An effective human immunodeficiency virus type 1 (HIV-1) vaccine is the best solution for halting the acquired immune deficiency syndrome epidemic. Here, we describe the design and preclinical immunogenicity of T-cell vaccine expressing novel immunogens tHIVconsvX, vectored by DNA, simian (chimpanzee) adenovirus, and poxvirus modified vaccinia virus Ankara (MVA), a combination highly immunogenic in humans. The tHIVconsvX immunogens combine the three leading strategies for elicitation of effective CD8(+) T cells: use of regions of HIV-1 proteins functionally conserved across all M group viruses (to make HIV-1 escape costly on viral fitness), inclusion of bivalent complementary mosaic immunogens (to maximize global epitope matching and breadth of responses, and block common escape paths), and inclusion of epitopes known to be associated with low viral load in infected untreated people (to induce field-proven protective responses). tHIVconsvX was highly immunogenic in two strains of mice. Furthermore, the magnitude and breadth of CD8(+) T-cell responses to tHIVconsvX-derived peptides in treatment-naive HIV-1(+) patients significantly correlated with high CD4(+) T-cell count and low viral load. Overall, the tHIVconsvX design, combining the mosaic and conserved-region approaches, provides an indisputably better coverage of global HIV-1 variants than previous T-cell vaccines. These immunogens delivered in a highly immunogenic framework of adenovirus prime and MVA boost are ready for clinical development.

Identification and characterization of CD8؉ T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8 ؉ T cells have been only partially identified. In this study, we sought to identify CD8 ؉ T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8 ؉ T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P ‫؍‬ 2.2 ؋ 10 ؊11 ) and positively associated with CD4 count (P ‫؍‬ 1.2 ؋ 10 ؊11 ), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8 ؉ T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. IMPORTANCEHLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52:01-restricted and 3 HLA-B*67:01-restricted CTLs, suggesting that these CTLs play a predominant role in HIV-1 control. The 13 CTLs showed synergistic effects on HIV-1 control. Twelve out of these 13 epitopes were recognized as conserved or cross-recognized ones. These findings strongly suggest that these 12 epitopes are candidates for antigens for AIDS vaccines.

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Peptides generated by proteasome-catalyzed splicing of noncontiguous amino acid sequences have been shown to constitute a source of nontemplated human leukocyte antigen class I (HLA-I) epitopes, but their role in pathogen-specific immunity remains unknown. CD8 + T cells are key mediators of HIV type 1 (HIV-1) control, and identification of novel epitopes to enhance targeting of infected cells is a priority for prophylactic and therapeutic strategies. To explore the contribution of proteasome-catalyzed peptide splicing (PCPS) to HIV-1 epitope generation, we developed a broadly applicable mass spectrometry-based discovery workflow that we employed to identify spliced HLA-I-bound peptides on HIV-infected cells. We demonstrate that HIV-1-derived spliced peptides comprise a relatively minor component of the HLA-I-bound viral immunopeptidome. Although spliced HIV-1 peptides may elicit CD8 + T cell responses relatively infrequently during infection, CD8 + T cells primed by partially overlapping contiguous epitopes in HIV-infected individuals were able to cross-recognize spliced viral peptides, suggesting a potential role for PCPS in restricting HIV-1 escape pathways. Vaccine-mediated priming of responses to spliced HIV-1 epitopes could thus provide a novel means of exploiting epitope targets typically underutilized during natural infection. peptide splicing | proteasome | immunopeptidome | T cell epitope | human immunodeficiency virus P eptides presented by human leukocyte antigen (HLA) class I and II molecules were originally thought to derive solely from contiguous protein sequences. Two seminal studies then reported T cell responses to noncontiguous (spliced/fusion) peptides in renal cell carcinoma and melanoma patients, although identification of these tumor-derived spliced peptides involved laborintensive in vitro approaches, and validation relied on the serendipitous availability of epitope-specific T cells (1, 2). Thus, only a handful of cis-spliced epitopes were described in more than a decade of subsequent research (3-7). In addition to epitopes generated by cis-splicing, which involves ligation of noncontiguous peptide fragments within the same polypeptide, recognition of HLA-II-restricted epitopes generated by fusion of peptide fragments from 2 different proteins (trans-splicing) by CD4 + T cells from patients with type I diabetes has also been reported (8), although whether the peptide fusion event leading to generation of these trans-spliced epitopes was catalyzed by the proteasome or other enzymes was not explored.More recently, the increased sensitivity of mass spectrometry (MS)-based approaches for characterizing the repertoire of HLAbound peptides (the immunopeptidome) has provided an opportunity for the discovery of spliced peptides on a much larger scale than was previously possible (9-11). Although proteasome-catalyzed peptide splicing (PCPS) had widely been assumed to be a relatively rare event, MS-based profiling of immunopeptidomic datasets has questioned this assumption. However, the proportion o...

BackgroundDevelopment of AIDS vaccines for effective prevention of circulating HIV-1 is required, but no trial has demonstrated definitive effects on the prevention. Several recent T-cell vaccine trials showed no protection against HIV-1 acquisition although the vaccines induced HIV-1-specific T-cell responses, suggesting that the vaccine-induced T cells have insufficient capacities to suppress HIV-1 replication and/or cross-recognize circulating HIV-1. Therefore, it is necessary to develop T-cell vaccines that elicit T cells recognizing shared protective epitopes with strong ability to suppress HIV-1. We recently designed T-cell mosaic vaccine immunogens tHIVconsvX composed of 6 conserved Gag and Pol regions and demonstrated that the T-cell responses to peptides derived from the vaccine immunogens were significantly associated with lower plasma viral load (pVL) and higher CD4+ T-cell count (CD4 count) in HIV-1-infected, treatment-naive Japanese individuals. However, it remains unknown T cells of which specificities have the ability to suppress HIV-1 replication. In the present study, we sought to identify more T cells specific for protective Gag epitopes in the vaccine immunogens, and analyze their abilities to suppress HIV-1 replication and recognize epitope variants in circulating HIV-1.ResultsWe determined 17 optimal Gag epitopes and their HLA restriction, and found that T-cell responses to 9 were associated significantly with lower pVL and/or higher CD4 count. T-cells recognizing 5 of these Gag peptides remained associated with good clinical outcome in 221 HIV-1-infected individuals even when comparing responders and non-responders with the same restricting HLA alleles. Although it was known previously that T cells specific for 3 of these protective epitopes had strong abilities to suppress HIV-1 replication in vivo, here we demonstrated equivalent abilities for the 2 novel epitopes. Furthermore, T cells against all 5 Gag epitopes cross-recognized variants in majority of circulating HIV-1.ConclusionsWe demonstrated that T cells specific for 5 Gag conserved epitopes in the tHIVconsvX have ability to suppress replication of circulating HIV-1 in HIV-1-infected individuals. Therefore, the tHIVconsvX vaccines have the right specificity to contribute to prevention of HIV-1 infection and eradication of latently infected cells following HIV-1 reactivation.Electronic supplementary materialThe online version of this article (10.1186/s12977-018-0429-y) contains supplementary material, which is available to authorized users.

BackgroundFine definition of targeted CD8+ T-cell epitopes and their human leucocyte antigen (HLA) class I restriction informs iterative improvements of HIV-1 T-cell vaccine designs and may predict early vaccine success or failure. Here, lymphocytes from volunteers, who had received candidate HIVconsv vaccines expressing conserved sub-protein regions of HIV-1, were used to define the optimum-length target epitopes and their HLA restriction. In HIV-1-positive patients, CD8+ T-cell responses predominantly recognize immunodominant, but hypervariable and therefore less protective epitopes. The less variable, more protective epitopes in conserved regions are typically subdominant. Therefore, induction of strong responses to conserved regions by vaccination provides an opportunity to discover novel important epitopes.MethodsCryopreserved lymphocytes from vaccine recipients were expanded by stimulation with 15-mer responder peptides for 10 days to establish short term-cell-line (STCL) effector cells. These were subjected to intracellular cytokine staining using serially truncated peptides and peptide-pulsed 721.221 cells expressing individual HLA class I alleles to define minimal epitope length and HLA restriction by stimulation of IFN-γ and TNF-α production and surface expression of CD107a.ResultsUsing lymphocyte samples of 12 vaccine recipients, we defined 14 previously unreported optimal CD8+ T-cell HIV-1 epitopes and their four-digit HLA allele restriction (6 HLA-A, 7 HLA-B and 1 HLA-C alleles). Further 13 novel targets with incomplete information were revealed.ConclusionsThe high rate of discovery of novel CD8+ T-cell effector epitopes warrants further epitope mining in recipients of the conserved-region vaccines in other populations and informs development of HIV-1/AIDS vaccines.Trial registrationClinicalTrials.gov NCT01151319

HIV‐1 mutants escaping from HLA‐A‐ or HLA‐B‐restricted CTL have been well studied, but those from HLA‐C‐restricted CTL have not. Therefore we investigated the ability of HLA‐C‐restricted CTL to select HIV‐1 escape mutants. In the present study, we identified two novel HLA‐Cw*1202‐restricted Pol‐specific CTL epitopes (Pol328‐9 and Pol463‐10). CTL specific for these epitopes were detected in 25–40% of chronically HIV‐1‐infected HLA‐Cw*1202+ individuals and had strong abilities to kill HIV‐1‐infected cells and to suppress HIV‐1 replication in vitro, suggesting that these CTL may have the ability to effectively control HIV‐1 in some HLA‐Cw*1202+ individuals. Sequence analysis of these epitopes showed that a V‐to‐A substitution at the 9th position (V9A) of Pol 463‐10 was significantly associated with the HLA‐Cw*1202 allele and that the V9A mutant was slowly selected in the HLA‐Cw*1202+ individuals. Pol 463‐10‐specific CTL failed both to kill the V9A virus‐infected cells and to suppress replication of the V9A mutant. These results indicate that the V9A mutation was selected as an escape mutant by the Pol463‐10‐specific CTL. The present study strongly suggests that some HLA‐C‐restricted CTL have a strong ability to suppress HIV‐1 replication so that they can select HIV escape mutants as in the case of HLA‐A‐restricted or HLA‐B‐restricted CTL.

HLA-B*52:01-C*12:02, which is found in approximately 20% of all Japanese persons, is well known to be associated with ulcerative colitis and Takayasu arteritis. This haplotype is also known to be protective in individuals infected with human immunodeficiency virus (HIV) type 1. Recent studies showed that HLA-B*52:01-restricted HIV-1-specific T cells suppress HIV-1 and that HLA-C*12:02 together with KIR2DL2 play an important role in natural killer cell-mediated control of HIV-1. However, the role of HLA-C*12:02-restricted cytotoxic T lymphocytes (CTLs) in suppressing HIV-1 replication remains unknown. In the present study, we demonstrated that HLA-C*12:02-restricted CTLs specific for 2 immunodominant epitopes, Pol IY11 and Nef MY9, contributed to the suppression of HIV-1 replication in HIV-1-infected individuals. Further analysis demonstrated that these 2 HLA-C*12:02-restricted CTLs together with 4 HLA-B*52:01-restricted ones effectively suppressed HIV-1 in individuals with the HLA-B*52:01-C*12:02 haplotype. Thus, both HLA-C*12:02 and HLA-B*52:01 alleles contribute to HIV-1 suppression via both HIV-1-specific CTLs and natural killer cells in individuals with this haplotype.