Although antiretroviral therapy (ART) has been demonstrated as an effective way to reduce morbidity, mortality and, more importantly, HIV transmission, its deleterious side-effects and cost also put a lifelong burden on its users. Prevention of HIV by vaccine is believed to be the most cost-effective and well tolerated intervention for HIV/AIDS epidemic. Decades of disappointing HIV vaccine development has asked us an important question ‘what are the immune correlates of protection against HIV infection?’.
Several immunological factors have been linked with HIV control in which HIV-specific CD8+ T cells are convincingly the most important component . Impacts of HIV-specific CD8+ T cells have been evidently demonstrated in a large number of studies, including reduction of peak viremia observed during primary infection, higher simian immunodeficiency virus load and rapid disease progression in CD8+-depleted macaques, direct killing of CD4+ T cells and suppression of HIV replication [2–8]. Recent evidence showed us that not all T cells were protective against HIV . Presence of HIV-specific T cells defined by gamma interferon assay is not indicative of T-cell-mediated immunity. Subsequent studies confirmed that only T cells with higher functional quality were protective and hence controlled HIV replication in HIV-infected individuals with good clinical outcome [9–11]. These high functional quality or ‘polyfunctional’ T cells are T cells which simultaneously produce multiple cytokines/chemokines, up-regulate surface cytotoxic function such as CD107a and are perhaps with proliferative capacity . Not only functional quality of the HIV-specific T cells, but also their specificity is undoubtedly essential to determine level of protection against HIV infection. T cells targeted at capsid p24 of HIV were seen to be associated with low HIV-RNA, whereas other T cells against other proteins seemed to relate with high HIV load .
Amongst associations between HIV control and CD8+ T-cell responses, the most unequivocal evidence is the protective effect observed with some certain human leukocyte antigen (HLA)-I alleles [14,15]. These HLA-I alleles are frequently presented in a unique group of infected individuals termed ‘HIV controllers’, who are able to control HIV naturally (lower than 2000 copies/ml HIV load). There are three HLA-I alleles frequently regarded as ‘protective alleles’: HLA-B*27, HLA-B*57 and HLA-B*58 [14–18]. These associations of HIV control might be hypothetically resulted from T-cell recognition of epitopes presented on these ‘protective’ HLA alleles [18–22]. However, possessing these protective alleles does not guarantee good clinical outcome; therefore it is very interesting to investigate T-cell quality in those protective allele-positive individuals with different HIV control.
In this study, we asked whether functional quality of the T cells directing against same p24 antigen in protective HLA-I allele-matched controllers and noncontrollers was different.
Materials and methods
Forty-five chronically HIV-infected individuals, from King Chulalongkorn Memorial Hospital and HIV Clinic, Thai Red Cross Society, were enrolled into this study. All were antiretroviral drug-naive with no opportunistic infections. Signed informed consents were obtained from all individuals. This study was approved by Ethic Committee of Faculty of Medicine, Chulalongkorn University.
Plasma HIV load (pVL) (Roche, Indianapolis, Indiana, USA), complete blood count (CBC), and CD4+ and CD8+ T-cell count (Beckman Coulter, Brea, California, USA) were determined at Department of Microbiology, King Chulalongkorn Memorial Hospital, Thai Red Cross Society.
Donors were categorized into two groups according to their pVL: ‘viremic controllers’ (pVL ≤2000 copies/ml) and ‘noncontrollers’ (pVL >2000 copies/ml) as defined elsewhere [23,24]. Only donors with consecutively 3 months apart pVL control were considered viremic controllers.
Peripheral blood mononuclear cell preparation
Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA blood by standard density-gradient centrifugation using Ficoll Hypaque (Amersham Biosciences, Uppsala, Sweden). Isolated PBMCs were either used freshly in an Enzyme-linked immunosorbent spot (ELISpot) assay or rested at 106 PBMC/ml overnight in R10 [RPMI1640 (Gibco, Carlsbad, California, USA) supplemented with 10% heat-inactivated fetal bovine serum (Lonza, Allendale, New Jersey, USA)] for an intracellular cytokines staining (ICS) assay performing on the following day. The rest were cryo-preserved at −80°C for future usage.
HLA class I alleles were typed using both PCR-sequence-specific oligonucleotides (PCR-SSOP) and PCR-sequence-specific primers (PCR-SSP) by Proimmune Ltd. (Oxford, UK).
HIV p24 sequencing
Viral RNA was extracted from 200 μl of fresh or frozen plasma. Reverse transcription-nested PCR was performed using the following primers: 5′-GAGGTGCA CACAGCAAGAGGCG-3′, 5′-CCCCCTATCATTTTTG GTTTCC-3′ (outer1), 5′-GCGRCTGGTGAGTACGCC-3′, 5′-RGGAAGGCCAGATYTTCC-3′ (outer2) and 5′-GGCGAGAGCGGCGACTGGTGAG-3′, 5′-CCCCTCTGT ATCATCTGCTCCTGTATC-3′ (inner) for p24. All sequences were analyzed using Bioedit Sequence Alignment Editor version 220.127.116.11 .
Design of currently circulating HIV Gag p24 overlapping peptides and epitopes
Twenty-three overlapping peptides (OLPs) (20 overlapped by 10 amino acids) and 7 HLA-B*27, B*57 and B*58 restricted epitopes (Supplement Table 1, http://links.lww.com/QAD/A268) spanning Gag p24 protein were designed based on a consensus sequence derived from 10 randomly selected HIV-infected individuals. The majority of these sequences (9/10) were CRF_01AE subtype (Supplement Fig. 1, http://links.lww.com/QAD/A268). All peptides were synthesized by Mimotopes (Clayton, Victoria, Australia).
Gamma interferon (IFN-γ) ELISpot was performed as follows. In brief, 2.5 × 105 freshly isolated PBMC was plated into each well of 96 wells polyvinylidene plate (Millipore, Billerica, Massachusetts, USA) which were manually coated for 3 h with 15 μg/ml of anti-IFN-γ mAb (D1K, Mabtech, Nacka Strand, Sweden) and incubated at 37°C, 5% CO2 for 15 h in the presence of 10 μg/ml of each peptide. Each peptide was tested individually and performed in duplicate. Phytohemagglutinin (Sigma–Aldrich, Munich, Germany) and R10 were used as positive and negative control. After discarding cell suspension, biotinylated secondary anti-IFN-γ mAb (7-B6–1; Mabtech) was added at a final concentration of 1 μg/ml and incubated in the dark for 3 h. Streptavidin-conjugated alkaline phosphatase (Mabtech) was then added and incubated for another hour. Plate was developed using alkaline phosphatase substrate kit (BioRad, Hercules, California, USA). Spot-forming units (SFUs) were determined using ELISpot reader (Carl-Zeiss, Maple Grove, Minnesota, USA) and calculated by subtracting with negative control. Only responses with more than 50 SFU/106 PBMCs and 10 times over the background were considered positive. Individual positive response (OLPs or epitopes) was used to calculate donors’ median which was subsequently used to calculated group median and compared.
P24-specific CD8+ T cells functional quality determination
Overnight-rested PBMCs were washed, resuspended and adjusted with R10 to a concentration of 107 PBMC/ml. One hundred microliters of cell suspension was cultured with antihuman CD28, antihuman CD49d, antihuman CD107a PE-Cy5 (Beckton Dickinson, Franklin Lakes, New Jersey, USA), Brefeldin-A (Sigma–Aldrich) and 10 μg/ml of each ELISpot-responding peptide and incubated at 37°C, 5% CO2 for 6 h. Streptococcus enterotoxin B (Sigma–Aldrich), irrelevant peptide and dimethyl sulfoxide (DMSO) (Sigma–Aldrich) were used as positive and negative control. PBMCs were then stained with antihuman CD3 APC-H7 (Beckton Dickinson) and antihuman CD8 Pacific Blue (Biolegend, San Diego, California, USA), and incubated for another 20 min. PBMCs were permeabilized using Cytofix/Cytoperm (Beckton Dickinson) according to user manual and stained with antihuman IL2 FITC, antihuman TNF-α APC, antihuman IFN-γ PE-Cy7 (Biolegend) and antihuman MIP1-β PE (Beckton Dickinson) for an additional 30 min. PBMCs were fixed with 1% paraformaldehyde in phosphate buffer saline (Sigma–Aldrich), kept at 4°C overnight and analyzed on FACS Aria II (Beckton Dickinson). All analyses were performed using FACSDiva software version 6.1.2 (Beckton Dickinson, Billerica, Massachusetts, USA). Only CD3+CD8+ T lymphocytes were taken into consideration as shown in gating strategy (Supplement Fig. 2, http://links.lww.com/QAD/A268). At least 1.5 × 106 events were recorded. Specific CD8+ T-cell responses were calculated by subtracting with negative control. Only responses above background level were considered positive. CBC and CD8+ T-cell count data were used to calculate absolute numbers of responding cells. Multiparameter analyses were performed using FCOM algorithm on WinList software (Verity Software House, Topsham, Maine, USA). Individuals’ absolute number of each functional phenotype was used to calculate group median which was subsequently compared to determine the differences between viremic controllers and noncontrollers.
Two-tailed, Mann–Whitney U test and Spearman R test were used to compare group median and determine a correlation, respectively (Prism version 5, Graphpad Software, La Jolla, California, USA). Principal component analysis (PCA) was used for a multivariate data analysis (Unscrambler version 9.7, CAMO Software, Oslo, Norway). P value less than 0.05 was considered statistically significant.
Protective HLA-I alleles were not associated with HIV control
We categorized donors in this study according to their pVL to reflect their HIV control ability. Indeed, viremic controllers (n = 13) were with better clinical outcome than noncontrollers (n = 32) as reflected by absolute CD4+ cell count (Table 1). This difference was not due to their stage of HIV infection since their median time after first diagnosis was similar (Table 1). HLA-B*27, HLA-B*57 and HLA-B*58 have been shown to be associated with HIV control [14–18]. We determined the influence of these ‘protective alleles’ by categorizing donors according to their HLA-I into two groups: individuals with protective allele(s) (n = 19) and individuals without protective allele(s) (n = 26). Surprisingly, both CD4+ cell count and pVL were not different between individuals with protective allele(s) and those without protective allele(s) (Table 1). This comparable clinical outcome was independent of HIV duration and other demographic characteristics (Table 1). Indeed, there was a similar proportion of individuals with protective alleles in both viremic controllers (70%) and noncontrollers (60%). It was apparent that the mere presence and absence of protective allele(s) was not associated with clinical outcome in this study. This finding suggested that possession of protective allele(s) per se might not be sufficient to arm HIV-infected individuals with protective HIV immunity, and additional factors would be required to confer an ability to control HIV.
Interferon-γ producing T cells were of similar characteristic between viremic controllers and noncontrollers
In order to determine the protective effect of HIV-specific T cells, we investigated p24-specific T-cell responses in fresh PBMCs using an IFN-γ ELISpot assay. There were four nonresponders, three viremic controllers and one noncontroller, who were excluded from further investigations. Overall, both noncontrollers and viremic controllers mediated the same breadth of responses (median = 3 OLPs). Although viremic controllers seemed to mount higher magnitude of responses than noncontrollers, it was not statistically significant (Table 2). Different HLA-I restriction might have an effect on both breadth and magnitude of T-cell responses. These differences were likely due to the frequency of HLA-I alleles, which, in turn, have an influence on immunodominance of epitopes in a certain population or patient. In order to exclude impacts of HLA-I restriction, p24 epitope-specific T-cell responses of viremic controllers and noncontrollers were analyzed in a protective allele-matched manner. There were eight HLA-B*27-positive donors in this study consisting of four viremic controllers and four noncontrollers. In this HLA-B*27-positive group, viremic controllers had significantly lower pVL than noncontrollers (1104.5 vs. 11 747 copies/ml; P < 0.05). Though CD4 cell count was higher in viremic controllers than noncontrollers, it did not reach statistic significance (Table 3). Similarly, in the HLA-B*57/58-positive group, there were three viremic controllers and nine noncontrollers in which viremic controllers had significantly lower pVL and higher CD4+ cell count than noncontrollers (Table 3). However, better clinical outcome observed in either HLA-B*27 and HLA-B*57/58-positive viremic controllers could not simply be explained by their better T-cell responses as estimated by IFN-γ ELISpot assay, since epitope-specific T-cell responses were similar. In addition, the overall p24-specific T-cell responses (as defined by both breadth and median magnitude of T-cell responses against 23 OLPs spanning Gag p24 protein) were also not different between these individuals with protective allele(s)-matched viremic controllers and noncontrollers (Table 3). These findings suggested that, at an epitope-specific level, analysis of IFN-γ-producing cells was insufficient to demonstrate the protective quality of the p24-specific T-cell responses . In order to precisely determine an effect of T-cell responses, we performed an intracellular cytokine staining (ICS) assay to investigate the functional quality of p24-specific CD8+ T-cell responses in these donors.
P24-specific-CD8+ T-cell responses of viremic controllers were of higher functional quality than those of noncontrollers
Total number of 20 individuals (8 viremic controllers and 12 noncontrollers) was included in this functional quality assessment by using multiparametric flow cytometry upon stimulation with each responding peptide previously defined by an IFN-γ ELISpot assay. Fresh PBMCs from the same time-point with ELISpot screening were used as results from our preliminary study showed an enhanced sensitivity of cytokine detection when using fresh PBMCs as compared to frozen samples (data not shown). Moreover, with the CBC data, we were able to calculate absolute number of responding CD8+ T cells, hence allowed us to investigate p24-specific CD8+ T-cell responses in their actual number, not the proportion of total lymphocyte.
Firstly, p24-specific CD8+ T-cell responses were determined as a whole (summation of every single OLP-specific response in each individual). Significantly larger number of high functional quality p24-specific CD8+ T cells (defined as having simultaneous four or five functions) was observed in viremic controllers as compared to noncontrollers (Fig. 1a). Subsequently, an absolute number of each possible functional phenotype was compared between noncontrollers and viremic controllers to determine its association with HIV control. Though many functional phenotypes were different between viremic controllers and noncontrollers, only three reached statistical significance; full five functions, IL-2+TNF-α+IFN-γ+CD107a+MIP1-β+, four functions TNF-α+IFN-γ+CD107a+MIP1-β+ and three functions IFN-γ+CD107a+MIP1-β+ (Fig. 1b). The absolute number of these subpopulations in viremic controllers and noncontrollers were 47 vs. 0 cell/μl (P = 0.01), 352 vs. 62 cell/μl (P = 0.0038) and 91 vs. 9 cell/μl (P = 0.01), respectively. In noncontrollers, p24-specific CD8+ T-cell responses were dominated by single MIP1-β-producing cells (Fig. 1b).
Discordant HIV control between protective-allele(s)-matched donors was attributed from the functional quality of CD8+ T-cell responses
Next, p24-specific CD8+ T-cell responses from viremic controllers and noncontrollers matching for the same protective HLA-B*27 or HLA-B*57/58 allele were subsequently analyzed to determine whether their diverse clinical outcome was resulted from their different quality of responses. In HLA-B*27 group, viremic controllers (n = 3) exhibited significantly more p24-specific CD8+ T cells with five functions than did noncontrollers (n = 4) (437 vs. 0 cell/μl; P < 0.05). Although several other functional phenotypes were also higher in viremic controllers than noncontrollers, these differences were not statistically significant (Fig. 2a). We next determined their quality of response against an HLA-B*27-restricted epitope, KRWIILGLNK (KK10). Significantly larger number of full five functions, KK10-specifc CD8+ T cells, were observed in viremic controllers than noncontrollers (Fig. 2b). Similarly, HLA-B*57/58-positive viremic controllers (n = 3) possessed significantly larger number of p24-specific CD8+ T cells with five functions than did noncontrollers (n = 5) (23 vs. 0 cell/μl; P < 0.05) (Fig. 2c). Due to the limited number of responders to assess each of the six HLA-B*57/58-restricted epitopes individually, they were accumulated as a summation of all the responding epitopes of each individual. Larger number of five functions CD8+ T cells was observed in viremic controllers than noncontrollers; however, it did not reach statistic significance (Fig. 2d).
Better clinical outcome could be explained by an absolute number of high functional quality p24-specific CD8+ T cells
Results from our previous experiments had demonstrated an association between high functional quality CD8+ T-cell responses and good clinical outcome at both total p24 protein and a single epitope-specific level (Figs 1 and 2). We next determined the relationship between the functional quality of p24-specific CD8+ T-cell responses and readouts of HIV clinical outcome (CD4+ cell count and pVL). Significantly lower level of pVL and higher level of CD4+ cell count were observed in HIV-1-infected donors possessing p24-specific CD8+ T cells with full five functions (n = 11) compared to those who did not (n = 9) (Fig. 3a and b). Results from a multivariate PCA roughly showed that absolute number of p24-specific CD8+ T cells with four and five functions were co-varying variables and in a negative and positive correlation with pVL and CD4+ cell count, respectively (Supplement Fig. 3, http://links.lww.com/QAD/A268). These relationships were confirmed with the Spearman's correlation coefficients. Indeed, an absolute number of CD8+ T cells with full five functions and four functions were significantly in a negative correlation with pVL (r = −0.6984, P = 0.0006 and r = −0.5729, P = 0.0083, respectively) (Fig. 3c and e) and in a positive correlation with CD4+ T-cell count (r = 0.5648, P = 0.0095 and r = 0.4567, P = 0.0429, respectively) (Fig. 3d and f). These data suggested that good clinical outcome observed in viremic controllers was strongly associated with their absolute number of high functional quality p24-specific CD8+ T cells.
In this study, we demonstrated that mere presence of some previously defined ‘protective alleles’ (HLA-B*27, HLA-B*57 and HLA-B*58) per se did not guarantee this controller status. Interestingly, ‘HLA-B*5801’ did not confer any protective effect in our study since all HLA-B*5801 volunteers were noncontrollers (Supplement Table 3, http://links.lww.com/QAD/A268). Moreover, we also demonstrated a strong association between an absolute number of Gag p24-specific CD8+ T cells with high functional quality and natural HIV control.
Presence or absence of an HLA-B*27-restricted KRWIILGLNK (KK10)-specific T-cell response was demonstrated to determine HIV load in HLA-B*27+ individuals [26–28]. In our study, despite 10-fold difference in HIV load, the comparable frequency and magnitude of the KK10 response was observed in viremic controllers and noncontrollers. In addition, both groups contained one HLA-B*27+-KK10 nonresponder. However, unlike previous studies [26,28,29], this was not due to KK10 escape mutation (all were conserved). A high-resolution HLA analysis revealed that HLA-B*27 of these nonresponders were HLA-B*2706 whilst KK10-responders were HLA-B*2704 and HLA-B*2705 alleles (Supplement Table 2, http://links.lww.com/QAD/A268). Different epitope-binding properties among HLA-B*27 subtypes might be related to this lack of KK10 response. HLA-B*2705-KK10 crystal structure has demonstrated that residues 77 and 116 are key residues in determining F-pocket binding affinity . Replacing wild-type aspartic acid at position 77 with nucleophilic serine (in HLA-B*2704) might have minor effect on KK10 binding, since HLA-B*2704+ individuals elicit KK10 responses equally well. On the contrary, substitution of aspartic acid at residue 116 with bulky aromatic-side-chained tyrosine (in HLA-B*2706) might abrogate binding of KK10 epitope and lead to failure of the T-cell response in HLA-B*2706+ individuals.
Due to the limited frequency of only 1.3% HLA-B*5701 carriers in Thai population  and since HLA-B*5701 and HLA-B*5801 are both members of HLA-B58 supertype which share the same binding specificity , study of these HLA-restricted T-cell responses was considered as a single group of HLA-B*57/58+ individuals. Unlike HLA-B*27, HLA-B*57/58 presents more than one HIV Gag p24 epitopes [18,21,22,32–35]. The strong association observed between HLA-B*57/58 and HIV control is hypothetically resulted from CD8+ T-cell responses against these high fitness cost epitopes . Indeed, sequential escape mutations of these epitopes resulting in narrowing the breadth of the HLA-B*57/58-restricted responses have been shown to be associated with loss of control [21,22]. In our study, though HLA-B*57/58+ viremic controllers were observed with less mutations, there were no noticeable differences, in term of responding epitopes, frequency and magnitude of IFN-γ responses, between viremic controllers and noncontrollers (Table 3 and Supplement Table 4, http://links.lww.com/QAD/A268).
In accordance with many previous studies, estimation of polyfunctional p24-specific CD8+ T cells in proportion to total CD8+ population or in absolute number (calculated with CBC) pointed out that viremic controllers were more polyfunctional than noncontrollers [1,9,12,36,37]. Additionally, most T-cell responses mediated by noncontrollers had only 2–3 functions [9,38]. Interestingly, some noncontrollers did possessed high proportion of polyfunctional T cells, but the absolute numbers were significantly lower than those of viremic controllers. The fact that total CD8+ cell count of noncontrollers and viremic controllers were similar emphasized that the absolute number of high functional quality T cells was associated with HIV control.
Detailed mechanisms to explain discrepancy of the T-cell quality in protective HLA-matched noncontrollers and viremic controllers remain unclear. Host factors such as T-cell antigen sensitivity, proliferative capacity, senescence and repertoire may also be important for determining quality of HIV-specific T-cell responses [39–43]. High antigen sensitivity and proliferative capacity with broader and cross-reactive T cells are favorable and likely to be characteristic of the viremic controllers [39,40]. Although T cells’ proliferative capacity was not directly investigated in this study, analysis of IL-2-producing CD8+ T cells could reflect this particular function of the T cells . In our study, the fact that IL-2-producing p24-specific CD8+ T cells were only observed in viremic controllers suggests that high-quality T cells in these individuals are continuously maintained by renewal of similar quality clones.
As viral escape from T-cell responses, unlike previously defined, might not be an all-or-none event. Virus might preferentially hinder the most potent antiviral function(s) whilst leaving another one(s) untouched. Variations within a given epitope, though maintaining certain affinity to HLA molecule and T-cell receptor (TCR), less-than-optimal interaction of TCR-HLA/peptide-complex, as cytokines release is sequential and time-dependent, might lead to an abrogation of T-cell functions . We speculated that IL-2 secretion is perhaps the most vulnerable function to be affected upon HIV escape? HLA-B*27+, 57/58+ controllers are able to maintain their high quality of HIV control as compared to their noncontroller counterparts by using different p24-specific T-cell clones which are more tolerant against this effect due to their higher antigen specificity. This speculation was supported by recent findings showing that HIV controllers used different T-cell clones and were able to generate de-novo responses against escape mutants [46,47]. A longitudinal study in a larger cohort of controllers is required in order to prove this hypothesis.
In conclusion, this study helps extend previous observations in subtype B-infected individuals that functional quality of p24-specific CD8+ T-cell response is also strongly associated with natural control of HIV in CRF01_AE-infected individuals. Our finding in HLA-B*2706+ and HLA-B*5801+ noncontrollers suggests that studies in diverse immunogenetic population infected with nonsubtype B HIV are warranted.
We thank Associate Professor Jintanart Ananworanich and Staff at Men Health Clinic, Thai Red Cross Society, for patient enrolment. We thank Dr Ittipon Techakriengkrai, Faculty of Science, Ramkhamhaeng University and Dr Napakkawat Buathong, Data Management Center, Faculty of Medicine, Chulalongkorn University for their assistance on statistical analysis. We also thank Ms Supranee Buranapraditkul and Mr Vittawat Jitnaitham for their technical assistance.
Authors’ contributions: N.T. designed and performed experiments, recruited volunteers, analyzed data and prepared the manuscript.
Y.T. performed experiments and recruit volunteers.
P.H. designed and supervised an experiment, obtained funding and prepared manuscript.
The work is supported by Government grant (through NRCT and Chulalongkorn University, Thailand). N.T. was funded by Chulalongkorn University Graduate Scholarship to commemorate the 72nd Anniversary of HM King Bhumibol Adulyadej.
Sequences: All sequences were submitted to GenBank with the following accession numbers: JN704002–JN704066.
Conflicts of interest
There were no conflicts of interest in this study.
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