosis and seroepidemiology. J Clin Microbiol 21 (2): 174-179 DOI 
10.1128/JCM.21.2.174-179.1985 

 
 
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20 
Lebens M, Shahabi V, Backstrom M, Houze T, Lindblad N, Holmgren J (1996) Synthesis 
of hybrid molecules between heat-labile enterotoxin and cholera toxin B subunits: 
potential for use in a broad-spectrum vaccine. Infect Immun 64 (6): 2144-2150 
21 
Clements JD, Norton EB (2018) The Mucosal Vaccine Adjuvant LT(R192G/L211A) or 
dmLT. mSphere 3 (4) DOI 10.1128/mSphere.00215-18 
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Holmgren J, Clemens J, Sack DA, Svennerholm AM (1989) New cholera vaccines. 
Vaccine 7 (2): 94-96 DOI 10.1016/0264-410x(89)90042-x 
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Freytag LC, Clements JD (2005) Mucosal adjuvants. Vaccine 23 (15): 1804-1813 DOI 
S0264-410X(04)00833-3 [pii] 
10.1016/j.vaccine.2004.11.010 
24 
Kotloff KL, Sztein MB, Wasserman SS, Losonsky GA, DiLorenzo SC, Walker RI (2001) 
Safety and immunogenicity of oral inactivated whole-cell Helicobacter pylori vaccine 
with adjuvant among volunteers with or without subclinical infection. Infect Immun 69 
(6): 3581-3590 DOI 10.1128/IAI.69.6.3581-3590.2001 
25 
Norton EB, Lawson LB, Freytag LC, Clements JD (2011) Characterization of a mutant 
Escherichia coli heat-labile toxin, LT(R192G/L211A), as a safe and effective oral 
adjuvant. Clin Vaccine Immunol 18 (4): 546-551 DOI CVI.00538-10 [pii] 
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El-Kamary SS, Cohen MB, Bourgeois AL, Van De Verg L, Bauers N, Reymann M, 
Pasetti MF, Chen WH (2013) Safety and immunogenicity of a single oral dose of 
recombinant double mutant heat-labile toxin derived from enterotoxigenic Escherichia 
coli. Clin Vaccine Immunol 20 (11): 1764-1770 DOI 10.1128/CVI.00464-13 
CVI.00464-13 [pii] 
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Harro C, Louis Bourgeois A, Sack D, Walker R, DeNearing B, Brubaker J, Maier N, Fix 
A, Dally L, Chakraborty S, Clements JD, Saunders I, Darsley MJ (2019) Live attenuated 
enterotoxigenic Escherichia coli (ETEC) vaccine with dmLT adjuvant protects human 
volunteers against virulent experimental ETEC challenge. Vaccine 37 (14): 1978-1986 
DOI 10.1016/j.vaccine.2019.02.025 
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Wenzel H, Shaw RM, Dyke AS Repeat dose oral toxicity study in Sprague- Dawley rats 
of double mutant labile toxin (dmLT). Study report No SVT09-20 Sykesville MD; 2010  
29 
Qadri F, Ahmed T, Ahmed F, Begum YA, Sack DA, Svennerholm AM, Group PTES 
(2006) Reduced doses of oral killed enterotoxigenic Escherichia coli plus cholera toxin B 
subunit vaccine is safe and immunogenic in Bangladeshi infants 6-17 months of age: 
dosing studies in different age groups. Vaccine 24 (10): 1726-1733 DOI 
10.1016/j.vaccine.2005.08.110 
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Qadri F, Ahmed T, Ahmed F, Begum YA, Sack DA, Svennerholm AM (2006) Reduced 
doses of oral killed enterotoxigenic Escherichia coli plus cholera toxin B subunit vaccine 
is safe and immunogenic in Bangladeshi infants 6-17 months of age: dosing studies in 
different age groups. Vaccine 24 (10): 1726-1733 DOI S0264-410X(05)01021-2 [pii] 
10.1016/j.vaccine.2005.08.110 

 
 
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31 
Lundgren A, Jertborn M, Svennerholm AM (2016) Induction of long term mucosal 
immunological memory in humans by an oral inactivated multivalent enterotoxigenic 
Escherichia coli vaccine. Vaccine Accepted for publication  
32 
Jertborn M, Svennerholm AM, Holmgren J (1993) Evaluation of different immunization 
schedules for oral cholera B subunit-whole cell vaccine in Swedish volunteers. Vaccine 
11 (10): 1007-1012 DOI 10.1016/0264-410x(93)90125-h 
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systematic review of the use of the non-inferiority margin in vaccine trials. Vaccine 33 
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Chakraborty S, Harro C, DeNearing B, Bream J, Bauers N, Dally L, Flores J, Van de 
Verg L, Sack DA, Walker R (2016) Evaluation of the Safety, Tolerability, and 
Immunogenicity of an Oral, Inactivated Whole-Cell Shigella flexneri 2a Vaccine in 
Healthy Adult Subjects. Clin Vaccine Immunol 23 (4): 315-325 DOI 
10.1128/CVI.00608-15 
35 
Riddle MS, Kaminski RW, Di Paolo C, Porter CK, Gutierrez RL, Clarkson KA, Weerts 
HE, Duplessis C, Castellano A, Alaimo C, Paolino K, Gormley R, Gambillara Fonck V 
(2016) Safety and Immunogenicity of a Candidate Bioconjugate Vaccine against Shigella 
flexneri 2a Administered to Healthy Adults: a Single-Blind, Randomized Phase I Study. 
Clin Vaccine Immunol 23 (12): 908-917 DOI 10.1128/CVI.00224-16 
36 
Carlin N, Lebens M Method of producing Thy-A-deficient strains of Vibrio cholerae, 
such strains and their use. Sweden: European patent office; EP1080211B1, 1999. .  
37 
Svennerholm AM, Tobias J (2008) Vaccines against enterotoxigenic Escherichia coli. 
Expert Rev Vaccines 7 (6): 795-804 DOI 10.1586/14760584.7.6.795 
38 
Tobias J, Svennerholm AM, Carlin NI, Lebens M, Holmgren J (2011) Construction of a 
non-toxigenic Escherichia coli oral vaccine strain expressing large amounts of CS6 and 
inducing strong intestinal and serum anti-CS6 antibody responses in mice. Vaccine 29 
(48): 8863-8869 DOI 10.1016/j.vaccine.2011.09.096 
39 
Carlin N, Lebens M Method of producing Thy-A-deficient strains of Vibrio cholerae, 
such strains and their use Sweden: European patent office; EP1080211B1, 1999  
 
 
 

 
 
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12.1 Signature pages 
 
 
Principal Investigator 
“I agree to the terms of this Study Protocol. I will conduct the study in accordance with 
the procedures specified in the protocol, the ethical principles in the latest version of the 
Declaration of Helsinki, ICH Good Clinical Practice and applicable regulatory 
requirements”. 
 
 
 
 
 
 
 
 
 
Investigator Name 
 
Signature 
 
Date 

 
 
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Sponsor 
“I agree to the terms of this Study Protocol.” 
 
 
 
 
Medical Advisor (Sponsor signatory) 
 
 
 
 
 
 
Name 
 
Signature 
 
Date 
 
 
 

 
 
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12.2 Declaration of Helsinki 
http://www.up.ac.za/media/shared/Legacy/sitefiles/file/45/2875/declarationofhelsinki_fortaleza_brazil
2013.pdf 
 
 
 
 
 

 
 
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12.3 Appendix 3: Clinical Toxicity Grading   
 
Event 
Reference Range of Normal 
Values 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
General Severity 
Grading 
 
An adverse event which is 
relatively mild and transient in 
nature, but can be an 
annoyance, and does not 
interfere with normal 
activities. 
An adverse event which may be 
uncomfortable but is not 
hazardous to health. It may be 
sufficiently discomforting to 
interfere with normal activities 
but does not completely prevent 
them. 
An adverse event which is 
incapacitating and/or it is a 
hazard to the subject. 
Systemic Reactogenicity 
 
Grade I – Mild 
Grade II – Moderate 
Grade III - Severe 
Diarrhea 
 
3 loose/liquid stools during 24 
hour period or loose/liquid 
stools totaling 200- 400 g 
4-5 loose/liquid stools during 24 
hour period, or loose/liquid 
stools totaling 401 - 800g 
6 or more loose/liquid stools 
during 24 hour period, or 
loose/liquid stools totaling  > 
800g 
Fever (Oral, Ear) 
 
≥100.4°F and ≤ 101.1°F (38.0-
38.4°C) 
>101.1°F and ≤ 102.0°F (38.5-
38.9°C) 
> 102.0°F (39.0°C) 
Vomiting 
 
1-2 episodes within a 24-hour 
period 
3-4 episodes within a 24-hour 
period 
>5 episodes within a 24-hour 
period