Summary of the publications
Yu Zhang, Takayuki Chikata, Nozomi Kuse, Hayato Murakoshi, Hiroyuki Gatanaga, Shinichi Oka, and Masafumi Takiguchi, Immunological control of HIV-1 disease progression by rare protective HLA allele. J. Virol. 96: e01248-22, 2022
Rare HLA alleles such as HLA-B57 are associated with slow progression to AIDS. However, the evidence for the advantage of rare protective alleles is limited, and the mechanism is still unclear. Although the prevalence of HLA-B*67:01 is only 1.2% in Japan, HLA-B*67:01-positive (HLA-B*67:01+) individuals had the lowest plasma viral load (pVL) and highest CD4 count in HIV-1 clade B-infected Japanese individuals. We investigated the mechanism of immunological control of HIV-1 by a rare protective allele, HLA-B*67:01. We identified six novel HLA-B*67:01-restricted epitopes and found that T cells specific for four epitopes were significantly associated with good clinical outcomes, pVL and/or CD4 count. The wild type or cross-reactive sequences of three protective and immunodominant Pol and Gag epitopes were found in around 95% of the circulating HIV-1, indicating that T cells specific for three conserved or cross-reactive epitopes contributed to good clinical outcomes. One escape mutation (Nef71K) in the Nef protective epitope, which was selected by T cells restricted by either HLA allele in the HLA-B*67:01-C*07:02 haplotype, affected the HLA-B*67:01-restricted RY11-specific T-cell recognition. These results imply that the further accumulation of the Nef71K mutation in the population will negatively affect the control of HIV-1 replication by RY11-specific CD8+ T cells in HIV-1-infected HLA-B*67:01+ individuals. The present study demonstrated that conserved or cross-reactive epitope-specific T cells mainly contribute to control of HIV-1 by a rare protective allele, HLA-B*67:01.
HLA-B57 is a relatively rare allele around world and the strongest protective HLA allele in Caucasians and African black individuals infected with HIV-1. Previous studies suggested that the advantage of this allele in HIV-1 disease progression is due to a strong functional ability of HLA-B57-restricted Gag-specific T cells and lower fitness of mutant viruses selected by the T cells. HLA-B*57 is a very rare allele and has not been reported as a protective allele in Asian countries, whereas a rare allele, HLA-B*67:01, was shown to be a protective allele in Japan. Therefore, the analysis of HLA-B*67:01-restricted T cells is important to clarify the mechanism of immunological control of HIV-1 by a rare protective HLA allele in Asia. We found that HLA-B*67:01-restricted T cells specific for three conserved or cross-reactive Gag and Pol epitopes are associated with good clinical outcomes in HLA-B*67:01+ individuals. It is expected that T cells specific for conserved or cross-reactive epitopes contribute to a curing treatment.
Nozomi Kuse,* Yu Zhang,* Takayuki Chikata,* Hung The Nguyen, Shinichi Oka, Hiroyuki Gatanaga, and Masafumi Takiguchi (*equal contribution), Long-term memory CD8+ T cells specific for SARS-CoV-2 in individuals who received the BNT162b2 mRNA vaccine, Nat. Commun.13: 5251, 2022
Long-term memory T cells have not been well analyzed in individuals vaccinated with a COVID-19 vaccine although analysis of these T cells is necessary to evaluate vaccine efficacy. Here, investigate HLA-A*24:02-restricted CD8+ T cells specific for SARS-CoV-2-derived spike (S) epitopes in individuals immunized with the BNT162b2 mRNA vaccine. T cells specific for the S-QI9 and S-NF9 immunodominant epitopes have higher ability to recognize epitopes than other epitope-specific T cell populations. This higher recognition of S-QI9-specific T cells is due to the high stability of the S-QI9 peptide for HLA-A*24:02, whereas that of S-NF9-specific T cells results from the high affinity of T cell receptor. T cells specific for S-QI9 and S-NF9 are detectable >30 weeks after the second vaccination, indicating that the vaccine induces long-term memory T cells specific for these epitopes. Because the S-QI9 epitope is highly conserved among SARS-CoV-2 variants, S-QI9-specific T cells may help prevent infection with SARS-CoV-2 variants.
Hung The Nguyen*, Nozomi Kuse*, Yu Zhang, Hayato Murakoshi, Yosuke Maeda, Yoshiko Tamura, Rie Maruyama, Giang Van Tran, Trung Vu Nguyen, Kinh Van Nguyen, Shinichi Oka, Takayuki Chikata, and Masafumi Takiguchi (*equal contribution), Control of HIV-1 replication by CD8+ T cells specific for two novel Pol protective epitopes in HIV-1 subtype A/E infection, J. Virol, 96: e00811-22, 2022
Although many HIV-1-specific CD8+ T cell epitopes have been identified and used in various HIV-1 studies, most of these epitopes were derived from HIV-1 subtypes B and C. Only 17 well-defined epitopes, none of which were protective, have been identified for subtype A/E infection. The roles of HIV-1-specific T cells have been rarely analyzed for subtype A/E infection. In this study, we identified six novel HLA-B*15:02-restricted optimal HIV-1 subtype A/E epitopes and then analyzed the presentation of these epitopes by HIV-1 subtype A/E virus-infected cells and the T cell responses to these epitopes in treatment-naive HIV-1 subtype A/E-infected HLA-B*15:02+ Vietnamese individuals. Responders to the PolTY9 or PolLF10 epitope had a significantly lower plasma viral load (pVL) than nonresponders among HLA-B*15:02+ individuals, whereas no significant difference in pVL was found between responders to four other epitopes and nonresponders. The breadth of T cell responses to these two Pol epitopes correlated inversely with pVL. These findings suggest that HLA-B*15:02-restricted T cells specific for PolTY9 and PolLF10 contribute to the suppression of HIV-1 replication in HLA-B*15:02+ individuals. The HLA-B*15:02-associated mutation Pol266I reduced the recognition of PolTY9-specific T cells in vitro but did not affect HIV-1 replication by PolTY9-specific T cells in Pol266I mutant virus-infected individuals. These findings indicate that PolTY9-specific T cells suppress replication of the Pol266I mutant virus even though the T cells selected this mutant. This study demonstrates the effective role of T cells specific for these Pol epitopes to control circulating viruses in HIV-1 subtype A/E infection.
IMPORTANCE It is expected that HIV-1-specific CD8+ T cells that effectively suppress HIV-1 replication will contribute to HIV-1 vaccine development and therapy to achieve an HIV cure. T cells specific for protective epitopes were identified in HIV-1 subtype B and C infections but not in subtype A/E infection, which is epidemic in Southeast Asia. In the present study, we identified six T cell epitopes derived from the subtype A/E virus and demonstrated that T cells specific for two Pol epitopes effectively suppressed HIV-1 replication in treatment-naive Vietnamese individuals infected with HIV-1 subtype A/E. One of these Pol protective epitopes was conserved among circulating viruses, and one escape mutation was accumulated in the other epitope. This mutation did not critically affect HIV-1 control by specific T cells in HIV-1 subtype A/E-infected individuals. This study identified two protective Pol epitopes and characterized them in cases of HIV-1 subtype A/E infection.
Takayuki Chikata, Wayne Paes, Nozomi Kuse, Thomas Partridge, Hiroyuki Gatanaga, Yu Zhang, Kimiko Kuroki, Katsumi Maenaka, Nicola Ternette, Shinichi Oka, Persephone Borrow, and Masafumi Takiguchi, Impact of micropolymorphism outside the peptide binding groove in the clinically-relevant allele HLA-C*14 on T cell responses in HIV-1 infection, J. Virol. 96: e00432-22, 2022
There is increasing evidence for the importance of human leukocyte antigen C (HLA-C)-restricted CD8+ T cells in HIV-1 control, but these responses are relatively poorly investigated. The number of HLA-C-restricted HIV-1 epitopes identified is much smaller than those of HLA-A-restricted or HLA-B-restricted ones. Here, we utilized a mass spectrometry-based approach to identify HIV-1 peptides presented by HLA-C*14:03 protective and HLA-C*14:02 nonprotective alleles. We identified 25 8- to 11-mer HLA-I-bound HIV-1 peptides from HIV-1-infected HLA-C*14:02+/14:03+ cells. Analysis of T cell responses to these peptides identified novel 6 T cell epitopes targeted in HIV-1-infected HLA-C*14:02+/14:03+ subjects. Analyses using HLA stabilization assays demonstrated that all 6 epitope peptides exhibited higher binding to and greater cell surface stabilization of HLA-C*14:02 than HLA-C*14:03. T cell response magnitudes were typically higher in HLA-C*14:02+ than HLA-C*14:03+ individuals, with responses to the Pol KM9 and Nef epitopes being significantly higher. The results show that HLA-C*14:02 can elicit stronger T cell responses to HIV-1 than HLA-C*14:03 and suggest that the single amino acid difference between these HLA-C14 subtypes at position 21, outside the peptide-binding groove, indirectly influences the stability of peptide-HLA-C*14 complexes and induction/expansion of HIV-specific T cells. Taken together with a previous finding that KIR2DL2+ NK cells recognized HLA-C*14:03+ HIV-1-infected cells more than HLA-C*14:02+ ones, the present study indicates that these HLA-C*14 subtypes differentially impact HIV-1 control by T cells and NK cells.
IMPORTANCE Some human leukocyte antigen (HLA) class I alleles are associated with good clinical outcomes in HIV-1 infection and are called protective HLA alleles. Identification of T cell epitopes restricted by protective HLA alleles can give important insight into virus-immune system interactions and inform design of immune-based prophylactic/therapeutic strategies. Although epitopes restricted by many protective HLA-A/B alleles have been identified, protective HLA-C alleles are relatively understudied. Here, we identified 6 novel T cell epitopes presented by both HLA-C*14:02 (no association with protection) and HLA-C*14:03 (protective) using a mass spectrometry-based immunopeptidome profiling approach. We found that these peptides bound to and stabilized HLA-C*14:02 better than HLA-C*14:03 and observed differences in induction/expansion of epitope-specific T cell responses in HIV-infected HLA-C*14:02+ versus HLA-C*14:03+ individuals. These results enhance understanding of how the microstructural difference at position 21 between these HLA-C*14 subtypes may influence cellular immune responses involved in viral control in HIV-1 infection.
Nozomi Kuse,* Hayato Murakoshi,* Tomohiro Akahoshi,* Takayuki Chikata, Katherine L James, Hiroyuki Gatanaga, Sarah L Rowland-Jones, Shinichi Oka, and Masafumi Takiguchi (*equal contribution), Collaboration of a detrimental HLA-B*35:01 allele with HLA-A*24:02 in co-evolution of HIV-1 with T-cells leading to poorer clinical outcomes, J. Virol. 95: e01259-21, 2021
Although mutant-specific T-cells are elicited in some individuals infected with HIV-1 mutant viruses, the detailed characteristics of these T-cells remain unknown. A recent study showed that the accumulation of strains expressing Nef135F, which were selected by HLA-A*24:02-restricted T-cells, was associated with poor outcomes in individuals with the detrimental HLA-B*35:01 allele, and that HLA-B*35:01-restricted NefYF9(Nef135-143)-specific T-cells failed to recognize target cells infected with Nef135F mutant viruses. Here we investigated HLA-B*35:01-restricted T-cells specific for the NefFF9 epitope incorporating the Nef135F mutation. Longitudinal TCR clonotype analysis demonstrated that 3 types of HLA-B*35:01-restricted T-cells (wild type-specific, mutant-specific, and cross-reactive) with different T-cell repertoires were elicited during the clinical course. HLA-B*35:01+ individuals possessing wild type-specific T-cells had a significantly lower pVL than those with mutant-specific and/or cross-reactive T-cells, even though the latter T-cells effectively recognized the mutant virus-infected cells. These results suggest that mutant-specific and cross-reactive T-cells could only partially suppress HIV-1 replication in vivo. Ex vivo analysis of the T-cells showed higher expression of PD-1 on cross-reactive T-cells and lower expression of CD160/2B4 on the mutant-specific T cells than other T-cells, implying that these inhibitory and stimulatory molecules are key to the reduced function of these T-cells. In the present study, we demonstrate that mutant-specific and cross-reactive T-cells do not contribute to suppression of HIV-1 replication in HIV-1-infected individuals, even though they have the capacity to recognize mutant virus-infected cells. Thus, the collaboration of HLA-A*24:02 with the detrimental allele HLA-B*35:01 resulted in the co-evolution of HIV-1 alongside virus-specific T-cells, leading to poorer clinical outcomes. Importance HIV-1 escape mutations are selected under pressure from HIV-1-specific CD8+ T-cells. Accumulation of these mutations in circulating viruses impairs control of HIV-1 by HIV-1-specific T-cells. Although it is known that HIV-1-specific T-cells recognizing mutant virus were elicited in some individuals infected with mutant virus, the role of these T-cells remains unclear. Accumulation of Phenylalanine at HIV-1 Nef135 (Nef135F), which is selected by HLA-A*24:02-restricted T-cells, led to poor clinical outcome in individuals carrying the detrimental HLA-B*35:01 allele. In the present study, we found that HLA-B*35:01-restricted mutant-specific and cross-reactive T-cells were elicited in HLA-B*35:01+ individuals infected with Nef135F mutant virus. These T-cells could not effectively suppress HIV-1 replication in vivo even though they could recognize mutant virus-infected cells in vitro. Mutant-specific and cross-reactive T-cells expressed lower levels of stimulatory molecules and higher levels of inhibitory molecules, respectively, suggesting a potential mechanism whereby these T-cells fail to suppress HIV-1 replication in HIV-1-infected individuals.
Nozomi Kuse, Tomohiro Akahoshi, and Masafumi Takiguchi, STING ligand-mediated priming of functional CD8+ T-cells specific for HIV-1 protective epitopes from naive T-cells, J. Virol. 95: e00699-21, 2021
Functional HIV-1-specific CD8+ T-cells primed from naive T-cells are expected to act as effector T-cells in a eeshock and killff therapeutic strategy for an HIV-1 cure since less functional HIV-1-specific CD8+ T-cells are elicited from memory T-cells in HIV-1-infected individuals on cART. CD8+ T-cells specific for HIV-1 conserved and protective epitopes are candidates of such T-cells. We here investigated the priming with STING ligand of CD8+ T-cells specific for HLA-B*52:01 or HLA-C*12:02-restricted protective epitopes from naive T-cells. STING ligand 3f3f-cGAMP effectively primed CD8+ T-cells specific for 3 of 4 HLA-B*52:01-restricted epitopes but failed to prime those specific for all 3 HLA-C*12:02-restricted epitopes from the naive T-cells of HIV-1-uninfected individuals having an HLA-B*52:01-C*12:02 protective haplotype. These HLA-B*52:01-restricted CD8+ T-cells had a strong ability to suppress HIV-1 replication and expressed a high level of cytolytic effector molecules. The viral suppression ability of these T-cells was significantly correlated with the expression level of perforin and showed a trend for a positive correlation with the expression level of CD107a. The present study highlighted the priming with STING ligand of functional CD8+ T-cells specific for protective epitopes, which T-cells would contribute as effector T-cells to a eeshock and killff therapy.
Importance Current therapeutic strategy eeshock and killff for HIV cure has been directed towards eliminating latent viral reservoirs by reactivation of latent reservoirs with latency-reversing agents followed by eradication of these cells by immune-mediated responses. Although HIV-1-specific T-cells are expected to eradicate viral reservoirs, the function of these T-cells is reduced in HIV-1-infected individuals with long-term cART. Therefore, priming of HIV-1-specific T-cells with high function from naive T-cells are to be expected in these individuals. We here demonstrated the priming with STING ligand 3f3f-cGAMP of CD8+ T-cells specific for HIV-1 protective epitopes from naive T cells. cGAMP primed CD8+ T-cells specific for 3 HLA-B*52:01-restricted protective epitopes, which cells expressed a high level of cytolytic effector molecules and effectively suppressed HIV-1 replication. The present study suggested that the priming with STING ligand of functional CD8+ T-cells specific for protective epitopes would be useful in a therapy for an HIV-1 cure.
Yu Zhang, Hayato Murakoshi, Takayuki Chikata, Tomohiro Akahoshi, Giang Van Tran, Trung Vu Nguyen, Hiroyuki Gatanaga, Kinh Van Nguyen, Shinichi Oka, Nozomi Kuse, and Masafumi Takiguchi, Effect of difference in consensus sequence between HIV-1 subtype A/E and subtype B viruses on elicitation of Gag-specific CD8+ T cells and accumulation of HLA-associated escape mutations, J. Virol. 95: e02061-20, 2021
The Gag280 mutation is associated with HLA-C*01:02 but not with HLA-B*52:01 in subtype A/E-infected individuals, whereas this mutation is associated with HLA-B*52:01 but not with HLA-C*01:02 in subtype B infections. Although it is known that the Gag280 mutant is selected by HLA-B*52:01-restricted GagRI8 (Gag275-282)-specific T-cells in subtype B infections, it remains unknown why this Gag280 mutation is associated with HLA-C*01:02 rather than HLA-B*52:01 in subtype A/E infections. The subtype B and A/E viruses have different consensus sequence, with Thr and Val at Gag280, respectively. To clarify the effect of this difference in Gag280 consensus sequence, we investigated the role of HLA-C*01:02-restricted GagYI9 (Gag277-285)-specific T cells in selection of Gag280 mutations in subtype A/E-infected Vietnamese and subtype B-infected Japanese individuals. GagYI9-4V-specific T-cells, which were frequently elicited in Vietnamese individuals infected with the consensus-type A/E virus, failed to recognize GagV280T mutant A/E virus-infected cells. GagYI9-4T mutant epitope-specific T-cells, which were weakly elicited in individuals infected with the mutant A/E virus, had weak or no ability to recognize the mutant virus. These results account for the mechanism for selection and accumulation of GagV280T mutants in the case of subtype A/E infections. In contrast, HLA-C*01:02-restricted GagYI9-4T-specific T-cells were weakly elicited in Japanese individuals infected with the subtype B virus, explaining why HLA-C*01:02-restricted Gag280 mutations are not accumulated in the case of a subtype B infection. The present study demonstrated that a difference in the Gag280 consensus sequence influenced the elicitation of the GagYI9-specific T-cells involved in the accumulation of HLA-C*01:02-associated Gag280 mutations.
IMPORTANCE HIV-1 mutations escaped from HIV-specific CD8+ T-cells are mostly detected as HLA-associated mutations. A diversity of HLA-associated mutations is somewhat distinct to each race and region, since HLA allele distribution differs among them. A difference in the consensus sequence among HIV-1 subtypes may also influence the diversity of HLA-associated mutations. HLA-C*01:02-associated GagV280T and HLA-B*52:01-associated GagT280A/S mutations were previously identified in HIV-1 subtype A/E-infected and subtype B-infected individuals, respectively, though these subtype viruses have a different consensus sequence at Gag280. We demonstrated that the GagV280T mutant virus was selected by HLA-C*01:02-restricted GagYI9-4V-specific T-cells in subtype A/E-infected Vietnamese but that HLA-C*01:02-restricted GagYI9-4T-specific T-cells were weakly elicited in subtype B-infected Japanese. Together with our recent study which demonstrated the mechanism for the accumulation of HLA-B*52:01-associated mutations, we clarified the mechanism for the accumulation of different Gag280 mutations and the effect of the difference in the consensus sequence on the accumulation of escape mutations.
Hayato Murakoshi, Takayuki Chikata, Tomohiro Akahoshi, Chengcheng Zou, Mohamed Ali Borghan, Giang Van Tran, Trung Vu Nguyen, Kinh Van Nguyen, Nozomi Kuse, and Masafumi Takiguchi, Critical effect of Pol escape mutations associated with detrimental allele HLA-C*15:05 on clinical outcome in HIV-1 subtype A/E infection, AIDS 35: 33-43, 2021
Objective: The mechanism explaining the role of detrimental HLA alleles in HIV-1 infections has been investigated in very few studies. HLA-A*29:01-B*07:05-C*15:05 is a detrimental haplotype in HIV-1 subtype A/E-infected Vietnamese individuals. The accumulation of mutations at Pol 653/657 is associated with a poor clinical outcome in these individuals. However, the detrimental HLA allele and the mechanism responsible for its detrimental effect remains unknown. Therefore, in this present study we identified the detrimental HLA allele and investigated the mechanism responsible for the detrimental effect.
Design and methods: A T-cell epitope including Pol 653/657 and its HLA restriction were identified by using overlapping HIV-1 peptides and cell lines expressing a single HLA. The effect of the mutations on the T-cell recognition of HIV-1-infected cells was investigated by using target cells infected with the mutant viruses. The effect of these mutations on the clinical outcome was analyzed in 74 HLA-C*15:05Vietnamese infected with the subtype A/E virus.
Results: We identified HLA-C*15:05-restricted SL9 epitope including Pol 653/657. PolS653A/T/L mutations within this epitope critically impaired the T-cell recognition of HIV-1-infected cells, indicating that these mutations had escaped from the T-cells. T-cell responders infected with these mutants showed significantly lower CD4 T-cell counts than those with the wild-type virus or Pol S653K/Q mutants, which are not associated with HLA-C*15:05.

Conclusions: The accumulation of Pol S653A/T/L escape mutants critically affected the control of HIV-1 by SL9-specific T-cells and led to a poor clinical outcome in the subtype A/E-infected individuals having the detrimental HLA-C*15:05 allele.