馬永莉++++++陳燕麗++++++郝金娟++++++阮玉華++++++邵一鳴++++++洪坤學
[摘要] 目的 探討人類免疫缺陷病毒Ⅰ型(HIV-1)B′亞型感染者人類白細胞抗原(HLA)-A、-B、-C位點等位基因的分布及其對病毒載量的影響。 方法 對146例HIV-1B′亞型感染者采用聚合酶鏈反應-序列特異性引物(PCR-SSP)擴增方法進行HLA-A、-B、-C位點等位基因檢測,計算各位點等位基因的頻率及其對感染者病毒載量的影響。 結果 在146例HIV-1B′亞型感染者中分別檢出HLA-A位點14個等位基因,-B位點24個等位基因,-Cw位點12個等位基因,其中頻率大于0.1的等位基因為HLA-A*02,-A*11,-A*24,-A*30,-B*13,-B*40,-B*51,-Cw*03,-Cw*06,-Cw*08;HLA-A、-B、-C等位基因純合子攜帶者的病毒載量高于雜合子(P=0.0013);HLA-A*03(P=0.0314)、-A*30(P=0.0072)、-B*13(P=0.0087)、-Cw*06(P=0.0145)等位基因攜帶者具有較低病毒載量。 結論 HIV-1B′亞型感染者HLA-A*03、-A*30、-B*13、-Cw*06等位基因與低病毒載量相關。
[關鍵詞] 人類免疫缺陷病毒;人類白細胞抗原;病毒載量;感染
[中圖分類號] R117[文獻標識碼] A[文章編號] 1673-7210(2014)06(c)-0009-04
Impact of HLA class Ⅰ alleles on viral load in HIV-1 B′ infected individuals
MA Yongli CHEN Yanli HAO Jinjuan RUAN Yuhua SHAO Yiming HONG Kunxue▲
National Center for AIDS/STD Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory for Infectious Disease Control and Prevention, Beijing 102206, China
[Abstract] Objective To study the distribution of HLA-A, -B, and C alleles in HIV-1 B′infected individuals and to analyze its impact on HIV-1 viral load. Methods 146 HIV-1 infected individuals were recruited and their HLA-A, -B, and C alleles were genotyped using PCR-SSP technique. Allele frequencies and association of HLA-A, -B, and C alleles with HIV-1 viral loads were also analyzed. Results 14 HLA-A allels, 24 HLA-B allels, and 12 HLA-Cw allels were detected in 146 HIV-1 B' infected individuals. Of them, HLA-A*02, -A*11, -A*24, -A*30, -B*13, -B*40, -B*51, - Cw*03, -Cw*06, -Cw*08 were among the most common alleles with frequencies above 0.1; subjects homozygous at HLA class Ⅰ loci had higher viral loads (P=0.0013), subjects carrying HLA-A*03 (P=0.0314), -A*30 (P=0.0072), -B*13 (P=0.0087) and -Cw*06 (P=0.0145) alleles had lower viral loads. Conclusion HLA-A*03, -A*30, -B*13 and -Cw*06 alleles may associate with lower viral loads in HIV-1 B′infected individuals.
[Key words] Human immunodeficiency virus; Human leukocyte antigen; Viral load; Infection
人類白細胞抗原(human leukocyte antigen,HLA)基因影響人類免疫缺陷病毒(human immunodeficiency virus,HIV)感染者的病毒復制,有研究報道攜帶HLA-B*27,-B*57等位基因的HIV感染者病毒載量較低,多表現為緩慢疾病進程,而HLA-B*35,-B*08,-B*56等位基因則與HIV-1感染者的疾病快速進展相關[1-2]。HLA不同等位基因對HIV病毒復制影響的差異可能與其提呈HIV保護性或非保護性抗原表位,從而激發(fā)具有細胞毒作用的CD8+T細胞(cytotoxic T lymphocyte,CTL)免疫應答的能力不同有關[3-4]。然而HLA基因對HIV-1病毒復制和感染者疾病進展的作用受不同人群特定的遺傳背景的影響,目前在不同地區(qū)不同種族人群中的研究結果并不完全一致[5]。本研究探討了146例未接受抗病毒治療的B′亞型HIV-1感染者的HLA Ⅰ類等位基因分布,并分析了該感染人群HLA Ⅰ類等位基因對HIV-1病毒載量的影響。
1 對象與方法
1.1 研究對象
根據知情同意原則從HIV-1B′亞型感染人群中招募146例未經抗病毒治療者,所有病例均經HIV抗體初篩和確證試驗檢測陽性,其中男82例,女64例,年齡22~60歲,平均(47.6±10.2)歲。采集EDTA抗凝靜脈血標本5 mL,常規(guī)分裝全血和血漿,-80℃保存?zhèn)溆?。研究方案經中國疾病預防控制中心性病艾滋病預防控制中心倫理委員會批準?;颊呔橥獠⒑炇鹬橥鈺?。
1.2 HLA基因分型
提取全血基因組DNA,并應用聚合酶鏈反應-序列特異性引物擴增(PCR-SSP)試劑盒(HLA-ABC SSP MorganTM)進行HLA基因分型。
1.3 病毒載量測定
使用羅氏公司HIV-1定量檢測試劑盒(COBAS AMPLICOR HIV-1 MONITOR Test)檢測血漿病毒載量,定量測定的檢出限為50拷貝/mL。
1.4 統(tǒng)計學方法
使用SigmaPlot 10.0和GraphPad Prism 5.0進行統(tǒng)計分析和作圖,攜帶或不攜帶HLA純合子、攜帶或不攜帶某HLA等位基因的感染者間病毒載量的差異分析用Mann-Whitney U檢驗進行,以P < 0.05為差異有統(tǒng)計學意義。
2 結果
2.1 研究對象中HLA-A、-B、-C位點等位基因的多態(tài)性分布
本研究在146例HIV-1感染者中分別檢出HLA-A位點14個等位基因,-B位點24個等位基因,-Cw位點12個等位基因,其中頻率大于0.1的高頻率的等位基因為HLA-A*02,-A*11,A*24,A*30,-B*13,-B*40,-B*51,- Cw*03,-Cw*06,-Cw*08,研究人群中HLA-A、B、C位點等位基因呈現多樣性分布(圖1)。
2.2HLA-Ⅰ類基因與病毒載量的相關性
分別對HLA-A、B、C位點等位基因和病毒載量關聯(lián)性進行分析,結果顯示攜帶或不攜帶HLA純合子的HIV感染者的病毒載量差異有高度統(tǒng)計學意義(P=0.0013),HLA純合子感染者的病毒載量高(圖2)。攜帶HLA-A*03(P=0.0314),-A*30(P=0.0072),-B*13(P=0.0087),和-Cw*06(P=0.0145)等位基因的HIV感染者病毒載量低于不攜帶相應基因者(圖3)。
3 討論
HLA等位基因控制HIV病毒復制的的保護性作用機制尚未完全清楚,以往有關的研究多集中于對攜帶HLA-B*27和HLA-B*57等位基因的HIV感染者進行分析,研究結果提示這種保護性效應可能與HLA-B*27和HLA-B*57限制識別的表位及其誘發(fā)的CTL應答特征相關[6],其中HLA-B*27識別的免疫顯性表位KK10(KRWIILLNK263~272)和HLA-B*57識別的免疫顯性表位TW10(TSTLQEQIGW240~249)位于序列相對保守的p24Gag區(qū),其所誘導的特異性CTL應答呈現多功能特征(CD107a,IFN-γ,TNF-α,IL-2,MIP-1β),這種多功能的CTL應答與病毒復制的控制顯著相關[7]。新近的研究發(fā)現HLA-B*27還能結合Leu268Met的KK10表位突變體,且所誘導的攜帶TRBV6-5 TRBJ1-1基序T細胞受體的CTL克隆能交叉識別野生型及突變型KK10表位,進一步說明HLA-B*27的保護性效應與其結合的KK10表位誘導的特異性CTL效應相關[8]。但本研究未觀察到HLA-B*27和HLA-B*57與病毒載量相關的保護性效應,這可能是這兩個等位基因在亞洲人群中的頻率低所致[5]。
本研究人群中檢出的高頻率等位基因為HLA-A*02、-A*11、-A*24、-A*30、-B*13、-B*40、-B*51、-Cw*03、-Cw*06、-Cw*08,符合我國漢族人群HLA-Ⅰ類等位基因的分布特點。對HIV-1B′亞型感染者的分析結果表明攜帶HLA-Ⅰ類等位基因純合子感染者的病毒載量較高(P=0.0013),這與以往的研究報道一致,表明HLA-Ⅰ類等位基因純合狀態(tài)不利于控制病毒的復制和疾病的發(fā)展。HLA雜合子則可能通過遞呈更多的CTL表位而誘導廣譜的CTL免疫反應,有利于控制病毒通過序列變異逃逸感染者體內CTL的識別與殺傷,因而在控制HIV感染者病毒復制和疾病進展中表現出一定的優(yōu)勢[9-10]。各等位基因與病毒載量相關性分析結果表明攜帶HLA-A*03、-A*30、-B*13、-Cw*06等位基因的感染者分別比不攜帶相應HLA等位基因的感染者的病毒載量低,其中HLA-B*13的保護性作用以往有報道,其與低病毒載量相關的保護性作用與其識別的表位誘導的CTL應答多功能特征密切相關[11]。HLA-A*03、-A*30、-Cw*06這三個等位基因與低病毒載量相關的保護性作用研究尚少,但有報道表明我國漢族人群中HLA-A*30-B*13-Cw*06處于連鎖不平衡狀態(tài),因而需要進一步的研究明確HLA- A*30、-Cw*06在HIV-1B′亞型感染者中與低病毒載量相關是否由于與HLA- B*13連鎖所致[12]。
本研究表明HIV-1B′亞型感染者HLA-A*03、-A*30、-B*13、-Cw*06等位基因攜帶者病毒載量較低,這種關聯(lián)可能與不同HLA-Ⅰ類分子提呈HIV保護性或非保護性抗原表位能力存在差異,從而激發(fā)具有細胞毒作用的CTL免疫應答的能力不同有關[13-14]。這為進一步深入鑒定相關HLA等位基因限制的CTL表位及探討其在控制HIV病毒感染復制中的作用機制提供了基礎。
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(收稿日期:2014-03-14本文編輯:衛(wèi)軻)
[9]Carrington M,O'Brien SJ. The influence of HLA genotype on AIDS [J]. Annu Rev Med,2003,54:535-551.
[10]Goulder PJ,Watkins DI. Impact of MHC class I diversity on immune control of immunodeficiency virus replication [J]. Nat Rev Immunol,2008,8:619-630.
[11]Honeyborne I,Prendergast A,Pereyra F,et al. Control of human immunodeficiency virus type 1 is associated with HLA-B13 and targeting of multiple gag-specific CD8+ T-cell epitopes [J]. Journal of virology,2007,81(7):3667-3672.
[12]Zhang H,Zhao B,Han X,et al. Associations of HLA class I antigen specificities and haplotypes with disease progression in HIV-1-infected Hans in northern China[J]. Hum Immunol,2013,74(12):1636-1642.
[13]Roider J,Kalteis AL,Vollbrecht T,et al. Adaptation of CD8 T Cell responses to changing HIV-1 sequences in a cohort of HIV-1 infected not selected for a certain HLA allele [J]. PLoS ONE,2013,8(12):e80045.
[14]McLaren PJ,Ripke S,Pelak K,et al. Fine-mapping classical HLA variation associated with durable host control of HIV-1 infection in African Americans [J]. Human Molecular Genetics,2012,21(19):4334-4347.
(收稿日期:2014-03-14本文編輯:衛(wèi)軻)
[9]Carrington M,O'Brien SJ. The influence of HLA genotype on AIDS [J]. Annu Rev Med,2003,54:535-551.
[10]Goulder PJ,Watkins DI. Impact of MHC class I diversity on immune control of immunodeficiency virus replication [J]. Nat Rev Immunol,2008,8:619-630.
[11]Honeyborne I,Prendergast A,Pereyra F,et al. Control of human immunodeficiency virus type 1 is associated with HLA-B13 and targeting of multiple gag-specific CD8+ T-cell epitopes [J]. Journal of virology,2007,81(7):3667-3672.
[12]Zhang H,Zhao B,Han X,et al. Associations of HLA class I antigen specificities and haplotypes with disease progression in HIV-1-infected Hans in northern China[J]. Hum Immunol,2013,74(12):1636-1642.
[13]Roider J,Kalteis AL,Vollbrecht T,et al. Adaptation of CD8 T Cell responses to changing HIV-1 sequences in a cohort of HIV-1 infected not selected for a certain HLA allele [J]. PLoS ONE,2013,8(12):e80045.
[14]McLaren PJ,Ripke S,Pelak K,et al. Fine-mapping classical HLA variation associated with durable host control of HIV-1 infection in African Americans [J]. Human Molecular Genetics,2012,21(19):4334-4347.
(收稿日期:2014-03-14本文編輯:衛(wèi)軻)