A High-throughput Bead-based Affinity Assay Enables Analysis of Genital Protein Signatures in Women At Risk of HIV Infection

Abstract

Women at high risk of HIV infection, including sex workers and those with active genital inflammation, have molecular signatures of immune activation and epithelial barrier remodeling in samples of their genital mucosa. These alterations in the local immunological milieu are likely to impact HIV susceptibility. We here analyze host genital protein signatures in HIV uninfected women, with high frequency of condom use, living in HIV-serodiscordant relationships. Cervicovaginal secretions from women living in HIV-serodiscordant relationships (n = 62) were collected at three time points over 12 months. Women living in HIV-negative seroconcordant relationships (controls, n = 25) were sampled at one time point. All study subjects were examined for demographic parameters associated with susceptibility to HIV infection. The cervicovaginal samples were analyzed using a high-throughput bead-based affinity assay. Proteins involved in epithelial barrier function and inflammation were increased in HIV-serodiscordant women. By combining several methods of analysis, a total of five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were consistently associated with this study group. Proteins analyzed using the affinity set-up were further validated by label-free tandem mass spectrometry in a partially overlapping cohort with concordant results. Women living in HIV-serodiscordant relationships thus had elevated levels of proteins involved in epithelial barrier function and inflammation despite low prevalence of sexually transmitted infections and a high frequency of safe sex practices. The identified proteins are important markers to follow during assessment of mucosal HIV susceptibility factors and a high-throughput bead-based affinity set-up could be a suitable method for such evaluation.

Keywords: Affinity proteomics; HIV; HIV-serodiscordant; Immunology*; Inflammation; Tandem Mass Spectrometry; cervix; female reproductive tract; reproductive immunology; vagina.

Graphical abstract

Fig. 1.

Overview of the study setup. A, In this study, 211 cervicovaginal secretion samples collected from 62 serodiscordant and 25 control women were analyzed to explore protein profiles associated to HIV infection susceptibility. The serodiscordant women were sampled at three time points and the control women were sampled once. B, Protein profiling was performed using 332 antibodies targeting 171 proteins in a suspension bead array set-up. C, Relative protein levels were obtained and sample profiles were subjected to statistical analysis. D, Based on univariate analysis, 24 proteins with altered levels in serodiscordant women compared with control women were identified. Seven proteins were designated as key features in a data driven multivariate model. By combining the uni- and multivariate analysis with immunohistological stainings of cervicovaginal tissues, five proteins (CAPG, KLK10, SPRR3, elafin/PI3, CSTB) were identified as consistently associated with the serodiscordant group.

Fig. 2.

Proteins with higher levels in serodiscordant than control women. Based on analysis of protein levels in genital secretions collected at three time points from serodiscordant women and controls, 24 proteins were identified as significantly differing between these groups. A, The result for the four most statistically significant proteins. KLK10: kallikrein-related peptidase 10, CAPG: actin filament gelsolin-like capping protein, TTR: transthyretin, SPRR3: small proline-rich protein 3. p values adjusted for multiple comparison corrections using Benjamini-Hochberg. B, Although all 24 proteins were found at higher levels in the serodiscordant group, clustering of correlation coefficients revealed generally low correlation between the protein levels.

Fig. 3.

Variation in protein levels over time for proteins in serodiscordant women. To assess variation of individual proteins over time, ratios of maximum and minimum protein levels for the three time points were calculated and subjected to hierarchical clustering. This analysis revealed that 10 of the 24 proteins showed relatively stable levels (mean cluster ratio of 0.6) whereas the remaining 14 indicated more changing profiles (mean cluster ratio of 1.2).

Fig. 4.

Proteomic features associated with serodiscordant status. A multivariate model based on least absolute shrinkage and selection operator (LASSO)-selected features and partial least-squares discriminant analysis classification. Left Panel: Scores plots of the 79 individual samples (serodiscordant participants = red, controls = blue) included in this study (excluding seminally-contaminated samples) from time point 1 (month 0, I), time point 2 (month 6, II), and time point 3 (month 12, III). Right Panel: Loadings plots of the LASSO-selected features. Features negatively loaded on LV1 indicate positive association with serodiscordant and negatively associated with control women, whereas positively loaded features on LV1 indicates negative association with serodiscordant and positive association with controls. Among the 17 features (proteins) identified, two were consistently associated with the serodiscordant phenotype over all three time points (SPRR3 and TTR) and three additional features (PI3, CSTB, and KLK10) were associated with the serodiscordant phenotype for two of three visits. Two proteins (KRT1 and SOD1) were negatively associated with the serodiscordant phenotype in two out of three time points.

Fig. 5.

Protein expression in cervix and vagina. Immunohistochemical analysis of cervical and vaginal tissue revealed distinct cytoplasmic expression of CAPG in macrophages present in connective tissue of both cervical and vaginal mucosa, whereas squamous epithelium was negative. CSTB, KLK10, PI3, and SPRR3 displayed most prominent expression in superficial layers of squamous epithelium of both cervix and vagina, with CSTB and SPRR3 being expressed in nucleus and cytoplasm, and KLK10 and PI3 showing cytoplasmic and membranous expression.

Fig. 6.

Proteins selected by bead-based affinity assay validated with mass spectrometry. Spearman's correlation coefficient (rho) calculated using the MFI per antibody (from the bead-based affinity assay) versus the normalized abundance of the corresponding protein (identified by MS). When several antibodies directed toward the same protein, we conducted one separate correlation analysis for each antibody/protein pair. A, Spearman's correlation coefficient (rho) of all antibody/protein pairs (n = 79) with MFI > 2 x median MFI of the bead with IgG from nonimmunized rabbits. Horizontal lines represent median and interquartile range. Proteins associated with the HIV-serodiscordant phenotype marked in color: SPPR3 (light green), CAPG (turquoise), CSTB (pink), KLK10 (red), elafin/PI3 (blue). Correlation plots for SPRR3 (B), KLK10 (C), PI3 (D), and CAPG (E) show normalized abundance (from MS) on x axis and MFI (in arbitrary units) on y axis. Antibody name, Spearman's rho and p value (adjusted for multiple comparison corrections with Benjamini-Hochberg) on right side of each graph. Normalized data was used for the bead-based affinity array and normalized abundance for the MS method. MFI: median fluorescence intensity; MS: mass spectrometry; SPPR3: small proline-rich protein 3; KLK10: anti-kallikrein-related peptidase 10; PI3: elafin; HPA: Human Protein Atlas; CAPG: actin filament gelsolin-like capping protein; AU: arbitrary units.