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

Abstract

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.

Cataloguing of Potential HIV Susceptibility Factors during the Menstrual Cycle of Pig-Tailed Macaques by Using a Systems Biology Approach

“…Other proteins significantly overexpressed in the follicular phase are vitronectin and apolipoproteins (A-I and D), which have HIV-1-inhibitory properties (23,30). It is intriguing to note that apoliprotein A-I (Apo A-I) was one of the abundantly expressed proteins (31) in the cervicovaginal mucosa of highly exposed seronegative (HESN) sex workers. Overexpressed proteins in these individuals included many serpins (31), several of which were also overexpressed in the follicular phase in our study, as determined by both proteomic and genomic analyses, consistent with earlier findings (32).…”

“…It is intriguing to note that apoliprotein A-I (Apo A-I) was one of the abundantly expressed proteins (31) in the cervicovaginal mucosa of highly exposed seronegative (HESN) sex workers. Overexpressed proteins in these individuals included many serpins (31), several of which were also overexpressed in the follicular phase in our study, as determined by both proteomic and genomic analyses, consistent with earlier findings (32). Serpins are reported to possess anti-inflammatory properties (33) and are important players in mucosal defense against HIV (32), especially in the eclipse phase (27).…”

A Systems Biology Examination of the Human Female Genital Tract Shows Compartmentalization of Immune Factor Expression

“…Tissue localization of factors associated with anti-HIV activity in different FGT compartments. Several soluble mucosal proteins have been associated with altered susceptibility to HIV in the cervical mucosa of HESN individuals, including secretory IgA, serpins, cystatins, elafin, and A2ML1 (10,(12)(13)(14)30). Many of these soluble factors may also inhibit HIV-1 replication in susceptible cell types (2,(30)(31)(32).…”

“…Complement components also play important roles in the innate and adaptive immune systems (7) and are of particular importance, as they can either inhibit or facilitate HIV-1 infection (8). Studies of HIV-exposed seronegative (HESN) individuals have shown that specific adaptive and innate factors are associated with HIV resistance, including HIVneutralizing IgA antibodies (9)(10)(11) and overexpression of serine/ cysteine antiproteases such as serpins, elafin, cystatins, and A2ML1 (12)(13)(14). However, little is known about the anatomical sites, spatial expression, or cell types that express these factors within the FGT.…”

“…We used a systems biology approach to characterize tissue sites of the lower and upper FGT, including the ectocervix, endocervix, and the endometrium from healthy women. The application of mass spectrometry-based proteomic techniques has allowed for a more in-depth examination of mucosal environments (13,15), and here we characterized individual expression patterns of Ͼ1,000 unique proteins, identifying anatomical differences in immune factor expression important for HIV pathogenesis.…”