An Experimental Medicine Study Modelling the Interaction Between Rationally-designed Synthetic Model Viral Protein Immunogens and the Breadth of the Induced B and T Cell Repertoires.
Phase: PHASE1
Conditions: HIV-1-infection
Sponsor: Imperial College London

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Study Protocol Title  
An experimental medicine study modelling the interaction between rationally-designed 
synthetic model viral protein immunogens and the breadth of the induced B and T cell 
repertoires. 
 
NCT Number: 
NCT03816137 
 
Protocol Version: 
10.0 
 
Protocol Date: 
14 Nov 2022 
 
 
 

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Study Protocol Title  
An experimental medicine study modelling the interaction between rationally-designed 
synthetic model viral protein immunogens and the breadth of the induced B and T cell 
repertoires. 
 
 
Investigators 
Dr Katrina Pollock – Chief Investigator: 
Clinical activities will be carried out at the NIHR 
Imperial CRF, Hammersmith Hospital, Imperial College Healthcare NHS Trust. 
Professor Robin Shattock – Scientific Co-Investigator: 
Immunogenicity 
laboratory 
investigations will be performed in the Department of Infection and Immunity at Imperial 
College London. 
 
Sponsor 
Imperial College London will act as the main Sponsor for this study. Delegated responsibilities 
will be assigned to Imperial College Healthcare NHS Trust as necessary.  
 
Manufacturer of Immunogens 
Polymun Scientific 
Immunbiologische Forschung GmbH 
Donaustr 
99, 3400 Klosterneuburg 
Austria 
www.polymun.com 
 
 
Protocol Version: 
10.0 
Protocol Date: 
14 Nov 2022 
 
 
 
THE CONFIDENTIAL INFORMATION IN THIS DOCUMENT IS PROVIDED TO YOU AS AN INVESTIGATOR, 
POTENTIAL INVESTIGATOR, OR CONSULTANT, FOR REVIEW BY YOU, YOUR STAFF, AND APPLICABLE 
INSTITUTIONAL REVIEW BOARDS (IRBs) AND/OR INDEPENDENT ETHICS COMMITTEES (IECs). IT IS 
UNDERSTOOD THAT THE INFORMATION WILL NOT BE DISCLOSED TO OTHERS, EXCEPT TO THE EXTENT 
NECESSARY TO OBTAIN ETHICAL AND REGULATORY APPROVAL FROM THE RESPECTIVE COMMITTEE’S 
AGENCIES AND INFORMED CONSENT FROM THOSE PERSONS TO WHOM THE IMMUNOGENS MAY BE 
ADMINISTERED. 
 
 

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Background 
One of the most effective arms of the human immune system is the ability of very low 
concentrations of antibody proteins to bind to viruses, bacteria and toxins and “neutralise” 
their activity or ability to infect. In contrast to cellular immunity, which may cause tissue 
destruction and pathology, antibody-mediated immunity can be very passive, while 
completely preventing infection. How antibodies bind their targets varies enormously, 
ranging from unhelpful “blocking” antibodies or narrowly focussed neutralising antibodies, to 
highly protective “broadly neutralising” antibodies (bNAbs) that can neutralise a wide range 
of strains of the same pathogen. Such bNAbs are especially sought after in virus infections 
such as HIV, influenza and others where the virus mutates to evade immune responses that 
are too narrow or focussed. Antibodies arise when an “immunogen” (an immunogen is 
anything that induces an immune response, typically a foreign protein) is taken up by the 
immune system and shown to white blood cells – T and B cells - by specialised immune cells. 
In some cases the T and B cells bind the immunogens to receptors on their surface, triggering 
an immune response in which T cells “help” B cells to manufacture specific antibodies. The 
events around how the protein is processed into manageable pieces, shown to the T and B 
cells, and the pattern of chemical signals produced by the immune cells is highly complex, but 
eventually determines how broad the antibody response will be (its breadth). For infections 
like HIV and influenza, decades of research and clinical vaccine trials have had limited or no 
success. To take HIV as an example, we have an almost complete lack of understanding of 
how immunogens interact with the naive human B cell receptor (BCR) repertoire and the 
pathways required to induce bNAbs during an infection or after an immunisation. Animal 
models have failed as the naïve, germline encoded, B cell antibody receptor repertoires of 
non-human species are sufficiently different from those of humans to render design and 
selection of vaccines based on non-human species problematic. Additionally, bNAbs isolated 
from HIV-1-infected individuals have structural features that occur rarely or not at all in other 
mammals, such as unusually long loop-binding regions (CDRH3 loops) required to penetrate 
past glycans on the surface of the envelope spike that shield key neutralising epitopes 
(Mascola JR 2013). There is therefore a critical need to better understand, in human 
experimental medicine models of immune challenge, how immunogens and B/T cells interact 
in the development of protective bNAb anti-viral responses. 
Our approach to resolving this impasse is to challenge the human immune system with 
rationally-designed model immunogens to determine the structural and other characteristics 
required to drive human B cell antibody responses towards neutralisation breadth. We have 
selected HIV as an experimental model as there is a reasonable understanding about the 
specificity and function of anti-HIV bNAbs, as well as an urgent need to identify novel 
immunisation approaches following decades of failed or poorly successful trials. There is also 
a huge database of safety using HIV proteins as immunogens, and the technological expertise 
to design and manufacture HIV viral proteins. Assays for HIV neutralising activity are also well 
established in our laboratories. Although focussed on HIV, our findings will be applicable to 
other viral infections. 

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The model immunogens we propose to use in these experimental medicine studies are 
unlikely to be suitable as vaccines, and any clinical development would require iterative cycles 
of design refinement and development based on immunological insights gleaned from these 
experimental investigations. Therefore, the focus is on in-depth characterisation of the 
elicited immune response to rationally-designed model immunogens that may inform the 
design process of actual vaccines. This experimental medicine approach is only now possible 
due to unprecedented progress in our abilities to study the human immune system and to 
obtain complete information on immune responses to vaccination, since performing research 
on the human immune system is now almost as easy as it has been in mice. The main focus 
of this study will be to determine which of the design strategies is able to prime human 
germline (naive) B cells and drive antibody responses towards induction of neutralising 
antibody breadth. 
Our range of model immunogens will be based on the envelope (Env) glycoprotein of HIV-1, 
which is the only target of neutralising antibodies, and therefore the only virally-encoded 
immunogen relevant for induction of such antibodies by immunisation. To ensure 
reproducibility of results and the highest level of volunteer safety, all immunogens will be 
manufactured under cGMP, using techniques applied to vaccine immunogens. 
Env has extensive amino acid variation, structural and conformational instability, and 
immunodominance of hypervariable regions (Kwong PD, 2011; Sattentau QJ, 2013). We will 
design soluble immunogens that closely mimic the native viral trimer in situ, but that 
incorporate design strategies that may alter the intrinsic viral immune evasion mechanisms 
(Sanders RW, 2013).  Env is made up of three identical complexes (trimers) each of which 
contains two molecules, gp120 and gp41 that can be modified to make a soluble molecule 
called gp140, upon which our immunogens are based.  We have developed model consensus 
gp140 Env trimers (consensus of all global strains) designed to prime B cell responses to 
common epitopes represented in all HIV-1 subtypes. We have utilised two design strategies 
to stabilise these in a native-like conformation: ConM SOSIP and ConS UFO. The ConM SOSIP 
trimer includes novel mutations that include the incorporation of a disulphide linkage 
between the gp120 and gp41 ectodomain (making up gp140) which prevents their 
disassociation into monomer subunits.  
The ConS UFO includes a short flexible amino-acid linker to tether the gp120 and gp41 
subunits together as an alternative strategy to prevent dissociation of the Env trimer. We wish 
to test both designs to determine the effect on B cell repertoire. To further stabilize global 
architecture we will employ an EDC crosslinking approach that has been shown to conserve 
bNAb epitopes, reduce non-antiviral antibody responses, and enhance overall immunogen 
stability (Schiffner et al., 2015). Thus, in Part 1 of this study we will test EDC ConM SOSIP and 
EDC ConS UFO versions in parallel.  
A critical adjunct to our consensus-based model design is to use cocktails of mosaic gp140 Env 
trimers designed to overcome the immunodominance of hypervariable regions of Env and to 
determine whether they will focus antibody responses towards conserved neutralisation 
epitopes. While designed using computer algorithms, these mosaics represent authentic Env 

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structures that are fully functional and native in their conformation. Our novel designs aim to 
eliminate unwanted immunodominant antibody responses and focus B cells towards highly 
conserved supersites of vulnerability on Env, with particular emphasis on quaternary bNAb 
epitopes (Julien, JP, 2013; Kong L, 2013; Lyumkis D, 2013). Like the ConM SOPSIP and ConS 
UFO trimers described above, the mosaic trimers have disulphide linkage which prevents 
disassociation of gp120 and gp41 into monomer subunits. 
In Groups A–E of this study, we will prime with one of the consensus immunogens (ConM or 
ConS), and boost with a cocktail of two mosaic immunogens (Mos3.1 and Mos3.2). 
In Groups F–I of this study, we will explore the use of three mosaic immunogens (Mos3.1, 
Mos3.2 and Mos3.3) used sequentially (in a series of different orders), or as a cocktail, to 
focus B cell responses towards conserved areas of Env. To amplify these responses we intend 
to give a final boosting immunization with both consensus immunogens (ConM and ConS). 
In Groups J–M we will use two HIV-1 envelope sequences (AMC011 and 763 SOSIP) which the 
EAVI2020 programme has found to be associated with the early induction of broadly 
neutralising antibodies. The sequences were identified from two HIV infected individuals that 
displayed HIV neutralisation breadth early in infection (less than 12 months). This is unusual 
as typically neutralisation breadth is only seen to develop in <5% of subjects and only after a 
period of more than 2 years. We seek to test the hypothesis that these two envelope 
sequences may display unique potential to prime and/or drive the induction of broadly 
neutralising antibody responses in HIV negative healthy volunteers. To test this hypothesis 
the EAVI2020 consortium has manufactured stabilised versions of these two envelope 
proteins to GMP. These will be used alone and in various combinations with consensus and 
mosaic immunogens to determine the extent to which they can drive the induction of broadly 
neutralising antibodies. Responses to these unique envelope sequences will be compared and 
contrasted to the consensus and mosaic immunogens evaluated in Groups A–I.  
The extent to which these different strategies may induce neutralising breadth, and the 
identification of the mechanisms and drivers involved, can only be determined empirically 
through human immunogen challenge studies. The results from these studies will help inform 
the future direction of additional refinement of HIV vaccine strategies for the induction of 
protective antibodies. 
 
Objectives 
The objective of this experimental medicine study is to determine the extent to which 
different prime-boost combinations influence serum neutralising antibody breadth and 
associated B and T cells responses. 
We hypothesise that the different prime-boost model immunogen combinations will have 
differential impact on: 
a) the magnitude and breadth of induced serum neutralising antibodies; 

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and 
b) the induced B- and T-cell responses in peripheral blood  
We will investigate this by challenging the immune system of healthy adults with various 
model immunogens based on HIV-1 Env (ConM and ConS, with and without EDC stabilisation; 
Mos3.1, Mos3.2 and Mos3.3, and AMC011 and 763 SOSIP) in different prime-boost 
combinations.  
In Groups A–I we will investigate the impact of a later immunisation challenge with either 
consensus or mosaic gp140 Envs to determine any additional effect on breadth of antibody 
neutralisation. 
The primary outcome will be measurement of specific viral neutralisation activity of serum 
antibodies. Exploratory outcomes will include titres of binding antibodies in serum and the 
characterisation of blood B and T cell responses. 
Data derived from these studies will help to inform how the structure of rationally-designed 
immunogens drives the immune system towards developing broadly neutralising antibodies 
that are rarely induced under natural conditions. 
 
Primary Outcomes 
 Serum titres of neutralising antibodies to virus expressing ConM and ConS envelopes 
 Serum titres of neutralising antibodies to virus expressing Mosaic envelopes (Mos3.1, 
Mos3.2 and Mos3.3) 
 Serum titres of neutralising antibodies to virus expressing AMC011 and 763 SOSIP 
envelopes 
 
 
Exploratory Outcomes 
 These will include: measurement of binding antibodies in serum; assessment of 
neutralisation breadth using a wider panel of globally circulating HIV strains; 
measurement of serum antibody function (ADCC, ADCP, FcR dimer binding); 
measurement of the magnitude and phenotype of B cell (plasmablast and memory B 
cells) and T cell responses (CD4 and CD8) in peripheral blood mononuclear cells 
(PBMC). Some endpoints will include only a subset of enrolled volunteers. Paxgene 
tubes for blood transcriptomics will be collected only from group A (see Table 2) 
because it is not expected that responses will differ at this level between groups. Blood 
transcriptomic profiles induced by immunisation will be correlated with the 
magnitude of induced serum antibody response.  
 

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Fine needle aspiration 
 In addition, the feasibility and safety of lymph node aspiration will be explored, and  
immunogen induced germinal centre B and T cell responses will be assessed, in 
volunteers from groups B-M willing to attend additional visits to have an ultrasound 
guided fine needle lymph node aspiration from axillary, cervical or inguinal lymph 
nodes.  Fine needle aspiration biopsies will occur in line with the sub-study protocol 
in Appendix 1.  
 This will be optional and participants will be provided with a separate fine needle 
aspiration information sheet and consent form. The participants will be reviewed by 
the medical team for any contraindications to fine needle aspiration prior to the 
procedure being carried out.  
 An additional blood sample of 42 ml of peripheral blood will be collected at each fine 
needle aspiration visit to compare the T- and B-cell populations by flow cytometry and 
to compare serum neutralising antibody activity.  
 
Statistics and Sample Size 
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, and 
the large number of experimental parameters in this experimental study. The sample size is 
based on the previous studies of HIV-1 gp140 immunogens (Joseph S, 2017, Cosgrove CA, 
2016, Kratochvil S, 2017, Lewis DJ, 2011). It is not the remit of this exploratory study to recruit 
a sufficient number of volunteers to be statistically confident about the differences between 
groups.  By the end of this study, 8–10 volunteers will have been exposed to each schedule 
and this provides confidence around the response/event proportions of 0–100% in Table 1. 
 
Table 1: 
Sample Size 
    Number of “responders” 
Proportion if n=10 
95% confidence 
interval1 
0 
0% 
0 – 31% 
2 
20% 
3 – 56% 
4 
40% 
12 – 74% 
6 
60% 
26 – 88% 
8 
80% 
44 – 98% 
10 
100% 
69 – 100% 
Number 
of 
“responders” 
Proportion if n=8 
95% 
confidence 
interval2 
0 
1 
2 
3 
4 
0% 
12.5% 
25% 
37.5% 
50% 
0–32% 
2–47% 
7–59% 
14–69% 
22–78% 

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5 
6 
8 
62.5% 
75% 
100% 
31–86% 
41–93% 
68–100% 
1, 2 Clopper-Pearson or Wilson method (suitable for small sample sizes) 
 
Number (percent) of responders, and median [IQR] and mean (range) of titre responses will 
be tabulated by group. Group comparisons of titre levels will be conducted via Kruskal-Wallis 
test or Wilson method suitable for small sample sizes. 
 
 
Volunteers & Withdrawal Criteria 
We will recruit 114 healthy male and females adults into five groups of 10 each (A-E) and eight 
groups of 8 each (F–M), see Table 2.  Recruitment will select potential volunteers who are 
willing to undergo serial blood draws. Health status will be determined at screening which 
will comprise a medical history, symptom-directed physical examination, vital signs, urine 
pregnancy test (women only), HIV screen, and routine safety blood draws (full blood count; 
renal, liver and bone panels).  
Additional volunteers may be enrolled to replace early withdrawals.  The reason(s) for 
withdrawal will be recorded in volunteer’s files. 
All volunteers are free to withdraw from the study at any time, for any reason, without 
affecting their future medical care. The PI may decide to withdraw a volunteer if the 
investigator deems that continuing might compromise the volunteer’s wellbeing or interfere 
with the achievement of the study’s objectives. 
 

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Table 2:  
Schedule of immunisations and doses of model immunogens 
 
Group 
 
N 
Months 
0 
3 
6 
12 
Part 1 
Part 2 
A 
10 
ConM SOSIP 
100 g 
ConM SOSIP 
100 g 
ConM SOSIP 
100 g 
Mos3.1 + Mos3.2 
100ug (2x50ug) 
B 
10 
EDC ConM SOSIP 
100 g 
EDC ConM SOSIP 
100 g 
EDC ConM SOSIP 
100 g 
Mos3.1 + Mos3.2 
100ug (2x50ug) 
C 
10 
ConS UFO 
100 g 
ConS UFO 
100 g 
ConS UFO 
100 g 
Mos3.1 + Mos3.2 
100ug (2x50ug) 
D 
10 
EDC ConS UFO 
100 g 
EDC ConS UFO 
100 g 
EDC ConS UFO 
100 g 
Mos3.1 + Mos3.2 
100ug (2x50ug) 
E 
10 
ConS UFO 
100 g 
ConS UFO 
100 g 
ConM SOSIP 
100 g 
Mos3.1 + Mos3.2 
100ug (2x50ug) 
 
Group 
 
N 
Months 
0 
2 
4 
6 
Part 1 
Part 2 
F 
8 
Mos3.1 
100 g 
Mos3.2 
100 g 
Mos3.3 
100 g 
ConM SOSIP + 
ConS UFO 
50 µg + 50 µg 
G 
8 
Mos3.2 
100 g 
Mos3.1 
100 g 
Mos3.3 
100 g 
ConM SOSIP + 
ConS UFO 
50 µg + 50 µg 
H 
8 
Mos3.3 
100 g 
Mos3.2 
100 g 
Mos3.1 
100 g 
ConM SOSIP + 
ConS UFO 
50 µg + 50 µg 
I 
8 
Mos3.1 
33 µg 
Mos3.2 
33 µg 
Mos3.3 
33 µg 
Mos3.1 
33 µg 
Mos3.2 
33 µg 
Mos3.3 
33 µg 
Mos3.1 
33 µg 
Mos3.2 
33 µg 
Mos3.3 
33 µg 
ConM SOSIP + 
ConS UFO 
50 µg + 50 µg 
Note: Participant 060, in Group F, received (in error) Mos3.3 100 µg at Month 2. She may continue to be 
challenged if she receives Mos3.2 100 µg at Month 4, and is challenged at Month 6 per the table above. 
 
 

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Table continues overleafTable 2 (continued):  
Schedule of immunisations and doses of 
model immunogens 
 
Group 
 
N 
 
Month 
 
 
0 
2 
4 
 
J 
 
8 
 
763 SOSIP 
100 µg 
 
763 SOSIP 
100 µg 
 
763 SOSIP 
100 µg 
 
K 
 
8 
 
AMC011 
100 µg 
 
AMC011 
100 µg 
 
AMC011 
100 µg 
 
L 
 
8 
763 SOSIP 
50 µg 
+ 
AMC011 SOSIP 
50 µg 
763 SOSIP 
50 µg 
+ 
AMC011 SOSIP 
50 µg 
 
763 SOSIP 
50 µg 
+ 
AMC011 SOSIP 
50 µg 
 
M 
 
8 
763 SOSIP 
AMC011 
ConM SOSIP 
Mos3.1 
Mos3.2 
(each at 20 µg) 
 
763 SOSIP 
AMC011 
ConM SOSIP 
Mos3.1 
Mos3.2 
(each at 20 µg) 
 
763 SOSIP 
AMC011 
ConM SOSIP 
Mos3.1 
Mos3.2 
(each at 20 µg) 
 
 
 
Inclusion Criteria 
1. Healthy male and female volunteers aged between 18 and 55 years.  
2. Available for ALL follow-up visits for the duration of the study.  
3. Entered and clearance obtained from The Over volunteering Prevention System (TOPS) 
database (to avoid impact of any co-administered investigational products or treatments 
on our outcomes). 
4. Women of childbearingi potential willing to use a highly effective method of 
contraceptionii for the duration of the study until a minimum of 12 weeks after the final 
injection. Periodic abstinence (calendar, symptothermal and post-ovulation methods) and 
withdrawal are not acceptable methods of contraception. 
5. Willing and able to give written informed consent. 
i 
A woman will be considered of childbearing potential following menarche and until becoming post-menopausal 
(no menses for 12 months without an alternative medical cause) unless permanently sterile. Permanent sterilisation 
methods include hysterectomy, bilateral salpingectomy and bilateral oophorectomy. A post-menopausal state is 
defined as no menses for 12 months without an alternative medical cause. 

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ii  
The following methods are considered highly effective: 
 
combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of 
ovulation – oral, intravaginal or transdermal; 
 
progestogen-only hormonal contraception associated with inhibition of ovulation – oral, injectable or 
implantable 
 
intrauterine device (IUD); 
 
intrauterine hormone-releasing system (IUS); 
 
bilateral tubal occlusion; 
 
vasectomised partner, where the vasectomised partner has received medical assessment of the surgical 
success; and 
 
sexual abstinence, defined as refraining from heterosexual intercourse – must be the preferred and usual 
lifestyle of the participant. 
 
Exclusion Criteria 
1. History of any medical, psychological or other condition, clinically significant laboratory 
result at screening, or use of any medications which, in the opinion of the investigators, 
would interfere with the study objectives or volunteers safety. 
2. Any history of angioedema 
3. History of urticaria deemed significant by the Chief Investigator 
4. HIV-1 or HIV-2 antibody positive or indeterminate upon screening, or history of receipt of 
Env-based HIV immunogens (which would render the volunteers non-naive to the model 
immunogens). 
5. Unable to read and/or speak English to a fluency level adequate for the full 
comprehension of study procedures and consent. 
 
Recruitment & Randomisation 
This is a single-blind study with volunteers being randomised into five groups of n=10 (Groups 
A-E) and eight groups of n=8 (Groups F–M), stratified by gender.  Volunteers will be blinded 
to their treatment regimes, and laboratory teams undertaking immunological analysis will be 
blinded to group and dosing regimen to prevent in-house analysis bias. The clinical team will 
remain un-blinded throughout.    
Volunteers will be recruited from the general population in London and the surrounding areas 
by means of paper and electronic advertising. We will also use a Healthy Volunteer database 
held at the Imperial Clinical Research Facility.  We will register volunteers as patients of 
Imperial College Healthcare NHS Trust before they are consented into the study. Volunteers 
will also be informed at screening and during the course of the experimental study protocol 
that the immune challenges will not provide protection against HIV acquisition, and that 
normal HIV prevention strategies should be maintained. 
Once all inclusion/exclusion criteria have been met, eligible volunteers will be enrolled into 
the study and randomly assigned into one of the nine treatment groups.  A study ID will be 
generated once randomisation has been completed. 
 
 

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Consent 
Signed written consent to enter the study must be obtained from each volunteer only after a 
full explanation has been given, an information leaflet provided, and at least 24-48 hours 
allowed for consideration. The right of the volunteer to refuse to participate and to withdraw 
consent at any time from the protocol without giving reasons will be respected. 
 
Model Immunogens 
Model immunogens will be obtained from Polymun Scientific, Austria, and stored in 
temperature controlled conditions, on-site at the Imperial CRF before administration.  To 
ensure reproducibility of results and the highest level of volunteer safety, all immunogens will 
be manufactured under cGMP, using techniques and specifications applied to vaccines. 
Recombinant HIV-1 trimeric gp140 Env proteins: ConM SOSIP gp140, ConS UFO gp140, and 
chemically fixed versions: EDC-ConM SOSIP and EDC-ConS UFO; the mosaic immunogens: 
Mos3.1, Mos3.2 and Mos3.3; and 763 SOSIP and AMC011, manufactured to EU cGMP quality 
standards by Polymun Scientific GmbH (Austria) will be mixed with adjuvant: MPLA liposomes 
manufactured to EU GMP quality standards by Polymun Scientific. All model immunogens will 
be used at the dosage of 100 g and will be admixed with 500 g MPLA formulated in 
liposomes and administered by intramuscular injection into the left or right deltoid muscle.   
 
Immunogens, adjuvant and dose justification 
Env glycoprotein and adjuvant vaccines have been administered without concerning adverse 
effect in numerous studies worldwide over many years.  Extensive safety data for the use of 
monomeric recombinant gp120 HIV-1 Env glycoprotein vaccines include the large Phase III 
Vaxgen and RV144 trials - albeit formulated with different adjuvants (Andersson et al. 2011; 
Su et al. 2000; UNIADS Report 2013). In the RV144 trial, the alum adjuvanted gp120 protein 
(AIDSVAX B/E) was injected along with ALVAC (canarypox expressing gag pro and Env genes) 
after two priming immunisations with ALVAC alone. There were no safety concerns in that 
trial of over 16,000 healthy volunteers in Thailand. 
Previous work by our own group, and others internationally, have used recombinant native 
HIV-1 Env glycoprotein gp140 in a number of CTIMPs, administered intramuscularly (I.M.) at 
the dose of 100μg adjuvanted with GLA-AF, an alternative MPLA (non-liposomal) formulation, 
as well as with other adjuvants such as alum and MF59. The dose of model gp140 
immunogens we propose to use in this protocol is therefore identical to the dose of native 
gp140 (100µg) used in three previous UK clinical trials: MUCOVAC2 (EudraCT 2010-019103-
27; Cosgrove CA, 2016); UKHVC Spoke 003 (EudraCT 2012-003277-26; Joseph S, 2017); X001 
(EudraCT 2013-001032-22; Kratochvil S, 2017). An intradermal dose of 50ug was acceptable 
when used in CUTHIVAC002 (EudraCT 2015-001023-23; Cheeseman HM, 2018). The proposed 
dose of native gp140 was safe, and volunteers made good binding antibody responses to the 
recombinant gp140, although very limited levels of neutralising antibodies. These previous 

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studies were performed with a single recombinant HIV-1 clade C trimeric gp140 Env 
glycoprotein derived from a Chinese viral isolate 97CN54. Additional studies have been 
performed with gp140 proteins at this dose (100 µg). A South African subtype C strain TV1 
trimeric gp140 protein (100 µg) combined with MF59 adjuvant was safely administered to 
114 volunteers primed with DNA/MVA (Churchyard G, 2016); a purified trimeric gp140 Env 
derived from the subtype B strain SF162 (100 µg) in MF59 was administered to 80 subjects 
with or without DNA priming (Spearman P, 2011); and a subtype D strain UG92005 trimeric 
gp140 Env (100 µg) combined with 500 µg alum was given twice to 9 volunteers (Hurwitz JL, 
2008).  
To adjuvant the protein this study will use MPLA in liposomal suspension. MPLA has been 
widely used in liposomal adjuvants for over 20 years (Alving CR, 2011). When given 
intramuscularly no systemic toxicity has been associated with doses up to 2200 µg (Fries LF 
1992, Heppner DG, 1996, Alving CR, 2011). We have selected a dose of 500 µg as optimal 
based on non-human primate immunogenicity studies of gp140, in which there was no 
toxicity. 
We also commissioned GLP-compliant preclinical toxicology testing in rabbits (RTC STUDY NO. 
A3063, Research Toxicology Centre S.p.A., Italy)  to mimic the human challenge studies, in 
which repeated injections of ConM SOSIP, EDC-ConM SOSIP, ConS UFO and EDC-ConS UFO 
with MPLA, and of ConM SOSIP and MPLA alone, were administered into the lateral surface 
of the quadriceps muscle of both legs. No relevant clinical signs were observed, and no 
reaction at injection sites was observed in any animal treated with the test item during the 
study.  In this model the challenge agents and schedule did not induce adverse effects at 
systemic or local levels.  
 
Reporting Procedures 
Any adverse events will be solicited at each attendance, reviewed by clinicians and recorded. 
In addition, participants will be asked to complete a symptom diary on the evening after each 
immunogen challenge, and every evening for 6 days thereafter, and entries will be reviewed 
by clinicians when they visit the investigator site. Any serious adverse events should be 
reported to the Sponsor and Research Ethics Committee where in the opinion of the Chief 
Investigator, the event was:  ‘related’, i.e. resulted from the administration of any of the 
research procedures; and ‘unexpected’, i.e. an event that is not listed in the protocol as an 
expected occurrence. Reports of related and unexpected SAEs should be submitted within 15 
days of the Chief Investigator becoming aware of the event, using the NRES SAE form for non-
IMP studies.  
 
Possible Adverse Reactions and Adverse Events monitoring 
As immediate anaphylaxis may rarely be associated with immunogen challenge (less than 1 
in 1 million doses) volunteers will remain in the investigation unit for at least one hour after 

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immunogen challenge. The symptom diary completed by participants after each immunogen 
challenge will support the completeness and accuracy of adverse events recording at each 
attendance. 
No serious adverse reactions are anticipated given the wide use of recombinant HIV-1 Env 
proteins in previous studies, and safe record of using MPLA as a common adjuvant. Various 
local, non-serious adverse reactions may be anticipated after immunogen challenge, 
expected to be of short duration, resolving within 1-7 days. These include pain, tenderness, 
erythema/discolouration, warmth, itching and swelling at the site of injection. Less frequent 
systemic non-serious adverse reactions associated with immunogen challenge may also occur 
and include: raised temperature, chills/rigors, myalgia/flu-like general muscle aches, malaise, 
excess fatigue, headache, nausea/vomiting; and, very rarely abdominal pain, diarrhoea 
and/or sore throat. These are also anticipated to resolve within a few days (<7 days). 
Adverse events associated with lymph node sampling are described in the sub-study protocol, 
in Appendix 1. 
It will be made clear to volunteers that they will be free to withdraw from the study at any 
time without providing a reason.  Any significant, severe adverse reactions will lead to 
withdrawal of the individual and any serious adverse reactions will terminate the whole study. 
Volunteers will be provided with 24 hour emergency contact numbers and clear instructions 
to call if they feel unwell following immunogen challenge.   
 
Schedule of visits, immunogen challenges, procedures and samples 
Volunteers will be screened, have their first immunogen challenge and will be followed for 7 
months (Groups J-M), 9 months (Groups F–I) or 13 months (Groups A–E) thereafter. It is 
expected to take approximately 12 months to enrol 114 volunteers.  
Volunteers will be enrolled in the groups described in Table 2 and blood sampling will be 
carried out at specific time points during the study (see Table 3).   
 
Avoidance of pregnancy during the study 
Women of childbearing potential are recommended to use highly effective contraception 
for the duration of the study until a minimum of 12 weeks after the final injection. The 
immunogen challenge contains 500 µg of MPLA, which is similar to the 50 µg of 3-O-desacyl-
4'-monophosphoryl lipid A in the licensed vaccine against Hepatitis B, Fendrix (GSK). The 
summary of medicinal product characteristics (SmPC) for this product states that although 
there are no data from the use of Fendrix in pregnant women, animal studies do not 
indicate direct or indirect harmful effects with respect to pregnancy, embryonal/foetal 
development, parturition or postnatal development. 
(https://www.medicines.org.uk/emc/product/137/smpc). The authorised product Cervarix 
(GSK) also contains MPLA. During clinical development 5,387 women who had received 

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Cervarix reported pregnancy. There was no evidence of increased risk to the pregnancy in 
these women (https://www.medicines.org.uk/emc/product/1177/smpc#gref). 
 
Study Management 
The day-to-day management of the study will be co-ordinated through Dr Katrina Pollock, the 
Chief Investigator, and the study team at the Hammersmith Hospital (clinical) and Imperial 
College London's St Mary’s Campus (laboratory).  Study management decisions will be 
discussed with the wider clinical team, and authorised by the Chief Investigator and Scientific 
Co-Investigator. 
A Data Safety Monitoring Committee will be formed and will meet to review the accumulated 
safety data after approximately half of the total number of immunogen challenges have been 
administered. The DSMC will be asked to make a recommendation such as: continuation of 
the study with no proposed changes to the study protocol; or early termination due to 
adverse reactions; or amendment of the study protocol. After the initial review meeting the 
DMSC will be sent periodic safety reports, approximately every 3 months, and all SAR reports. 
Meetings will be scheduled ad hoc, at the request of the CI or DMSC members. Further details 
are in the DSMC Charter. 
Reimbursement of Volunteers 
Volunteers will be paid for their time and effort to cover their travel expenses to the study 
site and any inconvenience caused due to study participation.  Volunteers will receive £100 
per completed study visit, and a £100 bonus if they attend all study visits on time and comply 
with all study requirements, as a lump sum at the end of their participation.   

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Table 3: 
Schedule of Study Events – Groups A–E 
Part 1 
Part 2 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 13 
14 
15 
16 
17 
 
19 
20 
21 
Visit Month 
M0 
 
 
 
 
M3 
 
 
 
 
M6 
 
 
 
 
 
M12 
 
 
 
 
Visit Week 
0 
0 
1 
2 
4 
12 
12 13 14 16 
24 
24 25 
26 
28 
36 
48 
 
49 
50 
52 
Visit Day 
0 
1 
7 
14 
28 
84 
85 91 98 112 
168 
169 175 182 196 252 
336 
 
343 
350 
364 
Visit Windows 
(Days) 
Screening 
0 
0 
 1  2  2 -7/+14 0 
 1  2  2 -7/+14 0 
 1  1  2  2 -7/+14 
 
 1 
 2 
 2 
Immunogen 
challenge(1) 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history/ 
demographics / 
height and weight 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Symptom-directed 
physical 
examination(2) 
 
X 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X) 
 
(X)  
 
(X) 
 
(X) 
 
 
(X) 
 
(X) 
 
X 
Vital Signs(3)  
X 
X 
 
X 
 
 
X 
 
X 
 
 
X 
 
X 
 
 
 
X 
 
X 
 
 
Full blood count, 
serum renal, liver, 
bone panels & HIV-1 
& 2 antibodies7 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X7 
Provide symptom 
diary 
 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
Review symptom 
diary 
 
 
X 
X 
 
 
 
X 
X 
 
 
 
X 
X 
 
 
 
 
 
X 
 
 
Urine Pregnancy 
Test (Women) 
X 
X 
 
 
 
 
X 
 
 
 
 
X 
 
 
 
 
 
X 
 
 
 
 
Blood for Serum – 
Immunogenicity 
 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
 
X 
X 
X 
X 
X 
 
X 
X 
X 
Blood for PBMC – 
Immunogenicity 
(*Group A only) 
 
X 
X* 
X 
 
 
X 
X* 
X 
 
 
X 
X* 
X 
X 
X 
X 
X 
 
X 
X 
X 
Blood RNA 
(Paxgene) 
(*Group A only) 
 
 
X* 
 
X* 
 
X* 
 
 
 
X* 
 
X* 
 
X* 
 
 
 
X* 
 
X* 
 
X* 
 
 
 
 
 
 
 
 
Lymph Node 
Aspirate 
(**Group B – E only) 
 
X**,
4 
 
 
X**,5 
 
 
 
 
X**
,6 
 
 
 
 
 
 
 
 
 
 
 
 
1 The second, third and fourth immunogen challenges (visits 6, 11 and 17) can be shifted by -7 or up to +14 days. 
Subsequent visits will be shifted by the same number of days to ensure the interval to subsequent visits are maintained   
2Symptom-directed physical examination maybe preformed at the discretion of the Chief Investigator/delegate  
3At screening and before immunogen challenge, and at 60 (+/-15) min and 7 (+/-1) days post-challenge 
4 FNA sample window 1:  from enrolment  to Day 0 (prior to  first  immunogen challenge).  
5FNA sample window 2: Day 7 to Day 84. Aim for FNA Samples to be obtained at Day 14 (2 weeks after first immunogen 
challenge) 
6FNA sample window 3: Day 85 to Day 364 (1 to 280 days post second challenge). Aim for FNA samples to be obtained 
at Day 98 (2 weeks after second immunogen challenge). If a participant has two sampling occasions during this window, 
the samplings should be at least 7 days apart. 
7HIV antibodies at screening only 
*Group A only  
**Group B-E only 
 

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Table 3 continued: 
Schedule of Study Events – Groups F–I 
 
Part 1 
Part 2 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
14 
15 
16 
17 
Visit Month 
M0 
 
 
 
M2 
 
 
 
M4 
 
 
 
M6 
 
 
 
M9 
Visit Week 
0 
1 
2 
4 
8 
9 
10 
12 
16 
17 
18 
20 
24 
25 
26 
28 
36 
Visit Day 
0 
7 
14 
28 
56 
63 
70 
84 
112 
119 
126 
140 
168 
175 
182 
196 
252 
Visit Windows 
(Days) 
Screening 
0 
 1 
 2 
 2 
-7/+14 
 1 
 2 
 2 
-7/+14 
 1 
 2 
 2 
-7/+14 
 1 
 2 
 2 
-7/+14 
Immunogen 
challenge(1) 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history/ 
demographics / 
height and weight 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Symptom-directed 
physical 
examination(2) 
X 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
X 
Vital Signs(3)  
X 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
 
Full blood count, 
serum renal, liver, 
bone panels & HIV-1 
& 2 antibodies4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
X4 
Provide symptom 
diary 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Review symptom 
diary 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
Urine Pregnancy 
Test (Women) 
X 
X 
 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Blood for Serum – 
Immunogenicity 
 
X 
 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Blood for PBMC – 
Immunogenicity 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
X 
X 
X 
X 
X 
X 
X 
Lymph Node 
Aspirate 
 
X* 
 
X** 
 
 
 
X*** 
 
 
 
 
 
 
 
 
 
 
1 The second, third and fourth immunogen challenges (Visits 5, 9 and 13) can be shifted up to -7 to +14 days. 
Subsequent visits will be shifted by the same number of days to ensure the interval to subsequent visits are maintained 
2 Symptom-directed physical examination maybe preformed at the discretion of the Chief Investigator/delegate  
3 At screening and before immunogen challenge, and at 60 (+/-15) min and 7 (+/-1) days post-challenge 
4 HIV antibodies at screening only 
* FNA sample window 1:  from enrolment to Day 0 (prior to first immunogen challenge). 
** FNA sample window 2: Day 7 to Day 56. Aim for  FNA Samples to be obtained at Day 14 (2 weeks after first 
immunogen challenge) 
***FNA sample window 3: Day 57 to Day 252 (1 to 196 days post challenge). Aim for FNA samples to be obtained at 
Day 70 (2 weeks after second immunogen challenge). If a participant has two sampling occasions during this window, 
the samplings should be at least 7 days apart. 
 
 

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Table 3 continued: 
Schedule of Study Events – Groups J–M 
Visit Number 
1 
2 
3 
4 
5 
6 
7 
8 
9 
10 
11 
12 
13 
Visit Month 
M0 
 
 
 
M2 
 
 
 
M4 
 
 
 
M7 
Visit Week 
0 
1 
2 
4 
8 
9 
10 
12 
16 
17 
18 
20 
28 
Visit Day 
0 
7 
14 
28 
56 
63 
70 
84 
112 
119 
126 
140 
196 
Visit Windows 
(Days) 
Screening 
0 
 1 
 2 
 2 
-7/+14 
 1 
 2 
 2 
-7/+14 
 1 
 2 
 2 
-7/+14 
Immunogen 
challenge(1) 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Informed consent 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Medical history/ 
demographics / 
height and weight 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
Symptom-directed 
physical 
examination(2) 
X 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
(X) 
X 
Vital Signs(3)  
X 
X 
X 
 
 
X 
X 
 
 
X 
X 
 
 
 
Full blood count, 
serum renal, liver, 
bone panels & HIV-1 
& 2 antibodies4 
X 
 
 
 
 
 
 
 
 
 
 
 
 
X4 
Provide symptom 
diary 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Review symptom 
diary 
 
 
X 
 
 
 
X 
 
 
 
X 
 
 
 
Urine Pregnancy 
Test (Women) 
X 
X 
 
 
 
X 
 
 
 
X 
 
 
 
 
Blood for Serum – 
Immunogenicity 
 
X 
 
X 
X 
X 
 
X 
X 
X 
X 
X 
X 
X 
Blood for PBMC – 
Immunogenicity 
 
X 
X 
 
 
X 
X 
 
 
X 
X 
X 
X 
X 
Lymph Node 
Aspirate 
 
X* 
 
X** 
 
 
 
X*** 
 
 
 
 
 
 
1 The second and third immunogen challenges (Visits 5 and 9) can be shifted up to -7 to +14 days. Subsequent visits 
will be shifted by the same number of days to ensure the interval to subsequent visits are maintained 
2 Symptom-directed physical examination maybe preformed at the discretion of the Chief Investigator/delegate  
3 At screening and before immunogen challenge, and at 60 (+/-15) min and 7 (+/-1) days post-challenge 
4 HIV antibodies at screening only 
* FNA sample window 1:  from enrolment to Day 0 (prior to first immunogen challenge). 
** FNA sample window 2: Day 7 to Day 56. Aim for  FNA Samples to be obtained at Day 14 (2 weeks after first 
immunogen challenge) 
***FNA sample window 3: Day 57 to Day 196 (1 to 140 days post challenge). Aim for FNA samples to be obtained at 
Day 70 (2 weeks after second immunogen challenge). If a participant has two sampling occasions during this window, 
the samplings should be at least 7 days apart. 
 
Transfer, sample storage and analysis 
All PBMC and Serum samples (for immunological analysis) obtained at the Imperial CRF will 
be transferred to the Shattock Laboratory at Imperial College London, St Mary’s Hospital 
campus, for analysis. Lymph node samples will also be transferred to the Shattock Laboratory. 

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Samples of cryopreserved PBMC, lymph node and serum will be stored and analysed 
internally as per the Shattock Laboratory working instructions for up to 10 years following 
study completion.  As part of our consortium (EAVI2020), our collaborators outside of the UK 
might also undertake further immunological analysis of data and / or samples generated from 
the study.  These samples will be pseudoanonymised so that our study collaborators are 
blinded to the volunteer’s personal identifiable information.   
Restarting participation following a pause due to COVID-19 or other reason 
Where participation of enrolled subjects was paused due to the COVID-19 pandemic, or other 
reason, we will restart affected participants at their next scheduled immunogen challenge 
visit. In order to check that their health status hasn’t changed significantly since we last saw 
them, they will attend the Imperial CRF for routine safety blood tests (full blood count; renal, 
liver and bone panels) before the challenge visit. The need for this additional visit is at the 
investigator’s discretion; it might not be required for participants who were paused for a 
relatively short time. 
For some affected participants, their most recent visit before restarting was an immunogen 
challenge visit at which follow-up assessments were performed but no immunogen was 
injected. These participants will repeat this visit, with immunogen challenge. 
Participants will be paid for extra visits. 
Concomitant medication 
We will record relevant concomitant medication taken by participants, including vaccines, in 
order to assess their potential impact on the study’s outcomes. Participants will be 
encouraged to have an authorised COVID-19 vaccine if they are offered one, but we will 
request that they have it at least 14 days before or after immunogen challenge in the study, 
and at least 28 days will be encouraged. For seasonal influenza vaccine we will request that 
they have it at least 7 days before or after immunogen challenge in the study, and at least 28 
days will be encouraged. Participants will be encouraged to receive NHS vaccines in the 
opposite arm from the immunogen challenge. 
Ethics Approval  
The Chief Investigator will obtain approval from the HRA and Research Ethics Committee. The 
study must also receive confirmation of capacity and capability from Imperial College 
Healthcare NHS Trust. The study will be conducted in accordance with the recommendations 
for physicians involved in research on human subjects adopted by the 18th World Medical 
Assembly, Helsinki 1964 and later revisions, and with ICH GCP. 
 
Confidentiality 
The Chief Investigator will preserve the confidentiality of volunteers taking part in the study. 
Readily identifiable volunteer data will be held securely at the NHS site hosting the study. All 
volunteer samples and data leaving the NHS site will be identified only by a unique study code, 

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and hence will be link-anonymised.  All data (results generated from the study) will be 
anonymised and stored on Imperial College London servers for 10 years. 
 
Indemnity 
Imperial College London holds negligent harm and non-negligent harm insurance policies, 
which apply to this study. For harm caused by staff with substantive NHS employment 
contracts, the NHS indemnity scheme or professional indemnity will apply. 
 
Sponsor 
Imperial College London will act as the main Sponsor for this study. Delegated responsibilities 
will be assigned to Imperial College Healthcare NHS Trust as necessary. 
 
Finance & Funding 
This study is funded by a grant from the European Commission in the H2020 Framework 
Programme. The investigators do not receive any payment for this study. 
 
Monitoring, Audits, Inspection & End of Study Definition 
The study may be subject to audit by Imperial College London under their remit as sponsor, 
the Study Coordination centre and other regulatory bodies to ensure adherence to GCP. On-
site monitoring will be delegated to a trained monitor  to ensure that the study is conducted 
in compliance with human subjects’ protection and other research regulations and guidelines, 
recorded and reported in accordance with the protocol, is consistent with SOPs, GCP 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 study will be closed when all 
volunteers have made their final visit.   
 
Storage of research data and archiving 
All data will be archived in accordance with the Imperial College London archiving policy 
implemented by the sponsor, Imperial College London. The archive facility is:   
ACRU Store 
Charing Cross Hospital 
London 
W6 8RP 
 

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Publication Policy 
Results from this study will be disseminated locally and to the wider scientific community at 
conferences and in published open-access peer-reviewed journals, as appropriate. Any data 
used for publication will be fully anonymised. We plan to publish the data and hold onto the 
data for 10 years. 
 
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CONFIDENTIAL 
 
EAVI2020_01 PROTOCOL 
 
 
IRAS ID:  251930 
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Appendix 1 
Sub-study Protocol