HIV CLINICAL TRIALS
Vaginal bacteria modify HIV
tenofovir microbicide efficacy
in African women
Nichole R. Klatt,
1
*† Ryan Cheu,
1
‡ Kenzie Birse,
2,3
‡ Alexander S. Zevin,
1
‡
Michelle Perner,
2,3
‡ Laura Noël-Romas,
2,3
‡ Anneke Grobler,
4
Garrett Westmacott,
5
Irene Y. Xie,
2,3
Jennifer Butler,
2,3
Leila Mansoor,
4
Lyle R. McKinnon,
3,4
Jo-Ann S. Passmore,
6,4
Quarraisha Abdool Karim,
4,7
Salim S. Abdool Karim,
4,7
Adam D. Burgener
2,3,8
*†
Antiretro viral-based strategies f or HIV pre v ention have sho wn inconsistent r esults in women.
We investigated w hether vaginal microbiota modulated tenofo vir g el microbicide efficacy
in the CAPRISA (Centre for the AIDS Progr am of Resear ch in South Africa) 004 trial.Tw o major
vaginal bacterial community types—one dominated by Lactobacillus (59.2%) and the other
where Gardnerella vaginalis predominated with other anaerobic bacteria ( 40 .8%)—were
identified in 688 women profiled. Tenofo vir reduced HIV incidence b y 61 % (P =0.013)in
Lactobacillus-dominant women but only 18% (P = 0.644) in women with non-Lactobacillus
bacteria, a threefold difference in efficacy . Detectible mucosal tenofovir was lower in
non-Lactobacillus women, negatively correlating with G. vaginalis and other anaerobic bacteria,
which depleted tenofovir by metabolism more rapidly than target cells convert to
pharmacologically active drug. This study pr o vides evidence linking vaginal bacteria to
microbicide efficacy through tenofovir depletion via bacterial metabolism.
M
ore than 1 million women are infected
with HIV annually, and the majority of
these new infections occur in young wom-
en in sub-Saharan Africa, with South Africa
having among the highest incidence rates
(1, 2). Antiretroviral-based clinical trials in men
who have sex with men have consistently dem-
onstrated effectiveness in preventing HIV infection
(3–5); however, studies in women have produced
widely varying results. In clinical trials of women,
the efficacy of antiretroviral drugs to prevent HIV
infection ranged from –49% [VOICE (Vaginal and
Oral Interventions to Control the Epidemic) study]
to 75% [TDF2 (Tenofovir Disoproxil Fumarate
Two) study] for daily oral tenofovir or tenofovir-
emtricitabine and from 0% [FACTS (Follow-on
African Consortium for Tenofovir Studies) 001]
to 39% [CAPRISA (Centre for the AIDS Program
of Research in South Africa) 004] for daily or co-
ital vaginally-applied tenofovir gel (fig. S1). Var-
iability in the levels of adherence (6) has been
shown to be a major contributing factor for the
diverse trial outcomes in women. However , little
is known about what biolo gical factors may also
contribute to the variability in these results and why
higheradherenceisrequiredforantiretroviral-
based prevention efficacy in women (7).
The vaginal compartment contains many mi-
crobial species critical for the health of the vaginal
mucosa, and dysbiosis of vaginal bacteria, clin-
ically known as bacterial vaginosis (BV), can re-
sult in negative reproductive health outcomes
(8, 9). The recent advent of advanced molecular
tools has redefined our understanding of vagi-
nal bacteria communities (10, 11), where the
most frequently observed community state types
(CSTs) have been described (11–14). Although
substantial heterogeneity exists, a key com-
monality is that CSTs fall into two clear groups:
(i) Lactobacillus-dominant, where one or more spe-
cies of Lactobacillus make up >90% of the t o-
tal copy number or sequencing reads (L. iners,
L. crispatus, L. jensenii, and L. gasseri), and (ii)
non–Lactobacillus-dominant, with Lactobacillus
making up <30% of the total copy number or se-
quencing reads. The non–Lactobacillus-dominant
group typically contains a high abundance of
Gardnerella vaginalis alone or codominant with
other facultative and obligate anaerobic bacte-
ria, including Prevotella ssp., Mobiluncus ssp.,
and/or several Clostridia species. BV occurs after
a shift from Lactobacillus dominance to these
more diverse communities (11, 12), is frequently
asymptomat ic, and can often go undetected using
traditional Amsel’s criteria (15) and/or the Nugent
’s
score
used to diagnose BV (16).
Bacterial vaginosis is associated with poor re-
productive health outcomes and increased HIV
infection risk in women (17 ), by as much as 60%
in some meta-analyses of women with BV (18).
BV likely increases HIV risk through multiple
mechanisms, including increased inflammation
and target cells, as well as vaginal epithelial bar-
rier disruption and wound-healing impairment;
however, the mechanisms are not entirely under-
stood (13, 19, 20). Given that women from sub-
Saharan Africa have high prevalence rates of BV
(21) and that HIV prevention strategies are being
targeted for women in these areas, we inves-
tigated whether vaginal microbial communities
may affect the efficacy of antiretroviral-based pre-
vention technologies, especially those that are
topically applied to the vaginal surface.
Characterization of the vaginal microbiome
using unbiased metagenomic, metatranscriptomic,
and metaproteomic approaches represents a po-
tential paradigm shift in understanding host-
microbial interactions at the mucosal surface
in vivo. We recently used metaproteomics to gain
insight into host-bacterial interactions in vaginal
microbial dysbiosis (20). This method simultane-
ously collects unbiased information on microbial
and host proteomes, thus providing systems-level
information on microbial communities and muco-
sal surfaces not available with other techniques.
In this work, we used a metaproteomic approach
to assess whether vaginal bacteria modulate the
efficacy of the topical microbicide tenofovir in pre-
venting HIV infection, and we also used in vitro
systems to determine mechanisms of microbiome
influence on tenofovir.
Vaginal microbial diversity in
women using tenofovir or placebo
microbicide gels
Samples from 688 HIV-negative women that were
assigned to either the tenofovir or the placebo-gel
armwereanalyzedbyproteinmassspectrometry,
as outlined in the materials and methods (fig. S2).
Proteomic analysis identified 3334 distinct bac-
terial proteins from 188 different species in the
cervicovaginal lavage (CVL) samples of 688 women.
Two major vaginal bacterial community groups
were identified: one in which Lactobacillus was
the predominant genus (group I) (n =423wom-
en, 61.5%) and the other dominated by non-
Lactobacillus microbiota (group II) (n = 265
women, 38.5%) (Fig. 1). Approximately 11% of indi-
viduals had no single dominant species (defined as
>50% community composition), and the majority
of these ind ividuals fell into group II.
In comparing the mass spectrometry proteo-
mic approach with 16S ribosomal RNA (rRNA)
sequencing, we found concordance with respect to
classifying women into groups I and II (91.5% agree-
ment) and measurements of bacterial abundance,
including major taxa Lactobacillus, G. vaginalis,
Prevotella,andothers(P<0.001) (fig. S3). A lim-
itation of this study was that clinical BV data
was not collected during the CAPRISA 004 trial,
RESEARCH
Klatt et al., Science 356, 938–945 (2017) 2 June 2017 1of7
1
Department of Pharmaceutics, Washington National Primate
Research Center, University of Washington, Seattle, WA, USA.
2
National HIV and Retrovirology Labs, J.C. Wilt Infectious
Diseases Research Centre, Public Health Agency of Canada,
Winnipeg, Manitoba, Canada.
3
Department of Medical
Microbiology and Infectious Diseases, University of Manitoba,
Winnipeg, Manitoba, Canada.
4
Centre for the AIDS Program of
Research in South Africa (CAPRISA), University of KwaZulu-
Natal, Durban, South Africa.
5
Mass Spectrometry and
Proteomics Core Facility, National Microbiology Laboratory,
Public Health Agency of Canada, Winnipeg, Manitoba, Canada.
6
Institute of Infectious Diseases and Molecular Medicine (IDM),
University of Cape Town and National Health Laboratory
Service, Cape Town, South Africa.
7
Department of Epidemiology,
Mailman School of Public Health, Columbia University, NY, USA.
8
Unit of Infectious Diseases, Department of Medicine Solna,
Center for Molecular. Medicine, Karolinska Institute, Karolinska
University Hospital, Stockholm, Sweden.
*Corresponding author. Email: adam.burgener@umanitoba.ca
(A.D.B.); klattnr@uw.edu (N.R.K.)
†These authors contributed equally to this work.
‡These authors contributed equally to this work.
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thus precluding comparisons to Nugent score or
other BV criteria.
Group I showed the lowest diversity [Shannon
diversity index (H-index) median: 0.05], with the
majority (70.5%) having L. iners as the dominant
species, followed by L. crispatus (15.1%) and other
lactobacilli, such as L. jensenii and L. gasseri
(3.0%).InFig.1B,thefirstsubgroup(a)washo-
mogeneous, with clear dominance of Lactobacillus
(54.7%) with very low diversity (H-index median:
0.035), whereas a minority of these individuals
(subgroup b, 6.8%) showed an intermediate amount
of diversity (H-index median: 0.87) where small
amounts of G. vaginalis, Pseudomonas,andother
bacteria were detected. A subanalysis of group
Ia at the Lactobacillus species level clearly illustrates
L. iners as predominant (fig. S4).
WomeningroupIIhadhigheroverallbacterial
diversity (H-index: 0.78) with several distinct sub-
groups. The largest subgroup (c) was dominated
by G. vaginalis (n = 163, 23.7%, H-index: 0.66) and
contained multiple taxa, including Prevotella and
minor amounts of Lactobacillus and Mobiluncus
(Fig. 1B). The second-largest subgroup (d) was
the most diverse and had no one clearly dominant
taxa (n = 78, 11.3%, H-index: 1.15); G. vaginalis,
Prevotella, and Mobiluncus were predominant.
Finally, the smallest subgroup (e) was low in diver-
sity, containing either Pseudomonas or Escherichia
(n = 24, 3.5%, H-index: 0.14) (Fig. 1B). Grouping
subgroups a and b, which showed relative homo-
geneity in a single group (group I), with the re-
maining three subgroups (c, d, and e), which had
more variability and diversity into a single group
(group II), was supported byprincipalcomponents
analysis (Fig. 1C). Overall, the majority (96.2%, n =
407) of women in group I had >50% Lactobacillus
by abundance.
For downstream comparisons of topical teno-
fovir efficacy, we chose a Lactobacillus-dominant
(>50%) (LD) or non–Lactobacillus-dominant (≤50%)
(non-LD) classification, as >50% Lactobacillus at
Klatt et al., Science 356, 938–945 (2017) 2 June 2017 2of7
Fig. 1. Bacterial profiling by mass spectrometry using cervicovaginal lavage samples from 688 women from the CAPRISA 004 trial. (A)Overall
bacterial diversity plot of the major genera of all women profiled. (B) Av er age bacterial community group structure for each of the two major profiles: group I
(subgr oups a and b) and gr oup II (subgro ups c, d, and e). (C) P rincipal components analysis of 688 women using bacterial proportion data showing the
five subgroups.
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the proteome level corresponds with a practical
clinical marker of vaginal pH below 4.5 [inter-
quartile range pH value of 4.0 to 4.5 (20)] and
accurately identified 96.2% of group I individuals.
The use of LD and non-LD definitions did not
appreciably change any outcomes reported on
the basis of group I versusgroupIIcomparisons.
Both LD and non-LD women had similar base-
line clinical, behavioral, and demographic char-
acteristics, as well as sexual behavior and gel
adherence during the trial (table S1). The presence
of a sexually transmitted infection (STI)—which
included Chlamydia trachomatis, Trichomonas
vaginalis, Neisseria gonorrhea, Mycoplasma gen-
italium, Treponema pallidum,andherpessimplex
virus 2 (HSV-2)—was comparable between groups.
Although not statistically significant, women within
the non-LD group were marginally younger (mean
age 23.6 versus 24.1 years, P =0.092).Withineach
of the groups of LD and non-LD women, the num-
ber of individuals assigned to tenofovir gel and
placebo gel was similar (table S2).
Vaginal microbial profiles and tenofovir
gel efficacy
In the LD group, the HIV incidence rate was 61%
lower [95% confidence interval (CI): 0.16 to 0.89]
in women assigned to tenofovir gel compared with
those assigned to the placebo gel [2.7 versus 6.9
per 100 women-years (where 1 woman-year is de-
fined as 1 year of study observation of one woman);
incidence rate ratio (IRR) = 0.39; P =0.013]
(Fig. 2A). In contrast, in non-LD women, the HIV
incidenceratewasonly18%lower(95%CI:0.37
to 1.77) in those assigned to tenofovir gel compared
with those assigned to placebo gel (6.4 versus 7.8
per 100 women-years; IRR = 0.82; P =0.644)(Fig.
2B). Adjusting for STIs (including HSV-2 infec-
tion), antibiotic usage, depot medroxyprogesterone
acetate (DMPA) usage, and sexual behaviors (fre-
quency of sex, number of partners, and condom
usage) did not affect these findings (table S3).
Lactobacillus,particularlyL. crispatus, has been
associated with reduced HIV infection. Although
the HIV incidence rate of 4.8 per 100 women-
years in LD women was 32% lower (95% CI: 0.4
to 1.12) than the 7.1 per 100 women-years in non-
LD women, this was not statistically significant
(P =0.127)(fig.S5).Comparingthesubgroup
of all 63 L. crispatus–dominant (>50% abun-
dance) to non-LD wo men yielded a 57% lower
HIV incidence (95% CI: 0.13 to 1.41) that was
not statistically significant (3.1 versus 7.1 per
100 women-years; P = 0.167) (fig. S6). Comparison
of L. crispatus–dominant women to all others as a
single group (L. iners–dominant and non-LD
women) produced similar findings (50% lower
HIV incidence; 3.1 versus 6.0 per 100 women-
years; 95% CI: 0.16 to 1.60, P=0.237). Similarly,
within the placebo group, the 31 L. crispatus–
dominant women compared with non-LD women
showed a 48% lower HIV incidence (95% CI: 0.12
to 1.27) that was not statistically significant (4.17
versus 7.8 per 100 women-years; P =0.387)(fig.S7).
This is likely attributed to insufficient power due
to the low numbers of women with L. crispatus
dominance. However, because the proportion of
women with L. crispatus dominance was similar
in women assigned to tenof ovir gel and placebo
gel(51%versus49%),theHIVincidencediffer-
ences between these two groups of women are
not due to
L. crispatus.
In addition, considering
just L. iners–dominant women, the efficacy of ten-
ofovir was maintained, where the HIV incidence
rate was 67% lower (95% CI: 0.13 to 0.83) in those
assigned to tenofovir gel compared with those
assigned to placebo gel (2.5 versus 7.7 per 100
women-years in the tenofovir and placebo arms,
respectively; P =0.0118)(fig.S8).
Microbicide gel adherence and tenofovir
efficacy in LD and non-LD women
Gel adherence, as assessed by monthly empty
applicator returns (22), was similar in both groups:
60.0% [244 of 407 (244/407)] of LD women
compared to 61.4% (172/280) of non-LD women
had >50% gel adherence (where >50% of sex acts
were covered by two applications of the gel, as
recommended in the trial) (Table 1). Stratifying
LD and non-LD women separately on adherence
demonstrates that gel adherence >50% was as-
sociated with higher efficacy in preventing HIV
inLDwomenthaninnon-LDwomen(Table1).
The efficacy of tenofovir gel in preventing HIV
infection in the subgroup of women with >50%
adherence was 78% (95% CI: 29%, 95%; P = 0.003)
intheLDgroupbutonly26%(95%CI:–98%,
73%; P = 0.558) in the non-LD group.
Vaginal tenofovir concentrations are
lower in non-LD versus LD women
Tenofovir concentrations (n = 270) were assessed
in a random sample of CVLs from HIV-negative
women and from the first postinfection visit CVL
from HIV seroconvertors. Although gel adherence
was not different between the LD and non-LD
groups and time since the last gel application was
similar (P=0.558) (table S1), tenofovir was only
detectable in CVL samples in 29.8% (34/114) of
non-LD women compared with 46.2% (72/156) of
LD women (P = 0.008). Genital tenofovir con-
centrations were also significantly lower in non-
LD (upper quartile = 24.3 ng/ml) compared
with LD women (upper quartile = 8020 ng/ml)
Klatt et al., Science 356, 938–945 (2017) 2 June 2017 3of7
Fig. 2. Cumulative HIV infection probability by treatment assignment in women with vaginal Lactobacillus dominance and non-Lactobacillus
bacterial dominance. Data for (A) Lactobacillus-dominant (Lactobacillus >50%)(n =407)and(B) non–Lactobacillus-dominant (Lactobacillus ≤ 50%)
(n = 281) w omen. The tables below each panel sho w the cumulative number of HIV infections in each study arm, corresponding HIV incidence rates, and
efficacy of tenofov ir gel to prevent HIV acquisition for each additional 6 months of follo w-up . The protectiv e efficacy of tenofovir gel was more than threefold
higher in women with Lactobacillus dominance (A) compare d with non-Lactobacillus dominance (B). HR, hazard ratio.
RESEARCH | RESEARCH ARTICLE
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(P=0.0077). A subanalysis showed that genital
tenofovir concentrations negatively correlated
with G. vaginalis protein abundance (correlation
coefficient r = –0.19, P = 0.0014) and other an-
aerobic bacteria (Prevotella, r = –0.14, P=0.023),
suggesting a relationship between BV-associated
bacteria and tenofovir levels.
Metabolism by G. vaginalis and
BV-associated bacteria leads
to tenofovir depletion
Given the decreased levels of mucosal tenofovir
in non–LD-dominant women with G. vaginalis
and other BV-associated bacteria versus LD wom-
en, we aimed to determine whether interactions
between microbes and tenofovir may underlie
altered drug levels. We used an in vitro culture
system to assess potential biodegradation of ten-
ofovir by the major bacterial species present in
this cohort. We found that tenofovir concentra-
tions in culture with G. vaginalis decreased
rapidly by 50.6% compared with marginal changes
in either L. iners (P=0.0037), L. crispatus (P =
0.0019), or abiotic (same NYCIII media without
bacteria) control (P < 0.0001) at 4 hours (Fig. 3A).
The differential decline continued; by 24 hours,
tenofovir concentrations in the culture mediu m
had dropped 67.4% with G. vaginalis but only
14.0% with L. iners (P < 0.0001) and 9.4% with
L. crispatus (P < 0.0001) (Fig. 3A). Of interest,
G. vaginalis used here was a subtype C strain
(ATCC type strain 14018), but repeating these
methods with G. vaginalis with Kenyan clinical
isolates from three different subtypes demon-
strated that all subtypes of G. vaginalis metab-
olized tenofovir (P < 0.005) (fig. S9). Concomitantly
to tenofovir loss, intracellular tenofovir concen-
trations rose sharply in G. vaginalis but not in
L. iners or L. crispatus cultures (P<0.0001) (Fig.
3B). Predicted metabolites at mass/charge ratios
(m/z) of 136.06, 206.10, 159.07, and 59.05 showed
a sharp increase at 136.06 m/z in G. vaginalis
cultures, indicating adenine production via cleav-
age
of oxy-methylphosphonic acid (Fig. 3C), the
side-chain component of tenofovir (P < 0.0001
compared with L. crispatus, L. iners, and abi-
otic). Residual tenofovir plus the intracellular
metabolite adenine made up >80% of recovered
products, indicating that adenine is the major
metabolite of tenofovir metabolism by G. vaginalis
(Fig. 3D). Finally, to determine whether other
major bacterial species in non-LD women were
capable of metabolizing tenofovir, we tested the
ability of P. amnii, P. bivia, Mobiluncus mulieris,
and Escherichia coli to deplete tenofovir . We found
that both Prevotella species and M. mulieris sig-
nificantly depleted tenofovir compared with abi-
otic (Wilkins-Chalgren media) controls (P = 0.0007
for all at 24 hours), and E. coli trended toward
depletion (P = 0.100 as compared to abiotic tryptic
soy media), though not to the same extent or as
rapidly as G. vaginalis (Fig. 3E).
Metabolism by vaginal bacteria affects
uptake and conversion of tenofovir to
active drug in target cells
We next asses sed whether tenofovir metabolism
by bacteria affects the kinetics or ability of target
cells to uptake tenofovir and convert to pharma-
cologically active tenofovir diphosphate. We per-
formed cocultures of Jurkat cells (HIV targets) in
the presence of tenofovir, with G. vaginalis,
L. iners, L. crispatus, or abiotic controls (both
tenofovir alone or tenofovir plus Jurkat cells),
to assess overall tenofovir depletion and uptake in
culture. In these Jurkat cell cultures, we found that
tenofovir is most rapidly depleted in the pres ence
of G. vaginalis, relative to Jurkat cells alone (P =
0.0001) or those with L. iners (P =0.0022)or
L. crispatus (P = 0.0238) (Fig. 4A). To assess mi-
crobial tenofovir metabolism, we measured ade-
nine levels in the cell pellets and found that adenine
was created only in cultures with G. vaginalis
(P = 0.0022 relative to all conditions) (Fig. 4B). A
Klatt et al., Science 356, 938–945 (2017) 2 June 2017 4of7
Fig. 3. Metabolism of tenofovir by G. vaginalis and BV-associated
bacteria. (A) Tenofovir fold change in supernatants after 1 mg/ml tenofovir
was added to G. vaginalis, L. iners, and L. crispatus cultures or abiotic
controls in NYCIII media. Tenofovir levels were measured by mass
spectrometry at 0, 4 and 24 hours. Data show average ± SEM (error bars)
of 18 replicate experiments for L. iners and G. vaginalis cultures compared
with 15 replicates of abiotic controls. (B) Total intracellular tenofovir
detected in cell pellets from cultures. (C) Predicted tenofovir metabolite
adenine was measured in cultures. (D) Total drug recovery of tenofovir
and adenine metabolite. (E) Tenofovir fold change in supernatants
after 1 mg/ml tenofovir was added to cultures of P. amnii, P. bivia,
M. mulieris, E. coli , or abiotic controls in Wilkins-Chalgren (WC) media
or tryptic soy (TS) broth (for E. coli). Data show average ± SEM of six
replicate experiments for P. amnii, P. bivia, and M. mulieris and four
replicates for E. coli, compared with five replicate abiotic controls.
P values are calculated by Mann-Whitney U test. ns, not significant.
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critical component is whether G. vaginalis can
metabolize tenofovir more rapidly than target
cells can convert tenofovir to pharmacologically
active tenofovir diphosphate. To assess this, we
measured tenofovir diphosphate in cell pellets
and found that although tenofovir diphosphate
was made at equal levels in Jurkat cells alone and
thepresenceofLactobacillus spp., it was signifi-
cantly decreased in the presence of G. vaginalis
(P = 0.0002, P =0.0022,andP = 0.0238 relative
to tenofovir + Jurkat alone, tenofovir + Jurkat +
L. iners, or tenofovir + Jurkat + L. crispatus,re-
spectively) (Fig. 4C). Total drug recovery mea-
surements (tenofovir + tenofovir diphosphate +
adenine) demonstrated that all components of
tenofovir were fully recovered (Fig. 4D). These
data demonstrate that G. vaginalis is capable of
decreasing pharmacologically active tenofovir di-
phosphate by metabolizing tenofovir before drug
uptake by target cells.
Implications for antiretroviral-based
HIV prevention
The efficacy of tenofovir-containing topical micro-
bicide to prevent HIV infection varied more than
threefold depending on vaginal bacterial profiles;
tenofovir gel reduced HIV incidence by 61% in LD
womenbutonlyby18%innon-LDwomen.These
efficacy differences between LD and non-LD wom-
en were consistently present in the most gel-
adherent women (78% versus 26%), as well as
in the least adherent women (17% versus 4%).
In vitro studies demonstrated that metabolism of
tenofovir occurred by G. vaginalis, P. bivia, P. amnii,
and M. mulieris, and slightly by E. coli, but not
by L. iners or L. crispatus, indicating a putative
mechanism for the observed differences in topi-
cal microbicide efficacy. The modifying effect of
Lactobacillus dominance on tenofovir gel efficacy
underscores the importance of both high adherence
and LD vaginal bacterial communities for women
to benefit maximally from topical microbicides for
HIV prevention.
In searching for an underlying cause for the
discordance between applicator adherence and
detectable vaginal tenofovir concentrations be-
tween the LD and non-LD groups, we found that
non–Lactobacillus-dominant bacteria assoc iated
with BV rapidly metabolized tenofovir, thereby
likely reducing extracellular drug availability. The
rapid loss of tenofovir by G. vaginalis and other
bacteria may also affect gel adherence estimates
based on vaginal drug levels in non-LD women,
particularly considering that Gardnerella metab-
olizes tenofovir more rapidly than target cells
uptake and convert the drug to active form. Thus,
although adherence i s importa nt for efficacy
(23, 24), the lack of Lactobacillus dominance
mayalsobeacontributingfactor.Thesedata
indicate that w omen with BV may have to be
more rigorous in their adherence to tenofovir
gel administration to be protected against HIV
infection, due to the rapid metabolism of tenofovir
by dysbiotic bacteria. Because of the potential in-
terplay between vaginal bacteria and adherence
in topical microbicide efficacy, a useful next step
could be to evaluate the proportion of non-LD
women in other topical microbicide trials of
tenofovir conducted in Africa such as VOICE and
FACTS (23, 24). These findings may have broader
implications for other topical antiretroviral deliv-
ery strategies, such as vaginal rings, for tenofovir-
based HIV prevention.
Theprevalenceof
Lactobacillus do
minance in
women in CAPRISA 004 (59%) was comparable
to that observed in self-described black women
from a North American study (58%) (12)butcon-
siderably lower than that reported in Caucasian
women (90%). However, the extent of Lactobacillus
dominance may differ even within the same
country or region. The Lactobacillus species and
overall CSTs in our study participants were sim-
ilar to those described in a previous South African
study (13, 25) but somewhat different from those
reported recently by the FRESH (Females Ris-
ing through Education, Support, and Health) co-
hort in KwaZulu-Natal in South Africa (13). The
reasons for these differences in the prevalence of
Lactobacillus dominance are not known. Some
notable differences were that the CAPRISA 004
women were slightly older (24 versus 21 years) and
had higher hormonal contraceptive usage (97%
versus 54% on DMPA, norethisterone oenan-
thate, or combined oral contraceptive pill), both
of which have previously been associated with
higher Lactobacillus (26, 27).
Although Lactobacillus has been associated
with lower HIV incidence in previous observa-
tional studies, we did not find a strong relation-
ship between LD status and HIV protection in
theplaceboarm.Thismaybeduetothepredom-
inance of L. iners in the women in our study,
rather than L. crispatus, which has been associ-
ated with lower HIV incidence (19, 28) [whereas
L. iners has been associated with increased HIV
risk (29)], or simply being underpowered with
few L. crispatus–dominant women. Nevertheless,
we did observe a nonsignificant trend of reduced
HIV incidence with L. crispatus–dominant women
compared with women who had non–Lactobacillus-
dominant profiles, regardless of tenofovir or placebo
gel assignment.
The ecological diversity in the vaginal micro-
biome has been previously linked to changes in
mucosal immunity in the female genital tract,
including elevated cytokine levels, increased
HIV target cells (CD4
+
CCR5
+
T cells), as well as
epithelial barrier disruption (20, 30, 31). Thus,
the presence of BV-associated inflammatory bac-
teria could have influences at the vaginal mucosa
that may add collectively to a multifactorial mech-
anism affecting microbicideefficacy.Therelative
contributions of drug depletion and these po-
tential host modulatory effects on microbicide
efficacy would be an important avenue of future
investigation.
Some limitations need to be taken into ac-
count when interpreting these data. The lack of
clinical BV measurements, such as Nugent score
or Amsel’s criteria, precludes our ability to associate
these findings with clinical BV criteria, an im-
portant factor in vaginal health. Instead, these
data are limited to associations with metapro-
teomic and metagenomic characterization of
BV. Another caveat of this study is the assumption
Klatt et al., Science 356, 938–945 (2017) 2 June 2017 5of7
Table 1. Effect of adherence on the HIV prevention efficacy of 1% tenofovir gel in women participating in the CAPRISA 004 trial, stratified by
Lactobacillus dominance in the female genital tract. One woman did not produce adherence data. n, number of women.
No. of HIV infections/women-years HIV incidence (95% CI)
Gel adherence Tenofovir Placebo n Tenofovir Placebo Incidence rate ratio Efficacy P value (log-rank)
All participants
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Lactobacillus-dominant 9/331 22/318 407 2.7 (1.2; 5.2) 6.9 (4.3; 10.5) 0.39 (0.16; 0.89) 61% 0.013
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Non–Lactobacillus-dominant 14/219 17/218 281 6.4 (3.5; 10.7) 7.8 (4.5; 12.5) 0.82 (0.37; 1.77) 18% 0.644
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Greater than 50% adherence 13/349 26/304 416 3.7 (2.0; 6.4) 8.6 (5.6; 12.5) 0.44 (0.21; 0.88) 56% 0.013
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Less than 50% adherence 10/200 13/232 271 5.0 (2.4; 9.2) 5.6 (3.0; 9.6) 0.89 (0.35; 2.21) 11% 0.771
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Lactobacillus-dominant
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Greater than 50% adherence 4/209 15/176 244 1.9 (0.5; 4.9) 8.5 (4.8; 14.1) 0.22 (0.05; 0.71) 78% 0.003
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Less than 50% adherence 5/122 7/142 163 4.1 (1.3; 9.6) 4.9 (2.0; 10.2) 0.83 (0.21; 3.05) 17% 0.735
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Non–Lactobacillus-dominant
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Greater than 50% adherence 9/141 11/128 172 6.4 (2.9; 12.2) 8.6 (4.3; 15.4) 0.74 (0.27; 1.98) 26% 0.558
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
Less than 50% adherence 5/78 6/90 108 6.4 (2.1; 15.0) 6.7 (2.4; 14.5) 0.96 (0.23; 3.79) 4% 0.935
............ ................ ................ ................ ............... ................ ................ ................ ............. ................ ............... ................ ................ ................ ................ ............... ................ ................ ................ ................ ............... ...........
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