Inhibition of HIV-1 Infection in Ex Vivo Cervical Tissue
Model of Human Vagina by Palmitic Acid; Implications
for a Microbicide Development
Xudong Lin1¤a, Elena E. Paskaleva1,3¤b, William Chang1, Alexander Shekhtman2,3, Mario Canki1,3*
1Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York, United States of America, 2Department of Chemistry, State University of New
York at Albany, Albany, New York, United States of America, 3Palm Biologicals, LLC. Albany, New York, United States of America
Background: Approximately 80% of all new HIV-1 infections are acquired through sexual contact. Currently, there is no
clinically approved microbicide, indicating a clear and urgent therapeutic need. We recently reported that palmitic acid (PA)
is a novel and specific inhibitor of HIV-1 fusion and entry. Mechanistically, PA inhibits HIV-1 infection by binding to a novel
pocket on the CD4 receptor and blocks efficient gp120-to-CD4 attachment. Here, we wanted to assess the ability of PA to
inhibit HIV-1 infection in cervical tissue ex vivo model of human vagina, and determine its effect on Lactobacillus (L) species
of probiotic vaginal flora.
Principal Findings: Our results show that treatment with 100–200 mM PA inhibited HIV-1 infection in cervical tissue by up to
50%, and this treatment was not toxic to the tissue or to L. crispatus and jensenii species of vaginal flora. In vitro, in a cell free
system that is independent of in vivo cell associated CD4 receptor; we determined inhibition constant (Ki) to be ,2.53 mM.
Significance: These results demonstrate utility of PA as a model molecule for further preclinical development of a safe and
potent HIV-1 entry microbicide inhibitor.
Citation: Lin X, Paskaleva EE, Chang W, Shekhtman A, Canki M (2011) Inhibition of HIV-1 Infection in Ex Vivo Cervical Tissue Model of Human Vagina by Palmitic
Acid; Implications for a Microbicide Development. PLoS ONE 6(9): e24803. doi:10.1371/journal.pone.0024803
Editor: Landon Myer, University of Cape Town, South Africa
Received April 11, 2011; Accepted August 22, 2011; Published September 19, 2011
Copyright: ? 2011 Lin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Institutes of Health grant number AT003371 to MC and GM085006 to AS. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: J. Craig Venter Institute, Rockville, Maryland, United States of America
¤b Current address: for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York, United States of America
Worldwide, sexual transmission remains the principal route of
HIV transmission accounting for approximately 80% of all new
infections, and women bear a disproportionate burden of people
living with HIV (UNAIDS, 2010) . In the absence of an
effective vaccine, there is an urgent need to supplement currently
available strategies with novel therapeutics including microbicides
that are aimed at preventing sexual transmission of HIV. Viral
entry inhibitors are ideally suited for use in microbicide
formulations , however currently there are no clinically
approved micobicide or trials that specifically include CD4
inhibitors of virus entry into the host (for a full list see www.
In our search for novel inhibitors of HIV-1, we investigated a
large number of natural products, and from Sargassum fusiforme we
isolated and identified palmitic acid (PA) as a natural small
molecule that blocked virus entry [3,4,5]. We reported that
palmitic acid is a specific CD4 fusion inhibitor of both X4 and R5
HIV-1 entry, which also efficiently inhibited virus-to-cell and cell-
to-cell fusion, and it did not internalize CD4 receptor or perturb
lipid rafts . PA bound to the CD4 receptor specifically, with
dissociation constant (Kd) of ,1.5 mM, and this binding was via
PA’s hydrophobic methyl and methelene groups located away
from the carboxyl end, which functions by blocking efficient
pgp120-to-CD4 attachment and fusion [6,7]. We also showed that
PA occupies a novel hydrophobic cavity on the CD4 receptor that
is constrained by amino acids Phe52-to-Leu70 , which
encompass residues that have been previously identified as a
region critical for gp120 binding [8,9].
In the present report we wanted to ascertain PA’s utility for
microbicide development by testing its ability to block HIV-1
infection in ex vivo model of vaginal mucosa, its effect on
Lactobacillus species present in normal vaginal flora, and PA’s in
vitro inhibition constant independent of variable in vivo CD4
Based on our previous results demonstrating that PA is a specific
HIV-1 entry inhibitor and a potential lead molecule for further
development [6,7], we wanted to examine the practical utility of
PA to inhibit HIV-1 infection in a human cervical tissue ex vivo
model of vaginal mucosa. This model closely resembles the vaginal
PLoS ONE | www.plosone.org1 September 2011 | Volume 6 | Issue 9 | e24803
epithelial layer that mimics in vivo conditions for HIV sexual
transmission and infection [10,11,12], and it has been established
for evaluating potential microbicide candidates [13,14,15]. Light
microscopy examination of the uninfected and paraffin embedded
sections from 3 mm3biopsy punches of the cervix revealed that
the tissue architecture was preserved (Fig. 1A). Although both X4
and R5 viruses are sexually transmitted, R5 viruses predominate
early in infection and may be more easily transmissible than X4
viruses [12,14]. Tissue was treated with 0, 100 or 200 mM PA, and
then tested for inhibition of productive HIV-1 R5-tropic BaL
infection by p24 ELISA (Fig. 1B). Measurements of HIV-1 p24
antigen levels in PA untreated cell-free tissue culture supernatants
(0 mM) revealed a peak of p24 production on day 7 that measured
1421 pg p24/ml. This represented an increase from 943 pg p24/
ml on day 4, which was followed by a gradual decline on day 10,
to 785 pg p24/ml. Increasing p24 values indicated productive and
ongoing HIV-1 infection, and de novo viral synthesis. In contrast,
treatment with 100 mM PA, significantly reduced HIV-1 replica-
tion to 604, 960, and 452 pg p24/ml on days 4, 7, and 10,
respectively (p=0.04, repeated measures ANOVA). Compared to
PA untreated tissue, this reduction in HIV-1 replication
corresponded to a calculated 36, 32, and 42% inhibition of
HIV-1 infection. Similar results were obtained for treatment with
200 mM PA, which also significantly reduced HIV-1 infection by
38, 48, and 43% on day 4, 7, and 10, respectively. However, there
appeared to be no significant difference between 100 and 200 mM
PA treatment. Tissue toxicity was measured on day 10 after
infection, by MTT viability assay, which demonstrated an absence
of toxicity (p=0.64, one-way ANOVA) (Fig. 1C). Tissue infection
with X4 HIV-1 was less productive, however inhibition by PA was
similar to that of R5 infection (not shown).
In vivo, normal vaginal flora consists predominantly of common
probiotic Lactobacillus bacterial species  that a successful
topical microbicide should not be toxic to, as was outlined in
screening algorithm for testing of preclinical topical microbicides
. To determine potential compound toxicity, we tested PA
treatment on common L. crispatus and jensenii species normally
present in vaginal tract (Fig. 2 A and B). Increasing concentra-
tions of PA were incubated for 24 hours in presence of either
L. crispatus (Fig. 2A) or jensenii (Fig. 2B), and % viability was
calculated from 0 mM PA (no treatment) that was taken as 100%
viability. L. crspatus showed viability over 96% with PA
concentrations of up to 50 mM, which decreased to 78.5% with
500 mM PA treatment. L. jensenii remained viable over 94% with
up to the highest treatment of 500 mM PA. Vehicle control (VC)
was also not toxic to either species that remained viable over
95%. Based on these results we conclude that PA may be
considered suitable for further topical microbicide evaluation and
Peripheral blood lymhocytes (PBL) and macrophages (Mw’s) are
primary targets for in vivo HIV-1 infection and replication in
systemic circulation as well as in vaginal submucosa, and
previously we demonstrated ability of PA treatment to inhibit
ongoing virus replication in these physiologically relevant cells .
However, PA inhibited HIV replication in PBL with approxi-
mately 10-fold greater efficacy as compared to inhibition in Mw’s
. Because CD4 cell surface HIV receptor expression varies
between different cell types, here we wanted to ascertain PA’s
inhibition efficacy that is independent of in vivo cell surface
expression. We utilized in vitro gp120-to-CD4 capture ELISA to
determine PA’s inhibition constant (Ki) (Figure 3). Envelope gp120
(IIIB) protein was captured on 96 well plates, washed, and
incubated in the presence of CD4-biotin alone or in the presence
of serial dilutions of PA as indicated. Percent CD4 binding was
calculated from gp120-CD4 complex formation in the absence of
any inhibitor. Inhibition constant, Ki, was calculated by using the
equation Ki=IC50/(1+[CD4]/Kd) , based on IC50concen-
tration of bound CD4, [CD4]=50 nM, and CD4 binding affinity
Figure 1. Inhibition of HIV-1 infection in human cervix model of vaginal mucosa. 3 mm3biopsy punches of the ectocervix tissue samples
from premenopausal women with conditions not involving the cervix were processed within 1–3 hours after surgery and directly cultured in 48 well
plates in 300 ml/well DMEM/F12 media. (A) Paraffin embedded, and hematoxylin and eosin (H&E) stained sections of the uninfected ectocervix tissue
were identified to be composed of (a) stratified squamous epithelial cell layer, (b) basal epithelial layer, and (c) submocosa, which was visualized on
an Olympus BX41 Altra 20 Soft Image System, 1006magnification. (B) Replicates (n=6) of tissue were treated for 24 h with 0, 100, or 200 mM PA, and
then infected with 26105p24/ml cell-free HIV-1 BaL in 300 ml for 16 h. Tissues was washed 3 times to remove the virus, and returned to culture with
each respective treatment for the duration of the experiment. At the indicated time points, HIV-1 replication was tested by p24 ELISA, and repeated
measures ANOVA was used to calculate statistical significance (*) between groups. (C) At day 10 after infection, tissue was collected and viability
determined by the MTT assay. Representative of three experiments, all data are mean 6 SD.
Palmitic Acid Microbicide
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for gp120, Kd=5 nM, which is consistent with and validated our
in vitro Kdvalue of 1.5 mM for CD4 binding .
In this ex vivo model of female genital mucosa that simulates
damaged epithelium and allows for maximal virus infection and
replication , our data clearly demonstrate that the 100–200 mM
PA treatment inhibits productive R5 HIV-1 infection by up to 48%,
and that the PA treatment is not toxic to tissue or to probiotic
Lactobacillus species tested here. Considering that PA was not
formulated into a topical delivery vehicle may have imposed
suboptimal tissue penetration and HIV inhibition in the underlying
submucosa containing PBL and Mw cells. Previously we demon-
strated ability of PA treatment to inhibit ongoing virus replication in
these physiologically relevant cells, however, inhibition was more
efficient inPBLascomparedtoMw’s . The reasonfor differences
in inhibition between tissue, PBL and Mw’s is not clear, however it
may indicate variable CD4 receptor cell surface expression, and
intrinsically different number of primary cells in each tissue biopsy.
the CD4 receptor to inhibit HIV-1 entry, and therefore quantita-
tively different CD4 expression will affect PA’s inhibition efficacy.
To avoid this degree of difference of in vivo primary cell number and
CD4 expression, we tested in vitro PA’s inhibition efficacy (Ki) to
block gp120-to-CD4 attachment (Figure 3). The measured Kiwas
,2.53 mM, which is in close agreement with previously published
CD4 dissociation constant of ,1.5 mM .
Although there are several candidate microbicides in clinical
trials , PA is a novel class of small molecules with bi-functional
mode of action that specifically targets and binds to the CD4
receptor via its hydrocarbon chain, and blocks efficient gp120-to-
CD4 fusion via its carboxyl end [6,7]. Targeting the human CD4
receptor as opposed to viral envelope or other viral proteins, offers
the advantage of removing selective pressure on the virus to
quickly mutate and bypass the imposed restriction.
Figure 2. Lactobacillus toxicity assay. L. crispatus (A) and jensenii (B) species were tested for toxicity against increasing concentrations of PA that
was diluted in vehicle control (VA), 0.5% DMSO highest concentration. Dilutions of PA or VC were plated in triplicate on 96-well plates in 100 ml
volume, and bacterial species grown in MRS medium were diluted and added to a final volume of 200 ml. After 24 hours incubation, bacterial cultures
were read in a spectrophotometer at OD490and % viability was calculated from untreated cultures (0 mg PA) that was taken as 100% viability. Bars
indicate 6 SD of three separate experiments.
Figure 3. PA inhibition constant for gp120-to-CD4 complex
formation. Inhibition of gp120-CD4 complex formation was
investigated by gp120 capture ELISA. Envelope gp120 (IIIB) protein
was captured on 96 well flat bottom plates, washed, and incubated
in the presence of CD4-biotin alone or in the presence of increasing
concentrations of PA, as indicated. Strepavidin-HRP was added, and
then developed by addition of o-Phenylenediamine dihydrochloride
(OPD) substrate. Colorimetric reaction was stopped by adding 1 N
HCl, and read at 490 nm. % CD4 binding was calculated from gp120-
CD4 complex formation in the absence of PA inhibitor, and
inhibition constant, Ki, was calculated utilizing previously published
equation . Representative of three experiments, all data are
mean 6 SD.
Palmitic Acid Microbicide
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Current results from The Center for the AIDS Program of
Research in South Africa (CAPRISA) clinical trial have demon-
strated that micobicide application of reverse transcriptase (RT)
tenofovir gel, prevented HIV infection by up to 39% overall, and
by 54% in women with high gel adherence . Considering that
nucleotide RT inhibitor prevents provirus transcription and not
virus entry, we postulate that combination with an entry inhibitor
such as PA may eliminate HIV infection with more robust efficacy.
The idea of combing therapeutics that target different stages of
virus life cycle is logical as it has been effectively demonstrated
with HAART therapy in AIDS patients.
PA’s toxicology screening, breadth of primary HIV inhibition,
and gel formulations are currently under investigation.
Materials and Methods
Palmitic acid (Sigma) was solublized at 100 mM in ethanol
(EtOH)  and stored at 220uC. Working aliquots were kept at
4uC for up to 4 weeks.
Written informed consent approved protocol by Albany
Medical College (AMC) Institutional Review Board (IRB) was
obtained from all participants involved in the study, which was
performed at the AMC, Albany New York. 3 mm3biopsy punches
of the ectocervix tissue samples were obtained from premeno-
pausal women with conditions not involving the cervix, and were
processed within 1–3 hours after surgery. Tissue was cultured in a
nonpolarized manner as previously described [10,19], in 48 well
plates in 300 ml/well DMEM/F12 media (Invitrogen) supple-
mented with 10% FBS for the duration of the experiment. Tissue
was treated with increasing concentrations of PA or 1026M ddC,
and infected with HIV-1 R5 BaL at 0.3 multiplicity of infection
Lactobacillus crispatus and jensenii species were purchased from
American Type Culture Collection (ATCC), number 33820 and
25258, respectively. Bacterial toxicity assay was performed under
facultative anaerobic conditions as previously described .
Briefly, bacterial species were grown in MRS medium and plated
on 96 well plates containing increasing concentrations of PA.
1.25 mg/ml, respectively, was used as positive toxicity control.
After 24 hours, bacterial culture growth was determined by
measurement at OD490using a 96 well SpectraMax L plate reader
HIV-1 X4-tropic molecular clone NL4-3, which expresses all
known HIV-1 proteins ; the R5-tropic molecular clone 81A
, which has R5 BaL Env sequences on the backbone of NL4-3;
and R5 BaL molecular clone were all obtained from the HIV
AIDS Research and Reference Reagent Program. Macrophage
HIV-1 R5-tropic isolate BaL was prepared as previously described
, and cell-free viral stock was quantified for HIV-1 p24 core
antigen content by enzyme-linked immunosorbent assay (ELISA,
AIDS Vaccine Program, NCI-Frederick). Potential tissue or cell
toxicity due to PA treatment was measured after infection by the
MTT assay (Promega), as previously described .
Percent CD4 binding was investigated by CD4-to-gp120
capture ELISA, in accordance with manufacturer’s instructions
(ImmunoDiagnostics, Inc., MA), and as previously described .
Briefly, envelope gp120 (IIIB) protein (ImmunoDiagnostics, Inc.,
MA) was captured on 96-well plates, washed, and incubated in the
presence of 50 nM biotin-conjugated sCD4 (Immunodiagnostics)
alone or in the presence of serial dilutions of PA, as indicated.
Strepavidin-HRP was added, and then developed by addition of
OPD substrate. Colorimetric reaction was stopped by adding 1 N
HCl, and read at 490 nm. Percent CD4 binding was calculated
utilizing previously published formula .
solution(Sigma) at 1.25 U/mland
The authors wish to thank Dr. Simon Hirschl for help with cervical tissue
preparation, histology, and valuable discussions.
Conceived and designed the experiments: MC AS EEP. Performed the
experiments: XL EEP WC AS. Analyzed the data: XL EEP AS MC.
Wrote the paper: MC.
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