Summary of the publications
Center for AIDS Research Best Paper Award 2016
Zhansong Lin
, Kimiko Kuroki, Nozomi Kuse, Xiaoming Sun, Tomohiro Akahoshi, Ying Qi, Takayuki Chikata, Takuya Naruto, Madoka Koyanagi, Hayato Murakoshi, Hiroyuki Gatanaga, Shinichi Oka, Mary Carrington, Katsumi Maenaka, Masafumi Takiguchi, HIV-1 control by NK cells via reduced interaction between KIR2DL2 and HLA-C*12:02/C*14:03, Cell Reports 17:2210-2220, 2016
 NK cells, which had been classified as a member of the innate immune system, exhibit specificity in the recognition of target cells. The interaction between Killer cell Immunoglobulin-like Receptors (KIRs) expressed on NK cells and HLA ligands on target cells plays an important role in the regulation of NK-cell functions which influences the clinical outcome of HIV-1 infection. In recent years, the effects of NK cells and KIR genes on HIV-1 control have been partially determined. However, few studies have shown the synergistic effect of KIR and HLA on HIV-1 infection and its mechanism. In our Cell Reports paper, we studied the effect of KIR-HLA interactions on NK-cell activation and eradication of HIV-1 infection in the Japanese population and its mechanism.
 We first analyzed the correlation between clinical outcomes of HIV-1 infection and the presence of different KIR genes or KIR-HLA combinations in 504 treatment-naive Japanese individuals who are chronically infected with HIV-1. We found that two HLA-C alleles, C*12:02 and C*14:03, showed a synergistic effect with KIR2DL2, which belongs to the inhibitory receptor family, on suppression of HIV-1 replication. To further uncover the protective mechanisms by the two KIR/HLA combinations, we investigated the function of KIR2DL2+ NK cells and performed molecular analysis.
 Interestingly, HLA-C*14:02 allele, which has only minor difference with -C*14:03, did not show such protective effect. Indeed, viral suppression assay showed that KIR2DL2+ NK cells suppressed wild type (WT) HIV-1 replication in HLA-C*14:03+ cells to a significantly greater extent than in -C*14:02+ cells. Functional analysis and peptide-HLA binding assay further demonstrated that the lower binding between HIV-1 derived peptides and C1403 alleles largely influenced antiviral function of KIR2DL2+ NK cells through reduced expression of peptide-HLA (pHLA) complex (i.e., lower presentation of KIR2DL2 ligand) on the cell surface.
 B52-C12 is a famous protective HLA haplotype in the Japanese population against HIV-1 infection. Our previous study identified a V to A mutation on the 9th amino acid (9A) within a C1202-restricted Pol-IY10 epitope peptide. This 9A mutation enables the virus to escape from the immune pressure exerted by C1202-restricted CTLs and thus longitudinally accumulates among C1202+ individuals. However, viral suppression assay in our present study showed that KIR2DL2+ NK cells suppressed replication of the mutant virus at a significantly higher level than that of the WT virus in HLA-C*12:02+ cells. Functional analysis and peptide-HLA binding assay suggested that the lower binding between 9A mutant peptide and HLA-C1202 allele largely facilitated antiviral function of KIR2DL2+ NK cells through reduced expression of pHLA complex on the cell surface.
 Surface plasmon resonance studies (BIAcore) revealed that peptide-HLA binding did not influence the binding of pHLA complex to KIR2DL2 molecules. Rather, it independently affected NK cell antiviral functions for both of the HLA/KIR combinations. The activating counterpart of KIR2DL2, KIR2DS2, did not play a role in either of the combinations.
 Taken together, our present study found that: 1) Two Japanese-specific HLA-C alleles combined with KIR2LD2 are associated with a synergistic protective effect on HIV-1 control; 2) Peptide-HLA binding directly influences NK-cell recognition of target cells and antiviral function through inhibitory receptor KIR2DL2; 3) CTL-selected escape mutations influence NK-cell antiviral function.
Xiaoming Sun, Yi Shi, Tomohiro Akahoshi, Mamoru Fujiwara, Hiroyuki Gatanaga, Christian Schonbach, Nozomi Kuse, Victor Appay, George F. Gao, Shinichi Oka, and Masafumi Takiguchi, Effects of a single escape mutation on T cell and HIV-1 co-adaptation, Cell Reports, 15:2279-2291, 2016
The mechanistic basis for the progressive accumulation of Y135F Nef mutant viruses in HIV-1-infected population remains poorly understood. Y135F viruses carry the 2F mutation within RW8 and RF10, which are two HLA-A*24:02-restricted superimposed Nef epitopes, recognized by distinct and adaptable CD8+ T-cell responses. We performed here comprehensive analyses of the T-cell receptor repertoire and cross-reactive potential of wild-type or 2F RW8- and RF10-specific CD8+ T-cells, together with peptide-MHC complex stability and crystal structure studies. We demonstrate that, by affecting critical direct hydrogen bond interactions and water molecule-mediated hydrogen bond networks within the peptide-MHC complex, the 2F mutation reduced both TCR and HLA bindings, thus granting definite advantages for the virus to decrease CD8+ T-cell efficacy and supporting the evolution of the virus towards the 2F variant. Our study provides refined understanding of the HIV-1 and CD8+ T-cell co-adaptation at the population level.
Giang Van Tran,* Takayuki Chikata,* Jonathan M. Carson, Hayato Murakoshi, Dung Hoai Nguyen, Yoshiko Tamura, Tomohiro Akahoshi, Nozomi Kuse, Keiko Sakai, Sachiko Sakai, Kyle Cobarrubias, Shinichi Oka, Zabrina L. Brumme, Kinh Van Nguyen, and Masafumi Takiguchi (*Equal contribution), A strong association of HLA-associated Pol and Gag mutations with clinical parameters in HIV-1 subtype A/E infection, AIDS 30:681-689, 2016
OBJECTIVES: Identification of human leukocyte antigen-associated HIV-1 polymorphisms (HLA-APs) in different global populations furthers our understanding of HIV-1 pathogenesis and may help identify candidate immunogens for HIV vaccines targeted to these populations. Although numerous population-based studies identifying HLA-APs have been conducted in HIV-1 subtype B- and subtype C-infected cohorts, few have focused on subtype A/E.
DESIGN: We investigated HLA-APs in a cohort of chronically HIV-1 subtype A/E-infected Vietnamese individuals.
METHODS: HLA-APs in HIV-1 Gag, Pol, and Nef regions from 388 treatment-naive individuals chronically infected with HIV-1 subtype A/E were analyzed using phylogenetically informed approaches.
RESULTS: A total of 303 HLA-APs were identified. HLA-APs occurring at six positions in Gag and six positions in Pol were significantly associated with higher plasma viral load (pVL), whereas HLA-APs occurring at two positions in Gag and 13 positions in Pol were significantly associated with lower CD4 T-cell counts. Furthermore, the proportion of Pol codons harboring an HLA-AP specific to the host's HLA correlated positively with HIV-1 pVL (R=0.22; P<0.0001) and inversely with CD4 T-cell counts (R=-0.32; P<0.0001). Similarly, the proportion of HLA-associated Gag codons harboring host-specific HLA-AP correlated inversely with CD4 T-cell counts (R=-0.13; P=0.01).
CONCLUSION: These significant associations between HIV-1 amino acids adapted to Vietnamese HLA alleles and higher pVL and lower CD4 T-cell counts suggests that accumulation of cytotoxic T cells escape mutations may influence clinical outcomes in HIV-1 subtype A/E-infected Vietnamese individuals.
Nozomi Kuse*, Mohammad Arif Rahman*, Hayato Murakoshi*, Giang Van Tran, Takayuki Chikata, Madoka Koyanagi, Kinh Van Nguyen, Hiroyuki Gatanaga, Shinichi Oka, and Masafumi Takiguchi, Different Effects of Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutations on Cytotoxic T Lymphocyte Recognition between HIV-1 Subtype B and Subtype A/E Infections (*Equal contribution). J. Virol. 89:7363-7372, 2015

The effect of antiretroviral drug resistance mutations on cytotoxic T lymphocyte (CTL) recognition has been analyzed in HIV-1 subtype B infections, but it remains unclear in infections by other HIV-1 subtypes that are epidemic in countries where antiretroviral drugs are not effectively used. We investigated the effect of nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistance mutations (Y181C, Y181I, and Y181V) on epitope recognition by CTLs specific for 3 different HIV-1 epitopes (HLAA*02:01-restricted IV10, HLA-B*35:01-restricted NY9, and HLA-C*12:02-restricted KY9) in subtype B and subtype A/E infections and the accumulation of these mutations in treatment-naive Japanese and Vietnamese. These NNRTI-resistance mutations critically affected NY9-specific and KY9-specific T cell responses in the subtype B infections, whereas they showed a different effect on IV10-specific T cell responses among the subtype B-infected individuals. These mutations affected IV10-specific T cell responses but weakly affected NY9-specific T cell responses in the subtype A/E infections. The substitution at position 3 of NY9 epitope which was found in the subtype A/E virus differently influenced the peptide binding to HLA-B*35:01, suggesting that the differences in peptide binding may result in the differences in T cell recognition between the subtype B virus and A/E virus infections. The Y181C mutation was found to be accumulating in treatment-naive Vietnamese infected with the subtype A/E virus. The present study demonstrated different effects of NNRTI-resistance RT181 mutations on CTL responses between the 2 subtype infections. The Y181C mutation may influence HIV-1 control by the CTLs in Vietnam, since this mutation has been accumulating in treatment-naive Vietnamese.

Center for AIDS Research Best Paper Award 2015
Hayato Murakoshi
, Tomohiro Akahoshi, Madoka Koyanagi, Takayuki Chikata, Takuya Naruto, Rie Maruyama, Yoshiko Tamura, Naoki Ishizuka, Hiroyuki Gatanaga, Shinichi Oka, and Masafumi Takiguchi, Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes. J. Virol. 89:5330-5339, 2015

Identification and characterization of CD8+ T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and the studies of AIDS pathogenesis although such CD8+ T cells have been only partially identified. We here 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 breadth of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (p = 2.1×10-8) and positively with CD4 counts (p = 5.3×10-8), 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 4 non-conserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates of antigens for 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.