In Depth Sampling and Subsequent Treatment Interruption to Identify the Anatomical Reservoir
Phase: NA
Conditions: HIV
Sponsor: University Hospital, Ghent

ID: IIS 52777    
Title:  
HIV Sequencing After Treatment Interruption to Identify the 
Clinically Relevant Anatomical Reservoir    
NCT02641756 
Date: 9/12/15 
 
 

 
Study protocol 
 
 
 
 
 
 
 
In depth sampling and subsequent treatment 
interruption to identify the HIV-1 anatomical 
reservoir.  
 
 
 
 
 
 
Sponsor: UZ Gent  
Principal Investigator: 
Prof Linos Vandekerckhove 
Department of General Internal Medicine 
 
 

Co investigators: 
 
Prof. Dr. Dirk Vogelaers 
Algemene inwendige ziekten,  UZGent 
 
Prof. Dr. Chris Verhofstede 
Aids Referentie labo UGent 
 
Prof Dr. Steven Callens  
Algemene inwendige ziekten,  UZGent 
 
Dr. Erica Sermijn  
Algemene inwendige ziekten,  UZGent 
 
Dr. Filip Van Wanzeele 
Algemene inwendige ziekten,  UZGent 
 
Dr. Beatrijs Van Der Gucht  
Algemene inwendige ziekten,  UZGent 
 
Dr. Jolanda Pelgrom 
Algemene inwendige ziekten,  UZGent 
 
Els Caluwé 
Algemene inwendige ziekten,  UZGent 
Study nurse 
 
Dr. De Scheerder Marie-Angélique 
Algemene inwendige ziekten, UZGent  
 
Prof. Dr. Annemieke Dhondt  
Nierziekten, UZGent  
 
Prof. Dr. Frank Vermassen 
Thoracale en vasculaire heelkunde, UZGent  
 
Prof. Dr. De Looze Danny 
Gastro-enterologie, UZGent  
 
Dr. Thomas Malfait 
Longziekten, UZGent 

 
Dr. Dimitri Hemelsoet 
Neurologie, UZGent  
 
Prof. Dr. Kerre 
Hematologie, UZGent  
 
Prof. Dr. Patrick Wouters 
Anesthesie, UZGent  
 
Prof. Dr. Katrien Roelens 
Verloskunde, UZ Gent 
 
Dr. Ward Despiegelaere 
HTRU, Ugent  
 
Financial support: Merck investigator Grant for independent scientific research.  
Departments/laboratories involved:  
Department of Internal Medicine, Infectious Diseases and Psychosomatic Medicine, Ghent 
University Hospital  
Department of Gastro-enterology, Ghent University Hospital  
Department of Anaesthesia, Ghent University Hospital  
Department of Haematology, Ghent University Hospital  
Department of Pneumology, Ghent University Hospital 
Department of thoracic and vascular surgery, Ghent University Hospital  
Department of Gynaecology, Ghent University Hospital  
Toxicology Lab, Ghent University Hospital  
 
 
 
 

1. Objective of the study:  
 
Goal of the study 
The main goal of this study is to identify and characterise the anatomical component of the 
replication competent HIV-1 reservoir.  
 
Hypothesis 
We hypothesize that the clinically relevant HIV-1 reservoir is hiding in various but specific 
anatomic compartments and is able to rebound when therapy is stopped.  
This reservoir is probably smaller than the HIV-1 reservoir hiding in the blood but could be 
more transcriptional active because of its specific environment, possibly influenced by lower 
concentrations of the antiretroviral therapy.  
 
Rationale 
With combined antiretroviral therapy (cART), HIV-1 infection can be suppressed, but not 
cured. A reservoir of HIV-1 infected cells remains impervious to treatment and reinitiates viral 
infection once treatment is stopped. The cellular spectrum of these HIV-1 cellular reservoirs 
is highly diversified, and its role varies according to the environment of the anatomical sites in 
which the virus hides (e.g. cell type, accessibility of cART). Hence, a key question in HIV-1 cure 
research is: Where in the body of patients receiving cART is the virus hiding? Current studies 
have mainly looked at the CD4 T cell compartment of the peripheral blood, which forms the 
easiest compartment to study, but may not be representing the actual replication competent 
HIV-1 reservoir. Although technologies like droplet digital PCR (ddPCR) (1-3) single HIV-1 
genome sequencing (4,5), Linear Amplification and target captured based HIV-1 integration 
site sequencing (6) have been introduced by us and others to investigate the viral reservoir, a 
comprehensive analysis that combines these tools to identify the source of the viral rebound 
in a relevant clinical setting is lacking. Previous studies that tried to answer this question were 
limited in scope as they only performed phylogenetic analysis on plasma (7) pre- and post- 
cART, or they only evaluated integration sites and performed phylogenetic analysis in the 
blood pre- and on cART (8). 
The current proposal will, for the first time, identify the source of the viral reservoir by 
phylogenetically backtracking the viral genome of the rebounding virus to the sequences of 
viral DNA in different anatomical compartments. The subsequent characterization of the viral 
reservoir markers (size, integration sites, methylation profile, stimulation and inhibition 
assays) will enable us to understand how this viral rebound occurred. 
This study proposal is unique as it combines new emerging technologies assessing highly 
relevant patient derived samples in a unique clinical and research setting.  
References:  
1.  Kiselinova M et al. Plos One (2014); 9: e85999 

2.  Malatinkova E et al. J Clin Microbiol (2014): doi:10.1128/JCM.03087-14 
3.  De Spiegelaere W et al. Analytical biochemistry (2013); 439: 201 
4.  Josefsson L et al. Proc Natl Acad Sci U S A (2011); 108: 11199 
5.  Josefsson L et al. Plos Pathog (2013); 9: e1003432 
6.  Schmidt M et al. Nature methods (2007); 4: 1051 
7.  Chalmet K et al. BMC infectious diseases (2010); 10: 262 
8. Maldarelli F et al. Science (2014); 345: 179 
2. The present study  
2.1 Study population 
 
2.1.1: Number of subjects  
 
10 patients with undetectable viral load under stable antiretroviral therapy with no potential 
contraindications for a temporary treatment interruption will be informed and further guided 
if interested.  
 
2.1.2: Inclusion and exclusion criteria: 
In order to be eligible, study participants must meet all the following criteria: 
 
 Documented HIV-1 infection, subtype B  
 Able and willing to provide written informed consent  
 Age = or >18 years < 65 years 
 Nadir CD4 count >=300/µl. CD4 count at screening > 500/μl  
 Patient should take ART for at least 2 years with no changes in the ART for at least 90 
days prior to study entry. Patients should be on an integrase inhibitor  + 2 nucleoside 
analogs based regimen. 
 Patient should have a viral load < 20 copies/ml determined by CobasTaqMan HIV-1 test 
v2.0 assay for at least 2 years. (Occasional “blips” will be permitted) (A blip is defined 
as an intermittent viremic episode with a viral load above detection level but below 
200 copies/ml and a return to an undetectable level in a next control, if more than six 
months prior to the study entry)   
 Last viral load undetectable 
 Ability to attend the complete schedule of assessments and patient visits. Ability and 
willingness to have blood and tissue samples collected and stored indefinitely and 
used for various research purposes. Women of childbearing potential or their partner 
should use double barrier contraception during the study. Females of reproductive 
potential (women who have not been post-menopausal for at least 24 consecutive 
months, i.e., who have had menses within the preceding 24 months, or women who 

have not undergone surgical sterilization, specifically hysterectomy, or bilateral 
oophorectomy and/or bilateral salpingectomy), will need a negative serum or urine 
pregnancy test within 48 hours prior to study entry. 
NOTE: Acceptable documentation of hysterectomy and bilateral oophorectomy, 
bilateral salpingectomy, tubal micro-inserts, partner who has undergone vasectomy, 
and menopause is participant-reported history. All participants must agree not to 
participate in the conception process (e.g., active attempt to become pregnant or to 
impregnate, sperm donation, or in vitro fertilization). Participants must agree to use 
barrier protection for all sexual activity and if participating in sexual activity that 
could lead to pregnancy, the participant/partner must use at least two reliable forms 
of contraceptives (condoms, with or without a spermicidal agent; a diaphragm or 
cervical cap with spermicide; an IUD; or hormone-based contraception) during the 
study.  
 
Potential participants meeting any of the following criteria will not be enrolled in the 
study: 
 
 Previous or current history of opportunistic infection. (AIDS defining events as 
defined in category C of the CDC clinical classification).  
 History of resistance to antiretroviral drugs, documented by genotyping.  
 Hepatitis B surface antigen positive or HBV viral load positive in the past and no 
evidence of subsequent seroconversion (=HBV antigen or viral load negative and 
positive HBV surface antibody). 
 HCV antibody positive result within 60 days prior to study entry or, if the HCV 
antibody result is negative, a positive  HCV RNA result within 60 days prior to 
study entry 
 Significant risk of HIV superinfection during treatment interruption.  
 Current or known history of cardiomyopathy or significant ischemic or 
cerebrovascular disease. 
 History of HIV-related thrombocytopenia.  
 Active renal disease (defined as a glomerular filtration rate (calculated by 
Cockroft Gault equation) below 50ml/min or the presence of HIVAN in the past 
medical history. 
 Current or known history of cancer (with the exception of in situ cervix carcinoma 
or squamous cell carcinoma of the skin) within five years prior to screening.  
 Pregnancy or breastfeeding.  
 Any conditions, including psychiatric and psychological disorders, which will in the 
opinion of the investigator interfere with the trial conduct or safety of the 
participant. 
 Previous participation in a trial evaluating an immune modulating agent  
 Abnormal laboratory tests results at screening:  
1. Confirmed Hemoglobin <11g/dl for women and <12 g:dl for men  
2. Confirmed platelet count < 100000/l  
3. Confirmed neutrophil count <1000/μl  
4. Confirmed AST and/or ALT > 2xULN  
The following treatment will be prohibited three months before screening and during the 
course of the study:  

1. immunosuppressive drugs (inclusive corticosteroids) with the exception of drugs used 
for topical use.  
2. Immunomodulatory drugs including but not limited to Granulocyte colony stimulating 
factors, Granulocyte-monocyte stimulating factor, interleukin 2, 7 & 15. 
 
 Active drug or alcohol use or dependence that, in the opinion of the site 
investigator, would interfere with adherence to study requirements. 
 Acute or serious illness, in the opinion of the site investigator, requiring systemic 
treatment and/or hospitalization within 60 days prior to entry. 
 
Criteria for ATI Discontinuation (ART resumption) 
Subjects will be advised to reinitiate ART after ATI for any of the following 
 HIV-1 RNA above 200 copies/ml on 2 consecutive determinations at least 3 days 
apart. 
 A single HIV-1 RNA above 1000 copies/ml 
 CD4+ T cell counts below 350 cells/mm3 on 2 consecutive determinations at least 
2 weeks apart 
 CD4+ T-cell count decline of > 50% from baseline prior to ATI 
 Clinical progression to CDC Category B or C disease 
 Diagnosis of Acute Retroviral Syndrome. 
 Pregnancy 
 Subject requests re-initiation of ART 
VL is done 2-3/week up to week 4 of ATI, then every week until treatment reinitiation.  
2.1.3 Prior and concomitant therapy: patients before starting the study will, if they are 
currently on another regimen, be switched to an integrase inhibitor based regimen, at the 
latest 90 days prior to study initiation (Current treatment options in infectious diseases, 
(2014)6:144-158).    
2.2 Study design and Intervention: 
 
This is a single arm mono-centric non-randomized (prospective) study.  
 
2.2.1 Primary objectives and outcomes:  
- 
Quantification of viral markers as total HIV DNA, episomal DNA (2LTR circles) and cell-
associated RNA in different anatomical reservoirs.  
- 
Genetically link the viral reservoir in different anatomical reservoirs to the rebounding 
virus found in the plasma after therapy-stop by doing phylogenetic analysis.  
- 
Integrations sites sequencing to look for clonal expansion.  
- 
Methylation profiles in the human genome where HIV-1 integrates. 
- 
Stimulation and inhibition assays to characterise the replication competent viral 
reservoir 

- 
TDM (therapeutic drug monitoring) studies to look at cART penetration in peripheral 
tissues. 
- 
Human markers to screen for inflammatory parameters related to HIV-1 infection 
especially in CSF.  
 
2.2.2 Procedures 
 
2.2.2.1: Eligibility screening and inclusion in the study: 
Study participants will be selected from patients in regularly follow-up at UZ Gent. Screening 
evaluation will be performed prior to study entry. The patient will be invited to participate in 
the trial and provided information about the aims and risks of the project. After obtaining 
written informed consent, clinical eligibility will be assessed. Patients will be eligible for 
enrolment if they fulfil all the inclusion criteria. Patients will not be eligible for the study if they 
meet any of the above described exclusion criteria. If patients are not on an integrase inhibitor 
based regimen, they should be switched 3 months prior to study initiation.  
 
The following will be assessed at this visit:  
- Assessment of subject eligibility according to the inclusion and exclusion criteria  
- Demographic and epidemiological data  
- Medical history (past and current), including HIV-associated conditions  
- Full antiretroviral history (including result of resistance assays if available)  
- Review of non-antiretroviral medication within the last 30 days with special attention for 
immunomodulatory and immunosuppressive drugs and anticoagulation therapy. 
- Physical examination including vital parameters, height and weight  
- HIV viral load and CD4 T cell count.  
- Laboratory test (non-fasting): hemoglobin, absolute neutrophil count (ANC), platelets, 
creatinine, electrolytes, liver enzymes AST & ALT, coagulation. Hepatitis HCV & HBV serology 
if necessary.  
- Pregnancy test among women. 
- ECG and lung X-ray 
- assessment of good peripheral venous access 
 
2.2.2.2: In depth sampling:  
An in depth sampling will be planned before therapy interruption. Together with our 
collaborators we will put in place a standardised trajectory, aiming for the highest possible 
comfort for the participants. The patients will therefore be admitted in the hospital for 24h. 
Bronchoscopy, gastroscopy and coloscopy will be performed under anaesthesia. Before the 
anaesthesia, the anaesthesiologist will perform a lumbar puncture. During anaesthesia a bone 
morrow aspirate and an inguinal biopsy will also be effectuated. Leukapheresis can be 
organised on ambulatory basis within one month of the in depth sampling but not the day 
before or after due to increased bleeding risk. If leucapheresis is contra-indicated this will be 
replaced by a peripheral blood draw of 6x 9ml. Sperm collection will be done by standardised 

protocol of the fertility clinic. Vaginal lavage will be done during hospitalisation or ambulatory 
at the gynaecology consultation.  
 
2.2.2.3: Treatment interruption to allow viral rebound and subsequent sampling:   
A second sampling will be foreseen after therapy-stop once viral rebound is detected and will 
consist on leukapheresis (or  peripheral blood draw if contraindicated), lumbar puncture and 
genital sampling. As before there should be a timespan of minimum 24h between 
leukapheresis and puncture due to increased bleeding risk. For this sampling an overnight stay 
in the hospital will be arranged.  
 
After the treatment interruption is initiated a follow-up of plasma viral load will be organized. 
Patients will be stopped at D1 (Friday) and a first control will be performed D4 (Monday) and 
D7 (Thursday). On an average we expect people to have rebounded before D12 and the 
second sampling will be planned like this. If the viral load control on D11 shows no signs of 
rebound, sampling will be postponed and further follow-up will be organized. If after 3 weeks 
the patient has not rebounded, we will discuss this result with the patient and we will propose 
weekly controls of viral loads and restart therapy after 2-3 months, after the second sampling. 
For patients without detectable viral load after 1 month, an additional sampling at that point 
(blood sampling, lumbar puncture and genital sampling) will be proposed.  
 
Sampling will consist of  
a: Leukapheresis 
Participants will undergo leukapheresis at the Ghent University Hospital. Leukapheresis runs 
will last approximately three hours for the collection of > 1x108 cells Peripheral Blood 
Mononucleair Cells. Leukapheresis is considered the gold-standard method for obtaining large 
volumes of peripheral cells from HIV-infected patients, and has been performed safely in 
individuals with and without hematologic malignancy and/or HSCT (1). Leukapheresis will be 
performed twice for each participant, with a minimum of three months interval following the 
initial collection. If contra-indicated this procedure will be replaced by a peripheral blood draw 
of 6x9ml.  
b: Bronchial lavage and endobronchial ultrasounds guided sampling 
Participants will undergo bronchial lavage and endobronchial ultrasound guided lymph node 
sampling at the pulmonary department of the University Hospital. The procedure is now 
routinely performed as part of clinical practice in patients with an unidentified lung disease 
(e.g. diagnosis for TB). This procedure will be performed only once for each participant.   
c: Lymph node resection 

The vascular surgery department will perform lymph node resection. Briefly, a small incision 
is made in the inguinal region (triangle of scarpa) allowing a lymph node resection. Lymph 
node resection will be performed once, before cART is stopped.   
d: Gastroscopy and duodenal biopsy 
Duodenal mucosal sampling will be obtained by gastroscopy with up to 10 mucosal biopsies 
obtained. This procedure will be performed only once for each participant.    
e: Colonoscopy 
We will take up to 10 mucosal biopsies, at the terminal ileum. We will also perform collection 
of gut-associated lymphoid tissue via biopsy of rectal mucosa. Rectal mucosal sampling will be 
obtained by anoscopy, collecting up to 10 mucosal biopsies.  
Subjects will be counselled to avoid rectal trauma and the use of anticoagulants (e.g. aspirin, 
NSAIDS) before and after the procedure. Mucosal biopsies will not be taken if the participant 
has an increased risk for complications, including receptive anal intercourse within 3 days of 
the procedure, an active anal infection, or recent use of anticoagulants. Gastroenterologists 
who have training and clinical certification in anoscopy and rectal mucosal biopsy will perform 
rectal biopsies. This procedure will be performed only once for each participant.   
f: Bone marrow biopsy 
We will collect bone marrow tissue from aspirates during the study performed by the 
haematology department. This procedure will be performed only once for each participant 
before treatment stop.   
g: Lumbar fluid 
Cerebrospinal fluid (CSF) will be obtained for study purposes and processed in standardized 
fashion as previously described in the study of central nervous system HIV-1 infection (2). A 
maximum volume of 3-5 ml will be collected. Patients, currently on systemic anticoagulation 
(e.g. warfarin therapy), with a platelet count < 50,000 per uL or with clinical signs of systemic 
infection won’t be included. Prior to lumbar puncture, a certified physician and study co-
investigator will perform a cranial nerve exam and fundoscopic exam; lumbar puncture will 
not be performed if evidence of intracranial pressure is identified. This procedure will be 
performed two times for each participant.   
h. Genital tract access 
Sperm will be collected according to the standard operating procedures from our fertility 
department. Cervicovaginal secretions will be harvest as described by Zara et al. (3). This 
procedure will be performed twice for each participant.  
References:  

1. Molina A et al. Blood reviews (2003); 17: 249 
2. Price RW et al. Aids (2001); 15: 1251 
3. Zara F et al. Sexually transmitted infections (2004); 80: 108 
2.2.3 Flowchart  
 
2.3 Description of the laboratory research techniques: 
2.3.1: Cell sorting of the different cell populations: to investigate the viral reservoir in 
different cell subsets, biopsies from each site will be dissociated to a single cell suspension 
and mechanical disruption as described by Shacklett (1). After collagenase I treatment and 
density gradient centrifugation, cell suspensions will be divided into fractions using magnetic 
bead selection and flowcytometer sorting with the FACSJazz, with antibody labelling based on 
cell surface markers. The immune cells can be divided in different fractions based on the 
presence or absence of cell specific markers like CD3, CD4, CD45RA, CCR7, CD27, CD62L, CD95, 
CD122, CD14, CD123, CD71, and CD206. Sorting will be based on available literature describing 
different tissue related cell types that might be important for HIV replication and latency.  
2.3.2 Phylogenetic analysis of the HIV-1 sequences present in the different cellular 
compartments:  
In order to perform phylogenetic tree analysis, acquiring adequate sequencing input is of 
paramount importance. Conventional PCR and subsequent sequencing has important 
limitations when used to analyse mixtures of genetically divergent templates. The taq 
polymerase is prone to switch templates during the amplification process, thereby generating 
recombinants not reflecting the initial sequence. This can be avoided by using single genome 
amplification, initially described by the group of S. Palmer (2). Single genome-sequencing uses 

Gag, Pol and Env sequences. Clonal sequences will be analysed using statistical phylogenetic 
techniques, including both maximum likelihood and Bayesian inference, in collaboration with 
Prof. Chris Verhofstede and Prof. Philippe Lemey (Rega Instituut Leuven, ERC 260864). This 
will allow to determine the compartmentalization structure of the viral population and to 
establish which reservoir sites contribute to the viral rebound upon therapy interruption.  
If virus upon rebound can be linked to other anatomical compartments (while patients are on 
cART), then we will further support the data by identifying if these cellular populations were 
mainly clonal expanded by doing additional integration site sequencing cfr 2.3.4. 
It might be important to approach the central nervous system differently compared to the 
other compartments for two reasons. First, due to the blood-brain barrier this reservoir might 
be more compartmentalized and secondly tissue sampling is not realistic. Most probably we 
will not be able to perform phylogenetic analysis on the lumbar fluid in patients on cART due 
to limited input material. Therefore, we will evaluate this reservoir mainly quantitatively in 
terms of viral rebound dynamics in the cerebrospinal fluid (CSF) compared to rebound in the 
peripheral blood (at day 12). An earlier or higher viral rebound in the CSF would then indicate 
that the replication competent reservoir is likely contained within the brain. 
2.3.3 Quantitative assessment of the HIV-1 viral reservoir in the plasma and in different 
cellular fractions of the different anatomical sites. 
To identify the nature of the replication competent HIV-1 reservoir (active cryptic replication 
versus dormant HIV-1 DNA) we will further characterize the viral reservoir in the body 
compartments containing viral DNA that is most closely related to the rebounding virus. The 
cell populations, sorted from the different tissues will be evaluated using a set of viral reservoir 
assays based on HIV-1 RNA/DNA intermediates, formed during each step of the replication 
cycle. The assays have been translated by my group from in vitro research towards the clinic, 
using state of the art droplet digital PCR technology, allowing absolute ultrasensitive 
quantification. The cellular blood fractions and cellular fractions from the anatomical 
compartments will be separated and subjected to quantification of cell-associated RNA, total 
HIV-1 DNA and 2 LTR circular HIV-1 DNA.  
- 
Our data show that HIV-1 is present in different anatomical compartments throughout 
the body. We will perform total HIV-1 DNA quantification on the different cellular 
fractions to understand the size of the potential reservoir in the different 
compartments. Furthermore, we will evaluate a potential correlation of the size of 
total HIV-1 DNA in the cellular compartment with the identified source of rebound.  
- 
Episomal HIV-2LTR circles are recognised as a marker of ongoing replication. 2LTR 
quantification assays have been transformed from in vitro to in vivo settings, mainly in 
PBMCs (3). Our current data show high levels of 2LTRs in PBMCs of recent 
seroconverters. For the first time we will perform quantification of 2LTR circles in 
different anatomical sites to detect ongoing replication.  

- 
Full-length cell associated RNA forms a marker for HIV-1 DNA transcription and has 
been used in the context of clinical trials aiming at activating the latent reservoir (4). 
We adapted these assays towards a clinical user-friendly ddPCR platform (5). We will 
be the first group to map the HIV-1 transcriptional active sources in the body in 
patients on cART. 
2.3.4: HIV-1 integration site analyses of the viral reservoir in different anatomical sites:  
Integration site analysis will be performed to identify if the replication competent viral 
reservoir mainly clonally expanded (indicating homeostatic proliferation as mechanism for 
viral persistence. CD4+ T cell populations, sorted from the different tissues will be evaluated 
using LAM-PCR/nrLAM-PCR and deep sequencing (6). The LAM-PCR strategy uses a linear 
amplification by PCR with an outward-bound HIV-1 specific primer. These linear amplicons 
containing part of the human genome sequence of the integration site are subsequently 
analyzed by deep sequencing. Our strategy is based on a combination of target capture (TC) 
sample enrichment and next-generation sequencing. Our preliminary data shows integrated 
HIV-1 genome over all the chromosomes. We are currently evaluating the presence of unique 
integration sites. This project will constitute the first study to evaluate integration sites in 
anatomical compartment to map HIV-1 integration in vivo. We have recently optimized an 
assay to identify HIV-1 integration site in the patient genome. Our assay has been validated 
both on HIV-1 infected cells and patient-derived samples. We analysed HIV-1 infected patient-
derived samples before treatment and analysed the integration site pattern.  
2.3.5 Analysis of the HIV-1 methylome in different anatomical sites:  
The HIV-1 methylome of proviral HIV-1 DNA will be assessed by bisulphite conversion and 
deep sequencing using an Illumina platform. Extracted DNA will be bisulphite treated to 
substitute all unmethylated Cytosines into Uracyls. Subsequently, a targeted PCR for the sense 
and antisense (as a control of the native sequence) strand will be performed to specifically 
isolate the HIV-1 CpG islands. Finally, methylation profiles are assessed by deep sequencing, 
enabling the analysis of separate HIV-1 DNA molecules within one sample. 
2.3.6 Therapeutic drug monitoring (TDM): To estimate the viral reservoir in the different 
anatomical compartments, the local concentrations of the administered drugs is of great 
interest. There is not sufficient knowledge on penetration in peripheral tissues of the current 
therapies in use. Some studies show that due to lower concentration of certain antiretrovirals 
in lymphnodes, there is persistent viral replication (7). This source of continuous viral 
replication could be in vivo the origin of relapsing virus at treatment interruption. To 
determine treatment concentrations, we will work in collaboration with Prof. A. Verstraete 
and different techniques as tandem mass spectrometry and ultra performance liquid 
chromatography will be used.  
2.3.7 Inflammatory parameters: we would like to look for biomarkers of axonal injury and 
neurodegeneration in the cerebrospinal fluid. We will collaborate with Prof Magnus Gisslen, 

a specialist in the field, doing additional testing of CSF measuring inflammatory parameters 
like neurofilament protein light, neopterin and CSF/plasma albumin ratio (8). Other markers 
of immune activation in blood and/or CSF can also be assessed for example IL-6, TNF-alpha, 
HLADR+CD38+, CD14s, CRP and beta2microglubuline.  
2.3.8 Stimulation and immunological inhibition assays: VOA (Viral Outgrowth Assay)/TILDA 
(Tat/rev Induced Limiting Dilution Assay).   
Infectious units per million cells- IUPM 
The principal approach for quantifying HIV-1 persistence during antiretroviral treatment (ART) 
is a viral outgrowth assay (VOA) performed on resting CD4 T cells. These cells do not produce 
virus without stimulation. Therefore, resting CD4 T cells are stimulated with 
phytohemagglutinin (PHA) in the presence of irradiated allogeneic peripheral blood 
mononuclear cells. These stimuli induce global T cell activation, which reverses latency at least 
in a fraction of cells carrying integrated HIV-1 genomes. The viruses released from these cells 
are expanded in MOLT4/CCR5 cell line and detected after 2 weeks in the supernatant. The 
frequency of latently infected cells, expressed in terms of infectious units per million (IUPM) 
resting CD4 T cells. In principle, this assay can also detect resting CD4+ T cells harboring labile 
unintegrated forms of HIV-1 DNA, although the frequency of cells containing unintegrated 
DNA during ART is low. Positive wells at each dilution were counted and the maximum 
likelihood method was used to calculate the frequency of cells with inducible replication 
competent HIV-1 (http://bioinf.wehi.edu.au/software/elda). 
 
Tat/rev Induced Limiting Dilution Assay – TILDA 
 Enriched CD4+ T cells are re-suspended at 2 x~ 106 cells/ml in media and rested for 3–5 h at 
37 °C, 5% CO2. CD4+ T cells are stimulated for 12 h with 100 ng/ml PMA and 1 μg/ml 
ionomycin (both from Sigma). The duration of 12 h was based on kinetic experiments 
demonstrating maximal production of tat/rev RNA and preserved cell viability at this time 
point. After stimulation, cells are serially diluted to 18 x~ 106 cells/ml, 9 x~ 106 cells/ml, 3 x~ 
106 cells/ml and 1 x~ 106 cells/ml in culture medium. 
The cell suspension from each serial dilution is distributed in 22 to 24 wells of a 96 well plate 
corresponding to 18,000, 9000, 3000 and 1000 cells per well. Pre-amplification was carried 
out. At the end of the pre-amplification, TE buffer was added to each well and the diluted 
PCR products was used as template for the tat/rev real-time PCR reaction (normal PCR). This 
reaction was performed using a real time PCR (Light Cycler 480, Roche Life Science). Positive 
wells at each dilution were counted and the maximum likelihood method was used to 
calculate the frequency of cells with inducible HIV msRNA. 
(http://bioinf.wehi.edu.au/software/elda). 
 
Biosafety:  All screening tests on patient material, isolation and sorting will be done in L2 
facilities. Stimulation and culture of prelevated cells will be effectuated in an L3 facility.  
 
References 
1. Shacklett BL et al. Methods in molecular biology (2009); 485: 359  
2. Svicher V et al. Current HIV/AIDS reports (2014); 11: 186  

3. Malatinkova E et al. J Clin Microbiol (2014): doi:10.1128/JCM.03087-14 
4. Rasmussen TA et al. Human vaccines & immunotherapeutics (2013); 9: 790  
5. Kiselinova M et al. Plos One (2014); 9: e85999  
6. Schmidt M et al. Nature methods (2007); 4: 1051  
7. Fletcher CV et al. Proceedings of the National Academy of Sciences of the United 
States of America 2014,111:2307-2312. 
8. Jan Jessen Krut et al., Plos One (2014) e88591 
 
3. Risks and advantages 
3.1 Risks  
In this project proposal, we have two important (risk related) ethical questions:  
1) Is it safe to select "healthy volunteers (seropositive people with a normal life)" and  
2) Can a treatment stop be carried out safely?  
These two questions will be further addressed in this document. Informed consent will be 
obtained from each eligible subject prior to each procedure and study staff will transport all 
material for immediate processing at the HIV research virology laboratory. Patients may 
refuse a procedure, but remain enrolled in the study and undergo other tissue sampling, 
and/or analytical treatment interruption. 
3.1.1 Risks related to patient sampling 
For all individual procedures, the individual risk will be discussed with the patient by an open 
communication. For each procedure, the reported individual risks are low (typically 1/1000 
for adverse events in all procedures), and are based on data from sick patients (e.g. lung 
disease for which bronchoscopy is needed). Also the patient's cumulative risk (all procedures 
taken together) will be discussed extensively. Risk estimation per procedure is shown below. 
Endobronchial ultrasound guided lymph node biopsy (EBUS) is considered a safe procedure 
(Rintoul RC et al. Thorax, 2014). A retrospective analysis involving 16.181 patients, reported 
23 serious adverse events (SAE) (0.14%) and 35 adverse events (AE) (by 0.22%) and no 
mortality. With EBUS, infectious SAE were most prevalent (down 0.07%), especially in patients 
with cystic lesions and sarcoidosis. 
Limited data is available for bronchoalveolar lavage (BAL) in patients with no existing 
pulmonary disease, and this technique is considered to be safe in healthy subjects. In the case 
of extensive lavage (immuno-BAL), in patients with severe pulmonary disease, a self-limited 
fever and hypoxemia are the most frequent complications (American Thoracic Society, 2012). 
Bone marrow aspirate: Large data series are published from the members of the British 
Society of Haematologists, reporting 54 890 bone marrow samples, between 1995 and 2001 

(Bain BJ Clinical and laboratory haematology (2004). 26: 315). Total of 26 AE were reported 
directly linked to the procedures. The most frequently reported SAE was bleeding, in 14 
patients, with emergency blood transfusion in 6 patients and leading to death in 1 patient 
(with underlying clotting problems). The most common risk factors associated with bleeding 
were myeloproliferative disease, aspirin use, or both; warfarin therapy, DIC and obesity. Other 
adverse effects are infection, long-lasting discomfort at the biopsy site, and penetration of the 
sternum, which can cause heart or lung problems.  
Possible complications from a lumbar puncture (LP) comprise of: local discomfort with pain at 
the puncture site, during or after the puncture, radicular pain, bleeding, infection and post-
puncture headache. A recent review (Wright et al J Neurol 2012; 259:. 1530-45) reported  
infections and bleeding, and each was seen in less than 0.01% of the cases. The incidence of 
post-puncture headache (PPH) is a fairly frequent complication and its incidence is highly 
dependent on the needle used. If an atraumatic needle is used, PPH is reported on average in 
3-9% of the cases (Wright et al J Neurol 2012; 259:. 1530-45; Lavi et al Neurology 2006; 67; 
1492-1494.). All the punctures within this study will be made with an atraumatic needle. 
Researchers investigated the prevention of PPH in an observational study with cross-sectional 
data analysis (Monteiro de Almeida et al Headache 2011; 51:. 1503-10). Total of 675 LPs were 
performed involving 477 volunteers (575 puncture in HIV-positive individuals and 100 in 
healthy volunteers). PPH was noted in only 0.9% of the subjects. Most cases of PPH (92%) 
recovered spontaneously or with medication. Three individuals were treated by an epidural 
blood patch. In UZ Gent an epidural blood patch is used with a protocol performed by an 
anaesthesiologist with specific expertise. 
Biopsy of the gastrointestinal tract: 
 
A colonoscopy with biopsy carries a risk of bowel perforation in 1/1500. If this occurs, 
the perforation is recognised immediately and treated during the same endoscopy. As 
a result of the administration of analgesics and/or narcotic drugs other side effects can 
occur, such as low blood pressure, nausea, or twists. These are short-lived side effects 
and after a colonoscopy, the patient is kept in observation overnight, with monitoring 
of the pulse and blood pressure. Subjects will be counselled to avoid rectal trauma and 
the use of anticoagulants (e.g. aspirin, NSAIDS) before and after the procedure. 
Mucosal biopsies will not be taken if the participant has an increased risk for 
complications, including receptive anal intercourse within 3 days of the procedure, an 
active anal infection, or recent use of anticoagulants. Biopsies will be performed by 
gastroenterologists who have training and clinical certification in coloscopy/ anoscopy.  
 
The major complication of a gastroscopy with biopsy is aspiration pneumonia 
(<1/1000). The risk of perforation is very small (<1/1500). 

Biopsy of the lymph node: Lymfocele is the main risk in removal of lymph nodes. This 
complication is infrequent (<1%) and can be surgically corrected. The infectious risk is <1% and 
easily treatable. 
Leukapheresis: is considered a safe procedure. All studies available were done in patients with 
hematologic or solid tumours malignancies (ex. Reik, 1997). The most frequent side effect 
(39%) was paresthesia due to citrate-related hypocalcaemia (1 in 10). This could easily be 
managed with oral calcium supplements and/ or slower flow rates. Other reactions included 
hypotension (4%) and headache (1,4%). Patients under antihypertensive treatment, will be 
asked to interrupt this treatment the day of the procedure. The bleeding risk after the 
procedure is transitory increased. There is also risk of catheter problems during procedure. 
Leukapheresis will not take place if screening beforehand reveals poor peripheral venous 
access or electrolyte disturbances. In these cases, the procedure will be replaced by a 
peripheral blood puncture (6x9ml).  
Semen sample and vaginal lavage: these are non-invasive procedures and not associated with 
serious adverse effects.  
Anaesthesia: Most healthy people don't have any problems with general anaesthesia. 
Although many people may have mild, temporary symptoms, general anaesthesia itself is 
exceptionally safe, even for the sickest patients. The risk of long-term complications, much 
less death, is very small. In general, the risk of complications is more closely related to the 
type of procedure, the general physical health, than to the anaesthesia itself. Some of the 
factors that can increase the risk of complications include: smoking, obstructive sleep 
apnoea, obesity, high blood pressure, diabetes, other medical conditions involving your 
heart, lungs or kidneys, medications, such as aspirin, that can increase bleeding, history of 
heavy alcohol use, drug allergies and history of adverse reactions to anaesthesia. Rare 
complications, which may occur more frequently in older adults or in people with serious 
medical problems, include: temporary mental confusion, lung infections, stroke, heart 
attack, and death.  
3.1.2 Risk due to intermittent stop of the medication 
All patients undergoing treatment interruption will be counselled on safer-sex practices as the 
treatment interruption may increase risk of HIV-1 transmission. Only patients that can stop 
therapy safely (high CD4 nadir, no pre-existing resistance, low cardiovascular risk profile,..) 
will be selected for the study. Patients will be restarted on cART (the same regimen as prior to 
interruption) if they have a confirmed viral load >200 copies/mL (two positive measurement 
>200 – the DHHS definition of viral rebound for patients on ART) or a single value greater than 
1000 copies/mL. 
Several recent follow-up studies consider a controlled intermittent stop in the medication as 
safe (Chun TW et al AIDS (2010). 24: 2803, Allard SD et al Clin Immunol (2012) 142:. 252, Good 

G et al AIDS (2013).; 27: 2679; Williams et al eLife JP (2014): e03821). Intermittent stop of the 
treatment was used extensively in the past in HIV research, for example, in the context of 
short-term treatment of acute (recent) HIV-1 infection in order to increase the therapy-free 
interval. In 2007, the SMART study was published in which an increased risk of cardiovascular 
mortality was demonstrated after stopping the medication. It was found that increased 
cardiovascular risk occurs in patients who suffer repetitive therapy stop with low CD4 cell 
counts (<100 CD4/ul) and with very high viral loads. In patients with high CD4 cell counts and 
low viral load before therapy, the stop in therapy is considered safe.  
Therapy stop is the only way to achieve a viral rebound. In addition, there is the perception 
that therapy stop may be associated with viral drug resistance. Viral resistance occurs when 
only sub-therapeutic drug concentrations are present in the body. Viral resistance does not 
occur if therapy is not taken, because there is no selective pressure present, unless the 
antiviral cocktail that was stopped consisted of individual HIV-1 inhibitors with a very different 
half-life. This phenomenon is well known, therefore, I will work together with the virologist 
Prof Chris Verhofstede, and each therapy stop will be guided so that no HIV resistance occurs. 
Patients are eligible only if no drug resistance has been detected in the past. With the current 
availability of different classes of antiretrovirals, an ART switch can help directly when a case 
of viral resistance occurs. Treatment history of the patients will be taken into account at 
inclusion, to be sure that a sufficient number of ART options remain, if necessary, to exchange 
the cART regime. 
3.1.3 Risk of not reaching enough study participants:  
This question rises whether this study will find enough motivated participants to participate. 
Therefore, an initial inquiry with several patients (N = 10) (on the advice of the Ethics 
Committee in UZ Gent) has already shown us that patients are indeed motivated to engage in 
such a study, provided that they are adequately informed about the scientific design and gain 
of the experiments. The burden of the intensive lifelong intake of ART medication causes HIV-
infected patients, more than healthy volunteers, to actively participate in studies that 
contribute to the search for an HIV cure. 
3.1.4 Risk of misperception of the study 
An important ethical conundrum lay in the false perception patients may have with regard to 
this project. These “therapeutic misconception and misestimation” are well known concepts 
in bioethics. Patients that participate in this study’s might perceive the wrong expectations 
from the study. Although there will be no medical intervention (no admission of a study drug), 
the patient might perceive this study as having an impact on his ‘HIV reservoir’. They lead 
potential participants and/or practitioner to over-consider the very theoretical advantage 
(participating in a Cure diagnostic study) and under-consider the side effects of the study. It 
will be a matter of explaining the study correctly and designing a good balanced informed 

consent that will be readily understandable to the potential participants. 
4. Study analysis  
4.1 Sample size calculation:  
The number of patients (N=10) that we will investigate exceeds by far the numbers reported 
in previous studies on HIV-1 reservoir dynamics, and should provide ample power to reveal 
consistent phylogenetic patterns while still adhering to the budget constraints.  
4.2 Data analysis:  
Samples and results will be analyzed together with Prof Linos Vandekerckhove and Ward 
Despiegelaere at Ghent University.  
For phylogenetic analysis we will collaborate with Prof. Chris Verhofstede and Prof. Philippe 
Lemey.  
For TDM we will work in collaboration with Prof. A. Verstraete.  
4.3 Statistical analysis:  
Because of the small sample size of this cohort, we will only be able to look at individual 
results and perform non-parametric statistics on the cohort. Statistical significance (P values) 
of the results will be calculated by two tailed student t-test using SPSS. Correlations among 
variables will be tested by regression analysis. Proportions will be compared with Fisher’s 
exact test using GraphPad Prism version 3.0 (GraphPad Software, San Diego, CA, USA). The 
level of significance will be set at P < 0.05 and Bonferroni adjustment of P-values will be 
applied in case of multiple testing. 
For phylogenetic analysis clonal sequences will be analysed using statistical phylogenetic 
techniques, including both maximum likelihood and Bayesian inference.  
4.4 Storage and Shipment of the samples 
All processed samples will be stored at a separate facility Biometra Biobank, UGent at -80 in 
FCS for further analysis. 
DNA and RNA isolates will be stored at -20°C.  
It is possible that samples will be shipped to internationally collaborating laboratories for 
assays described in this protocol.  
5. Duration and termination of study: 
The clinical study (including sampling and therapy-stop) will be performed over a timeframe 
of maximum 2 years. We expect to finish the analysis in 4 years. We hope to include patients 
starting from the 1st of January 2016 until the 1st of January 2018. Participation in this study is 
voluntary. Patients can refuse to participate or ask to end the participation before the final 
closure of the study, at any time. Patients themselves can be withdrawn from the study if they 
are not acting according to the procedures described or are not respecting the conditions.  

We will start recruiting patients when approval of EC has been obtained and aim to include 
patients in the study before the beginning of March 2016. Together with the patients and with 
the collaborating disciplines a date for admission in the hospital will be arranged. Patients will 
stay overnight in the hospital during the sampling phase.  
Approximately 3 months after initial sampling patients will be scheduled for a guided 
treatment interruption, followed by a smaller sampling after rebound, requiring again one 
night of observation in the hospital. After sampling the patient will restart therapy and will be 
followed-up closely until undetectable viral load.  
During therapy stop a 2-3 times a week visit will be necessary.  
 
6. Informed consent 
The participant must personally sign and date the latest approved version of the informed 
consent form before any study specific procedures are performed. Written and verbal versions 
of the Participant Information Sheet and Informed Consent will be presented to the 
participants detailing no less than: the exact nature of the study; the implications and 
constraints of the protocol; the known side effects and any risks involved in taking part. It will 
be clearly stated that the participant is free to withdraw from the study at any time for any 
reason without prejudice to future care, and with no obligation to give the reason for 
withdrawal. 
 
7. Costs and compensation for participating  
Participation in this trial will not result in any additional cost to the patients and an additional 
compensation for the patients will be foreseen (400 euro/d of sampling). 
 
8. Confidentiality 
In accordance with the Belgian law concerning the private life protection (08 Dec 1992) and 
the patient’s rights (22 Aug 2002) the information collected from participation in this study is 
protected. The participants have the right to request information of the existence of personal 
data held by the study coordinator and will have the right to rectify erroneous or inaccurate 
data. Representatives of the study coordinators, the Independent Ethical Committee and/or 
Regulatory Authorities will be granted direct access to the original medical records for 
verification of the clinical trial procedures and/or data, without violating the confidentiality 
according to the laws and regulations applicable in Belgium. By signing this informed consent 
form, participants are authorizing such access. 
From patients agreeing to participate in this study, personal data and clinical information will 
be collected and coded. No reports containing the personal data will be publicly available. 
9. ICH/GCP guidelines 
This trial will be conducted in accordance with the protocol, current ICH-GCP guidelines and 
applicable law(s).Good Clinical Practice (GCP) is an international ethical and scientific quality 

standard for designing, conducting, recording and reporting trials that involve the 
participation of human subjects. Compliance with this standard provides public assurance 
that the rights, safety and well being of trial subjects are protected, consistent with the 
principles that have their origin in the Declaration of Helsinki, and that the clinical trial data 
are credible. 
10. Insurance 
The coordinators of this study are liable, even without fault, for damage that the participants 
or legal successor has sustained and that has direct or indirect link with the trial. To that end 
the study has provided insurance coverage. 
Patients in follow-up at the Ghent University Hospital are covered by the insurance of the 
Ghent University Hospital.  
 
11. Dissemination 
The study outcome and data will be published in peer-reviewed journals, aiming the highest 
impact factor in the field.  
 
Contact person 
Prof. Dr. VANDEKERCKHOVE LINOS (09/3323398) 
Dr. De Scheerder Marie-Angélique (09/3321349) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Signature Page  
 
Investigator:  

 
Name: Prof. Dr. Linos Vandekerckhove  
 
Signature:  
 
Date: