Non-Cationic Proteins Are Associated with HIV Neutralizing Activity in Genital Secretions of Female Sex Workers

Abstract

Objective: Cationic proteins found in cervicovaginal secretions (CVS) are known to contribute to the early antiviral immune response against HIV-infection in vitro. We here aimed to define additional antiviral factors that are over-expressed in CVS from female sex workers at high risk of infection.

Methods: CVS were collected from Kenyan HIV-seronegative (n = 34) and HIV-seropositive (n = 12) female sex workers, and were compared with those from HIV-seronegative low-risk women (n = 12). The highly exposed seronegative (HESN) sex workers were further divided into those with less (n = 22) or more (n = 12) than three years of documented sex work. Cationic protein-depleted CVS were assessed for HIV-neutralizing activity by a PBMC-based HIV-neutralizing assay, and then characterized by proteomics.

Results: HIV neutralizing activity was detected in all unprocessed CVS, however only CVS from the female sex worker groups maintained its HIV neutralizing activity after cationic protein-depletion. Differentially abundant proteins were identified in the cationic protein-depleted secretions including 26, 42, and 11 in the HESN>3 yr, HESN<3 yr, and HIV-positive groups, respectively. Gene ontology placed these proteins into functional categories including proteolysis, oxidation-reduction, and epidermal development. The proteins identified in this study include proteins previously associated with the HESN phenotype in other cohorts as well as novel proteins not yet associated with anti-HIV activities.

Conclusion: While cationic proteins appear to contribute to the majority of the intrinsic HIV neutralizing activity in the CVS of low-risk women, a broader range of non-cationic proteins were associated with HIV neutralizing activity in HESN and HIV-positive female sex workers. These results indicate that novel protein factors found in CVS of women with high-risk sexual practices may have inherent antiviral activity, or are involved in other aspects of anti-HIV host defense, and warrant further exploration into their mode of action.

Fig 1. HIV neutralizing activity in unprocessed and cationic protein-depleted cervicovaginal secretions.

The HIV neutralizing activity of unprocessed cervicovaginal secretions (CVS) and cationic protein-depleted CVS, respectively, was assessed in pooled samples from the study groups. Green bars: HIV seronegative low-risk women (Low risk) [25]; Yellow bars: HIV seronegative women with less than 3 years of reported active sex work (HESN<3yr); Blue bars: HIV seronegative women with more than 3 years of reported active sex work (HESN>3yr); Red bars: HIV seropositive sex working women (HIV positive). The results shown are from two to three representative experiments using duplicate wells (median values ±SEM).

Fig 2. Biological processes of differentially abundant proteins between female sex worker and low-risk groups.

A: HIV-exposed seronegative female sex workers for a time greater than three years B: HIV-exposed seronegative female sex workers for a time less than three years C: HIV-seropositive female sex workers (DAVID Bioinformatics Resources 6.7, UniProt) (Blue bars represent overabundant proteins and red bars represent under abundant proteins compared to the low-risk, uninfected control group).