A Phase IIa Randomized, Placebo Controlled, Double Blinded Study to Evaluate the Safety and Immunogenicity of iHIVARNA-01 in Chronically HIV-infected Patients Under Stable Combined Antiretroviral Therapy
Phase: PHASE2
Conditions: HIV Infections
Sponsor: Rob Gruters

EudraCT 2016-002724-83   
phase IIa 
v3.1 
 
 
 
 
 
 
 
 
 
 
 
 
RESEARCH PROTOCOL  
iHIVARNA phase IIa 
A phase IIa randomized, placebo controlled, double blinded study to evaluate the safety and immunogenicity of 
iHIVARNA-01 in chronically HIV-infected patients under stable combined antiretroviral therapy. 
(14-APR-2017) 
 
NCT02888756 

EudraCT 2016-002724-83   
phase IIa 
v3.1 
 
 
 
PROTOCOL TITLE ‘iHIVARNA phase IIa’ 
Protocol ID 
A phase IIa randomized, placebo controlled, double blinded study to 
evaluate the safety and immunogenicity of iHIVARNA-01 in 
chronically HIV-infected patients under stable combined 
antiretroviral therapy 
Short title 
iHIVARNA phase IIa study 
EudraCT number  
2016-002724-83 
Version 
3.1 
Date 
14-APR-2017 
Coordinating investigator/ 
project leader 
 
 
Contact person for Sponsor 
Dr Felipe Garcia, Infectious Diseases Service, Hospital Clinic 
C/Villarroel 170, 08036 Barcelona, Spain  
Phone: +34 93 227 54 00 ext. 2884 Fax.: +34 93 451 44 38  
E-mail: fgarcia@clinic.ub.es  
Dr Rob Gruters, Erasmus MC, Wytemaweg 80, 3015 CN 
Rotterdam, The Netherlands, +31 613 508 033, 
r.gruters@erasmusmc.nl 
Principal investigator(s)  
 
 
 Erasmus MC, Rotterdam, the Netherlands,  Dr Eric van Gorp 
Hospital Clinic, Barcelona, Spain; Dr Lorna Leal 
Hospital Universitari Germans Trias i Pujol, Barcelona, Spain; Dr 
Beatriz Mothe 
UZ Brussel, Brussels, Belgium; Dr Sabine Allard 
ITM, Antwerp, Belgium; Dr Eric Florence 
Sponsor 
Erasmus MC, Rotterdam, The Netherlands 
Subsidising party 
European Commission FP7 
Independent expert (s) 
Dr P van Genderen, Havenziekenhuis, Rotterdam 
Prof Dr K Brinkman, OLVG, Amsterdam 
Dr D Rizopoulos, Erasmus MC, Rotterdam 
Laboratory sites  
Lab retrovirologia i immunopatogènia viral IDIBAPS 
Lab IRSICAIXA (immunology, biochemistry, hematology, microbiology) 
Lab ITM (HIV/STD reference laboratory, immunology, virology, CLKB) 
Lab Erasmus MC (Viroscience, immunology, chemistry) 
Lab VUB (Laboratory Molecular Cellular Therapy) 
Lab UZ Brussel (AIDS reference laboratory, clinical biology) 
Pharmacy  
Department of Pharmacy, Erasmus MC 
Partner 
eTheRNA immunotherapies NV 

EudraCT 2016-002724-83   
phase IIa 
v3.1 
 
 
 
PROTOCOL SIGNATURE SHEET 
 
Name 
Signature 
Date 
Sponsor or  
legal representative: 
 
 
 
Prof Dr M.P.G. Koopmans, DVM, 
Head, department of Viroscience, 
Rotterdam 
 
 
 
 
 
Coordinating Investigator: 
 
Dr Felipe Garcia MD, Head, 
Infectious Diseases Service 
Hospital Clinic, Barcelona 
 
 
 
 
 
 
 

EudraCT 2016-002724-83   
phase IIa 
v3.1 
 
 
 
TABLE OF CONTENTS 
1. 
INTRODUCTION AND RATIONALE ............................................................................... 12 
2. 
OBJECTIVES ................................................................................................................ 15 
3. 
STUDY DESIGN ........................................................................................................... 16 
4. 
STUDY POPULATION .................................................................................................. 17 
4.1 
Population (base) ........................................................................................................... 17 
4.2 
Inclusion criteria ............................................................................................................. 17 
4.3 
Exclusion criteria ............................................................................................................ 18 
4.4 
Sample size calculation ................................................................................................... 19 
5. 
TREATMENT OF SUBJECTS .......................................................................................... 20 
5.1 
Investigational product/treatment ................................................................................. 20 
5.2 
Use of co‐intervention .................................................................................................... 21 
5.3 
Escape medication ......................................................................................................... 21 
6. 
INVESTIGATIONAL PRODUCT ..................................................................................... 22 
6.1 
Name and description of investigational product(s) ........................................................ 22 
6.2 
Summary of findings from non‐clinical studies ............................................................... 22 
6.3 
Summary of findings from clinical studies ....................................................................... 25 
6.4 
Summary of known and potential risks and benefits ...................................................... 26 
6.5 
Description and justification of route of administration and dosage ............................... 27 
6.6 
Dosages, dosage modifications and method of administration ....................................... 28 
6.7 
Preparation and labelling of Investigational Medicinal Product ...................................... 28 
6.8 
Drug accountability ........................................................................................................ 29 
7. 
NON‐INVESTIGATIONAL PRODUCT ............................................................................. 30 
7.1 
Name and description of non‐investigational product(s) ................................................ 30 
7.2 
Summary of findings from non‐clinical studies ............................................................... 30 
7.3 
Summary of findings from clinical studies ....................................................................... 30 
7.4 
Summary of known and potential risks and benefits ...................................................... 30 
7.5 
Description and justification of route of administration and dosage ............................... 30 
7.6 
Dosages, dosage modifications and method of administration ....................................... 30 
7.7 
Preparation and labelling of Non Investigational Medicinal Product ............................... 30 
7.8 
Drug accountability ........................................................................................................ 30 
8. 
METHODS .................................................................................................................. 31 
8.1 
Study parameters/endpoints .......................................................................................... 31 
8.1.1 
Main study parameter/endpoint .................................................................................. 31 
8.1.2 
Secondary study parameters/endpoints ....................................................................... 31 
8.1.3 
Other study parameters (if applicable) ......................................................................... 32 
8.2 
Randomisation, blinding and treatment allocation ......................................................... 32 
8.3 
Study procedures ........................................................................................................... 34 
8.4 
Treatment discontinuation and study withdrawal of individual subjects ........................ 40 
8.4.1 
Specific criteria for withdrawal (if applicable) ............................................................... 40 

EudraCT 2016-002724-83   
phase IIa 
v3.1 
 
 
 
8.5 
Replacement of individual subjects after withdrawal ..................................................... 41 
8.6 
Follow‐up of subjects withdrawn from treatment .......................................................... 41 
8.7 
Premature termination of the study ............................................................................... 41 
9. 
SAFETY REPORTING .................................................................................................... 43 
9.1 
Temporary halt for reasons of subject safety .................................................................. 43 
9.2 
AEs, SAEs and SUSARs .................................................................................................... 43 
9.2.1 
Adverse events (AEs) ..................................................................................................... 43 
9.2.2 
Serious adverse events (SAEs) ....................................................................................... 43 
9.2.3 
Suspected unexpected serious adverse reactions (SUSARs) ......................................... 45 
9.3 
Annual safety report ...................................................................................................... 46 
9.4 
Follow‐up of adverse events ........................................................................................... 46 
9.5 
Data Safety Monitoring Board (DSMB) ........................................................................... 46 
10. 
STATISTICAL ANALYSIS ........................................................................................... 48 
10.1 
Primary study parameter(s) ............................................................................................ 48 
10.2 
Secondary study parameter(s) ........................................................................................ 48 
10.3 
Other study parameters ................................................................................................. 49 
10.4 
Interim analysis .............................................................................................................. 49 
11. 
ETHICAL CONSIDERATIONS ..................................................................................... 51 
11.1 
Regulation statement ..................................................................................................... 51 
11.2 
Recruitment and consent ............................................................................................... 51 
11.3 
Objection by minors or incapacitated subjects ............................................................... 51 
11.4 
Benefits and risks assessment, group relatedness .......................................................... 51 
11.5 
Compensation for injury ................................................................................................. 52 
11.6 
Incentives ....................................................................................................................... 52 
12. 
ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION ............................... 53 
12.1 
Handling and storage of data and documents ................................................................. 53 
12.2 
Monitoring and Quality Assurance ................................................................................. 53 
12.3 
Amendments.................................................................................................................. 54 
12.4 
Annual progress report ................................................................................................... 54 
12.5 
Temporary halt and (prematurely) end of study report .................................................. 54 
12.6 
Public disclosure and publication policy ......................................................................... 55 
13. 
STRUCTURED RISK ANALYSIS .................................................................................. 56 
13.1 
Potential issues of concern ............................................................................................. 56 
13.2 
Synthesis ........................................................................................................................ 58 
14. 
REFERENCES ........................................................................................................... 59 

 
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LIST OF ABBREVIATIONS AND RELEVANT DEFINITIONS  
 
ABR 
ABR form, General Assessment and Registration form (application form required for 
submission to accredited Ethics Committee; In Dutch, ABR = Algemene Beoordeling en Registratie) 
AE 
Adverse Event 
APC 
Antigen Presenting Cells 
AR 
Adverse Reaction 
ATI 
Antiretroviral Treatment Interruption 
CA 
Competent Authority 
CA-HIV 
HIV Capacity Assay 
CA RNA 
Cell-associated RNA 
cART 
Combined anti-retroviral therapy 
CCMO 
Central Committee on Research Involving Human Subjects  
(in Dutch: Centrale Commissie Mensgebonden Onderzoek) 
CD40L 
CD (cluster of differentiation) 40 ligand 
CD70 
Cluster of differentiation 70 
(e)CRF 
(electronic) Case Report Form 
CRO 
Clinical Research Organization 
CV 
Curriculum Vitae 
DC 
Dendritic Cells 
DSMB 
Data Safety Monitoring Board 
EDTA 
Ethylenediaminetetraacetic acid 
EU 
European Union 
EudraCT European drug regulatory affairs Clinical Trials  
Fluc 
GCP 
Firefly luciferase 
Good Clinical Practice 
GMP 
Good Manufacturing Practice 
HBsAg 
HCP 
Hepatitis B surface antigen 
Hospital Clinic Provincial 
HLA 
IB 
Human Leukocyte Antigen 
Investigator’s Brochure 
IC(F) 
Informed Consent (Form) 
ICS 
Intracellular cytokine-staining 
IMP 
Investigational Medicinal Product  
IMPD 
Investigational Medicinal Product Dossier  
ITT 
Intention to treat 

 
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IUD 
Intrauterine Device (contraceptive device containing copper or levonorgestrel) 
IUS 
Intrauterine System (see: IUD) 
METC  
Medical research ethics committee (MREC)  
(in Dutch: Medisch Ethische Toetsing Commissie (METC) 
mRNA 
Messenger Ribonucleic Acid 
NNRTI 
NSAID 
Non-Nucleoside Reverse Transcriptase Inhibitor 
Nonsteroidal anti-inflammatory drug 
PI 
Protease Inhibitor 
PrEP 
Pre-Exposure Prophylaxis 
PVL 
Plasma Viral Load 
(S)AE 
(Serious) Adverse Event  
SOP 
Standard Operating Procedure 
Sponsor 
Party that commissions the organisation or performance of the research, e.g. an 
academic hospital. A party that provides funding for a study but does not 
commission it is not regarded as the sponsor, but referred to as a subsidising 
party. 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
caTLR4 
Constitutively active Toll-Like Receptor 4 
UAR 
Unexpected Adverse Reaction 
WBP 
Personal Data Protection Act (in Dutch: Wet Bescherming Persoonsgevens) 
WFI 
Water for injection 
WMO 
Medical Research Involving Human Subjects Act  
(in Dutch: Wet Medisch-wetenschappelijk Onderzoek met Mensen) 

 
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SUMMARY 
 
Rationale: iHIVARNA-01 is a novel therapeutic vaccine for the treatment of HIV-1-infected 
patients based on in vivo modification of DCs. It consists of HIVACAT-TriMix: mRNA 
encoding a mixture of APC activation molecules (CD40L, a constitutively active variant of 
TLR4 and CD70) and the HIV target antigens contained in HIVACAT to be administered 
through the intranodal route. iHIVARNA-01 aims to achieve the ‘functional cure’ of HIV 
infection, i.e. controlling viral replication in the absence of anti-retroviral therapy. 
 
Objective: To evaluate the safety and immunogenicity of iHIVARNA-01 as a new therapeutic 
vaccine in HIV infected patients. 
 
Study design and duration: Phase IIa, multicentre double-blind placebo controlled 
intervention study. Each patient will be followed for 30 weeks. The study duration will be 38 
weeks from inclusion of the first patient. 
 
Sites: Erasmus MC, Rotterdam The Netherlands (sponsor), Hospital Clínic de Barcelona 
and Institut de Recerca de la Sida – Caixa, Barcelona, Spain, Instituut voor Tropische 
Geneeskunde Antwerp, Belgium and Vrije Universiteit Brussel/UZ Brussel, Belgium 
 
Study population: Chronically HIV-1- infected patients under stable cART with plasma viral 
load (pVL) ≤ 50 copies/ml and stable CD4+ T-cell counts ≥ 450/μl, aged 18 years or above. 
 
Sample size: after recruitment and screening, 70 patients will be included and randomized 
to one of the study-arms. 
 
Intervention: One group (n=40) receives the HIVACAT-TriMix (300 microgram TriMix + 900 
microgram HIVACAT) vaccine intranodally on three occasions with a two-week interval. One 
control group (n=15) receives TriMix only (300 microgram TriMix) and one group (n=15) 
receives saline intranodally on three occasions with a two-week interval. Two weeks after the 
last vaccination cART treatment will be interrupted. If plasma virus is detectable, cART will 
be re-initiated twelve weeks after treatment interruption. cART can always be re-initiated for 
medical reasons, as judged by the clinical investigator. 

 
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Figure; Study design and intervention scheme (arrows indicate intranodal vaccination) 
 
Main study parameters/endpoints:  
The primary endpoints of the study are: 
 
safety and tolerability, as measured by the total number of adverse events.  
 
immunogenicity of an immunization schedule with HIVACAT-TriMix as measured by 
changes in frequency of the HIV-specific T-cell responses between baseline and 2 
and 14 weeks after the last injection (i.e. weeks 6 and 18 of the study) as compared 
to both control groups (immunized with TriMix or placebo only) 
The secondary endpoints are: 
 
magnitude and the kinetics of the HIV-specific CD4+ and CD8+ T cell responses after 
immunization (ELISPOT) as defined by the increase in the number of spot forming 
units from baseline and if positive the number of poly-functional T cells as determined 
by intracellular cytokine staining (ICS) at weeks 6 and 30. 
 
time until viral rebound after discontinuation at week 6. 
 
plasma viral load in vivo after analytical treatment interruption (ATI) 
 
functional cure: proportion of patients with viral load below detectable level after ATI 
 
primary immune response against vaccine: Change in frequency of at least 0.7log10 
HIV-specific T-cell responses between baseline and week 6 
 
the in vivo vaccine-induced capacity of ex-vivo CD8 T cells to suppress HIV growth in 
autologous CD4 T cells i.e. “CD8 T cell mediated viral suppression”   
 
effect on reservoir as measured by changes in the proviral DNA copy numbers per 
million cells and the intracellular viral RNA copy numbers per million cells during and 
after immunization.  

 
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 
viral immune escape: change in % mutated epitopes from pre-cART to post-ATI 
(week 18) 
 
host protein mRNA expression profiles in whole blood  
 
 
Nature and extent of the burden and risks associated with participation, benefit and 
group relatedness:  Participation to the trial will consist of 15 visits to the hospital for a 
estimated total of 7 hours for vaccinations and exams, during a 30-week period. During these 
visits blood will be drawn for various assays. A total of 926ml blood will be taken over a 30-
week period (see appendix B).  
 
Before inclusion a physical screening will be performed. The patient will be asked to fill in a 
diary card to record local and systemic adverse events during one week after each 
vaccination.  
Discomfort can be experienced as a result of intranodal vaccination including: local injection 
site reactions: pain, itching, redness/discoloration or fluid/blood filled blisters at the injection 
site. Hard swelling in skin surface at or close to site. General/ systemic reactions may 
include: temperature rise, chills/rigors, malaise, tiredness muscle aches, headache, nausea 
or vomiting. 
 
As a result of treatment interruption a viral rebound syndrome may be experienced, with 
symptoms similar to those of acute HIV infection. Symptoms may include fever, fatigue, 
pharyngitis, lymphadenopathy, rash and weight loss. 
 
The administration of the investigational medicinal product (IMP) has been tested in the 
phase I clinical trial. Based on interim analysis of July 26, 2016: twenty-one volunteers were 
recruited in Hospital Clinic Provincial (HCP) in Barcelona from June 23, 2015 until March 29, 
2016. No patients have discontinued the trial. 
 
ARM I 
n=3 
100µg TriMix mRNA 
ARM II 
n=3 
300µg TriMix mRNA 
ARM III 
n=3 
600µg (300µg HIV mRNA +300µg TriMix mRNA) 
ARM IV 
n=6 
900µg (600µg HIV mRNA + 300µg TriMix mRNA) 
ARM V 
n=6 
1200 (900μg HIV mRNA + 300μg TriMix mRNA) 
 
The mean and median age was 46 years (range 28-59). Five (24%) were female and 16 
were male (76%). Overall, the vaccine was well tolerated. A total of 17 adverse events (AE) 
were reported during follow-up until July 12th, 2016 (4, 4, 5, 4 and 0 in each ARM 
respectively). Ten AE were grade 1, 6 grade 2 and 1 grade 3. Of these AE, 2 were definitely 
related (2 grade 1) and 5 were possibly related (2 grade 1, 3 grade 2) to the IMP. 

 
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Data handling and storage: According to international regulations, all data collected in the 
case report form (CRF) should be verifiable against the source documents. In addition, the 
sponsoring investigator will keep the trial master file that will contain all case report forms, 
data correction forms, templates, monitoring records and visit scheduling, regulatory filings 
(e.g. signed protocol, amendments, correspondence with Ethics Committee, approval, 
approved version of patient information sheet, signed patient consents, investigator 
agreement, regulatory agency authorization, etc.). These files must be kept in accordance 
with local regulations in each country. 
 
Data monitoring: Data will be monitored by a CRO: CR2O (Clinical Research Rotterdam 
B.V., Rotterdam, the Netherlands), based on the results of the monitoring plan. Visits to the 
clinical sites in The Netherlands, Belgium and Spain will be organized regularly. 
 
Data safety monitoring board: A DSMB has been installed that will overview the results 
from safety monitoring of all 5 study sites.  
 
Statistical analysis: Proportions will be compared between groups using logistic regression 
and continuous variables using linear or quantile regression. For longitudinal data, mixed 
effects models will be fitted. Time to event data will be presented by Kaplan-Meier curves 
and analysed using Cox proportional hazards models. 
 

 
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1. INTRODUCTION AND RATIONALE 
 
The human immunodeficiency virus (HIV) is a single-stranded positive-sense RNA virus 
belonging to the lentivirus genus. It mainly infects human dendritic cells (DCs), macrophages 
and CD4+T cells where it integrates into the host genome after reverse transcription to 
double-stranded DNA. In the absence of antiretroviral therapy, infection progresses to 
acquired immunodeficiency syndrome (AIDS) as defined by progressive depletion of CD4+ T 
cells. Patients with advanced HIV infection have a CD4+ T-cell count below 50/mm3 and 
their median survival is between 12 to 18 months in the absence of antiretroviral therapy (1).  
The HIV pandemic has caused as much as 35 million deaths worldwide. As of December 
2013, 35 million people were estimated to be infected with HIV, from which 23.5 million were 
living in Sub-Saharan regions (2).  
Current therapy for HIV infection is based on combined anti-retroviral treatment (cART). HIV 
infected patients, which are currently treated with cART show a stabilized condition with no 
clinical progression. However, cART has to be maintained for life, since it is not able to 
eradicate the infection by itself (3). This represents very high costs for administrations 
together with difficulties for treatment adherence and widespread distribution, mainly in 
developing countries, but also in the developed world. Therefore, there is a clear unmet need 
regarding cost-effective and viable treatments for HIV-infected patients. Therapeutic 
vaccination has emerged as an attractive strategy aiming at achieving a ‘functional cure’, a 
situation in which the immune system is able to control viral replication without the need for 
cART.  
In this regard, several antigenic molecules, administration routes and strategies have been 
employed, so far with limited success. The most promising results to date were obtained by 
Garcia et al. (4) (partner of the consortium iHIVARNA). In this clinical trial, HIV-infected 
patients were administered autologous dendritic cells pulsed with HIV viral particles, a 
strategy known as ‘ex vivo modification of DCs’. The results of this clinical trial indicate that 
DC-based vaccines offer a promising strategy for therapeutic vaccination. DC-based 
vaccines rely on the ability of DCs to uptake and present antigens to CD4+ and CD8+ T 
cells, eliciting an immune response that should ultimately control viral replication by lysing 
infected cells. In fact, in a small percentage of individuals who are known to efficiently 
suppress viral replication in the absence of cART (known as ‘elite controllers’), control of viral 
replication has been  associated with effective CD8+T cell responses (5).  
Although results obtained by Garcia et al. (4) demonstrated a reduction of 90-95% of pVL in 
HIV-infected patients, ‘functional cure’ (a 100% reduction of pVL) was not achieved. In 
addition, the use of ex vivo modification of DCs is a technical challenge for widespread use 
and a very costly strategy.  

 
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For that reason, iHIVARNA-01, which is the current product under development, is based on 
the so-called ‘in vivo modification of DCs’, in which the product is directly administered to the 
patient without the need to modify DCs ex-vivo. 
The intended indication for iHIVARNA-01 is the therapeutic vaccination of HIV-infected 
patients. iHIVARNA-01 is a biological product that consists of naked mRNA to be 
administered through the intranodal route. 
The product is a combination of mRNA sequences that fulfil two main objectives: 1) providing 
effective HIV antigens for specific T cell activation that will lead to protective immunity 
(HIVACAT mRNA sequence) and 2) providing adequate stimuli required for the activation of 
antigen presenting cells (APCs) (DCs) and co-activation of specific T cells (TriMix mRNA 
sequences) (6). 
 
Thus, iHIVARNA-01 has been developed as a combination of two active substances:  
 
HIVACAT immunogen: confers specificity to key HIV regions designed to provide 
beneficial T cell immunity associated with viral control and ensuring broad HLA class I 
and class II coverage (7).  
 
TriMix: provides the necessary activation molecules to enhance antigen presentation 
by DCs and T cell activation. It is a combination of mRNA sequences corresponding 
to human CD40L, constitutively active TLR4 (caTLR4) and human CD70 (8) 
 
The iHIVARNA-01 investigational product, specified as ratio TriMix:HIVACAT = 1:3 (300μg 
TriMix + 900μg HIVACAT in 200 μl water for injection (WFI), is the result of the Phase 1 
iHIVARNA trial where iHIVARNA-01 was tested as three IMPs with mixed ratios of 
TriMix:HIVACAT (study) versus TriMix solution (control).   
 
Data based on interim analysis of July 26, 2016: twenty-one volunteers were recruited in 
Hospital Clinic Provincial (HCP) in Barcelona from June 23, 2015 until March 29, 2016. No 
patients have discontinued the trial. 
 
ARM I 
n=3 
100µg TriMix mRNA 
ARM II 
n=3 
300µg TriMix mRNA 
ARM III 
n=3 
600µg (300µg HIV mRNA +300µg TriMix mRNA) 
ARM IV 
n=6 
900µg (600µg HIV mRNA + 300µg TriMix mRNA) 
ARM V 
n=6 
1200 (900μg HIV mRNA + 300μg TriMix mRNA) 
 
 
 

 
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The mean and median age was 46 years (range 28-59). Five (24%) were female and 16 
were male (76%). Overall, the vaccine was well tolerated. A total of 17 adverse events (AE) 
were reported during follow-up until July 12th, 2016 (4, 4, 5, 4 and 0 in each ARM 
respectively). Ten AE were grade 1, 6 grade 2 and 1 grade 3. Of these AE, 2 were definitely 
related (1 grade 1 and 1 grade 2) and 5 were possibly related (2 grade 1, 3 grade 2) to the 
IMP. 
Table: Overview of all adverse events in phase I trial. 
 
 
 

 
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2. OBJECTIVES 
Primary objectives:  
 
 
To evaluate the safety and tolerability of intranodal iHIVARNA-01 vaccination 
compared with TriMix or placebo, focusing on the nature, frequency and severity of 
local adverse events (pain, cutaneous reactions including induration) and systemic 
adverse events (temperature, chills, headache, nausea, vomiting, malaise, and 
myalgia). 
 
To evaluate the immunogenicity of an immunization schedule with HIVACAT-TriMix 
(iHIVARNA-01) to increase the frequency of HIV-specific T-cell responses between 
baseline and 2 and 14 weeks after the last injection (i.e. weeks 6 and 18 of the study) 
as compared to the control groups, immunized with TriMix-mRNA only or WFI only. 
 
Secondary Objectives:  
 
 
To evaluate the magnitude and the kinetics of the HIV-specific CD4+ and CD8+ T cell 
responses generated by the immunization schedule in the 3 groups by two methods 
(ELISPOT, intra-cellular cytokine staining - ICS) at baseline and at weeks 6 and 30. 
 
To evaluate the ability of the immunization schedules to prolong time until viral 
rebound after discontinuation at W6 as compared to control groups TriMix or 
placebo. 
 
To evaluate the suppressive effect on plasma viral load in vivo after ATI from W6 
to W18 compared to two control groups, receiving TriMix only or placebo. 
 
To assess the proportion of patients with control of viral load below detectable level 
12 and 24 weeks after start ATI (functional cure). 
 
To evaluate the percentage of patients who generate a primary immune response 
to previously not-recognized HIV peptides (as defined in 8.1.2)  
 
To analyze induction or enhancement of the CD8+ T-cell HIV suppressive capacity. 
 
To evaluate the effect on reservoir as measured by proviral DNA and the 
intracellular viral RNA (unspliced and multiple spliced viral RNA). 
 
To detect viral immune escape by sequencing of the HIV and conducting sieve 
effect analyses in rebounding virus after cART interruption. 
 
To evaluate host protein mRNA expression profiles in whole blood at baseline and 
W6 and W18. 
 
To store samples for future research to relate gut microbiota composition and 
diversity to HIV immune status 

 
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3. STUDY DESIGN 
 
This is a phase IIa, randomized, double-blind, placebo-controlled outpatient clinic based 
multicentre study in chronically HIV-1 infected patients under stable cART. Enrolled and 
randomized patients will be treated in 1 of the 3 study-arms, comprising 2 stages. 
Randomization will be stratified per center, in order for all centers to have an equal number 
of cases and controls. 
 
Stage I: Immunization period from W0 to W6, while on cART. The immunization will be 
administered by intranodal injection. Patients will be randomized to receive: 
 
Arm A (study treatment: HIVACAT-TriMix)   
one injection of HIVACAT-TriMix at W0, W2 and W4 while on cART  
(n= 40) 
 
Arm B (controls TriMix) 
one injection of TriMix at W0, W2 and W4 while on cART   
 
(n= 15) 
 
Arm C (controls buffer solution/placebo) 
one injection of buffer solution at W0, W2, and W4 while on cART 
(n= 15) 
Stage II: Post immunization and analytical treatment interruption (ATI) period from W6 to 
W18, off cART, patients with undetectable level of viral load at week 18 will remain off-cART 
up to week 30. At week 30 the study ends, if a patient still has an undetectable viral load, he 
or she may continue cART interruption in consultation with the treating physician. cART can 
be re-initiated for medical reasons at any time, as judged by the clinical investigator. 
 
Figure: phase IIa treatment arms 
Figure: study flowchart  

 
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4. STUDY POPULATION 
4.1 Population (base)  
The study will be conducted in chronically HIV-1 infected patients under stable cART with 
plasma viral load ≤ 50 copies/ml and stable CD4+ T-cell counts ≥ 450 cells. Patients will 
be recruited via the outpatient clinic in the five hospitals that participate in the study. 
Patients will be screened for eligibility after obtaining informed consent. A total of 70 
patients will be randomized into 3 different groups (one study group (n=40) and two 
control groups (both n=15)). Every participating hospital will include 14 patients from their 
outpatient clinic-visiting group of 700-4000 HIV-1 infected patients.  
 
4.2 Inclusion criteria 
In order to be eligible to participate in this study, a patient must meet all of the following 
criteria: 
1. ≥ 18 years of age; 
2. Voluntarily signed informed consent; 
3. Proven HIV-1 infection (with documented antibodies against HIV-1 and a detectable 
plasma HIV-1 RNA before initiation of therapy); 
4. On stable treatment with cART regimen (antiretroviral therapy consisting of at least 
three registered antiretroviral agents) for at least 3 years; 
5. Nadir CD4+ ≥ 350 cells/μl (up to 2 occasional determinations ≤ 350 cells/μl are 
allowed); 
6. Current CD4+ cell count ≥ 450 cells/μl; 
7. HIV-RNA below 50 copies/mL in the last 6 months prior to randomization, during at 
least two measurements (occasional so called ‘blips’ ≤ 500 copies/mL are permitted); 
8. If sexually active, willing to use a reliable method of reducing the risk of transmission 
to their sexual partners during treatment interruption (including PrEP).  
a. For heterosexually active female, using an effective method of contraception 
with partner (combined oral contraceptive pill; injectable or implanted 
contraceptive; IUD/IUS; consistent record with condoms; physiological or 
anatomical sterility (in self or partner) from 14 days prior to the first vaccination 
until 4 months after the last vaccination.  
b. For heterosexually active male, using an effective method of contraception with 
their partner from the first day of vaccination until 4 months after the last 
vaccination. 
 

 
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4.3 Exclusion criteria 
A potential participant who meets any of the following criteria will be excluded from 
participation in this study: 
1. Treatment with non-cART regimen prior to cART regimen; 
2. Previous failure to antiretroviral and/or mutations conferring genotypic resistance to 
antiretroviral therapy; 
3. Non-subtype B HIV infection; 
4. Active Hepatitis B virus and/or Hepatitis C virus co-infection; 
5. History of a CDC class C event (see appendix A); 
6. Pregnant female (screened with a positive pregnancy test), lactating or intending to 
become pregnant during the study; 
7. History of malignancy ≤ 30 days (extended period on the clinical assessment of the 
investigator) prior to screening; 
8. Active infection with fever (38°C or above) ≤ 10 days of screening and/or first 
vaccination; 
9. Therapy with immunomodulatory agents (e.g. systemic corticosteroids), including 
cytokines (e.g. IL-2), immunoglobulins and/or cytostatic chemotherapy ≤ 90 days prior 
to screening. This does not include seasonal influenza, hepatitis B and/or other travel 
related vaccines; 
10. Congenital, acquired or induced coagulation disorders, such as thrombocytopenia 
(thrombocytes < 150x109/L) and/or current use of anti-coagulant medication (e.g. 
coumarins, inhibitors of Xa); Usage of NSAIDs (including acetylsalicylic acid) is 
allowed, however it is advised to interrupt therapy 10 days ahead of vaccination;  
11. Usage of any investigational drug ≤ 90 days prior to study entry; 
12. An employee of the investigator or study site, with direct involvement in the proposed 
study or other studies under the direction of that investigator or study site, or is a 
family member of an employee or the investigator 
13. Any other condition, which, in the opinion of the investigator, may interfere with the 
evaluation of the study objectives 

 
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4.4 Sample size calculation 
Assuming a standard deviation of 0.5 log10 in the change from baseline of cumulative 
frequencies of HIV-specific PBMC, a type 1 error of 5%, a power of 90%, as determined 
by a two-sided non parametric test (15% additional subjects), taking into account the 
weighting for case group ratio of more than 2:1, a sample of 40 in the HIVACAT-TriMix 
arm and 15 in each control arm will allow to detect a difference of at least 0.7 log10 
between any HIVACAT-TriMix arm and the controls arms, allowing for 10% of non-
assessable patients. 
  
 
 

 
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5. TREATMENT OF SUBJECTS 
5.1 Investigational product/treatment 
The Investigational Medicinal Product (IMP) under development is designated as 
iHIVARNA-01, and is a combination of two mRNA active substances, TriMix and 
HIVACAT to be administered through the intranodal route. 
 
HIVACAT immunogen: confers specificity to key HIV regions designed to provide 
beneficial T cell immunity associated with control of viral replication and ensuring broad 
HLA class I and class II coverage. 
 
TriMix: provides the necessary activation molecules to enhance antigen presentation by 
DCs and T cell activation. It is a combination of mRNA sequences corresponding to 
human CD40L, constitutively active TLR4 (caTLR4) and human CD70. 
The optimal combination of HIVACAT and TriMix has been tested in a phase I clinical trial 
Combination of TriMix and HIVACAT within the manufacturing process of the IMP ensures 
it is done in adequate conditions to avoid mRNA degradation, avoiding extensive 
manipulation before use.  
 
Table: IMP 
Drug Substances 
Comments 
TriMix 
Mixture of  mRNAs  of  human  CD40L,  TLR4ca  and human CD70 
HIVACAT 
mRNA encoding different HIV antigens 
Drug Products 
Group 
Comments 
iHIVARNA-01 
solution for injection 
(Arm V of phase I 
Study product 
ARM A 
300 µg TriMix + 900 µg HIVACAT in 200 µl WFI  
(ratio TriMix:HIVACAT = 1:3) 
TriMix_300 solution 
for injection 
Control: TriMix 
ARM B 
Containing 300 µg of TriMix in 200 µl WFI 
WFI for injection 
Control: WFI 
ARM C 
Containing 200 µl WFI 
WFI: water for injection 
 
Patients will receive 3 immunizations of the vaccine at weeks 0, 2 and 4. 
The study treatment period will be 4 weeks. The total follow-up period will be 30 weeks. 
 

 
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5.2 Use of co-intervention  
During the vaccination phase, i.e. until week 6, the current cART regimen should be 
continued. Sexually active subjects, are demanded to use a reliable method of reducing 
the risk of transmission to their sexual partners during treatment interruption (including 
PrEP). Heterosexually active female should use an effective method of contraception with 
their partner (combined oral contraceptive pill; injectable or implanted contraceptive; 
IUD/IUS; consistent record with condoms; physiological or anatomical sterility (in self or 
partner) from 14 days prior to the first vaccination until 4 months after the last. 
Heterosexually active male should use an effective method of contraception with their 
partner from the first day of vaccination until 4 months after the last vaccination 
 
5.3 Escape medication  
In the first 12 weeks of ATI, personal cART has to be re-started if warranted by a drop in 
CD4+ T cell counts below 50% of baseline or below 350 cells/µl and/or on the decision of 
the treating physician. If there are reasons to believe that the patient has had unprotected 
sex during the trial, it is advised that cART will not be interrupted or that cART will be re-
started during ATI. At week 18, all patients with a pVL ≥50 cop/mL will be asked to restart 
cART. We have observed a rapid drop of PVL to undetectable level after cART re-
initiation, in previous studies with treatment interruption, including the DCV-2 study and 
the DCTRN study. In a previous study Andres et al reported an increase in the viral 
reservoir after 12 weeks off cART (9). However, the viral reservoir drops to the same 
levels that the patients had before cART discontinuation, when cART is re-initiated 
subsequently (10,11) .   
 

 
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6. INVESTIGATIONAL PRODUCT  
 
6.1 Name and description of investigational product(s) 
The main characteristics of iHIVARNA-01 are detailed in the Investigator’s Brochure (IB). 
The drug products correspond to the active substance TriMix (control), a combination of 
TriMix and HIVACAT or a placebo; suspended in water for injection (WFI) and aliquoted in 
single dose vials. During aseptic vialing, for HIVACAT-TriMix and TriMix an extra volume 
of 10% is aliquoted per single dose at 220 µl per vial. Prior to injection, the vialed IMP 
should be reconstituted in 880 µl of Hartmann solution. 1mL of the product is 
administered. The IMP is produced according to the GMP guidelines. Briefly, the main 
steps in the aseptic production of the IMPs are: 
1. mixing of the active pharmaceutical products (APIs) at pre-defined ratio’s (where 
applicable, for iHIVARNA-01 products only), 
2. purification of the mixed APIs and resuspension and 
3. aseptic vialing and storage of the IMP. 
 
More details on the manufacturing process of the active substances and drug products 
are given in the Investigator’s Brochure. 
Quality of production batches of active substances and drug products is ensured by 
evaluating its compliance with specifications as detailed in sections the Investigator’s 
Brochure. eTheRNA is responsible for manufacture and supply of all clinical material 
according to Good Manufacturing Practice. iHIVARNA-01 will be packed and labelled by 
eTheRNA in accordance with European regulations. 
 
6.2 Summary of findings from non-clinical studies 
A detailed description of the pharmacodynamics for iHIVARNA-01 is provided in the IMPD 
section 4, Non Clinical studies, table 7 and IB.  
 
Local biodistribution studies have been conducted in mice with eGFP and Fluc mRNAs 
to allow easy tracking of the product. Results from these studies indicate that intranodal 
injection of mRNA leads to efficient uptake and translation of injected mRNA by resident 
DCs (refer to IB; 4.2.1.1 Uptake and translation study of intranodally injected mRNA by 
resident DCs in mice), and that mRNA is stable for up to 12 hours after its administration, 
leading to efficient expression of functional protein up to 9 days. The only cell type 
observed to express functional protein derived from administered mRNA was resident DC 
(refer to IB; 4.2.1.2 Stability study of mRNA in vivo in mouse  lymph nodes). 

 
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Immunogenicity studies were conducted in vitro and in vivo either with HIVACAT alone 
(refer to IB; 4.3.1 Studies with HIVACAT DNA), TriMix alone (refer to IB; 4.3.2 Studies 
with TriMix) or the combination of TriMix plus HIVACAT, designated as iHIVARNA-01 
(refer to IB; 4.3.3 Studies with iHIVARNA-01).  
Immunogenicity studies with pDNA-HIVACAT: some preclinical studies to test 
immunogenicity of HIVACAT were conducted with HIVACAT encoded in a DNA vector 
(named here as pDNA-HIVACAT) that was administered by the intramuscular route. 
These were initial studies that supported the immunogenicity of the antigens coded by the 
HIVACAT sequence. These studies were conducted in mice and Indian Rhesus 
macaques, and results indicated that HIVACAT immunogen is able to induce high 
frequencies of T cell responses, broadly distributed among all HIV-1 proteins included in 
the immunogen with central an effector memory phenotype (12) (refer to IB; sections 
4.3.1.1 In vivo immune activation study of T cells by pDNA-HIVACAT in mice; 4.3.1.2 In 
vivo immune activation study of T cells by pDNA-HIVACAT in Indian Rhesus macaques).  
Immunogenicity studies with TriMix have been performed in vitro and in vivo in mice. 
Results indicate that TriMix efficiently activates human DCs in vitro as observed by the 
upregulation of DCs maturation markers and IL-12 secretion, and promotes subsequent 
allogeneic T cell activation as measured by IFNγ secretion. (refer to IB; 4.3.2.1 In vitro 
maturation study of human DCs with TriMix (8). 
In addition, it was shown in mice that intranodal administration of mu_TriMix does not 
impair in vivo uptake and subsequent translation of a co-administered mRNA (FLuc 
mRNA) by resident DCs (refer to IB; 4.3.2.2 In vivo maturation study of DCs with TriMix in 
mice (6), suggesting that co-administration of TriMix with a desired antigen is a valid 
strategy for adequate antigen presentation. 
On the other hand, the enhancement effect of TriMix on the immunogenic potential of a 
given mRNA antigen has been demonstrated in a transgenic mice model bearing 
ovoalbumin-specific T cells. Results showed that intranodal co-administration of TriMix 
with OVA mRNA antigen enhances activation of specific T cell responses as compared to 
administration of the mRNA antigen alone (refer to IB; 4.3.2.3 In vivo effect of TriMix on 
specific T cell activation study in mice (6). 
Immunogenicity studies with iHIVARNA-01 were conducted in vitro with peripheral blood 
mononuclear cells (PBMCs) derived from HIV infected patients, in vivo in mice and ex vivo 
in lymph node explants. Results from in vitro studies indicate that human DCs derived 
from HIV-infected patients are able to efficiently uptake and translate iHIVARNA in vitro 
and activate CD8+T cell responses (refer to IB; 4.3.3.3 In vitro human CD8+ T-cell 
stimulation study with iHIVARNA-01 loaded DCs; 4.3.3.1 In vitro human PBMC stimulation 

 
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with iHIVARNA-01; 4.3.3.2 In vitro human PBMC stimulation with iHIVARNA-01 loaded 
DCs).  
Results in mice intranodally administered with mu_iHIVARNA-01 demonstrate that HIV-1 
specific T-cell responses are induced after three intranodal administrations of 50 µg of 
iHIVARNA-01 (12.5 µg Trimix + 37.5 µg HIVACAT, ratio 1:3) which were broadly 
distributed among all HIV-1 proteins included in the immunogen (refer to IB; 4.3.3.4 In vivo 
immunogenicity study of mu-iHIVARNA-01 in mice). 
Results in lymph node explants showed that exposure to iHIVARNA mRNA contributed to 
DC maturation. Further, both IFNγ and IP-10 were significantly increased 24h after 
iHIVARNA exposition. Moreover, proinflammatory cytokines such as IL-1β were also 
increased whereas inflammation antagonists such as IL-305 1RA showed an opposite 
tendency. 
Additional studies demonstrated specific lysis of splenocytes loaded with HIVACAT-
derived peptides in mice treated with mu_iHIVARNA-01, indicating that T cells activated 
by the product are cytotoxic and capable of killing targets expressing HIV-1 derived 
peptides (4.3.3.5 In vivo cytotoxicity assay in mice treated with mu_iHIVARNA-01).  
Efficacy studies in viral infection models have been conducted in mice models of 
acute influenza infection. The rational for conducting these studies was to demonstrate 
the efficacy of intranodal administration of mRNA-vaccines against infectious diseases, 
since previous published data were available only in cancer models. Because there are 
currently no well-established mice models of HIV infection, and because iHIVARNA-01 is 
species-specific, models of alternative viral infections have been used, i.e. influenza virus. 
Studies in mice challenged with influenza virus after vaccination with a TriMix-based 
mRNA influenza vaccine (TriMix-NP) showed that this was as effective as a previously 
described effective intramuscular DNA-based vaccine (DNA-NP) (13) in inducing T cell 
responses, and that TriMix-NP was able to substantially reduce mortality due to acute 
influenza infection in mice (from a ~20% survival to a ~95% as compared to controls) 
(4.4.1.1 Immunization of mice with an mRNA-based vaccine for influenza infection). 
Toxicity studies were performed to assess systemic toxicity and local tolerance of 
iHIVARNA-01 and included a repeat-dose study in mice consisting in intranodal 
administration of mu_TriMix or mu_iHIVARNA-01 (3 administrations 1 week apart) (see 
section 4.5.2 Repeat-dose toxicity study by intranodal administration in C57BL/6 in mice). 
The proposed starting dose for the clinical trial bears more than an 8x safety margin with 
respect to the human equivalent of the dose used in this toxicity analysis (see 4.6 Calculation 
of the maximum recommended starting dose in humans). No treatment-related effects were 
noted in the toxicity study, and thus the no-observed-adverse-effect level (NOAEL) was 
considered to be above 10 μg/animal for Trimix and above 40 μg/animal for iHIVARNA-01.3 

 
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after repeated intranodal injection to the inguinal lymph nodes. In paragraph 4,6,3 from the IB 
it is concluded: The dose of iHIVARNA-01 tested in toxicity studies in mice (40 µg) can be 
approximated to be equivalent to a human dose of between 10 mg and 40 mg. Taking into 
account the lower dose for additional safety (i.e, 10 mg), and applying an 8-fold safety factor, 
a maximal dose of 1200 µg of iHIVARNA-01 (300 µg TriMix + 900 µg HIVACAT) would be 
recommended in phase IIa clinical trials. This dose is equivalent to the dose used in mRNA-
based cancer treatment as is used in RBL001/RBL002 Phase I Clinical Trial (available from 
ClinicalTrials.gov identifier NCT01684241), no published data available yet 
6.3 Summary of findings from clinical studies 
Therapeutic vaccines using mRNA electroporated dendritic cells have been used in HIV 
infected patients with a very good safety profile (14–16). In addition, naked or protamin 
mRNA has been used directly in 3 clinical trials in cancer without any important side effect 
(17–19). 
Additionally, TriMix has been authorized in Belgium for its use in a Phase I clinical trial in 
combination with mRNAs encoding for hepatocellular carcinoma (HCC) antigens, in 
patients with metastatic HCC. The trial was initiated in 2014 (EudraCT 2012-005572-74) 
and is still ongoing. TriMix has also been authorized in Belgium for its use in a Phase I 
clinical trial in combination with mRNAs encoding for human papillomavirus (HPV) 
antigens and for treatment of patients with hr-HPV positive cervical intraepithelial 
neoplasia (CIN grade 2-3) (EudraCT 2013-005136-22). In Europe, electroporation of 
TriMix in DCs has also been approved in 4 other clinical trials for the treatment of 
myeloma or melanoma (EudrraCT 2009-015737-73, NL; 2010-023058-35, BE; 2011-
001410-33, BE; 2013-000795-15, BE).  
As mentioned above, therapeutic vaccinations are considered as one of the most 
promising strategies that could restore HIV-specific T-cell responses in HIV infected 
patients and help them control viral replication without cART. Expected potential risks for 
iHIVARNA-1 are non-serious and related to the administration of the product. Considering 
this, the Applicant considers that expected benefits outweigh the risks and justify the use 
of this novel vaccine chronic HIV-infected patients under stable cART. 
iHIVARNA-01 was tested in the phase I clinical trial (iHIVARNA Study, EudraCT: 2014-
004591-32). Fifteen volunteers were recruited in Hospital Clinic Provincial (HCP) from 
June 23, 2015 until March 29, 2016. The mean and median age was 46 years (range 28-
59). Five (24%) were female and 16 were male (76%). Overall the vaccine was well 
tolerated. A total of 17 adverse events (AE) were reported during follow-up until July 12th, 
2016 (4, 4, 5, 4 and 0 in each ARM respectively). Ten AE were grade 1, 6 grade 2 and 1 

 
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grade 3. Of these AE, 2 were definitely related (1 grade 1 and 1 grade 2) and 5 were 
possibly related (2 grade 1, 3 grade 2) to the IMP. 
 
6.4 Summary of known and potential risks and benefits 
iHIVARNA-01 has been tested in the iHIVARNA Phase I dose escalating trial. Overall the 
vaccine was well tolerated. A total of 16 AE were reported during follow-up (4, 6, 5 and 1 
in the first four ARMs respectively). An increase in potential toxicity compared with 
standard therapy is not expected and was not observed in the phase I dose escalating 
trial. In addition, the assessment of safety will follow a comprehensive approach through 
clinical evaluation, lab analysis, biochemical, immunological and virological testing during 
the whole study. 
The risk of virological failure during the vaccination phase in patients with inclusion criteria 
similar to those in the study is very small. No viral blips were observed during the phase I 
clinical trial. In addition, virological monitoring in this study will be more frequent than in 
clinical practice and the results of HIV-RNA measurements in plasma will be available two 
days after blood sampling. The product has been developed based on several distinctive 
features, which provide several advantages over previous therapeutic vaccines:  
 
Direct administration of nude mRNA: allows feasible widespread use and ease of 
production as well as a more favourable safety profile as compared to DNA 
vaccines or adoptive transfer of in vitro-modified autologous DCs. 
 
Intranodal administration: provides higher immunogenicity as compared to 
intradermal administration.  
 
Rational design of HIV antigens (HIVACAT): contains HIV regions shown to confer 
beneficial T cell immunity to control viral replication in the absence of cART. 
 
Key DC and T cell activation molecules (TriMix): allows efficient antigen 
presentation and T cell co-activation. 
 
As mentioned, the product is a combination of mRNA sequences designed to specifically 
target DCs and activate HIV-specific T cells. Of note, direct administration of nude mRNA 
poses several important advantages with respect to administration of other antigenic 
molecules (i.e., DNA or peptides) and with respect to ‘ex vivo modification of DCs’: 
 
in contrast to autologous ex vivo modified DCs, mRNA can be relatively easily 
produced, which facilitates for feasible large-scale production and widespread use 
 
as compared to DNA vaccines, mRNA is safer as it does not integrate into the host 
genome and the expression of the delivered transgenes is transient 

 
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 
mRNA is degraded very rapidly by endogenous RNases, limiting exposure to the 
product and associated side-effects as compared to DNA and protein antigens 
 
the expression of mRNA is efficient as there is no need, in contrast to DNA, to 
cross the nuclear envelope 
 
Temporarily interruption of cART during ATI, may result in viral rebound, immune 
decompensation, and clinical progression. There is a sub study of SMART that suggests 
that with our inclusion criteria, to discontinue therapy for 12 weeks is safe for the patients 
(20). When all cART regimen components have a similar half-life; all components can be 
stopped and started simultaneously. However, when drugs have a different half-life 
(typically a non-nucleoside reverse transcriptase inhibitor [NNRTI]), it may increase the 
risk of selection of NNRTI-resistant mutations. It is required to stop the NNRTI 2 to 4 
weeks in advance of the start of ATI. Alternatively, the NNRTI may be replaced with a 
ritonavir (or cobicistat)-boosted protease inhibitor (PI/r or PI/c) or integrase inhibitor for 
more than 4 weeks prior to ATI.  
 
6.5 Description and justification of route of administration and dosage 
iHIVARNA-01 administered through the intranodal route activates specific T cell 
responses after antigen presentation by DCs. This vaccination strategy is so-called ‘in 
vivo modification of DCs’ (see Investigator’s Brochure for more information on previous 
experience with this strategy). 
When administered into the lymph node, resident DCs take up the mRNA sequences 
contained in iHIVARNA-01 translating them to the corresponding proteins or peptides: 
 
Molecules encoded by TriMix sequences (CD40L, caTLR4 and CD70) are expressed 
on DCs cell surface. CD40L and caTLR4 function by activating immature DCs to 
become mature DCs, which is essential for subsequent antigen presentation. CD70 
expressed on DCs’ cell surface engages CD27 on T cells, a co-activation signal that 
is essential for efficient T cell activation and survival. 
 
Antigens encoded by the HIVACAT sequence are presented by DCs to T cells 
through MHC-I and MHC-II complexes (thus including antigen cross-presentation). 
Overall, T cells specific for the antigens encoded in the HIVACAT sequence become 
activated, which should ultimately lead to the lysis of HIV-infected cells. 
Of note, several studies suggest that a strong HIV-specific T cell-mediated immunity can 
indeed limit virus replication and protect against CD4 depletion and disease progression 
(21). In fact, the best-defined correlates of protection against disease progression as 
observed in patients who are able to control viral replication in the absence of cART 

 
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(known as ‘elite controllers’) are low proviral load, low to absent levels of intracellular 
spliced and unspliced viral mRNA and potent HIV-suppressive CD8+ T-cell responses 
(5,22). The critical role of the CD8+T cell responses in controlling viral replication was also 
shown in both the infection model with macaques devoid of CD8+ T lymphocytes (23,24) 
and in an immune deficient murine model (25). For that reason, current therapeutic 
vaccines under development, such as iHIVARNA-01, are based on activation of HIV-
specific T cell responses by dendritic cells (DCs) presenting target antigens. 
 
6.6 Dosages, dosage modifications and method of administration 
The study treatment period will last 5 weeks, with a total follow-up period of 30 weeks. 
Patients will receive 3 ultrasound guided inguinal intranodal injections of iHIVARNA-01 
(HIVACAT/TriMix), or TriMix control or placebo on weeks 0, 2 and 4. The injections will be 
alternated between the right and the left inguinal lymph node. The dosage has been 
determined in the phase I dose-escalating trial of iHIVARNA and is equal to the mRNA 
dosage that has been used previously in the Ribological melanoma study, in which mRNA 
was also administered intranodally (available from ClinicalTrials.gov identifier 
NCT01684241), no published data available yet).  
The main characteristics of iHIVARNA-01 are detailed in the Investigator’s Brochure. More 
details on the manufacturing process of the active substances and drug products are 
given in the Investigator’s Brochure. Quality of production batches of active substances 
and drug products is ensured by evaluating its compliance with specifications as detailed 
in sections the Investigator’s Brochure. 
 
6.7 Preparation and labelling of Investigational Medicinal Product 
eTheRNA is responsible for manufacturing and supplying all clinical material according to 
Good Manufacturing Practice. iHIVARNA-01 will be packed and labelled by eTheRNA in 
accordance with European regulations (as set in annex 13 of the Good Manufacturing 
Practice Guideline (Regulation EU no 536/2014)). Each delivered ampoule has a unique 
blinded label that has been generated during randomization. Batch-/lot numbers coupled 
to randomization numbers will be kept at eTheRNA, in order to prevent potential 
unblinding based on batch-/lot-numbers.  The IMP is formulated in water for injection 
(WFI) and aliquoted in single dose vials and stored at -20ºC. During aseptic vialing, an 
extra volume of 10% is aliquoted per single dose, totaling at 220 µl per vial. Prior to 
injection the vialed IMP should be reconstituted with 880 µl of Hartmann solution (80% 
final). Hartman solution is provided in single-dose vials. Prior to use the vials should be 
thawed at room temperature. When completely thawed the vials should be gently swirled. 

 
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Care must be taken not to invert the vials (refer to IMPD and SOP provided by eTheRNA 
for full details). Only 1 ml of the IMPs will be administered by ultrasound-guided intranodal 
injection. Example labels of the IMP are given in document D3 for the METC. 
 
6.8 Drug accountability 
During the trial, product accountability will be monitored by the dispensing log, the 
returns, the trial register and data collected on the case report forms. The individual who 
administers the injection will be responsible for ensuring that the return of the used vials 
is recorded in the dispensing log at the end of the clinical session, and that they are 
placed in the participant’s carton. At the end of the trial, all used and unused vials will be 
checked against the inventory by staff from the local clinical research organization before 
returning to the supplier or disposal on site according to local pharmacy guidelines and 
applicable regulations. Documentation of disposal will be provided to CRO and the 
supplier. 
 
 

 
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7. NON-INVESTIGATIONAL PRODUCT 
Not applicable 
7.1 Name and description of non-investigational product(s) 
7.2 Summary of findings from non-clinical studies 
7.3 Summary of findings from clinical studies 
7.4 Summary of known and potential risks and benefits 
7.5 Description and justification of route of administration and dosage 
7.6 Dosages, dosage modifications and method of administration 
7.7 Preparation and labelling of Non Investigational Medicinal Product 
7.8 Drug accountability 

 
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8. METHODS 
8.1 Study parameters/endpoints 
8.1.1 Main study parameter/endpoint 
 
Safety, number of adverse events and serious adverse events defined as: 
 
 
Grade 3 or above local adverse event (pain, cutaneous reactions including 
induration). 
 
Grade 3 or above systemic adverse event (temperature, chills, headache, 
nausea, vomiting, malaise, and myalgia). 
 
Grade 3 or above other clinical or laboratory adverse event confirmed at 
examination or on repeat testing respectively. 
 
Any event attributable to vaccination leading to discontinuation of the 
immunisation regimen. 
 
Immunogenicity defined as: 
Immunogenicity as measured by ELISPOT at baseline and weeks 6 and 18, i.e. two 
and twelve weeks after the last immunization compared to both control groups 
 
8.1.2 Secondary study parameters/endpoints 
 
o magnitude and the kinetics of the HIV-specific CD4+ and CD8+ T cell 
responses after immunization (ELISPOT) as defined by the increase in the 
number of spot forming units and if positive the number of poly-functional T 
cells as determined by intracellular cytokine staining, (ICS) at baseline and 
weeks 6 and 30. 
o time until viral rebound (defined as two consecutive measurements of plasma 
viral load > 1000 copies/mL separated by at least 15 days) after 
discontinuation at week 6. 
o plasma viral load in vivo after ATI, between week 6 and 18 
o functional cure: proportion of patients with viral load below detectable level of 
50 copies/mL in plasma after ATI, week 18 and 30 
o primary immune response against vaccine: Change in frequency of at least 
0.7log10 HIV-specific T-cell responses between baseline and week 6 

 
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o the in vivo vaccine-induced capacity of ex-vivo CD8 T cells to suppress HIV 
growth in autologous CD4 T cells i.e. “CD8 T cell mediated viral suppression”   
o effect on reservoir as measured by changes in the proviral DNA copy numbers 
per million cells and the intracellular viral RNA copy numbers per million cells 
during and after immunization (week 4 and 6).  
o viral immune escape: change in % mutated epitopes from pre-cART to post-
ATI (week 18) 
o host protein mRNA expression profiles in whole blood (week 6 and 18).  
8.1.3 Other study parameters (if applicable) 
Not applicable 
 
 
8.2 Randomisation, blinding and treatment allocation 
Please mind: in this paragraph the terms “randomisation code” and “random code” are 
used in close relationship. They serve a different purpose. 
 
After signing the Informed Consent Form (ICF), subjects will undergo a screening to verify 
that they fulfil all inclusion and none of the exclusion criteria. If a patient is eligible, he or 
she will be randomly assigned to one of the study arms and included in the intention to 
treat (ITT) analysis. 
 
It is chosen to implement central randomisation and blinded labelling of IMP. I.e. a 
sponsor-designated statistician will generate code lists in advance.  
 
Six versions of the randomisation code list will be generated: 
1. Unblinded: a list to both eTheRNA and the pharmacy of the sponsor (the latter only 
for emergency unblinding purposes). This list includes names of study sites, 
randomisation codes and treatment arms. 
2. Blinded: five lists to the clinical investigators of the participating study sites 
(including site of the sponsor). These lists are site-specific and only contain the 
name of the study site and randomisation codes.  
Furthermore, one accident random code list is generated: 
1. Unblinded: a list to both eTheRNA and the pharmacy of the sponsor. This list 
includes names of study sites, random codes and treatment arms. 
 
The randomisation code list is computer generated (R: A language and environment for 
statistical computing. R Foundation for Statistical Computing, Vienna, Austria) and will be 

 
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balanced using randomly permuted blocks and is stratified by site location in order for 
each site to have an equal amount of subjects per treatment arm (40:15:15 ratio of 
HIVACAT-TriMix : TriMix alone : placebo). Every randomisation code is a unique, random 
3-letter code (i.e. ABC). One randomisation code couples to one study subject. Extra 
random codes (accident random code list) are generated for accidents (see below). 
Refer to section 5.7 for labelling of the vials. Labels on vials, boxes etc. will only contain 
blinded information (refer to section 5.7 and document D3), in order to keep the blind for 
investigators after dispatching the IMP from eTheRNA until study-end. 
 
Upon successful screening, the investigator assigns his/her study subject to the next free 
randomisation code of his/her study site. The randomisation code will be both 
documented in the patient files as well as in the eCRF. After assignment, no notification 
of the sponsor, eTheRNA or pharmacy of the sponsor is needed. In case a subject 
terminates study participation before the first vaccination, but after assignment of a 
randomisation code, the investigator may not reuse the randomisation code for another 
study subject. It is agreed to recruit 14 patients per site. Therefore, each site will have 
eight subjects assigned to the iHIVARNA vaccine arm, three subjects to the TriMix control 
arm and three subjects to the placebo arm. If any site fails to include 14 patients, the 
sponsor will decide to re-allocate part of the randomisation code list for that site to 
another site and demand shipping of accompanying vials to another site.  
 
eTheRNA will supply each site with 8 boxes (3 vials each) of iHIVARNA vaccine, 3 boxes 
(3 vials each) of TriMix alone and 3 boxes (3 vials each) of placebo. Furthermore, backup 
vials are supplied (see below). 
 
An accident random code list will aid in assigning the correct vials to the correct study 
subjects / study arms in case of accidental loss, breakage, expiration of vials etc. The list 
will only be available to eTheRNA and the pharmacy of the sponsor. It contains study 
sites, treatment arms and a unique, random 3-letter code preceded by an ‘b’ (backup) 
(i.e. bDEF). Each study site will be supplied with 9 extra: 3 vials for HIVACAT-TriMix, 3 
vials for TriMix control and 3 vials for control. In contrast to the boxes of 3 vials with the 
same randomisation code per study subject (as mentioned above); these backup vials 
will have one random code (i.e. bDEF) per vial as labelled by eTheRNA (see above). In 
case of accidental loss, breakage or expiration of a vial, the investigator has to contact 
the pharmacy of the sponsor to request a new random code per expired vial. The 
investigator needs to inform the pharmacy about the name of the study site and the 
original randomisation code. The (unblinded) designated person at the pharmacy will 

 
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check this code with the study arm and pick a code from the accident random code list 
corresponding to the same study site and study arm. The pharmacy of the sponsor and 
the local investigator register the usage of this accident random code in their logs. Since 
there is a one by one replacement of vials, the investigator should not alter or re-assign 
the original randomisation code. When the pharmacy of the sponsor notices that all 
random codes (bDEF) of one study arm for a site are used; they will notify eTheRNA 
directly, in order to deliver extra backup vials.  
 
For reconstitution of all vials, 51 vials of Hartmann solution of the same lot/batch will be 
provided per site. 
 
After immunization, the empty vial will be stored at room temperature, until final 
monitoring and drug accountability has been performed by the CRO. Thereafter the IMP 
may be destroyed as per local SOP. Non-used vials should be stored at the conditions as 
indicated on the vials/packages until monitoring.  
 
Under normal circumstances the blind should not be broken until all subjects have 
completed and the database is locked after data completion and verification. Otherwise, 
the blind should be broken only if specific emergency treatment/course of action would be 
dictated by knowing the treatment status of the subject. In such cases, the investigator or 
sponsor may determine the identity of the treatment by contacting the pharmacy of the 
sponsor. It is recommended that the local investigator first contacts the sponsor, if 
possible, to discuss the particular situation, before breaking the blind. Telephone contact 
with the pharmacy of the sponsor will be available 24 hours per day, 7 days per week. In 
the event the blind is broken, the sponsor must be informed as soon as possible. The 
date, time, and reason for the unblinding must be documented by the clinician, in the 
appropriate section of the electronic case report form (eCRF), and in the source 
documents. The documentation received from the pharmacy indicating the code break 
must be retained with the subject's source documents in a secure manner. 
 
 
8.3 Study procedures 
Recruitment 
Their treating physician will first inform patients about the phase II clinical trial and confirm 
their eligibility. The study team then pick patients who have indicated their willingness to 

 
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participate. Patients will receive a hardcopy/digital version of the PIF and are invited for 
the W-4 screening visit (informed consent, eligibility and laboratory).  
 
Informed consent and Screening  
Screening visit  
Screening should be performed at most 8 weeks prior to enrolment 
(randomization/baseline visit at week 0), an interval of 4 to 2 weeks is however advised. 
 
At screening, the study will be discussed in detail and any questions about the study will 
be answered. For heterosexually active female, using an effective method of 
contraception with partner from 14 days prior to the first vaccination until 4 months after 
the last vaccination will be demanded. For heterosexually active male, using an effective 
method of contraception with their partner from the first day of vaccination until 4 months 
after the last vaccination will be demanded. 
Specific attention will be paid to risks of viral rebound syndrome. Adequate measures for 
risk reduction of virus transmission during ATI (in order to prevent potential HIV 
transmission will be discussed. A potential subject for the study will be offered an optional 
two-week consideration period before signing informed consent. If the patient wants to 
make use of this consideration period, a new appointment will be scheduled for obtaining 
written informed consent and performing study assessments specific to the study 
protocol.  
 
The principal investigator or designated collaborator will then obtain written informed 
consent. Genetic analysis is specifically mentioned in the patient information and consent 
forms. Hereafter, assessments and procedures will be undertaken according to the 
schedule in Appendix B. Demographic data, medical history, complete physical 
examination and laboratory tests will be obtained and assessed to confirm the eligibility of 
the patient. Patients will be monitored for active HBV/HCV infection. The patient will be 
given a container for collecting stool (to be brought upon inclusion at week 0). After 
obtaining informed consent and screening, the participant will also be registered in the 
eCRF database and any AE needs to be recorded into the eCRF. 
 
Study enrolment  
Review data screening visit  
Review of the screening data will we performed between -4 and week 0. If, after 
completing the Informed consent and screening procedures, a patient is found to be 

 
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eligible, the investigator will notice and reconfirm their study participation with the patient. 
The patient may be enrolled to the study.  
 
Screening failure 
A screening failure is considered if a potential subject did not meet to one or more of the 
criteria required for participation in the trial in between the period of signing informed 
consent and prior to study enrolment/randomization. Several reasons for a screening 
failure may be present, such as inclusion/exclusion criteria not met, non-compliance and 
consent withdrawn. Again: all study procedures after informed consent need to be 
entered in the eCRF. A screening failure including reason will be documented in the 
eCRF. A screening failure might be reconsidered for further enrolment/randomization to 
be eligible in agreement with the sponsor, principle investigator and CRO; in case the 
failure was due to temporarily factors (i.e. a laboratory error). 
 
Study visits for enrolled and randomized subjects 
Baseline visit (trial enrolment) 
During the baseline visit, the investigator will ensure that all patient selection procedures 
were performed and the results confirm that the patient is eligible for participation in the 
study. During the visit, a complete physical exam will be performed and blood will be 
drawn (see: Appendix B). Female patients will undergo a urine pregnancy test. A negative 
test allows for further study continuation. If all criteria for study participation are met, the 
subject will be randomized. The investigator picks the next free randomisation code on 
the site specific randomisation code list and assigns the study subject. The randomisation 
code should also be noted in the patient record and eCRF. Explicit notification of an 
assignment to the sponsor, pharmacy of the sponsor is not needed. Hereafter, the patient 
will be included to the intention to treat (ITT) analysis. 
 
The participant will receive the first immunization. If immunization within the same visit is 
not possible due to logistic reasons, it is preferred to let immunization occur within 24 
hours of the first visit, but always after all blood samples of the baseline visit were taken. 
The treating physician(s) and/or general practitioner of the patient will be notified about 
the study participation. 
 
Immunization 
Patients will receive 3 inguinal intranodal injections of iHIVARNA-01 on weeks 0, 2 and 4 
in the Radiology Service of participating hospitals. Prior to each immunization, women of 
childbearing age will undergo a pregnancy rest (urine test) and will only be allowed to 

 
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receive intranodal vaccination if they obtained a negative pregnancy result within the last 
48 hours prior to vaccination. Injections will be performed with ultrasound guidance using 
a multifrequency gauge for soft tissue. This procedure should be done by a radiologist 
trained both in ultrasound imaging and tissue biopsy.  The injections will be given 
alternated between the right and the left inguinal lymph region, as described in SOP030-
iHIVARNA. Date, time and location of the injection will be recorded in the CRF and on the 
patient diary card. The visits of week 0, 2, 4, including drawing of blood, should be 
performed prior to vaccination. If a female participant becomes pregnant along study, she 
will discontinue the study, but will be asked for consent for the pregnancy and delivery 
follow up.  
 
Follow-up after immunization 
Subjects will be closely monitored during the first hour after immunization, at which point 
vital signs (pulse, blood pressure and respiratory rate) will be recorded on the case record 
form, as well as any local reactions and systemic events. A diary card will be given to all 
the subjects, with instructions and a full verbal explanation to record local and systemic 
adverse events following one week after immunization. 
 
24 hours and 1 week after the second immunization (week 2+1day and week 3) and 24 
hours and 1 week after the third immunization (week 4+1day and week 5) a short visit for 
drawing blood for the cell-associated HIV RNA (CA-HIV) assay will be scheduled. 
 
Telephone contact 
The clinical team will hold a telephone contact with the study subject 7 days (+/- 2 days) 
after the first immunization. This contact will inquire about adverse events, up to 
resolution of solicited local and systemic events. Additional visits may be recommended 
at the discretion of the clinical and principal investigators, if clinically indicated or in order 
to clarify observations. 
 
Follow-up visits 
Patients will return to the clinical centre in weeks 6, 8, 10, 14, 18, 24 and 30 of the study. 
At each follow-up visit, information on concomitant medication, a complete physical 
examination, and specific laboratory tests (see: appendix B) will be obtained. Subjects 
that have not resumed cART at week 30, because their pvl remains undetectable and it is 
their preference not to re-initiate cART, will be closely monitored for PVL and CD4 T cells 
(every 8 weeks) in the follow-up period until cART is resumed.    
 

 
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Treatment interruption at week 6 
Two weeks after the third vaccination (week 6 visit), cART will be interrupted. Female 
subjects will be allowed interruption after a negative pregnancy test. 
 
Assessment of safety 
Safety assessments will take place at fixed time points during the follow-up visits and 
telephone consultation. The patients will be informed of potential safety issues. Patients 
are able to contact the investigator or a member of the investigational team 24 hours a 
day, 7 days a week. 
 
During all visits, the following will be assessed as a minimum: vital signs (blood pressure, 
heart rate, temperature) inspection of lymph nodes and in particular the site of injection. 
Full physical examination (general appearance, eyes, ears/nose/throat, cardiovascular, 
respiratory, gastrointestinal, neurological, musculoskeletal, skin) will be performed at 
screening and baseline (W0) and will only be repeated at follow-up visits on clinical 
indication (thus when a patient has systemic or local complaints). 
 
Special attention to immunization related signs and symptoms: 
After each immunization, close attention must be paid to the following immunization 
related signs and symptoms: 
 
Local injection site reactions: 
• 
Pain at injection site 
• 
Itching at injection site 
• 
Redness/discoloration 
• 
Fluid filled blisters 
• 
Blood filled blisters 
• 
Hard swelling in skin surface at or close to site 
 
General/systemic reactions: 
 
Temperature; 
 
Chills/rigors; 
 
Malaise/ tiredness; 
 
General muscle aches; 
 
Headache; 
 
Nausea; 

 
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 
Vomiting. 
 
Viral rebound during Analytical treatment interruption (ATI): 
cART interruption (ATI) is needed in the search for HIV cure (26). During ATI subjects will 
be monitored for adverse events of plasma viral rebound such as fever, fatigue, 
lymphadenopathy, pharyngitis, rash and/or weight loss. If the CD4+ T cell counts drop 
below 50% of baseline level or below 350 cells/ml, an extra CD4+ T cell count will be 
performed within 7 days. If the results are below 50% of baseline or below 350 cells/ml, 
cART will be re-initiated, immediately. cART will also be re-initiated if the physician or 
investigator judges that this is in the best interest of the patient.  
Safety laboratory (all visits):  
Haematology (except W0): red blood cell count, leukocytes, haemoglobin, haematocrit, 
thrombocytes. Required amount of blood: 4 ml EDTA blood; 
  
Chemistry (except W0): creatinine, ASAT, ALAT, alkaline phosphatase, GGT, total 
bilirubin, amylase or lipase, CPK, glucose. 
Required amount of blood: 9 ml serum. 
 
Immunology (except W0): Lymphocyte subsets (CD4+ and CD8+ T cells) (all visits).  
Required amount of blood: 4 mL EDTA blood.  
 
HLA typing (only at inclusion).  
Required amount of blood: 2-10 ml of EDTA blood. 
 
Elispot (week -4, weeks 0, 6, 10, 18, 30) and intracellular cytokine staining (if Elispot is 
positive).  
Required amount of blood:  27 ml EDTA blood. 
 
CD8+T-cell suppressive capacity assay (week -4, week 0, week 4)  
45 ml EDTA blood 
 
HIV-1 plasma viral load (except week 0)  
9ml EDTA blood 
 
Systems biology (week -4, week 6 and week 18)  
3 ml blood in Tempus tube 
 

 
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Virus sequencing (week week 10, week 18)  
one ml EDTA plasma 
 
Other laboratory 
Faeces microbiota (week week 0, week 6, week 18) one sample 
 
To give the clinical sites some flexibility in visit planning the following visit windows are 
allowed: W2 until W6 +/-1day, W8 until W10 +/- 2days, W14 until W30 +/- 1 week. 
See appendix B for scheme of sampling 
 
8.4 Treatment discontinuation and study withdrawal of individual subjects 
 
Treatment discontinuation: A patient will prematurely discontinue the study in 
case of withdrawal of Informed Consent or if the investigator considers it in the best 
interest of the patient to withdraw. All terminations of study patients and the reasons 
for them must be reported immediately to monitor and sponsor and be duly 
documented in both the medical records and the case report form. 
 
Study withdrawal: The patient may discontinue study participation if he/she is 
unwilling or unable to meet the protocol requirements in terms of the visit schedule 
or if the patient or the investigator considers it best to end his/her participation in the 
study. All patients have the right to withdraw their consent at any time during the 
study without prejudice to them. 
If a female participant becomes pregnant along study, she will discontinue the study 
treatment (i.e. intranodal vaccination), but will be asked for consent for the 
pregnancy and delivery follow up.  
In case a patient withdraws the study, all efforts will be made to complete the week 
30 procedures as termination visit. 
This will be reported immediately to the study monitor and sponsor. 
 
8.4.1 Specific criteria for withdrawal (if applicable) 
A patient will not receive the first, second and/or third immunization if: 
- there is a need to start concomitant treatment with medication that is not 
compatible with the use of the study immunization (e.g. corticosteroids, as it is an 
exclusion criterium). 

 
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- a  severe  adverse  event  occurs  which  is  considered  as  a  (possible)  result  of  
the immunization and/or SAE not resolved at time of vaccination. 
- a female participant becomes pregnant along study, she will discontinue the study, 
but will be asked for consent for the pregnancy and delivery follow up. 
- it is in her or his best interest in the opinion of the investigator. 
 
8.5 Replacement of individual subjects after withdrawal 
Subjects who do not receive the second or third immunization (for any reason) will be 
asked to stay in follow up for safety and immunological assessments according to the 
protocol whenever possible. In case of an adverse event, subjects will be followed until 
the adverse event has been resolved or stabilized. Safety data of these patients will be 
collected according to the protocol where possible.  
The date that the participant is discontinued from further immunizations and the reason 
will be recorded in the eCRF.  
Subjects, who have not received any of the study vaccinations, after randomization, will 
not be replaced. Unblinding of replaced participants will occur after the database lock, or 
earlier if necessary (see also: 8.2). 
Patients that have received 1 or more immunizations will not be replaced. All patients that 
were randomized will be analyzed in the ITT approach. 
 
8.6 Follow-up of subjects withdrawn from treatment 
All randomized subjects, including those who do not receive the second and/or third 
immunization (for any reason), will be asked to stay in follow up for safety and 
immunological assessments according to the protocol where possible. In case of an 
adverse event subjects will be followed until the adverse event has been resolved or 
stabilized. Safety data of these patients will be collected according to the protocol 
whenever possible. Subjects that have not received all three immunizations will not 
interrupt cART. The date that the participant is discontinued from further immunizations 
and the reason will be recorded in the eCRF. 
 
8.7 Premature termination of the study 
Premature termination of the study will occur when two or more grade 3 or above SAE’s, 
related to the vaccine or SUSARs occur or when subjects are exposed to unacceptably 
high risk in the investigators opinion or when advised by the DSMB and accepted by the 
sponsor. Premature termination will be reported to the local EC and competent authorities 
within 15 days. 

 
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9. SAFETY REPORTING 
9.1 Temporary halt for reasons of subject safety 
In accordance to section 10, subsection 4, of the WMO, the sponsor will suspend the 
study if there is sufficient ground that continuation of the study will jeopardise patient 
health or safety. The sponsor will notify the accredited METC with undue delay of a 
temporary halt including the reason for such an action. The study will be suspended 
pending further review by the accredited METC. The investigator will take care that all 
subjects are kept informed.  
 
9.2 AEs, SAEs and SUSARs 
9.2.1 
Adverse events (AEs) 
Adverse events are defined as any undesirable experience occurring to a subject 
during the study, whether or not considered related to the IP. The study period is 
defined as the time from inclusion, after signing the informed Consent Form (ICF) 
until the end of the study at week 30 after the first vaccination. Any medical 
condition that was present before the study treatment and that remains unchanged 
or improves should not be considered or recorded as an AE. A worsening of that 
medical condition will be considered as an AE. 
All adverse events reported spontaneously by the subject or observed by the 
investigator or his staff will be recorded. Notes of the attributability and causal 
relationship to the IP or other study related procedures should be made. 
An adverse reaction (AR) is any noxious and unintended reaction to an 
investigational drug, regardless of the dose administered. 
An unexpected adverse reaction (UAR) is an adverse reaction, the nature or 
severity of which is not consistent with the information about the medicinal product 
set out in the Investigator Brochure (IB). 
 
9.2.2 Serious adverse events (SAEs) 
A serious adverse event is any untoward medical occurrence or effect that  
- 
results in death; 
- 
is life threatening (at the time of the event); 
- 
requires hospital admission or prolongation of existing inpatients’ admission; 
- 
results in persistent or significant disability or incapacity; 
- 
is a congenital anomaly or birth defect; 
- 
requires medical or surgical intervention to preclude of 

 
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An elective hospital admission will not be considered as a serious adverse event. 
 
AEs will be recorded at each visit based on careful clinical observation of the 
patient, laboratory tests and spontaneous reports by the patient and also by open-
ended questioning by the investigator. 
All AEs (serious or not) occurring during the study must be noted in the medical 
history and recorded in the CRF. The investigator will also decide whether the 
adverse event is, based on his/her judgment, related or not to the study drug—this 
decision should also be noted in the medical history and CRF. 
At each visit, all AEs experienced by the patient since the previous visit should be 
recorded in the specific adverse event form of the CRF. 
The following will be recorded for each event: description, severity (grade 1, 2, 3, 4 
and 5), duration (start and end dates), causal relationship with the drug (according 
to the previously attributed criteria) and study drug(s) for which this causal 
relationship is suspected, need for treatment (if applicable) or the actions taken, 
possible alternative explanations, predisposing factors, and outcome. For a pre-
existing AE that has worsening in terms of severity or frequency, the meaning of the 
change should be specified. 
The degree of severity of an adverse event provides a qualitative assessment of the 
extent or intensity of an adverse event elicited by the investigator or reported by the 
patient. Severity does not reflect the clinical seriousness of the event, only the grade 
or extent of the complaint or incidence. 
 
The study will utilize the Division of AIDS Table for grading the severity of adverse 
events (CTCAE table v4, dd14-JUN-2010) (see Appendix C). 
 
The investigator will report SAEs to the sponsor within 24 hours of the investigator 
becoming aware of the events. The investigator must complete the SAE form and 
send the form BOTH by email to r.gruters@erasmusmc.nl AND by fax to +31 10 70 
33441 AND by mail to CR2O: ihivarnasafety@cr2o.nl AND 
by phone to CR2O: +31 6 2061 0123 (safety physician). It is advised to contact the 
sponsor by phone (+31 613 50 80 33) as well, to avoid any unnecessary delay in the 
notification. 
 
The sponsor will report these SAEs through the accredited METC that approved the 
protocol through the web portal ToetsingOnline and this, within 7 days for SAEs that 
result in death or are life threatening. Thereafter, a period of maximum 8 days is 

 
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allowed to complete the initial preliminary report. All other SAEs will be reported 
within a period of maximum 15 days after the sponsor has first knowledge of the 
serious adverse events. 
 
9.2.3  Suspected unexpected serious adverse reactions (SUSARs) 
Adverse reactions are all untoward and unintended responses to an investigational 
product related to any dose administered. 
 
Unexpected adverse reactions are SUSARs if the following three conditions are met: 
1. the event must be serious (see chapter 9.2.2); 
2. there must be a certain degree of probability that the event is a harmful and an 
undesirable reaction to the medicinal product under investigation, regardless of 
the administered dose; 
3. the adverse reaction must be unexpected, that is to say, the nature and severity 
of the adverse reaction are not in agreement with the product information as 
recorded in: 
- 
Investigator’s Brochure for an unauthorised medicinal product. 
 
The sponsor will report the following SUSARs to the METC through the web portal 
ToetsingOnline: 
 SUSARs that have arisen in the clinical trial that was assessed by the METC; 
 SUSARs that have arisen in other clinical trials of the same sponsor and with the 
same medicinal product, and that could have consequences for the safety of the 
subjects involved in the clinical trial that was assessed by the METC. 
The remaining SUSARs are recorded in an overview list (line-listing) that will be 
submitted once every half year to the METC. This line-listing provides an overview 
of all SUSARs from the study medicine, accompanied by a brief report highlighting 
the main points of concern.  
The expedited reporting of SUSARs through the web portal Eudravigilance or 
ToetsingOnline is sufficient as notification to the competent authority. 
 
The sponsor will report expedited all SUSARs to the competent authorities in other 
Member States, according to the requirements of the Member States.  
 
The expedited reporting will occur not later than 15 days after the sponsor has first 
knowledge of the adverse reactions. For fatal or life threatening cases the term will 

 
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be maximal 7 days for a preliminary report with another 8 days for completion of the 
report.  
 
9.3 Annual safety report 
 
In addition to the expedited reporting of SUSARs, the sponsor will submit a safety report 
to the accredited METC, competent authority, and competent authorities of the concerned 
Member States, once a year throughout the clinical trial. 
This safety report consists of: 
 a list of all suspected (unexpected or expected) serious adverse reactions, along with 
an aggregated summary table of all reported serious adverse reactions, ordered by 
organ system, per study; 
 a report concerning the safety of the patients, consisting of a complete safety analysis 
and an evaluation of the balance between the efficacy and the harmfulness of the 
medicine under investigation. 
 A line listing of all SAE’s which occurred in the 2 Belgian sites will be sent to all the 
Belgian EC’s and FAGG on a yearly basis. 
 
9.4 Follow-up of adverse events 
All AEs will be followed until they have abated, or until a stable situation has been 
reached. Depending on the event, follow up may require additional tests or medical 
procedures as indicated, and/or referral to the general physician or a medical specialist. 
SAEs need to be reported until the end of study within the Netherlands, as defined in the 
protocol. 
 
9.5 Data Safety Monitoring Board (DSMB) 
The DSMB is an independent group of experts that advises the sponsor of this study. The 
advice(s) of the DSMB will only be sent to the sponsor of the study. Should the sponsor 
decide not to fully implement the advice of the DSMB, the sponsor will send the advice to 
the reviewing METC, including a note to substantiate why (part of) the advice of the 
DSMB will not be followed. 
 
DSMB composition: 
Dr. P.J.J. (Perry) van Genderen (president), dept. of Internal Medicine, Havenziekenhuis 
Rotterdam 
Dr. C (Kees) Brinkman, Onze Lieve Vrouwe Gasthuis, Amsterdam 

 
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Dr. D. (Dimitris) Rizopoulos, dept. of Biostatistics, Erasmus MC, Rotterdam 
 
The DSMB will meet at least on four occasions, preferably face to face, or alternatively by 
teleconference. These meetings are scheduled  
- 
before the start of the phase II clinical trial  
- 
after completion of immunization of the first two patients at each clinical site,  
- 
after the last immunization of the last study subject and  
- 
at the end of the clinical trial. 
  
The DSMB will review at least: 
- 
this protocol prior to study start 
- 
cumulative study data to evaluate safety, study conduct, and scientific validity and 
integrity of the trial.  
- 
interim/cumulative data for evidence of study-related adverse events 
- 
interim/cumulative data for evidence of efficacy according to pre-established 
statistical guidelines, if appropriate. 
- 
factors that might affect the study outcome or compromise the confidentiality of the 
trial data (such as protocol violations, unmasking, etc.). 
- 
factors external to the study such as scientific or therapeutic developments that 
may impact participant safety or the ethics of the study. 
A more detailed description of the DSMB tasks is available in form K5 from the METC 
submission documents. 
 
 

 
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10. STATISTICAL ANALYSIS 
In general, analyses will be performed using an “intent-to-treat” approach. All subjects 
randomized to either group will be included in the analysis under the group they are 
randomized to, even if they didn’t receive all vaccinations. All analyses will be adjusted by 
center. 
Every effort will be made to minimize the amount of missing data in the trial. However it is 
realistic to assume a priori that missingness may be informative. Sensitivity analyses, 
including multiple imputations, will be undertaken to assess the robustness of the 
conclusions to the missing data. 
 
10.1 Primary study parameter(s) 
 
 All non-serious and serious adverse events will be grouped according to a pre-
specified side-effect coding system and tabulated. The number (and percentage) of 
patients experiencing any grade 3 or above local adverse event, any grade 3 or 
above systemic adverse event, any grade 3 or above other clinical or laboratory 
adverse event, any adverse event attributable to vaccination leading to 
discontinuation of the immunization regimen will be compared between treatment 
groups using logistic regression. Safety will be analyzed using the all-patients-treated 
approach. 
 
Difference in frequency of log10 HIV-specific T-cell responses as determined by 
SFU/10ˆ6*cells between baseline and 2 and 14 weeks after the last injection will be 
compared between the three study arms (HIVACAT-TriMix; TriMix; placebo) using 
linear or quantile regression as appropriate. 
 
 
10.2 Secondary study parameter(s)  
 
The magnitudes and kinetics of the HIV-specific CD4+ and CD8+ T cell responses at 
week 6 and week 30 will be compared between the three study arms (HIVACAT-
TriMix; TriMix; placebo) using linear or quantile regression as appropriate. 
 
Time from discontinuation at W6 until viral rebound is compared between the three 
intervention arms using a Cox proportional hazards model 
 
Linear mixed effects model to compare the evolution of log10 plasma viral load over 
time between the three intervention groups 
 
Logistic regression to compare the proportion of patients with functional cure at week 
18 and week 30 between the three intervention groups 

 
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 
The proportion of patients with a primary immune response against vaccine, at week 
6, after 3 vaccinations, will be compared between the three intervention groups using 
logistic regression 
 
Mixed effects regression to compare the evolution of CD8+ T-cell HIV suppressive 
capacity from baseline to two weeks after the last vaccination between the three 
intervention groups 
 
Mixed effects regression to compare the evolution of log10 proviral DNA and log10 
intracellular viral RNA from baseline to week 30 after re-start cART, between the 
three intervention groups 
 
Compare viral immune escape (change in % mutated epitopes from pre-cART to 
post-ATI) between the three intervention groups using linear or quantile regression as 
appropriate 
 
Principal component analysis will be applied on the mRNA expression profiles. The 
evolution over time (baseline, after 3 vaccinations and after ATI) of selected principal 
components will be compared between the three intervention groups using linear 
mixed effects models 
 
10.3 Other study parameters 
Not applicable 
 
10.4 Interim analysis  
 
Rationale: 
 
The phase I clinical trial has proven safe (primary outcome). However, the secondary 
outcome, immunogenicity as measured by peptide Elispot showed limited effect of the 
vaccine on the number of spot forming units. This was also due to high baseline values.  
Based on these results the consortium wants to be cautious in the phase II trial and an 
interim analysis has been proposed. 
 
Proposal 
 
We will include 35 patients into the phase II trial (this is half of the total study population). 
The interim analysis is the same as the primary analysis in the protocol, i.e. comparison 
of change from baseline to week 6 of cumulative frequencies of HIVACAT-specific PBMC 

 
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(on log10 scale) between HIVACAT-TriMix arm and placebo arm. Thus we test H0: 
mu1=mu2 vs H1: mu1≠mu2. We assume a standard deviation of 0.5 in both arms.) 
 
Time 
point 
N 
Z_l 
Z_f 
Probability 
stopping under 
H0 
Probability 
stopping 
under H1 (p1) 
Power  
Type I 
error 
0.5 
20,8 2,23 
1.71 99% 
15% 
85% 
1.6% 
 
Column ‘N’ shows the number of patients included in the interim analysis in both arms. If the 
test statistic at the interim analysis is smaller than ‘Z_l’ the trial is stopped for futility, 
otherwise the rest of the patients will be enrolled and the test statistic of the final analysis will 
be compared to cut-off value ‘Z_f’. The power is calculated under the alternative that the 
difference in means is 0.7. 
 
The analysis will be performed by an unblinded statistician, who is not a member of the 
study consortium. 
 

 
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11. ETHICAL CONSIDERATIONS 
11.1 Regulation statement 
The clinical trial will be conducted in accordance with the principles contained in the 
Declaration of Helsinki. 
 
For participating Dutch sites the following is applicable as well: 
The clinical trial will be conducted in accordance with the Medical Research Involving 
Human Subjects Act (WMO, BWBR0009408, 26-FEB-1998).  
 
For participating Spanish sites the following is applicable as well: 
Royal Decree 1090/2015, CREC, authorization from the Spanish Agency for Medicinal 
Products and Medical Devices, and approval by the Director of the Institution. The 
investigator agrees to comply with the rules set forth in the applicable clinical trial 
regulations: Medicines Act 29/2006 (Official State Journal No. 178, 27-07-06) and Royal 
Decree 1090/2015 on Clinical Trials. For participating Belgian sites, the law on 
experiments on the human person of May 7th, 2004 is applicable. 
 
11.2 Recruitment and consent 
Patients will be informed verbally by their treating doctor and in writing via the patient 
information form. All relevant information will be reported to the subjects adapted to their 
level of understanding. After informed consent they will have a minimum of two weeks 
before they will be asked to sign the form for study participation. 
 
11.3 Objection by minors or incapacitated subjects  
Not applicable  
 
11.4 Benefits and risks assessment, group relatedness 
Vaccination in HIV-infected patients against other pathogen (e.g. influenza virus) is 
common practice and has not shown extra risk for these patients. cART although effective 
requires lifelong medication with side effects and long-term toxicities including 
cardiovascular and renal problems. Therapeutic vaccination may complement or even 
replace daily treatment thus alleviating the burden of cART. 
The use of TriMix may activate the immune system a-specifically. This may provide target 
cells for HIV replication and in the case of ATI, increased viral replication. 

 
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11.5 Compensation for injury 
The sponsor has liability insurance, for all subjects, which is in accordance with article 7 
of the WMO for the study at all clinical sites. 
  
The sponsor (also) has an insurance, which is in accordance with the legal requirements 
in the Netherlands (Article 7 WMO). This insurance provides cover for damage to 
research subjects enrolled at all clinical sites through injury or death caused by the study. 
The insurance applies to the damage that becomes apparent during the study or within 4 
years after the end of the study. 
 
11.6 Incentives 
Reimbursement of subject travel expenses and parking costs will follow on a per center 
base, after receiving an original receipt. Car travel expenses will be reimbursed based on 
a fixed price per kilometer according to local regulations. 
 
Subjects will not receive any other payment or compensation for participation in the study. 
 
 

 
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12. ADMINISTRATIVE ASPECTS, MONITORING AND PUBLICATION 
12.1 Handling and storage of data and documents 
The study will be conducted in accordance with the International Conference on 
Harmonization (ICH) for Good Clinical Practice (GCP) and the appropriate regulatory 
requirement(s). Essential clinical documents will be maintained per study site to 
demonstrate the validity of the study and the integrity of the data collected. Master files 
should be established at the beginning of the study, maintained for the duration of the 
study, and retained for at least 15 years after terminating the study, or longer according to 
regulations by local authorities. The source data has to be kept at the study site and is 
accessible for the study team. It is also available for the monitor and authorities on 
request – in order to fulfil adequate monitoring, source data verification and to perform 
study site audits.  
 
 
OpenClinica, running in a GCP/ICH compliant environment within Erasmus MC, will be 
used as Clinical Data Management System (CDMS). All data will be entered coded as 
required by the Personal Data Protection Act (WBP in Dutch). All study procedures in 
OpenClinica are traceable by the build in study audit log and by using personal, non-
transferable accounts.  The principal investigator per site is responsible for administering 
the list with personal patient data and coded study numbers (hereafter: code list). Study 
numbers should not contain any data, which might identify a person. The code list should 
be locked away in such a manner that unauthorized persons cannot access it.  
 
Blood, urine and stool samples for routine laboratory diagnostics (i.e. red blood cell count, 
liver function) will be labelled with non-coded patient information. All samples for non-
routine laboratory diagnostic (i.e. for intracellular cytokine staining) and all samples being 
processed at another site will be labelled with coded information alone.  
 
12.2 Monitoring and Quality Assurance  
Monitoring will occur according to an intermediate risk profile based on monitoring 
guidelines of METC Erasmus MC/NFU (Dutch Federation of University Medical Centres) 
and in compliance with GCP/ICH guidelines. 
 
Every monitor visit will be reported in a written report. The head of the department has the 
final responsibility for filing the monitoring reports for at least 15 years after terminating 
the study, or longer according to regulations by local authorities. Monitoring visit reports 

 
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and other documents related to iHIVARNA phase IIa will be available on request of the 
Board of Directors and their authorised representatives. 
 
12.3 Amendments  
A ‘substantial amendment’ is defined as an amendment to the terms of the METC 
application, or to the protocol or any other supporting documentation, that is likely to 
affect to a significant degree: 
- the safety or physical or mental integrity of the subjects of the trial; 
- the scientific value of the trial; 
- the conduct or management of the trial; or 
- the quality or safety of any intervention used in the trial. 
 
All substantial amendments will be notified to the METC and to the competent authority. 
 
Non-substantial amendments will not be notified to the accredited METC and the 
competent authority, but will be recorded and filed by the sponsor.  
 
12.4 Annual progress report 
The sponsor/investigator will submit a summary of the progress of the trial to the 
accredited METC once a year. Information will be provided on the date of inclusion of the 
first subject, numbers of subjects included and numbers of subjects that have completed 
the trial, serious adverse events/ serious adverse reactions, other problems, and 
amendments.  
 
12.5 Temporary halt and (prematurely) end of study report 
The sponsor will notify the accredited METC and the competent authority of the end of 
the study within a period of 90 days. The end of the study is defined as the last patient’s 
last follow-up visit. 
 
The sponsor will notify the METC immediately of a temporary halt of the study, including 
the reason of such an action. 
 
In case the study is ended prematurely, the sponsor will notify the accredited METC and 
the competent authority within 15 days, including the reasons for the premature 
termination. 
 

 
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Within one year after the end of the study, the investigator/sponsor will submit a final 
study report with the results of the study, including any publications/abstracts of the study, 
to the accredited METC and the Competent Authority.  
 
12.6 Public disclosure and publication policy 
The investigators, in agreement with the Coordinating Center for the study, will publish the 
results of the trial in internationally indexed journals. Authorship will take into account 
members of the study management, participating investigators and persons responsible 
for coordination, data analysis and article writing. 

 
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13. STRUCTURED RISK ANALYSIS  
 
13.1 Potential issues of concern 
 
a. Level of knowledge about mechanism of action. 
Control of HIV viral replication in the absence of cART (known as 'elite controllers), has 
been attributed to the ability to develop effective HIV-specific CD8+T cell responses (5,6).  
The HIVACAT immunogen confers this specific immunity to key HIV regions. Furthermore, 
the TriMix substance provides the necessary activation molecules to enhance antigen 
presentation by DC's and T-cell activation (mRNA sequences of CD40L, caTLR4 and 
CD70). The intranodal vaccination route is based on the knowledge of the strategy to ex-
vivo modify DC's with HIV-particles and successful intranodal vaccination strategies with 
mRNA-optimized dendritic cells in melanoma patients (6,27). These methods have 
already proven to be both safe and immunogenic.  
 
b. Previous exposure of human beings with the test product(s) and/or products with a 
similar biological mechanism. 
To date, clinical experience with HIVACAT and TriMix in the iHIVARNA phase I trial 
showed that the vaccination is well tolerated with limited adverse events obviously or 
possibly related to the product. TriMix has been used with tumor-associated antigens in 
mRNA-based antitumor therapy studies and has proven to be safe (6). 
 
c. Can the primary or secondary mechanism be induced in animals and/or in ex-vivo 
human cell material? 
Pre-clinical studies in mice have shown a strong induction of vaccine specific immune 
responses (ELISPOT) after administration of the TriMix HIVACAT combination. Ex vivo 
stimulation of PBMC with electroporated DC from HIV-1 infected patients showed a strong 
induction of vaccine specific responses (see IMPD document 
4_iHIVARNA_NonClinical_ASP_01). 
 
d. Selectivity of the mechanism to target tissue in animals and/or human beings. 
Targeting of DC’s by intranodal administration was shown in mice (section 2.2.4..8.1 of 
MPD doc 4). eGFP localizes in CD11c positive cells. Luciferase activity was eliminated by 
depletion of DC by diphtheria toxin in CD11c-DTR transgenic mice. Bioluminescence was 
shown after intranodal administration of luciferase mRNA in mice (section 2.2..3.2.3.d of 
IMPD doc 4) (6) 

 
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e. Analysis of potential effect. 
Intranodal vaccination experiments with murine TriMix and HIVACAT in C57BL/6 mice 
showed a strong induction of cellular immune responses as measured by IFNy ELISPOT 
and in vivo CTL. 
 
f. Pharmacokinetic considerations 
According to the Note for guidance on preclinical pharmacological and toxicological 
testing of vaccines (CPMP/SWP/465/96), pharmacokinetic studies are normally not 
needed. The need for specific studies should be considered in a case-by-case basis and 
may include considerations such as local deposition studies, which would assess the 
retention at the site of injection and its further distribution; histopathological studies of the 
draining lymph nodes, which might illustrate depot characteristics of the vaccine. 
Distribution studies should be considered in case of new formulations, novel adjuvants or 
alternative routes of administration (eg. oral or intranasal). 
Local deposition of intranodally administered mRNA is supported by studies conducted by 
the Applicant with Fluc mRNA and GFP mRNA (2.2.4.8.1 Local deposition studies). As 
demonstrated in these studies, intranodal injection of mRNA allows for its uptake and 
translation by resident DCs in mice. mRNA is stable up to 12 hours in lymph nodes and 
functional protein can be observed up to 9 days. The only cell type observed to express 
functional protein derived from administered mRNA was resident DCs (see study details 
and results in section 2.2.4.8.1 Local deposition studies). 
 
g. Study population 
Only patients who fulfill their regular outpatient clinic visits with their treating physician will 
be selected. All patients are on stable cART treatment and CD4 counts should be above 
450/ml, with an undetectable PVL. Women of child-bearing potential are tested for 
pregnancy and are required to use contraceptives. Regular pregnancy testing is included 
in the protocol. 
 
h. Interaction with other products 
Vaccination is topical and pharmaco-active compounds are however not included. 
Therefore no interactions with cART or any other medication and products or other 
treatment is expected. 
 
 
 

 
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i. Predictability of effect 
Biomarkers for measuring the effectiveness of HIVACAT/TriMix are T cell immune 
responses as measured with two assays. Indirectly PVL may reflect effectiveness of 
vaccination. 
 
j. Can effects be managed? 
Direct management of adverse immune responses is not foreseen, however, immune 
suppressive medication (prednisone) may be administered at the discretion of the 
physician. Any unforeseen allergic/anaphylactic reactions can be managed with standard 
treatment (e.g. with epinephrine, steroids and antihistamine – according to local 
guidelines). During antiretroviral treatment interruption (ATI) frequent monitoring allows to 
follow the viral rebound. Criteria for re-start of cART are defined in the study protocol. 
 
13.2 Synthesis 
Risks on vaccination as strategy in preventing infectious diseases (e.g. influenza) are well 
known and generally regarded as safe. Recent studies on intranodal vaccination and the 
usage of TriMix proved the procedures and product being safe and well tolerated in 
general. Knowledge of intranodal vaccination with TriMix and HIVACAT is limited to 
iHIVARNA Phase I trial and showed that the vaccine was well tolerated, with few non-
serious AE obviously or possible related to the vaccination. Lastly, ATI is used regularly in 
clinical studies. To further limit the risks; patients are followed up until one hour after 
vaccination, regular telephone follow-ups are scheduled and multiple visits including 
safety laboratory analysis are planned (see: chapter 8.3, chapter 9). Furthermore, the ATI 
is limited to a period of 12 weeks; while PVL monitoring takes place on a regular base in 
order to detect HIV replication. The investigator will review the physical examination, 
safety laboratory results and the patient diary before continuing any study activities. 
Lastly, a DSMB is appointed. 
 
 
 
 
 

 
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