Project Title
Interaction of vaginal virome and bacteriome in pregnant women living with HIV in sub-Saharan Africa and risk of preterm birth
EDCTP Project
TMA2020CDF-3192
EDCTP Program
EDCTP2
EDCTP Project Call
Career Development Fellowship (CDF)
Project Objectives
1. To compare vaginal virome diversity and composition of pregnant WLHIV versus pregnant WNLHIV. 2. Evaluate the relationship between vaginal virome and bacteriome. 3. To compare vaginal virome diversity and composition in WLHIV experiencing PTB versus WLHIV with healthy birth outcomes.
Study Design
Observational
Project Summary
Pregnant women living with HIV (WLHIV) are more likely to experience adverse birth outcomes, including preterm birth, than pregnant women not living with HIV (WNLHIV), thereby substantially contributing to maternal and infant morbidity in sub-Saharan Africa. Although alterations in vaginal bacteriome have been linked to preterm birth, they cannot fully explain the increased risk. Yet the role of vaginal pro-and eukaryotic viral communities, collectively referred to as “virome”, has not been rigorously evaluated. As the enteric virome is expanded in individuals living with HIV and modulates their gut bacteriome, we hypothesized that HIV infection in women of childbearing age would lead to an expanded vaginal virome, including the increased number and diversity of bacteriophages, which either directly or indirectly (through alteration of the bacterial microbiome) is associated with a higher risk of preterm birth in women with HIV. To test this hypothesis, we (1) compared the composition and diversity of the vaginal virome in pregnant women with HIV versus pregnant women without HIV, both with healthy birth outcomes, using shotgun metagenomic sequencing; (2) identified in vivo associations between vaginal viruses associated with HIV infection and bacteriome and validated key bacterial-viral interactions in vitro; and (3) compared vaginal virome diversity and composition of women with HIV experiencing preterm birth versus age-, parity- and antiretroviral regimen-matched women with HIV with healthy birth outcomes. This project leveraged the infrastructure and rigorous metadata of an ongoing pregnancy cohort study in Khayelitsha, Cape Town, South Africa. Pregnant women with and without HIV were enrolled during routine antenatal care visits and vaginal swabs, clinical and obstetric data are collected throughout pregnancy. At delivery, birth outcomes and other clinical data were collected. We found that vaginal DNA virome profiles were distinct between pregnant WLHIV and pregnant WNLHIV. The observed participant richness of viral operational taxonomic units (vOTUs) was lower in pregnant WLHIV compared to pregnant WNLHIV. Papillomavirus vOTUs were uniquely detected in pregnant WLHIV, while herpesvirus vOTUs were noted in pregnant WLHIV and pregnant WNLHIV to a similar extent. Strikingly, in both groups of women, the vaginal viral community was dominated by bacteriophages, making up almost 97% of the total vOTUs. Bacteriophage vOTUs with the predicted bacterial hosts Prevotella and Fannyhessea vaginae were more prevalent in pregnant WLHIV than pregnant WNLHIV, while those predicted to target Peptoniphilus were only present in pregnant WNLHIV. Bacteriophages with a predicted temperate lifestyle were more common than those predicted to be lytic in both groups of women. Predicted hosts of virulent phages in both groups included Lactobacillus mulieris, Prevotella bivia, Fannyhessea vaginae, Bifidobacterium swidsinskii, Dialister microaerophilus, and Sneathia vaginalis. Differences in vaginal viral diversity and composition suggest that, like the bacterial microbiome, the vaginal virome might also differ between pregnant WLHIV and pregnant WNLHIV. Future research is needed to understand its impact on reproductive health outcomes. Understanding the interplay between vaginal viruses, bacteria and preterm birth is relevant for developing novel diagnostics and interventions for improving birth outcomes among women with HIV, and to potentially decrease infant morbidity and mortality. Project website: https://idm.uct.ac.za/contacts/anna-ursula-happel-0/full?subsite=1506
Host Organisation
| Department | Institution | Country |
|---|---|---|
| Pathology | University of Cape Town | ZA |
Current Organisation
University of Cape Town
Current Job Title
Senior Research Officer
Biography
Translational scientist with experience in clinical and laboratory sciences with a vision to actively contribute to the development of locally implementable intervention strategies and policies to improve maternal and child health in Southern Africa.
Publications
Objectives
Young women in sub-Saharan Africa are at high risk of STIs and unintended pregnancies, yet hormonal contraceptive (HC) use may affect STI risk. We compared the influence of three HCs on the incidence and prevalence of STIs and bacterial vaginosis (BV) in South African adolescents.
Methods
One hundred and thirty adolescents between 15 and 19 years were randomised to the injectable norethisterone enanthate (Net-En), combined oral contraceptives (COC) (Triphasil or Nordette) or a combined contraceptive vaginal ring (CCVR; NuvaRing) for 16 weeks (clinicaltrials.gov/NCT02404038). Vaginal samples were collected at baseline and 16 weeks post contraceptive initiation for STI and BV testing.
Results
In an intention-to-treat analysis, no significant differences in BV prevalence were found between study arms. The overall incidence of any STI at follow-up was high: 16.2% in the COC arm; 25.7% in the Net-En arm; and 37.1% in the CCVR arm. The incidence rate (IR) of any STI was similar between Net-En (IR 0.74 (95% CI 0.34 to 1.41)) and the oestrogen-containing contraceptives (IR 0.78 (95% CI 0.47 to 1.22)). A lower IR of
Chlamydia trachomatis
(incidence rate ratio (IRR) 0.68 (95% CI 0.19 to 1.99)) and
Neisseria gonorrhoeae
(IRR 0.25 (95% CI 0.01 to 1.35)) but a higher IR of
Mycoplasma genitalium
(IRR 16.0 (95% CI 2.96 to 400)), was observed in the Net-En arm compared with the oestrogen-containing contraceptives, although the overall incidence of
M. genitalium
was low (4.7%). In an exploratory analysis, the risk of any STI and
N. gonorrhoeae
was lower in the COC arm compared with CCVR. A per-protocol analysis yielded similar results.
Conclusion
Our results suggest that use of Net-En may be associated with increased risk of
M. genitalium
compared with oestrogen-containing contraceptives but not with overall STI risk. COC use may decrease STI risk relative to CCVR.
Young women in sub-Saharan Africa are disproportionally affected by HIV infection and unintended pregnancies. However, hormonal contraceptive (HC) use may influence HIV risk through changes in genital tract microbiota and inflammatory cytokines. To investigate this, 130 HIV negative adolescent females aged 15-19 years were enrolled into a substudy of UChoose, an open-label randomized crossover study (NCT02404038), comparing acceptability and contraceptive product preference as a proxy for HIV prevention delivery methods. Participants were randomized to injectable norethisterone enanthate (Net-En), combined oral contraceptives (COC) or etonorgesterol/ethinyl estradiol combined contraceptive vaginal ring (CCVR) for 16 weeks, then crossed over to another HC for 16 weeks. Cervicovaginal samples were collected at baseline, crossover and exit for characterization of the microbiota and measurement of cytokine levels; primary endpoints were cervical T cell activation, vaginal microbial diversity and cytokine concentrations. Adolescents randomized to COCs had lower vaginal microbial diversity and relative abundance of HIV risk-associated taxa compared to Net-En or CCVR. Cervicovaginal inflammatory cytokine concentrations were significantly higher in adolescents randomized to CCVR compared to COC and Net-En. This suggests that COC use may induce an optimal vaginal ecosystem by decreasing bacterial diversity and inflammatory taxa, while CCVR use is associated with genital inflammation.
Background
Cervicovaginal inflammation, bacterial microbiota and hormonal contraceptives all influence sexual and reproductive health. To date, the effects of intramuscular depo-medroxyprogesterone acetate (DMPA-IM) versus injectable norethisterone enanthate (NET-EN) on vaginal microbiota or cytokines have not been compared back-to-back, although
in-vitro
data suggest that DMPA-IM and NET-EN have different pharmacokinetic and biologic activities. This study aimed at comparing the effects of DMPA-IM versus NET-EN initiation on cervicovaginal cytokines and microbiota in women at high risk for sexually transmitted infections (STIs) assigned to the respective contraceptives.
Methods
We collected socio-demographic characteristics and vaginal samples from women initiating DMPA-IM (ECHO Trial; n = 53) and NET-EN (UChoose Trial; n = 44) at baseline and after two consecutive injections to assess cytokine concentrations by Luminex, vaginal microbiota by 16S rRNA gene sequencing, STIs, bacterial vaginosis (BV) and candidiasis.
Results
Cytokine concentrations did not change significantly after initiating DMPA-IM or NET-EN, although NET-EN versus DMPA-IM-associated profiles were distinct. While the abundance of bacterial taxa associated with optimal and non-optimal microbiota fluctuated with DMPA-IM use, overall community composition did not significantly change with either contraceptive. HSV-2 serology, chlamydial infection, gonorrhoea and candidiasis did not influence the associations between contraceptive type and cervicovaginal cytokines or microbiota.
Conclusions
Both DMPA-IM and NET-EN use did not lead to broad inflammatory or microbiota changes in the female genital tract of sub-Saharan African women. This suggests that NET-EN is likely a viable option for contraception in African women at high risk of BV and STIs.
Introduction
Young women in Southern Africa have extremely high HIV incidence rates necessitating the availability of female-controlled prevention methods. Understanding adolescent preference for seeking contraception would improve our understanding of acceptability, feasibility and adherence to similar modes of delivery for HIV prevention.
Methods
UChoose was an open-label randomized crossover study over 32 weeks which aimed to evaluate the acceptability and preference for contraceptive options in healthy, HIV-uninfected, female adolescents aged 15 to 19 years, as a proxy for similar HIV prevention methods. Participants were assigned to a contraceptive method for a period of 16 weeks in the form of a bi-monthly injectable contraceptive, monthly vaginal Nuvaring
®
or daily combined oral contraceptive (COC) and then asked to state their preference. At 16 weeks, participants crossed over to another contraceptive method, to ensure that all participants tried the Nuvaring
®
(least familiar modality) and additionally, either the injection or COC. Primary outcomes were contraceptive acceptability and preference. At the end of the 32 weeks they were also asked to imagine their preference for an HIV prevention modality. Secondary endpoints included changes in sexual behaviour, contraceptive adherence and preference for biomedical and behavioural HIV prevention methods.
Results
Of the 180 participants screened, 130 were enrolled and randomized to the Nuvaring
®
(n = 45), injection (n = 45) or COC (n = 40). Significantly more Nuvaring
®
users (24/116; 20.7%) requested to change to another contraceptive option compared to injection (1/73; 1.4% p = 0.0002) and COC users (4/49; 8% p = 0.074). Of those that remained on the Nuvaring
®
, adherence was significantly higher than to COC (p < 0.0001). Significantly more injection users (77/80; 96.3%) thought this delivery mode was convenient to use compared to Nuvaring
®
(74/89; 83.1%; p = 0.0409) or COC (38/50; 76.0%; p = 0.0034). Overall, the preferred contraceptive choice was injection, followed by the ring and lastly the pill.
Conclusions
Adherence to daily COC was difficult for adolescents in this cohort and the least favoured potential HIV prevention option. While some preferred vaginal ring use, these data suggest that long-acting injectables would be the preferred prevention method for adolescent girls and young women. This study highlights the need for additional options for HIV prevention in youth.
Cervicovaginal inflammation, nonoptimal microbiota, T-cell activation, and hormonal contraceptives may increase HIV risk, yet associations between these factors and subclinical
Candida
colonization or hyphae are unknown. We collected cervicovaginal samples from 94 South African adolescents, aged 15 to 19 years, who were randomized to injectable norethisterone enanthate (Net-En), an etonorgesterol/ethinyl estradiol vaginal ring (NuvaRing), or oral contraceptives in the UChoose trial (NCT02404038) at baseline and 16 weeks post-randomization. We assessed cervicovaginal samples for subclinical
Candida
colonization (by quantitative PCR [qPCR]), hyphae (by Gram stain), microbiota composition (by 16S rRNA gene sequencing), cytokine concentrations (by Luminex), and cervical T-cell phenotypes and activation (by multiparameter flow cytometry). While hormonal contraceptive type did not influence incidence of
Candida
colonization or hyphae, hyphae presence was associated with significantly elevated concentrations of IL-22, IL-17A and IL-17F, all produced by Th17 cells, but not of other cytokines, such as IL-1β or IL-6, after adjustment for confounders. Subclinical
Candida
colonization was associated with reduced frequencies of Th17-like cells and elevated frequencies of CCR6-CCR10 T cells. Women with
Candida
hyphae were less likely to have bacterial vaginosis (BV). Persistent, subclinical colonization with
Candida
over 16 weeks was associated with significant increases in Th17-related cytokine concentrations and highly activated Th17-like and CCR6-CCR10 T-cell frequencies. These data suggest that vaginal
Candida
colonization and hyphae increase Th17-related cytokines, but not overall female genital tract inflammation in Sub-Saharan African adolescents. Persistent
Candida
colonization, even when asymptomatic, may increase Th17 cell frequencies and related cytokines and thereby could subsequently increase HIV risk, although the causal relationship requires confirmation.
IMPORTANCE
Sub-Saharan African female adolescents are globally at the highest risk of HIV acquisition, and genital inflammation, microbial dysbiosis, and cervical HIV target cell activation are thought to contribute to this risk. Previously, the relationship between these mucosal factors and subclinical vaginal
Candida
colonization or hyphae has not been described, and the role of HIV-susceptible Th17 cells in mediating anti-
Candida
immunity in the human female genital tract has not been clearly established. We show that presence of yeast hyphae was associated with increases in Th17 cell-related cytokines and the absence of microbial dysbiosis, and that persistent
Candida
colonization resulted in significant increases in Th17-related cytokines and highly activated Th17-like cell frequencies. Our results suggest that Th17 cells are important for anti-
Candida
immunity in the human female genital tract and that prolonged vaginal
Candida
colonization may contribute to increased HIV risk in Sub-Saharan African adolescents by increasing HIV target cell frequencies and activation.
Determinants of the acquisition and maintenance of maternal microchimerism (MMc) during infancy and the impact of MMc on infant immune responses are unknown. We examined factors that influence MMc detection and level across infancy and the effect of MMc on T cell responses to bacillus Calmette-Guérin (BCG) vaccination in a cohort of HIV-exposed, uninfected and HIV-unexposed infants in South Africa. MMc was measured in whole blood from 58 infants using a panel of quantitative PCR assays at day 1, and 7, 15, and 36 weeks of life. Infants received BCG at birth, and selected whole blood samples from infancy were stimulated in vitro with BCG and assessed for polyfunctional CD4+ T cell responses. MMc was present in most infants across infancy, with levels ranging from 0 to 1,193/100,000 genomic equivalents and was positively impacted by absence of maternal HIV, maternal and infant HLA compatibility, infant female sex, and exclusive breastfeeding. Initiation of maternal antiretroviral therapy prior to pregnancy partially restored MMc level in HIV-exposed, uninfected infants. Birth MMc was associated with an improved polyfunctional CD4+ T cell response to BCG. These data emphasize that both maternal and infant factors influence the level of MMc, which may subsequently affect infant T cell responses.
The ECHO Trial randomized women to intramuscular depot medroxyprogesterone acetate (DMPA-IM), levonorgestrel implant (LNG-implant), or copper intrauterine device (Cu-IUD). In a sub-study of the ECHO Trial, we tested the hypothesis that contraceptives influence genital inflammation by comparing cervicovaginal cytokine changes following contraception initiation. In addition, we compared cytokine profiles in women who acquired HIV (cases) versus those remaining HIV-negative (controls). Women (n=251) from South Africa and Kenya were included. Twenty-seven cervicovaginal cytokines were measured by Luminex at baseline, 1- and 6-month post-contraceptive initiation. In addition, cytokines were measured pre-seroconversion in HIV cases (n=25) and controls (n=100). At 6-months post-contraceptive initiation, women using Cu-IUD had increased concentrations of 25/27 cytokines compared to their respective baseline concentrations. In contrast, women initiating DMPA-IM and LNG-implant did not experience changes in cervicovaginal cytokines. Pre-seroconversion concentrations of IL-1β, IL-6, and TNF-α, previously associated with HIV risk, correlated with increased HIV risk in a logistic regression analysis, although not significantly after correcting for multiple comparisons. Adjusting for contraceptive arm did not alter these results. Although Cu-IUD use broadly increased cervicovaginal cytokine concentrations at 6-months post-insertion, these inflammatory changes were found not to be a significant driver of HIV risk.
Background
Adolescents in sub-Saharan Africa are at risk for human immunodeficiency virus (HIV) infection and unintended pregnancies. Observational studies suggest that injectable hormonal contraceptives (HCs) increase the HIV risk, although their effects on genital inflammation, particularly HIV-susceptible T-helper 17 (Th17) cells, are unknown. In a randomized crossover study, the effect of injectable norethisterone oenanthate (NET-EN), combined contraceptive vaginal rings (CCVR; NuvaRing), and combined oral contraceptive pills (COCPs) on cervical Th17 cells and cytokines were compared.
Methods
Adolescents (n = 130; 15-19 years) were randomly assigned 1:1:1 to NET-EN, CCVR, or COCPs for 16 weeks, then subsequently crossed over to another HC for 16 weeks. Estrogen, follicular stimulating hormone (FSH), and luteinizing hormone (LH) levels were measured. Chemokine receptor 5 (CCR5), human leukocyte antigen (HLA) DR isotope, and cluster of differentiation 38 (CD38) expression by cervical cytobrush-derived CD4+ T cells was assessed by fluorescence-activated cell sorting. Th17 cells were defined as CCR6+ and CCR10-. Cervicovaginal Th17-related cytokines were measured by Luminex.
Results
CCVR use for the first 16 weeks was associated with reduced Th17 frequencies and lower FSH and LH concentrations, as compared to NET-EN and COCPs, with FSH concentrations and Th17 frequencies correlating significantly. However, Th17-related cytokine concentrations (interleukin [IL]-21, IL-1β, tumor necrosis factor-α, interferon-γ) and CCR5, HLA-DR, CD38, and Th17 frequencies were significantly higher in CCVR than NET-EN and COCP. At crossover, CCVR users changing to COCPs or NET-EN did not resolve activation or cytokines, although switching from COCP to CCVRs increased cytokine concentrations.
Conclusions
CCVR use altered endogenous hormone levels and associated cervical Th17 cell frequencies to a greater extent than use of NET-EN or COCPs, although Th17 cells were more activated and Th17-related cytokine concentrations were elevated. While CCVRs may impact the HIV risk by regulating Th17 numbers, increased activation and inflammation may balance any risk gains.