Comprehensive Proteomic Study Identifies Serpin and Cystatin Antiproteases as Novel Correlates of HIV-1 Resistance in the Cervicovaginal Mucosa of Female Sex Workers

Not all individuals exposed to HIV-1 become infected, and evidence from HIV-1 highly exposed seronegative women (HIV-1-resistant) suggests that mucosal factors in the female genital tract, the first site of contact for the virus, are playing a role. To better understand factors mediating protection from HIV-1, we performed a large clinical study using the tools of systems biology to fully characterize the cervicovaginal mucosa proteome in HIV-1-resistant women. Cervicovaginal lavage fluid was collected from 293 HIV-1-resistant, uninfected, and infected sex workers and analyzed by 2D-LC LTQ-FT-MS. Of the more than 360 unique proteins identified, 41 were differentially abundant (>3-fold cutoff) in HIV-1-resistant women. The majority of over-abundant proteins were antiproteases (>40%), some with described anti-inflammatory and anti-HIV-1 activity. Quantification of specific anti-HIV-1 antiproteases Serpin A1, Serpin A3, and Cystatin B and an epithelial antiprotease A2ML1 found them to be significantly over-abundant in HIV-1-resistant women (p = 0.004; p = 0.046; p = 0.0003; and p = 0.04, respectively). Expression levels were not correlated to sexual practices or other epidemiological factors. Mucosal antiprotease levels correlated with pro-inflammatory cytokine concentration (p = <0.0001), but independently of pro-inflammatory cytokine levels in HIV-1-resistant women including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, and IL-8. This comprehensive systems biology approach identifies mucosal serpins and cystatins as novel correlates of HIV-1-resistance. This represents the first study characterizing these factors in the female genital tract.

并非所有接触人类免疫缺陷病毒1型（HIV-1）的个体都会发生感染，针对HIV-1高暴露血清阴性女性（即HIV-1抗性人群）的研究证据表明，作为病毒初次接触位点的女性生殖道黏膜因素在抵抗感染中发挥了关键作用。为深入阐明介导HIV-1抵抗的保护因子机制，我们开展了一项大型临床研究，借助系统生物学（systems biology）工具对HIV-1抗性女性的宫颈阴道黏膜蛋白质组（cervicovaginal mucosa proteome）进行全面表征。研究共纳入293名HIV-1抗性、未感染及已感染的性工作者，采集其宫颈阴道灌洗液（cervicovaginal lavage fluid），并通过二维液相色谱-线性离子阱傅里叶变换质谱（2D-LC LTQ-FT-MS）进行分析。在鉴定得到的360余种独特蛋白质中，有41种蛋白质在HIV-1抗性女性体内的丰度差异达到3倍以上的筛选阈值。其中高丰度蛋白质大多为抗蛋白酶类物质（占比超40%），部分已被证实具有抗炎及抗HIV-1活性。针对特定抗HIV-1抗蛋白酶类物质丝氨酸蛋白酶抑制剂A1（Serpin A1）、丝氨酸蛋白酶抑制剂A3（Serpin A3）、半胱氨酸蛋白酶抑制剂B（Cystatin B）以及上皮细胞抗蛋白酶A2ML1进行定量分析后发现，它们在HIV-1抗性女性体内的丰度显著升高（对应p值分别为0.004、0.046、0.0003及0.04）。上述蛋白质的表达水平与性行为习惯或其他流行病学因素无显著相关性。黏膜抗蛋白酶水平与促炎细胞因子（pro-inflammatory cytokine）浓度呈显著正相关（p<0.0001），但在HIV-1抗性女性群体中，该相关性独立于肿瘤坏死因子-α（TNF-α）、白细胞介素-1α（IL-1α）、白细胞介素-1β（IL-1β）、白细胞介素-6（IL-6）及白细胞介素-8（IL-8）等促炎细胞因子的表达水平。本研究采用的综合性系统生物学策略，将黏膜丝氨酸蛋白酶抑制剂及半胱氨酸蛋白酶抑制剂鉴定为HIV-1抗性的新型关联因子。这也是首个针对女性生殖道内此类保护因子的系统性表征研究。