Cationic Polypeptides Are Required for Anti-HIV-1 Activity of Human Vaginal Fluid

Department of Molecular Biology and Microbiology, Biomolecular Science Center, University of Central Florida, Orlando, FL 32816-2364, USA.
The Journal of Immunology (Impact Factor: 4.92). 01/2006; 175(11):7560-7. DOI: 10.4049/jimmunol.175.11.7560
Source: PubMed


Mucosal surfaces of the vagina are the portals for heterosexual transmission of HIV-1 and therefore play a fundamental role in the pathogenesis of primary infection. In the search for direct biological evidence for the role of human vaginal fluid in innate host defense, we characterized the anti-HIV-1 function of cationic polypeptides within minimally manipulated vaginal fluid. In the current study we revealed that vaginal fluid confers intrinsic anti-HIV-1 properties against both X4 and R5 strains of HIV-1 and could protect against HIV-1 infection and reduce proviral genome integration in organotypic cultures of human cervicovaginal tissue. The majority of this activity was contained in the cationic polypeptide fraction, and the depletion of cationic polypeptides using a selective cation exchange resin ablated most of the intrinsic activity against HIV-1. By adding the cationic polypeptide fraction to depleted vaginal fluid, we were able to restore activity against HIV-1. Using a proteomic approach, we identified 18 cationic polypeptides within vaginal fluid, nearly all of which are either known antimicrobials or have other purported roles in host defense. Interestingly, physiologic concentrations of 13 of the cationic polypeptides were not active alone against HIV-1, yet in concert they partially restored the anti-HIV-1 activity of cation-depleted vaginal fluid. These results suggest that synergism between cationic polypeptides is complex, and full anti-HIV-1 activity probably involves the aggregate of the cationic peptides and proteins in vaginal fluid.


Available from: Jan Pohl
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    • "Previous characterization of the CVF proteome has relied on sampling methods such as Dacron tipped swabs [15,17,19,20], sponges or gauze [11,16], or direct collection of CVF [29,30] or cervical washings [13,14]. Only in the analysis by Zegels [13], who used cervical washings collected during colposcopy, were routine clinical samples utilized for proteomics. "
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    ABSTRACT: Background The proteomic analysis of body fluids is a growing technology for the identification of protein biomarkers of disease. Given that Papanicolaou tests (Pap tests) are routinely performed on over 30 million women annually in the U.S. to screen for cervical cancer, we examined the residual Pap test fluid as a source of protein for analysis by mass spectrometry (MS). In the liquid-based Pap test, cervical cells are collected from the ectocervix and placed into an alcohol-based fixative prior to staining and pathologic examination. We hypothesized that proteins shed by cells of the female genital tract can be detected in the Pap test fixative by MS-based proteomic techniques. We examined the feasibility of using residual fluid from discarded Pap tests with cytologically “normal” results to optimize sample preparation for MS analysis. The protein composition of the cell-free Pap test fluid was determined by silver staining of sodium dodecyl sulfate -polyacrylamide gels, and the abundance of serum proteins was examined by Western immunoblot using an antibody against human serum albumin. Both pooled and individual samples were trypsin digested and analyzed by two-dimensional MS/MS. Proteins were identified by searching against the Human Uniprot database, and characterized for localization, function and relative abundance. Results The average volume of the residual Pap test fluid was 1.5 ml and the average protein concentration was 0.14 mg/ml. By Western immunoblot we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. By MS/MS, we identified 714 unique proteins in pooled Pap test samples and an average of 431 proteins in individual samples. About 40% of the proteins identified were extracellular or localized to the plasma membrane. Almost 20% of the proteins identified were involved in immunity and defense, characteristic of the healthy cervical-vaginal proteome. By merging the protein sets from the individual and pooled Pap test samples, we created a “Normal Pap test Core Proteome” consisting of 153 proteins. Conclusions Residual Pap test fluid contains a sufficient amount of protein for analysis by MS and represents a valuable biospecimen source for the identification of protein biomarkers for gynecological diseases.
    Clinical Proteomics 07/2014; 11(1):30. DOI:10.1186/1559-0275-11-30
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    • "Information from qualitative comprehensive proteomics studies on CVF showed that this biological fluid contains proteins/ peptides with intrinsic anti-HIV activity such as defensins, lactoferrin , lysozyme, cathelicidin and SLPI (Cole and Cole, 2008; Hirbod and Broliden, 2007; Kazmi et al., 2006; Zegels et al., 2009). In addition, (Venkataraman et al., 2005) demonstrated that the cationic fraction of CVF has inherent anti-HIV activity and hypothesized that this activity is the result of a complex synergism between different proteins in CVF (Levinson et al., 2012). Later, a study of Levinson et al. (2012) confirmed these findings and pointed to HNP1-3 and LL-37 as possible mediators. "
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    ABSTRACT: HIV-exposed seronegative individuals (HESNs) are persons who possess in vivo resistance against HIV. Using iTRAQ, we compared protein abundance profiles from cervicovaginal fluid (CVF) samples obtained from HESNs with samples from low-risk seronegative and from seropositive persons. Serpin A5 and myeloblastin were up- and downregulated, respectively. We hypothesize that a balance between the downregulation of serine proteinases and the upregulation of their inhibitors may contribute to HIV-resistance Figure optionsDownload full-size imageDownload high-quality image (155 K)Download as PowerPoint slide
    Virology 06/2014; s 458–459(1):11–21. DOI:10.1016/j.virol.2014.04.015 · 3.32 Impact Factor
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    • "We traced the mechanism of the anti-HIV activity of HD5 under serum-deprived conditions to defensin-mediated cell death, which is not known to occur in the milieu of the genital mucosa. Since abundant and diverse proteins are present in cervico-vaginal fluid [19], [20], [21] and lymphocytes are viable at the genital mucosa despite the enrichment of antimicrobial peptides including HD5 [1], [11], [22], [23], primary CD4+ T cells cultured under serum deprived conditions are unlikely to represent mucosal CD4+ T cells. "
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    ABSTRACT: We have previously shown that human defensin 5 (HD5) promotes HIV infectivity in both primary CD4+ T cells and HeLa cells expressing CD4 and CCR5. HD5 is induced in response to sexually transmitted infections (STIs) such as Chlamydia trachomatis and Neisseria gonorrhoeae, suggesting it plays a role in STI-mediated enhancement of HIV transmission. In contrast to our findings, a recent study reports that HD5 has an anti-HIV effect in primary CD4+ T cells under serum-deprived conditions. To resolve these apparently contradictory observations, we investigated experimental parameters that might contribute to contrasting effects of HD5. Serum-deprived culture conditions were associated with anti-HIV activity. In contrast to the dependence of the HIV enhancing effect on HD5 structure, the anti-HIV activity in serum-deprived primary CD4+ T cells was independent of HD5 structure as the linear peptide [Abu] HD5 exhibited similar anti-HIV activity. Under serum deprived conditions, HD5 blocked CD4-receptor-independent HIV-1vsv infection before or after viral entry. We found that HD5 and its linear form induced significant cell death in primary CD4+ T cells under serum-deprived culture conditions. HD5-mediated apoptosis was observed as early as 2 h after addition of defensins to serum-deprived primary CD4+ T cells. In contrast to primary CD4+ T cells, HD5 did not induce cytotoxicity and promote HIV infectivity of HeLa-CD4-CCR5 cells under serum-deprived conditions. These results indicate that under serum-deprived culture conditions HD5 is toxic for primary CD4+ T cells, warranting caution in data interpretation.
    PLoS ONE 09/2013; 8(9):e76038. DOI:10.1371/journal.pone.0076038 · 3.23 Impact Factor
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