1

Adenovirus based HIV-1 vaccine candidates tested in efficacy

trials elicit CD8 T-cells with limited breadth of HIV-1 inhibition

1Peter J. HAYES, 1Josephine H. COX, 1Adam R. COLEMAN, 1Natalia FERNANDEZ,

1Philip J. BERGIN, 1Jakub T. KOPYCINSKI, 2Sorachai NITAYAPHAN, 3Punnee

PITISUTTIHUM, 4Mark de SOUZA, 5Ann DUERR, 5Cecilia MORGAN, 1Jill W.

GILMOUR.

1IAVI Human Immunology Laboratory, Imperial College London, UK

2Armed Forces Research Institute of Medical Science (AFRIMS), Dept. of

Retrovirology, Humoral Immunology and Assessment Laboratory, 315/6 Rajvithi Road,

Bangkok 10400, Thailand.

3Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road,

Ratchathewi, Bangkok 10400, Thailand.

4SEARCH, Thai Red Cross AIDS Research Centre, Bangkok 10330, Thailand.

5Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Corresponding author and reprint requests: Peter Hayes, IAVI Human Immunology

Laboratory, 2nd floor Lift bank D, Chelsea & Westminster Hospital, 369 Fulham Road,

London, SW10 9NH, UK.

E-mail: p.hayes@imperial.ac.uk

Running title: Vaccine-elicited CD8 T-cell HIV-1 inhibition (40 characters)

Main text word count: 3299 (including subheadings and reference numbers)

There are no known commercial or other conflicts of interest.

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Sources of funding: IAVI’s work is made possible by generous support from

many donors including: the Bill & Melinda Gates Foundation; the Ministry of

Foreign Affairs of Denmark; Irish Aid; the Ministry of Finance of Japan in

partnership with the World Bank; the Ministry of Foreign Affairs of the

Netherlands; the Norwegian Agency for Development Cooperation (NORAD);

the United Kingdom Department for International Development (DFID) and the

United States Agency for International Development (USAID). The full list of

IAVI donors is available at www.iavi.org . This study is made possible by the

generous support of the American people through USAID. The contents are the

responsibility of the International AIDS Vaccine Initiative and do not necessarily

reflect the views of USAID or the United States Government.  

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Abstract

Objectives: The ability of HIV-1 vaccine candidates; MRKAd5, VRC DNA/Ad5 and

ALVAC/AIDSVAX to elicit CD8 T-cells with direct anti-viral function was assessed and

compared with HIV-1 infected subjects.

Design: Adenovirus serotype 5 (Ad5)-based regimens MRKAd5 and VRC DNA/Ad5

designed to elicit HIV-1 specific T-cells, are immunogenic but failed to prevent infection

or impact on viral loads in subjects infected subsequently. Failure may be due in part to

a lack of CD8 T-cells with effective anti-viral functions.

Methods: An in vitro viral inhibition assay (VIA) tested the ability of bi-specific antibody

expanded CD8 T-cells from peripheral blood mononuclear cells (PBMC) to inhibit

replication of a multi-clade panel of HIV-1 isolates in autologous CD4 T-cells. HIV-1

proteins recognized by CD8 T-cells were assessed by IFN ELISpot assay.

Results: Ad5-based regimens elicited CD8 T-cells that inhibited replication of HIV-1

IIIB isolate with more limited inhibition of other isolates. IIIB isolate Gag and Pol genes

have high sequence identities (>96%) to vector HIV-1 gene inserts and these were the

predominant HIV-1 proteins recognized by CD8 T-cells. Virus inhibition breadth was

greater in antiretroviral naïve HIV-1 infected subjects naturally controlling viremia

(plasma viral load (pVL) <10000/mL). HIV-1 inhibitory CD8 T-cells were not elicited by

the ALVAC/AIDSVAX regimen.

Conclusions: The Ad5-based regimens, although immunogenic, elicited CD8 T-cells

with limited HIV-1 inhibition breadth. Effective T-cell based vaccines should presumably

elicit broader HIV-1 inhibition profiles. The VIA can be used in vaccine design and to

prioritise promising candidates with greater inhibition breadth for further clinical trials.

Abstract word count: 248 words (including subheadings)

Key words: CD8-Positive T-lymphocytes, HIV-1, vaccines, genetic vectors

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Introduction

There is reasonable consensus that both humoral and T-cell responses will be required

for an effective HIV vaccine [1-4] and vaccine candidates designed to elicit these

responses have undergone efficacy trials. Two trials (Step/Phambili) tested an

adenovirus serotype-5 (Ad5) vector encoding HIV-1 Gag/Pol/Nef (MRKAd5 HIV-1)

designed to induce T-cells to reduce viremia [5-7]. HVTN505 trial employed DNA-prime

and Ad5-boost encoding Gag/Pol/Nef/Env (Virus Research Centre (VRC DNA/Ad5)),

designed to elicit T-cells and antibody [8]. RV144 trial tested a canarypox vector

encoding Gag/Pol/Env (ALVAC-HIV) and gp120 protein-boost (AIDSVAX) designed to

elicit T-cells and antibody [9]. Interferon- (IFN) Enzyme-linked Immunospot (ELISpot)

and flow cytometry assays demonstrated that both Ad5-based regimens are

immunogenic, eliciting HIV-1 specific T-cells in 70 to 80% of subjects, which comprised

both CD4 and CD8 polyfunctional T cells [6,10-12]. However, these regimens tested in

efficacy trials failed to protect against infection with limited impact on viremia in

vaccinees infected subsequently [5-8], although T-cell responses to 3 or more Gag

peptides in infected STEP vaccinees were associated with half-log lower viremia,

compared with subjects without Gag responses [13,14]. The ALVAC/AIDSVAX regimen

elicited CTL responses in 24% of vaccinees with binding and neutralising antibody

responses in 100% and 98% of subjects respectively [15] and demonstrated a 31.2%

reduction in HIV acquisition, the most plausible correlate of protection being antibody-

based [16].

Despite the lack of efficacy of vaccine candidates designed to elicit T-cells, there is

ample evidence that T-cells, in particular CD8 T-cells, can control viral replication in

vivo in both HIV-1 [17-30] and SIV infection, including vaccinated SIV-challenged non-

human primates (NHP) [31-35]. The induction of broadly cross-reactive anti-viral T-cells

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is likely to be a crucial facet of efficacious vaccines, complementing humoral responses

containing virus at the infection site.

ELISpot and flow cytometry assays, commonly employed to assess immunogenicity of

vaccine candidates, measure cytokine production by T-cells in response to exogenous

synthetic HIV peptides, functions that do not necessarily mediate direct inhibition of

viral replication [36-38] and did not predict vaccine efficacy. The present report

addressed whether CD8 T-cells elicited in phase I/II trials of the MRKAd5 HIV-1, VRC

DNA/Ad5 and ALVAC/AIDSVAX regimens mediate direct anti-viral function by inhibition

of HIV-1 replication in an in vitro viral inhibition assay (VIA). Vaccine candidate HIV-1

gene insert-matched and mismatched HIV-1 isolates were tested, including

transmitted/founder (T/F) viruses responsible for establishing infection [39]. Such

assays would allow CD8 T-cells to recognise naturally processed HIV-1 epitopes in

infected CD4 T-cells and correlate with virus control in humans [40-45] and protection

in NHP [32,35]. Our initial qualification studies [43] in HIV-1 infected subjects

demonstrated a significant negative correlation of pVL and virus inhibition, with no

correlation between IFN ELISpot magnitude and pVL. Therefore, as a benchmark, the

breadth of any inhibition elicited by vaccination was compared with that of HIV-1

infected subjects demonstrating some natural control of HIV-1 replication in vivo,

evidenced by lower plasma viral loads (pVL)(<10000/mL) in the absence of anti-

retroviral therapy.

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Methods

Subjects: Frozen PBMC were obtained from phase I/II clinical trials HVTN 071

(MRKAd5 HIV-1 regimen), V001 (VRC DNA/Ad5 regimen) and RV135

(ALVAC/AIDSVAX regimen) (table 1). Samples were obtained from 16 anti-retroviral

(ARV)-naive chronically HIV-1-infected subjects from Chelsea and Westminster

Hospital, London, UK and from HIV-1-uninfected South African blood transfusion

volunteers. HIV-1 infected subjects were selected with pVL <10000 copies/mL (median

1265, range 49-9287). Median CD4 count was 628 (range 247-1249). PBMC were

isolated by density centrifugation, frozen at 10 million per vial in 10%

dimethylsulphoxide/90% heat-inactivated foetal calf serum (HIFCS) (Sigma, UK) and

stored in vapour phase liquid nitrogen [47]. For all studies, informed consent and local

ethical approval was obtained.

Viruses: RV135 and HIV-1 infected subjects’ samples were tested in VIA with HIV-1

IIIB and CRF01_AE CM235 isolates, the latter provided by Victoria Polonis, Walter

Reed Army Institute of Research, USA. For other vaccinees and HIV-1 infected

subjects, VIA was applied using the following six HIV-1 isolates (Genbank accession

numbers where known and subtype); IIIB (HXB2 K03455, B), ELI (K03454, A/D) and

97ZA012 (AF286227, C) provided by the NIH AIDS Research and Reference Reagent

Program. Virus stocks of T/F isolates CH77 (JN944909, B), CH106 (JN944897, B) and

247FV2 (C) were generously donated by Prof. George Shaw, University of

Pennsylvania, Philadelphia, USA. Virus and Ad5-based regimen HIV-1 gene insert

sequence homologies were determined using “Align sequences protein blast” tool

available at http://blast.ncbi.nlm.nih.gov/blast.cgi (table 2). CM235 and 97ZA012

isolates were cultured in 3 day phytohaemagglutinin (PHA)-treated PBMC followed by

interleukin-2 (IL-2) (Roche Diagnostics, UK, 20 units/mL). IIIB and ELI were cultured in

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H9 cell line. Viral titres were determined using the TZM-bl cell line [49]. Cell lines were

obtained from the NIH AIDS Research and Reference Reagent Program.

Luciferase viruses: Two HIV-1 infectious molecular clones (IMC) engineered to

contain Renilla reniformis luciferase gene (LucR) (GenBank AF362549) were

generously provided by Dr. Ochsenbauer, University of Alabama, Birmingham, USA;

NL-LucR.T2A and CH077.t-LucR.T2A. The construction of these from NL4-3 (M19921,

subtype B) and CH077.t (JN944909, B) respectively, has been described [50,51].

These viruses were applied to a sample subset to compare inhibition of CD8 T-cells

isolated directly from PBMC or following culture with CD3/4 bi-specific antibody.

CD4 and CD8 T-cells: PBMC were re-suspended at 1-1.5x106/mL in RPMI/10%

HIFCS/50 units IL-2 (R10/50) and 0.5g/mL CD3/CD4 or CD3/CD8 bi-specific antibody

for generation of CD8 or CD4 T-cells, respectively [52]. Culture volumes were doubled

at days 3 and 6 with R10/50 medium. Typical purities of 7-day cultures are 97% and

87% of CD3+ T-cells for CD4 and CD8 T-cells respectively [43]. Expanded CD8 T-cell

memory phenotypes were assessed by flow cytometery.

Viral inhibition assay: 7-day expanded CD4 T-cells were infected for 4 hours/370C at

multiplicity-of-infection (MOI) of 0.01, washed and 0.5x106 infected cells cultured

with/without 0.5x106 autologous CD8 T-cells in 1 mL R10/50 in 48 well plates. Half of

the supernatant was replaced with R10/50 on days 3, 6 and 10. Day 13 supernatant

p24 content was measured by ELISA (Perkin Elmer, UK). CD8 T-cell-mediated

inhibition was expressed as log10 reduction in p24 content (pg/mL) of CD4/CD8 co-

cultures compared with CD4 T-cells alone. A positive inhibition threshold of p24

reduction >1.5 logs was determined in previous studies from the distribution of

inhibition observed in placebo and pre-vaccination trial subjects [43,53-55]. Similar

levels of replication of all HIV-1 isolates were demonstrated in day 13 CD4 T-cell only

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cultures by median log10 p24 pg/mL of 5.69, 5.66, 5.56, 6.06, 5.58, 5.70 and 5.62 for

isolates IIIB, ELI, CH77, CH106, 247Fv2, ZA97012 and CM235 respectively.

Luciferase virus VIA: T-cells were generated as above. 1.3x106 CD4 T-cells were

spinoculated (2 hours, 1800g) with LucR HIV-1 (MOI 0.1), re-suspended at 1x106

cells/mL in R10/50 (without phenol red) and cultured at 370C. Expanded CD8 T-cells

adjusted to 1.5x106 cells/mL in R10/50 were cultured separately. After 3 days, T-cells

were re-suspended at 1x106 cells/mL. 1x105 CD4 T-cells were added to wells of a 96

well, white, flat-bottomed plate and co-cultured with either 1x105 CD8 T-cells or 100µl

R10/50 (CD4 T-cells only). After 5 days, 100µl supernatant was removed, 100µl

Renilla-Glo luciferase buffer (Promega, Madison, WI, USA) added according to

manufacturer’s instructions and luminescence read at 100ms on a Tecan Infinite m200

pro Microplate reader using Magellan 7 software (Tecan, Maennedorf, Switzerland).

Data are expressed as log10 reduction in relative light units measured in CD4 and CD8

T-cell co-cultures, with assay positive cut-off value of 0.6 log10 [56].

Comparison of antibody-expanded and directly-isolated CD8 T-cells: LucR VIA

experiments used expanded CD4 T-cells co-cultured with expanded CD8 T-cells or

CD8 T-cells obtained by direct isolation from PBMC thawed one day prior to co-culture

and rested overnight in RPMI 20% HIFCS. CD8 T-cells were negatively-selected using

magnetic beads (Miltenyi-Biotec, Bergisch Gladbach, Germany).

IFN-γ ELISpot assay: To identify which HIV-1 proteins were targeted by CD8 T-cells

tested in VIA, expanded CD8 T-cells were washed three times in PBS and rested for

9

24 hours in RPMI 20% HIFCS without IL-2 before IFN- ELISpot as previously

described [47] using pools of overlapping 15mer peptides of HIV-1 consensus clade B

Gag, Pol, Nef, Env, Vif, Vpu and Vpr (peptide sets from NIH AIDS Reagent Program).

Expanded cell peptide responses were considered positive if they were twice wells

without peptide (background) and background-subtracted responses were ≥50 spot-

forming units (SFU) per million cells.

Statistics: Linear regressions and significance values were generated using GraphPad

Prism software. Non-parametric Kruskal-Wallace test was used to determine significant

differences within multiple data groups followed by Dunn’s multiple comparison to

identify differences between individual groups.

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Results

Comparison of bi-specific antibody-expanded and directly-isolated CD8 T-cells:

LucR VIA compared HIV-1 inhibitory abilities of expanded and directly isolated CD8 T-

cells from HIV-1 infected subjects, selected with known ability to inhibit LucR viruses.

From 8 subjects, between 0.8 and 2.8 million CD8 T-cells (median 1.7 million) were

recovered by direct isolation from 10 million cryopreserved PBMC. Bi-specific antibody

expansion achieved a median 4.7 and 4.9-fold (minimum 3-fold) increase from starting

PBMC for CD4 and CD8 T-cells respectively (data not shown). Expanded CD8 T-cell

phenotypes were predominantly central memory (CD45RO+/CD27+) with some

effector memory (CD45RO+/CD27-) (supplementary Fig 1). There was a significant

positive correlation between log10 inhibition for expanded and isolated CD8 T-cells (Fig

1A, r2 = 0.72, p<0.0001) with median inhibition of 1.250 and 1.248 respectively (Fig

1B). Therefore, antibody-expanded and directly isolated CD8 T-cells demonstrated

similar HIV-1 inhibition. Subsequent data in this report utilised expanded cells.

Inhibition breadth of Ad5-based regimen vaccinees and HIV-1 infected subjects:

CD8 T-cells from 15 HIV-1 uninfected subjects did not inhibit any HIV-1 isolate above

the positive cut-off (Fig 1C). Sixteen MRKAd5 vaccinees (26 weeks post-final

vaccination, visit 16) inhibited a median 2.0 isolates (range 0-5). Twelve VRC DNA/Ad5

vaccinees inhibited with slightly narrower breadth with median of 1 isolate at both 6

(visit 12) and 24 weeks (visit 14) post-final vaccination (range 0-4). CD8 T-cells from 16

ARV-naive HIV-1 infected subjects with pVL <10000/mL, inhibited a median of 3

isolates (range 0-5) (Fig 1C). Within this group, there was no correlation between pVL

and breadth of inhibition (r2 =0.012, p=0.69, data not shown). Kruskal-Wallace analysis

indicated a significant difference in inhibition breadth between the Ad5-based regimen

vaccinees and HIV-1 infected subject groups (p=0.032). Dunn’s multiple comparison

identified inhibition breadth at both VRC DNA/Ad5 time-points to be significantly lower

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than HIV-1 infected subjects (p<0.05). There were no significant differences between

other subject groups (p>0.05).

Lack of inhibition with ALVAC/AIDSVAX regimen: CD8 T-cells from 21 RV135

subjects 4 weeks post final vaccination did not inhibit isolates IIIB (clade B) and CM235

(AE) above the assay positive value (Fig 1D). HIV-1 gene inserts of this regimen were

derived from these two isolates. Other unmatched viruses were not tested. 69% and

56% of the HIV-1 infected subjects inhibited IIIB and CM235 isolates respectively (Fig

1D).

Inhibition of HIV-1 isolates elicited by Ad5-based regimens: The MRKAd5 and

VRC DNA/Ad5 candidates were engineered with HIV-1 gene inserts derived from or

with high sequence identities (≥96% for Gag/Pol, 85-97% for Nef) to clade B HIV-1

isolate IIIB (table 2) and this isolate was the most commonly inhibited with 69%

MRKAd5 subjects inhibiting (Fig 1D). 67% of VRC DNA/Ad5 subjects inhibited this

isolate at one or more of the two post-vaccination time-points. The other clade B T/F

isolates CH77 and CH106 (91-95% identities for Gag/Pol, 79-82% for Nef) were

inhibited to a lesser extent: 38% and 50% of MRKAd5 subjects inhibited these two

viruses, respectively, whilst only 17% of VRC DNA/Ad5 subjects inhibited. Non-clade

B isolates (84-95% identities for Gag/Pol, 75-82% for Nef) were inhibited the least with

19% and 8% of MRKAd5 and VRC DNA/Ad5 subjects inhibiting clade AD ELI isolate

respectively. 44% of MRKAd5 subjects inhibited clade C 247Fv2 isolate but VRC

DNA/Ad5 subjects did not inhibit this isolate. No subjects from either trial inhibited

clade C isolate ZA97012 despite the VRC DNA/Ad5 regimen encoding the Env gene

derived from this isolate.

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Clade B and non-clade B isolate inhibition in a clade B infected cohort: The HIV-1

infected subjects were from a predominantly clade B cohort (84% for UK, HIV

sequence database at www.hiv.lanl.gov). The three clade B isolates were the most

commonly inhibited with 69% of subjects inhibiting isolates IIIB and CH77 and 64%

inhibiting CH106 (Fig 1D). Within this lower pVL group there were no correlations

between pVL and log10 magnitude of inhibition for any of these clade B isolates (r2

0.001-0.024, p=0.56-0.91, data not shown). Inhibition of non-clade B isolates ELI,

247Fv2 and ZA97012 was less common at 25%, 38% and 13% of subjects,

respectively (Fig 1D).

IFN-γ ELISpot identified HIV-1 genes targeted by expanded CD8 T-cells: 56% of

MRKAd5 subjects (regimen containing only clade B HIV-1 Gag/Pol/Nef genes)

responded to at least one HIV-1 peptide pool (median 1.0, range 0-3) and all

responding subjects targeted at least one Gag or Pol pool with two subjects (13%) also

responding to Nef (table 3). 42% of VRC DNA/Ad5 subjects (regimen containing clade

B Gag/Pol/Nef and multi-clade Env genes) responded to at least one HIV-1 peptide

pool (median 0, range 0-1) with responses restricted to Gag or Pol.

CD8 T-cells from 81% of HIV-1 infected subjects responded in ELISpot to at least one

clade B HIV-1 peptide pool (median 1.0, range 0-6) with the predominant response

being to Gag (63% of subjects). 25 to 38% of subjects responded to other pools.

Combining all subject groups there was no correlation between ELISpot response

magnitude (summed SFU) and VIA breadth (r2=0.003, p=0.72) (Fig 2A). There was a

significant correlation between the number of ELISpot pool responses and VIA breadth

(r2=0.15, p=0.01) (Fig 2B).

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Conclusions

MRKAd5 and VRC DNA/Ad5 Ad5-based regimens although immunogenic, eliciting

HIV-1-specific T-cells detected by IFN ELISpot and flow cytometry assays [10-12],

failed to protect against infection or reduce viremia [5-8]. The present study

demonstrated that CD8 T-cells elicited by these Ad5-based regimens are directly anti-

viral, inhibiting HIV-1 replication in vitro although with limited inhibition breadth, HIV-1

IIIB being the most frequently inhibited followed by other clade B isolates (Fig 1C,D).

Vector encoded Gag and Pol genes for both regimens had high sequence identity

(>96%, table 2) to HIV-1 IIIB Gag and Pol genes with these proteins being the

predominant targets recognized in ELISpot (table 3), explaining the efficient inhibition

of this isolate. HIV-1 isolate inhibition frequencies following these Ad5-based regimens

can be summarised as: vector insert sequence matched isolate IIIB > vector insert

clade matched isolates (clade B T/F isolates CH77 and CH106) > non-clade B isolates.

No inhibition of vaccine insert matched isolates IIIB or CM235 was detected in

ALVAC/AIDSVAX vaccinees, although these isolates were readily inhibited by HIV-1

infected subjects (Fig 1D). This would be expected as this regimen induces minimal

CD8 T-cell responses [15] and a subsequent efficacy trial demonstrated a significant

31% protection from infection [9] was associated with humoral responses [16].

HIV-1 inhibition breadth of ARV-naive HIV-1 infected subjects with pVL <10000/mL

was assessed as a benchmark to represent subjects where CD8 T-cell-mediated

control of HIV-1 replication may be expected [17-30]. Inhibition breadth was superior to

that elicited by either Ad5-based regimen (Fig 1C) but was significant only for the VRC

DNA/Ad5 regimen. There was no significant difference between Ad5 regimens. These

presumed clade B infected subjects commonly inhibited the three clade B isolates;

laboratory-adapted IIIB and T/F isolates CH77 and CH106. Non-clade B isolates were

15

inhibited to a lesser extent (Fig 1D). IFN ELISpot demonstrated CD8 T-cells from HIV-

1 infected subjects primarily targeted Gag amongst other HIV-1 proteins (table 3).

The narrower breadth of vaccine-elicited inhibition compared with HIV-1 infected

subjects could be explained by a report [57] demonstrating that both MRKAd5 and

VRC Ad5/DNA regimens elicit T-cell responses to far fewer HIV-1 epitopes than natural

infection and that these epitopes were often clustered in highly variable regions. The

finding that CD8 T-cells from infected subjects controlling viremia in vivo, can inhibit a

range of HIV-1 isolates, including T/F viruses, demonstrates that inhibitory T-cells can

be elicited with sufficient breadth to recognise both intra- and inter-clade HIV-1 isolates

and supports the development of regimens designed to elicit T-cells. It would be

desirable for vaccine candidates to induce CD8 T-cells capable of inhibition breadth at

least similar or superior to that seen in naturally controlled HIV-1 infection. At a

minimum such regimens could encode HIV-1 genes for the predominant clade

circulating within geographic regions, with Gag and Pol being included due to greater

sequence conservation of these genes. The present report demonstrated that Gag and

Pol were the predominant HIV-1 proteins recognised by CD8 T-cells in both Ad5

regimen vaccinees and HIV-1 infected subjects. The correlation between the number

of peptide pools recognized by CD8 T-cells and breadth of VIA (Fig 2B) supports the

strategy that T-cell based vaccines should aim to increase the number of HIV-1

epitopes targeted by T-cells thereby potentially enhancing vaccine efficacy [58].

The present report agrees with a report [59] describing inhibitory abilities of CD8 T-cells

isolated directly from PBMC from HIV-1 infected STEP vaccinees (MRKAd5) where

inhibition would be due to a combination of vaccination and natural infection. Inhibition

breadth was narrow and inferior to chronically infected viremic controllers (pVL

<5000/mL). However, PBMC availability limited the number of isolates tested in the

16

majority of vaccinees to 3 or 4. As blood volumes from multiple clinical trial time-points

are limited, expansion of T-cells would often be the only practical approach to assess

inhibition breadth of multiple HIV-1 isolates on a consistent basis. One concern would

be that T-cell expansion results in T-cell specificities that do not reflect those in vivo.

The present report demonstrated that inhibition values of expanded and directly

isolated CD8 T-cells were almost identical (Fig 1A,B), demonstrating antibody

expansion is a valid approach for assessing CD8 T-cell mediated HIV-1 inhibition. The

expansion approach allows testing of at least 12 HIV-1 isolates and ELISpot analysis in

parallel from a single frozen PBMC vial. The luciferase VIA under development uses

fewer cells, allowing even more HIV-1 isolates to be tested [56]. Another report

demonstrated the VRC DNA/Ad5 regimen elicited inhibitory CD8 T-cells although the

virus isolates tested only differed in Env sequence, being isogenic for other HIV-1

genes. Inhibition was associated with central and effector memory CD8 T-cells

expressing CD107a and MIP1[60]. The present report demonstrated expanded CD8

T-cells were of central and effector memory phenotypes (Fig 1S).

Present VIA and other immunogenicity data from phase I/II clinical trials [36-38] have

demonstrated that Ad5-based regimens elicit T-cells with limited breath of recognition

of HIV-1 isolates and this may be a factor in the lack of efficacy demonstrated in

subsequent trials. A full assessment of immunogenicity profiles elicited by candidate

regimens in early phase trials, including inhibition breadth, allows development and

selection of the most promising regimen combinations for further trials. The challenge

for development of T-cell based vaccine candidates is to elicit CD8 T-cells with broader

inhibition than MRKAd5 and VRC DNA/Ad5 regimens and at least as broad or superior

to ARV-naive HIV-1 infected subjects. Approaches designed to enhance the breadth of

vaccine-elicited T-cell responses have been proposed [58]. Although the benefit of

eliciting CD8 T-cells with inhibitory breadth equivalent to HIV-1 infected subjects may

17

be questionable, as such individuals usually progress to AIDS without treatment, the

hope would be that eliciting such T-cells prior to HIV-1 exposure would contain initial

viral replication to prevent infection establishment or reduce viremia. Such a possibility

has been demonstrated for SIV infection where a replicating cytomegalovirus vector

[31,61] or a combination of yellow fever and Ad5 vectors [34] elicit potent SIV-specific

CD8 T-cell responses and control of SIV replication in NHP. VIA can also characterise

beneficial CD8 T-cell responses in infection, determining which HIV-1 genes and

epitopes targeted are associated with control and informing on regimen design and has

demonstrated the potential benefit of enhancing subdominant CD8 T-cell responses as

part of cure strategies aimed at elimination of the latent viral reservoir in HIV-1 infected

subjects [62].

18

Acknowledgements (not already included in main text): We thank the staff and

volunteers of the clinical trials and clinics. We thank Merck, the NIH National Institute of

Allergy and Infectious Diseases (NIAID) and the NIAID-funded HIV Vaccine Trials

Network (HVTN) for providing samples from the HVTN 071 clinical trial. RV135 clinical

trial samples were provided by Mark de Souza, SEARCH, Thai Red Cross AIDS

Research Centre and Sorachai Nitayaphan, Armed Forces Research Institute of

Medical Science (AFRIMS), Bangkok, Thailand. IAVI V001 clinical trial samples were

provided by the Kenya AIDS Vaccine Initiative (KAVI), University of Nairobi, Kenya and

Projet San Francisco, Kigali, Rwanda. The following reagents were obtained through

the NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,

NIH; HIV-1 ELI from Dr. Jean-Marie Bechet and Dr. Luc Montagnier; HIV-1 IIIB and H9

cell line from Dr. Robert Gallo; TZM-bl from Dr. John C. Kappes, Dr. Xiaoyun Wu and

Tranzyme Inc; HIV-1 97ZA012 reagent was obtained through the NIH AIDS Reagent

Program, Division of AIDS, NIAID, NIH: from The UNAIDS Network for HIV Isolation

and Characterization. We thank Professor Johnson Wong and Dr. Galit Alter,

Massachusetts General Hospital, Boston, USA for providing CD3/4 and CD3/8 bi-

specific antibodies.

Author roles

PH, JC, AC, PB and JG designed the study. PH, AC, NF and JK acquired and

analysed data. PH, JC, AC, JK and JG interpreted data. PH, JC, AC, NF, PB, JK, SN,

PP, MdS, AD, CM and JG drafted, reviewed, revised and approved the manuscript and

agree to be accountable for all aspects of the work.

19

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27

Table 1: Phase I/II HIV-1 vaccine candidate trials and subsequent efficacy studies

Samples tested

from phase I/II

trial

(NCT number)

Regimen

Regimen

administration

& sample time

point

weeks (W)

Equivalent

efficacy or

proof-of

concept trial

Reference

HVTN 071

NCT00486408

n = 16

Merck Ad5 HIV-1

Gag/Pol/Nef (clade B)

(MRKAd5)

Ad5: W0, 4, 26

Sample: W52

STEP

NCT00095576

& Phambili

NCT00413725

[5]

IAVI V001 group

C

NCT00124007*

n = 12

VRC multiclade HIV-1

DNA plasmid (clade B

Gag/Pol/Nef, clades

ABC Env) & multiclade

Ad5 vector (expressing

same genes without

Nef)

(VRC DNA/Ad5)

DNA: W0, 4, 8

Ad5: W24

Sample: W30,

48

HVTN 505

NCT00865566[8,11,12]

RV135

NCT Not

available

n = 21

ALVAC-HIV (vCP1521)

& VaxGen gp120

(clades B/E )

(ALVAC/AIDSVAX)

vCP1521: W0,

4, 12, 24

AIDSVAX: W12,

24

Sample: W28

RV144

NCT00223080[15,16,46]

*Equivalent regimen also tested in clinical trial HVTN 204, NCT00125970

AbbreviationsNCT: National clinical trial, information at https://clinicaltrials.gov/, a service of the U.S. National Institutes of HealthPU: Particle units

28

Table 2: Percentage sequence identities between genes of HIV-1 isolate used in VIA and Ad5-based regimen gene inserts

Vaccine candidate and HIV-1 gene insert VIA HIV-1 isolate (clade), gene accession and % identity with vaccine gene insert

HIV-1

gene

Insert

isolateClade

Insert

accessionIIIB (B) CH77 (B) CH106 (B) ELI (AD) 247FV2 (C) 97ZA012 (C)

MRKAd5

Gag CAM-1 B BAA00992AAB50258 AET76070 AET75990 AAA44324 ** AAK30990

96% 92% 92% 91% 84% 84%

Pol IIIB (HXB2) B AAB50259AAB50259 AET76071 AET75991 AAA44325 ** AAK30991

100% 94% 95% 95% 92% 92%

Nef JRFL B [48]AAB50263 ** AET75998 AAA44330 ** AAK30998

85% 82% 79% 75% 77% 80%

VRC

DNA/Ad5Gag HXB2 B AAB50258

AAB50258 AET76070 AET75990 AAA44324 ** AAK30990

100% 92% 91% 89% 84% 84%

Pol NL4-3 B AAA44988AAB50259 AET76071 AET75991 AAA44325 ** AAK30991

97% 93% 95% 95% 92% 90%

Nef NY5/BRU B AAA44993 AAB50263 NA AET75998 AAA44330 ** AAK30998

29

LAV1 NL43 97% 77% 79% 79% 78% 82%

Env 92RW020 A AAT67478AAB50262 AET76077 AET75997 AAA44329 ** AAK30997

76% 74% 73% 74% 73% 76%

Env HXB2 / Bal* B AAB50262AAB50262 AET76077 AET75997 AAA44329 ** AAK30997

100% 79% 79% 76% 70% 72%

Env 97ZA012 C AAK30997AAB50262 AET76077 AET75997 AAA44329 ** AAK30997

72% 69% 70% 70% 78% 100%

*amino acids 275-361 derived from HIV-1 Bal isolate

**sequence provided by George Shaw, University of Pennsylvania, Philadelphia, personal communication

30

Table 3: Proportion of subjects in each group with expanded CD8 T-cell IFN ELISpot responses to HIV-1 peptide pools

(percent responders)

Subject group

HIV-1 consensus clade B peptide pool

(Consensus set overlapping peptide numbers in pool)

Gag

(1-123)

Pol1

(1-125)

Pol2

(126-249)

Gag or Pol

Nef

(1-49)

Env1

(1-106)

Env2

(107-211)

Vpu/Vif/Vpr

(1-87)

Response to

any pool

MRKAd5

7/16

(44%)

4/16

(25%)

4/16

(25%)

9/16

(56%)

2/16

(13%)

ND ND ND

9/16

(56%)

VRC

DNA/Ad5

3/12

(25%)

1/12

(6%)

1/12

(6%)

5/12

(42%)

0/12 0/12 0/12 ND

5/12

(42%)

HIV+

10/16

(63%)

5/16

(31%)

4/16

(25%)

11/16

(69%)

6/16

(38%)

4/16

(25%)

2/16

(13%)

4/16

(25%)

13/16

(81%)

ND: not done (HIV-1 genes not encoded by regimen vectors)

31

Figure 1: CD8 T-cell mediated inhibition of HIV-1 replication.

(a) Linear regression of magnitude (log10) inhibition of HIV-1 replication mediated by

directly-isolated or bi-specific antibody expanded CD8 T-cells. The slope of best fit

and 95% confidence intervals are displayed.

(b) Magnitude (log10) inhibition of HIV-1 replication mediated by directly-isolated or bi-

specific antibody expanded CD8 T-cells. Bar represents median of the group.

Horizontal line represents the inhibition positive cut-off value (0.6 log10).

(c) Number of HIV-1 isolates (maximum = 6) inhibited by subjects within each group.

Bar represents the median of the group.

(d) Proportion of subjects in each group inhibiting each HIV-1 isolate. VRC V12 and

V14 are 6 and 24 weeks post final administration of VRC DNA/Ad5. MRKAd5 is 16

weeks post final administration. Inhibition of six HIV-1 isolates (clades AD, B & C)

was assessed in Ad5 regimen and HIV-1 infected subjects. ALVAC/AIDSVAX is 4

weeks post final administration where vector insert-matched HIV-1 isolates IIIB

and CM235 were tested (clades B & AE).

Figure 2: ELISpot responses and the number of HIV-1 isolates inhibited.

(a) Linear regression of magnitude of ELISpot responses (summed background

subtracted SFU for each peptide pool) and the number of HIV-1 isolates inhibited

for each subject. The slope of best fit and 95% confidence intervals are displayed.

(b) Linear regression of the number of peptide pools responses and the number of

HIV-1 isolates inhibited for each subject. The slope of best fit and 95% confidence

intervals are displayed.

Supplementary Figure 1: Flow cytometry profiles of expanded CD8 T-cells. Dot

plots show expression of CD27 (y-axis) and CD45RO (x-axis) for bi-specific

antibody expanded CD8 T-cells from six representative HIV-1 infected subjects.

Cells were stained with aqua viability dye (Invitrogen, UK) and anti-CD3 APC-H7

32

(SK7), CD4 BV605 (RPA-T4), CD8 PerCP Cy5.5 BV421 (RPA-T8), CD45RO

PeCF594 (UCHL1) and CD27 PeCy7 (M-T271) fluorescently conjugated

antibodies (all BD Biosciences, UK), washed and fixed for >1hour at 4°C in BD

Cytofix solution containing >4% paraformaldehyde. Events were acquired on a 4

laser BD Fortessa II flow cytometer and analysed using FlowJo v9.9 software.

Viable singlet CD8 T-cells were identified as CD3+, CD8+ and CD4- lymphocytes.

Values in the quadrants are the percentage events within that quadrant.