Effective control of HIV type 1 (HIV-1) infection will likely require concerted and balanced actions of broadly neutralizing antibodies (bnAbs) and T cells [1,2]. Neutralizing antibodies slow the incoming virus infection and killer T cells eliminate any breakthrough virus-infected cells. Even if bnAbs are present at high concentrations and prevent most of HIV-1 infection, it will be very difficult to prevent all infection and some may also occur through direct cell–cell transmission. Therefore, it is likely that to prevent and eliminate incoming HIV-1, both effective bnAbs and effective T-cell-mediated immune responses will always be required. Using rational design of novel immunogens and their more potent heterologous prime-boost vector deliveries [1,2], the prospect of inducing the desired effectors of adaptive immunity by active immunization is increasingly promising. Therefore, it is timely to start laying down guiding principles for optimal combinations of the antibody- and T-cell-inducing vaccine components into a single immunization regimen without compromising each other's induction.
HIV-1 genome plasticity results in cocirculation of diverse viruses in the population and escape of transmitted/founder viruses from mounted immune responses in already-infected individuals. Several immunogen design approaches have been proposed to tackle escape from HIV-1-specific T-cell responses [3,4]. We designed immunogen HIVconsv derived from the 14 most-conserved subprotein regions of the HIV-1 proteome . A heterologous vector combination for HIVconsv delivery employing plasmid DNA, non-replicating simian chimpanzee adenovirus of origin ChAdV-63 and non-replicating poxvirus modified vaccinia virus Ankara (MVA) induced high frequencies of HIV-1 conserved region-specific T cells in mice [5–7], rhesus macaques [6,8,9] and human volunteers . The same ChAdV and MVA vectors delivering a close HIVconsv derivative, designated tHIVc, were used here as vaccine components inducing broadly reactive T cells focused on the conserved HIV-1 regions.
PG9 and PG16 are glycan-dependent human monoclonal antibodies, which were isolated from an HIV-1 clade A-infected individual in International AIDS Vaccine Initiative Protocol G [11,12]. In the same individual, a bioinformatics approach was used to identify viruses that were potentially sensitive to PG9 and PG16 inhibition. When a most-recent common ancestor sequence for Env was determined and aligned with 99 clade A gp160 sequences from the Los Alamos National Laboratory HIV Sequence Database (LANL-HSD), virus BG505.W6 M.ENV.C2 (BG505) was found to have the highest degree of sequence identity (73%) to the most-recent common ancestor sequence . Pseudoviruses prepared with the BG505 Env were sensitive to neutralization with a broad panel of bnAbs including PG9 and PG16. Thus, for the antibody-inducing vaccine components, immunogens were derived from the BG505 Env. These were presented to the immune system as furin-uncleaved gp140 (BG505u), which is not in the native configuration because of lack of cleavage , delivered by ChAdV-63 and MVA. BG505 sequences were also presented as adjuvanted gp120 glycoprotein.
In natural infection and often in vaccination, Env-specificity dominates anti-HIV-1 T-cell responses. However, Env-specific CD8+ T cells were not beneficial for control of HIV-1 replication [15–18] likely because Env is the most variable HIV-1 protein [19,20] and replicating viruses rapidly escape . Therefore, it has been suggested that Env should be avoided in the designs of T-cell immunogens [1,22]; this creates a challenge of inducing high titres of Env-specific antibodies in the absence of Env-specific CD8+ T cells.
Here, we report on rational combination of T-cell and B-cell vaccine components into one immunization regimen. First, heterologous BG505 Env regimens were assessed for maximum antibody titre induction while minimizing elicitation of Env-specific CD8+ T-cell responses. Next, the same BG505 Env regimens were administered along with the tHIVc vaccines inducing conserved region-focused CD8+ T cells. Finally, the levels of tier-1 and tier-2 virus neutralization induced by all these protocols were determined. These results will inform initial designs of similar studies in human volunteers.
Construction and production of virus-vectored vaccines
For antibody induction, the BG505u gp140 immunogen coupled to the tissue plasminogen activator (tPA) leader sequence and ending at the C-terminus with amino acids …KWAS (HXB2 amino acid positions 31–668) was designed with the furin-target site REKR (amino acids 508–511) mutated to uncleavable SEKS and the corresponding synthetic gene was made (Life Technologies). The tHIVc immunogen gene was PCR amplified from plasmid pSG2.HIVconsv DNA  using primers designed to introduce a tPA leader sequence to the N-terminus and remove epitopes included for preclinical development from the C-terminus of the HIVconsv protein. For the generation of recombinant ChAdV-63s, both the BG505u and tHIVc genes were subcloned under the control of the human cytomegalovirus immediate early promoter in plasmid pENTR4_Mono and inserted at the E1 locus of the ChAdV-63 genome by GalK recombineering . Recombinant ChAdV-63 vaccines were grown in suspension culture of HEK 293 cells. To generate recombinant MVAs, the genes were subcloned under the control of the modified H5 promoter  in plasmid MVAgfpTD and inserted into the thymidine kinase region of the MVA genome to generate marker-less vaccines , which were expanded by growth in chicken embryo fibroblasts. Vaccines were titred and maintained at −80°C until use.
Production and purification of the BG505 gp120 protein
For the upstream process, the BG505 gp120 gene was first mutated to introduce the L111A substitution, which makes the expressed protein less prone to aggregation . BG505 L111A gp120 protein was then produced by transient transfection of HEK 293 cells using the FuGENE 6 kit (Promega, Madison, Wisconsin, USA) according to the vendor's protocol. The cell supernatant was harvested after 4–5 days, centrifuged for 10 min at 500g to remove cells, filtered through 0.2 μm Nalgene Rapid-Flow filter (Thermo Scientific, Waltham, Massachusetts, USA), protease inhibitors (Roche, Indianapolis, Indiana, USA) were added and the medium was stored at 4°C until purification. For the downstream process, protein preparations were purified by affinity chromatography with a mannose-specific Galanthus nivalis lectin agarose column (Sigma-Aldrich, St. Louis, Missouri, USA) . The lectin column was equilibrated with phosphate-buffered saline (PBS), pH 7.4, elution was performed with 0.5 mol/l methyl-alpha-D-mannopyranoside (Sigma-Aldrich), the elution buffer was exchanged for PBS by diafiltration and the gp120 protein was stored at −80°C.
Mice and immunization regimens
Six-week-old female BALB/c mice were purchased from Harlan Laboratories (Bicester, UK) and housed at the Functional Genomics Facility, University of Oxford. Groups of seven mice were immunized intramuscularly under general anaesthesia either with 108 infectious units of ChAdV63.BG505u, 5 × 106 plaque-forming units (PFU) of MVA.BG505u or 10 μg of BG505 gp120 protein adjuvanted using AddaVax (InvivoGen, Toulouse, France) according the vendor's instructions, alone or in combination with ChAdV63.tHIVc and MVA.tHIVc (Table 1). In the combined regimens, the BG505 and tHIVc vaccines were always injected into the left and right hind quadriceps, respectively. Blood was obtained by venepuncture of the lateral tail vein at weeks 0, 5, 8, 12 and 14. All procedures and care were approved by the local Research Ethics Committee, University of Oxford and conformed strictly to the United Kingdom Home Office Guidelines under the Animals (Scientific Procedures) Act 1986. Experiments were conducted under Project License 30/2833 held by T.H.
15-mer peptides overlapping by 11 amino acids (15/11) spanning the entire BG505 gp140 and tHIVc open reading frames were obtained from Pepscan (Lelystad, The Netherlands) and AnaSpec (Cambridge Biosciences, Cambridge, UK), respectively. Peptides were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 20 mg/ml and stored at −80°C. Working stocks of 4 mg/ml were prepared by diluting 20 mg/ml stocks with PBS. Peptides were used in assays at a final concentration of 2 μg/ml.
Serum was isolated from blood collected by venepuncture of the lateral tail vein and stored at −80°C. A standardised mouse immunoglobulin G (IgG) ELISA was performed as previously described . Briefly, a reference serum stock was created by pooling 10 μl fractions of the week 12 (peak response) serum samples from 20 mice. Sera were diluted 1 in 100 (week 0 samples), 1 in 500 (week 5 samples), 1 in 5000 (week 8 samples) or 1 in 10 000 (weeks 12 and 14 samples) in Casein blocking buffer (Thermo Scientific). The reference serum was diluted 1 in 800 in blocking buffer, and 1 in 2 serial dilutions prepared for the construction of a standard curve. Casein block alone was used as a negative control. Nunc Immuno F96 Maxisorp plates (Thermo Fisher, Waltham, Massachusetts, USA) were coated overnight at room temperature with BG505 gp120 protein (see above) at 2 μg/ml and then treated with Casein blocking buffer for 1 h at room temperature to prevent non-specific binding. Standards and samples were added to plates in duplicate and triplicate, respectively, and incubated at room temperature for 2 h. The plates were washed with PBS/0.05% Tween-20 (Sigma-Aldrich) and incubated with secondary antibody (goat antimouse IgG; Sigma-Aldrich) diluted 1 : 5000 in Casein blocking buffer for 1 h at room temperature. After washing, plates were developed using 4-nitrophenyl phosphate (Sigma-Aldrich) dissolved in deionized water. Absorbance was measured using an ELx800 Microplate Reader with Gen5 ELISA software (BioTek). Data were expressed as absorbance units.
Interferon-γ ELISPOT assay
Splenocytes were harvested and isolated 4 weeks after the final immunization (week 14) and interferon (IFN) γ production in response to BG505 and tHIVc peptides was determined by enzyme-linked immunospot (ELISPOT) assay as previously .
Intracellular cytokine staining
Cytokine production by splenocytes from immunized mice was assessed by intracellular cytokine staining (ICS) as described previously . Samples were acquired on an LSR II flow cytometer (BD Biosciences, Oxford, UK) and data analysed using FlowJo version 9.5.2 (Tree Star, Ashland, Oregon, USA).
HIV-1 neutralization assay
A validated TZM-bl assay was used against MN.3 and MW965.26 (tier 1) and BG505/T332N and Ce1176_A3 (tier 2) viruses as described previously to assess the neutralization of potency of the vaccine-elicited sera at various time points through out the regimens .
Statistical analyses were performed using Graph Pad Prism version 5 (GraphPad Software, San Diego, California, USA). Simple comparisons were performed using two-way Student's t-test. Multiple comparisons were performed using one-way ANOVA with Bonferroni's multiple comparison posttest for parametric data and the Kruskal–Wallis test with Dunn's multiple comparison posttest for non-parametric data. A P < 0.05 was considered significant.
CMP, CPP and PPP induce similar antibody titres
Three vaccine modalities, namely non-replicating simian adenovirus ChAdV63.BG505u (C) and non-replicating MVA.BG505u (M) and protein BG505 gp120 formulated with MF59-like [30,31] squalene-based oil-in-water emulsion AddaVax (P), were combined into three sequential regimens to assess the magnitude of antibody induction (Table 1). Both virus-vectored vaccines expressed uncleaved BG505 gp140 (BG505u; Fig. 1). Groups of BALB/c mice received regimens CMP, CPP and PPP, and BG505 gp120-specific IgG antibody titres were determined prior to, and at time points during and after the immunizations. While baseline (week 0) BG505 gp120-specific antibody titres were below the level of detection of the assay in all groups, all three regimens elicited medium-high antibody titres to the BG505 gp120 and were not statistically separable at any of the analyzed time points (Fig. 2a). Using peak values for individual mice, the antibody responses reached mean ± SD titres of 4573 ± 2489, 4300 ± 1783 and 6861 ± 1549 for the CMP, CPP and PPP regimens, respectively, and the peak values for the PPP regimen were significantly higher than for the CPP delivery (P = 0.02)(Fig. 2b).
PPP elicits a fewer CD8+ T cells than CMP and CPP
We argue that the induction of immunodominant, yet unprotective CD8+ T-cell responses to highly variable regions of HIV-1 Env by vaccines should be avoided. To assess vaccine-elicited frequencies of T cells specific for BG505u, we examined IFN-γ production by splenocytes stimulated with six peptide pools P11-P16 spanning the entire BG505 gp140 4 weeks after the last vaccine administration (week 14). The total frequencies of BG505u-specific T cells reached mean ± SD of 2611 ± 407, 1263 ± 555 and 60 ± 57S FU/106 splenocytes for the CMP, CPP and PPP regimens, respectively, thus a clear hierarchy of CMP>CPP>PPP for T-cell immunogenicity was detected (P < 0.001)(Fig. 2c). The region of gp120 spanned by peptide pool P14 (amino acids 298–413) dominated the T-cell response and concurred with the overall regimen hierarchy (Fig. 2d). Collectively, these observations indicated that the three regimens delivering the BG505 Env induced comparable medium level anti-BG505 antibody responses, while differing dramatically in the frequencies of vaccine-elicited, Env-specific T cells.
Coinduction of BG505 Ab and conserved region T-cell responses
Next, we assessed whether high anti-Env antibody titres can be induced together with robust T-cell responses focused on the conserved regions of the HIV-1 proteome and with a minimal distraction from T cells recognizing the highly variable Env regions. We previously showed that recombinant ChAdV63 followed by recombinant MVA elicited strong T-cell responses to immunogen HIVconsv in humans . Here, for the induction of T cells against the conserved regions, a close derivative of the HIVconsv immunogen, designated tHIVc (Fig. 1), was delivered using regimen ChAdV63.tHIVc followed by 2x MVA.tHIVc (CMM) injecting the T-cell vaccines into an anatomically separate site from the BG505 Env (right and left hind legs for tHIVc and BG505, respectively). Thus, groups of BALB/c mice were immunized with the CMP+CMM, CPP+CMM and PPP+CMM regimens expressing the BG505 Env and tHVc immunogens (Table 1), and BG505 gp120-specific antibodies and BG505u-specific and tHIVc-specific T cells were determined. All three groups showed high frequencies of T cells directed to tHIVc with mean ± SD of 4820 ± 1376, 5692 ± 1249 and 3947 ± 1337 SFU/106 splenocytes, respectively, with CMP+CMM and CPP+CMM being statistically significantly higher than PPP+CMM (P = 0.04 and P = 0.02, respectively) (Fig. 3a). This suggested that the immunogenicity of the CMM tHIVc regimen was negatively affected by the AddaVax-adjuvanted gp120 co-delivery. In all groups, the highest magnitude responses were detected in response to stimulation with tHIVc peptide pools P2 and P4 (Fig. 3b). Responses to individual 15/11 peptides were mapped to peptides numbers 9 and 15 (P1), 42 and 55 (P2), 112 (P4), and 151 and 164 (P5) . Second, there was a clear hierarchy of the CMP>CPP>PPP regimens for the induction of BG505-specific T cells (P < 0.001) (Fig. 2c). For the strongest CMP+CMM regimen, the frequencies of Env-specific T cells reached 42% of those recognizing tHIVc (Fig. 3a and c) with an overall trend of lower induction of Env-specific T cells in combined regimens relative to the BG505 Env-alone delivery (Figs 3c and 2c). Vaccine-elicited antibody titres at each of the measured time points were not statistically separable among the regimens (Fig. 3e). The peak BG505-specific antibody titres for individual mice elicited by the CMP+CMM, CPP+CMM and PPP+CMM regimens reached mean ± SD titres of 3858 ± 1519, 3005 ± 960 and 3157 ± 1797, respectively, and only the PPP+CMM regimen induced significantly lower peak levels of gp120-specific antibodies compared with the PPP alone (P = 0.02) (Figs 3f and 2b), possibly because the AddaVax adjuvant was optimized for Ab rather than T-cell induction. These data clearly demonstrated that regimens matter and that the levels of antibodies and T cells can be manipulated by the choice of the combined immunogens’ delivery.
Combined regimens induced oligofunctional tHIVc-specific CD8+ T cells
Next, we investigated the functionality of the Env-specific CD4+ T helper (Th1/Th2) and tHIVc-specific CD8+ T-cell responses. In particular, in-vitro production of IFN-γ, interleukin-2 and interleukin-4 upon specific peptide stimulation was assessed using a polychromatic ICS assay. Thus, comparison between the CMP+CMM and CPP+CMM regimens showed that the percentages of CD4+ T cells recognizing BG505 Env were very low, but similar for both combined regimens (Fig. 4a). Relative frequencies among regimens of BG505u-specific, IFN-γ-producing CD8+ T cells corresponded well to the ELISPOT results (Fig. 4b). Whereas the BG505 Env- and tHIVc CD4+ T cell were of similar frequencies (Fig. 4c), the tHIVc-specific CD8+ T cells producing IFN-γ and tumour necrosis factor-α reached levels of 3–5% of the total CD8+ cells particularly in responses to peptide pools P2 and P4 (Fig. 4d) and were again statistically inseparable between the CMP+CMM and CPP+CMM regimens. Thus, the tHIVc immunogen induced a strong, mostly at least bi-functional CD8+ T-cell response regardless of the codelivered BG505 regimen.
BG505 vaccination induced neutralizing antibodies
Pooled prevaccination and postvaccination sera from all regimens were analyzed in the TMZ-bl HIV-1 neutralization assay  against two tier 1 (easy-to-neutralize) clade B MN.3 and clade C MW965.26, and two tier 2 (harder-to-neutralize) clade A BG505CT/T332N and clade C Ce1176_A3 viruses. A low- to moderate-titre neutralization of the tier 1 viruses with little background signals in the preimmune sera were detected for all regimens (Table 2). Although the 50% neutralization titres did not benefit from the third vaccine administration, two to 10-fold lower neutralization titres were induced by the PPP regimen. Very weak neutralization against the tier 2 viruses was detected for the CMP, CPP and CMP+CMM regimens. Thus, rather encouragingly, neutralization activity was induced by uncleaved gp140-prime gp120-boost regimens of the BG505 Env.
Both immunogen design and delivery are likely to be critical for eliciting protective responses to HIV-1 . The two immunogens employed in this study were BG505 Env  derivatives for antibody induction and chimaeric tHIVc protein based on the most conserved HIV-1 regions for CD8+ T-cell induction . These immunogens were vectored by simian (chimpanzee) adenovirus ChAdV-63 and poxvirus MVA or delivered as AddaVax-adjuvanted gp120 protein. Both T-cell and B-cell immunogens had been developed with a strong rationale and independently from each other by specialized laboratories [5,13]. Here we explored in mice several strategies for combining BG505 Env and tHIVc into single regimens for maximum induction of binding antibody titres and CD8+ T cells focused on the conserved HIV-1 regions with minimum interference from hypervariable BG505 Env-specific T cells.
A number of observations were made, which will guide at least initial designs of similar regimens in humans. First, the tested vaccinations elicited medium-to-strong Env-specific antibody responses. Although the homologous PPP regimen induced significantly higher peak titres of anti-BG505 gp120 antibodies than CPP when administered without the T-cell components (P = 0.02), this difference was lost upon tHIVc coadministration. This was mainly because of the significantly higher antibody induction by the PPP alone regimen compared with PPP+CMM (P = 0.002). Second, BG505u vector prime-gp120 boost induced antibody responses capable of neutralizing two tier 1 and marginally two tier 2 HIV-1 species. This was encouraging given that our secreted uncleaved BG5050u immunogen will not form a correctly configured trimer like the BG505.SOSIP.R6.664 [33,34], indicating a conformational and antigenic superiority of the cleaved and stabilized gp140 form . Also adjuvanted BG505 gp120 alone (P) induced tier 1 neutralization as previously reported in rabbits . Thus, BG505 Env is a promising HIV-1 Env immunogen [14,33,35]. In the next stage, similar antibody-inducing regimens, but delivering BG505 SO SIP.R6.664 gp140 gene and protein trimers [14,33–35], will be assessed in animal models more relevant for the human antibody repertoires for the breadth of vaccine-elicited HIV-1-neutralization.
Third, the frequencies of T cells specific for the HIV-1 most conserved protein regions induced by the CMM-delivered tHIVc immunogen were high in all combined regimens, though lower when coadministered with the BG505 PPP regimen. The magnitude of Env-specific T cells was 2-fold to 10-fold decreased by avoiding the use of virus-vectored vaccines. The best regimen for achieving the desired balance of T-cell and B-cell responses on one hand and T-cell responses to tHIVc and high titres to Env on the other was the combination of ChAdV63.BG505u, gp120, ChAdV63.tHIVc and MVA.tHIVc (CPP+CMM). Such sequential prime-boost regimen can easily accommodate delivery of a series of Env immunogens guiding the affinity maturation from that of a B-cell germline to a broad neutralization of HIV-1 isolates [36–38] when available.
The marginal 31% protection from acquisition of HIV-1 observed in trial RV144 [39–42] was an enormous boost to the HIV-1 vaccine development field and is inevitably being followed and built upon by an effort co-ordinated under the Pox Protein Public-Private Partnership (P5) (www.niaid.nih.gov). The RV144 vaccine regimen consisted of four administrations of non-replicating canary poxvirus ALVAC vCP1521 expressing immunogens Gag/Rt/Nef/Env coadministered on the last two deliveries with alum-adjuvanted gp120 protein . Although no or marginal CD8+ T-cell responses were detected, thorough exploratory analyses of the correlates of risk pointed to the association between high titres of non-neutralizing antibodies against variable regions 1 and 2 of gp120 and protection from HIV-1-acquisition, which was abolished by immunoglobulin A responses [39,43–45]. As it is likely that RV144 vaccines would have benefitted from strong CD8+ T-cell responses targeting protective epitopes and/or neutralizing antibodies, the rational and empirical vaccine approaches synergize and inform each other of the most promising designs for future development.
In conclusion, here we report a rational attempt to combine separately developed T-cell and B-cell vaccine components into a single vaccination regimen for a maximum induction of the two main arms of immune effectors. The main observations are that T-cell and B-cell responses can be induced in parallel to different immunogens, and that AddaVax-adjuvanted protein and CM delivery of tHIVc interfere mutually with optimal induction of the other. Although both BG505 and tHIVc immunogens and their delivery will continue to be improved through iterative preclinical and clinical studies, the results provide an initial guidance as for what can be achieved using these three frequently used vaccine modalities.
The authors have contributed to the work in the following way: T.H. conceived the experiments, T.H., G.C., H.J.D., C.L.P. and D.M. designed the experiments; C.R.K., C.L.P., H.J.D. and A.C. designed and produced the Env immunogens; G.C. and C.L. carried out the experiments; T.H. and G.C. wrote the article.
The work is jointly funded by the UK Medical Research Council (MRC) and the UK Department for International Development (DFID) under the MRC/DFID Concordat agreements, and the International AIDS Vaccine Initiative: this study was also made possible by the generous support of the American people through the United States Agency for International Development (USAID). The contents are the responsibility of the International AIDS Vaccine Initiative and do not necessarily reflect the views of USAID or the United States Government.
Conflicts of interest
The authors have no conflicts of interest.
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Keywords:© 2014 Lippincott Williams & Wilkins, Inc.
antibodies; BG505; conserved regions; heterologous prime-boost regimen; HIV type 1 vaccine; HIVconsv; modified vaccinia virus Ankara MVA; simian adenovirus; T cells