Original Research Article
VIRAL IMMUNOLOGY Volume 29, Number 3, 2016 ª Mary Ann Liebert, Inc. Pp. 1–8 DOI: 10.1089/vim.2015.0057
Analysis of HIV-1c-Specific CTL Responses with HIV-1 Reservoir Size and Forms Wenzhen Kang,1,* Weijun Zhu,2,* Yuan Li,1 Yanmei Jiao,3 Yan Zhuang,1 Yumei Xie,1 Ke Zhao,1 Bianli Dang,1 Qingquan Liu,1 Ye Zhang,1 and Yongtao Sun1
Abstract
Cytotoxic T lymphocytes (CTL) are critical in cellular immune responses; therefore the study of CTL responses is profound in HIV-1 eradication. We aim to dissect the relationship between HIV-1 reservoir size and the magnitude and recognition of viral-specific CTL responses. An IFN-g ELISpot assay with peptides spanning the HIV-1 clade C consensus sequences were designed to analyze HIV-1c-specific CTL responses. HIV-1 DNA, integrated HIV-1 DNA, and 2-LTR HIV-1 DNA were quantitated by real-time PCR. We observed significant increases in total HIV-1 DNA and integrated HIV-1 DNA after highly active antiretroviral treatment (HAART) compared with naive patients. Total HIV-1 DNA had a significant negative correlation with HIV-1c-specific CTL response magnitude. Baseline CD4+ T lymphocyte counts and antiretroviral treatment affected the size of the HIV-1 reservoirs. Taken together, HIV-1-specific CTL responses correlated with the size of HIV-1 reservoir. In addition, HIV-1-specific CTL response against p17 was associated with low integral efficiency of HIV-1, which might be a biomarker to evaluate the efficacy of HAART.
role of HIV-1-specific CTL response in the latent HIV infection is still not extensively studied. Previously Chun et al. investigated the suppression of HIV-specific CD8+ T cells on the HIV-1 replication in the resting CD4+ T-cell reservoir (10), and their results indicated that CD8+ T cells provided a potent suppressive activities against HIV replication in the latent viral reservoir through direct cellular contacts in patients who are naturally LTNPs and treated with HAART, but this antiviral activity was independent of CLT response. Furthermore, they found that early initiation of HAART in HIV patients may provide a sustaining antiviral activity to contain viral replication in patients with interrupted therapy (10, 29, 35). An IFN-g ELISpot assay with a total of 426 overlapping peptides spanning the HIV-1 clade C consensus sequences were applied to comprehensively analyze HIV-1-specific CTL responses. Moreover, an optimized real-time (RT)-PCR was utilized to measure integrated, 2-LTR circular, and total HIV-1 DNA in patients with HIV-1 infection. Our study may help to understand the relationship between HIV-1 reservoir size and cellular immune responses.
Introduction
H
IV infection has seen a dramatic decrease in mortality and morbidity worldwide after the great success of highly active antiretroviral treatment (HAART) (6, 18, 20, 27). However, HAART is a lifelong treatment because the antiretroviral drug is not effective in the elimination of latent HIV1-infected cells. Latent infected cells are predominantly resting CD4+ T cells (9, 12, 30), but also include other long-lived cells, such as monocyte/macrophages (25) and astrocytes (11, 13). Latency represents one of the biggest challenges of HIV-1 eradication. Cellular immune responses are believed to play crucial roles in antiviral defense. It is well known that cytotoxic T lymphocytes (CTL) are crucial to cellular immune responses; therefore the study of CTL responses is profound toward HIV-1 eradication. A previous study observed strong HIV-1-specific CTL responses in HIV-1-infected individuals with long-term nonprogression (LTNP) (16), and another study also indicated an inverse correlation between HIV-1specific CTL responses and viral load (22). However, the
1 Department of Infectious Diseases, Tangdu Hospital Affiliated to the Fourth Military Medical University, Xi’an, People’s Republic of China. 2 AIDS Research Center, MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China. 3 Center for Infectious Diseases, Beijing You-An Hospital, Capital Medical University, Beijing, People’s Republic of China. *These authors contributed equally to this work.
1
2 Materials and Methods Study objectives
Ninety two HIV-1-infected individuals (58 males and 34 females) were recruited from the Department of Infectious Disease of Tangdu Hospital in Xi’an, China. Sixty six out of these patients were antiretroviral-treatment naive, and the other 26 subjects were undergoing HAART. The test subjects had a median age of 37 years (36.65 – 9.52), and their median CD4+ T-lymphocyte count was 286 cells/lL (285.91 – 260.39) and their median plasma viral load (log10 copies/mL) was 4.08 (4.08 – 2.11). All 92 subjects were infected with HIV for at least 12 months. All studies were conducted with the approval of the ethics review board of Tangdu hospital, and each individual gave written informed consent for this study. Viral load and lymphocyte subset test
HIV plasma viral load was analyzed by an immunofluorescence quantifying PCR assay (Roche Corporation) according to the manufacturer’s instructions. T-lymphocyte subsets were enumerated by flow cytometry using TriTEST (CD4-FIITC/CD8-PE/CD3-PerCP, Becton Dickison) according to the ‘‘lyse-no-wash’’ procedure. Synthetic HIV-1-peptides
A total of 426 synthetic 15–20 amino acid peptides overlapping by 10 amino acids spanning the entire HIV-1 clade C consensus sequences were synthesized at the Massachusetts General Hospital Peptide Core Facility on an automated peptide synthesizer using Fmoc technology. ELISpot assays
Fresh peripheral blood mononuclear cells (PBMCs) were separated from whole blood by Ficoll–Hypaque (Sigma) density gradient centrifugation. 1 · 105 of cells/well were plated in 96-well polyvinylidene difluoride-backed plates (MAIP S45; Millipore), which had been previously coated with 100 lL/well of anti-gamma-interferon (IFN-g) MAb 1D1k (0.5 lg/mL; Mabtech) overnight at 4C. Peptides were added directly to the wells at a final concentration of 10 lM with a final volume of 130 lL/well in RPMI 1640 with 10% of FBS. Wells containing PBMCs only were used as negative controls and were run in triplicate on each plate. Wells containing PBMCs stimulated with phytohemagglutinin (PHA) served as positive controls. The plates were incubated at 37C with 5% CO2 overnight (14–16 h) and then washed six times with phosphate-buffered saline (PBS) before 100 lL of biotinylated anti-IFN-g MAb 7-B6-1 (1 lg/mL; Mabtech) was added for 90 min of incubation. After being washed again with PBS, 100 lL/well of 1:20,000-diluted streptavidin–alkaline phosphatase conjugate (Mabtech) was added to all wells of the plates. The plates were incubated at room temperature for 45 min. Wells were washed with PBS again and incubated in dark for 20–30 min with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Bio-Rad Laboratories). IFN-gproducing cells were identified as darkly stained cells and were counted by an Elispot reader (AID EliSpot reader system; Autoimmune Diagnostika GmbH). The results were expressed as the number of spot-forming cells (SFC) per million PBMCs. The numbers of specific IFN-g-secreting T cells were calculated
KANG ET AL.
by subtracting the negative control value from the calculated SFC count. Responses were considered positive if there were >50 SFC/106 PBMCs and had at least thrice more than the mean number of SFC of the negative controls. The overall specificity of using the IFN-g ELISpot to test responses of CD8+ T cells to synthetic peptides was demonstrated previously by others in a series of CD8+ and CD4+ depletion and enrichment experiments (28, 31, 33, 34). Generation of standards for real-time PCR assay
The primers and probes for real-time PCR were optimized based on published sequences of geographical variants in the Los Alamos HIV databases (www.hiv.lanl.gov/) and previous reports (7). The primer, probe sequences, and their targets of detection were used as previously described (8, 35). As the standard for RT-PCR, we used an M13 bacteriophage as a vector to express the appropriate HIV-1 DNA and cellular sequences. The chemokine receptor CCR5 was chosen as a surrogate to estimate the input number of cells for cell containing only one single copy of CCR5 (4, 32). The absolute number of target sequences could be calculated based on the number of plaque-forming units (PFU) of M13 phage (26). To generate standards for the various forms of HIV-1 DNA and the cellular CCR5, the corresponding gene fragments were inserted into the M13 genome. The recombinant M13 phages were propagated in Escherichia coli JM109 and harvested by centrifugation. The titer of recombinant phage in the supernatant was estimated by serial dilution, followed by the counting of PFU (26). HIV DNA quantification
The total DNA from patients’ PBMCs was extracted using a QIAamp DNA blood mini kit (Qiagen) according to the manufacturer’s protocol. Modified real-time PCR was performed in 25 lL solution containing 2.5 lL of DNA target, 12.5 lL of Gene Expression Master Mix (Applied Biosystems 4369016), 1 lM of primers, and 0.2 lM of the probe under the following conditions: 95C for 10 min, followed by 95C for 15 s, and 60C for 1 min (40 cycles) in an ABI 7500 PCR machine (Applied Biosystems). For the integrated HIV-1 DNA, the first round of PCR was conducted with the following conditions: 95C for 8 min, and then 12 cycles of amplification: 95C for 10 s, 60C for 10 s, and 72C for 170 s. One-tenth of the first round PCR product was used for following real-time PCR detection. The PCR signal from first round PCR without the Alu primers was subtracted from the total signals before the estimate of copy numbers. A standard curve was created for each run in a 7log-unit range by 1:10 serial dilutions. Statistical analysis
Statistical analysis and graphical presentation were performed using SPSS 13.0 and Sigma Plot 10.0. Student’s t-test was used for data that fulfilled normal distribution. Data were expressed as the mean – standard deviation (SD). The nonparametric Mann–Whitney U test was used to perform the comparison between Naive and On-HAART adults. Data were expressed as median values and interquartile range. Wilcoxon rank sum tests were used to compare the distribution of cumulative CTL responses to a particular peptide or protein. A
ANALYSIS OF HIV-1C-SPECIFIC CTL RESPONSES
Table 1. Baseline Demographic and Immunological Parameters of Study Participants Parameters
Naive
On-HAART
Number 66 26 Sex ratio (male/female) 42/14 16/10 Age (years) 36.52 – 9.51 37.00 – 9.70 CD4+ T cell count 293.09 – 278.05 260.88 – 194.98 (cells/lL) HIV-1 Viral load 4.09 – 2.17 4.04 – 1.99 (log10 copies/mL) HAART, highly active antiretroviral treatment.
3
rank-based test was used for this comparison because it appropriately accounts for the censoring of within-well Elispot responses of >50 spots/well. A response was considered positive if the probability of a cell forming a spot was higher in the test well compared to the negative control well. Spearman rank correlation was used for correlation analysis. All tests were twotailed, and p-values of 0.05). HIV-1 viral load was lower in the On-HAART group than in naive group, but this difference was not statistically significant ( p > 0.05). We assessed HIV-1-specific CTL responses in the PBMCs of two study groups using 426 overlapping peptides spanning all expressed HIV-1 proteins by Elispot assay. To identify responses with both high magnitude and high frequency, a normalized cumulative CTL response was used by combining data on CTL magnitude and frequency (21, 34). HIV-1c-specific CTL responses directed against all the HIV-1c proteins were detectable in the two groups (Fig. 1). The most immunogenic Nef, P24, and RT were targeted by 80.30%, 71.21%, and 72.73% of the tested naive patients, respectively. The HIV-1c-
KANG ET AL.
specific CTL responses against each HIV-1c protein was greatly reduced in the On-HAART group, especially in P15, Rev, and Vpu, whose CTL responses magnitude dropped by 92.55%, 90.45%, and 90.36%, respectively (Fig. 2A). However, there was less difference in the percentage of HIV protein recognition between the naive and On-HAART group (Fig. 2B). Compared to the naive group, recognition of P15, P17, and Rev in the On-HAART group were decreased by 42.89%, 32.05%, and 23.61%, respectively. Overall, P15 and Rev were the two proteins that showed the greatest change in response between naive patients and those on HAART. Patients receiving HAART have higher total HIV-1 DNA and integrated HIV-1 DNA
After HIV-1 binding and entry into the host cells, the viral genome is reverse transcribed into the DNA, transported into the nucleus, and integrated into the host genome for viral replication (15). Cellular HIV-1 reservoirs are fundamental to
FIG. 2. Magnitude and breadth of HIV-1c-specific CTL responses in protein levels. HIV-1-specific CTL responses were directed in the PBMCs of two study groups using 426 overlapping peptides spanning all expressed HIV-1 proteins by IFN-g-ELISpot assay (x-axis). (A) Cumulative Elispot-based CTL responses were measured as a sum of the naive or OnHAART of HIV-1 group to a particular protein and expressed as the number of cumulative SFC/106 PBMC (y-axis). (B) Percentage of positive IFN-g lymphokine responders of the naive or On-HAART of HIV-1 group reacting to a particular protein. The x-axis of each graph was scaled according to the name of protein or protein subunits. The peptide concentration was 10 lM with a final volume of 130 lL/well in RPMI 1640 with 10% of FBS.
ANALYSIS OF HIV-1C-SPECIFIC CTL RESPONSES
5
FIG. 3. Comparison of HIV-1 DNA copies in PBMCs between naive and On-HAART of HIV-1-infected patients. Medians and p-values from Mann–Whitney test are shown. (A) The quantity of total HIV-1 DNA of the On-HAART group was significantly higher than the naive group. (B) The difference of 2-LTR HIV-1 DNA between naive and On-HAART of HIV-1 group was not significant. (C) The quantity of integrated HIV-1 DNA of the On-HAART group was significantly higher than the naive group. The Mann–Whitney U test was used to perform the comparison between Naive and OnHAART adults, p-values of 350
Total HIV-1 DNA 2-LTR HIV-1 DNA/Total HIV-1 DNA Integrated HIV-1 DNA/Total HIV-1 DNA
-0.265 0.100 0.160
-0.632a -0.401b -0.762a
0.243 0.120 0.026
-0.024 -0.683b -0.119
a
Correlation is significant at the 0.01 level (two-tailed). Correlation is significant at the 0.05 level (two-tailed). However, the naive group had strong statistically significant correlations, while the On-HAART group presented no statistical significance. These correlations disappeared after HAART which suggested that different immune cells could influence the HIV-1 reservoirs and reassured the necessary consideration of basic immune cell levels of study subjects. CTL, cytotoxic T lymphocytes. b
ANALYSIS OF HIV-1C-SPECIFIC CTL RESPONSES
7
Table 3. Number of HIV-1 Peptides with Strong Correlation of Different HIV-1 Reservoir Sizes Number of HIV-1 peptides with strong correlation (p < 0.05) Total HIV-1 DNA 2-LTR HIV-1 DNA/Total HIV-1 DNA Integrated HIV-1 DNA/Total HIV-1 DNA
Naive
On-HAART
CD4 £ 350
CD4 > 350
CD4 £ 350
CD4 > 350
0 0 0
5 (P17, P24, Nef, Pro, RT, Gp41) 2 (P17, Nef) 6 (P17, P24, Nef, Pro, Int, RT, Gp41)
0 0 0
0 1(P17) 0
circulating HIV-1 DNA, and total HIV-1 DNA) and thus tried to further explore the relationship of HIV-1-specific CTL responses with HIV-1 reservoirs. Consistent with previous studies (31, 33, 34), we found that HIV-infected patients had a wide range of cumulative CTL responses against HIV-1 peptides and recognition of these peptides appeared to be extensive in Gag, Nef, and Env regions. Moreover, both the cumulative CTL responses and the recognition of HIV peptides were significantly weaker in the On-HAART group compared with the naive group. HIV-specific CTL responses became weaker with the initiation of HAART, but accompanied with more amounts of HIV reservoirs. Unexpectedly, our results are contradictory with those of Graf et al. (14). They showed that total and integrated HIV-1 DNAs both decreased in patients on HAART. We assumed that such discrepancy might be caused by the characteristics of study subjects or length on HAART. In their studies, the off-HAART group usually had a median of about 4,000 copies of total HIV DNA per mL PBMCs, but in our naive group, only a median of 53 copies of total HIV DNA was detected. Thus, their off-HAART group may represent the patients in rapid HIV progression, but our naive patients are more like ES with low total or integrated HIV DNAs. In our study, CD4+ T-lymphocyte counts between the two study groups did not demonstrate statistical significance, which excluded the potential effect of CD4+ T-cell levels on the lower HIV-1 reservoirs in the On-HAART group. However, high CD4+ T-lymphocyte counts may affect HIV reservoirs. HIV-1 reservoirs became more strongly correlated with CTL cumulative responses when CD4+ T-lymphocyte counts were above 350 cells/lL. However, these correlations disappeared after HAART. These results suggested that different immune cells could influence the HIV-1 reservoirs and reassured the necessary consideration of basic immune cell levels of study subjects. Accordingly, CTL responses against different peptides are correlated with HIV-1 reservoirs. Also, the CTL responses were more remarkable when CD4+ T-lymphocyte counts were above 350 cells/lL, but less notable after HAART. Interestingly, P17 peptide positively correlated with the amount of integrated HIV-1 DNA, but negatively correlated with the amount of 2-LTR DNA and total HIV-1 DNA. We propose that HIV-specific CTL responses against p17 may be associated with the low integrate efficiency of HIV-1, which might be a biomarker to evaluate the efficacy of HAART. In summary, we analyzed the association of HIV-1-specific CTL responses and HIV reservoirs in patients with or without HAART. We observed an increase in total HIV-1 DNA and integrated HIV-1 DNA after HAART compared with naive patients. HIV-1c-specific CTL responses may be suppressed after HAART rather than HIV-1b-specific CTL responses. Moreover, we found that the baseline CD4+ T-lymphocyte
counts and antiretroviral treatment affected the size of HIV-1 reservoirs. In addition, HIV-specific CTL responses against p17 were shown to correlate with low integrate efficiency of HIV-1, which can possibly be monitored to evaluate the efficacy of HAART. Acknowledgments
This work was supported by the grant from National Key Technologies Research and Development Program of China (2008ZX10001-006, 2008ZX10001-002, and 2012ZX10001-003), Natural Science Foundation of China (grant 31000084 and 81202305), and Tangdu Technology Innovation and Development Program (2012LCYJ002). Ethics Approval
This work was approved by the Ethics board of Tangdu Hospital. The Judgement reference number was TDLL2012016. Author Disclosure Statement
No competing financial interests exist. References
1. Agosto LM, Liszewski MK, Mexas A, et al. Patients on HAART often have an excess of unintegrated HIV DNA: implications for monitoring reservoirs. Virology 2011;409: 46–53. 2. Alatrakchi N, Duvivier C, Costagliola D, et al. Persistent low viral load on antiretroviral therapy is associated with T cell-mediated control of HIV replication. AIDS 2005;19: 25–33. 3. Alexaki A, Liu Y, and Wigdahl B. Cellular reservoirs of HIV-1 and their role in viral persistence. Curr HIV Res 2008;6:388–400. 4. Beloukas A, Paraskevis D, Haida C, et al. Development and assessment of a multiplex real-time PCR assay for quantification of human immunodeficiency virus type 1 DNA. J Clin Microbiol 2009;47:2194–2199. 5. Betts MR, Ambrozak DR, Douek DC, et al. Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection. J Virol 2001;75:11983– 11991. 6. Braitstein P, Brinkhof MW, Dabis F, et al. Mortality of HIV-1-infected patients in the first year of antiretroviral therapy: comparison between low-income and high-income countries. Lancet 2006;367:817–824. 7. Brussel A, and Sonigo P. Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus. J Virol 2003;77:10119–10124.
8
8. Chomont N, El-Far M, Ancuta P, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 2009;15:893–900. 9. Chun TW, Carruth L, Finzi D, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997;387:183–188. 10. Chun TW, Justement JS, Moir S, et al. Suppression of HIV replication in the resting CD4+ T cell reservoir by autologous CD8+ T cells: implications for the development of therapeutic strategies. Proc Natl Acad Sci U S A 2001;98: 253–258. 11. Churchill MJ, Wesselingh SL, Cowley D, et al. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia. Ann Neurol 2009;66: 253–258. 12. Finzi D, Blankson J, Siliciano JD, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 1999;5:512–517. 13. Gorry P, Purcell D, Howard J, et al. Restricted HIV-1 infection of human astrocytes: potential role of nef in the regulation of virus replication. J Neurovirol 1998;4:377– 386. 14. Graf EH, Mexas AM, Yu JJ, et al. Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2LTR circular HIV DNA compared to HIV+ patients on and off HAART. PLoS Pathog 2011;7:e1001300. 15. Greene WC, and Peterlin BM. Charting HIV’s remarkable voyage through the cell: basic science as a passport to future therapy. Nat Med 2002;8:673–680. 16. Harrer T, Harrer E, Kalams SA, et al. Strong cytotoxic T cell and weak neutralizing antibody responses in a subset of persons with stable nonprogressing HIV type 1 infection. AIDS Res Hum Retroviruses 1996;12:585–592. 17. Hersperger AR, Martin JN, Shin LY, et al. Increased HIVspecific CD8+ T-cell cytotoxic potential in HIV elite controllers is associated with T-bet expression. Blood 2011; 117:3799–3808. 18. Luo L, and Li TS. Overview of antiretroviral treatment in China: advancement and challenges. Chin Med J (Engl) 2011;124:440–444. 19. Migueles SA, Laborico AC, Imamichi H, et al. The differential ability of HLA B*5701+ long-term nonprogressors and progressors to restrict human immunodeficiency virus replication is not caused by loss of recognition of autologous viral gag sequences. J Virol 2003;77:6889– 6898. 20. Mocroft A, Ledergerber B, Katlama C, et al. Decline in the AIDS and death rates in the EuroSIDA study: an observational study. Lancet 2003;362:22–29. 21. Novitsky V, Rybak N, McLane MF, et al. Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic Tlymphocyte responses for AIDS vaccine design. J Virol 2001;75:9210–9228. 22. Ogg GS, Jin X, Bonhoeffer S, et al. Quantitation of HIV-1specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 1998;279:2103–2106.
KANG ET AL.
23. Ogg GS, Kostense S, Klein MR, et al. Longitudinal phenotypic analysis of human immunodeficiency virus type 1specific cytotoxic T lymphocytes: correlation with disease progression. J Virol 1999;73:9153–9160. 24. Sa´ez-Cirio´n A, Sinet M, Shin SY, et al. Heterogeneity in HIV suppression by CD8+ T cells from HIV controllers: association with Gag-specific CD8+ T cell responses. J Immunol 2009;182:7828–7837. 25. Sonza S, Mutimer HP, Oelrichs R, et al. Monocytes harbour replication-competent, non-latent HIV-1 in patients on highly active antiretroviral therapy. AIDS 2001;15:17–22. 26. Sum SS, Wong DK, Yuen MF, et al. Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA. J Clin Microbiol 2004;42:3438–3440. 27. Tuboi SH, Schechter M, McGowan CC, et al. Mortality during the first year of potent antiretroviral therapy in HIV1-infected patients in 7 sites throughout Latin America and the Caribbean. J Acquir Immune Defic Syndr 2009;51:615– 623. 28. Wang S, Sun Y, Zhai S, et al. Identification of HLA-A11restricted HIV-1-specific cytotoxic T-lymphocyte epitopes in China. Curr HIV Res 2007;5:119–128. 29. Watanabe D, Ibe S, Uehira T, et al. Cellular HIV-1 DNA levels in patients receiving antiretroviral therapy strongly correlate with therapy initiation timing but not with therapy duration. BMC Infect Dis 2011;11:146. 30. Wong JK, Hezareh M, Gunthard HF, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 1997;278:1291–1295. 31. Zhai S, Zhuang Y, Song Y, et al. HIV-1-specific cytotoxic T lymphocyte (CTL) responses against immunodominant optimal epitopes slow the progression of AIDS in China. Curr HIV Res 2008;6:335–350. 32. Zhang L, Ramratnam B, Tenner-Racz K, et al. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med 1999;340:1605– 1613. 33. Zhao S, Zhai S, Zhuang Y, et al. Inter-clade cross-reactivity of HIV-1-specific T cell responses in human immunodeficiency virus type 1 infection in China. Curr HIV Res 2007; 5:251–259. 34. Zhuang Y, Sun Y, Zhai S, et al. Relative dominance of Env-gp41-specific cytotoxic T lymphocytes responses in HIV-1 advanced infection. Curr HIV Res 2008;6:239–245. 35. Zhu W, Jiao Y, Lei R, et al. Rapid turnover of 2-LTR HIV1 DNA during early stage of highly active antiretroviral therapy. PLoS One 2011;6:e21081.
Address correspondence to: Dr. Wenzhen Kang Department of Infectious Diseases Tangdu Hospital Affiliated to the Fourth Military Medical University Xi’an 710038 People’s Republic of China E-mail: [email protected]
VIRAL IMMUNOLOGY Volume 29, Number 3, 2016 ª Mary Ann Liebert, Inc. Pp. 1–8 DOI: 10.1089/vim.2015.0057
Analysis of HIV-1c-Specific CTL Responses with HIV-1 Reservoir Size and Forms Wenzhen Kang,1,* Weijun Zhu,2,* Yuan Li,1 Yanmei Jiao,3 Yan Zhuang,1 Yumei Xie,1 Ke Zhao,1 Bianli Dang,1 Qingquan Liu,1 Ye Zhang,1 and Yongtao Sun1
Abstract
Cytotoxic T lymphocytes (CTL) are critical in cellular immune responses; therefore the study of CTL responses is profound in HIV-1 eradication. We aim to dissect the relationship between HIV-1 reservoir size and the magnitude and recognition of viral-specific CTL responses. An IFN-g ELISpot assay with peptides spanning the HIV-1 clade C consensus sequences were designed to analyze HIV-1c-specific CTL responses. HIV-1 DNA, integrated HIV-1 DNA, and 2-LTR HIV-1 DNA were quantitated by real-time PCR. We observed significant increases in total HIV-1 DNA and integrated HIV-1 DNA after highly active antiretroviral treatment (HAART) compared with naive patients. Total HIV-1 DNA had a significant negative correlation with HIV-1c-specific CTL response magnitude. Baseline CD4+ T lymphocyte counts and antiretroviral treatment affected the size of the HIV-1 reservoirs. Taken together, HIV-1-specific CTL responses correlated with the size of HIV-1 reservoir. In addition, HIV-1-specific CTL response against p17 was associated with low integral efficiency of HIV-1, which might be a biomarker to evaluate the efficacy of HAART.
role of HIV-1-specific CTL response in the latent HIV infection is still not extensively studied. Previously Chun et al. investigated the suppression of HIV-specific CD8+ T cells on the HIV-1 replication in the resting CD4+ T-cell reservoir (10), and their results indicated that CD8+ T cells provided a potent suppressive activities against HIV replication in the latent viral reservoir through direct cellular contacts in patients who are naturally LTNPs and treated with HAART, but this antiviral activity was independent of CLT response. Furthermore, they found that early initiation of HAART in HIV patients may provide a sustaining antiviral activity to contain viral replication in patients with interrupted therapy (10, 29, 35). An IFN-g ELISpot assay with a total of 426 overlapping peptides spanning the HIV-1 clade C consensus sequences were applied to comprehensively analyze HIV-1-specific CTL responses. Moreover, an optimized real-time (RT)-PCR was utilized to measure integrated, 2-LTR circular, and total HIV-1 DNA in patients with HIV-1 infection. Our study may help to understand the relationship between HIV-1 reservoir size and cellular immune responses.
Introduction
H
IV infection has seen a dramatic decrease in mortality and morbidity worldwide after the great success of highly active antiretroviral treatment (HAART) (6, 18, 20, 27). However, HAART is a lifelong treatment because the antiretroviral drug is not effective in the elimination of latent HIV1-infected cells. Latent infected cells are predominantly resting CD4+ T cells (9, 12, 30), but also include other long-lived cells, such as monocyte/macrophages (25) and astrocytes (11, 13). Latency represents one of the biggest challenges of HIV-1 eradication. Cellular immune responses are believed to play crucial roles in antiviral defense. It is well known that cytotoxic T lymphocytes (CTL) are crucial to cellular immune responses; therefore the study of CTL responses is profound toward HIV-1 eradication. A previous study observed strong HIV-1-specific CTL responses in HIV-1-infected individuals with long-term nonprogression (LTNP) (16), and another study also indicated an inverse correlation between HIV-1specific CTL responses and viral load (22). However, the
1 Department of Infectious Diseases, Tangdu Hospital Affiliated to the Fourth Military Medical University, Xi’an, People’s Republic of China. 2 AIDS Research Center, MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, People’s Republic of China. 3 Center for Infectious Diseases, Beijing You-An Hospital, Capital Medical University, Beijing, People’s Republic of China. *These authors contributed equally to this work.
1
2 Materials and Methods Study objectives
Ninety two HIV-1-infected individuals (58 males and 34 females) were recruited from the Department of Infectious Disease of Tangdu Hospital in Xi’an, China. Sixty six out of these patients were antiretroviral-treatment naive, and the other 26 subjects were undergoing HAART. The test subjects had a median age of 37 years (36.65 – 9.52), and their median CD4+ T-lymphocyte count was 286 cells/lL (285.91 – 260.39) and their median plasma viral load (log10 copies/mL) was 4.08 (4.08 – 2.11). All 92 subjects were infected with HIV for at least 12 months. All studies were conducted with the approval of the ethics review board of Tangdu hospital, and each individual gave written informed consent for this study. Viral load and lymphocyte subset test
HIV plasma viral load was analyzed by an immunofluorescence quantifying PCR assay (Roche Corporation) according to the manufacturer’s instructions. T-lymphocyte subsets were enumerated by flow cytometry using TriTEST (CD4-FIITC/CD8-PE/CD3-PerCP, Becton Dickison) according to the ‘‘lyse-no-wash’’ procedure. Synthetic HIV-1-peptides
A total of 426 synthetic 15–20 amino acid peptides overlapping by 10 amino acids spanning the entire HIV-1 clade C consensus sequences were synthesized at the Massachusetts General Hospital Peptide Core Facility on an automated peptide synthesizer using Fmoc technology. ELISpot assays
Fresh peripheral blood mononuclear cells (PBMCs) were separated from whole blood by Ficoll–Hypaque (Sigma) density gradient centrifugation. 1 · 105 of cells/well were plated in 96-well polyvinylidene difluoride-backed plates (MAIP S45; Millipore), which had been previously coated with 100 lL/well of anti-gamma-interferon (IFN-g) MAb 1D1k (0.5 lg/mL; Mabtech) overnight at 4C. Peptides were added directly to the wells at a final concentration of 10 lM with a final volume of 130 lL/well in RPMI 1640 with 10% of FBS. Wells containing PBMCs only were used as negative controls and were run in triplicate on each plate. Wells containing PBMCs stimulated with phytohemagglutinin (PHA) served as positive controls. The plates were incubated at 37C with 5% CO2 overnight (14–16 h) and then washed six times with phosphate-buffered saline (PBS) before 100 lL of biotinylated anti-IFN-g MAb 7-B6-1 (1 lg/mL; Mabtech) was added for 90 min of incubation. After being washed again with PBS, 100 lL/well of 1:20,000-diluted streptavidin–alkaline phosphatase conjugate (Mabtech) was added to all wells of the plates. The plates were incubated at room temperature for 45 min. Wells were washed with PBS again and incubated in dark for 20–30 min with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium (Bio-Rad Laboratories). IFN-gproducing cells were identified as darkly stained cells and were counted by an Elispot reader (AID EliSpot reader system; Autoimmune Diagnostika GmbH). The results were expressed as the number of spot-forming cells (SFC) per million PBMCs. The numbers of specific IFN-g-secreting T cells were calculated
KANG ET AL.
by subtracting the negative control value from the calculated SFC count. Responses were considered positive if there were >50 SFC/106 PBMCs and had at least thrice more than the mean number of SFC of the negative controls. The overall specificity of using the IFN-g ELISpot to test responses of CD8+ T cells to synthetic peptides was demonstrated previously by others in a series of CD8+ and CD4+ depletion and enrichment experiments (28, 31, 33, 34). Generation of standards for real-time PCR assay
The primers and probes for real-time PCR were optimized based on published sequences of geographical variants in the Los Alamos HIV databases (www.hiv.lanl.gov/) and previous reports (7). The primer, probe sequences, and their targets of detection were used as previously described (8, 35). As the standard for RT-PCR, we used an M13 bacteriophage as a vector to express the appropriate HIV-1 DNA and cellular sequences. The chemokine receptor CCR5 was chosen as a surrogate to estimate the input number of cells for cell containing only one single copy of CCR5 (4, 32). The absolute number of target sequences could be calculated based on the number of plaque-forming units (PFU) of M13 phage (26). To generate standards for the various forms of HIV-1 DNA and the cellular CCR5, the corresponding gene fragments were inserted into the M13 genome. The recombinant M13 phages were propagated in Escherichia coli JM109 and harvested by centrifugation. The titer of recombinant phage in the supernatant was estimated by serial dilution, followed by the counting of PFU (26). HIV DNA quantification
The total DNA from patients’ PBMCs was extracted using a QIAamp DNA blood mini kit (Qiagen) according to the manufacturer’s protocol. Modified real-time PCR was performed in 25 lL solution containing 2.5 lL of DNA target, 12.5 lL of Gene Expression Master Mix (Applied Biosystems 4369016), 1 lM of primers, and 0.2 lM of the probe under the following conditions: 95C for 10 min, followed by 95C for 15 s, and 60C for 1 min (40 cycles) in an ABI 7500 PCR machine (Applied Biosystems). For the integrated HIV-1 DNA, the first round of PCR was conducted with the following conditions: 95C for 8 min, and then 12 cycles of amplification: 95C for 10 s, 60C for 10 s, and 72C for 170 s. One-tenth of the first round PCR product was used for following real-time PCR detection. The PCR signal from first round PCR without the Alu primers was subtracted from the total signals before the estimate of copy numbers. A standard curve was created for each run in a 7log-unit range by 1:10 serial dilutions. Statistical analysis
Statistical analysis and graphical presentation were performed using SPSS 13.0 and Sigma Plot 10.0. Student’s t-test was used for data that fulfilled normal distribution. Data were expressed as the mean – standard deviation (SD). The nonparametric Mann–Whitney U test was used to perform the comparison between Naive and On-HAART adults. Data were expressed as median values and interquartile range. Wilcoxon rank sum tests were used to compare the distribution of cumulative CTL responses to a particular peptide or protein. A
ANALYSIS OF HIV-1C-SPECIFIC CTL RESPONSES
Table 1. Baseline Demographic and Immunological Parameters of Study Participants Parameters
Naive
On-HAART
Number 66 26 Sex ratio (male/female) 42/14 16/10 Age (years) 36.52 – 9.51 37.00 – 9.70 CD4+ T cell count 293.09 – 278.05 260.88 – 194.98 (cells/lL) HIV-1 Viral load 4.09 – 2.17 4.04 – 1.99 (log10 copies/mL) HAART, highly active antiretroviral treatment.
3
rank-based test was used for this comparison because it appropriately accounts for the censoring of within-well Elispot responses of >50 spots/well. A response was considered positive if the probability of a cell forming a spot was higher in the test well compared to the negative control well. Spearman rank correlation was used for correlation analysis. All tests were twotailed, and p-values of 0.05). HIV-1 viral load was lower in the On-HAART group than in naive group, but this difference was not statistically significant ( p > 0.05). We assessed HIV-1-specific CTL responses in the PBMCs of two study groups using 426 overlapping peptides spanning all expressed HIV-1 proteins by Elispot assay. To identify responses with both high magnitude and high frequency, a normalized cumulative CTL response was used by combining data on CTL magnitude and frequency (21, 34). HIV-1c-specific CTL responses directed against all the HIV-1c proteins were detectable in the two groups (Fig. 1). The most immunogenic Nef, P24, and RT were targeted by 80.30%, 71.21%, and 72.73% of the tested naive patients, respectively. The HIV-1c-
KANG ET AL.
specific CTL responses against each HIV-1c protein was greatly reduced in the On-HAART group, especially in P15, Rev, and Vpu, whose CTL responses magnitude dropped by 92.55%, 90.45%, and 90.36%, respectively (Fig. 2A). However, there was less difference in the percentage of HIV protein recognition between the naive and On-HAART group (Fig. 2B). Compared to the naive group, recognition of P15, P17, and Rev in the On-HAART group were decreased by 42.89%, 32.05%, and 23.61%, respectively. Overall, P15 and Rev were the two proteins that showed the greatest change in response between naive patients and those on HAART. Patients receiving HAART have higher total HIV-1 DNA and integrated HIV-1 DNA
After HIV-1 binding and entry into the host cells, the viral genome is reverse transcribed into the DNA, transported into the nucleus, and integrated into the host genome for viral replication (15). Cellular HIV-1 reservoirs are fundamental to
FIG. 2. Magnitude and breadth of HIV-1c-specific CTL responses in protein levels. HIV-1-specific CTL responses were directed in the PBMCs of two study groups using 426 overlapping peptides spanning all expressed HIV-1 proteins by IFN-g-ELISpot assay (x-axis). (A) Cumulative Elispot-based CTL responses were measured as a sum of the naive or OnHAART of HIV-1 group to a particular protein and expressed as the number of cumulative SFC/106 PBMC (y-axis). (B) Percentage of positive IFN-g lymphokine responders of the naive or On-HAART of HIV-1 group reacting to a particular protein. The x-axis of each graph was scaled according to the name of protein or protein subunits. The peptide concentration was 10 lM with a final volume of 130 lL/well in RPMI 1640 with 10% of FBS.
ANALYSIS OF HIV-1C-SPECIFIC CTL RESPONSES
5
FIG. 3. Comparison of HIV-1 DNA copies in PBMCs between naive and On-HAART of HIV-1-infected patients. Medians and p-values from Mann–Whitney test are shown. (A) The quantity of total HIV-1 DNA of the On-HAART group was significantly higher than the naive group. (B) The difference of 2-LTR HIV-1 DNA between naive and On-HAART of HIV-1 group was not significant. (C) The quantity of integrated HIV-1 DNA of the On-HAART group was significantly higher than the naive group. The Mann–Whitney U test was used to perform the comparison between Naive and OnHAART adults, p-values of 350
Total HIV-1 DNA 2-LTR HIV-1 DNA/Total HIV-1 DNA Integrated HIV-1 DNA/Total HIV-1 DNA
-0.265 0.100 0.160
-0.632a -0.401b -0.762a
0.243 0.120 0.026
-0.024 -0.683b -0.119
a
Correlation is significant at the 0.01 level (two-tailed). Correlation is significant at the 0.05 level (two-tailed). However, the naive group had strong statistically significant correlations, while the On-HAART group presented no statistical significance. These correlations disappeared after HAART which suggested that different immune cells could influence the HIV-1 reservoirs and reassured the necessary consideration of basic immune cell levels of study subjects. CTL, cytotoxic T lymphocytes. b
ANALYSIS OF HIV-1C-SPECIFIC CTL RESPONSES
7
Table 3. Number of HIV-1 Peptides with Strong Correlation of Different HIV-1 Reservoir Sizes Number of HIV-1 peptides with strong correlation (p < 0.05) Total HIV-1 DNA 2-LTR HIV-1 DNA/Total HIV-1 DNA Integrated HIV-1 DNA/Total HIV-1 DNA
Naive
On-HAART
CD4 £ 350
CD4 > 350
CD4 £ 350
CD4 > 350
0 0 0
5 (P17, P24, Nef, Pro, RT, Gp41) 2 (P17, Nef) 6 (P17, P24, Nef, Pro, Int, RT, Gp41)
0 0 0
0 1(P17) 0
circulating HIV-1 DNA, and total HIV-1 DNA) and thus tried to further explore the relationship of HIV-1-specific CTL responses with HIV-1 reservoirs. Consistent with previous studies (31, 33, 34), we found that HIV-infected patients had a wide range of cumulative CTL responses against HIV-1 peptides and recognition of these peptides appeared to be extensive in Gag, Nef, and Env regions. Moreover, both the cumulative CTL responses and the recognition of HIV peptides were significantly weaker in the On-HAART group compared with the naive group. HIV-specific CTL responses became weaker with the initiation of HAART, but accompanied with more amounts of HIV reservoirs. Unexpectedly, our results are contradictory with those of Graf et al. (14). They showed that total and integrated HIV-1 DNAs both decreased in patients on HAART. We assumed that such discrepancy might be caused by the characteristics of study subjects or length on HAART. In their studies, the off-HAART group usually had a median of about 4,000 copies of total HIV DNA per mL PBMCs, but in our naive group, only a median of 53 copies of total HIV DNA was detected. Thus, their off-HAART group may represent the patients in rapid HIV progression, but our naive patients are more like ES with low total or integrated HIV DNAs. In our study, CD4+ T-lymphocyte counts between the two study groups did not demonstrate statistical significance, which excluded the potential effect of CD4+ T-cell levels on the lower HIV-1 reservoirs in the On-HAART group. However, high CD4+ T-lymphocyte counts may affect HIV reservoirs. HIV-1 reservoirs became more strongly correlated with CTL cumulative responses when CD4+ T-lymphocyte counts were above 350 cells/lL. However, these correlations disappeared after HAART. These results suggested that different immune cells could influence the HIV-1 reservoirs and reassured the necessary consideration of basic immune cell levels of study subjects. Accordingly, CTL responses against different peptides are correlated with HIV-1 reservoirs. Also, the CTL responses were more remarkable when CD4+ T-lymphocyte counts were above 350 cells/lL, but less notable after HAART. Interestingly, P17 peptide positively correlated with the amount of integrated HIV-1 DNA, but negatively correlated with the amount of 2-LTR DNA and total HIV-1 DNA. We propose that HIV-specific CTL responses against p17 may be associated with the low integrate efficiency of HIV-1, which might be a biomarker to evaluate the efficacy of HAART. In summary, we analyzed the association of HIV-1-specific CTL responses and HIV reservoirs in patients with or without HAART. We observed an increase in total HIV-1 DNA and integrated HIV-1 DNA after HAART compared with naive patients. HIV-1c-specific CTL responses may be suppressed after HAART rather than HIV-1b-specific CTL responses. Moreover, we found that the baseline CD4+ T-lymphocyte
counts and antiretroviral treatment affected the size of HIV-1 reservoirs. In addition, HIV-specific CTL responses against p17 were shown to correlate with low integrate efficiency of HIV-1, which can possibly be monitored to evaluate the efficacy of HAART. Acknowledgments
This work was supported by the grant from National Key Technologies Research and Development Program of China (2008ZX10001-006, 2008ZX10001-002, and 2012ZX10001-003), Natural Science Foundation of China (grant 31000084 and 81202305), and Tangdu Technology Innovation and Development Program (2012LCYJ002). Ethics Approval
This work was approved by the Ethics board of Tangdu Hospital. The Judgement reference number was TDLL2012016. Author Disclosure Statement
No competing financial interests exist. References
1. Agosto LM, Liszewski MK, Mexas A, et al. Patients on HAART often have an excess of unintegrated HIV DNA: implications for monitoring reservoirs. Virology 2011;409: 46–53. 2. Alatrakchi N, Duvivier C, Costagliola D, et al. Persistent low viral load on antiretroviral therapy is associated with T cell-mediated control of HIV replication. AIDS 2005;19: 25–33. 3. Alexaki A, Liu Y, and Wigdahl B. Cellular reservoirs of HIV-1 and their role in viral persistence. Curr HIV Res 2008;6:388–400. 4. Beloukas A, Paraskevis D, Haida C, et al. Development and assessment of a multiplex real-time PCR assay for quantification of human immunodeficiency virus type 1 DNA. J Clin Microbiol 2009;47:2194–2199. 5. Betts MR, Ambrozak DR, Douek DC, et al. Analysis of total human immunodeficiency virus (HIV)-specific CD4(+) and CD8(+) T-cell responses: relationship to viral load in untreated HIV infection. J Virol 2001;75:11983– 11991. 6. Braitstein P, Brinkhof MW, Dabis F, et al. Mortality of HIV-1-infected patients in the first year of antiretroviral therapy: comparison between low-income and high-income countries. Lancet 2006;367:817–824. 7. Brussel A, and Sonigo P. Analysis of early human immunodeficiency virus type 1 DNA synthesis by use of a new sensitive assay for quantifying integrated provirus. J Virol 2003;77:10119–10124.
8
8. Chomont N, El-Far M, Ancuta P, et al. HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nat Med 2009;15:893–900. 9. Chun TW, Carruth L, Finzi D, et al. Quantification of latent tissue reservoirs and total body viral load in HIV-1 infection. Nature 1997;387:183–188. 10. Chun TW, Justement JS, Moir S, et al. Suppression of HIV replication in the resting CD4+ T cell reservoir by autologous CD8+ T cells: implications for the development of therapeutic strategies. Proc Natl Acad Sci U S A 2001;98: 253–258. 11. Churchill MJ, Wesselingh SL, Cowley D, et al. Extensive astrocyte infection is prominent in human immunodeficiency virus-associated dementia. Ann Neurol 2009;66: 253–258. 12. Finzi D, Blankson J, Siliciano JD, et al. Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy. Nat Med 1999;5:512–517. 13. Gorry P, Purcell D, Howard J, et al. Restricted HIV-1 infection of human astrocytes: potential role of nef in the regulation of virus replication. J Neurovirol 1998;4:377– 386. 14. Graf EH, Mexas AM, Yu JJ, et al. Elite suppressors harbor low levels of integrated HIV DNA and high levels of 2LTR circular HIV DNA compared to HIV+ patients on and off HAART. PLoS Pathog 2011;7:e1001300. 15. Greene WC, and Peterlin BM. Charting HIV’s remarkable voyage through the cell: basic science as a passport to future therapy. Nat Med 2002;8:673–680. 16. Harrer T, Harrer E, Kalams SA, et al. Strong cytotoxic T cell and weak neutralizing antibody responses in a subset of persons with stable nonprogressing HIV type 1 infection. AIDS Res Hum Retroviruses 1996;12:585–592. 17. Hersperger AR, Martin JN, Shin LY, et al. Increased HIVspecific CD8+ T-cell cytotoxic potential in HIV elite controllers is associated with T-bet expression. Blood 2011; 117:3799–3808. 18. Luo L, and Li TS. Overview of antiretroviral treatment in China: advancement and challenges. Chin Med J (Engl) 2011;124:440–444. 19. Migueles SA, Laborico AC, Imamichi H, et al. The differential ability of HLA B*5701+ long-term nonprogressors and progressors to restrict human immunodeficiency virus replication is not caused by loss of recognition of autologous viral gag sequences. J Virol 2003;77:6889– 6898. 20. Mocroft A, Ledergerber B, Katlama C, et al. Decline in the AIDS and death rates in the EuroSIDA study: an observational study. Lancet 2003;362:22–29. 21. Novitsky V, Rybak N, McLane MF, et al. Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic Tlymphocyte responses for AIDS vaccine design. J Virol 2001;75:9210–9228. 22. Ogg GS, Jin X, Bonhoeffer S, et al. Quantitation of HIV-1specific cytotoxic T lymphocytes and plasma load of viral RNA. Science 1998;279:2103–2106.
KANG ET AL.
23. Ogg GS, Kostense S, Klein MR, et al. Longitudinal phenotypic analysis of human immunodeficiency virus type 1specific cytotoxic T lymphocytes: correlation with disease progression. J Virol 1999;73:9153–9160. 24. Sa´ez-Cirio´n A, Sinet M, Shin SY, et al. Heterogeneity in HIV suppression by CD8+ T cells from HIV controllers: association with Gag-specific CD8+ T cell responses. J Immunol 2009;182:7828–7837. 25. Sonza S, Mutimer HP, Oelrichs R, et al. Monocytes harbour replication-competent, non-latent HIV-1 in patients on highly active antiretroviral therapy. AIDS 2001;15:17–22. 26. Sum SS, Wong DK, Yuen MF, et al. Real-time PCR assay using molecular beacon for quantitation of hepatitis B virus DNA. J Clin Microbiol 2004;42:3438–3440. 27. Tuboi SH, Schechter M, McGowan CC, et al. Mortality during the first year of potent antiretroviral therapy in HIV1-infected patients in 7 sites throughout Latin America and the Caribbean. J Acquir Immune Defic Syndr 2009;51:615– 623. 28. Wang S, Sun Y, Zhai S, et al. Identification of HLA-A11restricted HIV-1-specific cytotoxic T-lymphocyte epitopes in China. Curr HIV Res 2007;5:119–128. 29. Watanabe D, Ibe S, Uehira T, et al. Cellular HIV-1 DNA levels in patients receiving antiretroviral therapy strongly correlate with therapy initiation timing but not with therapy duration. BMC Infect Dis 2011;11:146. 30. Wong JK, Hezareh M, Gunthard HF, et al. Recovery of replication-competent HIV despite prolonged suppression of plasma viremia. Science 1997;278:1291–1295. 31. Zhai S, Zhuang Y, Song Y, et al. HIV-1-specific cytotoxic T lymphocyte (CTL) responses against immunodominant optimal epitopes slow the progression of AIDS in China. Curr HIV Res 2008;6:335–350. 32. Zhang L, Ramratnam B, Tenner-Racz K, et al. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med 1999;340:1605– 1613. 33. Zhao S, Zhai S, Zhuang Y, et al. Inter-clade cross-reactivity of HIV-1-specific T cell responses in human immunodeficiency virus type 1 infection in China. Curr HIV Res 2007; 5:251–259. 34. Zhuang Y, Sun Y, Zhai S, et al. Relative dominance of Env-gp41-specific cytotoxic T lymphocytes responses in HIV-1 advanced infection. Curr HIV Res 2008;6:239–245. 35. Zhu W, Jiao Y, Lei R, et al. Rapid turnover of 2-LTR HIV1 DNA during early stage of highly active antiretroviral therapy. PLoS One 2011;6:e21081.
Address correspondence to: Dr. Wenzhen Kang Department of Infectious Diseases Tangdu Hospital Affiliated to the Fourth Military Medical University Xi’an 710038 People’s Republic of China E-mail: [email protected]
