HLA class I Sanger sequences data of Honduras HIV cohort

HLA polymorphisms represent the strongest genetic modifier of HIV disease progression. Diverse HLA distribution can lead to distinct HIV control landscapes at the population level. We aimed to describe HLA allele and haplotype frequencies (linkage disequilibrium, LD), CCR5-Δ32 frequency and the impact of these variants on HIV disease outcome. HLA class I (cI) loci were typed at 4-digit resolution, and CCR5 variants were determined in 402 HIV clade B-infected, ART-naïve individuals from Honduras. HLA LD were assessed using Fisher’s exact test. Using univariable and multivariable analyses we evaluated HLA associations with HIV pVL and CD4 counts. We did not find any effect on HIV control between CCR5 genotypes. Previously defined HLA associations were found: B*57:01/03, B*42:01, A*25:01 and C*12:03 (protective), and B*53:01 and A*68:01 (risk). Being consistent with our previous research in a Mesoamerican HIV cohort, Amerindian B*35:12 was associated to poor HIV control. Other HLA-HIV associations not previously described were C*03:04 and B*08:01 that were associated with higher pVL. Overall, this first report highlights the immunogenetic uniqueness admixture of the Honduras population that express Amerindian, Caucasian and African HLA subtypes. These findings not only support this cohort as ideal for identifying HLA correlates of HIV control but also may improve future research regarding allotransplantation and disease association.

Methods
HLA class I loci (HLA-A, HLA-C and HLA-B) were resolved at second field resolution (i.e., subtype or protein level) using a previously described protocol. Briefly, a nested PCR with locus-specific primers were used to amplify a ~1000 bp region spanning exons 2 and 3. These PCR products were directly sequenced using a set of sequencing primers as previously described (Cotton LA, et al. 2012. HLA class I sequence-based typing using DNA recovered from frozen plasma. J Immunol Methods 382:40–47) on a 3730xl Genetic Analyzer (Applied Biosystems, Foster City). HLA subtypes were assigned using UType v7.1 RUO (Applied Biosystems) using the IPD-IMGT/HLA Database (release 3.31.0, 2018-01). Primer (allelic) and phase resolution ambiguities were resolved as previously explained (Valenzuela-Ponce H, et al. 2018. Novel HLA class I associations with HIV-1 control in a unique genetically admixed population. Sci Rep 8:17.).

人类白细胞抗原（Human Leukocyte Antigen，HLA）多态性是影响HIV疾病进展的最强遗传修饰因子。不同的HLA分布格局可在群体层面形成各异的HIV病毒控制特征。本研究旨在描述HLA等位基因与单倍型频率（即连锁不平衡（linkage disequilibrium，LD））、CCR5-Δ32频率，以及上述变异体对HIV疾病转归的影响。研究对来自洪都拉斯的402名HIV B亚型感染者（均为抗逆转录病毒治疗（antiretroviral therapy，ART）初治患者），完成了HLA I类（HLA class I，cI）位点的4位分辨率分型，并检测了CCR5变异体。采用Fisher精确检验评估HLA连锁不平衡程度；通过单变量与多变量分析，评估了HLA与HIV血浆病毒载量（plasma viral load，pVL）及CD4计数的相关性。本研究未发现CCR5基因型对HIV病毒控制存在显著影响。已被报道的HLA关联得到验证：B*57:01/03、B*42:01、A*25:01及C*12:03具有保护作用，而B*53:01与A*68:01则为疾病风险因素。与此前中美洲HIV队列的研究结果一致，美洲原住民来源的B*35:12与不良HIV控制结局相关。此外本研究还发现了此前未被报道的HLA-HIV关联：C*03:04与B*08:01与更高的血浆病毒载量相关。总体而言，本首项研究凸显了洪都拉斯人群的免疫遗传独特性与遗传混合特征——该人群携带有美洲原住民、白种人及非洲来源的HLA亚型。上述发现不仅证实本队列可作为鉴定HIV病毒控制相关HLA关联因子的理想模型，同时也可为未来同种异体移植（allotransplantation）及疾病关联相关研究提供参考价值。方法本研究采用已发表的实验方案，对HLA I类（HLA class I）位点（HLA-A、HLA-C及HLA-B）以第二场分辨率（即亚型或蛋白水平）完成分型。简言之，使用位点特异性引物进行巢式PCR扩增，扩增片段为覆盖外显子2与3的约1000bp区域。参照此前发表的研究（Cotton LA等，2012年，"HLA class I sequence-based typing using DNA recovered from frozen plasma", "J Immunol Methods" 382:40–47），将上述PCR产物在3730xl基因分析仪（Applied Biosystems，福斯特城）上，采用一组测序引物完成直接测序。采用UType v7.1 RUO软件（Applied Biosystems）结合IPD-IMGT/HLA数据库（版本3.31.0，2018-01）进行HLA亚型的分型。引物（等位基因）及相位解析歧义的处理，参照此前的研究方案（Valenzuela-Ponce H等，2018年，"Novel HLA class I associations with HIV-1 control in a unique genetically admixed population", "Sci Rep" 8:17.）。