Effects of the levonorgestrel-containing intrauterine device, copper intrauterine device, and levonorgestrel-containing oral contraceptive on susceptibility of immune cells from cervix, endometrium and blood to HIV-1 fusion measured ex vivo
Effects of the levonorgestrel-containing intrauterine device, copper intrauterine device, and levonorgestrel-containing oral contraceptive on susceptibility of immune cells from cervix, endometrium and blood to HIV-1 fusion measured ex vivo
Editor: Pierre Roques 3
Marielle Cavrois 0 3
Joan F. Hilton 1 3
Nadia R. Roan 0 3
Margaret Takeda 3
Dominika Seidman 3
Sarah Averbach 2 3
Eric Chang 0 3
Nandhini Raman 0 3
Ruth Greenblatt 1 3
Barbara L. Shacklett 3
Karen Smith-McCuneID 3
0 Gladstone Institute of Virology and Immunology , San Francisco, California , United States of America
1 Department of Epidemiology and Biostatistics, University of California San Francisco , San Francisco , California, United States of America, 3 Department of Urology, University of California San Francisco , San Francisco , California, United States of America, 4 Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco , San Francisco, California , United States of America
2 Department of Obstetrics, Gynecology and Reproductive Sciences, University of California San Diego, San Diego, California, United States of America, 6 Departments of Clinical Pharmacy and Medicine, University of California San Francisco , San Francisco , California, United States of America, 7 Department of Medical Microbiology and Immunology, School of Medicine, University of California Davis , Davis, California , United States of America
3 Current address: Gilead Sciences Inc , Foster City, California , United States of America
Globally, HIV/AIDS is a leading cause of morbidity worldwide among reproductive-aged cisgender women, highlighting the importance of understanding effects of contraceptives on HIV-1 risk. Some observational studies suggest there may be an increased risk of HIV-1 acquisition among women using the long-acting injectable progestin contraceptive, depomedroxyprogesterone acetate. The potential mechanism of this susceptibility is unclear. There are few data on the role of the upper female reproductive tract in HIV-1 transmission, and the mechanisms of HIV-1 infection are likely to differ in the upper compared to the lower reproductive tract due to differences in tissue composition and variable effects of sex steroids on mucosal immune cell distribution and activity. In this study, we measured the susceptibility of mucosal immune cells from the upper female reproductive tract to HIV-1 entry using the virion-based HIV-1 fusion assay in samples from healthy female volunteers. We studied 37 infectious molecular clones for their ability to fuse to cells from endometrial biopsies in three participants and found that subtype (B or C) and origin of the virus (transmitted founder or chronic control) had little influence on HIV-1 fusion susceptibility. We studied the effect of contraceptives on HIV-1 susceptibility of immune cells from the cervix, endometrium and peripheral blood by comparing fusion susceptibility in four groups: users of the copper intrauterine device (IUD), levonorgestrel-containing oral contraceptive,
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
Funding: This study was funded by the National
Institute of Allergy and infectious Diseases,
National Institutes of Health, through R01AI111925
(PI Smith-McCune) and by the National Center for
Advancing Translational Sciences, National
Institutes of Health, through UCSF-CTSI Grant
Number UL1 TR991872 to support the collection
of clinical samples. Its contents are solely the
responsibility of the authors and do not necessarily
represent the official views of the NIH. The funders
had no role in study design, data collection and
analysis, decision to publish, or preparation of the
Competing interests: SA is on the Board of
Directors of One Heart World Wide, an NGO that
does safe motherhood work in Nepal, is on the
Advisory Board of Center for AIDS Research at
University of California San Diego and has a
research grant from the Society of Family Planning
to study early initiation of postpartum
contraception. BLS receives funding from the
National Institutes of Health (NIAID R01 AI057020;
NIDDK R01 DK108350; NCI P30 CA093373) and
research contracts from Gilead Sciences. This does
not alter our adherence to PLOS ONE policies on
sharing data and materials.
levonorgestrel-containing IUD and unexposed controls (n = 58 participants). None of the
contraceptives was associated with higher rates of HIV-1 entry into female reproductive
tract cells compared to control samples from the mid-luteal phase.
An estimated 14.3% of women of reproductive age use intrauterine devices (IUDs) globally
]. However, little is known about the impact of IUD use on mucosal immunity of the female
reproductive tract, and whether it influences risk of HIV-1 infection. Most literature on HIV-1
risk in IUD users was published mainly in the 1990s and focused on the copper IUD, before
the now commonly used levonorgestrel (LNG)-containing IUD was widely available. In 2007
and 2012, the World Health Organization (WHO) convened technical panels to discuss
hormonal contraceptives, IUD use and HIV-1 risk [2, 3]. They concluded that none of the existing
prospective studies found an association between IUD use and HIV-1 acquisition, but the
numbers of studies, and of observations of IUD-users, were small [
]. The available
crosssectional studies were mainly focused on the copper IUD and were limited by methodological
issues such as failure to control for confounding factors, and unclear timing between IUD use
and HIV-1 acquisition . The panel concluded: ?Current evidence suggests that the use of
the copper IUD does not increase the risk of HIV-1 acquisition. However, this evidence is
limited and weak.? The panels also concluded that most available research assessed hormonal
contraceptives or progestin-only injectable contraceptives such as depo-medroxyprogesterone
acetate, whereas there is little evidence about the potential relationship between HIV-1 risk
and other contraceptive methods such as IUDs. The 2012 panel stressed the need for ongoing
research to evaluate the effects of hormonal contraceptives on HIV-1 acquisition risk .
Understanding the effects of contraceptives on HIV-1 acquisition is essential given that
HIV/AIDS is a leading cause of morbidity and mortality in women in their reproductive years
. In addition, observational studies suggest an increased risk of HIV-1 acquisition among
women using hormonal contraceptives, specifically the long-acting injectable progestin
contraceptive, depo-medroxyprogesterone acetate [
]. A recent randomized trial compared rates
of HIV acquisition among women using depo-medroxyprogesterone acetate, a copper IUD
and a levonorgestrel implant, and showed no significant differences in HIV risk between the
groups; these results are reassuring about the safety of each of these methods [
]. This trial
however did not study oral contraceptives or the LNG-IUD, as was done in this study.
There are few data on the risk of HIV-1 acquisition relating to upper female reproductive
tract (FRT), which includes the endocervix and endometrium. The mechanisms of HIV-1
infection are likely to differ in the upper compared to the lower FRT due to cyclic effects of sex
hormones on relevant characteristics of mucosal immunity [
]. Additionally, the upper
FRT is lined by a single layer of columnar epithelium which is more susceptible to injury and
absorption of exogenous substances than the vagina and ectocervix, which are lined with a
multi-layered squamous epithelium that functions effectively as a barrier to systemic access.
The parallels between the immunological characteristics of the upper FRT and the
gastrointestinal tract highlight the importance of studying the upper FRT as a portal of HIV-1 acquisition
. Indeed, studies in primates confirm that SIV infection can occur in the upper FRT [
We previously reported that the LNG-IUD created both inflammatory and
immunosuppressive changes in the mucosal microenvironment in the upper FRT [
]. Samples from the
endometrium and endocervix of women using the LNG-IUD showed higher proportions of
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CD4+ T-cells expressing both CXCR4 and CCR5, the two main HIV-1 coreceptors, compared
to controls. Activated CD4+ T-cells (i.e. CD4+CD38+HLA-DR+ cells) were also more abundant
in endometrial samples from LNG-IUD users than controls. These phenotyping experiments
suggested that the upper FRT of LNG-IUD users may be more sensitive to HIV-1 acquisition
as compared to non-users. However, in that prior study we did not test susceptibility of FRT
cells to HIV-1 infection. Furthermore, given that the LNG-IUD combines a hormonal
treatment (levonorgestrel) with a device (the IUD), the effects observed in our previous study could
result from either or both interventions. In this study, we compared immune cells of women
exposed to levonorgestrel systemically using LNG-based combined oral contraceptives
(COCs), locally using the LNG-IUD, to a hormonally inert IUD (copper IUD), or to neither
IUD nor hormone, for their susceptibilities to HIV-1 entry using the virion fusion assay. We
chose the following anatomic sites as sources of immune cells to study HIV-1 susceptibility:
peripheral blood, as it is a commonly used reference source of cells for HIV-1 infection studies;
the cervical transformation zone, as it constitutes the junction between the upper and lower
reproductive tracts, is enriched for immune cells [
] and is the site at which
HIV-contaminated semen would first make contact with the upper FRT; and the endometrium, as it is the
primary site of contraceptive effects. Using samples from these sites obtained from healthy
female participants, we measured ex vivo the HIV-1 fusion susceptibility of immune cells as
a surrogate marker for the potential risk of HIV-1 susceptibility in women using these
Materials and methods
This cross-sectional study compares HIV-1 fusion to immune cells from the blood,
endometrium and cervix from samples donated by 4 groups of HIV-negative women: women using no
hormonal or intrauterine contraception (controls), women using copper IUDs, women using
LNG-IUDs, and women using LNG-containing COCs. The UCSF Human Research
Protection Program & IRB approved the study protocol, recruiting and consent materials.
Recruitment and screening of human volunteers
Healthy women volunteers age 18?45 years from San Francisco and the greater Bay Area were
recruited via flyers placed in a variety of venues, local publications, and social media.
Volunteers were pre-screened by telephone to ensure they were eligible for the control group or
were using one of the designated methods of contraception for the past 6?48 months, with a
goal of recruiting an equal number of women per group. Participants in the COC group were
included if they were using a 28-day pill pack of combined contraceptive (estrogen plus LNG)
containing either 0.10 or 0.15 mg of LNG per tablet on a cyclic schedule; participants taking
the pills continuously were excluded. Participants in the control and copper IUD groups were
included if they had regular periods every 21?35 days. Potential participants were ineligible if
they had undergone hysterectomy, were breast-feeding, were within 6 months of parturition,
had abnormal cervical cytology in the past year, used systemic corticosteroids or
immunemodulating therapies or used non-steroidal anti-inflammatory agents daily, were unwilling/
unable to refrain from vaginal intercourse for 3 days prior to specimen collection, or were
unwilling to use non-lubricated condoms throughout the duration of the study. Candidates
were scheduled for a screening visit, at which time study personnel explained procedures in
detail; obtained written informed consent and demographic information; collected urine to
test for pregnancy, Chlamydia trachomatis and Neisseria gonorrheae, and collected blood for
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HIV-1 serology. Candidates were ineligible if they had a positive result on any of those tests or
had clinical evidence of vaginitis, vaginosis or pelvic inflammatory disease.
Participants were taught how to use urine testing kits for detection of luteinizing hormone
(LH) (ClearBlue Ovulation test Digital, Proctor and Gamble, Cincinnati, OH) and were asked
to phone study personnel when the test showed a positive result. Women in the control and
copper IUD groups were asked to present for biopsies 7 to 11 days after a positive home urine
LH test. Women using LNG-IUDs were asked to present for biopsies 7 to 11 days after a
positive home urine LH test or at their convenience after testing for 2 months with no positive
result, whichever came first. COC users were asked to present for biopsies on day 12?16 of
their pill pack. All participants had a blood sample collected for measurement of plasma
progesterone level on the day of biopsy, and in the COC and LNG-IUD groups, for measurement
of LNG levels. All participants were confirmed to have a negative urine pregnancy test on the
day of biopsy.
For sample collection, a speculum was inserted into the vagina, the cervix was visualized
and the posterior vaginal fornix was swabbed with a Q-tip for determination of pH on pH
paper (VWR, Visalia, CA) and a second Q-tip for measurement prostate specific antigen
(Abacus Diagnostics, West Hills, CA), a marker of recent vaginal intercourse. If cervicitis or
vaginitis was noted, a wet mount was performed, and the specimen collection visit was canceled if
bacterial vaginosis, candidiasis or trichomoniasis was diagnosed. Blood was obtained for
isolation of peripheral blood mononuclear cells and for measurement of progesterone (Quest
Diagnostics, West Hill, CA) and levonorgestrel (University of Southern California Reproductive
Endocrinology Laboratory, Los Angeles, CA). A speculum was inserted into the vagina and
the cervix was washed with Lugol?s iodine solution and an endometrial biopsy was obtained
with a 3 mm biopsy cannula (Miltex brand Softflex) inserted through the cervical os. If
necessary, the ectocervix was injected with 1% lidocaine and a tenaculum was placed for retraction.
If the amount of endometrial tissue was assessed to be inadequate, a second pass of the cannula
was made. The cervical transformation zone (TZ) was identified as the junction between the
Lugol?s staining and non-staining epithelium and 2 biopsies at separate locations were taken
using a Tischler biopsy forceps; if the TZ could not be identified because the entire ectocervix
was stained, the biopsies were taken with one of the biopsy prongs inside the os. Some
participants could not provide biopsies from both anatomical sites (endometrium and cervix) due to
intolerance of the procedure and some participants were unable to tolerate phlebotomy. Of 58
participants studied, 40 (69%) donated samples from all three sites (endometrium, cervix and
blood), 17 (29%) from two sites and 1 (2%) from the cervix only.
Preparation of single cell suspensions
Sample preparation, the HIV-1 fusion assay, and FACS analysis were conducted by a
researcher blinded as to the group assignment of the women who donated the samples.
Endometrial and cervical biopsies were placed in a 15 ml centrifuge tube containing 5 ml of RPMI
supplemented with 2% fetal bovine serum and were processed and frozen within 2?3 h of
collection. When needed, the cervical biopsies were cut into smaller pieces (2 x 2 mm). To
generate single cell suspensions, the biopsies were first centrifuged at 365 x g for 5 min. The
supernatant was removed by aspiration and the sample resuspended in 1 ml of phosphate buffered
saline. After addition of 1 ml of the 2 X digestion buffer containing collagenase Type I,
hyaluronidase, and penicillin/streptomycin as described [
]; digestion was allowed to proceed for
1.5 h at 37?C. On a few occasions, when tissue pieces were small, the digestion was stopped
4 / 23
earlier when the entire tissue was visibly digested. The cells were washed, pelleted and frozen
in 1ml of fetal bovine serum containing 10% dimethylsulfoxide. An aliquot was taken from the
freshly digested suspension and stained with an immunostaining panel that included an
antiCD45 antibody conjugated to APC (BD biosciences, reference 555485) and anti-CD235a
conjugated to FITC (BD biosciences, reference 559943), a marker of red blood cells (RBCs).
The ratio of RBCs to white blood cells (WBCs) in blood is typically 700:1, and we excluded
samples with >7000 RBC per WBC as those samples had high levels of blood contamination
( 10%). This resulted in exclusion of one endometrial and 2 cervical biopsies. Samples with
total numbers of white blood cells < 200 were also excluded. The median yield of white blood
cells from endometrial biopsies was 1.3 x 106 (range 0.033?47 x 106), from cervical
transformation zone biopsies was 110,000 (range 0.005?4.2 x 106) and from blood was 2.4 x106 (range
0.3?26 x 106).
BlaM-Vpr containing HIV-1 virions were produced by transfection of 293T cells with the
indicated molecular clones as described [
] and the p24Gag content measured with the FlaQ assay
] to allow normalization of virus input to 500 ng p24Gag in all assays. The fusion assay was
conducted as previously described [
]. We had previously shown that triplicate infections
were not needed and that the major source of variation was the donor of target cells [
Single cell suspensions were thawed and counted, and aliquots containing at least 5 x 104 cells
from cervical biopsies, 4 x 105 cells from endometrial biopsies or 3 x 106 cells from peripheral
blood were infected for 1.5 h with BlaM-Vpr containing HIV-1 virions (500 ng p24Gag/ml).
After infection the cells were washed and loaded with CCF2, the BlaM substrate, and incubated
overnight at room temperature to allow cleavage by BlaM. Cells from FRT biopsies were
stained with the FRT immunophenotyping panel composed of 11
antibodies:anti-CD3BUV737 (BD Biosciences #564307), anti-CD4-BUV395 (BD Biosciences #563550),
antiCD14-BV650 (Biolegend # 301836), anti-HLA-DR-PerCP-Cy5.5 (Biolegend #307630),
antiCD45-BV605 (BD Biosciences #564047), anti-CD207 (Biolegend #352204),
anti-CD45-ROECD (Beckman coulter #IM2712U), anti-CD163-PE-Cy7 (Biolegend #333614),
anti-CD1aA700 (Biolegend #300120), anti-CD69-APC7 (BD Biosciences #560737), anti-LIN [i.e.
antiCD56-APC (BD Biosciences #555518), anti-CD20-APC (BD Biosciences #559776) and
antiCD19-APC (BD Biosciences # 3404370)] and LIVE/DEAD Green (Invitrogen). We noticed
some inconsistencies in fluorescence measurements for CD45RO-ECD marker for the cervical
and endometrial samples. We elected not to use this marker and instead to phenotype the
CD4+ T-cells using only CD69. Given that most of the cells in these FRT tissues are memory
cells (Fig 1A), this omission had minimal impact on the phenotyping).
Cells from peripheral blood were stained with the immunophenotyping panel composed of
8 antibodies: anti-CD3-BUV737 (BD biosciences #564307), anti-CD4-BUV395 (BD
biosciences #563550), anti-CD14-BV650 (Biolegend # 301836), anti-HLA-DR-PerCP5.5 (Biolegend
#307630), anti-CD1c-PE (Affymetrix #12-0015-42), anti-CD303-PE-Cy7 (Biolegend #354214),
anti-CD141-APC-Cy7 (Miltenyi # 130-098-217), anti-LIN (same as above) and LIVE/DEAD
Cells from all sample types were fixed in phosphate-buffered saline containing 1.2%
paraformaldehyde and acquired on a FACS Aria. Compensation beads were stained and acquired
in parallel with the fusion assay. Compensation and gating were performed on Flow Jo Version
X. The number of cells within each gate (immune cells present and number fused, by
phenotype) was exported to an Excel spreadsheet for further analysis.
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Fig 1. Fusion assay combined with immunostaining to identify and quantify phenotypes of immune cells from endometrium
that support HIV-1 fusion. (A) Gating strategy: The first 4 multicolor FACS plots allow the identification of the ?Live CD45+?,
which represent the total number of immune present in the endometrial sample. From the ?Live CD45+? gate, the ?BlaM+ cells? can
be identified and represent the immune cells that supported NL-109F4 viral fusion. The FACS plots on the gray background show
the gating strategy to identify 11 cellular subsets among the ?all immune cells? (dark gray) and among ?fused immune cells? (light
gray). Note that LIN+DR- or LIN+DR+ can be used as internal control for gating the fused cells (bottom left inset). (B) For an
endometrial biopsy sample from a single participant, paired bar graphs show the distribution of immune cell phenotypes (left) and
prevalence of fusion susceptibility by phenotype (right). In this sample our immunophenotyping panel recognized the phenotype of
more than 90% of the immune cells present in the biopsies, and overall fusion susceptibility was 13.6%.
Within contraceptive groups we describe characteristics of the 58 healthy women participants
who donated samples for this study (Table 1). For categorical characteristics we report counts
and proportions and compare groups using Chi-square tests of independence with 3x(R-1)
6 / 23
a Kruskall Wallis test
b Chi square test of association
c S1 Fig: For virus ZM247Fv2, N = 51 samples infected. For viruses REJO and RHPA, N = 40 samples infected.
degrees of freedom (df), where R is the number of categories of the variable. For continuous
characteristics we report median (and IQR or range) and Kruskal-Wallis tests with 3 df. We
also report the number of samples available for analysis by type and group. Most experiments
included multiple samples per woman, with replicates arising in two ways: 1) multiple
singlecell suspensions created from a single endometrial sample, each infected with a distinct virus;
and 2) distinct samples from three anatomic sites, all infected with the same virus.
7 / 23
To convey methods for analyses of immune cells and their fusion to viruses, it is helpful to
introduce some replicate-level notation. In biopsy and blood samples, staining by
immunophenotyping panels allowed identification of J specific immune cell phenotypes; we grouped
all other phenotypes as ?other.? We denote the J immune cell counts per replicate by Cj,
j = 1,2,. . .,J, which sum to the total, Ctotal, and the J quantities of fused immune cells by Fj,
j = 1,2,. . .,J, which sum to the total, Ftotal. We used these quantities in two ways. When
describing individual replicates, we calculated the observed phenotypic distribution of immune cells
by Cj / Ctotal, j = 1,2,. . ., J, and of fused immune cells by Fj / Ftotal, j = 1,2,. . .,J, and calculated
the prevalence of HIV-1 fusion susceptibility (FS) per phenotype, Fj / Ctotal, j = 1,2,. . .,J, and
overall, Ftotal / Ctotal. Therefore, all summaries of interest are proportions.
When evaluating groups of replicates, we used a generalized estimating equation (GEE)
model with a logit link and binomial distribution to estimate a mean proportion from the
components that form each ratio (e.g., Ftotal / Ctotal) as a function of covariate(s) (e.g.,
phenotype X contraceptive group), and back-transform the logit-scale results to percentiles. All
estimates of 95% confidence intervals (CI) are based on robust standard error (SE) and GEE score
tests with 3 degrees of freedom (df) were used to assess statistically significant variation among
contraceptive groups. To minimize inflation of estimates, participants were excluded from
calculation of fused-cell distributions if Ftotal <6 (1 endometrial sample and 2 cervical samples).
Statistical analyses were conducted using SAS version 9.4, with plots created using Excel.
To illustrate replicate-level results generated by the fusion assay, for an endometrial biopsy
sample and a blood sample, each from a single control participant not included in subsequent
analyses, we present the observed distribution of immune cells (Cj / Ctotal, j = 1,2,. . .,12) and
FS prevalence by phenotype (Fj / Ctotal, j = 1,2,. . .,12) using bar graphs. For Fig 2, using
endometrial biopsy samples from n = 3 participants, immune cells and fused cells were quantified
by phenotype (Cj and Fj, j = 1, 2,. . .,12, respectively) for each of 37 distinct viruses. In Fig 2A,
for each virus, we estimated the mean (95% confidence interval) (CI) of the sampling
distribution of fusion susceptibility prevalence. These CIs use the standard deviation (SD) rather than
the standard error (SE), and the coefficient of Student?s t distribution with 2 degrees of
freedom (df). Mean FS prevalence and robust SEs were generated via a generalized estimating
equation (GEE) model of the ratio Ftotal / Ctotal as a function of virus, using a logit link and
assuming a binomial distribution, accounting for correlated outcomes among viruses within
participant. We then calculated the virus-specific standard deviation (SD) from the SE,
calculated t-based confidence limits, and back-transformed the logit-scale mean (95% CI) to
To describe variation in FS prevalence across viruses and participants, 37 replicates from
endometrial samples donated by three participants were each infected with a distinct virus.
We used control participants not using hormonal or IUD contraceptives to avoid measuring
variation due to contraceptive effects; we used samples from three participants in order to get
an appreciation for the variability between participant samples; the specific samples used were
chosen at the beginning of the study based on high yield of tissue from the endometrial
biopsies, since the assay required a large number of cells. For each virus, we estimated the sampling
distribution of overall FS prevalence by: generating the mean and SD-based 95% CI via a GEE
model of Ftotal/Ctotal as a function of virus; calculating the SD from the robust SE and
calculating SD-based confidence limits using the coefficient of Student?s t distribution with 2 df. To
examine virulence by TF/CC status and B/C subtype, we estimated mean (95% CI) overall FS
prevalence as a function of these strata, accounting for correlated outcomes among viruses
within participant. (C) For each replicate, we calculated the observed immune cell distribution
and FS prevalence by phenotype, then calculated arithmetic means across viruses for each
8 / 23
Fig 2. Fusion of 37 primary HIV-1 viruses to single cell suspensions from endometrial biopsies from 3 participants.
Thirtyseven viruses containing BlaM-Vpr were obtained by co-transfection of 293T cells with infectious molecular clones and
pcDNA43-BlaM-Vpr. Viral supernatants were concentrated and 500 ng p24Gag was used to infect single cell suspensions generated from
EMBs from three participants not using a contraceptive (EMB-3064, EMB-3041 and EMB-3782). The fusion assay was combined
with immunostaining and gating as performed in Fig 1. (A) For each of 37 viruses, we display the sampling distribution of the
prevalence of immune cells supporting fusion (i.e. % Live CD45+BlaM+ cells) based on n = 3 participants. Here, the horizontal bar
represents the mean while the vertical bar indicates the 95% confidence interval (CI) based on Student?s t-distribution (2 df). (B)
Mean (95% CI) fusion susceptibility of 37 viruses grouped by HIV-1 status at the time of viral isolation (founder [TF] vs. chronic
[CC]) and subtype (B vs. C). (C) For each participant, distribution of immune cells and fusion susceptibility prevalence by
phenotype, averaged over 37 viruses.
To estimate immune cell abundance and susceptibility to HIV-1 infection, one replicate per
available sample type was infected with virus ZM247Fv2 for each participant. All analyses were
stratified by sample type (cervix, endometrium or blood). By phenotype, we summarized
median counts and mean proportions of immune cells (Cj / Ctotal), fused cells (Fj / Ftotal), and
FS prevalence (Fj / Ctotal), where medians were generated via descriptive statistics and means
were generated via GEE models of the respective ratio as a function of phenotype. In addition,
we use boxplots to describe heterogeneity among participants in cell counts by phenotype.
To estimate the effect of contraceptive exposure on HIV-1 susceptibility, we reduced
phenotype categories of interest to four, and grouped remaining phenotypes as ?other.? The
first set used the samples described above, and stratified analysis by sample type. The second
set used three replicates per participant from endometrial samples, each infected with one
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virus (ZM247Fv2, REJO, or RHPA) and stratified by virus. Using stratified GEE models, we
estimated mean FS prevalence (Fj / Ctotal) as a function of phenotype, contraceptive group,
and their interaction; from the same models we estimated mean (95% CI) overall FS
prevalence (Ftotal / Ctotal) per contraceptive group. FS prevalence estimates are presented via bar
Fusion assay adapted to cells from endometrial biopsies
The fusion assay allows quantification of HIV-1 entry into target cells [
] including those
from the FRT [
] and relies on the transfer of BlaM-Vpr chimera from the HIV-1 virus to the
target cells. This transfer can then be revealed by loading the cells with CCF2, the fluorescent
substrate of BlaM. To profile the cells that supported fusion in endometrial and cervical TZ
biopsies, we developed an immunostaining panel that allows identification of the various cell
populations that support HIV-1 entry. The staining panel includes markers for CD4+ T-cells
(CD3, CD4, CD45RO, CD69), macrophages (CD14, HLA-DR and CD163), DCs (CD1a) and
Langerhans cells (CD207). Additionally, a set of lineage markers (CD56, CD19 and CD20) and
a live/dead cell marker were included to facilitate the gating strategy.
Development of the assay using a laboratory-adapted HIV-1 strain. NL4-3 based
provirus encodes the CCR5-tropic envelope found in transmitted founder virus 109F4 herein
named NL-109F4 [
] (Fig 1). Single cells suspensions (4 x 105 cells) from endometrial
biopsies were infected for 1.5 h at 37?C with NL-109F4 containing BlaM-Vpr (500 ng of p24Gag).
The fusion assay was conducted as previously described and combined with the
immunostaining panel described above. Our gating strategy, presented in Fig 1A, was designed to (1) profile
the phenotype of immune cells (CD45+) present in the biopsies (2) measure the total
percentage of immune cells that supported HIV-1 fusion and (3) phenotype the immune cells that
supported HIV-1 fusion. The fusion gate was defined using a FACS plot showing the ratio of
fluorescence of cleaved to uncleaved substrate on the x axis and uncleaved substrate on the y
axis. As expected, the fused cells were almost exclusively in cell populations known to express
HIV-1 receptors (CD4+ T-cells, macrophages, dendritic cells [DCs] and Langerhans cells). As
expected, LIN+ cells (mainly B and NK cells) did not support fusion and could be used as an
internal control to establish the fusion gate (Fig 1A, bottom left inset). Our immunostaining
panel could phenotype most of the immune cells that supported NL-104F4 fusion as evidenced
by the very low number of fused cells not identified by our panel (classified as ?other?) (Fig 1B).
In the sample shown, ~13.6% of the total immune cells (i.e. CD45+ live cells) supported fusion
of NL-109F4; we henceforth refer to the ratio of fused cells to total immune cells as the fusion
Investigating fusion using a panel of 37 primary HIV-1 viral isolates. Given the high
degree of diversity among HIV-1 isolates, we next investigated fusion mediated by a set of 37
circulating HIV-1 isolates that were previously cloned and characterized [
]. This set
includes Subtype B and C HIV-1 species that were found either in the chronic phase of HIV-1
infection (CC) or were transmitted founder viruses (TF) found early in the course of infection.
More details on these molecular clones are given in Table 2. BlaM-Vpr containing virions
corresponding to these isolates were produced by transfection of 293T as described in Methods.
Endometrial biopsies from 3 healthy women participants not on hormones or IUDs were used
as the source of target cells for the set of 37 viruses (Fig 2). The estimated sampling
distributions of FS varied within and between viruses (Fig 2A). For example, virus RHPA, REJO, and
ZM247Fv2 had mean (95% CI) FS of 3.6% (range 1.6?7.5), 11.3% (range 1.2?57), and 15.3%
(range 2.4?57), respectively.
10 / 23
Rows in bold text are the viruses used for comparison of fusion in the different contraceptive groups.
a TF: transmitted founder; CC: chronic control
b MSM: men who have sex with men; HSX: heterosexual exposure.
Country of origin
Although the FS was lowest in EMB-3064 and highest in EMB-3782 for most viruses, the
values fell within the respective 95% confidence intervals of the sample mean (Fig 2A). When
viruses were classified according to their subtype B or C and whether they were identified in
early (TF) or in the chronic phase of infection (CC), there were no major differences in mean
FS prevalence by virus subtypes or phases of infection. Fig 2B shows the mean and 95%
11 / 23
confidence interval for each stratum. While the TF viruses and subtype C viruses appeared to
be more fusogenic, the high degree of variability among viruses in a sample of 3 participants
precluded reliable comparison via a statistical test.
When we investigated the prevalence of FS across the major cellular subsets (Fig 2C), we
found that M2 macrophages (CD14+CD163+) and activated CD4+ T-cells (RO+CD69+)
accounted for the majority of the fused cells in cervix and endometrium. DCs and Langerhans
cells accounted for small portions of fused cells likely because of their low abundance in the
samples. As expected, fusion to CD4- T-cells, B cells (i.e., LIN+HLA-DR+ cells) and NK cells
(LIN+ HLA-DR- cells) was minimal.
Fusion assay adapted to cells from peripheral blood
To profile the cells that supported fusion in peripheral blood mononuclear cells samples, we
tailored the immunostaining panel to allow for phenotyping of immune cells known to be
present in peripheral blood, which differ from those in the endometrium and cervix. The
staining panel includes markers for CD4+ T-cells (CD3, CD4, CD45RO, CD69), monocytes (CD14,
HLA-DR), plasmacytoid DC (pDC) (CD303), and myeloid DC (CD1c, D141); DC
characterization was performed as previously recommended [
]. Additionally, a set of lineage markers
(CD56, CD19 and CD20) and a live/dead cell marker (Invitrogen) were included to facilitate
Given that peripheral blood cells are known to harbor relatively low numbers of cells
susceptible to HIV-1 entry [
], the fusion assay was conducted using more cells than used for the
endometrial and cervical samples (3 x 106 cells per sample). These cells were infected for 1.5 h
with 500 ng p24Gag of ZM247v2 and the fusion assay was conducted as described above. Fig
3A illustrates the gating strategy employed to identify the major subsets of HIV-1 susceptible
cells in the blood (CD4+ T-cells, monocytes, pDC, and myeloid DC). As for the endometrial
and cervical cell analysis, this gating strategy was also designed to (1) profile the phenotype of
immune cells (2) measure the total percentage of immune cells that supported HIV-1 fusion;
and (3) phenotype the immune cells that supported HIV-1 fusion. As expected, LIN+ cells
(mostly B and NK cells) did not support fusion and could be used as an internal control to set
the fusion gate (Fig 3A, bottom left plot). PBMCs are known to harbor few activated CD4+ T
memory cells (CD45RO+CD69+) in healthy individuals, and the CD69- memory CD4+ T-cells
accounted for the majority of fusion-susceptible cells in the blood (Fig 3B). Monocytes and
pDC were also susceptible to entry by ZM247v2.
Characterization of participants in the four contraceptive groups
To investigate the effects of contraceptive use on susceptibility to HIV-1 infection, we studied
samples from women using LNG-containing IUDs, LNG-containing COCs, copper IUDs, and
women not on hormonal or intrauterine contraceptives (control group) as described in
Methods. Table 1 shows the numbers and demographic characteristics of participants in each group
who contributed samples to this study. Although we planned to balance the sample size by
group, for logistical reasons we closed accrual after 11 to 17 eligible participants per group
were enrolled. The number of participants available for specific analyses depended on sample
type (endometrium n = 51, cervix n = 50, and blood n = 53) and the virus studied (Table 1
Controls differed from the contraceptive-exposed groups with respect to age, body mass
index, race, and frequency of sexual intercourse (Table 1). With regards to biologic variables,
the median progesterone levels at the time of sample collection was 9.5 ng/ml (range 0.5?18.6)
in the control group and 10.9 ng/ml (range 0.8?27.8) in the copper IUD users, demonstrating
12 / 23
Fig 3. Fusion assay combined with immunostaining allows measurement of the percentage and phenotypes of immune cells
that support HIV-1 fusion in cells from peripheral blood. (A) Gating strategy: The first 4 multicolor FACS plots allow the
identification of the ?Live cells?. From this gate, the ?BlaM+ cells? can be defined and represent the immune cells that supported
ZM247v2 viral fusion. The FACS plot on the gray background show the gating strategy to identify 11 cellular subsets among the ?all
immune cells? (dark gray) and among ?fused immune cells? (light gray). Note that LIN+ can be used as internal control for gating the
fused (bottom left inset). (B) For a blood sample from a single participant, paired bar graphs show the distribution of immune cell
phenotypes (left) and prevalence of prevalence of fusion susceptibility by phenotype (right). In this sample our immunophenotyping
panel recognized the phenotype of more than 85% of the immune cells present in blood, and overall prevalence of fusion
susceptibility was 2.6%.
PLOS ONE | https://doi.org/10.1371/journal.pone.0221181
13 / 23
that the majority of women in those groups had ovulated. The median progesterone level in
the LNG-IUD group was much lower at 1.8 ng/ml (range 0.5?14.5) and is consistent with our
finding that the majority of women in the LNG-IUD group did not detect an LH surge over a
2-month testing period. The median progesterone level in the COC group was 0.5 ng/ml
(range 0.5?1.0), consistent with suppression of ovulation by COC use. We verified that the
women on LNG-containing contraceptives had measurable LNG levels in their blood; the
median LNG level for OC users was 4.23 ng/ml (range 0.7?6.8) and for LNG-IUD users was
0.18 (range 0.1?0.3). The median vaginal pH was 4.4 in all groups, with a range of 3.6?6.1.
One participant in the control group had a positive test for PSA, indicating recent sexual
Immune cell phenotypes and HIV-1 fusion susceptibility with ZM247Fv2
to cells from 3 anatomic locations
Samples collected over a 2-year period were frozen in liquid nitrogen. To limit experimental
variability, the fusion assay was conducted at a single time for all samples from the same
anatomic site. Fusion was tested with ZM247Fv2, the highly fusogenic TF subtype C virus, for all
Endometrium and cervix samples. We used the gating strategy described in Fig 1 to
assess the distributions of immune cells across all participants for endometrium (n = 51) and
cervix (n = 50). We found higher proportions of macrophages and dendritic cells in the
endometrium and more CD4+ T-cells and Langerhans cells in the cervix (Fig 4 and Table 3), but
collectively, the sum of these four HIV-1 target cells amongst all immune cells was similar for
the 2 sites (33% for cervix and 40% for endometrium). Fig 4 reveals substantial inter-individual
variability in the proportion of each cell type, both among all immune cells and among cells
that fused to HIV-1 virus ZM247Fv2.
Regarding the types of cells that supported fusion of ZM247Fv2, approximately 60% and
80% of fused cells were CD4+ T-cells for endometrium and cervix, respectively, with most of
these cells expressing CD69 (Fig 4 and Table 3). The CD4+CD69- T-cells accounted for a lower
proportion of fused immune cells than the CD4+CD69+ cells, despite being present at roughly
similar mean quantities. In the endometrial samples, macrophages were the next most
common cell supporting fusion, at 18.2% of fused cells with the majority being M2 macrophages
(11.8% M2 and 6.3% M1); in cervix the proportion of macrophages amongst fused cells was
much lower at 2.2%.
The overall susceptibility of immune cells to ZM247Fv2 fusion was approximately 9% in
both endometrium and cervix (Table 3), with CD4+ T-cells accounting for the majority of
susceptible cells (5.80 and 7.04% respectively). In endometrium, M2 macrophages were the next
most susceptible cell type (1.07%). In cervix, given that the numbers of macrophages were
much lower, the measurements were therefore less reliable, and prevalence of FS was <1% for
macrophages and for all other immune cell phenotypes.
Peripheral blood cells. We used the gating strategy described in Fig 3 to determine the
distributions of immune cells in peripheral blood across all participants (n = 53). Of note,
direct comparisons to the FRT sites could not be made due to differences in the phenotype
panels. As seen in Fig 4, there was substantial variability among individuals in the quantity of
each phenotype, similar to what was seen in FRT samples. Memory CD4+ T-cells accounted
for 67% of the cells that supported fusion of ZM247v2 (Table 4). Monocytes, despite their
relatively low abundance in blood (5.5%), accounted for 12.2% of fused cells; pDCs represented
only 0.2% of immune cells in blood but 2.3% of fused immune cells. These results indicate a
high HIV-1 susceptibility of monocytes and pDCs to HIV-1 entry. CD4- T-cells and LIN+ cells
14 / 23
Fig 4. Distribution of cellular subsets in total and fused immune cells from 3 anatomic locations. Single-cell suspensions from
endometrium (n = 51), cervix (n = 50), and peripheral blood (n = 53) were infected with ZM247v2 HIV-1 virions containing
BlaM-Vpr. All infections were performed simultaneously for a given anatomical site. After 1.5 h of infection the fusion assay was
conducted. Box plots are used to describe the distributions of immune cells (left) and fused immune cells (right) for 11 cellular
subsets per sample location. The line through the box represents the median, and the x represents the mean; whiskers on the
box plots represent the 255h and 75th percentiles; outliers are indicated with black circles.
were not represented among the fused cells, as expected, despite their high representation in
the samples, demonstrating the specificity of the assay.
Overall, FS was higher in endometrial and cervical samples than in peripheral blood (9.11%
versus 8.70% versus 1.29% respectively, Tables 3 and 4). FS was highest in CD4+ T-cells in all 3
anatomic sites (5.9, 7.0 and 0.98% respectively). None of the subsets of blood cells had >1%
FS, demonstrating the overall low susceptibility of blood cells to HIV-1 entry.
The effect of contraceptives on HIV-1 entry into target cells from
endometrium, cervix and blood
We investigated whether the type of contraceptive used by the women participants influenced
FS overall or by cell phenotype in endometrial, cervical, and blood samples. To limit the
15 / 23
a Denominator is total number of immune cells per sample type; all phenotypes have Ctotal > 200.
b Denominator is total number of fused immune cells per sample type. Column excludes three samples with Ftotal <6 (one endometrium, 2 cervix).
c Counts (#) are medians across n = 51 (endometrium) and n = 50 (cervix) participants. For medians, sum of components can differ from total. Percents (%) are means
estimated from generalized linear models (binomial distribution with logit link).
d Other = All?(Macrophages + Langerhans + Dendritic cells + CD4- + CD4+ + LIN+)
a Denominator is the total number of immune cells.
b Denominator is the total number of fused cells.
c Counts (#) are medians across 53 participants. For medians, sum of components can differ from total.
Percents (%) are means estimated from generalized linear models (binomial distribution with logit link).
16 / 23
Fig 5. Effect of contraceptives on HIV-1 acquisition as measured by prevalence of fusion susceptibility of cells from 3 anatomic
locations to HIV-1 fusion ex vivo. Bar graphs depict the mean prevalence of immune cells supporting ZM247v2 fusion (overall
height of the bar, with confidence limits) by donor contraception group. Contributions of four major cellular subsets to the overall
prevalence are identified by color clade. Note that the vertical axis is 10-fold greater for biopsy samples than blood samples. OCP is
combined oral contraceptive.
influence of subsets represented at low levels, we grouped activated and non-activated CD4+
T-cells under the category of CD4+ T-cells, and M1 and M2 macrophages under the category
of macrophages. Likewise, all the CD4+ T subtypes from peripheral blood were grouped under
the larger category of CD4+ T-cells.
Fusion susceptibility with ZM247Fv. For all sample types, viral fusion was higher among
contraceptive non-users than contraceptive users (Fig 5); the differences were significant for
the endometrial samples (Table 5), discussed in detail below. Fusion varied among immune
cell phenotypes (p<0.001), with CD4+ T-cells accounting for the majority of cells that
supported ZM247Fv2 fusion. The proportions of ?other? cells, which are the immune cells that fell
into the fusion gate but for which the gating and immunophenotyping did not lead to a clear
classification, were similarly low across the groups, indicating that the immunostaining panel
allowed phenotyping of the majority of fused immune cells in all of the participant groups.
In endometrial samples, FS varied significantly among contraceptive groups both overall
(P = 0.026; Table 5) and within the four major cell phenotypes (CD4+ T cells, macrophages,
dendritic cells and Langerhans cells). In particular, significantly higher FS in the non-IUD
groups as compared to the IUD groups (Control and COC versus IUDs, P = 0.010) was
associated with above-average fusion of three subsets of cells (macrophages, Langerhans and
dendritic cells; P = 0.007), whereas significantly higher FS in non-hormone groups as compared to
17 / 23
a In this table, overall estimates of fusion susceptibility are adjusted for contraceptive group.
b P-values comparing exposure groups, adjusted for cell phenotypes shown, are based on 3-df score statistics for Type 3 generalized estimating equation analysis using
c Macrophages = M1+M2 macrophages combined
the hormone groups (Control and Copper IUD versus COC and LNG-IUD p = 0.007) was not
associated with a particular cell type.
In cervical and blood samples, the percent of immune cells supporting fusion varied little
among contraceptive groups (overall P = 0.70 and 0.14, respectively; Fig 5 and Table 5). In
cervical samples, FS of macrophages varied by contraceptive group (P = 0.032), being highest in
the LNG-IUD group; however, very few cells were available for analysis (Table 3). In blood
samples, FS of pDC varied by contraceptive group (P = 0.01); although the cell count was
sufficient for analysis (Table 4), no clear association with either hormone or IUD exposure
emerged (Table 5).
Fusion susceptibility with additional HIV-1 isolates in endometrial samples. All results
above apply to ZM247Fv2 entry. We also compared FS by contraceptive group in those
endometrial samples that had sufficient cell numbers using 2 additional HIV-1 isolates: REJO, a
subtype B TF virus found in a newly infected male (n = 40 samples), and RHPA, a subtype B
TF virus found in a newly infected woman (n = 40 samples) (S1 Fig). This analysis was limited
18 / 23
to endometrial samples as insufficient cell numbers were available from the cervix. ZM247Fv2
and RHPA were the most and least fusogenic, respectively, consistent with the patterns seen
from the three participants shown in Fig 2. In each case, CD4+ T-cells explained most of the FS
and the proportions explained by ?other? cells were low.
For viruses ZM247Fv2, REJO, and RHPA, FS was highest in each control group (14%, 10%,
and 3.5%, respectively) and lowest in the LNG-IUD group (6.9%, 5.7%, and 1.3%), yielding
relative Control:LNG-IUD ratios of 2.0, 1.8, and 2.7. In turn, the 3-df p-values testing for
variation in FS were P = 0.026, P = 0.12, and P = 0.024, respectively. As shown in S1 Fig, FS of
CD4+ T-cells was substantially higher in the groups not exposed to LNG (Control and copper
IUD). FS of macrophages was higher in groups not exposed to a device and decreased across
groups in the order displayed (Control > COC > Cu-IUD > LNG-IUD).
Our study was designed to assess the effects of hormonal and intrauterine contraceptives,
separately and combined, on susceptibility to HIV-1 fusion. The analysis of 154 samples from 58
participants and 3 anatomical sites (endometrium, cervical transformation zone and
peripheral blood) demonstrated that CD4+ T cells are the most susceptible cell type in all 3 anatomic
locations, and that fusion was significantly higher in cells from cervix and endometrium than
in blood. Our results confirm other reports that endometrial macrophages are highly
susceptible to HIV-1 infection [
]. We also found that cells from women using LNG-containing
COCs, LNG-IUDs and copper IUDs are not associated with increased susceptibility to HIV-1
fusion. In fact, the control group had the highest susceptibility to ex vivo fusion in samples
from both the FRT and peripheral blood, a finding that was statistically significant and
consistent amongst 3 different viral isolates. These results contribute to the knowledge base about
hormonal contraceptives and IUDs in light of recent concerns about possible adverse effects of
hormonal contraceptives on HIV-1 susceptibility, and are consistent with recent results from a
randomized trial in women that showed no difference in HIV risk amongst women using
depo-medroxyprogesterone acetate, a copper IUD and an levonorgestrel implant [
Our results show that different HIV-1 viruses have high variation in fusogenicity to primary
cells from the cervix and endometrium. While TF subtype C viruses seemed to enter targets
cells more efficiently than the other subtypes (Fig 2B), the sample size was too small to draw
definitive conclusions regarding the effects of HIV-1 subtype or the phase of infection at the
time when the virus was cloned (transmitted/founder virus and chronic infection) on HIV-1
fusogenicity to endometrial cells. Our data confirms the findings of a previous report that
indicated that envelopes of transmitted/founder or control/reference viruses have similar infection
patterns of CD4+ T-cells in human cervical tissue ex vivo [
We found a wide range of distribution of immune cell types susceptible to HIV-1 fusion,
and variations in the extent of fusion depending on the participant. This range is indicated by
the wide confidence intervals surrounding the medians in Figs 2 and 4. Fig 2C directly
compares the distributions of immune cells and of fused cells in endometrial samples from 3
women and shows that the sample from participant EMB-3782 had a much higher proportion
of macrophages and a correspondingly higher proportion of fusion events. Seminal fluid
introduced into the vagina from coitus induces an influx of macrophages and dendritic cells into
the cervix [
], and presumably a similar event could occur in the endometrium. However,
our participants were instructed to use condoms throughout the study period and to refrain
from intercourse for 72 hours prior to sample collection. We also collected a vaginal swab for
prostate specific antigen detection at the time of sample collection as a marker for recent
intercourse, which was negative for all of the samples in Fig 2, indicating that the high proportion
19 / 23
of macrophages in sample EMB-3782 is unlikely to result from recent intercourse. To reduce
the impact of infections on study samples, we screened women for sexually transmitted
infections at study entry, and performed vaginal pH and when indicated, a wet mount, to exclude
women with bacterial vaginosis at the time of sample collection. We conclude that the
variation in immune cellular composition that we observed in endometrial and cervical samples
may be attributable to the natural variation between women.
Our study has several strengths. This is the first direct comparison of HIV-1 fusion events
in samples from women on hormonal and non-hormonal IUDs and COCs. We restricted the
type of oral contraceptives to those containing LNG in a 28-day pill pack. Our collection of
samples synchronized to the luteal phase (for cycling women), or to the latter part of the pill
pack (for women on COCs), is meant to reduce biological variability due to hormonal
fluctuations across the menstrual cycle. We used clones of primary HIV-1 isolates from different
times in the infectious life cycle and of different subtypes. The fusion assay is performed on
target cells that have not been externally activated or cultured, and hence are more likely to
reflect the status of cells within their local environment than assays that measure downstream
events in the HIV-1 viral lifecycle. We used state-of-the-art statistical methods to look for
contraceptive effects on fusion susceptibility. Our study also has limitations. The control
group differed from the other groups in age and other demographic characteristics
representative of women who choose different methods. The relatively small sample sizes precluded
rigorous statistical analyses of some comparisons. We were unable to time sample collection
to precise times in the menstrual cycle in women on LNG-IUD because the majority were
anovulatory. Participants had used contraceptive methods for varying lengths of time. Finally,
the fusion assay is a surrogate for HIV-1 infection and therefore these results may not
accurately reflect susceptibility in vivo. For example, epithelial cells could respond to the local
hormonal environment by producing cytokines which could in turn affect HIV replication.
Likewise, the microbiota could be influenced by the hormonal context and subsequently
influence HIV replication [
]. These factors were not directly addressed by the ex vivo fusion
In summary, we found that the use of LNG-containing COCs, LNG-IUDs and the copper
IUD were not associated with increased HIV-1 fusion susceptibility of immune cells from
the endometrium, cervix or peripheral blood. These findings are reassuring given the
high numbers of women worldwide using such devices, and in light of our previous work
showing an increase in the numbers of activated CD4+ T-cells in endometrial samples from
LNG-IUD users compared to controls [
]. In addition, our results demonstrate that upper
FRT tissues contain cells that are highly susceptible to HIV-1 fusion, supporting the
hypothesis that the cervical transformation zone and the endometrium are potential sites of HIV-1
acquisition. The fact that our results for the copper IUD are congruent with recent results
from a randomized trial in women [
] is reassuring that the HIV-1 fusion assay may have
relevance for predicting contraceptive safety, suggesting that HIV-1 risk should not be
increased in IUD users and women on COCs compared to those not on these forms of
S1 Fig. Effect of contraceptives on HIV-1 acquisition as measured by prevalence of fusion
susceptibility of endometrial cells to HIV-1 fusion ex vivo mediated by three different viral
clones. Single cell suspension from EMBs were infected for 1.5h with M247Fv2, REJO and
RHPA (500 ng p24Gag). Bar graphs depict the mean prevalence of immune cells supporting
ZM247v2 fusion (overall height of the bar, with confidence limits) by donor contraception
20 / 23
group. Contributions of four major cellular subsets to the overall prevalence are identified by
color clade. OCP is combined oral contraceptive.
The authors would like to thank all study participants.
Conceptualization: Marielle Cavrois, Joan F. Hilton, Nadia R. Roan, Dominika Seidman,
Sarah Averbach, Ruth Greenblatt, Barbara L. Shacklett, Karen Smith-McCune.
Data curation: Marielle Cavrois, Joan F. Hilton, Karen Smith-McCune.
Formal analysis: Marielle Cavrois, Joan F. Hilton, Barbara L. Shacklett, Karen
SmithFunding acquisition: Marielle Cavrois, Joan F. Hilton, Ruth Greenblatt, Barbara L. Shacklett,
Investigation: Marielle Cavrois, Nadia R. Roan, Margaret Takeda, Eric Chang, Nandhini
Raman, Ruth Greenblatt, Barbara L. Shacklett, Karen Smith-McCune.
Methodology: Marielle Cavrois, Nadia R. Roan, Margaret Takeda, Dominika Seidman, Sarah
Averbach, Ruth Greenblatt, Barbara L. Shacklett, Karen Smith-McCune.
Project administration: Marielle Cavrois, Margaret Takeda, Karen Smith-McCune.
Resources: Marielle Cavrois, Dominika Seidman, Sarah Averbach, Karen Smith-McCune.
Supervision: Marielle Cavrois, Karen Smith-McCune.
Visualization: Marielle Cavrois, Joan F. Hilton.
Writing ? original draft: Marielle Cavrois, Joan F. Hilton, Karen Smith-McCune.
Writing ? review & editing: Marielle Cavrois, Joan F. Hilton, Nadia R. Roan, Margaret
Takeda, Dominika Seidman, Sarah Averbach, Eric Chang, Nandhini Raman, Ruth
Greenblatt, Barbara L. Shacklett, Karen Smith-McCune.
21 / 23
World Health Organization. Programmatic and research considerations for hormonal contraception for
women at risk of HIV and women living with HIV. May 2012.
22 / 23
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