A Phase I Clinical Trial to Assess the Safety and Immunogenicity of HIV DNA-C CN54ENV Immunisations Administered Via the IM and ID Methods With and Without Electroporation Followed by Boosting With Recombinant HIV CN54gp140 in Healthy Male and Female Volunteers
Phase: PHASE1
Conditions: HIV
Sponsor: Imperial College London

CUTHIVAC002 
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CUTHIVAC002 
 
A Phase I clinical trial to assess the safety and immunogenicity of HIV 
DNA-C CN54ENV immunisations administered via the Intramuscular and 
Intradermal methods with and without electroporation followed by 
boosting with recombinant HIV CN54gp140 in healthy male and female 
volunteers 
 
EUDRACT: 2015-001023-23  
 
Protocol version number 4.0 
 
Protocol date:  10_APR_2017 
 
 
 
 
Authorised by: 
Name:     
Dr David Lewis 
Role: 
Chief Investigator
NIHR/Wellcome Trust Imperial Clinical 
Research Facility 
Hammersmith Hospital 
Signature: 
 
 
 
Date: 
 
Name: 
Mr Gary Roper  
Role: 
Head of Regulatory Compliance 
Joint Research Compliance Office 
Imperial College London (Sponsor) 
Signature: 
 
 
 
Date: 
 
 
 

CUTHIVAC002 
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GENERAL INFORMATION 
 
This document describes the CUTHIVAC002 trial and provides information about 
procedures for entering participants into it. Every care was taken in drafting the protocol, 
but corrections or amendments may be necessary.  
 
Compliance 
 
The trial will be conducted in compliance with the approved protocol, the Declaration of 
Helsinki (1994) the principles of Good Clinical Practice (GCP), Commission Directive 
2005/28/EC with the implementation in national legislation in the UK by Statutory 
Instrument 2004/1031 and subsequent amendments, the UK Data Protection Act (DPA 
number: Z5886415), and the National Health Service (NHS) Research Governance 
Framework for Health and Social Care (RGF).  
 
Sponsor 
 
Joint Research Compliance Office 
 
Imperial College London 
 
Room 221 
 
Medical School Building 
 
St Mary's Campus 
 
Norfolk Place 
 
London W2 1PG 
    Tel:  0207 594 6245 
 
 
 
Funder 
European Commission under the 7th Framework Programme – CUT’HIVAC Grant 
Agreement 241904 
 
 
Trial Management 
Chief Investigator 
Dr David Lewis MD 
Head Of Clinical Studies, NIHR/Wellcome Trust Imperial CRF 
Imperial Centre for Translational and Experimental Medicine, Hammersmith  Hospital 
Du Cane Road  
London W12 0NN 
Office:  +44 2033136195    
Reception: +44 2033138070      
Email: d.lewis@imperial.ac.uk 
Website:http://imperial.crf.nihr.ac.uk 

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Principal Investigator 
Dr Sonya Abraham MBBS MRCP PhD 
Consultant Research Physician, NIHR/Wellcome Trust Imperial CRF 
Imperial Centre for Translational and Experimental Medicine, Hammersmith  Hospital 
Du Cane Road  
London W12 0NN 
Office:  +44 2033136104 
Mobile: 07714 761 224 
Reception: +44 2033138070      
Email: sonya.abraham@imperial.nhs.uk 
Website:http://imperial.crf.nihr.ac.uk 
 
Co- Investigator 
Professor Sheena McCormack, MSc FRCP 
Clinical Trials Centre 
Winston Churchill Wing 
St.Mary’s Hospital, Praed Street 
London W2 1NY 
Tel: 020 7670 4807  
Fax: 020 7670 4659 
Email: s.mccormack@ucl.ac.uk 
 
Project Lead 
Professor Robin Shattock PhD 
St Mary’s Campus 
Imperial College London 
Wright-Fleming Institute 
Norfolk Place 
London W2 1PG 
Tel: 020 7594 3611 
Fax: 020 7706 0094 
Email: r.shattock@imperial.ac.uk 
 
Immunology Laboratory 
Professor Robin Shattock 
GCLP lab 463-C 
St Mary's Campus  
Imperial College London  
Norfolk Place  
London W2 1PG 
Tel: 020 7594 3611  
Fax: 020 7706 0094 
Email: r.shattock@imperial.ac.uk 
 
Pharmacy 
Regina Storch 
Pharmacist Clinical Trials 
Hammersmith Hospital 
Du Cane Road 
London W12 0HS 
Tel: 0203 313 4333 
Fax: 0203 313 4704 
Email: regina.storch@imperial.nhs.uk 
 
Trial Statistician 
Mr Adrian Cook 
MRC Clinical Trials Unit at UCL 
Institute of Clinical Trials & Methodology  
Aviation House 
125 Kingsway 
London WC2B 6NH 
Tel: 020 7670 4893 
Email: Adrian.cook@ucl.ac.uk 
 
Clinical Projects Manager 
Dr Tom Cole 
NIHR/Wellcome Trust Imperial CRF 
Imperial Centre for Translational and 
Experimental Medicine (L-Block) 
Hammersmith Hospital 
Du Cane Road 
London W12 0HS  
Tel: 020 3313 6198 
Fax: 020 3313 1763 
Email: t.cole@imperial.ac.uk 
 
 
Clinical Trial Manager / Monitor 
Dr Sue Fleck 
MRC Clinical Trials Unit at UCL 
Institute of Clinical Trials & Methodology  
Aviation House 
125 Kingsway 
London WC2B 6NH 
Tel: 020 7670 4893 
Email: s.fleck@ucl.ac.uk 
 
 
ICHOR:  TriGrid Devices 
Mr Drew Hannaman 
Vice President – R&D 
Ichor Medical Systems 
6310 Nancy Ridge Drive. #107 
San Diego CA, 92121 
Tel: +(858) 550-2022 ext. 103 
Fax: +(858) 550-2092 
Email: dhannaman@ichorms.com 
 

 
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IMP Manufacturer and Supply of the Vaccines for the Trial 
 
Vaccines will be supplied by Imperial College London and Manufactured as 
below : 
DNA-C CN54ENV 
Althea, 11040 Roselle Street, San Diego, CA 92121, USA 
CN54gp140 
Polymun Scientific Immunbiologische Forschung GmBH, Honaustrasse 
99, 3400 Klosterneuburg, Austria 
 
GMP Storage, Labelling, Packaging, QP release and Distribution of Vaccine to 
Sites 
PCI 
Biotech House 
Central Park 
Western Avenue 
Bridgend Industrial Estate, Bridgend 
CF31 3RT; UK 
 
 

 
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SAE AND IMPORTANT AE NOTIFICATION 
 
 
Within 24 hours  of becoming aware of an SAE, please email the Imperial 
College London Joint Research Compliance Office at 
 
 
jrco.ctimp.team@imperial.ac.uk and the MRC CTU at UCL at 
mrcctu.cuthivac002safety@ucl.ac.uk using the template provided 
 
 
 
POSSIBLE SERIOUS BREACH 
NOTIFICATION 
 
 
Within 24 hours of becoming aware of a serious breach, please  
e-mail details to the Imperial College London Joint Research 
Compliance Office at jrco.ctimp.team@imperial.ac.uk and the MRC 
CTU at UCL on mrcctu.cuthivac002safety@ucl.ac.uk 
 
 
 
 

 
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CONTENTS 
1. 
Summary ................................................................................. 9 
1.1 
Summary of trial design ................................................................................... 9 
1.2 
Flow Diagram ................................................................................................ 13 
2. 
Background ........................................................................... 14 
2.1 
Background................................................................................................... 14 
2.2 
Rationale and Objective ................................................................................. 22 
3. 
Selection of Centres/Clinicians ............................................. 32 
4. 
Selection of Participants ....................................................... 32 
4.1 
Participant Inclusion Criteria .......................................................................... 32 
4.2 
Participant Exclusion Criteria .......................................................................... 33 
4.3 
Number and Source of Participants ................................................................. 34 
4.4 
Screening Procedures and Pre-Randomisation Investigations ........................... 34 
5. 
Randomisation & Enrolment Procedure ................................ 35 
5.1 
Randomisation Practicalities ........................................................................... 35 
5.2 
Randomisation Codes and Un-blinding ............................................................ 35 
5.3 
Co-Enrolment Guidelines ................................................................................ 35 
6. 
Treatment of Participants ..................................................... 36 
6.1 
Vaccine Products ........................................................................................... 36 
6.2 
Clinical Management of Adverse Events .......................................................... 38 
6.3 
Non-Trial Treatment ...................................................................................... 38 
6.4 
Issues Related to HIV .................................................................................... 38 
7. 
Assessments and follow-up ................................................... 40 
7.1 
Duration of Follow-up and Schedule ............................................................... 40 
7.2 
Screening Assessments .................................................................................. 41 
7.3 
Immunogenicity Assessments ........................................................................ 42 
7.4 
Procedures for Assessing Safety and Tolerability ............................................. 43 
7.5 
Criteria for Stopping Treatment Groups or Whole Trial ..................................... 45 
7.6 
Procedures at the end of the trial ................................................................... 46 
8. 
Safety Reporting ................................................................... 49 
8.1 
Definitions .................................................................................................... 49 
8.2 
Reporting Adverse Events .............................................................................. 50 
8.3 
Pregnancy ..................................................................................................... 51 
9. 
Withdrawal of participants .................................................... 52 
10. 
STATISTICAL CONSIDERATIONS .......................................... 54 
10.1 
Method of Randomisation .............................................................................. 54 
10.2 
Endpoints ..................................................................................................... 54 
10.3 
Sample Size .................................................................................................. 55 
10.4 
Data analyses and Presentations .................................................................... 55 
11. 
Data Management ................................................................. 56 
11.1 
Data management at the Clinical Centre ......................................................... 56 
11.2 
Data Management in the Immunology Laboratories ......................................... 57 
11.3 
Data management at the MRC CTU at UCL ..................................................... 57 

 
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12. 
Trial Monitoring ..................................................................... 58 
12.1 
Risk Assessment ............................................................................................ 58 
12.2 
 Monitoring by Medical Research Council Clinical Trials Unit at University College 
London (MRC CTU at UCL) ............................................................................................ 58 
12.3 
Monitoring by the Imperial CRF ...................................................................... 58 
12.4 
Clinical Site Monitoring .................................................................................. 58 
12.5 
Monitoring by the Trial Management Group .................................................... 59 
12.6 
Confidentiality ............................................................................................... 59 
13. 
Ethical Considerations and Approval ..................................... 60 
13.1 
Ethical Issues ................................................................................................ 60 
13.2 
Ethical Considerations .................................................................................... 60 
14. 
Indemnity .............................................................................. 61 
15. 
Finance .................................................................................. 61 
16. 
Trial Committees ................................................................... 62 
16.1 
Trial Management Group (TMG) ..................................................................... 62 
16.2 
CUT’HIVAC Consortium Governing Board ........................................................ 62 
16.3 
Trial Management Team (TMT) ...................................................................... 62 
17. 
Publications ........................................................................... 62 
18. 
Protocol Amendments ........................................................... 62 
19. 
References ............................................................................ 64 
Appendices ...................................................................................... 67 
 
 
Appendices 
APPENDIX 1: ELECTROPORATION .................................................................... 67 
APPENDIX 2: TOXICITY TABLE ......................................................................... 70 
 
 
LIST OF TABLES 
Table 1:       Schedule of Doses and Methods of Immunisation 
Table 2: 
Solicited Adverse Events 
Table 3: 
Summary of completed trials using the TDS-IM device 
Table 4: 
Summary of trials in which the TDS-IM device is currently being 
evaluated  
Table 5: 
Schedule of Visits, Immunisations and Assessments 
Table 6: 
Sample Size  
 
 
LIST OF FIGURES 
Figure 1: Screening, Randomisation, Immunisation and Follow-up Flow Diagram 
Figure 2: 
Safety Reporting Flowchart  
 
 

 
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ABBREVIATIONS AND GLOSSARY 
Ab 
Antibody 
ADCC 
Antibody-dependent cell-mediated cytotoxicity 
AE 
Adverse event 
Ag 
Antigen 
APC 
Antigen presenting cells 
AR 
Adverse reaction 
ASC 
Ab secreting cells 
CF 
Consent form 
CFSE 
Carboxyfluorescein succinimidyl ester 
CRF     
Case Report Form 
CTA 
Clinical Trial Authorisation 
CTL 
Cytotoxic T-lymphocyte 
dDC 
Dermal dendritic cell 
DC 
Dendritic cell  
 
 
EP 
Electroporation 
EUDRACT 
European Union Drug Regulatory Agency Clinical Trial 
GTU 
Gene Transfer Unit 
HIV 
Human immunodeficiency virus 
IB 
Investigator’s Brochure 
ICL 
 
Imperial College London 
ICS 
Intracellular cytokine staining 
ID 
Intradermal 
IM 
Intramuscular 
JRCO 
Joint Research Compliance Office 
LC 
Langerhans cell 
MHRA 
Medicines and Healthcare Regulatory Agency 
MRC 
Medical Research Council 
MRC CTU at 
UCL 
Medical Research Council Clinical Trials Unit at University College London (UCL) 
NHS 
National Health Service 
PBMC 
Peripheral Blood Mononuclear Cell 
PI 
Principal Investigator 
PIS 
Participant Information Sheet 
R&D 
Research and Development 
REC 
Research Ethics Committee 
SAE 
Serious adverse event 
SAR 
Serious adverse reaction 
SFC 
Spot forming cell 
SIV 
Simian immunodeficiency virus 
SOP 
Standard operating procedures 
SUSAR 
Suspected unexpected serious adverse reaction 
TDS 
TriGrid Delivery System 
Tfh 
T follicular helper 
Th 
T-helper 
TLR 
Toll-like receptor 
TMG 
Trial Management Group 
TOPS 
The Overvolunteering Prevention System 
UAR 
Unexpected adverse reaction 

 
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1. SUMMARY   
1.1 Summary of trial design 
1.1.1 
Type of design 
CUTHIVAC002 is an open-labelled randomised Phase I study aimed at exploring the safety 
and immunogenicity of two different modes of delivery of a deoxyribonucleic acid (DNA) 
vaccine (DNA-C CN54ENV) via combined intramuscular and intradermal methods with and 
without electroporation, and boosted with recombinant HIV CN54gp140 administered by 
intradermal injection in healthy volunteers.  
 
The aim of this study is to identify optimal DNA delivery conditions for promoting enhanced 
antibody responses to boosting with recombinant protein by the intradermal method. 
 
1.1.2 
Disease/participants studied 
Healthy male and female volunteers 18 to 50 years old who are at low risk of HIV infection 
are to be recruited. For more details refer to Section 4. 
 
1.1.3 
Trial interventions 
The trial interventions are: 
 
• 2.6mg DNA/dose: plasmid encoding clade C gp140 envelope derived from HIV-1 isolate 
97CN54 
 
• 50ug CN54gp140/dose: trimeric recombinant clade C envelope protein derived from 
97CN54 
DNA immunisations will be administered by the combined intramuscular (IM) and 
intradermal (ID) methods as described in Table 1 below. The CN54gp140 will be delivered 
by ID injection.  
 
The doses, methods and schedule of immunisation are described in Table 1.  
 
 

 
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Table 1:   Schedule of Doses and Methods of Immunisation 
 
Group 
Method of Immunization; Dose of DNA 
Method of 
Immunization; Dose of 
CN54gp140 
Dose 1 at WK 0 
Dose 2 at WK 4
Dose 3 at WK 8 
Dose 4 at WK 20
1  
N=8 
0.6mg ID*/ EP 
2mg IM** 
0.6mg ID*/ EP
2mg IM** 
0.6mg ID*/ EP
2mg IM** 
50ug ID***
2  
N=8 
0.6mg ID*
2mg IM**/ EP 
0.6mg ID*
2mg IM**/ EP 
0.6mg ID*
2mg IM**/ EP 
50ug ID***
3 
N=8 
0.6mg ID*/ EP 
2mg IM**/ EP 
0.6mg ID*/ EP
2mg IM**/ EP 
0.6mg ID*/ EP
2mg IM**/ EP 
50ug ID***
* 
1 x 0.15ml injections ID via a needle – into the upper arm with or without 
electroporation (EP) 
**  
1 x 0.5ml injections IM – into the upper thigh with or without electroporation (EP) 
*** 
1 x 0.1ml injections ID via a needle - into the upper arm 
 
1.1.4 
Aim 
This study will test the hypothesis that DNA-C CN54ENV vaccination administered via the 
combined intramuscular and intradermal methods with and without electroporation can 
maximise responses on subsequent intradermal boosting with recombinant CN54gp140 
protein. 
 
1.1.5 
Objectives  
Primary 
 
To evaluate the safety and immunogenicity of an HIV-1 DNA vaccine delivered via 
combined intramuscular and intradermal methods with and without electroporation, 
and boosted with CN54gp140 administered by intradermal injection with the aim of 
indentifying optimal conditions capable of promoting enhanced antibody responses 
to HIV. 
 
 
 
Secondary  
 
To compare the safety of the electroporation devices to standard intramuscular or 
intradermal injections  
 
 
Exploratory: 
 
To describe qualitative differences between the different methods of delivery in 
terms of changes from baseline in overall magnitude and functionality of antibody 

 
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responses to the vaccines, and quantitative differences in cellular responses (CD4+ 
and CD8+ T cells) 
 
To evaluate the tolerability of vaccination using the electroporation devices 
 
1.1.6 
Endpoints 
Primary 
Safety 
 Grade 3 or above local solicited adverse event (Table 2) 
 Grade 3 or above systemic clinical and laboratory solicited adverse event (Table 2) 
 Any grade of adverse event that results in a clinical decision to discontinue further 
immunisations 
 Any grade of adverse event within 7 days of receiving intradermal and standard 
intramuscular vaccinations with or without electroporation  
 
Immunogenicity 
 The magnitude of antigen specific systemic IgG antibody binding responses (µg/ml) 2 
weeks after the final vaccination. 
 
Secondary  
Safety 
 Any grade of adverse event, local to the ID and IM injection sites that start within 7 days 
after Doses 1-3.   
 
Exploratory:  Immunogenicity 
 Frequency and magnitude of HIV-gp140 specific B-cell-mediated responses in the 
systemic compartment measured by B-cell ELISPOT 
 The magnitude of vaccine specific systemic T cell responses by T cell ELISpot assay  
 The magnitude of antigen specific systemic IgA antibody responses (ug/ml) 
 Frequency, titre and avidity of serum binding antibodies to other HIV Env antigens 
(alternative clades) by ELISA or other assays. 
 Mapping of serum binding antibodies using  Env subunit constructs  (e.g., V2 scaffolds 
and hotspots) by ELISA. 
 Frequency and magnitude of mucosal IgG and IgA antibody responses to CN54gp140 
measured four weeks after the final immunisation. 
 Frequency and titre of serum neutralising antibodies to homologous virus, and, if 
warranted a wider  panel of viruses representing different clades.  
 Frequency and magnitude of HIV-specific T-cell mediated responses measured by T-cell 
CFSE, and ICS (Intracellular Cytokine Staining). 
 Frequency and magnitude of T-cell chemokine and cytokine release following ex-vivo 
antigen stimulation quantified by Luminex. 
 Isolation and characterization of Env-specific monoclonal antibodies (IgG) from memory 
B cells in the systemic compartments (dependent upon elicited specific memory B-cell 
numbers). 
 Characterisation of non-neutralising antibody function using ADCC/ADCVI, viral capture 
and aggregation assays.  
 Epitope mapping of B- and T-cell responses. 
 

 
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Exploratory: Tolerability 
 Pain scores at 0, 10 and 30 minutes following vaccination with EP 
Table 2  
Solicited Adverse Events 
 
Type 
Event 
Local AEs 
(immunisation 
site) 
Discomfort 
Redness 
Swelling (soft) 
Induration (hard) 
Blisters 
Systemic 
Clinical AEs 
Abnormally raised temperature 
Chills 
Myalgia/flu-like general muscle aches 
Malaise (excess fatigue) 
Headache 
Nausea 
Vomiting 
Systemic 
Laboratory AEs 
Abnormalities in: 
Creatinine, AST, ALT, alkaline phosphatase, total bilirubin, glucose         
Hb, total WCC, neutrophils, lymphocytes, platelets 
 
1.1.7 
Duration 
It is anticipated that enrolment will take 12 months. Each subject’s participation in the trial, 
from screening until final visit (a total of 12 visits), will be approximately 12 months. 
Therefore the end of the trial, defined here as the final visit of the last participant, should 
be achieved approximately 24 months after the start. 
 
1.1.8 
Data collection for the study 
All participants will be registered as patients at Hammersmith Hospital. Data will be 
recorded in the medical notes which will contain the handwritten clinical notes and 
additional information such as the results of pregnancy tests and urinalysis. Data will be 
entered onto the case report form (CRF) and (randomised subjects only) sent to MRC CTU 
at UCL for data entry into a secure database. The CRF data will include medical history 
related to eligibility, dates of visits including immunisation, results of pregnancy tests, 
solicited adverse events, a description of non-solicited adverse events, and concomitant 
medication. Participants will record information on solicited adverse events in a diary card 
which will act primarily as an aide memoire to be checked at the next clinic visit and 
(randomised subjects only) sent to MRC CTU at UCL for data entry when complete. 
 
1.1.9 
Organisation 
Imperial College London (ICL) will act as sponsor for the CUTHIVAC002 trial and the MRC 
CTU at UCL will manage the data (randomised subjects only) on their behalf. The clinical 
site will be the Hammersmith Hospital (Imperial College Healthcare NHS Trust). The project 
is funded by the European Commission FP7. 
 

 
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1.2 Flow Diagram 
Figure 1 
Screening, Randomisation, Immunisations and Follow-up Flow 
Diagram 
 
 
 
Screen wk -6 to 0 
 
 
 
 
 
 
Randomise wk 0 
 
 
 
 
 
 
 
 
 
 
 
Group 1 
 
Group 2 
 
Group 3 
0.6mg ID+EP 
2mg IM 
DNA  
Wk 0 
0.6mg ID 
2mg IM+EP 
DNA  
Wk 0 
0.6mg ID+EP 
2mg IM+EP  
DNA 
 
 
 
 
 
 
 
 
0.6mg ID+EP 
2mg IM 
DNA 
Wk 4 
0.6mg ID 
2mg IM+EP 
DNA 
Wk 4 
0.6mg ID+EP 
2mg IM+EP  
DNA 
 
 
 
 
 
 
 
 
0.6mg ID+EP 
2mg IM 
DNA 
Wk 8 
0.6mg ID 
2mg IM+EP 
DNA 
Wk 8 
0.6mg ID+EP 
2mg IM+EP  
DNA 
 
 
 
 
 
 
 
 
50ug ID 
CN54gp140 
Wk 20 
50ug ID 
CN54gp140 
Wk 20 
 
50ug ID 
CN54gp140 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Primary immunogenicity at wk 22 
and follow-up to final visit at wk 24 (non-
responders) 44 (responders only) 
 
 
 
 
 
 
 
 
 

 
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2. BACKGROUND 
2.1 Background 
2.1.1 
The global HIV-1 situation and the need for a 
vaccine 
The global AIDS epidemic continues to grow. In its 2013 Report on the global AIDS 
epidemic, UNAIDS published the following estimates for 2012.  
 
35.3 million people living with HIV 
 
There were 6301 new infections per day 
 
Gains in expanding access to HIV treatment cannot be sustained without a reduction 
in the rate of new HIV infections 
 
2.1.2 
The status of the field 
A prophylactic HIV vaccine is considered to be the most effective and sustainable way of 
reducing the rate of new infections. The International AIDS Vaccine Initiative (IAVI) has 
estimated that a vaccine has the potential to prevent over 70 million infections in 15 years 
[1]. The six different HIV vaccine efficacy trials which have been conducted to date have 
suggested an important role for both humoral and cellular immune responses and both are 
considered central to vaccine design, but to date there has been no robust correlate of 
protection against infection.  The first vaccine efficacy trials (Vax 003 and Vax 004) 
focussed on the humoral response to the viral envelope and when they failed to show 
efficacy [2] [3] the focus of design shifted to T-cell responses. There was no envelope 
immunogen included in the vaccine used in the Step and Phambili trials which both made 
use of an adenovirus (Ad5) derived vaccine encoding gag, pol and nef. These two trials 
were both stopped early for futility and there was also a worrying suggestion of increased 
infection risk in vaccinated individuals [4, 5]. The HVTN505 trial also included an attenuated 
Ad 5 vaccine – this time given as a single boost after 3 priming immunisations with DNA 
encoding matched HIV surface and internal proteins (VRC-HIVDNA016-00VP). Although this 
trial was also stopped early for futility, there was no evidence of increased risk of infection 
in those who had received the vaccine, providing some reassurance about the platform [6]. 
RV144 is the only Phase III trial carried out to date to demonstrate (partial) efficacy, where 
IgG antibodies to the V1/V2 region of gp120 correlated with decreased risk of infection. 
Nevertheless, both non-efficacious vaccine and partially effective vaccine trials have 
provided immune correlates of infection risk. Three have described correlates of infection 
risk/incidence per se (VAX004, Step and RV144) and two have identified potential sites of 
selective immune pressure on the virus (Step and RV144), reviewed in [7]. 

 
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2.1.3 
The immune response to HIV  
2.1.3.1        The role of Antibodies 
Neutralizing antibodies (NAb) are the dominant correlate of protection in most prophylactic 
vaccines, and a successful vaccine against HIV-1 will probably be no exception. Studies in 
animals have suggested that pre-existing neutralising antibodies can prevent HIV infection, 
but it is not clear how to induce such responses with a vaccine. A significant minority of HIV 
infections result in heterogeneous mixtures of polyclonal broadly neutralizing antibodies 
(bNAbs) with broad specificity for the virus envelope [8].  Passive transfer studies in 
macaques using cloned bNAbs have shown that realistically achievable levels of such 
antibodies are able to block infection following low dose intravaginal challenge with SHIV -
providing proof of concept for the strategy [9, 10]. Four of the completed efficacy trials 
induced envelope-specific antibody responses and whilst conducted in very different 
populations with varying risks of infection, some lessons can be drawn. The Vax 003 trial 
which was conducted in high risk injecting drug users showed that antibodies to pre-
specified and vunerable regions on the viral envelope (V2, V3 and gp120) were not 
correlates of infection risk and neither were those which blocked CD4 binding or neutralised 
certain strains of HIV-1 (MN).  
 
These results contrasted with those reported in Vax 004 which enrolled high risk men 
(predominantly MSM) and women. Follow-up studies in this trial reported that higher 
neutralising antibody responses to tier 1 viruses, those that blocked CD4 binding and those 
with antibody-dependent cellular virus inhibitory functions correlated with lower levels of 
HIV infection. An ideal HIV vaccine should induce potent and sustained bNAb responses. It 
is assumed that the breadth and magnitude of responses induced in Vax 004 were not 
sufficient to be protective. The RV144 trial reported an efficacy of 31% and so provided the 
first ever opportunity to look for correlates of protection. An international consortium of 
scientists worked together on a retrospective case control study. Six primary immunological 
variables were selected and analysis revealed that only two correlated with the risk of HIV-1 
infection. IgG antibodies to the V1/V2 region of gp120 correlated with decreased risk and 
levels of envelope-specific IgA in plasma correlated with increased risk of infection in 
vaccinated individuals (ie decreased vaccine efficacy). Although neutralising antibodies were 
one of the six primary variables included in the primary analysis, levels were not correlated 
with risk of infection. Nevertheless, this does not exclude the possibility that the low but 
detectable neutralising responses may have had an impact on the risk of transmission [11].  
 
The envelope protein immunogen used (A244 gp120) had an 11 amino acid deletion 
leading to better exposure of certain epitopes. This protein was antigenic for both linear V2 
epitopes as well as for V1V2-glycan epitopes bound by V1V2 broadly neutralising 
antibodies. Understanding why the immune responses in this population were so dominated 
by antibodies to non-glycan epitopes remains an area of intense interest.  
 
 
2.1.3.2        The role of T cells 
A direct role for CD8+ T-cells in the control of viral replication in humans has been 
demonstrated during acute infection and also in those rare individuals who are able to 
naturally control infection. Non-human primate (NHP) models have provided compelling 
evidence that Class I restricted CD8+ T-cells can be sufficient for highly effective control of 
viral replication and eventual viral clearance. More recently Picker and colleagues have 
described a protective role for a novel subset of Class II restricted CD8+ T-cells with broad 
specificity for HIV-1 which develop following vaccination with this CMV derived vaccine [12, 
13]. Step and Phambili were the first two Phase III trials which focussed solely on the 
CD8+ T-cell response. The trials were both designed to test the concept that a replication 

 
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defective adenovirus serotype 5 virus could decrease viral load in those who became 
infected- a hypothesis also tested in the HVTN505 trial which used the same Ad5 vaccine 
but this time as a single boost after priming three times with DNA. All three trials were 
stopped prematurely for lack of efficacy and there were increased infections seen in those 
who received vaccine relative to placebo in the Step trial – which was not seen in HVTN505. 
Post-hoc analysis revealed that the increased susceptibility correlated with levels of pre-
existing antibody to Ad5 in uncircumcised individuals, although the effect could not be 
disentangled from other confounders [14]. Nevertheless, “sieve analysis” of the viruses 
circulating in infected vaccinated individuals revealed that CD8+ T-cells were exerting a 
direct impact on viral replication -arguing the case for continued investment in this strategy. 
However, proof of concept that CD8 responses can provide protection for infection in 
human is still needed.  
 
2.1.4 
       Current Vaccine Strategies 
In an effort to induce balanced immune responses several groups are focussed on the 
optimisation of heterologous prime boost vaccination regimens with DNA and modified viral 
vectors [15] [16] [17]. In light of the results of the RV144 trial, there is also an interest in 
combining recombinant proteins with viral vaccines. The rationale for these strategies 
initially stemmed from the results of pre-clinical studies which showed that priming with 
DNA followed by boosting with modified viruses induced stronger cellular immunity than 
either DNA or virus alone. The first HIV-based DNA vaccines have been somewhat 
disappointing in man in terms of their immunogenicity- but DNA has repeatedly been shown 
to play a significant role in priming B and T-cell responses. The approach has proved 
partially protective in animal models and early phase trials in humans have reported 
particularly strong immunogenicity within the T-cell compartment with varying levels of 
success within the B-cell compartment [15] [16]. However, importantly for this study DNA 
prime-protein boost strategies have been shown to increase antibody functional activity and 
avidity [18, 19] [20]. A vaccine able to induce neutralising antibodies remains elusive- but 
the antibody responses in RV144 which correlated with risk of infection has brought the role 
of binding antibodies and those with other non-neutralising functions such as antibody-
dependent cell-mediated cytotoxicity (ADCC) into the spotlight.  
 
2.1.5 
DNA Vaccines  
DNA plasmid vaccines showed great promise in small animals, resulting in the licensing of 
three veterinary DNA-based vaccines but these successes failed to translate in human trials 
and this is probably because the uptake of DNA is very inefficient with as much as 91%-
99% degraded within 90 minutes [21] [22]. DNA is particularly well suited to priming T-cell 
responses (CD4+ & CD8+) and does not carry the disadvantages associated with modified 
viral vectors and so is an attractive candidate for HIV vaccines [23-25]. Preclinical studies 
suggested a clear dose dependency in immune responses to DNA, but there is relatively 
limited clinical data available. When comparisons have been made, results have actually 
been somewhat inconsistent with some showing dose dependency [19] [26] and others not 
[27] [28]. In the study of Bansal and colleagues 100% (n=6) of healthy volunteers given 
7.2mg DNA (5 plasmids encoding gp120 and 1 encoding gag) three times over 12 wks 
made CD4+ ELISPOT responses to Env peptides, whereas only ~30% responded to 6-fold 
lower dose of vaccine  [26]. Interestingly, CD8+ T-cell responses to gag and poly [26] 
functional CD4+ T-cell responses (2 or more cytokines) were seen only in response to the 
higher dose of DNA. Although early approaches to DNA vaccination failed to induce 
significant antibody responses in humans, more recent trials have shown that DNA vaccines 
can induce neutralizing antibodies against a number of viruses [29-31]  and detectable 
antibody responses to HIV-1 [32]. A dose relationship for DNA delivered with EP in humans 
has recently been shown for an immunotherapeutic vaccine against HPV16/18 in humans 

 
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[33]. This demonstrated a dose response trend where mid dose (1mg) appeared to be 
marginally better in inducing antibody responses than low (0.3mg) or high (3mg) doses. 
Various strategies can now be used to improve and augment the immunogenicity of DNA 
vaccines including: promoter selection and codon optimization; the use of electroporation 
(EP); and the method of administration (intramuscular (IM) or intradermal [34, 35]. 
 
2.1.6 
     Increasing the potency of DNA vaccination 
2.1.6.1        Electroporation 
When administered via a needle, relatively high doses of DNA are required, typically in the 
range of milligrams per dose. Physical methods of delivering the DNA such as via in vivo 
electroporation (EP) or gene gun have been shown to enhance immunogenicity [36] [37, 
38]. In addition to increasing uptake directly, EP also results in increased local inflammation 
and recruitment of antigen-presenting cells (APC) which has been shown to augment 
immunogenicity and increase the duration and levels of antigen expression. EP has been 
shown to increase the potency of HIV DNA vaccines in animal models [39-41]. T-cell 
responses to the GTU®MultiHIV vaccine were enhanced when tweezer electrodes were used 
in association with ID immunisation of macaques and mice [42]. The same TriGrid device 
from Ichor which is advocated for use has been used to deliver EP with IM for 
administration of the ADVAX DNA vaccine to rabbits and mice. In an immunogenicity study 
carried out in mice, the magnitude of ELISPOT and intracellular cytokine responses was 
increased by EP without increased risks of integration of the DNA [43]. The first use of EP 
with an HIV DNA vaccine has been published and showed that the method was safe and T-
cell immunogenicity was enhanced [44]. More recently EP has been used to augment the 
potency of HIV vaccines in early phase trials and has shown to be well tolerated and 
effective and is being assessed for feasibility in larger Phase II trials by several different 
networks [45]. 
 
2.1.6.2         DNA  
Volunteers will receive 3 injections of 0.6 mg, ID with or without electroporation in a 
volume of 0.15ml at 0, 4 and 8 weeks together with 2mg of DNA in 0.5ml delivered via IM 
with or without electroporation. The DNA-C CN54ENV plasmid encodes clade C envelope 
derived from the HIV-1 97CN54 coding sequences (Geneart). Sequenced optimised inserts 
were introduced into the VRC8400 CMV/R vector (NIAID/NIH) for GMP manufacture. This 
vaccine has been manufactured by Althea Technology, Inc (USA).  The DNA is formulated 
in 1 x phosphate buffered saline (PBS; pH 7.2) and will be supplied at a concentration of 
4.0 mg/mL in vials containing 1.2 mL. 
 
2.1.6.3           Recombinant CN54gp140 
CN54gp140 is a recombinant GP140 derived from the HIV-1 97CN54 coding sequence and 
has been manufactured by Polymun, Austria. CN54gp140, is a trimeric recombinant C-clade 
ENV protein, derived from the 97CN54 Chinese viral isolate. The protein comprises a 
sequence of 670 amino acids, and has been shown to be immunogenic in humans. 
MucoVac 1 (EudraCT number 2007-000781-20) was the first human clinical trial to have 
used the trimeric CN54gp140 although the protein was not administered systemically but 
topically. Mucovac 2 generated the first safety data on the systemic administration of the 
CN54gp140 in healthy volunteers (see section 2.8.3). In this trial the protein will be 
supplied at 0.5mg/ml in a volume of 0.3mls. 50ug of protein will be administered ID at 
Week 20 (see below). 

 
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2.1.7 
The relevance of the method of vaccination 
Vaccine success depends upon the targeting and activation of dendritic cells (DCs). 
Vaccines are typically injected into the skeletal muscle (IM) or subcutaneous (SC) tissues 
for reasons of convenience. They are also typically given with adjuvants which augment 
recruitment of immature DC, stimulating their maturation and migration to the draining 
lymph nodes where they interact with naive T-cells. There is increasing interest in 
cutaneous vaccination (ID, TC and epidermal) because in comparison to muscle and SC 
tissues, the skin has relatively high numbers of resident APC. The relatively polarised 
location and function of the two major subsets of DC found in skin (Langerhans and dermal 
DC) has provided biological insights into the outcomes of dermal vaccination. The human 
epidermis is particularly rich in LC whereas dermal DC (dDC) are found primarily in the 
dermis. Both these cell types are able to migrate to the draining lymph nodes or the spleen 
via lymphatic drainage where they initiate immune responses following interaction with T- 
and B-cells [45]. Klechevsky et al., purified these two subsets from human skin and 
analysed their functions in vitro [46]. dDC (CD14+) interacted preferentially with CD4+ T 
follicular helper (Tfh) cells, inducing naive T- and B-cells to make antibody, and were 
relatively poor at stimulating CD8+ T-cell responses, primarily because they were unable to 
induce granzyme A/B or perforin in those cells.  
By contrast, epithelial LC were not able to induce Tfh development or induce naive B-cells 
to make antibody but were efficient at processing antigens via the Class I pathway and 
were relatively good at stimulating lytic CD8+ T-cells. These findings might explain the  
immunological effects of scarification as used with small pox vaccination.  This results in 
injury to both the epidermis and dermis thereby mobilising both dDC and epidermal LC -
resulting in the stimulation of both humoral and cellular immune responses. The two cell 
subsets have also been shown to differ in their expression of Toll-like receptor (TLR), with 
dDC expressing a broader repertoire than LC, which might account for the relative 
unresponsiveness of LC to (commensal) bacteria [47] [48] . In a study directly supporting 
the design of this trial, concomitant DNA priming ID and IM followed by boosting SC was 
shown to optimise antibody responses in the mouse. EP delivered by tweezer electrodes IM 
and ID was also shown to significantly augment T-cell and B-cell responses and to increase 
antibody avidity [41]. 
 
(i) 
Dermal Immune Responses 
It thought that cells activated in the dermis are able to home back both to the skin and the 
mucosa linking these two immunological compartments and thus making dermal vaccination 
attractive for targeting mucosal immune responses [49] [50].  The volumes which can be 
delivered ID are much smaller than can be delivered IM/SC which is a practical constraint 
when considering this route. The dose sparing effects of ID vaccination has been shown 
using a variety of vaccines including rabies, hepatitis and influenza, typically, sparing 80-
90% of the dose which would be required via IM/SC routes [16, 45, 51]. Using the hepatitis 
B vaccine, the ID route is also able to overcome the lack of responsiveness seen in patients 
on hemodialysis to IM vaccination [52]. Several explanations have been suggested for the 
relatively increased efficacy of ID vaccination; ID may result in increased local inflammation 
which, in turn, might result in the recruitment of APC such as LC and DC, resulting in the 
increased cross presentation of antigens. ID vaccination has also been shown to favour the 
direct migration of antigens through the lymphatic ducts. Soluble proteins injected this way 
are able to reach the lymph nodes within 2 hrs which is ~12 hrs before the arrival DCs. This 
reduced transit time is thought to be purely a consequence of the mechanics of fluid 
injection. The increase in interstitial pressure caused by the injected fluid probably results in 
increased permeability in the capillaries and so also increased lymphatic flow.   

 
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2.1.8 
    Research Leading up to the Proposed Trial  
2.1.8.1     The vaccine  
DNA-C CN54ENV 
The DNA vaccine will utilize the pVRC8400 plasmid expression vector containing the 
optimized pCMVR promotor [53]encoding a codon-optimized sequence for clade C gp140. 
The sequence was derived from a Chinese viral isolate, 97CN54. The C-clade HIV subtype is 
believed to cause more than 50% of worldwide HIV-1 infections, and is predominant in 
southern and eastern Africa and India [54]. Previous clinical studies using this plasmid 
backbone to express similar HIV-1 Env sequences generated low but detectable antibody 
responses to HIV-1 Env and achieved 70% seroconversion [32] following intramuscular 
injection without EP. Transgene codon optimization for CN54gp140 and insertion into the 
pVRC8400 vector have been performed by GeneArt and GMP product has been 
manufactured by Althea Technology, Inc (USA). There are no specific nonclinical 
pharmacology studies with the DNA-C CN54ENV vaccine alone. A murine study carried out 
to inform the design UKHVC_003 trial included a group which received the CN54ENV DNA 
alone.  Whilst a relatively much higher concentration of DNA was used, the DNA was well 
tolerated and was clearly serving to prime and influence immune responses to the other 
boosting vaccines used. The env encoding plasmid vaccine (CN54ENV) is very similar in 
structure and HIV insert sequence to the equivalent plasmid previously tested, in 
combination with a NYVAC-C boost, in clinical trials EV02 and EV03 [55, 56].  In these 
studies, priming with DNA at a dose of 4mg on two or three occasions was shown to be 
well-tolerated and effective in contributing to overall immunogenicity. Identical CN54EENV 
DNA plasmid is currently in use in the UKHVC spoke 3 (EUDRACT 2012-003277-26) trial 
initiated in July 2013, which is exploring priming with DNA followed by boosting with MVA 
combined with CN54gp140 or the same immunogens given sequentially.  
 
 
CN54gp140 
The CN54gp140 is a recombinant gp140 derived from the HIV-1 97CN54 coding sequence 
and will be manufactured by Polymun, Austria. CN54rGP140, is a trimeric recombinant C-
clade envelope protein. The protein comprises a sequence of 670 amino acids, and has 
been shown to be immunogenic in humans. To date Mucovac 2 (EUDRACT 2010-019103-
27) is the only completed trial to have generated safety data on the systemic administration 
of the CN54gp140 in GLA-AF in healthy volunteers. 100g of protein was administered IM 
after bedside mixing with GLA-AF (an aqueous formulation of synthetic MPLA). Analysis of 
the (presumed) peak antibody responses measured after the last immunisation 
demonstrated induction of robust antibody responses, with moderate neutralization activity 
against easy to neutralize tier 1 viral isolates. 
 
 
Preclinical Studies 
A Single Dose Biodistribution Study (No 525574) of CN54ENV DNA delivered intramuscularly 
with electroporation has been performed in rats. The results from this study show that 
biodistribution of the plasmid is restricted to the site of administration and that there is no 
persistence of the plasmid in these tissues beyond 60 days. A good laboratory practice 
(GLP) rabbit tolerance and toxicity study (CR 525569) has been performed to directly 
support this study, where animals received vaccinations with a 2mg dose of CN54DNA given 
intramuscularly three times at 3 weekly intervals with electroporation, followed by 
intradermal or intramuscular boosting with CN54gp140 (100ug).  There were no adverse 

 
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clinical signs that were considered to be related to treatment with DNA-C-CN54ENV, DNA-C-
ZM96GPN or CN54 gp140. There was transient very slight local irritation noted after 
intradermal injection of CN54gp140.  There were no worrying local side effects or 
indications of systemic toxicity. Analysis of the (presumed) peak antibody responses 
measured 2 weeks after the last immunisation corroborated what had been seen previously 
in mouse and rabbit studies. 
 
 
Clinical Studies 
In the TaMoVac 1 trial which was carried out in two sites in Tanzania, 40 subjects who had 
previously been given three doses of HIVIS-DNA followed by two with MVA-CMDR were 
given two further boosts with 100g CN54gp140, adjuvanted with 5g GLA-AF.  The 
rationale for boosting with CN54gp140 in TaMoVac 1 was informed by the results of RV144 
and other trials using recombinant protein vaccines and a desire to complement the T-cell 
response with a strong B-cell response to HIV envelope. The adjuvanted protein was well 
tolerated and systemic CN54gp140-specific antibody responses significantly boosted in 
those primed with DNA and MVA to levels in the range seen in the RV144 trial (A.Bauer 
personal communication). The neutralising antibody responses were, however disappointing 
(A.Joachim personal communication) CN54gp140 has also been given to human subjects in 
two completed trials: MUCOVAC 1 and MUCOVAC 2 and is part of the regimen in 3 ongoing 
trials: UKHVC-003  which is a two centre Phase I trial running in the UK; TaMoVac 02, a 
two centre Phase II trial which is running in two sites in Tanzania and one in Mozambique; 
and X001 which is a single centre trial in the UK. 
In MUCOVAC 1, which was conducted in the UK in 2008, CN54gp140 formulated without 
adjuvant was administered to healthy female volunteers by the IVAG route.  Subjects 
received nine doses over a 3-week period over the course of one menstrual cycle. The 
vaccine was well tolerated and there were no serious adverse events. The immune 
responses were disappointing and there were no specific antibody responses detected. One 
possible explanation given was that the vaccinations were given too close together for 
optimal priming but there was no adjuvant included in the formulation which may also have 
contributed [57].  
MUCOVAC 2 (EudraCT 2010-019103-27) built directly on MUCOVAC 1 with a continued 
focus on mucosal immune responses. The trial was conducted in 36 healthy women in two 
centres in the UK who were randomised to one of four groups to receive CN54gp140 with 
or without GLA-AF/Chitosan and by a variety of methods.  The first (reference) group 
received three doses of 100g CN54gp10 adjuvanted with 5g GLA-AF (IM high dose), the 
second received three doses at a lower dose of 20g also adjuvanted with GLA-AF (IM low 
dose). A third group received a single priming dose of 100g CN54gp140/GLA-AF IM 
followed by two doses of 500g given intra-vaginally formulated in carbopol gel (IM IVAG) 
and a fourth received three doses of 100ug intranasally formulated in chitosan (IN). 
Following a protocol amendment, a group of 8 individuals (5 from the high dose IM group 
and 3 from the IN group) went on to receive 2 further systemic immunisations with GLA-AF 
adjuvanted CN54gp140 IM. The lower dose (20g) of CN54gp140 was as potent as the 
higher dose (100g) when given IM with GLA-AF- eliciting systemic binding IgG responses 
in the majority of individuals in both groups (9/11 and 9/9 respectively). 4/11 and 4/9 of 
the same women also had cervico-vaginal IgG responses. By contrast, there were no 
mucosal immune responses detected in the groups of women who received mucosal 
immunisations (IN or IVAG). Only 1/11 and 0/5 women in the IM IVAG or IN groups made 
systemic IgG. Interestingly after a single IM boost of the IN group this increased to 3/3, 
suggestive of amnestic responses and some effect of priming via the IN route. There were 

 
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no vaccine related serious adverse events although participants experienced at least one 
mild or moderate solicited adverse event but there were no safety concerns of note 
attributable to the study products and all participants completed all their allocated 
immunisations. 
Studies directly supporting the design of CUTHIVAC002 
Preclinical studies 
To directly support the design of this trial Mann and colleagues carried out studies in mice 
and rabbits using a similar DNA plasmid encoding env for priming and the same CN54gp140 
protein for boosting. The aims of the studies were (i) to assess the impact of DNA priming 
via different methods on T-cell and B-cell responses and (ii) to assess the impact of EP on 
each route after boosting with CN54gp140 [41]. The first part of the murine study was 
focussed on the optimisation of DNA priming. Vaccine specific antibody and cellular 
responses could be seen after DNA priming alone, which is one advantage of the model.  
Results showed that priming via the ID route favoured T-cell responses whilst the IM route 
favoured B-cell responses and that both were significantly augmented by EP.  They also 
clearly showed that in addition to increasing the magnitude of the antibody response to 
CN54gp140, when given with both ID and IM priming , EP also increased the avidity of the 
antibody response. The greatest impact upon the avidity of the response was seen only 
when EP was given with both ID and IM priming suggesting that both the T-cell and B-cell 
responses were required for optimal avidity.  
The same study also examined the optimal route for boosting with CN54gp140 DNA priming 
ID/IM with or without EP. The effect of DNA priming was striking and antibody responses 
were generally poor without it. Of the methods tested, subcutaneous (SC) and intranasal 
(IN) boosting was the best for generating systemic IgG responses, with IM boosting 
performing almost as well. IN boosting was significantly better than any other method in 
terms of IgA responses followed by the SC and IM methods. The transcutaneous route (TC) 
was able to boost systemic antibody responses but IgA responses were disappointing. In 
contrast to what was seen for antibody responses, the route of boosting with CN54gp140 
did not have much impact on the magnitude of cellular responses seen after DNA priming 
(ID and IM) in the absence of EP but without priming, the responses were negligible. 
Studies in rabbits also clearly showed that priming by combined ID and IM immunization 
delivered with electroporation, followed by boosting SC resulted in antibody responses with 
neutralising activity (Tier 1) and that both were required for optimal responses. Increasing 
antibody affinity could also be seen after repeated protein boosts. Additional animal studies 
have shown that the route of DNA administration (IM vs. ID) can also impact on the quality 
of antibody induction. Recent studies in macaques suggest that both ID/EP and IM/EP DNA 
delivery promoted robust antibody responses to SIV Envs (reciprocal endpoint titre log 4.5–
5) but that ID/EP administration induced higher cross-reactive neutralizing responses than 
IM/EP administration [58].This is further supported by previous studies using DNA encoding 
H5 hemaglutinin where concomitant IM/EP plus ID/EP induced significantly stronger 
responses than either route individually [59].These data suggest that split dose vaccination 
when given at different anatomical sites may have a positive impact on the magnitude of 
induced antibody responses. While these preclinical data are promising, differences in skin 
structure and resident immune cell populations between species mean the relative merits of 
ID/EP and IM/EP in the context of Env DNA can only be accurately determined in humans. 
Therefore we will also assess the impact of concurrent ID and IM administration in this 
study. 

 
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Clinical studies:   CUT*HIVAC_001 
This Phase I trial, which is fully recruited and volunteers are currently in follow up in 
London, is part of the same programme of research and is also exploring different methods 
of vaccination with a similar HIV DNA plasmid vaccine developed by FitBiotech and 
encoding a polypeptide containing peptides derived from envelope and core proteins.  
The primary objective of this study is the induction of CD8 T cell responses to the vaccine 
immunogen. 30 healthy volunteers were randomised to receive 13.2 mg of GTU® MultiHIV 
B clade DNA plasmid given in three immunisations over 12 weeks via (i) IM and ID (ii) IM 
and Transcutaneously  (TC) or (iii) IM with EP (Ichor TriGrid device). The transcutaneous 
route has been shown to favour the development of CD8+ T-cell responses in animal 
models and also in one Phase I clinical trial of a licensed influenza vaccine.  
 
2.2 Rationale and Objective 
2.2.1 
Study Rationale  
We will explore three combination regimens with and without EP with the overall aim of (i) 
optimising humoral and cellular immune responses (ii) developing safe and well tolerated 
vaccination strategies.  We propose to combine the previously used IM and ID methods 
because clinical and preclinical data suggest that the combination will favour the 
development of balanced immune responses.  All groups will receive 3 doses of 2.0mg of 
the  CN54ENV plasmid DNA (CN54ENV) vaccine in 0.5ml volume IM together with 3 doses 
of 0.6mg of the CN54ENV vaccine in 0.15ml ID each given at 4 weekly intervals over 8 
weeks followed by 1 dose of 50g CN54gp140 ID at Week 20. A directly supportive murine 
study using a similar DNA vaccine, the same CN54gp140 protein and following the same 
design (albeit using a different EP device) demonstrated that priming via the combined 
IM+ID route favoured the development of high avidity humoral immune responses. This is 
further supported by the preclinical toxicology study (CR 525569) performed in rabbits 
using one of the Ichor devices that will be used in this study.  
We are interested to see whether such concomitant priming performs similarly well in man 
resulting in optimal balanced immune responses. DNA vaccines have been relatively 
disappointing in man but there is still great interest in this platform because of its intrinsic 
flexibility, the relatively low cost of production of GMP material and the speed at which 
immunogens can be modified. We will be able to assess the effect of EP on each different 
route of vaccination and also to make a direct assessment of the translation of the very 
clear results seen in the murine study to the human which would be of general benefit to 
the field [34]. The trial is not powered to carry out formal statistical comparisons between 
the three groups and the immunological component is intended to be primarily descriptive. 
 
2.2.2 
Investigational Product / Intervention(s) 
2.2.2.1  Investigational product DNA 
The plasmid (DNA-C CN54ENV) encodes the HIV-1 clade C gp140 (env) derived from 
97CN54.  The IM dose will be 2mg which will be given in 0.5ml and the ID dose 600ug 
given in 150ul. The codon-optimised HIV-1 insert was introduced into an E coli VRC8400 
CMV/R vector for manufacture. For the proposed clinical study, CN54ENV DNA will be 
manufactured in accordance with GMP by Ajinomoto Althea Technologies.  Manufacture of 
the plasmid at Ajinomoto Althea is based on starting material containing synthetic genes 

 
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assembled by GENEART AG. The manufacturing process for CN54ENV DNA is based on 
established procedures as follows: fermentation and harvest of E coli host, cell lysis and 
diafiltration; purification by multiple steps (including endotoxin removal, anion exchange 
chromatography, ethanol precipitation, sterile filtration); formulation and filling. The 
plasmid is formulated in phosphate buffered saline, pH 7.2 and supplied as a sterile solution 
for injection at a concentration of 4.0 mg/mL in a 1.2ml volume.  The DNA preparation is 
presented as a clear to opalescent, colourless liquid in 2 mL glass vials with rubber stoppers 
and flip-off seals. 
 
2.2.2.2  Recombinant CN54gp140 
For the proposed clinical study, the CN54gp140 will be manufactured by Polymun Scientific. 
CN54gp140 is a trimeric recombinant HIV-1 clade C envelope glycoprotein derived from a 
Chinese viral isolate 97CN54 [60] [61] . CN54gp140 solution comprises CN54gp140 
recombinant glycoprotein formulated in an aqueous dilution buffer. The HIV-derived amino 
acid sequence of CN54gp140, as predicted from the primary DNA sequence of the clone, 
comprises 634 residues.  The molecular mass predicted by the polypeptide sequence alone 
is approximately 70 kD. However, the protein is heavily glycosylated and has a mass of 
approximately 140 kD as determined by SDS-PAGE and size-exclusion chromatography. 
Furthermore, the CN54gp140 secreted by CHO cells is oligomeric, and following purification 
is essentially trimeric, with a projected mass of 420 kD. CN54gp140 solution is provided at 
a concentration of 0.50 mg/mL, in a volume of 0.3ml, as a clear, colourless, sterile liquid, 
presented in translucent polypropylene vials.  
 
 
2.2.2.3      Device Specific Interventions  
(i) Electroporation: The Ichor TriGrid™ Delivery System for Intramuscular 
Delivery (TDS-IM) 
Background 
Ichor developed the first integrated, automated application system called the TriGrid 
Delivery System (TDS) for the intramuscular administration of DNA vaccines with EP. A 
comprehensive technical dossier providing detailed descriptions of the device, its function, 
manufacturing procedures, as well as relevant verification, validation, and safety studies will 
be submitted by Ichor in the form of a Clinical Investigation Application for a Medical device 
submitted to the MHRA which references the corresponding Medicinal product application. 
The TDS-IM device has not been used to deliver this DNA plasmid vaccine, but has been 
used for the delivery of several other similar HIV DNA vaccines (summarised in tables 3 and 
4) 
 
The TriGridTM array consists of four electrodes arranged in two equilateral triangles 
arranged to form a diamond shape around a central injection needle. Technical details of 
the device can be found in Appendix 1. This ensures effective and reliable “co-localisation” 
of the electrical fields and the agent to be delivered. Over 600 individuals have now 
received a variety of DNA vaccines in the clinic including ten clinical trials which have been 
completed and ten more which are ongoing. Extensive non-clinical studies of plasmid 
biodistribution, persistence, and integration have shown that, consistent with conventional 
IM injection,  EP based DNA delivery using the TDS-IM device is associated with localised 
uptake of the DNA at the injection site in surrounding tissues and draining lymph nodes 
within one week of administration. By 30-90 days there is substantial reduction in the levels 

 
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of DNA present in the injection site tissues and no evidence of significant integration of the 
vaccine candidates into genomic DNA. A single dose biodistribution study (Charles River 
Laboratories, UK Study No 525574) to directly support the proposed trial, demonstrated 
that the distribution of CN54 ENV expression plasmid when delivered using the TDS IM 
device was restricted to the site of administration and that there is no persistence of the 
plasmid in these tissues beyond 60 days. 
 
 
Preclinical assessment of the device:  Safety  
The TDS IM device has been shown to enhance cellular and humoral immune responses to 
a viral, parasitic and tumor associated antigens in a wide variety of animal species including 
rodent, rabbit, non-human primate and cattle. The device has been assessed in multiple 
GLP compliant repeat dose/toxicity studies.  
 
Formal safety studies have been conducted with nine individual DNA vaccines: 
 
Melanoma DNA vaccine encoding a xenogenic form of the tyrosinase antigen. 
 
Multi-antigen DNA vaccine encoding the gag pol nef tat vif and env antigens of HIV 
subtype C 
 
A malaria DNA vaccine encoding multiple epitopes isolated from malaria antigens. 
 
An epitope based melanoma DNA vaccine encoding epitopes isolated from the TRP-
2 and gp-100 antigens. 
 
HIV-1 DNA vaccine encoding the subtype B gag, pol, nef, tat vif, env antigens 
coformulated with plasmid DNA encoding the human IL12 cytokine. 
 
HBV DNA vaccine coformulated with plasmid DNA encoding the human IL-12 
cytokine. 
 
DNA vaccines encoding the M antigen of the Hantaan and Puumala hantaviruses. 
 
A DNA  vaccine encoding HPV E7 antigen with calrecticulin based adjuvant. 
 
A DNA vaccine encoding multiple antigens from the Venezuelan equine encephalitis 
virus. 
 
A single dose Biodistribution Study (N0 525574) of CN54ENV DNA delivered 
intramuscularly with electroporation preformed in rats 
 
A 
 rabbit 
tolerance 
and 
toxicity 
study 
(CR 
525569) 
where 
animals 
received vaccinations with a 2mg dose of CN54DNA given intramuscularly three 
times at 3 weekly intervals with electroporation, followed by intradermal or 
intramuscular boosting with CN54gp140 (100ug). 
 
The evaluations included repeated-dose safety/toxicology studies with as many as 5 
immunisations of doses of DNA up to 4.0 mg as well as biodistribution and 
persistence/integration studies which were conducted in rabbits- with the exception of the 
epitope based melanoma vaccine (which was conducted in HLA-A2 transgenic mice) and 
the HPV E7 vaccine (which was conducted in C57Bl6 mice). The data from the studies of 
ADVAX have been published [43] . Extensive analysis of tissues at necroscopy reveal EP 
delivery of DNA to be limited to localised inflammatory responses of mild to moderate 
severity at the site of administration which resolves over a few weeks. Extensive 
biodistribution and integration studies conducted after 7 and 30-90 days showed negligible 
systemic uptake of the DNA. The low levels of residual plasmid (i.e <1000 copies/mg 
genomic DNA) for all DNA vaccines suggested a low level risk of potential integration. The 
only difference between EP based delivery and conventional IM delivery was the detection 
of DNA in the draining lymph node. 
 
 

 
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Clinical Experience to Date 
Ten clinical trials of the TDS-IM device have been completed and are summarised below: 
 
Table 3:  Summary of completed trials using the TDS-IM device 
 
Clinical trial.gov or 
EudraCT study reference 
Vaccine candidate 
Subject population 
NCT00545987 
Multigene HIV-1 DNA vaccine 
(ADVAX) 
Healthy, HIV uninfected adult 
volunteers 
NCT00471133 
Xenogeneic tyrosinase DNA 
vaccine (pINGmuTYR) 
Patients with Stage IIB-IV 
melanoma 
NCT01169077 
Multi-epitope malaria DNA 
vaccine (EP-1300) 
Healthy adult volunteers 
NCT01502345 
Multi-antigen hantavirus DNA 
vaccine (pWRG/HTN-M9x) and 
pWRG/PUUVM9s2)) 
Healthy adult volunteers 
NCT01641536 
Multi-antigen HBVDNA vaccine 
with DNA-based IL12 adjuvant 
(HB-110) 
HBV infected adult volunteers 
NCT01496989 
Multi-antigen HIV DNA vaccine 
(HIV-MAG) with or without DNA 
based IL-12 (GENEVAX) prior to 
adenovirus vector (Ad35GRIN) 
Healthy, HIV uninfected 
volunteers 
NCT01634503 
Multi-antigen HPV DNA vaccine 
administered with a DNA-based 
human FLT3 adjuvant (GX-188E) 
Patients with HPV16 or 18 
associated Grade 3 Cervical 
Intraepithelial Neoplasia 
NCT01266616 
Multi-antigen HIV DNA vaccine 
(HIV-MAG) administered with or 
without a DNA-based human IL-
12 adjuvant (GENEVAX) 
HIV infected adult volunteers 
NCT01578889 
Multi-antigen HIV DNA vaccine 
(HIV-MAG) administered with or 
without a DNA-based human IL-
12 adjuvant (GENEVAX) prior to 
administration of an vesicular 
stomatitis virus vector (rVSVgag) 
Healthy adult volunteers 
NCT01493154 
HPV DNA vaccine administered 
with a DNA-based calreticulin 
adjuvant (pNGVL-4a-CRT/E7 
(detox)) 
HPV-associated squamous cell 
carcinoma of the head and neck 

 
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The TDS-IM device is currently being evaluated in ten clinical trials which are summarised 
below: 
 
Table 4: Summary of trials in which the TDS-IM device is currently being 
evaluated 
 
Clinical trial.gov or 
EudraCT study reference 
Vaccine candidate 
Subject population 
NCT01138410 
Epitope-based TRP-2 melanoma 
vaccine (SCIB-1) 
NCT01138410 
NCT01859325 
Multi-antigen HIV DNA vaccine 
(HIV-MAG) administered with 
or without a DNA-based human 
IL-12 adjuvant (GENEVAX) prior 
to administration of an vesicular 
stomatitis virus vector 
(rVSVgag) 
HIV infected adult 
volunteers 
NCT02075983 
GTU®-multiHIV B clade DNA 
vaccine 
Healthy adult volunteers 
NCT01984983 
VEEV DNA vaccine candidate 
Healthy adult volunteers 
NCT02099994 
HIV DNA vaccine 
(pSG2.HIVconsv DNA) 
administered prior to 
administration with an 
adenovirus vector (Ad35GRIN) 
and a vaccinia virus vector 
(MVA.HIVconsv) 
Healthy adult volunteers 
NCT02116205 
Multi-antigen hantavirus DNA 
vaccine (pWRG/HTN-M(x) and 
pWRG/PUUV-M(s2)) 
Healthy adult volunteers 
NCT02139267 
Multi-antigen HPV DNA vaccine 
administered with a DNA-based 
human FLT3 adjuvant (GX-
188E) 
Patients with HPV16 or 
18 associated Grade 3 
Cervical Intraepithelial 
Neoplasia 
NCT02204098 
Mammaglobin DNA vaccine 
Breast cancer patients 
undergoing neoadjuvant 
endocrine therapy 
2011-003171-11 
CUTHIVAC_001 
Healthy adult volunteers 
2014-001997-33 
DNAVAC 
Healthy adult volunteers 

 
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Safety 
To date, the 20 clinical trials that have been completed or are currently ongoing have 
enrolled over 600 subjects in the electroporation arms of the studies (including subjects 
receiving either the DNA vaccine candidate or placebo). The device has been used for 
administration of DNA injections of up to 1.0 ml volume and 4.0 mg DNA dose per injection 
site. Subjects have received the vaccine candidate either as a single injection in one muscle 
site (total DNA dose up to 4.0 mg per administration time point) or as 2 injections in 2 
separate muscle sites (total DNA dose up to 8.0 mg per administration time point). Subjects 
given the DNA dose as a single injection have received up to 5 TDS-IM injections and up to 
4.0 mg DNA, and subjects administered with the DNA dose in 2 injections received up to 10 
administrations (i.e., 20 total TDS-IM injections). 
Acute adverse responses associated with the use of the device include transient pain 
associated with electroporation-induced localised muscle contractions reported by virtually 
all subjects. This has occasionally been accompanied by mild paresthesia in the 
administered limb lasting for several seconds to minutes after application of electroporation. 
Mild, transient bleeding at the sites of electrode/needle penetration is commonly observed 
following removal of the device from the administration site. Local site reactogenicity, 
including injection site soreness, erythema, and/or induration of mild to moderate severity, 
has been commonly reported following electroporation-mediated DNA delivery.  Mild to 
moderate bruising at the administration site has been observed occasionally. The local site 
reactions typically resolve within 24-72 hours following administration, but, in rare 
instances, local site soreness has been reported to persist for up to one week.  
Transient local site tenderness of Grade 3 has been reported in several subjects.  Vasovagal 
reactions comprising mild dizziness, light-headedness, and/or hypotension occurring 
immediately after procedure application have been reported in approximately 1% of 
subjects. A more pronounced vasovagal reaction including a brief syncopic episode (of 
approximately 30 seconds duration) following procedure application was observed in one 
subject in the tyrosinase study. Systemic adverse events reported during the studies and 
judged to be possibly related to the study product and/or delivery device have been 
generally mild to moderate in severity and include headache, fatigue, fever, dizziness, 
malaise, , arthralgia, myalgia, and aphthous stomatits. Minor serological and hematological 
abnormalities observed in these studies have included transient Grade 1-3 elevation in 
serum creatine phosphokinase.  Two subjects (one in the malaria vaccine study, one in an 
HIV study) have reported a single instance of severe fatigue within 24 hours of dosing that 
was resolved by the following day. No serious events attributed to the device or 
administration procedure have been observed to date. 
 
Immunogenicity 
The completed study of the multi-antigen HIV DNA candidate was a randomised placebo-
controlled comparison of EP relative to conventional IM. There were no cellular immune 
responses after two immunisations IM with 4.0mg of DNA given without EP- but there was a 
dose dependent increase in both the frequency of responders and the magnitude of the 
response seen with EP  [44]. 6/8 individuals made a cellular immune response after 2 
immunisations with 4.0mg DNA given with EP and this increased to 87% after a third dose. 
The completed study of the tyrosinase DNA vaccine was a single arm dose escalation study 
(0.2, 0.5 and 1.5 mg DNA) of the vaccine in HLA-A1 and A24 positive individuals with Stage 
IIB-IV melanoma. The immune responses of 21/24 were assessed and positive anti-
tyronsinase responses were observed in 6/15 patients who received 1.5mg doses of DNA. 

 
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(ii) The Ichor TriGrid™ Delivery System:  For Intradermal Delivery (TDS-ID) 
Background 
The TDS-ID device has not been used to deliver this HIV DNA vaccine to healthy volunteers 
in man before, and there is not as much clinical data available as there is for the TDS-IM 
device. To date, the device has been used in 20 healthy volunteers. Building on the initial 
data generated with the TDS-IM device, Ichor has adapted the TDS platform to enable 
assessment of intradermal DNA delivery. The objective of this development effort is to 
provide the means to evaluate both intramuscular and intradermal delivery for a given 
vaccine candidate, thereby allowing identification of the route of administration best suited 
for further clinical development.  The TriGrid Delivery System for intradermal administration 
(TDS-ID) is a device developed for EP mediated intradermal DNA delivery in the setting of 
early phase clinical trials. To accommodate testing of multiple product candidates with the 
platform as well as the range of parameters likely to be tested in these early phase clinical 
studies, the TDS-ID has been designed to utilize an "off-the-shelf" needle free injector (the 
Medi-Jector Vision [Antares Pharma]) that utilizes a standard vial interface for loading the 
syringe and is capable of administering a range of injection volumes. If warranted by 
interim clinical trial results, a refined TDS-ID design incorporating an integrated needle free 
injection apparatus suitable for use in late stage clinical studies and commercial deployment 
will be implemented.  
 
The procedure for EP mediated intradermal DNA delivery with the TDS-ID comprises three 
principal steps: electrode insertion, agent administration, and EP application. Briefly, the 
procedure is initiated with the placement of the device against the skin at the target site of 
administration. User activation of the device results in the automated insertion of an array 
of four conductive electrodes positioned around the injection orifice of the Medi-Jector 
Vision needle-free injection device. The electrode insertion step is concluded with the 
application of a brief electrical signal to the electrodes in order to determine the impedance 
between the electrodes. If the impedance value indicates an ineffective insertion into the 
tissue or the potential for a safety hazard, the procedure is halted and the array removed 
from the tissue prior to the administration of DNA. If the impedance is within the range 
consistent with an acceptable insertion, the procedure proceeds with the injection of the 
agent of interest through the central injection orifice. The injection results in the formation 
of a circular bleb at the site of injection and within the skin tissue circumscribed by the 
deployed electrodes. Immediately following distribution of the agent to the tissue, the 
procedure concludes with the propagation of the electroporation inducing electrical fields at 
the site of agent administration. Once the electroporation sequence is completed, the 
electrodes and injection needle are immediately removed from the recipient. The entire 
procedure requires approximately ten seconds to complete from the time of device 
placement against the recipient's skin until removal of the device. Technical details of the 
device can be found in Appendix 1. 
 
 
Summary of Experience with Device 
 
The TDS-ID has been authorized for use as the means of DNA vaccine administration in a 
human clinical study of a DNA vaccine candidate for Venezuelan equine encephalitis virus. 
The study has enrolled 20 subjects in the TDS-ID arm of the study. The device was used to 
administer a series of three injections at DNA doses of up to 0.3 mg.  Adverse responses 
reported in association with use of the device include acute discomfort / pain during EP 
application and minor cutaneous bleeding at the site of injection.  Pinpoint eschar formation 
at the sites of needle formation has been commonly observed as well as transient injection 
site reactogenicity (erythema, induration, and/or soreness) of mild to moderate severity, 

 
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typically resolving within 24-72 hours following administration. Systemic adverse events 
reported during the study and judged to be possibly related to the study product and/or 
delivery device have been generally mild in severity and have included fatigue, headache, 
sore throat, and flushing.  The findings are largely consistent with the adverse events 
observed with the TDS-IM device which has been utilized for the delivery of a wide range of 
DNA vaccines intended for use in oncology and infectious disease applications.  
 
The TDS-IM has been used as the means for administration in 20 completed or ongoing 
clinical studies totalling over 600 subjects. Adverse reactions associated with the use of the 
device include acute pain with localized muscle contractions in almost all subjects.  
Occasionally this is accompanied by mild, transient paresthesia. Mild, transient bleeding at 
the sites of electrode/needle penetration is commonly observed following removal of the 
device.  Soreness, erythema, and/or induration of mild to moderate severity are commonly 
reported at the administration site. Transient vasovagal reactions, comprising mild 
dizziness, skin pallor, diapheresis, light-headedness, and/or hypotension occurring 
immediately after device application, have been reported in approximately 1% of subjects.  
A more pronounced vasovagal reaction including a brief syncopic episode (of approximately 
30 seconds duration) following device application was observed in one subject. Systemic 
adverse events reported during the studies and judged to be possibly related to the study 
product and/or delivery device have been generally mild to moderate in severity and include 
headache, fever, dizziness, malaise, fatigue, arthralgia, myalgia, influenza-like symptoms, 
and aphthous stomatitis.  Transient elevation in serum creatinine phophokinase of mild to 
moderate severity and judged to have at least a potential association with candidate 
administration have been reported in a small minority of subjects. Two subjects have 
reported a single instance of severe fatigue within 24 hours of dosing that was resolved by 
the following day.   
 
To date, TDS-ID associated malfunctions observed in clinical trials to date have been limited 
to reports of device initiated interruption of the procedure prior to its completion. As 
described above, the device performs an impedance evaluation following electrode array 
insertion but prior to agent administration. In the event of an improper insertion of the 
electrodes (due to, for example, an inadequate depth of penetration into the skin or 
theoretically, distortion of the electrodes) is detected, the TDS-ID system will interrupt the 
administration procedure prior to the injection of the agent and notify the user. At that 
point, the device is withdrawn from the subject. The overall design of the TDS-ID system 
and the specific implementation of the impedance check are intended to minimize the 
occurrence of DNA administration without the subsequent application of EP. 
 
Subject to the specific procedures described in the clinical protocol, following an incomplete 
procedure application in which the DNA was not delivered to the subject, the user may 
obtain a new Application Cartridge and agent dose to attempt re-administration of the 
procedure. In its investigation of these occurrences, Ichor has identified several causes for 
the interruption of the procedure application.  
 
These include: Improper loading, placement, and/or activation of the device. In several 
cases, investigation indicates that user error has contributed to the occurrence of an 
interrupted procedure applications. Specific circumstances have included improper device 
set up and suboptimal positioning of the device relative to the target skin site. Since their 
identification, these issues have been addressed as points of emphasis during the conduct 
of the training seminar used to qualify clinical site personnel prior to use of the TDS-ID 
device.  
 

 
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2.2.3 
Rationale for Interventions 
DNA Doses 
There are very few published studies of dose response to DNA vaccination delivered with 
electroporation in humans, thus it is hard to directly extrapolate the results above to the 
human setting. A dose relationship for DNA delivered with EP in humans has recently been 
shown for an immunotherapeutic vaccine against HPV16/18 in humans [33]. This 
demonstrated a dose response trend where mid dose (1mg) appeared to be marginally 
better in inducing antibody responses than low (0.3mg) or high (3mg) doses.  
To the best of our knowledge the dose relationship for a single plasmid expressing an Env 
transgene delivered with electroporation has not been defined in humans. Using a similar 
design to that performed for the Human Papilloma Virus (HPV), and taking into account our 
own preclinical studies, we propose to assess a mid-dose 2mg (500ul) for intramuscular 
injection. As we are constrained by the volume that can be administered we will use a low 
dose 
600ug 
(150ul) 
for 
intrademal 
immunization. 
The 
difference 
in 
DNA concentrations between IM (2mg) and ID (0.6mg) doses mirrors that modelled in 
supportive preclinical studies [41]. Vaccinations will be delivered by IM or ID electroporation 
using the Ichor TriGrid™ Delivery System (http://www.ichorms.com) - see appendix 1 for 
additional detail.  
All volunteers will receive 3 immunisations of 2.0mg DNA IM and 0.6mg DNA ID and the 
immunisations will be administered with or without EP as described in table 1. In a 
supportive rabbit GLP toxicity study which was carried out with the same vaccine, the 
individual IM dose was 2mg, this was given for up to six administrations was shown to be 
well tolerated in rabbits (CR 525569) and lack of DNA persistence or distribution beyond the 
site of administration was demonstrated in rats (CR525574). Furthermore, a previous Phase 
I trial to use electroporation (EP) for the delivery of a DNA vaccine for HIV demonstrated 
increased immunogenicity in the absence of associated safety concerns [44] [62]. 
 
CN54gp140 Doses 
We will give 1 immunisation of 50g CN54gp140. In MucoVac 2 there was no significant 
impact on the magnitude of the responses after reducing the dose from 100g to 20g 
although this was in the presence of adjuvant. 
 
2.2.4 
Possible Next Steps  
 
The following hypotheses will be explored with a view to informing the design of future 
trials both in terms of size and selection of methods: 
 That each of the methods and combination regimens delivered with and without EP will 
be safe and acceptable 
o If any of the methods or regimens have unacceptable safety defined in terms 
of the proportion of participants that experience a grade 3 or above solicited 
adverse event or an event that leads to an investigator decision to 
discontinue immunisations, then there will be no further exploration of the 
route with the product/regimen. 
 That immunisation via the combined IM+ID methods will elicit balanced immune 
responses. 
 That EP will augment antibody responses to both ID and IM. 

 
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o That the greatest proportion of individuals will respond and make 
significantly greater antibody responses in group 3 (ID/EP + IM/EP) relative 
to the other groups. If EP is shown to impact upon the proportion of 
individuals making systemic IgG without compromising safety/tolerability, the 
strategy would be taken forward into future trials. 
 
2.2.5 
Risks and Benefits 
This is a Phase I exploratory study in healthy male and female volunteers.  
HIV DNA plasmid-based vaccines are widely used alone and in combination regimens, 
including the multicentre EuroVacc trials EV01-3 in which we have been involved and which 
volunteers were given up to 12mg DNA over 12 weeks with no safety concerns. There is 
currently limited data available on the systemic use of CN54ENV DNA but our group is 
directly involved in the only trial using CN54ENV and CN54gp140 delivered IM in DNA-MVA-
protein strategy (UKHVC SPOKE 003; EUDRACT: 2012-003277-26) initiated in July 2013 and 
now completed, for which our group have direct access to safety data as it becomes 
available.  
 
While electroporation has been used in multiple studies, it has not been used to deliver this 
paricular vaccine in humans before, but the supporting preclinical study (CR 525569), was 
shown to be well tolerated in rabbits. Furthermore, a previous Phase I trial to use 
electroporation (EP) for the delivery of a DNA vaccine for HIV demonstrated increased 
immunogenicity in the absence of associated safety concerns [44] [62]. A more recent 
study (HVTN 080) assessed a mixture of 3 expression plasmids encoding HIV-1 Clade B 
Env, Gag, and Pol delivered together with a DNA expressing IL-12 followed by EP with no 
associated safety concerns [63]).  The vaccination schedules in this study are complex and 
on each visit volunteers will receive at least one vaccination with EP. There is an increased 
risk that retention to the trial will suffer as a result.  The first Phase I trial to use EP for the 
delivery of a DNA vaccine for HIV has recently reported and observed an increase in 
immunogenicity.  All the volunteers completed their immunisations, and adverse reactions 
were in line with licensed vaccines.  In the ongoing healthy volunteer study (CUTHIVAC 
001, EUDRACT 2011-003171-11), one participant elected to discontinue further 
immunisations due to pain and eight participants were subsequently randomised to EP and 
preceded without a problem. 
  
There is no direct benefit to the volunteers. They will be reimbursed for their time and 
travel. Volunteers also derive the benefits of clinical screening and any follow up care for 
the time that they are enrolled in the study and afterwards where appropriate.   As there is 
no placebo group, there is a risk that adverse events will be over-reported, but this should 
not influence grade and is not a concern at this stage of evaluation.  
 
The laboratory endpoints will be analysed in laboratories at Imperial College London, and 
the staff blinded to the regimen, although not the time point. Samples may also be shipped 
to specialist laboratories in Europe and the USA for wider exploratory immunological 
measurements of interest. Procedures will be put in place to ensure the chain of custody of 
samples when transporting from clinic to laboratory and between laboratories; therefore we 
consider that the risk of loss or compromise of samples is low. 

 
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3.  SELECTION OF CENTRES/CLINICIANS 
The Principal Investigator, Sonya Abraham (Imperial Clinical Research Facility, 
Hammersmith Hospital) is an experienced clinical trialist.  Volunteers will be seen by clinical 
staff from the Imperial Clinical Research Facility, Imperial College Healthcare NHS Trust, 
who have experience of running Phase 1 clinical trials.  
 
4. SELECTION OF PARTICIPANTS 
There will be no exceptions to eligibility requirements at the time of randomisation. 
Questions about eligibility should be addressed prior to attempting to randomise the 
participant.  
The eligibility criteria for this trial have been carefully considered. The eligibility criteria are 
the standards used to ensure that only appropriate participants are considered for this 
study. Participants not meeting the criteria should not join the study. For the safety of the 
participants it is important that no exceptions be made to these criteria for admission to the 
study. 
Participants will be considered eligible for enrolment in this trial if they fulfil all the inclusion 
criteria and none of the exclusion criteria as defined below.  
 
4.1 Participant Inclusion Criteria 
1. Men and women aged between 18 and 50 years (inclusive) on the day of screening 
2. BMI between 18-30 kg/m2 (inclusive) 
3. Available for follow-up for the duration of the study (up to ~12 months from 
screening) 
4. Willing and able to give written informed consent 
5. At low risk of HIV and willing to remain so for the duration of the study defined as: 
 
no history of injecting drug use in the previous ten years 
 
no gonorrhoea or syphilis in the last six months 
 
no high risk partner (e.g. injecting drug use, HIV positive partner) either 
currently or within the past six months 
 
no unprotected anal intercourse in the last six months, outside a relationship 
with a regular partner known to be HIV negative 
 
no unprotected vaginal intercourse in the last six months outside a 
relationship with a regular known/presumed HIV negative partner 
6. Willing to undergo a HIV test 
7. Willing to undergo a genital infection screen 
8. Must agree to require male sexual partner to use condoms, from at least 14 days 
before the first vaccination until at least 4 months after the last 
9. If heterosexually active female capable of becoming pregnant, must (in addition to 
requiring male partner to use condoms) agree to use hormonal contraception, or to 

 
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complete abstinence, from at least 14 days before the first vaccination until at least 4 
months after the last. [Note: Periodic abstinence (e.g. calendar, ovulation, 
symptothermal, post-ovulation methods) and withdrawal, and IUD/IUS, are not 
acceptable methods of contraception.] If sexually active male, must agree to use 
condoms from the day of first vaccination until at least 4 months after the last. [Note: 
Additional use of an effective method of contraception is recommended for any non-
pregnant female partner over the same period.]   
10. Agree to abstain from donating blood for three months after the end of their 
participation in the trial, or longer if necessary 
11. Registered with a GP for at least the past three months 
12. Entered and clearance obtained from The Over-volunteering Prevention System (TOPS) 
database.  
 
4.2 Participant Exclusion Criteria 
1. Pregnant or lactating 
2. History of cardiac arrhythmia or palpitations [e.g., supraventricular tachycardia, atrial 
fibrillation, frequent ectopy, or sinus bradycardia prior to study entry (sinus arrhythmia 
is not excluded) 
3. History of syncope or fainting episodes within 1 year of study entry 
4. History of grand-mal epilepsy, seizure disorder or any history of prior seizure 
5. Individuals in which a skin-fold measurement (cutaneous and subcutaneous tissue) of 
the upper right and left thigh exceeds 40 mm 
6. Clinically relevant abnormality on history or examination  
7. Known hypersensitivity to any component of the vaccine formulations used in this trial, 
or have severe or multiple allergies to drugs or pharmaceutical agents 
8. History of severe local or general reaction to vaccination defined as 
 
local: extensive, indurated redness and swelling involving most of the antero-
lateral thigh or the major circumference of the arm, not resolving within 72 
hours 
 
general: fever ≥39.5oC within 48 hours; anaphylaxis; bronchospasm; laryngeal 
oedema; collapse; convulsions or encephalopathy within 72 hours 
9. Receipt of live attenuated vaccine within 60 days or other vaccines within 14 days of 
enrolment 
10. Receipt of an experimental vaccine containing HIV envelope components at any time 
in the past 
11. Receipt of blood products or immunoglobulin within 4 months of screening 
12.  
13. Has received treatment with immunosuppressive agents within 3 months of screening 
e.g. oral, inhaled, nasal or injected corticosteroids. (Topical steroids are allowed, 
unless applied to the IM or ID injection sites.)Participation in another trial of a 
medicinal product, completed less than 30 days prior to enrolment. 
14.  HIV 1 or 2 positive or indeterminate on screening. 

 
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15. Positive for hepatitis B surface antigen, hepatitis C antibody or serology indicating 
active syphilis requiring treatment  
16. Grade 1 (or above) clinically significant routine laboratory parameters (see appendix 2 
for definitions). Hyperbilirubinaemia to be considered an exclusion criterion only when 
confirmed to be conjugated bilirubinaemia 
17. Current use of any electronic stimulation device, such as cardiac demand pacemakers, 
automatic implantable cardiac defibrillator, nerve stimulators, or deep brain 
stimulators.  
18. Presence of any surgical or traumatic metal implants at the sites of administration  
19. Unable to read and speak English to a fluency level adequate for the full 
comprehension of procedures required in participation and consent. 
20. Women with a history of toxic shock syndrome.  
21. Women using an intrauterine device for contraception (as incompatible with softcup 
sampling) 
22. Unlikely to comply with protocol. 
 
4.3 Number and Source of Participants 
Healthy volunteers will be recruited through advertising. They will be provided with further 
information about the study and asked to complete a short interview (by telephone or in 
person) to assess their suitability. They will be given or sent an information sheet. The 
target is for 24 participants to complete the trial. 
 
4.4 Screening Procedures and Pre-Randomisation Investigations 
At the screening visit the volunteer will be allocated a number from the register. The trial 
will be discussed in detail, and a brief check of eligibility conducted. Any questions about 
the study will be answered. If volunteers appear potentially eligible, and willing and 
interested, they will be asked to sign the informed consent form. 
 
After written informed consent has been collected, assessments and investigations will be 
undertaken according to the schedule in Table 5 , section 7. These include demographic, 
sexual and medical histories and general examination, and collection of urine and blood 
samples for routine laboratory investigations. Samples for screening sexually transmitted 
infections including HIV will be collected (see section 7.2). An ECG will be performed. 
 
As soon as all required test results are available: 
 
Data will be entered onto the screening CRF and the results of the screening 
investigations will be reviewed and eligibility signed off by a physician. 
 
 
 
 

 
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5. RANDOMISATION & ENROLMENT PROCEDURE 
5.1 Randomisation Practicalities 
The enrolment visit will take place within 6 weeks (42 days) of the screening visit and will 
be the same day as the first vaccination. Assessments and procedures will be undertaken 
according to the schedules (Section 7 and Table 5), and data entered on the case report 
forms.  
Volunteers who are eligible and willing will be randomised when all inclusion/exclusion 
criteria have been met and the appropriate CRF completed. The PI or team member will log 
into a web-based system managed by MRC CTU at UCL to obtain the regimen allocated to 
the participant.  
 
Further details on the process of randomisation can be found in Section 10. 
 
5.2 Randomisation Codes and Un-blinding 
The master randomisation list linking subject numbers to allocation will be generated by the 
trial statistician.  Randomisation will be carried out on the day of the first dosing visit.   
Randomisation arm will be known to participants and clinical staff.  
The laboratory staff conducting the assays will be blind to the regimen throughout. 
The trial is open-label so no unblinding will be necessary. 
 
5.3 Co-Enrolment Guidelines 
Participants will be advised that they cannot enrol in any other trials that would interfere 
with the study endpoints during the period from screening to the final visit in 
CUTHIVAC002. All participants will be entered onto the TOPS (The Overvolunteering 
Prevention System) database as a measure to prevent over volunteering. 
If staff discover that a participant is enrolled on another study during this period, they must 
immediately contact the Chief Investigator for advice. Decisions will be reviewed on a case 
by case basis and participant safety will be the primary concern. 

 
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6. TREATMENT OF PARTICIPANTS  
6.1 Vaccine Products 
Participants will be vaccinated in a schedule of doses and methods as described below. 
Each participant will receive three DNA immunisations, at wk 0, 4 and 8, boosted with 
recombinant HIV CN54gp140 administered by intradermal injection at wk 20. 
A vaccine accountability log will be kept throughout the study. This should be used to 
record the Trial ID of the subject to whom the study vaccine was dispensed. This will be 
verified by the study monitor. The date and time of administration will be recorded on the 
CRF. 
 
6.1.1 
Products and Administration 
The individual IMP vials will be labelled, and packaged in cartons which will also be labelled.  
The Principal Investigator will ensure that staff administering the vaccines have been 
appropriately trained in the written procedures for ID, IM and EP. 
 
All individuals will be given one IM injection of 2.0mg CN54ENV DNA and one ID injection of 
0.6mg of CN54ENV DNA at Weeks 0, 4 and 8, and one ID injection of 50 µg CN54gp140 at 
Week 20.  See table 1, section 6.1.3.  
 
6.1.2 
Accountability for Used and Unused Supplies 
The PI will ensure that the IMPs are dispensed in accordance with the protocol and local 
procedures, and that records are maintained of receipt, dispensing and return/destruction of 
all supplies. 
 
The PI must ensure that all IMP supplies are kept in a secure area accessible only to 
authorised individuals, and maintained in storage that guarantees the following 
temperatures:  
 
CN54ENV DNA at -25 to -15oC 
 
CN54gp140 at 2-8oC 
 
Upon receipt of supplies, a designated member of staff will conduct an inventory and 
acknowledge receipt to the supplier.  
A record must be kept of all CN54ENV DNA and CN54gp140 used during the trial. This will 
include the description (lot numbers and expiry dates) and quantity of IMP received at the 
trial site and date of receipt, as well as a record of when (date of administration) and to 
whom (Trial ID) it was dispensed. 
At the end of the trial, IMP accountability will be checked by the designated member of 
staff responsible for the inventory, and by the trial monitor. The Sponsor and the PI will 
retain copies of the complete IMP accountability. 
All supplies (used and unused) will be retained at the trial site until the Sponsor gives 
instructions for their return or destruction. 
 
 
 
 

 
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6.1.3 
Immunisation schedule 
The doses, methods and schedule of immunisation are described in Table 1 below. 
Table 1:   Schedule of Doses and Methods of Immunisation 
 
Group 
Method of immunization; Dose of DNA 
Method of 
immunization; Dose 
of CN54gp140 
Dose 1 at WK 0 
Dose 2 at WK 4
Dose 3 at WK 8
Dose 4 at WK 20
1  
N=8 
0.6mg ID*/ EP 
2mg IM** 
0.6mg ID*/ EP
2mg IM** 
0.6mg ID*/ EP
2mg IM** 
50ug ID***
2  
N=8 
0.6mg ID* 
2mg IM**/ EP 
0.6mg ID*
2mg IM**/ EP 
0.6mg ID*
2mg IM**/ EP 
50ug ID***
3 
N=8 
0.6mg ID*/ EP 
2mg IM**/ EP 
0.6mg ID*/ EP
2mg IM**/ EP 
0.6mg ID*/ EP
2mg IM**/ EP 
50ug ID***
* 
1 x 0.15ml injections ID via a needle - into the upper arm with or without 
electroporation (EP). 
**  
1 x 0.5ml injections IM – into the upper thigh with or without electroporation (EP). 
*** 
1 x 0.1ml injections ID via a needle - into the upper arm.  
 
6.1.4 
Compliance and Adherence 
All immunisations will be administered by site staff according to written procedures.  
 
6.1.5 
Dose Modifications and Discontinuation 
There are no planned modifications to dose, other than discontinuation. 
The schedule may be modified if a participant has symptoms or signs on the day of 
scheduled immunisation, and the investigator considers it best to defer the immunisation. 
The participant will be asked to return for review within the window period of the scheduled 
immunisations (-3/+7 days from week 4, 8 and 20 for the second, third and fourth 
immunisations respectively).  
A clinical investigator may decide to permanently discontinue dosing in a participant who 
has received one or more immunisations, if the investigator deems that continuing might 
compromise participant wellbeing or interfere with the achievement of the trial’s objectives. 
Such a decision should only be taken in consultation with the Chief Investigator. 
Participants will be encouraged to continue to attend trial visits for sampling and safety 
monitoring.   
Discontinuation of dosing is essential following a grade 3 or 4 clinical or laboratory event 
(confirmed on examination or repeat testing respectively) which is considered possibly, 
probably or definitely related to the vaccine and which did not resolve within 72 hours. 

 
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Provided the participant agrees they will continue to be followed up for the duration of the 
study and samples will be collected at the allotted times. 
Dosing must be discontinued in participants who become pregnant or HIV infected. 
Participants may decide to discontinue dosing. They will be encouraged to provide a reason, 
and to remain in follow-up.  The appropriate CRF should be completed as soon as possible 
if a patient chooses to stop receiving vaccines or to withdraw from the trial, and within 24 
hours of a decision being taken to discontinue a participant when that decision is informed 
by an adverse event (section 8.1). 
 
6.2 Clinical Management of Adverse Events 
Events will be managed by the clinical trial team who will assess and treat the event as 
appropriate, including referral to an independent physician and/or the participant’s General 
Practitioner if required.  
See also section 7.4 
 
6.3 Non-Trial Treatment 
As stated in the exclusion criteria in Section 4.2, participants should not have received 
other immunisations, immunosuppressive agents, blood products, immunoglobulin or other 
trial medication within specified periods prior to enrolment. This applies during the trial 
through to the final safety visit 22 weeks after enrolment, 2 weeks after the last scheduled 
immunisation, unless the treatment is required for an emergency. 
Should a participant require immunisation for the purposes of travel, occupation or other 
clinical need during the trial, the request will be reviewed by the Trial Management Group 
who will advise on timing and whether or not the trial immunisation schedule needs to be 
amended. 
Participants will be allowed to continue with hormonal contraception if this forms part of 
their regular appropriate contraception plan. The precise method will be recorded on the 
screening CRF and any changes on the concomitant medication CRF.  
All concomitant medication will be recorded on the CRF, including any dispensed by the 
investigators in the management of adverse events or reactions. 
 
6.4  Issues Related to HIV 
Only volunteers with a negative 4th generation HIV antibody/antigen result will be enrolled 
onto the study.  
A 4th generation HIV antibody/antigen assay, the standard laboratory method for 
diagnosing HIV infection, will be used to screen volunteers after they have received 
appropriate counselling. An HIV test will be performed at two time-points in the study: the 
screening visit, and the visit 4 weeks after the final vaccination, unless clinically indicated at 
other times.  
At each immunisation visit a risk assessment will be conducted and an additional HIV test 
will be performed if there has been a change in risk status.  Participants will be counselled 
by study personnel about the importance of condoms and reminded on the day of each 
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In the event that any blood specimen is positive, participants will be invited for 
confirmatory tests to distinguish between vaccine-induced seropositivity and genuine HIV 
infection. Participants with vaccine-induced seropositivity will be provided with appropriate 
certification of their HIV status, and retested at the visit 6 months after the final 
vaccination. If that test is positive also, they will be invited to return to the clinical centre 
for re-testing until such time as the test becomes negative. 
 
6.4.1 
Verification of HIV status of participants 
In the event of an equivocal or positive result, a specimen will be processed through a 
range of assays according to the local laboratory operating procedures to establish the HIV 
status of the individuals. A confirmatory specimen will be collected at a later date, if the 
first result suggests that the participant is HIV infected. 
If a participant requires certification independent of the local laboratory, then this can be 
arranged through the clinic team. 
 
6.4.2 
HIV infection 
In the unexpected circumstances that a participant in the trial acquires HIV infection, they 
will be referred for clinical care and counselling. 

 
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7. ASSESSMENTS AND FOLLOW-UP 
7.1 Duration of Follow-up and Schedule 
The assessments that will be performed at each visit are described in Table 5.  
Participants will be required to make a minimum of 11 scheduled outpatient visits over the 
course of up to 30 weeks. Time 0 will start on the day of enrolment, which is also the day 
of randomisation and first immunisation. 
Screening can take place up to 6 weeks (42 days) before time 0. 
Immunisations will take place at weeks 0, 4, 8 and 20. 
A 12th scheduled outpatient visit, at Week 44, will only take place for participants known to 
have responded to the vaccine. 
Serum will be collected for immunogenicity at enrolment, and weeks 4, 5, 8, 9, 20, 21, 22 
(the primary endpoint), 24 and 44. Peripheral blood mononuclear cells (PBMC) will be 
collected at enrolment, and weeks 4, 8, 20, 21, 22, 24 and 44.   
Adverse events will be assessed during the enrolment visit before and after immunisation 
and at every visit thereafter up to (and including) Week 22. Routine laboratory safety 
parameters will be collected at screening, enrolment, and weeks 1, 4, 5, 8, 9, 20, 21 and 
22, and at any other visit if clinically indicated. 
 
7.1.1 
Additional Visits 
Additional visits and assessments may be required to evaluate an adverse event, and/or 
identify a diagnosis. These are compatible with the protocol.  
 
Referral to an independent specialist with the appropriate expertise will be arranged if there 
is uncertainty about the relationship of an adverse event to study vaccine. 
 
7.1.2 
Visit Windows 
The second, third and fourth immunisation visits scheduled for weeks 4, 8 and 20 will be 
compliant with the protocol if they take place ±3 days either side of the target date 
determined by the date of the previous vaccination. If there is a delay >4 weeks a decision 
will be made by the trial management group (TMG see section 16.1) as to whether 
immunisation will continue. 
The post-immunisation safety visits scheduled for weeks 1, 5 and 9 will be compliant with 
the protocol if they take place -3 to +7 days of the target date determined by the date of 
the previous vaccination. The safety visit at Week 21 will be compliant with the protocol if it 
takes place -3 to +3 days of the target date. 
The primary endpoint and follow-up visits scheduled for weeks 22 and 24 will be compliant 
with the protocol if they take place ±3 days either side of the target date determined by the 
date of the fourth vaccination.  
The follow-up visit scheduled for week 44 will be compliant with the protocol if it takes 
place ±14 days either side of the target date determined by the date of the fourth 
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7.2     Screening Assessments 
7.2.1 
Demographics, Medical History and Examination 
Demographic information such as age, and ethnic origin will be collected at screening and 
entered onto the screening CRF. 
A past and current medical history will be collected at screening during a face to face 
structured interview using a case report form, including details of any previous reaction to 
vaccination, allergies, history of epileptic fit, reproductive and respiratory symptoms, 
contraceptive practices (current method and the length of time using the method), and 
smoking, alcohol, steroid use and illicit drug history. 
The general examination will include weight (kg), height (cm), calculation of BMI, 
temperature, arm and thigh circumference, blood pressure, inspection of the skin to 
exclude severe eczema and respiratory, cardio-vascular, abdominal, and neurological 
examination. Skin fold thickness will be assessed within the upper right and left thigh. An 
assessment of cervical, axillary and inguinal lymph nodes will also be undertaken.  
An ECG will be done to exclude cardiac arrhythmia or palpitations [e.g., supraventricular 
tachycardia, atrial fibrillation, frequent ectopy, or sinus bradycardia (i.e., <50 beats per 
minute on exam)] 
 
7.2.2 
Sexual History and Genital Infection Screen 
A sexual history will be taken for the preceding 6 month period, and participants will be 
asked whether they have ever been tested for sexually transmitted infections in the past 
and if so, whether any were found.   
The following will be collected in all participants 
 
serology for syphilis  
 
serology for markers of hepatitis B surface antigen and hepatitis C antibody 
 
urine for Chlamydia and gonorrhoea at screening.  
 
 Other gonorrhoea and chlamydia tests will be collected if indicated on account of risk and 
symptoms, at the discretion of the PI. 
 
7.2.3 
Routine Laboratory Parameters 
Peripheral blood will be collected by clinical staff experienced in phlebotomy into the 
appropriate containers and transported to the local laboratory.  
Mid-stream urine will be collected into a sterile container and either tested on site (urine 
dipstick and pregnancy tests) or transported to the local laboratory. All laboratory 
assessments will be performed by Imperial College Healthcare NHS Trust. 
 
The following parameters will be collected in all participants: 
 
Hb, total WBC, neutrophils, lymphocytes and platelets 
 
Creatinine, total and conjugated bilirubin (conjugated only if indicated) , alkaline 
phosphatase, AST, ALT and glucose 
 
Urinalysis using a dipstick for protein, ketones, blood, leukocyte esterase, and nitrites 

 
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and for women of child-bearing potential only 
 
Urine test to exclude pregnancy 
A urine specimen will be sent to the laboratory if the level of protein, blood, leukocyte 
esterase or nitrites is considered clinically relevant. 
 
 
7.3 Immunogenicity Assessments 
Samples will be collected at the timepoints specified in Table 5 and transferred to the 
Immunology Core Laboratory for analysis.  
7.3.1    Mucosal Specimens 
Rectal secretions will be collected from male and female volunteers using an ophthalmic 
sponge. The sponge will be pre-moistened with sterile normal saline and inserted into the 
rectum using a proctoscope. Sponges will be held against the rectal wall for up to for 2 min. 
Should a participant not wish to have proctoscopy the sponge may be inserted by the nurse 
or doctor 3 cm into the anal canal for 2 minutes.  
Vaginal secretions will be collected from female volunteers using the INSTEAD Softcup, a 
commercially available, self-inserted menstrual cup made of polyethylene. Mucosal 
secretion collection will occur at the time points indicated in Table 5, except at visits when 
female volunteers are menstruating in which case vaginal secretions will not be collected. 
7.3.2     Blood and Serology Specimens 
For measurement of antibodies in serum, 6ml peripheral venous blood will be collected into 
an appropriate blood collection tube, and then processed to obtain serum. 
For assessment of T-cell responses, 40-50ml ml of peripheral venous blood will be collected 
into tubes containing sodium heparin as an anti-coagulant, mixed by inverting gently 
several times and then processed to obtain PBMC which will be frozen and stored for future 
batch immunogenicity analysis. 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
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7.4 Procedures for Assessing Safety and Tolerability 
7.4.1 
Adverse Event Assessment 
Information on adverse events will be collected through a structured interview at scheduled 
visits as indicated in Table 5.  
The investigator will record the diagnosis or the symptoms if a diagnosis is not apparent, 
the date of onset and the date of resolution if appropriate. For events not specified in the 
tables in study specific toxicity grading table (Appendix 2) the severity will be determined 
according to the FDA guidance (2007) 
http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInfor
mation/Guidances/Vaccines/ucm091977.pdf 
The seriousness of the event and its relationship to vaccine will be determined by the 
investigator according to the definitions provided in section 8. All of this information will 
be recorded in the adverse event CRF. 
 
7.4.2 
General Examination and Vital Signs 
General examinations will be done at screening and on dosing days, as per section 7.2.1 
(height and weight at screening only). General examinations at other visits per Table 5 will 
be done only if clinically indicated. Assessment of vital signs (blood pressure, pulse, 
respiratory rate and oral temperature) will be performed at all visits up to Week 22.  
 
 
7.4.3 
Routine Laboratory Parameters and Urinalysis  
A pregnancy test will be performed by analysis of a urine sample for Human Chorionic 
Gonadotrophin (HCG) collected from women of child-bearing potential at screening, the day 
of each immunisation and at the final safety visit (Week 22). The analysis will be conducted 
by a member of the study team. 
Peripheral blood and urine will be collected and analysed, as described in section 7.2.3, 
for the following parameters at the time points specified in Table 5, and at additional time 
points if indicated to further evaluate or follow up adverse events: 
 
7.4.3.1 Blood  
 
Creatinine, AST, ALT, alkaline phosphatase, total bilirubin, glucose 
 
Hb, total WCC, neutrophils, lymphocytes, platelets 
 
7.4.3.2 Urine  
 
protein, ketones, blood, leukocyte esterase, and nitrites – by dipstick 
 
A urine specimen will be sent to the laboratory if the level of protein, blood, leukocyte 
esterase or nitrites is considered clinically relevant. 
 
 
 
 
 
 
 
 
 
 

 
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7.4.4 
Solicited Local and Systemic Clinical and 
Laboratory Adverse Events 
Various local and systemic adverse events are known to be associated with licensed 
vaccines, and are referred to as ‘solicited adverse events’. These include disturbances in 
routine laboratory parameters, and are described in table 2 below.  
Information on solicited adverse events will be collected on the day of each immunisation 
(pre-immunisation, and at 10 and 60 minutes post-immunisation), at a safety call the day 
after each immunisation and for the following 7 days through a structured interview and 
examination according to the schedule.  
In addition systemic laboratory adverse events will be collected through routine laboratory 
testing according to the schedule. These will be recorded on the appropriate CRF page.  
Participants will also be asked to complete a diary card recording solicited adverse events 
that start within 7 days of each immunisation. 
 
 
 
Table 2:   
Solicited Adverse Events 
 
Type 
Event 
Local AEs 
(immunisation 
site) 
Discomfort 
Redness 
Swelling (soft) 
Induration (hard) 
Blisters 
Systemic 
Clinical AEs 
Abnormally raised temperature 
Chills 
Myalgia/flu-like general muscle aches 
Malaise (excess fatigue) 
Headache 
Nausea 
Vomiting 
Systemic 
Laboratory AEs 
Abnormalities in: 
Creatinine,  AST, ALT, alkaline phosphatase, total bilirubin, glucose 
Hb, total WCC, neutrophils, lymphocytes, platelets 
 
The events will be graded according to the study specific toxicity table (see Appendix 2). 
 
Relationship to study product, which is defined in section 8, will be recorded in the 
immunisation CRF but not on the diary card, on the assumption that any of these events 
starting within 7 days of an immunisation are at least possibly related. If the onset is 
beyond 7 days, the event will be recorded on the adverse event CRF and a relationship 
determined by the investigator reviewing the event. 
 

 
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7.4.5 
Non-solicited Adverse Events 
Non-solicited AEs starting from the first vaccination until the final safety visit (Week 22) will 
be captured and reported on the adverse event CRF. When making a non-solicited AE 
enquiry, staff should ask a non-leading question such as 'how have you been feeling?' 
 
7.4.6 
Follow-up of Adverse Events and Pregnancy 
The Investigator will make every effort to monitor all adverse events, regardless of severity, 
until resolution or stabilisation, and to obtain documentary evidence of the outcome of 
pregnancy, in order to report this on the CRF during the trial. 
 
7.4.7 
Serious adverse events 
A serious adverse event is defined in section 8. 
All serious adverse events should be reviewed by the Principal Investigator for the clinical 
centre, and discussed at the next Trial Management Group call (section 16.1).  
The assessment should include consideration of whether or not to discontinue dosing or to 
withdraw a participant from the trial. 
 
7.4.8 
Tolerability of vaccination using the electroporation 
devices 
A tolerability questionnaire will be given to subjects to complete after each vaccination with 
EP. They will score pain at the time of vaccine injection, the time of electrical stimulation, 
and 10 and 30 min afterwards, using a 10-point scale. They will also be asked general 
questions about acceptability, and asked for their comments. 
 
7.5 Criteria for Stopping Treatment Groups or Whole Trial 
In the event of a SUSAR, the Chief Investigator should notify the sponsor within 24 hours of 
the event taking place, and the Chief Investigator will arrange for any necessary expert 
reviews to take place within 3 working days.  
Further immunisations will be put on hold until the review is completed. 
A component of any expert review will be to consider whether or not further immunisations 
should be discontinued in the individual, and/or the trial. 
If 3 or more participants experience a grade 3 or 4 clinical or laboratory event (confirmed 
on attendance or repeat testing) not resolved within 72 hours and considered possibly, 
probably or definitely related to vaccine product, further immunisations will be put on hold 
and the Chief Investigator will call a meeting with the Trial Management Group (TMG) to 
review the safety data.  If upon review of the safety data it is deemed appropriate to 
restart dosing, a substantial amendment with relevant data has to be submitted to the 
MHRA for approval.  If an unscheduled TMG meeting is warranted, the Sponsor will be 
informed and the TMG asked to make a recommendation to the Chief Investigator and the 
Sponsor about continuing further immunisations.  
 
At any time if the study is put on hold (for example following the occurrence of a SUSAR) 
the Regulatory authority has to be informed of the temporary halt and a substantial 
amendment with relevant data has to be submitted to the MHRA for approval in case a 

 
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decision to resume dosing is taken.  The Sponsor reserves the right to stop the whole trial 
at any time. 
 
7.6 Procedures at the end of the trial 
The trial will be closed when all participants have made their final follow-up visit (visit 12), 
the data collected on randomised participants entered into the database and the database 
locked. There will be a final monitoring/closeout visit to the clinical site between the last 
visit and the database lock.

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Table 5:         Schedule of visits, Immunisations and Assessments  
Visit number 
1 
2 
 
3 
4 
 
5 
6 
 
7 
8 
 
9 
10 
11 
12 
Nature of visit 
Screen 
Dosin
g 
visit 
Safety 
call 
Safety 
visit 
Dosing 
visit 
Safety 
call 
Safety 
visit 
Dosing 
visit 
Safety 
call 
Safety 
visit 
Dosing 
visit 
Safety 
call 
Safety 
visit 
Final 
safety 
visit 
Follow up to 
assess 
response 
Follow up to 
assess 
response 
Week6 
-6 
0 
0+1d 
1 
4 
4+1d 
5 
8 
8+1d 
9 
20 
20+1d 
21 
22 
24 
44 
Visit window 
(days) 
N/A 
N/A 
Vaccine 
1+3d 
-3d+7d
±3d 
Vaccin
e 
2+3d 
-3d+7d
±3d 
Vaccine 
3+3d 
-
3d+7d
±3d 
V4+3d 
-3d+3d 
-3d+3d 
-3d+3d 
–14d to +14d 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Demographics/ 
Medical history/ 
screening exam 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
General exam  
X 
X 
 
(X) 
X 
 
(X) 
X 
 
(X) 
X 
 
(X) 
(X) 
 
 
ECG 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Vital signs 
X 
X7 
 
X 
X7 
 
X 
X7 
 
X 
X7 
 
X 
X 
 
 
Inspection of 
administration sites 
X 
X7 
X8 
X 
X7 
X8 
X 
X7 
X8 
X 
X7 
X8 
X 
(X) 
 
 
Adverse event and 
conmeds assessment 
 
X7 
X 
X 
X7 
X 
X 
X7 
X 
X 
X7 
X 
X 
X 
 
 
Urine pregnancy 
test4 
X 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
Routine safety 
bloods1 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
 
Urinalysis 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
X 
X 
 
 
HIV test2 
X 
(X) 
 
 
(X) 
 
 
(X) 
 
 
(X) 
 
 
 
X 
(X) 
HBV, HCV, syphilis, 
urine for chlamydia, 
and gonnorhoea4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 Other gonnorhoea 
and chlamydia tests4 
(X) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Blood for serum – 
Immunogenicity 
 
X 
 
 
X 
 
X 
X 
 
X 
X 
 
X 
X 
X 
X 
Mucosal sampling  
 
X 
 
 
 
 
 
 
 
 
X 
 
 
X 
 
 

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X means mandatory, (X) means if clinically indicated 
1 The parameters are detailed in section 7.2.3 
2 At each dosing visit clinic staff will determine whether the risk status for HIV has changed and repeat the test if necessary 
3 Diary card given to subjects at each vaccination visit, and reviewed with subject at time-points indicated 
4 Additional HIV/STI and pregnancy tests will be performed if indicated by a change in risk status or menstrual history respectively 
5 Immunisation should be after completion of the other procedures scheduled for that visit except for the AE assessment and injection site inspection which will be conducted before and after each immunisation 
6 After the first vaccination the timing of all visits is determined by the date of the preceding vaccination 
7 Pre-dose and again at 10 (+/–2) min and 60 (+/–10) min post-dose; subjects are free to leave after the 60 min post-dose assessment unless otherwise indicated by AEs etc. 
8 Volunteer invited back to clinic for assessment if reporting grade 3 AE or above  
9 The tolerability questionnaire should be completed post-vaccinations 1–3 only 
 
Blood for PBMC –  
Immunogenicity  
 
X 
 
 
X 
 
 
X 
 
 
X 
 
X 
X 
X 
X 
Immunisation5 
 
V1 
 
 
V2 
 
 
V3 
 
 
V4 
 
 
 
 
 
Tolerability 
questionnaire9 
 
X 
 
 
X 
 
 
X 
 
 
 
 
 
 
 
 
Diary card3 
 
 
 
X 
 
 
X 
 
 
X 
 
 
X 
 
 
 

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8. SAFETY REPORTING 
UK clinical trials regulations require that both investigators and sponsors follow specific procedures when 
notifying and reporting adverse events/reactions in clinical trials. These procedures are described in this 
section of the protocol. Section 8.1 lists definitions, section 8.2 describes details of the responsibilities 
of the institutions/investigators. 
 
8.1 Definitions 
The definitions of the EU Directive 2001/20/EC Article 2 based on the principles of ICH GCP apply to 
this protocol. These definitions are given below: 
 
Adverse event 
Any untoward medical occurrence in a subject to whom a medicinal product has been administered, 
including occurrences which are not necessarily caused by or related to that product.  
The investigator will use the following criteria when deciding whether to report a laboratory parameter 
that falls outside the normal range according to the local laboratory guidelines as an adverse event: 
 
The test result is associated with relevant accompanying symptoms 
 
Additional diagnostic tests or medication are indicated 
 
As a consequence of the test result, an immunisation is delayed or further immunisations are 
discontinued 
 
The investigator considers the result to constitute an adverse event for any other reason 
 
Adverse reaction 
Any untoward and unintended response in a subject to an investigational medicinal product which is 
related to any dose administered to that subject. 
‘Related’ means possibly, probably or definitely as defined below. 
All solicited adverse events which start within 7 days of a vaccination will be automatically classified as 
an adverse reaction. 
Unexpected adverse reaction 
An adverse reaction, the nature or severity of which is not consistent with the information about the 
medicinal product in question set out in the Investigator’s Brochure. 
 
Serious Adverse Events (SAEs), Serious Adverse Reaction (SARs), Suspected Unexpected 
Serious Adverse Reactions (SUSARs) 
Any adverse event, adverse reaction, or unexpected adverse reaction is considered to be a “serious” if it: 
 
Results in death 
 
Is life-threatening  
 
Requires hospitalisation or prolongation of existing hospitalisation 
 
Results in persistent or significant disability or incapacity 
 
Consists of a congenital anomaly or birth defect 
 
Any other medically important event 
 
Notes: 
 ‘A threat to life’ refers to an event or reaction in which the patient was at risk of death at the 
time of the event; it does not refer to an event or reaction which hypothetically might have 
caused death had it been more severe. 

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If an AE is serious and unexpected and considered possibly, probably or definitely related to 
study product according to the classification below then it meets the criteria for a SUSAR 
and should be reported accordingly.  
 
Relationship to study product 
This can be classified as: 
Unrelated 
adverse events that can be clearly explained by extraneous causes and for which 
there is no plausible association with study product, or adverse events for which 
there is no temporal relationship 
Unlikely 
adverse events that may be temporally linked, but which are much more likely to be 
due to other causes than study product and which do not get worse with continuing 
use of product  
Possibly 
adverse events that could equally well be explained by study product or other 
causes, which are usually temporally linked and may improve when not using study 
product but do not reappear when using study product 
Probably 
adverse events that are temporally linked and for which the study product is more 
likely to be the explanation than other causes, which may improve when not using 
study product 
Definitely 
adverse events that are temporally linked and for which the study product is the 
most likely explanation, which disappear or decrease when not using study product 
and reappear when using study product 
 
8.2  Reporting Adverse Events 
Adverse events should be recorded on the appropriate CRF. 
 
All SAEs should be reported to the sponsor within 24 hours of the Clinical Investigator becoming aware 
of the event fulfilling the criteria. The  SAE form should be completed and e-mailed to the Project 
Manager/Monitor and the Joint Research Compliance Office. The minimum criteria required in reporting 
a SAE are the participant identifiers (trial number/date of birth), reporting source (name of Investigator), 
and why the adverse event is identifiable as serious. 
 
Other important adverse events that should be reported to the sponsor within 24 hours of the 
Clinical Investigator becoming aware of the event, include 
 
allergic bronchospasm requiring intensive emergency treatment 
 
a seizure 
 
any adverse event that results in discontinuation of the immunisation schedule 
 
any adverse event that requires intervention to prevent a threat to life or death 
 
 
 
 
 
SAE AND IMPORTANT AE NOTIFICATION 
 
 
Within 24 hours of becoming aware of an  SAE or Important AE, 
please enter onto the SAE CRF and indicate as serious and email the 
MRC CTU at UCL mrcctu.cuthivac002safety@ucl.ac.uk and the 
Imperial College London Joint Research Compliance Office at 
jrco.ctimp.team@imperial.ac.uk 
 
 
SAE AND IMPORTANT AE NOTIFICATION 
 
 
 
 
 
 

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Faxes and emails should be acknowledged by the MRC CTU immediately on receipt. If the Clinical 
Investigator does not receive an acknowledgement they will assume that MRC CTU is not aware of the 
event and use an alternative method of notification. 
 
The Principal Investigator will ensure that the SAE form is sent within 24 hours of an event. The Chief 
Investigator will assess whether the event qualifies in seriousness and relationship as a Suspected 
Unexpected Serious Adverse Reaction (SUSAR). Fatal and life-threatening SUSARs must be 
reported to the MHRA within 7 days of day 0 which is defined as the day the sponsor became aware of 
the event. Relevant follow-up information should be sought and a further report completed as soon as 
possible and submitted within 8 additional days. SUSARs which do not result in death or a threat to life 
should be reported within 15 days of day 0. Ultimate responsibility for classification resides with the 
study Sponsor. 
 
Investigators should notify the JRCO of all SAEs occurring from the time of randomisation until 
30 days after the last protocol treatment administration. SARs and SUSARs must be notified to the 
JRCO until trial closure.  
The MRC CTU Project Manager/Monitor will be responsible for coordination of the report review and 
filing with the JRCO at Imperial College London, and for ensuring the REC and MHRA are informed. From 
01 May 2017 this responsibility will transfer to the study team at the Imperial CRF. 
 
8.3  Pregnancy 
Pregnancy is not an adverse event. However, it is a reportable event in a Phase I trial, and should be 
reported to the Chief Investigator within 24 hours of the Clinical Investigator becoming aware of the 
pregnancy by email, fax or phone.  
All pregnancies will be followed up to collect information about the outcome which will be recorded in 
the clinical study report. 

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No 
Figure 2: Safety Reporting Flowchart 
  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
AE: Adverse Event 
AR: Adverse Reaction 
CRF: Case report form 
SAE: Serious adverse event 
SUSAR: Suspected unexpected serious adverse reaction 
Adverse Event/Adverse Reaction
Was the event serious or important? 
‐resulted in death 
‐is life‐threatening 
‐required hospitalisation or prolongation of existing hospitalisation 
‐persistent or significant disability/incapacity 
‐consists of a congenital anomaly/birth defect 
‐allergic bronchospasm requiring intensive emergency treatment 
‐a seizure  
‐any event resulting in discontinuation of the immunisation schedule 
‐any event that requires intervention to prevent death or a threat to life  
 
SAE 
Record on the ‘AE and SAE 
CRFs’ and securely fax/e‐
mail CRF to the MRC CTU 
Project Manager/Monitor 
and JRCO within 24 hours of 
notification of the event.  
Yes
No 
Yes 
SAE 
No further action required 
after reporting as above 
Is it expected according to the Investigator Brochure? 
Yes 
Is  it  reasonably  causally  related  to  the  study  intervention?  I.e.  the 
relationship to the product is considered definite, probable or possible 
SUSAR 
MRC CTU Project 
Manager/Monitor sends 
expedited reports to 
MHRA/REC. From 01 May 
2017 this responsibility will 
transfer to the study team 
at the Imperial CRF. 
No 
AE/AR 
Record on the ‘AE CRF' 
No 
Yes 

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9. WITHDRAWAL OF PARTICIPANTS 
Withdrawal from the trial means stopping all further visits.  
The reason(s) for withdrawal should be recorded on the Status CRF. 
All participants are free to withdraw from the trial at any time, for any reason, without affecting their 
future medical care. An investigator may decide to withdraw a participant if the investigator deems that 
continuing might compromise participant wellbeing or interfere with the achievement of the trial’s 
objectives. 
Participants who are withdrawn due to an adverse event (AE) will be followed-up until the event has 
stabilised. 
Withdrawn participants who have received any immunisations should be asked to undergo the 
procedures scheduled for the primary endpoint visit (Visit 10, week 22).  
 
Policy for replacing withdrawals 
Additional volunteers will be enrolled to replace early withdrawals or those who have discontinued their  
immunisations early, with the aim of having 24 subjects complete the study with no major protocol 
deviations.  However, no more than 30 individuals in total will be exposed to the vaccine. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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10. STATISTICAL CONSIDERATIONS 
10.1   Method of Randomisation 
Participants will be block-randomised centrally using a computer-generated algorithm with a back-up 
manual procedure.  Randomisation will be stratified on the basis of gender. 
 
10.2       Endpoints 
10.2.1 
    Primary Endpoints 
10.2.1.1   Safety 
 Grade 3 or above local solicited adverse event (Table 2) 
 Grade 3 or above systemic clinical and laboratory solicited adverse event (Table 2) 
 Any grade of adverse event that results in a clinical decision to discontinue further immunisations 
 Any grade of adverse event within 7 days of receiving intradermal and standard intramuscular 
vaccinations with or without electroporation  
 
10.2.1.2 Immunogenicity 
 The magnitude of antigen specific systemic IgG antibody binding responses (µg/ml) 2 weeks after the 
final vaccination. 
 
10.2.2 
  Secondary Endpoints 
10.2.2.1  Safety 
 Any grade of adverse event, local to the ID and IM injection sites, that starts within 7 days after 
Doses 1-3.   
 
10.2.3 
 Exploratory immunogenicity endpoints 
 Frequency and magnitude of HIV-gp140 specific B-cell-mediated responses in the systemic 
compartment measured by B-cell ELISPOT 
 The magnitude of vaccine specific systemic T cell responses by T cell ELISpot assay 
 The magnitude of antigen specific systemic IgA antibody responses (ug/ml) 
 Frequency, titre and avidity of serum binding antibodies to other HIV Env antigens (alternative 
clades) by ELISA or other assays. 
 Mapping of serum binding antibodies using  Env subunit constructs  (e.g., V2 scaffolds and hotspots) 
by ELISA. 
 Frequency and magnitude of mucosal IgG and IgA antibody responses to CN54gp140 measured four 
weeks after the final immunisation. 
 Frequency and titre of serum neutralising antibodies to homologous virus, and, if warranted a wider 
panel of viruses representing different clades.  
 Frequency and magnitude of HIV-specific T-cell mediated responses measured by T-cell CFSE, and 
ICS (Intracellular Cytokine Staining). 
 Frequency and magnitude of T-cell chemokine and cytokine release following ex-vivo antigen 
stimulation quantified by Luminex. 
 Isolation and characterization of Env-specific monoclonal antibodies (IgG) from memory B cells in the 
systemic compartments (dependent upon elicited specific memory B-cell numbers). 
 Characterisation of non-neutralising antibody function using ADCC/ADCVI, viral capture and 
aggregation assays.  
 Epitope mapping of B- and T-cell responses. 
 
10.2.4 
Exploratory tolerability endpoints 
 Pain scores at 0, 10 and 30 minutes following vaccination with EP 
 

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10.3   Sample Size 
It is not the remit of this Phase I trial to recruit a sufficient number of participants to be statistically 
confident about the differences between groups. By the end of this study 8 participants will have been 
exposed to each schedule in groups 1, 2, and 3 and this provides confidence around the response/event 
proportions of 0–60% in table 6. 
 
Table 6:  Sample Size 
 
Number of 
“responders” 
Proportion 
if n=8 
95% confidence interval1 
0 
0% 
0 – 28% 
1 
10% 
2 – 40% 
2 
20% 
6 – 51% 
3 
30% 
11 – 60% 
4 
40% 
17 – 69% 
5 
50% 
24 – 76% 
6 
60% 
31 – 83% 
 
1 Wilson interval (suitable for small sample sizes) 
 
It is difficult to give an estimate of the power of group comparisons using quantitative antibody titre 
outcomes at this stage as this is dependent on the number of responders.  
 
10.4 Data analyses and Presentations 
A full statistical analysis plan will be developed before the final analysis. It will be based on the following 
summary: 
 
10.4.1 
  Participant Populations 
 
Intention-to-treat (ITT) population: all participants randomised and given at least one immunisation 
in the trial. 
 
Replacement participant population: participants who were not randomised but did receive at least 
one immunisation. 
 
Per-protocol (PP) population: all participants randomised and non-randomised who were immunised 
with all scheduled immunisations, and who complete the trial. 
 
10.4.2 
   Immunogenicity 
10.4.2.1  Primary Immunogenicity Outcomes 
All serum samples will be screened for antigen specific antibodies IgG (and IgA). The absolute levels of 
antibody in samples that are found to be positive will be determined using a standardised and 
quantitative ELISA developed in Robin Shattock’s  laboratory at Imperial College London. In this 
sandwich capture ELISA, the Ab of interest is captured by the relevant target antigen  and then detected 
using a labelled isotype specific secondary Ab. An estimate of the concentration of Ab in the sample is 
calculated by interpolation relative to a standard curve based on titration of purified human standards 
IgG or IgA captured by anti-human kappa/lambda-specific antibodies. The number of ‘responders’ in 
each assay will be presented by time-point and group as a proportion with 95% confidence interval. A 
‘responder’ will be defined as a participant in whom a response was detected in at least one post 
treatment immunogenicity sample. Titres of antigen specific antibodies will be described by time-point 
and group, and compared using rank tests where appropriate. Although all samples will be processed, 
the analyses will be for the per-protocol population and conducted in Robin Shattock’s laboratory.  

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10.4.3 
  Exploratory Immunogenicity Outcomes 
The proportion of individuals mounting a B cell response using frozen PBMC will be determined by B cell 
Elispot assay for antigen induced IgG production from antibody secreting cells (ASC’s). CN54gp140 
specific responses will be analysed as well as total IgG and compared between vaccine groups.  
In addition vaccine induced T cell responses will be analysed by T cell Elispot assay for IFN- production 
on frozen PBMC samples and the proportion of individuals mounting a response quantified as SFU (spot 
forming units) compared between vaccine groups. These exploratory analyses will use descriptive 
statistics and will be conducted in Robin Shattock’s laboratory.   
 
10.4.4 
  Safety Outcomes 
The AEs for the randomised subjects will be coded by MRC CTU staff using MedDRA. If a participant 
reports the same event more than once then the worst severity and worst relationship to trial vaccine 
will be taken. Discrepancies between diary card and CRF reports will be queried by the monitor if 
unclear. It is assumed that the grade assigned by the clinician is more accurate, and this will be the 
grade reported in the ITT analysis tables, prepared by MRC CTU. If the diary card grade is worse, this 
will be foot noted.  
All safety end-points will be graded by the Clinical Investigators.  
Safety outcomes for the randomised participants will be reported overall with proportion and 95% 
confidence interval, and by group and time-point, and by relationship to study product, and method. 
For the primary ITT analysis of safety endpoints (as defined in section 10.2), results will be expressed 
as a proportion with confidence interval, and groups compared using Fisher’s exact test. 
Safety outcomes for the non-randomised participants will be reported separately. 
 
10.4.5 
  Tolerability Outcomes 
The tolerability data from the questionnaires will not be part of the SAP or reported in the Clinical Study 
Report. 
 
11. DATA MANAGEMENT  
Data management, analysis and reporting of trial data collected from all randomised subjects will be 
prepared by the MRC CTU at UCL according to the Data Management Plan and Statistical Analysis Plan. 
 
11.1 Data management at the Clinical Centre 
Staff at the clinical centre will be responsible for: 
 Creating medical notes for each participant which includes paper copies of the consent 
documents and the blood results 
 Entering information relevant to eligibility and emergent adverse events and documenting the 
result of any pregnancy tests and urinalysis in the medical notes 
 The accurate completion of the CRFs 
 Collection and review of the diary cards from participants 
 Notification of SAEs within 24 hours of becoming aware of the event to the MRC CTU Project 
Manager/Monitor and the JRCO 
 Notification of pregnancy within 24 hours of becoming aware of the pregnancy to the Chief 
Investigator 
 From 1st May 2017, notification of SUSARs within the timelines defined in Section 8 and 
maintaining the Trial Master File. 
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significant adverse events and concomitant medication) will be documented in the medical notes. 
The CRFs will not bear the participant’s name. The participant’s initials, date of birth and trial number 
(which will have been given at screening) will be used for identification. 
A member of the clinical trial team must sign the laboratory and ECG reports. In the event of an 
abnormality, an indication should be given whether or not the abnormality is clinically significant, the 
date of review and the signature of the clinician reviewing the result. 
The database and medical notes should be kept in a secure location for 2 years after the last approval of 
a marketing application or until 2 years have elapsed since formal discontinuation of product 
development, and at least 15 years after the clinical trial has ended. 
11.2 Data Management in the Immunology Laboratories 
Standard operating procedures will be followed in all laboratories to ensure the quality of the data. Data 
will be stored electronically and transferred in an agreed format for analysis.  
 
11.3 Data management at the MRC CTU at UCL 
Staff at the MRC CTU will be responsible for: 
 Designing the CRFs 
 Creating a database  
 Training staff at the clinical centre in data collection and overseeing data entry which may 
include data entry at the clinical centre directly into the database  
 Notification of SUSARs within the timelines defined in section 8  
 Drafting the Data Management and Statistical Analysis Plans and conducting the analyses 
 Clinical site monitoring 
 Maintaining the Trial Master File on behalf of the sponsor until 1st May 2017 
 Coordination of the Trial Management Group  

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12. TRIAL MONITORING 
12.1 Risk Assessment 
The MRC will perform a risk assessment to assess the risks and benefits of trial participation to individual 
participant safety, as well as the risks that underlie the validity of the trial results with respect to safety 
and immunogenicity outcome measurements.  
The risk assessment will be reviewed by the Chief Investigator. 
This assessment will be used to guide the development of procedures with respect to informed consent, 
confidentiality, trial monitoring and audit.    
 
12.2  Monitoring by Medical Research Council Clinical Trials Unit at University 
College London (MRC CTU at UCL)  
Monitoring will be performed according to ICH-GCP.  A monitoring plan will be written based on the risk 
assessment.  
 
On-site monitoring will be delegated to a trained monitor by the MRC CTU at UCL to ensure that the 
study is conducted in compliance with the protocol, is consistent with SOPs, the principles of GCP, 
applicable regulatory requirements and locally accepted practices. The investigators, as well as 
volunteers through consenting to the study, agree that the monitor may inspect study facilities and 
source records (e.g., informed consent forms, clinic and laboratory records, other source documents), as 
well as observe the performance of study procedures. Such information will be treated as strictly 
confidential and will under no circumstances be made publicly available. 
 
The Principal Investigator will permit inspection of the facilities and all study-related documentation by 
authorised representatives of the Sponsor, and Regulatory Authorities responsible for this study. 
 
Prior to the first volunteer being screened, a site initiation visit will be made by the MRC CTU and will 
consist of review of protocol and trial documents, training with respect to trial procedures (informed 
consent, SAE reporting, inclusion and exclusion criteria), review of recruitment strategy, review of site 
facilities and equipment, essential document receipt, collection and filing, and archiving and inspection. 
Copies of the trial specific documents will be given to the investigators. The approved version of the 
protocol should be followed at all times, and any significant protocol deviations will be documented on a 
Protocol Deviation Form and submitted by MRC CTU to the Sponsor as soon as possible.  The 
investigators will allow the monitors to:  
 
Inspect the site, the facilities, IMP management and materials used for the trial 
 
Meet all members of the team involved in the trial, and ensure all staff working on the trial are 
experienced and appropriately trained, and have access to review all of the documents relevant 
to the trial  
 
Have access to the case report forms and source data  
 
Discuss with the investigator and site staff trial progress and any issues on a regular basis  
 
12.3 Monitoring by the Imperial CRF 
From 01 May 2017, MRC CTU at UCL will cease to perform monitoring and that responsibility will 
transfer to the Imperial CRF. 
 
12.4 Clinical Site Monitoring 
The trial site will be monitored to ensure that: 
 
All participant records exist; participants are eligible and informed consents signed 
 
There is adherence to the protocol, including consistency with inclusion/exclusion criteria 

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 
There is compliance with the principles of GCP and regulatory requirements  
 
Trial Documentation is complete and up to date (e.g. correct versions of documents being used, 
source data captured) and relevant documents are collected for the Trial Master File (TMF)  
 
The CRFs have been completed correctly and accurately, and all entries correspond to data 
captured in source documents  
 
The IMP accountability records are in order (receipt, dispensing and return/destruction), storage 
is under appropriate conditions and secure, expiry dates are being checked and adhered to, and 
dispensing is according to the protocol and trial procedures.   
 
All information dealt with during such visits will be treated as strictly confidential.  At the end of the trial, 
a close out visit will be performed by the monitor after the final participant visit has been completed and 
prior to database lock. During this visit the monitor will verify that all trial close out activities are 
completed – all queries resolved, missing data completed, monitoring completed, archiving 
arrangements in place, IMP accountability complete and all used and unused IMP returned/destroyed, 
ISF (investigator site file) completed and TMF documents collected, and end of trial notified.  Each 
investigator will also be notified that an audit or inspection may be carried out - by the sponsor; 
sponsor's representatives or the regulatory authorities - at any time, before, during or after the end of 
the trial.  The investigator must allow the representatives of the audit or inspection team:  
 
To inspect the site, facilities and material used for the trial 
 
To meet all members of his/her team involved in the trial 
 
To have direct access to trial data and source documents, to consult all of the documents 
relevant to the trial  
 
12.5 Monitoring by the Trial Management Group 
The Trial Management Group (see section 16.1) will monitor the following: 
 
Screening and enrolment numbers 
 
Immunisations completed and any missed or outside the window 
 
Adverse events of note 
 
Missed visits and loss to follow-up 
 
Logistical difficulties at the clinical centre 
 
Data management issues (timeliness of CRFs, completeness) 
 
Immunology core lab issues (completeness of specimen collection, next batch transfer or 
analysis) 
 
GCP issues (minor or other breaches) 
 
12.6 Confidentiality 
The principles for the UK DPA will be followed. All personal details of the participants and the results of 
the trial will be kept strictly confidential. The Sponsor, as represented by the ICL JRCO, will not keep any 
material on file containing the volunteers’ full names; this information will be kept by trial team in the 
clinical trial facilities in a secure location. The confidentiality of volunteers will be respected and 
maintained at all times. 
Each participant’s GP will be notified of their patient’s participation in the trial.  
 
 
 
 
 
 
 
 
 
 

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13. ETHICAL CONSIDERATIONS AND APPROVAL 
13.1 Ethical Issues 
There are three aspects of this trial that raise ethical issues  
First, safety: DNA plasmid vaccines have been widely used in man at similar doses to those employed in 
this trial to vaccinate against a wide range of infectious diseases such as HIV as well as non infectious 
diseases such as cancer. To date, there have been no safety concerns. Also such vaccines have been 
administered via conventional methods such as IM and ID as well as using EP. 
Second, issues particular to HIV: because the product under investigation is a candidate HIV vaccine, 
and HIV is transmitted sexually. The nature of the product may lead volunteers to erroneously conclude 
they are protected against HIV and to engage in riskier behaviour as a consequence. It is possible that 
following immunisations, participants may have equivocal results in the standard laboratory tests for 
HIV. However, any accredited laboratory would be able to distinguish between a response to the vaccine 
and the occurrence of a natural infection using routine assays.  
Third, financial: the reimbursement to compensate for the intense follow-up schedule, which is a 
feature of healthy volunteer trials, could be sufficient incentive for individuals to take part against their 
better judgement. However, the rate of reimbursement in this trial is in line with those of similar healthy 
volunteer trials. 
 
13.2 Ethical Considerations 
The trial will be conducted in compliance with the approved protocol, UK Clinical Trial Regulations and 
any amendments, which include compliance with the principles of Good Clinical Practice (GCP) and will 
abide by the principles of the Declaration of Helsinki, the UK Data Protection Act (DPA number: 
Z5886415), and the National Health Service (NHS) Research Governance Framework for Health and 
Social Care (RGF). 
The trial proposal including the trial specific information to be provided to volunteers will be reviewed by 
a recognised Research Ethics Committee (REC) and by the Medicines and Healthcare products 
Regulatory Authority (MHRA), The trial will not proceed unless the sponsor obtains approvals from these 
two authorities. 
All volunteers must give written consent to participate in this trial, before any screening evaluation. 
Before giving consent, volunteers will be asked to read the information sheet about the trial and raise 
questions. They must also read the consent form. They will have the opportunity to discuss the trial with 
the Principal Investigator or delegate, and be asked to explain what the trial involves in their own words, 
to ensure the volunteer understands the intensity of the schedule and the issues associated with taking 
part in a trial of a candidate HIV vaccine.  
The safety assessments are intense. Participants will be asked to remain in clinic for about 1 hour 
following each immunisation, to complete a diary card for at least 7 days thereafter, respond to a 'safety 
[telephone] call' the day after each immunisation, and return for a safety visit a week (-3 days/+ 7 days) 
after each immunisation. They will be advised to call the clinic staff if they are concerned, and 24 hour 
cover will be available.  
 
 

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14. INDEMNITY 
The Sponsor for the trial is Imperial College London (ICL). 
The Sponsor undertakes to compensate any volunteers for injuries which are considered, on the balance 
of probabilities to have arisen as a result of their participation in the trial regardless of whether the 
injuries were caused by negligence or not. 
ICL holds insurance to cover participants for injury caused by their participation in the clinical trial. 
Participants may be able to claim compensation if they can prove that ICL has been negligent. However, 
as this clinical trial is being carried out in a hospital, the hospital continues to have a duty of care to the 
participant in the clinical trial. ICL does not accept liability for any breach in the hospital’s duty of care, 
or any negligence on the part of the hospital employees. This applies whether the hospital is an NHS 
Trust or not. This does not affect the participant’s right to seek compensation via the non-negligence 
route. 
Participants may also be able to claim compensation for injury caused by participation in this clinical trial 
without the need to prove negligence on the part of ICL or another party. Participants who sustain injury 
and wish to make a claim for compensation should do so in writing in the first instance to the Principal 
Investigator, who will pass the claim to the Sponsor’s Insurers, via the Joint Research Compliance Office.  
 
15. FINANCE 
The clinical trial activities, acquisition of product, data management and analysis are funded by the EC 
under the FP7 framework.   
 
Participants will receive recompense for their time and travel, £200 per vaccination visit and £100 per 
screening/safety/follow-up visit up to a total of £1600 over the course of the study. Only enrolled 
participants will be paid for the screening visit.  
 
There are no bonuses or per participant incentives paid to staff.  
 

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16. TRIAL COMMITTEES 
16.1 Trial Management Group (TMG) 
The Trial Management Group (TMG) will be formed of the Chief, Principal and other lead Investigators 
(clinical and non-clinical) from the clinical centre and immunology laboratory as well as members of MRC 
CTU.  
The TMG will be responsible for the day-to-day running and management of the trial and will be 
accountable to the Sponsor. 
The TMG will also be responsible for the composition of the expert panel to review any emergent 
SUSARs. 
 
16.2 CUT’HIVAC Consortium Governing Board 
This Governing Board has reviewed the design of the trial. 
The Governing Board will be notified by the Project Lead of the progress of the trial, and provided with 
the final Clinical Study Report. 
The CUT’HIVAC Consortium funding ended in December 2015. Subsequently the accountability has been 
to the Project Coordinator at INSERM, France.  
 
16.3 Trial Management Team (TMT) 
Weekly clinical TMT meetings are held at the Imperial CRF, Hammersmith Hospital. 
 
17. PUBLICATIONS 
The preparation of a manuscript for publication in a peer-reviewed professional journal or an abstract for 
presentation, oral or written, to a learned society or symposium will be discussed on the Trial 
Management Group calls. The Sponsor will be notified of this intention through the Chief Investigator 
and the TMG notes. Every effort will be made to allow the Sponsor and other relevant parties involved in 
the clinical trial and named in the clinical trial agreement prepared by the Sponsor, 30 days to comment 
before any results are submitted. This timeline will be strictly observed for peer-review journals, but may 
be more difficult to adhere to for conference presentations. Approval from the Chief Investigator, the 
clinical centre Principal Investigator and Project Lead must be obtained as a minimum before submission 
to a conference.  
Authorship should reflect work done by the investigators and personnel of the sponsor, in accordance 
with generally recognised principles of scientific collaboration. 
 
 
 
18. PROTOCOL AMENDMENTS 
After the protocol has been approved by the main REC and the MHRA, no changes may be made 
without the written agreement of the Chief Investigator and the sponsor.   
The MHRA and main REC do not need to approve any substantial change to the protocol that needs to 
be implemented urgently to avoid an immediate hazard to trial participants. The sponsor will ensure that 
the MHRA and main REC are informed of urgent amendments in accordance with UK clinical trials 
regulatory guidance. 
The REC and/or MHRA must approve substantial amendments before they are implemented.  

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19. REFERENCES 
1.  IAVI, IAVI: ESTIMATING THE GLOBAL IMPACT OF AN AIDS VACCINE. POLICY BRIEF, 2006.  
2.  FLYNN, N.M., ET AL., PLACEBO-CONTROLLED PHASE 3 TRIAL OF A RECOMBINANT GLYCOPROTEIN 
120 VACCINE TO PREVENT HIV-1 INFECTION. J INFECT DIS, 2005. 191(5): P. 654-65. 
3.  PITISUTTITHUM, P., ET AL., RANDOMIZED, DOUBLE-BLIND, PLACEBO-CONTROLLED EFFICACY 
TRIAL OF A BIVALENT RECOMBINANT GLYCOPROTEIN 120 HIV-1 VACCINE AMONG INJECTION 
DRUG USERS IN BANGKOK, THAILAND. J INFECT DIS, 2006. 194(12): P. 1661-71. 
4.  BUCHBINDER, S.P., ET AL., EFFICACY ASSESSMENT OF A CELL-MEDIATED IMMUNITY HIV-1 
VACCINE (THE STEP STUDY): A DOUBLE-BLIND, RANDOMISED, PLACEBO-CONTROLLED, TEST-OF-
CONCEPT TRIAL. LANCET, 2008. 372(9653): P. 1881-93. 
5.  GRAY, G.E., ET AL., SAFETY AND EFFICACY OF THE HVTN 503/PHAMBILI STUDY OF A CLADE-B-
BASED HIV-1 VACCINE IN SOUTH AFRICA: A DOUBLE-BLIND, RANDOMISED, PLACEBO-
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22.  LU, S., S. WANG, AND J.M. GRIMES-SERRANO, CURRENT PROGRESS OF DNA VACCINE STUDIES IN 
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31.  LEDGERWOOD, J.E., ET AL., A WEST NILE VIRUS DNA VACCINE UTILIZING A MODIFIED 
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PHASE I CLINICAL TRIAL. J INFECT DIS, 2011. 203(10): P. 1396-404. 
32.  CATANZARO, A.T., ET AL., PHASE I CLINICAL EVALUATION OF A SIX-PLASMID MULTICLADE HIV-1 
DNA CANDIDATE VACCINE. VACCINE, 2007. 25(20): P. 4085-92. 
33.  BAGARAZZI, M.L., ET AL., IMMUNOTHERAPY AGAINST HPV16/18 GENERATES POTENT TH1 AND 
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34.  SARDESAI, N.Y. AND D.B. WEINER, ELECTROPORATION DELIVERY OF DNA VACCINES: PROSPECTS 
FOR SUCCESS. CURR OPIN IMMUNOL, 2011. 23(3): P. 421-9. 
35.  YIN, J., ET AL., HIGH ANTIBODY AND CELLULAR RESPONSES INDUCED TO HIV-1 CLADE C 
ENVELOPE FOLLOWING DNA VACCINES DELIVERED BY ELECTROPORATION. VACCINE, 2011. 
29(39): P. 6763-70.  
36.  DOBANO, C., ET AL., ENHANCEMENT OF ANTIBODY AND CELLULAR IMMUNE RESPONSES TO 
MALARIA DNA VACCINES BY IN VIVO ELECTROPORATION. VACCINE, 2007. 25(36): P. 6635-45.  
37.  DRAPE, R.J., ET AL., EPIDERMAL DNA VACCINE FOR INFLUENZA IS IMMUNOGENIC IN HUMANS. 
VACCINE, 2006. 24(21): P. 4475-81. 
38.  VAN DRUNEN LITTEL-VAN DEN HURK, S. AND D. HANNAMAN, ELECTROPORATION FOR DNA 
IMMUNIZATION: CLINICAL APPLICATION. EXPERT REV VACCINES, 2010. 9(5): P. 503-17.  
39.  OTTEN, G., ET AL., ENHANCEMENT OF DNA VACCINE POTENCY IN RHESUS MACAQUES BY 
ELECTROPORATION. VACCINE, 2004. 22(19): P. 2489-93.  
40.  OTTEN, G.R., ET AL., POTENT IMMUNOGENICITY OF AN HIV-1 GAG-POL FUSION DNA VACCINE 
DELIVERED BY IN VIVO ELECTROPORATION. VACCINE, 2006. 24(21): P. 4503-9.  
41.  MANN, J.F., ET AL., ENHANCED IMMUNOGENICITY OF AN HIV-1 DNA VACCINE DELIVERED WITH 
ELECTROPORATION VIA COMBINED INTRAMUSCULAR AND INTRADERMAL ROUTES. J VIROL, 
2014. 88(12): P. 6959-69.  
42.  MARTINON, F., ET AL., PERSISTENT IMMUNE RESPONSES INDUCED BY A HUMAN 
IMMUNODEFICIENCY VIRUS DNA VACCINE DELIVERED IN ASSOCIATION WITH 
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1291-307.  
43.  DOLTER, K.E., ET AL., IMMUNOGENICITY, SAFETY, BIODISTRIBUTION AND PERSISTENCE OF 
ADVAX, A PROPHYLACTIC DNA VACCINE FOR HIV-1, DELIVERED BY IN VIVO ELECTROPORATION. 
VACCINE, 2011. 29(4): P. 795-803.  
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DNA VACCINE CANDIDATE IN HEALTHY VOLUNTEERS. PLOS ONE, 2011. 6(5): P. E19252.  

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45.  NICOLAS, J.F. AND B. GUY, INTRADERMAL, EPIDERMAL AND TRANSCUTANEOUS VACCINATION: 
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CELLS AND CD14+ DERMAL DENDRITIC CELLS. IMMUNITY, 2008. 29(3): P. 497-510. ..  
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177(11): P. 7959-67.  
48.  VAN DER AAR, A.M., ET AL., LOSS OF TLR2, TLR4, AND TLR5 ON LANGERHANS CELLS ABOLISHES 
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ENGL J MED, 2004. 351(22): P. 2295-301.  
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APPENDICES 
APPENDIX 1: ELECTROPORATION ICHOR TRIGRID™ DELIVERY SYSTEM 
FOR 
INTRAMUSCULAR 
(TDS-IM) 
AND 
INTRADERMAL 
(TDS-ID) 
DELIVERY 
 
TDS Delivery Technology 
 
EP is an efficient DNA delivery method that has been shown to significantly increase DNA vaccine 
potency by up to several orders of magnitude compared to delivery by conventional injection.  EP is a 
technique for intracellular delivery based on the brief application of electrical fields in a target region of 
tissue. This process induces a transient state of membrane destabilization/permeability, during which 
time normally impermeant substances present in the interstitial space at the site of EP application can be 
taken up into the affected cells. Shortly after EP, the cell membrane stabilizes and the cells resume 
normal function. EP has been demonstrated to be a potent method for DNA delivery in a variety of 
tissues including skeletal muscle, liver, lung, skin, and various tumour types, enhancing intracellular DNA 
uptake and gene expression by 2-3 orders of magnitude compared to conventional methods of 
administration. 
 
Properties of the TDS Technology 
 
The TDS devices are designed for intramuscular or intradermal administration of DNA using EP. The TDS 
technology utilizes the in vivo application of electrical fields to enhance the intracellular delivery of 
agents of interest in a targeted region of tissue. Specifically, the device is designed applying electrical 
fields at the site of administration to induce the EP effect in the presence of the DNA to be delivered. 
The device has been designed for use in human clinical studies and complies with the applicable safety 
and electromagnetic compatibility requirements of the International Electrotechnical Commission (IEC) 
60601-1. 
 
TDS device components 
 
Both the TDS-IM and TDS-ID consist of the following components: 
 
Pulse Stimulator  
 
Integrated Applicator  
 
Single use Application Cartridge  
In addition, the TDS-ID device has a  
 
Medi-Jector Vision needle free injection device 
 
The configuration of the TDS device is designed for the 
administration of investigational agents in the context of early 
phase human clinical studies.  
 
Device Components 
 
Pulse Stimulator  
The Pulse Stimulator is an electronic device that controls the 
administration sequence, generates the electrical signals 
necessary to enhance the intracellular delivery of the agent, 
and monitors the administration sequence for safety hazards.  
The Pulse Stimulator performs a comprehensive self-diagnostic to ensure that the device and all internal 
safety systems are functioning properly before an administration procedure can be initiated. The device 
has been designed to comply with the applicable safety and electromagnetic compatibility requirements 
of IE. 
 

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Integrated Applicator 
 
The Integrated Applicator is a reusable hand-held device that contains mechanisms to automatically 
deploy the electrodes into the tissue and administer the biologic agent. The device is configured so that 
the entire procedure is applied in an automated fashion following the activation the procedure initiation 
button. This ensures that the prescribed administration parameters (e.g. site of agent injection relative 
to EP application, rate of agent injection, and time interval between agent injection and EP application) 
will be implemented for every recipient in a uniform fashion, thereby minimizing variability in the 
application of the procedure arising as result of operator skill or level of training. A user activated 
mechanical safety switch reduces the possibility of an inadvertent discharge of the device during set up. 
 
                         TDS-IM 
 
 
     
                TDS-ID 
 
 
 
 
Application Cartridge 
 
 
Each Application Cartridge is packaged sterile for single use and is the only component of the system 
that contacts the recipient 
 
 
TDS-IM 
 
The TDS-IM Application Cartridge is used 
to house the agent to be delivered (in a 
standard syringe) and the electrodes 
used for EP application. It is comprised of 
a plastic, injection molded body that 
encloses the four electroporation 
electrodes.  
The TDS electrode array consists of four 
electrodes arranged in two equilateral triangles to form a diamond configuration around a central 
injection needle. With the long axis of the diamond placed in parallel with the direction of the muscle 
fibres this configuration corresponds with the ellipsoid fluid distribution characteristic of an intramuscular 
injection.  
 
 
 
 
 
 
 
 
 
 
 

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TDS-ID 
 
The TDS-ID Application Cartridge consists of a plastic body that encloses the four electrode TriGridTM 
array and interfaces with a Medi-Jector Vision needle free injection device containing the agent to be 
administered. It is attached to the Medi-Jector Vision syringe once it has been loaded with the dose to 
be administered. 
Prior to administration, the electrodes remain recessed within the sterile Application Cartridge body. A 
plastic safety cap located on the tip of the cartridge protects the operator from accidental stick injury 
and ensures that the electrodes remain sterile prior to administration. Each Application Cartridge also 
incorporates a spring loaded stick shield that deploys over the electrodes as the device is retracted from 
the recipients skin. This facilitates safe and simple disposal of the single use Application Cartridge and 
ensures that the electrodes are never visible to the recipient 
 
 
TDS-ID Medi-Jector Vision needle free injection device 
 
The Medi-Jector Vision injection device and needle free syringe is filled to the prescribed dosage and 
inserted into rear aperture of the application cartridge. Once the syringe is inserted, tabs located on the 
cartridge body lock it into place to facilitate safe disposal. 
 
 
 
 
 
 
 
 
 
 
 
 

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APPENDIX 2: TOXICITY TABLE 
Based on systems in use at the MRC CTU, IAVI and NIH Division of AIDS  
 
Abbreviations:   
    ULN      Upper Limit of Normal 
   LLN   Lower Limit of Normal 
 
Rx 
Therapy 
Req 
Required 
 
Mod 
Moderate 
IV 
Intravenous 
 
ADL 
Activities of Daily Living 
Dec 
Decreased 
For other events not specified in the tables below the severity will be determined according 
to the US department of health and human services: Guidance for Industry – Toxicity 
grading scale for Healthy adult and adolescent volunteers enrolled in Preventative Vaccine 
Clinical trials. 
http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInfor
mation/Guidances/Vaccines/ucm091977.pdf 
 
LABORATORY PARAMETERS 
 
PARAMETER 
 
GRADE 1 
MILD 
GRADE 2 
MODERATE 
GRADE 3 
SEVERE 
GRADE 4 
EXTREME 
HAEMATOLOGY 
Hb 
10.0-10.9 g/dL 
9.0-9.9 g/dL 
7.0-8.9 g/dL 
<7.0 g/dL 
White Blood Count 
13.0 – 14.9 x109/l   
or 2 – 2.5 x109/l 
15.0 – 19.9 x109/l 
or 1.5 – <2.0 x109/l 
20.0 – 29.9 x109/l 
or 1 – <1.5 x109/l 
≥30.0  
or <1.0 x109/l 
Absolute Neutrophils 
1.3-1.0 x109/l 
<1.0->0.75 x109/l 
<0.75->0.5 x109/l 
<0.5 x109/l 
Percent neutrophils 
>80% 
90% 
>95% 
--- 
Lymphocytes 
0.7- 0.899 x109/l 
0.5-0.699 x109/l 
0.35-0.499 x109/l 
<0.35 x109/l 
Platelets 
100 –124.999 x 109/l 
50 – 99.999 x 109/l 
25 – 49.999 x109/l 
<25.0 x109/l 
CD4 Count 
300-400/mm3 
<300mm3 
<200/mm3 
<100/mm3 
BIOCHEMISTRY 
Potassium 
 
Hyperkalemia 
 
Hypokalemia 
 
5.6 – 6.0 meq/L 
3.0 – 3.4 meq/L 
 
6.1-6.5 meq/L 
2.5 – 2.9 meq/L 
 
6.6-7.0 meq/L 
2.0– 2.4 meq/L 
 
>7.0 meq/L 
<2.0 meq/L 
Bilirubin 
 
Hyperbilirubinemia 
 
>1.25 – 2.0 x ULN 
 
>2.0 – 2.5 x ULN 
 
>2.5 – 5 x ULN 
 
>5 x ULN 
Glucose 
 
Hypoglycaemia 
 
Hyperglycaemia 
nonfasting; no prior 
diabetes 
 
2.3-2.4 mmol/l 
7.0-10.0 mmol/l 
 
2.1-2.2 mmol/l 
10.1-15.0 mmol/l 
 
1.5-2.0 mmol/l 
15.1-25.0 mmol/l 
 
<1.5 mmol/l 
>25.0 mmol/l 
Transaminases 
 
AST (SGOT) 
 
ALT (SGPT) 
 
GGT 
 
Alk Phos 
 
1.25 – 2.5 x ULN 
1.25 – 2.5 x ULN 
1.25 – 2.5 x ULN 
1.25 – 2.5 x ULN 
 
>2.5 – 5.0 x ULN 
>2.5 – 5.0 x ULN 
>2.5 – 5.0 x ULN 
>2.5 – 5.0 x ULN 
 
>5.0 – 10.0 x ULN 
>5.0 – 10.0 x ULN 
>5.0 – 10.0 x ULN 
>5.0 – 10.0 x ULN 
 
> 10.0 x ULN 
> 10.0 x ULN 
> 10.0 x ULN 
> 10.0 x ULN 
Amylase 
>1.0 – 1.5 x ULN 
>1.5 – 2.0 x ULN 
>2.0 – 5.0 x ULN 
>5.0 x ULN 
Creatinine 
130-180mol/l 
181-360mol/l 
361-720mol/l 
>720mol/l 
URINALYSIS 
Proteinuria: 
24 hour urine 
200 mg - 1 g loss/day 
OR <0.3% OR <3 g/l 
1 – 2 g loss/day OR 
0.3 – 1.0% OR 3 - 
10 g/l 
2 – 3.5 g loss/day 
OR 
>1.0% OR > 10 g/l 
Nephrotic syndrome 
OR >3.5 g loss/day 
Haematuria 
Microscopic only <10 
RBC/HPF 
>10 RBC/HPF 
Gross, 
with 
or 
without 
clots 
OR 
RBC casts 
Obstructive OR transfusion 
req 

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SOLICITED VACCINE REACTIONS 
GENERAL 
Fever 
Oral>12 hours 
37.7 - 38.9°C 
(100.0 – 101.5°F) 
39.0 – 39.7°C 
(101.6 – 102.9°F) 
39.8 – 40.5°C 
(103 - 105°F) 
>40.5°C (105°F) 
OR max temp of >105°F 
Chills/rigors 
Mild 
hot/cold 
flush 
requires 
blanket 
or 
occasional 
over 
the 
counter treatment  
Limiting daily activity 
>6 
hours, 
or 
need 
regular over the counter 
treatment 
Uncontrollable 
shaking, prescription 
treatment needed 
Hospitalisation 
Malaise/abnormal 
tiredness 
Normal 
activity 
reduced – not bad 
enough to go to bed  
Fatigue such that ½ 
day in bed for 1 or 2 
days 
Fatigue such that in 
bed all day or ½ day 
for more than 2 days 
Hospitalisation 
General (all over) muscle 
aches and pains 
No limitation of activity 
Muscle 
tenderness, 
aches/pains 
limiting 
activity 
e.g. 
difficulty 
climbing stairs 
Severe limitation e.g. 
can’t climb stairs 
Hospitalisation 
Headache 
No 
treatment 
or 
responds to over the 
counter treatment 
Regular 
over 
the 
counter or occasional 
prescription 
treatment 
needed 
Regular 
prescription 
treatment needed 
Hospitalisation 
Nausea 
Intake maintained 
Intake 
reduced 
less 
than 3 days 
Minimal intake 3 days 
or more 
Hospitalisation 
Nausea/vomiting  
Less than 4 x a day or 
lasting less than 1 
week 
At least 4 x day or 
lasting 1 week or more 
Unable to keep any 
food or fluids down 
Hospitalisation 
 
 
 
 
 
CUTANEOUS 
Discomfort/pain in injected 
muscle (including ache) or 
overlying skin 
Mild itch or ache that 
responds to over the 
counter treatment, if 
needed 
Pain requiring regular 
over 
the 
counter 
treatment or occasional 
prescription treatment  
Pain requiring regular 
prescription treatment 
Hospitalisation 
Immediate 
reactions 
(within 
6 
hours 
of 
injection) 
Symptoms of irritation 
locally (usually itching 
at the injection site) 
OR 
Erythema +/- swelling 
at the injection site 
 
 
 
 
 
Laryngeal oedema 
insufficient to require 
intubation; diarrhoea 
insufficient to require 
IV fluids, or asthma 
nsufficient to require 
hospitalisation 
OR Urticaria, angio-
oedema OR 
Generalised pruritus 
Anaphylactic shock 
Erythema at injection site 
 
Erythema up to and 
including 50% of 
baseline arm 
circumference 
OR 
Symptoms of irritation 
that are easily 
tolerated and do not 
require repeated 
medication 
OR 
Both 
Erythema greater than 
50% of the arm 
circumference at 
baseline With or without 
Symptoms of irritation 
that do not require 
repeated medication 
OR 
Symptoms of irritation 
that require repeated 
medication AND 
erythema up to and 
including 50% 
Erythema greater 
than 50% of the arm 
circumference at 
baseline AND 
symptoms of irritation 
requiring repeated 
medication 
Hospitalisation 
Blistering or ulceration at 
injection site 
 
Fluid filled vesicles or 
superficial 
disruption 
of epithelium covering 
an area < 1cm 
Fluid filled vesicles or 
superficial disruption of 
epithelium, area 1 - 
2cm 
OR 
Blood filled vesicles  
OR 
Full thickness disruption 
of 
epithelium 
healed 
within 2 weeks 
Full thickness 
disruption of 
epithelium not healed 
within 2 weeks 
Necrosis 
Soft swelling – local 
 
Swelling <25% of arm  
Swelling 25-50%of arm  
Swelling >50% of 
arm  
Or 
Induration/hardened 
swelling  (when 
considered by the 
 

CUTHIVAC002 
 
 
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10_APR_2017 
 
 
72
clinician to be 
associated with a 
process arising in the 
muscle)