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1 Modern Virology Research Center (MVRC), State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, China
2 Zhongnan Hospital, Wuhan University, Wuhan 430072, China
3 Molecular Virology Department (MVD), Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China
Correspondence
Po Tien
tienpo{at}sun.im.ac.cn
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). According to the China Center for Disease Control and Prevention survey supported by WHO, UNAIDS and the US Centers for Disease Control and Prevention, China had 840 000 people living with HIV/AIDS in 2003, among which 80 000 were AIDS patients. The number of people living with HIV in East Asia rose by almost 50 % between 2002 and 2004, an increase that is attributable largely to China's swiftly growing epidemic (http://www.unaids.org/wad2004/report.html). If the current spread of HIV infection is not controlled effectively, experts predict a rise to over 10 million infections within the next decade.
The most frequent modes of HIV-1 transmission have been sharing of contaminated needles among drug users in southern and western China and unsafe practices among paid blood donors (PBDs). The infection is moving into the general population and is causing an increasing number of deaths due to AIDS, particularly in the Henan and Hubei provinces, where many people became infected through unsafe blood collections in the 1990s. The vast majority of HIV-1 infections in central China are caused by subtype B' (Su et al., 2003).
The alarming rate of infection, together with the expense of effective antiviral therapies, has called for the urgent development of a vaccine to control the AIDS epidemic in China. The design of a vaccine for the Chinese population would be impossible without a comprehensive characterization of HIV-1 B' at the full-length genome level. Full-length genome sequencing of the HIV-1 B' Henan isolate from HIV-1 B'-infected PBDs was performed by the Modern Virology Research Center of Wuhan University (Su et al., 2003). Thirty-five HIV-1 B' Gag-specific synthetic peptides corresponding to the sequence of the Henan isolate were analysed in the present study.
HIV-1-specific cytotoxic T-lymphocyte (CTL) responses are thought to be important components of the immune system in the course and control of HIV-1 infection (Altfeld & Rosenberg, 2000; Brander & Walker, 1999; Chouquet et al., 2002; Goulder et al., 2000; Kalams & Walker, 1998; Letvin, 1998; Picker & Maino, 2000). Despite the fact that a large number of CTL epitopes across the HIV-1 B' subtype have been identified (http://www.hiv.lanl.gov/content/immunology/index.html/), relatively little is known about the HIV-1-specific CTL epitopes that are presented by human leukocyte antigen (HLA) class I molecules prevalent in the Chinese population. This study therefore focused on epitopes restricted by HLA-A2 and -A11, the two predominant alleles in China (Lin et al., 1999; Zou & Gou, 1999).
Traditionally, the magnitude and frequency of CTL responses have been studied on a per-patient basis. We attempted to analyse the cumulative magnitude of enzyme-linked immunospot (ELISPOT)-based CTL responses within a cohort of HIV-1 B'-infected PBDs, reasoning that profiles of HIV-1 B'-specific ELISPOT-based CTL responses could be extrapolated to the population of potential vaccinees. Cumulative analysis of ELISPOT-based CTL responses performed in this study was expressed as a sum of individual responses or as a mean response per study subject.
The results from the study presented here indicate that, in the subgroup analysis, significantly lower-magnitude responses were found with the infected treated subgroup [median, 93 spot-forming cells (SFCs) per 106 peripheral blood mononuclear cells (PBMCs)] than in the chronically infected untreated subgroup (median, 221 SFCs per 106 PBMCs), and HLA-A2-restricted treated PBDs had a response of much higher frequency and magnitude than HLA-A11-restricted treated PBDs. Moreover, some novel peptides restricted by the HLA-A2 and -A11 molecules were identified.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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HLA class I typing.
For the molecular HLA class I typing, genomic DNA was extracted from PBMCs by using GenomicPrep cells and a Tissue DNA isolation kit (Amersham Biosciences). HLA typing was performed in the Immunology Department of Tongji Medical College, Wuhan, China. ‘Low-resolution’ HLA class I typing was performed by sequence-specific primer PCR as described by Bunce et al. (1995).
HIV-1 B' viral load measurement.
Plasma viral loads were analysed by the fluorescence quantification PCR monitor assay according to the manufacturer's instructions (PG Biotech); this method has a detection limit of 100 HIV-1 RNA copies ml–1.
HIV-1 B' Gag peptide synthesis.
Thirty-five HIV-1 B' Gag-specific peptides corresponding to the sequence of the Henan isolate were synthesized. Design of the peptides was based on the HLA Peptide Binding Predictions program (http://bimas.dcrt.nih.gov/molbio/hla_bind/). Of the 35 peptides, 20 were predicted as potential epitopes restricted by HLA-A11 and 15 peptides were predicted to be restricted by HLA-A2. Peptides were synthesized by Shenzhen Hybio Engineering Co. on an automated peptide synthesizer by using Fmoc chemistry. Purity (>95 %) of the peptides was confirmed by high-performance liquid chromatography and mass spectrometry.
Designing of peptide pools.
Thirty-five peptides were included in 17 different peptide pools, with each peptide being included in two different peptide pools, which allowed for the identification of the respective peptide by responses in two corresponding pools. The number of pools and the number of peptides per pool are summarized in Table 2. The final concentration of each peptide within a peptide pool was 100 µg ml–1.
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) mAb 1-D1K (Mabtech AB). PBMCs were added at 50 000–100 000 cells per well in a volume of 100 µl R10 medium (RPMI 1640, 10 % fetal calf serum; Sigma) with antibody (2 mM L-glutamine, 50 U penicillin/streptomycin ml–1). The final concentration of the peptides in the wells was 10 µg ml–1. Plates were incubated overnight at 37 °C, 5 % CO2. Wells containing PBMCs and phytohaemagglutinin served as positive controls and wells containing PBMCs and R10 medium were used as negative controls. The number of spots per well was counted by using an automated ELISPOT plate reader (Bioreader 3000 PRO) and the number of specific T cells was calculated by subtracting the negative-control values. A positive response was defined as >100 SFCs per 106 PBMCs and more than three times the background control. The background was <30 per 106 PBMCs in all cases.
Tetramer staining of peripheral blood T lymphocytes.
Tetrameric HLA-A2–
2m–peptide and HLA-A11–2m–peptide complexes were produced as described by Altman et al. (1996). Fresh unstimulated PBMCs were washed twice with PBS buffer. In a 100 µl volume, cells were stained in the dark for 20 min at 37 °C with the tetramer (5 µg ml–1 for fresh PBMCs) along with anti-human CD8 antibody. The cells were then washed three times with PBS buffer and fixed by adding 500 µl 2 % polyformaldehyde. Controls included HLA-A2- and -A11-negative individuals. Sample data were acquired on a Beckman Coulter EPICS XL flow cytometer.
Statistical analysis.
Statistical analysis and graphical presentation were done by using SigmaPlot 11.5 (SPSS Inc.) and Microsoft Excel. Results were given with SD or medians with ranges.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We tested HLA-A allele distribution in the PBDs (Fig. 1). Based on cumulative allele frequency, the A2 and A11 supertypes had cumulative frequencies of 53·3 and 47·2 %, respectively; the A24 and A33 supertypes followed with frequencies of about 27·3 and 10·3 %. Three other HLA class I supertypes, A1, A3 and A68, were seen at relatively low frequencies in Chinese PBDs (cumulative frequency, <10 %). These HLA-A frequencies were similar to the frequencies described previously (Lin et al., 1999; Zou & Gou, 1999).
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Efficient assessment of HIV-1 B' T-cell responses by using a peptide matrix-based IFN- ELISPOT assay
Comprehensive analysis of immune responses to individual HIV-1 B'-specific peptides requires a large number of PBMCs. The use of peptide pools for initial screening offers the possibility of a lower level of specimen usage. Previous studies have validated the peptide-pools approach (Addo et al., 2003). In this study, we first evaluated the peptide-based approach by using five individual peptides (pep6–pep10) that responded to 14 infected PBDs, in comparison to peptide pools (pools B and E–I) that responded to 20 infected PBDs. The number of T-cell responses to individual peptides detected via the peptide-pools approach correlated well with the number of responses to individual peptides detected by a peptide-based approach (Fig. 2).
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, pools A–I) and 15 peptides were predicted to be restricted by HLA-A2 (Table 2, pools J–Q). Among the 26 treated HIV-1 B' chronically infected PBDs, 21 were HLA-A2- or -A11-positive, 12 (of 21, 57 %) of which responded to the 35 synthetic peptides in this study.
These studies revealed that all 35 synthetic peptides could serve as targets for HIV-1 B'-specific CD8+ T-cell responses (Fig. 3a, b). Individual peptides were targeted at different frequencies. Some peptides were recognized by only one study subject, whereas others were targeted by several subjects (Fig. 3b).
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). In order to clarify the relationship of response magnitude with the HLA frequencies, we listed the total frequencies of virus-specific responses restricted by HLA-A2 and -A11 in Fig. 3(b). The data indicated that treated HIV-1 B' chronically infected PBDs had higher-magnitude responses to peptides restricted by HLA-A2 than to peptides restricted by HLA-A11. The five most frequently recognized peptides were located in p2p7p1p6gag [FLQSRPEPTA (76·9 %) and VLAEAMSQVT (69·2 %)] and p24gag [AEWDRLHPV (69·2 %), WMTNNPPIPV (69·2 %) and NLQEQIGWM (69·2 %)], which were all restricted by the HLA-A2 subtype.
Differences in peptide targeting in treated and untreated PBDs
Twenty-eight untreated and 26 treated HIV-1 B'-infected PBDs were screened for HIV-1 B' Gag-specific CD8+ T-cell responses with 20 peptides (peptide pools A–I, shown in Table 2) in the ELISPOT assay. The magnitude of the responses ranged from 60 SFCs per 106 PBMCs, in a PBD treated during HIV-1 B' infection, to 2650 SFCs per 106 PBMCs in a PBD without treatment. A profile of cumulative HIV-1 B' Gag-specific CTL responses is shown in Fig. 4(a). In the subgroup analysis, significantly (P=0·000) lower-magnitude responses on the single-peptide level were found in the subgroup with treatment (median, 93 SFCs per 106 PBMCs; range, 47–175 SFCs per 106 PBMCs) than in the chronically infected untreated subgroup (median, 221 SFCs per 106 PBMCs; range, 152–268 SFCs per 106 PBMCs).
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). These data also revealed that lower-frequency responses were found among the HLA-A11 treated subgroup than among the untreated subgroup. The two most frequently recognized peptides restricted by HLA-A11 were CQGVGGPGHK (58·8 %) and VQNSNPDCK (54·5 %), located in p24gag. The CQGVGGPGHK peptide was located in the previously identified HLA-A11-restricted epitope ACQGVGGPGHK (http://www.hiv.lanl.gov/content/immunology/tables/ctl_summary.html). Taken together, these data indicate that T-cell responses in PBDs who received antiretroviral treatment were significantly lower and more narrowly directed than in PBDs without antiretroviral treatment. These also demonstrated that CD8+ T-cell responses can be very broadly directed in the setting of uncontrolled viraemia.
Recognized peptides restricted by HLA-A2 and HLA-A11
The distribution of HIV-1 B' Gag-specific ELISPOT-based CTL responses was analysed for subsets of common HLA-A alleles that were seen at two frequencies. The inclusion criterion was set to 
50 %, which means that at least 50 % of study PBDs in the subset of carriers of a particular HLA class I allele should demonstrate peptide-specific CTL responses in the analysis. The most significant responses were in carriers of HLA-A2: 10 of 13 PBDs (77 %) demonstrated CTL responses to the peptide FLQSRPEPTA (P=0·001, Fig. 5a); in carriers of HLA-A11, 10 of 17 PBDs (58·8 %) responded to the epitopes p24gag(350–359) CQGVGGPGHK (P=0·01, Fig. 5b) and p24gag(323–331) VQNSNPDCK (P=0·004, Fig. 5c). The p2p7p1p6gag(362–370) CTL epitope VLAEAMSQV was described previously to have the strongest CTL response in HLA-A2-positive individuals. However, in this study, eight of 13 (61·5 %) HLA-A2-positive PBDs demonstrated moderate CTL responses to the peptide VLAEAMSQV. Interestingly, peptides VLAEAMSQVT(362–371) and RVLAEAMSQV(361–370) were seen in nine of 13 PBDs (69·2 %) and six of 13 PBDs (46 %) who were HLA-A2-positive. The non-anchor motif mutants p24gag(77–85) SLYNTVAVL and p24gag(242–250) NLQEQIGWM were recognized by 61·5 % of HLA-A2-positive PBDs in this study.
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) and -A11-negative (Fig. 6d) PBDs. The median CD4 count of the six HIV-1 B'-infected PBDs was 301 cells mm–3. Plasma viral loads were <500 copies ml–1. Fig. 6 shows examples of HLA tetrameric staining: HLA-A2 tetramer refolded around Gag peptide VLAEAMSQV (Gag 362–370; Fig. 6b), HLA-A2 tetramer refolded around Gag peptide FLQSRPEPTA (Gag 448–457; Fig. 6c), HLA-A11 tetramer refolded around Gag peptide CQGVGGPGHK (Gag 350–359; Fig. 6e) and HLA-A11 tetramer refolded around Gag peptide VQNSNPDCK (Gag 323–331; Fig. 6f).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, 2002). Nevertheless, the induction of HIV-1-specific CTL responses remains a central component of vaccine development. Although the precise targets of HIV-1-specific CTL responses have been well characterized over the years, it has become increasingly clear that, besides viral genetic diversity encountered in different geographical locations, the genetic variability in human populations is yet another stumbling block for the development of an effective HIV-1 vaccine.
Much work on the characterization of HIV-1-specific immunity has been performed in HIV-1-infected individuals of Caucasian and African descent (Brander & Walker, 2003; Goulder, 2000). Studies performed in individuals of African descent with clade C or clade B infection demonstrated that individuals infected with different viral clades had different response patterns, at least for the responses against the HIV-1-encoded Gag protein. Although similarities in the response pattern towards conserved regions of the viral genome exist, some recent studies have also shown that, even among individuals with the same clade B infection, ethnicity-dependent response patterns can also be identified (Bansal et al., 2003; Frahm et al., 2004). This suggests that a potential vaccine needs to be tailored not only to local viral sequence diversity, but also to the local HLA-allele distributions.
A comparative analysis of HLA-A frequencies within the general population in China (Lin et al., 1999; Zou & Gou, 1999) and within the HIV-1 B'-infected PBDs in this study (Fig. 1) revealed similar distributions of HLA alleles. Based on the cumulative allele-frequency analysis, the A2 and A11 supertypes of HLA-A alleles are the two most common alleles in Chinese PBDs, followed by the A24 and A33 supertypes. Three other HLA-A supertypes, A1, A3 and A68, were seen at relatively low frequencies in Chinese PBDs. Taken in the context of the nature of HLA-A restriction of CD8+ T-cell responses, similarities in the HLA-allele frequencies between HIV-1 B'-infected PBDs and the epitopes identified across the HIV-1 B' genome should represent the overall cell-mediated immune responses in the HIV epidemic in China fairly well.
In this study, we addressed the magnitude of, frequency of and normalized cumulative HIV-1 B' Gag-specific CTL responses in China, a country with a very high prevalence of HIV-1 B' infection (Su et al., 2003). CTL responses were analysed in 54 infected PBDs from the Henan and Hubei provinces, a cohort that represents the general population in China fairly well. We employed 35 synthetic peptides corresponding to the Henan isolate sequence and characterized the T-cell responses to these peptides with an ELISPOT assay. Previous studies were validated this approach and it was shown that the responses detected by this method are, with few exceptions, the result of CD8+ T-cell activities (Addo et al., 2003).
Marked differences in the frequency and magnitude of targeting of individual peptides (Fig. 3a) were revealed; 35 peptides were targeted by at least one PBD and 15 peptides were targeted by 30 % of PBDs. For the 35 targeted peptides, we noted a strong association with specific HLA-A-allele expression: 25 peptides exhibited a strong allele-specific association (P<0·05), of which two remained significant (P=0·001) following correction for multiple comparisons. These include one newly identified HLA-A2-restricted epitope, p2p7p1p6gag FLQSRPEPTA (76·9 %), and one newly identified HLA-A11-restricted epitope, p24gag VQNSNPDCK (54·5 %). In each case, the restricting allele was predicted precisely by the statistical association. This analysis therefore affords a stringent approach to define the HLA class I restriction for a large majority of CD8+ T-cell responses detected in this population. We next used this approach to determine the relative contributions of the different HLA-A alleles to immune recognition of HIV-1 B'. Much stronger responses toward treated PBDs were found in HLA-A2-restricted epitopes than in HLA-A11-restricted epitopes (Fig. 3b).
In the subgroup analysis, significantly lower-magnitude responses were found in the subgroup with treatment than in the chronically infected untreated subgroup. In the detailed analysis of epitopic regions, HIV-1 B' p24gag was the most frequently recognized protein and contributed most to the total HIV-1 B' Gag-specific responses in untreated PBDs.
Taken together, by using a highly sensitive and specific peptide-pools system, this study provided a comprehensive analysis of HIV-1 B'-specific T-cell responses and a detailed dissection of the novel epitopes and the magnitude of the total HIV-1 B'-specific CD8+ T-cell responses.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 12 July 2005;
accepted 4 October 2005.
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) and individual-peptide (
) approaches. The five individual peptides are represented on the x axis; the corresponding percentage of the study subjects that responded to the five peptides detected by the individual-peptide approach (n=14) and the peptide-pool approach (n=20) are represented on the y axis.
