Summary of the publications

Summary of the publications

2019-2020

Tomohiro Akahoshi, Hiroyuki Gatanaga, Nozomi Kuse, Takayuki Chikata, Madoka Koyanagi, Naoki Ishizuka, Chanson J. Brumme, Hayato Murakoshi, Zabrina L. Brumme, Shinichi Oka, and Masafumi Takiguchi, T-cell responses to sequentially emerging viral escape mutants shape long-term HIV-1 population dynamics, PLoS Pathog. 16(12): e1009177, 2020

HIV-1 strains harboring immune escape mutations can persist in circulation, but the impact of selection by multiple HLA alleles on population HIV-1 dynamics remains unclear. In Japan, HIV-1 Reverse Transcriptase codon 135 (RT135) is under strong immune pressure by HLA-B*51:01-restricted and HLA-B*52:01-restricted T cells that target a key epitope in this region (TI8; spanning RT codons 128-135). Major population-level shifts have occurred at HIV-1 RT135 during the Japanese epidemic, which first affected hemophiliacs (via imported contaminated blood products) and subsequently non-hemophiliacs (via domestic transmission). Specifically, threonine accumulated at RT135 (RT135T) in hemophiliac and non-hemophiliac HLA-B*51:01+ individuals diagnosed before 1997, but since then RT135T has markedly declined while RT135L has increased among non-hemophiliac individuals. We demonstrated that RT135V selection by HLA-B*52:01-restricted TI8-specific T-cells led to the creation of a new HLA-C*12:02-restricted epitope TN9-8V. We further showed that TN9-8V-specific HLA-C*12:02-restricted T cells selected RT135L while TN9-8T-specific HLA-C*12:02-restricted T cells suppressed replication of the RT135T variant. Thus, population-level accumulation of the RT135L mutation over time in Japan can be explained by initial targeting of the TI8 epitope by HLA-B*52:01-restricted T-cells, followed by targeting of the resulting escape mutant by HLA-C*12:02-restricted T-cells. We further demonstrate that this phenomenon is particular to Japan, where the HLA-B*52:01-C*12:02 haplotype is common: RT135L did not accumulate over a 15-year longitudinal analysis of HIV sequences in British Columbia, Canada, where this haplotype is rare. Together, our observations reveal that T-cell responses to sequentially emerging viral escape mutants can shape long-term HIV-1 population dynamics in a host population-specific manner.

Yu Zhang, Nozomi Kuse, Tomohiro Akahoshi, Takayuki Chikata, Hiroyuki Gatanaga, Shinichi Oka, Hayato Murakoshi, and Masafumi Takiguchi,　Role of escape mutant-specific T cells in suppression of HIV-1 replication and co-evolution with HIV-1, J. Virol. 94:e01151, 2020

The accumulation of HIV-1 escape mutations affects HIV-1 control by HIV-1-specific T cells. Some of these mutations can elicit escape mutant-specific T-cells, but it still remains unclear whether they can suppress the replication of HIV-1 mutant viruses. It is known that HLA-B*52:01-restricted RI8 (Gag 275-282, RMYSPTSI) is a protective T cell epitope in HIV-1 subtype B-infected Japanese individuals, though 3 Gag280A/S/V mutations are found in 26% of them. Gag280S and Gag280A were HLA-B*52:01-associated mutations; whereas Gag280V was not, implying a different mechanism for the accumulation of Gag280 mutations. We here investigated the co-evolution of HIV-1 with RI8-specific T-cells and suppression of HIV-1 replication by its escape mutant-specific T-cells both in vitro and in vivo The HLA-B*52:01+ individuals infected with Gag280A/S mutant viruses failed to elicit these mutant epitope-specific T-cells, whereas those with the Gag280V mutant one effectively elicited RI8-6V mutant-specific T-cells. These RI8-6V-specific T cells suppressed the replication of Gag280V virus and selected wild-type virus, suggesting a mechanism affording no accumulation of the Gag280V mutation in the HLA-B*52:01+ individuals. The responders to wild-type (RI8-6T) and RI8-6V mutant peptides had significantly higher CD4 counts than non-responders, indicating that the existence of not only RI8-6T-specific T cells but also RI8-6V-specific ones was associated with a good clinical outcome. The present study clarified the role of escape mutant-specific T cells in HIV-1 evolution and in the control of HIV-1.Importance Escape mutant-specific CD8+ T-cells were elicited in some individuals infected with escape mutants, but it is still unknown that these CD8+ T-cells can suppress HIV-1 replication. We clarified that Gag280V mutation were selected by HLA-B*52:01-restricted CD8+ T-cells specific for GagRI8 protective epitope whereas the Gag280V virus could frequently elicit GagRI8-6V mutant-specific CD8+ T-cells. GagRI8-6V mutant-specific T-cells had a strong ability to suppress the replication of the Gag280V mutant virus both in vitro and in vivo In addition, these T cells contributed to the selection of wild-type virus in HLA-B*52:01+ Japanese individuals. We for the first time demonstrated that escape mutant-specific CD8+ T-cells can suppress HIV-1 replication and play an important role in the coevolution with HIV-1. Thus, the present study highlighted an important role of escape mutant-specific T-cells in the control of HIV-1 and co-evolution with HIV-1.

Takayuki Chikata, Wayne Paes, Tomohiro Akahoshi, Thomas Partridge, Hayato Murakoshi, Hiroyuki Gatanaga, Nicola Ternette, Shinichi Oka, Persephone Borrow, and Masafumi Takiguchi, Identification of immunodominant HIV-1 epitopes presented by HLA-C*12:02, a protective allele, using an immunopeptidomics approach, J. Virol. 93: e00634-19, 2019

Despite the fact that the cell surface expression level of HLA-C on both uninfected and HIV-infected cells is lower than those of HLA-A and -B, increasing evidence suggests an important role for HLA-C and HLA-C-restricted CD8+ T cell responses in determining the efficiency of viral control in HIV-1-infected individuals. Nonetheless, HLA-C-restricted T cell responses are much less well studied than HLA-A/B-restricted ones, and relatively few optimal HIV-1 CD8+ T cell epitopes restricted by HLA-C alleles have been defined. Recent improvements in the sensitivity of mass spectrometry (MS)-based approaches for profiling the immunopeptidome present an opportunity for epitope discovery on a large scale. Here, we employed an MS-based immunopeptidomic strategy to characterize HIV-1 peptides presented by a protective allele, HLA-C*12:02. We identified a total of 10,799 unique 8- to 12-mer peptides, including 15 HIV-1 peptides. The latter included 2 previously reported immunodominant HIV-1 epitopes, and analysis of T cell responses to the other HIV-1 peptides detected revealed an additional immunodominant epitope. These findings illustrate the utility of MS-based approaches for epitope definition and emphasize the capacity of HLA-C to present immunodominant T cell epitopes in HIV-infected individuals, indicating the importance of further evaluation of HLA-C-restricted responses to identify novel targets for HIV-1 prophylactic and therapeutic strategies.IMPORTANCE Mass spectrometry (MS)-based approaches are increasingly being employed for large-scale identification of HLA-bound peptides derived from pathogens, but only very limited profiling of the HIV-1 immunopeptidome has been conducted to date. Notably, a growing body of evidence has recently begun to indicate a protective role for HLA-C in HIV-1 infection, which may suggest that despite the fact that levels of HLA-C expression on both uninfected and HIV-1-infected cells are lower than those of HLA-A/B, HLA-C still presents epitopes to CD8+ T cells effectively. To explore this, we analyzed HLA-C*12:02-restricted HIV-1 peptides presented on HIV-1-infected cells expressing only HLA-C*12:02 (a protective allele) using liquid chromatography-tandem MS (LC-MS/MS). We identified a number of novel HLA-C*12:02-bound HIV-1 peptides and showed that although the majority of them did not elicit T cell responses during natural infection in a Japanese cohort, they included three immunodominant epitopes, emphasizing the contribution of HLA-C to epitope presentation on HIV-infected cells.

Center for AIDS Research Best Paper Award 2019
Nozomi Kuse, Xiaoming Sun, Tomohiro Akahoshi, Anna Lissina, Takuya Yamamoto, Victor Appay, and Masafumi Takiguchi, Priming of HIV-1-specific CD8+ T cells with strong functional properties from naive T cells, EBioMedicine 42:109-119, 2019

BACKGROUND: HIV-1-specific CD8+ T cells are required for immune suppression of HIV-1 replication and elimination of the associated viral reservoirs. However, effective induction of functional HIV-1-specific CD8+ T cells from naive cells remains problematic in the setting of human vaccine trials. In this study, we investigated priming of functional HIV-1-specific CD8+ T cells from naive cells.
METHODS: HIV-1-specific CD8+ T cells were primed from naive T cells of HIV-1-seronegative individuals using TLR4 ligand LPS or STING ligand 3'3'-cGAMP in vitro. We established HIV-1-specific CD8+ T cell lines from primed T cells and then investigated functional properties of these cells.
FINDINGS: HIV-1-specific CD8+ T cells primed with LPS failed to suppress HIV-1. In contrast, 3'3'-cGAMP effectively primed HIV-1-specific CD8+ T cells with strong ability to suppress HIV-1. 3'3'-cGAMP-primed T cells had higher expression levels of perforin and granzyme B than LPS-primed ones. The expression levels of granzyme B and perforin and viral suppression ability of 3'3'-cGAMP-primed T cells were positively correlated with the production level of type I IFN from PBMCs stimulated with 3'3'-cGAMP.
INTERPRETATION: The present study demonstrates the potential of 3'3'-cGAMP to induce HIV-1-specific CD8+ T cells with strong effector function from naive cells via a strong type I IFN production and suggests that this STING ligand may be useful for AIDS vaccine and cure treatment.

Chengcheng Zou,* Hayato Murakoshi,* Nozomi Kuse, Tomohiro Akahoshi, Takayuki Chikata, Hiroyuki Gatanaga, Shinichi Oka, Tomas Hanke, and Masafumi Takiguchi (*Equal contribution), Effective suppression of HIV-1 replication by CTLs specific for Pol epitopes in conserved mosaic vaccine immunogens, J. Virol. 93:e02142-18, 2019

Cytotoxic T lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize circulating HIV-1 could be key for both HIV-1 cure and prophylaxis. We recently designed conserved mosaic T-cell vaccine immunogens (tHIVconsvX) composed of 6 Gag and Pol regions. Since the tHIVconsvX vaccine targets conserved regions common to most global HIV-1 variants and employs a bivalent mosaic design, it is expected that it could be universal if the vaccine works. Although we recently demonstrated that CTLs specific for 5 Gag epitopes in the vaccine immunogens had strong ability to suppress HIV-1 replication in vitro and in vivo, it remains unknown whether the Pol region-specific CTLs are equally efficient. In this study, we investigated CTLs specific for Pol epitopes in the immunogens in treatment-naive Japanese patients infected with HIV-1 clade B. Overall, we mapped 20 reported and 5 novel Pol conserved epitopes in tHIVconsvX. Responses to 6 Pol epitopes were significantly associated with good clinical outcome, suggesting that CTLs specific for these 6 Pol epitopes had a strong ability to suppress HIV-1 replication in HIV-1-infected individuals. In vitro T-cell analyses further confirmed that the Pol-specific CTLs could effectively suppress HIV-1 replication. The present study thus demonstrated that the Pol regions of the vaccine contained protective epitopes. T-cell responses to the previous 5 Gag and present 6 Pol protective epitopes together also showed a strong correlation with better clinical outcome. These findings support the testing of the conserved mosaic vaccine in HIV-1 cure and prevention in humans.IMPORTANCE It is likely necessary for an effective AIDS vaccine to elicit CD8+ T cells with the ability to recognize circulating HIV-1 and suppress its replication. We recently developed novel bivalent mosaic T-cell vaccine immunogens composed of conserved regions of the Gag and Pol proteins matched to at least 80% globally circulating HIV-1 isolates. Nevertheless, it remains to be proven if vaccination with these immunogens can elicit T cells with the ability to suppress HIV-1 replication. It is well known that Gag-specific T cells can suppress HIV-1 replication more effectively than T cells specific for epitopes in other proteins. We recently identified 5 protective Gag epitopes in the vaccine immunogens. In this study, we identified T cells specific for 6 Pol epitopes present in the immunogens with strong abilities to suppress HIV-1 in vivo and in vitro This study further encourages clinical testing of the conserved mosaic T-cell vaccine in HIV-1 prevention and cure.

Hayato Murakoshi,* Nozomi Kuse,* Tomohiro Akahoshi, Yu Zhang, Takayuki Chikata, Mohamed Ali Borghan, Hiroyuki Gatanaga, Shinichi Oka, Keiko Sakai, and Masafumi Takiguchi (*Equal contribution), Broad recognition of circulating HIV-1 by HIV-1-specific CTLs with strong ability to suppress HIV-1 replication, J. Virol. 93: e01480-18, 2019

HIV-1-specific cytotoxic T-lymphocytes (CTLs) with strong abilities to suppress HIV-1 replication and recognize most circulating HIV-1 strains are candidates for effector T cells for cure treatment and prophylactic AIDS vaccine. Previous studies demonstrated that the existence of CTLs specific for 11 epitopes was significantly associated with good clinical outcomes in Japan, although CTLs specific for one of these epitopes select for escape mutations. However, it remains unknown whether the CTLs specific for the remaining 10 epitopes suppress HIV-1 replication in vitro and recognize circulating HIV-1. Here, we investigated the abilities of these CTLs to suppress HIV-1 replication and to recognize variants in circulating HIV-1. CTL clones specific for 10 epitopes had strong abilities to suppress HIV-1 replication in vitro The ex vivo and in vitro analyses of T-cell responses to variant epitope peptides showed that the T cells specific for 10 epitopes recognized mutant peptides which are detected in 84.1% to 98.8% of the circulating HIV-1 strains found in HIV-1-infected Japanese individuals. In addition, the T cells specific for 5 epitopes well recognized target cells infected with 7 mutant viruses that had been detected in >5% of tested individuals. Taken together, these results suggest that CTLs specific for the 10 epitopes effectively suppress HIV-1 replication and broadly recognize the circulating HIV-1 strains in the HIV-1-infected individuals. This study suggests the use of these T cells in clinical trials.
IMPORTANCE In recent T-cell AIDS vaccine trials, the vaccines did not prevent HIV-1 infection, although HIV-1-specific T cells were induced in the vaccinated individuals, suggesting that the T cells have a weak ability to suppress HIV-1 replication and fail to recognize circulating HIV-1. We previously demonstrated that the T-cell responses to 10 epitopes were significantly associated with good clinical outcome. However, there is no direct evidence that these T cells have strong abilities to suppress HIV-1 replication and recognize circulating HIV-1. Here, we demonstrated that the T cells specific for the 10 epitopes had strong abilities to suppress HIV-1 replication in vitro Moreover, the T cells cross-recognized most of the circulating HIV-1 in HIV-1-infected individuals. This study suggests the use of T cells specific for these 10 epitopes in clinical trials of T-cell vaccines as a cure treatment.

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