ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, May 2007, p. 1770–1779
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 5
Preclinical Testing of Candidate Topical Microbicides for Anti-Human
Immunodeficiency Virus Type 1 Activity and Tissue Toxicity in a
Human Cervical Explant Culture?
James E. Cummins, Jr.,1* Jeannette Guarner,2Lisa Flowers,3Patricia C. Guenthner,1Jeanine Bartlett,2
Timothy Morken,2† Lisa A. Grohskopf,4Lynn Paxton,4and Charlene S. Dezzutti1‡
Laboratory Branch1and Epidemiology Branch,4Division of HIV/AIDS Prevention, National Center for HIV, STD,
and TB Prevention, and Infectious Disease Pathology Activity, Division of Viral and Rickettsial Diseases,2
National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,
and Emory University School of Medicine, Atlanta, Georgia 303223
Received 7 September 2006/Returned for modification 7 November 2006/Accepted 27 February 2007
A human cervical explant culture was utilized for the preclinical assessment of anti-human immunodefi-
ciency virus type 1 (HIV-1) activity and tissue toxicity of formulated, candidate topical microbicides. Products
tested included cellulose acetate 1,2-benzene dicarboxylate (CAP), a carrageenan-based product (PC-515), a
naphthalene sulfonate polymer (PRO 2000), a lysine dendrimer (SPL7013), a nonnucleoside reverse tran-
scriptase inhibitor (UC781), and an antimicrobial peptide (D2A21), along with their placebos. Cervical
explants were cultured overnight with HIV-1 with or without product, washed, and monitored for signs of HIV-1
infection. HIV-1 infection was determined by p24gag levels in the basolateral medium and by immunohisto-
chemical analysis of the explant. Product toxicity was measured by the MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-
diphenylformazan] assay and histology. CAP, PRO 2000, SPL7013, and UC781 consistently prevented HIV-1
infection in all explants tested. PC-515 and D2A21 prevented HIV-1 infection in 50% or fewer of the explants
tested. Placebos did not prevent infection in any of the explants tested. With the exception of PRO 2000 (4%),
the MTT assay and histological analysis of the other products and placebos showed minimal toxicity to the
epithelium and submucosa. Collectively, these data suggest that this culture system can be used for evaluating
the safety and efficacy of topical microbicides designed for vaginal use.
Sexual transmission of human immunodeficiency virus type
1 (HIV-1) fuels the HIV/AIDS pandemic, with more than 90%
of all adolescent and adult HIV-1 infections resulting from
heterosexual intercourse (23). Worldwide, approximately half
of the 42 million people living with HIV/AIDS are women, and
that proportion is growing (23, 37). A variety of biological and
socioeconomic factors contribute to women’s vulnerability to
infection. Studies in discordant couples indicate that the virus
is more easily transmitted from male to female than from
female to male (30, 33, 34). Anatomic and histological differ-
ences in the genital mucosa between men and women may
account for the increased susceptibility of women to HIV-1
infection (14). The presence of sexually transmitted diseases,
the exchange of sex for drugs or money, or limited control of
their sexuality places many women at increased risk for acquir-
ing HIV-1. Despite evidence suggesting that proper use of
condoms is effective at decreasing sexual transmission of
HIV-1 (11), they are not used consistently. As a result, women
are at risk for acquiring HIV-1 due to exposure to infectious
seminal fluid during intercourse with high-risk sex partners
Significant advances have been made in the development of
antiretroviral drugs, resulting in enhanced quality of life and
reduced morbidity and mortality. However, fueled by the lack
of access to these drugs and the lack of a preventative vaccine,
the spread of HIV-1 continues unabated. Over the past de-
cade, an alternative strategy focused on the development of
low-cost intravaginal formulations of anti-HIV-1 agents to
curb mucosal and perinatal HIV-1 transmission, termed mi-
crobicides, has emerged (15, 21). While topical formulations of
reverse transcriptase inhibitors (RTIs) such as N-[4-chloro-3-
(UC781, a nonnucleoside RTI [NNRTI]) and 9-[2-(phospho-
nomethoxy)propyl]adenine (PMPA, a nucleoside RTI) are be-
ing considered, the majority of candidate topical microbicides
are designed to disrupt the virus or block adherence of HIV-1
to target genital tissues (4). More novel approaches include
plant-derived antibodies and lactobacilli engineered to secrete
potential microbicides (4) to augment the vaginal environ-
The first product to be evaluated as a topical microbicide
was one containing the surfactant nonoxynol-9 (N9), originally
developed as a spermicide with known in vitro anti-HIV-1
activity. The results from the clinical trial (41) demonstrated
the toxic effects of N9. Moreover, HIV-1 infection of the women
using the N9 product was higher than that of the women
receiving the placebo. This work has emphasized the need for
improved preclinical testing of microbicides. Although several
* Corresponding author. Mailing address: Southern Research Insti-
tute, 431 Aviation Way, Frederick, MD 21701. Phone: (301) 694-3232.
Fax: (301) 694-7223. E-mail: firstname.lastname@example.org.
† Present address: Lab Vision-Neomarkers, 47790 Westinghouse
Drive, Fremont, CA 94539.
‡ Present address: University of Pittsburgh, Department of Obstet-
rics, Gynecology, and Reproductive Sciences, Magee-Womens Re-
search Institute, 204 Craft Avenue, Pittsburgh, PA 15213.
?Published ahead of print on 12 March 2007.
groups have used epithelial cell lines, peripheral blood mono-
nuclear cells, and blood-derived macrophages to evaluate
product toxicity and efficacy (5, 16), these cell types are not
representative of the primary cells, both epithelial and immune
target cells, present in the genital mucosa.
To better approximate the in vivo setting, we have developed
a polarized cervical explant culture system to comparatively
evaluate the toxicity and efficacy of topical microbicides that
are formulated for use in humans. Six candidate topical micro-
bicides and N9 were evaluated for their ability to prevent
HIV-1 infection and for their possible toxicity on cervical tis-
sue. The products include those that (i) maintain or enhance
normal vaginal defense mechanisms (e.g., an antimicrobial
peptide), (ii) disrupt or inactivate the pathogen (e.g., cellulose
acetate 1,2-benzene dicarboxylate [CAP]), (iii) block binding
and fusion of pathogens (e.g., a naphthalene sulfonate poly-
mer, a lysine dendrimer, and CAP), and (iv) affect the patho-
gen life cycle (e.g., an NNRTI). The results presented here
demonstrate that this cervical explant culture provides addi-
tional information for the preclinical assessment of topical
microbicides to prevent HIV-1 transmission during vaginal
(This work was presented in part at the following meetings:
Microbicides 2002, May 2002, Antwerp, Belgium, and Micro-
bicides 2004, March 2004, London, England.)
MATERIALS AND METHODS
Tissues. Unless otherwise stated, all tissue culture reagents were obtained
from Invitrogen Corp. (Carlsbad, CA). After obtaining informed consent, nor-
mal ectocervical tissue (i.e., with no clinically observed disease) was acquired
from premenopausal women undergoing routine hysterectomy. Tissues were
transported on ice in L15 medium containing 10% fetal calf serum, 100 U/ml
penicillin, 100 ?g/ml streptomycin, and 2.5 ?g/ml fungizone (amphotericin B).
The cervix was washed twice with phosphate-buffered saline (PBS). Excess sub-
mucosal tissue was removed to produce a flat piece containing both epithelium
and stromal tissue. Dermal biopsy punches (5-mm diameter) (Miltex Instrument
Company, Inc., Bethpage, NY) were used to cut full-thickness tissue specimens.
Each explant was inserted through a 4-mm-diameter hole in a transwell insert
(12-mm diameter, polyester membrane; Corning, New York, NY) with the epi-
thelium oriented upward in the apical chamber. The epithelial surface of the
explant was surrounded with 2% agarose to maintain the tissue orientation. In
the basolateral chamber, the stroma was cultured in 0.6 ml of Dulbecco’s min-
imal essential medium containing 10% human AB serum, 100 U/ml penicillin,
100 ?g/ml streptomycin, and nonessential amino acids (cDMEM) (Fig. 1A).
Tissues were cultured at 37°C under 7% CO2in a humidified incubator.
Tissue permeability. To evaluate the integrity of the explants, the transmission
of fluorescent polymer microspheres (0.026-?m diameter; Duke Scientific Corp.,
Palo Alto, CA) was tested. After removal of the suspension solution, the micro-
spheres were washed and suspended in PBS (1 ? 1014microspheres/ml). On the
first day of culture, 0.5 ml of the microspheres was added to the epithelial
surface. As controls, the same volume of microspheres was added to a blank well
and a well containing 2% agarose. The basolateral medium was sampled at 1, 3,
and 7 days. The fluorescence in the basolateral supernatant was measured using
a Fluoroskan Ascent FL fluorometer (Thermo Electron Corp., Waltham, MA)
using 485-nm- (excitation) and 538-nm- (emission) wavelength filters. The per-
cent transmission was calculated by dividing the fluorescent signal in the baso-
lateral supernatant from the explants and agarose control by that from the blank
Topical microbicides. Six candidate microbicides were evaluated in formula-
tions designed for use in humans (Table 1). Four percent N9 (Ortho-McNeil
Pharmaceuticals, Inc., Raritan, NJ), an approved over-the-counter product, was
used as a control for tissue toxicity. PC-515, a carrageenan-based product, was
provided by the Population Council (New York, NY). PRO 2000, a naphthalene
sulfonate polymer, was provided by Procept, Inc. (Cambridge, MA). CAP, a
lysine dendrimer (SPL7013), an NNRTI (UC781), and an antimicrobial peptide
(D2A21) were provided in response to a solicitation placed in the Federal
Register by the Division of HIV/AIDS Prevention, CDC, which requested pro-
posals for collaboration in the evaluation of potential microbicide agents (9, 10).
CDC remains interested in testing new agents using these methods; potential
agents should have demonstrated in vitro anti-HIV-1 activity and have been
formulated for vaginal or rectal application (8).
HIV-1 infection of cervical explants. Explants were activated for 2 days in
cDMEM containing 5 ?g/ml phytohemagglutinin-P (PHA; Difco Laboratories,
Detroit, MI) and 100 U/ml human interleukin-2 (Roche, Indianapolis, IN).
HIV-1BaLwas purchased from ABI (Columbia, MD) and supplied with a known
endpoint titer calculated by the Karber method (17). On day 3, 0.5 ml of
HIV-1BaL(5 ? 10450% tissue culture infective doses [TCID50]) in cDMEM was
applied to the epithelium and incubated for 18 h. Afterwards, the epithelium was
washed five times with PBS to remove residual virus, and the basolateral medium
was replaced with cDMEM containing interleukin-2 without PHA. The culture
medium was harvested twice weekly over a 14- to 17-day period and stored at
?70°C. Viral replication was determined by using a p24gag enzyme-linked im-
munosorbent assay kit (Beckman Coulter, Miami, FL). To demonstrate growth
of primary HIV-1 subtypes, 104TCID50of 96USSN20/A (CCR5/CXCR4 using),
97USSN54/A (CCR5 using), and 92US660/B (CCR5 using), obtained from the
AIDS Research and Reference Reagent Program, Division of AIDS, NIAID,
NIH, were used to infect cervical explants from one tissue donor. Endpoint titers
for the primary HIV-1 stocks were determined using the Reed and Muench
Efficacy of microbicides. To facilitate the even spread of products over the
tissues, microbicides or placebos were diluted 1:10 (vol/vol) in 0.5 ml cDMEM
containing 5 ? 104TCID50HIV-1BaLand mixed. After 18 h of exposure to the
product and virus, the epithelium was washed five times with PBS to remove
residual product and virus, and the tissues were cultured as described above.
Virus replication was determined in harvested culture supernatants by Coulter
p24gag enzyme-linked immunosorbent assay. HIV-1 p24 levels were determined
from standard curves using a four-parameter logistic algorithm (DeltaSoft mi-
croplate analysis software; BioMetallics, Princeton, NJ). Samples with concen-
trations that were too high to measure accurately were diluted in cDMEM to
produce values in the linear range of the assay. Each product was tested in tissues
from at least two donors. Tissues from a total of 16 donors were used in this
FIG. 1. Cervical explant culture. (A) Circular tissue explants were
inserted through a hole in a transwell insert with the epithelium ori-
ented upward in the apical chamber. The epithelial surface of the
explant was surrounded with 2% agarose to maintain tissue orientation
and minimize seepage of microbicides and/or virus around the tissue
edges. The stroma was cultured in cDMEM in the basolateral cham-
ber. HIV-1 alone in medium or mixed with microbicide was applied to
the apical surface to simulate mucosal exposure in vivo. (B to D)
Histology of a representative hematoxylin-and-eosin-stained normal
human cervix at day 0 (day of surgery; B), day 3 (C), and day 7 (D) of
culture. Original magnification, ?50.
VOL. 51, 2007MICROBICIDE TESTING IN CERVICAL EXPLANTS1771
study. For each treatment condition (virus alone, microbicide plus virus, and
placebo plus virus), one replicate per donor was tested in tissues from at least two
donors. The following final concentrations were tested for efficacy and toxicity:
(i) microbicides: 0.4% N9, 0.3% PC-515, 1.3% CAP, 0.05% and 0.4% PRO 2000,
0.5% SPL7013, 0.01% and 0.1% UC781, and 0.1% D2A21, and (ii) placebos:
0.25% methyl cellulose (for PC-515 and CAP), 0.2% cross-linked polyacrylic acid
polymer (for PRO 2000), 0.5% cross-linked polyacrylic acid polymer (for
SPL7013), 0.1% cross-linked polyacrylic acid polymer (for UC781), and 0.3%
hydroxyethylcellulose (for D2A21).
Histology and IHC. Tissues were fixed in formalin and processed to evaluate
general tissue architecture and product toxicity. Following routine paraffin em-
bedding, sections were cut and stained with hematoxylin and eosin. Normal
ectocervical tissue contains ?15 layers of epithelium, including the basal layer
and stratified layers of epithelial cells that mature as they reach the surface. In
tissue that is cultured ex vivo, there is a loss of the outer stratified layers and a
retention of the basal layer. Toxicity was determined by examining the loss of this
remaining basal layer of epithelium and necrosis in the submucosa. Epithelial
necrosis, sloughing, and regeneration of the epithelium and necrosis of the
submucosa were evaluated as indicators of toxicity. For evaluation of the tissues
described above in the efficacy studies, at the study endpoint tissues were fixed
and processed for histology to determine product toxicity and immunohisto-
chemical (IHC) analysis for p24 antigen to determine product efficacy. For each
tissue donor, one section from each treated explant was analyzed for p24 antigen
Four-micron sections of paraffin-embedded explants were used for IHC assays.
Sections were deparaffinized, rehydrated, digested in 0.1 mg/ml proteinase K
(Roche Molecular Biochemicals, Indianapolis, IN) in 0.6 M Tris-HCl (pH 7.5)–
0.1% CaCl2for 15 min, washed, and incubated at room temperature for 1 h with
anti-p24 (Dako Corp., Carpinteria, CA). Slides were then washed, and swine
anti-mouse biotinylated link antibody, alkaline phosphatase-labeled streptavidin,
and naphthol fast red chromogenic substrate (LSAB2 universal alkaline phos-
phatase system; Dako Corp.) were sequentially applied. Sections were then
counterstained in Mayer’s hematoxylin (Fisher Scientific, Pittsburgh, PA). Neg-
ative controls were tissue sections from each explant incubated with normal
mouse ascitic fluid.
To determine which cells were HIV-1 infected, double-staining was performed
by using the Envision double-stain system (Dako Corp.). Sections were pre-
treated as previously described and incubated for 1 h with anti-S-100, anti-CD3,
or anti-CD68 (Dako Corp.). A horseradish peroxidase-labeled polymer was
applied for 30 min, and detection was performed by incubating the specimens for
5 min with liquid diaminobenzidine plus substrate-chromogen. A double-stain
block for endogenous alkaline phosphatase activity was applied for 3 min. Tissue
sections were then incubated for 1 h with the antibody against p24 and followed
by a 30-min application of an alkaline phosphatase-labeled polymer. Fast red
substrate (15 min of incubation) was used to detect the second antigen. Sections
were counterstained in Meyer’s hematoxylin.
MTT assay. Viability of the explants and assessment of microbicide toxicity
was quantified by the reduction of the tetrazolium salt 1-(4,5-dimethylthiazol-2-yl)-
3,5-diphenylformazan (MTT) by viable tissue into a methanol-soluble formazan
product. Cervical explants (4-mm diameter) were incubated with or without
product diluted 1:10 in cDMEM. For comparison, a tissue control that had been
incubated in medium alone was used. After culturing for 18 h, explants were
washed five times in PBS. For determination of toxicity, tissues were immediately
cultured in cDMEM containing MTT (250 ?g/ml) for an additional 3 h at 37°C.
Tissue viability was determined by dividing the optical density of the formazan
product (570 nm) by the dry weight of the explant. The effect of each microbicide
on tissue viability was determined by comparing the viability of the treated
explants to the untreated tissue control. Tissue from a minimum of two donors
was tested for each product. A Tukey’s multiple comparison test (GraphPad
software, version 4) was used to determine significant differences in MTT levels.
Validation of the cervical explant culture system. (i) Tissue
morphology, cellular distribution, and permeability. The de-
sign of the culture is presented in Fig. 1A. Under these con-
ditions, the outer stratified layers of epithelium had sloughed
off by day 3 of culture (Fig. 1C; compare to day of surgery [Fig.
1B]). Although the basal epithelium showed signs of prolifer-
ation at day 7 (Fig. 1D), the normal thickness and differenti-
ation of the stratified epithelium were not recuperated. The
cells and architecture of the submucosa remained intact
throughout the culture period, as shown by histology. Activa-
tion of the tissue with PHA did not adversely affect the tissue’s
architecture (data not shown).
IHC was used to determine the presence of target cells for
HIV-1 (CD3?T cells, S100?dendritic cells, and CD68?mac-
rophages) in the submucosa of the explants (Fig. 2A). CD3?
cells were the most abundant while S100?cells were the least
abundant. CD3?cells showed small round nuclei and little
cytoplasm, CD68?cells had round nuclei with opened chro-
matin and abundant cytoplasm, and S100?cells had round
TABLE 1. Topical microbicide products and placebos tested
Product descriptionPlaceboPlacebo description Product manufacturer
product (PC-515) (25)
Linear sulfated polysaccharide
(lambda- and kappa-
polycarboxylic polymer with
Methyl cellulose2.5% aqueous gel Population Council (New
13% cellulose acetate
Methyl cellulose2.5% aqueous gelLindsley F. Kimball Research
Institute (New York, NY)
and Dow Pharmaceutical
Sciences (Petaluma, CA)
Indevus Pharmaceuticals, Inc.
0.5% and 4% naphthalene
sulfonate (PRO 2000) (28)
Sulfonated polymer with
0.5% and 4% formulations
contain 1.35% and 2%
gelling agent, respectively,
with pH buffering
5% gelling agent with pH
5% lysine dendrimer
(SPL7013) (7, 22)
Surface has been derivatized
with sodium naphthalene
disulfonate groups with
Tightly binding NNRTI;
anti-HIV-1 activity only
0.1% and 1% NNRTI
1% gelling agent with pH
Biosyn, Inc. (Huntingdon
1% antimicrobial peptide
amphipathic peptide with
3.25% aqueous gel Demegen, Inc. (Pittsburgh, PA)
aThe percent concentration of each formulated product is shown.
1772 CUMMINS ET AL.ANTIMICROB. AGENTS CHEMOTHER.
nuclei with opened chromatin and cytoplasm with fingerlike
To determine the integrity of the agar/explant interface,
fluorescent microspheres one-fourth of the size of HIV-1 viri-
ons were applied to the epithelium on day 0 of culture. The
basolateral supernatant was sampled at days 1, 3, and 7 of
culture and tested for fluorescence (Fig. 2B). Although in-
creased permeability was observed by day 7 (15 to 22% trans-
mission), the amount of microspheres transmitted at day 3 was
?10% of that in the blank well. The agar control well had
?3% transmission by day 3 compared to the blank well.
(ii) HIV-1 infection. Initial experiments were performed to
determine whether activation of the tissue was necessary for
productive HIV-1 infection. Dilutions (103to 106TCID50) of
HIV-1BaLwere added to tissues with or without PHA activa-
tion. Reproducible virus replication was observed in all ex-
plants exposed to ?5 ? 104TCID50in PHA-activated tissues.
Overnight infection with 5 ? 104TCID50HIV-1BaLresulted in
a steady rise of virus replication that typically peaked by day 12
to 15 of culture (Fig. 3 and 4A). Despite reproducible virus
replication in PHA-activated tissues, inconsistent levels of vi-
rus replication (?500 pg/ml) were detected in resting tissues
(data not shown). Therefore, to facilitate productive infection
in subsequent experiments, all tissues were pretreated with
PHA (48 to 72 h) and exposed to 5 ? 104TCID50HIV-1 on
day 3 of culture. To demonstrate the ability to infect cervical
FIG. 2. (A) Immunohistochemical analysis of cervical tissue infected with HIV-1BaL. Colocalization of HIV-1 p24 antigen (red) with cell
markers (brown) for T cells (CD3, left), macrophages (CD68, middle), and dendritic cells (S100, right) in human cervical tissues. Arrows point to
macrophages (CD68?cells) with brown cytoplasmic staining that also stain red, denoting the presence of p24 antigen (original magnification, ?50).
(B) The integrity of the cervical explant model system was evaluated by examining the transmission of fluorescent microspheres (0.026-?m
diameter) across the tissues. On the first day of culture, 0.5 ml of the fluorescent microspheres (1 ? 1014/ml) was added to the epithelial surface
of three explants (1, 2, and 3; same tissue donor) and a well filled with 2% agarose. The basolateral medium was sampled at 1, 3, and 7 days, and
the percent transmission was calculated by dividing the fluorescent signal in the basolateral supernatant from the explants and agarose control by
that from the blank well.
FIG. 3. Growth kinetics of HIV-1 in cervical tissues. After an over-
night incubation with 104TCID50HIV-1BaLor primary HIV-1 isolates
(96USSN20/A CCR5/CXCR4-using, 97USSN54/A CCR5-using, and
92US660/B CCR5-using), the explants were washed and maintained in
culture for 21 days. Basolateral supernatants were sampled every 3 to
4 days and assayed for p24gag protein.
VOL. 51, 2007MICROBICIDE TESTING IN CERVICAL EXPLANTS 1773
tissues with primary HIV-1 isolates, explants were successfully
infected with two clade A isolates (96USSN20/A [CCR5/
CXCR4] and 97USSN54/A [CCR5]) and one clade B isolate
(92US660/B [CCR5]) (Fig. 3). These clades were chosen to
show that primary HIV-1 isolates grow in a manner similar to
that of laboratory-adapted HIV-1 and that there was no core-
ceptor preference in this explant culture system. Virus levels at
day 14 postinfection were comparable to those observed after
infection with HIV-1BaL. Further, HIV-1 replication occurred
regardless of the coreceptor used (Fig. 3).
To determine the phenotypes of infected cells, tissues in-
fected with HIV-1 were analyzed by IHC for colocalization of
p24 antigen with target cell markers (Fig. 2A). Although p24
antigen and T cells were observed in the same microscopic
field, infected T cells were difficult to discern because of color
deposition and their low cytoplasmic volume. p24 antigen did
not colocalize with dendritic cells. Macrophages were the only
infected cell type detected in these tissue sections (Fig. 2A).
Assessment of microbicide efficacy. Microbicides and place-
bos were tested for their efficacy against HIV-1 by determining
inhibition of p24 levels in explant supernatants and p24 antigen
in endpoint tissues compared to infection controls. Microbi-
cides that demonstrated efficacy in all tissue explants included
CAP, SPL7013, PRO 2000, and UC781 (Table 2 and Fig. 4).
Virus replication waned over time as demonstrated by a ?80%
inhibition of p24 levels (compared to the virus control in me-
dium) by the end of culture. Moreover, these explants did not
have any detectable HIV-1-infected cells at the study endpoint
(Fig. 4B). Efficacy was only demonstrated by PC-515 in tissue
from one of two donors and by D2A21 in tissue from one of
three donors (Table 2 and Fig. 4). Although a reduction of p24
levels was demonstrated by the placebos (0.25% methyl cellu-
lose, 42% inhibition; polyacrylic acid polymer [0.2%, 59% in-
hibition; 0.5%, 32% inhibition; 0.1%, 44% reduction]; 0.3%
hydroxyethylcellulose, 63% inhibition), all placebos were con-
sidered nonefficacious since endpoint tissues tested positive for
p24 antigen by IHC (Table 2 and Fig. 4B).
Assessment of microbicide toxicity. (i) Acute toxicity (MTT
assay). After overnight treatment with microbicide or placebo,
the tissues were immediately assayed using the MTT assay. No
toxicity was observed in tissues treated with the placebos (95 to
138%) (Fig. 5A). With the exception of CAP, 0.4% PRO 2000,
and the N9 toxic control, treatment of tissues with PC-515,
0.05% PRO 2000, SPL7013, UC781, and D2A21 resulted in
MTT levels similar to or above (82 to 105%) those of the
untreated tissue controls after treatment (Fig. 5A). While re-
duced viability, as determined by reduced levels of MTT, was
observed in tissues treated with CAP (59%), 0.4% PRO 2000
(64%), and N9 (44%), MTT levels were significantly lower only
in the N9-treated tissues compared to the other microbicides
and their placebos (P ? 0.05).
(ii) Endpoint toxicity (histology). Histology was used to
evaluate product toxicity at the study endpoint (Fig. 5B). Tis-
sues treated with CAP, PC-515, 0.05% PRO 2000, SPL7013,
UC781, and D2A21 showed regenerated epithelium and an
intact lamina propria. Denuded epithelium and focal necrosis
of the lamina propria were observed in tissues treated with N9
and 0.4% PRO 2000. The tissue architecture appeared normal
after treatment with the placebos. With the exception of the
reduced MTT viability observed with CAP, the toxicity ob-
served by histology confirmed the MTT results for N9 and
0.4% PRO 2000 (Fig. 5B and Table 2).
Ex vivo organotypic cultures offer a valuable link between in
vitro culture systems and the clinic by providing a controlled
format in which microbicides can be comparatively evaluated
for (i) anti-HIV-1 activity in target cells present within the
submucosa of human tissues and (ii) toxicity against the mu-
FIG. 4. The effects of microbicides and placebos on HIV-1BaLinfection in representative cervical explant cultures. (A) After overnight
incubation with HIV-1BaLalone (5 ? 104TCID50) in medium or a 1:10 dilution of microbicide or placebo (final concentrations are indicated on
the figure) with virus, the explants were washed and maintained in culture. Basolateral supernatants were sampled twice a week for up to 17 days
and assayed for p24gag protein. (B) Comparison of HIV-1-infected cervical explant cultures treated with a representative placebo or each of the
tested microbicides. Products were diluted 1:10 in culture medium (final concentrations are indicated on the figure) containing HIV-1 (5 ? 104
TCID50) and added to the apical surface of the tissue. After overnight incubation, the tissues were washed and maintained in culture. At the study
endpoint, tissues were fixed and examined by immunohistochemical analysis for the presence of p24 antigen (red). Representative photographs
from an explant treated with a placebo (polyacrylic acid) (top left) or each of the microbicides are shown (original magnification, ?50).
TABLE 2. Summary of toxicities and efficacies of products and
placebos in cervical explant cultures
Methyl cellulose (CAP
and PC-515 placebo)
Polyacrylic acid (PRO
aFinal concentration of each product and placebo.
bHistology was used to evaluate tissue sections from treated explants. Prod-
ucts were considered toxic if damage (necrosis) to the epithelium or submucosa
cIHC was used to detect p24 antigen in tissue sections from treated explants.
Values represent numbers of tissue explants with detectable p24?cells per
number of explants tested.
dProducts were considered efficacious if tissues tested negative for p24 antigen
by IHC. Values represent numbers of tissue explants with no detectable p24?
cells per number of explants tested.
VOL. 51, 2007 MICROBICIDE TESTING IN CERVICAL EXPLANTS1775
cosal epithelium. Two other ex vivo ectocervical explant sys-
tems have been described (13, 19). In one method, tissues are
completely embedded/submerged and HIV-1 with or without
microbicide is applied apically (13). In the other method, the
tissue is exposed to HIV-1 with or without microbicide in a
nonpolarized manner, and the explants are cultured while sub-
merged in medium (19). We describe a cervical explant culture
for which tissue is maintained in a polarized state with the
FIG. 5. (A) Effects of diluted microbicides and placebos on cervical explant viability as determined using the MTT assay. After an overnight
(18-h) incubation, the effect of each product on tissue viability was determined by comparing the viability of the treated explants to that of the
untreated tissue control (same donor). Data are expressed as the average percent viabilities (? standard deviations) for each treatment tested in
two donors (*, P ? 0.05). (B) Histopathologic comparison of the effect of the different microbicides in cervical tissues. Microbicides or their
placebos were diluted 1:10 in culture medium and added to the apical surface of the tissue. After overnight incubation, the tissues were washed
and maintained in culture for 21 days. At the study endpoint, tissues were fixed and examined histologically for morphological changes.
Representative photographs for one placebo (polyacrylic acid) and each of the microbicides are shown (hematoxylin and eosin stain; original
magnification, ?50). The final concentrations of each product and placebo are indicated.
1776CUMMINS ET AL.ANTIMICROB. AGENTS CHEMOTHER.
epithelial surface positioned at the air/tissue interface and the
submucosa (stroma) submerged in medium. Unlike the other
explant culture systems (13, 19), this positioning of the cervical
tissue allows application of virus and candidate topical micro-
bicides directly to the epithelium and allows access to the cells
in the submucosa. As indicated in the present study and in
descriptions of other published explant systems (13, 19), cer-
vical explant cultures have several limitations. These include
lack of hormone modulation, lack of recruitment of immune
cells, loss of epithelium, and inability to regenerate/repair.
However, the last two attributes make this system sensitive to
any potential toxic effect by the topical microbicide. Further,
this explant culture demonstrates the capacity to be infected
with HIV-1 with the subsequent evaluation of efficacy of sev-
eral topical microbicide compounds. It is important to note
that there is currently no consensus among the laboratories
using tissue explants regarding appropriate efficacy and toxicity
endpoints. A multisite effort is currently under way to deter-
mine the optimum endpoints and to address tissue replicates
As reported elsewhere (19), there was a loss of the outer
stratified layers of epithelium during the initial 24 to 72 h of
culture of untreated tissues. Although the basal layer remained
intact with gradual regeneration over the culture period, the
epithelium of these explants did not recapitulate to that of the
normal cervix. Since leakiness of the explant culture is a con-
cern with embedded tissues, other investigators have analyzed
the integrity of the system by measuring the transmission of
blue dextran or fluorescent beads (7.2-?m diameter) across the
mucosa (13, 20). Given the size of HIV-1 virions (?100-nm
diameter), smaller fluorescent beads (26-nm diameter) were
used for the permeability studies and allowed to remain for 7
days. Because of the smaller bead size and the modifications to
the embedded culture system, a higher rate of transmission of
the fluorescent beads compared to the 7.2-?m-diameter beads
was observed (20). Despite this, ?10% transmission of the
beads was detected by day 3 of culture. Although a true po-
larized tissue orientation may be difficult to achieve ex vivo,
this does not negate use of this system. Tears and disruptions
to the mucosal epithelium are likely to occur during sexual
intercourse (39). Moreover, sexually transmitted pathogens
can result in ulceration of the mucosa and increased acquisi-
tion of HIV-1 (24, 25). An ideal microbicide should have the
capacity to prevent HIV-1 infection when the virus has its best
opportunity to achieve infection: during the presence of a
compromised epithelium and activated immune targets (i.e.,
inflammation). The cervical explant culture described here
represents a unique system for the preclinical evaluation of
candidate microbicides that takes into account the optimal
environment for HIV-1 infection.
In this cervical explant culture, T cells, macrophages, and
dendritic cells were detected by IHC within cervical tissues.
However, HIV-1 p24 antigen only colocalized with macro-
phages. Macrophages were the primary target for infection in
one culture system (19) while CD4?T cells were the primary
infected cell type in the other culture (20). Since the sensitivity
of the IHC technique and the timing after infection can affect
the ability to detect infected cells, the inability to detect T-cell
infection based on the IHC data does not exclude infection of
these or other nonmacrophage cell types in this cervical ex-
plant culture. Regardless of which cell type was infected,
HIV-1 infection and replication were consistently observed
after infection with HIV-1BaL, thus allowing for the compara-
tive analysis of six topical microbicides for their anti-HIV-1
activity. Microbicides with significant anti-HIV-1 activity typi-
cally demonstrated ?90% (1 to 2 log10) reduction in p24 levels
at day 14. In instances where ?80% reduction of p24 levels was
observed, no p24 antigen could be detected in tissue sections
by IHC. While the carrageenan (PC-515)- and peptide (D2A21)-
based products showed some anti-HIV-1 activity, the CAP,
naphthalene sulfonate (PRO 2000), lysine dendrimer (SPL7013),
and NNRTI (UC781) products consistently blocked HIV-1 infec-
In previously described explant cultures, the toxicities of
candidate topical microbicides were assessed using either bio-
chemical (MTT) (19) or histopathological (43) methods. In the
present study, both methods were employed. While MTT rep-
resents a convenient method for measuring tissue toxicity, it is
a limited approach since it measures general toxicity. Con-
versely, histopathology allows determination of toxicity specific
to the mucosal epithelium or the underlying submucosa. With
the exception of CAP, both methods detected tissue toxicity
after treatment with N9 and 4% PRO 2000. After treatment
with CAP, reduced cell viability was observed by MTT assay,
yet histology revealed no physical damage to the epithelium or
submucosa. The reduced viability observed in the MTT assay
may result from interference by CAP, suggesting a potential
artifact of this biochemical assay. Alternatively, CAP could be
affecting the mitochondrial metabolism without affecting the
overall tissue structure/integrity. While the MTT assay may
prove beneficial for initial toxicity testing in tissues, these data
suggest that it should be used in conjunction with histology to
In the explant culture presented here, virus growth was eval-
uated by monitoring p24 levels over time (at multiple time
points) and determining the presence of p24 antigen by IHC. It
should be noted that efficacy data obtained for several other
explant cultures are limited to one time point (19, 42), are
sometimes presented as percentages of a virus control (thus
not depicting actual p24 levels) (17–18), and/or rely on that
one endpoint (without confirmation by a secondary method,
such as IHC) (42). In the present study, compounds that were
found to be active in both donors showed a ?80% decrease in
culture supernatant p24 levels at the study endpoint and no
p24 staining by IHC.
Six topical microbicides were evaluated for toxicity and ef-
ficacy in a modified cervical explant culture system. With the
exception of 4% PRO 2000, the remaining microbicides were
relatively nontoxic, consistent with toxicity results from other
explant cultures (19, 43), animal studies (5, 31), and safety and
acceptability trials (3, 6, 27, 31, 35, 36, 42). With the exception
of PC-515 and D2A21, the microbicides were efficacious
against HIV-1 infection, consistent with efficacy data from
other cervical explant cultures (1, 18, 19, 43) and preclinical
and animal studies (16, 26, 32). The low toxicity and high
anti-HIV-1 activity of CAP, 0.5% PRO 2000, SPL7013, and
UC781 suggest the need for further testing of these products.
Despite the limitations described above, our study shows
that the cervical explant culture is valuable for evaluating the
efficacy and toxicity of potential microbicides. In addition,
VOL. 51, 2007MICROBICIDE TESTING IN CERVICAL EXPLANTS 1777
when used as a secondary confirmatory assay for compounds
demonstrating activity in cell-based assays (14b, 15), this cer-
vical explant culture can be a valuable tool for selecting priority
candidates to advance to clinical trials.
We thank Ira Horowitz (Emory University) for his support as well as
Rita Lloyd for coordinating collection of the cervical tissues at Grady
Memorial Hospital (Atlanta, GA). We acknowledge Nicola Richard-
son-Harman at BioStat Solutions (Mt. Airy, MD) for performing the
statistical analysis. We also thank Lisa Rohan and Robin Shattock for
J.E.C. was supported by a National Research Service Award (5 F32
HD40727) from the National Institute of Child Health and Human
Development, National Institutes of Health.
None of the authors has a significant financial interest in any of the
commercial products used in this study. The findings and conclusions
in this report are those of the authors and do not necessarily represent
the views of the U.S. Department of Health and Human Services, the
Public Health Service, or the Centers for Disease Control and Preven-
tion. In addition, use of trade names is for identification only and does
not imply endorsement by these government agencies.
1. Abner, S. R., P. C. Guenthner, J. Guarner, K. A. Hancock, J. E. Cummins,
Jr., A. Fink, G. T. Gilmore, C. Staley, A. Ward, O. Ali, S. Binderow, S. Cohen,
L. A. Grohskopf, L. Paxton, C. E. Hart, and C. S. Dezzutti. 2005. A human
colorectal explant culture to evaluate topical microbicides for the prevention
of HIV infection. J. Infect. Dis. 192:1545–1556.
2. Bader, J. P., J. B. McMahon, R. J. Schultz, V. L. Narayanan, J. B. Pierce,
W. A. Harrison, O. S. Weislow, C. F. Midelfort, S. F. Stinson, and M. R.
Boyd. 1991. Oxathiin carboxanilide, a potent inhibitor of human immuno-
deficiency virus reproduction. Proc. Natl. Acad. Sci. USA 88:6740–6744.
3. Balzarini, J., L. Naesens, E. Verbeken, M. Laga, L. Van Damme, M. Parniak,
L. Van Mellaert, J. Anne, and E. De Clercq. 1998. Preclinical studies on
thiocarboxanilide UC-781 as a virucidal agent. AIDS 12:1129–1138.
4. Beer, B. E., and J. E. Cummins, Jr. 2005. Novel strategies in HIV preven-
tion—development of topical microbicides, p. 277–290. In A. M. Doherty
(ed.), Annual reports in medicinal chemistry, vol. 40. Academic Press, San
5. Beer, B. E., G. F. Doncel, F. C. Krebs, R. J. Shattock, P. S. Fletcher, R. W.
Buckheit, Jr., K. Watson, C. S. Dezzutti, J. E. Cummins, E. Bromley, N.
Richardson-Harman, L. A. Pallansch, C. Lackman-Smith, C. Osterling, M.
Mankowski, S. R. Miller, B. J. Catalone, P. A. Welsh, M. K. Howett, B.
Wigdahl, J. A. Turpin, and P. Reichelderfer. 2006. In vitro preclinical testing
of nonoxynol-9 as potential anti-human immunodeficiency virus microbicide:
a retrospective analysis of results from five laboratories. Antimicrob. Agents
6. Bernstein, D. I., L. R. Stanberry, S. Sacks, N. K. Ayisi, Y. H. Gong, J.
Ireland, R. J. Mumper, G. Holan, B. Matthews, T. McCarthy, and N.
Bourne. 2003. Evaluations of unformulated and formulated dendrimer-
based microbicide candidates in mouse and guinea pig models of genital
herpes. Antimicrob. Agents Chemother. 47:3784–3788.
7. Bourne, N., L. R. Stanberry, E. R. Kern, G. Holan, B. Matthews, and D. I.
Bernstein. 2000. Dendrimers, a new class of candidate topical microbicides
with activity against herpes simplex virus infection. Antimicrob. Agents Che-
8. Centers for Disease Control and Prevention. 2004. Opportunity to collabo-
rate in the evaluation of topical microbicides to reduce sexual transmission
of human immunodeficiency virus. Fed. Regist. 69:29138–29139.
9. Centers for Disease Control and Prevention. 2001. Opportunity to collabo-
rate in the evaluation of topical microbicides to reduce heterosexual trans-
mission of human immunodeficiency virus (HIV). Fed. Regist. 66:34453.
10. Centers for Disease Control and Prevention. 2001. Opportunity to collabo-
rate in the evaluation of topical microbicides to reduce transmission of
human immunodeficiency virus (HIV) among men who have sex with men
(MSM). Fed. Regist. 66:34452.
11. Centers for Disease Control and Prevention. 1993. Update: barrier protec-
tion against HIV infection and other sexually transmitted diseases. Morb.
Mortal. Wkly. Rep. 42:589–597.
12. Coggins, C., F. Alvarez, V. Brache, I. S. Fraser, M. Lacarra, P. Lahteenmaki,
R. Massai, D. R. Mishnell, D. M. Phillips, A. M. Salvatierra, and C. J. Elias.
1997. Female-controlled methods to prevent sexual transmission of HIV.
13. Collins, K. B., B. K. Patterson, G. J. Naus, D. V. Landers, and P. Gupta.
2000. Development of an in vitro organ culture model to study transmission
of HIV-1 in the female genital tract. Nat. Med. 6:475–479.
14. Cummins, J. E., and C. S. Dezzutti. 2000. Sexual HIV-1 transmission and
mucosal defense mechanisms. AIDS Rev. 2:144–154.
14a.Cummins, J., N. Richardson-Harman, J. Bremer, P. Anton, C. Dezzutti, P.
Gupta, N. Lurain, L. Margolis, R. Shattock, and P. Reichelderfer. 2006.
Abstr. 13th Conf. Retrovir. Opportun. Infect., Denver, CO, p. 378.
14b.Cummins, J., M. Jones, C. Lackman-Smith, and B. Beer. 2006. Abstr. Mi-
crobicides 2006, Cape Town, South Africa, p. 212.
15. D’Cruz, O. J., and F. M. Uckun. 2004. Clinical development of microbicides
for the prevention of HIV infection. Curr. Pharm. Des. 10:315–336.
16. Dezzutti, C. S., V. N. James, A. Ramos, S. T. Sullivan, A. Siddig, T. J. Bush,
L. A. Grohskopf, L. Paxton, S. Subbarao, and C. E. Hart. 2004. In vitro
comparison of topical microbicides for prevention of human immunodefi-
ciency virus type 1 transmission. Antimicrob. Agents Chemother. 48:3834–
17. Finney, D. J. 1952. Probit analysis: a statistical treatment of the sigmoid
response curve. Cambridge University Press, Cambridge, United Kingdom.
18. Fletcher, P., Y. Kiselyeva, G. Wallace, J. Romano, G. Griffin, L. Margolis, and
R. Shattock. 2005. The nonnucleoside reverse transcriptase inhibitor UC-781
inhibits human immunodeficiency virus type 1 infection of human cervical tissue
and dissemination by migratory cells. J. Virol. 79:11179–11186.
19. Greenhead, P., P. Hayes, P. S. Watts, K. G. Laing, G. E. Griffin, and R. J.
Shattock. 2000. Parameters of human immunodeficiency virus infection of
human cervical tissue and inhibition by vaginal virucides. J. Virol. 74:5577–
20. Gupta, P., K. B. Collins, D. Ratner, S. Watkins, G. J. Naus, D. V. Landers,
and B. K. Patterson. 2002. Memory CD4?T cells are the earliest detectable
human immunodeficiency virus type 1 (HIV-1)-infected cells in the female
genital mucosal tissue during HIV-1 transmission in an organ culture system.
J. Virol. 76:9868–9876.
21. International Working Group on Vaginal Microbicides. 1996. Recommen-
dations for the development of vaginal microbicides. AIDS 10:1–6.
22. Jiang, Y. H., P. Emau, J. S. Cairns, L. Flanary, W. R. Morton, T. D.
McCarthy, and C. C. Tsai. 2005. SPL7013 gel as a topical microbicide for
prevention of vaginal transmission of SHIV89.6P in macaques. AIDS Res.
Hum. Retrovir. 21:207–213.
23. Joint United Nations Program on HIV/AIDS (UNAIDS). 2005. AIDS epi-
demic update, December 2004. Joint United Nations Program on HIV/AIDS
(UNAIDS), Geneva, Switzerland.
24. Lushbaugh, W. B., A. C. Blossom, P. H. Shah, A. K. Banga, J. M. Jaynes,
J. D. Cleary, and R. W. Finley. 2000. Use of intravaginal microbicides to
prevent acquisition of Trichomonas vaginalis infection in Lactobacillus-pre-
treated, estrogenized young mice. Am. J. Trop. Med. Hyg. 63:284–289.
25. Maguire, R. A., N. Bergman, and D. M. Phillips. 2001. Comparison of
microbicides for efficacy in protecting mice against vaginal challenge with
herpes simplex virus type 2, cytotoxicity, antibacterial properties, and sperm
immobilization. Sex. Transm. Dis. 28:259–265.
26. Manson, K. H., M. S. Wyand, C. Miller, and A. R. Neurath. 2000. Effect of
a cellulose acetate phthalate topical cream on vaginal transmission of simian
immunodeficiency virus in rhesus monkeys. Antimicrob. Agents Chemother.
27. Mayer, K. H., S. A. Karim, C. Kelly, L. Maslankowski, H. Rees, A. T. Profy,
J. Day, J. Welch, and Z. Rosenberg. 2003. Safety and tolerability of vaginal
PRO 2000 gel in sexually active HIV-uninfected and abstinent HIV-infected
women. AIDS 17:321–329.
28. Mohan, P., R. Singh, and M. Baba. 1991. Potential anti-AIDS agents. Syn-
thesis and antiviral activity of naphthalenesulfonic acid derivatives against
HIV-1 and HIV-2. J. Med. Chem. 34:212–217.
29. Neurath, A. R., N. Strick, Y. Y. Li, K. Lin, and S. Jiang. 1999. Design of a
“microbicide” for prevention of sexually transmitted diseases using “inac-
tive” pharmaceutical excipients. Biologicals 27:11–21.
30. Nicolosi, A., M. L. Correa Leite, M. Musicco, C. Arici, G. Gavazzeni, A.
Lazzarin, et al. 1994. The efficiency of male-to-female and female-to-male
sexual transmission of the human immunodeficiency virus: a study of 730
stable couples. Epidemiology 5:570–575.
31. Niruthisard, S., R. E. Roddy, and S. Chutivongse. 1991. The effects of
frequent nonoxynol-9 use on the vaginal and cervical mucosa. Sex. Transm.
32. Otten, R. A., D. R. Adams, C. N. Kim, E. Jackson, J. K. Pullium, K. Lee, L. A.
Grohskopf, M. Monsour, S. Butera, and T. M. Folk. 2005. Multiple vaginal
exposures to low doses of R5 simian-human immunodeficiency virus: strat-
egy to study HIV preclinical interventions in nonhuman primates. J. Infect.
33. Padian, N., L. Marquis, D. P. Francis, R. E. Anderson, G. W. Rutherford,
P. M. O’Malley, and W. Winkelstein, Jr. 1987. Male-to-female transmission
of human immunodeficiency virus. JAMA 258:788–790.
34. Padian, N. S., S. C. Shiboski, S. O. Glass, and E. Vittinghoff. 1997.
Heterosexual transmission of human immunodeficiency virus (HIV) in
northern California: results from a ten-year study. Am. J. Epidemiol.
35. Patton, D. L., Y. T. Cosgrove Sweeney, L. K. Rabe, and S. L. Hillier. 2002.
Rectal applications of nonoxynol-9 cause tissue disruption in a monkey
model. Sex. Transm. Dis. 29:581–587.
1778 CUMMINS ET AL.ANTIMICROB. AGENTS CHEMOTHER.
36. Phillips, D. M., C. L. Taylor, V. R. Zacharopoulos, and R. A. Maguire. 2000. Download full-text
Nonoxynol-9 causes rapid exfoliation of sheets of rectal epithelium. Contra-
37. Quinn, T. C., and J. Overbaugh. 2005. HIV/AIDS in women: an expanding
epidemic. Science 308:1582–1583.
38. Reed, L. J., and J. Muench. 1938. A simple method of estimating fifty per
cent endpoints. Am. J. Hyg. 27:493–497.
39. Shattock, R. J., and J. P. Moore. 2003. Inhibiting sexual transmission of
HIV-1 infection. Nat. Rev. Microbiol. 1:25–34.
40. Stein, Z. A. 1990. HIV prevention: the need for methods women can use.
Am. J. Public Health 80:460–462.
41. Van Damme, L., G. Ramjee, M. Alary, B. Vuylsteke, V. Chandeying, H. Rees,
P. Sirivongrangson, L. Mukenge-Tshibaka, V. Ettiegne-Traore, C. Uaheow-
itchai, S. S. Karim, B. Masse, J. Perriens, and M. Laga. 2002. Effectiveness
of COL-1492, a nonoxynol-9 vaginal gel, on HIV-1 transmission in female
sex workers: a randomised controlled trial. Lancet 360:971–977.
42. Van Damme, L., A. Wright, K. Depraetere, I. Rosenstein, V. Vandersmissen,
L. Poulter, M. McKinlay, E. Van Dyck, J. Weber, A. Profy, M. Laga, and V.
Kitchen. 2000. A phase I study of a novel potential intravaginal microbicide,
PRO 2000, in healthy sexually inactive women. Sex. Transm. Infect. 76:126–
43. Zussman, A., L. Lara, H. H. Lara, Z. Bentwich, and G. Borkow. 2003.
Blocking of cell-free and cell-associated HIV-1 transmission through human
cervix organ culture with UC781. AIDS 17:653–661.
VOL. 51, 2007 MICROBICIDE TESTING IN CERVICAL EXPLANTS1779