Hayato Murakoshi

Novel Conserved-region T-cell Mosaic Vaccine With High Global HIV-1 Coverage Is Recognized by Protective Responses in Untreated Infection

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.

Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes

ABSTRACT 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. IMPORTANCE HLA-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.

Host-Specific Adaptation of HIV-1 Subtype B in the Japanese Population

ABSTRACT The extent to which HIV-1 clade B strains exhibit population-specific adaptations to host HLA alleles remains incompletely known, in part due to incomplete characterization of HLA-associated HIV-1 polymorphisms (HLA-APs) in different global populations. Moreover, it remains unknown to what extent the same HLA alleles may drive significantly different escape pathways across populations. As the Japanese population exhibits distinctive HLA class I allele distributions, comparative analysis of HLA-APs between HIV-1 clade B-infected Japanese and non-Asian cohorts could shed light on these questions. However, HLA-APs remain incompletely mapped in Japan. In a cohort of 430 treatment-naive Japanese with chronic HIV-1 clade B infection, we identified 284 HLA-APs in Gag, Pol, and Nef using phylogenetically corrected methods. The number of HLA-associated substitutions in Pol, notably those restricted by HLA-B*52:01, was weakly inversely correlated with the plasma viral load (pVL), suggesting that the transmission and persistence of B*52:01-driven Pol mutations could modulate the pVL. Differential selection of HLA-APs between HLA subtype members, including those differing only with respect to substitutions outside the peptide-binding groove, was observed, meriting further investigation as to their mechanisms of selection. Notably, two-thirds of HLA-APs identified in Japan had not been reported in previous studies of predominantly Caucasian cohorts and were attributable to HLA alleles unique to, or enriched in, Japan. We also identified 71 cases where the same HLA allele drove significantly different escape pathways in Japan versus predominantly Caucasian cohorts. Our results underscore the distinct global evolution of HIV-1 clade B as a result of host population-specific cellular immune pressures. IMPORTANCE Cytotoxic T lymphocyte (CTL) escape mutations in HIV-1 are broadly predictable based on the HLA class I alleles expressed by the host. Because HLA allele distributions differ among worldwide populations, the pattern and diversity of HLA-associated escape mutations are likely to be somewhat distinct to each race and region. HLA-associated polymorphisms (HLA-APs) in HIV-1 have previously been identified at the population level in European, North American, Australian, and African cohorts; however, large-scale analyses of HIV-1 clade B-specific HLA-APs in Asians are lacking. Differential intraclade HIV-1 adaptation to global populations can be investigated via comparative analyses of HLA-associated polymorphisms across ethnic groups, but such studies are rare. Here, we identify HLA-APs in a large Japanese HIV-1 clade B cohort using phylogenetically informed methods and observe that the majority of them had not been previously characterized in predominantly Caucasian populations. The results highlight HIVs unique adaptation to cellular immune pressures imposed by different global populations.

Selection of escape mutant by HLA-C-restricted HIV-1 Pol-specific cytotoxic T lymphocytes carrying strong ability to suppress HIV-1 replication

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.

Distinct HIV-1 Escape Patterns Selected by Cytotoxic T Cells with Identical Epitope Specificity

ABSTRACT Pol283-8-specific, HLA-B*51:01-restricted, cytotoxic T cells (CTLs) play a critical role in the long-term control of HIV-1 infection. However, these CTLs select for the reverse transcriptase (RT) I135X escape mutation, which may be accumulating in circulating HIV-1 sequences. We investigated the selection of the I135X mutation by CTLs specific for the same epitope but restricted by HLA-B*52:01. We found that Pol283-8-specific, HLA-B*52:01-restricted CTLs were elicited predominantly in chronically HIV-1-infected individuals. These CTLs had a strong ability to suppress the replication of wild-type HIV-1, though this ability was weaker than that of HLA-B*51:01-restricted CTLs. The crystal structure of the HLA-B*52:01-Pol283-8 peptide complex provided clear evidence that HLA-B*52:01 presents the peptide similarly to HLA-B*51:01, ensuring the cross-presentation of this epitope by both alleles. Population level analyses revealed a strong association of HLA-B*51:01 with the I135T mutant and a relatively weaker association of HLA-B*52:01 with several I135X mutants in both Japanese and predominantly Caucasian cohorts. An in vitro viral suppression assay revealed that the HLA-B*52:01-restricted CTLs failed to suppress the replication of the I135X mutant viruses, indicating the selection of these mutants by the CTLs. These results suggest that the different pattern of I135X mutant selection may have resulted from the difference between these two CTLs in the ability to suppress HIV-1 replication.

Naturally selected rilpivirine-resistant HIV-1 variants by host cellular immunity

BACKGROUND Rilpivirine is listed as an alternative key drug in current antiretroviral therapy (ART) guidelines. E138G/A/K in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rilpivirine resistance-associated mutations and can be identified in a few ART-naive patients, although at low frequency. The 138th position in HIV-1 RT is located in one of the putative epitopes of human leukocyte antigen (HLA)-B*18-restricted cytotoxic T lymphocytes (CTLs). CTL-mediated immune pressure selects escape mutations within the CTL epitope. Here we tested whether E138G/A/K could be selected by HLA-B*18-restricted CTLs. METHODS The amino acid variation at the 138th position was compared between ART-naive HIV-1-infected patients with and without HLA-B*18. The optimal epitope containing the 138th position was determined and the impact of E138G/A/K on CTL response was analyzed by epitope-specific CTLs. The effect of E138G/A/K on drug susceptibility was determined by constructing recombinant HIV-1 variants. RESULTS The prevalence of E138G/A/K was 21% and 0.37% in 19 and 1088 patients with and without HLA-B*18, respectively (odds ratio, 72.3; P = 4.9 × 10(-25)). The CTL response was completely abolished by the substitution of E138G/A/K in the epitope peptide. E138G/A/K conferred 5.1-, 7.1-, and 2.7-fold resistance to rilpivirine, respectively. CONCLUSIONS E138G/A/K can be selected by HLA-B*18-restricted CTLs and confer significant rilpivirine resistance. We recommend drug resistance testing before the introduction of rilpivirine-based ART in HLA-B*18-positive patients.

Effective recognition of HIV-1-infected cells by HIV-1 integrase-specific HLA-B∗4002-restricted T cells.

HLA-B∗4002 is one of the common HLA-B alleles in the world. All 7 reported HLA-B∗4002-restricted HIV epitopes are derived from Gag, Nef, and Vpr. In the present study we sought to identify novel HLA-B∗4002-restricted HIV epitopes by using overlapping 11-mer peptides of HIV-1 Nef, Gag, and Pol, and found that 6 of these 11-mer Pol peptides included HLA-B∗4002-restricted epitopes. Analysis using truncated peptides of these 6 peptides defined 4 optimal Pol (integrase) epitopes. All epitopes previously reported had Glu at position 2 (P2), suggesting that Glu at P2 is the anchor residue for HLA-B∗4002; whereas only 2 of the integrase epitopes that we here identified had Glu at P2. CTL clones specific for the 2 epitopes effectively recognized HIV-1-infected cells whereas those for other 2 epitopes only weakly recognized them. The antigen sensitivity of the former clones for the epitope peptide was much higher than that of the latter clones, suggesting 2 possibilities: 1) the former T cells have high-affinity TCRs and/or 2) the epitope peptides recognized by the former T cells are highly presented by HLA-B∗4002 in HIV-1-infected cells. These integrase-specific T cells with high antigen sensitivity may contribute to the suppression of HIV-1 replication in HIV-1-infected HLA-B∗4002+ individuals.

Different In Vivo Effects of HIV-1 Immunodominant Epitope-Specific Cytotoxic T Lymphocytes on Selection of Escape Mutant Viruses

ABSTRACT HIV-1 escape mutants are well known to be selected by immune pressure via HIV-1-specific cytotoxic T lymphocytes (CTLs) and neutralizing antibodies. The ability of the CTLs to suppress HIV-1 replication is assumed to be associated with the selection of escape mutants from the CTLs. Therefore, we first investigated the correlation between the ability of HLA-A*1101-restricted CTLs recognizing immunodominant epitopes in vitro and the selection of escape mutants. The result showed that there was no correlation between the ability of these CTLs to suppress HIV-1 replication in vitro and the appearance of escape mutants. The CTLs that had a strong ability to suppress HIV-1 replication in vitro but failed to select escape mutants expressed a higher level of PD-1 in vivo, whereas those that had a strong ability to suppress HIV-1 replication in vitro and selected escape mutants expressed a low level of PD-1. Ex vivo analysis of these CTLs revealed that the latter CTLs had a significantly stronger ability to recognize the epitope than the former ones. These results suggest that escape mutations are selected by HIV-1-specific CTLs that have a stronger ability to recognize HIV-1 in vivo but not in vitro.

Novel, in-natural-infection subdominant HIV-1 CD8+ T-cell epitopes revealed in human recipients of conserved-region T-cell vaccines

Background Fine 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. Methods Cryopreserved 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. Results Using 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. Conclusions The 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 registration ClinicalTrials.gov NCT01151319

Identification of cross-clade CTL epitopes in HIV-1 clade A/E-infected individuals by using the clade B overlapping peptides

Identification of cross-clade T cell epitopes is one of key factors for the development of a widely applicable AIDS vaccine. We here investigated cross-clade CD8(+) T cell responses between clade B and A/E viruses in chronically HIV-1 clade A/E-infected Japanese individuals. CD8(+) T cell responses to 11-mer overlapping peptides derived from Nef, Gag, and Pol clade B consensus sequences were at a similar level to those to the same peptides found in clade B-infected individuals. Fifteen cross-clade CTL epitopes were identified from 13 regions where the frequency of responders was high in the clade A/E-infected individuals. The sequences of 6 epitopes were conserved between the clade B and clade A/E viruses whereas 9 epitopes had different amino acid sequences between the 2 viruses. CD8(+) T cells specific for the 6 conserved epitopes recognized cells infected with the clade A/E virus, whereas those for 8 diverse epitopes recognized both the clade A/E virus-infected and clade B-infected cells. All of the cross-clade CD8(+) T cells specific for conserved and diverse epitopes were detected in chronically HIV-1 clade A/E-infected individuals. These results show that in addition to conserved regions polymorphic ones across the clades can be targets for cross-clade CTLs.