Immunogenicity and Safety of Heterologous Combinations of COVID-19 Vaccines Available Under Emergency Use Authorization in Pakistan: A Randomized Phase II Trial
Phase: PHASE2
Conditions: COVID 19 Vaccine
Sponsor: Aga Khan University Hospital, Pakistan

 
 
 
 
 
 
 
Study Title: Immunogenicity and safety of heterologous combinations of COVID-19 vaccines 
available under Emergency Use Authorization in Pakistan: A randomized phase II trial 
 
Short Title: Combination assessment trial of COVID-19 vaccines (COMBAT-COVID) 
Ethics Ref: 6739 
Date and Version: 22.10.2021 and 1.1 
 
 
Sponsor:                                     The Aga University Hospital  
Funder:                                       Coalition for Epidemic Preparedness Innovations (CEPI)  
Program:                                     CfP CCT 
Principal Investigator:              Farah Naz Qamar 
                                                     Associate Professor 
                                                     Dept: Paediatrics and Child Health 
                                                    The Aga Khan University Hospital, Karachi, Pakistan. 
 
 
 

INVESTIGATORS AND ROLES 
  
Role 
Name 
Affiliation(s) 
Responsibilities 
Principal 
Investigator 
(PI)  
Farah Naz Qamar 
Aga Khan 
University 
Hospital, Pakistan 
(AKU) 
Project design, protocol development, site 
visits and project supervision, data 
monitoring and analysis, report writing. 
Dissemination of results (reports, 
manuscripts). Overall project coordination. 
Co-
Investigator 
(Co-I) 
M. Tahir Yousafzai 
AKUH 
Project design, protocol development, field 
supervision and monitoring, guided data 
analysis, site supervision, report, and 
manuscript writing. 
Co-I 
Zahra Hassan 
AKUH 
Project design, protocol development, 
standardizing lab protocols, guided data 
analysis, supervision of lab analysis, report, 
and manuscript writing 
Co-I 
Junaid Iqbal 
AKUH 
Project design, protocol development, 
standardizing lab protocols, guided data 
analysis, supervision of lab analysis, report, 
and manuscript writing 
Co-I 
Kiran Iqbal 
AKUH 
Standardizing lab protocols, guided data 
analysis, supervision of lab analysis, report, 
and manuscript writing 
Co-I 
Sonia Qureshi  
AKUH 
Project design, protocol development, field 
supervision and monitoring, guided data 
analysis, report, and manuscript writing 
Co-I 
Maria Fletcher  
AKUH 
Project design, protocol development, 
standardizing lab protocols, guided data 
analysis, supervision of lab analysis, report, 
and manuscript writing 
Co-I 
Najeeha Iqbal 
AKUH 
Standardizing lab protocols, guided data 
analysis, supervision of lab assays, report, 
and manuscript writing 
Co-I 
 
 
 
Momin Kazi 
AKUH 
Field supervision and monitoring, and 
eCRFs designing. 
Co-I 
Shazia Sultana 
AKUH 
Field supervision and monitoring, site 
supervision, and CRFs development 
National and International Collaborators 
Co-I 
Andrew Pollard 
Matthew Snape 
Teresa Lambe 
Xinxue Liu 
University of 
Oxford 
Scientific, technical, and clinical support, 
Biostatistics and data management support, 
and clinical samples testing support. 
Co-I 
Anh Wartel 
Jean-Louis Excler 
Deok-Ryun Kim  
International 
Vaccine Institute 
(IVI), Korea 
Scientific, technical, and clinical support. 
Biostatistics and data management support. 
 
Co-I 
Galit Alter 
Ragon Institute, 
Harvard School of 
Medicine, USA 
Support in system serology assays. 
Co-I 
Maj. Gen. Aamir Ikram 
Ghazala Parveen 
Firdous Nawaz Khan 
Omera Naseer 
National Institute 
of Health (NIH) 
Pakistan 
Central Vaccine storage and management. 
Technical, advisory, and laboratory support. 
Support in obtaining regulatory approvals.  

1. Table of Contents 
 
1. Table of Contents ...................................................................................................................................... 3 
2        ABBREVIATIONS ................................................................................................................................... 6 
3         BACKGROUND ..................................................................................................................................... 8 
3.1 
COVID-19 Situation and Vaccination in Pakistan ................................................................................ 9 
4         STUDY RATIONALE ............................................................................................................................... 9 
5         STUDY OBJECTIVES ............................................................................................................................ 10 
5.1 
Primary Objectives: .......................................................................................................................... 10 
5.2 
Secondary objectives: ....................................................................................................................... 10 
5.3 
Exploratory objectives: ..................................................................................................................... 11 
6 
STUDY ENDPOINTS .............................................................................................................................. 11 
6.1 
Primary Endpoints ............................................................................................................................ 11 
6.2 
Secondary Endpoints ........................................................................................................................ 11 
6.3 
Exploratory Endpoints ...................................................................................................................... 12 
7 
METHODOLOGY ................................................................................................................................... 12 
7.1 
Study Setting..................................................................................................................................... 12 
7.2 
Study Design ..................................................................................................................................... 13 
7.2.1    Trial duration .......................................................................................................................... 13 
7.2.2    Study Groups ........................................................................................................................... 13 
7.3      Study Participants and Eligibility Criteria ........................................................................................... 14 
7.3.1   Trial participants ...................................................................................................................... 14 
7.3.2   Inclusion criteria ...................................................................................................................... 14 
7.3.3    Exclusion criteria .................................................................................................................... 15 
7.3.4   Temporary exclusion criteria ................................................................................................... 16 
7.4 
Participants’ recruitment and screening .......................................................................................... 16 
7.4.1    Screening ................................................................................................................................. 16 
7.4.2    Informed consent ..................................................................................................................... 16 

7.5 
Randomisation .................................................................................................................................. 17 
7.6 
Details of Study Follow-ups/Visits .................................................................................................... 17 
7.7 
Eligibility Screening: .......................................................................................................................... 19 
7.7.1    Informed Consent Process: ..................................................................................................... 19 
7.8      Randomization:......................................................................................................................... 19 
7.9    Blood sample collection: ............................................................................................................ 19 
7.10 
First dose of vaccine: ............................................................................................................... 19 
7.11    Interview and CRF-2 filling: .................................................................................................... 19 
7.12       Phone Calls/ Home Visits for Recording AE And Safety Concerns: ................................................. 20 
7.13     Subsequent Visits: ............................................................................................................................. 20 
7.14      Adverse Events: ................................................................................................................................ 21 
7.15    Participants under quarantine ........................................................................................................... 22 
7.16     Participants with Confirmed SARS-CoV-2 Infection (COVID-19 Pathway) ........................................ 23 
7.16.1     Whole genome and targeted genome sequencing for regional VOCs .................................. 23 
7.17 
Sample Handling ............................................................................................................................... 24 
7.17.1 
Blood sampling, transportation, and storage ...................................................................... 24 
7.17.2 
Nasal swabs ......................................................................................................................... 25 
7.18 
Early Discontinuation/Withdrawal of Participants ........................................................................... 25 
7.19 
Contra-Indications to Receipt of Second/Booster Dose of Vaccine ................................................. 26 
7.20 
Definition of End of Trial .................................................................................................................. 26 
7.21 
Trial Intervention .............................................................................................................................. 27 
7.22 
Storage of Study Vaccines ................................................................................................................ 28 
7.23 
Compliance with Trial Treatment ..................................................................................................... 28 
7.23.1   Accountability of the trial treatment ....................................................................................... 28 
7.23.2   Concomitant medication ......................................................................................................... 28 
7.23.3   Post-trial treatment ................................................................................................................ 28 
7.24 
Sample Size ....................................................................................................................................... 28 
7.25 
Plan of Statistical Analysis ................................................................................................................ 29 

8 
DATA MANAGEMENT .......................................................................................................................... 31 
8.1 
Missing Data ..................................................................................................................................... 31 
8.2 
Access to data & data protection ..................................................................................................... 31 
8.3 
Data collection and recording .......................................................................................................... 31 
8.4 
Record keeping ................................................................................................................................. 32 
8.5 
Source data and case report forms (CRFs) ....................................................................................... 32 
8.6 
Data quality ...................................................................................................................................... 32 
8.7 
Quality Assurance Procedures .......................................................................................................... 32 
8.7.1 
Risk assessment ................................................................................................................... 33 
8.7.2 
Monitoring ........................................................................................................................... 33 
9 
TRIAL COMMITTEES ............................................................................................................................. 33 
9.1 
Joint steering committee .................................................................................................................. 33 
9.2 
Independent data monitoring committee (IDMC) ........................................................................... 34 
10 PROTOCOL DEVIATIONS ...................................................................................................................... 34 
11 ETHICAL AND REGULATORY CONSIDERATIONS ................................................................................... 35 
12 TRIAL REGISTRATION ........................................................................................................................... 35 
13 EXPENSES AND BENEFITS .................................................................................................................... 35 
14      EXPERIENCE IN CONDUCTING VACCINE RESEARCH (38-46) .............................................................. 35 
15       POTENTIAL CHALLENGES: ................................................................................................................. 36 
16       REFERENCES ...................................................................................................................................... 37 
17       APPENDICES ...................................................................................................................................... 39 
17.1    Appendix A: Blood Sampling .............................................................................................................. 39 
17.2     Appendix B: Flow of Sample Processing ........................................................................................... 43 
 
 
 

2        ABBREVIATIONS 
ADEM 
Acute Disseminated Encephalomyelitis 
AE 
Adverse event 
AESI 
Adverse Event of Special Interest 
AKUH 
Aga Khan University Hospital 
ALT 
Alanine Aminotransferase 
ARDS 
Acute Respiratory Distress Syndrome 
AST 
Aspartate aminotransferase 
BIBP 
Beijing Institute of Biological Products 
C-19 
COVID-19 
CBC 
Complete Blood Count 
CEPI 
Coalition for Epidemic Preparedness Innovations 
ChAdOx1 
Chimpanzee adenovirus 1 
CNBG 
China National Biotec Group 
CoVs 
Coronaviruses 
CRF 
Case Report Form 
CTU 
Clinical Trial Unit 
DRAP 
Drug Regulatory Authority of Pakistan 
eCRF 
Electronic Case Report Form 
EDC 
Electronic Data Capture 
ELISA 
Enzyme Linked Immunosorbent Assay 
ELISPOT 
Enzyme-Linked Immune Absorbent Spot 
EPI 
Expanded Program of Immunization 
EUA 
Emergency Use Authorization 
GCP 
Good Clinical Practice 
GMR 
Geometric Mean Ratio 
GTCH 
Ghurki Trust Secondary Care Hospital 
hACE 
Human Angiotensin Converting Enzyme 2 
hADV 
Human Adenovirus 
HIV 
Human Immunodeficiency virus 
HPB 
Heterologous Prime Boost 
ICS 
Intracellular Cytokine Staining 
IHITC 
Isolation Hospital and Infection Treatment Centre 
IM 
Intramuscular 
IPD 
Invasive Pneumococcal Disease 
IPV 
Inactivated Polio Vaccine 
LGH 
Lahore General Hospital 
LHR 
Lahore 
LMIC 
Low- and Middle-Income Countries 
MERS 
Middle East Respiratory Syndrome 
NBC 
National Bioethics Committee 
NIH 
National Institute of Health 

NRL 
Nutrition Research Laboratory 
PCR 
Polymerase Chain Reaction 
PHCs 
Primary Healthcare Centers 
PHC Global 
Precision Health Consultants Global 
PI 
Principal Investigator 
PID 
Primary Immune Deficiency 
SAE 
Serious Adverse Event 
SARS-CoV 
Severe Acute Respiratory Syndrome Coronavirus 
SD 
Standard Deviation 
SMC 
Safety Monitoring Committee 
SOP 
Standard Operating Procedure 
SS 
Sample Size 
WHO 
World Health Organization 
XDR 
Extensively Drug-Resistant 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

3         BACKGROUND  
On March 11, 2020 Coronavirus Disease 2019 (COVID-19) was declared a pandemic by World 
Health Organization (WHO), and within a year of its emergence, this disease with pneumonia like 
symptoms spread over 220 countries of the world (1).  With millions of laboratory-confirmed cases, 
deaths (WHO), and widespread social and economic disruption, this ever-increasing outbreak has 
become the biggest challenge of this century (2).  
Coronaviruses (CoVs) were first discovered in 1960s (3) and classified as order Nidovirus, family 
Coronaviridae, subfamily Coronavineae which is finally divided into α, β, ϒ and δ genera (4). In last 
two decades, three CoVs of β genus, SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), 
MERS (Middle East Respiratory Syndrome Coronavirus) and SARS-CoV-2 (Severe Acute 
Respiratory Syndrome Coronavirus-2), have crossed specie barriers and transmitted to humans from 
animals (5).  
All CoVs displays crown-shaped peplomers/spikes on their envelope with large positive-sense single 
stranded RNA (+ssRNA) (6).  One fourth of viral genome is responsible for coding structural proteins, 
such as the spike (S) glycoprotein, envelope (E), membrane (M) and nucleocapsid (N) proteins. E, M, 
and N proteins are mainly responsible for virion assembly whilst the S protein is involved in receptor 
binding that mediates virus entry into host cells during infection (7).  The S-protein divides into two 
subunits, namely, S1 and S2. S1 subunit further subdivided into three domains, particularly A, B, and 
C. SARS-CoV-2 enters the host cell through direct attachment of domain B (aka receptor binding 
domain) to the angiotensin converting enzyme-2 (ACE-2) receptor (4).  
SARS-CoV-2 became a global threat last year and measures such as social distancing, lockdown and 
wearing masks were undertaken in countries across the world to prevent the spread of the virus (8). 
Currently there is no definitive treatment for COVID-19 though Dexamethasone has improved 
mortality in those with confirmed disease and requiring oxygen support (9).  
Cases are rising and many countries are facing second and third waves of infections. Pakistan is also 
experiencing the third wave of COVID-19 since March 2021 (10). The Drug Regulatory Authority of 
Pakistan (DRAP) has evaluated and granted emergency use authorization (EUA) to four vaccines 
against COVID-19. These vaccines were imported from China (Sinopharm/BIBP and CanSino), 
Russia (Sputnik V, Gamaleya), and United Kingdom (AstraZeneca) (11-13). All these vaccines are 
approved for use as two-dose regimens except CanSino, which is administered as a single dose (details 
of the types of the vaccines included in the study and their dosing regimens is detailed in Table 7).  
Pakistan formally launched the coronavirus vaccination campaign on February 3rd, 2021 and till 
March 3rd, 2021, 72,882 individuals have received doses of Sinopharm (14). The first round of 
vaccination was targeted towards health workers. Pakistan is the world’s fifth most-populous country 

with a population exceeding 220 million, and programmatic challenges are anticipated in immunising 
such a large population. It would be advantageous to have a permissive approach of using 
heterologous prime/boost strategy for vaccination, where the second vaccine is not necessarily the 
same as the first one. This strategy is already approved and in practice for some vaccines that may be 
used interchangeably during a vaccination series (from different manufacturers), according to 
recommendations from the AAP or ACIP, include diphtheria and tetanus toxoids, hepatitis A, 
hepatitis B, and rabies vaccines (15).  
3.1       COVID-19 Situation and Vaccination in Pakistan  
Pakistan, world's fifth-most populous country with an estimated 220 million population. The country 
spreads over an area of 881,913 square kilometres (km2) (16) and continues to face challenges 
including poverty, illiteracy, unemployment, and poor vaccination coverage (17, 18). 
As of September 06, 2021, around 1,182,918confirmed COVID-19 cases are reported in Pakistan, of 
which 1,064,319have recovered and 26,232deaths (case fatality rate of 2.2%) have been reported (19). 
Daily over 4000 new confirmed COVID-19 cases are reported from Pakistan. Majority of the cases 
are reported from Sindh province of Pakistan. In Pakistan, coronavirus vaccinations started on 
February 3rd, 2021. Chinese government has donated 500,000 doses of Sinopharm vaccine. 
Furthermore, through the COVAX initiative to ensure the supply of vaccines to all countries, Pakistan 
has received first batch of 1.2 million doses of AstraZeneca. Pakistan in collaboration with China is 
also trying to procure 20 million doses of CanSino vaccine, provided that the results of ongoing phase 
3 clinical trial show the vaccine to be effective. With the successful completion of Phase 1 of COVID-
19 vaccination program in which vaccine is provided to the frontline healthcare workers, current 
counts report that more than 19,427,419 individuals are fully vaccinated. Though different strategies 
being employed by Pakistan to acquire enough vaccines for its enormous population of 220 million, 
considering the costs, far flung areas, and huge population, healthcare officials predict that it is going 
to be very difficult to immunise a critical mass of individuals which could lead to herd immunity (21, 
22). 
4         STUDY RATIONALE 
The urgency for the development of a COVID-19 vaccine has led to a paradigm shift in process 
development, where many steps of vaccine development and testing have been performed in parallel 
to save time. As a result, different types of COVID-19 vaccines emanating from multiple platforms 
and formulations (23) have progressed rapidly to advanced stages of development. In line with this 
paradigm shift, the availability of several COVID-19 vaccine candidates could be tested for 
heterologous prime-boost vaccination strategies (24-27) to elicit higher and broader putative 
protective immune responses (both antibody and T-cell responses, possibly against circulating 

variants of concerns (VOCs)) of longer duration, and for improved safety profiles. It is estimated that 
over 80% of the adult population have been naturally exposed to the human Adenovirus (hAdV) 
serotypes which is a component of some viral-vectored COVID-19 vaccine candidates (28). Although 
not addressed in this study, people previously vaccinated with the standard regimen could be offered 
a booster immunization with a different vaccine, a different platform. 
Circumstances such as vaccine shortages, contraindication to a specific vaccine, changes in product 
availability, or migration across jurisdictions may necessitate giving vaccines from different 
manufacturers to the same individual over time. Such data are ascertained and interpreted more readily 
for diseases with known correlates of protective immunity (e.g., specific serologic markers). For 
diseases without such surrogate laboratory markers, prelicensure field vaccine efficacy (phase III) 
trials or post-licensure surveillance generally are required to determine protection (29). Currently, 
there is a global shortage of COVID-19 vaccines, Pakistan and other Low- and Middle-Income 
Countries (LMIC) will face a more acute shortage of the vaccines compared to High-Income (HIC) 
and upper Middle-Income Countries. With the current arrangements full vaccination of all eligible 
Pakistani population would be challenging. The heterologous prime boost (HPB) approach will help 
to mitigate the shortage of available COVID-19 vaccines (26, 27). HPB will allow combination of 
different vaccines in case the same vaccine is not available at the time of boosting (follow-up dose) 
and longer interval between the two doses could be more pragmatic and practical in real-life settings. 
Furthermore, the HPB strategy might prove to be more effective against the VOCs of SARS CoV-2. 
Such a matrix of HPB possibilities has triggered major interest and consideration by manufacturers, 
funders, and regulators however, data on potential benefits and adverse effects of the HPB matrix of 
various COVID-19 vaccines is not available. Therefore, we aim to evaluate the safety and 
immunogenicity of HPB for vaccines approved for use in Pakistan. This exploration meets emergency 
needs and may assist in the formulation of public health policies. 
5         STUDY OBJECTIVES 
5.1      Primary Objectives: 
1. To determine whether the serum anti-spike IgG concentration against SARS-COV-2 four weeks 
post boost in COVID seronegative participants immunized with HPB COVID-19 vaccines 
regimens is non-inferior to that observed following homologous immunization (second dose at 
Day 70).  
5.2 
Secondary objectives: 
1. To assess the safety and reactogenicity of different combinations of heterologous and homologous 
COVID-19 vaccines.   

2. To characterize the neutralizing antibody response (pseudo-virus neutralization assay) against 
SARS-CoV-2 (as per schedule of events) in COVID seronegative participants immunized with 
HPB and homologous COVID-19 vaccines regimens.  
3. To assess trend in immunogenicity (anti-spike IgG, anti-nucleocapsid) against SARS-COV-2 as 
per schedule events in COVID seronegative participants immunized with HPB and homologous 
CVODI-19 vaccines regimens.  
4. To assess safety and immunogenicity of homologous full dose versus fractional dose homologous 
booster as compared to no booster at 6 months following the second dose of primary series among 
the participants in homologous arms.  
5.3 
Exploratory objectives: 
1. To characterize SARS-CoV-2 infections (breakthrough infections) and immune responses in 
participants immunized with COVID-19 vaccines (Whole Genome Sequencing of viral strains). 
2. To measure neutralizing antibody response (pseudo-virus neutralization assay) against SARS-
CoV-2 VOCs circulating in Pakistan. 
3. To assess phenotypic characteristics of CD4 and CD8 T cells by Intracellular Cytokine Staining 
(ICS) on Immunology cohort 
4. To assess the functionality of vaccine-induced antibodies against SARS-CoV2 by performing a 
panel of systems serology on Immunology cohort. 
6       STUDY ENDPOINTS 
6.1       Primary Endpoints 
1. Anti-spike IgG antibodies by ELISA at Week 14 (4 weeks post booster dose)  
6.2       Secondary Endpoints 
1. Safety and reactogenicity endpoints are described as below: 
a. Serious adverse events (SAE) and adverse events of special interest (AESI) throughout the 
study  
b. Solicited local and systemic reactions within 7 days post each dose  
c. Unsolicited reactions within 28 days post each dose  
d. Medically attended adverse reactions up to 3 months post booster dose  
e. Changes from baseline to 4 weeks post each dose in biochemical and hematological tests for 
safety measures  
2. Neutralizing antibody response (pseudo-virus neutralization assay) against SARS-CoV-2 at day 
0, and weeks 4, 14, 24, 28, 48, 60 and 96 as per schedule of events (only immunology cohort) and 
at baseline and 4 weeks post booster dose in general cohort. 
3. Immunogenicity of HPB COVID-19 vaccine regimens and homologous immunization, including:  

a. Anti-spike IgG responses measured by ELISA at Day 0, and Weeks 4, 10, 14, 24, 28, 48, 60 
and 96 as per schedule of events (in full cohort) 
b. Anti-nucleocapsid IgG, responses will be measured at Day 0, and Week 2, 14, 24, 48, 60, and 
96 as per schedule of events (in full cohort).  
c. ELISpot assays will be performed at Day 0, and week 2, 4, 10, 12, 14, 24, 48, 96 (immunology 
cohort)  
6.3     Exploratory Endpoints   
1. Neutralizing antibody response (pseudo-virus neutralization assay) against SARS-CoV-2 and 
anti-spike IgG responses measured by ELISA within 1 week of breakthrough infection among the 
participants. 
2. Sequencing of SARS-CoV-2 viruses of breakthrough infections in vaccine recipients. 
3. Neutralizing antibody response (pseudo-virus neutralization assay) against SARS-CoV-2 VOCs 
circulating in Pakistan. 
4. ICS will be performed to assess the phenotypic characteristics of CD4 and CD8 T cells 
recognizing the antigen by high throughput multicolor flow cytometry. It will measure cytokine 
secretion after stimulation with Ag and T-cell epitope mapping (optional). 
5. Strategic stage-gated Systems Serology analysis will be performed that will not only objectively 
profile all vaccine induced immune responses but because of this, we will have the opportunity to 
deprioritize assays and antigens based on rapidly emerging data.  
7    METHODOLOGY 
7.1    Study Setting 
This study will be conducted in Karachi, Lahore, and Islamabad for rapid recruitment and 
generalizability. Karachi is the capital of Sindh province in Pakistan with a total population of 14.9 
million. Karachi is administratively divided into 18 towns. (27). These communities are coastal 
settlements, with a total area of 8.1 square miles under surveillance.  
Volunteers will be identified actively through community mobilization and door to door activity in 
Ibrahim Hyderi and Ali Akbar Shah Goth extensions of Karachi. Volunteers will be screened for 
eligibility by the study physicians/research associates during household visits and those who found 
eligible and consent for participation will be taken to the clinical trial unit of The Aga Khan University 
for vaccine administration. Volunteers will also be identified actively through community 
mobilization and door to door activity in sites adjacent to AKUH secondary care Hospitals in Karachi, 
including The Aga Khan Hospital for Women, Karimabad and Garden. Those who are found eligible 
and consent for participation will be brought to these two sites for vaccine administration. Other 
primary health care centers in Karachi are to be decided. 

 In Islamabad and Lahore volunteers from adjacent areas of Infection treatment center (IHITC) in 
Islamabad and clinics of Chughtai labs in Lahore will be identified after social mobilization, 
advertisement and communication and will be brought to these two sites for vaccine administration.  
Islamabad is the capital city of Pakistan located in the north-eastern part of the country between 
Rawalpindi and Margalla hills national park to the north. The population of Islamabad is about one 
million. Isolation hospital and infection treatment centre (IHITC) is 250 bedded hospitals in 
Islamabad providing free of cost treatment to the people of Islamabad and adjacent cities. IHITC is 
equipped with central HVAC system, ensuring negative pressure throughout the facility to optimize 
infection control. Ambulatory services are also available for patients needing transfer to or from 
IHITC. Additionally, it ensures optimal management for telemedicine and home quarantine patients.  
Lahore is situated in the north-eastern part of Punjab. The district consists of ten administrative strata, 
including nine towns and one cantonment area. We will enrol participants at clinics of Chughtai labs. 
Chughtai labs have a network of vaccination centres, clinics where enrolment procedures, vaccine 
administration and blood sample collection can be performed, and they are also affiliated with 
hospitals which can provide care for patients needing in case of any adverse events. Volunteers from 
adjacent areas after social mobilization, advertisement, and communication with those visiting the 
vaccination centres/ clinics will be offered invitation for participation in trial. We estimate to enrol 8-
10 participants per site /day. There are six trial sites, and we will enrol approximately 120 participants 
/week and aim to complete the enrolments within approximately 14-16 weeks at this pace. 
7.2      Study Design  
This will be a randomized, phase II trial, conducted among volunteers aged 18 years and above in 
Karachi, Lahore, and Islamabad, Pakistan. The study groups are mentioned in Table 1. 
7.2.1    Trial duration 
The total duration of the trial will be approximately 2 ½ years (14-16 weeks for complete enrolments 
and two years follow up for each participant following the day of enrolment).    
7.2.2    Study Groups 
The study will enroll a total of 1680 participants (160 per heterologous group and 240 in each 
homologous group) which will be divided into 2 cohorts, one for more detailed immunological 
assessment (immunology cohort, N=225, 25 per group) and one for main immunology endpoints 
(general cohort N=1455, 135 per heterologous group and 215 per homologous group). We will ensure 
that the participants of all age groups are represented including less than 40 years and above. The 
study will include 9 study groups (2:2:2:2:2:2:3:3:3) with different combinations of COVID-19 
vaccines schedule as shown in Table 1. This is a pragmatic trial where the interval between the two 
doses will be kept longer than the conventional recommendations of 21/28 days.  Further, we will 

include any further homologous vs heterologous groups as more vaccines will receive emergency use 
approval (EUA) by the drug regulatory authority of Pakistan. The homologous arm will be further 
sub-divided into three groups to receive either no booster, fractional dose of booster or full dose of 
booster after six months of second dose.  
Table 1: Description of study groups showing interval of vaccine doses and their sample sizes 
(SS) (SS calculations are provided on page 26) 
Group 
First Dose 
Second Dose 
Interval 
Booster 
Dose 
SS* 
1 
BIBP (CNBG, Sinopharm) 
WIV 
CanSinoBIO 
70±7 days 
(10 wks±2) 
- 
1680 
(160 in each 
heterologous group and 
240 in each homologous 
group)  
2 
BIBP (CNBG, Sinopharm) 
WIV 
AstraZeneca ChAdOx 
3 
CanSinoBIO 
BIBP (CNBG, Sinopharm) 
WIV 
4 
CanSinoBIO 
AstraZeneca ChAdOx 
5 
AstraZeneca ChAdOx 
BIBP (CNBG, Sinopharm) 
WIV 
6 
AstraZeneca ChAdOx 
CanSinoBIO 
7(ref)** 
BIBP (CNBG, Sinopharm) 
WIV 
BIBP (CNBG, Sinopharm) 
WIV 
6 months 
post 
second 
dose 
8(ref)** 
CanSinoBIO 
CanSinoBIO  
9(ref)** 
AstraZeneca ChAdOx 
AstraZeneca ChAdOx 
*SS: Sample Size; a subset of each group (25 per group) will be allocated to the immunology sub cohort 
** 240 participants each will be enrolled in the reference/homologous groups 
7.3      Study Participants and Eligibility Criteria 
7.3.1   Trial participants 
Adult male and female volunteers aged 18 years and above and volunteers with well controlled mild 
or moderate comorbidities will be enrolled to participate in trial.  
7.3.2   Inclusion criteria 
• Participant is willing and able to give written informed consent for participation in the trial. 
• Male or Female aged 18 years or above and in good health as determined by a trial clinician. 
Participants may have well controlled mild-moderate comorbidity. 
• In the Investigator’s opinion, is able and willing to comply with all trial requirements. 
• Residing in the study areas. 

7.3.3    Exclusion criteria 
The participant may not enter in the trial if ANY of the following apply: 
• Pregnant women or those who are planning to conceive within next 70 days.  
• Women who are breast feeding 
• Already received any COVID-19 vaccine or any other vaccine likely to impact on interpretation 
of the trial data (e.g., Adenovirus vectored vaccines). 
• Administration of immunoglobulins and/or any blood products within the three months preceding 
the planned administration of the vaccines. 
• Any confirmed or suspected immunosuppressive or immunodeficient state; asplenia; recurrent 
severe infections and use of immunosuppressant medication within the past 6 months, except 
topical steroids or short-term oral steroids (course lasting ≤14 days) 
• History of allergic disease or reactions likely to be exacerbated by any component of study 
vaccines (e.g., hypersensitivity to the active substance of the COVID-19 vaccines included in the 
study groups 
• Any history of anaphylaxis. 
• Current diagnosis of or treatment for cancer (except basal cell carcinoma of the skin and cervical 
carcinoma in situ)  
• Bleeding disorder (e.g., factor deficiency, coagulopathy, or platelet disorder), or prior history of 
thrombotic events and/or significant bleeding or bruising following IM injections or venipuncture. 
• Continuous use of anticoagulants, such as coumarins and related anticoagulants (i.e., warfarin)  
• Any other significant disease, disorder or finding which may significantly increase the risk to the 
volunteer because of participation in the study, affect the ability of the volunteer to participate in 
the study or impair interpretation of the study data. 
• Severe and/or uncontrolled cardiovascular disease, respiratory disease, gastrointestinal disease, 
liver disease, renal disease, endocrine disorder, and neurological illness (mild/moderate well 
controlled comorbidities are allowed) 
• History of active or previous auto-immune neurological disorders (e.g., multiple sclerosis, 
Guillain-Barre syndrome, transverse myelitis). Bell’s palsy will not be an exclusion criterion. 
• History of laboratory confirmed COVID-19 within 6 months prior to enrolment (history of SARS-
CoV-2 detection by PCR or antibody to SARS-CoV-2). 
• Scheduled elective surgery during the trial. 
• Participants enrolled in any other research trial. 
• Participants planning to migrate out of the study area within 2 years of the study.  

7.3.4   Temporary exclusion criteria 
If the volunteer has any of the following, they will not be enrolled that day.   
- Acute respiratory illness (moderate or severe illness with or without fever) 
- Fever (temperature greater than 38°C) 
They may be considered for enrolment later in the trial if they recover in sufficient time. 
7.4 
Participants’ recruitment and screening  
Potential participants will be invited to participate in the trial by door-to-door home visitation in the 
selected communities. There will also be publicizing via flyers at the enrolment sites i.e., vaccination 
centres and clinics. A study information leaflet will be provided to the volunteers so that they can 
understand the trial and procedure of participation. A written version and verbal explanation of the 
study information leaflet will be presented to the participant, detailing: 
• The exact nature of the study 
• What it will involve for the participant 
• The implications and constraints of the protocol 
• The known side effects and any risks involved in taking part. 
• The sample handling protocol. 
The Study information leaflet will be made available to the participant for an appropriate amount of 
time (where possible this will be a minimum of 24 hours) prior to consent being obtained. 
7.4.1    Screening 
Once volunteers express an interest in joining the trial after going through the information leaflet, 
they will be directed to the designated sites for screening and eligibility assessment by the study staff 
including study physician. If volunteer is found eligible for participation, informed consent will be 
collected. 
7.4.2    Informed consent 
The participant will personally sign and date the latest approved version of the informed consent form. 
A witness or interpreter will be arranged for those who cannot read or understand our language. 
Consent form will be available in Urdu and English languages. Participants will have the opportunity 
to individually question an appropriately trained and delegated researcher before signing consent. 
The following general principles will also be emphasised: 
• Participation in the study is entirely voluntary. 
• Refusal to participate involves no penalty or loss of medical benefits. 
• The participant may withdraw from the study at any time. 
• The participant is free to ask questions at any time to allow him or her to understand the 
purpose of the study and the procedures involved. 

• Those participants will not be sure whether they have received an approved COVID-19 
vaccine schedule. This may have implications for any travel or other activities that may require 
individuals to be considered ‘fully immunised’.  
• Participants, like the general population, will not be exempt from following the 
contemporaneous government COVID-19 guidance to minimise viral transmission. 
• Samples taken as part of the study may be sent outside of Pakistan to the laboratories for 
testing. Samples will be de-identified. Volunteers will be asked if they consent to indefinite 
storage of any leftover samples for use in other ethically approved research, this will be 
optional. 
The person who obtained the consent must be suitably qualified and experienced and have been 
authorised to do so by the Chief/Principal Investigator and listed on the delegation log. A copy of the 
signed informed consent will be given to the participant. The original signed form will be retained at 
the research study office. 
7.5 
Randomisation 
Computer generated randomisation list will be prepared and participants will be individually 
randomized using the block randomization technique. A block size of 21 will be used to randomize 
nine heterologous/homologous groups. The initial 25 participants in each group will be included in 
the immunology cohort.  All participants enrolled in each of the three homologous groups will be 
randomized for booster dose at 6 months following the second dose of vaccine administration to 
receive either full dose of the homologous booster, or fractional dose intradermal booster or no 
booster. Since each homologous group has 240 participants, about 80 participants in each group will 
receive full dose booster, 80 will receive fractional dose and 80 will receive no booster. 
7.6 
Details of Study Follow-ups/Visits 
The study visits and procedures will be undertaken by study team. The procedures to be included in 
each visit are documented in the schedule of events (Table 2). Each visit is assigned a time-point and 
a window period, within which the visit will be conducted. If a participant cannot attend a visit, where 
possible, this will be re-arranged to an in-person visit within the time window. A phone call will be 
made to collect as much relevant information as possible if the participant is unable to attend the visit 
in person, because of quarantine or self-isolation restrictions, and the participant will be out of window 
period if the visit is further postponed.

Table 2: Schedule of Events 
Variables 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
Additional visit only 
for homologous arms 
V9 
V10 
V11 
C-19 
(COVID-19) 
pathway§ 
Study timeline (Days) 
D0 
D14 
D28 
D70 
D84 
D98 
D168 
D196 
D238 
D266 
D336 
D420 
D672 
D0-D672 
Study timeline (Weeks) 
W0 
W2  
(2 wks. post 
first dose) 
W4 
W10 
W12 
(2 wks. post 
2nd dose) 
W14 
 
W24 
(6 months post 
first dose) 
W28 
 
W34 
W38 
(4wks post 
booster dose) 
W48 
(1 yr. post 
first dose) 
W60 
W96 
(2 yrs. post 
first dose) 
ASAP after 
the COVID-
19 test report 
Window period 
 
±7 
±7 
±7 
±7 
±7 
±7 
±7 
±7 
±7 
±7 
±7 
±7 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Interim medical history 
 
 
X 
X 
 
X 
 
X 
 
 
X 
X 
X 
X 
Physical examination 
(including vital signs etc. as 
required) 
X 
 
(X) 
(X) 
 
(X) 
 
(X) 
 
 
(X) 
X 
(X) 
(X) 
COVID-19 vaccination 
X 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
Home Visit 
 
X 
X 
(X) 
X 
X 
X 
X 
(X) 
X 
X 
X 
X 
X 
Safety Bloods (*Biochemistry 
& **Hematology) BoC 
X 
 
X 
 
 
X 
 
 
 
X 
 
 
 
X 
Phone calls for AE 
 
Weekly till 14 days after each dose in addition to Monthly phone calls 
 
Physician Teleclinic Visit 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Pseudovirus neutralization 
assay(BoC) 
X 
 
X 
 
 
X 
X 
 
X 
 
X 
X 
X 
X 
X 
Anti-spike IgG (BoC) 
X 
 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Anti-nucleocapsid IgG (BoC) 
X 
 
X 
 
 
X 
X 
 
 
X 
X 
X 
X 
X 
SARS-CoV-2 viral swab 
(Nasal swab) 
 
 
 
 
 
 
 
 
 
 
 
 
 
X 
Systems Serology (IC) 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
Blood sample for ICS & 
ELISpot (IC) 
X 
X 
X 
X 
X 
X 
X 
 
X 
X 
X 
 
X 
X 
V: Visit, D: Day     X: Additional measurements only for immunology cohort.  
§ Whole genome sequencing for all breakthrough cases and targeted sequencing for regional VOCs.   BoC: Both cohorts      IC:  Immunology cohort 
*Biochemistry: Na, K, Urea, Creatinine, Bilirubin, ALT, ALT Phosphatase, Albumin, and AST   
**Hematology: CBC    

Visit 1 (D0): Baseline assessment, eligibility check, enrolment, and vaccination visit  
7.7 
Eligibility Screening: 
At the initial visit participants will be assessed at the study site, by study staff including study 
physician for eligibility to participate. A structured questionnaire (CRF 1) will be used to evaluate the 
volunteer for all eligibility criteria. If any of the exclusion criteria will be found volunteer will be 
declared as not eligible. If some temporary illness or condition is found, the volunteer will be declared 
as temporary not eligible and will be called for another screening session in due time, if feasible 
according to study timelines. 
7.7.1    Informed Consent Process: 
If volunteer is found eligible, he/she will be offered participation in the trial and informed consent 
will be administered. Complete informed consent document will be read and explained to each 
potential participant despite the fact that study information leaflet had already been handed over to 
them. A trained study team member will conduct the consent taking process and will get the form 
signed, or thumb impression taken with date by participant and witness, or interpreter as required. 
One signed copy of consent form will be handed over to the participant and original form will be kept 
in study office. 
7.8      Randomization: 
Once consent is obtained, enrolment procedure will be initiated. Enrolment will be done on the day 
of vaccine first dose administration. Participants will be randomized in one of the study groups and 
will be enrolled in immunology or general cohort. Participants in homologous group will be further 
randomized for booster doses as well.  
7.9    Blood sample collection: 
Blood sample will be taken as per laboratory specifications to assess immunogenicity and serological 
assays including baseline serology to confirm seronegative against SARS-CoV2 at the time of first 
dose administration.  
7.10  First dose of vaccine: 
Volunteers will be considered enrolled to the trial at the point of vaccination. All vaccines will be 
administered intramuscularly according to specific SOPs. The participant will stay in the trial site for 
observation for at least 15 minutes, for any immediate adverse events. 
7.11    Interview and CRF-2 filling: 

CRF 2 will be filled on the day of enrolment which will record information on their demographic and 
basic clinical data, including co-morbidities and concomitant medications and their weight height will 
also be measured.   
7.12       Phone Calls/ Home Visits for Recording AE And Safety Concerns: 
All participants will be given an emergency 24-hour telephone number to contact the on-call study 
physician if needed.  Participants will be instructed on how to self-assess the severity of the adverse 
events (AEs).  Paper tape will be provided to each participant to measure the erythema or swelling (if 
any) following vaccination. Participants will be contacted via phone calls/home visit weekly until 
D14 post vaccination and monthly thereafter to record AE or any serious medical illnesses or 
unplanned hospital/visits and adverse events of special interest. These follow-ups will collect 
information on the timing and severity of the following solicited AEs (Table 3). Adverse events of 
special interest (AESI) will be collected and recorded in the AE reporting form in electronic data 
capturing system throughout the duration of this study. These should also be reported as SAEs if they 
fulfil the definition criteria for SAEs (definitions of adverse events provided in Table 4). All AESI’s 
not already reported as SAEs should be included in the reports to the IDMC. 
7.13     Subsequent Visits:   
There will be home visits, in addition to 2 or 3 clinic visits for vaccine doses. During each call and 
visit, CRF will be filled and collected information will be recorded. 
At each visit: 
• 
Participant identification will be verified by vaccination card/ trail registration card. 
• 
Brief medical history will be taken. 
• 
All married women of childbearing age will be enquired about pregnancy. 
• 
Physical examination will be performed (if indicated). 
• 
Well-being, adverse reactions, or side-effects will be assessed and managed (if applicable). 
• 
Information on symptoms compatible with COVID-19, testing for and results of testing for  
            the infection will be collected. 
• 
Blood sample will be collected as per laboratory specifications to assess AEs, and 
immunogenicity assays. 
• 
Vaccine dose will be administered (if applicable) 
Prior to administering the second dose and booster dose of the vaccine, any newly available and 
relevant safety data will be reviewed from animal studies or clinical trials of coronavirus vaccines 
included in this study being tested elsewhere and discussed with the Independent Data Monitoring 
Committee (IDMC), as necessary. While there will be no planned safety pause, a review of safety 
data will be conducted after the initial 50 - 60 participants have received a second dose at the day 

70±7 days post first time-point. This will assess safety in the first 48 hours after immunisation. Further 
details about the IDMC, its mandate, frequency of meetings, and its charter is provided in section 9.2 
on page 38.  
   7.14      Adverse Events: 
Table 3: Solicited Adverse Events to Record Post Vaccination  
Local solicited AEs 
Pain, Tenderness, Redness, Warmth, Itch, Swelling/ Induration 
Systemic solicited AEs 
Fever, Feverishness, Chills, Joint pains, Muscle pains, Fatigue, 
Headache, Malaise, Nausea, Vomiting, Diarrhoea 
 
Table 4: Adverse Event Definitions 
AE 
Any untoward medical occurrence in a participant to whom a medicinal product 
has been administered, including occurrences which are not necessarily caused by 
or related to that product. 
AESI 
Adverse events identified as being of relevance to the IMP’s. These will also be 
reported as an SAE, if meeting SAE criteria (e.g., hospitalisation) 
 SAE 
SAE is any untoward medical occurrence that: 
• Results in death 
• Is life-threatening 
• Requires inpatient hospitalization or prolongation of existing hospitalization 
• Results in persistent or significant disability/incapacity 
• Consists of a congenital anomaly or birth defect* 
Other ‘important medical events’ may also be considered a SAE when, based 
upon appropriate medical judgement, the event may jeopardise the participant and 
may require medical or surgical intervention to prevent one of the outcomes listed 
above. 
NOTE: The term "life-threatening" in the definition of "serious" refers to an event 
in which the participant was at risk of death at the time of the event; it does not 
refer to an event which hypothetically might have caused death if it were more 
severe. 
 
Table 5: Adverse events of special interest  
Immunologic 
Anaphylaxis 
Neurological 
Isolated anosmia/ageusia* 
Guillain-Barre Syndrome 
Acute disseminated 
encephalomyelitis (ADEM) 
Meningoencephalitis 
Peripheral facial nerve palsy 
Generalized convulsion 
Myelitis 

Aseptic meningitis 
Hematological 
Thrombosis** 
Stroke 
Thrombocytopenia*** 
Eosinophilia**** 
Coagulation disorder (includes 
coagulopathy, thrombosis, 
thromboembolism, 
internal/external bleed, and stroke) 
Thrombosis with 
Thrombocytopenia Syndrome 
(TTS) 
Cardiac 
Acute cardiovascular injury 
(includes myocarditis, 
pericarditis, arrhythmias, heart 
failure, infarction) 
Multisystem Inflammatory 
Syndrome in Adults (MIS-A) 
 
Dermatological 
Chilblain-like lesions 
Single organ cutaneous 
vasculitis 
Erythema multiforme 
Alopecia 
Gastrointestinal 
Acute liver injury ⴕⴕ ⴕ 
Appendicitis 
Respiratory 
ARDSⴕⴕ 
 
Renal 
Acute kidney injury 
 
Other 
COVID-19 diseaseⴕ 
SARS-CoV2 positivity on a 
validated test 
*In the absence of COVID-19 
** Excluding superficial thrombophlebitis (including line-associated) 
*** G3 or above 
**** This will be used as a marker of skewed Th2 responses and will be routinely monitored 
in participants attending the COVID-19 Pathway and follow-up visits. Only G2 and above. 
ⴕ In particular, any occurrence of suspected vaccine associated enhanced disease (VAED) as 
defined by most recent Brighton Collaboration Case Definition  
ⴕⴕ In the absence of an infective etiology (including COVID-19) 
ⴕⴕ ⴕ As defined in Hy’s Law (see Error! Reference source not found.) 
 
 
7.15    Participants under quarantine 
Given the evolving epidemiological situation both globally and in Pakistan, when a participant will 
be unable to attend any of their scheduled or unscheduled visits, a telephone consultation will be 
arranged to obtain core study data where possible. Participants should not attend for in-person visits 
if they are in their period of self-isolation/quarantine – the exception to this is the COVID-19 Pathway. 

If blood sample collection is involved, then the trained study staff in complete PPE as per 
recommendation by WHO will visit the home. 
7.16     Participants with Confirmed SARS-CoV-2 Infection (COVID-19 Pathway) 
Participants will be counselled at enrolment that if they receive a positive SARS-CoV-2 test (e.g., an 
antigen detection or nucleic acid amplification test, for example, via test and trace or occupational 
health services) they should contact the trial team on receipt of the positive result. Participants will be 
reminded of this with a phone call which will commence after the first vaccine dose.  
Once the participant has conveyed his/her result to the study team, and the study team confirm an 
appropriate test has been used (verbal discussion with participant as to how testing was obtained, 
confirmatory documentation will not be sought), a physician tele-clinic appointment will be arranged 
within 7 days of reporting by the study staff to assess the participants’ clinical severity at the relevant 
study site and a sample collection at home will be arranged. At this visit nasal swab for storage and 
subsequent viral isolation and whole genome sequencing (WGS) and targeted sequencing for VOCs 
will be collected and a blood sample for safety assessment (if clinically indicated). Vital signs (if 
possible) and other clinical data will be recorded. The window period for performing this visit is 
mentioned in Table 6. 
Table 6. COVID-19 Pathway for the enrolled participants during the study period. 
Variables 
Margin 
Reporting of participants to Study staff about COVID-19 
status 
As soon as possible after the 
COVID-test report 
Physician Teleclinic 
Within 7 days of reporting to study 
staff 
Nasal Swab for COVID-19 PCR, WGS and targeted 
sequencing and/or blood sampling (if clinically indicated) 
Within 7 days of Teleclinic 
Participants will be screened for severity of disease during their visit and referred to the nearby tertiary 
care hospital/government facility if there will be any shortness of breath or chest tightness. 
Participants will be counselled to follow national/local guidelines/SOPs for isolation/infection 
prevention measures. 
 
 
7.16.1     Whole genome and targeted genome sequencing for regional VOCs 
As RNA viruses are known to mutate at higher rates compare to DNA viruses, SARS-CoV-2 has also 
been mutated significantly over time compared to the first reported Wuhan strain and has resulted in 
the generation of different variants. Some of these variants because of higher transmissibility or 
infection rate and escape from neutralizing antibodies could become dominant variants and are known 

as VOCs. Genomic surveillance played a pivotal role in identification of these variants to understand 
the evolution and genomic diversity of SARS-CoV-2 and is helping us to design better diagnostic 
tests and vaccines against the virus. Therefore, in the proposed study we are aiming to perform 
targeted whole genome sequencing (WGS) of SARS-CoV-2 isolates using MinION platform (Oxford 
Nanopore Technologies) at AKUH for the identification of VOCs of SARS-CoV-2 for all 
breakthrough infections during this trial.  
7.17      Sample Handling 
7.17.1 Blood sampling, transportation, and storage 
For subsequent visits, appointment will be taken through telephone at least 24-48 hrs prior and the 
team will visit the participant at home to collect the blood sample. A trained staff will collect blood 
sample according to requirement of study cohort using aseptic technique and will transfer these 
samples to respective sites in temperature-controlled environment. Blood samples will be processed, 
transport and stored according to the SOPs of respective laboratories for required tests.  
Safety monitoring tests for AEs will be performed at clinical lab AKUH Karachi and AKUH stat 
laboratories Lahore and Islamabad. PBMCs from whole blood will be separated at AKUH Karachi 
for ELISpot and ICS. ELISpot will be performed at AKUH with the assistance of our collaborator, 
Oxford University who have fully validated protocol to perform ELISpot. ICS will be performed at 
Oxford university. NIH Laboratory Islamabad will also separate PBMCs for ELISpot and ICS. At all 
sites PBMCs will be stored in liquid nitrogen till further use. The Infectious Disease Research 
Laboratory (IDRL) at AKUH Karachi will archive and ship all samples to respective labs for 
particular assays.  
Humoral immunogenicity will be assessed for antibodies to SARS-CoV-2-Spike and nucleocapsid 
antigens by ELISA and pseudovirus neutralization assay. Samples will be processed for Anti-Spike 
IgG, Anti-Nucleocapsid IgG and Pseudo-neutralizing antibody responses at AKUH laboratory. 
AKUH laboratory is accredited by the College of American Pathologists (CAP). A subset will be sent 
to the CEPI laboratory network for analysis which will also be serve as External Quality Assessment 
(EQA). Ragon Institute, Harvard Medical School, Boston MA, USA, will provide training to 
researchers from AKUH, Pakistan to perform system serology assay to assess functional antibodies, 
using the Luminex based technology which will allow rapid profiling of the overall levels of antibody 
isotypes, subclasses, Fc-receptor binding antibodies (to 6 different Fc-receptors), complement binding 
antibodies, and Fc-glycan changes (using lectins) and  to profile the humoral immune response across 
the vaccine Spike antigen (RBD, NTD, S1, S2) as well as RBD and full Spike VOCs (alpha, beta, 
delta, gamma, etc). ELISA for Anti-spike IgG will also be performed at Nutrition Research 
Laboratory (NRL) AKUH. At AKUH blood samples will be processed in biosafety level three (BSL-

3) facility at Juma Research Laboratory as per COVID-19 biosafety guidelines. All time points, 
volume of blood collected at each time point and designated assays have mentioned in Appendix A 
and Appendix B of protocol.  
Subjects will be informed that there may be leftover samples of their blood (after all testing for this 
study is completed), and that such samples may be stored indefinitely for possible future research 
(exploratory immunology), including genotypic testing of genetic polymorphisms potentially relevant 
to vaccine immunogenicity. Subjects will be able to decide if they will permit such future use of any 
leftover samples. With the participants’ informed consent, any leftover cells and plasma will be frozen 
indefinitely for future analysis of COVID-19 and other coronaviruses related diseases or vaccine-
related responses. If the subjects elect not to permit this, all those participants’ leftover samples will 
be discarded at the end of the trial. 
7.17.2 Nasal swabs 
Nasal swabs will be collected from study participants with breakthrough infection and subjected to 
RT-PCR assay for the detection of SARS-CoV-2 viral RNA. Remaining RNA samples from PCR 
confirmed-cases will be stored at -80°C until subject to WGS.  WGS will be performed for the 
identification of VOCs of SARS-CoV-2 for all breakthrough infections during this trial.  
7.18 Early Discontinuation/Withdrawal of Participants  
In accordance with the principles of the current revision of the Declaration of Helsinki and any other 
applicable regulations, a participant has the right to withdraw from the study at any time and for any 
reason and is not obliged to give his or her reasons for doing so. The investigator may withdraw the 
participant at any time in the interests of the participants’ health and well-being. In addition, the 
participant may withdraw/be withdrawn for any of the following reasons: 
• Administrative decision by the Investigator 
• Ineligibility (either arising during the study or retrospectively, having been overlooked at 
screening). 
• Significant protocol deviation 
• Participant non-compliance with study requirements 
• An AE, which requires discontinuation of the study involvement or results in inability to 
continue to comply with study procedures 
The reason for withdrawal will be recorded in the CRF. If withdrawal is due to an AE, appropriate 
follow-up visits or medical care will be arranged, with the agreement of the volunteer, until the 
AE has resolved, stabilised or a non-trial related causality has been assigned. The IDMC chair 
may recommend withdrawal of participants. 

If a participant withdraws from the study, storage of samples will continue unless the participant 
specifically requests otherwise. Any data collected before their withdrawal will still be used in the 
analysis for trial integrity; if the participant requests this could be de-identified following the end of 
the study. 
7.19    Contra-Indications to Receipt of Second/Booster Dose of Vaccine 
The following AEs associated with any vaccine, identified on or before the day of vaccination constitu 
te absolute contraindications to further administration of a study vaccine to the participant in question. 
If any of these events occur during the study, the subject will not be eligible to receive a second or 
booster dose and will be followed up by the clinical team or their physician until resolution or 
stabilisation of the event: 
• Anaphylactic reaction following administration of vaccine 
• Any vaccine-related SAE, AESI, particularly any thrombotic event 
• Any AE that in the opinion of the Investigator may affect the safety of the participant or the 
interpretation of the study results 
Participants who develop COVID-19 symptoms and have a positive SARS-CoV-2 antigen test/ PCR 
after the first vaccination can only receive a booster dose after a minimum 4-week interval from their 
first positive test, provided their symptoms have significantly improved. The decision to proceed with 
booster vaccinations in those cases will be at clinical discretion of the investigators. For participants 
who are asymptomatic and have a positive SARS-CoV-2 test, a minimum of 2 weeks from first test 
positivity will be required before boosting provided they remain asymptomatic. Married women of 
childbearing age who report to be pregnant before the administration of the booster dose will not be 
offered the heterologous vaccine booster. They will be referred to an obstetrician for pregnancy 
follow-up and to the nearest government vaccination team for the administration of the homologous 
vaccine dose. The data of these women will be excluded from the final analysis. However, we do not 
expect losing more than a couple of women who might get pregnant after the first dose (despite of 
counselling and clear assurance from their side to avoid pregnancy at the time of enrolment).   
7.20      Definition of End of Trial 
The en d of the trial is the last participant last visit. 

7.21    Trial Intervention 
Below are the details of the types of the vaccines included in the study and their dosing regimens.  
Table 7: Details of Trial Intervention 
Vaccine 
Type 
Vector 
Vial 
Volume 
of  
Per 
Dose 
(ml) 
Site of 
administrati
on 
No. of  
Doses 
Time 
Interval 
Route 
Storage 
Temp. 
Recommended  
Age group 
(years) 
Efficacy 
References 
AstraZeneca-
University of 
Oxford (UK) 
Viral 
Vector 
Chimpanzee Adenovirus 
encoding the SARS-CoV-
2 Spike glycoprotein 
(ChAdOx1-S) ChAdOx1-
S (recombinant) 
Multidose 
contains 5 
ml 
0.5 ml 
Deltoid 
muscle   
(upper arm) 
10 
4-12 weeks 
apart 
IM 
2-8°C 
> 18 
Single dose: 76% 
Two doses (4 weeks apart): 
54.9% 
Two doses (12 weeks 
apart): 82.4% 
(34, 35) 
CanSinoBio 
(China) 
Viral 
Vector 
Ad5-nCoV 
Prefilled 
syringe 
0.5 ml 
Deltoid 
muscle   
(upper arm) 
1 
NA 
IM 
2-8°C 
> 18 
Single dose: 65.7% 
(moderate disease), 91% 
(severe disease) 
(36) 
Sinopharm/BIBP 
(China) 
Whole 
Inactivated 
Virus 
NA 
Prefilled 
syringe 
0.5 ml 
Deltoid 
muscle   
(upper arm) 
1 
3 weeks 
apart 
IM 
2-8°C 
>18 
Two doses: 79-86% 
(37) 

 
 
7.22     Storage of Study Vaccines 
Vaccines will be stored in accordance with manufacturers’ recommendations at the NIH, Islamabad, 
Pakistan and shipped in batches to the study sites in Karachi, and Lahore. In Lahore, the vaccines will 
be stored at Chughtai lab facility according to manufacturer’s recommended temperatures and doses 
for daily usage will be received from the cold storage facility in Coleman. Similarly, At AKUH 
vaccines will be stored at clinical trial unit (CTU) and will be dispatched to respective sites. All 
movements of the study vaccines will be documented in accordance with existing standard operating 
procedure (SOP). Vaccine accountability, storage, shipment, and handling will be in accordance with 
relevant SOPs and forms.  
7.23      Compliance with Trial Treatment 
All vaccinations will be administered by the research team and recorded in the CRF. The study 
vaccine will be at no time in the possession of the participant There will be reminders and facilitation 
for clinic visit for vaccination to maximize compliance. 
7.23.1   Accountability of the trial treatment 
The vaccine supplies will be supervised at all times by study teams. Vaccine counts will occur to 
ensure that no vaccines are missing. Dispensation and return of study vaccines will be recorded in the 
Study Vaccine Accountability Log according to the standards. 
7.23.2   Concomitant medication 
Concomitant medications taken at enrolment will be recorded, as will new medications have taken 
within the 28 days after each immunisation. Subsequently only new medications taken in response to 
a medically attended adverse event up until 3 months post second and booster dose will be recorded. 
7.23.3   Post-trial treatment 
If any heterologous boost regimen is not found to be immunogenic, decisions regarding the need for 
a booster dose, the nature of the booster dose and mode of delivery (e.g., national program vs. study 
site) will be made in consultation with the IDMC and study management group.  
7.23.4   Other treatments  
Participants will be advised that they may take paracetamol prophylactically after vaccine 
administration.  This will be from the participants own supplies rather than supplied by the study 
team. 
7.23.5   Other interventions 
There are no additional investigations other than those specified in this protocol.  
7.24     Sample Size 
The participants will be divided into two cohorts one for detailed immunological assessments 
(immunology cohort) and one for main immunology endpoints (General cohort). We choose the 

 
 
sample size of 160 participants per heterologous group and 240 per homologous group (total N=1680) 
who will be boosted at D70, including 25 participants per group in the immunology cohort and 135 
per heterologous and 215 per homologous group of the general cohort.  
The below sample size calculation is based on the primary endpoints of anti-spike IgG and 
neutralizing antibody responses to live SARS-CoV-2. The current available data from COMCOV at 
University of Oxford suggest the standard deviation (SD) of anti-spike IgG (Nexelis) at 4 weeks post 
boost is around 0.3-0.4 at log10 scale among participants received different vaccine schedules (4 
weeks first boost interval), and the SD of neutralizing antibody response to live SARS-CoV-2 
(normalised MNA50) is 0.4 at log10 scale among 194 participants in COV001 and COV002 (prime-
boost interval of 4-12 weeks).  
The assumptions for sample size calculation are:   
1. The non-inferiority margin for geometric mean ratio (GMR) is 0.63 between heterologous and 
homologous boost groups.  
2. The standard deviation is 0.4 on log10 scale.  
3. The true GMR is 1.  
Based on the above assumptions, the study will need to recruit 100 participants who are seronegative 
at baseline in each group to achieve 90% of power to detect the non-inferiority of immunogenicity of 
heterologous group compared to homologous group at one-sided 1.25% significance level to preserve 
a family-wise type 1 error rate of one-sided 2.5% since there will be two formal comparisons within 
each of the group administered with 3 different vaccines.  After accounting for ~25% seropositivity 
rate at baseline and ~15% attrition rate, the study will need to recruit 160 participants per group. This 
means that total sample size will be N=160 x 9 = 1440. In order to be able to assess the immune 
response of any potential heterologous booster (3rd dose) introduced among the homologous groups 
in future, we will enrol more than the expected number of participants in the homologous group 
(instead of 160 per homologous group, 240 will be enrolled), thereby increasing the required SS to 
N=1680.  With the expanded size in the homologous arms, the power for the primary endpoint 
analyses will be >90%. 
The immunogenicity cohort will used for exploratory analyses to generate hypothesis, and thus no 
formal sample size calculation was carried out for this cohort. The sample size of 25 per group was 
therefore chosen based on practical constraints. This means we will have around 20 seronegative 
participants in each group for the analysis. 
7.25     Plan of Statistical Analysis 

 
 
The primary analysis of this study will be a non-inferiority comparison between heterologous and 
homologous boost schedules, i.e., Groups 1/2 vs. 7, 3/4 vs. 8, and 5/6 vs. 9 as defined in Table 1. All 
the primary analyses will be done among the seronegative population at baseline. For the primary 
endpoint analyses, the GMC of anti-spike IgG and neutralizing antibody responses will be compared 
between different heterologous boost groups and their corresponding homologous group at Day 98 (4 
weeks post booster dose).  We will claim heterologous boost group is non-inferior to homologous 
boost group if the lower limit of two-sided 97.5% CI of GMR between heterologous boost group and 
their corresponding homologous group lies above 0.63. The sensitivity analysis will be done to 
estimate the adjusted GMR with two-sided 97.5% CI after controlling for the randomisation design 
factor of study site. The GMC will be transferred using logarithmic transformations (base 10) to render 
a normal distribution. Trend in the levels of GMC over the period in each group will be plotted and 
comparison between heterologous groups and their corresponding homologous group at several 
timepoints will be performed. The primary analysis will be carried out when the primary endpoints of 
D98 anti-spike IgG and/or neutralizing antibody response data become available. The two primary 
endpoints analyses will be considered separately. The success of one endpoint will not be contingent 
on the success of the other. Therefore, either or both the primary endpoints could be successful for 
the study. No multiplicity adjustment will be made for the two primary endpoints as they are likely to 
be highly correlated. The formal test of non-inferiority for two different heterologous groups 
compared to homologous group will be conducted with significance level of one-sided 1.25% using 
Bonferroni approach. There will no adjustment for multiple testing for secondary analyses and a two-
sided 0.05 will be considered statistically significant. All the other secondary immunogenicity 
endpoints analysis in terms of further immunogenicity characteristics or different timepoints will be 
descriptively summarized.  
The analysis population for the primary endpoints will be on the per-protocol basis as the intent-to-
treat analysis is no longer a conservative estimate for non-inferiority comparisons. Sensitivity 
analyses for primary endpoints on the modified intent-to-treat basis will be also conducted.  
For safety analysis, we will descriptively summarize the number of event and number of participants 
and proportion of participants who experienced SAE, AESI, solicited and unsolicited AE, medically 
attended adverse reactions by vaccine group. The safety analysis population will be the participants 
who received at least one dose of the study vaccines.   
An interim analysis will be carried out when the serology data at 4 weeks post second dose become 
available for the first 300 participants at baseline. If the seropositivity rate is higher than 25%, we will 
re-evaluate the study power and increase the sample size if needed.  

 
 
Further interim analyses for primary and secondary endpoints will be carried out when the data 
becomes available to support policy making.  
A fully detailed statistical analysis plan will be prepared and will be signed off by the PI prior to 
database lock for conducting any data analyses.   
8 
DATA MANAGEMENT 
The PI will be responsible for all data that accrues from the study. 
8.1 
Missing Data  
The level and pattern of the missing data in the baseline variables and outcomes will be reported. The 
potential causes of any missing data will be investigated and documented as far as possible. Any 
missing data will be dealt with using methods appropriate to the conjectured missing mechanism and 
level of missing.   
8.2 
Access to data & data protection 
Direct access will be granted to authorised representatives from the funder, host institution and the 
regulatory authorities to permit trial-related monitoring, audits, and inspections. The study protocol, 
documentation, data, and all other information generated will be held in strict confidence. No 
information concerning the study, or the data will be released to any unauthorised third party, without 
prior written approval of the funder. 
8.3 
Data collection and recording 
 All study data including participant diary will be recorded directly into an electronic CRF using 
electronic data capture (EDC) system (e.g., REDCap or any other validated EDC system) or onto a 
paper source document for later entry into EDC if direct entry is not available.  This includes 
sociodemographic, safety, and laboratory and outcome data.  All documents will be stored safely and 
securely in confidential conditions. The eCRF data will be stored at data management unit server 
located at AKUH, Pakistan.  
The participants will be identified by a unique trial specific number and code in any database. The 
name and any other identifying detail will NOT be included in any trial data electronic file, except for 
the electronic data records for AEs, for which consent will be obtained to store the participant’s name 
and phone number for quality control purposes. The diaries (with identifiers) will only be 
visible/accessible to the investigator(s) and relevant study staff (not funder) and is to serve the purpose 
of being able to query data from the subject if needed. The sponsor has well defined SOPs which 
describe how only deidentified data will be shared with the funder.  
The data backups will be maintained according to the set standards of the data management unit of 
the Department of Paediatrics and Child Health, AKUH, Karachi. Access to the study's database will 
be restricted to members of the study team by username and password. 

 
 
8.4 
Record keeping 
The Investigators will maintain appropriate medical and research records for this trial, in compliance 
with GCP and regulatory and institutional requirements for the protection of confidentiality of 
volunteers. The PI and Co-Is will have access to records. The Investigators will permit authorised 
representatives of the funder and Host institution, as well as ethical and regulatory agencies to 
examine (and when required by applicable law, to copy) clinical records for the purposes of quality 
assurance reviews, audits and evaluation of the study safety and progress. 
Identifiable information such as contact details will be stored for a minimum of 5 years from the end 
of the study. This includes storage of consent forms. Storage of these data will be reviewed every 5 
years and files will be confidentially destroyed if storage is no longer required. Considerations at the 
time of this review will include the value of retaining this information for participant safety (e.g., to 
inform participants of unexpected safety signals emerging from post-licensing surveillance), as a 
resource for the participants (e.g., if they wish to check which vaccines they have received in the 
study) and any regulatory requirements. De-identified research data maybe be stored indefinitely. If 
volunteers consent to be contacted for future research, a record of this consent will be recorded, 
retained, and stored securely and separately from the research data. If volunteers consent to have their 
samples stored and used in future research, information about their consent form will be retained and 
stored securely as per Biobanking procedures and SOP. 
8.5 
Source data and case report forms (CRFs) 
All protocol-required information will be collected in CRFs designed by the Investigator. All source 
documents will be filed in the participant file. Source documents are original documents, data, and 
records from which the participant CRF data are obtained. For this study, these will include, but are 
not limited to, volunteer consent form, blood results, laboratory records, diaries, medical records. In 
most cases, CRF entries will be considered source data as the CRF is the site of the original recording 
(i.e., there is no other written or electronic record of data). In this study this will include, but is not 
limited to medical history, medication records, vital signs, physical examination records, adverse 
event data and details of vaccinations. All source data and participant files will be stored securely.   
8.6 
Data quality 
Data collection tools will undergo appropriate validation to ensure that data are collected accurately 
and completely. Datasets provided for analysis will be subject to quality control processes to ensure 
analysed data is a true reflection of the source data. Trial data will be managed in compliance with 
local data management SOPs. If additional, study specific processes are required; an approved Data 
Management Plan will be implemented. 
8.7 
Quality Assurance Procedures 

 
 
8.7.1 Risk assessment  
The trial will be conducted in accordance with the current approved protocol, GCP, relevant 
regulations and standard operating procedures. A risk assessment and monitoring plan will be 
prepared before the study opens and will be reviewed as necessary over the course of the trial to reflect 
significant changes to the protocol or outcomes of monitoring activities. 
8.7.2 Monitoring  
Monitoring will be performed according to Good Clinical Practice (GCP) guidelines by an external 
monitor Precision Health Consultants (PHC) Global. PHC Global, registered in Pakistan since 2012 
is an organization lead by public health experts and professionals with extensive experience and in 
depth understanding of research conduct and public policy formulation based on internationally 
accepted frameworks in health and education. PHC Global functions under a board of directors 
following robust standards of financial, and institutional policies. In addition, PHC Global’s in house 
team comprises of social scientists, anthropologists, economists, and biostatisticians with their 
branches available in Karachi, Lahore, and Rawalpindi in Pakistan as well as the USA, Canada, and 
Australia and support multi-disciplinary projects. In this project PHC global will monitor and verify 
that the clinical trial is conducted as per the SOPs, and data are generated, documented, and reported 
in compliance with the protocol, GCP and the applicable regulatory requirements. The investigator 
sites will provide direct access to all trial related source data/documents and reports for the purpose 
of monitoring and auditing by the funder or the Host institution and inspection by local and regulatory 
authorities. In the presence of travel restrictions having a CRO from within the country is in the best 
interest of the study. 
9 TRIAL COMMITTEES 
9.1 
Joint steering committee 
A Joint Steering Committee (JSC) will be formed to oversee the study and advise the Study 
Management Committee on key issues of study conduct, including, but not limited to, study pauses 
due to safety concerns on the advice of the independent data monitoring committee (IDMC). The JSC 
will be comprised of independent (external) technical experts with relevant research experience from 
CEPI & consortium members (investigators) and nominated focal person from CEPI. The study 
management committee will be comprised of only study investigators and research managers. The 
JSC will be referred for deliberation and expert guidance related to the technical issues regarding the 
study protocol, amendments, design etc. They will also be informed regularly about the progress of 
the study and challenges being faced. The study management committee is internal body which will 
meet frequently (every week in the initial 2 months, followed by fortnightly meetings & need based) 
to discuss the study progress, enrolment, challenges, and issues. They will take decisions regarding 

 
 
day-to-day issues however, high level decision related to the change in study protocol will be referred 
to the JSC. 
9.2 
Independent data monitoring committee (IDMC)  
An independent data monitoring committee (IDMC) will be convened. All collaborators/consortium 
members will be non-voting observers along with a SPEAC Meta-DSMB expert (also non-voting 
observer). The IDMC will evaluate frequency of adverse events, safety data as specified in the IDMC 
charter (to be developed later). The IDMC will make recommendations concerning the conduct, 
continuation, or modification of the study for safety reasons to the JSC. The IDMC will review SAEs 
or AESIs deemed possibly, probably, or definitively related to study interventions. The IDMC will be 
notified within 24 hours of the Investigators’ being aware of their occurrence. The IDMC can 
recommend placing the study on hold if deemed necessary following a study intervention-related 
SAE.IDMC will conduct the review of safety data after the initial 50 - 60 participants have received 
a second dose at the day 70±7 days post first time-point. Further, the IDMC will meet quarterly to 
evaluate data for any safety signals, SAEs and any other issues.  
10      PROTOCOL DEVIATIONS  
A trial related deviation is a departure from the ethically approved trial protocol or other trial 
document or process (e.g., consent process or IMP administration) or from Good Clinical Practice 
(GCP) or any applicable regulatory requirements. Necessary trainings will be provided to the study 
staff and logs will be maintained to avoid deviations however, in case of any deviation from the 
protocol, it will be documented in a protocol deviation form and filed in the trial master file. Some of 
the major deviations are defined below: 
• Schedule of events: for each event or visit, there is a specified timepoint and a window period 
during which the visit should be performed. Any deviation from that window will be 
considered as protocol deviation. 
• Study vaccines: for each group the first, second and booster dose of the vaccine is pre-specified 
as per protocol. Once the participants are randomized to the assigned group, the vaccine as per 
protocol in the given group should be administered. If the given vaccine is not administered 
or first and second vaccines (in heterologous arms) are exchanged, this will be considered as 
protocol deviation.  
• missing any exclusion criteria during screening and finding after enrolment. 
• Randomization error 
• Missing any sample 
 
 

 
 
11       ETHICAL AND REGULATORY CONSIDERATIONS 
The Investigator will ensure that this trial is conducted in accordance with the principles of the 
Declaration of Helsinki and in accordance with Good Clinical Practice. Ethical approval will be 
obtained from the national bioethics committee of Pakistan (NBC), AKUH Ethical Review 
Committee, IRB approvals from IVI and Oxford University and approval from Drug Regulatory 
Authority of Pakistan (DRAP). The submissions to the respective IRBs/ECs and regulatory authorities 
will be made in parallel and we expect 6-8 weeks of time for receiving the complete set of approvals.  
12       TRIAL REGISTRATION  
Prior to the recruitment of the first participant, the trial will have been registered on a publicly 
accessible database and the trial information will be kept up to date during the trial, and the PI or their 
delegate will upload results to the trial registry within 12 months of the completion of the trial follow-
ups.  
13        EXPENSES AND BENEFITS 
Volunteers will be compensated with 2000 Pakistani rupees per visit for their time and travel 
expenses. If the patient suffered from any adverse event causally related to the given vaccine, the cost 
of treatment including laboratory investigations will be provided from the study.  
 
14      EXPERIENCE IN CONDUCTING VACCINE RESEARCH (38-46) 
The AKUH has well-developed infrastructure for conducting clinical trials. A Clinical Trials Unit 
supports drug/vaccination importation and management, GCP training and compliance. The study 
team have recently successfully conducted complex polio vaccine trials including; 1) Poliovirus type 
2 challenge study, a community based randomized controlled trial, 2) Assessment of intradermal 
adaptors in administration of fractional dose IPV among children, 3) Polio endgame strategies: Polio 
Virus Type 2 Challenge study in Pakistan: five groups randomized control trial, 4) Intradermal 
Administration of Fractional Dose of Inactivated Poliovirus Vaccine Using Intradermal Adaptors vs. 
BCG syringe: Community Based Randomized Control Trial in Pakistan, 5) Intradermal 
Administration of Fractional Dose of Inactivated Poliovirus Vaccine (fIPV) Using Tropis® 
Intradermal Needle Free Injection System, Karachi Pakistan, 6) Comparison of IPV and fractional 
dose IPV in routine immunization: A community based Randomized Control Trial in Pakistan, 7) Pilot 
Surveillance of poliovirus excretion among persons with primary immune deficiency in Pakistan & 
Assessment of Sensitivity of Jeffery Modell Foundation Signs for screening of Suspected PID 
Children. In addition, the team has also successfully completed impact evaluation of pneumococcal 
conjugate vaccine against invasive pneumococcal disease (IPD) in Sindh, impact evaluation of typhoid 
conjugate vaccine in an outbreak setting of XDR typhoid in Hyderabad and Lyari town Karachi, and 

 
 
a five-year longitudinal cohort study evaluating sero-efficacy of typhoid conjugate vaccine among 
children in Hyderabad is currently in progress.  
15       POTENTIAL CHALLENGES: 
• 
Study delays/timelines may be affected in the event of lockdowns due to COVID-19. 
• 
Vaccine hesitancy in general population will be a challenge. 
• 
Regulatory approvals from DRAP (Drug Regulatory Authority of Pakistan) may affect the 
study timelines. 
• 
Unexpected disruptions to the supply of the Project Vaccines, recalls due to safety issues or 
withdrawals of the Project Vaccines can be a risk for the trial 

 
 
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17       APPENDICES   
17.1    Appendix A: Blood Sampling 
General Cohort 
 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
Additional visit only for 
homologous arms 
V8 
V9 
V10 
COVID-19 
Pathway 
Timeli
ne 
D0 
D14 
D28 
D70 
D98 
D168 
D196 
D238 
D266 
D336 
D420 
D672 
D0-D672 
W0 
W2 
(2wks 
post first 
dose) 
W4 
W10 
W14 
(4wks post 
2nd dose) 
W24 
(6months 
post first 
dose) 
W28 
 
W34 
W38 
(4wks post 
booster dose) 
W48 
(1 yr. 
post first 
dose) 
W60 
 
W96 
(2 yrs. 
post first 
dose) 
W0-W96 
Safety 
Tests 
AKUH/NIH/LHR 
Biochemistry 
Hematology 
- 
AKUH/NIH/
LHR 
Biochemistry 
Hematology 
- 
AKUH/NIH/
LHR 
Biochemistry 
Hematology 
 
 
 
- 
 
 
- 
AKUH/NIH/
LHR 
Biochemistry 
Hematology 
- 
- 
- 
AKUH/NIH/LHR 
Biochemistry 
Hematology 
Safety 
Blood 
1xGel top (~5ml) 
1xEDTA (~3ml) 
- 
1xGel top 
(~4ml) 
1xEDTA 
(~3ml) 
- 
1xGel top 
(~5ml) 
1xEDTA 
(~3ml) 
 
 
- 
- 
1xGel top 
(~5ml) 
1xEDTA 
(~3ml) 
- 
- 
- 
1xGel top (~5ml) 
1xEDTA (~3ml) 
Prima
ry 
Endpo
int 
(PE) 
- 
- 
- 
- 
AKUH/ 
CEPI 
Anti-spike 
IgG 
Neut. Abs 
 
 
 
 
 
 
 
 
Secon
dary 
Endpo
int 
(SE) 
AKUH/ CEPI 
Anti-spike IgG 
Neut. Abs 
Anti-
Nucleocapsid IgG 
 
 
 
 
AKUH/ 
CEPI 
Anti-spike 
IgG 
Anti-
Nucleocapsid 
IgG 
AKUH 
Anti-
spike 
IgG 
 
AKUH 
      CEPI 
Anti-
Nucleocapsid 
IgG 
AKUH 
Anti-spike 
IgG 
Nucleocap
sid IgG 
AKUH 
Anti-spike 
IgG 
 
AKUH 
Anti-
spike 
IgG 
 
AKUH 
Anti-spike 
IgG 
Nucleocapsi
d IgG 
Neut. Abs 
AKUH 
Anti-
spike 
IgG 
Nucleoca
psid IgG 
AKU
H 
Anti-
spike 
IgG 
Anti-
Nucleo
capsid 
IgG  
 
AKUH 
Anti-spike 
IgG 
Anti-
Nucleocap
sid IgG 
AKUH/ CEPI 
Anti-spike IgG 
Neut. Abs 
Anti-
Nucleocapsid IgG 
Blood 
for PE 
+ SE 
2xEDTA 
(~3ml each) 
 
2xEDTA 
(~3ml each) 
2xEDTA 
(~3ml 
each) 
2xEDTA 
(~3ml each) 
2xEDTA 
(~3ml each) 
2xEDTA 
(~3ml 
each) 
2xEDTA 
(~3ml 
each) 
2xEDTA 
(~3ml each) 
2xEDTA 
(~3ml 
each) 
2xEDT
A 
(~3ml 
each) 
2xEDTA 
(~3ml 
each) 
2xEDTA 
(~3ml each) 

 
 
Total 
Blood/
visit 
~14ml 
(1xGel top ~5ml) 
(3xEDTA ~3ml 
each) 
~3ml 
(1xEDT
A) 
 
~13ml 
(1xGel top 
~4ml) 
(3xEDTA 
~3ml each) 
6ml 
(2xEDT
A ~3ml 
each)) 
~14ml 
(1xGel top 
~5ml) 
(3xEDTA 
~3ml each) 
6ml 
(2xEDTA 
~3ml each) 
 
~6ml 
(2xEDTA 
~3ml 
each) 
6ml 
(2xEDT
A ~3ml 
each)) 
~14ml 
(1xGel top 
~5ml) 
(3xEDTA 
~3ml each) 
~6ml 
(2xEDT
A ~3ml 
each) 
 
 
~6ml 
(2xED
TA 
~3ml 
each) 
~6ml 
(2xEDTA 
~3ml 
each) 
~14ml 
(1xGel top ~5ml) 
(3xEDTA ~3ml 
each) 

 
 
Immunology Cohort 
 
V1 
V2 
V3 
V4 
V5 
V6 
V7 
V8 
Additional visit only 
for homologous arms 
V9 
V10 
V11 
COVID-19 
Pathway 
 Timeline  
D0 
D14 
D28 
D70 
D84 
D98 
D168 
D196 
D238 
D266 
D336 
D420 
D672 
D0-D672 
W0 
W2 
(2wks 
post 
first 
dose) 
W4 
W10 
W12 
(2wks 
post 
boost) 
W14  
(4wks 
post 2nd 
dose) 
W24  
(6months 
post first 
dose) 
W28 
 
W34 
W38 
(4wks 
post 
booster 
dose) 
W48  
(1 yr. post 
first dose) 
W60  
 
W96 
(2 yrs. post 
first dose) 
Safety Tests 
AKUH/NI
H/LHR 
Biochemis
try 
Hematolo
gy 
- 
AKUH/N
IH/LHR 
Biochemi
stry 
Hematolo
gy 
- 
- 
AKUH/NI
H/LHR 
Biochemis
try 
Hematolo
gy 
- 
- 
- 
- 
- 
- 
- 
AKUH/NIH/L
HR 
Biochemistry 
Hematology 
Safety 
Blood 
1xGel top 
(~5ml) 
1xEDTA 
(~3ml) 
- 
1xGel top 
(~5ml) 
1xEDT
A 
(~3ml) 
- 
- 
1xGel top 
(~5ml) 
1xEDTA 
(~3ml) 
- 
- 
- 
1xGel top 
(~5ml) 
1xEDTA 
(~3ml) 
- 
- 
- 
1xGel top 
(~5ml) 
1xEDTA 
(~3ml) 
Primary 
Endpoint 
(PE) 
 
 
 
 
 
AKUH/ 
CEPI 
Anti-spike 
IgG 
Neut. Abs 
 
 
 
 
 
 
 
 
Secondary 
Endpoint 
(SE) 
AKUH/ 
CEPI 
Anti-spike 
IgG 
Neut. Abs 
Anti-
Nucleocap
sid IgG  
Oxford/ 
AKUH 
ELISpot 
ICS 
 
Oxford/ 
AKUH 
ELISpot 
ICS 
AKUH/ 
CEPI 
Anti-
spike IgG 
Nucleoca
psid IgG  
Neut. Abs 
Oxford/ 
AKUH 
ELISpott 
ICS 
AKUH 
Anti-
spike IgG 
Oxford 
U/ 
AKUH 
ELISpot 
ICS 
 
 
 
 
Oxford/ 
AKUH 
ELISpot 
ICS 
AKUH/ 
CEPI 
Anti-
Nucleocap
sid IgG 
Oxford/ 
AKUH 
ELISpot 
ICS 
AKUH 
Anti-spike 
IgG 
Neut. Abs 
Anti-
Nucleocap
sid IgG  
Oxford/ 
AKUH 
ELISpot 
ICS 
AKU
H 
Anti-
spike 
IgG 
Neut. 
Abs 
AKUH 
Anti-spike 
IgG 
Oxford/ 
AKUH 
ELISpot 
ICS 
AKUH 
Anti-spike 
IgG Anti-
Nucleocap
sid IgG 
Neut. Abs 
Oxford/ 
AKUH 
ELISpot 
ICS 
AKUH 
Anti-spike 
IgG 
Neut. Abs 
Anti-
Nucleocap
sid IgG  
Oxford/ 
AKUH 
ELISpot 
ICS 
AKUH 
Anti-
spike 
IgG 
Neut. 
Abs 
Anti-
Nucleoca
psid IgG  
 
AKUH 
Anti-spike 
IgG 
Neut. Abs 
Anti-
Nucleocapsi
d IgG  
Oxford/ 
AKUH 
ELISpot 
ICS 
AKUH/ 
CEPI 
Anti-spike 
IgG 
Neut. Abs 
Anti-
Nucleocapsid 
IgG  
Oxford/ 
AKUH 
ELISpot 
ICS 

 
 
Explorator
y Endpoint 
(EE) 
Harvard/ 
AKUH 
Systems 
Serology 
Harvard
/ AKUH 
Systems 
Serolog
y 
Harvard/ 
AKUH 
Systems 
Serology 
Harvard/ 
AKUH 
Systems 
Serology) 
Harvard/ 
AKUH 
Systems 
Serology 
Harvard/ 
AKUH 
Systems 
Serology 
Harvard/ 
AKUH 
Systems 
Serology 
 
Harvard/ 
AKUH 
Systems 
Serology 
Harvard/ 
AKUH 
Systems 
Serology 
Harvard/ 
AKUH 
Systems 
Serology 
 
Harvard/ 
AKUH 
Systems 
Serology 
Harvard/ 
AKUH 
Systems 
Serology 
Blood for 
 PE 
+SE+EE 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDT
A 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
1xEDTA 
(~3ml) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDT
A 
(~3ml 
each) 
 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDTA 
(~3ml 
each) 
2x Na 
Heparin 
(~10ml 
each) 
1xGel 
top 
(~3ml) 
2xEDTA 
(~3ml 
each) 
 
2xEDTA 
(~3ml each) 
2x Na 
Heparin 
(~10ml 
each) 
2xEDTA 
(~3ml each) 
2x Na 
Heparin 
(~10ml each) 
Total 
Blood/visit 
~34ml 
(1xGel top 
~5ml) 
(3xEDTA 
~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each) 
~26ml 
(2xEDT
A ~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each) 
~34ml 
(1xGel 
top ~5ml) 
(3xEDTA 
~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each) 
~26ml 
(2xEDTA 
~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each 
~23ml 
1xEDTA 
(~3ml) 
2x Na 
Heparin 
(~10ml 
each) 
~34ml 
(1xGel top 
~5ml) 
(3xEDTA 
~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each) 
~26ml 
(2xEDTA 
~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each) 
~6ml 
(2xEDT
A) 
 
~26ml 
(2xEDTA 
~3ml each) 
(2x Na 
Heparin 
~10ml each 
~34ml 
(1xGel top 
~5ml) 
(3xEDTA 
~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each) 
~26ml 
(2xEDTA 
~3ml 
each) 
(2x Na 
Heparin 
~10ml 
each) 
~9ml 
(1xGel 
top 
~3ml) 
(2xEDT
A ~3ml 
each) 
 
~26ml 
(2xEDTA 
~3ml each) 
(2x Na 
Heparin 
~10ml each) 
~34ml 
(1xGel top 
~5ml) 
(3xEDTA 
~3ml each) 
(2x Na 
Heparin 
~10ml each) 
 
 
 
 
 
 
 
 
 

 
 
17.2     Appendix B: Flow of Sample Processing 
 
BoC: Both cohorts   GC: General cohort    IC: Immunology cohort   CL: clinical lab PNA: Pseudo neutralization assay    S: Anti-spike IgG   N: 
Anti-nucleocapsid IgG    SS: System serology