Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a Cohort of HIV Positive Girls and Women
Conditions: Cervical Cancer, Human Papillomavirus Infection, Human Immunodeficiency Virus, Cervical Dysplasia, Genital Warts
Sponsor: University of British Columbia

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
 
RESEARCH PROTOCOL 
 
 
 
 
 
 
 
TITLE 
Long Term Follow-up Study of CTN 236 –  
 
 
A Study of an HPV VLP Vaccine in a  
 
 
Cohort of HIV Positive Girls and Women 
 
Abbreviated Title 
Long Term Follow-up of  
 
 
HPV Vaccine in HIV (CTN 236) 
 
Protocol Version Date  
13 October  2015 
 
Funding Agency 
Canadian Institutes of Health Research (CIHR) 
 
Study Principal Investigator 
Deborah Money, MD 
 
 
Women’s Health Research Institute (WHRI) 
 
 
B327-4500 Oak Street 
 
 
Vancouver, BC V6H 3N1 
 
 
Tel: (604) 875-3459 
 
 
Fax: (604) 875-3895 
 
Study Coordinating Centre 
Women’s Health Research Institute 
 
 
Project Manager: Nancy Lipsky 
 
 
Tel: (604) 875-2424, ext 4877 
 
Data Management Centre 
CIHR Canadian HIV Trials Network (CTN) 
 
 
Data Manager: Hector Leon 
 
 
Tel: (604) 806-8761 
 
Supported by 
CIHR Canadian HIV Trials Network (CTN) 
 
 
Women’s Health Research Institute 
 
 
 SAE Fax 
(604) 875-3895

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 1 of 44 
 
 
Clinical Study Sites and Investigators 
 
 
Study Site 
Local Investigators 
 
British 
Columbia 
Oak Tree Clinic - Children and Women’s 
Health Centre of BC, Vancouver 
 
Deborah Money Ariane Alimenti/ 
Jan Christilaw/ Julie van 
Schalkwyk/ Neora Pick/ 
Gina Ogilvie  
 
St. Paul’s Infectious Disease Clinic, Vancouver  
 
Marianne Harris/ Sarah Stone 
Ontario 
Toronto General Hospital, Toronto 
 
Maple Leaf Medical Clinic, Toronto 
 
St. Michaels Hospital, Toronto 
 
Hospital for Sick Children, Toronto 
 
McMaster University Hospital, Hamilton 
 
Kingston General, Kingston 
 
Children’s Hospital of Eastern Ontario, Ottawa 
 
HIV Care Program, Windsor 
Sharon Walmsley 
 
Mona Loutfy 
 
Mark Yudin 
 
Ari Bitnun 
 
Fiona Smaill/ Sandi Seigel 
 
Wendy Wobeser 
 
Lindy Samson 
 
Jeff Cohen 
 
 
Quebec 
McGill University Health Centre, Montreal 
 
Montreal Children’s Hospital, Montreal 
 
CHU Sainte Justine, Montréal 
 
Centre Hospitalier de l’Université Laval, 
Québec City 
 
Marina Klein 
 
Christos Karatzios 
 
Normand Lapointe 
 
Sylvie Trottier 
 
 
 
 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 2 of 44 
 
Additional Investigators/Collaborators 
 
Gynaecological Oncology 
Andy Coldman 
 
 
BC Cancer Agency, Vancouver BC 
Marette Lee 
 
 
 
Dept of Obstetrics and Gynaecolology, UBC, Vancouver BC 
Dianne Miller  
 
 
Dept of Obstetrics and Gynaecolology, UBC, Vancouver BC 
Dirk van Niekerk 
 
 
BC Cancer Agency, Vancouver BC 
Gavin Stuart 
 
 
 
Dept of Obstetrics and Gynaecolology, UBC, Vancouver BC 
 
Vaccine Evaluation 
Simon Dobson 
 
 
Vaccine Evaluation Centre, Vancouver BC 
David Scheifele 
 
 
Vaccine Evaluation Centre, Vancouver BC 
 
Laboratory Expertise 
Francois Coutlée 
 
 
Dept of Oncology, McGill University, Montreal PQ 
Janet Hill 
 
 
 
Dept of Veterinary Microbiology, U of SK, Saskatoon SK 
Mel Krajden 
 
 
 
BC Centre for Disease Control, Vancouver BC 
 
Statistical Analysis, Study Design and Population Health 
Janet Raboud  
 
 
University Health Network Research Institutes, Toronto ON 
Joel Singer 
 
 
 
Clinical Trials Network 
 
Community Partners 
Marcie Summers 
 
 
Positive Women’s Network, Vancouver BC 
 
International Consultant 
Catherine Hankins 
 
 
AIGHD, Amsterdam, the Netherlands 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 3 of 44 
 
TABLE OF CONTENTS 
SUMMARY ........................................................................................................................................................... 5 
LIST OF ABBREVIATIONS ............................................................................................................................ 7 
1.0 Background ..................................................................................................................................................... 8 
1.1 
Epidemiology and Burden of Disease ...................................................................................... 8 
1.2 
Clearance and persistence of HPV ........................................................................................... 9 
1.3 
Natural HPV Immunology ........................................................................................................ 9 
1.4 
HPV/ HIV Interaction ............................................................................................................10 
1.5 
HPV Prevalence is high in HIV-positive cohort studies .....................................................10 
1.6 
HPV, Other STI’s and the Vaginal Microbiome ..................................................................11 
1.7 
HPV Vaccines ............................................................................................................................12 
1.8 
HPV Vaccine Implementation ................................................................................................12 
1.9 
Global HPV Vaccine Implementation Plans ........................................................................13 
1.10 HPV Vaccine in HIV Positive Studies ...................................................................................13 
2.0 HYPOTHESIS .............................................................................................................................................15 
3.0 OBJECTIVES ..............................................................................................................................................15 
3.1 
Primary Objective ......................................................................................................................15 
3.2 
Secondary Objective .................................................................................................................15 
3.3 
Exploratory Objective ..............................................................................................................15 
4.0 STUDY DESIGN & METHODS ...........................................................................................................15 
4.1 
Design .........................................................................................................................................15 
4.2 
Rationale .....................................................................................................................................15 
5.0 STUDY COHORT......................................................................................................................................16 
5.1 
Population ..................................................................................................................................16 
5.2 
Delays and Contraindications to Subsequent Study Procedures ........................................17 
5.2.1 
Indications for a Delay in Genital Sampling ............................................................17 
6.0 STUDY CONDUCT ..................................................................................................................................17 
6.1 
Adherence to Industry Guideline and Regulations ..............................................................17 
6.2 
Ethical Conduct / Research Ethics Board (REB) ................................................................17 
6.3 
Informed Consent/ Assent ......................................................................................................18 
6.4 
Source Documentation .............................................................................................................19 
6.5 
Monitoring ..................................................................................................................................19 
7.0 STUDY PROTOCOL ................................................................................................................................20 
7.1 
Participant identification number (PID#) assignment ........................................................20 
7.1.1 
Table of Site Numbers ................................................................................................20 
7.2 
Considerations for Special Groups .........................................................................................20 
7.2.1 
Females aged 11-18 who are pre-menarchal and not sexually active ...................20 
7.2.2 
Changes to Study Procedures in Pregnancy ............................................................21 
7.3 
Summary Table of Study Visits ...............................................................................................21 
7.4 
Study Visits .................................................................................................................................22 
7.4.1 
Ordering of Study Procedures ...................................................................................22 
7.4.2 
Study Visit Windows ...................................................................................................22 
7.4.3 
Outline of Study Visits ................................................................................................22 
7.5 
Study Procedures in Detail .......................................................................................................25 
7.5.1 
Informed Consent/Assent .........................................................................................25 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 4 of 44 
7.5.2 
Medical History ............................................................................................................25 
7.5.3 
Height and Weight .......................................................................................................26 
7.5.4 
Clinical laboratory investigations ...............................................................................26 
7.5.5 
Study Blood Work .......................................................................................................26 
7.5.6 
Pelvic examinations .....................................................................................................26 
7.5.7 
Study Genital Samples ................................................................................................26 
7.5.7.1 Cervical Cytology and HPV DNA .............................................................26 
7.5.7.2 Vaginal Samples for Microbiota Analyses ................................................27 
7.5.7.3 Concomitant Medications and Exposures ................................................27 
7.5.7.4 Lower Urinary Tract Symptoms………………………..…..……….27  
8.0 
MANAGEMENT OF CERVICAL CYTOLOGIC ABNORMALITIES .....................................28 
9.0 
SERIOUS ADVERSE EVENTS ..........................................................................................................29 
10.0 PREGNANCY .........................................................................................................................................29 
11.0 SUBJECT COMPLETION AND WITHDRAWAL ........................................................................30 
12.0 LABORATORY METHODS AND ANALYSES .............................................................................30 
12.1 Blood Test ..................................................................................................................................30 
12.1.1 HPV Serotesting ..........................................................................................................30 
12.1.2  Merck Laboratory Serotesting Methods ...................................................................31 
12.1.3  HSV2 Serology Testing ...............................................................................................31 
13.0 GENITAL SAMPLING ..........................................................................................................................31 
13.1 HPV DNA Testing - Coutlée Laboratory .............................................................................31 
13.2 Cervical Cytology - Cervical Cancer Screening Laboratory of BC.....................................32 
13.3 Vaginal Microbiota Analyses - Hill Lab, University of Saskatchewan ...............................32 
14.0 TRANSPORTATION OF LABORATORY SAMPLES ...................................................................32 
15.0 DATA SAFETY MONITORING BOARD/COMMITTEE ...........................................................33 
16.0 STATISTICAL ANALYSIS .....................................................................................................................33 
16.1 Statistical Analysis Plan .............................................................................................................33 
16.2 Primary Analysis ........................................................................................................................33 
16.3 Exploratory Analyses ................................................................................................................34 
17.0 DATA MANAGEMENT ........................................................................................................................35 
18.0 ADMINISTRATIVE MATTERS ...........................................................................................................36 
18.1 Protocol amendment procedure .............................................................................................36 
18.2 Records Retention .....................................................................................................................36 
18.3 Clinical Study Report ................................................................................................................36 
18.4 Publication Plan .........................................................................................................................36 
19.0 REFERENCE LIST ..................................................................................................................................37 
 
 
 
 
 
 
 
 
 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 5 of 44 
SUMMARY 
 
Title 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine 
in a Cohort of HIV Positive Girls and Women 
 
Goal 
We hypothesize that HPV antibody levels in HIV-positive girls and women 
will decline more rapidly and more significantly than in HIV-negative girls and 
women and that this decline will be determined by HIV parameters.  
 
Study population 
HIV-positive girls and women (greater than, or equal to, age 11) attending the 
HIV treatment clinic that are currently enrolled in CTN 236 study will be 
invited to participate in the 5 year long term follow-up study. 
 
Objectives 
 
Primary 
To measure the antibody response to each genotype contained in the qHPV 
vaccine out to 96 months post-vaccination regimen.  
 
Secondary 
 
 
 
 
 
 
 
 
Exploratory 
1) To determine the incidence rate and nature of ‘breakthrough’ HPV 
incident and persistent (2 sequential positive HPV DNA in > 6 months) 
infections of vaccine- non-vaccine-containing high-risk types;  
2) To determine the incidence rate of cervical dysplasia (LSIL or greater) 
and/or vulvar and vaginal dysplasia associated HPV genotypes (both 
with and without vaccine- types); and 
3) To determine the incidence rate of external genital warts.  
 
 
1) To examine the relationship between HSV-2 serostatus and peak HPV 
antibody response as well as HPV incident and persistent infections; 
and 
2) To relate vaginal microbiome profiles to HPV acquisition/persistence 
and cervical dysplasia. 
 
Timeline 
First Visit, First Subject (FVFS): 10 June 2015 
Last Visit, Last Subject (LVLS): 30 March 2019 
 
Study design 
A longitudinal cohort follow up study of girls and women aged 11 years of age 
and older who participated in the study, “A Study of an HPV VLP Vaccine in 
a Cohort of HIV Positive Girls and Women (CTN 236)” 
 
Inclusion Criteria 
 Enrolled in CTN 236 study 
 Able to give fully informed consent or assent 
 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 6 of 44 
 
Exclusion Criteria 
 Did not receive at least one vaccination via CTN 236, phase 1 
 Cannot provide fully informed consent or assent 
 
Duration of study 
for each subject 
 
Up to 60 months 
Subjects will participate in up to 3 possible study visits over a total of 5 years. 
Duration of time since vaccination as part of CTN 236 varies depending on 
when they were enrolled; therefore, to ensure all subjects are offered 
participation in the long term follow up, 6 possible visit times have been 
created. 
Visit schedule 
Main Study Procedures (no standard clinical or sub-study procedures) 
 
 
 
Informed 
Consent/ 
Assent 
 
Health 
Questionnaire  
 
Study Blood 
Work 
 
Cytology and  HPV 
DNA & vaginal 
swabs* 
 
SAE and Con Med Assessment  
Month 36 
(Visit 8) 
X 
X 
X 
X 
X 
Month 48 
(Visit 9) 
X 
X 
X 
X 
X 
Month 60 
(Visit 10) 
X 
X 
X 
X 
X 
Month 72 
(Visit 11) 
X 
X 
X 
X 
X 
Month 84 
(Visit 12) 
X 
X 
X 
X 
X 
Month 96 
(Visit 13) 
X 
X 
X 
X 
X 
* Those girls and women who are pre-menarchal or never sexually active will not have any pelvic 
examinations, clinical Pap smears, or study genital sampling until post-menarchal and sexually active. 
Complete outline of all procedures found in table 7.3 
 
Data collection 
 
Template recruitment documents, consent and source documents will be 
provided by the Study Coordinating Centre. Source data will be transferred to 
paper case report forms and sent to the CIHR Canadian HIV Trials Network 
(CTN) where it will be entered into an electronic database. 
 
Monitoring 
This study is being conducted according to the following: International 
Committee on Harmonization (ICH) Guidance Document E6: Good Clinical 
Practice (GCP): Consolidated Guidelines. Site monitoring will be performed to 
ensure compliance with the above. 
 
Data Safety 
Monitoring Board 
(DSMB) 
The established CTN data safety monitoring committee with additional 
expertise in pediatrics and gyne-oncology will provide oversight and 
monitoring of the conduct of the study to ensure the safety of the subjects. 
Additionally, the DSMB will ensure the validity and integrity of the study data. 
 
 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 7 of 44 
LIST OF ABBREVIATIONS 
 
AE  
 
Adverse event 
AIDS  
Acquired immunodeficiency syndrome 
ART  
Antiretroviral therapy 
ARV  
Antiretroviral 
BCCDC 
British Columbia Centre for Disease Control 
CPARG 
Canadian Pediatrics AIDS Research Group 
CIN 
 
Cervical intraepithelial neoplasia 
CIS 
 
Carcinoma in situ 
CRF  
Case report form 
DAIDS 
Division of AIDS Table for Grading the Severity of Adult and Pediatric Adverse Events 
DNA  
Deoxyribonucleic acid 
DSMB  
Data Safety Monitoring Board 
CTN  
CIHR Canadian HIV Trials Network 
GCP  
Good clinical practices 
HAART 
Highly active antiretroviral therapy 
hCG  
Human Chorionic Gonadotropin 
HIV 
 
Human Immunodeficiency Virus 
HPV  
Human Papillomavirus 
ICH 
 
International Conference on Harmonization 
IM  
 
Intramuscular 
NIH  
National Institutes of Health 
Pap  
 
Papanicolaou cervical smear 
PCR  
Polymerase chain reaction 
PID#  
Primary identification number 
PRN  
When necessary 
Q-HPV 
Quadrivalent Human Papillomavirus Vaccine 
REB  
Research ethics board 
RNA  
Ribonucleic acid 
SAE 
 
Serious adverse event 
SOP 
 
Standard operating procedure 
STI 
 
Sexually transmitted infection 
STM  
Specimen transport media 
VLP 
 
Virus-like particles 
WHRI  
Women’s Health Research Institute 
 
 
 
 
 
 
 
 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 8 of 44 
1.0 Background 
1. 
Epidemiology and Burden of  Disease 
Worldwide, cervical cancer is the fourth leading cause of cancer-related death in 
women with 528,000 new cases and 266,000 deaths yearly.1
 
Cervical cancer is the single greatest cause of death from cancer in women in sub-
Saharan Africa, where HIV prevalence is exceptionally high particularly among 
young women.1,2,3 In countries with wide access to quality Pap smear screening, the 
burden of disease has shifted to pre-invasive dysplastic lesions. In Canada, for 
example, there is at least 50 times more pre-invasive disease in women than there is 
cervical cancer, which still represents a substantial economic, social and 
psychological burden to women.4,5 In spite of the pap screening program, there are 
over 350 deaths per year in Canada due to invasive cervical cancer (approximately 1 
death every day)6, often as a result of late diagnosis due to inadequate Pap smear 
screening, especially in vulnerable populations including immigrant and Aboriginal 
women.7 This underlines a clear need to develop and evaluate preventative options. 
 
The link between HPV and cervical cancer is well-established. HPV DNA is 
detected in 96.6% of cervical cancer tissue and the vast majority of cervical cancers 
can be attributed to HPV infection.8 Based on both epidemiologic and phylogenetic 
data, the high-risk HPV types, for cervical cancer, in frequency order, are: 16, 18, 33, 
45, 31, 58, 52 and 35, and probably high risk are: 51, 56, 39 and 59.9 Overall, 70% 
of cervical cancer cases are caused by the two most common HPV types; 16 and 18 
(high risk), and 90% of genital warts are caused by HPV 6 and 11 (low risk).10  
 
The burden of disease in HPV infection outside the cervix is also important. HPV 
has been implicated in most non-cervical genital tract cancers, including penile and 
anal cancer, and in head and neck cancers. Cervical cancer incidence in a Canadian 
population is seen at 199.54 for 100,000 person/years (PY) in HIV positive women, 
compared to 47.5 per 100,000 PY in HIV negative women.11 Fortunately, anal cancer 
has not been frequent in women, even women with HIV infection, with rates of 
15.96 per 100,000 PY.11 Research on the efficacy of this vaccine in preventing these 
cancers is also required.12 As we did not conduct evaluations of baseline anal and oral 
HPV we are not in a position to conduct research into the efficacy of the HPV 
vaccine in this cohort to these types. 
 
The affliction of disease from genital warts is also substantial. Data from the US 
suggests that genital warts are the most common sexually transmitted infection.13 
HPV 6 and 11 cause up to 90% of genital warts. In women, genital warts may 
develop anywhere in the lower genital tract including the cervix, with multiple sites 
being common. Clinically visible warts are present in approximately 1% of sexually 
active adults.14 In persons with normal immune function, visible warts and evidence 
of viral replication are typically gone by approximately 18 months after initial 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 9 of 44 
infection.15 However, in HIV-positive individuals the lesions can be significantly 
larger and very problematic to treat, highlighting the benefits of HPV primary 
prevention in this population.16,17 Of note, vaccination programs in HIV negative 
healthy populations are already showing decrease in genital warts,18 but no data is 
available on this response in HIV positive vaccinated persons.  
 
2. 
Clearance and persistence of HPV 
The role of the host immune system in regression of HPV infection is not fully 
understood but in regressing genital warts an infiltration can be detected of CD4 
cells, CD8 cells and macrophages that likely represents a T-cell response to specific 
early proteins of HPV (E2 and E6).19 In HIV-negative women, most of these 
infections (80%) appear to ‘clear’; that is, HPV DNA can no longer be detected on 
the mucosal or epithelial surface. The time required for this ‘clearance’ appears to 
vary with different HPV genotypes; ranging from 5-6 months for low risk types to 8-
14 months for high-risk types.20,21,22 It is unclear whether the virus is completely 
eliminated, or if it remains latent in basal cells and could reactivate under immune 
permissive conditions. Risk of carcinogenesis appears to be directly related to the 
persistence of oncogenic (high risk) virus. The definition of transient or persistent 
infection has been defined in most studies as the presence of HPV DNA by 
polymerase chain reaction (PCR) at two consecutive assessments 6 months apart.23  
 
3. 
Natural HPV Immunology 
Many nuances of HPV immunology are poorly understood, in particular the ability 
of the virus to evade the immune system. Natural immunity is generally not effective 
in protecting against re-infection with the same genotype.24 Our current 
understanding is that the host’s immune response to natural infection initially 
involves a local innate response, which then triggers circulating humoral (antibody) 
and cell mediated responses (e.g. CD4 and CD8 cells). The cell-mediated immune 
response is required for HPV containment and lesion regression.13,25 Persons with 
cell-mediated immune dysfunction, such as HIV-positive individuals, have higher 
rates of HPV infection. HIV-positive women have much higher rates of cervical 
dysplasia and cancer than HIV-negative women and progress more rapidly 
from low grade to high grade lesions and to invasive cancer.26,27 Of note, our 
recent study of cervical cancer in HIV-positive women in British Columbia has 
determined that HIV-positive women have 4.2 fold (2.72-6.20) higher rate of HPV 
associated female genital cancer than the general population.11 The baseline rate of 
low-grade cervical dysplasia is approximately 13% in HIV-positive women compared 
to 4% in HIV-negative women.27,28 The problem is exacerbated in HIV endemic 
areas such as sub-Saharan Africa where Pap smear screening programs are largely 
lacking.29  

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 10 of 44 
 
4. 
HPV/ HIV interactions 
The T cell immune dysfunction caused by HIV infection offers a unique opportunity 
to study and better understand the interrelationship between the neutralizing 
antibody protection of the humoral immune system triggered by the vaccine, and 
HPV elimination that requires adequate T cell function. The most valuable 
knowledge gained in this area will be a definition of an immune correlate of 
protection, i.e. a level of circulating antibody above which the majority of 
persons will be protected from acquisition of the infection. This new 
knowledge will be highly relevant to vaccine programming in Canada and 
globally. 
 
The interactions between HIV and HPV are not fully understood. HIV-mediated 
immune suppression appears to facilitate HPV persistence and its oncogenic 
potential. In part this appears to be due to direct enhancement of HPV integration in 
the presence of HIV.30 The effect of HPV on HIV replication and viral load in the 
circulation and in the genital tract is not well characterized. Of note, HPV studies 
have typically involved interval evaluations of HPV shedding which has led to a poor 
understanding of the longitudinal process of infection, persistence, clearance, and 
reinfection or reactivation.23 In some studies to date, combination antiretroviral 
therapy (cART), presumably through immune function reconstitution, results in 
higher rates of clearance compared to those not on cART.31,32,33 However, in other 
studies, cART and CD4 counts have not been well correlated with clearance and in 
fact HIV suppression and inversely HIV replication (higher viral load) has been 
associated independently.34 Some of the difficulty has been the prior study designs. 
Data from our study group prior to the availability of HPV vaccine showed that 
there was a differential response to immune suppression and infection with HPV 16 
in that perhaps HPV 16 is such a potent oncogenic genotype that it can cause disease 
regardless of immune function.24,31 
 
5. 
HPV prevalence is high in HIV-positive cohort studies 
Our studies and others have shown that HPV is highly prevalent in HIV positive 
populations. Even in a post HAART era, clearance is still diminished compared with 
HIV negative populations and this underscores the need to better understand the 
role of HIV replication and HPV infection and the HPV immune response. Data 
from the Canadian Women’s HIV study group found high risk HPV genotypes, as 
detected by genital tract sampling in HIV-infected women, in the following order of 
frequency: 52, 84, 16 and 83.35 In this cohort (n=198, samples=437), 62.1% of 
women were HPV positive at least once for one or more of the 27 HPV types tested, 
with 7.6% positive for 6/11, 15.7% positive for 16/18, and 1.0% positive for 6/11 & 
16/18. In an international study, 77% of HIV-positive women had HPV detected at 
least once (incident infection). HPV 16 was the most frequent genotype, with mixed 
infection seen in 26%.36  Persistent infection was seen in 41.5% of women but there 
was no relationship between HPV infection status and CD4 cell count or HIV viral 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 11 of 44 
load. This compares to a 2.6% HPV 16 persistence seen in the HIV-negative women 
in the placebo arm of the large HPV 16/18 trial.37 Furthermore, an improved 
response to treatment for cervical dysplasia was observed for women on highly 
active antiretroviral therapy (HAART).38 Similar data were seen in other cohorts 
including a South African study demonstrating rapid increase in HPV infection after 
HIV seroconversion39 and in a US cohort with relatively high HPV prevalence and 
higher rates of cervical dysplasia in women with lower CD4 cell counts.40 Some 
studies have shown a similar 16, 18 genotype dominance in HIV-positive women 
compared to HIV-negative women in causing cervical dysplasia and cancer.41,42,43,44 
However in other studies multiple type infections and non 16/18 oncogenic HPV 
were associated with persistence, cervical dysplasia and cancer, highlighting the 
importance of cross-protection, and/or multivalent vaccines in HIV positive 
populations.45,46,47 
 
6. 
HPV, other STI’s and the vaginal microbiome 
Elucidating the role of HSV-2 in contributing to HPV persistent infection and 
oncogenic progression is important because HSV-2 co-infection is common, with an 
estimated seroprevalence of 55% in Canadian HIV-infected populations.48 Safe and 
inexpensive antiviral drugs can be used both for direct treatment but also for 
treatment as prevention by suppressing HSV viral shedding in an infected person 
and substantially decreasing risk of transmission to an uninfected person.49,50 Existing 
data on the role of HSV-2 in cervical dysplasia are unclear.  While several studies 
have observed an association between HSV-2 co-infection (defined either 
serologically or by the presence of HSV-2 DNA in cervical samples) and cervical 
cancer, this relationship has sometimes51,52,53,54 but not always55,56 persisted after 
adjustment for HPV DNA.  However, most studies were limited by their use of 
HSV-2 serology assays with poor specificity (e.g. based on whole virus or crude 
lysate57), and to our knowledge there are no prior studies in the setting of HIV 
infection. The rich clinical and microbiologic data within our HPV vaccine in HIV 
positive girls and women study provides a unique opportunity to evaluate the role of 
herpes simplex virus type 2 (HSV-2) as a cofactor in progression to cervical dysplasia 
among HIV-infected women.  
 
There has been increasing evidence of the interrelationship between the composition 
of the endogenous lower genital tract microbiota and the risk of acquisition and/or 
transmission of genital pathogens, including HIV and HPV.58,59,60,61,62 These findings 
suggest there are relationships between microorganisms in a given niche, and provide 
evidence to support the hypothesis that a healthy vaginal microbiome may be 
protective against pathogens. Dysbiosis of the vaginal microbiome is associated with 
increased susceptibility to human papillomavirus (HPV) infection, and may also 
influence progression of HPV to cervical cancer.63,64 Although we understand that 
HPV infection is a prerequisite for the development of cancer of the uterine cervix, 
we are also beginning to understand the ability of other infections or perturbations 
of the vaginal ecology to affect acquisition of HPV, persistence of HPV, and the 
ability of this virus to cause cervical dysplasia and cancer. The complexity of this 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
Page 12 of 44 
interplay is intricate and only understood in a very limited fashion. A microbial 
ecology approach is required to fully understand these phenomena. Our investigator 
team, which includes members funded through the CIHR Canadian Microbiome 
Initiative to study the vaginal microbiome utilizing a metagenomic approach, is well 
equipped to explore these issues. This collective expertise will permit the profiling of 
the bacteriome associated with oncogenic HPV persistence and/or integration into 
the host genome, and will enable future personal profiling of cervical cancer risk 
based on microbiome profiles.  
 
7. 
HPV Vaccines 
The two currently licensed vaccines designed to prevent HPV infection, have been 
tested in large clinical trials. There is a bivalent vaccine against HPV 16 and 18 
(GlaxoSmithKline, GSK) and a quadrivalent vaccine against HPV 6, 11, 16 and 18 
(Merck). In addition, a 9-valent vaccine is currently being investigated in large scale 
clinical trials, with positive safety, immunogenicity and efficacy data under review by 
the FDA. These vaccines are produced as virus-like particles (VLP’s) based on the LI 
capsid protein gene of the virus inserted in insect or yeast vectors. These proteins 
self-assemble into protein shells with identical configuration to the native virus, but 
without DNA, and so are deemed subunit, protein vaccines. Safety, efficacy, and 
immunogenicity data on these vaccines in HIV-negative populations of young 
women are based on clinical trials conducted by GSK and Merck, respectively, with 
some follow up post-licensure trials now in progress. Both have shown remarkable 
safety profiles in otherwise healthy young HIV-negative women, with limited local 
injection site reactions as the primary side-effect and no evidence for differences in 
serious adverse events between vaccine and placebo arms.65  
 
Immunogenicity to the quadrivalent vaccine has been well studied in HIV-negative 
populations, showing that anti-HPV 6, 11, 16 and 18 geometric mean titers (GMT) 
were highest at month 7 (1 month following the final dose 3). Anti-HPV GMT’s 
declined thereafter but reached a plateau at month 24 and remained stable through 5 
years.66,67,68 In the original immunogenicity study by Villa et al.,67 100% of the subjects 
who were antibody negative to the relevant HPV type at baseline had a detectable 
antibody response at month 7 to HPV 6, 11, 16 and 18 respectively. These levels 
were 10-to 104-fold higher than natural antibody levels in placebo recipients who 
were vaccine type positive at baseline.  
 
8. 
HPV vaccine implementation 
Currently, Canada has established a population level HPV vaccination strategy 
through school-based publicly-funded programming. As yet, the impact of this is 
limited by uptake and by the relatively young age of the public based program. 
Individuals who are not eligible in the public program and who have the means are 
able to pay for the vaccine, but vulnerable populations who cannot afford the 
vaccine lack access and continue to acquire HPV infection unnecessarily. It is vital to 
understand how best to protect our most vulnerable persons from HPV infection 

Research Protocol 
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and the risk of invasive cervical cancer. The implications of the vaccine programme 
for Pap smear screening is yet to be fully understood and therefore current screening 
schedules have not been adjusted. It is hoped that the vaccine will be particularly 
useful in preventing disease in women who have difficulty accessing Pap smear 
screening, e.g. Aboriginal women and Asian immigrant women who have shown low 
access to and uptake of Pap smear screening.69,70,71,72,73 
 
9. 
Global HPV vaccine implementation plans 
Globally 266,000 women die every year, and without significant changes, this is 
estimated to be 416,000 by 2035. In resource-limited areas such as Africa, with little 
or no access to Pap smear screening, cervical cancer not only kills women, but its 
incidence is greatly exacerbated by the high prevalence of HIV infection. Cervical 
cancer incidence in South Africa is 174.8 per 100,000. In India, it is even higher at 
186.5 per 100,000.74 Safe and effective HPV vaccines would be of immense value in 
countries with such high rates of cervical cancer, especially in the absence of widely 
implemented screening programs. The World Health Organization (WHO) has 
demonstrated the capability of implementing large-scale vaccine programs.  For 
example in 2003, global coverage of three doses of the diphtheria-tetanus-pertussis 
combination vaccine reached 78%.75 Even complicated, comprehensive antiretroviral 
therapy rollout has been impressive with UNAIDS estimating that almost 10 million 
people in resource-constrained countries were on antiretroviral therapy at the end of 
2012.76 The Global Alliance for Vaccine and Immunisation (GAVI) is negotiating 
affordable three-tiered pricing for countrywide roll-out now with a number of low-
income countries. The rational implementation of HPV vaccination programs in 
these areas is dependent on a sound understanding of the impact, safety, and efficacy 
of vaccination in HIV-positive persons who may be at varying stages of immune 
dysfunction.77 As of February 4 2014, 10 additional countries were added as 
recipients of HPV vaccine rollout (to a total of 21 countries). This is to be primarily 
implemented using a 3 dose schedule but some pilot projects have opted for a 2 dose 
schedule, based on Canadian data from some of our investigator group. Although, 
we are confident in healthy Canadian HIV-negative girls, 2 doses of the vaccine 
provides equivalent antibody levels to 16-26 year old women who are well protected 
with these levels, this dosing regimen has not been tested in HIV positive 
populations. This greatly increases the urgency to determine the immunogenicity and 
efficacy for areas with high rates of HIV prevalence.  
 
10. 
HPV vaccine in HIV positive studies 
Our study group was one of the first groups globally to study one of these vaccines 
in HIV-positive, immune compromised populations. As the qHPV vaccine 
GARDASILTM (Merck), which contains subtypes 6 and 11 (responsible for 90% of 
genital warts) and subtypes 16 and 18 (responsible for 70% of cervical cancer) was 
the first to be licensed and available in Canada for post-licensure studies, we engaged 
with Merck to permit an initial study of the safety and immunogenicity of this 
vaccine in a cohort of HIV-positive girls and women. Data from this cohort has 

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Cohort of HIV Positive Girls and Women 
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provided valuable information about the health of Canadian women and girls living 
with HIV. At the initial screening visit more than 80% of subjects were negative to 
HPV genotypes 6, 11, 16 and 18, found in the qHPV vaccine. This was a higher 
seronegative rate than seen in a US based study in which only 55.6% were HPV 16 
negative and 73.7% HPV 18 negative at baseline, despite being a younger cohort 
overall (N=99, mean age 21.4 years).78 Preliminary data outlined that in our study, 17 
% of HIV-positive women had low-grade squamous intraepithelial lesions (LSIL) or 
greater at screening and 29% had at least one strain of oncogenic HPV present. It 
was observed that greater numbers of oncogenic HPV types were present in those 
women with cervical dysplasia.79  
Antibody response to HPV types 6, 11, 16 and 18 has been collected and analyzed 
on over 1500 serology samples for months 0, 7, 12, 18 and 24. Seroconversion rates 
at month 7 were approximately 93-98% in this HIV-positive population, much 
higher than anticipated.  However, peak Geometric Mean Titres (GMT) were lower 
than in HIV negative populations. In addition this was associated with a significant 
decline in antibody levels by month 24, particularly to HPV18. Similar to our 
findings, Kahn et al., also showed that level of antibody was lower in individuals not 
on cART in their open label 16-23 year old study of 99 women given the q HPV 
vaccine.80 Of note, we also observed statistically significantly higher levels of incident 
and persistent infections of vaccine containing types in a short term follow up 
period. This preliminary data outlines the importance of following this established 
cohort for long-term immunogenicity. When directly comparing the school based 
vaccine target age of 9-13 years, the HIV positive girls had a statistically significantly 
lower peak GMT than age matched HIV comparators.81,82 Similar studies conducted 
by other investigator groups have preliminary data available as well. Weinberg et al, 
showed that in HIV infected 7-12 year old children, there was a good seroconversion 
rate at > 94% but noted that the level of antibody to HPV18 was less than for the 
other genotypes. They also added a fourth dose of the vaccine which demonstrated 
an appropriate anamnestic response suggesting that should a booster be deemed 
necessary it should function to appropriately enhance the antibody response.83 A 
South African based study of the bivalent vaccine demonstrated safety and 100% 
seroconversion to the vaccine containing serotypes regardless of CD4, HIV viral 
load and HIV clinical stage of disease.84 
Our study will ensure careful assessment and scrutiny of the health outcomes in 
HIV-positive girls and women who were able to access the HPV quadrivalent 
vaccine (GARDASILTM) in our previous study. We will be able to determine the 
value of this vaccine for sub-groups of girls and women with CD4 T cell depletion 
and variable HAART uptake. Most studies published to date on HPV vaccines have 
been, by necessity, supported by the vaccine manufacturing companies, and have 
excluded HIV-infected subjects. This study remains one of the few investigator-
driven, peer-reviewed, independently funded vaccine studies, allowing for open 
scrutiny of vaccine efficacy and safety in a typically understudied population.  
  

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Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
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2.0 HYPOTHESIS  
It is hypothesized that HPV antibody levels in HIV-positive girls and women will decline more 
rapidly and more significantly than in HIV-negative girls and women and this decline will be 
determined by HIV parameters. 
 
3.0 OBJECTIVES 
The overall goal of the study is to determine the immunogenicity and efficacy of the HPV 
vaccine out to 4 years in an established cohort of HIV-positive girls and women in Canada. 
 
1. 
Primary 
To measure the antibody response to each genotype contained in the qHPV vaccine 
out to 96 months post vaccination regimen.  
 
2. 
Secondary 
1) To determine the incidence rate and nature of ‘breakthrough’ HPV incident 
and persistent (2 sequential positive HPV DNA in > 6 months) infections of 
vaccine- non-vaccine-containing high-risk types;  
2) To determine the incidence rate of cervical dysplasia (LSIL or greater) 
and/or vulvar and vaginal dysplasia associated HPV genotypes (both with 
and without vaccine- types); and 
3) To determine the incidence rate of external genital warts.  
 
3. 
Exploratory 
1) To examine the relationship between HSV-2 serostatus and peak HPV 
antibody response as well as HPV incident and persistent infections; 
2) To relate vaginal microbiome profiles to HPV acquisition/persistence and 
cervical dysplasia. 
 
4.0  STUDY DESIGN & METHODS 
1. 
Design 
This prospective longitudinal cohort study will evaluate the effectiveness of a 
quadrivalent HPV VLP vaccine in an HIV-positive cohort of girls and women in 
multiple centres across Canada. 
 
2. 
Rationale 
This study presents a unique opportunity to understand the long term impact of 
HPV vaccination in immune compromised women. Our prior study demonstrated 
that although women and girls living with HIV in Canada have complicated lives and 
have a high burden of socioeconomic disadvantages, they are very interested and 
committed to participating in research. This is clearly demonstrated by the degree of 
engagement of positive women in the National Consensus Statement on Women, 
Trans People and Girls and HIV Research in Canada. Our study sites across the 
country have been highly effective at engaging with this challenging population 

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Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
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through their long-standing, dedicated clinics. We have a cohort of over 400 girls and 
women who continue to live with their HIV disease and will require ongoing 
specialized HIV care for the rest of their lives. As they are just completing the first 
protocol out to 24 months and are for the most part still attending for care, with 
94% remaining engaged with our study groups. We are confident that we can 
effectively re-engage them in our follow-up study. It is vital that this study be 
launched now while most participants are at 24-36 months from vaccine course, and 
key follow up timed sampling can ascertain the trajectory of antibody responses as 
well as the course of HPV disease.  
 
Parallel studies in HIV-negative women, some being conducted by members of our 
investigator group, in Canadian cohorts, are ideal for comparison purposes. For 
example, there are over 1,000 girls enrolled in Quest, the long term follow up of the 
2 dose/3 dose study of the qHPV vaccine in healthy volunteers in Canada 
(Dobson/Ogilvie). This permits patient level data to be compared directly with 
inclusion of known confounders in the analysis. We have support from Merck (see 
appended letter of support) for the competitive Luminex immunoassay (cLIA) assay 
to ensure consistency and comparability to both our prior study antibody titres and 
to the published literature on qHPV antibody responses. To date, all qHPV vaccine 
studies in the published literature, including those conducted by our investigator 
group, have had the antibody assays conducted through Merck laboratories. This 
provides an extraordinary ability to conduct robust comparisons of immune 
responses across study populations who have been given the qHPV vaccine.   
 
5.0  STUDY COHORT 
1. 
Population 
Four hundred and seven (407) HIV-positive girls and women (greater than, or equal 
to, age 11) attending the HIV treatment clinics in each of the 13 sites across Canada 
and who have enrolled in the original study “A Study of an HPV VLP Vaccine in a 
Cohort of HIV Positive Girls and Women (CTN 236)” will be offered participation 
in the study. 
 
 
 
Inclusion Criteria 
 Enrolled in CTN 236 study 
 Able to give fully informed consent or assent 
 
Exclusion Criteria 
 Did not receive at least one vaccination via CTN 236, phase 1 
 Cannot provide fully informed consent or assent 
 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
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HPV Vaccine in HIV (CTN 236) 
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2. 
Delays and contraindications to subsequent study procedures 
5.2.1 Indications for a delay in genital sampling 
 Pre-menarchal and not sexually active – no genital sampling until 
post-menarchal and sexually active or at the discretion of the 
investigator 
 Subjects must not be menstruating at the time of a genital sampling. 
A study visit may need to be delayed until menses has stopped or the 
subject may have all other study procedures performed and the 
subject will have to be brought back to the clinic for genital sampling. 
 
6.0  STUDY CONDUCT 
1. 
Adherence to Industry Guideline and Regulations    
This trial is to be conducted in accordance with the protocol and ICH GCP. 
 
All study staff should be familiar with the following document: 
ICH GCP: http://www.ich.org/cache/compo/276-254-1.html  
 
All investigators and their investigative sites are obligated to comply with 
investigator responsibilities, monitoring, and archiving data, audits, confidentiality, 
and publication requirements. 
 
The Study Coordinating Centre shall also accept the responsibilities of the Sponsor 
as outlined in ICH GCP. 
 
2. 
Ethical conduct / research ethics board (REB) 
Each site will be required to utilize a REB that conforms to section 3 of the GCPs: 
3.1 Responsibilities, 3.2 Composition, Functions and Operations, 3.3 Procedures, 3.4 
Records.  
 
Prior to the conduct of this trial, the protocol, consent form, and all related 
documents must be submitted to the REB at each site. Approval for this study must 
be granted before the trial can be started at that site.  
 
The Principal Investigator at each site will forward a copy of the REB approval to 
the Study Coordinating Centre before the first subject is enrolled in the study at that 
site. All amendments to the protocol that affect the safety and/or welfare of the 
study participants shall be submitted to the site REB and approved prior to 
implementation. 
 
All study staff should be familiar with the Tri-Council Policy Statement on Ethical 
Conduct for Research Involving Humans: 
http://www.pre.ethics.gc.ca/english/policystatement/policystatement.cfm 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
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3. 
Informed Consent/ Assent  
Typically, each subject and parent/legally authorized representative (if applicable) 
must participate in the informed consent process and sign and date the informed 
consent form before any procedures specific to this protocol are performed on that 
subject.  
 
A modified recruitment method may be utilized if clinical care flow for a patient 
would be excessively interrupted by research. To ensure clinical history and pelvic 
examination occur in a timely manner, a HPV in HIV researcher may not always 
have the opportunity to speak with a potential study participant prior to a pelvic 
examination. In these circumstances prior to the routine pelvic exam, the attending 
clinician will request verbal permission to collect two additional vaginal swab samples 
and a cytology sample during the exam for the purposes of this study. Ability to 
verbally consent, in these instances, will better respect both clinician and patient time 
and reduce participation burden by allowing for study sample collection to align with 
clinical sample collection.   
 
After the exam, the HPV in HIV researcher will immediately follow up and meet 
with the patient to go over the full consent form, and answer any questions or 
concerns prior to their signing the informed consent document. If written informed 
consent is not provided, samples will be discarded. 
 
Consenting a posteriori will confer benefits to the participating women as their clinical 
care flow will not be interrupted by research. Also, by obtaining verbal consent at the 
initial pelvic exam to obtain samples, we will minimize the discomfort, stress and 
inconvenience experienced by women that could otherwise be subjected to a second 
pelvic exam.   
 
In the absence of a site SOP for informed consent, the site will write a note to file 
explaining the procedures followed while obtaining informed consent/assent. 
 
At each subsequent study visit, subjects and parent/legally authorized representative 
(if applicable) will be asked if they wish to continue in the study and give their 
ongoing consent/assent to participate. This will be documented in the source 
documentation. 
 
In accordance with the ICH Harmonized Tripartite Guidelines for GCP for those 
subjects who can be enrolled in the trial with the consent of the subject’s legally 
acceptable representative (e.g. minors), the subject should be informed about the trial 
to the extent compatible with the subject’s understanding and, if capable, the subject 
should sign and personally date a written informed assent. It is required that the 
assent be signed by each subject, if capable, in addition to the informed consent that 
is to be signed by her legal representative. 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
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Page 19 of 44 
A young subject who originally signed an assent form who becomes menarchal and 
sexually active will be required to sign a consent form that includes the study genital 
sampling prior to performing any of these procedures. 
 
Study consent forms and assent forms and above outlined use of verbal consent 
must be approved by a REB at the particular study site. 
 
An informed consent form template and assent form template are included in the 
protocol appendices and may be amended as per site and REB requirements. REB 
approved consent and assent forms for each site are to be sent to the Study 
Coordinating Centre and updated as necessary. 
 
4. 
Source Documentation 
Study source documentation should be kept for each subject at the study site. Source 
data is raw data such as original laboratory records, documentation, documentation 
of original observations and activities. Source data is the first time and place an 
observation is documented, for example the documentation of a blood pressure. 
 
Study source documentation should be captured separately in the subject study chart; 
however, when the original source data is such that it cannot be placed with the 
subject source document chart, for example, the source data is contained in the 
hospital patient chart, sites may take a photocopy of the document and indicate on 
the photocopy where the data was first documented.   
 
When source data is in electronic form, for example, laboratory results, a printout of 
the electronic data is to be printed and placed in the subjects source document chart. 
 
A source document template will be provided to sites by the study coordinating 
centre. However, sites are not required to use the template if other approved 
methods of capturing source data are preferred. Alternate methods of capturing 
source data must be approved by the Study Coordinating Centre. 
 
5. 
Monitoring 
A representative of the Study Coordinating Centre will visit investigative sites during 
the first months of enrollment to ensure that all procedures are consistent with the 
protocol and that appropriate Standard Operating Procedures (SOPs) and essential 
study documentation are on file.  
 
The sites will be monitored to ensure that the trial is being conducted in accordance 
with the protocol and ICH GCP. 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
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Consent documentation will be reviewed at 100%. Source documentation/CRF 
reconciliation will occur at 20% at each site. Monitoring at each site will be based on 
enrollment at that site. A close-out of the study will occur after the Last Patient Last 
Visit has been conducted at each site. 
 
Enrollment and study progress reports will be requested of the study sites at a 
frequency to be determined by enrollment rates. 
 
7.0 STUDY PROTOCOL 
1. 
Participant identification number (PID#) assignment  
 
For this long term follow-up study of CTN 236, subjects will not be assigned new 
study numbers and will remain with the study number assigned as part of CTN 236. 
 
7.1.1 Table of Site Numbers  
Site 
Number 
 
Study Site 
01 
05 
06 
07 
09 
10 
11 
12 
13 
14 
15 
17 
21 
Oak Tree Clinic - Children and Women’s Health Centre of BC, Vancouver 
Toronto General Hospital, Toronto 
Maple Leaf Medical Clinic, Toronto 
St. Michael’s Hospital, Toronto 
Hospital for Sick Children, Toronto 
McMaster University Hospital, Hamilton 
Kingston General, Kingston 
Children’s Hospital of Eastern Ontario, Ottawa 
McGill University Health Centre, Montreal 
CHU Sainte Justine, Montreal 
Centre Hospitalier de l’Université Laval, Québec City 
HIV Care Program, Windsor 
St Paul’s Hospital, Vancouver 
 
2. 
Considerations for special groups  
7.2.1 Females aged 11-18 who are pre-menarchal and not sexually 
active 
Adolescents and young women will be offered enrollment in the setting of 
established and experienced pediatric clinics. There are young girls with HIV 
infection, primarily perinatally acquired, who may be unaware of their 
diagnosis and the actual terminology of HIV. This is standard practice for 
care of children up to age 12 with a complex diagnosis with profound social 
stigmatization issues.  
 
Those that are aware of the HIV terminology will receive the information 
and consents with HIV specific information. The term 
“immunocompromised” has been used in place of HIV in documents and 

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templates that may be viewed by these young women (e.g. consent forms). 
Each site may use the standard terminology used at their site. 
 
Those girls and women who are pre-menarchal and not sexually active will 
not have the same degree of disease endpoint evaluations (i.e. will not have 
any pelvic examinations or Pap smears until post menarchal and sexually 
active or at the discretion of the site investigator).  
 
A young subject who originally signed an assent form who becomes 
menarchal and sexually active will be required to sign a consent form that 
includes the study genital sampling prior to performing any of these 
procedures. 
 
7.2.2 Changes to study procedures in pregnancy 
In the event of pregnancy Pap testing and genital study sampling will 
continue; however, an Ayre spatula will be used to collect cervical samples in 
place of the cytobroom until 3 months post-pregnancy.   
 
3. 
Summary table of study visits 
 
 
Visit Window 
Ongoing 
Consent/Assent 
 
Incl/Excl or 
Contraindication 
Criteria 
Clinical 
Laboratory 
Investigations 
Study Blood- 
Work (serology) 
Pelvic Exam& 
Vaginal Sample 
Collection * 
Study Cervical 
Cytology  and 
HPV  DNA * 
SAE/Med Hx 
and Con 
Med/Exposure 
Update  
 
Month 36 
(Visit 8) 
 
First Dose + 36 
months (+/- 2 
months) 
X 
X 
X 
X 
X 
X 
X 
 
Month 48 
(Visit 9) 
 
First Dose + 48 
months (+/- 2 
months) 
X 
X 
X 
X 
X 
X 
X  
 
Month 60 
(Visit 10) 
 
First Dose + 60 
months (+/- 2 
months) 
X 
X 
X 
X 
X 
X  
X 
 
Month 72 
(Visit 11) 
 
First Dose + 72 
months (+/- 2 
months) 
X 
X 
X 
X 
X 
X 
X 
 
Month 84 
(Visit 12) 
First Dose + 84 
months (+/- 2 
months) 
X 
X 
X 
X 
X 
X 
X 

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Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
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HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
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Month 96 
(Visit 13) 
 
First Dose + 96 
months (+/- 2 
months) 
X 
X 
X 
X 
X 
X 
X 
* Those girls and women who are pre-menarchal and not sexually active will not have any pelvic 
examinations or Pap smears until post-menarchal and sexually active or at discretion of the site 
investigator. 
Due to variation in duration of time since first dose of vaccine, some subjects are out of window for 
Month 36 and Month 48, e.g. it has been more than 48 months since first dose of vaccine was 
received. Therefore, we have extended the study visit timeline up to and including Month 96 post-
vaccine, also designated Visit 13. Visits will occur annually until project grant is completed (March 
31, 2019). 
 
 
4. 
Study Visits 
7.4.1 Ordering of Study Procedures 
At all visits - Study blood for serum antibody and cervical cytology/HPV 
DNA test and vaginal samples are to be done within a 24 hour period.  Any 
deviations to the above exceptions must be documented in the source 
document and reported to the Study Coordinating Centre. 
 
One time point - Lower Urinary Tract Symptoms Survey 
This survey is a one-time cross sectional measure of lower urinary tract 
symptoms in participants above age 18.  Questionnaires will be completed 
varying months post-vaccination, in line with varying enrollment dates in the 
precursor study. 
 
 
7.4.2 Study Visit Window 
 
Interval 
Length of interval 
(months) 
Recommended interval 
between scheduled visits 
(months) 
First dose to Month 36/Visit 8 
(36 ± 2) 
12 
First dose to Month 48/Visit 9 
(48 ± 2) 
12 
First dose to Month 60/Visit 10 
(60 ± 2) 
12 
First dose to Month 72/Visit 11 
(72 ± 2) 
12 
First dose to Month 84/Visit 12 
(84 ± 2) 
12 
First dose to Month 96/Visit 13 
(96 ± 2) 
12 
 
7.4.3 Outline of study visits 
 
MOTNH 36/VISIT 8 (36 months from first vaccine):   
 Assess ongoing consent/assent 
 Health history update, Pap testing/treatment outside study, and any new/resolved health 
issues 
 Update concomitant medication and exposure usage 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
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 Review study blood work/genital sampling delay and contraindication criteria 
 Height and weight 
 Clinical laboratory investigations including resistance testing 
 Study serology  
 Assess menarchal and sexually active status 
Post menarchal and sexually active girls and women and/or at the discretion of the 
site investigator ONLY: 
 Pelvic examination, including 
o external genital examination (including genital warts assessment) 
o inspection of vagina and cervix 
o additional assessments as per local practice and/or clinically indicated (e.g. 
sexually transmitted infection screening samples, bacterial vaginosis, clinical Pap 
smear, etc.) 
 Liquid based study cervical cytology and HPV DNA sample 
 Vaginal microbiota swabs 
 
MONTH 48/VISIT 9 (48 months from first vaccine):   
 Assess ongoing consent/assent 
 Health history update, Pap testing/treatment outside study, and any new/resolved health 
issues 
 Update concomitant medication and exposure usage 
 Review study blood work/genital sampling delay and contraindication criteria 
 Height and weight 
 Clinical laboratory investigations including resistance testing 
 Study serology  
 Assess menarchal and sexually active status 
Post menarchal and sexually active girls and women and/or at the discretion of the 
site investigator ONLY: 
 Pelvic examination, including 
o external genital examination (including genital warts assessment) 
o inspection of vagina and cervix 
o additional assessments as per local practice and/or clinically indicated (e.g. 
sexually transmitted infection screening samples, bacterial vaginosis, clinical Pap 
smear, etc.) 
 Liquid based study cervical cytology and HPV DNA sample 
 Vaginal microbiota swabs  
 
MONTH 60/VISIT 10 (60 months from first vaccine):   
 Assess ongoing consent/assent 
 Health history update, Pap testing/treatment outside study, and any new/resolved health 
issues 
 Update concomitant medication and exposure usage 
 Review study blood work/genital sampling delay and contraindication criteria 

Research Protocol 
Long Term Follow-up Study of CTN 236 - A Study of an HPV VLP Vaccine in a  
Cohort of HIV Positive Girls and Women 
Long Term Follow-up of  
HPV Vaccine in HIV (CTN 236) 
Version 3:  13 Oct 2015 
Confidential 
 
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 Height and weight 
 Clinical laboratory investigations including resistance testing 
 Study serology  
 Assess menarchal and sexually active status 
Post menarchal and sexually active girls and women and/or at the discretion of the 
site investigator ONLY: 
 Pelvic examination, including 
o external genital examination (including genital warts assessment) 
o inspection of vagina and cervix 
o additional assessments as per local practice and/or clinically indicated (e.g. 
sexually transmitted infection screening samples, bacterial vaginosis, clinical Pap 
smear, etc.) 
 Liquid based study cervical cytology and HPV DNA sample 
 Vaginal microbiota swabs 
 
MONTH 72/VISIT 11 (72 months from first vaccine):   
 Assess ongoing consent/assent 
 Health history update, Pap testing/treatment outside study, and any new/resolved health 
issues 
 Update concomitant medication and exposure usage 
 Review study blood work/genital sampling delay and contraindication criteria 
 Height and weight 
 Clinical laboratory investigations including resistance testing 
 Study serology  
 Assess menarchal and sexually active status 
Post menarchal and sexually active girls and women and/or at the discretion of the 
site investigator ONLY: 
 Pelvic examination, including 
o external genital examination (including genital warts assessment) 
o inspection of vagina and cervix 
o additional assessments as per local practice and/or clinically indicated (e.g. 
sexually transmitted infection screening samples, bacterial vaginosis, clinical Pap 
smear, etc.) 
 Liquid based study cervical cytology and HPV DNA sample 
 Vaginal microbiota swabs 
 
MONTH 84/VISIT 12 (84 months from first vaccine):   
 Assess ongoing consent/assent 
 Health history update, Pap testing/treatment outside study, and any new/resolved health 
issues 
 Update concomitant medication and exposure usage 
 Review study blood work/genital sampling delay and contraindication criteria 
 Height and weight 

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 Clinical laboratory investigations including resistance testing 
 Study serology  
 Assess menarchal and sexually active status 
Post menarchal and sexually active girls and women and/or at the discretion of the 
site investigator ONLY: 
 Pelvic examination, including 
o external genital examination (including genital warts assessment) 
o inspection of vagina and cervix 
o additional assessments as per local practice and/or clinically indicated (e.g. 
sexually transmitted infection screening samples, bacterial vaginosis, clinical Pap 
smear, etc.) 
 Liquid based study cervical cytology and HPV DNA sample 
 Vaginal microbiota swabs 
 
MONTH 96/VISIT 13 (96 months from first vaccine):   
 Assess ongoing consent/assent 
 Health history update, Pap testing/treatment outside study, and any new/resolved health 
issues 
 Update concomitant medication and exposure usage 
 Review study blood work/genital sampling delay and contraindication criteria 
 Height and weight 
 Clinical laboratory investigations including resistance testing 
 Study serology  
 Assess menarchal and sexually active status 
Post menarchal and sexually active girls and women and/or at the discretion of the 
site investigator ONLY: 
 Pelvic examination, including 
o external genital examination (including genital warts assessment) 
o inspection of vagina and cervix 
o additional assessments as per local practice and/or clinically indicated (e.g. 
sexually transmitted infection screening samples, bacterial vaginosis, clinical Pap 
smear, etc.) 
 Liquid based study cervical cytology and HPV DNA sample 
 Vaginal microbiota swabs 
 
5. 
Study Procedures in Detail 
7.5.1 Informed Consent/Assent 
Please refer to section 6.3 for detailed information on Informed 
Consent/Assent. 
 
7.5.2 Medical History 
General Medical Systems History 
A review of health history including current medications will be conducted.  

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7.5.3 Height and Weight 
Height and weight will be documented. 
 
7.5.4 Clinical Laboratory Investigations 
Clinical blood samples will be collected, handled, processed, and analyzed at 
site local laboratories as per local practices. At each visit in which clinical 
laboratory investigations are to be performed, results for the following 
laboratory tests will be collected as part of the study: Hgb, WBC, platelets, 
ALT, AST, ALP, bilirubin, BUN, serum creatinine, CD4, CB8 and HIV 
RNA quantitative PCR (viral load). Results of any Hepatitis B or C and 
resistance testing, including clade, will be updated whenever additional 
resistance testing is performed. 
 
Clinical labs for study purposes may be collected within 3 months (+ or – 12 
weeks) of the study visit date. 
 
7.5.5 Study Blood Work 
Ensure all blood tubes and cryovials are clearly labeled with the 5 digit PID#, 
collection date, and visit month. Study blood work must be collected within 
24 hours of the study cervical cytology/HPV DNA sample. 
 
7.5.6 Pelvic Examinations 
 
Pelvic examinations will include: 
 Inspection of external genital exam including a standardized assessment 
of genital  warts using a tool provided by the Study Coordinating Centre, 
 Speculum exam including clinical Pap smear (if clinically indicated), 
 Clinical standard Pap tests will be collected, handled, and processed as 
per local practices. The clinical standard Pap test sample is to be collected 
PRIOR to study cervical cytology collection, 
 If clinical Pap is not done at study visit, obtain the result of most recent 
clinical Pap performed, 
 If clinically indicated, samples are to be collected for sexually transmitted 
infection screening samples, bacterial vaginosis, etc. 
 Vaginal microbiota swabs 
 
7.5.7 Study Genital Samples 
Ensure samples are clearly labeled with the 5 digit PID#, collection date, and 
visit month. Cervical cytology/HPV DNA samples must be collected within 
24 hours of the study blood work sample. 
 
7.5.7.1 Cervical Cytology and HPV DNA 
Cervical cells will be obtained using a broom shaped endocervical 
cytobrush. Insert brush into the endocervical canal until the smaller 
bristles meet the ectocervix. Press gently and rotate clockwise 5 times 

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to ensure adequate cell sampling. The brush is then immediately 
rinsed in the PreservCyt solution by pushing the brush into the 
bottom of the vial 10 times to force the bristles apart. To ensure all 
cells are released, swirl the brush vigorously. Securely seal the vial and 
discard the collection device.  
 
Samples may be kept at ambient to 4 degrees Celsius at the local sites 
until recalled monthly by the Study Coordinating Centre. The 
samples will then be shipped to the Coordinating Laboratory for 
Cytology/HPV DNA (BCCDC) where the samples will be processed 
and forwarded to the appropriate lab for cervical cytology (Cervical 
Cancer Screening Laboratory of BC) and for HPV DNA testing 
(Coutlée lab). 
 
HPV DNA types and cervical cytology results may not be available to 
sites until the end of the study. Clinical Paps are being done as per 
standard of care and follow up to abnormal cytology will be at the 
discretion of the Site Investigator in cases where the liquid based 
cytology samples are available. 
 
7.5.7.2 Vaginal Samples for Microbiota Analyses 
Vaginal samples should be collected during the speculum exam. 
Samples will be obtained using a Dacron swab. Remove study swab 
from kit and unscrew cap from red top tube. Insert swab into the 
vaginal canal and gently brush the swab against the surface of the 
vaginal wall. Rotate the swab clockwise approximately 3 times during 
collection to ensure adequate cell sampling. Remove swab and place 
into empty tube. Break off the swab shaft at the score even with the 
top of the tube. Align the shaft with the hole inside the tube cap and 
push down tightly over the shaft, sealing the tube. Screw red cap on. 
Repeat process with second swab. Refrigerate at 4°C and transfer to -
20°C as soon as possible. Complete the sample collection log to 
indicate date and time swabs are transferred to refrigerator and/or 
freezer. 
 
7.5.7.3 Concomitant Medications and Exposures 
 
Concomitant medications for the purpose of this study are to 
include: antiretroviral therapy, other vaccines,; exposures will include: 
use of drugs of addiction, tobacco, and alcohol. 
 
At each visit, subjects will be asked about their medication 
usage/exposures. Information collected as part of the original CTN 
236 will be reviewed with the subject and any changes to medications 
will be documented. Information to be collected is to include: name 
of 'medication' (generic name or trade name for combination 
medications), medical indication, and total daily dose, route, start and 

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stop dates. PRN medications may be listed as PRN for total daily 
dose.  
 
If a subject is on and off a treatment throughout the study, please 
ensure that the start and stop date indicate the time the subject was 
on and off treatment. Additionally if the dosages change a stop date 
should be entered for the previous dosage and a new entry made for 
the start of the new dosage. 
 
For exposure to drugs of addiction, tobacco, and alcohol, use the 
most basic description, for example, THC, heroin, cocaine 
etc., tobacco, ETOH. For route, indicate whether smoked, snorted, 
injected, oral etc. For frequency, have subject estimate usage per day, 
week, or month. 
 
7.5.7.4 Lower Urinary Tract Symptoms Survey 
The objective of this survey is to further investigate the prevalence 
and impact of lower urinary tract symptoms in cohort participants 
above the age of 18.  This survey is a one-time cross sectional 
measure comprised of 14 questions, to be administered at one study 
visit per participant; questions may be answered at any of the 6 
possible study visits and will take 10 – 15 minutes to complete.   
 
The survey is comprised of 14 questions and includes two validated 
instruments. 85   The Urogenital Distress Inventory 6 (UDI-6) asks 
questions about certain bladder symptoms and how much 
participants are bothered by them.  The Urinary Incontinence 
Questionnaire 7 (UIQ-7), ask questions about how much these 
symptoms affect participants' activities of daily living.  This study 
questionnaire concludes with one supplementary question regarding 
prevalence of urinary tract infections in the past year.  Inclusion of 
this survey will provide a greater understanding of gynaecological 
health in this cohort and speak to any relation between HPV 
infection and urogenital distress. 
 
 
8.0 MANAGEMENT OF CERVICAL CYTOLOGIC ABNORMALITIES 
All participants (except those pre-menarchal and not yet sexually active) will undergo clinical 
standardized Pap smear testing as per local site standards. Cytologic abnormalities will be 
managed according to local algorithms. Management of cervical cytologic abnormalities will be 
decided by the Site Investigator and may be based on the clinical standardized Pap smear or 
study cervical cytologies when available. It is suggested to manage abnormal results based on the 
higher grade of dysplasia.  
 

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Participants who have high grade or persistent low grade changes will be referred for follow-up 
evaluation by colposcopists involved with local HIV treatment centres. After obtaining consent 
to release medical records, all colposcopic, colpophotographic and histopathologic (biopsy) 
results will be added to the study record; slides from biopsies will be requested by the BC Cancer 
Agency for central confirmation of histopathologic diagnoses. Invasive cancers diagnosed during 
the study will be carefully documented and recorded once local, appropriate clinical management 
is provided. As in previous multicentre studies of high risk HIV positive women, we 
acknowledge variation in local colposcopic and treatment algorithms and anticipate imperfect 
compliance with referral for colposcopy; similarly, cervical disease endpoints (squamous 
intraepithelial neoplasia or dysplasia) in this study are cytologically and/or histologically defined 
as appropriate. Histopathology will be reported as cervical intraepithelial neoplasia grades, 1, 2 or 
3 (CIN 1, 2 or 3), or carcinoma in situ. 
 
9.0 SERIOUS ADVERSE EVENTS 
The investigator is responsible for documentation and reporting of any Serious Adverse Event 
that results in death.   
 
1. 
Reporting/ Time Period 
All SAEs resulting in death occurring during the study must be recorded and 
reported to the Study Coordinating Centre whether or not they are considered 
vaccine-related. Sites should continue to use the SAE reporting forms utilized in 
CTN236. 
 
SAE reports will be faxed to Dr. Deborah Money at the Study Coordinating 
Centre within 24 hours of discovery or notification to the study centre of an 
SAE resulting in death and within 48 hours of discovery to the Study Data 
Management Centre (CTN). The Study Coordinating Centre will promptly notify 
Health Canada as appropriate. Site investigators are obligated to notify their REB as 
per local reporting requirements. 
 
SAE Notification: 
Dr. Deborah Money (fax#) 604-875-3895 (24 hours) 
AND CTN (fax#) 604-806-8005 (48 hours) 
 
The Study Coordinating Centre will acknowledge receipt of the faxed SAE form.   
 
Site Investigators should also report SAEs to their provincial adverse event reporting 
centre as would occur when administering any routine licensed vaccine. 
 
10.0 PREGNANCY 
All pelvic examinations, including Pap testing and study sampling will be continue during 
pregnancy; however, an Ayre spatula will be used to collect cervical samples in place of the 
cytobroom until 3 months post resolution of the pregnancy. 

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11.0 SUBJECT COMPLETION AND WITHDRAWAL 
Investigators or their delegates will attempt to contact those study subjects who do not 
return for study visits.  Study personnel will make at least three attempts to contact the 
subject using various communication means.  Each attempt should be documented in the 
subject source documents.   
 
A subject completing three annual visits up to and including Month 96/Visit 13 will be 
considered to have completed the study.   
 
Withdrawals will not be replaced.  All withdrawals will be documented including the date of 
the withdrawal and the reason.   
 
12.0 LABORATORY METHODS AND ANALYSES 
Please consult the Research Laboratory Manual for detailed information on the requirements 
for supplies, collection, and processing, and shipping of laboratory specimens. 
 
Blood Testing 
 
Whole blood by venous route is to be collected observing aseptic conditions into one 10 mL 
red top Serum Vacutainer (non-heparinized, non-EDTA, non-serum separator) tube. 
Specimens should be placed upright and allowed to clot for 30 to 60 minutes. After the 
sample has clotted is should then be refrigerated at 2-8 degrees Celsius until centrifuged. 
Samples are to be centrifuged within 24 hours of collection and according to manufactures 
specification to allow for complete separation of serum. Serum is to be aliquoted using a 
plastic pipette into 3 cryovials, each containing a minimum of 1.0 mL and then frozen 
between -60 and -80 degrees Celsius at the investigative site until recalled by the Study 
Coordinating Centre. Samples will then be shipped frozen to the Coordinating Laboratory 
for serum samples. 
 
The Coordinating Laboratory will arrange for transportation of the samples to the individual 
laboratories performing serotesting, specifically , 1) BC Centre for Disease Control, Virology 
research laboratory, Vancouver, BC, and 2) Merck & Co., Wayne, Pennsylvania and 3) to 
remain at the BC Women’s Research Laboratory at BC Women’s Hospital & Health Centre. 
 
12.1.1 HPV Serotesting 
Each serum sample will be divided into 3 aliquots with one going to the 
BCCDC Virology research laboratory, Vancouver, BC, the second being sent 
to the Merck laboratory for in house assay assessments, and the third being 
sent to the BC Women’s Research Laboratory for storage. The Merck 
laboratory will be conducting the immune assays on the same platform as 
those performed for the pre-licensure published antibody. 

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12.1.2 Merck laboratory Serotesting Methods 
Serum anti-HPV 6, 11, 16, and 18 immunoglobulin levels will be measured 
using a competitive Luminex immunoassay (cLIA), and reported in arbitrary 
units (milli-Merck Units per millilitre or mMU/ml) relative to the standard 
curves generated for each individual HPV type.86  The HPV cLIA uses yeast-
derived HPV L1 6, 11, 16 and 18 VLPs that have been covalently conjugated 
to Luminex microspheres. The type specific HPV-VLP antibody responses 
are associated with specific Luminex microspheres that are identified by their 
distinct fluorescent dye spectral properties. Antibody titers are determined in 
a competitive format, where known, with type-specific labeled neutralizing 
antibodies for each type competing with patient serum antibodies for binding 
to conformation-sensitive neutralizing epitopes on the VLPs. For each HPV 
type, relative inhibition is compared to a pooled standard reference serum. 
Seropositivity will be defined in this assay as > 20 mMU/ml for HPV 6, 16 
mMU/ml for type 11, 20 mMU/ml for 16 and 24 mMU/ml for 18. Of note, 
lower limits of detection for this assay are 8.0, 8.0, 12.0, and 8.0 mMU/ml 
for 6, 11, 16 and 18 respectively.67 
 
12.1.3 HSV2 Serology Testing 
Samples will be sent to the BCCDC Virology research laboratory, 
Vancouver, BC for analysis. The HerpeSelect ELISA (Focus Technologies) 
will be used but will employ a high cutoff index value of 3.5, which has been 
shown to offer improved specificity in the setting of HIV co-infection.87,88,89 
Previously stored blood will be pulled as per protocol for baseline testing and 
at each study visit, participants will have serum stored for batched testing of 
HSV type-specific antibody. After baseline determination of serostatus for all 
participants, follow-up testing will be performed only on previously 
seronegative participants to assess for interval seroconversion.  
 
 
13.0 Genital sampling 
All PreservCyt samples will be sent monthly by the local sites to the Coordinating 
Laboratory for Cytology/HPV DNA. Samples will be re-suspended in the Preservcyt and a 4 
ml aliquot will be taken and sent to the Coutlée Laboratory for analysis of HPV DNA.  The 
remainder of the sample will be sent to the Cervical Cancer Screening Laboratory of BC for 
cervical cytology. 
 
1. 
HPV DNA testing – Coutlée Laboratory 
The 4 ml endocervical samples in PreservCyt will be centrifugation at 13000xg for 15 
min at 22°C, the supernatant will be discarded, the cell pellet will be left to dry and 
re-suspended in 300 µl of 20 mM Tris buffer, pH 8.3.63,64 DNA will be purified with 
Master pure.65 ß-globin and HPV DNA amplification and typing. Extracted DNA 
will be tested in batches for ß-globin with PC04 and GH20 to screen for inhibitors 
or inadequate quantities of cellular DNA and for HPV DNA with previously 
described PGMY primers.66,67 Samples negative for ß-globin are considered 

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inadequate. PCR L1 consensus tests are well standardized and have been validated 
on large numbers of clinical samples, as reviewed by members of this team.68,69   HPV 
DNA will be first amplified with PGMY09-PGMY11 and typed with the quality-
controlled Linear array assay commercialized by Roche Molecular Systems.66,67,70-73 
This specific PCR assay reliably detects 10 HPV DNA copies.66,71 This test permits 
testing and typing for 37 genital types of HPV (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 
42, 45, 51-59, 61, 62, 64, 66-73, 81, 82, 83, 84, 89 and IS39 (a subtype of HPV 82)). 
The genotypes relevant for this proposal including 6, 11, 16 and 18 are all detected 
by these specific PCR protocols. The Coutlée laboratory has extensive experience 
with PCR for HPV detection. 66-68,70-74,74-76, 78-80 ,82,83 and has participated in the 
evaluation and validation of the Linear array and PGMY09-PGMY11 primers. 
66,67,70,71 ,83  Weak (10 HPV18 DNA copies), and strong positive HPV controls, will be 
included in each amplification run as internal control and precautions to avoid 
contamination will be in place at all times.68,74,76,78,80,82 
 
2. 
Cervical Cytology – Cervical Cancer Screening Laboratory of BC  
The PreservCyt vial will be processed for cytologic evaluation following the 
ThinPrep protocol (Cytyc Incorporated). Slides will be read by cytotechnologists at 
the Cervical Cancer Screening Laboratory (CCSL) of BC. Reporting rules for normal 
and abnormal results will be the same as for routine specimens processed at the 
CCSL, a facility that processes all cervical smears performed in British Columbia (up 
to 600,000 per year). Standard reporting of Pap smears will be according to the 
Bethesda criteria e.g. low grade and high grade squamous intraepithelial neoplasia 
(SILs) 
 
3. 
Vaginal Microbiota Analyses – Hill Lab, University of Saskatchewan 
Vaginal samples will be collected for microbiome analysis as per the VOGUE 
Emerging Team Grant protocol (CIHR FRN 108030; PI D Money). Samples will be 
transported to the Hill laboratory at the University of Saskatchewan, where total 
DNA will be extracted from vaginal swabs using the MagMAX magnetic DNA 
binding platform (Applied Biosystems). Chaperonin 60 (cpn60) amplicons130 will be 
then prepared as previously described utilizing multiplexed ID sequences (MID) to 
pool samples during sequencing.90 Sequencing will be performed on the Roche GS 
Junior pyrosequencer in the Hill lab. Raw sequence data will be processed initially 
using the standard on-rig software tools for quality control and filtering. Quality 
filter-passed read data will then be analyzed using the mPUMA bioinformatics 
pipeline developed by the Hill lab.91  
 
 
14.0 TRANSPORTATION OF LABORATORY SAMPLES 
All shipments of serum samples and vaginal microbiota swabs are to be shipped frozen and 
prepared by qualified individuals ensuring that all shipments are packed and shipped 
according to IATA regulations and are to be packed using dry ice. 

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All shipments of liquid based cytology are to be shipped ambient and prepared by qualified 
individuals ensuring that all shipments are packed and shipped according to IATA 
regulations.  
 
All samples are to be clearly labeled with subjects’ 5 digit PID#, date of collection, 
and visit month. 
 
All shipments must have an accompanying biological shipment listing, with a copy being 
retained at the site. A biological shipment listing template will be provided to the sites by the 
Study Coordinating Centre. 
 
Prior to the shipment of any samples, the sites must inform the Study Coordinating Centre 
regarding the date of shipment to ensure that there is someone at the Coordinating 
Laboratory to receive the shipment. Confirmation must be received from the Study 
Coordinating Centre prior to the shipment of any samples. Sites should plan to ship Monday 
through Wednesday to ensure that samples arrive within 72 hours of packaging to ensure 
that thawing does not occur before processing. 
 
The Coordinating Laboratories will arrange for shipment of all samples (serum, PreservCyt, 
microbiota swabs) to the individual laboratories performing the analysis/testing.  
 
15.0 DATA SAFETY MONITORING BOARD/COMMITTEE 
The established CTN data safety monitoring board (DSMB) will be used for this study with 
additional representation by pediatrics and gynecology-oncology. The DSMB will provide 
oversight and monitoring of the conduct of the study to ensure the safety of the subjects. 
Additionally, the DSMB will ensure the validity and integrity of the study data. The DSMB 
meets twice a year. 
 
16.0 STATISTICAL ANALYSIS 
1. 
Statistical Analysis Plan  
Demographics: Characteristics of the study population will be described with 
frequencies and proportions for categorical variables, means and standard deviations 
for normally distributed continuous variables and medians and interquartile ranges 
for skewed continuous variables.  
 
2. 
Primary Analysis 
For each of the four vaccine containing HPV types, antibody response will be 
summarized using GMTs calculated for age specific groups within the HIV-positive 
cohort. Specific age groupings have been chosen to permit age matched HIV 
comparisons. 
a) Antibody response for subjects aged 9-13 and those aged 16-26 will be 
compared to the individual patient level data from an HIV-negative cohort 
86 using Wilcoxon rank-sum tests. The age adjusted fold-difference in GMT 

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between HIV-positive and HIV-negative populations will be calculated using 
generalized linear models. For women over age 26, the type specific HPV 
GMT will be compared to GMTs in the published literature using a sign test.  
 
b) Within the HIV-positive cohort, predictors of decline from peak antibody 
response among HIV-positive girls and women will be determined using 
generalized linear models. Potential covariates to be considered in these 
models are age, HSV-2 status, substance use, CD4 count, HIV viral load and 
antiretroviral status at the time of first vaccination, and CD4 nadir. 
 
c) Longitudinal analyses will be conducted to describe the effect of HIV on 
antibody response at the visits at 36, 48, 60, 72, 84, and 96 months post first 
vaccine dose. Generalized estimating equation (GEE) models with a unity 
link will be used to adjust for correlation among repeat observations within 
study participants. Individual level data on HIV-negative girls and young 
women will be available at 36, 48, 60, 72, 84 and 96 months post first vaccine 
dose. After adjusting for age, an interaction between HIV and time since first 
dose will be included in the model to test for a difference in the degree of 
waning of response in addition to the strength of the initial response. GEE 
models will also be fit for the HIV-positive girls and women alone to 
examine the associations. 
 
d) Predictors of HPV antibody seroresponse in our study population will be 
identified using logistic regression. This analysis is considered to be of vital 
importance as other vaccines have been shown to not be effective in 
subgroups of HIV positive individuals and could permit targeting of the 
proper population allowing for cost-effective use of this vaccine. The 
primary predictor of interest is CD4 count and justification for this is based 
on hepatitis B vaccination data described above. The secondary predictors of 
interest will include the variables: age, presence and number of strains of 
HPV, HIV viral load, use of HAART, use of injection drugs, and ethnicity. 
These predictors were chosen as ones that make the most clinical sense for 
potent impact on vaccine antibody response.  
 
3. 
Exploratory Analyses 
HSV analyses will employ linear or logistic regression models as appropriate, with 
HSV-2 serostatus as a time- updated primary predictor variable. To address these 
issues, we will quantify the impact of both incident (acquired within the past year) 
and prevalent (present at the time of diagnosis) HSV-2 co-infection on each of the 
primary HPV-related outcomes as described in preceding section (GMT, 
breakthrough HPV infections, genital tract dysplasia, and external genital warts).  
 
Vaginal Microbiota Analysis. The output of the bioinformatics pipeline, including 
inventory of unique operational taxonomic unit (OTU) sequences, their taxonomic 
lineage and abundance, will be used to visualize and characterize the vaginal 
microbiota. Using various community-level analyses available as packages in R: 133 

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notably the BiodiversityR, 134 vegan, 135 and ALDeX2136 packages, we will apply 
statistical and graphical techniques to identify patterns of bacterial diversity that are 
associated with HPV infection and disease. Specifically, we will use compositional 
analysis as implemented in ALDeX2 to test for relationships between specific 
bacterial taxa and HPV infection and cervical dysplasia in addition to other clinical 
variables at each time point. Taxa thus identified will be examined for changes in 
relative abundance across time using mixed-effects models to control for multiple 
measures on each subject, and comparing to HPV infection status, HPV type and 
other clinical and HIV correlates. We will also use hierarchical clustering methods to 
determine if the bacterial communities form identifiable and robust clusters, with 
particular species occurring more often together. We can then test if these clusters 
are associated with HPV infection, burden of infection (single vs. multiple types) and 
evidence of cervical disease using standard statistical tests such as logistic regression 
and ANOVA. Finally, we will use mixed-effects models to test for changes in 
bacterial diversity and species richness that are associated with HPV infection, 
burden of infection (i.e. infection with multiple HPV types) and evidence of cervical 
disease while controlling for repeated measures on each subject. 
 
HIV and Lower Urinary Tract Symptoms Analysis. Descriptive statistics will be used 
for baseline patient characteristics. Logistic regression analysis will be performed to 
determine the effect of:  severity of HIV (CD4 count, viral load, AIDS criteria met), 
current HIV medications, time since HIV diagnosis, and vaginal microbiome on 
lower urinary tract symptoms, while also considering the effects of age, parity, 
ethnicity, sexual practices (from various questions in the HPV in HIV questionnaire), 
history of STI, history of UTI, smoking status, alcohol use, drug use.   
 
17.0 DATA MANAGEMENT  
 
 
Data management will be performed by the CIHR Canadian HIV Trials Network (CTN).  
 
 
 
Source document data will be transferred to a case report form by study personnel at the 
investigative sites. The participant identification number (PID) and a unique 3 letter code 
that was assigned in CTN 236 will be used to label the case report form and all study 
samples.   
 
Data will be entered into a relational database with pre-programmed quality assurance 
checks. Queries will be resolved with the site study coordinator, with appropriate 
documentation. Data edits will checked by the CTN data manager before being added to the 
master database. Backup copies of the database will be made daily and stored securely at a 
second location. No personal identifiers will be included in the database. Analysis will be 
conducted using statistical software (e.g. SAS or SPSS). 
 
All personal information pertaining to participants will be securely stored at sites in a locked 
room, under the supervision of the site study coordinator(s) and accessible to study staff 
only. Where electronic databases are used as mechanisms for subject tracking etc. these 
databases will be password protected and available only to specified study staff and site 
investigators.   

Research Protocol 
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18.0 ADMINISTRATIVE MATTERS 
1. 
Protocol amendment procedure 
No modification of this protocol will be allowed unless discussed and approved with 
the Study Coordinating Centre and a filed and approved change to the protocol is 
made with local REBs. 
 
Any administrative changes or amendments to this protocol will be adhered to by all 
participating centres and will apply to all subjects.  Documentation of the 
amendments and REB certification will be maintained at each investigative site and 
are required prior to implementation. 
 
2. 
Records Retention 
Data and study documents (all sites) will be stored securely for 25 years, after which 
they will be destroyed in keeping with privacy and confidentiality regulations and 
guidelines. 
 
All personal health information pertaining to subjects will be securely stored in a 
locked area, under the supervision of the site study coordinator(s) and accessible to 
study staff only.  The location of storage of the study files must be documented and 
revised if the location changes.  The Study Coordinating Centre must be aware of the 
storage location at each site and changes to the location must be provided in writing. 
 
3. 
Clinical Study Report 
Interim study progress reports, as well as the final report, will be submitted to the 
Canadian Institutes of Health Research.   
 
4. 
Publication Plan 
Manuscripts of the study findings will be prepared for submission to peer reviewed 
publications. Study findings will also be presented at appropriate scientific meetings.  
 

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19.0 REFERENCE LIST 
 
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4 British Columbia Cancer Agency, 2005 Annual Report, Cervical Cancer Screening. Vancouver, 
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7 Canadian Task Force on Preventive Health Care. Recommendations on screening for cervical 
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12 Frisch M, Fenger C, van den Brule A, et al. Variants of squamous cell carcinoma of the anal canal 
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17 Silverberg M, Ahdieh L, Munoz A, et al. The impact of HIV infection and immunodeficiency on 
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23 Trottier H, Franco E. The epidemiology of genital human papillomavirus infection. Vaccine. 
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24 Silverberg MJ, Schneider MF, Silver B, et al. Serological detection of human papillomavirus type 
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26 Sun X, Kuhn L, Ellerbrock T, Chiasson M, Bush T, Wright T. Human papillomavirus infection in 
women infected with the human immunodeficiency virus. N Engl J Med. 1997;337:1343-1349. 

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Cohort of HIV Positive Girls and Women 
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27 Wright T, Ellerbrock T, Chiasson M, et al. Cervical Intraepithelial Neoplasia in Women Infected 
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30 Palefsky J, Minkoff H, Kalish L, et al. Cervicovaginal human papillomavirus infection in human 
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31 Blitz S, Baxter J, Raboud J, et al. Evaluation of HIV and Highly Active Antiretroviral Therapy on 
the Natural History of Human Papillomavirus Infection and Cervical Cytopathologic Findings in 
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32 Minkoff H, Zhong Y, Burk RD, et al. Influence of Adherent and Effective Antiretroviral Therapy 
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33 Konopnicki D, Manigart Y, Gilles C, et al. Sustained Viral Suppression and Higher CD4(+) T-Cell 
Count Reduces the Risk of Persistent Cervical High-Risk Human Papillomavirus Infection in HIV-
Positive Women. J Infect Dis. 2013;207:1723-1729. 
34 Rositch AF, Gravitt PE, Tobian AAR, et al. Frequent Detection of HPV before and after 
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35 Petignat P, Hankins C, Walmsley S, et al. Self-sampling is associated with increased detection of 
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like-particle vaccine against infection with human papillomavirus types 16 and 18 in young women: 
an interim analysis of a phase III double-blind, randomised controlled trial. Lancet. 2007;369:2161-
2170. 
 

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Cohort of HIV Positive Girls and Women 
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HPV Vaccine in HIV (CTN 236) 
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38 Del Mistro A, Bertorelle R, Franzetti M, et al. Antiretroviral therapy and the clinical evolution of 
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Infection Soon After Incident HIV Infection Among South African Women. J Infect Dis. 
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40 Harris T, Burk R, Palefsky J, et al. Incidence of cervical squamous intraepithelial lesions associated 
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American Medical Association. 2005;293:1471-1476.  
 
41 Canadas MP, Videla S, Darwich L, et al. Human papillomavirus HPV-16, 18, 52 and 58 integration 
in cervical cells of HIV-1-infected women. Journal of Clinical Virology. 2010;48:198-201. 
 
42 Dartell M, Rasch V, Kahesa C, et al. Human Papillomavirus Prevalence and Type Distribution in 
3603 HIV-Positive and HIV-Negative Women in the General Population of Tanzania: The 
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43 Naucler P, da Costa FM, da Costa JL, Ljungberg O, Bugalho A, Dillner J. Human papillomavirus 
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44 Darwich L, Canadas M, Sirera G, et al. Human Papillomavirus Genotype Distribution and Human 
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45 Melgaco FG, Rosa MLG, Augusto EF, et al. Human papillomavirus genotypes distribution in 
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46 Garbuglia AR, Piselli P, Lapa D, et al. Frequency and multiplicity of human papillomavirus 
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Cohort of HIV Positive Girls and Women 
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49 Warren T, Harris J, Brennan CA. Efficacy and safety of valacyclovir for the suppression and 
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Cohort of HIV Positive Girls and Women 
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HPV Vaccine in HIV (CTN 236) 
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62 Ugwumadu A, Hay P, TaylorRobinson D. HIV-1 infection associated with abnormal vaginal flora 
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uninfected women. J Infect Dis. 2005;191:1129-1139.  
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lesions (SILs) and bacterial vaginosis. Eur J Gynaecol Oncol. 2007;28:310-312. 
 
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66 Olsson S, Villa LL, Costa RLR, et al. Induction of immune memory following administration of a 
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79 Moses E, Blitz S, Raboud J, et al. HPV Infection and Cervical Dysplasia in HAART-Treated HIV 
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80 Kahn JA, Xu J, Kapogiannis BG, et al. Immunogenicity and Safety of the Human Papillomavirus 
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Research Protocol 
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Cohort of HIV Positive Girls and Women 
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HPV Vaccine in HIV (CTN 236) 
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87 Ashley-Morrow R, Nollkamper J, Robinson NJ, Bishop N, Smith J. Performance of focus ELISA 
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88 Biraro S, Mayaud P, Morrow RA, Grosskurth H, Weiss HA. Performance of Commercial Herpes 
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Review and Meta-analysis. Sex Transm Dis. 2011;38:140-147. 
89 Mujugira A, Morrow RA, Celum C, et al. Performance of the Focus HerpeSelect-2 enzyme 
immunoassay for the detection of herpes simplex virus type 2 antibodies in seven African countries. 
Sex Transm Infect. 2011;87:238-241. 
90 Schellenberg J, Links MG, Hill JE, Hemmingsen SM, Peters GA, Dumonceaux TJ. 
Pyrosequencing of chaperonin-60 (cpn60) amplicons as a means of determining microbial 
community composition. Methods in Molecular Biology. 2011;733:143-158.  
91 Links MG, Chaban B, Hemmingsen SM, Muirhead K, Hill JE. mPUMA: a computational 
approach to microbiota analysis by de novo assembly of OTUs based on protein-coding barcode 
sequences. Microbiome. 2013;1:23.