EClinicalMedicine 11 (2019) 65–80 Contents lists available at ScienceDirect EClinicalMedicine journal homepage: https://www.journals.elsevier.com/ eclinicalmedicine Research Paper Therapeutic Vaccination Refocuses T-cell Responses Towards Conserved Regions of HIV-1 in Early Treated Individuals (BCN 01 study) Beatriz Mothe a,b,c,⁎, Christian Manzardo d, Alvaro Sanchez-Bernabeu a, Pep Coll a,b, Sara Morón-López a, Maria C. Puertas a, Miriam Rosas-Umbert a,e, Patricia Cobarsi b, Roser Escrig b, Núria Perez-Alvarez b,f, Irene Ruiz d, Cristina Rovira d, Michael Meulbroek g, Alison Crook h, Nicola Borthwick h, Edmund G. Wee h, Hongbing Yang i, Jose M. Miró d, Lucy Dorrell i, Bonaventura Clotet a,b,c,e, Javier Martinez-Picado a,c,j, Christian Brander a,c,j,1, Tomáš Hanke h,k,1, for theBCN 01 study group a IrsiCaixa AIDS Research Institute-HIVACAT, Badalona, Spain b Fundació Lluita contra la Sida, Hospital Universitari Germans Trias i Pujol, Badalona, Spain c Universitat de Vic - Universitat Central de Catalunya (UVIC-UCC), Vic, Spain d Hospital Clinic-IDIBAPS, University of Barcelona, Barcelona, Spain e Universitat Autònoma de Barcelona (UAB), Barcelona, Spain f Technical University of Catalonia, Barcelona, Spain g Projecte del Noms, BCN Checkpoint, Barcelona, Spain h The Jenner Institute, University of Oxford, Oxford, UK i Nuffield Department of Medicine and Oxford NIHR Biomedical Research Centre, University of Oxford, Oxford, UK j ICREA, Pg. Luis Companys 23, Barcelona, Spain k International Research Center for Medical Sciences, Kumamoto University, Kumamoto, Japan a r t i c l e i n f o a b s t r a c t Article history: Background: Strong and broad antiviral T-cell responses targeting vulnerable sites of HIV-1 will likely be a critical Received 22 January 2019 component for any effective cure strategy. Received in revised form 21 May 2019 Methods: BCN01 trial was a phase I, open-label, non-randomized, multicenter study in HIV-1-positive individuals Accepted 21 May 2019 diagnosed and treated during early HIV-1 infection to evaluate two vaccination regimen arms, which differed in Available online 5 June 2019 the time (8 versus 24 week) between the ChAdV63.HIVconsv prime and MVA.HIVconsv boost vaccinations. The primary outcome was safety. Secondary endpoints included frequencies of vaccine-induced IFN-γ+ CD8+ T cells, in vitro virus-inhibitory capacity, plasma HIV-1 RNA and total CD4+ T-cells associated HIV-1 DNA. (NCT01712425). Findings: No differences in safety, peak magnitude or durability of vaccine-induced responses were observed be- tween long and short interval vaccination arms. Grade 1/2 local and systemic post-vaccination events occurred in 22/24 individuals and resolved within 3 days. Weak responses to conserved HIV-1 regions were detected in 50% of the individuals before cART initiation, representing median of less than 10% of their total HIV-1-specific T cells. All participants significantly elevated these subdominant T-cell responses, which after MVA.HIVconsv peaked at median (range) of 938 (73-6,805) IFN-γ SFU/106 PBMC, representing on average 58% of their total anti-HIV-1 T cells. The decay in the size of the HIV-1 reservoir was consistent with the first year of early cART initiation in both arms. Interpretation: Heterologous prime-boost vaccination with ChAdV63-MVA/HIVconsv was well-tolerated and refocused pre-cART T-cell responses towards more protective epitopes, in which immune escape is frequently as- sociated with reduced HIV-1 replicative fitness and which are common to most global HIV-1 variants. Funding: HIVACAT Catalan research program for an HIV vaccine and Fundació Gloria Soler. Vaccine manufacture was jointly funded by the Medical Research Council (MRC) UK and the UK Department for International Devel- opment (DFID) under the MRC/DFID Concordat agreements (G0701669. Research in Context: Evidence Before this Study: T cells play an important role in the control of HIV infection and may be particularly useful for HIV-1 cure by killing cells with reactivated HIV-1. Evidence is emerging that not all T-cell responses are protective and mainly only those targeting conserved regions of HIV-1 proteins are effective, but typically immunologically subdominant, while those recognizing hypervariable, easy-to-escape immunodominant ‘decoys’ do not control viremia and do not protect from a loss of CD4 T cells. We pioneered ⁎ Corresponding author at: IrsiCaixa AIDS Research Institute, Hospital Universitari Germans Trias i Pujol, Crta Canyet s/n, 08916, Badalona, Barcelona, Spain. E-mail address: bmothe@irsicaixa.es (B. Mothe). 1 Contributed equally https://doi.org/10.1016/j.eclinm.2019.05.009 2589-5370/© 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 66 B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 a vaccine strategy focusing T-cell responses on the most conserved regions of the HIV-1 proteome using an im- munogen designated HIVconsv. T cells elicited by the HIVconsv vaccines in HIV-uninfected UK and Kenyan adults inhibited in vitro replication of HIV-1 isolates from 4 major global clades A, B, C and D. Added Value of this Study: The present study demonstrated the concept that epitopes subdominant in natural infection, when taken out of the context of the whole HIV-1 proteome and presented to the immune system by a potent simian adenovirus prime-poxvirus MVA boost regimen, can induce strong responses in patients on antiretroviral treatment and efficiently refocus HIV-1-specific T-cells to the protective epitopes delivered by the vaccine. Implications of all the Available Evidence: Nearly all HIV-1 vaccine strategies currently emphasize induction of broadly neutralizing Abs. The HIVconsv vaccine is one of a very few approaches focussing exclusively on elicita- tion of T cells and, therefore, can complement antibody induction for better prevention and cure. Given the cross- clade reach on the HIVconsv immunogen design, if efficient, the HIVconsv vaccines could be deployed globally. Effective vaccines will likely be a necessary component in combination with other available preventive measures for halting the HIV-1/AIDS epidemic © 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 1. Introduction low-level viral reservoir: the “Mississippi baby” [40,41], and individuals treated in Fiebig I from the SEARCH 010/RV254 Acute HIV Infection co- Cytotoxic T lymphocytes (CTL) play a key role in the control of hort [42]. Thus, future HIV-1 cure approaches are likely to require a po- chronic HIV-1 and simian immunodeficiency virus infections [1–3]. tent T-cell vaccine component [43] that is able to stimulate CTL HIV-1-specific CD8+ T cells in acute infection appear to be uniquely responses capable of eliminating early cells, in which the virus is able to efficiently suppress viral replication and force escape mutations reactivated [44], thereby containing viral rebound after treatment [4], whereas CD8+ T-cell responses generated in the chronic infection interruption. often lack this capacity [5]. Multiple strategies to achieve optimal im- The BCN 01 trial evaluated for the first time the safety and immuno- mune control of HIV-1 infection in the absence of cART have been eval- genicity of a heterologous prime/boost regimen of the ChAdV63. uated, including a wide range of therapeutic T-cell vaccines [6–11]. The HIVconsv and MVA.HIVconsv vaccines in a cohort of 24 individuals, largest, although still suboptimal, clinical effect demonstrated to date by who were diagnosed with well-documented acute/recent HIV-1 infec- active vaccination was obtained by transfer of antigen-pulsed autolo- tion and were treated immediately with cART including Tenofovir/ gous dendritic cells, which resulted in a 10-fold, but only transient re- Emtricitabine/Raltegravir. In this population, we demonstrate a marked duction in the viral set-point after treatment discontinuation [12]. shift in immunodominance profiles of HIV-1-specific T-cell responses The failures of past therapeutic vaccines to control HIV-1 replication towards conserved T-cell epitopes, along with high in vitro viral inhibi- in the absence of cART might have been caused by suboptimal specific- tion capacity without signs of immune exhaustion. These findings may ity and limited breadth of vaccine-induced T-cell responses, due to ei- prove critical for future successful combination ‘kick and kill’ eradica- ther inadequate immunogen design, its delivery strategy or both [13]. tion strategies [44–46]. In particular, inclusion of full-length HIV-1 proteins in vaccine immuno- gens may drive CTL responses towards immunodominant, but often 2. Methods variable and therefore non-protective ‘decoy’ epitopes similar to those elicited by natural HIV-1 infection [14]. Thus, some groups have refined 2.1. Trial Design and Study Participants immunogen design by selecting protein segments able to focus T-cell immunodominance towards more beneficial and conserved determi- Trial BCN 01 was a phase I, open-label, non-randomized, multicenter nants [15–17] in order to tackle HIV-1 viral diversity and escape prime/boost therapeutic vaccination study in acute and recently HIV-1- [18–22] and circumvent the effect of host-genetics in natural response infected individuals to evaluate the safety and immunogenicity of a vac- to HIV-1 [23] (reviewed in [1]). cination regimen of the ChAdV63.HIVconsv and MVA.HIVconsv vac- The HIVconsv T-cell immunogen was constructed by assembling 14 cines using two intervals between the prime and boost. Study regions that are highly conserved among the four major HIV-1 clades A, participants were recruited at two HIV-1 units (Fundació Lluita contra B, C and D into one chimeric protein based on alternating clade consen- la Sida at Hospital Universitari Germans Trias i Pujol, Badalona and Hos- sus sequences [19]. The HIVconsv immunogen delivered by combined pital Clínic, Barcelona). Individuals with confirmed acute/recent HIV-1 plasmid DNA, non-replicating simian adenovirus and non-replicating infection (b6 months from estimated HIV-1 acquisition), who fulfilled poxvirus MVA regimens was tested in European and African trials in all eligibility criteria, were enrolled and started on cART with an INSTI- HIV-1-negative adult volunteers, and demonstrated a good safety pro- based regimen (Tenofovir/Emtricitabine plus Raltegravir). Complete file and induction of high frequencies of CD8+ T cells capable of strong list of inclusion/exclusion criteria is available at NCT01712425. Patients in vitro HIV-1 inhibition [22,24,25], especially when compared to were sequentially allocated into the Long arm first (the first participants those reported in the STEP [26] and RV144 [27] studies. For the 1–12) followed by the Short (participants 13–24) arm later. These two HIVconsv delivery, modified simian (chimpanzee) adenovirus serotype arms had a 24- or 8-week interval between the ChAdV63.HIVconsv 63 (ChAdV-63) was chosen for its low pre-existing seroprevalence in prime and MVA.HIVconsv boost administrations in the Long and Short human subjects [28], thus avoiding limitations associated with some arm, respectively, and were chosen to assess whether or not the MVA. of the human adenovirus vectored vaccines [29,30]. MVA was chosen HIVconsv boost given 24 weeks after the ChAdV63.HIVconsv vaccine in- as the subsequent boost vaccination, since poxviruses were shown to stead of the previously reported 8-week interval [25] was able to induce strongly boost existing CD8+ T-cell responses [31]. higher and/or longer-lasting HIVconsv-specific responses. The rational Several studies demonstrated that early cART in acute/recent HIV-1 for this is that ChAd vaccines may persist for longer period of time com- infection improved immune recovery [32,33], reduced the incidence of pared to other (viral) vectored vaccines and could thus induce a more AIDS and non-AIDS-related diseases [34], restricted immune escape robust memory T cell responses. Sequential recruitment was performed [35] and limited the size of latent reservoir [36–39]. However, recent to reduce the total duration of the trial and ensure that participants clinical examples suggested that in the absence of a targeted immune were suppressed for a similar period of time when entering into future therapy, HIV-1 could rebound even from an undetectable and/or very roll-over study BCN 02-Romi (NCT02616874) involving analytical cART B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 67 interruption. An amendment was approved to enroll 24 additional indi- enzyme-linked immunoabsorbent spot (ELISpot) assay. The IFN-γ viduals who commenced the same cART regimen and were followed for Mabtech kit was used according to manufacturer's instructions. All pep- 60 weeks, but did not receive any vaccine (Controls). This group was tides used in the study were 15-mer peptides overlapping by 11 amino only introduced to address potential differences in viral reservoir acid residues covering the full HIV-1 proteome consensus for subtype B decay between any of the two vaccination arms and the natural decay and were obtained through the NIH AIDS Reagent Program, except for of the 1st year of INSTI-based cART when started at acute/recent stages the HIVconsv immunogen sequence, for which a matching 199- of HIV-1 infection. No safety or immunological analyses were conducted peptide set (Ana Spec, San José, CA; 95,131) was generously donated in the control arm as HIV-1-specific T-cell responses were expected to by the International AIDS Vaccine Initiative. Peptides were combined contract once cART mediated viral suppression was achieved. Flow into 6 pools P1-P6 of 32–33 peptides per pool corresponding to the chart of the study design and patient disposition is depicted in Fig. 1a, HIVconsv vaccine insert (IN pools for ‘inside’ the immunogen) and 12 and the chronogram of the study is shown in Fig. 1b. pools of 39–67 peptides per pool spanning the rest of the HIV-1 viral Criteria for documented acute and recent infection included plasma protein sequences (OUT pools for outside the immunogen). All peptides HIV-1 RNA with Ab (EIA) negativity, Gag p24 antigen assay positivity, an pools were tested in duplicates. The final concentration of individual indeterminate Western immunoblot signal or the absence of p31 band peptides in the ELISpot assay was 1.57 μg/mL. Medium only was used in a positive Western blot. Days since estimated HIV-1 acquisition and as no-peptide negative control in quadruplicate wells, and PHA (1 Fiebig staging at treatment initiation time point were defined according μg/mL) and a CEF peptide pool (2 μg/mL) consisting of 23 previously de- to the time since known exposure, acute antiretroviral syndrome and/or fined human CD8+ T-cell epitopes from cytomegalovirus, Epstein–Barr results of clinical laboratory tests at the time of HIV-1 diagnosis [47]. virus and influenza virus (C.T.L. OH, USA) were added as positive con- The study protocol as well as patient information sheet and consent trols. All peptide stocks were stored at − 80 °C until use. Spots were forms were discussed, reviewed and approved by the Community Advi- counted using an automated Cellular Technology Limited (C.T.L., OH, sory Committee of the HIVACAT program. All patients provided written USA) ELISpot Reader Unit. The threshold for positive responses was de- informed consent before enrolment. The study was approved by the in- fined as at least 50 spot-forming units (SFU)/106 PBMC (5 spots per stitutional ethical review board of both Hospital Universitari Germans well) and responses exceeding the mean number of SFU in negative Trias i Pujol and Hospital Clinic (Reference Nr AC-11-027) and by the control wells plus 3 SD of the negative control wells, or 3x the mean Spanish Regulatory Authorities (EudraCT 2011-000846-39, IND ap- of negative control wells, whichever was higher. provals with Nr 12-016 & 12-017). Risk of Genetically Modified Organ- For flow-cytometry and CD8+ T-cell suppression assays, cryopre- ism release to the environment was evaluated by the Ministry of served PBMCs were used from the vaccination time point (C0), at peak Environment (B/ES/12/10 & B/ES/12/09). ClinicalTrials.gov identifier: of vaccine-induced T cells (M1 or M4) and at the end of trial visit NCT01712425 (M24). PBMC were CD8+ T-cell depleted by magnetic bead separation (MACS Milteny Biotec) and stimulated with PHA (5 μg/mL) in RPMI 2.2. Procedures 10% fetal bovine serum (R10). After 3 days of stimulation, the CD4- enriched fraction was infected by spinoculation with HIV-1 BaL and Vaccinations started after 6 months on stable cART and with plasma IIIB isolates at a multiplicity of infection (MOI) of 0.01. HIV-1-infected virus load (pVL) b 50 copies/mL (week 24 of trial = C0). The ChAdV63. cells were cultured in duplicates or triplicates in R10 medium with HIVconsv and MVA.HIVconsv vaccines were GMP manufactured at the 20 IU/mL of interleukin 2 in 96-well round-bottomed plates, alone or to- Clinical Biomanufacturing Facility, University of Oxford, UK and IDT gether with unstimulated CD8+ T cells obtained by positive magnetic Biologika GmbH, Germany, respectively [19,24,48]. All vaccines were bead separation from an additional vial of PBMC thawed on the same stored below − 65 °C until use. Vaccines were thawed no more than day. Cultures at different CD8+ effector:CD4+ target ratios (E:T of 1:1, 30 min prior to injection. Vaccination with 5 × 10 [10] virus particles 1:2 and 1:10) were harvested after 6 days. Cells were stained first (vp) of ChAdV63.HIVconsv at week 0 (C0) was followed by 2 × 10 [8] with Aqua Live/Dead and fixed with 1% paraformaldehyde/20 μg/mL ly- plaque-forming units (PFU) of MVA.HIVconsv at weeks 24 or 8 (M0). solecithin at room temperature, permeabilized with cold 50% methanol Vaccines were administered by intramuscular needle injection into followed by 0.1% Nonidet P-40, and finally stained with anti-Gag p24 the deltoid region of both arms (each dose was divided between two (KC-57-FITC; Beckman Coulter), anti-CD3 (APC-Cy7, BD Biosciences), arms). The study entailed a total of 24 weeks of clinical and laboratory anti-CD4 (PerCP, BD Biosciences), and anti-CD8 (APC, BD Biosciences) follow-up after the last vaccination. Visits at 1, 4, 12 and 24 weeks mAbs. CD8+ T-cell antiviral activity was expressed as percentage of in- after MVA.HIVconsv boost are designated M1, M4, M12 and M24, respec- hibition calculated as: [(fraction of p24+ cells in CD4+ T cells cultured tively. Antiretroviral treatment was maintained during the trial in all 48 alone) – (fraction of p24+ in CD4+ T cells cultured with CD8+ T individuals. cells)]/(fraction of p24+ cells in CD4+ T cells cultured alone) × 100. Data previously generated from cART-suppressed individuals treated 2.3. Outcomes during chronic HIV-1 infection [49] and from a longitudinal observa- tional cohort of elite and viremic controller individuals (EO-09-042) The primary objective of the study was to evaluate the safety of were used for comparison of CD8+ T-cell antiviral activity from early ChAdV63.HIVconsv and MVA.HIVconsv vaccines administered sequen- cART-treated BCN 01 participants. Elite and viremic controller individ- tially in a heterologous prime-boost regimen with two intervals to uals were defined as individuals with sustained levels of pVL b50 or HIV-1-positive adults. Local and systemic events were solicited prospec- b2000 copies/mL, respectively, for more than 10 years in the absence tively for a minimum of 7 days following each immunization. Both local of cART. Total CD8-depleted cells from day 0 and CD4-enriched fraction and systemic events were graded according to NIH Division of AIDS. Ad- from day 3 were stained for the exhaustion marker PD-1 using mAb verse Events (AEs) were specified as unrelated, unlikely, probably or conjugated to PE (BioLegend). definitely related to the vaccination. To quantify the size of the peripheral blood proviral reservoir, lysed Secondary endpoints included several immunological and virologi- extracts from 2.5 × 106 CD4+ T cells were used to measure total cell- cal readouts. Total HIV-1 and HIVconsv-specific T cells were assessed associated HIV-1 DNA by ddPCR with primers and probes binding using cryopreserved peripheral blood mononuclear cells (PBMC) ob- within 5′-LTR and Gag [50]. The RPP30 cellular gene was quantified in tained at week 0 as baseline (BL: at day of cART initiation); week 24 parallel to normalize sample input [39,50,51]. (C0: pVL b 50 copies/mL, before vaccination); weeks 1 and 4 after To evaluate HIV-1 RNA below 40 copies/mL, 4–8 mL of plasma sam- ChAdV63.HIVconsv (C1 and C4); and at and 1, 4 and 24 weeks after ples were ultracentrifugated at 170,000g at 4 °C for 30 min before quan- MVA.HIVconsv (M0, M1, M4 and M24) using an IFN-γ-detecting tification using the Abbott Real-Time HIV-1 assay (Abbott Molecular 68 a Detection of acute/early HIV infections b HGTIP HClinic Sequential allocation B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 Long Arm Short Arm Control Arm n=14 n=13 n=27 2 withdraw consent 1 withdraw consent 2 withdraw consent before Vax at wk4 before Vax at wk4 before w4 1 moved at wk24 VACCINATIONS VACCINATIONS n=12 n=12 wk24 post wk24 post wk60 MVA.HIVconsv Last MVA.HIVconsv Last Last Follow-Up Follow-Up Follow-Up n=24 n=12 n=12 Safety, Safety, Immunogenicity & Immunogenicity & Reservoir Analysis Reservoir Analysis Reservoir Analysis n=24 n=12 n=12 Fig. 1. Disposition of participants and flow chart of the study. Consolidated Standards of Reporting Trials (CONSORT) flow diagram for the trial. Trial BCN 01 was a non-randomized, open label, sequence allocation study. CONSORT diagram delineates the study enrollment of 54 subjects who underwent sequential allocation to the long, short vaccination arms and the control group. Three subjects withdrew from the study before vaccination and/or week 24 and were replaced during enrollment period. All the participants in the Long and Short arms (b) received the ChAdV63.HIVconsv and MVA.HIVconsv vaccines, while 24 individuals in the control arm did not receive any vaccine. All 48 subjects completed the study as per protocol. The 24 vaccinated individuals were included in the safety and immunological analyses. Latent viral reservoir was measured in all 48 participants. B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 69 Inc.). Serial dilutions of a positive control down to 5 HIV-1 RNA diagnosed and younger population over the recruitment periods. All copies/mL were used as a standard curve to calculate quantitative the 48 participants showed over 99% visit adherence and all completed values from raw qPCR 51CT data [39]. the study. Study participants' demographics are given in Table 1. Plasma viral loads b 50 copies/mL were reached within median of 12 weeks in 2.4. Statistical Analysis all groups as expected for an integrase inhibitor-based regimen (INSTI) (Fig. 2a). Baseline CD4+ T-cell counts (Fig. 2b) and CD4+/ Trial BCN 01 was an exploratory pilot study and, because of the small CD8+ T-cell ratio (Fig. 2c) significantly increased during the trial during sample size of the study, was not powered to detect significant differ- the first year of cART. There was a tendency in older individuals towards ences between arms and only allowed to detect trends in safety, immu- lower CD4+ T-cell counts at baseline (Spearman r = −0.27; p = 0.06) nological and virological effects, which collectively inform the design of and poorer CD4+ T-cell recovery over the first 60 weeks after treatment future studies. The safety endpoints were described in the Long and initiation (Spearman r = −0.24; p = 0.09) (Appendix Fig. 1). Short arms and summarized by the number and percentage of AEs and their grading. Immunogenicity endpoints were described in the 3.2. Both ChAdV63.HIVconsv and MVA.HIVconsv vaccines were safe and Long and Short arms. The minimum breadth of the T-cell response well tolerated was estimated as the number of peptide pools eliciting a positive re- sponse. The total frequency of HIV-1-specific IFN-γ+ T cells was calcu- All 24 subjects from the Long and Short arms were included in the lated as the sum of the SFU/106 PBMC stimulated with individual safety analysis. Overall, the vaccines were well tolerated. One partici- peptide pools. pant experienced a severe adverse event (SAE) prior to the administra- Differences in the breadth and frequency of the HIV-1-specific re- tion of the ChAdV63.HIVconsv vaccine (acute pancreatitis requiring sponses and virologic determinations between two longitudinal deter- hospitalization). No other serious adverse reactions or suspected unex- minations in the same individual were assessed using a Wilcoxon pected adverse reactions occurred during the study. A total of 334 AE signed-rank test. Differences between groups were compared using a were recorded during the study (182 in the Long Arm and 152 in the Mann–Whitney U-test or Kruskal-Wallis, ANOVA tests as indicated. Short Arm, Chi-square, p = 0.25), the majority of which (300, 90%) Only the reservoir measurement (proviral DNA) was performed in the were mild or moderate (Grade 1–2). The number of AEs per patient Control arm. Correlations between the HIV-1-specific responses, provi- was not significantly different between the Long and Short vaccination ral DNA and clinical parameters were performed using non-parametric Arm (Chi-square, p = 0.590). Spearman's rank correlation. Missing data due to technical problems The summary of local and systemic AEs related to vaccination is were censored from the analysis, and no imputation techniques were shown in Table 2. Local and systemic events after vaccination occurred used. Censored matched pairs of ultrasensitive viral load were analyzed in 22/24 individuals. Peak of reactogenicity was observed at 24–48 h using the paired Prentice-Wilcoxon test. Statistical significance was set after vaccination and resolved spontaneously within 3 days. The most at 5% for all the univariate tests. The analyses were performed with R frequently reported local reactogenicity AE was a Grade-1 pain reported (v3.0.2) and GraphPad Prism (v5.01) for Mac OS X (San Diego, CA). in both the Long and Short arms, and more frequently reported after the Flow data were analyzed using FlowJo software. MVA.HIVconsv vaccination than after the ChAdV63.HIVconsv. The most frequently reported systemic reactogenicity event was malaise. As for 3. Results laboratory abnormalities, no Grade 3 or 4 abnormalities were observed in the performed hematological and biochemical tests (data not 3.1. Participants Enrolled in the Study shown). Overall, the vaccines were safe and consistent with safety and tolerability data previously observed in HIV-1-negative volunteers [22, Between October 5, 2012 and April 15, 2013, 27 acute/early HIV-1- 25]. infected individuals were sequentially enrolled into the Long and Short arms of BCN 01. Three participants withdrew consent before vac- 3.3. ChAdV63.HIVconsv-MVA.HIVconsv regimen induced high frequencies cinations due to suboptimal compliance expectations for follow-up of T cells against conserved regions of HIV-1 in all vaccine recipients visits or reasons unrelated to the trial. Between May 27, 2013 and April 10, 2014 individuals were enrolled into the no-vaccine arm Peptide pools P1-P6 covering the HIVconsv immunogen (778 amino (Fig. 1b). Recruitment into the BCN 01 trial was enhanced by an intensi- acids) contained 15-mer peptides overlapping by 11 amino acids fied MSM testing campaign, which resulted in a gradually earlier- (Fig. 3a). Out of the 8 time points analyzed for each vaccine recipient Table 1 Clinical characteristics of the patients included in the BCN 01 study. Long arm (A) Short arm (B) Controls 0–24 weeks 0–8 weeks No vaccine (n = 12) (n = 12) (n = 24) Median age at HIV-1 diagnose 41 (30–54) 38 (27–48) 34 (19–62) Sex (M/F) 11/1 12/0 24/0 MSM/HTSa 9/3 12/0 24/0 Days since HIV-1 to cART, median (range) 91 (28–203) 82 (32–116) 73 (17–130) Fiebig stage at cART initiation, I (1, 4%) (numer and Percent of individuals) IV (1, 4%) V (6, 50%) V (8, 66%) V (18, 75%) VI (6, 50%) VI (4, 33%) VI (4, 17%) Log10 of pVL before cART, median (range) 5.04 (3.20–5.84) 4.80 (3.35–5.48) 5.18 (2.91–6.39) CD4 (cells/mm3) before cART, median (range) 574 (299–785) 519 (309–990) 482 (224–1014) CD4 (cells/mm3) at week 60, median (range) 664 (291–986) 684 (437–1161) 785 (451–1876) CD4/CD8 ratio before cART, median (range) 0.54 (0.17–1.26) 0.60 (0.27–2.15) 0.68 (0.14–2.38) CD4/CD8 ratio at week 60, median (range) 1.10 (0.68–1.66) 1.29 (0.73–2.34) 1.12 (0.53–2.34) Number of individuals with B*27/B*57/B*58 3 (3/0/0) 0 (0,0,0) 3 (1/3/0) a MSM (men who have sex with men)/HTS (heterosexual). 70 B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 ChAdV63.HIVconsv and the remaining 9 individuals (37%) after MVA. HIVconsv. There was no difference between the Long and Short arms, whereby 8 and 7 subjects responded to ChAdV63.HIVconsv, and 4 and 5 responded only after MVA.HIVconsv, respectively. For all individuals, the peak of total T-cell frequencies (sum of frequencies to HIVconsv P1-P6 pools) was detected after MVA.HIVconsv. Individuals in the Long arm showed a tendency towards reaching peak responses more slowly than subjects in the Short Arm with 2 vs 6 individuals peaking at 1 week (M1) and 9 vs 6 individuals peaking at 4 weeks after MVA. HIVconsv (M4), respectively (Chi-square, p = 0.08). Altogether, median (range) total peak total frequencies of HIVconsv-specific T cells reached 938 (73-6,805) SFU/106 PBMC, which represented an absolute increase in the frequencies of 750 (124-1,948) and 1,015 (73-6,535) SFU/106 PBMC from the time point before vaccination (C0) in the Long and Short arm, respectively (Wilcoxon signed-rank, p b 0.0001) (Fig. 3d). The longevity of vaccine-elicited responses was not statistically differ- ent between the Long and Short Arms as frequencies of total HIVconsv-specific T cells were 390 (60-1,701) and 140 (0-1,567) SFU/ 106 PBMC at 24 weeks after the MVA.HIVconsv administration in these arms (M24), respectively (Fig. 3e). 3.4. ChAdV63.HIVconsv-MVA.HIVconsv vaccination elicited responses to multiple epitopes within the HIVconsv immunogen Effector CD8+ T-cell recognition of multiple conserved epitopes will likely be one of the key features of a successful HIV-1 vaccine. Vaccine recipients responded to median (range) of 4 [1–6] peptide pools out of 6 vaccine peptide pools P1-P6 (Appendix Fig. 2). There was no signif- icant difference in the specific T-cell frequencies for the different pools (ANOVA test, p = 0.3211, Fig. 4a), indicating balanced and polyspecific vaccine-induced responses across the HIVconsv immunogen. Most (83%) of the HIVconsv peptide pool-specific T cells detected at peak im- munogenicity were considered de novo responses as they were not de- tectable before cART initiation (BL), when high levels of plasma viremia were present. This was the case for peptide pools P4-P6 (Fig. 4b), which covered the conserved regions of polymerase, integrase, Vif, and Env gp41 and gp120 regions included in the HIVconsv immunogen. The highest proportion of pre-vaccination responses was directed against pool P1 covering partial Gag sequences (Fig. 4b). Finally, in individuals who showed response to HIVconsv peptide pools at the time of HIV-1 diagnosis (12 individuals, 16 responses in total), vaccination effectively boosted their pre-existing responses. Indeed, these presumably ex- panded T cells reached higher frequencies of median (range) of 497 (87–3250) SFU/106 PBMC than de novo vaccine-induced T cells of me- dian (range) of 156 (0–1440) SFU/106 PBMC (Mann–Whitney U-test, p = 0.0029) (Fig. 4c). Fig. 2. Immune recovery after early treatment initiation. Evolution of pVL (a) and CD4 T- cell counts (b) and CD4/CD8 ratio (c) over the first 60 weeks after early-cART start with 3.5. ChAdV63.HIVconsv-MVA.HIVconsv vaccination shifts immuno- TDF/FTC/RAL in study participants. dominance T-cell patterns towards conserved regions of the HIV-1 prote- ome without induction of responses against junctional regions Next, we assessed whether or not the HIVconsv vaccinations led to a (total of 192 time points), 4 samples (2% of evaluated samples) were non-specific expansion of T cells targeting HIV-1 regions not present in censored due to low positive controls and/or high background. T-cells the HIV-1 immunogen or T cells specific for unrelated co-pathogens. to the HIVconsv immunogen of median (range) frequency of 286 (55- The non-HIVconsv HIV-1-specific responses referred to as the OUT pep- 2,640) SFU/106 PBMC were detected in 50% of study participants in tide pools and those directed to the CEF peptide pool were determined the Long (Fig. 3b) and Short (Fig. 3c) arms equally at HIV-1 diagnosis in an IFN-γ ELISPOT assay. The dominance of HIVconsv-specific re- and before cART (BL). Individuals with and without HIVconsv pre- sponses was calculated at each time point as the percentage of existing responses did not differ in duration with untreated infection HIVconsv-specific T-cell frequencies divided by the total HIV-1 or pre-cART viremia. All HIV-1-specific T cells were significantly re- proteome-specific (HIVconsv + OUT) T-cell frequencies. A significant duced after initiation of cART and viral suppression to frequencies of reduction in total HIV-1-specific T cells after viral suppression by cART median of 75 (0–476) SFU/106 PBMC (BL vs C0; Wilcoxon signed-rank, and elimination of HIV-1 antigenemia was observed (Wilcoxon p = 0.0173). All vaccine recipients completed ChAdV63.HIVconsv- signed-rank, p b 0.001 at C0 (Fig. 5a). No expansion of T cells targeting MVA.HIVconsv immunizations and all 24 (100%) showed an absolute HIV-1 regions outside of the immunogen was noted over the vaccina- increase in HIVconsv-specific IFN-γ-producing T cells during the tion time points, indicating that the vaccines selectively expanded study; in 15 individuals (63%) showed increase already after HIVconsv-specific T cells. An effective shift of response patterns towards B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 71 Table 2 Number and proportion of volunteers suffering local or systemic side effects related to vaccination. ChAdV63.HIVconsv MVA.HIVconsv Grade 1 Grade 2 Grade 3 Any Grade 1 Grade 2 Grade 3 Any Local reactogenicity Redness/Erythema (≥2.5 cm diameter)a Left arm 0 0 0 0 (0%) 1 0 0 1 (4%) Right arm 0 0 0 0 (0%) 1 1 0 2 (8%) Induration (≥2.5 cm diameter)b Left arm 0 0 0 0 (0%) 0 0 0 0 (0%) Right arm 0 0 0 0 (0%) 0 0 0 0 (0%) Local pain Left arm 13 1 1 15 (62%) 13 9 1 23 (96%) Right arm 12 1 0 13 (54%) 11 9 0 20 (83%) Systemic adverse events Fever 0 0 0 0 (0%) 2 0 1 3 (12%) Headache 8 2 1 11 (46%) 7 5 1 13 (54%) Malaise 5 5 1 11 (46%) 8 8 2 18 (75%) Nausea 0 0 0 0 (0%) 0 0 0 0 (0%) Diarrhea 1 1 0 2 (8%) 3 0 1 4 (17%) Sweating 2 2 0 4 (17%) 7 0 0 7 (29%) Myalgia 2 5 0 7 (29%) 8 5 2 15 (62%) Anorexia 2 0 0 2 (8%) 1 2 0 3 (12%) Abdom pain 0 1 0 1 (4%) 3 3 0 6 (25%) Note to local AE: a Injection-site erythema not reaching Grade 1 DAIDS criteria (b 2.5 cm in diameter) was observed in 4 (17%) and 4 (17%) participants in the left/right arm, respectively after the ChAdV63.HIVconsv vaccination, and in 12 (50%) and 9 (37%) participants on the left/right arms, respectively after the MVA.HIVconsv vaccination. b Injection-site induration not reaching Grade 1 DAIDS criteria (b 2.5 cm in diameter) was observed in 2 (8%) and 3 (12%) participants in the left/right arm, respectively after the ChAdV63.HIVconsv vaccination, and in 17 (71%) and 14 (58%) participants on the left/right arm, respectively after the MVA.HIVconsv vaccination. conserved T-cell epitopes reached its peak after the MVA.HIVconsv vac- test, p b 0.0001) and closer to levels observed in cohorts of elite and vi- cination with median (range) of 58% (7%–100%) of the total anti-HIV-1 remic controllers (median 84%, Mann–Whitney U-test, p = 0.0074, immune responses. Despite some decay of vaccine-elicited T-cell fre- Fig. 6a). This high suppressive capacity was maintained during the en- quencies, this focus was maintained over time and was still 33% (0%– tire vaccination period, was also observed at lower E:T ratios and was 100%) of all detectable HIV-1 responses 24 weeks after the last vaccina- confirmed with both 2 clade B viruses tested (Fig. 6b). In line with an ef- tion (M24) (Fig. 5b). Similarly, no increases in the frequency of re- fective antiviral T-cell response, the levels of the PD-1 exhaustion sponses to the CEF peptides were observed over the vaccination time markers on total CD8+ T cells were maintained at low levels throughout points (Fig. 5c). the study period with median (range) of 23% (10–45%) of CD8+ T cells Additionally, we determined the immunogenicity of potential junc- suggesting that early cART initiation might preserve CD8+ T-cell func- tional neo-epitopes located across the adjacent segment junctions of tion and consequently allow for an effective vaccine response (Fig. 6c). the HIVconsv immunogen [19] by comparing response rates to two sets of peptide pools with and without junctional peptides. In contrast to what was observed in an HIV-1-negative cohort where ~ 20% of in- 3.7. Early cART, but not HIVconsv vaccination limits the size of latent viral duced responses were specific for junctions [24], responses to both reservoir sets of peptide pools were of comparable frequency all individuals (Fig. 5d), indicating that responses against junctional regions were min- To determine whether vaccine-induced immune activation resulted imal if induced at all. in an increased HIV-1 replication despite cART, pVL was measured just prior to and at 1 week after the MVA.HIVconsv administration (M0 and M1) in all vaccinated subjects using both standard and ultrasensi- 3.6. Early cART preserves antiviral function of CD8+ T cells and might tive assays. Overall, 6 participants had plasma HIV-1 RNA “blips” of me- contribute to an efficacious vaccine response dian (range) of 200 (37-119) RNA copies/mL, whereby 40 copies/mL is the detection limit, while on reported optimal cART adherence along To determine CD8+ T-cell mediated viral inhibitory capacity against the entire trial follow-up period (data not shown). Using the ultrasensi- HIV-1 BaL and IIIB isolates at different CD8:CD4 ratios (E:T = 1:1, 1:2 tive pVL assay, plasma viremia was detectable in 92% of individuals at and 1:10), longitudinal cryopreserved-and-thawed samples obtained M0 or M1 (Fig. 7a). We observed 8 patients with a 2-fold increase in ul- at 6 months on stable cART and just before vaccination (C0), at the trasensitive pVL 1 week after vaccination (M1), but changes in the ultra- peak of vaccine-induced immunogenicity (M1 or M4, collectively sensitive pVL in the overall cohort were not statistically significant Mpeak) and at the end of trial in all individuals from the Long and between the two tested time points (Fig. 7a). In addition, there was Short arms (M24) were used in in vitro viral replication inhibition as- no correlation between the frequencies of HIVconsv-specific IFN-γ- says. Data previously generated from individuals virologically sup- producing cells at peak and the detected increases in plasma viremia pressed, who were treated during chronic HIV-1 infection [49] and (Spearman r 0.3122, p = 0.1375; Fig. 7b). from eight long-term controller individuals were used for comparison To determine the proportion of total circulating CD4+ T cells harbor- with the CD8+ T-cell antiviral activity from early-cART-treated BCN 01 ing proviral DNA and to identify potential changes in the viral reservoir participants. CD8+ T-cell viral inhibition of the BaL virus was higher in size induced by vaccination, total HIV-1 DNA copies in purified PBMC- vaccinated individuals at C0 at median (range) of 68% (37–87%) viral in- derived CD4+ T cells were quantified longitudinally. Total HIV-1 DNA hibition when compared to the same day levels described in individuals was measured just prior to vaccination when all subjects had achieved treated during chronic HIV-1 infection (median 20%, Mann–Whitney U- pVL b 50 copies/mL after 24 weeks under stable cART and at 12 and 72 B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 Fig. 3. Vaccination immunogenicity. Cryopreserved, unexpanded PBMC were stimulated with pools P1–P6 of overlapping 15-mer peptides across the HIVconsv immunogen in an IFN-γ ELISPOT assay. (a) Schematic representation of the employed conserved regions in the HIV proteome from different HIV-1 clades included in the HIVconsv immunogen and distribution of the set of 6 peptide pools used for immunogenicity studies. Magnitude of total HIVconsv-specific responses (sum of SFU/106 PBMC to pools P1-P6) over trial duration in the Long (b) and Short (c) vaccination arms are shown. (d) Total magnitude of HIVconsv-specific responses before and at peak immunogenicity in all vaccinated individuals. Median total frequency for the entire cohort is shown in red. Wilcoxon signed-rank p value is shown (e) Comparison of total magnitude of HIVconsv-specific responses between Long and Short vaccination arms at different time points of the clinical study. Mann–Whitney U-test is used for comparisons between Long and Short arms, and Wilcoxon signed-rank for comparisons within timepoints in the same individual. 24 weeks post MVA.HIVconsv for both the Long and Short arms (~1 year suppressed individuals who started cART during chronic HIV-1 infection after treatment initiation). Proviral DNA from 24 early treated, but not (median of 1065 copies/106 CD4+ T cells) [49]. The proviral DNA decay vaccinated individuals (Control) was measured for comparison with was consistent with a decay over the first year of early initiated cART the natural decay due to cART initiation. At pre-vaccination, total HIV- [50], but was not further accelerated by the ChAdV63.HIVconsv-MVA. 1 DNA was not statistically different between the Long, Short and Con- HIVconsv vaccinations (Fig. 7d). trol arms with median (range) of 815 (134-5,660), 639 (24-3,289) Levels of proviral DNA in PBMC-derived CD4+ T cells measured at and 358 (54-7,665) copies of total HIV-1 DNA/106 CD4+ cells, respec- pre-vaccination (BL) and at the end of study (M24) did not correlate tively (Fig. 7c). Peripheral blood CD4+ T-cell proviral reservoir showed with estimated days since HIV-1 acquisition to cART initiation and similar decay kinetics in all groups. Overall, in the compiled BCN 01 co- were not associated with either the HIVconsv-specific nor the total hort (n = 48), we observed median (range) of 342 (5.4-4,565) copies of HIV-1-specific T-cell frequencies (data not shown). Overall, the fre- HIV-1 DNA/106 CD4+ cells at 1 year after treatment initiation. These quency of CD4+ T cells harboring HIV-1 DNA correlated well with pre- levels of proviral DNA were 3-fold lower than values in virally cART viremia in acute/recent infection (Spearman r = 0.54, p b B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 73 Fig. 4. Breadth of vaccine-elicited HIVconsv-specific T-cells. (a) Comparison (ANOVA p-value) of the frequency of each participant's response to individual peptide pools at the peak immunogenicity time point after MVA.HIVconsv booster vaccination and (b) percentages of participants showing a detectable response (‘responders’) to HIVconsv peptide pools either before any vaccination (white bars) or at the peak immunogenicity time point (gray bars). (c) Comparison of the frequency of responses detected at the peak immunogenicity time point reflecting either de-novo induced or vaccine-boosted (‘pre-existing’) responses. Mann–Whitney U-test p value is shown. 0.0001 at C0, and Spearman r = 0.59, p b 0.0001, at M24; see Appendix side effects limited to Grade 1 and 2, whereby most AEs judged to Fig. 3). be related to the vaccination were transient and resolved spontane- ously within 3 days. These data support the previously reported 4. Discussion safety profile of the combined ChAdV63.HIVconsv and MVA. HIVconsv vaccination in HIV-1-negative individuals immunized The present BCN 01 trial data show that a heterologous regimen with the same regimen [25]. using the ChAdV63.HIVconsv and MVA.HIVconsv vaccines in a cohort Our analyses show that T-cell responses to conserved regions of HIV- of early-treated individuals with acute or recent HIV-1 infection was a 1 were sub-dominant in natural HIV-1 infection [53,54]. Administration safe and effective strategy to shift the immunodominance of the total of HIVconsv vaccines was able to induce broad and potentially novel T- HIV-1-specific T-cell response towards conserved regions of HIV-1. Vac- cell responses by strongly refocusing the HIV-1-specific T cells to con- cination led to a marked and highly selective expansion of pre-existing served epitopes contained in the immunogen insert. These responses as well as induction of new T-cell responses to HIVconsv, but failed to were of high frequency, although lower than vaccine responses seen impact the size of the in vivo latent viral reservoir. in the HIV-1-negative individuals included in the earlier HIV-CORE 02 A successful therapeutic vaccination may ultimately be achieved by trial [24]. In the HIV-CORE 02 trial, median (range) frequency of an optimal T-cell immunogen and the use of highly immunogenic vac- HIVconsv-induced peak responses induced in the ChAdV63.HIVconsv- cine vector or vector combination, which can refocus HIV-1-specific MVA.HIVconsv arm was 5150 (1475–16,510) SFC/106 PBMC24. How- CTL response to vulnerable sites of the virus [15,19–21,52]. Here, we ever, it is important to note that HIV-CORE 02 used freshly isolated tested the immunogenicity of two heterologous prime-boost regimens PBMC IFN-γ ELISPOT assays, which has been shown by reference labs with simian adenovirus and MVA-vectored vaccines expressing the and some of the co-authors using HIV-CORE 02 samples to lead to ap- first-generation conserved immunogen HIVconsv in a population of proximately 5-times higher frequency when compared to frozen-and- early-diagnosed and early-treated HIV-1-positive individuals in Barce- thawed cells (Spearman r = 0.9156; P b 0.0001). Therefore, peak lona, Catalonia, Spain. Overall, the vaccines were well tolerated with vaccine-induced T-cell levels observed in BCN 01 were comparable to 74 B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 Fig. 5. Changes in T-cell dominance patterns. (a) Schematic representation of the average distribution of total HIV-1 T-cells among different HIV-1 proteins at baseline (BL), its decrease during viral suppression and its expansion at peak responses. HIVconsv-specific responses are shown in purple. Sizes of pie charts are to scale with total frequencies of responses. Acc - Accessory proteins. (b) Median frequency of total HIVconsv-specific T cells (green bars) and changes in median HIVconsv immunodominance (red line) are shown over time. (c) Mean ± SD frequencies of T cells specific for OUT and CEF peptide pools are shown over time. (d) Comparison of the frequency of individual HIVconsv peptide pool responses detected in 15 study subjects using two sets of 15-mer peptides covering (P1-P6, + junctions) or avoiding (P1-P6, − junctions) the junctional regions is shown. Wilcoxon signed-rank t-test is used. those observed in HIV-1-negative individuals in HIV-CORE 02 and con- limited by the non-randomized nature of the study and the small siderably stronger than responses reported in past therapeutic vaccine size of the clinical trial, we did not observe statistically separable dif- trials such as the ones using canarypox ALVAC-HIV vaccine [6] or ho- ferences in the frequency or breadth of induced responses, function- mologous vaccinations with MVA-B [55] in chronically suppressed indi- ality or longevity between the longer and the shorter vaccination viduals. Thus, our data strongly suggest that potent heterologous prime- intervals. This does not exclude the possibility that larger, random- boost vaccination regimens including an adenovirus-vectored prime as ized trials may reveal some differences between different dosing in- well as early cART initiation contribute critically to the effective refocus tervals though, and that these differences could have a potential and boost of T-cell responses to relevant epitopes in BCN 01. Although effect on viral control. B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 75 Fig. 6. High CD8+ T-cell viral inhibitory capacity and low levels of PD-1-expressing CD8+ T cells in early-treated individuals. (a) Comparison (Mann–Whitney U-test) of levels of CD8+ T- cell viral inhibition is shown for HIV-1Bal (E:T 1:1) in individuals, who started cART during chronic infection (Chronic), participants in BCN 01 (Early_cART) 24 weeks after cART initiation and before any vaccination (C0), and elite and viremic controllers (EC/VC). (b) Levels of CD8+ T-cell viral inhibition are shown for HIV-1Bal (E:T ratio 1:1, 1:2 and 1:10) for individuals in the Long and Short Arms before vaccination (C0), at peak of vaccine-induced immunogenicity (Mpeak) and at the end of trial (M24). (c) Expression of PD-1 by CD8+ T cells for the same time points. The frequency and kinetics of CD8+ T cell activation during acute immune responsiveness to the therapeutic vaccination strategy tested HIV-1 infection has recently been related to viral setpoint [4], suggest- in this population. ing that early treatment initiation could help maintain necessary HIV- Aside from the non-randomized nature of the trial, one potential ca- 1-specific CD8+ T-cell responses associated with subsequent control veat of the present study was that the relative contribution of HIVconsv of viremia. In line with this hypothesis, we observed also in our cohort vaccine-induced T cells to the total in vitro CD8+ T-cell inhibition activ- a more marked immune restoration (normal CD4/CD8 T-cell ratios) ity could not be accurately estimated. Thus, although we document a [56], a reduced expression of immune exhaustion markers and stronger strong refocus of the specificity of the T-cell response after vaccination, CD8+ T-cell viral inhibitory capacity than those reported for individuals it remains unclear how much these responses added to the observed in- who started treatment in the chronic phase of HIV-1 infection [49]. hibition of viral replication in vitro. The increase in viral inhibition ca- Some or all these factors might have contributed to an increased pacity could be limited due to the continuous presence of CD8+ T cells 76 B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 Fig. 7. Changes in ultrasensitive pVL after MVA.HIVconsv booster vaccination and proviral DNA decay dynamics. (a) Copies of HIV-1 RNA per mL of plasma are shown for each vaccine group just before MVA.HIVconsv (M0) and 1 week later (M1) with censored values below the limit of detection shown in gray. Prentice-Wilcoxon p values are shown. (b) Correlation (Spearman r) between total frequency of HIVconsv-specific T cells at the peak immunogenicity timepoint and the absolute increase in pVL. (c) Total HIV-1 DNA copies/106 CD4+ T cells for each vaccination group are shown at week 24 (before vaccination) and at week 56/60 after treatment initiation (Wilcoxon signed-rank p values are shown for comparisons within timepoints in same individual) (d) Comparison (ANOVA) of fold change of proviral DNA over 1 year after viral suppression from week 24 of cART in all groups (mean ± SE). targeting non-protective epitopes, the existence of other vulnerable non-vaccinated individuals. However, the same was reported in other viral epitopes not covered by HIVconsv immunogen and/or insufficient therapeutic vaccine trials, where less immunogenic regimens were T-cell help stimulation, which improved vaccine immunogen designs tested in chronic cART-suppressed subjects [49,55]. In that sense, the re- may overcome [15,19,21,57]. On the other hand, it is likely that the vac- sults may not be surprising as no latency reversing agent was adminis- cination contributed to the prolonged maintenance of responses able to tered in BCN-01 and vaccination did not induce robust signals of virus suppress HIV-1 replication in vitro, which would have been gradually reactivation either. In addition, the effect of vaccine-induced T-cells on lost if no vaccine were administered. While in trial BCN 01, the non- residual replicating cells in the tissue is unknown. Whether or not if ef- vaccinated control arm could not be tested for antiviral activity, this sce- fective, such elimination of HIV-1-producing cells in tissues would be nario concurs with recently reported data from the London-based RIVER detectable by reservoir analyses in the peripheral blood remains to be trial (NCT02336074 [58]). In that trial, using the same vaccine combina- assessed. tion along with vorinostat, the vaccinees maintained in vitro inhibitory Importantly, a change in T-cell immunodominance towards targets activity over time whereas placebo controls showed indeed a progres- of interest would not occur if the vaccination regimen led to a broad, sive decay, suggesting that also in BCN 01, the vaccine was responsible non-specific expansion of all HIV-1-specific T cells that existed before for the continuously high levels of antiviral activity observed. vaccination. We assessed this by measuring longitudinally T-cell re- Despite a strong expansion of T-cell responses to HIV-1, a prolonged sponses to regions of HIV-1 that both were and were not covered by maintenance of antiviral activity of these responses with vaccination the HIVconsv immunogen, by documenting T cells to epitopes from un- and relatively low reservoir levels at the end of the trial [38,59], we related viruses such as CMV, EBV and influenza virus as well as by deter- did not observe a more pronounced reduction in the size of the HIV-1 mining potential responses to junctional regions of the HIVconsv reservoir associated with vaccination when compared to non- chimeric protein. These analyses showed no signs of expansion of pre- vaccinated individuals. Moreover, no differences in the viral reservoir existing T-cell responses to HIV-1 regions not covered by the immuno- decay dynamics between the longer and the shorter vaccination regi- gen or of responses to other viral infections. Interestingly, a marked ab- men were noted. This does not exclude however the possibility that sence of responses to junctional regions contained in the HIVconsv more sensitive assays or larger, randomized trials and specially with sequence was also noted, which is in contrast to findings with the longer follow-up could reveal differences between vaccinated and same vaccination regimen in HIV-1-uninfected subjects [24] and B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 77 which may suggest that de novo responses may be primed differently in 2016 meeting (Abstract Nr. 320). We especially thank Rafaela Ayen HIV-1-negative versus already HIV-1-positive individuals. Alternatively, and Carmen Ligero for their technical assistance. We thank the opera- another possible explanation could be that low-levels of HIVconsv tional support of Okairos on the management with ChAdV63 vaccines immunogen-specific T-cell responses already existed at pre- given under the clinical trial agreement. After acquisition of Okairos, vaccination time points in the already HIV-1-infected subjects and GSK now co-owns the ChAdV63.HIVconsv vaccine and allowed the that vaccination expanded these responses rather than induced them BCN 01 trial to be completed. Special thanks to all the volunteers partic- de novo. Detailed T-cell receptor analyses on total T cells and epitope- ipating in this study for their perseverance and dedication, without specific populations will be needed to address this question. Neverthe- whom this trial would not have been possible. less, regardless of whether vaccination induced de novo responses or expanded T cells from very subdominant populations, the results in Role of the Funding Source BCN 01 document for the first time an impressive plasticity in the HIV-1-specific T-cell response patterns upon potent vaccination in BCN01 study was funded by the HIVACAT Catalan research program early-treated individuals. Thus, and to the best of our knowledge, this for an HIV vaccine and Fundació Gloria Soler. The GMP manufacture of is the first therapeutic HIV-1 vaccine trial able to demonstrate a shift the ChAdV63.HIVconsv and MVA.HIVconsv vaccines was jointly funded in the immunodominance of the virus-specific T cells towards con- by the Medical Research Council (MRC) UK and the UK Department for served regions of HIV-1 in a cohort of early-treated individuals. The International Development (DFID) under the MRC/DFID Concordat data provide strong rationale for the further development of this vac- agreements (G0701669). The funders had no role in the study design, cine strategy and incorporation of this regimen into future kick-and- data collection, data analysis, data interpretation, writing of the manu- kill approaches to address whether or not these responses can control script, or in the decision to submit the results for publication. rebounding virus after cART interruption. Members of the BCN 01 Study group Declaration of Competing Interest IrsiCaixa AIDS Research Institute-HIVACAT, Hospital Universitari BM, PC, ASB, MR, MCP, SML, BC, JMP and CB report grants from the Germans Trias i Pujol, Badalona, Spain: Beatriz Mothe, Pep Coll, Alvaro HIVACAT Catalan research program for an HIV vaccine and Fundació Sanchez-Bernabeu, Miriam Rosás, Maria C. Puertas, Sara Morón-López, Gloria Soler. BM holds a post-doctoral fellowship grant from ISCIII (JR Bonaventura Clotet, Javier Martinez-Picado and Christian Brander. 13/00024) from 2014 to 2016 during the conduct of the study and is a Fundació Lluita contra la Sida, Hospital Universitari Germans Trias i consultant for AELIX THERAPEUTICS, S.L., outside the submitted work. Pujol, Badalona, Spain: Patricia Cobarsi, Jordi Puig, Isabel Bravo, Roser SML holds a PhD grant from DGR (2013FI_B 00275) from 2013 to Escrig, Jessica Toro, Aintzane Ayestaran-Loinaz, Silvia Gel, Nuria Perez- 2016 during the conduct of the study. JMM received a personal 80:20 re- Alvarez, Arelly Ornelas, Cristina Perez-Reche and Ana Maria Barriocanal. search grant from the Institut d'Investigacions Biomèdiques August Pi i Hospital Clinic-HIVACAT, IDIBAPS, University of Barcelona, Spain: Sunyer (IDIBAPS), Barcelona, Spain, during 2017–19 and reports grants Christian Manzardo, Juan Ambrosioni, David Nicolas, Irene Ruiz, Cristina and personal fees from Abbvie, Angelini, Contrafect, Genentech, Gilead, Rovira, Carmen Ligero, Josep M. Gatell and Jose M. Miro. Jansen, Medtronic, MSD, Pfizer, ViiV Healthcare, outside the submitted Projecte dels noms, BCN Checkpoint, Barcelona, Spain: Jorge Saz, Mi- work. LD reports being employer of Immunocore. TH reports grants chael Meulbroek, Ferran Pujol. from Medical Research Council UK, during the conduct of the study, The Jenner Institute, The Nuffield Department of Medicine, Univer- and has a patent US 7981430B2 issued. CB is founder, CSO and share- sity of Oxford, UK: Nicola Borthwick, Alison Crook, Edmund G. Wee, holder of AELIX THERAPEUTIC, S.L. BC is founder, consultant and share- Lucy Dorrell and Tomáš Hanke. holder of AELIX THERAPEUTICS, S.L. TH reports grants from Medical Research Council UK, during the conduct of the study, and has a patent Author's Contributions US 7981430B2 issued. SML hold a PhD grant from DGR (2013FI_B 00275) from 2013 to 2016 during the conduct of the study. CM, ASB, BM, LD, TH, BC and CB conceived and designed the study. BM, CM, PCo, PCo, RE, NP, IR, CR, MM, AC, NB, EW, HY have nothing to disclose. PC, RE, IR, MM, AC, JA, DN, JMM, LD, BC and CB contributed to the study design and data management. BM, ASB, CR, MR, MCP, SML performed Acknowledgments the experiments. NP, AO, BM, MCP and SML undertook the statistical anal- ysis. NB, AC, EW, MR, LD, TH contributed with reagents/materials/analysis This study was presented in part at the IAS 2015 Conference, Van- tools. BM, SML, CB and TH drafted the manuscript. CM, PCo, MCP, JMM, couver, 2015 (Abstract Nr. MOPEA036); at the 7th International Work- JMG, LD, JMP and BC participated in study analyses and revised the man- shop on HIV-1 Persistence, HIV-1 Reservoirs and Strategies Towards uscript critically for important intellectual content. All authors reviewed HIV-1 Cure, Miami, 8-11th Dec 2015 (Abstract Nr. OP 9.3) and at CROI and approved the final version of the manuscript. Appendix A Appendix Fig. 1. Correlation between baseline CD4 T-cell counts (left), CD4 T-cell counts increase at week 60 (right) and age of individuals at study entry is shown. 78 B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 Appendix Fig. 2. Net frequencies of cells specific for each pool (color-coded) are shown for each participant. Participant's numbers are shown above the graphs, A and B corresponding to the Long and Short vaccination arms, respectively. Appendix Fig. 3. Correlation between the baseline plasma viremia (log10 HIV-1 RNA copies/mL of plasma) and proviral HIV-1 DNA levels (log10 copies/106 CD4+ T cells) 24 (left) and 60 weeks (right) after treatment initiation in all vaccinated and non-vaccinated individuals (Spearman r). B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 79 References [27] Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, et al. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009;361(23): [1] Mothe B, Ibarrondo J, Llano A, Brander C. Virological, immune and host genetics 2209–20. https://doi.org/10.1056/NEJMoa0908492. markers in the control of HIV infection. Dis Markers 2009;27(3):105–20. https:// [28] Colloca S, Barnes E, Folgori A, et al. Vaccine vectors derived from a large collection of doi.org/10.3233/DMA-2009-0655. simian adenoviruses induce potent cellular immunity across multiple species. Sci [2] Schmitz JE, Kuroda MJ, Santra S, et al. Control of viremia in simian immunodeficiency Transl Med 2012;4(115):115ra2. https://doi.org/10.1126/scitranslmed.3002925. virus infection by CD8 + lymphocytes. Science 1999;283(5403):857–60http:// [29] Buchbinder SP, Mehrotra DV, Duerr A, et al. Efficacy assessment of a cell-mediated www.ncbi.nlm.nih.gov/pubmed/9933172, Accessed date: 25 November 2015. immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo- [3] Cartwright EK, Spicer L, Smith SA, et al. CD8(+) lymphocytes are required for main- controlled, test-of-concept trial. Lancet 2008;372(9653):1881–93. https://doi.org/ taining viral suppression in SIV-infected macaques treated with short-term antire- 10.1016/S0140-6736(08)61591-3. troviral therapy. Immunity 2016;45(3):656–68. https://doi.org/10.1016/j.immuni. [30] Gray G, Buchbinder S, Duerr A. Overview of STEP and Phambili trial results: two 2016.08.018. phase IIb test of concept studies investigating the efficacy of MRK ad5 gag/pol/nef [4] Ndhlovu ZM, Kamya P, Mewalal N, et al. Magnitude and kinetics of CD8+ T cell ac- sub-type B HIV vaccine. Curr Opin HIV AIDS 2010;5(5):357–61. https://doi.org/10. tivation during hyperacute HIV infection impact viral set point. Immunity 2015;43 1097/COH.0b013e32833d2d2b.Overview. (3):591–604. https://doi.org/10.1016/j.immuni.2015.08.012. [31] Hanke T, Goonetilleke N, McMichael AJ, Dorrell L. Clinical experience with plasmid [5] Streeck H, Jolin JS, Qi Y, et al. Human immunodeficiency virus type 1-specific CD8+ DNA- and modified vaccinia virus Ankara-vectored human immunodeficiency T-cell responses during primary infection are major determinants of the viral set virus type 1 clade a vaccine focusing on T-cell induction. J Gen Virol 2007;88(Pt point and loss of CD4+ T cells. J Virol 2009;83(15):7641–8. https://doi.org/10. 1):1–12. https://doi.org/10.1099/vir.0.82493-0. 1128/JVI.00182-09. [32] Fidler S, Porter K, Ewings F, et al. Short-course antiretroviral therapy in primary HIV [6] Autran B, Murphy RL, Costagliola D, et al. Greater viral rebound and reduced time to infection. N Engl J Med 2013;368(3):207–17. https://doi.org/10.1056/ resume antiretroviral therapy after therapeutic immunization with the ALVAC-HIV NEJMoa1110039. vaccine (vCP1452). AIDS 2008;22(11):1313–22. https://doi.org/10.1097/QAD. [33] Le T, Wright EJ, Smith DM, et al. Enhanced CD4+ T-cell recovery with earlier HIV-1 0b013e3282fdce94. antiretroviral therapy. N Engl J Med 2013;368(3):218–30. https://doi.org/10.1056/ [7] Schooley RT, Spritzler J, Wang H, et al. AIDS clinical trials group 5197: a placebo- NEJMoa1110187. controlled trial of immunization of HIV-1-infected persons with a replication- [34] Lundgren JD, Babiker AG, Gordin F, et al. Initiation of antiretroviral therapy in early deficient adenovirus type 5 vaccine expressing the HIV-1 core protein. J Infect Dis asymptomatic HIV infection. N Engl J Med 2015;373(9):795–807. https://doi.org/ 2010;202(5):705–16. https://doi.org/10.1086/655468. 10.1056/NEJMoa1506816. [8] Harrer T, Plettenberg A, Arastéh K, et al. Safety and immunogenicity of an [35] Deng K, Pertea M, Rongvaux A, et al. Broad CTL response is required to clear latent adjuvanted protein therapeutic HIV-1 vaccine in subjects with HIV-1 infection: a HIV-1 due to dominance of escape mutations. Nature 2015;517(7534):381–5. randomised placebo-controlled study. Vaccine 2014;32(22):2657–65. https://doi. https://doi.org/10.1038/nature14053. org/10.1016/j.vaccine.2013.10.030. [36] Ananworanich J, Puthanakit T, Suntarattiwong P, et al. Reduced markers of HIV per- [9] Caskey M, Schoofs T, Gruell H, et al. Antibody 10-1074 suppresses viremia in HIV-1- sistence and restricted HIV-specific immune responses after early antiretroviral infected individuals. Nat Med 2017. https://doi.org/10.1038/nm.4268 January. therapy in children. AIDS 2014;28(7):1015–20. https://doi.org/10.1097/QAD. [10] Scheid JF, Horwitz JA, Bar-On Y, et al. HIV-1 antibody 3BNC117 suppresses viral re- 0000000000000178. bound in humans during treatment interruption. Nature 2016. https://doi.org/10. [37] Hocqueloux L, Avettand-Fènoël V, Jacquot S, et al. Long-term antiretroviral therapy 1038/nature18929 June. initiated during primary HIV-1 infection is key to achieving both low HIV reservoirs [11] Sneller MC, Justement JS, Gittens KR, et al. A randomized controlled safety/efficacy and normal T cell counts. J Antimicrob Chemother 2013;68(5):1169–78. https://doi. trial of therapeutic vaccination in HIV-infected individuals who initiated antiretrovi- org/10.1093/jac/dks533. ral therapy early in infection. Sci Transl Med 2017;9(419):eaan8848. https://doi.org/ [38] Hey-Cunningham WJ, Murray JM, Natarajan V, et al. Early antiretroviral therapy 10.1126/scitranslmed.aan8848. with raltegravir generates sustained reductions in HIV reservoirs but not lower T- [12] García F, Climent N, Guardo AC, et al. A dendritic cell-based vaccine elicits T cell re- cell activation levels. AIDS 2015. https://doi.org/10.1097/QAD.0000000000000625 sponses associated with control of HIV-1 replication. Sci Transl Med 2013;5(166): February. 166ra2. https://doi.org/10.1126/scitranslmed.3004682. [39] Martínez-Bonet M, Puertas MC, Fortuny C, et al. Establishment and replenishment of the [13] Hanke T. Conserved immunogens in prime-boost strategies for the next-generation viral reservoir in perinatally HIV-1-infected children initiating very early antiretroviral HIV-1 vaccines. Expert Opin Biol Ther 2014;14(5):601–16. https://doi.org/10.1517/ therapy. Clin Infect Dis 2015;61(7):1169–78. https://doi.org/10.1093/cid/civ456. 14712598.2014.885946. [40] Luzuriaga K, Gay H, Ziemniak C, et al. Viremic relapse after HIV-1 remission in a peri- [14] Kulkarni V, Valentin A, Rosati M, et al. Altered response hierarchy and increased T- natally infected child. N Engl J Med 2015;372(8):786–8. https://doi.org/10.1056/ cell breadth upon HIV-1 conserved element DNA vaccination in macaques. PLoS NEJMc1413931. One 2014;9(1):e86254. https://doi.org/10.1371/journal.pone.0086254. [41] Persaud D, Gay H, Ziemniak C, et al. Absence of detectable HIV-1 viremia after treat- [15] Mothe B, Llano A, Ibarrondo J, et al. Definition of the viral targets of protective HIV-1- ment cessation in an infant. N Engl J Med 2013;369(19):1828–35. https://doi.org/10. specific T cell responses. J Transl Med 2011;9:208. https://doi.org/10.1186/1479- 1056/NEJMoa1302976. 5876-9-208. [42] Colby DJ, Trautmann L, Pinyakorn S, et al. Rapid HIV RNA rebound after antiretroviral [16] Pereyra F, Heckerman D, Carlson JM, et al. HIV control is mediated in part by CD8+ treatment interruption in persons durably suppressed in Fiebig I acute HIV infection. T-cell targeting of specific epitopes. J Virol 2014;88(22):12937–48. https://doi.org/ Nat Med 2018;24(7):923–6. https://doi.org/10.1038/s41591-018-0026-6. 10.1128/JVI.01004-14. [43] Anderson JL, Fromentin R, Corbelli GM, Østergaard L, Ross AL. Progress towards an [17] Murakoshi H, Akahoshi T, Koyanagi M, et al. Clinical control of HIV-1 by cytotoxic T HIV cure: update from the 2014 International AIDS Society Symposium. AIDS Res cells specific for multiple conserved epitopes. J Virol 2015. https://doi.org/10.1128/ Hum Retroviruses 2015;31(1):36–44. https://doi.org/10.1089/AID.2014.0236. JVI.00020-15 March. JVI.00020-15-. [44] Shan L, Deng K, Shroff NS, et al. Stimulation of HIV-1-specific cytolytic T lymphocytes [18] Hancock G, Yang H, Yorke E, et al. Identification of effective subdominant anti-HIV-1 facilitates elimination of latent viral reservoir after virus reactivation. Immunity CD8+ T cells within entire post-infection and post-vaccination immune responses. 2012;36(3):491–501. https://doi.org/10.1016/j.immuni.2012.01.014. PLoS Pathog 2015;11(2):e1004658. https://doi.org/10.1371/journal.ppat.1004658. [45] Søgaard OS, Graversen ME, Leth S, et al. The depsipeptide romidepsin reverses HIV-1 [19] Létourneau S, Im E-J, Mashishi T, et al. Design and pre-clinical evaluation of a universal latency in vivo. PLoS Pathog 2015;11(9):e1005142. https://doi.org/10.1371/journal. HIV-1 vaccine. PLoS One 2007;2(10):e984. https://doi.org/10.1371/journal.pone. ppat.1005142. 0000984. [46] Leth S, Schleimann MH, Nissen SK, et al. Combined effect of Vacc-4x, recombinant [20] Rolland M, Nickle DC, Mullins JI. HIV-1 group M conserved elements vaccine. PLoS human granulocyte macrophage colony-stimulating factor vaccination, and Pathog 2007;3(11):e157. https://doi.org/10.1371/journal.ppat.0030157. romidepsin on the HIV-1 reservoir (REDUC): a single-arm, phase 1B/2A trial. Lancet [21] Ondondo B, Murakoshi H, Clutton G, et al. Novel conserved-region T-cell mosaic vac- HIV 2016;3(10):e463–72. https://doi.org/10.1016/S2352-3018(16)30055-8. cine with high global HIV-1 coverage is recognized by protective responses in un- [47] Cohen MS, Chen YQ, McCauley M, et al. Prevention of HIV-1 infection with early an- treated infection. Mol Ther 2016;24(4):832–42. https://doi.org/10.1038/mt.2016.3. tiretroviral therapy. N Engl J Med 2011;365(6):493–505. https://doi.org/10.1056/ [22] Mutua G, Farah B, Langat R, et al. Broad HIV-1 inhibition in vitro by vaccine-elicited NEJMoa1105243. CD8(+) T cells in African adults. Mol Ther Methods Clin Dev 2016;3:16061. https:// [48] Rosario M, Bridgeman A, Quakkelaar ED, et al. Long peptides induce polyfunctional T doi.org/10.1038/mtm.2016.61. cells against conserved regions of HIV-1 with superior breadth to single-gene vac- [23] Pereyra F, Jia X, McLaren PJ, et al. The major genetic determinants of HIV-1 control cines in macaques. Eur J Immunol 2010;40:1973–84. https://doi.org/10.1002/eji. affect HLA class I peptide presentation. Science 2010;330(6010):1551–7. https:// 201040344. doi.org/10.1126/science.1195271. [49] Hancock G, Morón-López S, Kopycinski J, et al. Evaluation of the immunogenicity [24] Borthwick N, Ahmed T, Ondondo B, et al. Vaccine-elicited human T cells recognizing and impact on the latent HIV-1 reservoir of a conserved region vaccine, MVA. conserved protein regions inhibit HIV-1. Mol Ther 2014;22(2):464–75. https://doi. HIVconsv, in antiretroviral therapy-treated subjects. J Int AIDS Soc 2017;20(1): org/10.1038/mt.2013.248. 1–11. https://doi.org/10.7448/IAS.20.1.21171. [25] Hayton E-J, Rose A, Ibrahimsa U, et al. Safety and tolerability of conserved region [50] Puertas MC, Massanella M, Llibre JM, et al. Intensification of a raltegravir-based reg- vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus imen with maraviroc in early HIV-1 infection. AIDS 2014;28(3):325–34. https://doi. ankara administered to human immunodeficiency virus type 1-uninfected adults org/10.1097/QAD.0000000000000066. in a randomized, single-blind phase I trial. PLoS One 2014;9(7):e101591. https:// [51] Hindson BJ, Ness KD, Masquelier DA, et al. High-throughput droplet digital PCR sys- doi.org/10.1371/journal.pone.0101591. tem for absolute quantitation of DNA copy number. Anal Chem 2011;83(22): [26] McElrath MJ, De Rosa SC, Moodie Z, et al. HIV-1 vaccine-induced immunity in the 8604–10. https://doi.org/10.1021/ac202028g. test-of-concept Step Study: a case-cohort analysis. Lancet 2008;372(9653): [52] Mothe B, Hu X, Llano A, et al. A human immune data-informed vaccine concept 1894–905. https://doi.org/10.1016/S0140-6736(08)61592-5. elicits strong and broad T-cell specificities associated with HIV-1 control in mice 80 B. Mothe et al. / EClinicalMedicine 11 (2019) 65–80 and macaques. J Transl Med 2015;13(1):60. https://doi.org/10.1186/s12967-015- [57] Ahmed T, Borthwick NJ, Gilmour J, Hayes P, Dorrell L, Hanke T. Control of HIV-1 rep- 0392-5. lication in vitro by vaccine-induced human CD8(+) T cells through conserved sub- [53] Bansal A, Gough E, Sabbaj S, et al. CD8 T-cell responses in early HIV-1 infection are dominant Pol epitopes. Vaccine 2016;34(9):1215–24. https://doi.org/10.1016/j. skewed towards high entropy peptides. AIDS 2005;19(3):241–50http://www.ncbi. vaccine.2015.12.021. nlm.nih.gov/pubmed/15718834, Accessed date: 12 August 2018. [58] Fidler S, Stohr W, Pace M, et al. A randomised controlled trial comparing the impact [54] Yang OO, Daar ES, Ng HL, Shih R, Jamieson BD. Increasing CTL targeting of conserved of antiretroviral therapy (ART) with a “kick-and-kill” approach to ART alone on HIV sequences during early HIV-1 infection is correlated to decreasing viremia. AIDS Res reservoirs in individuals with primary HIV infection (PHI); RIVER trial. 22nd Interna- Hum Retroviruses 2011;27(4):391–8. https://doi.org/10.1089/aid.2010.0183. tional AIDS Conference; Amsterdam, July 23–27; 2018. [55] Mothe B, Climent N, Plana M, et al. Safety and immunogenicity of a modified vac- [59] Ananworanich J, Chomont N, Fletcher JL, et al. Markers of HIV reservoir size and im- cinia Ankara-based HIV-1 vaccine (MVA-B) in HIV-1-infected patients alone or in mune activation after treatment in acute HIV infection with and without raltegravir combination with a drug to reactivate latent HIV-1. J Antimicrob Chemother Febru- and maraviroc intensification. J Virus Erad 2017;1(2):116–22http://www.ncbi.nlm. ary 2015. https://doi.org/10.1093/jac/dkv046. nih.gov/pubmed/26835516.nnnnJanuary15. [56] Thornhill J, Inshaw J, Kaleebu P, et al. Brief report. JAIDS J Acquir Immune Defic Syndr 2016;73(1):69–73. https://doi.org/10.1097/QAI.0000000000001013.