- Open Access
CD8+ T cells specific for conserved, cross-reactive Gag epitopes with strong ability to suppress HIV-1 replication
- Hayato Murakoshi†1,
- Chengcheng Zou†1,
- Nozomi Kuse1,
- Tomohiro Akahoshi1,
- Takayuki Chikata1,
- Hiroyuki Gatanaga1, 3,
- Shinichi Oka1, 3,
- Tomáš Hanke2, 4 and
- Masafumi Takiguchi1Email author
© The Author(s) 2018
Received: 9 May 2018
Accepted: 25 June 2018
Published: 3 July 2018
Development 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.
We 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.
We 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.
Development of effective vaccines against HIV-1 is the best hope for controlling the AIDS epidemic, but no trials has yet showed definitive effect on prevention of HIV-1 infection. Although the RV144 trial in Thailand showed weak protection against HIV-1 most likely through generation of non-neutralizing antibodies [1–7], the outcome of this vaccine trial remains to be reproduced [8, 9]. The STEP study of a candidate T-cell vaccine induced low frequency responses in 77% of vaccine recipients and showed no protection against HIV-1 acquisition [10, 11]. Although a sieve effect of break-through viruses was described, overall, the vaccine-elicited CD8+ T cells had insufficient capacity to suppress HIV-1 replication. There is no simple functional or phenotypic T-cell marker consistently associated with HIV-1 control. The protective capacity of CD8+ T cells likely comes from multiple attributes including specificity, breadth, quality, quantity and being at the right time at the right place. Targeting protective epitopes is one of the key traits.
Our strategy for induction of effective responses is to focus the CD8+ T cells on the highly functionally conserved regions of the HIV-1 proteome [12, 13]. To further improve their efficacy, vaccine immunogens tHIVconsvX consist of 6 protein regions from Gag and Pol with high coverage of known protective epitopes and employ a bi-valent mosaic (two versions of each region, which differ in approximately 1/10 amino acids) to maximize the match of the vaccine to the global circulating viruses. Indeed, using overlapping 15-mer peptides derived from the tHIVconsvX immunogens, we demonstrated the correlation of both the total magnitude and breadth of the tHIVconsvX-specific T-cell responses to lower plasma viral load (pVL) and higher CD4+ T-cell count (CD4 count) in a cohort of 120 treatment-naïve, HIV-1-positive patients in Japan .
Numerous studies have showed that CD8+ T cell targeting of HIV-1 Gag linked to viral load or disease outcome in HIV-1 infection [15–17]. The mechanism of this effect may involve that the viral genome is delivered into the cell in a ribonucleoprotein complex composed largely of Gag after infection, so that Gag epitopes can be processed, presented on the cell surface and finally recognized by Gag-specific CD8+ T cells within a few hours of infection before Nef-mediated down-regulation takes place [18–20].
We previously showed association of CD8+ T cells specific for several Gag and Pol epitopes with significantly lower pVL and higher CD4 count in chronically HIV-1-infected Japanese patients . The majority of these CD8+ T cells recognized conserved or cross-recognized mutated epitopes mostly on the Gag protein. Half of these T cells were restricted by 2 protective alleles, HLA-B*52:01 or HLA-B*67:01, while the remaining recognized peptides presented by HLA-A*02:06, HLA-B*40:02, or HLA-B*40:06.
In the present study, we determined fine specificities and HLA-restriction of CD8+ T-cell responses specific for the two Gag conserved regions of the candidate tHIVconsvX vaccine and extended the protective correlations to clinical outcome in 221 treatment-naïve HIV-1-positive patients. The study identified additional CD8+ T cells specific for Gag conserved epitopes with strong ability to suppress HIV-1 replication. Thus, Gag-specific CD8+ T cells induced by the tHIVconsvX vaccine have the potential to significantly contribute to prevention of HIV-1 infection and eradication of latently infected cells.
T-cell responses to the conserved regions of Gag were protective
Mapping the CD8+ T-cell Gag-specificity to optimal epitopes
We next narrowed the optimal epitopes by using overlapping 11-mer and further truncated peptides. Both the C052/053 STCL and C054/055 STCL recognized both Gag 11-121 and Gag 11-122 peptides (Fig. 4c), suggesting that all the STCL were specific for the same epitope. Analysis using truncated peptides showed that the optimal peptide was TLQEQIGWM (TM9) (Fig. 4d) restricted by HLA-A*26:02. This epitope has not been previously reported. The C113/114 STCL and C125/126 STCL recognized Gag 11-200 and Gag 11-214/215, respectively (Fig. 4c). Again using truncated peptides, we identified previously unreported epitopes HIAKNCRAPR (HR10) and RQANFLGKI (RI9) as the optimal peptides presented by HLA-A*33:03 and HLA-A*02:06, respectively (Fig. 4d). Thus, we identified three novel candidate peptides for the Los Alamos National Laboratory HIV Sequence Database (LANL-HSD; www.hiv.lanl.gov) ‘A’ list of well-defined epitopes.
CD8+ T cells recognizing Gag conserved epitopes are protective in vivo
Association of CTL responses to Gag epitopes with pVL and CD4 count
Median of pVL
Median of CD4
TL8- and HR10-specific T cells suppress HIV-1 replication in vitro
Gag-specific CD8+ T cells cross-recognize epitope variants
Unique vaccine immunogens tHIVconsvX were assembled, which use 2 Gag and 4 Pol conserved regions to focus T-cell responses on the most vulnerable regions (not full-size proteins and not epitopes) of HIV-1 with the bi-valent mosaic design providing the vaccine with a high match to global HIV-1 variants. To date, the immunogenicity of these regions was demonstrated in preclinical studies [14, 26]. Importantly, we were previously able to show that CD8+ T-cell responses to 10 peptide pools derived from the two tHIVconsvX mosaic immunogens correlated significantly with low pVL and high CD4 count in 120 treatment-naïve, HIV-1-infected Japanese patients . In the present study, we focused on Gag-specific CD8+ T-cells because of their strong contribution to favorable clinical outcome. We defined a number of optimal epitopes and their HLA restriction, which were recognized by CD8+ T cells specific for the Gag conserved regions of tHIVconsvX. Furthermore, we demonstrated that 9 Gag-specific CD8+ T-cell responses were significantly associated with low pVL and/or high CD4 count in an extended 221-patient cohort of treatment-naïve patients, implying that T cells specific for these 9 epitopes may contribute to suppression of HIV-1 replication in these individuals. To increase the analysis sensitivity and further endorse the ability of these CD8+ T-cell responses to suppress HIV-1 replication, we confirmed the association of these T-cells with good clinical parameters among the individuals carrying the appropriate HLA restriction molecules. Finally, we identified 5 Gag epitope-specific protective CD8+ T-cell responses and showed a strong association of the magnitude and the breadth of the responses to the 5 epitopes with favorable pVL or CD4 count, indicating again that the responses to 5 Gag epitopes are beneficial. Although escape mutants were selected by the RI8-specific T cells and these HIV-1 mutants accumulated in the Japanese population , 70% of circulating HIV-1 still contain variants of the RI8 epitope recognized by RI8-elicited T cells.
It has now been well documented that not all CD8+ T-cell responses contribute to the suppression of HIV-1 replication equally [14, 15, 21, 27]. Mothe et al. showed that the T-cell responses to forty-eight 18-mer overlapping peptides associated with low viral load in South Africa, Peru, and Spain cohorts . These 18-mer overlapping peptides covering Gag, Pol, Nef, and Vif regions include 59 optimal defined epitopes in the LANL-HSD. The tHIVconsvX immunogen contains most of these; 19 in Gag and 14 in Pol. In the present study, we identified CD8+ T cells specific for 5 Gag conserved epitopes with the ability to suppress HIV-1 replication in Japanese individuals. These CD8+ T cells are restricted by HLA-B*52:01, HLA-A*02:06, HLA-A*33:03, and HLA-B*40:02, and at least one of these four alleles are shared in approximately 70% of Japanese population . Since at least one of these alleles is also found in 45% of Chinese , 35% of Vietnamese , 40% of Thai , 50% of Indian , and 65% of Korean , we hypothesize that if indeed effective, the tHIVconsvX vaccine could be deployed in all these countries; the vaccine would be universal.
To confirm whether the 5 Gag epitopes were recognized by specific T cells as conserved epitopes, we analyzed cross-recognitions of mutant epitopes among the subtype B using the IFN-γ ELISPOT assay. Notably, the circulating viruses carry epitopes with a perfect 9/9 amino acid match or cross-recognized variations in 70–97% isolates from the Japanese population and 46–97% of subtype B viruses (LANL-HSD) and were recognized by T cells specific for 4 epitopes WV8, AA9, TL8, and HR10, though escape mutants selected by the RI8-specific T cells were detected in 27% of the circulating virus in the Japanese cohort . These results suggest that the 4 epitopes are functionally conserved at least among the subtype B viruses. We further analyzed HLA-associated mutations within these six epitopes and found HLA-B*40:02-associated Gag mutation T427N within the HLA-B*40:02/TL8 epitope , suggesting that this mutant is accumulated in the HLA-B*40:02+ Japanese individuals. However, since mutant Gag epitope TL8-1N was cross-recognized by CD8+ T cells in 5 individuals with HLA-B*40:02 (Fig. 7b), TL8-1N may elicit a new T-cell repertoire specific for the 1N mutant or cross-recognizing this mutant. CD8+ T-cells responses specific for TL8 and HR10 epitopes can recognize 70–75% of the circulating HIV-1 isolates in Japan while those specific for WV8, AA9, and RI8 did recognized > 70% of them.
We previously demonstrated that three HLA-B*52:01-, two HLA-B*67:01-, and one HLA-B*02:06-restricted CD8+ T cells specific for Gag epitopes had a strong ability to suppress HIV-1 replication in the Japanese population . The present study demonstrated that CD8+ T cells specific for additional 2 Gag epitopes also contribute to the suppression of HIV-1. Although contained in the tHIVconsvX vaccines, T cells specific for MI8 restricted by HLA-B*52:01 , and TL9 and NL11 restricted by HLA-B*67:01  were not identified in this study. This may be explained by the fact that MI8 and TL9 are included in Pool 1 and that the HLA-B*67:01 is a rare allele found in only 4 out of 221 patients involved in the present study. These findings suggest that Japanese individuals vaccinated with the tHIVconsvX vaccines may respond to 8 Gag epitopes and suppress better HIV-1 replication by killer T cells (Additional file 5: Fig. S5).
The present work supports the hypothesis that the CD8+ T-cell responses, which the tHIVconsvX vaccines aim to elicit, have a real chance to contribute to the suppression of HIV-1 replication in vivo in Japan and other Asian populations both in the context of prevention of HIV-1 infection complementing neutralizing antibodies and in HIV cure by eradicating latently infected cells after HIV-1 reactivation. These results warrantee timely testing of this target immunogen strategy in the clinic.
All treatment-naïve Japanese individuals chronically infected with HIV-1 subtype B were recruited from the National Center for Global Health and Medicine. This study was approved by the ethics committees of Kumamoto University and the National Center for Global Health and Medicine. Informed consent was obtained from all individuals according to the Declaration of Helsinki. Plasma and PBMCs were separated from whole blood. HLA types of the individuals were determined by standard sequence-based genotyping.
The mosaic proteins maximize the coverage of potential T-cell epitopes for the global circulating viruses. We generated three pools containing pairs of 15-mer Gag peptides overlapped by 11 amino acids covering two mosaic regions in the tHIVconsvX. Each pool contains 17 to 23 pairs of the 15-mer peptides. Pool 1, 2, and 3 cover Gag 133-231, Gag 221-327, and Gag 317-363/391-459, respectively . The sequences of the 15-mer peptides in each Pool are shown in Additional file 6: Fig. S6. The 15-mer peptides derived from the tHIVconsvX mosaics were generously provided by the International AIDS Vaccine Initiative. Shorter mapping peptides were synthesized by utilizing an automated multiple peptide synthesizer and purified by high-performance liquid chromatography (HPLC). Purity of all peptides (> 90%) was examined by HPLC and mass spectrometry.
C1R cells expressing HLA-A*26:01 (C1R-A2601), HLA-A*26:02 (C1R-A2602), HLA-A*33:03 (C1R-A3303), HLA-B*07:02 (C1R-B0702), HLA-B*15:01 (C1R-B1501), HLA-B*39:01 (C1R-B3901), or HLA-B*44:03 (C1R-B4403) were previously generated by transfecting these genes into C1R cell lines [21, 34–39]. 721.221 cells expressing CD4 molecules and HLA-C*03:03 (721.221-C0303) were generated by transfecting the genes into the 721.221 cell line. 721.221 cells expressing CD4 molecules and HLA-A*02:06 (721.221-A0206), HLA-A*24:02 (721.221-A2402), HLA-A*33:03 (721.221-A3303), HLA-B*40:02 (721.221-B4002), HLA-C*14:03 (721.221-C1403), or HLA-C*07:02 (721.221-C0702) were previously generated [21, 40–43]. All cell lines were cultured in RPMI 1640 medium containing 10% FCS medium (R10) with 0.15 mg/ml hygromycin B.
Expansion of HIV-1-specific T cells from HIV-1-infected individuals
PBMCs from KI-1020, KI-1102, and KI-1114 were incubated with 1 μM 15-mer peptide pairs (C052/053 or C054/055, C113/114, and C125/126, respectively) and cultured for 12-14 days to induce peptide-specific short-term cell lines (STCL).
Intracellular cytokine staining (ICS) assay
C1R and 721.221 cells prepulsed with each peptide or 721.221 cells infected with the HIV-1, strain NL4-3, were added to the effector STCLs in a 96-well plate and incubated for 2 h at 37 °C. Brefeldin A (10 μg/ml) was then added and the cells were incubated further for 4 h, fixed with 4% paraformaldehyde and incubated in permeabilization buffer [0.1% saponin–10% FBS–phosphate-buffered saline (PBS)] after staining with allophycocyanin (APC)-labeled anti-CD8 monoclonal antibody (mAb) (Dako, Glostrup, Denmark). Thereafter, the cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-interferon γ (IFN-γ) mAb (BD Bioscience, CA). The percentage of IFN-γ-producing cells among the CD8+ T-cell population was determined by flow cytometry.
IFN-γ enzyme-linked immunospot (ELISPOT) assay
1×105 PBMCs from HIV-1-positive individuals and peptides were added to 96-well polyvinylidene plates (Millipore, Bedford, MA) that had been precoated with 5 μg/ml anti-IFN-γ mAb; 1-D1K (Mabtech, Stockholm, Sweden). Peptide pools or 15-mer peptide pairs were used at a concentration of 1 µM whereas optimal epitope peptides at a concentration of 100 nM in this assay. The plates were then incubated for 16 h at 37 °C before the addition of biotinylated anti-IFN-γ mAb (Mabtech) at 1 μg/ml at room temperature for 90 min, streptavidin-conjugated alkaline phosphatase (Mabtech) for at room temperature 60 min. Individual cytokine-producing cells were visualized as dark spots after a 20-min reaction with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium in the presence of an alkaline phosphatase-conjugated substrate (Bio-Rad, Richmond, CA, USA). The spots were counted with an Eliphoto-Counter (Minerva Teck, Tokyo, Japan). The frequencies of the responding cells were represented as spot-forming units (SFU)/106 CD8+ T cells by measuring frequency of CD8+ T cells using a flow cytometry. A mean+5 SD of the SFUs of samples (N = 3) from 12 HIV-1-naïve individuals for the peptide pool was 115 SFU/106 CD8+ T cells. Therefore, we defined a positive ELISPOT response as larger than 200 SFU/106 CD8+ T cells to exclude false positive.
Establishment of T-cell lines specific for TL8 and HR10 peptides using HLA/peptide tetramer complexes
To establish T-cell lines specific for TL8 and HR10 peptides, HLA-B*40:02/TL8 or HLA-A*33:03/HR10 tetrameric complexes (tetramers) were synthesized as previously described . Briefly, PBMCs of HLA-B*40:02+ individual (KI-1391) and HLA-A*33:03+ one (KI-1320) were stained with PE-conjugated specific tetramers at a concentration of 100 nM at 37 °C for 30 min. The cells were then washed twice with R10, followed by staining with FITC-conjugated anti-CD8 mAb and 7-AAD at 4 °C for 30 min. The CD8+ T cells specific for the TL8 and HR10 peptides were then sorted by the FACSAria. The sorted cells were stimulated with 100 nM of the corresponding epitope peptides and then cultured for 12–14 days to induce T cell lines specific for TL8 and HR10. To confirm purities of the specific T cells, the cell lines were analyzed by using the specific tetramers.
In vitro virus inhibition assay
The ability of HIV-1-specific CTLs to suppress HIV-1 replication was examined as previously described [45, 46]. CD4+ T cells isolated from PBMCs of healthy donors carrying HLA-B*40:02 or -A*33:03 were infected with NL4-3 and then the infected cells were co-cultured with epitope-specific T-cell lines at E:T ratios of 1:1 and 0.1:1. On day 3–4 post infection, the concentration of p24 Ag in the culture supernatant was measured by using an enzyme-linked immunosorbent assay.
Two-tailed Mann–Whitney’s test was performed for comparison of two groups. Correlations between magnitudes and breadths of T cell responses and pVL or CD4 count were statistically analyzed using Spearman rank test. P values < 0.05 were considered to be statistically significant.
HM wrote the manuscript, performed assays, and analyzed assay results. CZ performed assays and analyzed assay results. NK, TA and TC performed assays. HG and SO collected samples and clinical data from patients. TH provided the overlapping 15-mer peptides derived from the tHIVconsvX immunogens and contributed to writing the manuscript. MT designed the study, oversaw the experiments, analyzed all of the data, and wrote and edited the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Availability of data and materials
Consent for publication
Ethics approval and consent to participate
This study was approved by the ethics committees of Kumamoto University and the National Center for Global Health and Medicine. Informed consent was obtained from all individuals according to the Declaration of Helsinki.
This research was supported by grants-in-aid (15fk0410019h0001, 16fk0410202h0002, and17fk0410302h0003) for AIDS Research from AMED and by grants-in-aid (26293240, 17K10021) for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan. C.Z. was supported by the China Scholarship Council (CSC) scholarship. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
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