Cationic Polypeptides Are Required for Anti-HIV-1 Activity of
Human Vaginal Fluid1
Nitya Venkataraman,2* Amy L. Cole,2* Pavel Svoboda,†Jan Pohl,†and Alexander M. Cole3*
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. The Journal of Immunology, 2005, 175: 7560–7567.
increase in the global spread of HIV-1, especially via the hetero-
sexual mode of transmission (2, 3). At present, nearly 60% of
infected individuals are women (4, 5). The natural sexual trans-
mission of HIV occurs through mucosal surfaces, such as vaginal
or rectal mucosa (6). Vaginal and rectal subepithelial stromal tis-
sues 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 immuno-
pathogenetic research on HIV-1 has been focused on acquired im-
munity, 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
pproximately 40 million people have been infected with
HIV-1 worldwide according to the 2004 World Health
Organization estimates (1). There has been a dramatic
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 in-
hibitor (SLPI),4human neutrophil peptides (HNP-1, -2, and -3),
and human ?-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 (16–19). Human ?-defensins have been shown to inhibit
HIV-1 replication (20) through modulation of the CXCR4 core-
ceptor as well by interacting directly with the virions. Several re-
ports 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 internal-
ization 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 vi-
rus 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 (25–27).
In the current report we explore the biological role of cationic
antimicrobial polypeptides in protecting the vaginal mucosa from
*Department of Molecular Biology and Microbiology, Biomolecular Science Center,
University of Central Florida, Orlando, FL 32816; and†Microchemical and Proteom-
ics Facility, Winship Cancer Institute, Emory University School of Medicine, Atlanta,
Received for publication July 20, 2005. Accepted for publication September 23, 2005.
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.
1This work was supported by Grants AI052017 and AI065430 (to A.M.C.) from the
National Institutes of Health. Emory Microchemical and Proteomics Facility is sup-
ported by National Institutes of Health-National Center for Research Resources
Grants 02878, 12878, 13948, and 016692.
2N.V. and A.L.C. contributed equally to this work.
3Address 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: firstname.lastname@example.org
4Abbreviations used in this paper: SLPI, secretory leukocyte protease inhibitor; AU,
acid urea; CM, carboxymethyl; HBD, human ?-defensin; HNP, human neutrophil
peptide; NET, neutrophil extracellular trap; VFB, vaginal fluid buffer.
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc.0022-1767/05/$02.00
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 an-
timicrobial properties. Although individual polypeptides at phys-
iological 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. Col-
lectively, these studies suggest that the intrinsic anti-HIV-1 activ-
ity of vaginal fluid is an aggregate effect of all its active cationic
Materials and Methods
Human neutrophil lysozyme and human milk lactoferrin were purchased
from Sigma-Aldrich. Recombinant calgranulin A (S100A8) and calgranu-
lin 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 ?-defensins, HBD-1 and -2, were gifts from Dr. T. Ganz
(David Geffen School of Medicine, University of California, Los Angeles,
CA). The ?-defensins, HNP-1, -2, and -3, were purified from human leu-
kocytes 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 Flor-
ida. Donors with current or recent vaginal infections and those under an-
tibiotic treatment for any reason were excluded from the study using a
questionnaire. To collect undiluted vaginal fluid, an Instead SoftCup (Ul-
trafem) was inserted into the vagina according to the manufacturer’s in-
structions and was removed after 30 min. The SoftCup was then centri-
fuged for 10 min at 1000 ? 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 mini-
mally manipulated, whole vaginal fluid samples were stored in aliquots at
?20°C. This method enabled us to collect ?200 ?l 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 ex-
tracted using 5% acetic acid, vacuum dried, and resuspended in 0.1% hexa-
decyl trimethyl ammonium bromide/10% acetic acid/3? 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 ? 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 ? 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 sub-
sequent 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 orig-
inal 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 ?g/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 ?
106/ml in RPMI 1640 supplemented with 100 U/ml penicillin, 100 ?g/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 Ref-
erence 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-?m 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
Assays to determine anti-HIV-1 activity of vaginal fluid and
TZM-bl cells were seeded in 96-well dishes (4000 cells/well). After 24 h,
cells were treated in triplicate with 50 ?l 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 me-
dium (2 ng/ml p24 for BaL and 5 ng/ml p24 for IIIB) in 50 ?l was im-
mediately 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).
Two-dimensional gel electrophoresis of vaginal fluid
Acid-extracted vaginal fluid samples were electrophoresed on a 12.5% na-
tive 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 0.1? Amido Black
(0.04% napthol blue-black, 2.5% isopropanol, and 1% acetic acid) to vi-
sualize 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 spectro-
metric 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
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 mm2of tissue adhered tightly atop a mi-
croporous 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 ?l of PBS or 50 ?l of vaginal fluid
diluted 1/1 with PBS for 30 min and then rinsed twice with warm PBS.
Tissues were topically applied with 100 ?l of PBS (control), PBS contain-
ing 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 re-
moved, and tissues were washed with 100 ?l of warm PBS, then vaginal
fluid (50%) or PBS vehicle was reapplied in 50 ?l. A one-time dose of 1 ?
106HIV-1 BaL-infected PM1 promyelocytic cells was included under-
neath 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
7561 The Journal of Immunology
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
by ELISAfor HIV-1p24gag
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 ?-actin controls) in
total tissue DNA isolated 9 days after infection. The HIV-1 BaL primers
used were 5?-AACACCTCAGTCATTACAC-3? and 5?-TACATTGC
TCTTCCTACTTC-3?, which amplify a 700-bp region of BaL gp120. The
?-actin primers used were 5?-CCTTCCAGCAGATGTG-3? and 5?-GGT
GTAACGCAACTAAG-3?, which amplify a 105-bp region of human ?-ac-
tin. Two hundred nanograms of DNA was mixed with 2? SYBR Green
Supermix (Bio-Rad), 200 nM of each primer, and dH2O. Triplicate 20-?l
reactions were conducted using the MyiQ real-time PCR detection system
(Bio-Rad), and HIV-1 BaL levels were normalized to ?-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
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).
Human vaginal fluid is intrinsically active against HIV-1
The mucosal layer lining the vaginal epithelial cells is rich in an-
timicrobial polypeptides that provide a crucial barrier against in-
vading microbial and viral pathogens (15). Although some of these
polypeptides have been shown to exhibit antiviral properties (19,
23, 25, 36–38), detailed analysis of the intrinsic anti-HIV-1 activ-
ity 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, vag-
inal 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 cyto-
toxic (data not shown). These results indicate that human vaginal
fluid intrinsically inhibits the entry of HIV-1 into host cells.
Anti-HIV-1 activity resides in the cationic fraction of vaginal
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, un-
diluted 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 pro-
teins and electrolytes. We pioneered the CM-resin-mediated de-
pletion 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 polypep-
tides 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 com-
pared 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.
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 sep-
arates polypeptides based on cationic charge density (31, 39). A
slice from the AU-PAGE was inserted into a Tricine-SDS-PAGE
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 differ-
ent pools of vaginal fluid, each in triplicate. ??, p ? 0.0002; ?, p ? 0.0005
(compared with controls). Error bars represent the SEM.
Human vaginal fluid inhibits HIV-1 infection. TZM-bl cells
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 in-
fected 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 per-
formed with four different pools of vaginal fluid, each repeated three times.
?, p ? 0.0002.
Depletion of cationic proteins from vaginal fluid increases
7562CATIONIC POLYPEPTIDES IN VAGINAL HOST DEFENSE
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 se-
quenced 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 frag-
ments of albumin were also detected, which are indicated by un-
labeled 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).
Comparison of proteomic profiles of whole and CM-depleted
vaginal fluids reveals cationic polypeptides that contribute to
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 polypep-
tides extracted from the resin. Spots without arrows were not iden-
tified. 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.
Whole vaginal fluid (10 ?l) was subjected to one-dimensional electro-
phoresis in an AU-PAGE, followed by Tricine-SDS-PAGE as the second
dimension. The SYPRO Ruby-stained two-dimensional gel of whole vag-
inal 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
Identification of cationic polypeptides in vaginal fluid.
Table I. Cationic polypeptides identified in vaginal fluid by two-dimensional analysis and MALDI-TOF MS/MS analysis
Mass (Da) Scoreb
Role in Host Defense
1 Albumingi?23241675 45,130.4 90100 Transport of metabolites, drugs and toxins,
transcytosis of myeloperoxidase (56)
Bacteriostatic, sequesters iron siderophores (57, 58)2 Neutrophil Gelatinase
Cathepsin-G (Chain A)
gi?4261868 20,534.551 100
3 gi?2066422026,740.9227100 Serine protease, chemoattractant, lymphocyte
activator, inflammation. (55, 59)
Antibacterial, sequesters iron siderophores (57, 58)4 Neutrophil Gelatinase
Unnamed protein product
H2A histone family
Fatty acid binding
protein 5 E-FABP
gi?7767000 19,976.3 41100
Antibacterial (51, 52, 60, 61)
Antibacterial (51, 52, 60, 61)
Intracellular fatty acid trafficking, stabilization of
leukotrienes, skin inflammation (62, 63)
S-lectin involved in cell-adhesion (64, 65)
migration immune response (66, 67)
Antibacterial and antiviral (17, 29, 68, 69)
Artifact obtained after extraction from CM-resin
Cysteine proteinase inhibitor, immunomodulator,
activates NO synthesis in macrophages (70, 71)
Antibacterial (73, 74)
Antibacterial (51, 52, 60, 61)
Antibacterial (51, 52, 60, 61)
Antibacterial (51, 52, 60, 61)
Cysteine proteinase inhibitor, immunomodulator
Antibacterial (72, 73)
Antimicrobial (25, 27, 75–77)
9gi?3891470 14,934.871 100
Histone H2A family
Histone H4 family
H2A histone family
Unnamed protein product
aThe numbers correspond to the labeled spots in Fig. 3.
bIon score of one or more peptide fragments that match a protein in the database.
cConfidence index percentage.
dNote that this spot has a C.I.% index of ?85% but was considered positive because it was identified in multiple samples.
7563The Journal of Immunology
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 recombi-
nantly. 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 indi-
vidual 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 in-
hibited viral entry, as shown by the absence of reduction in lucif-
erase 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
The abundance of antimicrobial peptides in vaginal fluid with of-
ten 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 physiolog-
ical concentrations do not prevent entry of HIV-1 into host cells.
We therefore hypothesized that these polypeptides must act in con-
cert to prevent HIV-1 infection. To test our hypothesis, we pre-
pared 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 in-
fectivity ?40%, this was not significant compared with the vehi-
cle-only control. Moreover, the addition of the polypeptide mix to
CM-depleted fluid was not completely restorative. Due to avail-
ability, 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
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 primar-
ily contained in the cationic fraction, and that the activity is com-
plex and requires the collective polypeptides.
Vaginal fluid protects against HIV-1 infection of human
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 depleted vaginal fluid. Two-dimensional gel electrophoresis of 10 ?l 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 corre-
sponding cationic proteins in A are absent in depleted vaginal fluid.
Comparison of proteomic profiles of whole vaginal fluid
Table II. Physiological concentration of cationic proteins that contribute to anti-HIV-1 activity of vaginal fluid
(?g/ml) Method of Detection Reference
34 ? 7
13 ? 2
0.9 ? 0.2
0.7 ? 0.1
0.57 ? 0.13
0.35 ? 0.07
0.04 ? 0.02
Semiquantitative Western blot and densitometry
Semiquantitative Western blot and densitometry
Semiquantitative Western blot and densitometry
Semiquantitative Western blot and densitometry
Semiquantitative Western blot and densitometry
Semiquantitative Western blot and densitometry
(15, 72, 73)
(15, 16, 19)
(15, 22, 23, 78)
(15, 27, 38, 79)
aRepresents the arrows indicated in Fig. 4, A and C.
bCalprotectin, heterodimer of calgranulin A and calgranulin B, was tested as individual peptides.
cHNP-1, HNP-2, and HNP-3 were tested as individual polypeptides.
7564 CATIONIC POLYPEPTIDES IN VAGINAL HOST DEFENSE
and vagina, containing a full-thickness epithelia composed of vag-
inal-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, cervico-
vaginal 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 infec-
tion, and the proviral DNA levels were assessed by real-time quan-
titative 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, al-
though this trend was not statistically significant due to variability
in the untreated condition (Fig. 6A). However, viral titer, as quan-
tified by p24gagELISA 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
The mechanisms by which vaginal mucosa protects against sexu-
ally transmitted and other infections are not completely under-
stood. Although several studies have focused on the adaptive im-
mune system of mucosal surfaces of the female reproductive tract,
scant attention has been focused on the innate immune factors in
vaginal secretions (41–44). 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 vag-
inal fluid against HIV-1 infection. Proteomic analysis of the cat-
ionic polypeptide fraction of vaginal fluid revealed numerous cat-
ionic antimicrobial and host defense polypeptides. Polypeptides
with known microbicidal effects that have been identified in our
study and previously identified in mucosal secretions include ly-
sozyme, lactoferrin, cathelicidin (47, 48), ?-defensins, ?-de-
fensins, 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 physi-
ological 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 reflec-
tive of the following. 1) Some of the recombinant proteins may not
exhibit the same anti-HIV-1 activity as that of the purified or nat-
ural 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 het-
erodimeric 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 collec-
tive cationic polypeptide fraction is responsible for anti-HIV-1 ac-
tivity of vaginal fluid. Indeed, when the extracted cationic
polypeptide fraction (bound to the CM-resin) was used to recon-
stitute the CM-depleted fluid, anti-HIV-1 activity was restored
Whole vaginal fluid was collected from healthy donors using a
diaphragm-like device (Instead SoftCup), which enabled the col-
lection 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 In-
stead 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 representa-
tion of the condition in vivo.
Lactic and other organic acids that result in the low pH of hu-
man vaginal fluid (normally pH 3.8–4.5) as well as volatile com-
pounds, such as H2O2, are thought to contribute to microbial host
defense (13, 14). Our studies were designed to minimize or elim-
inate 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
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). Lu-
ciferase was then measured as described in the text, and the percent infec-
tion was calculated. ?, p ? 0.00015. Experiments were performed in trip-
licate, and error bars represent the SEM.
Cationic polypeptides of vaginal fluid synergize to inhibit
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 anal-
ysis. A, Real-time quantitative PCR of HIV-1 BaL proviral DNA corre-
sponding 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.
Vaginal fluid inhibits HIV-1 infection of human vaginal
7565 The Journal of Immunology
contained in the collective polypeptide extract, the procedures re-
quired for extraction would inactivate or remove H2O2and 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 com-
ponents 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 ap-
proach, we identified histone fragments in every vaginal fluid sam-
ple tested. Histones have been shown to possess antibacterial prop-
erties and are released from activated neutrophils (51, 52). Why
are histones present in mucosal secretions? Do these proteins con-
fer an active function, or are they merely byproducts of cellular
decay? Until recently, the latter was the most plausible explana-
tion. However, a current study by Brinkmann et al. (52) provided
an alternative mechanism behind the presence of extracellular hi-
stones. 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-nega-
tive 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 his-
tones 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 ?-defensins using sensitive Abs were able to immu-
nodetect 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 ?-defensins have been shown to inhibit HIV-1
infection (50% inhibition at 20 ?g/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 phys-
Surprisingly, vaginal fluid contains components that are permis-
sive to the transmission of HIV-1. For example, samples of cer-
vicovaginal 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 neu-
trophil-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 devel-
opment 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
The authors have no financial conflict of interest.
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