Conditions: Pertussis/Whooping Cough Bordetella Pertussis, Whooping Cough
Keywords: Bordetella Pertussis BPZE1 Vaccine Live Attenuated Vaccine Challenge Study
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 53
Healthy volunteers: yes

Brief summary:
This is a randomised, double-blinded, placebo-controlled trial of BPZE1 that includes virulent B. pertussis challenge followed by a safety follow-up.

Detailed description:
This Phase 2b challenge study will investigate colonisation rates, immunologic response, and the safety of BPZE1 vaccination to potentially protect against colonising, virulent wild-type B. pertussis infection in healthy adults using a virulent challenge model. Consenting, eligible participants will receive a single dose of BPZE1 or placebo. 2-4 months later they will be challenged with B. pertussis and admitted to a challenge unit. Participants will remain in the challenge unit for a total of 17 days and 16 nights during which time they will be monitored closely. If a participant develops symptoms of pertussis (per investigator discretion), antibiotic (azithromycin) will be started and the participant will remain in the unit for 3 additional days of observation before discharge. If symptoms of pertussis do not develop, then participants will receive antibiotic (azithromycin) from Days 14-16 of the challenge unit stay. Participants will undergo safety follow-up for at least 6 months post-vaccination and at least 3 months post-challenge, for a total follow-up of 6-7 months.

Arms:
arm 1: Participants will receive an intranasal dose of BPZE1 via the mucosal atomization device followed by a dose of the challenge strain (B. Pertussis strain 1917) approximately 60-120 days later. Participants will receive azithromycin for 3 days beginning 14 days after administration of the challenge strain. arm 2: Participants will receive an intranasal dose of placebo via the mucosal atomization device followed by a dose of the challenge strain (B. Pertussis strain 1917) approximately 60-120 days later. Participants will receive azithromycin for 3 days beginning 14 days after administration of the challenge strain.

Interventions: intervention 1: BPZE1 intervention 2: Placebo intervention 3: Azithromycin intervention 4: Bordetella Pertussis Challenge Strain
Intervention descriptions: intervention 1: Live attenuated vaccine intervention 2: Placebo intervention 3: Antibiotic intervention 4: Challenge Strain

Protocol document:
Prot_000.pdf: 
 
 
Study Number: IB-202P 
Compound No.: BPZE1 
Protocol 
Version: 5 
 
Page 1 of 90 
 
 
CONFIDENTIAL 
 
Title: 
A Phase 2b, Placebo-Controlled, Randomized Study of BPZE1 
Intranasal Pertussis Vaccine in Healthy Adults to Assess 
Protection Against Colonization Following Challenge with 
Virulent Wild-Type Bordetella pertussis 
Effective Date: 
06 March 2023 
 
Short Title: Pertussis challenge study in adults vaccinated with BPZE1 
 
The information contained in this document is the property of ILiAD Biotechnologies, 
LLC and may not be produced, published, or disclosed to others without written 
authorisation from ILiAD Biotechnologies, LLC. 
ILiAD Biotechnologies, LLC  
4581 Weston Road, Suite 260,  
Weston, FL 33331, USA 
 
 
 
 


 
 
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Compound No.: BPZE1 
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CONFIDENTIAL 
SPONSOR SIGNATURE PAGE 
 
Sponsor Signatory: 
 


 
 
Study Number: IB-202P 
Compound No.: BPZE1 
Protocol 
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CONFIDENTIAL 
 
INVESTIGATOR SIGNATURE PAGE 
I agree to conduct this Study in accordance with the requirements of this document (the 
Clinical Study Protocol), the Study Reference Manual, and in accordance with the 
following:  
• Declaration of Helsinki (revised version of Fortaleza, Brazil, 2013). 
• The International Council on Harmonisation harmonised tripartite guideline 
regarding Good Clinical Practice (E6 R2, November 2016). 
• The United Kingdom Statutory Instrument (UKSI) 2004 No. 1031 and UKSI 
2006 No.1928 
• All applicable local and global regulatory requirements 
• Any amendments to these regulations  
 


 
 
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SPONSOR INFORMATION PAGE 
Sponsor Contact Information:  
ILiAD Biotechnologies, LLC  
4581 Weston Road, Suite 260,  
Weston, FL 33331, USA 
 
 


 
 
Study Number: IB-202P 
Compound No.: BPZE1 
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CONFIDENTIAL 
TABLE OF CONTENTS 
PAGE 
ABBREVIATIONS ........................................................................................................... 9 
PROTOCOL SYNOPSIS ............................................................................................... 11 
1. 
INTRODUCTION .................................................................................................... 26 
1.1. 
Background ................................................................................................ 26 
1.1.1. 
Name and Description of the Investigational Product ................... 27 
1.1.2. 
Name and Description of Challenge Agent .................................. 28 
1.1.3. 
Non-clinical Studies ..................................................................... 28 
1.1.4. 
Clinical Studies ............................................................................ 29 
1.1.5. 
Study Conduct ............................................................................. 29 
1.2. 
Rationale .................................................................................................... 30 
1.3. 
Potential Risks and Benefits to Human Subjects ......................................... 31 
1.3.1. 
Potential Risks ............................................................................. 31 
1.3.2. 
Risks of Study Participation ......................................................... 31 
1.3.3. 
Risks Relating to BPZE1 .............................................................. 31 
1.3.4. 
Risks to the Environment or Potential for Interaction with 
Wild-type B. pertussis Strains ...................................................... 32 
1.3.5. 
Risks Relating to the Challenge Agent ......................................... 33 
1.3.6. 
Known Potential Benefits ............................................................. 34 
2. 
OBJECTIVES ......................................................................................................... 34 
2.1. 
Primary Objective ....................................................................................... 34 
2.2. 
Secondary Objectives ................................................................................. 35 
2.3. 
Exploratory Objectives ................................................................................ 35 
3. 
ENDPOINTS .......................................................................................................... 36 
3.1. 
Primary Endpoint ........................................................................................ 36 
3.2. 
Secondary Endpoints .................................................................................. 36 
3.3. 
Exploratory Endpoints ................................................................................. 36 
4. 
STUDY DESIGN .................................................................................................... 37 
4.1. 
Summary of Study Design .......................................................................... 37 
4.1.1. 
Study Design ............................................................................... 37 
4.1.1.1. 
Vaccination Phase ...................................................... 38 
4.1.1.2. 
Challenge Phase ........................................................ 39 
4.1.1.3. 
Follow-up Phase ......................................................... 40 
4.1.2. 
Randomisation and Blinding ........................................................ 40 
4.1.3. 
Duration of Subject Participation .................................................. 40 
4.2. 
Stopping Rules ........................................................................................... 40 
4.2.1. 
Study Stopping Rules/Vaccination or Challenge Pause 
Rules ........................................................................................... 40 
4.2.2. 
Individual Stopping Rules ............................................................ 41 
5. 
STUDY POPULATION ........................................................................................... 41 


 
 
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5.1. 
Number of Subjects .................................................................................... 41 
5.2. 
Eligibility Criteria ......................................................................................... 41 
5.2.1. 
Inclusion Criteria .......................................................................... 41 
5.2.2. 
Exclusion Criteria ......................................................................... 43 
6. 
STUDY ASSESSMENTS AND PROCEDURES ..................................................... 46 
6.1. 
Screening Procedures ................................................................................ 46 
6.1.1. 
Volunteer Information Sheet/Pre-Consent 
Questionnaire/Informed Consent Form/The 
Overvolunteering Prevention System Registration ....................... 46 
6.1.2. 
Inclusion and Exclusion Criteria ................................................... 47 
6.1.3. 
Demographic and Baseline Data ................................................. 47 
6.1.4. 
Medical History ............................................................................ 48 
6.2. 
Efficacy Procedures .................................................................................... 48 
6.2.1. 
Nasopharyngeal Swabs and Nasal Washes ................................ 48 
6.2.2. 
Nasal Mucosal Secretion Samples............................................... 49 
6.2.3. 
Blood Samples............................................................................. 49 
6.2.3.1. 
Serum Immunogenicity ............................................... 49 
6.2.3.2. 
Whole Blood and Peripheral Blood 
Mononuclear Cell Samples ......................................... 49 
6.3. 
Safety Procedures ...................................................................................... 49 
6.3.1. 
Infection Control Rules................................................................. 49 
6.3.2. 
Vital Signs.................................................................................... 51 
6.3.3. 
Physical Examination ................................................................... 51 
6.3.4. 
Laboratory Assessments ............................................................. 52 
6.3.4.1. 
Haematology .............................................................. 52 
6.3.4.2. 
Clinical Chemistry ....................................................... 52 
6.3.4.3. 
Additional Laboratory Parameters .............................. 53 
6.3.5. 
Twelve-Lead Electrocardiogram .................................................. 53 
6.3.6. 
Pregnancy Testing ....................................................................... 54 
6.3.7. 
Post-Vaccination Evaluation ........................................................ 54 
6.3.8. 
Post-Challenge Evaluation ........................................................... 55 
6.3.9. 
Azithromycin ................................................................................ 55 
7. 
LIFESTYLE AND/OR DIETARY RESTRICTIONS .................................................. 55 
8. 
INVESTIGATIONAL PRODUCT ............................................................................. 56 
8.1. 
Dosage and Administration ......................................................................... 56 
8.2. 
Dose Rationale ........................................................................................... 56 
8.3. 
Maintaining the Blind................................................................................... 57 
8.4. 
Treatment Assignment ................................................................................ 57 
8.5. 
Packaging and Labelling ............................................................................. 57 
8.6. 
Preparation ................................................................................................. 58 
8.7. 
Handling and Storage ................................................................................. 58 
8.8. 
Product Accountability and Assessment of Compliance .............................. 58 
8.9. 
Treatment of Investigational Product Overdose .......................................... 58 
8.10. 
Occupational Safety .................................................................................... 59 
9. 
CHALLENGE AGENT ............................................................................................ 59 


 
 
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9.1. 
Manufacturing of the Bordetella pertussis Inoculum .................................... 59 
9.2. 
Quality Assessment of the Inoculum ........................................................... 59 
9.3. 
Transport of the Inoculum ........................................................................... 60 
9.4. 
Storage of the Bordetella pertussis Inoculum .............................................. 60 
9.5. 
Dilution of the Inoculum .............................................................................. 60 
9.6. 
Administration of the Challenge Agent ........................................................ 60 
9.7. 
Monitoring of the Bordetella pertussis Dose Given to the Subject ............... 60 
9.8. 
Disposal of the Inoculum ............................................................................. 61 
10. CONCOMITANT MEDICATIONS AND NON-DRUG THERAPIES ......................... 61 
10.1. 
Permitted Medications ................................................................................ 61 
10.2. 
Prohibited Medications................................................................................ 61 
11. SUBJECT COMPLETION AND WITHDRAWAL ..................................................... 61 
11.1. 
Subject Completion ..................................................................................... 61 
11.2. 
Subject Withdrawal ..................................................................................... 62 
11.3. 
Treatment after the End of the Study .......................................................... 62 
11.4. 
Screen Failures........................................................................................... 62 
12. ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS ..................................... 62 
12.1. 
Definitions ................................................................................................... 62 
12.1.1. 
Adverse Events............................................................................ 62 
12.1.2. 
Treatment-Emergent Adverse Event ............................................ 63 
12.1.3. 
Adverse Events of Special Interest .............................................. 63 
12.1.4. 
Serious Adverse Events ............................................................... 63 
12.1.5. 
Suspected Unexpected Serious Adverse Reactions .................... 63 
12.2. 
Safety Reporting ......................................................................................... 64 
12.2.1. 
Reporting of Adverse Events ....................................................... 64 
12.2.2. 
Reporting of Serious Adverse Events and Adverse Events 
of Special Interest ........................................................................ 64 
12.3. 
Classification of Adverse Events ................................................................. 65 
12.3.1.1. Assessment of Severity .............................................. 65 
12.3.1.2. Assessment of Causality ............................................ 66 
12.4. 
Pregnancy .................................................................................................. 66 
13. DATA ANALYSIS AND STATISTICAL CONSIDERATIONS ................................... 67 
13.1. 
Study Design Considerations ...................................................................... 67 
13.1.1. 
Sample Size Assumptions ........................................................... 67 
13.1.2. 
Sample Size Re-estimation .......................................................... 69 
13.1.3. 
Study Stopping Criteria ................................................................ 69 
13.2. 
Data Analysis Considerations ..................................................................... 69 
13.2.1. 
Analysis Populations .................................................................... 69 
13.2.2. 
Treatment Comparisons .............................................................. 70 
13.2.3. 
Interim Analysis ........................................................................... 70 
13.2.4. 
Key Elements of Analysis Plan .................................................... 70 
13.2.4.1. Efficacy Analyses ....................................................... 70 
13.2.4.2. Safety Analyses .......................................................... 71 
13.2.4.2.1. Adverse Events ...................................... 71 
13.2.4.2.2. Concomitant Medication ......................... 71 


 
 
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13.2.4.2.3. Clinical Laboratory Tests ........................ 71 
13.2.4.2.4. Vital Signs .............................................. 72 
13.2.5. 
Missing, Unused and Spurious Data ............................................ 72 
13.2.6. 
Reporting Deviations from the Statistical Plan.............................. 72 
14. STUDY ADMINISTRATION .................................................................................... 72 
14.1. 
Independent Data Safety Monitoring Board ................................................ 72 
14.2. 
Regulatory and Ethical Considerations, Including the Informed 
Consent Process ........................................................................................ 73 
14.3. 
Study Monitoring ......................................................................................... 73 
14.3.1. 
Access to Source Data ................................................................ 74 
14.3.2. 
Data Handling and Record Keeping ............................................. 74 
14.4. 
Data Management ...................................................................................... 75 
14.5. 
Provision of Study Results and Information to Investigators ........................ 75 
14.6. 
Insurance, Indemnity and Finance .............................................................. 76 
14.7. 
Publishing ................................................................................................... 76 
15. REFERENCES ....................................................................................................... 77 
16. APPENDICES PROVIDED FOR STUDY IB-202P .................................................. 81 
16.1. 
Appendix 1: Schedule of Events ................................................................. 81 
16.2. 
Appendix 2: Table for Reactogenicity Grading ............................................ 85 
16.3. 
Appendix 3: Table for Laboratory Grading .................................................. 87 
16.4. 
Appendix 4: Table for Vital Signs Grading................................................... 89 
16.5. 
Appendix 5: Process to be followed in cases of suspected 
symptomatic Bordetella pertussis infection ................................................. 90 
 
 
 
 


 
 
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CONFIDENTIAL 
ABBREVIATIONS 
aPVs 
Acellular pertussis vaccines 
AE 
Adverse event  
AESI 
Adverse event of special interest 
BMI 
Body mass index 
B. pertussis 
Bordetella pertussis 
CFU 
Colony forming unit  
CI 
Confidence interval 
CRO 
Contract research organisation  
CSR 
Clinical study report 
DNT 
Dermonecrotic toxin 
DSMB 
Data safety monitoring board 
ECG 
Electrocardiogram 
eCRF 
Electronic case report form 
ELISA 
Enzyme-linked immunosorbent assay 
EOS 
End of study 
FDA 
Food and Drug Administration  
FHA 
Filamentous hemagglutinin 
FIM2/3 
Fimbriae types 2 and 3  
FSH 
Follicle stimulating hormone  
GMC 
Geometric mean concentration 
GMFR  
Geometric mean fold rise 
GMP 
Good manufacturing practice  
GMT 
Geometric mean titer 
ICF 
Informed consent form 
ICH-GCP 
International Council on Harmonisation Good Clinical Practice  
IEC 
Independent ethics committee 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
MAD 
Mucosal atomization device 
MedDRA 
Medical dictionary for Regulatory Activities  
NCS 
Not clinically significant 
PBMC 
Peripheral blood mononuclear cell 
PCR 
Polymerase chain reaction  
PRN 
Pertactin 
PT 
Pertussis toxin 
SAE 
Serious adverse event 
SAM 
Synthetic absorptive matrix 
SAP 
Statistical analysis plan 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SAS 
Statistical analysis system 
SBA 
Serum bactericidal assay 
S-IgA 
Secretory immunoglobulin A  


 
 
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SOP 
Standard operating procedures 
SUSAR 
Suspected unexpected serious adverse reaction 
TCT 
Tracheal cytotoxin 
TEAEs 
Treatment-emergent adverse events 
Th 
T helper cell 
TOPS 
The Over-volunteering Prevention System 
UK 
United Kingdom 
ULN 
Upper limit of normal 
VIS 
Volunteer information sheet 
WCE 
Whole cell extract 
WFI 
Water for injection 
WHO 
World Health Organization 
 
 


 
 
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PROTOCOL SYNOPSIS 
Study Information 
Protocol 
Number: 
IB-202P 
Title: 
A Phase 2b, Placebo-Controlled, Randomized Study of BPZE1 
Intranasal Pertussis Vaccine in Healthy Adults to Assess Protection 
Against Colonization Following Challenge with Virulent Wild-
Type Bordetella pertussis 
Sponsor: 
ILiAD Biotechnologies 
4581 Weston Road, Suite 260 
Weston, FL 33331 
Study Phase: 
2b 
Study Sites: 
Two centres in the UK 
Product 
Indication: 
Pertussis immunization booster via mucosal barrier in adults and 
adolescents (≥10 years old) 
 
Rationale 
The resurgence of Bordetella pertussis, despite high vaccination rates in childhood, is 
hypothesized to be linked to expanding use of acellular pertussis vaccines (aPVs), which 
do not prevent B. pertussis transmission or acquisition and induce anti-pertussis 
immunity of short duration. The availability of a non-injectable pertussis vaccine that 
affects colonisation, provides long-term protection, and has the potential to reduce or 
eliminate transmission would present a breakthrough in the prevention of B. pertussis 
disease.  
Current acellular pertussis vaccine strategies do not induce mucosal immunity or prevent 
B. pertussis acquisition that fuels human-to-human transmission, which occurs during the 
initial 30-day period of B. pertussis infection. As humans are the only known natural 
reservoir for B. pertussis, targeting colonisation would provide a novel approach through 
mucosally-induced immunity to reduce the acquisition of B. pertussis, reduce the 
duration of infection, reduce transmission and therefore, ultimately reduce, and 
potentially eliminate, the B. pertussis reservoir in the population. Adolescents and adults 
are the largest reservoir of community B. pertussis, thus controlling transmission and 
outbreaks requires an approach that targets these groups. Recent developments in human 
challenge models for pertussis now allow a pathway forward to assess the degree of 
protection against early infection by way of colonisation assessments. Mucosally-induced 
immunity and protection from infection is expected to ultimately lead to control of 
transmission and reduction of epidemic cycles.  


 
 
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Intranasally administered BPZE1, a live attenuated pertussis vaccine, provides an 
opportunity to generate locally effective mucosal antibodies and T cell responses at the 
site of potential exposure. It mimics the route of entry of the wild-type pathogen and 
induces a broader immune response as measured by cellular, mucosal, and serum indices, 
compared to currently used aPVs. Inducing mucosal immunity with a live attenuated 
vaccine delivered through intranasal vaccination (e.g., BPZE1 vaccine) could halt 
pathogen progression from upper airway infection to toxin-mediated damage. With fewer 
upper airway infections, transmission will be curtailed, and herd immunity will more 
likely be achieved.  
In a recent Phase 2b adult attenuated challenge model, subjects were vaccinated with 
either BPZE1 or an active control acellular pertussis vaccine (Boostrix™) and challenged 
3 months later with BPZE1. The protective efficacy of BPZE1 vaccination against re-
infection was 85% compared with Boostrix (10% and 70% colonisation rates following 
BPZE1 attenuated challenge, respectively).  
This Phase 2b challenge study will investigate colonisation rates, immunologic response, 
and the safety of BPZE1 vaccination to potentially protect against colonising, virulent 
wild-type B. pertussis infection in healthy adults using a virulent challenge model. The 
comparator will be placebo. In addition, evidence of BPZE1-directed induction of 
mucosal immunity will be confirmed. The ability of BPZE1 to induce rapid and sustained 
anti-pertussis systemic immunity (Immunoglobulin A [IgA] and Immunoglobulin G 
[IgG]) along with functional and cellular immunity will also be assessed. This study will 
assist in determining an adequate sample size, appropriate procedures and focused 
objectives and endpoints for a future pivotal Phase 3 virulent challenge study.  
Objectives and Endpoints 
Primary 
Objective 
Endpoint 
To demonstrate that prior immunization with 
BPZE1 protects against colonisation as 
evidenced by a negative B. pertussis culture 
following virulent B. pertussis challenge 2–4 
months after vaccination 
The proportion of subjects by treatment group 
(BPZE1 and placebo) colonised on any day 
(Challenge Day 9, 11 or 14) following virulent 
challenge as determined by culture 
Secondary 
Objectives 
Endpoints 
To demonstrate BPZE1 induction of anti-
pertussis mucosal secretory IgA (S-IgA) 
antibody is improved from baseline to Day 28 
The geometric mean fold rise (GMFR) of 
mucosal anti-pertussis S-IgA antibody (whole 
cell extract [WCE], filamentous hemagglutinin 
[FHA], pertactin [PRN], pertussis toxin [PT] 
and fimbriae types 2 and 3 [FIM2/3]) from 
baseline to Day 28 (BPZE1 and placebo). 


 
 
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Secretory IgA to be normalized ([specific S-
IgA]/[total S-IgA]) 
To demonstrate BPZE1 induction of systemic 
IgA is improved from baseline to Day 28 
The GMFR of serum IgA antibody (WCE, FHA, 
PRN, PT and FIM2/3) from baseline to Day 28 
(BPZE1 and placebo) 
To demonstrate BPZE1 induction of systemic 
IgG is improved from baseline to Day 28 
The GMFR of serum IgG antibody (WCE, 
FHA, PRN, PT and FIM2/3) from baseline to 
Day 28 (BPZE1 and placebo) 
To assess reactogenicity profiles (by toxicity 
scoring) through 7 days following study 
vaccination 
Occurrence and intensity of solicited AEs for 
nasal/respiratory and systemic reactogenicity 
through 7 days following vaccination by 
treatment group (BPZE1 and placebo) 
To describe all unsolicited treatment-emergent 
adverse events (TEAEs) through 28 days 
following study vaccination and following 
virulent challenge 
Occurrence and intensity of TEAEs through 
28 days following study vaccination and 
following challenge by treatment group 
(BPZE1 and placebo) 
To describe all unsolicited TEAEs related to 
vaccination from time of vaccination to 
challenge or related to challenge for 3 months 
after challenge 
Occurrence and intensity of TEAEs related to 
vaccination from time of vaccination to 
challenge or related to challenge for 3 months 
after challenge by treatment group (BPZE1 
and placebo) 
To describe any adverse events (AEs) of 
special interest (AESI) and serious adverse 
events (SAEs) through end of study (EOS) 
Occurrence, intensity, and relationship to study 
vaccine of AESIs and SAEs from vaccination 
through EOS by treatment group (BPZE1 and 
placebo) 
Exploratory 
Objectives 
Endpoint 
To demonstrate prior immunization with 
BPZE1 reduces overall bacterial load following 
virulent B. pertussis challenge 
Absolute counts (colony forming unit; CFU) in 
nasal wash samples on Challenge Days 9, 11 
and 14 following virulent challenge by 
treatment group (BPZE1 and placebo) 
To demonstrate BPZE1 induction of mucosal 
secretory immunity (S-IgA) and systemic 
immunity (IgA and IgG) 
Geometric mean concentration 
(GMC)/geometric mean titre (GMT) of mucosal 
anti-pertussis S-IgA antibody, serum IgA and 
serum IgG antibody (WCE, FHA, PRN, PT and 
FIM2/3) throughout the study, by treatment 
group (BPZE1 and placebo). S-IgA to be 
normalized ([specific S-IgA]/[total S-IgA]) 


 
 
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To demonstrate BPZE1 induction of immunity 
is similar to that observed following virulent 
challenge 
The GMC/GMT and GMFR of mucosal anti-
pertussis S-IgA antibody, serum IgA and 
serum IgG antibody (WCE, FHA, PRN, PT and 
FIM2/3) post-vaccination and post-challenge 
by treatment group (BPZE1 and placebo). S-
IgA to be normalized ([specific S-IgA]/[total S-
IgA]) 
To compare functional antibody response by 
serum bactericidal assay (SBA) 
Proportion of subjects by treatment group 
(BPZE1 and placebo) with an increase in SBA 
50% killing titre 
Correlation and threshold analyses by 
individual and combination anti-pertussis IgG 
antibodies to SBA 
Analysis (GMC/GMT) by treatment group 
(BPZE1 and placebo) for each B. pertussis 
strain studied by SBA and by anti-pertussis 
IgG antibody 
To assess cytokine and T helper cell 
(Th)1/Th2/Th17 dominance response using 
peripheral blood mononuclear cells (PBMCs), 
whole blood and/or nasopharyngeal samples 
Cell-mediated immunity using PBMCs, whole 
blood and/or nasopharyngeal samples, 
including but not limited to cytokines, 
Th1/Th17 and Th2 responses 
Study Population 
A suitable number of subjects (approximately 60) will be screened, randomized and 
vaccinated so that approximately 44 subjects (for a minimum of 20 evaluable subjects per 
arm with culture results during the challenge phase) are enrolled and challenged in the 
study. Subjects who withdraw, are withdrawn due to an unrelated AE as determined by 
the Investigator, are lost to follow-up prior to the challenge phase, or subjects who miss 
all 3 culture results (Challenge Days 9, 11 and 14) during the challenge phase may be 
replaced at the discretion of the Sponsor.  
Inclusion Criteria 
Subjects must meet all the following inclusion criteria to be enrolled in the study: 
1. Is a male or female between 18–50 years of age, inclusive on Day 0 
2. Able to correctly answer all questions in the questionnaire provided during the 
consent process to ensure understanding of the study 
3. Is capable of understanding the written informed consent, provides signed and 
witnessed written informed consent, and agrees to comply with protocol 
requirements 
4. Is fully conversant in the English language 
5. Female subjects must not be pregnant nor breast-feeding and meet one of the 
following criteria: 


 
 
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a. Female subjects must be post-menopausal (defined as 12 consecutive months with 
no menses without an alternative medical cause) or surgically sterile (i.e., 
hysterectomy, bilateral tubal ligation, or bilateral oophorectomy) 
(NOTE: These procedures must be confirmed by physical examination, or by 
subject recall of specific date and hospital/facility of procedure, or by medical 
documentation of the procedure. A blood sample for testing follicle-stimulating 
hormone [FSH] level may be drawn to confirm a post-menopausal state in women 
not using hormonal contraception or hormonal replacement therapy (when 
appropriate) after history of 12 months of no menses has been taken.) 
OR 
b. Female subjects of childbearing potential (defined as any female who has 
experienced menarche and is not yet in menopause) agree to true heterosexual 
abstinence (when this is in line with the preferred and usual lifestyle of the 
subject) from at least 21 days prior to enrolment through to 3 months after the 
virulent challenge or agree to consistently use any of the following methods of 
contraception from at least 21 days prior to enrolment through to 3 months after 
challenge:  
• Condoms (male or female) with spermicide 
• Diaphragm with spermicide 
• Cervical cap with spermicide 
• Intrauterine device 
• Oral or patch contraceptives 
• Other approved pharmaceutical contraceptive methods that are designed to inhibit 
ovulation and protect against pregnancy 
(NOTE: Periodic abstinence [e.g., calendar, ovulation, symptom-thermal, post 
ovulation methods], declaration of abstinence for the duration of exposure to 
study vaccines, and withdrawal are not acceptable methods of contraception.) 
6. Willing to refrain from any nasal sprays (including intranasal steroid sprays) and 
nasal washes not part of the study for 14 days prior to vaccination (Day 0) and for 28 
days following vaccination and challenge  
7. Is a non-smoker at the time of enrolment, has not smoked (or vaped) in the past 
7 days prior to vaccination (including marijuana), and is willing not to smoke (or 
vape; including marijuana) from the time of vaccination throughout the in-unit 
challenge phase 
8. Has a stable health status as assessed by the Investigator inclusive of medical history, 
vital sign assessments and physical examination 


 
 
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9. Has been sufficiently vaccinated (per site and local guidelines) against severe acute 
respiratory syndrome coronavirus 2 (SARS-CoV-2; proof of vaccination required) 
>14 days prior to study vaccination 
10. Has access to a consistent and reliable means of telephone, text, and email contact, 
by personal mobile electronic device 
11. Is able to understand and comply with planned study procedures including admission 
for virulent challenge for 17 days and willingness to take the curative antibiotic 
regimen (azithromycin after inoculation with B. pertussis) 
12. Lives a reasonable distance from the clinical site(s) to be able to travel to and from 
the clinical site(s) for follow-up visits 
13. Agrees to stay in contact with the clinical site for the duration of the study, has no 
current plans to move from the study area and provides updated contact information 
as necessary 
14. Willing to provide written agreement to and abide by infection control rules from 
challenge until 1 week following completion of azithromycin eradication 
Exclusion Criteria 
Subjects must not be enrolled if they meet any of the following exclusion criteria: 
1. Body mass index <17 kg/m2 or >35 kg/m2 
2. History of being vaccinated against pertussis within 5 years of enrolment  
3. History of never being vaccinated for pertussis in lifetime 
4. A diagnosis of pertussis by laboratory confirmation or by physician diagnosis in the 
past 5 years  
5. Serum anti-pertussis toxin IgG >20 IU/mL conducted locally during Screening  
6. Serum anti-pertussis PRN IgG >30 IU/mL conducted locally during Screening  
7. Previously inoculated with B. pertussis in a pertussis challenge study 
8. Screening laboratory values outside of the local laboratory’s normal ranges 
(chemistry or haematology excluding white blood cell differential) and clinically 
significant, except for documented Gilbert’s syndrome with direct bilirubin ≤ upper 
limit of normal. (Laboratory samples may be repeated once during the screening 
period)  
9. Positive test for human immunodeficiency virus, hepatitis B or hepatitis C at 
Screening 
10. Use of illicit drugs (excluding marijuana), evidenced by urine toxicology at 
Screening or a history of drug/alcohol abuse within the past 2 years 


 
 
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11. Any chronic illness being treated actively with evidence of recent intervention for 
worsening or fluctuating symptoms such that a stable baseline of disease is not 
possible (at the discretion of the Investigator) 
12. The subject has a history of active cancer (malignancy) in the last 10 years 
(exception is subjects with adequately treated non-melanomatous skin carcinoma, 
who may participate in the study) 
13. Existing chronic disorders inclusive of lung (e.g., asthma, chronic obstructive 
pulmonary disease), kidney, heart (including screening QT interval corrected 
according to Fridericia’s formula ≥ 440 msec on the 12-lead ECG), liver, diabetes, 
immunodeficiency (acquired or congenital), autoimmune (exception is Hashimoto’s 
thyroid disease) or significant neurologic condition (including, but not limited to, 
facial paralysis or Bell’s palsy). Note, well-controlled chronic disorders including 
hypertension and hypercholesterolemia are permitted 
14. History of Guillain-Barré syndrome (genetic/congenital or acquired) 
15. History of head trauma with potential of cribriform plate fracture within 1 year prior 
to Day 0 
16. History of nasal or sinus surgery within 6 months or receipt of facial cosmetic fillers 
within 3 months prior to Day 0 or diagnosis of nasal polyps 
17. Has taken immunosuppressive therapy or other immune-modifying drugs (including 
but not limited to systemic corticosteroids, biologics, and methotrexate) in the past 
6 months, is on scheduled immunosuppressive therapy or is planning to start 
immunosuppressive therapy during the trial. For systemic corticosteroids this means 
prednisone or equivalent for 10 days or more. The use of corticosteroids (inhaled, 
topical, ophthalmologic or localized injections in joints) are permitted  
18. Receipt of immunoglobulins or any blood products within 3 months prior to study 
vaccine administration or planned receipt during the study 
19. The subject resides in a residence or has routine contact (face to face <2 meters) with 
persons with known immunodeficiency, including persons on immunosuppressant 
therapy from study vaccination to challenge and for 1 week after exiting the 
challenge unit  
20. The subject resides in a residence, works regularly with, or has contact (face to face 
<2 meters) with, infants less than 1 year of age, partially immunised infants or 
pregnant women, adults >65 years of age who have not received a dose of acellular 
pertussis vaccine (e.g. Tdap) within the past 10 years, from study vaccination to 
challenge and for 1 week after exiting the challenge unit 
21. Known hypersensitivity to any component of the study vaccine 
22. Contraindications or allergic to azithromycin, erythromycin or other macrolide 
antibiotics 


 
 
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23. Taking medication that may interact with azithromycin (e.g., nelfinavir, warfarin, 
digoxin and phenytoin) 
24. Inability to adhere to the protocol, visit schedule or sample collection needs 
(including housing in the challenge unit) 
25. Any medical condition, including previous serious event due to a vaccination that, in 
the opinion of the Investigator, might interfere with the evaluation of the study 
objectives or might affect the safety of the individual (e.g., frequent migraines, major 
depression or history of recent suicidal attempt) 
26. Participation in any other clinical trial for the testing of an unlicensed product during 
the previous 3 months or planned during the study conduct 
(NOTE: ‘Testing’ is intended to mean receipt of an investigational product. Subjects in 
long term safety follow-up studies where unlicensed product has completed 
administration >3 months prior to this study enrolment are allowed. Allowance is given 
for subjects who have received a product under emergency use authorization if the 
product has been given >14 days before study enrolment.) 
27. Subject is a first-degree relative of study team member 
Additional Exclusion from Entry into Challenge Unit 
Subjects meeting any of the following criteria will be excluded from the challenge phase:  
28. Has received any form of immunotherapy determined to potentially affect the 
immune response or increase the risk of severe disease in a virulent challenge setting 
(as determined by medical consultation with Sponsor, when necessary)  
29. Has a B. pertussis positive nasal sample (by culture) within 7 days of admission to 
challenge unit 
Other Considerations for Temporarily Holding Vaccination or Entry into Challenge 
Unit 
Subjects meeting any of the following criteria may have a planned study vaccination or 
challenge admission deferred to a later date, but these criteria are not exclusionary for 
study enrolment. Subjects may be enrolled on a return visit if they remain within 45 days 
of consenting. Otherwise, subjects must be re-consented and reassessed per the inclusion 
and exclusion criteria (i.e., re-enrolled). Subjects may be admitted for virulent challenge 
until Day 120 post-vaccination; if a subject is unable to be admitted for virulent challenge 
by Day 120 post-vaccination, the subject should continue to be followed-up for safety per 
the Schedule of Events.  
• 
Has recent symptoms or illness (documented by AE) that requires deferment of a 
virulent challenge due to confounding effects or new/recent disease onset such that a 
virulent challenge is not medically acceptable for safety reasons. Therefore, the 
Investigator will determine the appropriate allowance of time with the subject either 


 
 
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resolving recent illness or medical condition determined to be stable (although within 
the timelines noted above) 
• 
The subject has, or reports, a body temperature of 100.4°F (38°C) or higher within 
the past 3 days. The subject may be vaccinated/challenged once the fever resolves 
and body temperature (subject measured) remains below 100.4°F (38°C) for at least 
3 days 
• 
The subject has, or reports, any infection, acute respiratory tract symptoms or 
rhinorrhoea within the past 3 days. Subject may be vaccinated/challenged once all 
symptoms have been resolved for at least 7 days 
• 
The subject has used antibiotics (including systemic antibiotics, antibiotic lozenges 
and topical antibiotics) within 10 days prior to study vaccination or within 30 days 
prior to challenge  
• 
Has a SARS-CoV-2 positive sample within 3 days prior to time of vaccination or 
challenge unit entry. Requires retest with negative results prior to vaccination or 
prior to entering challenge unit  
Study Design 
This is a randomised, double-blinded, placebo-controlled trial of BPZE1 that includes 
virulent B. pertussis challenge followed by a safety follow-up.  
Consenting eligible subjects will receive a single dose of BPZE1 or placebo. 2–4 months 
later, they will be challenged with B. pertussis and admitted to a challenge unit. Subjects 
will remain in the challenge unit for a total of 17 days and 16 nights during which time 
they will be monitored closely. If a subject develops symptoms of pertussis (per 
Investigator discretion), antibiotic (azithromycin) will be started, and the subject will 
remain in the unit for 3 additional days of observation before discharge. If symptoms of 
pertussis do not develop, then subjects will receive antibiotic (azithromycin) from 
Days 14–16 of the challenge unit stay. Subjects will undergo safety follow-up for at least 
6 months post-vaccination and at least 3 months post-challenge, for a total follow-up of 
6–7 months post-vaccination. 
The exact visits, and procedures to be performed at each visit, can be found in the 
Schedule of Events. 
An independent Data Safety Monitoring Board (DSMB) will be established by the 
Sponsor prior to the study commencing. Refer to the DSMB charter for specific 
responsibilities.  
The primary database lock will occur after the last subject’s virulent challenge visit (Visit 
C16). The longer-term safety and durability responses at the end of the study will be 
included in the final database lock after all planned study procedures through EOS are 
complete. 


 
 
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Vaccination Phase 
After signing the informed consent form (ICF) and meeting all inclusion and none of the 
exclusion criteria, a suitable number of eligible subjects (approximately 60) will be 
randomised 1:1 to receive BPZE1 (109 CFU) or placebo (formulation buffer) via an 
intranasal atomization device (Table 1) so that approximately 44 subjects are enrolled and 
challenged in the study for a minimum of 20 evaluable subjects per arm with culture 
results during the challenge phase. It is estimated that an approximate dropout rate of 
10% during the challenge phase may occur, allowing for a minimum of 20 evaluable 
subjects in each arm.  
 
Table 1 
Dosing Scheme 
Treatment Arms 
N=22a 
Nasal Vaccination (Day 0) 
B. pertussis 
Virulent Challenge (Day 60c) 
BPZE1 
Placebob 
A 
X 
- 
X 
B 
- 
X 
X 
a. 
Evaluable subjects estimated at 20 per treatment arm with two additional to account for dropouts 
b. 
Intranasal application of 0.4 mL per nostril (0.8 mL total volume delivered) formulation buffer placebo 
c. Subject may enter virulent challenge unit between Days 60 to 120 
Unblinded clinical staff will manage vaccine logistics and preparation, but they will not 
be involved in study-related assessments or have subject contact for data collection. 
Blinded individuals will administer the study vaccine.  
On Day 0, nasal/respiratory and systemic reactogenicity will be assessed approximately 
30 minutes after vaccination prior to release from clinical observation. Then, all subjects 
will record maximum daily reactogenicity in a subject diary for 7 consecutive days 
starting the day following study vaccination (retrospective of the highest value in the 
previous 24 hours). Both nasal/respiratory, and general systemic reactogenicity events 
including actual measurements as needed (i.e., body temperature) will be recorded. 
Trained clinical staff will review the information from the subject diary to ensure 
completeness and accuracy. A standard toxicity grade will be assigned to each 
reactogenicity event. Should subjects experience a reactogenicity event with a potential 
toxicity of Grade 3 at any time they should contact the site and be seen or referred to a 
qualified medical facility within 24 hours for further evaluation. 
Challenge Phase 
Between Days 60 and 120 after vaccination (to accommodate in-unit capacity) subjects 
will plan to enter the challenge unit, but they must first be reviewed to be confirmed 
suitable to proceed to virulent challenge (e.g., vitals, clinical exam, culture for B. 
pertussis, additional eligibility criteria). Once subjects are cleared for virulent challenge, 
a dose of the B. pertussis strain B1917 at 105 CFU dose will be administered while lying 


 
 
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supine and using the procedures established within the challenge unit. The inoculum will 
be instilled into each nostril with a Gilson pipette by droplet. Subjects will remain supine 
for 15 minutes. Subjects will remain in the unit for a total of 17 days for observation. 
Daily monitoring will occur including vital signs, physical exam (as needed), 
signs/symptoms of pertussis infection (e.g., catarrhal symptoms), and select clinical 
laboratory testing will occur on pre-specified days. Nasal washes will occur at the 
baseline of the challenge and on Challenge Days 9, 11, and 14, or prior to initiating 
antibiotics should persistent symptoms of pertussis (as per the Investigator’s discretion) 
occur as well as after receiving antibiotics. If persistent symptoms suggestive of pertussis 
disease occur, then subjects will be started on azithromycin and remain in the unit for 
approximately 3 days from the initiation of antibiotics for observation before discharge. 
The remaining subjects will receive azithromycin daily from Day 14 of their challenge 
unit stay and will be discharged from the Challenge Unit on Day 16, after the third dose 
of azithromycin is administered. The 3-day course of azithromycin will be dosed at 
500 mg orally once daily (as indicated by UK Health Security Agency for post-exposure 
prophylaxis for respiratory infections) and subjects must abide by stipulated infection 
control rules. If a B. pertussis culture collected on the last day of the in-unit stay is 
positive, the subject will return to the site to receive continued out-patient antibiotic 
treatment (azithromycin). Nasopharyngeal swabs, mucosal samples and blood samples 
for immunogenicity testing, cell-mediated immunity, and safety (IgA, IgG, functional and 
cell-mediated) will be collected as per the Schedule of Events.  
Follow-up Phase 
Subjects will be followed for safety approximately 6 months following vaccination or 
3 months following challenge, whichever is longer. Adverse events will be collected as 
follows: 
• 
All AEs will be collected through 28 days after study vaccination and 28 days after 
virulent challenge  
• 
All AEs related either to vaccination or to challenge will be collected from time of 
vaccination to challenge or for 3 months after challenge, respectively  
• 
All AESIs and SAEs will be monitored from signing of informed consent through 
EOS  
Study Duration 
The subjects will each be enrolled in the study for up to 8 months. This includes a 
screening phase of up to 30 days prior to enrolment, a vaccination phase lasting up to 
120 days post-vaccination, a challenge phase of 17 days and a safety follow-up lasting 
until either 180 days post-vaccination or 90 days post-challenge (whichever is longer). 


 
 
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Study Assessments 
Nasal wash samples will be evaluated by culture for B. pertussis colonization to 
determine the colonization status (Yes/No) and colonization burden (bacterial counts). 
Immune measurements will be conducted on serum (IgA and IgG) and nasal mucosal 
secretion samples (e.g., nasosorption; S-IgA) for anti-pertussis antibodies of WCE, PT, 
FHA, PRN, and FIM2/3. Serum bactericidal activity (50% killing titre) will be 
determined. Nasopharyngeal swab samples and blood samples (whole blood and PBMCs) 
will be collected to be evaluated for cell-mediated immunity. Additional testing for 
antibodies specific to B. pertussis may be performed at a later date as BPZE1 and virulent 
wild-type B. pertussis induce broad immune responses to many B. pertussis antigens, 
since they do not only contain the purified antigens found in Tdap booster vaccines 
(standard of care). Safety laboratory testing for study entry and additional testing during 
the challenge unit stay will be conducted according to the Schedule of Events and using 
FDA toxicity scoring parameters.  
Subjects will consent for the use of samples for further anti-pertussis antibody testing or 
other assay development as part of the standard consenting process. Aliquots of collected 
samples from this study may be retained for additional testing of biological responses 
(i.e., antibodies, T-cell responses, microbiome) specific to future development of BPZE1 
and ILiAD Biotechnologies’ B-Tech program for a maximum of 15 years (starting from 
the date at which the last subject had the last study visit), unless local rules, regulations, 
or guidelines require different time frames or different procedures, and in accordance 
with subject consent. 
Safety assessments for all subjects will include the following: vital sign measurements 
(heart rate, systolic/diastolic blood pressure, temperature, respiratory rate, oxygen 
saturation), physical examinations (including weight, height, and derived BMI at 
baseline), solicited 7-day reactogenicity, and unsolicited AEs (including AESIs and 
SAEs). Past medical history and medications/vaccines will be recorded, as well as any 
vaccination that occurs outside of the trial during the trial period. Additional collection of 
data elements (vital signs, symptoms, significant physical exam findings during challenge 
unit stay) will also be included. Specific daily evaluations for pertussis-related symptoms 
will be collected. 
Study Vaccination 
• BPZE1, reconstituted with sterile water to provide 109 CFU per 0.8 mL; 
administered intranasally, with approximately 0.4 mL per nostril (0.8 mL total 
volume delivered), by mucosal atomization device (MAD) 
• Intranasal placebo (lyophilized BPZE1 buffer reconstituted with sterile water); 
administered intranasally, with approximately 0.4 mL per nostril (0.8 mL total 
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Statistics 
Sample Size 
Table 3 depicts the power for the challenge study as computed under the following 
assumptions: two-sided significance level of 0.05, use of a likelihood ratio test for testing 
equality of two proportions, a sample size of 20 in each of the two groups, a colonisation 
rate of 60%, 70%, and 80% in the unvaccinated group and colonisation rates of 10%, 
20%, 30%, and 40% in the vaccinated group.  
Group sample sizes of 20 evaluable subjects in each group achieve a power of at least 
90% to detect differences between colonisation rates of at least 50% in six scenarios. 
These scenarios include the following colonisation rates for the placebo versus BPZE1 
groups, respectively: 60% versus 10%, 70% versus 10%, 70% versus 20%, 80% versus 
10%, 80% versus 20% and 80% versus 30%, which are bolded in Table 3.  
 
Table 3 
Power for Sample Sizes of 20 in Each Group for Varying 
Colonisation Rates Based on a Likelihood Ratio Test 
Colonisation Rate 
 
 
Placebo 
BPZE1 
N/group 
Power 
 
 
 
 
60% 
10% 
20 
94% 
 
20% 
 
75% 
 
30% 
 
48% 
 
40% 
 
24% 
 
 
 
 
70% 
10% 
20 
99% 
 
20% 
 
92% 
 
30% 
 
73% 
 
40% 
 
48% 
 
 
 
 
80% 
10% 
20 
100% 
 
20% 
 
99% 
 
30% 
 
92% 
 
40% 
 
75% 
 
Table 4 includes results based on vaccine efficacy computed from the colonisation rates 
in each population, rather than comparing the two colonisation rates and testing the null 
hypothesis that they are equal in the two populations. The computations presented in the 
table below are based on a normal approximation. The table includes the required sample 


 
 
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size for a power of 80% and for lower bounds of vaccine efficacy of 0%, 10%, 20%, and 
30%. For a colonisation rate of 70% in the placebo group and a lower bound of efficacy 
of 20%, the study will be powered for efficacy rates of 71% or higher. Focusing on an 
efficacy rate greater than 50% and a lower bound of 0% (sample sizes in bold), the study 
is well powered to detect efficacy rates of 57% or higher. 
 
Table 4 
Estimated Sample Size Based on Assumed Colonisation rates and 
Power of 80% and Lower Bounds of 0%, 10%, 20% and 30% 
Colonisation rate 
Sample size/group 
Control 
Vaccine 
Efficacy 
LB 0% 
LB 10% 
LB 20% 
LB 30% 
60% 
10% 
0.833 
11 
12 
14 
18 
 
20% 
0.667 
18 
22 
29 
42 
 
30% 
0.500 
33 
47 
75 
149 
 
40% 
0.333 
77 
141 
390 
5675 
 
 
 
 
 
 
 
70% 
10% 
0.857 
8 
9 
10 
12 
 
20% 
0.714 
12 
14 
18 
24 
 
30% 
0.571 
19 
25 
36 
60 
 
40% 
0.429 
33 
51 
95 
268 
 
 
 
 
 
 
 
80% 
10% 
0.875 
6 
6 
7 
9 
 
20% 
0.750 
8 
9 
12 
16 
 
30% 
0.625 
12 
15 
20 
31 
 
40% 
0.500 
18 
25 
40 
82 
 
For the endpoint of absolute counts, the standard deviation was computed to be 2.2 for 
the natural log of the counts in the BPZE1 group and 4.2 for the natural log of the counts 
in the control group. Assuming a sample size of 20 evaluable subjects in each group, a 
two-sample t-test accounting for unequal variances, and a two-sided significance level of 
0.05, the study has approximately 80% power to detect differences of the order of 3.1 
units between the BPZE1 group and the control group on the natural log scale. 
Statistical Methods 
Statistical analysis will be performed using SAS (Statistical Analysis System) software 
Version 9.4 or later. Continuous variables will be summarized using the mean, standard 
deviation, median, first quartile, third quartile, minimum value, and maximum value. 
Categorical variables will be summarized using frequency counts and percentages, as 


 
 
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well as a 2-sided 95% confidence interval (CI) for proportions computed using a Agresti-
Coull method. All statistical tests will be 2-sided at 0.05 significance level.  
For the primary analysis of the between treatment groups difference in the proportion of 
subjects colonized on any day (Challenge Day 9, 11 or 14) following challenge, a 
likelihood ratio test and the corresponding 95% confidence interval will be computed 
with colonization defined as a positive result on any of the 3 days. Additional summaries 
for each of the days will be presented including the results of the likelihood ratio test and 
confidence interval. 
Immunogenicity endpoints (GMC/GMT, GMFR, and/or seroconversion) will be 
summarized at baseline and each time point by treatment group. Reverse cumulative 
distribution curves, which are also known as survival curves, will be presented for each 
measure. Comparison of the BPZE1 and control groups will use a two-sample t-test. 
Confidence intervals will be back-transformed from the log scale to the original scale.  
Exploratory analyses include analyses to assess whether colonization status has a 
relationship to pre-challenge mucosal immunity levels (by individual or by combination 
of anti-pertussis antibodies) or SBA activity. Analyses will include the GMCs/GMTs 
with 95% CIs and GMFRs with 95% CIs and p-values testing as per Statistical Analysis 
Plan (SAP). Pearson’s and Spearman correlations and repeated measures analyses will be 
conducted to assess for correlations and for SBA and protection against colonization. For 
immunogenicity analysis, it is assumed that the natural log of the data is normally 
distributed. All statistical tests will be two-sided at 0.05 significance level and nominal p-
values will be reported.  
The SAP will provide additional details of all analyses. 
 
 
 


 
 
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1. 
INTRODUCTION 
1.1. 
Background 
Bordetella pertussis is a gram negative, aerobic coccobacillus that causes a non-invasive 
pathogenic respiratory tract infection (known commonly as ‘pertussis’ or ‘whooping 
cough’). Since B. pertussis is endemic only to human beings and does not survive for a 
meaningful duration in the air or on surfaces, disease transmission typically occurs 
between infected humans and their close contacts via water droplets and aerosolized 
spray expelled by coughing, sneezing, talking, or breathing during the early infectious 
period. Bordetella pertussis induces a two-stage disease process which may last up to 3 
months: a) the infectious stage where upper respiratory tract colonisation/ infection is 
established and symptoms manifest; and b) the toxin-mediated stage in which damage to 
the lower respiratory tract has occurred due to B. pertussis-specific toxins and is 
irreversible with available medical treatments. The infectious stage begins with non-
specific catarrhal symptoms similar to other upper respiratory infections. Shortly after 
bacteria colonise the upper respiratory tract, physiological changes to the body (i.e., 
disease) have already begun with early catarrhal signs and symptoms often misconstrued 
as the “common cold” (e.g., viral upper respiratory tract infection), and going 
unrecognised/untested, resulting in significant delays in diagnosis. Even in the earliest 
period of infection, damage to epithelial cells and paralysis of the mucosal cilia, which 
mediates the clearance of mucus, has already started and toxin-mediated effects in the 
lower respiratory tract ensue [Babu et al, 2001; Mattoo et al, 2005]. Early use of 
antibiotics to treat pertussis is important, especially as the treatment is unlikely to help 
after 3 weeks of illness [Centers for Disease Control and Prevention]. 
Based on emerging evidence, a review by the Scientific Advisory Group for Emergencies 
through the World Health Organization (WHO) has led to the conclusion that resurgence 
of B. pertussis infections is potentially linked to the use of acellular pertussis vaccines 
(aPVs) and recent analysis by the Advisory Committee on Immunization Practices 
concurs [World Health Organisation, 2015; Liang, 2018]. Furthermore, current position 
and review papers raise the arguments that the combination of limited durability and 
unconstrained disease transmission are playing increasing roles in B. pertussis resurgence 
[Bolotin et al, 2015, Decker et al, 2021; Plotkin et al, 2014; Schwartz et al, 2016; Warfel 
and Edwards, 2015; Witt et al, 2013; Zerbo et al, 2019].  
Multiple studies now support the notion that persistent epidemiological cycles of B. 
pertussis outbreaks are the result of aPVs not controlling the infectious period or disease 
transmission. Instead, aPVs exert their main protective effect on later-stage pertussis 
disease, which is protection against toxin-mediated damage, but with limited durability. 
With B. pertussis infectivity rates similar to that of other childhood diseases in the pre-
vaccination era (e.g., polio, smallpox, measles) and greater than seasonal respiratory 
diseases such as influenza and respiratory syncytial virus, a focus on controlling the first 
stage of infection (e.g., colonising infection) is paramount to controlling recurrent 
epidemics [McGirr et al, 2013; Biggerstaff et al, 2014; Van Boven et al, 2020; 


 
 
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Kretzschmar et al, 2010]. The prolonged period and high bacterial burden of B. pertussis 
upper respiratory infection results in highly contagious individuals who are the source of 
person-to-person B. pertussis transmission, irrespective of aPV vaccination status. A 
systematic review has identified high levels of transmission in humans, including 
asymptomatic individuals. In 14 studies, 46.2% of household contacts testing positive for 
B. pertussis reported having mild/atypical pertussis, whereas in 24 studies, 55.6% of 
household contacts tested positive for B. pertussis but were reported to be asymptomatic 
[Craig et al, 2020]. The mean serial interval (average time between the time of symptom 
onset of a primary case [infector] and that of a secondary case [infectee]) in a systematic 
review was 22.8 days (95% CI 10–35 days) [Vink et al, 2014]. During colonising 
infections, when the disease transmission potential is at its highest, modelling has 
identified secondary attack rate estimates up to 80% [Althouse et al, 2015]. 
In 2015, due to increasing severity and extent of B. pertussis outbreaks, the National 
Institute for Allergy and Infectious Diseases added B. pertussis to the emerging infectious 
disease pathogen list [National Institute of Allergy and Infectious Diseases] and the 
Center for Disease Control and Prevention listed B. pertussis on the watch list 
for Antibiotic Resistance Threats in the United States [Centres for Disease Control and 
Prevention].  
For the next generation of pertussis vaccines, there is a need to combat both transmission 
and durability issues that currently contribute to epidemic outbreaks. To achieve this 
goal, next generation vaccines must ideally have durable protection at the mucosal barrier 
by (1) restricting B. pertussis acquisition and (2) achieving rapid clearance of the 
organism, thereby preventing (a) the long infectious period, (b) the later stage toxin-
mediated damage and (c) halting transmission potential. BPZE1 has demonstrated the 
ability to prevent B. pertussis colonising infections, induce measurable mucosal secretory 
immunoglobulin A (S-IgA) responses, and induce systemic anti-pertussis antibodies after 
a single vaccination (both immunoglobulin A [IgA] and immunoglobulin G [IgG]). The 
demonstration of systemic anti-pertussis antibody responses is important in case B. 
pertussis should ever elude the mucosal barrier of protection established by BPZE1.  
1.1.1. 
Name and Description of the Investigational Product 
BPZE1 has been developed as a live attenuated pertussis vaccine candidate using genetic 
inactivation or removal of three major virulence factors from the B. pertussis Tohama I 
strain: pertussis toxin (PT), tracheal cytotoxin (TCT) and dermonecrotic toxin (DNT) 
[Mielcarek et al, 2006]. The pertussis toxin (pt) gene is modified by altering two codons, 
resulting in the replacement of Arg-9 with lysine and Glu-129 with glycine in the 
substrate binding and catalytic centre of the S1 subunit of PT, respectively. The S1 
subunit mutations prevent adenosine diphosphate-ribosylation of G-proteins of the host 
cell that are involved in signal transduction processes. Both mutations separately abolish 
the enzymatic activity [Locht and Antoine, 1995]. The ampG gene of B. pertussis is 
replaced by the ampG gene of Escherichia coli. This gene substitution results in an 
enhanced internalization of the peptidoglycan breakdown products by the AmpG 


 
 
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transport protein, and the breakdown products are reused for the biosynthesis of the 
bacterial cell wall. The gene substitution results demonstrated an over 99% decrease in 
the excretion of peptidoglycan degradation product TCT by BPZE1 [Mielcarek et al, 
2006]. The B. pertussis dnt gene (the structural gene coding for DNT), which shows 
strong homology with the B. bronchiseptica gene, is deleted. The deletion avoids 
cytoplasmic expression of the toxin in the BPZE1 strain. Dermonecrotic toxin causes 
necrotic lesions after intradermal injection in adult mice, and intravenous administration 
of microquantities is lethal in newborn mice [Locht et al, 1999]. Recently the cellular 
receptor of DNT was identified, which is mainly expressed on cells of the nervous 
system, implicating DNT as a potential neurotoxin [Teruya et al, 2020]. 
1.1.2. 
Name and Description of Challenge Agent 
The PERISCOPE consortium, formed to facilitate the development and licensing of the 
next generation of improved pertussis vaccines, developed a B. pertussis human virulent 
wild-type challenge (virulent challenge) model to predict and evaluate vaccine efficacy 
[PERISCOPE project]. The human model included the use of the same B. pertussis 
isolate to be used in this study, strain B1917, which is representative of current isolates in 
Europe [de Graaf et al, 2020; Bart et al, 2015] and nearly genetically identical to the US 
clinical isolate D420. The strain, isolated in 2000 from a Dutch patient with B. pertussis 
disease, is characterised by ptxP3-ptxA1-prn2-fim3-2, fim2-1 MLVA27, PFGE BpSR11 
and expresses pertactin (PRN), pertussis toxin (PT) and filamentous hemagglutinin 
(FHA). This strain has been extensively characterised in the mouse model as well as by 
proteomics and transcriptomics and has a closed genome available. It is fully sensitive to 
azithromycin in vitro. 
1.1.3. 
Non-clinical Studies 
Direct evidence that aPVs do not alter the B. pertussis infectious period (e.g., 
colonisation of the upper airway) has been demonstrated in the non-human primate 
baboon challenge model. In this model, a full aPV series given to baboons failed to 
prevent upper respiratory tract colonisation from a virulent B. pertussis challenge (strain 
D420), and neither colony counts (burden) nor duration of colonisation was reduced 
compared to naïve baboons [Warfel et al, 2014]. In addition, when aPV-vaccinated 
baboons were challenged and then co-housed with unchallenged naïve baboons, 
transmission was observed up to 40 days later, despite infected baboons being 
asymptomatic [Warfel et al, 2014]. The baboon model that was established demonstrated 
that exposure to wild-type B. pertussis resulted in classical symptoms of pertussis and 
induced leukocytosis, lending credibility that this is a fitting disease model [Warfel et al, 
2012]. Using the same virulent B. pertussis challenge model, when baboons were 
vaccinated with BPZE1 (109 or 1010 colony forming units [CFU]) and then subsequently 
challenged (1.5 × 1010 CFU of B. pertussis, strain D420), protection against both 
colonisation and disease was evident [Locht et al, 2017]. 


 
 
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Further details on non-clinical studies can be found in the Investigator’s Brochure 
[Investigator’s Brochure 2021]. 
1.1.4. 
Clinical Studies 
Success with the baboon model allowed for the next stage in virulent pertussis challenge: 
a controlled human challenge model. This model was established at University of 
Southampton, in the United Kingdom (UK) utilising an isolate of virulent B. pertussis, 
strain B1917. Healthy adults (age 18–45 years) were enrolled if they had not been 
vaccinated with B. pertussis vaccine within 5 years and had low anti-pertussis toxin IgG 
antibodies (≤20 IU/mL) and negative B. pertussis culture to ensure no recent community 
exposure. In this dose escalation study an optimal challenge dose of 105 CFU wild-type 
B. pertussis, the B1917 strain was shown to induce colonisation in 80% of subjects [de 
Graaf et al, 2020]. Some subjects with colonisation had increases in serum anti-pertussis 
toxin IgG levels corresponding to having been infected.  
In addition to virulent challenge models, an attenuated human challenge model has been 
developed using BPZE1 (Tohama I derivative, parental strain BPSM) in the recent 
Phase 2b adult study conducted by ILiAD Biotechnologies [Keech, 2020a; Keech, 
2020b]. The Phase 2b study corroborates what has been observed in the virulent 
challenge models: most individuals (70%) who received an aPV vaccination (Boostrix™) 
just 3 months earlier were unable to avert colonisation with BPZE1 attenuated challenge 
(1x109 CFU). In contrast, prior vaccination 3 months earlier with BPZE1 resulted in 90% 
protection against re-colonisation following attenuated challenge (estimated 85% efficacy 
versus aPVs). In the same study, BPZE1 demonstrated induction of anti-pertussis 
mucosal antibodies, which were not consistently evident following Boostrix vaccination. 
BPZE1 also induced systemic anti-pertussis antibodies with a balanced immunoglobulin 
(Ig)A:IgG response. More recently, in a post-hoc, proof-of-concept study, using a 
randomised convenience sample from the Phase 2b study, BPZE1 vaccination was shown 
to have similar protection in a serum bactericidal assay (SBA) as Boostrix using a B. 
pertussis PRN(+) strain; however, when tested in a B. pertussis PRN(-) strain, BPZE1 
maintained protective antibody levels, whereas Boostrix did not [Keech et al 2021]. This 
suggests that BPZE1 induces a broad functional antibody response. Lastly, BPZE1 has 
been shown to be well-tolerated with acceptable solicited reactogenicity (systemic and 
nasal/mucosal), mainly mild in severity and of limited duration (mean <3 days). Prior 
Boostrix or BPZE1 vaccination followed by attenuated challenge with BPZE1 3 months 
later also resulted in no concerning safety findings.  
Further details on clinical studies can be found in the Investigator’s Brochure 
[Investigator’s Brochure 2021]. 
1.1.5. 
Study Conduct 
The study will be performed in accordance with the ethical principles that have their 
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harmonised tripartite guideline regarding Good Clinical Practice (ICH GCP), the protocol 
and all applicable regulations. 
1.2. 
Rationale 
The resurgence of B. pertussis, despite high vaccination rates, is hypothesized to be 
linked to the expanding use of aPVs, which do not prevent B. pertussis transmission or 
acquisition and induce immunity against pertussis disease of limited duration. The 
availability of a non-injectable pertussis vaccine that affects colonisation, provides 
prolonged protection, and has the potential to reduce or eliminate transmission would 
present a breakthrough in the prevention of colonising B. pertussis infections.  
Current acellular pertussis vaccine strategies do not induce mucosal immunity, nor 
prevent B. pertussis acquisition that fuels human-to-human transmission, which occurs 
during the initial 30-day period of B. pertussis infection. As humans are the only known 
natural reservoir for B. pertussis, targeting colonisation would provide a novel approach 
through mucosally-induced immunity to reduce the acquisition of B. pertussis, reduce the 
duration of infection, ultimately reduce the B. pertussis reservoir in the population and 
potentially eradicate pertussis. Adolescents and adults are the largest reservoir of 
community B. pertussis; controlling transmission and outbreaks requires an approach that 
targets these groups. Recent developments in human challenge models for pertussis now 
allow a pathway forward to assess the degree of protection against early infection by way 
of colonisation assessments. Mucosally-induced immunity and protection from infection 
is expected to ultimately lead to control of transmission and reduction of epidemic cycles.  
The intranasally administered BPZE1 live attenuated vaccine provides an opportunity to 
generate locally effective mucosal antibodies and T cell responses at the site of potential 
exposure, mimic the route of entry of the wild-type pathogen, and induces a broader 
immune response as measured by cellular, mucosal, and serum indices, compared to 
currently used aPVs. Inducing mucosal immunity with a live attenuated vaccine delivered 
through intranasal vaccination (e.g., BPZE1 vaccine) could halt pathogen progression 
from upper airway infection to toxin-mediated damage. With fewer upper airway 
infections, transmission will be curtailed, and herd immunity will more likely be 
achieved.  
In a recent Phase 2b adult attenuated challenge model, subjects were vaccinated with 
either BPZE1 or an active control acellular pertussis vaccine (Boostrix™) and challenged 
3 months later with BPZE1. The protective efficacy of BPZE1 vaccination against re-
infection was 85% compared to Boostrix (10% and 70% colonisation rates following 
BPZE1 attenuated challenge, respectively).  
This Phase 2b virulent challenge study will investigate colonisation rates, immunologic 
response and the safety of BPZE1 vaccination as well as looking at how BPZE1 
potentially protects against colonising wild-type B. pertussis infection in healthy adults 
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evidence of BPZE1-directed induction of mucosal immunity will be confirmed. The 
ability of BPZE1 to induce rapid and sustained anti-pertussis systemic immunity (IgA 
and IgG) along with functional and cell-mediated immunity will be assessed. This study 
will assist in determining an adequate sample size, appropriate procedures and focused 
objectives and endpoints for a future pivotal Phase 3 virulent challenge study.  
1.3. 
Potential Risks and Benefits to Human Subjects 
1.3.1. 
Potential Risks 
Potential risks include risks of study participation (in general), specific risks to study 
subjects and theoretic risks to the environment through the introduction of an attenuated 
B. pertussis strain into human hosts. 
1.3.2. 
Risks of Study Participation 
The risks of study participation include exposure to the study product, exposure to the 
challenge agent, maintenance of confidentiality and side effects of nasal sample 
collections and phlebotomy. All risks will be minimized to every extent possible.  
1.3.3. 
Risks Relating to BPZE1 
The risks of BPZE1 administration are expected to be minimal and clinically 
manageable. Bordetella pertussis colonisation is strictly limited to respiratory epithelium 
without dissemination of the bacteria outside the respiratory tract, which also excludes 
systemic bacteraemia of the BPZE1 strain. Clearance of BPZE1 follows kinetics of wild-
type clearance over the course of 28 days with most subjects clearing in the first 2 weeks 
(data on file). In the recent Phase 2b study, systemic and nasal/respiratory reactogenicity 
events were mainly none to mild in severity with mean duration of 3 days, consistent with 
the safety profile seen in the Phase 1 studies. A second dose of BPZE1 did not induce 
greater reactogenicity events. No Grade 3 reactogenicity events occurred without a 
secondary cause (unrelated to vaccination). Treatment-emergent AEs were mainly mild. 
There were no SAEs attributed to vaccination in any studies to date. 
Bordetella pertussis is spread mainly via aerosol formed by coughing of infected persons. 
The coughing is induced by the TCT, which is more than 99% reduced in BPZE1. The 
BPZE1 strain is not expected to induce coughing (as demonstrated in two adult Phase 1 
and two Phase 2 studies), and thus, transmission is highly unlikely. Bordetella spp. have 
fastidious growth requirements and have limited survival time outside the human body.  
Bordetella pertussis has not been shown to be allergenic in any preclinical or clinical 
studies to date, nor have any of the excipients in the lyophilized formulation. BPZE1 has 
been shown to protect against airway inflammation induced by allergens or viral 
infections in a murine model [Cauchi et al, 2018; Kavanagh et al, 2010; Li et al, 2010; Li 
et al, 2012; Schnoeller et al, 2014]. BPZE1 has also been shown to protect against wild-
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2006]. However, there remains a theoretical risk of allergic reaction, as is present with 
any vaccine product and therefore subjects will remain in clinic for 30 minutes of 
observation following vaccination. 
Either the BPZE1 vaccine or the placebo will be administered nasally via the intranasal 
mucosal atomization device (MAD) attached to a syringe to healthy adult volunteers 
under strictly controlled conditions. The MAD atomizes the liquid vaccine as it exits the 
syringe, and could induce local nasal reactogenicity such as sneezing, irritated nasal 
passage, cough, sore throat or in very rare cases, epistaxis. These reactogenicity 
parameters will be assessed daily for 7 days following study vaccination. 
To minimize the risk of transmission, the subjects will stay at the study centre for at least 
30 minutes after administration on Day 0. In addition, although no cross-contamination 
between subjects has been observed in the previous clinical studies of BPZE1, and no 
risk to the family members of study subjects been reported, as a precaution, subjects with 
frequent contact with children less than 1 year of age (parent, childcare worker, nurse, 
etc.), partially immunized infants, pregnant women, subjects who live in the same 
household as individuals with known immunodeficiency or individuals on 
immunosuppressant therapy, will be excluded from participation in the study.  
No reproductive toxicology studies have been performed with BPZE1 to date; however, 
when female mice were vaccinated with BPZE1 shortly before mating, no negative effect 
on either the pregnancy or their offspring was observed, and the offspring were protected 
against B. pertussis challenge [Feunou et al, 2016]. 
In summary, the risk assessment for this study shows a very low potential risk for the 
study subjects and impact associated with administering BPZE1.  
1.3.4. 
Risks to the Environment or Potential for Interaction with 
Wild-type B. pertussis Strains 
To avoid accidental exposure, actions should be taken to minimize generation of aerosols, 
since the bacterium is strictly a respiratory tract organism. The clinical staff members 
should wear eye-protective glasses and masks during the vaccination. Persons handling 
the BPZE1 bacteria should wear gloves and must wash their hands with a suitable 
disinfecting soap before touching their skin and eyes. Effective antibiotic treatment with 
azithromycin (or an appropriate antibiotic if the subject is allergic to azithromycin) 
should be given in case of accidental transmission to other humans. 
The attenuated strain of B. pertussis (BPZE1) was engineered by genetically altering or 
removing three toxins from B. pertussis: PT, TCT and DNT. The genetic modifications 
(replacement of the ampG gene, deletion of the DNT, and the mutations of the PT) are 
not expected to alter the host range of the B. pertussis BPZE1 strain compared to the 
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The genetic modifications in BPZE1 strongly increase the in vivo safety: 
• 
The double nucleotide mutation in the substrate binding and the active site of the PT 
results in a strong reduction of the enzyme activity 
• 
The replacement of the B. pertussis ampG gene by the E. coli ampG gene results in 
an over 99% reduction in release of the TCT in the medium 
• 
The DNT is not expressed in the BPZE1 strain 
• 
BPZE1 is not invasive and has no selective advantage in the environment. The 
potential for exchange of genetic material is virtually non-existent since B. pertussis 
does not harbour plasmids or conjugative transposons. In addition, B. pertussis 
Tohama I (origin of BPZE1) does not harbour intact prophage genomes and is 
incapable of producing functional phage particles 
Chronic carriage of B. pertussis has not been reported and is not expected. No cross-
contamination between the subjects was observed in the previous Phase 1 or 2 clinical 
trials of BPZE1, nor was any risk to the family members of study subjects observed. In 
case of transmission to other humans, that is, those who are accidentally exposed, an 
efficient treatment against B. pertussis is commercially available and is based on 
administering erythromycin/azithromycin. BPZE1 has been shown to be as sensitive to 
erythromycin as wild-type B. pertussis. 
Due to robust preclinical safety data, BPZE1 has been classified as a Biosafety 
Level 1 organism by French authorities République Française Ministère de 
l’Enseignement Supérieur, de la Recherche et de l’Innovation (French Ministry of Higher 
Education, Research and Innovation). Germany, Belgium, Spain, Sweden and the United 
States have accepted the Biosafety Level 1 rating for the purpose of manufacturing and 
clinical studies.  
In summary, the preliminary risk assessment for this study suggests there is an extremely 
low risk for potential environmental impact associated with administering BPZE1 
vaccine to study subjects. 
1.3.5. 
Risks Relating to the Challenge Agent 
Approximately 60–120 days after inoculation with BPZE1, subjects will be admitted to 
the clinical research challenge unit and challenged with the B. pertussis, B1917 strain at 
105 CFU. Subjects will remain in the unit for a total of 17 days for observation and daily 
monitoring. If persistent symptoms occur, then subjects will be started on azithromycin 
and remain in the unit for an additional 3 days for observation before discharge. The 
remaining subjects will receive azithromycin on Day 14 of their challenge unit stay and 
will be discharged from the Challenge Unit on Day 16 after the third dose of 
azithromycin is administered. The 3-day course of azithromycin will be dosed at 500 mg, 
by mouth, once daily (as indicated by UK Health Security Agency for post-exposure 
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control rules. If the B. pertussis culture collected on the last day of the in-unit stay is 
positive, the subject will return to the site to receive additional antibiotic treatment 
(azithromycin). 
Potential risks of azithromycin include abdominal discomfort, nausea, vomiting, diarrhea, 
rash, liver dysfunction and in rare instances antibiotic-associated colitis, heart rhythm 
problems, pancreatitis, and severe skin/mucosal reactions. See current package insert for 
listing of risks due to use of azithromycin [Package insert 2020]. 
This challenge model has previously been used in a study in which 34 subjects were 
enrolled and approximately 80% of inoculated subjects were infected at the dose level to 
be used in this study. There were no serious adverse events (SAEs) during the course of 
the challenge study, no subjects received rescue-eradication therapy and no subjects 
withdrew due to study-related adverse events (AEs). Minor AEs were reported 
(specifically cough, rhinorrhoea and nasal congestion) but overall, controlled human 
pertussis infection was safe with no significant safety concerns in any subject [de Graaf et 
al, 2020]. 
The challenge element of the study appears to pose no environmental risk as no shedding 
of B. pertussis was detected in any of the systematically collected environmental samples 
(such as those taken from masks, air samples collected during aerosol-provoking 
procedures, bedroom air samples, contact cultures and fingertip cultures). 
1.3.6. 
Known Potential Benefits 
The benefits of the study lie primarily in the opportunities to science and humanity. No 
direct personal benefit from participation in the study can be guaranteed, as the vaccine 
may or may not confer protection in humans. It is possible, though, that taking part in this 
study will result in the subject having a degree of immunity to whooping cough, but we 
cannot be certain that the subject will benefit directly from this study. Subjects will 
receive information about their general health status during the study. 
In summary, although there is no personal benefit to the subjects, the opportunities for 
science and potential future vaccines recipients and the low-risk nature of the study 
procedures mean that, in the opinion of the Sponsor, the risk-benefit profile is favourable 
for performing this study. 
2. 
OBJECTIVES 
2.1. 
Primary Objective 
The primary objective is to demonstrate that prior immunization with BPZE1 protects 
against colonisation as evidenced by a negative B. pertussis culture following virulent 
B. pertussis challenge 2–4 months after vaccination. 


 
 
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2.2. 
Secondary Objectives 
The secondary immunogenicity objectives are: 
• 
To demonstrate BPZE1 induction of mucosal anti-pertussis secretory IgA (S-IgA) 
antibody is improved from baseline to Day 28 
• 
To demonstrate BPZE1 induction of systemic IgA is improved from baseline to 
Day 28 
• 
To demonstrate BPZE1 induction of systemic IgG is improved from baseline to 
Day 28 
The secondary safety objectives are: 
• 
To assess reactogenicity profiles (by toxicity scoring) through 7 days following study 
vaccination 
• 
To describe all unsolicited treatment-emergent AEs (TEAEs) through 28 days 
following study vaccination and following virulent challenge 
• 
To describe all unsolicited TEAEs related to vaccination from time of vaccination to 
challenge or related to challenge for 3 months after challenge 
• 
To describe any AEs of special interest (AESI) and SAE through end of study (EOS) 
2.3. 
Exploratory Objectives 
The exploratory objectives are: 
• 
To demonstrate prior immunization with BPZE1 reduces overall bacterial load 
following virulent B. pertussis challenge 
• 
To demonstrate BPZE1 induction of mucosal secretory immunity (S-IgA) and 
systemic immunity (IgA and IgG) 
• 
To demonstrate BPZE1 induction of immunity is similar to that observed following 
virulent challenge 
• 
To compare functional antibody response by SBA 
• 
Correlation and threshold analyses by individual and combination anti-pertussis IgG 
antibodies to SBA 
• 
To assess cytokine and T helper cell (Th)1/Th2/Th17 dominance response using 
whole blood, peripheral blood mononuclear cells (PBMCs) and/or nasopharyngeal 
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3. 
ENDPOINTS 
3.1. 
Primary Endpoint 
The primary endpoint is the proportion of subjects by treatment group (BPZE1 and 
placebo) colonised on any day (Challenge Day 9, 11 or 14) following virulent challenge 
as determined by culture. 
3.2. 
Secondary Endpoints 
The secondary immunogenicity endpoints are: 
• 
The geometric mean fold rise (GMFR) of mucosal anti-pertussis S-IgA antibody 
(whole cell extract [WCE], FHA, PRN, PT and fimbriae types 2 and 3 [FIM2/3]) 
from baseline to Day 28 (BPZE1 and placebo). Secretory IgA to be normalized 
([specific S-IgA]/[total S-IgA]) 
• 
The GMFR of serum IgA antibody (WCE, FHA, PRN, PT and FIM2/3) from 
baseline to Day 28 (BPZE1 and placebo) 
• 
The GMFR of serum IgG antibody (WCE, FHA, PRN, PT and FIM2/3) from 
baseline to Day 28 (BPZE1 and placebo) 
The secondary safety endpoints are: 
• 
Occurrence and intensity of solicited AEs for nasal/respiratory and systemic 
reactogenicity through 7 days following vaccination by treatment group (BPZE1 and 
placebo) 
• 
Occurrence and intensity of TEAEs through 28 days following study vaccination and 
following challenge by treatment group (BPZE1 and placebo) 
• 
Occurrence and intensity of TEAEs related to vaccination from time of vaccination 
to challenge or related to challenge for 3 months after challenge by treatment group 
(BPZE1 and placebo) 
• 
Occurrence, intensity, and relationship to study vaccine of AESIs and SAEs from 
vaccination through EOS by treatment group (BPZE1 and placebo) 
3.3. 
Exploratory Endpoints 
The exploratory endpoints are: 
• 
Absolute counts (CFU) in nasal wash samples on Challenge Days 9, 11 and 14 
following virulent challenge by treatment group (BPZE1 and placebo) 
• 
Geometric mean concentration (GMC)/geometric mean titre (GMT) of mucosal anti-
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and FIM2/3) throughout the study by treatment group (BPZE1 and placebo). 
Secretory IgA to be normalized ([specific S-IgA]/[total S-IgA]) 
• 
The GMC/GMT and GMFR of mucosal anti-pertussis S-IgA antibody, serum IgA 
and serum IgG antibody (WCE, FHA, PRN, PT and FIM2/3) post-vaccination and 
post-challenge by treatment group (BPZE1 and placebo). S-IgA to be normalized 
([specific S-IgA]/[total S-IgA]) 
• 
Proportion of subjects by treatment group (BPZE1 and placebo) with an increase in 
SBA 50% killing titre 
• 
Analysis (GMC/GMT) by treatment group (BPZE1 and placebo) for each B. 
pertussis strain studied by SBA and by anti-pertussis IgG antibody 
• 
Cell-mediated immunity using whole blood, PBMC and/or nasal pharyngeal 
samples, including but not limited to cytokines, Th1/Th17 and Th2 responses 
4. 
STUDY DESIGN 
4.1. 
Summary of Study Design 
4.1.1. 
Study Design 
This is a randomised, double-blinded, placebo-controlled trial of BPZE1 that includes 
virulent B. pertussis challenge followed by a safety follow-up.  
Consenting, eligible subjects will receive a single dose of BPZE1 or placebo. 2–4 months 
later they will be challenged with B. pertussis and admitted to a challenge unit. Subjects 
will remain in the challenge unit for a total of 17 days and 16 nights during which time 
they will be monitored closely. If a subject develops symptoms of pertussis (per 
Investigator discretion), antibiotic (azithromycin) will be started and the subject will 
remain in the unit for 3 additional days of observation before discharge. If symptoms of 
pertussis do not develop, then subjects will receive antibiotic (azithromycin) from 
Days 14–16 of the challenge unit stay. Subjects will undergo safety follow-up for at least 
6 months post-vaccination and at least 3 months post-challenge, for a total follow-up of 
6–7 months. 
A schematic diagram can be seen in Figure 1. 


 
 
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Figure 1 
Schematic Diagram 
 
The exact visits, and procedures to be performed at each visit, can be found in the 
Schedule of Events (Appendix 1: Schedule of Events). 
An independent Data Safety Monitoring Board (DSMB) will be established by the 
Sponsor prior to the study commencing. Refer to the DSMB charter for specific 
responsibilities. Further information can be found in Section 14.1. 
The primary database lock will occur after the last subject’s virulent challenge visit (Visit 
C16). The longer-term safety and durability responses will be included in the final 
database lock after all planned study procedures through EOS are complete. 
4.1.1.1. 
Vaccination Phase 
After signing the informed consent form (ICF) and meeting all inclusion and none of the 
exclusion criteria, a suitable number of eligible subjects (approximately 60) will be 
randomized 1:1 to receive BPZE1 (109 CFU) or placebo (formulation buffer) via an 
intranasal MAD (Table 1). Approximately 44 subjects will be enrolled and challenged in 
the study for a minimum of 20 evaluable subjects per arm, with culture results during the 
challenge phase. It is estimated that an approximate dropout rate of 10% during the 
challenge may occur, thereby allowing for a minimum of 20 evaluable subjects in each 
arm.  
Table 1 
Dosing Scheme 
Treatment Arms 
N=22a 
Nasal Vaccination (Day 0) 
B. pertussis 
Virulent Challenge (Day 60c) 
BPZE1 
Placebob 
A 
X 
- 
X 
B 
- 
X 
X 
a. 
Evaluable subjects estimated at 20 per treatment arm with two additional to account for dropouts 
b. 
Intranasal application of 0.4 mL per nostril (0.8 mL total volume delivered) formulation buffer placebo 
c. Subject may enter virulent challenge unit between Days 60 to 120 


 
 
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Unblinded clinical staff will manage vaccine logistics and preparation, but they will not 
be involved in study-related assessments or have subject contact for data collection. 
Blinded individuals will administer the study vaccine.  
On Day 0, nasal/respiratory and systemic reactogenicity will be assessed approximately 
30 minutes after vaccination prior to release from clinical observation. Then, all subjects 
will record maximum daily reactogenicity in a subject diary for 7 consecutive days 
starting the day following study vaccination (retrospective of the highest value in the 
previous 24 hours). Both nasal/respiratory and general systemic reactogenicity events 
including actual measurements as needed (i.e., body temperature) will be recorded. 
Trained clinical staff will review the information from the subject diary to ensure 
completeness and accuracy. A standard Food and Drug Administration (FDA) toxicity 
grade will be assigned to each reactogenicity event (see Appendix 2: Table for 
Reactogenicity Grading). Should subjects experience a reactogenicity event with a 
potential toxicity of Grade 3 at any time they should contact the site and be seen or 
referred to a qualified medical facility within 24 hours for further evaluation. 
4.1.1.2. 
Challenge Phase 
Between Days 60 and 120 after vaccination (to accommodate challenge unit capacity) 
subjects will plan to enter the challenge unit, but they must first be confirmed suitable to 
proceed to virulent challenge (e.g., vitals, clinical exam, culture for B. pertussis, 
additional eligibility criteria). Once subjects are cleared for virulent challenge, a dose of 
the B. pertussis strain B1917 at 105 CFU will be administered while lying supine and 
using the procedures established within the challenge unit. The inoculum will be instilled 
into each nostril with a Gilson pipette by droplet. Subjects will remain supine for 15 
minutes. Subjects will remain in the unit for a total of 17 days for observation. Daily 
monitoring will occur including vital signs, physical exam (as needed), signs/symptoms 
of pertussis infection (e.g., catarrhal symptoms), and select clinical laboratory testing will 
occur on pre-specified days (see Schedule of Events [Appendix 1: Schedule of Events] 
and Appendix 3: Table for Laboratory Grading for grading requirements). Nasal washes 
will occur at the baseline of the challenge and on Challenge Days 9, 11, and 14, or prior 
to initiating antibiotics should persistent symptoms of pertussis (as per the Investigator’s 
discretion) occur as well as after receiving antibiotics. If persistent symptoms occur, then 
subjects will be started on azithromycin and remain in the unit for approximately 3 days 
from the initiation of antibiotics for observation before discharge. The remaining subjects 
will receive azithromycin daily from Day 14 of their challenge unit stay and will be 
discharged from the Challenge Unit on Day 16, after the third dose of azithromycin is 
administered. The 3-day course of azithromycin will be dosed at 500 mg orally once 
daily (as indicated by UK Health Security Agency for post-exposure prophylaxis for 
respiratory infections) and subjects must abide by stipulated infection control rules. If a 
B. pertussis culture collected on the last day of the in-unit stay is positive, the subject will 
return to the site to receive continued out-patient antibiotic treatment (azithromycin). 
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cell-mediated immunity, and safety (IgA, IgG, functional and cell- mediated) will be 
collected as per the Schedule of Events (Appendix 1: Schedule of Events).  
4.1.1.3. 
Follow-up Phase 
Subjects will be followed for safety for 6 months following vaccination or 3 months 
following challenge, whichever is longer. Adverse events will be collected as follows: 
• 
All AEs will be collected through 28 days after study vaccination and 28 days after 
virulent challenge  
• 
All AEs related either to vaccination or to challenge will be collected from time of 
vaccination to challenge or for 3 months after challenge, respectively  
• 
All AESIs and SAEs will be monitored from signing of informed consent through 
EOS  
4.1.2. 
Randomisation and Blinding 
This is a double-blind study. Neither the subject nor the Investigator/site staff will know 
what study vaccine the subject is receiving with the exception of site staff involved in the 
preparation of the vaccination. These members of staff will take no further part in the 
study. 
4.1.3. 
Duration of Subject Participation 
The subjects will each be enrolled in the study for up to 8 months. This includes a 
screening phase of up to 30 days prior to enrolment, a vaccination phase lasting up to 
120 days post-vaccination, a challenge phase of 17 days and a safety follow-up lasting 
until either 180 days post-vaccination or 90 days post-challenge (whichever is longer). 
4.2. 
Stopping Rules 
4.2.1. 
Study Stopping Rules/Vaccination or Challenge Pause Rules 
There are no study specific stopping rules.  
Further enrolment and study vaccinations will be paused for DSMB review/ 
recommendation if any of the following are reported: 
• 
Any subject experiences ulceration, abscess, or necrosis in the nose that is 
considered related to study vaccination, through 28 days following study vaccination 
• 
Any subject experiences severe respiratory symptoms of laryngospasm, shortness of 
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• 
Any subject experiences an SAE considered related to study vaccination from the 
time of the vaccination or related to challenge from the time of the challenge through 
to the subject’s last study visit 
• 
Suspected pertussis disease for which early antibiotic therapy is given to >20% of 
subjects administered virulent challenge 
Grading scales for solicited reactogenicity events are included in Appendix 2: Table for 
Reactogenicity Grading. 
If any of the vaccination pause rules are met following any subject receipt of study 
vaccination, then this study will not continue with the remaining enrolments or future 
study vaccinations or admissions to the challenge unit without a review by the DSMB 
and a recommendation from the DSMB to proceed (with or without alteration of 
protocol). Vaccination windows and challenge windows will be adjusted for any such 
pause to allow subjects to remain within the expected schedule of events. All events that 
classify for vaccination or challenge pause rules will be entered as AEs. 
The Sponsor retains the authority to suspend additional enrolment and study interventions 
or administration of study vaccine or challenge agent during the study, as applicable. 
4.2.2. 
Individual Stopping Rules 
There are no individual stopping rules. 
Situations in which a subject may be withdrawn are outlined in Section 11.2. 
5. 
STUDY POPULATION 
5.1. 
Number of Subjects 
A suitable number of subjects (approximately 60) will be screened, randomized and 
vaccinated so that approximately 44 subjects (for a minimum of 20 evaluable subjects per 
arm with culture results during the challenge phase) are enrolled and challenged in the 
study. 
Subjects who withdraw, are withdrawn due to an unrelated AE as determined by the 
Investigator or are lost to follow-up prior to the challenge phase, or subjects who miss all 
3 culture results (Challenge Days 9, 11 and 14) during the challenge phase, may be 
replaced at the discretion of the Sponsor.  
5.2. 
Eligibility Criteria 
5.2.1. 
Inclusion Criteria 
Subjects must meet all the following criteria in order to be enrolled in the study: 


 
 
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1. Is a male or female between 18–50 years of age, inclusive on Day 0  
2. Able to correctly answer all questions in the questionnaire provided during the 
consent process to ensure understanding of the study  
3. Is capable of understanding the written informed consent, provides signed and 
witnessed written informed consent, and agrees to comply with protocol 
requirements 
4. Is fully conversant in the English language 
5. Female subjects must not be pregnant nor breast-feeding and meet one of the 
following criteria: 
a. 
Female subjects must be post-menopausal (defined as 12 consecutive months 
with no menses without an alternative medical cause) or surgically sterile (i.e., 
hysterectomy, bilateral tubal ligation, or bilateral oophorectomy) 
(NOTE: These procedures must be confirmed by physical examination, or by subject 
recall of specific date and hospital/facility of procedure, or by medical 
documentation of said procedure. A blood sample for testing follicle-stimulating 
hormone [FSH] level may be drawn to confirm a post-menopausal state in women 
not using hormonal contraception or hormonal replacement therapy (when 
appropriate) after history of 12 months of no menses has been taken.) 
OR 
b. Female subjects of childbearing potential (defined as any female who has 
experienced menarche and is not yet in menopause) agree to true heterosexual 
abstinence (when this is in line with the preferred and usual lifestyle of the 
subject) from at least 21 days prior to enrolment through to 3 months after the 
virulent challenge or agree to consistently use any of the following methods of 
contraception from at least 21 days prior to enrolment through to 3 months 
after challenge:  
• Condoms (male or female) with spermicide 
• Diaphragm with spermicide 
• Cervical cap with spermicide 
• Intrauterine device 
• Oral or patch contraceptives 
• Other approved pharmaceutical contraceptive methods that are designed to inhibit 
ovulation and protect against pregnancy 
(NOTE: Periodic abstinence [e.g., calendar, ovulation, symptom-thermal, post-
ovulation methods], declaration of abstinence for the duration of exposure to study 
vaccines, and withdrawal are not acceptable methods of contraception.) 


 
 
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6. Willing to refrain from any nasal sprays (including intranasal steroid sprays) and 
nasal washes not part of the study for 14 days prior to vaccination (Day 0) and for 28 
days following vaccination and challenge  
7. Is a non-smoker at the time of enrolment, has not smoked (or vaped) in the past 
7 days prior to vaccination (including marijuana), and is willing not to smoke (or 
vape; including marijuana) from the time of vaccination throughout the in-patient 
challenge phase 
8. Has a stable health status as assessed by the Investigator inclusive of medical history, 
vital sign assessments and physical examination  
9. Has been sufficiently vaccinated (per site and local guidelines) against severe acute 
respiratory syndrome coronavirus 2 (SARS-CoV-2; proof of vaccination required) 
>14 days prior to study vaccination 
10. Has access to a consistent and reliable means of telephone, text, and email contact, 
by personal mobile electronic device 
11. Is able to understand and comply with planned study procedures including admission 
for virulent challenge for 17 days and willingness to take the curative antibiotic 
regimen (azithromycin after inoculation with B. pertussis) 
12. Lives a reasonable distance from the clinical site(s) to be able to travel to and from 
the clinical site(s) for follow-up visits 
13. Agrees to stay in contact with the clinical site for the duration of the study, has no 
current plans to move from the study area and provides updated contact information 
as necessary 
14. Willing to provide written agreement to and abide by infection control rules from 
challenge until 1 week following completion of azithromycin eradication 
5.2.2. 
Exclusion Criteria 
Subjects must not be enrolled if they meet any of the following criteria: 
1. Body mass index <17 kg/m2 or >35 kg/m2 
2. History of being vaccinated against pertussis within 5 years of enrolment  
3. History of never being vaccinated for pertussis in lifetime 
4. A diagnosis of pertussis by laboratory confirmation or by physician diagnosis in the 
past 5 years  
5. Serum anti-pertussis toxin IgG >20 IU/mL conducted locally during Screening  
6. Serum anti-pertussis PRN IgG >30 IU/mL conducted locally during Screening  
7. Previously inoculated with B. pertussis in a pertussis challenge study 
8. Screening laboratory values outside of the local laboratory’s normal ranges 
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significant, except for documented Gilbert’s syndrome with direct bilirubin ≤ upper 
limit of normal. (Laboratory samples may be repeated once during the screening 
period) 
9. Positive test for human immunodeficiency virus, hepatitis B or hepatitis C at 
Screening 
10. Use of illicit drugs (excluding marijuana), evidenced by urine toxicology at 
Screening or a history of drug/alcohol abuse within the past 2 years 
11. Any chronic illness being treated actively with evidence of recent intervention for 
worsening or fluctuating symptoms such that a stable baseline of disease is not 
possible (at the discretion of the Investigator) 
12. The subject has a history of active cancer (malignancy) in the last 10 years 
(exception is subjects with adequately treated non-melanomatous skin carcinoma, 
who may participate in the study) 
13. Existing chronic disorders inclusive of lung (e.g., asthma, chronic obstructive 
pulmonary disease), kidney, heart (including screening QT interval corrected 
according to Fridericia's formula ≥440 msec on the 12-lead ECG), liver, diabetes, 
immunodeficiency (acquired or congenital), autoimmune (exception is Hashimoto’s 
thyroid disease) or significant neurologic condition (including, but not limited to, 
facial paralysis or Bell’s palsy). Note, well-controlled chronic disorders including 
hypertension and hypercholesterolemia are permitted 
14. History of Guillain-Barré syndrome (genetic/congenital or acquired) 
15. History of head trauma with potential of cribriform plate fracture within 1 year prior 
to Day 0 
16. History of nasal or sinus surgery within 6 months or receipt of facial cosmetic fillers 
within 3 months prior to Day 0 or diagnosis of nasal polyps 
17. Has taken immunosuppressive therapy or other immune-modifying drugs (including 
but not limited to systemic corticosteroids, biologics and methotrexate) in the past 
6 months, is on scheduled immunosuppressive therapy or is planning to start 
immunosuppressive therapy during the trial. For systemic corticosteroids this means 
prednisone or equivalent for 10 days or more. The use of corticosteroids (inhaled, 
topical, ophthalmologic, or localized injections in joints) are permitted  
18. Receipt of immunoglobulins or any blood products within 3 months prior to study 
vaccine administration or planned receipt during the study 
19. The subject resides in a residence or has routine contact (face to face <2 meters) with 
persons with known immunodeficiency including persons on immunosuppressant 
therapy, from study vaccination to challenge and for 1 week after exiting the 
challenge unit 
20. The subject resides in a residence, works regularly with, or has contact (face to face 
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pregnant women, adults >65 years of age who have not received a dose of acellular 
pertussis vaccine (e.g. Tdap) within the past 10 years from study vaccination to 
challenge and for 1 week after exiting the challenge unit 
21. Known hypersensitivity to any component of the study vaccine  
22. Contraindications or allergic to azithromycin, erythromycin or other macrolide 
antibiotics 
23. Taking medication that may interact with azithromycin (e.g., nelfinavir, warfarin, 
digoxin and phenytoin) 
24. Inability to adhere to the protocol, visit schedule or sample collection needs 
(including housing in the challenge unit) 
25. Any medical condition, including previous serious event due to a vaccination that, in 
the opinion of the Investigator, might interfere with the evaluation of the study 
objectives or might affect the safety of the individual (e.g., frequent migraines, major 
depression or history of recent suicidal attempt) 
26. Participation in any other clinical trial for the testing of an unlicensed product during 
the previous 3 months or planned during the study conduct  
(NOTE: ‘Testing’ is intended to mean receipt of an investigational product. Subjects in 
long term safety follow-up studies where unlicensed product has completed 
administration >3 months prior to this study enrolment are allowed. Allowance is given 
for subjects who have received a product under emergency use authorization if the 
product has been given >14 days before study enrolment.) 
27. Subject is a first-degree relative of study team member 
Additional Exclusion from Entry into Challenge Unit 
Subjects meeting any of the following criteria will be excluded from the challenge phase:  
28. Has received any form of immunotherapy determined to potentially affect the 
immune response or increase the risk of severe disease in a virulent challenge setting 
(as determined by medical consultation with Sponsor, when necessary)  
29. Has a B. pertussis positive nasal sample (by culture) within 7 days of admission to 
challenge unit 
Other Considerations for Temporarily Holding Vaccination or Entry into Challenge 
Unit 
Subjects meeting any of the following criteria may have a planned study vaccination or 
challenge admission deferred to a later date, but these criteria are not exclusionary for 
study enrolment. Subjects may be enrolled on a return visit if they remain within 45 days 
of consenting. Otherwise, subjects must be re-consented and reassessed per the inclusion 
and exclusion criteria (i.e., re-enrolled). Subjects may be admitted for virulent challenge 
until Day 120 post-vaccination; if a subject is unable to be admitted for virulent challenge 


 
 
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by Day 120 post-vaccination, the subject should continue to be followed-up for safety per 
the Schedule of Events.  
• 
Has recent symptoms or illness (documented by AE) that requires deferment of a 
virulent challenge due to confounding effects or new/recent disease onset such that a 
virulent challenge is not medically acceptable for safety reasons. Therefore, the 
Investigator will determine the appropriate allowance of time with the subject either 
resolving recent illness or medical condition determined to be stable (although within 
the timelines noted above)  
• 
The subject has, or reports, a body temperature of 100.4°F (38°C) or higher within 
the past 3 days. The subject may be vaccinated/challenged once the fever resolves 
and body temperature (subject measured) remains below 100.4°F (38°C) for at least 
3 days 
• 
The subject has, or reports, any infection, acute respiratory tract symptoms or 
rhinorrhoea within the past 3 days. Subject may be vaccinated/challenged once all 
symptoms have been resolved for at least 7 days 
• 
The subject used antibiotics (including systemic antibiotics, antibiotic lozenges and 
topical antibiotics) within 10 days prior to study vaccination or within 30 days prior 
to challenge 
• 
Has a SARS-CoV-2 positive sample within 3 days prior to time of vaccination or 
challenge unit entry. Requires retest with negative results prior to vaccination or 
prior to entering challenge unit  
6. 
STUDY ASSESSMENTS AND PROCEDURES 
Study procedures will be performed at the times outlined in the Schedule of Events 
(Appendix 1: Schedule of Events). No procedures should be performed prior to the 
subject providing written informed consent. 
6.1. 
Screening Procedures 
6.1.1. 
Volunteer Information Sheet/Pre-Consent Questionnaire/Informed 
Consent Form/The Overvolunteering Prevention System Registration 
A volunteer information sheet (VIS) will be available on the study website and may be 
given to potential subjects before the screening visit. The VIS will include all risks and 
safety measures that are involved in the study.  
Individuals who have expressed an interest in taking part in the study will be invited to 
attend a screening session after a short telephone screening. During the screening visit the 
study will be explained. If the subject has any questions they can be addressed during this 
visit.  


 
 
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After their understanding of the study aims and level of involvement required has been 
established and if they wish to participate, subjects will be asked to complete a 
questionnaire testing their understanding of the challenge phase of the trial. Subjects who 
fail to answer all questions correctly on their first attempt will be allowed to re-take the 
questionnaire following further discussion with the investigator. If the subject is not able 
to answer all questions correctly within three attempts, he/she will be asked to read the 
VIS again and come back at least 24 hours later for a screening visit and repeat the pre-
consent questionnaire.  
After the questionnaire has been completed and any questions answered, a written 
informed consent in compliance with regulatory authority regulations shall be obtained 
from each subject before entering the study or performing any non-routine procedure that 
involves risk to the subject. Both the VIS and ICF will be reviewed and approved by the 
sponsor and site(s), prior to independent ethics committee (IEC) review and approval. If 
the VIS and ICF are revised during the course of the study, the all active participating 
subjects must sign the revised forms unless advised by the sponsor, and subject to 
approval by the IEC. 
Once the investigator is assured that the subject understands the implications of 
participating in the study, the subject will be asked to give written informed consent by 
signing and dating the ICF. The authorised person obtaining the informed consent will 
also sign and date the ICF. 
The ICF/VIS will contain separate sections that address the use of remaining mandatory 
samples for exploratory research (if applicable) and explain/address the exploratory 
research portion of the study. Subject medical records must state that written informed 
consent was obtained. 
The investigator shall retain the signed original ICF(s) and provide copies of the signed 
original form(s) to the subject. 
As part of the informed consent process, subjects will also consent to registration in The 
Overvolunteering Prevention Systems (TOPS) database (www.tops.org.uk). Registration 
in TOPS is required to help ensure the health and safety of the subjects during the study, 
by tracking prior study participation, timing of recent sample collections and/or potential 
medication interactions.  
6.1.2. 
Inclusion and Exclusion Criteria 
Inclusion and exclusion criteria will be reviewed for each potential subject. Eligibility 
will be documented in the electronic case report form (eCRF). 
6.1.3. 
Demographic and Baseline Data 
The following data will be collected at the times outlined in the Schedule of Events 
(Appendix 1: Schedule of Events). 


 
 
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• 
Age/date of birth 
• 
Sex 
• 
Race/ethnicity 
• 
Weight 
• 
Height 
• 
Body mass index (electronically derived from weight and height) 
6.1.4. 
Medical History 
The following data will be collected from the subjects at the times outlined in the 
Schedule of Events (Appendix 1: Schedule of Events). General practitioner’s records are 
not required; however, any available site medical records and/or histories should be 
reviewed before randomization. 
• 
Prior medical conditions 
• 
Ongoing medical conditions 
• 
Surgeries 
• 
Significant, non-screening, procedures (e.g., endoscopy, uterine ablation, lithotripsy) 
Ideally, the subject will provide a vaccination card (or equivalent) or the subject’s 
vaccination history can be accessed via a centralised vaccination register to record their 
last known pertussis vaccination. If the subject’s vaccination history is not available, the 
timing of prior pertussis vaccine may be estimated based on the national vaccination 
schedule appropriate for the age of subject and past pregnancies. 
6.2. 
Efficacy Procedures 
Subjects will consent for the use of samples for further anti-pertussis antibody testing or 
other assay development as part of the standard consenting process. Aliquots of collected 
samples from this study may be retained for additional testing of biological responses 
(i.e., antibodies, T-cell responses, microbiome) specific to future development of BPZE1 
and ILiAD Biotechnologies’ B-Tech program for a maximum of 15 years (starting from 
the date at which the last subject had the last study visit), unless local rules, regulations, 
or guidelines require different time frames or different procedures, and in accordance 
with subject consent. 
6.2.1. 
Nasopharyngeal Swabs and Nasal Washes 
Nasopharyngeal swabs and nasal washes should be collected at the times outlined in the 
Schedule of Events (Appendix 1: Schedule of Events) with the nasopharyngeal swab(s) 
(up to two) collected before the nasal wash. 


 
 
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Nasopharyngeal swabs will be used to test for cell-mediated immunity. Nasal washes will 
be used for B. pertussis colonisation by culture and PCR testing. 
Further details on sampling, processing, and shipping procedures can be found in the 
study laboratory manual. 
6.2.2. 
Nasal Mucosal Secretion Samples 
Secretion samples should be collected at the times outlined in the Schedule of Events 
(Appendix 1: Schedule of Events). Samples will be used for testing levels of 
immunogenicity. 
Further details on sampling, processing, and shipping procedures can be found in the 
study laboratory manual. 
6.2.3. 
Blood Samples 
6.2.3.1. 
Serum Immunogenicity 
Samples for serum immunogenicity should be collected at the times outlined in the 
Schedule of Events (Appendix 1: Schedule of Events). The processed samples will be 
shipped to an analytical laboratory where the tests (including enzyme-linked 
immunosorbent assay [ELISA] and SBA) will be performed. 
Further details on sampling, processing, and shipping procedures can be found in the 
study laboratory manual. 
6.2.3.2. 
Whole Blood and Peripheral Blood Mononuclear Cell Samples 
Blood samples for analysis of whole blood and PBMCs should be collected at the times 
outlined in the Schedule of Events (Appendix 1: Schedule of Events). The samples will 
be used to test for cell mediated immunity at a central laboratory. 
Further details on sampling, processing, and shipping procedures can be found in the 
study laboratory manual. 
6.3. 
Safety Procedures 
6.3.1. 
Infection Control Rules 
Subjects will be asked to adhere to infection control measures to limit possible onward 
transmission of B. pertussis. Subjects must sign the infection control agreement during 
Screening and again prior to the virulent challenge (Appendix 1: Schedule of Events).  
Infection control rules are in place beginning with the virulent challenge and ending one 
week after discharge from the challenge unit facility: 


 
 
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During admission: 
• 
Volunteers must wear a surgical mask unless alone in their own personal room or 
bathroom facilities 
• 
Volunteers should wash their hands before leaving their room 
• 
Volunteers must not enter the rooms of other volunteers. 
• 
Volunteers must not have any contact with high risk of transmission with any 
individuals – such contact includes: 
a. Intimate/sexual contact 
b. Contact that may involve transfer of respiratory secretions e.g., kissing 
c. Sharing cutlery or drinking vessels 
• 
The volunteer may receive a maximum of two guests at a time between 8.00 and 
22.00, who must wear masks covering nose and mouth while in close proximity to 
the volunteer and must adhere to strict infection control procedures. These guests 
must not be:  
o Unimmunised or partially immunised children and infants aged <1 year 
o Pregnant women >32 weeks who have not received pertussis vaccination at least 
a week prior to contact  
o Immunosuppressed or frail individuals 
o Healthcare workers regularly working with vulnerable individuals as above  
• 
Volunteers are allowed to leave the challenge unit during daytime for a maximum of 
2 hours twice a day during daytime with the agreement of the study team 
While outside the challenge unit and for 1 week following discharge: 
• 
Volunteers must wash their hands before leaving the challenge unit or their home 
• 
Volunteers must be contactable by mobile phone, which has the study emergency 
phone number programmed in, and contact the clinical study team if they have any 
symptoms suggestive of early pertussis disease.  
• 
Volunteers must be able to return to the challenge unit within 120 minutes 
• 
Volunteers must avoid crowded social environments 
• 
Volunteers must wear a surgical mask during any face to face contact with other 
individuals 
• 
Volunteers must avoid contact with: 
o Unimmunised or partially immunised children and infants aged <1 year 


 
 
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o Pregnant women >32 weeks who have not received pertussis vaccination at least 
a week prior to contact  
o Immunosuppressed or frail individuals 
o Healthcare workers regularly working with vulnerable individuals as above  
• 
Volunteers must not have any contact with high risk of transmission with any 
individuals – such contact includes: 
o Bedroom sharing 
o Intimate/sexual contact 
o Contact that may involve transfer of respiratory secretions e.g., kissing 
o Sharing cutlery or drinking vessels 
For safety, all the subjects will receive antibiotic eradication treatment with a macrolide 
(azithromycin) after the virulent challenge.  
6.3.2. 
Vital Signs 
Vital signs (heart rate, systolic/diastolic blood pressure, temperature, respiratory rate, 
oxygen saturation) will be assessed at the times outlined in the Schedule of Events 
(Appendix 1: Schedule of Events), pre-vaccination and pre-challenge and approximately 
30 minutes following vaccination and challenge, respectively. During the challenge unit 
stay, vital signs will be collected at least twice daily (once in the morning and once in the 
evening). Additional vital signs may be performed at any time point at the Investigator’s 
discretion. 
Subjects should rest in a supine position for 10 minutes before the vital signs are 
assessed. 
Any vital signs with results falling outside normal ranges may be repeated at the 
discretion of the Investigator. After randomisation, if any results falling outside the 
normal ranges are deemed clinically significant by the Investigator or appropriately 
qualified designee, they should be recorded as an AE/SAE. 
6.3.3. 
Physical Examination 
A full or targeted physical examination will be performed at the times outlined in the 
Schedule of Events (Appendix 1: Schedule of Events). During the full physical 
examination, the following areas will be assessed by the Investigator or medically-
qualified designee: general appearance; eyes, ears, nose and throat, head and neck; chest 
and lungs; cardiovascular; abdomen; musculoskeletal; lymphatic; dermatologic; 
neurologic and extremities. 
Additional targeted physical examinations will be symptom-directed and performed at the 
discretion of the investigator, including unscheduled visits, if necessary, to evaluate AEs. 


 
 
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6.3.4. 
Laboratory Assessments 
Samples for safety laboratory tests should be taken at the times outlined in the Schedule 
of Events (Appendix 1: Schedule of Events). Additional samples may be collected at the 
Investigator’s discretion. Any clinically significant results and all Grade 3 or higher 
results should be discussed with the CRO. 
A single repeat sample will be permitted during the screening process to allow inclusion. 
6.3.4.1. 
Haematology 
For safety, haematology will be assessed during the study.  
• 
Total blood count consisting of: 
• 
Haemoglobin concentration 
• 
Platelets 
• 
White blood cells 
• 
Neutrophils 
• 
Lymphocytes 
• 
Monocytes 
• 
Eosinophils 
• 
Basophils 
Blood samples will be analysed locally and samples will be destroyed following database 
lock (at the latest). Further details on processing can be found in the study laboratory 
manual. 
6.3.4.2. 
Clinical Chemistry 
For safety, the following parameters will be assessed during the study:  
• 
Sodium 
• 
Potassium 
• 
Urea 
• 
Creatinine 
• 
Total protein 
• 
Albumin 
• 
Total bilirubin 
• 
Alanine aminotransferase 


 
 
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• 
Alkaline phosphatase 
• 
Random glucose 
• 
C-reactive protein 
Blood samples will be analysed locally and samples will be destroyed following database 
lock (at the latest). Further details on processing can be found in the study laboratory 
manual. 
6.3.4.3. 
Additional Laboratory Parameters 
The following parameters will be assessed, at a local or central laboratory, at specified 
time points, depending on the parameter (see Laboratory Manual): 
• 
Serology 
• Human immunodeficiency virus 
• Hepatitis B 
• Hepatitis C 
• Anti-PT antibodies (local laboratory results to be used for screening purposes. 
Central laboratory results to be used for analyses) 
• Anti-PRN antibodies (local laboratory results to be used for screening purposes. 
Central laboratory results to be used for analyses) 
• 
Urine toxicity screen 
• 
Severe acute respiratory syndrome coronavirus 2 testing (SARS-CoV-2 test for 
active infection (an approved local test accepted by public health authorities and 
readily available) will be administered to subjects within 72 hours prior to 
vaccination and challenge unit admission. Should the subject have a positive SARS-
CoV-2 test result, regardless of symptomatology, then vaccination/admission to the 
challenge unit will be temporarily held until the subject has a negative test result) 
• 
Respiratory PCR panel 
6.3.5. 
Twelve-Lead Electrocardiogram  
A standard 12-lead electrocardiogram (ECG) will be performed at the times outlined in 
the Schedule of Events (Appendix 1: Schedule of Events). Additional ECGs may be 
performed at any time point at the Investigator’s discretion. QT interval corrected 
according to Fridericia’s formula will be calculated. The ECG measurements will be 
made with the subject in a semi-supine position and having rested in this position for at 
least 10 minutes before each time point. 
Any ECG with results falling outside the normal ranges may be repeated at the discretion 
of the Investigator. If any results falling outside the normal ranges are deemed not 


 
 
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clinically significant by the Investigator, or appropriately qualified designee, this should 
be clearly stated on the hard copies of the ECG and signed and dated by the Investigator. 
If an ECG trace indicates an abnormality that is measured by the equipment but is 
deemed normal by the Investigator, this should be clearly stated on the ECG trace as 
normal and signed and dated by the Investigator or appropriately qualified designee. If 
the ECG trace indicates an abnormality that is present but deemed as not clinically 
significant by the Investigator or appropriately qualified designee, then this should be 
clearly stated on the ECG trace as “NCS” and signed and dated by the Investigator or 
appropriately qualified designee. After randomisation, any results falling outside the 
normal ranges and deemed clinically significant by the Investigator or appropriately 
qualified designee should be recorded in the electronic case report form (eCRF) as an 
AE/SAE. 
6.3.6. 
Pregnancy Testing 
Serum and/or urine will be collected from female subjects of childbearing potential at the 
times outlined in the Schedule of Events (Appendix 1: Schedule of Events) to enable 
pregnancy tests to be performed. Serum and urine samples will be analysed locally, and 
samples will be destroyed following analysis. A positive urine pregnancy test during the 
study will be confirmed by a serum test. 
Female subjects who are identified as being pregnant during the study will be withdrawn 
from further participation in study vaccination and/or challenge, but they will continue to 
attend safety follow-up visits.  
6.3.7. 
Post-Vaccination Evaluation 
All subjects will be monitored for at least 30 minutes after study vaccination. The 
following evaluations will be performed: 
1. Obtain vital signs 
2. Assess for reactogenicity based on nasal/respiratory and systemic (including oral 
temperature) FDA toxicity grading scales  
Immediate reactogenicity (including grade any actual measurements) will be recorded at 
30 minutes following study vaccination. All subjects will record maximum daily 
reactogenicity in a subject diary for 7 consecutive days starting the day following any 
study vaccination (retrospective of the highest value in the previous 24 hours). A 
thermometer for measuring body temperature (oral or tympanic) will also be distributed 
to subjects. The use of anti-pyrogenic medication will also be specifically queried by the 
subject diary during the 7 days following study vaccination. 
Nasal/respiratory and general systemic reactogenicity events will be recorded, including 
start and stop dates and actual measurements of body temperature. Trained clinical staff 
will review the information from the subject diary with the subjects at the following visit 
and apply a standard toxicity grade (refer to Appendix 2: Table for Reactogenicity 


 
 
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Grading) to any reactogenicity event. Subjects will be instructed that should they have 
reactogenicity with a potential toxicity Grade 3 at any time, they should contact the site 
on the same day and be seen or referred to a qualified medical facility within 24 hours. 
Any discrepancies between the Investigator- and the subject reported outcomes of 
reactogenicity will be noted in the source documents prior to entry into the clinical 
database. 
6.3.8. 
Post-Challenge Evaluation 
During the in-unit period the volunteers will be reviewed at least twice per day (once in 
the morning and once in the evening) following a standardised checklist including body 
temperature, respiratory rate, heart rate, and early symptoms of pertussis: rhinorrhoea, 
nasal congestion, epistaxis, sneezing, ear pain, eye pain, sore throat, cough, dyspnoea, 
feeling generally unwell, tiredness and headache. Actions to be taken when symptoms of 
early B. pertussis disease are suspected are included in Appendix 5: Process to be 
followed in cases of suspected symptomatic Bordetella pertussis infection. All AEs are to 
be reported in the eCRF. 
6.3.9. 
Azithromycin 
If persistent symptoms suggestive of pertussis disease occur, then subjects will be started 
on azithromycin and remain in the unit for approximately 3 days from the initiation of 
antibiotics for observation before discharge. The remaining subjects will receive 
azithromycin daily from Day 14 of their challenge unit stay and will be discharged from 
the Challenge Unit on Day 16 after the third dose of azithromycin is administered. The 3-
day course of azithromycin will be dosed at 500 mg orally once daily (as indicated by UK 
Health Security Agency for post-exposure prophylaxis for respiratory infections) and 
subjects must abide by stipulated infection control rules. If a B. pertussis culture collected 
on the last day of the in-unit stay is positive, the subject will return to the site to receive 
continued out-patient antibiotic treatment (azithromycin).  
7. 
LIFESTYLE AND/OR DIETARY RESTRICTIONS 
The following lifestyle and dietary restrictions apply throughout the study: 
• 
Subjects should not use drugs of abuse unless prescribed by a physician (e.g., 
benzodiazepines) 
• 
Subjects should not consume more than 14 units of alcohol per week. A unit is 
defined as one shot (25 mL) of spirits, half a pint (236 mL) of standard-strength beer 
or 1 small glass (125 mL) of wine 
• 
Subjects should not consume alcohol within 24 hours prior to receiving study 
vaccination or receiving virulent challenge, and throughout the challenge in-unit stay 
• 
Subjects should not smoke or vape from 1 week prior to vaccination until after the 
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• 
Subjects should refrain from any nasal sprays and washes not part of the study for 
28 days following vaccination and the challenge phase 
• 
Female subjects who are of child-bearing potential should observe the contraceptive 
guidelines, outlined in the inclusion criteria, from 21 days prior to enrolment until 
3 months after the challenge. 
8. 
INVESTIGATIONAL PRODUCT 
8.1. 
Dosage and Administration 
BPZE1 investigational vaccine is for intranasal administration and is an off-white 
lyophilized cake that contains genetically modified, live B. pertussis BPZE1 bacterial 
strain in lyophilisation buffer.  
The Sponsor will ensure the investigational vaccine and placebo (lyophilised buffer) is 
properly labelled and distributed to the clinical site(s). 
The supplies which will be used for vaccination in the study are outlined in Table 2. 
Table 2 
Study Investigational Medicinal Products 
Product 
Dosage and Route of Administration 
BPZE1 
2 x 0.4 mL (0.4 mL per nostril containing half 
the dose (5 x 108 CFU) bacteria to give a total 
dose of 109 CFU) administered intranasally via 
a MAD 
Placebo 
2 x 0.4 mL (0.4 mL per nostril) reconstituted 
lyophilized buffer administered intranasally via 
a MAD 
CFU=colony-forming units; MAD=mucosal atomization device 
 
The intranasal placebo consists of lyophilised buffer diluted with sterile water for 
injection (WFI), which are the same constituents in the same quantities as the BPZE1 
investigational vaccine, except for the attenuated B. pertussis cells.  
Each vaccination with BPZE1 or placebo will utilise a single vial/syringe of product for 
each single use (1:1 assignment for study vaccine accountability).  
Further details on administration are provided in the Pharmacy/Challenge Manual. 
8.2. 
Dose Rationale 
BPZE1 in a liquid formulation has been studied at various doses from 103 CFU to 
109 CFU in two Phase 1 clinical studies in Sweden with no vaccine-related SAEs and 
comparable general AEs to placebo controls [Jahnmatz et al, 2020; Thorstensson et al 
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liquid formulation. The Phase 1b study demonstrated the ability for a 0.4 mL (400 μL) 
per nostril dose of 107, 108, and 109 CFU BPZE1 to achieve transient nasopharyngeal 
colonization in >80% of subjects. Furthermore, 92% of the subjects in the 107 and 108 
CFU/dose groups and 100% of the 109 CFU group had a positive serological response 
(immunoglobulin G [IgG] or immunoglobulin A [IgA]) to any of the four B. pertussis 
antigens tested, and 100% of the 109 CFU subjects had a positive serological response 
(IgG or IgA) to two or more of the antigens tested. The 109 CFU dosage appears to be the 
most promising to elicit the needed immune response in the majority of adults. 
The Phase 2a and 2b studies assessed the 109 CFU dose of BPZE1 and showed that this 
dosage is safe and well-tolerated. 
8.3. 
Maintaining the Blind 
This is a double-blind study. 
The site(s) being used will identify an unblinded individual(s) who will be provided with 
a randomisation list. This will be used to allocate the subjects to either BPZE1 or 
placebo. To maintain the blind, unblinded individuals will be involved in vaccine 
logistics and preparation. The unblinded team will not be involved in any study-related 
assessments or have subject contact for data collection following study vaccination. 
Blinded individuals will administer the study vaccine with further details in the 
Pharmacy/Challenge Manual. 
Code break envelopes will be provided to the site(s) so that they can unblind an 
individual subject in the event of a medical emergency. Where possible they should 
discuss the situation with the Sponsor’s medical monitor prior to unblinding; however, 
the subject’s safety must always come first. Reasons for treatment unblinding must be 
clearly explained and justified in the eCRF. The date on which the code was broken 
together with the identity of the person responsible must also be documented. 
8.4. 
Treatment Assignment 
Subjects will be assigned to a study vaccine based on the order in which they are 
enrolled. This allocation will be performed by an unblinded individual at the site who 
will be independent of blinded staff determining eligibility of subjects. 
Subjects will be randomised in a 1:1 ratio to receive a single dose of either BPZE1 
(109 CFU) or placebo. 
8.5. 
Packaging and Labelling 
Vaccine will be provided to the sites in an open-label format. The labels will contain all 
information required to meet the applicable local regulatory requirements. The labels 
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Further information on the study vaccine packaging, labelling, and dispensing are 
included in the Pharmacy/Challenge Manual. 
8.6. 
Preparation 
The dose of BPZE1 or placebo will be administered intranasally. A total of 0.8 mL of 
vaccine will be withdrawn for administration. Following reconstitution, the MAD will be 
attached to the syringe, and 0.4 mL volume will be administered to each nostril. In the 
case of placebo nasal vaccination, a similar volume of WFI will be reconstituted into a 
vial containing only lyophilised buffer, followed by 0.4 mL volume administered into 
each nostril with the vaccinator. The mucosal atomiser device provides a uniform, 
controlled delivery, which allows the vaccinator to accurately deliver 0.4 mL of vaccine 
to the initial nostril and then administer the remaining 0.4 mL to the opposite nostril.  
Further details on preparation are provided in the Pharmacy/Challenge Manual. 
8.7. 
Handling and Storage 
BPZE1 and the placebo must be stored in a secure area (e.g., locked room or locked 
refrigerator), protected from light and moisture as required by manufacturer. The vaccine 
will be shipped from the Sponsor’s European contract manufacturing organization to the 
CRO’s repository and distribution centre and should be stored at -20°C or below prior to 
shipping to the clinical sites, where the vaccine will be stored at 8°C or below. 
Reconstituted BPZE1 should not be kept at room temperature prior to vaccination for 
more than 4 hours. 
A detailed description regarding the storage and handling the study vaccine will be in the 
Pharmacy/Challenge Manual. 
8.8. 
Product Accountability and Assessment of Compliance 
In accordance with International Council on Harmonisation Good Clinical Practice (ICH-
GCP), the study centre will account for all supplies of study vaccine. Details of receipt, 
storage, assembly, and return will be recorded. The unit of accountability will be one vial. 
All unused supplies will either be destroyed or returned to the study Sponsor at the end of 
the study in accordance with instructions by the Sponsor. 
All study vaccines will be administered by site staff. If the complete vaccination is not 
administered as planned this will be documented in the eCRF. 
8.9. 
Treatment of Investigational Product Overdose 
An overdose is any dose of study treatment given to a subject or taken by a subject that 
exceeds the dose described in the protocol. If the subject receives an overdose of the 


 
 
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study vaccine, then the Sponsor should be notified, in writing, within 24 hours of the 
Investigator becoming aware. This will be classified as a protocol violation. 
The Investigator will treat the overdose according to their clinical judgment depending on 
the extent of the overdose and the clinical signs and symptoms exhibited by the subject. 
In case of any AEs associated with the overdose, these should be reported on relevant 
AE/SAE sections in the eCRF. BPZE1 is a live attenuated B. pertussis bacterium and is 
susceptible to erythromycin/azithromycin antibiotic treatment. 
8.10. 
Occupational Safety 
To avoid accidental exposure actions should be taken to minimize generation of aerosols, 
since the bacterium is strictly a respiratory tract organism. Clinical staff members 
performing reconstitution should wear eye-protective glasses, gloves and masks, and 
clean surfaces prior to and after performing reconstitution activities with 70% ethanol 
solution. Persons handling the BPZE1 bacteria for vaccination should wear gloves, 
goggles and face mask, and must wash their hands with a suitable disinfecting soap after 
administration. Effective antibiotic treatment with azithromycin could be given in case of 
accidental transmission to other humans, however, BPZE1 is an attenuated live organism 
and is not pathogenic.  
The Material Safety Data Sheets will be made available where required by local 
regulations. 
9. 
CHALLENGE AGENT 
9.1. 
Manufacturing of the Bordetella pertussis Inoculum 
The inoculum is prepared to good manufacturing practice (GMP) standard in licensed 
cGMP facilities and using a process free of animal-derived products. The identity and 
purity of the cell bank is confirmed, in addition to any other quality specifications agreed 
with the consortium and needed for compliance with regulatory requirements. There will 
be no culture of the challenge inoculum at the clinical site, other than to assess the dose 
and purity of the inoculum after inoculation and quality assessment.  
More information regarding the challenge agent will be in the Pharmacy/Challenge 
Manual. 
9.2. 
Quality Assessment of the Inoculum 
Each vial used for inoculation will be thawed and tested for culture on Bordetella 
selective medium (charcoal blood agar with cephalexin) for determination and viable 
counts of B. pertussis and on a Plate Count Agar to assess the purity of the inoculum. 
Culture identity will be confirmed by visual appearance of colonies and Gram stain. Full 
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Ionisation Time-of-Flight Mass Spectrometry may be used to identify isolates to species 
level. UK standards for identification of Bordetella species [UK Standard for 
Microbiology Investigations] will be followed.  
9.3. 
Transport of the Inoculum 
The inoculum is transferred to the site under temperature-monitored conditions. For 
transport within the site, containers of inoculum are placed into secondary containers, 
which are transported in leak and shock resistant transport boxes with secured lids. 
9.4. 
Storage of the Bordetella pertussis Inoculum 
The cell banks are stored at -80oC (±20oC) in a locked, dedicated, temperature monitored 
freezer at the site. 
9.5. 
Dilution of the Inoculum 
One vial of inoculum will be removed from the freezer, thawed, and diluted to the 
required inoculum dose following the challenge specific standard operating procedures 
(SOP). Dilutions will be carried out by one staff member and checked by another 
member. The inoculum will be administered to the volunteer within an hour of removal 
from the freezer following the specific SOP: Administering the B. pertussis inoculum to 
healthy volunteers. 
9.6. 
Administration of the Challenge Agent 
Once subjects are cleared for virulent challenge of a dose of the B. pertussis B1917 strain 
at 105 CFU, the dose will be administered while lying supine and using the procedures 
established within the challenge unit. The inoculum will be instilled into each nostril with 
a Gilson pipette by droplet. Subjects will remain supine for 15 minutes. 
9.7. 
Monitoring of the Bordetella pertussis Dose Given to the 
Subject 
After the inoculum is administered, the tube residuum will be cultured for 5 days on 
Bordetella selective medium (charcoal blood agar with cephalexin) for isolation and 
purity-checking and viable counts of B. pertussis will be measured using a standard 
dilution technique. If the inoculum CFU count measured is more than five times the 
inoculum CFU count intended for the volunteer, they will be given oral azithromycin 
500 mg once a day for 3 days, observed for an additional 48 hours in-unit, and excluded 
from the study. Safety follow-up in this case of overdosing will take place at Days 14 and 
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9.8. 
Disposal of the Inoculum 
All materials used during the challenge, including any remaining inoculum will be 
disposed of following site policy.  
10. 
CONCOMITANT MEDICATIONS AND NON-DRUG 
THERAPIES 
All medications (including over the counter products, herbals, vitamins, supplements, and 
vaccinations) taken from 7 days prior to Screening until the EOS visit should be 
recorded. Information recorded should include the generic name, dose, route of 
administration, frequency, date started and stopped, and reason the medication was taken. 
Intake will include whether there was knowledge of past pertussis vaccination and (when 
available) the year of receipt. Use of new medication(s) should prompt evaluation for the 
presence of a new diagnosis of chronic medical disease or condition. 
10.1. 
Permitted Medications 
Subjects are permitted to take paracetamol at doses up to 4 g per day. The use of anti-
pyrogenic medication will be specifically queried by diary during the 7 days following 
study vaccination. 
Female subjects of childbearing potential are permitted to use contraception (e.g., 
contraceptive pill, contraceptive patch). 
All subjects who are challenged should take azithromycin orally, once daily, for a 
minimum of three doses prior to being discharged from the challenge unit. 
10.2. 
Prohibited Medications 
Immunosuppressive therapy or other immune-modifying drugs (including but not limited 
to systemic corticosteroids, biologics and methotrexate) during the trial is prohibited. 
Corticosteroids (inhaled, topical, ophthalmologic or localized injections in joints) are 
permitted. 
Antibiotics are not permitted during the challenge unit stay with the exception of 
azithromycin, as described in Section 6.3.9. 
Medications mentioned in the inclusion/exclusion criteria are restricted as outlined in 
those criteria. 
11. 
SUBJECT COMPLETION AND WITHDRAWAL 
11.1. 
Subject Completion 


 
 
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A subject is classified as having completed the study when he/she completes the EOS 
visit. 
11.2. 
Subject Withdrawal 
Subjects may withdraw/discontinue from the study any time and for any reason without 
prejudice to their future medical care by the Investigator or at the study centre. The 
reason for subject withdrawal/discontinuation will be recorded. 
A subject may be withdrawn from treatment or challenge for any of the following 
reasons: 
• 
Withdrawal of consent to continue in the study (a subject may withdraw his or her 
consent at any time) 
• 
The study Investigator or Sponsor, for any reason, decides the subject should be 
withdrawn from the treatment 
• 
Adverse events, which cannot be tolerated by the subject 
• 
Pregnancy  
• 
Significant noncompliance with the protocol, as determined by the medical 
monitor(s)  
• 
Sponsor decides to discontinue the subject’s participation in the study or to terminate 
the study 
If a subject is withdrawn from the study, where possible, he/she should continue to attend 
the safety follow-up visits so that data can be collected for the Intention-to-Treat 
Population. If subjects will not agree to this, then the subject should complete the EOS 
visit. 
11.3. 
Treatment after the End of the Study 
No treatment/vaccination will be provided after the EOS. 
11.4. 
Screen Failures 
Information on screening failures will be collected. 
12. 
ADVERSE EVENTS AND SERIOUS ADVERSE EVENTS  
12.1. 
Definitions 
12.1.1. 
Adverse Events 
An AE is any untoward medical occurrence in a patient or clinical investigation subject 
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relationship with this treatment. An AE can be any unfavourable and unintended sign 
(including an abnormal laboratory finding), symptom, or disease temporally associated 
with the use of a medicinal (investigational) product, whether or not related to the 
medicinal (investigational) product. 
12.1.2. 
Treatment-Emergent Adverse Event 
A TEAE is an AE that occurs after the subject has been vaccinated. 
12.1.3. 
Adverse Events of Special Interest 
The following AEs are classified as AESIs and will be collected on a unique eCRF 
through to the EOS: 
• Adverse events related to infection with or vaccination for SARS-CoV-2 
12.1.4. 
Serious Adverse Events 
An SAE is any untoward medical occurrence that at any dose:  
• 
Results in death  
• 
Is life-threatening  
• 
Requires inpatient hospitalisation or prolongation of existing hospitalisation  
• 
Results in persistent or significant disability/incapacity  
OR  
• 
Is a congenital anomaly/birth defect  
Medical and scientific judgement should be exercised in deciding whether reporting is 
appropriate in other situations, such as important medical events that may not reach the 
above definition but may jeopardise the subject or may require medical or surgical 
intervention to prevent one of the other outcomes listed in the above definition. These 
should also be considered serious. Examples of such events are invasive or malignant 
cancers, intensive treatment in an accident and emergency department or at home 
treatment for allergic bronchospasm, blood dyscrasias or convulsions that do not result in 
hospitalisation, or development of drug dependency or drug abuse. 
12.1.5. 
Suspected Unexpected Serious Adverse Reactions 
Suspected unexpected serious adverse reactions (SUSARs) are SAEs, classified as 
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12.2. 
Safety Reporting  
12.2.1. 
Reporting of Adverse Events 
Adverse events will be collected as follows: 
• 
All AEs will be collected through 28 days after study vaccination and 28 days after 
virulent challenge. Note that between screening (signing of ICF) and Day 0, AEs will 
be collected only if classified as serious or if considered related to study procedure or 
study involvement. Any event that results in vaccination delay (e.g., temporary 
exclusions due to acute upper respiratory symptoms/illness or fever, or investigator 
determined medical condition) during screening or during study conduct will also be 
recorded as an AE. Should any reactogenicity event extend beyond 7 days following 
vaccination, it will be recorded as an AE with the same start date as the 
reactogenicity event and followed until considered resolved 
• 
All AEs related either to vaccination or to challenge will be collected from time of 
vaccination to challenge and for 3 months after challenge, respectively 
Adverse events will be recorded in the eCRF. The CRO should be notified within 
24 hours if the AE leads to withdrawal of the subject. 
Any medical condition that is present at the time that the subject is screened will be 
considered as baseline (recorded as medical history) and not reported as an AE; however, 
if the severity of any pre-existing medical condition increases, it will be recorded as an 
AE. Adverse events must be graded for severity and assessed for relationship to study 
vaccination. Adverse events characterised as intermittent require documentation of onset 
and duration of each episode. The start and stop date of each reported AE will be 
recorded on the appropriate data collection form and eCRF. 
All subjects will record maximum daily reactogenicity/solicited AEs in the subject diary 
starting for the subsequent 7 days after vaccination. Subjects will be instructed that 
should they have a reactogenicity event with a potential toxicity Grade 3 at any time after 
vaccination, they should contact the site on the same day and be seen or referred to a 
qualified medical facility. 
12.2.2. 
Reporting of Serious Adverse Events and Adverse Events of 
Special Interest 
All AESIs and SAEs will be monitored from signing of informed consent through to the 
EOS visit.  
All SAEs/AESIs occurring during the clinical study must be reported to the appropriate 
CRO contact person by investigational staff within 24 hours of their knowledge of the 
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Information regarding SAEs/AESIs will be transmitted to the CRO using the SAE Form, 
which must be completed and signed by a member of the investigational staff and 
transmitted to the CRO within 24 hours of knowledge of the event.  
All SAEs/AESIs that have not resolved by the EOS, or that have not resolved upon 
discontinuation of the subject's participation in the study, must be followed until any of 
the following occurs: 
• 
The event resolves 
• 
The event stabilises 
• 
The event returns to baseline, if a baseline value/status is available 
• 
The event can be attributed to agents other than the study vaccine/challenge or to 
factors unrelated to study conduct 
• 
It becomes unlikely that any additional information can be obtained (subject or 
health care practitioner refusal to provide additional information, lost to follow-up 
after demonstration of due diligence with follow-up efforts) 
• 
Thirty days have elapsed after the EOS (last subject, last visit) 
Any event requiring hospitalisation (or prolongation of hospitalisation) that occurs during 
the course of a subject's participation in a clinical study must be reported as an SAE, 
except hospitalisations for the following: 
• 
Social reasons in the absence of an AE 
• 
Surgery or procedure planned before entry into the study (must be documented in the 
source documents and eCRF) 
The Sponsor will promptly evaluate any new SUSARs and expeditiously communicate 
possible new safety findings to investigators, institutional review boards or independent 
ethics committees, and applicable health authorities based on applicable legislation. Any 
SAE considered to be related to BPZE1 will be classified as a SUSAR. The Investigator 
(or Sponsor where required) must report these events to the appropriate IEC that 
approved the protocol unless otherwise required and documented by the IEC. 
12.3. 
Classification of Adverse Events 
12.3.1.1. 
Assessment of Severity 
The severity, or intensity, of an AE refers to the extent to which an AE affects the 
subject’s daily activities. The intensity of the AE will be rated as mild, moderate, or 
severe using the following criteria: 
• 
Mild (Grade 1): these events require minimal or no treatment and do not interfere 
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• 
Moderate (Grade 2): these events result in a low level of inconvenience or concern 
with the therapeutic measures. Moderate events may cause some interference with 
normal functioning 
• 
Severe (Grade 3): these events interrupt a subject’s usual daily activity and may 
require systemic drug therapy or other treatment. Severe events are usually 
incapacitating 
Changes in the severity of an AE should be documented to allow an assessment of the 
duration of the event at each level of intensity to be performed. Adverse events 
characterized as intermittent require documentation of onset and duration of each 
episode. 
Laboratory assessments and vital signs will be classified according to the FDA severity 
scales see Appendix 3: Table for Laboratory Grading and Appendix 4: Table for Vital 
Signs Grading. 
12.3.1.2. 
Assessment of Causality 
The Investigator’s assessment of an AE’s relationship to study vaccination is part of the 
documentation process, but may not be a factor in determining what is or is not reported 
in the study. If there is any doubt as to whether a clinical observation is an AE, the event 
should be reported. 
The relationship or association of the test article in causing or contributing to the AE will 
be characterised using the following classification and criteria: 
• 
Related to vaccination: there is a reasonable possibility that the study vaccination 
caused the AE. Reasonable possibility means that there is evidence to suggest a 
causal relationship between the study vaccination and the AE 
• 
Not related to vaccination: there is not a reasonable possibility that the 
administration of the study vaccination caused the event 
• 
Related to challenge agent: there is a reasonable possibility that the challenge agent 
caused the AE. Reasonable possibility means that there is evidence to suggest a 
causal relationship between the study vaccination and the AE 
• 
Not related to challenge agent: there is not a reasonable possibility that the 
administration of the challenge agent caused the event 
12.4. 
Pregnancy 
Pregnancy is not regarded as an AE unless there is a suspicion that an investigational 
vaccine may have interfered with the effectiveness of a contraceptive medication. Any 
pregnancy that occurs during study participation must be reported using a clinical study 
pregnancy form. To ensure subject safety, each pregnancy must be reported to the 
Sponsor within 2 weeks of learning of its occurrence. If pregnancy occurs, neither further 
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outcome (including spontaneous miscarriage, elective termination, normal birth, or 
congenital abnormality) and status of mother and child, even if the subject is discontinued 
from the study. Pregnancy complications and elective terminations for medical reasons 
must be reported as an AE or SAE. Spontaneous miscarriages must be reported as an 
SAE. 
Any pregnancy brought to the Investigator’s attention after the subject has completed the 
study, but occurring while the subject was in the study, must be promptly reported to the 
Sponsor. 
13. 
DATA ANALYSIS AND STATISTICAL CONSIDERATIONS 
Further information on the data analysis and statistical considerations is provided in the 
SAP. 
13.1. 
Study Design Considerations 
13.1.1. 
Sample Size Assumptions 
Table 3 depicts the power for the challenge study as computed under the following 
assumptions: two-sided significance level of 0.05, use of a likelihood ratio test for testing 
equality of two proportions, a sample size of 20 in each of the two groups, a colonisation 
rate of 60%, 70%, and 80% in the unvaccinated group and colonisation rates of 10%, 
20%, 30%, and 40% in the vaccinated group.  
Group sample sizes of 20 evaluable subjects in each group achieve a power of at least 
90% to detect differences between colonisation rates of at least 50% in six scenarios. 
These scenarios include the following colonisation rates for the placebo and BPZE1 
groups, respectively: 60% versus 10%, 70% versus 10%, 70% versus 20%, 80% versus 
10%, 80% versus 20% and 80% versus 30%, which are bolded in Table 3.  
 


 
 
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Table 3 
Power for Sample Sizes of 20 in Each Group for Varying 
Colonisation Rates Based on a Likelihood Ratio Test 
Colonisation Rate 
 
 
Placebo 
BPZE1 
N/group 
Power 
 
 
 
 
60% 
10% 
20 
94% 
 
20% 
 
75% 
 
30% 
 
48% 
 
40% 
 
24% 
 
 
 
 
70% 
10% 
20 
99% 
 
20% 
 
92% 
 
30% 
 
73% 
 
40% 
 
48% 
 
 
 
 
80% 
10% 
20 
100% 
 
20% 
 
99% 
 
30% 
 
92% 
 
40% 
 
75% 
 
Table 4 includes results based on vaccine efficacy computed from the colonisation rates 
in each population, rather than comparing the two colonisation rates and testing the null 
hypothesis that they are equal in the two populations. The computations presented in the 
table below are based on a normal approximation. The table includes the required sample 
size for a power of 80% and for lower bounds of vaccine efficacy of 0%, 10%, 20%, and 
30%. For a colonisation rate of 70% in the placebo group and a lower bound of efficacy 
of 20%, the study will be powered for efficacy rates of 71% or higher. Focusing on an 
efficacy rate greater than 50% and a lower bound of 0% (sample sizes in bold), the study 
is well powered to detect efficacy rates of 57% or higher. 
 


 
 
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Table 4 
Estimated Sample Size Based on Assumed Colonisation rates and 
Power of 80% and Lower Bounds of 0%, 10%, 20% and 30% 
Colonisation rate 
Sample size/group 
Control 
Vaccine 
Efficacy 
LB 0% 
LB 10% 
LB 20% 
LB 30% 
60% 
10% 
0.833 
11 
12 
14 
18 
 
20% 
0.667 
18 
22 
29 
42 
 
30% 
0.500 
33 
47 
75 
149 
 
40% 
0.333 
77 
141 
390 
5675 
 
 
 
 
 
 
 
70% 
10% 
0.857 
8 
9 
10 
12 
 
20% 
0.714 
12 
14 
18 
24 
 
30% 
0.571 
19 
25 
36 
60 
 
40% 
0.429 
33 
51 
95 
268 
 
 
 
 
 
 
 
80% 
10% 
0.875 
6 
6 
7 
9 
 
20% 
0.750 
8 
9 
12 
16 
 
30% 
0.625 
12 
15 
20 
31 
 
40% 
0.500 
18 
25 
40 
82 
 
For the endpoint of absolute counts, the standard deviation in the BPZE1 group was 
computed to be 2.2 for the natural log of the counts and 4.2 for the natural log of the 
counts in the control group. Assuming a sample size of 20 evaluable subjects in each 
group, a two-sample t-test accounting for unequal variances, and a two-sided significance 
level of 0.05,the study has approximately 80% power to detect differences of the order of 
3.1 units between the BPZE1 group and the control group on the natural log scale. 
13.1.2. 
Sample Size Re-estimation 
Sample size re-estimation will not be performed in this study. 
13.1.3. 
Study Stopping Criteria 
There are no study-specific stopping criteria. 
13.2. 
Data Analysis Considerations 
13.2.1. 
Analysis Populations 
The ‘Intention-to-treat’ and ‘Safety’ Populations will both be defined as all subjects 
randomised to treatment who are vaccinated. 


 
 
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The ‘modified intent-to-treat’ (mITT) analysis population will be defined as all subjects 
randomized to treatment who are vaccinated, challenged, and have at least one culture 
result at Challenge Day 9, 11, or 14. Subjects will be classified according to the 
randomized treatment group. 
The ‘Per-Protocol’ Population is defined as all subjects who are included in mITT 
population and completed the study with no major protocol deviations. 
13.2.2. 
Treatment Comparisons 
Comparisons will be made between the two treatment arms: BPZE1 and placebo. 
13.2.3. 
Interim Analysis 
No interim analysis will be performed. 
13.2.4. 
Key Elements of Analysis Plan 
Statistical analysis will be performed using SAS software Version 9.4 or later.  
13.2.4.1. 
Efficacy Analyses 
Statistical analysis will be performed using SAS software Version 9.4 or later. 
Continuous variables will be summarized using the mean, standard deviation, median, 
first quartile, third quartile, minimum value, and maximum value. Categorical variables 
will be summarized using frequency counts and percentages, as well as a 2-sided 95% 
confidence interval (CI) for proportions computed using the Agresti-Coull method. All 
statistical tests will be 2-sided at 0.05 significance level.  
For the primary analysis of the between treatment groups difference in the proportion of 
subjects colonized on any day (Challenge Day 9, 11 or 14) following challenge, a 
likelihood ratio test and the corresponding 95% confidence interval will be computed 
with colonization defined as a positive result on any of the 3 days. Primary estimand is 
described in the SAP. Additional summaries for each of the days will be presented 
including the results of the likelihood ratio test and associated confidence intervals.  
Immunogenicity endpoints (GMC/GMT, GMFR, and/or seroconversion) will be 
summarized at baseline and each time point by treatment groups. Reverse cumulative 
distribution curves, which are also known as survival curves, will be presented for each 
measure. Comparison of the BPZE1 and control groups will use a two-sample t-test. 
Confidence intervals will be back-transformed from the log scale to the original scale.  
Exploratory analyses include analyses to assess whether colonization status has a 
relationship to pre-challenge mucosal immunity levels (by individual or by combination 
of anti-pertussis antibodies) or SBA activity. Analyses will include the GMCs/GMTs 
with 95% CIs and GMFRs with 95% CIs and p-values testing as per the SAP. Pearson’s 


 
 
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and Spearman correlations and repeated measures analyses will be conducted to assess 
for correlations and for SBA and protection against colonization. For immunogenicity 
analysis, it is assumed that the natural log of the data is normally distributed. All 
statistical tests will be 2-sided at 0.05 significance level and nominal p-values will be 
reported.  
The SAP will provide additional details of all analyses. 
13.2.4.2. 
Safety Analyses 
All safety analyses will be performed using the safety population. Safety and tolerability 
will be assessed by clinical review of all safety parameters including AEs, laboratory 
values, and vital signs. The safety analyses will include results collected from 
randomization through Day 28 post-challenge as well as EOS. All safety presentations 
will be presented by treatment group. 
13.2.4.2.1. Adverse Events 
The verbatim terms used in the eCRF by Investigators to identify AEs will be coded 
using the Medical Dictionary for Regulatory Activities (MedDRA) version 24.1 or above. 
All reported TEAEs will be included in the analysis. For each AE, the percentage of 
subjects who experience at least one occurrence of the given event will be summarised by 
treatment group. Summaries of any AESI will also be included as part of the safety 
analysis. 
Summaries, listings, datasets, or subject narratives may be provided, as appropriate, for 
those subjects who die, who discontinue treatment due to an AE or who experience an 
AESI or a serious AE. 
13.2.4.2.2. Concomitant Medication 
The verbatim terms used in the eCRF by the Investigators to identify concomitant 
medication will be coded using the WHO Drug Dictionary. A listing of the medications 
taken will be provided. 
13.2.4.2.3. Clinical Laboratory Tests 
Laboratory data will be summarised by type of laboratory test. Reference ranges and 
markedly abnormal results (specified in the SAP) will be used in the summary of 
laboratory data. Descriptive statistics will be calculated for each laboratory parameter at 
baseline and at each scheduled time point and boxplots will be created. Changes from 
baseline results will be presented in pre- versus post-treatment cross-tabulations (with 
classes for below, within, and above normal ranges). A listing of subjects with any 
laboratory results outside the reference ranges will be provided. A listing of subjects with 
values meeting FDA toxicity criteria (Appendix 3: Table for Laboratory Grading) will be 
summarised. 


 
 
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13.2.4.2.4. Vital Signs 
Descriptive statistics of temperature, pulse rate, blood pressure (systolic and diastolic) 
values, and changes from baseline will be summarised at each scheduled time point. The 
percentage of subjects with values meeting FDA toxicity criteria (Appendix 4: Table for 
Vital Signs Grading) will be summarised. 
13.2.5. 
Missing, Unused and Spurious Data 
Missing data will be accounted for in all summaries by time, with reasons provided 
where possible (e.g., discontinuation). If any data are excluded from analyses the reason 
for their exclusion will be documented in the clinical study report (CSR). 
13.2.6. 
Reporting Deviations from the Statistical Plan 
Any deviations from the planned analyses will be described and justified in the final 
CSR. 
14. 
STUDY ADMINISTRATION 
14.1. 
Independent Data Safety Monitoring Board 
Safety oversight will be conducted by an independent DSMB. The DSMB members will 
be separate and independent of study personnel participating in this study and should not 
have scientific, financial, or other conflict of interest related to this study. The DSMB 
will consist of members with appropriate expertise to contribute to the interpretation of 
the data from this study. 
The DSMB will convene prior to study initiation, if a pause is triggered, or upon the 
request of the medical monitor and/or sponsor. The medical monitor and sponsor are 
empowered to request a review by the DSMB for any safety reason. 
The DSMB will operate under the rules of a sponsor-approved charter that will be 
approved at the organizational meeting of the DSMB. Procedures for DSMB 
reviews/meetings will be defined in the charter. The DSMB will review applicable data to 
include, but not limited to subject’s clinical course or safety results (e.g., AEs, 
reactogenicity). Reports may include enrolment and demographic information, medical 
history, concomitant medications, physical assessments, and solicited and unsolicited 
AE/SAEs. Additional data may be requested by the DSMB, and interim statistical reports 
may be generated as deemed necessary and appropriate by the sponsor. The DSMB may 
receive data in aggregate and presented by treatment group. 
The DSMB will review grouped and unblinded data in the closed session only. As an 
outcome of each review/meeting, the DSMB will make a recommendation as to the 
advisability of continuing, modifying or terminating the study. 


 
 
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Further information on the composition, meeting schedule and responsibilities of the 
DSMB can be found in the DSMB Charter. 
14.2. 
Regulatory and Ethical Considerations, Including the 
Informed Consent Process 
Before initiation of the study site(s), the Sponsor, or their representatives, will obtain 
approval from the appropriate regulatory agency to conduct the study in accordance with 
ICH-GCP and applicable country-specific regulatory requirements. 
The study will be conducted in accordance with all applicable regulatory requirements 
such as ICH-GCP, all applicable subject privacy requirements, and the ethical principles 
that are outlined in the Declaration of Helsinki 2013, including, but not limited to: 
• 
An IEC review and approval of study protocol and any subsequent amendments and 
all ICFs or other information given to the subject 
• 
Subject informed consent 
• 
Investigator reporting requirements 
The Sponsor will provide full details of the above procedures, either verbally, in writing, 
or both. 
Written informed consent must be obtained from each subject before participation in the 
study. Written informed consent will be collected following a review of the volunteer 
information sheet by the potential subject and a discussion between the subject and the 
Investigator or suitably qualified designee. 
The Investigator will cooperate with all regulatory inspections and will notify the 
Sponsor as soon as they are aware of an inspection which may involve this study. With 
the exception of statutory regulatory authority inspections, the Sponsor will be consulted 
in the event of inspection of the clinical site(s) by an outside authority before the 
Inspectors are permitted access to any of the study records or the study areas. 
14.3. 
Study Monitoring 
In accordance with applicable regulations, ICH-GCP, the monitoring plan and the 
Sponsor’s and/or delegate procedures, monitors will contact the site before the start of the 
study to review with the site staff the protocol, study requirements, and their 
responsibilities to satisfy regulatory, ethical, and the Sponsor’s requirements. When 
reviewing data collection procedures, the discussion will include identification, 
agreement, and documentation of data items for which the CRF will serve as the source 
document. 


 
 
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The Sponsor and/or delegated monitors will visit the Investigator and study site(s) at 
periodic intervals (including remote monitoring activities), in addition to maintaining 
necessary telephone and letter contact to ensure that: 
• 
The data are authentic, accurate and complete 
• 
The subject’s safety and rights are being protected 
• 
The study is conducted in accordance with the currently approved protocol and any 
other study agreements, ICH-GCP and all applicable regulatory requirements 
14.3.1. 
Access to Source Data 
The Investigator and the head of the medical institution (where applicable) agrees to 
allow the monitor, Sponsor-appointed auditors, and regulatory inspectors direct access to 
all relevant documents. 
14.3.2. 
Data Handling and Record Keeping 
Following closure of the study, the Investigator or head of the medical institution (where 
applicable) must maintain all site study records (except for those required by local 
regulations to be maintained elsewhere) in a safe and secure location. The records must 
be easily accessible when needed (e.g., for a Sponsor audit or regulatory inspection) and 
must be available for review in conjunction with assessment of the facility, supporting 
systems, and relevant site staff. 
Where permitted by local laws/regulations or institutional policy, some or all of the 
records may be maintained in a format other than hard copy (e.g., microfiche, scanned, 
electronic); however, caution must be exercised before such action is taken. The 
Investigator must ensure that all reproductions are legible and are a true and accurate 
copy of the original. In addition, they must meet accessibility and retrieval standards, 
including regeneration of a hard copy, if required. The Investigator must also ensure that 
an acceptable back-up of the reproductions exists and that there is an acceptable quality 
control procedure in place for creating the reproductions. 
The Sponsor will inform the Investigator of the time period for retaining the site records 
in order to comply with all applicable regulatory requirements. The minimum retention 
time will meet the strictest standard applicable to a particular site, as dictated by local 
laws/regulations, the Sponsor SOPs and/or institutional requirements. 
The Investigator must notify the Sponsor of any changes in the archival arrangements, 
including, but not limited to archival of records at an off-site facility or transfer of 
ownership of the records in the event that the Investigator is no longer associated with the 
site. 


 
 
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14.4. 
Data Management 
For this study, subject data will be collected using an eCRF and combined with data 
provided from other sources in a validated data system. 
Management of clinical data will be performed in accordance with the applicable 
Sponsor/Sponsor’s representative standards and data cleaning procedures to ensure the 
integrity of the data, e.g., removing errors and inconsistencies in the data. Adverse events 
and concomitant medications terms will be coded using the MedDRA and the WHO 
Drug Dictionary, respectively.  
When using electronic trial data handling and/or remote electronic trial data systems, the 
Sponsor or designee will:  
a. 
Ensure and document that the electronic data processing system(s) conforms to 
the Sponsor’s established requirements for completeness, accuracy, reliability, 
and consistent intended performance (i.e., validation) 
b. Maintain SOPs for using these systems 
c. 
Ensure that the systems are designed to permit data changes in such a way that the 
data changes are documented and that there is no deletion of entered data (i.e., 
maintain an audit trail, data trail, edit trail) 
d. Maintain a security system that prevents unauthorised access to the data 
e. 
Maintain a list of the individuals who are authorised to make data changes 
f. 
Maintain adequate backup of the data 
g. Safeguard the blinding, if any (e.g., maintain the blinding during data entry and 
processing) 
Training on the use of the electronic data collection system will be provided to all 
relevant study site staff. 
14.5. 
Provision of Study Results and Information to Investigators 
Where required by applicable regulatory requirements, an Investigator signatory will be 
identified for the approval of the clinical study report. The Investigator will be provided 


 
 
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reasonable access to statistical tables, figures and relevant reports and will have the 
opportunity to review the complete study results at a mutually agreeable location. 
The Sponsor will also provide the Investigator with the full summary of the study results. 
The Investigator is encouraged to share the summary results with the study subjects, as 
appropriate. 
14.6. 
Insurance, Indemnity and Finance 
The Sponsor maintains appropriate insurance coverage for clinical studies and will follow 
applicable local compensation laws. 
The Sponsor will indemnify all Investigators participating in this study against future 
claims by study subjects; the terms of this will be detailed within a separate letter of 
indemnification. The indemnity will only apply where all study procedures have been 
carried out according to this protocol. 
The financial aspects of the study are addressed in a separate agreement. 
14.7. 
Publishing 
The Sponsor retains ownership of all data. When the study is complete the sponsor shall 
arrange the development of a CSR. All proposed publications based on this study must be 
approved by the Sponsor. 
The study will be registered in an international registry(ies) and the results will be loaded 
into the registry(ies) at the end of the study. 
 
 


 
 
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15. 
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g. 
All subjects will record maximum daily reactogenicity/solicited AEs in the subject diary for the subsequent 7 days after vaccination. Subjects will be instructed that should they 
have reactogenicity with a potential toxicity grade 3 at any time after vaccination, they should contact the site on the same day and be seen or referred to a qualified medical 
facility 
h. 
Immediate reactogenicity will be assessed 30 minutes after the study vaccination, prior to release from clinical observation 
i. 
Site staff reviews the information from the subject diary/checklist with the subjects to confirm accuracy. For reactogenicity and post-challenge checklist, the appropriate FDA 
toxicity grading should be applied. If any reactogenicity event extended beyond 7 days post-vaccination and is deemed clinically significant by AE classification, then it should be 
entered as an AE (graded by AE criteria) with the same start date as the reactogenicity event and followed to resolution. All AEs identified by the post-challenge checklist should 
be entered as an AE 
j. 
Prior to Study visit C14, azithromycin may be initiated if subjects have symptoms consistent with pertussis, although assessments listed for C14 should be carried out prior to 
starting azithromycin. On Study visit C14, all subjects who have not yet started azithromycin should begin a 3-day course of azithromycin 500 mg per day after all study samples 
have been collected; the third dose of azithromycin must be taken prior to the subject exiting the challenge unit on Study visit C16 
k. 
Subjects with positive culture or PCR sampled on C16 will be required to return to the site to receive additional antibiotic treatment 
l. 
Collect prior to vaccination/challenge 
m. All unsolicited AEs to be collected from time of vaccination to 28 days after vaccination and from time of challenge to 28 days after challenge 
n. 
Treatment-related AEs due to vaccination and due to challenge to be collected from time of vaccination to challenge and then for 3 months after challenge, respectively 
o. 
All AESIs and SAEs to be collected from time of signed informed consent to EOS 
p. 
Maybe performed at the discretion of the Investigator throughout the challenge phase 
q. 
This visit will occur at least 6 months post-vaccination or at least 3 months post-challenge 
r. 
Visit may occur up to 8 days before C0 or as close to C0 as possible to allow availability of test results. 


 
 
Study Number: IB-202P 
Compound No.: BPZE1 
Protocol 
Version: 4 
 
Page 85 of 90 
 
 
CONFIDENTIAL 
16.2. Appendix 2: Table for Reactogenicity Grading 
Nasal/Respiratory/Systemic Reactogenicity Grading 
 Reaction 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Runny nose 
Noticeable but does not interfere with daily 
activity 
Moderate discomfort that interferes 
with daily activity 
Significant discomfort/prevents daily 
activity or seeks medical care 
Stuffy nose/congestion 
Noticeable but does not interfere with daily 
activity 
Moderate discomfort that interferes 
with breathing from nose 
Unable to breathe through nose or seeks 
medical care 
Nasal pain/irritation 
Noticeable but does not interfere with daily 
activity 
Moderate discomfort that interferes 
with daily activity 
Significant discomfort that prevents daily 
activity or seeks medical care 
Epistaxis 
Total duration of all episodes in a 24-hour 
period <30 minutes 
Total duration of all episodes in a 
24-hour period >30 minutes 
Any bleeding that required visit for medical 
encounter 
Sneezing 
Noticeable but does not interfere with daily 
activity 
Moderate discomfort that interferes 
with daily activity 
Significant discomfort; prevents daily 
activity 
Sinus pressure/pain 
Noticeable but does not interfere with daily 
activity 
Moderate discomfort that interferes 
with daily activity 
Significant discomfort that prevents daily 
activity or seeks medical care 
Sore/irritated throat 
Noticeable but does not interfere with eating 
or drinking 
Moderate discomfort that interferes 
with eating or drinking 
Significant discomfort that prevents eating 
or drinking or seeks medical care 
Cough 
Noticeable but does not interfere with daily 
activity or sleeping 
Frequent cough that interferes with 
daily activity or sleeping 
Prevents daily activity, prevents sleep, or 
seeks medical care 
Shortness of 
breath/wheezing 
Noticeable but does not interfere with daily 
activity 
Moderate discomfort that interferes 
with daily activity 
Significant discomfort/ prevents daily 
activity or seeks medical encounter 
Fever  
100.4–101.1°F  
(38.0–38.4°C) 
101.2–102.0°F 
(38.5–38.9°C) 
>102°F  
(>39.0°C) 
Fatigue (tiredness) 
No interference with daily activity 
Some interference with daily activity 
Significant interference, prevents daily 
activity 
Malaise (general unwell 
feeling) 
No interference with daily activity 
Some interference with daily activity 
Significant interference, prevents daily 
activity 


 
 
Study Number: IB-202P 
Compound No.: BPZE1 
Protocol 
Version: 4 
 
Page 86 of 90 
 
 
CONFIDENTIAL 
Nasal/Respiratory/Systemic Reactogenicity Grading 
 Reaction 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Myalgia (body 
aches/muscular pain) 
No interference with daily activity 
Some interference with daily activity 
Significant interference, prevents daily 
activity 
Arthralgia (joint pain) 
No interference with daily activity 
Some interference with daily activity 
Significant interference, prevents daily 
activity 
Headache 
No interference with daily activity 
Some interference with daily activity 
Significant interference, prevents daily 
activity 
Rash/hypersensitivity 
Pruritus OR local rash 
Diffuse rash 
Diffuse rash with blisters or mouth 
ulcerations, anaphylaxis, or angioedema 
Source: Modified based on Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical 
Trials (fda.gov). Accessed 26 April 2022.Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, US 
Department of Health and Human Services. Division of AIDS (DAIDS) table for grading the severity of adult and pediatric adverse events. July 2017 [cited26 April 
2022]. Available from: https://rsc.niaid.nih.gov/sites/default/files/daidsgradingcorrectedv21.pdf 
 
 


 
 
Study Number: IB-202P 
Compound No.: BPZE1 
Protocol 
Version: 4 
 
Page 87 of 90 
 
 
CONFIDENTIAL 
16.3. 
Appendix 3: Table for Laboratory Grading 
Seruma 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Potentially Life 
Threatening (Grade 4)b 
Glucose – hyperglycemia  
Random (mg/dL; mmol/L)  
 
110–125 (6.1–<7.0) 
 
126–200 (7.0–11.1) 
 
>200 (>11.1) 
Insulin requirements or 
hyperosmolar coma 
Blood Urea Nitrogen (mg/dL; mmol/L)  
23–26 (8.2–9.5) 
27–31 (9.6–11.1) 
>31 (>11.1) 
Requires dialysis 
Creatinine (mg/dL; umol/L) 
1.5–1.7 (133–154) 
1.8–2.0 (155–180) 
2.1–2.5 (181–225) 
>2.5 (>225) or requires 
dialysis 
Albumin – hypoalbuminemia (g/dL; g/L)  
2.8–3.1 (28–31) 
2.5–2.7 (25–27) 
<2.5 (<25) 
– 
Total Protein – hypoproteinemia (g/dL; g/L)  
5.5–6.0 (55–60) 
5.0–5.4 (50–54) 
<5.0 (<50) 
– 
Liver Function Tests – ALT, AST increase by 
factor 
1.1–2.5 x ULN 
2.6–5.0 x ULN 
5.1–10 x ULN 
>10 x ULN 
Bilirubin – when accompanied by any increase 
in liver function test; increase by factor 
1.1–1.25 x ULN 
1.26–1.5 x ULN 
1.51–1.75 x ULN 
> 1.75 x ULN 
Bilirubin – when liver function test is normal; 
increase by factor 
1.1–1.5 x ULN 
1.6–2.0 x ULN 
2.0-3.0 x ULN 
> 3.0 x ULN 
Haematologya 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Potentially Life 
Threatening (Grade 4)b 
Haemoglobin (Female) (g/dL; g/L) 
11.0–12.0 (110–120) 
9.5–10.9 (95–109) 
8.0–9.4 (80–94) 
<8.0 (<80) 
Haemoglobin (Male) (g/dL; g/L) 
12.5–13.5 (125–135) 
10.5–12.4 (105–124) 
8.5–10.4 (85–104) 
<8.5 (<85) 
WBC elevation (cell/mm3; x109/L) 
10,800–15,000 (10.8–
15) 
15,001–20,000 
(>15–20) 
20,001–25,000 (>20–
25) 
>25,000 (>25) 
WBC suppression (cell/mm3; x109/L) 
2500–3500 (2.5–3.5) 
1500–2499 (1.5–
<2.5) 
1000–1499 (1.0–<1.5) 
<1,000 (<1.0) 
Platelets Decreased (cell/mm
3; x109/L) 
125,000–140,000 (125–
140) 
100,000–124,000 
(100–124) 
25,000–99,000 (25–
99) 
<25,000 (<25) 


 
 
Study Number: IB-202P 
Compound No.: BPZE1 
Protocol 
Version: 4 
 
Page 88 of 90 
 
 
CONFIDENTIAL 
ALT=alanine aminotransferase; AST=aspartate aminotransferase; ULN=upper limit of normal; WBC=white blood cell. 
a. 
The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal parameters. Institutional normal reference ranges should be provided 
to demonstrate that they are appropriate  
b. The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the laboratory abnormalities as Potentially Life Threatening (Grade 4). 
For example, a low sodium value that falls within a Grade 3 parameter (125–129 mEq/L) should be recorded as a Grade 4 hyponatremia event if the subject had a new seizure 
associated with the low sodium value 
Note: Screening laboratory samples with toxicity scores greater than 1 are exclusion criteria for vaccination enrolment. Laboratory sampling while in the challenge unit will comply with 
challenge unit standard protocol with the proviso that concurrent evidence of illness requires a daily complete blood count with differential to be conducted. 
Source: Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials (fda.gov). Accessed April 26, 2022. 
 
 


 
 
Study Number: IB-202P 
Compound No.: BPZE1 
Protocol 
Version: 4 
 
Page 89 of 90 
 
 
CONFIDENTIAL 
16.4. 
Appendix 4: Table for Vital Signs Grading 
Vital signs 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Potentially life threatening (Grade 
4) 
Fever (°C) 
38.0–38.4 
38.5–38.0 
>39.0 
>40.0 
Tachycardia (bpm) 
101–115 
116–130 
>130 
Emergency room visit or hospitalised 
for arrhythmia 
Bradycardia (bpm) 
50–54 
45–49 
<45 
Emergency room visit or hospitalised 
for arrhythmia 
Hypertension (systolic) mmHg 
141–150 
151–155 
>155 
Emergency room visit or hospitalised 
for malignant hypertension 
Hypertension (diastolic) mmHg 
91–95 
96–100 
>100 
Emergency room visit or hospitalised 
for malignant hypertension 
Hypotension (systolic) mmHg 
85–89 
80–84 
<80 
Emergency room visit or hospitalised 
for hypotensive shock 
Bpm=beats per minute 
a. 
Subject should be at rest for all vital sign measurements  
b. 
Oral temperature; no recent hot or cold beverages or smoking  
c. 
When resting heart rate is between 60 to 100 bpm. Use clinical judgement when characterizing bradycardia among some healthy subject populations, for example, conditioned 
athletes 
Note: Grade 0 will be the classification if the observation is less than a Grade 1. Respiratory rate was removed from the FDA table for vital sign grading as this is not a parameter being 
measured in this study 
Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials (fda.gov). Accessed April 26, 2022. 
 


 
 
Page 90 of 90 
 
 
CONFIDENTIAL 
16.5. 
Appendix 5: Process to be followed in cases of suspected 
symptomatic Bordetella pertussis infection 
 

SAP_001.pdf: 
 
Confidential 
 
 
 
 
 
 
 
 
 
ILiAD Biotechnologies 
 
IB-202P 
 
A Phase 2b, Placebo-Controlled, Randomized Study of BPZE1 Intranasal 
Pertussis Vaccine in Healthy Adults to Assess Protection Against Colonization 
Following Challenge with Virulent Wild-Type Bordetella pertussis 
 
30Jun2023 
 
Statistical Analysis Plan 
 
Final Version 1.0 
 
Prepared by: 
 
PPD 
929 N Front St,  
Wilmington, NC 28401 USA 
 
 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 II
TABLE OF CONTENTS 
LIST OF ABBREVIATIONS ...................................................................................................... V 
1. 
INTRODUCTION ............................................................................................................ 1 
2. 
OBJECTIVES ................................................................................................................... 1 
2.1. PRIMARY OBJECTIVE .......................................................................................................... 1 
2.2. SECONDARY OBJECTIVES ................................................................................................... 1 
2.2.1. 
Secondary Immunogenicity Objectives ....................................................................... 1 
2.2.2. 
Secondary Safety Objectives ....................................................................................... 1 
2.3. EXPLORATORY OBJECTIVES ............................................................................................... 2 
3. 
INVESTIGATIONAL PLAN .......................................................................................... 2 
3.1. OVERALL STUDY DESIGN AND PLAN .................................................................................. 2 
3.1.1. 
Vaccination Phase ...................................................................................................... 2 
3.1.2. 
Challenge Phase ......................................................................................................... 3 
3.1.3. 
Follow-up Phase ......................................................................................................... 3 
3.2. STUDY ENDPOINTS ............................................................................................................. 4 
3.2.1. 
Primary Endpoint........................................................................................................ 4 
3.2.2. 
Secondary Endpoints .................................................................................................. 4 
3.2.3. 
Exploratory Endpoints ................................................................................................ 4 
3.3. TREATMENTS ...................................................................................................................... 5 
4. 
GENERAL STATISTICAL CONSIDERATIONS ....................................................... 5 
4.1. SAMPLE SIZE ...................................................................................................................... 6 
4.2. RANDOMIZATION AND BLINDING ....................................................................................... 7 
4.3. ANALYSIS SET .................................................................................................................... 8 
4.3.1. 
Safety Analysis Set ...................................................................................................... 8 
4.3.2. 
Intent-to-Treat (ITT) Analysis Set ............................................................................... 8 
4.3.3. 
Modified Intent-to-Treat (mITT) Analysis Set ............................................................. 8 
4.3.4. 
Adequate Inoculum Analysis Set ................................................................................. 8 
4.3.5. 
Per Protocol (PP) Analysis Set ................................................................................... 9 
4.4. HANDLING OF MISSING DATA ............................................................................................ 9 
4.5. ANALYSIS WINDOW/VISIT ................................................................................................ 11 
5. 
SUBJECT DISPOSITION ............................................................................................. 11 
5.1. DISPOSITION ..................................................................................................................... 11 
5.2. PROTOCOL DEVIATIONS ................................................................................................... 13 
6. 
DEMOGRAPHICS AND BASELINE CHARACTERISTICS .................................. 13 
6.1. DEMOGRAPHICS ................................................................................................................ 13 
6.2. MEDICAL HISTORY ........................................................................................................... 14 
7. 
TREATMENTS AND MEDICATIONS ....................................................................... 14 
7.1. PRIOR AND CONCOMITANT MEDICATIONS ........................................................................ 14 
7.1.1. 
Prior Medications ..................................................................................................... 14 
7.1.2. 
Concomitant Medications ......................................................................................... 14 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
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Date Issued: 30Jun2023 
 
  III 
7.1.3. 
Concomitant Vaccination.......................................................................................... 14 
7.2. STUDY TREATMENTS ........................................................................................................ 15 
8. 
EFFICACY ANALYSIS................................................................................................. 15 
8.1. PRIMARY EFFICACY ANALYSIS ......................................................................................... 15 
8.1.1. 
Sensitivity Analysis for the Primary Efficacy Analysis ............................................. 18 
8.2. SECONDARY EFFICACY ANALYSIS .................................................................................... 18 
8.2.1. 
GMFR of Mucosal Anti-pertussis S-IgA Antibody from Baseline to Day 28 ............ 18 
8.2.2. 
GMFR of Serum IgA Antibody from Baseline to Day 28 .......................................... 19 
8.2.3. 
GMFR of Serum IgG Antibody from Baseline to Day 28 ......................................... 19 
8.2.4. 
Sensitivity Analysis for the Secondary Efficacy Analysis ......................................... 19 
8.3. EXPLORATORY EFFICACY ANALYSIS ................................................................................ 19 
8.3.1. 
Absolute Counts (CFU/mL) in Nasal Wash Samples on Challenge Days C9, C11 and 
C14 Following Virulent Challenge by Treatment Group ...................................................... 19 
8.3.2. 
GMC/GMT/GMR of Mucosal anti-pertussis S-IgA Antibody, Serum IgA and Serum 
IgG Antibody Throughout the Study by treatment group ....................................................... 20 
8.3.3. 
GMC/GMT/GMR and GMFR of Mucosal Anti-pertussis S-IgA Antibody, Serum IgA 
and Serum IgG Antibody Post-vaccination and Post-challenge by Treatment Group .......... 20 
8.3.4. 
Proportion of Subjects by Treatment Group with a ≥2x Increase in SBA 
Bactericidal Titer ................................................................................................................... 21 
8.3.5. 
Analysis of Treatment Group for Each B. pertussis Strain Studied by SBA and by 
Anti-pertussis IgG Antibody ................................................................................................... 21 
8.3.6. 
Cell-mediated Immunity Using Whole blood, PBMC and/or Nasal Pharyngeal 
Samples .................................................................................................................................. 21 
9. 
SAFETY ANALYSIS ..................................................................................................... 21 
9.1. SOLICITED ADVERSE EVENTS ........................................................................................... 21 
9.2. UNSOLICITED ADVERSE EVENTS ...................................................................................... 23 
9.2.1. 
Incidence of Adverse Events ..................................................................................... 23 
9.2.2. 
Relationship of Adverse Events to Study Vaccination/Challenge ............................. 24 
9.2.3. 
Severity of Adverse Event.......................................................................................... 24 
9.2.4. 
Serious Adverse Events ............................................................................................. 25 
9.2.5. 
Adverse Events of Special Interest ............................................................................ 25 
9.2.6. 
Adverse Events Leading to Study Discontinuation ................................................... 25 
9.2.7. 
Death ......................................................................................................................... 25 
9.2.8. 
Pre-defined Post-Challenge Checklist ...................................................................... 26 
9.3. CLINICAL LABORATORY EVALUATIONS ........................................................................... 26 
9.3.1. 
Pregnancy Test.......................................................................................................... 28 
9.3.2. 
COVID-19 Testing .................................................................................................... 28 
9.4. VITAL SIGN MEASUREMENTS ........................................................................................... 28 
9.5. PHYSICAL EXAMINATION ................................................................................................. 29 
10. 
PRIMARY ANALYSIS .................................................................................................. 29 
11. 
CHANGES IN THE PLANNED ANALYSIS .............................................................. 29 
12. 
REFERENCES ................................................................................................................ 30 
13. 
APPENDICES ................................................................................................................. 31 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
  IV 
13.1. SCHEDULE OF EVENTS ...................................................................................................... 31 
 
TABLES 
Table 1. Dosing Scheme ............................................................................................................... 3 
Table 2. Power for Sample Sizes of 20 in Each Group for Varying Colonization Rates Based on 
a Likelihood Ratio Test ................................................................................................................... 6 
Table 3. Estimated Sample Size Based on Assumed Colonization rates and Power of 80% and 
Lower Bounds of 0%, 10%, 20% and 30% .................................................................................... 7 
Table 4. Analysis windows ........................................................................................................ 11 
Table 5. Estimand attributes for the primary efficacy analysis .................................................. 14 
Table 6. Visit windows ............................................................................................................... 14 
Table 7. Reactogenicity Grading Scale ...................................................................................... 18 
Table 8. Table for Laboratory Grading ...................................................................................... 23 
Table 9. Table for Vital Sign Grading ........................................................................................ 24 
 
FIGURE 
Figure 1. 
Dosing Scheme ........................................................................................................... 2 
 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 V
List of Abbreviations 
Abbreviation 
Definition 
AE 
Adverse event  
AESI 
Adverse event of special interest  
B. pertussis 
Bordetella pertussis 
CFU 
Colony-forming units 
CI 
Confidence interval 
eCRF 
Electronic case report form 
ELISA 
Enzyme-linked immunosorbent assay 
EOS 
End of study 
FDA 
Food and Drug Administration  
FHA 
Filamentous hemagglutinin 
FIM2/3 
Fimbriae types 2 and 3  
GMC 
Geometric mean concentration 
GMFR  
Geometric mean fold rise 
GMR 
Geometric mean ratio 
GMT 
Geometric mean titer 
ICE 
Intercurrent event 
ICF 
Informed consent form 
IgA 
Immunoglobulin A 
IgG 
Immunoglobulin G 
IQR 
Interquartile range 
MAD 
Mucosal atomization device 
MedDRA 
Medical dictionary for Regulatory Activities  
OTC 
Over-the-counter 
PBMC 
Peripheral blood mononuclear cell 
PCR 
Polymerase chain reaction  
PRN 
Pertactin 
PT 
Pertussis toxin 
PT 
Preferred term 
SAP 
Statistical analysis plan 
SARS-CoV-2 
Severe acute respiratory syndrome coronavirus 2 
SBA 
Serum bactericidal assay 
S-IgA 
Secretory immunoglobulin A  
SOC 
System organ class 
TEAEs 
Treatment-emergent adverse events 
Th 
T helper cell 
TOPS 
The Overvolunteering Prevention System 
ULN 
Upper limit of normal 
VIS 
Volunteer information sheet 
 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 1
1. Introduction 
This statistical analysis plan (SAP) describes the analyses and data presentations for ILiAD’s 
protocol IB-202P “A Phase 2b, Placebo-Controlled, Randomized Study of BPZE1 Intranasal 
Pertussis Vaccine in Healthy Adults to Assess Protection Against Colonization Following 
Challenge with Virulent Wild-Type Bordetella pertussis” which was issued on 03Feb2022 
(amended 02Mar2022, 30May2022, 27Jul2022, and 06Mar2023). It contains definitions of 
analysis populations, derived variables, and statistical methods for the analysis of colonization, 
immunogenicity, and safety.  
The analyses for this study include one primary analysis after the last subject completes 
Challenge Day 16 (Visit C16) and one final analysis at the end of study (EOS). The purpose of 
the SAP is to ensure the credibility of the study findings by pre-specifying the statistical 
approaches to the analysis of study data prior to database lock and any data analysis for the 
primary/final analyses. This SAP will be finalized and signed prior to the clinical database lock 
for the primary analyses. All statistical analyses detailed in this SAP will be conducted using 
SAS® Version 9.4 or higher. 
This SAP is to be interpreted in conjunction with the protocol. Should the SAP and protocol be 
inconsistent with respect to the planned analyses, the language of the SAP is governing. If the 
final clinical study report (CSR) contains changes to any planned statistical analyses, the 
justification for any such differences will be fully documented in the CSR. 
2. Objectives 
2.1.Primary Objective 
The primary objective is to demonstrate that prior immunization with BPZE1 protects against 
colonization as evidenced by a negative B. pertussis culture following virulent B. pertussis 
challenge 2–4 months after vaccination. 
2.2.Secondary Objectives 
2.2.1. Secondary Immunogenicity Objectives 
• To demonstrate BPZE1 induction of mucosal anti-pertussis secretory IgA (S-IgA) 
antibody is improved from baseline to Day 28 
• To demonstrate BPZE1 induction of systemic IgA is improved from baseline to Day 28 
• To demonstrate BPZE1 induction of systemic IgG is improved from baseline to Day 28 
2.2.2. Secondary Safety Objectives 
• To assess reactogenicity profiles (by toxicity scoring) through 7 days following study 
vaccination 
• To describe all unsolicited treatment-emergent AEs (TEAEs) through 28 days following 
study vaccination and following virulent challenge 
• To describe all unsolicited TEAEs related to vaccination from time of vaccination to 
challenge or related to challenge for 3 months after challenge 
• To describe any AEs of special interest (AESI) and SAEs through end of study (EOS) 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
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Date Issued: 30Jun2023 
 
 
 2
2.3.Exploratory Objectives 
• To demonstrate prior immunization with BPZE1 reduces overall bacterial load following 
virulent B. pertussis challenge 
• To demonstrate BPZE1 induction of mucosal secretory immunity (S-IgA) and systemic 
immunity (IgA and IgG) 
• To demonstrate BPZE1 induction of immunity is similar to that observed following 
virulent challenge 
• To compare functional antibody response by serum bactericidal assay (SBA) 
• Correlation and threshold analyses by individual and combination anti-pertussis IgG 
antibodies to SBA 
• To assess cytokine and T helper cell (Th)1/Th2/Th17 dominance response using whole 
blood, peripheral blood mononuclear cells (PBMCs) and/or nasopharyngeal samples 
3. Investigational Plan 
3.1.Overall Study Design and Plan 
This is a randomized, double-blinded, placebo-controlled trial of BPZE1 that includes virulent B. 
pertussis challenge followed by a safety follow-up. 
Consenting, eligible subjects will receive a single dose of BPZE1 or placebo; 2–4 months later 
they will be challenged with B. pertussis and admitted to a challenge unit. Subjects will remain 
in the challenge unit for a total of 17 days and 16 nights during which time they will be 
monitored closely. If a subject develops symptoms of pertussis, antibiotic will be started, and the 
subject will remain in the unit for 3 additional days of observation before discharge. If symptoms 
of pertussis do not develop, then subjects will receive antibiotic (azithromycin) from Days 14–16 
of the challenge unit stay. Subjects will undergo safety follow-up for at least 6 months 
post-vaccination and at least 3 months post-challenge, for a total follow-up of 6–7 months.  
Figure 1. Dosing Scheme 
 
3.1.1. Vaccination Phase 
After signing the informed consent form (ICF) and meeting all inclusion and none of the 
exclusion criteria, a suitable number of eligible subjects (approximately 60) will be randomized 
1:1 to receive BPZE1 (109 CFU) or placebo (formulation buffer) via an intranasal mucosal 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 3
atomization device (MAD) (Table 1) so that approximately 44 subjects are enrolled and 
challenged in the study for a minimum of 20 evaluable subjects per arm with culture results 
during the challenge phase, i.e., a minimum of 20 subjects per arm included in the mITT analysis 
set.  
Table 1. 
Dosing Scheme 
Treatment Arms 
 
Nasal Vaccination (Day 0) 
B. pertussis 
Virulent Challenge (Day 60c) 
BPZE1 
placebob 
A (N=22a) 
X 
 
X 
B (N=22a) 
 
X 
X 
a  
Evaluable subjects estimated at 20 per treatment arm with two additional to account for dropouts 
b  
Intranasal application of 0.4 mL per nostril (0.8 mL total volume delivered) formulation buffer placebo 
c  
Subject may enter virulent challenge unit between Days 60 to 120 
On Day 0, nasal/respiratory and systemic reactogenicity will be assessed approximately 
30 minutes after vaccination prior to release from clinical observation. Then, all subjects will 
record maximum daily reactogenicity in a subject diary for 7 consecutive days starting the day 
following study vaccination (retrospective of the highest value in the previous 24 hours). Both 
nasal/respiratory and general systemic reactogenicity events including actual measurements as 
needed (i.e., body temperature) will be recorded. 
3.1.2. Challenge Phase 
Between Days 60 and 120 after vaccination, subjects will plan to enter the challenge unit after 
confirmation to proceed to virulent challenge. Once subjects are cleared for virulent challenge, a 
dose of the B. pertussis strain B1917 at 105 colony-forming units (CFU) will be administered 
while lying supine and using the procedures established within the challenge unit. Subjects will 
remain supine for 15 minutes. Subjects will remain in the unit for a total of 17 days for 
observation. Daily monitoring will occur including vital signs, physical exam (as needed), 
signs/symptoms of pertussis infection (e.g., catarrhal symptoms), and select clinical laboratory 
testing will occur on pre-specified days. Nasal washes will occur at the baseline of the challenge 
(Pre-Challenge) and on Challenge Days C9, C11, and C14, or prior to initiating antibiotics as 
well as after receiving antibiotics. If a B. pertussis culture collected on the last day of the in-unit 
stay (C16) is positive, the subject will return to the site to receive continued outpatient antibiotic 
treatment (azithromycin).  
3.1.3. Follow-up Phase 
Subjects will be followed for safety for 6 months following vaccination or 3 months following 
challenge, whichever is longer. 


ILiAD Biotechnologies 
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 4
3.2.Study Endpoints 
3.2.1. Primary Endpoint 
The primary endpoint is the proportion of subjects by treatment group (BPZE1 and placebo) 
colonized on any day (Challenge Day C9, C11 or C14) following virulent challenge as 
determined by culture. 
3.2.2. Secondary Endpoints 
Secondary Immunogenicity Endpoints 
• The geometric mean fold rise (GMFR) of mucosal anti-pertussis S-IgA antibody (whole 
cell extract [WCE], Filamentous hemagglutinin [FHA], Pertactin [PRN], Pertussis toxin 
[PT] and fimbriae types 2 and 3 [FIM2/3]) from baseline to Day 28 (BPZE1 and 
placebo). S-IgA to be normalized ([specific S-IgA]/[total S-IgA]) 
• The GMFR of serum IgA antibody (WCE, FHA, PRN, PT and FIM2/3) from baseline to 
Day 28 (BPZE1 and placebo) 
• The GMFR of serum IgG antibody (WCE, FHA, PRN, PT and FIM2/3) from baseline to 
Day 28 (BPZE1 and placebo) 
Secondary Safety Endpoints 
• Occurrence and intensity of solicited AEs for nasal/respiratory and systemic 
reactogenicity through 7 days following vaccination by treatment group (BPZE1 and 
placebo) 
• Occurrence and intensity of TEAEs through 28 days following study vaccination and 
following challenge by treatment group (BPZE1 and placebo) 
• Occurrence and intensity of TEAEs related to vaccination from time of vaccination to 
challenge or related to challenge for 3 months after challenge by treatment group (BPZE1 
and placebo) 
• Occurrence, intensity, and relationship to study vaccine of AESIs and SAEs from 
vaccination through EOS by treatment group (BPZE1 and placebo) 
3.2.3. Exploratory Endpoints 
• Absolute counts (CFU/mL) in nasal wash samples on Challenge Days C9, C11 and C14 
following virulent challenge by treatment group (BPZE1 and placebo) 
• Geometric mean concentration (GMC)/geometric mean titer (GMT)/geometric mean ratio 
(GMR) of mucosal anti-pertussis S-IgA antibody, serum IgA and serum IgG antibody 
(WCE, FHA, PRN, PT and FIM2/3) throughout the study by treatment group (BPZE1 
and placebo). S-IgA to be normalized ([specific S-IgA]/[total S-IgA]) 
• The GMC/GMT/GMR and GMFR of mucosal anti-pertussis S-IgA antibody, serum IgA 
and serum IgG antibody (WCE, FHA, PRN, PT and FIM2/3) post-vaccination and 
post-challenge by treatment group (BPZE1 and placebo). S-IgA to be normalized 
([specific S-IgA]/[total S-IgA]) 
• Proportion of subjects by treatment group (BPZE1 and placebo) with a ≥2x increase in 
SBA 50% killing titer 
• Analysis (GMC/GMT) by treatment group (BPZE1 and placebo) for each B. pertussis 
strain studied by SBA and by anti-pertussis IgG antibody 


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• Cell-mediated immunity using whole blood, PBMC and/or nasal pharyngeal samples, 
including but not limited to cytokines, Th1/Th17 and Th2 responses 
3.3.Treatments 
Subjects in the BPZE1 group will receive intranasal application of 2 x 0.4 mL, with 0.4 mL per 
nostril containing half the dose (5 x 108 CFU) bacteria to give a total dose of 109 CFU, via a 
MAD on Day 0 (Visit 1). 
Subjects in the placebo group will receive an intranasal application of 2 x 0.4 mL, with 0.4 mL 
per nostril, reconstituted lyophilized buffer via a MAD on Day 0 (Visit 1). 
The intranasal placebo consists of lyophilized buffer diluted with sterile water for injection 
(WFI), which are the same constituents in the same quantities as the BPZE1 investigational 
vaccine, except for the attenuated B. pertussis cells. 
Once subjects are cleared for virulent challenge of a dose of the B. pertussis B1917 strain at 
105 CFU, the dose will be administered while lying supine and using the procedures established 
within the challenge unit. The inoculum will be instilled into each nostril with a Gilson pipette 
by droplet. Subjects will remain supine for 15 minutes. 
4. General Statistical Considerations 
Continuous variables will be summarized using the mean, standard deviation (SD), median, first 
quartile, third quartile, interquartile range (IQR), minimum value, and maximum value. 
Categorical variables will be summarized using frequency counts and percentages. All statistical 
tests will be two-sided at 0.05 significance level. 
Baseline is defined as the last non-missing value before the vaccination on Day 0 (Visit 1). 
Pre-Challenge for the challenge phase is defined as the last non-missing value before the 
challenge on Challenge Day C0 (Visit C0).  
For summary precision, mean and median will have one more decimal place than the reported 
value, SD will have two more decimal places than the reported value, minimum and maximum 
will have the same decimal place as the reported value. Percentages will have one decimal place 
and the 95% confidence interval (CI) will have one more decimal place than the point estimate. 
For each antibody, if results are reported as lower than the minimal limit of assay detection, a 
value equal to half of the minimal limit of assay detection will be imputed in the calculation. The 
minimal limit of assay detection for each of the antibody concentrations will be established 
following assay qualification or validation (depending on the assay) and prior to database lock. 
GMC/GMT/GMR will be calculated as anti-logarithm of ∑ (log-transformed concentration, titer, 
or ratio/number of subjects with concentration, titer or ratio information). The 95% CI for 
GMC/GMT/GMR will be calculated as the anti-log transformation of lower and upper limits for 
a two-sided 95% CI of the mean of the log-transformed titers based on normal approximation.  
GMFR will be calculated as anti-logarithm of ∑[log-transformed titer ratio of Yi/Bi)/number of 
subjects with titer information], where Yi is the post-dose titer and Bi is the baseline titer for 
subject i. GMFR over baseline and GMFR over Pre-Challenge will be calculated, if available. 
The same data summaries will be provided as GMC/GMT/GMR. 


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For immunogenicity analysis, it is assumed that the natural log of the data is normally 
distributed.  
All data collected will be listed in data listings.  
4.1.Sample Size 
Table 2 depicts the power for the challenge study as computed under the following assumptions: 
two-sided significance level of 0.05, use of a likelihood ratio test for testing equality of two 
proportions, a sample size of 20 in each of the two groups, a colonization rate of 60%, 70%, and 
80% in the unvaccinated group and colonization rates of 10%, 20%, 30%, and 40% in the 
vaccinated group.  
Sample sizes of 20 evaluable subjects in each group achieve a power of at least 90% to detect 
differences between colonization rates of at least 50% in six scenarios. These scenarios include 
the following colonization rates for the placebo and BPZE1 groups, respectively: 60% versus 
10%, 70% versus 10%, 70% versus 20%, 80% versus 10%, 80% versus 20% and 80% versus 
30%, which are bolded in Table 2.  
Table 2. 
Power for Sample Sizes of 20 in Each Group for Varying Colonization Rates 
Based on a Likelihood Ratio Test 
Colonization Rate 
 
 
Placebo 
BPZE1 
N/group 
Power 
 
 
 
 
60% 
10% 
20 
94% 
 
20% 
 
75% 
 
30% 
 
48% 
 
40% 
 
24% 
 
 
 
 
70% 
10% 
20 
99% 
 
20% 
 
92% 
 
30% 
 
73% 
 
40% 
 
48% 
 
 
 
 
80% 
10% 
20 
100% 
 
20% 
 
99% 
 
30% 
 
92% 
 
40% 
 
75% 
 
Table 3 includes results based on vaccine efficacy computed from the colonization rates in each 
population, rather than comparing the two colonization rates and testing the null hypothesis that 
they are equal in the two populations. The computations presented in the table below are based 
on a normal approximation. The table includes the required sample size for a power of 80% and 
for lower bounds of vaccine efficacy of 0%, 10%, 20%, and 30%. For a colonization rate of 70% 
in the placebo group and a lower bound of efficacy of 20%, the study will be powered for 


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efficacy rates of 71% or higher. Focusing on an efficacy rate greater than 50% and a lower bound 
of 0% or higher (sample sizes in bold), the study is well powered to detect efficacy rates of 57% 
or higher. 
Table 3. 
Estimated Sample Size Based on Assumed Colonization rates and Power of 80% 
and Lower Bounds of 0%, 10%, 20% and 30% 
Colonization rate 
Sample size/group 
Control 
Vaccine 
Efficacy 
LB 0% 
LB 10% 
LB 20% 
LB 30% 
60% 
10% 
0.833 
11 
12 
14 
18 
 
20% 
0.667 
18 
22 
29 
42 
 
30% 
0.500 
33 
47 
75 
149 
 
40% 
0.333 
77 
141 
390 
5675 
 
 
 
 
 
 
 
70% 
10% 
0.857 
8 
9 
10 
12 
 
20% 
0.714 
12 
14 
18 
24 
 
30% 
0.571 
19 
25 
36 
60 
 
40% 
0.429 
33 
51 
95 
268 
 
 
 
 
 
 
 
80% 
10% 
0.875 
6 
6 
7 
9 
 
20% 
0.750 
8 
9 
12 
16 
 
30% 
0.625 
12 
15 
20 
31 
 
40% 
0.500 
18 
25 
40 
82 
 
For the endpoint of absolute counts, the standard deviation in the BPZE1 group was computed to 
be 2.2 for the natural log of the counts and 4.2 for the natural log of the counts in the control 
group. Assuming a sample size of 20 evaluable subjects in each group, a two-sample t-test 
accounting for unequal variances, and a two-sided significance level of 0.05, the study has 
approximately 80% power to detect differences of 3.1 units or larger between the BPZE1 group 
and the control group on the natural log scale.  
4.2.Randomization and Blinding 
Subjects will be randomized in a 1:1 ratio to receive BPZE1 or placebo. Unblinded individuals at 
the site will use the randomization list generated by the Biostatistics randomization team to 
allocate the subjects to either BPZE1 or placebo. All vaccinated subjects will be invited to 
Challenge phase. Per protocol, subjects who withdraw, are withdrawn due to an unrelated AE as 
determined by the Investigator, are lost to follow-up prior to the challenge phase, or subjects who 
miss all 3 culture results (Challenge Days C9, C11 and C14) during the challenge phase may be 
replaced. The randomization schedule with leading number “1” (1001-1088) is the main 
randomization number. The randomization schedule with leading number “2” (2001-2088) is the 
replacement randomization number. Replacement randomization schedule is the identical 
treatment arm with the main randomization schedule (i.e., the treatment arm of 1001 and 2001 


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are identical). If a randomized subject is deemed to be replaced, the Investigator will assign the 
replacement randomization number to the subject to ensure the replacement subject is in the 
same treatment arm with the subject being replaced.  
Subjects who decline to receive the challenge agent can remain in the study to participate in 
safety follow-up visits after vaccination. An unblinded statistician not associated with the study 
team will review the randomization assignment to ensure that a minimum of 20 evaluable 
subjects per arm are included in the mITT analysis set.  
This is a double-blind study. Neither the subject nor the Investigator/site staff will know what 
study vaccine the subject is receiving with the exception of site staff involved in the preparation 
of the vaccination. These members of staff will take no further part in the study. 
The site(s) will identify an unblinded individual(s) who will be provided with a randomization 
list. This list will be used to allocate the subjects to either BPZE1 or placebo. To maintain the 
blind, unblinded individuals will only be involved in vaccine logistics and preparation. The 
unblinded team will not be involved in any study-related assessments or have subject contact for 
data collection following study vaccination. Blinded individuals will administer the study 
vaccine with further details in the Pharmacy/Challenge Manual. 
Code break envelopes will be provided to the site(s) so that they can unblind an individual 
subject in the event of a medical emergency. Where possible they should discuss the situation 
with the Sponsor’s medical monitor prior to unblinding; however, the subject’s safety must 
always come first. Reasons for treatment unblinding must be clearly explained and justified in 
the eCRF. The date on which the code was broken together with the identity of the person 
responsible must also be documented. 
4.3.Analysis Set 
4.3.1. Safety Analysis Set 
The safety analysis set will consist of all subjects randomized to treatment who are vaccinated. 
Subjects will be classified according to the actual vaccine received. The safety analysis will be 
done on this analysis set. 
4.3.2. Intent-to-Treat (ITT) Analysis Set 
The intent-to-treat analysis set will consist of all subjects randomized to treatment who are 
vaccinated. Subjects will be classified according to the randomized treatment group. 
4.3.3. Modified Intent-to-Treat (mITT) Analysis Set 
The modified intent-to-treat analysis set will consist of all subjects randomized to treatment who 
are vaccinated, challenged, and have at least one culture result at Challenge Day C9, C11, or 
C14. Subjects will be classified according to the randomized treatment group. 
4.3.4. Adequate Inoculum Analysis Set 
The adequate inoculum analysis set will consist of all subjects who are included in the mITT 
analysis set and received a challenge inoculum of ≥5 x 104 CFU on Challenge Day C0. 


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4.3.5. Per Protocol (PP) Analysis Set 
The per-protocol analysis set will consist of all subjects who are included in mITT population 
with no major protocol deviations. This wording has been updated from the protocol definition. 
4.4.Handling of Missing Data 
Colonization data will be presented in the listings as reported. For summaries and analysis, the 
following conventions apply. 
When only one day out of three days (Challenge Days C9, C11 and C14) is missing: 
• If Day C9 and Day C11 are negative but Day C14 is missing, Day C14 will be imputed as 
negative; 
• If Day C9 and Day C14 are negative but Day C11 is missing, Day C11 will be imputed as 
negative; 
• Because participants are closely monitored in the challenge unit, it is deemed highly 
unlikely that they will miss Day C9 but come back for Days C11 and Day C14, therefore 
the scenario of missing Day C9 only is not considered; 
• If any day is positive, the remaining missing data will not be imputed. 
 
When two days out of three days (Challenge Days C9, C11 and C14) are missing: 
• If Day C9 is negative but Day C11 and Day C14 are missing, Day C11 and Day C14 will 
be imputed as negative; 
• As described above, it is deemed highly unlikely that participants will miss an earlier 
assessment but come back for later day assessment, including the following two 
scenarios:  
− Day C11 is negative but Day C9 and Day C14 are missing  
− Day C14 is negative but Day C9 and Day C11 are missing 
therefore both scenarios are not considered. 
• Data may also be missing due to the sample being corrupt. 
• If any day is positive, the remaining missing data will not be imputed. 
Impute Missing Adverse Events/ Prior or Concomitant Medications 
A. Incomplete Start Date: 
Missing day and month 
• If the year is the same as the year of the first dosing date, then the day and month of the 
first dosing date will be assigned to the missing fields. 
• If the year is prior to the year of first dosing date, then December 31 will be assigned to 
the missing fields. 


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• If the year is after the year of first dosing, then January 1 will be assigned to the missing 
fields. 
Missing day only  
• If the month and year are the same as the year and month of first dosing date, then the 
first dosing date will be assigned to the missing day. 
• If either the year of the partial date is before the year of the first dosing date or the years 
of the partial date and the first dosing date are the same but the month of partial date is 
before the month of the first dosing date, then the last day of the month will be assigned 
to the missing day. 
• If either the year of the partial date is after the year of the first dosing date or the years of 
the partial date and the first dosing date are the same but the month of partial date is after 
the month of the first dosing date, then the first day of the month will be assigned to the 
missing day. 
• If the stop date is not missing, and the imputed start date is after the stop date, the start 
date will be imputed by the stop date, i.e. set to the stop date.  
Missing day, month, and year 
• No imputation is needed; the corresponding AE will be included as TEAE if the end date 
of the AE is after the first dosing date or the end date is also missing.  
B. Incomplete End Date: 
If the imputed stop date is before the start date, then the imputed stop date will be equal to the 
start date. 
Missing day and month 
• If the year of the incomplete stop date is the same as the year of the last dosing date, then 
the day and month of the last dosing date will be assigned to the missing fields.  
• If the year of the incomplete stop date is prior to the year of the last dosing date, then 
December 31 will be assigned to the missing fields.  
• If the year of the incomplete stop date is prior to the year of the last dosing date but is 
the same as the year of the first dosing date, then the first dosing date will be assigned to 
the missing date.  
• If the year of the incomplete stop date is after the year of the last dosing date, then 
January 1 will be assigned to the missing fields. 
Missing day only  
• If the month and year of the incomplete stop date are the same as the month and year of 
the last dosing date, then the day of the last dosing date will be assigned to the missing 
day.  
• If either the year of the partial date is not equal to the year of the last dosing date or the 
years of the partial date and the last dosing date are the same but the month of partial date 


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is not equal to the month of the last dosing date, then the last day of the month will be 
assigned to the missing day.  
4.5.Analysis Window/Visit 
Visit windows are defined in Section 13.1. Additionally, analysis windows will be derived using 
the following rules: 
Table 4. 
Analysis windows 
Study Phase 
Study Visit/Study Day 
Analysis Window 
Vaccination Phase 
Visit 1/Day 0 
No analysis window allowed 
Visit 2/Day 7  
Day 5 to Day 21 
Visit 3/Day 28 
Day 22 to Day 45 
Visit 4/Day 52a 
Day 46 to Day 120 
Challenge Phase 
Visit C0 to C16/Day 0-16b 
No analysis window allowedd 
Safety Follow-up 
Visit 5/Day 28b 
Challenge Day 21 to Challenge 
Day 89 
EOS/Day 180c 
Challenge Day 90 to Challenge 
Day 120 (EOS) 
a  
Initiate screening for eligibility for the Challenge Phase; admit to challenge unit on Study Visit C0 
b  Days relative to Challenge Phase 
c  
This visit will occur at least 6 months post-vaccination or at least 3 months post-challenge 
d  The analysis window for Challenge Day 9, 11 and 14 refers to table 5. 
5. Subject Disposition 
5.1.Disposition 
The number of subjects who were randomized and screen failed will be presented based on all 
screened subjects.  
Disposition of all randomized subjects (ITT Analysis Set and mITT Analysis Set) will be 
summarized by treatment groups and overall including: 
• 
Number of subjects vaccinated 
• 
Number of subjects never vaccinated 
• 
Number of subjects received challenge agent 
• 
Number of subjects who completed challenge unit 
• 
Reasons for not completing challenge unit 
o 
Adverse Event 


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o 
Withdrawal of Consent 
o 
Other 
• 
Number of subjects never challenged 
o 
Number of subjects discontinued from the study before challenge 
o 
Primary reasons for study discontinuation before challenge 
• 
Lost to Follow-Up 
• 
Withdrawal of Consent by Subject 
• 
Investigator Decision 
• 
Adverse Event 
• 
Death 
• 
Significant Noncompliance with Protocol 
• 
Sponsor Decision 
• 
Other 
o 
Number of subjects who did not receive challenge but participated in safety follow-up 
o 
Reasons for not receiving challenge agent after receiving study vaccine 
• 
Adverse Event 
• 
Withdrawal of Consent 
• 
Sponsor Decision 
• 
Other 
• 
Number of subjects who completed study (EOS) 
• 
Number of subjects who prematurely discontinued study 
• 
Primary reason for premature discontinuation of study 
Primary reasons for study discontinuation collected on the Study Termination eCRF will be 
summarized with the following categories: 
• 
Lost to Follow-Up 
• 
Withdrawal of Consent by Subject 
• 
Investigator Decision 
• 
Adverse Event 
• 
Death 
• 
Significant Noncompliance with Protocol 
• 
Sponsor Decision 
• 
Other 


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Number of subjects in each analysis set will also be summarized in disposition table.  
Reasons for ITT subject excluded from mITT analysis set will be summarized with the following 
categories: 
• 
Missing Colonization Culture (Challenge Days C9, C11, or C14) 
• 
Not Challenged 
The subjects who did not receive challenge but completed the safety follow-up will be 
considered as Completed Study.  
A by-subject disposition listing will be provided based on the ITT Analysis set. Eligibility 
criteria met/failed for vaccination phase and challenge phase will be listed based on all screened 
subjects. 
5.2.Protocol Deviations 
A deviation from the protocol is an unintended or unanticipated departure from the agreed 
procedures or processes. Deviations will be assessed with Sponsor involvement and deemed 
significant or non-significant prior to database lock. 
Protocol deviations will be documented by the clinical monitor throughout the course of 
monitoring visits. Significant protocol deviations will be summarized using the ITT Analysis Set.  
A by-subject listing will be provided based on the ITT Analysis Set including both the 
significant and non-significant protocol deviations.  
6. Demographics and Baseline Characteristics 
6.1.Demographics 
Demographic and baseline characteristics data to be analyzed will include age, sex, ethnicity, 
weight, height, and body mass index (BMI) on the ITT analysis set, mITT analysis set, Adequate 
Inoculum analysis set, and Per-Protocol analysis set by treatment group and overall. 
The following characteristics will be summarized and presented as continuous variables: 
• 
Age (years) 
• 
Weight (kg) 
• 
Height (cm) 
• 
BMI (kg/m2) 
The following characteristics will be summarized and presented as categorical variables: 
• 
Sex (Male, Female) 
• 
Ethnicity (White, Black, Asian, Mixed or Multiple ethnic groups, Other) 
A by-subject listing will be provided based on the ITT Analysis set. 


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6.2.Medical History 
Medical history will be summarized based on the ITT Analysis Set and mITT Analysis Set and 
will be coded according to latest version of Medical Drug Regulatory Activities (MedDRA). 
Medical history will be summarized by system organ class (SOC) and preferred term (PT), with 
SOCs sorted alphabetically and PTs within each SOC in descending order of frequency.  
A by-subject listing will be provided for medical history based on the ITT Analysis set. 
7. Treatments and Medications 
7.1.Prior and Concomitant Medications 
The Anatomical Therapeutic Chemical (ATC) coding scheme of the latest World Health 
Organization Drug Dictionary (WHODrug) will be used to group medications into relevant 
categories. 
7.1.1. Prior Medications 
Prior medications are defined as non-study medications with a start date before the date of 
vaccination. Prior medications that continue on and after the date of vaccination will be reported 
as both prior and concomitant medications. Prior medications will be summarized based on the 
ITT Analysis Set and mITT Analysis Set by ATC2 level and preferred medication names, with 
ATC2 level sorted alphabetically and preferred names within each ATC2 level in descending 
order of frequency.  
A by-subject listing will be provided for prior medications based on the ITT Analysis Set. 
7.1.2. Concomitant Medications 
Concomitant medications are defined as all medications and over-the-counter (OTC) products, 
including herbals, supplements, vaccines, and multivitamins, taken by the subject from the time 
of vaccination through the EOS visit, or through an early termination visit, if prior to these time 
points.  
Concomitant medications will be summarized by ATC2 level and standardized medication 
names, with ATC2 level sorted alphabetically and standardized names within each ATC2 level in 
descending order of frequency. Summaries will be provided based on the Safety Analysis Set.  
A by-subject listing will be provided for concomitant medications based on the Safety Analysis 
set. 
7.1.3. Concomitant Vaccination 
Vaccines received outside of the study will be specifically queried during each visit and recorded 
through EOS. Concomitant vaccinations will be summarized based on Safety Analysis Set. 
A by-subject listing will be provided for concomitant vaccinations based on the Safety Analysis 
set. 


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7.2.Study Treatments 
Number of days from vaccination to receiving challenge agent will be summarized using 
descriptive statistics based on the mITT Analysis Set.  
A by-subject listing will be provided based on the All Randomized subjects for receiving 
vaccination and receiving challenge agent.  
8. Efficacy Analysis 
Unless otherwise specified, efficacy analysis will be performed based on the mITT Analysis Set. 
All summaries and analyses will be presented over all subjects and by treatment groups.  
By subject listings will be provided for pertussis antigen specific antibody titer using mucosal 
samples, pertussis antigen specific antibody titer using serum sample, colonization B. pertussis 
Bacterial Culture, and cellular response based on ITT Analysis Set. 
The visit windows to be used for the definition of the primary endpoint of colonization are 
provided in Table 5. 
 
Table 5. Visit windows for endpoint of colonization 
Visit 
Window 
Challenge Day 9 
Challenge Day 8, 9 or 10 
Challenge Day 11 
Challenge Day 11, 12 
Challenge Day 14 
Challenge Day 13, 14 
 
8.1.Primary Efficacy Analysis 
 
The estimand description for the primary efficacy analysis is provided in Table 6. 
 
 


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Table 6. 
Estimand attributes for the primary efficacy analysis 
Estimand Label 
Estimand  
Estimand Description  
The difference in the proportion of colonization on any day 
(Challenge Day C9, C11, or C14) following virulent 
Challenge agent (B. pertussis B1917 strain at 105 CFU) on 
Challenge Day C0 between BPZE1 and placebo administered 
intranasally via a MAD in healthy adult subjects 2-4 months 
prior to challenge 
Target population 
All healthy adult subjects who receive vaccination, are 
challenged, and have at least one culture result at Challenge 
Day C9, C11, or C14. 
Treatment Condition 
Trial arm: BPZE1 (2 x 0.4 mL (0.4 mL per nostril containing 
half the dose (5 x 108 CFU) of bacteria to give a total dose of 
109 CFU) administered intranasally via a MAD) on Day 0 
Control arm: Placebo (2 x 0.4 mL (0.4 mL per nostril) 
reconstituted lyophilized buffer administered intranasally via 
a MAD) on Day 0 
Challenge: a 105 CFU dose of the B. pertussis strain B1917 
on Challenge Day C0 
Endpoint 
The proportion of subjects by treatment group (BPZE1 and 
placebo) colonized on any day (Challenge Day C9, C11 or 
C14) following virulent challenge as determined by culture. 
Population-level summary 
Difference between the proportion of subjects colonized on 
any day (Challenge Day C9, C11, or C14) in the BPZE1 
group and the placebo group. 
Intercurrent Events and 
Strategies 
 
ICE1 (discontinued prior to 
challenge or prior to 
Challenge Day C9 culture) 
Subjects may be replaced. 


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ICE2 Missing culture(s) 
(a) Single missing culture 
• If Day C9 and Day C11 are negative but Day C14 is 
missing, Day C14 will be imputed as negative; 
• If Day C9 and Day C14 are negative but Day C11 is 
missing, Day C11 will be imputed as negative; 
Because participants are closely monitored in the 
challenge unit, it is deemed highly unlikely that they 
will miss Day C9 but come back for Days C11 and 
Day C14; the scenario of missing Day C9 only will 
not be considered 
(b) Two missing cultures 
• If Day C9 is negative but Day C11 and Day C14 are 
missing, Day C11 and Day C14 will be imputed as 
negative; 
• As described above, it is deemed highly unlikely that 
participants will miss an earlier assessment but come 
back for later day assessment, including the following 
two scenarios:  
a. Day C11 is negative, but Day C9 and Day 
C14 are missing  
b. Day C14 is negative, but Day C9 and Day 
C11 are missing 
Both Scenarios will not be considered. 
(c) Positive culture at any of Challenge Days C9, C11, and 
C14 in the presence of missing cultures 
When this is the case, the result will be considered 
positive. 
 
ICE3 (protocol deviations) 
Treatment Policy 
ICE4 (prohibited 
medications) 
Treatment Policy 
 
A subject is defined to be colonized if the culture result is positive. The number and proportion 
of subjects colonized on any of the three days - Challenge Days C9, C11, or C14 will be 
summarized by treatment group. The two-sided 95% CIs for each treatment group will be 
computed using the Agresti-Coull method, and the 95% CIs for the difference in proportions 
between the BPZE1 group and the placebo group will be computed using Agresti-Caffo method. 
The colonization counts and proportions in the BPZE1 and placebo groups will be summarized 
for each of Challenge Days C9, C11, and C14 using same method. 


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To assess the primary objective, the following superiority hypothesis will be tested on the 
proportion of subjects colonized on any of the days (Challenge Days C9, C11 or C14) following 
challenge.  
Test hypothesis for superiority: 
𝐻𝐻0: 𝑃𝑃1 ≥𝑃𝑃2 
𝐻𝐻1: 𝑃𝑃1 < 𝑃𝑃2 
where, 
     P1 = proportion of subjects colonized on any of the days (Challenge Days C9, C11 or C14) 
following challenge in BPZE1 group 
     P2 = proportion of subjects colonized on any of the days (Challenge Days C9, C11 or C14) 
following challenge in placebo group 
Superiority of BPZE1 will be declared if P1 < P2 and the two-sided P-value is less than 0.05 for 
the likelihood ratio chi-square test. If 25% of the cells have expected counts that are less than 5, a 
Fisher’s exact test will be performed. 
Analysis will be conducted on mITT Analysis Set.  
8.1.1. Sensitivity Analysis for the Primary Efficacy Analysis 
Sensitivity analyses will be performed on the Adequate Inoculum Analysis Set and Per Protocol 
Analysis Set separately following the same analysis method described above in Section 8.1.  
An additional sensitivity analysis will use a logistic regression model with treatment group, 
denoted as 1 for BPZE1 and 0 for placebo, and inoculum count as covariates, with colonization 
status (any day of Challenge Day C9, C11, or C14) as the outcome (modelling the response of 
not being colonized). The null hypothesis will be tested using the coefficient associated with 
treatment group. The odds and 95% CI, and odds Ratio (BPZE1 vs. placebo) and 95% CI and p-
value will be provided. 
Results for the mITT, Adequate Inoculum, and Per Protocol analysis sets will be summarized in 
a forest plot. Results from the logistic regression will also be summarized in a forest plot.  
8.2.Secondary Efficacy Analysis 
8.2.1. GMFR of Mucosal Anti-pertussis S-IgA Antibody from Baseline to Day 28 
The GMFR and GMC/GMR, as well as the corresponding 95% CIs, for mucosal S-IgA for each 
of the anti-pertussis antibodies (WCE, FHA, PRN, PT and FIM2/3) at Visit 3/Day 28 will be 
provided by treatment group. A paired t-test will be used to test the null hypothesis of no change 
from baseline to Visit 3/Day 28 in log-transformed anti-pertussis mucosal secretory IgA (S-IgA) 
antibody levels within both the BPZE1 group and placebo group and a two-sided p-value will be 
provided. The 95% CI for the GMFRs within both the BPZE1 group and placebo group for the 
changes from baseline to Visit 3/Day 28 of the log-transformed mucosal S-IgA will be 
constructed based on normal approximation. The 95% CI will be transformed back to the 
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Mucosal S-IgA will be normalized as [specific S-IgA]x100/[Total S-IgA] and it will also be 
summarized using same method. 
Analyses will be based on mITT Analysis Set.  
8.2.2. GMFR of Serum IgA Antibody from Baseline to Day 28  
The GMFR and GMC, as well as the corresponding 95% CIs for serum IgA for each of the anti-
pertussis antibodies (WCE, FHA, PRN, PT and FIM2/3) at Visit 3/Day 28, will be provided by 
treatment group. The same approach described in Section 8.2.1 for the GMFR of Mucosal Anti-
pertussis S-IgA Antibody from baseline to Visit 3/Day 28 will be applied.  
Analyses will be based on mITT Analysis Set. 
8.2.3. GMFR of Serum IgG Antibody from Baseline to Day 28  
The GMFR and GMC, as well as the corresponding 95% CIs for Serum IgG for each of the anti-
pertussis antibodies (WCE, FHA, PRN, PT and FIM2/3) at Visit 3/Day 28, will be provided by 
treatment group. The same approach described in Section 8.2.1 for the GMFR of Mucosal Anti-
pertussis S-IgA Antibody from baseline to Visit 3/Day 28 will be applied.  
Analyses will be based on mITT Analysis Set. 
8.2.4. Sensitivity Analysis for the Secondary Efficacy Analysis 
Sensitivity analyses will be performed on the ITT Analysis Set, Adequate Inoculum Analysis Set 
and Per Protocol Analysis Set separately following the same analysis method described in 
Sections 8.2.1, 8.2.2, and 8.2.3.  
An additional ANCOVA model will be fit with the outcome being the change from baseline to 
Day 28 in natural log scale (i.e., GMFR in natural log scale), baseline value in natural log scale, 
treatment group, and inoculum count in natural log scale as covariates.  
Results for the mITT, ITT, Adequate Inoculum, and Per Protocol analysis sets will be 
summarized in a forest plot.  
Results from the ANCOVA models will also be summarized in a forest plot. 
8.3.Exploratory Efficacy Analysis 
Unless otherwise stated, all analyses described in this Section will be performed on the mITT 
analysis set and Adequate Inoculum Analysis Set. 
8.3.1. Absolute Colony Counts in Nasal Wash Samples on Challenge Days C9, C11 and 
C14 Following Virulent Challenge by Treatment Group 
Absolute colony counts (colony forming unit per mL, CFU/mL) in nasal wash samples on 
Challenge Days C9, C11, and C14 following virulent challenge will be summarized using 
descriptive statistics by treatment group and by timepoint. Geometric means and corresponding 
95% CIs will be provided. 
Reverse cumulative distribution curves will be provided for nasal wash samples on Challenge 
Days C9, C11 and C14 Following Virulent Challenge by Treatment Group. 


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8.3.2. GMC/GMR of Mucosal Anti-pertussis S-IgA Antibody, Serum IgA and Serum IgG 
Antibody Throughout the Study by treatment group  
The GMC/GMR with the 95% CI for mucosal S-IgA, Serum IgA, and Serum IgG for each of 
anti-pertussis antibodies (WCE, FHA, PRN, PT and FIM2/3) throughout the study will be 
provided by treatment group by timepoint. S-IgA will be provided both as specific mucosal 
S-IgA and normalized using [specific S-IgA]x100/[total S-IgA]. 
A two-sample t-test will be used to compare the log-transformed GMCs/GMRs between the 
BPZE1 group and placebo group at each timepoint and a two-sided p-value will be provided. In 
addition, the difference of the log-transformed GMCs/GMRs between the BPZE1 and placebo 
groups will be calculated and the 95% CIs for the differences will be constructed based on the 
normal approximation. To transform back to the original data scale as the 95% CI for ratio of 
two GMCs/GMRs, the upper and lower limits of the 95% CI will be exponentiated.  
Reverse cumulative distribution curves will be provided for Mucosal anti-pertussis S-IgA 
Antibody at baseline, Visit 3/Day 28, Visit 4/C-1, Visit 5/C28, and Visit 6/EOS based on ITT 
and mITT analysis sets.  
Reverse cumulative distribution curves will be provided for Serum IgA and Serum IgG Antibody 
at baseline, Visit 3/Day 28, Visit C0, Visit 5/C28, and Visit 6/EOS based on ITT and mITT 
analysis sets. 
8.3.3. GMC/GMR and GMFR of Mucosal Anti-pertussis S-IgA Antibody, Serum IgA and 
Serum IgG Antibody Post-vaccination and Post-challenge by Treatment Group  
The GMC/GMR with the 95% CI for mucosal S-IgA, Serum IgA, and Serum IgG for each of the 
anti-pertussis antibodies (WCE, FHA, PRN, PT and FIM2/3) at baseline, Visit 3/Day 28, Visit 
4/C-1 or Visit C0, Visit 5/C28, and Visit 6/EOS, will be provided by treatment group and 
timepoint. S-IgA will be provided both as specific mucosal S-IgA and normalized using [specific 
S-IgA]x100/[total S-IgA]. Visit 3/Day 28 will be considered as post-vaccination. Visit 5/C28 
will be considered as post-challenge.  
A two-sample t-test will be used to compare the log-transformed GMCs/GMRs between the 
BPZE1 and placebo groups at each timepoint and a two-sided p-value will be provided. In 
addition, the difference of the log-transformed GMCs/GMRs between the BPZE1 and placebo 
groups will be calculated and the 95% CI for the difference will be constructed based on the 
normal approximation. To transform back to the original data scale as the 95% CI for ratio of 
two GMCs/GMRs, the lower and upper limits of the 95% CI will be exponentiated.  
The GMFR for mucosal S-IgA, Serum IgA, and Serum IgG for each of the anti-pertussis 
antibodies (WCE, FHA, PRN, PT and FIM2/3) at Visit 3/Day 28, Visit 4/C-1 or Visit C0, Visit 
5/C28, and Visit 6/EOS will be provided by treatment group and timepoint.  
The 95% CI for the GMFRs in the BPZE1 group and placebo group of the log-transformed 
mucosal S-IgA, Serum IgA, and Serum IgG for each of the anti-pertussis antibodies (WCE, 
FHA, PRN, PT, and FIM2/3) will be constructed based on normal approximation. To transform 
back to the original data scale, the lower and upper limits of the 95% CI will be exponentiated.   
Two-sample t-tests will be used to compare the log-transformed GMFRs post-vaccination 
(Visit 3/Day 28 over baseline) and post-challenge (Visit 5/C28 over Visit 4/C-1 or Visit C0) 
between the BPZE1 and placebo groups and two-sided p-values will be provided. In addition, the 


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95% CI will be constructed based on normal approximation. To transform back to the original 
data scale as the 95% CI for ratio of two GMFRs, the lower and upper limits of the 95% CI will 
be exponentiated.  
8.3.4. Proportion of Subjects by Treatment Group with a ≥2x Increase in SBA 
Bactericidal Titer  
The number and proportion of subjects with a ≥2x increase from baseline in SBA bactericidal 
titer (50% killing titre) will be summarized by treatment group and by strain. The 95% CIs for 
each treatment group will be computed using the Agresti-Coull method and the 95% CIs for the 
difference in proportions between the BPZE1 group and the placebo group will be computed 
using Agresti-Caffo method. A chi-square test will be performed to compare the proportion of 
subjects between two treatment groups with a ≥2x increase in SBA bactericidal titer and 
two-sided p-value will be provided. 
8.3.5. Analysis of Treatment Group for Each B. pertussis Strain Studied by SBA and by 
Anti-pertussis IgG Antibody 
The GMT with the 95% CI for SBA and Anti-pertussis IgG Antibody for each B. pertussis strain 
will be provided by treatment group. Correlation analyses will be conducted between SBA 
(PRN+ and PRN-) and anti-pertussis IgG antibody (WCE, PRN, FHA, PT, and FIM2/3) levels 
by B. pertussis strain and timepoint using either Pearson’s Correlation coefficient or Spearman’s 
Rho depending on the normality of the sample distribution.  
Reverse cumulative distribution curves will be provided for SBA (PRN+ and PRN-) at baseline, 
Visit 3/Day 28, Visit C0, Visit 5/C28, and Visit 6/EOS based on ITT and mITT analysis sets. 
8.3.6. Cell-mediated Immunity Using Whole blood, PBMC and/or Nasal Pharyngeal 
Samples 
The mean with 95% CI for cell-mediated immunity using PBMC, whole blood, and/or nasal 
pharyngeal samples, including but not limited to cytokines, Th1/Th17 and Th2 responses will be 
provided by treatment group and timepoint. 
Reverse cumulative distribution curves will be provided for cell-mediated immunity using 
PBMC, whole blood, and/or nasal pharyngeal samples by treatment group based on ITT and 
mITT analysis sets.  
9. Safety Analysis 
Unless otherwise specified, safety analysis will be performed based on the Safety Analysis Set. 
All summaries and analyses will be presented over all subjects and by treatment groups (BPZE1, 
placebo, and Total).  
9.1.Solicited Adverse Events 
The number and percentage of subjects with any solicited AEs (nasal/respiratory and systemic 
reactogenicity events) through 7 days following study vaccination will be calculated by treatment 
group.  


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Solicited reactogenicity events occurring through 7 days following study vaccination, including 
nasal/respiratory reactions and systemic reactions, will be summarized as number and frequency 
by reactogenicity grades (Table 7). 
If subjects have fever in 7 days following vaccination, the oral temperature will be measured and 
will be summarized using descriptive statistics. 
Duration through 7 days following study vaccination and duration through resolution following 
study vaccination will be summarized using descriptive statistics. Duration through 7 days is 
defined as days with the reactogenicity event onset through 7 days following vaccination per 
patient reported daily diary. Duration beyond 7 days is defined as days with the reactogenicity 
event continuing beyond 7 days as captured in adverse events.  
Solicited nasal/respiratory and systemic adverse events will be presented in data listings. 
Table 7. 
Reactogenicity Grading Scale 
Nasal/Respiratory/Systemic Reactogenicity Grading 
Reaction 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
Runny nose 
Noticeable but does not 
interfere with daily 
activity 
Moderate discomfort 
that interferes with 
daily activity 
Significant 
discomfort/prevents 
daily activity or seeks 
medical care 
Stuffy 
nose/congestion 
Noticeable but does not 
interfere with daily 
activity 
Moderate discomfort 
that interferes with 
breathing from nose 
Unable to breathe 
through nose or seeks 
medical care 
Nasal pain/irritation 
Noticeable but does not 
interfere with daily 
activity 
Moderate discomfort 
that interferes with 
daily activity 
Significant discomfort 
that prevents daily 
activity or seeks medical 
care 
Epistaxis 
Total duration of all 
episodes in a 24-hour 
period <30 minutes 
Total duration of all 
episodes in a 24-hour 
period >30 minutes 
Any bleeding that 
required visit for 
medical encounter 
Sneezing 
Noticeable but does not 
interfere with daily 
activity 
Moderate discomfort 
that interferes with 
daily activity 
Significant discomfort; 
prevents daily activity 
Sinus pressure/pain 
Noticeable but does not 
interfere with daily 
activity 
Moderate discomfort 
that interferes with 
daily activity 
Significant discomfort 
that prevents daily 
activity or seeks medical 
care 
Sore/irritated throat 
Noticeable but does not 
interfere with eating or 
drinking 
Moderate discomfort 
that interferes with 
eating or drinking 
Significant discomfort 
that prevents eating or 
drinking or seeks 
medical care 
Cough 
Noticeable but does not 
interfere with daily 
activity or sleeping 
Frequent cough that 
interferes with daily 
activity or sleeping 
Prevents daily activity, 
prevents sleep, or seeks 
medical care 
Shortness of 
breath/wheezing 
Noticeable but does not 
interfere with daily 
activity 
Moderate discomfort 
that interferes with 
daily activity 
Significant discomfort/ 
prevents daily activity or 
seeks medical encounter 
Fever  
100.4–101.1°F  
101.2–102.0°F 
>102°F  


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Nasal/Respiratory/Systemic Reactogenicity Grading 
Reaction 
Mild (Grade 1) 
Moderate (Grade 2) 
Severe (Grade 3) 
(38.0–38.4°C) 
(38.5–38.9°C) 
(>39.0°C) 
Fatigue (tiredness) 
No interference with daily 
activity 
Some interference 
with daily activity 
Significant interference, 
prevents daily activity 
Malaise (general 
unwell feeling) 
No interference with daily 
activity 
Some interference 
with daily activity 
Significant interference, 
prevents daily activity 
Myalgia (body 
aches/muscular pain) 
No interference with daily 
activity 
Some interference 
with daily activity 
Significant interference, 
prevents daily activity 
Arthralgia (joint 
pain) 
No interference with daily 
activity 
Some interference 
with daily activity 
Significant interference, 
prevents daily activity 
Headache 
No interference with daily 
activity 
Some interference 
with daily activity 
Significant interference, 
prevents daily activity 
Rash/hypersensitivity Pruritus OR local rash 
Diffuse rash 
Diffuse rash with 
blisters or mouth 
ulcerations, anaphylaxis, 
or angioedema 
Source: Modified based on Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent 
Volunteers Enrolled in Preventive Vaccine Clinical Trials (fda.gov). Accessed 26 April 2022.Division of AIDS 
(DAIDS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, US Department of 
Health and Human Services. Division of AIDS (DAIDS) table for grading the severity of adult and pediatric adverse 
events. July 2017 [cited 26 April 2022]. Available from: 
https://rsc.niaid.nih.gov/sites/default/files/daidsgradingcorrectedv21.pdf 
9.2.Unsolicited Adverse Events 
Unsolicited AEs will be coded according to Medical Dictionary for Regulatory Activities 
(MedDRA) latest version. Unless otherwise specified, unsolicited AEs will be summarized by 
System Organ Class (SOC) and Preferred Term (PT), with SOCs sorted in the alphabetical order 
and PTs within each SOC in descending order of subject incidence. 
A TEAE is defined as an AE that occurs after the subject has been vaccinated. AEs will be 
summarized by treatment groups and overall and by study phase (vaccination phase and 
challenge phase). All AESIs and SAEs will be monitored from signing of informed consent 
through to the EOS visit. All AEs regardless of causality will be recorded for 28 days after 
vaccination and 28 days after challenge. All AEs related either to vaccination or to challenge will 
be collected from time of vaccination to challenge and for 3 months after challenge, respectively.  
9.2.1. Incidence of Adverse Events 
An overview summary of the following TEAE categories by treatment group and total will be 
provided: 
• 
Any TEAE 
• 
Any TEAE from Vaccination Phase Day 29 to prior to Challenge 
• 
Any TEAE from Challenge Phase Day 29 to EOS 
• 
Any TEAE related to vaccination 
• 
Any TEAE related to MAD 
• 
Any TEAE related to challenge 


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• 
Any severe TEAE 
• 
Any serious TEAE 
• 
Any serious TEAE related to vaccination 
• 
Any serious TEAE related to MAD 
• 
Any serious TEAE related to challenge 
• 
Any AESI 
• 
Any AESI related to vaccination 
• 
Any AESI related to MAD 
• 
Any AESI related to challenge 
• 
Any TEAE leading to study discontinuation 
• 
Any TEAE leading to death 
9.2.2. Relationship of Adverse Events to Study Vaccination/Challenge 
The relationship of vaccination to TEAE will be characterized using the following criteria: 
• 
Related to vaccination: there is a reasonable possibility that the study vaccination caused the 
AE. Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study vaccination and the AE 
• 
Not related to vaccination: there is not a reasonable possibility that the administration of the 
study vaccination caused the event 
• 
Related to challenge agent: there is a reasonable possibility that the challenge agent caused 
the AE. Reasonable possibility means that there is evidence to suggest a causal relationship 
between the study vaccination and the AE 
• 
Not related to challenge agent: there is not a reasonable possibility that the administration of 
the challenge agent caused the event 
All TEAEs through 28 days following study vaccination and following challenge will be 
summarized by relationship to study vaccination/challenge/MAD, SOC/PT and by treatment 
group. 
9.2.3. Severity of Adverse Event 
The severity of the AE will be rated as mild, moderate, or severe using the following criteria: 
• 
Mild/Grade 1: these events require minimal or no treatment and do not interfere with the 
subject’s daily activities. 
• 
Moderate/Grade 2: these events result in a low level of inconvenience or concern with the 
therapeutic measures. Moderate events may cause some interference with normal 
functioning. 
• 
Severe/Grade 3: these events interrupt a subject’s usual daily activity and may require 
systemic drug therapy or other treatment. Severe events are usually incapacitating. 


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If a subject reports multiple occurrences of a specific event, the subject will be counted only 
once by the maximum severity. If the severity is missing for one or more of the occurrences, the 
maximum severity of the remaining occurrences will be used. 
All TEAEs through 28 days following study vaccination and following challenge will be 
summarized by SOC/PT and severity, and by treatment group. 
TEAEs related to vaccination from time of vaccination to challenge and TEAEs related to 
challenge for 3 months after challenge will be summarized by severity and SOC/PT, and by 
treatment group.  
9.2.4. Serious Adverse Events 
An SAE is defined as any event that: 
• 
results in death 
• 
is life threatening 
• 
requires inpatient hospitalization or prolongation of existing hospitalization 
• 
results in persistent or significant disability/incapacity 
• 
is a congenital anomaly/birth defect 
Important medical events that may not result in death, be life threatening, or require 
hospitalization may be considered SAEs when, based upon appropriate medical judgment, they 
may jeopardize the subject or may require medical or surgical intervention to prevent one of the 
outcomes listed in this definition.  
Serious TEAE will be summarized by severity and SOC/PT and by treatment group, and by 
relationship separately.  
A by-subject listing will be provided. 
9.2.5. Adverse Events of Special Interest 
An AESI is defined as adverse events related to infection with or vaccination for SARS-CoV-2 
and will be collected on a unique eCRF through to the EOS.  
All AESIs will be summarized as number and percentage by severity and SOC/PT and by 
treatment group, and by relationship separately. 
A by-subject listing will be provided. 
9.2.6. Adverse Events Leading to Study Discontinuation 
TEAEs leading to study discontinuation will be summarized by SOC/PT and by treatment group.  
A by-subject listing will also be provided. 
9.2.7. Death 
A by-subject listing will also be provided for all AEs leading to death. 


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9.2.8. Pre-defined Post-Challenge Checklist  
During the in-unit period the volunteers will be reviewed at least twice per day (once in the morning 
and once in the evening) following a standardised checklist including body temperature, respiratory 
rate, heart rate, and early symptoms of pertussis: rhinorrhoea, nasal congestion, epistaxis, sneezing, 
ear pain, eye pain, sore throat, cough, dyspnoea, feeling generally unwell, tiredness and headache. 
The pre-defined post-challenge checklist will be summarized using descriptive statistics by 
treatment group and overall. 
A by-subject listing will be provided for all pre-defined post-challenge checklist.  
9.3.Clinical Laboratory Evaluations 
Subjects with values meeting FDA toxicity criteria (Table 8) will be summarized by grade and 
treatment group. Only abnormal laboratory values were collected. 
A by-subject listing will be provided for abnormal laboratory results.  


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Table 8. 
Table for Laboratory Grading 
Seruma 
Mild (Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4)b 
Glucose – hyperglycemia  
Random (mg/dL; mmol/L)  
 
110–125 (6.1–
<7.0) 
 
126–200 (7.0–
11.1) 
 
>200 (>11.1) 
Insulin 
requirements or 
hyperosmolar 
coma 
Blood Urea Nitrogen (mg/dL; 
mmol/L)  
23–26 (8.2–9.5) 
27–31 (9.6–
11.1) 
>31 (>11.1) 
Requires dialysis 
Creatinine (mg/dL; umol/L) 
1.5–1.7 (133–
154) 
1.8–2.0 (155–
180) 
2.1–2.5 (181–
225) 
>2.5 (>225) or 
requires dialysis 
Albumin – hypoalbuminemia 
(g/dL; g/L)  
2.8–3.1 (28–31) 
2.5–2.7 (25–
27) 
<2.5 (<25) 
– 
Total Protein – hypoproteinemia 
(g/dL; g/L)  
5.5–6.0 (55–60) 
5.0–5.4 (50–
54) 
<5.0 (<50) 
– 
Liver Function Tests – ALT, AST 
increase by factor 
1.1–2.5 x ULN 
2.6–5.0 x ULN 
5.1–10 x ULN 
>10 x ULN 
Bilirubin – when accompanied by 
any increase in liver function test; 
increase by factor 
1.1–1.25 x ULN 
1.26–1.5 x 
ULN 
1.51–1.75 x 
ULN 
> 1.75 x ULN 
Bilirubin – when liver function 
test is normal; increase by factor 
1.1–1.5 x ULN 
1.6–2.0 x ULN 
2.0-3.0 x ULN 
> 3.0 x ULN 
Haematologya 
Mild (Grade 1) 
Moderate 
(Grade 2) 
Severe 
(Grade 3) 
Potentially Life 
Threatening 
(Grade 4)b 
Haemoglobin (Female) (g/dL; 
g/L) 
11.0–12.0 (110–
120) 
9.5–10.9 (95–
109) 
8.0–9.4 (80–94) 
<8.0 (<80) 
Haemoglobin (Male) (g/dL; g/L) 
12.5–13.5 (125–
135) 
10.5–12.4 
(105–124) 
8.5–10.4 (85–
104) 
<8.5 (<85) 
WBC elevation (cell/mm3; x109/L) 
10,800–15,000 
(10.8–15) 
15,001–20,000 
(>15–20) 
20,001–25,000 
(>20–25) 
>25,000 (>25) 
WBC suppression (cell/mm3; 
x109/L) 
2500–3500 (2.5–
3.5) 
1500–2499 
(1.5–<2.5) 
1000–1499 
(1.0–<1.5) 
<1,000 (<1.0) 
Platelets Decreased (cell/mm
3; 
x109/L) 
125,000–140,000 
(125–140) 
100,000–
124,000 (100–
124) 
25,000–99,000 
(25–99) 
<25,000 (<25) 
ALT=alanine aminotransferase; AST=aspartate aminotransferase; ULN=upper limit of normal; WBC=white blood 
cell. 
a  The laboratory values provided in the tables serve as guidelines and are dependent upon institutional normal 
parameters. Institutional normal reference ranges should be provided to demonstrate that they are appropriate  
b  The clinical signs or symptoms associated with laboratory abnormalities might result in characterization of the 
laboratory abnormalities as Potentially Life Threatening (Grade 4). For example, a low sodium value that falls 
within a Grade 3 parameter (125–129 mEq/L) should be recorded as a Grade 4 hyponatremia event if the subject had 
a new seizure associated with the low sodium value 
Note: Screening laboratory samples with toxicity scores greater than 1 are exclusion criteria for vaccination 
enrolment. Laboratory sampling while in the challenge unit will comply with challenge unit standard protocol with 
the proviso that concurrent evidence of illness requires a daily complete blood count with differential to be 
conducted. 
Source: Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in 
Preventive Vaccine Clinical Trials (fda.gov). Accessed April 26, 2022. 
 
 


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9.3.1. Pregnancy Test 
Serum and/or urine will be collected from female subjects of childbearing potential at the times 
outlined in the Schedule of Events (Section 13.1) to enable pregnancy test. 
Only a by-subject listing will be provided for serum/urine pregnancy test. 
9.3.2. COVID-19 Testing 
COVID-19 testing will be performed at the times outlined in the Schedule of Events (Section 
13.1). 
Only a by-subject listing will be provided for COVID-19 testing. 
9.4.Vital Sign Measurements 
Vital sign measurements will include body temperature (oral, tympanic, or noncontact), heart 
rate, systolic and diastolic blood pressure, respiratory rate, and oxygen saturation, and will be 
collected at the times outlined in the Schedule of Events (Section 13.1), pre-vaccination and 
pre-challenge and approximately 30 minutes following vaccination and challenge, respectively, 
and any unscheduled visits. During the challenge unit stay, vital signs will be collected at least 
twice daily (once in the morning and once in the evening). Temperature will also be recorded 
daily in a subject diary for 7 consecutive days starting the day following study vaccination. 
Vital sign measurements will be summarized using descriptive statistics for reported values and 
change from baseline at each scheduled visit. Shift from baseline results will be presented in 
pre- versus post-treatment cross-tabulations, with classes for mild, moderate, and severe, as 
indicated in Table 9. 
A by-subject listing will be provided. 
 
 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
  29 
 
Table 9. 
Table for Vital Sign Grading 
Vital signs 
Mild (Grade 
1) 
Moderate 
(Grade 2) 
Severe (Grade 
3) 
Potentially life 
threatening (Grade 4) 
Fever (°C) 
38.0–38.4 
38.5–38.0 
>39.0 
>40.0 
Tachycardia (bpm) 
101–115 
116–130 
>130 
Emergency room visit or 
hospitalised for arrhythmia 
Bradycardia (bpm) 
50–54 
45–49 
<45 
Emergency room visit or 
hospitalised for arrhythmia 
Hypertension (systolic) 
mmHg 
141–150 
151–155 
>155 
Emergency room visit or 
hospitalised for malignant 
hypertension 
Hypertension (diastolic) 
mmHg 
91–95 
96–100 
>100 
Emergency room visit or 
hospitalised for malignant 
hypertension 
Hypotension (systolic) 
mmHg 
85–89 
80–84 
<80 
Emergency room visit or 
hospitalised for 
hypotensive shock 
Bpm=beats per minute 
a  
Subject should be at rest for all vital sign measurements  
b  Oral temperature; no recent hot or cold beverages or smoking  
c  
When resting heart rate is between 60 to 100 bpm. Use clinical judgement when characterizing bradycardia 
among some healthy subject populations, for example, conditioned athletes 
Note: Grade 0 will be the classification if the observation is less than a Grade 1. Respiratory rate was removed from 
the FDA table for vital sign grading as this is not a parameter being measured in this study 
Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive 
Vaccine Clinical Trials (fda.gov). Accessed April 26, 2022. 
9.5.Physical Examination 
A full physical examination will be done at Screening visit and pre-challenge. A targeted 
physical examination will be assessed at the times outlined in the Schedule of Events 
(Section 13.1), including unscheduled visits. Height and weight will be measured at the 
screening and pre-challenge visits only.  
Height, weight, and BMI will be summarized using descriptive statistics for reported values and 
change from baseline at each scheduled visit. 
A by-subject listing will be provided for all physical examination data. 
10. Primary Analysis 
Primary analysis will be performed at the time when all subjects completed Challenge phase 
(Challenge Day C16).  
11. Changes in the Planned Analysis 
Not applicable.  


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
  30 
12. References 
Modified based on Guidance for Industry: Toxicity Grading Scale for Healthy Adult and 
Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials (fda.gov). Accessed 26 
April 2022.Division of AIDS (DAIDS), National Institute of Allergy and Infectious Diseases, 
National Institutes of Health, US Department of Health and Human Services. Division of AIDS 
(DAIDS) table for grading the severity of adult and pediatric adverse events. July 2017 [cited26 
April 2022]. Available from: 
https://rsc.niaid.nih.gov/sites/default/files/daidsgradingcorrectedv21.pdf 
 
Guidance for Industry: Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers 
Enrolled in Preventive Vaccine Clinical Trials (fda.gov). Accessed April 26, 2022. 
 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 31 
13. Appendices 
13.1. 
Schedule of Events 
 
 
Vaccination Phase 
Challenge Phase 
Safety Follow-
up 
Study visit 
Screenin
g 
1 
2 
3 
4/C-
1a 
C
0 
C1-
6b 
C7 
C8, C10, 
C12 & 
C13b 
C9, C11 
& C14b 
C1
5 
C1
6 
5 
6 
Days Relative to 
Vaccinationc 
-30 to -7 
0 
7 
28 
52 
--- 
--- 
--- 
--- 
--- 
--- 
--- 
--- 
180/EO
Sq 
Days Relative to 
Challenge 
 
 
 
 
-7 r 
0 
1-6 
7 
8, 10, 12, 
and 13 
9, 11 and 
14 
15 
16 
28 
90q 
Window Allowancec 
30 
NA 
+3 
+7 
+60 
0 
0 
0 
0 
0 
0 
0 
+7 
+30 
VIS/Informed 
consent/TOPS registration 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Inclusion/exclusion 
criteria 
X 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
Infection control 
agreement 
X 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Demographic and baseline 
data 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Concomitant medication 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Vital signs  
X 
Xd 
X 
X 
 
Xd 
X 
X 
X 
X 
X 
X 
X 
X 
Physical examination 
Xe 
X 
X 
X 
 
Xe 
X 
X 
X 
X 
X 
X 
X 
X 
Serology and anti-PT and 
anti-PRN Ab (local and 
central lab) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Safety laboratory samples 
X 
 
 
 
X 
 
 
X 
 
X (C9 
and C14 
only) 
 
 
 
 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 32 
 
 
Vaccination Phase 
Challenge Phase 
Safety Follow-
up 
Study visit 
Screenin
g 
1 
2 
3 
4/C-
1a 
C
0 
C1-
6b 
C7 
C8, C10, 
C12 & 
C13b 
C9, C11 
& C14b 
C1
5 
C1
6 
5 
6 
Days Relative to 
Vaccinationc 
-30 to -7 
0 
7 
28 
52 
--- 
--- 
--- 
--- 
--- 
--- 
--- 
--- 
180/EO
Sq 
Days Relative to 
Challenge 
 
 
 
 
-7 r 
0 
1-6 
7 
8, 10, 12, 
and 13 
9, 11 and 
14 
15 
16 
28 
90q 
Window Allowancec 
30 
NA 
+3 
+7 
+60 
0 
0 
0 
0 
0 
0 
0 
+7 
+30 
Respiratory PCR panel 
 
 
 
 
 
Xp 
 
 
 
 
 
 
 
 
Urine toxicity screen  
X 
 
 
 
X 
 
 
 
 
 
 
 
 
 
Serum pregnancy test 
(FCBP only) 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Urine pregnancy test 
(FCBP only) 
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
12-lead electrocardiogram 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
SARS-CoV-2 test for 
active infectionf  
 
X 
 
 
 
X 
 
 
 
 
 
 
 
 
Randomization 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Vaccination 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Challenge  
 
 
 
 
 
X 
 
 
 
 
 
 
 
 
Subject diary dispensingg 
 
X 
 
 
 
 
 
 
 
 
 
 
 
 
Reactogenicity/Solicited 
AEsg 
 
Xg,h 
X 
 
 
 
 
 
 
 
 
 
 
 
Post-challenge checklist 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Subject diary or checklist 
reviewedi 
 
 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
 
 
Azithromycin 
 
 
 
 
 
 
 
 
 
X 
(C14 
only)j 
X 
Xj 
 
 
Nasal wash for B. 
pertussis colonization by 
culture ± PCR 
 
 
 
 
X 
 
 
 
 
X 
 
Xk 
X 
 


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 33 
 
 
Vaccination Phase 
Challenge Phase 
Safety Follow-
up 
Study visit 
Screenin
g 
1 
2 
3 
4/C-
1a 
C
0 
C1-
6b 
C7 
C8, C10, 
C12 & 
C13b 
C9, C11 
& C14b 
C1
5 
C1
6 
5 
6 
Days Relative to 
Vaccinationc 
-30 to -7 
0 
7 
28 
52 
--- 
--- 
--- 
--- 
--- 
--- 
--- 
--- 
180/EO
Sq 
Days Relative to 
Challenge 
 
 
 
 
-7 r 
0 
1-6 
7 
8, 10, 12, 
and 13 
9, 11 and 
14 
15 
16 
28 
90q 
Window Allowancec 
30 
NA 
+3 
+7 
+60 
0 
0 
0 
0 
0 
0 
0 
+7 
+30 
Nasopharyngeal swab(s) 
for cell-mediated 
immunity  
X 
 
X 
X 
X 
 
 
 
 
X  
(C9 only) 
 
 
X 
 
Nasal mucosal secretion 
sample (SAM) for 
immunogenicity test  
X 
 
 
X 
X 
 
 
 
 
 
 
 
X 
X 
Serum for immunogenicity 
(including ELISA and 
SBA assays) 
 
Xl 
 
X 
 
Xl 
 
 
 
 
 
 
X 
X 
Blood sampling for 
cellular-mediated 
immunity 
 
Xl 
X 
X 
 
Xl 
 
X 
 
 
 
 
X 
 
All unsolicited AEsm 
 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
 
TEAEs related to 
vaccinationn 
 
X 
X 
X 
X 
 
 
 
 
 
 
 
 
 
TEAE related to 
challengen 
 
 
 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AESIs and SAEso 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
AE=adverse event; AESI=adverse event of special interest; ELISA=enzyme-linked immunosorbent assay; EOS=end of study; FCBP=female(s) of 
childbearing potential; PCR=polymerase chain reaction; SAE=serious adverse event; SBA=serum bactericidal assay; TEAE=treatment emergent 
adverse event 
a. 
Initiate screening for eligibility for the Challenge Phase; admit to challenge unit on Study Visit C0 
b. Each day has the same assessments, unless specified  
c. 
Days relative to vaccination are only estimates as the window allowances are not inclusive. Subjects will remain in the study until either 6 months 
after vaccination or 3 months after challenge, whichever is longer. If a study pause occurs, the visits/windows will be adjusted to allow for 
subjects to continue without protocol deviation.  


ILiAD Biotechnologies 
Statistical Analysis Plan, Final Version 1.0 
IB-202P 
Date Issued: 30Jun2023 
 
 
 34 
d. Pre-vaccination and approximately 30 minutes following vaccination and challenge; additional vital sign measurements to be taken per challenge 
unit as needed 
e. 
Full physical examination including height and weight at Screening and Study Visit C0. Symptom-directed (targeted) physical examination at all 
other scheduled time points. Interim physical examinations will be performed at the discretion of the Investigator, if necessary  
f. 
SARS-CoV-2 testing should be performed within approved national guidelines with tests that are approved through a (minimum) of emergency 
use authorization. Minimally invasive testing should be considered for the comfort of subjects. Subjects need to have negative test results within 
72 hours prior to vaccination and admission to the challenge unit. Repeat testing is allowed on multiple occasions, if needed  
g. All subjects will record maximum daily reactogenicity/solicited AEs in the subject diary for the subsequent 7 days after vaccination. Subjects will 
be instructed that should they have reactogenicity with a potential toxicity grade 3 at any time after vaccination, they should contact the site on the 
same day and be seen or referred to a qualified medical facility 
h. Immediate reactogenicity will be assessed 30 minutes after the study vaccination, prior to release from clinical observation 
i. 
Site staff reviews the information from the subject diary/checklist with the subjects to confirm accuracy. For reactogenicity and post-challenge 
checklist, the appropriate FDA toxicity grading should be applied. If any reactogenicity event extended beyond 7 days post-vaccination and is 
deemed clinically significant by AE classification, then it should be entered as an AE (graded by AE criteria) with the same start date as the 
reactogenicity event and followed to resolution. All AEs identified by the post-challenge checklist should be entered as an AE 
j. 
Prior to Study visit C14, azithromycin may be initiated if subjects have symptoms consistent with pertussis, although assessments listed for C14 
should be carried out prior to starting azithromycin. On Study visit C14, all subjects who have not yet started azithromycin should begin a 3-day 
course of azithromycin 500 mg per day after all study samples have been collected; the third dose of azithromycin must be taken prior to the 
subject exiting the challenge unit on Study visit C16 
k. Subjects with positive culture or PCR sampled on C16 will be required to return to the challenge unit to receive additional antibiotic treatment 
l. 
Collect prior to vaccination/challenge 
m. All unsolicited AEs to be collected from time of vaccination to 28 days after vaccination and from time of challenge to 28 days after challenge 
n. Treatment-related AEs due to vaccination and due to challenge to be collected from time of vaccination to challenge and then for 3 months after 
challenge, respectively 
o. All AESIs and SAEs to be collected from time of signed informed consent to EOS 
p. Maybe performed at the discretion of the Investigator throughout the Challenge Phase 
q. This visit will occur at least 6 months post-vaccination or at least 3 months post-challenge 
r. 
Visit may occur up to 8 days before C0 or as close to C0 as possible to allow availability of test results. 
 




Statistical Analysis Plan (SAP) Client Approval Form 
 
Document Number: FORM-02894 v8.0 
Page 1 of 1 
Effective Date: 22 Nov 2021 
Printed on: 1 July 2023 
 
Client: 
ILiAD Biotechnologies, LLC 
Protocol Number: 
IB-202P 
 
Document Description: 
Final Statistical Analysis Plan 
SAP Title: 
A Phase 2b, Placebo-Controlled, Randomized Study of 
BPZE1 Intranasal Pertussis Vaccine in Healthy Adults to 
Assess Protection Against Colonization Following 
Challenge with Virulent Wild-Type Bordetella pertussis 
SAP Version Number: 
1.0 
Effective Date: 
30JUN2023 
 
Author(s): 
For PPD: Pu Huang, Biostatistician II 
 
Approved by: 
 
 
 
 
 
Xinayi Kong, Biostatistics Director 
Biostatistics & Programming, CRG 
 
Date (DD-MMM-YYYY) 
 
 
 
 
 
Stephanie Noviello, Global Clinical Lead, 
ILiAD Biotechnologies    
 
Date (DD-MMM-YYYY) 
 
 
 


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