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The Journal of Immunology, 2005, 175: 7560-7567.
Copyright © 2005 by The American Association of Immunologists

Cationic Polypeptides Are Required for Anti-HIV-1 Activity of Human Vaginal Fluid1

Nitya Venkataraman2,*, Amy L. Cole2,*, Pavel Svoboda{dagger}, Jan Pohl{dagger} and Alexander M. Cole3,*

* Department of Molecular Biology and Microbiology, Biomolecular Science Center, University of Central Florida, Orlando, FL 32816; and {dagger} Microchemical and Proteomics Facility, Winship Cancer Institute, Emory University School of Medicine, Atlanta, GA 30322


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Approximately 40 million people have been infected with HIV-1 worldwide according to the 2004 World Health Organization estimates (1). There has been a dramatic increase in the global spread of HIV-1, especially via the heterosexual mode of transmission (2, 3). At present, nearly 60% of infected individuals are women (4, 5). The natural sexual transmission of HIV occurs through mucosal surfaces, such as vaginal or rectal mucosa (6). Vaginal and rectal subepithelial stromal tissues are densely populated with dendritic cells, macrophages, and T cells that express both CD4 and the HIV-1 coreceptors, CXCR4 and CCR5 (7, 8). The mechanisms by which HIV-1 journeys across the mucosal epithelia are not completely understood, but may directly involve the epithelial cells (9). Once the virus reaches the lamina propria, it can either directly infect macrophages or T lymphocytes or adhere to (or infect) dendritic cells, whose traffic to the regional lymph nodes converts them into sites of vigorous viral replication (10, 11). Although considerable attention in immunopathogenetic research on HIV-1 has been focused on acquired immunity, only recently has the role of innate immunity surfaced.

A layer of mucosal fluid covers the vaginal epithelium and is composed of secretions from the cervical vestibular glands, plasma transudate, and endometrial and oviductal fluids (12, 13). The fluid covering the vaginal mucosa protects against entry of pathogens into deeper tissues, including periodic sloughing of mucus and underlying cells to remove adherent microbes. The vagina is a host for numerous commensal microorganisms, which release organic acids and antimicrobial peptides to kill pathogenic invaders (13, 14). The vaginal epithelial cells, cervical glands, and neutrophils contribute antimicrobial peptides to the milieu of the vaginal fluid, including lysozyme, lactoferrin, secretory leukocyte protease inhibitor (SLPI),4 human neutrophil peptides (HNP-1, -2, and -3), and human {beta}-defensins (HBDs) (15). We hypothesized that the sum total of these and probably other antimicrobial peptides and proteins contribute to the innate host defense of the vagina.

To date, evidence for the role of antimicrobial polypeptides in vaginal anti-HIV-1 host defense has been largely circumstantial. Lysozyme and lactoferrin have been shown to inhibit the infection by HIV-1 in vitro by preventing the adsorption and penetration of the virus (16, 17, 18, 19). Human {beta}-defensins have been shown to inhibit HIV-1 replication (20) through modulation of the CXCR4 coreceptor as well by interacting directly with the virions. Several reports have shown that the level of SLPI is reduced in vaginal fluid of HIV-infected persons (21, 22). SLPI has been shown to block HIV-1 infection in monocytes and T cells by preventing internalization of the virus before RT (22, 23). However, the action of SLPI is still debatable, because other reports suggest that SLPI by itself has no effect on HIV-1 replication (24). HNP-1 to -3 inhibit HIV-1 replication in vitro by two mechanisms: in the absence of serum, they inhibit HIV-1 replication before integration of the virus in CD4+ T cells, and in the presence of serum, they interfere with the signaling pathways on target cells and block the nuclear import and transcription of the HIV-1 genome (25, 26, 27).

In the current report we explore the biological role of cationic antimicrobial polypeptides in protecting the vaginal mucosa from infection by HIV-1. We reveal that the cationic proteins in human vaginal fluid inhibit the entry of HIV-1 in human epithelial cell lines and organotypic cervicovaginal tissues. We used a proteomic approach to identify 18 different cationic polypeptides in vaginal fluid, most of which have been previously reported to exhibit antimicrobial properties. Although individual polypeptides at physiological concentration did not exhibit antiviral activity against HIV-1 infection, a combination of the peptides partially restored the antiviral activity. Selective depletion of cationic polypeptides from whole vaginal fluid reduced the intrinsic anti-HIV-1 activity. Most importantly, the anti-HIV-1 activity of depleted fluid was restored upon repletion with the cationic polypeptide extract. Collectively, these studies suggest that the intrinsic anti-HIV-1 activity of vaginal fluid is an aggregate effect of all its active cationic polypeptide components.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Human neutrophil lysozyme and human milk lactoferrin were purchased from Sigma-Aldrich. Recombinant calgranulin A (S100A8) and calgranulin B (S100A9) were purchased from Abnova. Recombinant cystatin B and SLPI were purchased from R&D Systems. Histone H2A was purchased from Upstate USA. Cathepsin G was purchased from Bachem Bioscience. Recombinant {beta}-defensins, HBD-1 and -2, were gifts from Dr. T. Ganz (David Geffen School of Medicine, University of California, Los Angeles, CA). The {alpha}-defensins, HNP-1, -2, and -3, were purified from human leukocytes and were gifts from Drs. Ganz and R. I. Lehrer (David Geffen School of Medicine).

Collection and processing of vaginal fluid

Vaginal fluid was collected from postmenarcheal, but premenopausal, healthy female donors after obtaining informed consent according to the guidelines of the institutional review board of University of Central Florida. Donors with current or recent vaginal infections and those under antibiotic treatment for any reason were excluded from the study using a questionnaire. To collect undiluted vaginal fluid, an Instead SoftCup (Ultrafem) was inserted into the vagina according to the manufacturer’s instructions and was removed after 30 min. The SoftCup was then centrifuged for 10 min at 1000 x g in a 50-ml sterile conical tube to collect the fluid sample (28). Retrieved samples were homogenized by sonication on ice using a microtip ultrasound probe (10 2- to 3-s pulses). These minimally manipulated, whole vaginal fluid samples were stored in aliquots at –20°C. This method enabled us to collect ~200 µl to 1 ml of vaginal fluid/collection. For most antiviral cell culture assays, the vaginal fluid was not manipulated further. For antiviral cell culture assays, the vaginal fluids were extracted with 5% acetic acid for 2 h with gentle agitation, and the clarified supernatant was vacuum dried and resuspended to the original volume in 5 mM sodium phosphate (pH 7.4). To prepare the samples for two-dimensional proteomic analyses, the undiluted vaginal fluids were extracted using 5% acetic acid, vacuum dried, and resuspended in 0.1% hexadecyl trimethyl ammonium bromide/10% acetic acid/3x acid urea loading dye (9 M urea, 5% acetic acid, and methyl green).

Selective depletion of cationic polypeptides from vaginal fluid

Carboxymethyl weak cation exchange resin (CM resin; Bio-Rad) was used to deplete cationic polypeptides from vaginal fluid (29). The CM resin was pre-equilibrated with vaginal fluid buffer (VFB; 60 mM NaCl and 20 mM KH2PO4 (pH 6)), which has been reported to be similar in electrolyte composition to vaginal fluid (15). The CM resin was washed with VFB and centrifuged at 10,000 x g for 10 min, and the overlying VFB was removed. Equal volumes of vaginal fluid from 10–23 donors were pooled, added to an equal bed volume of CM resin pre-equilibrated with VFB, and incubated overnight at 4°C with gentle agitation. The CM resin was sedimented by centrifugation (16,000 x g, 5 min) and the cationic polypeptide-depleted supernatant was collected (hereafter termed CM-depleted vaginal fluid). The cationic polypeptides bound to the CM resin were extracted in subsequent 2- and 24-h extractions using 5 resin volumes of 5% acetic acid at 4°C. The extracts were pooled, vacuum dried, and resuspended to the original volume of vaginal fluid.

Cell lines and viruses

PM1, TZM-bl, and H9 cells were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. TZM-bl cells are a HeLa-derived cell line that stably expresses CD4 and CCR5 and contains the luciferase gene under control of the HIV-1 promoter (30). TZM-bl cells were grown in high glucose DMEM (Mediatech) supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS. Passages 5–15 were used for experiments, and no change in cell behavior was observed between passages. PM1 cells were maintained at a density of 0.4–0.8 x 106/ml in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 100 mM HEPES, and 20% FBS (Gemini Bio-Products). H9 cells were cultured in the same manner as PM1 cells, except that 10% FBS was used. The HIV-1 laboratory strains BaL (R5) and IIIB (X4) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. HIV-1 BaL was propagated in PM1 cells over 16 days. Supernatants containing virus were collected every other day starting 5 days after infection, passed through a 0.45-µm pore size filter, and stored in aliquots at –80°C. HIV-1 IIIB was propagated similarly using H9 cells. Virus was quantitated by a sensitive commercial ELISA for p24gag (PerkinElmer).

Assays to determine anti-HIV-1 activity of vaginal fluid and cationic polypeptides

TZM-bl cells were seeded in 96-well dishes (4000 cells/well). After 24 h, cells were treated in triplicate with 50 µl of culture medium containing whole vaginal fluid, CM-depleted vaginal fluid, peptides recovered from CM resin, vehicle control (5 mM sodium phosphate (pH 7.4)), individual recombinant or purified peptides at physiological concentration (Table II), or combinations thereof. Culture medium or virus diluted in culture medium (2 ng/ml p24 for BaL and 5 ng/ml p24 for IIIB) in 50 µl was immediately added to each well and allowed to incubate at 37°C in 5% CO2 for 24 h. Subsequently, luciferase activity was measured with Bright-Glo reagents (Promega) according to the manufacturer’s instructions using an LMax luminometer (Molecular Devices). Cytotoxicity and the metabolic activity of the cells were verified by a tetrazolium-based (MTT) assay according to the manufacturer’s instructions (R&D Systems).


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  		Table II. Physiological concentration of cationic proteins that contribute to anti-HIV-1 activity of vaginal fluid

 
Two-dimensional gel electrophoresis of vaginal fluid

Acid-extracted vaginal fluid samples were electrophoresed on a 12.5% native acid urea-polyacrylamide gel (AU-PAGE) in the first dimension at 75 V for 16–18 h (29, 31). The gel was then stained with 0.1x Amido Black (0.04% napthol blue-black, 2.5% isopropanol, and 1% acetic acid) to visualize the protein bands. The entire lane of the first dimension AU gel was excised, washed twice for 5 min each time in dH2O, followed by two 5-min washes with 50 mM Tris (pH 8.8), and soaked for 10 min in equilibration buffer (50 mM Tris, 6 M urea, 2% SDS, 20% glycerol, and bromophenol blue ad libitum (pH 8.8)) containing 10 mg/ml DTT. The gel strips were electrophoresed in a 16% Tricine-SDS-PAGE as the second dimension for 20 h at 40 mA (32). Protein spots were visualized by SYPRO Ruby gel stain (Bio-Rad), excised, and stored at 4°C in 1% acetic acid until analyzed by mass spectrometry.

Identification of cationic polypeptides of vaginal fluid

The proteins were then subjected to trypsin digestion and mass spectrometric analysis (MALDI-TOF-MS/MS analysis) (33) at the microchemical and proteomics facility at Emory University as described previously (34, 35). GPS Explorer 2.0 software (Applied Biosystems) and a MASCOT (<www.matrixscience.com/>) search engine were used for identification of peptide fragments. The National Center for Biotechnology Information nonredundant database and the Mammalia taxonomy were used for the searches.

Human cervicovaginal tissue model

Organotypic EpiVaginal cultures of normal human vaginal-ectocervical epithelial cells and immunocompetent dendritic cells were propagated as suggested by MatTek. Each 60 mm2 of tissue adhered tightly atop a microporous membrane insert and was maintained at the air-liquid interface using 5 ml of maintenance medium (MatTek). Tissues (three per treatment condition) were pretreated in with 50 µl of PBS or 50 µl of vaginal fluid diluted 1/1 with PBS for 30 min and then rinsed twice with warm PBS. Tissues were topically applied with 100 µl of PBS (control), PBS containing 25 ng p24 of HIV-1 BaL, or PBS containing BaL and vaginal fluid (equivalent to 50% of whole fluid) for 24 h. Treatments were then removed, and tissues were washed with 100 µl of warm PBS, then vaginal fluid (50%) or PBS vehicle was reapplied in 50 µl. A one-time dose of 1 x 106 HIV-1 BaL-infected PM1 promyelocytic cells was included underneath the microporous insert to sustain the initial HIV-1 infection, and these were removed after 2 days. Basal maintenance medium was changed every other day, and the topical (apical) treatments were removed and reapplied on days 3 and 6 after infection. On day 9 after infection, DNA was extracted from two tissues per treatment condition using Qiagen’s DNA Micro Kit. Total protein was extracted from the third tissue per treatment condition and assayed by ELISA for HIV-1 p24gag (PerkinElmer).

Detection of HIV-1 provirus in human cervicovaginal tissue

HIV-1 infection of cervicovaginal tissues was assessed by real-time PCR quantitation of the HIV-1 BaL env gene (relative to {beta}-actin controls) in total tissue DNA isolated 9 days after infection. The HIV-1 BaL primers used were 5'-AACACCTCAGTCATTACAC-3' and 5'-TACATTGCTCTTCCTACTTC-3', which amplify a 700-bp region of BaL gp120. The {beta}-actin primers used were 5'-CCTTCCAGCAGATGTG-3' and 5'-GGTGTAACGCAACTAAG-3', which amplify a 105-bp region of human {beta}-actin. Two hundred nanograms of DNA was mixed with 2x SYBR Green Supermix (Bio-Rad), 200 nM of each primer, and dH2O. Triplicate 20-µl reactions were conducted using the MyiQ real-time PCR detection system (Bio-Rad), and HIV-1 BaL levels were normalized to {beta}-actin. Data were analyzed with iCycler iQ Optical System software. Melt-curve analysis and gel electrophoresis revealed that single PCR products were amplified for each gene. Moreover, the env PCR product was verified by subcloning into pCR4-TOPO (Invitrogen Life Technologies), followed by DNA sequence analysis (Biomolecular Sciences Genomics Core Laboratory, University of Central Florida).

Statistics

Luciferase assays were performed in triplicate for each treatment condition in each experiment, with relative light units in vehicle-only control wells set at 100% infection. Each treatment condition was analyzed by one-way ANOVA, followed by Tukey’s pairwise comparison. Mass spectrometric analysis for each polypeptide identified was performed in duplicate, and protein spots with a confidence index >85% combined with ion scores of ≥40 for one or more peptides matched to each protein were considered positively identified (33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Human vaginal fluid is intrinsically active against HIV-1

The mucosal layer lining the vaginal epithelial cells is rich in antimicrobial polypeptides that provide a crucial barrier against invading microbial and viral pathogens (15). Although some of these polypeptides have been shown to exhibit antiviral properties (19, 23, 25, 36, 37, 38), detailed analysis of the intrinsic anti-HIV-1 activity of vaginal fluid has not been reported. In this study we explored the activity of the cationic polypeptide components of vaginal fluid against HIV-1. TZM-bl cells were treated with either PBS (vehicle control) or vaginal fluid diluted in DMEM/high glucose medium with 10% FBS and infected with both R5 (HIV-1 BaL; Fig. 1A) and X4 (HIV-1 IIIB; Fig. 1B) strains of HIV-1. After 24 h, excess virus was removed, and infection was quantitated as a measure of luciferase expression. Compared with vehicle-only controls, vaginal fluid extracts significantly reduced the infection of both viral strains in a dose-dependant manner. As measured by a standard MTT tetrazolium assay, the vaginal fluid extracts were not cytotoxic (data not shown). These results indicate that human vaginal fluid intrinsically inhibits the entry of HIV-1 into host cells.



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  		FIGURE 1. Human vaginal fluid inhibits HIV-1 infection. TZM-bl cells were treated with PBS (vehicle control) or vaginal fluid diluted in DMEM/high glucose medium to the final concentration indicated in the figure and were subsequently infected with HIV-1 BaL (A; 2 ng/ml p24) or HIV-1 IIIB (B; 5 ng/ml p24) for 24 h. Infection was measured as the percent reduction in luciferase activity compared with infected vehicle-only control (RLU, relative light units). Experiments were performed using four different pools of vaginal fluid, each in triplicate. **, p < 0.0002; *, p < 0.0005 (compared with controls). Error bars represent the SEM.

 
Anti-HIV-1 activity resides in the cationic fraction of vaginal fluid

Experiments were designed to selectively remove the cationic polypeptides from whole vaginal fluid to determine whether this depletion reduced the anti-HIV-1 activity of the fluid. Whole, undiluted vaginal fluid was collected from healthy donors using an Instead SoftCup. A weak cation exchange resin, CM-Prep (Bio-Rad), was used to deplete the cationic peptides and proteins from vaginal fluid while sparing the concentrations of remaining proteins and electrolytes. We pioneered the CM-resin-mediated depletion technique and have characterized the selective depletion of cationic polypeptides from nasal fluid (29). The activities of whole vaginal fluid extract, CM-depleted vaginal fluid, and the polypeptides extracted from the CM resin were tested individually against HIV-1 BaL (Fig. 2A) and HIV-1 IIIB (Fig. 2B) in TZM-bl cells for 24 h. Cells treated with whole vaginal fluid showed a significant reduction in infection compared with the PBS-treated control (p < 0.0002; n = 13), whereas the CM-depleted fluid did not inhibit infection. Similar to whole vaginal fluid, polypeptides extracted from the CM resin exhibited significant anti-HIV-1 activity compared with both the PBS control and CM-depleted vaginal fluid (p < 0.0002; n = 14). Taken together, these data indicate that the anti-HIV-1 activity of vaginal fluid is contained in the cationic fraction. Whole vaginal fluid, CM-depleted vaginal fluid, and the extracted cationic polypeptides were used in subsequent proteomic and reconstitution assays.



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  		FIGURE 2. Depletion of cationic proteins from vaginal fluid increases HIV-1 infection. TZM-bl cells were treated with vehicle control, whole vaginal fluid extract (Whole-VF), CM-depleted vaginal fluid (CM-depl-VF), or the recovered peptides from the CM-resin (CM-extract) and infected for 24 h with HIV-1 (A) BaL p24 (2 ng/ml; B), or IIIB p24 (B; 5 ng/ml). Luciferase levels were measured, and the percent infection was calculated as described in Fig. 1. The results are from experiments performed with four different pools of vaginal fluid, each repeated three times. *, p < 0.0002.

 
Identification of cationic polypeptides of vaginal fluid

We next used a novel proteomic technique to identify cationic polypeptide components in vaginal fluid. The cationic polypeptide fraction from whole, undiluted vaginal fluid was subjected to AU-PAGE (the first dimension of a two-dimensional gel), which separates polypeptides based on cationic charge density (31, 39). A slice from the AU-PAGE was inserted into a Tricine-SDS-PAGE gel (the second dimension) to separate low m.w. polypeptides by size (32). Gels were stained with SYPRO Ruby (Fig. 3) or silver (not shown). Spots representing single polypeptides were excised from the gel, and the sequence was identified by tandem mass spectrometry (MALDI-TOF/TOF). Each polypeptide spot was sequenced from samples excised from both silver-stained and SYPRO Ruby-stained gels. Table I lists the corresponding protein spots labeled in Fig. 3 and indicates how each spot was identified in this study as well as the previously reported biological activity for each identified polypeptide. Spots with a confidence index >85% were considered positive. Note that several smaller fragments of albumin were also detected, which are indicated by unlabeled arrows. We also discovered two unnamed polypeptide fragments (Fig. 3 and Table I, spots 5 and 19) in vaginal fluid. In total, we have positively identified 18 unique cationic polypeptides in vaginal fluid, many of which have reported roles in host defense against HIV-1 infection (16, 20, 23, 25, 38, 40).



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  		FIGURE 3. Identification of cationic polypeptides in vaginal fluid. Whole vaginal fluid (10 µl) was subjected to one-dimensional electrophoresis in an AU-PAGE, followed by Tricine-SDS-PAGE as the second dimension. The SYPRO Ruby-stained two-dimensional gel of whole vaginal fluid extract shows the protein spots identified by MALDI-TOF mass spectrometric analysis. Each polypeptide spot and their reported roles in host defense are listed in Table I. Unlabeled arrows are fragments of human albumin.

 

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  		Table I. Cationic polypeptides identified in vaginal fluid by two-dimensional analysis and MALDI-TOF MS/MS analysis

 
Comparison of proteomic profiles of whole and CM-depleted vaginal fluids reveals cationic polypeptides that contribute to anti-HIV-1 activity

Two-dimensional gel electrophoresis was next used to characterize the cationic polypeptides that remained in the vaginal fluid after CM depletion as well as those that were extracted with the CM resin. Fig. 4 compares two-dimensional gel electrophoretograms of whole vaginal fluid, CM-depleted vaginal fluid, and the polypeptides extracted from the resin. Spots without arrows were not identified. Among the polypeptides that were absent in CM-depleted fluid yet recovered from the resin include lysozyme, cystatin B, calgranulin B, histone H2A, HNP1–3, lipocalin-2, and cathepsin G (indicated by arrows in Fig. 4, A and C). Some components are reportedly active against HIV-1 (lysozyme and HNP1–3), whereas the anti-HIV-1 activities of the others have not been reported. We next explored which of the cationic polypeptide components of vaginal fluid were the principal effectors active against HIV-1.



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  		FIGURE 4. Comparison of proteomic profiles of whole vaginal fluid and depleted vaginal fluid. Two-dimensional gel electrophoresis of 10 µl of whole vaginal fluid (A), CM-depleted vaginal fluid (B), and the extract of bound proteins from CM-resin (C) was performed. The arrows in A and C indicate protein spots that were recovered in the polypeptide extract and are listed in Table II. The circles in B indicate the regions where the corresponding cationic proteins in A are absent in depleted vaginal fluid.

 
Individual cationic peptides and proteins at physiologic concentrations are not active against HIV-1

We tested the anti-HIV-1 activity of 13 cationic polypeptides that were either purified from natural sources or generated recombinantly. Table II lists the physiologic concentrations of cationic polypeptides in vaginal fluid from healthy donors as identified in this study and in that by Valore et al. (15). Very little is known about the anti-HIV-1 activity of most of these polypeptides. Each polypeptide was tested for anti-HIV-1 activity at its measured physiological concentration. TZM-bl cells were treated with individual polypeptides at the final concentrations given in Table II, then infected with HIV-1 BaL or HIV-1 IIIB; at 24 h, anti-HIV-1 activity was measured by quantifying luciferase expression. At physiological concentrations, none of the polypeptides alone inhibited viral entry, as shown by the absence of reduction in luciferase expression compared with the control (data not shown). These data suggest that the ability of vaginal fluid to prevent HIV-1 entry may be due to two or more cationic antimicrobial polypeptides acting in synergy.

Cationic polypeptides of vaginal fluid synergize to inhibit HIV-1 infection

The abundance of antimicrobial peptides in vaginal fluid with often overlapping roles in host defense suggests that the anti-HIV-1 activity is not a result of actions of individual peptides. Moreover, our studies indicate that the individual polypeptides at physiological concentrations do not prevent entry of HIV-1 into host cells. We therefore hypothesized that these polypeptides must act in concert to prevent HIV-1 infection. To test our hypothesis, we prepared a mixture of 13 available recombinant or natural peptides at physiological concentrations, as shown in Table II. TZM-bl cells were treated with the polypeptide mix, either alone or added to CM-depleted vaginal fluid, and were subsequently infected with HIV-1 (Fig. 5). Although the polypeptide mix alone reduced infectivity ~40%, this was not significant compared with the vehicle-only control. Moreover, the addition of the polypeptide mix to CM-depleted fluid was not completely restorative. Due to availability, not every polypeptide identified was represented in the mixture, which may have contributed to the incomplete restoration of CM-depleted fluid. This hypothesis was supported in our next experiment.



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  		FIGURE 5. Cationic polypeptides of vaginal fluid synergize to inhibit HIV-1 infection. TZM-bl cells were treated as indicated in the figure and infected with BaL (p24, 2 ng/ml) for 24 h or with IIIB (not shown). Luciferase was then measured as described in the text, and the percent infection was calculated. *, p < 0.00015. Experiments were performed in triplicate, and error bars represent the SEM.

 
Interestingly, the cationic polypeptide extract (cleaved from the CM resin) was highly restorative to CM-depleted fluid (p = 0.00012; n = 14), and the combined anti-HIV-1 activity was equivalent to the activity of whole vaginal fluid (Fig. 5). These data suggest that the anti-HIV-1 activity of vaginal fluid is primarily contained in the cationic fraction, and that the activity is complex and requires the collective polypeptides.

Vaginal fluid protects against HIV-1 infection of human cervicovaginal tissue

We next tested whether vaginal fluid could protect organotypic human cervicovaginal tissues against HIV-1 infection. This ex vivo model closely resembles the native mucosae of the ectocervix and vagina, containing a full-thickness epithelia composed of vaginal-ectocervical cells that are interspersed with immunocompetent dendritic (Langerhans) cells in the basal and suprabasal layers. To study the role of vaginal fluid in reducing HIV-1 infection and integration of the proviral DNA into the host genome, cervicovaginal tissues infected with HIV-1 BaL in the absence or the presence of an apical film of vaginal fluid were compared. The tissues were treated with PBS (vehicle control) or vaginal fluid diluted 1/1 in PBS for 30 min before infection with HIV-1 BaL (p24, 25 ng/tissue) diluted in PBS (control) or in 50% vaginal fluid. Twenty-four hours after infection, excess virus was removed, and PBS control or 50% vaginal fluid was reapplied to the apical tissue surface. Total tissue DNA was extracted 9 days after infection, and the proviral DNA levels were assessed by real-time quantitative PCR of the env gene of HIV-1 BaL. Compared with tissues topically infected with HIV-1 BaL alone, cervicovaginal tissues that were treated with vaginal fluid for 30 min before the addition of HIV-1 BaL had ~4-fold fewer copies of proviral DNA, although this trend was not statistically significant due to variability in the untreated condition (Fig. 6A). However, viral titer, as quantified by p24gag ELISA was significantly lower in cervicovaginal tissues treated with vaginal fluid compared with control tissues (p = 0.0091; n = 2; Fig. 6B). These studies imply that vaginal fluid plays an important role in preventing HIV-1 transmission in cervicovaginal mucosa.



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  		FIGURE 6. Vaginal fluid inhibits HIV-1 infection of human vaginal tissues. Human vaginal organotypic cultures were treated with PBS control or 50% vaginal fluid and infected with HIV-1 BaL or IIIB (not shown). Nine days after infection, tissues were harvested for DNA or protein analysis. A, Real-time quantitative PCR of HIV-1 BaL proviral DNA corresponding to a 700-bp region of the env gene was performed in BaL- vs BaL- plus vaginal fluid (VF)-infected tissues (n = 4). B, HIV-1 p24 protein levels in BaL- vs BaL- plus V- infected tissues. *, p = 0.0091. Error bars represent the SEM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The mechanisms by which vaginal mucosa protects against sexually transmitted and other infections are not completely understood. Although several studies have focused on the adaptive immune system of mucosal surfaces of the female reproductive tract, scant attention has been focused on the innate immune factors in vaginal secretions (41, 42, 43, 44). Evidence is accumulating that vaginal epithelia are more than simple physical barriers to protect against invading pathogens (15, 45, 46). On the contrary, this surface and its overlying fluid are replete with antimicrobial polypeptides that act as effectors of innate host defense.

The current study provides evidence that cationic polypeptides contribute significantly to the intrinsic biological activity of vaginal fluid against HIV-1 infection. Proteomic analysis of the cationic polypeptide fraction of vaginal fluid revealed numerous cationic antimicrobial and host defense polypeptides. Polypeptides with known microbicidal effects that have been identified in our study and previously identified in mucosal secretions include lysozyme, lactoferrin, cathelicidin (47, 48), {beta}-defensins, {alpha}-defensins, and SLPI (15, 45). Although each of these polypeptides reportedly prevented HIV-1 infection, their activities were realized only when assayed at supraphysiologic concentrations, and they were not active against HIV-1 when tested individually at physiological concentrations. However, a mixture of the peptides added back to CM-depleted vaginal fluid partially restored the activity. Partial (rather than complete) restoration of activity may be reflective of the following. 1) Some of the recombinant proteins may not exhibit the same anti-HIV-1 activity as that of the purified or natural proteins in the secretions. 2) Although we created the mixture with individual polypeptides, the full activity of certain proteins (e.g., calgranulins A and B) may be best realized in their heterodimeric form. 3) Due to availability, several polypeptides that we identified could not be included in the polypeptide mixture. Any or all of these conditions support the premise that the collective cationic polypeptide fraction is responsible for anti-HIV-1 activity of vaginal fluid. Indeed, when the extracted cationic polypeptide fraction (bound to the CM-resin) was used to reconstitute the CM-depleted fluid, anti-HIV-1 activity was restored completely.

Whole vaginal fluid was collected from healthy donors using a diaphragm-like device (Instead SoftCup), which enabled the collection of whole undiluted cervicovaginal fluid (28). In contrast, other commonly used methods of cervicovaginal fluid collection, such as extraction from preweighed tampons or vaginal lavage (15, 49, 50), can suffer from protein adhesion to the tampon or a dilute lavage of unknown protein concentration. Unlike lavage, the Instead SoftCup is convenient and can be self-inserted; thus, women are more receptive to donating cervicovaginal fluid. Although no one method of collection is perfect, approaches that enable the retrieval of whole, undiluted fluid may afford the best representation of the condition in vivo.

Lactic and other organic acids that result in the low pH of human vaginal fluid (normally pH 3.8–4.5) as well as volatile compounds, such as H2O2, are thought to contribute to microbial host defense (13, 14). Our studies were designed to minimize or eliminate the effects of these factors, because the acidity of the vaginal fluid was neutralized (pH 7.4) before subjecting the fluid to anti-HIV-1 assays. Moreover, although all the anti-HIV-1 activity was contained in the collective polypeptide extract, the procedures required for extraction would inactivate or remove H2O2 and other volatile compounds. Both whole vaginal fluid and the collective cationic polypeptide fraction were equally active against HIV-1; thus, the activity against HIV-1 was not a result of ancillary components of the cervicovaginal fluid.

Histones and their fragments were some of the more abundant polypeptides identified in vaginal fluid. Valore et al. (15) identified histone H2B in the vaginal fluid of a healthy donor using a specific, yet quite insensitive, Ab. Using a more sensitive proteomic approach, we identified histone fragments in every vaginal fluid sample tested. Histones have been shown to possess antibacterial properties and are released from activated neutrophils (51, 52). Why are histones present in mucosal secretions? Do these proteins confer an active function, or are they merely byproducts of cellular decay? Until recently, the latter was the most plausible explanation. However, a current study by Brinkmann et al. (52) provided an alternative mechanism behind the presence of extracellular histones. They reported that activated neutrophils release neutrophil extracellular traps (NETs), long elaborations of chromatin and neutrophil elastase that are independent of apoptosis or necrosis. NETs bound and inactivated both Gram-positive and Gram-negative bacteria and prevented their dispersal. Moreover, NETs were abundant in experimental dysentery and in spontaneous human appendicitis. It is not known whether inflammatory cells in the cervicovaginal mucosa elaborate NETs and their associated histones as a host defense mechanism, or if histones are released simply as a result of cellular damage.

Other studies of vaginal fluid that specifically searched for the presence of {beta}-defensins using sensitive Abs were able to immunodetect low levels in vaginal fluid (53). However, we did not uncover these peptides in our proteomic search, probably due to the scant concentration of these peptides and thus the low chance that they would be identified as a major spot on two-dimensional PAGE. Although {beta}-defensins have been shown to inhibit HIV-1 infection (50% inhibition at 20 µg/ml concentration) in human oral epithelial cells (20), the concentrations in vivo are 35- to 500-fold lower (Table II), suggesting that they may not play a major role in antiretroviral host defense. Moreover, our studies revealed that HBD-1 and HBD-2 alone failed to inhibit HIV-1 infection at physiological concentration.

Surprisingly, vaginal fluid contains components that are permissive to the transmission of HIV-1. For example, samples of cervicovaginal lavage fluid that contained higher concentrations of calgranulin A have been shown to exhibit greater activation of HIV-1 in latently infected monocytic cells (54). The human neutrophil-derived serine protease cathepsin G has also been shown to increase the susceptibility of macrophages to HIV-1 infection in vitro (55). Although the mechanism is not known, insights into the roles of mucosal polypeptides that increase the probability of transmission and infection of HIV-1 could be critical in the development of effective antiretroviral treatments and preventatives. Taken together, the above studies reveal that human vaginal fluid plays a crucial role in innate host defense against HIV-1 transmission.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Grants AI052017 and AI065430 (to A.M.C.) from the National Institutes of Health. Emory Microchemical and Proteomics Facility is supported by National Institutes of Health-National Center for Research Resources Grants 02878, 12878, 13948, and 016692. Back

2 N.V. and A.L.C. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Alexander M. Cole, Department of Molecular Biology and Microbiology and the Biomolecular Science Center, 4000 Central Florida Boulevard, Building 20, Room 136, University of Central Florida, Orlando, FL 32816-2364. E-mail address: acole{at}mail.ucf.edu Back

4 Abbreviations used in this paper: SLPI, secretory leukocyte protease inhibitor; AU, acid urea; CM, carboxymethyl; HBD, human {beta}-defensin; HNP, human neutrophil peptide; NET, neutrophil extracellular trap; VFB, vaginal fluid buffer. Back

Received for publication July 20, 2005. Accepted for publication September 23, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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