Despite recent advances in vaginal microbicides and pre-exposure prophylaxis , it is unlikely that a profound fall in the incidence of HIV-1 infections will occur without an effective prophylactic vaccine . The major challenge that both antibody and T-cell-eliciting vaccines face is the extreme variability of the HIV-1 genome: a successful vaccine has to effectively target diverse HIV-1 strains circulating in the population and then must deal with ongoing virus escape in infected individuals. To address these issues, we assembled vaccine immunogen HIVconsv from the functionally most conserved regions (not epitopes) of the HIV-1 proteome with the underlying working hypothesis that early focus of vaccine-elicited immune responses on these regions will lead to a better recognition and control of transmitting viruses .
Plasmid DNA is a highly attractive vaccine modality because of its simplicity and stability; however, in its ‘naked’ form, it has not been sufficiently immunogenic in humans. Thus, a variety of physical, chemical and immunological strategies are under development to improve its immunogenic efficiency. One of these strategies is electroporation, which has been shown to increase the transgene expression and immunogenicity of its product. Since the first trial in 2004 , a number of human clinical studies have been initiated investigating both intramuscular and intradermal DNA electroporation mainly for cancer patients (reviewed [5–9]), but also to stimulate HIV-1-specific responses [10,11]. Thus, electroporation represents a potentially important development, which improves efficacy of genetic subunit vaccines vectored by conventional plasmid DNA.
Synthetic long peptides (SLPs) are a novel vaccine modality, which showed promising results in the clinic by induction of efficacious T-cell responses against human papillomavirus serotype 16 (HPV-16) [12,13]. SLPs are designed as approximately 30-mer peptides overlapping by 10 to 15 amino acids, whereby the length strongly favours peptide processing by ‘professional’ antigen-presenting cells to direct binding to major histocompatibility complex (MHC) class I on the cell surface; this provides a parallel stimulation of both CD4+ helper and CD8+ cytotoxic T cells [14,15]. Nevertheless, SLP immunogenicity requires optimal peptide adjuvantation [12,16]. Previously, we demonstrated in rhesus macaques that sub-pools of adjuvanted SLP.HIVconsv delivered as a boosting modality to anatomically separate sites broadened T-cell responses induced by single-gene genetic vaccines . However, the efficiency of T-cell priming in naïve animals by SLP.HIVconsv remained untested.
Heterologous prime-boost regimens are on the forefront of the current sub-unit vaccine development [17–19]. These regimens employ sequential administrations of distinct vaccine modalities, which all share a common immunogen. Because CD8+ T cells compete for priming by ‘professional’ antigen-presenting cells [20–24], simple modalities such as plasmid DNA and SLP, which may be less immunogenic, but induce no responses to vector antigens and therefore focus T cells on the transgene product, are preferentially used for priming. In contrast, boosting vectors can be more complex because they stimulate already expanded transgene-specific T cells, and are typically derived from attenuated, nonreplicating viruses. Assembling heterologous vaccines into more complex regimens remains largely empirical and will require fine-tuning in humans for each immunogen and vector combination [3,25–27]. In addition, an effective vaccine regimen against HIV-1 may have to combine various vaccine modalities to induce both T cells and antibodies. Whereas ultimately the only vaccine-induced protection that matters is that of humans against HIV-1, nonhuman primate experiments can provide helpful information on various aspects of vaccination regimens and save time and money relative to phase I/IIa human trials.
Here, we aimed to assess the induction of HIV-1-specific responses using a combination of four heterologous vaccines, electroporated pSG2.HIVconsv DNA, adjuvanted SLP.HIVconsv, nonreplicating modified vaccinia virus Ankara MVA.HIVconsv and nonreplicating adenovirus of chimpanzee origin ChAdV63.HIVconsv [3,16], with a particular attention to the priming by the two antigenically ‘pure’ formulations. Both T cells and Env-specific antibodies were induced. These results complement on going phase I/IIa clinical trials of the genetic HIVconsv vaccines and encourage translational studies in humans of the SLP platform.
Preparation of pSG2.HIVconsv, ChAdV63.HIVconsv, MVA.HIVconsv and SLP.HIVconsv vaccines was describe previously [3,16].
Animals, vaccination and isolation of PBMC
Fifteen Indian rhesus macaques (Macaca mulatta) just weaned and all younger than 2 years were assigned into three groups of five animals, of which two were Mamu-A*01-positive and 3 Mamu-A*01-negative, receiving DDDCMS, DSSCMS, or SSSCMS vaccines, where D is 900 μg total (s.q.) electroporated over six sites (two upper arms, upper and lower back, two thighs), C is 1010 infectious units of nonreplicating ChAdV63.HIVconsv i.m., M is 108 plaque-forming units of MVA.HIVconsv i.m. and S represents 46 synthetic 25–28-amino acid-long SLPs, 0.3 mg each adjuvanted by topical imiquimod and montanide ISA-51 emulsification, divided into six sub-pools and injected s.q. into six anatomically separate sites as above. Monkey blood was drawn from superficial veins and peripheral blood mononuclear cells (PBMC) were isolated using the Lymphoprep cushion centrifugation and either used fresh, or were frozen and stored in liquid nitrogen until use. Frozen cells were thawed at 37°C, washed with Roswell Park Memorial Institute medium (GIBCO, UK) and rested overnight before use. Animals were anaesthetized solely for the purpose of immobilization using ketamine sedation (10 mg/kg) with optional isoflurane (5 mg/kg). All procedures and care strictly conformed to the UK Home Office Guidelines.
Peptides and antigens
For immunological assays, 199 15-mer peptides overlapping by 11 amino acids (15/11) and spanning the entire HIVconsv protein and 80% pure were generously provided by the International AIDS Vaccine Initiative. Individual peptides were dissolved in dimethyl sulfoxide (DMSO; Sigma–Aldrich) to yield a stock of 40 mg/ml and stored at –80°C until use. Peptide pools 1 to 6 consisted of 32 to 35 peptides and, in the IFN-γ enzyme-linked immunospot (ELISPOT) assay, were used at final concentration of 2 μg/ml for each peptide. Negative or ‘mock’ controls contained 0.5% DMSO in culture media. Phytohemagglutinin (PHA) 5 μg/ml (Sigma SL4144) containing wells served as positive controls.
IFN-γ ELISPOT assay
The frequencies of cells releasing IFN-γ upon re-stimulation using HIVconsv-derived peptides or peptide pools were assessed in an ELISPOT assay. The procedures and reagents of MABTECH (Cat. No. 3420M-2A) were used throughout as describe previously . Assays were carried out in triplicates and the spots were counted using the AID ELISPOT Reader System (Autoimmun Diagnostika). For CD4+ and CD8+ T-cell depletions, the procedures and reagents of Dynal UK (magnetic beads) were used throughout .
Carboxy-fluorescein diacetate succinimidyl ester proliferation assays
Thawed and overnight-rested PBMCs were labelled with 0.8 μmol/l carboxy-fluorescein diacetate succinimidyl ester (CFSE; Invitrogen, Paisley, UK) for 8 min in the dark. Cells were washed and cultured in R-10 at 37°C, 5% CO2 for 5 days with mock or peptide Pool 1. Cells were stained with CD3-APC clone SP34–2 (BD Biosciences) and CD8 PerCP clone RPA-T8 (BD Biosciences), CD4 APC-Cy7 clone OKT4 and at least 40 000 events were acquired using a CyAn ADp LX 9 flow cytometer. Analysis was performed using Summit 4.3 (Dako) software. Results are expressed with the following formula: proliferation index (PI) x (sum of the cells in all generations)/(computed number of original parent cells theoretically present at the start of the experiment) – (results of a nonstimulated control for each time point and animal).
Intracellular cytokine staining
One million of thawed and overnight-rested PBMCs were washed, incubated with no peptide or 2 μg/ml peptides (Pool1) in 5% CO2 at 37°C. After 90 min, Golgiplug (BD Biosciences) containing brefeldin A was added. After a further 8-h incubation, cells were washed with fluorescence-activated cell sorting wash buffer [PBS and 1% fetal calf serum (FCS)], stained with CD3-APC-Cy7 clone SP 34–2 (BD Biosciences), CD8-Pac blue (clone RPA-T8, BD) or CD4-Pac blue (clone OKT4), CD 28-Per-CP (clone L293), and CD 95-PE (clone DX2) at 4°C for 30 min and permeabilized with Cytofix/Cytopermsolution (BD Biosciences) at room temperature for 20 min. Cells were then washed with Perm wash buffer (PBS and 1% FCS, 10% BD Perm/wash) and stained with anti-IFN-γ-FITC mAb clone B27 (BD Biosciences), anti-TNF-α-PE-Cy7 mAb clone MAb11 (BD Biosciences) and anti-IL-2-APC clone MQ1–17H12 mAb (BD Biosciences) at 4°C for 30 min and washed. Cells were analysed by flow cytometry using the CellQuest software (BD Biosciences). The gating strategy involved selecting CD3+CD4+ or CD3+CD8+ then CD95+CD28+ or CD95+CD28−. Therefore, there is CD4+ (CM and EM) and CD8+ (CM and EM) information post DDD, DDDCM and DDDCMS. All tested samples were individually controlled with a mock DMSO and this number was subtracted from the test sample.
Six pools of 15/11 HIVconsv peptides (0.5 μg/well) or recombinant proteins (1 μg/ml) were coated onto flat 96 well plates and kept overnight a 4°C. Recombinant proteins were heat treated at 100°C for 5 min. Wells then were blocked with PBS blocking buffer (Thermo Scientific) for 1 h, washed and dilutions of sera were added. After 2 h at 37°C, wells were washed and a 1/2000 dilution of conjugate antirhesus IgG ALP conjugate antibody (Southern Biotech) in PBS was added. After 1 h, wells were washed and substrate ALP ELISA substrate (Sigma) was added at room temperature (RT) for 1 h. The reaction was stopped with 25 μl of 3 mol/l NaOH and plates were read at 405 nm in an FLX800 microplate fluorescence reader BIO-TEKinc. The endpoint titre is based on the reactivity of the preimmune sample at 1 : 50 dilution + 2 SDs.
Statistical significance was determined using an unpaired Student's t-test with a two-tailed distribution on group immunization data. Differences were considered as significant at P 0.05 or less.
DDD, but not SSS or DSS, primes consistently low T cell responses
Three groups of five rhesus macaques received combinations of electroporated pSG2.HIVconsv DNA (D), adjuvanted SLP.HIVconsv peptides (S) , MVA.HIVconsv (M) and chimpanzee adenovirus ChAdV63.HIVconsv (C) and the vaccine-induced, HIV-1-specific T-cell frequencies were measured regularly prior to and throughout the vaccinations in an ex-vivo IFN-γ ELISPOT assay employing six pools of 15/11 peptides across the entire HIVconsv immunogen.
First, we assessed three different priming regimens. Following DDD, DSS and SSS immunizations, five, five and two macaques in each group, respectively, responded with detectable T-cell responses (Fig. 1a). The respective group frequencies peaked at medians of 245 (range 53–707) on day 104, 112 (range 23–233) on day 70 and 30 (range 0–619) on day 35 of HIV-1-specific spot-forming units (SFUs)/106 PBMCs. Thus, priming protocols involving electroporated pSG2.HIVconsv DNA induced more consistently HIV-1-specific T-cell responses detectable in ex-vivo IFN-γ ELIPSOT assay than the adjuvanted SLP.HIVconsv vaccine alone. Furthermore, electoporation afforded over a six-fold dose-sparing of the vaccine DNA amount relative to historic experiments [16,28].
DDD primes for robust T-cell expansions by CM
Next, macaques in all three groups were administered ChAdV63.HIVconsv followed by MVA.HIVconsv boosts, therefore receiving overall regimens of DDDCM, DSSCM or SSSCM. In the DDD-primed animals, ChAdV63.HIVconsv increased efficiently HIV-1-specific responses to median 1039 (range 680–3225) SFUs/106 PBMCs (Fig. 1a, group 1, and b) and so did the MVA.HIVconsv administration reaching 1828 (range 304–3194) SFUs/106 PBMCs, although with exception of one initially lower-responding macaque, the frequencies did not supersede those induced by ChAdV63.HIVconsv. Thus, three deliveries of pSG2.HIVconsv DNA aided by electroporation provided a basis for potent, repeated expansions of the HIV-1-specific T cells by subsequent administrations of the virus-vectored vaccines.
In contrast, regimens DSSCM and SSSCM elicited lower frequencies of specific T-cell responses (Fig. 1b) with overall peaks ranging 146–1295 and 128–721 SFUs/106 PBMCs at different time points, respectively (Fig. 1a, groups 2 and 3). It is not clear whether the lack of robust boosts by the recombinant virus vaccines observed previously  and in humans (T.H., unpublished) is particular to the SLP.HIVconsv adjuvantation and/or timing of the two vaccine boosts used in this study or whether the animals were simply low responders. Nevertheless, these results are compatible with our previous observations, which indicated that single-open reading frame genetic vaccines cannot maintain the breadth of responses elicited by the SLP vaccine delivered to multiple sites .
SLP.HIVconsv boost broadens mainly CD4+ T-cell responses
Next, we estimated the breadth of CD8+ and CD4+ T-cell responses induced by the three regimens using 199 individual 15/11 HIVconsv peptides. The average numbers of epitopes stimulating in-vitro IFN-γ production post DDDCM, DSSCM and SSSCM regimens were 6.4, 2.6 and 4.6 for CD8+ and 7.6, 3.0 and 1.2 for CD4+ T cells, respectively (Fig. 2). Therefore, SLP.HIVconsv vaccination did not increase the breath of T-cell recognition when used as a prime.
To confirm the previously observed capacity of SLP.HIVconsv to expand existing responses , all animals received one SLP.HIVconsv vaccination. This administration re-boosted the overall HIV-1-specific T-cell frequencies to median levels of 2209 (range 683–3798), 263 (range 0–859) and 270 (range 0–476) SFUs/106 PBMCs for groups 1–3, respectively (Fig. 1a and b), that is similar frequencies to those induced by MVA.HIVconsv. The average numbers of detected response specificities induced by DDDCMS, DSSCMS and SSSCMS regimens were 6.8, 3.0 and 6.8 for CD8+ and 20.6, 7.8 and 4.6 for CD4+ T-cell responses, respectively (Fig. 2a and b). Only broadening of the CD4+ T-cell specificities by the SLP.HIVconsv boost achieved statistical significance for groups 1 (P = 0.01) and 3 (P = 0.02) and group 1 had significantly broader CD4+ responses that the other two groups (P = 0.001 and 0.007); no statistically separable increase in the number of CD8+ T-cell-recognized epitopes was detected.
HIVconsv-specific T cells proliferate and are oligofunctional
Proliferation and oligofunctionality of HIV-1-specific T cells in response to cognate antigen were associated with a good control of chronic HIV-1 infection [29–31]. First, proliferation indices before and after SLP.HIVconsv boost were estimated using peptides derived from the conserved regions of Gag incorporated into the HIVconsv immunogen. For CD3+CD8+ PBMC of groups 1, 2 and 3, these were 2.9, 2.6 and 3.0, respectively, and did not increase by the SLP.HIVconsv administration. In contrast for CD3+CD4+ PBMCs, proliferation indices were about ½ of those of the CD8+ T cells prior to SLP.HIVconsv, but increased significantly for all three regimens after SLP.HIVconsv boost to respective 2.3 (P = 0.02), 9.1 (P = 0.04) and 7.2 (P = 0.01). In addition, regimen SSSCMS displayed significantly higher CD4+ cell proliferation relative to DDDCMS (P = 0.03) (Fig. 3a). Second, we investigated production of INF-γ, TNF-α, and IL-2 peptide re-stimulation. We found in all three groups large proportions of Gag-responsive PBMCs producing two or three cytokines (Fig. 3b, top). For the strongest responding group 1, functional analysis was performed separately for central (CD28+CD95+) and effector memory (CD28−CD95+) CD8+ and CD4+ PBMC subpopulations at various stages of immunization. Overall, following the DDD and DDDCM vaccination, both CD4+ and CD8+ T cells of the central and effector memory subsets produced predominantly a single cytokine with less than 17% of T cells displaying two or three effector functions (Fig. 3b, bottom).
SLP.HIVconsv induces antibodies to conserved regions of Env
Because the HIVconsv immunogen also contains two highly conserved regions of Env (residues 88–124 of gp120 and 522–575 of gp41) in addition to a BALB/c mouse CD8+ T-cell epitope RGPGRAFVTI of the hypervariable V3 loop, we also assessed the HIV-1 Env-specific humoral responses. Thus, an end-point titration ELISA assay using pooled 15/11 peptides corresponding the Env regions (without peptide RGPGRAFVTI) indicated, that of the three priming protocols, the strongest antibody responses were generated by the combined DSS regimen (Fig. 4a). DSS responses peaked at median anti-Env titre of 6120 (range 1870–6823), whereas titres for DDD and SSS reached medians 316 (range 272–562) and 620 (range 526–853), respectively. Animals in groups 2 and 3 had similar antibody responses after the final SLP.HIVconsv boost. Only the DSS-elicited sera were tested against CHO-expressed trimeric CN54 gp120 and gp140 antigens and bound these at median titres of 829 (range 452–1346) and 485 (range 299–882), respectively (Fig. 4b). This binding was not affected by heat treatment suggesting recognition of linear antigenic sites (Fig. 4c). No clear or consistent neutralization of MN.3, MW965.26 or TH023.6 pseudoviruses produced in 293T cells in the TZM-bl cell assay was detected (not shown). Thus, the HIVconsv immunogen elicited antibodies binding to regions of trimeric Env highly conserved among the four major HIV-1 clades A, B, C and D.
In the rhesus macaque study presented here, three vaccination regimens DDDCMS, DSSCMS and SSSCMS were compared for their immunogenic potencies. It was found that DDD primed for the largest subsequent expansions of HIV-1-specific T cells, whereas DSS primed for the highest titres of Env-specific antibodies. It was also affirmed that electroporation spares the DNA vaccine dose and SLP broadens the specificity of T cells induced by single-gene genetic vaccines.
Previously, we showed that imiquimod/montanide ISA-51 adjuvanted SLP.HIVconsv divided into six sub-pools and administered as a boost to rhesus macaques into anatomically separated sites greatly enhanced the breath and overall magnitude of the HIVconsv-induced CD4+ and CD8+ T-cell responses . Here, we established that the same SLP.HIVconsv peptides using the same adjuvantation are not an efficient primer of T cells and, as a boost following the DDDCM regimen, efficiently expanded mainly CD4+ T cells. The present observations concur with the HPV-16 E6/E7 SLP clinical trial data, in which SLPs alone induced effector T-cell-mediated clinical benefit in patients with vulvar intraepithelial neoplasia and mainly CD4+ T-cell expansions were detected [12,13]. In these women, HPV-specific T cells had already been primed and expanded by HPV infection. A strong correlation was seen between vaccine prompted HPV-specific effector T cells and complete clinical regression of the HPV-associated lesions [12,32].
To further characterize the HIVconsv vaccine-induced T-cell responses, we mapped the T-cell specificities in all animals in an INF-γ ELISPOT assay before and after the SLP.HIVconsv boost using 15/11 peptides across the entire HIVconsv protein. We found that the responding macaques in all groups had multispecific CD4+ and CD8+ T-cell responses before the SLP.HIVconsv vaccination and that in the DDDMC group, the SLP.HIVconsv administration enhanced the response breath in the CD4+ T-cell compartment. Whereas the relevance of IFN-γ production for protection against HIV-1-infection is uncertain [33,34], it remains a useful, highly standardized first-line way of enumerating vaccine-elicited T cells, which may express other effector functions. Although detailed analysis of T-cell subpopulations throughout the DDDCM regimen indicated predominantly production of only one cytokine IFN-γ, TNF-α or IL-2 upon HIVconsv Gag peptide stimulation, majority of PBMCs in all 15 animals following the final SLP.HIVconsv administration produced at least two of these intercellular signalling molecules. CFSE proliferation results confirmed that the vaccine-elicited T cells expanded in response to the HIVconsv Gag peptides. Although T-cell responses directed against the conserved regions of HIV-1 proteins are mainly subdominant during natural HIV-1 infection , in the murine model they can induce broad, functional responses when taken away from the domination by highly variable epitopes of HIV-1 and contribute to protection against the whole virus [36–38]. Thus, in the absence of simple functional correlates of T-cell protection , the optimal vaccine should aim to induce broad and polyfunctional T cells, and the vaccines and schedules tested here satisfy these requirements.
SLP.HIVconsv induced the strongest antibody responses to conserved regions of the HIV-1 Env when used in the DSS regimen. These sera were able to bind trimeric Env glycoproteins. This is encouraging in the light of the 31% vaccine protection against HIV-1 acquisition achieved by attenuated poxvirus prime-Env protein boost regimen in 2009 trial RV144 . Although the correlates of this marginal protection may never be firmly identified, the reduced level of HIV-1 acquisition without a significant effect on initial virus load or CD4+ T-cell counts and the absence of significant antibody neutralization generated a hypothesis about a possible protective role of non-neutralizing antibody-dependent effector functions [41,42]. Thus, regimens involving the SLP.HIVconsv modality can elicit both T cells and Env-binding antibodies directed to the conserved regions of the HIV-1 proteome and demand evaluation in humans.
To date, not very many clinical trials of HIV-1 vaccines employed peptide-based formulations and the few published studies showed little immunogenicity in healthy HIV-1-negative individuals [43–45]. As an immunotherapy, initial study employing recombinant poxvirus ALVAC 1433 combined with HIV-1 lipopeptide vaccine impacted on the control of viral replication following highly active antiretroviral treatment (HAART) interruption in chronically HIV-1-infected patients ; however, attempts to replicate these findings were unsuccessful [47,48]. Immunizations with peptide pulse dendritic cells is the most immunogenic peptide strategy [45,49,50], but it is not practical for HIV-1 prophylaxis in the developing world. Critical to the success of peptide vaccines is their adjuvantation. Whereas only a handful of adjuvants have been licensed for human use predominantly focusing on enhancing antibody induction, there has been a recent flurry of research on adjuvanting systems optimizing T-cell induction . As we demonstrated here, one of the challenges for subunit HIV-1 vaccines is maximizing induction of both T-cell and antibody responses at the same time by a combination of novel heterologous prime-boost regimens and/or adjuvantation.
In conclusion, given the proven ability of SLP to induce in humans protective, antiviral T-cell effectors at the mucosal portal of entry [12,13], focus T and B cells on invariable regions of the HIV-1 proteome  and potential protective role of non-neutralizing antibodies highlighted by the RV144 Thai trial , SLP.HIVconsv is a highly promising anti-HIV-1 vaccine strategy, which merits evaluation on its own right as immunotherapy and/or in combination with other heterologous vaccines as immunoprophylaxis in clinical trials in humans.
Authors substantially contributed to conception and design (T.H., C.J.M.M., M.R., J.W.D., N.B.), acquisition of data, or analysis and interpretation of data (M.R., N.B., D.M., A.M.-M.); writing the article or revising it critically for important intellectual content (T.H., M.R., C.J.M.M., A.J.M.) or provided essential materials (G.B.S.-J., S.C., A.N., J.W.D., A.B., D.M.).
Conflicts of interest
Source of funding: C.J.M.M. is 75% employed by ISA Pharmaceuticals and 25% by LUMC. He has a 1% stock appreciation share in ISA Pharmaceuticals. ISA Pharmaceuticals has licensed from LUMC the technology for application of synthetic peptide vaccines against high-risk HPV and several other targets. C.J.M.M. does not receive income now or in the future from the patents on targets that ISA has licensed from LUMC. A.N. is an author of filed patents on the ChAdV-63 vector. T.H. and A.J.McM. are authors on patent filed on the HIVconsv immunogen. For the remaining authors none were declared.
Sources of support: The work was supported by Medical Research Council UK.
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