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Female genital tract (FGT) inflammation increases HIV infection susceptibility. Non-optimal cervicovaginal microbiota, characterized by depletion of Lactobacillus species and increased bacterial diversity, is associated with increased FGT cytokine production. Lactobacillus species may protect against HIV partly by reducing FGT inflammation. We isolated 80 lactobacilli from South African women with non-optimal (Nugent 4–10; n = 18) and optimal microbiota (Nugent 0–3; n = 14). Cytokine production by vaginal epithelial cells in response to lactobacilli in the presence and absence of Gardnerella vaginalis was measured using Luminex. Adhesion to vaginal epithelial cells, pH, D/L-lactate production and lactate dehydrogenase relative abundance were assessed. Lactobacilli from women with non-optimal produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1α, IL-1β and MIP-1α/β production significantly elevated. Overall, lactobacilli suppressed IL-6 (adjusted p G. vaginalis. Cytokine responses to the lactobacilli were inversely associated with lactobacilli adhesion to epithelial cells and D-lactate dehydrogenase relative abundance. Thus, while cervicovaginal lactobacilli reduced the production of the majority of inflammatory cytokines in response to G. vaginalis, isolates from women with non-optimal microbiota were more inflammatory and produced less lactic acid than isolates from women with optimal microbiota.

Background Cervicovaginal CD4+ T cells are preferential targets for human immunodeficiency virus (HIV) infection and have consequently been used as a proxy measure for HIV susceptibility. The ECHO randomized trial offered a unique opportunity to consider the association between contraceptives and Th17-like cells within a trial designed to evaluate HIV risk. In a mucosal substudy of the ECHO trial, we compared the impact of initiating intramuscular depot medroxyprogesterone acetate (DMPA-IM), copper-IUD, and the levonorgestrel (LNG) implant on cervical T cells. Methods Cervical cytobrushes from 58 women enrolled in the ECHO trial were collected at baseline and 1 month after contraceptive initiation. We phenotyped cervical T cells using multiparameter flow cytometry, characterized the vaginal microbiome using 16s sequencing, and determined proteomic signatures associated with Th17-like cells using mass spectrometry. Results Unlike the LNG implant or copper-IUD, DMPA-IM was associated with higher frequencies of cervical Th17-like cells within 1 month of initiation (P = .012), including a highly susceptible, activated population co-expressing CD38, CCR5, and α4β7 (P = .003). After 1 month, women using DMPA-IM also had more Th17-like cells than women using the Cu-IUD (P = .0002) or LNG implant (P = .04). Importantly, in women using DMPA-IM, proteomic signatures signifying enhanced mucosal barrier function were associated with the increased abundance of Th17-like cells. We also found that a non–Lactobacillus-dominant microbiome at baseline was associated with more Th17-like cells post–DMPA-IM (P = .03), although this did not influence barrier function. Conclusions Our data suggest that DMPA-IM–driven accumulation of HIV-susceptible Th17-like cells might be counteracted by their role in maintaining mucosal barrier integrity. Clinical Trials Registration NCT02550067.

While live biotherapeutics offer a promising approach to optimizing vaginal microbiota, the presence of functional prophages within introduced

Background The ECHO trial randomized women to intramuscular depot medroxyprogesterone acetate (DMPA-IM), levonorgestrel implant (LNG-implant), or copper intrauterine device (Cu-IUD). In a substudy 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). Methods Women (n = 251) from South Africa and Kenya were included. Twenty-seven cervicovaginal cytokines were measured by Luminex at baseline, and 1 and 6 months after contraceptive iTanko et alnitiation. In addition, cytokines were measured preseroconversion in HIV cases (n = 25) and controls (n = 100). Results At 6 months after 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. Preseroconversion 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. Conclusions Although Cu-IUD use broadly increased cervicovaginal cytokine concentrations at 6 months postinsertion, these inflammatory changes were found not to be a significant driver of HIV risk. Clinical Trials Registration NCT02550067.

Background Female genital tract (FGT) inflammation is an important risk factor for HIV acquisition. The FGT microbiome is closely associated with inflammatory profile; however, the relative importance of microbial activities has not been established. Since proteins are key elements representing actual microbial functions, this study utilized metaproteomics to evaluate the relationship between FGT microbial function and inflammation in 113 young and adolescent South African women at high risk of HIV infection. Women were grouped as having low, medium, or high FGT inflammation by K-means clustering according to pro-inflammatory cytokine concentrations. Results A total of 3186 microbial and human proteins were identified in lateral vaginal wall swabs using liquid chromatography-tandem mass spectrometry, while 94 microbial taxa were included in the taxonomic analysis. Both metaproteomics and 16S rRNA gene sequencing analyses showed increased non-optimal bacteria and decreased lactobacilli in women with FGT inflammatory profiles. However, differences in the predicted relative abundance of most bacteria were observed between 16S rRNA gene sequencing and metaproteomics analyses. Bacterial protein functional annotations (gene ontology) predicted inflammatory cytokine profiles more accurately than bacterial relative abundance determined by 16S rRNA gene sequence analysis, as well as functional predictions based on 16S rRNA gene sequence data (p < 0.0001). The majority of microbial biological processes were underrepresented in women with high inflammation compared to those with low inflammation, including a Lactobacillus-associated signature of reduced cell wall organization and peptidoglycan biosynthesis. This signature remained associated with high FGT inflammation in a subset of 74 women 9 weeks later, was upheld after adjusting for Lactobacillus relative abundance, and was associated with in vitro inflammatory cytokine responses to Lactobacillus isolates from the same women. Reduced cell wall organization and peptidoglycan biosynthesis were also associated with high FGT inflammation in an independent sample of ten women. Conclusions Both the presence of specific microbial taxa in the FGT and their properties and activities are critical determinants of FGT inflammation. Our findings support those of previous studies suggesting that peptidoglycan is directly immunosuppressive, and identify a possible avenue for biotherapeutic development to reduce inflammation in the FGT. To facilitate further investigations of microbial activities, we have developed the FGT-DB application that is available at http://fgtdb.org/.

Bacterial vaginosis (BV) and periodontal disease (PD) are characterised as bacterial dysbioses. Both are associated with an increased risk of poor pregnancy outcomes, yet it is unknown whether PD and BV are related. We characterised the oral microbiota of young South African females with a high prevalence of BV and investigated the association between oral communities and vaginal microbiota. DNA was extracted from vaginal lateral wall, saliva and supragingival plaque samples from 94 adolescent females (aged 15&ndash, 19 years). 16S rRNA gene sequencing of the V4 hypervariable region was performed for analysis of the oral and vaginal microbiota and BV status was determined by Nugent scoring. The core oral microbiota was predominately comprised of Firmicutes followed by Proteobacteria and Bacteroidetes. The salivary microbiota of participants with BV was more diverse than those with lactobacillus-dominated communities (p = 0.030). PD-associated bacterial species, including Prevotella intermedia and Porphyromonas endodontalis were enriched in the supragingival microbiota of women with non-optimal vaginal communities compared to those with Lactobacillus-dominant communities, while Pseudomonas aeruginosaand Prevotella intermedia were enriched in the saliva of women with non-optimal vaginal microbiota. These data suggest a relationship between oral and vaginal dysbiosis, warranting further investigation into whether they are casually related.

Antibiotics continue to be the standard-of-care for bacterial vaginosis (BV), although recurrence rates are high. Vaginal probiotics may improve durability of BV treatment, although few probiotics for vaginal health contain Lactobacillus spp. that commonly colonize the lower female genital tract. Characteristics of vaginal Lactobacillus strains from South African women were evaluated for their probiotic potential in vitro compared to strains from commercial vaginal products, including growth at varying pHs, ability to lower pH, produce D-/L-lactate and H2O2, influence growth of BV-associated Gardnerella vaginalis and Prevotella bivia, adherence to cervical cells and susceptibility to antibiotics. Fifty-seven Lactobacillus strains were purified from cervico-vaginal fluid, including L. crispatus, L. jensenii, L. gasseri, L. mucosae, and L. vaginalis. L crispatus strains grew better at pHs below 4.5 and lowered pH more effectively than other strains. Production of D-/L-lactate and H2O2 varied between Lactobacillus species and strains. Lactobacillus strains generally inhibited P. bivia more uniformly than G. vaginalis isolates. All vaginal Lactobacillus isolates were resistant to metronidazole while susceptibility to clindamycin varied. Furthermore, vaginal Lactobacillus strains tended to be broadly susceptible to penicillin, amoxicillin, rifampicin and rifabutin. Whole-genome-sequencing of five of the best-performing vaginal Lactobacillus strains confirmed their likely safety, due to antimicrobial resistance elements being largely absent, while putative intact prophages were present in the genomes of two of the five strains. Overall, vaginal Lactobacillus strains largely performed better in these in vitro assays than probiotic strains currently used in probiotics for vaginal health. Including the best-performing vaginal Lactobacillus isolates in a region-specific probiotic for vaginal health may result in improved BV treatment options., Author summary Lactobacillus species in the lower reproductive tract of healthy women lower vaginal pH and protect against sexually transmitted infections. However, women commonly suffer from bacterial vaginosis, a disruption in the optimal Lactobacillus-dominated genital microbiota to a more diverse one marked by higher pH, vaginal discharge and inflammation. Bacterial vaginosis is associated with adverse pregnancy outcomes and increased risk of sexually transmitted infections, including HIV. While antibiotics are the standard-of-care for bacterial vaginosis, most cases recur within six-months. Probiotics that include lactobacilli have been explored to improve treatment, although the majority of products do not contain species commonly found in the vagina. Here, we characterised a large panel of vaginal Lactobacillus strains from young African women (including growth, adhesion to host cells, ability to produce antimicrobial products, and pathogen inhibition), compared to isolates from commercial probiotic products for vaginal health. We also sequenced the genomes of top-performing isolates. Several vaginal Lactobacillus strains exhibited better probiotic profiles than commercial strains, suggesting that they would be beneficial in the development of probiotic treatment of bacterial vaginosis. A wider range of well-characterized Lactobacillus-containing probiotics may improve bacterial vaginosis treatment outcomes, which would hopefully lower the burden of adverse pregnancy outcomes and sexually transmitted infections.

ObjectivesYoung 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.MethodsOne 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.ResultsIn 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.ConclusionOur 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.

ObjectivesVaginal dysbiosis and STIs are important drivers of the HIV epidemic and reproductive complications. These conditions remain prevalent, partly because most cases are asymptomatic. We have shown that inflammatory cytokines interleukin (IL)-1α, IL-1β and interferon-γ-induced protein (IP)-10 are biomarkers for detecting asymptomatic STIs and vaginal dysbiosis (bacterial vaginosis (BV) or intermediate microbiota). This study aimed to validate the performance of these biomarkers in African women recruited regardless of symptoms.MethodsIL-1α, IL-1β and IP-10 were measured in menstrual cup secretions, endocervical, lateral vaginal wall and vulvovaginal swabs from 550 women from Pretoria, Soweto and Cape Town, South Africa and Bondo, Kenya using Luminex and ELISA. STIs were assessed by PCR, BV by Nugent scoring and vaginal microbiota by 16S rRNA sequencing.ResultsAcross four study populations and four types of genital specimens, the performance of IL-1α, IL-1β and IP-10 for identification of women with STIs, BV or intermediate microbiota was consistent. Of the genital samples assessed, biomarkers measured in lateral vaginal wall swabs performed best, correctly classifying 76%(95% CI 70% to 81%) of women according to STI, BV or intermediate microbiota status (sensitivity 77%, specificity 71%) and were more accurate than clinical symptoms (sensitivity 41%, specificity 57%) (p=0.0003). Women incorrectly classified as STI/BV positive using the biomarkers had more abundant dysbiosis-associated bacteria, including Prevotella bivia and Gardnerella sp, detected by 16S rRNA sequencing, but not Nugent scoring. Including vaginal pH with the cytokine biomarkers improved the accuracy of the test (82% (95% CI 75% to 88%) correctly classified), although pH alone had poor specificity (61%).ConclusionsAn inexpensive, point-of-care screening test including IL-1α, IL-1β and IP-10 (and potentially pH) could be used in resource-limited settings to identify women with asymptomatic STIs and dysbiosis. These women could then be referred for aetiological testing, followed by specific treatment.

Adolescents in Africa are at high risk for HIV infection, other sexually transmitted infections (STIs) and bacterial vaginosis (BV). Since behavior and burden of STIs/BV may influence HIV risk, behavioral risk factors and prevalence of STIs/BV were compared in HIV-seronegative adolescent females (n = 298; 16–22 years) from two South African communities (Soweto and Cape Town). STIs ( Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, herpes simplex virus (HSV)-1, HSV-2, Treponema pallidum, and Haemophilus ducreyi) were detected by multiplex polymerase chain reaction, human papillomavirus (HPV) by Roche Linear Array, and BV by Nugent scoring. Rates of BV (Nugent ≥7; 46.6%) and HPV (66.8%) were high in both communities. Prevalence of C. trachomatis and N. gonorrhoeae were >2-fold higher in Cape Town than Soweto (Chlamydia: 42% [62/149] versus 18% [26/148], p 70% of young women with treatable conditions that could enhance HIV risk would have been missed because they lacked symptoms associated with syndromic management.

Table S2. List of protein BLAST results against UniProt and NCBI nr databases (DBs). Liquid chromatography-tandem mass spectrometry was used to evaluate the metaproteome in lateral vaginal wall swabs from 113 women from Cape Town, South Africa. Primary assigned taxa, obtained based on MaxQuant version 1.5.7.4, are presented in the second column. The curated taxa (third column) were obtained after considering the top similar and frequent BLAST hits for each protein. IDs: UniProt Identity codes. Table S3. List of differentially abundant taxa distinguishing women with low and high inflammation. Liquid chromatography-tandem mass spectrometry was used to evaluate the metaproteome in lateral vaginal wall swabs from 113 women from Cape Town, South Africa. The false discovery rate (FDR) corrected p-values were obtained based on the aggregated log2-transformed intensity-based absolute quantification of all proteins assigned to the same taxa applying the moderated t-test (limma R package), after adjusting for confounding variables including age, contraceptives, prostate specific antigen (PSA) and sexually transmitted infections (STIs). Taxa with FDR adjusted p-values 1.2 or < -1.2 were considered as differentially abundant taxa. Table S4. List of differentially abundant proteins distinguishing women with low, medium and high inflammation. Liquid chromatography-tandem mass spectrometry was used to evaluate the metaproteome in lateral vaginal wall swabs from 113 women from Cape Town, South Africa. The false discovery rate (FDR) corrected p-values were obtained based on the log2-transformed intensity-based absolute quantification of each protein applying the moderated t-test (limma R package), after adjusting for confounding variables including age, contraceptives, prostate specific antigen (PSA) and sexually transmitted infections (STIs). Proteins with FDR adjusted p-values 1.2 or < -1.2 were considered as differentially abundant proteins. IDs: UniProt Identity codes. Table S5. List of differentially abundant molecular function (MF) gene ontology terms distinguishing women with low and high inflammation. Liquid chromatography-tandem mass spectrometry was used to evaluate the metaproteome in lateral vaginal wall swabs from 113 women from Cape Town, South Africa. The false discovery rate (FDR) corrected p-values were obtained based on the aggregated log2-transformed intensity-based absolute quantification of all proteins assigned to the same MF term applying the moderated t-test (limma R package), after adjusting for confounding variables including age, contraceptives, prostate specific antigen (PSA) and sexually transmitted infections (STIs). Taxa with FDR adjusted p-values 1.2 or < -1.2 were considered as significantly abundant MFs. (PDF 6094 kb)

Table S1. Demographic and clinical characteristics of study participants stratified by female genital tract pro-inflammatory cytokine levels. Table S6. Details of MaxQuant version 1.5.7.4 parameters used for analysis of metaproteomic data obtained from lateral vaginal wall swabs from 113 women from Cape Town, South Africa. Figure S1. Clustering of women according to overall metaproteomic profiles. Figure S2. Identification of women with low, medium and high levels of pro-inflammatory cytokines and chemokines. Figure S3. Human biological processes, microbial taxa and microbial biological processes in individual women with low, medium and high inflammation. Figure S4. Differentially abundant proteins in women with high versus low inflammatory cytokine profiles. Figure S5. Top 50 significantly differentially abundant human biological processes in women with low, and high inflammation. Figure S6. Relative abundance of gene ontologies in BV negative women with Lactobacillus dominant communities. Figure S7. Longitudinal changes in FGT microbial biological process and cellular component gene ontologies. (PDF 6091 kb)

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., Hormonal contraception may alter women’s susceptibility to HIV. Here, the authors report the results of a randomized clinical trial substudy assessing the effects of injectable Net-En, oral contraceptives (COC) and Nuvaring on vaginal microbiota and cytokines, associating COC with lower microbial diversity and Nuvaring with increased inflammation.

Background Young women in sub-Saharan Africa are at high risk for sexually transmitted infections (STIs) and often rely on hormonal contraception (HC) to prevent unwanted pregnancies. Some observational data suggest that HC might affect STI risk. We examined the impact of three HC methods on the adolescent STI incidence and BV prevalence in a randomized trial. Methods 130 adolescent females aged 15 to 19 from Cape Town were enrolled and randomized into three study arms: 1. injectable norethisterone enanthate (NET-EN), 2. combined oral contraceptives (COCs) or 3. combined contraceptive vaginal ring (CCVR) for 16 weeks. Participants then switched to a second HC for a final four months. Vaginal samples were collected at baseline, crossover and exit for STI (chlamydia, gonorrhoea, mycoplasma and trichomoniasis) and bacterial vaginosis (BV) testing by Nugent scoring. Results At baseline, the BV and STI prevalence was 44% and 42%, respectively. There were no significant differences in STI incidence between study arms at crossover, however in an according to protocol analyses, participants using COCs were significantly less likely to present with any STI than participants using either NET-EN (OR 0.22, 95% CI 0.06–0.71, p=0.017) or CCVR (OR 0.21, 95% CI 0.05–0.69, p=0.015). Specifically, participants on CCVR were more likely to be infected with N. gonorrhoea (OR 11.7, 95% CI 2.0–224, p=0.025). These associations stayed significant after adjusting for sexual risk behaviour, including condom use. There was a decreased prevalence of BV in the COC arm and an increased BV prevalence in the NET-EN and CCVR arms at crossover, but these differences were not statistically significant. Conclusion Use of COCs was associated with lower BV prevalence and STI incidence compared to NET-EN and CCVR use. Disclosure No significant relationships.

Studies of seronegative individuals in HIV discordant relationships provide important insights into the effects of HIV exposure on the seronegative partner, but few have examined the impact of partner serostatus on disease progression in seropositive individuals. We investigated the impact of HIV serostatus on clinical and biological factors influencing HIV disease progression in 337 HIV-infected heterosexual individuals in stable long-term HIV-seroconcordant or HIV-serodiscordant relationships. Seroconcordant individuals had significantly higher plasma viral loads (pVLs) than HIV-infected partners in serodiscordant partnerships [4.4 log10 copies RNA/mL (interquartile range 3.7-5.0) versus 3.9 (3.3-4.5), P < 0.0001], irrespective of gender. pVLs correlated inversely with CD4 T-cell counts, although CD4 counts did not differ significantly between seroconcordant and serodiscordant individuals. HIV+ seroconcordant individuals had higher frequencies of CCR5 CD4 and CD8 T cells (P = 0.03 and P = 0.02, respectively) than HIV+ individuals in serodiscordant relationships and higher concentrations of plasma IL-1β (P = 0.04), TNF-α (P = 0.02), and IL-10 (P = 0.02). Activated CD4 T-cell frequencies and TNF-α were the most influential in determining variation in pVLs, independently of CD4 counts. In addition, HIV+ seroconcordant women had significantly higher genital VLs (gVLs) than HIV+ women in serodiscordant relationships (P < 0.001), with pVLs correlating significantly with gVLs (Rho = 0.65, P < 0.0001). Cervical and blood T-cell activation tended to correlate positively, although partner seroconcordance did not influence genital T-cell activation. We conclude that HIV+ seroconcordant individuals have higher frequencies of activated, CCR5-expressing T cells in blood and higher pVLs and gVLs than their HIV+ counterparts in discordant relationships, which could translate to faster disease progression or larger viral reservoir.

BACKGROUND: Untreated sexually transmitted infections (STIs) and bacterial vaginosis (BV) cause genital inflammation and increased risk of HIV infection. WHO-recommended syndromic STI and BV management is severely limited as large numbers of women with asymptomatic infections go untreated. The purpose of this cross-sectional study was to evaluate genital cytokine profiles as a biomarker of STIs and BV to identify women with asymptomatic, treatable infections. METHODS: Concentrations of 42 cytokines in cervicovaginal lavages (CVL) from 227 HIV-uninfected women were measured using Luminex. All women were screened for BV by microscopy and STIs using molecular assays. Multivariate analyses were used to identify cytokine profiles associated with STIs/BV. RESULTS: A multivariate profile of seven cytokines [interleukin (IL)-1α, IL-1β, tumor necrosis factor (TNF)-β, IL-4, fractalkine, macrophage-derived chemokine (MDC), and interferon (IFN)-γ] most accurately predicted the presence of a treatable genital condition, with 77% classification accuracy and 75% cross-validation accuracy [sensitivity 72%; specificity 81%, positive predictive value (PPV) 86%, negative predictive value (NPV) 64%]. Concomitant increased IL-1β and decreased IP-10 concentrations predicted the presence of a treatable genital condition without a substantial reduction in predictive value (sensitivity 77%, specificity 72%, PPV 82% and NPV 65%), correctly classifying 75% of the women. This approach performed substantially better than clinical signs (sensitivity 19%, specificity 92%, PPV 79% and NPV 40%). CONCLUSION: Supplementing syndromic management with assessment of IL-1β and IP-10 as biomarkers of genital inflammation may improve STI/BV management for women, enabling more effective treatment of asymptomatic infections and potentially reducing their risk of HIV infection.

Several host factors have been implicated in resistance to HIV infection in individuals who remain HIV-seronegative despite exposure. In a cohort of HIV-serodiscordant heterosexual couples, we investigated interactions between systemic inflammation and T-cell activation in resistance to HIV infection. Males and females in stable long-term relationships with either HIV-infected or uninfected partners were recruited, blood T-cell activation (CD38, HLA-DR, CCR5 and Ki67) and plasma cytokine concentrations were evaluated. The HIV-negative exposed individuals had significantly lower frequencies of CCR5+ CD4+ and CD8+ T cells than unexposed individuals. Mean fluorescence intensity of CCR5 expression on CD4+ T cells was significantly lower in HIV-negative exposed than unexposed individuals. Protective CCR5 haplotypes (HHA/HHF*2, HHF*2/HHF*2, HHC/HHF*2, HHA/HHA, HHA/HHC and HHA/HHD) tended to be over-represented in exposed compared with unexposed individuals (38% versus 28%, P = 0·58) whereas deleterious genotypes (HHC/HHD, HHC/HHE, HHD/HHE, HHD/HHD and HHE/HHE) were under-represented (26% versus 44%; P = 0·16). Plasma concentrations of interleukin-2 (P = 0·02), interferon-γ (P = 0·05) and granulocyte-macrophage colony-stimulating factor (P = 0·006) were lower in exposed compared with unexposed individuals. Activation marker expression and systemic cytokine concentrations were not influenced by gender. We conclude that the dominant signature of resistance to HIV infection in this cohort of exposed but uninfected individuals was lower T-cell CCR5 expression and plasma cytokine concentrations.

OBJECTIVE: In sub-Saharan Africa HIV-exposed uninfected (HEU) infants have higher morbidity and mortality than HIV-unexposed infants. To evaluate whether immune dysfunction contributes to this vulnerability of HEU infants we conducted a longitudinal observational cohort study to assess T-cell immune responses to infant vaccines (Mycobacterium bovis BCG and acellular pertussis) and staphylococcal enterotoxin B (SEB). In total 46 HEU and 46 HIV-unexposed infants were recruited from Khayelitsha Cape Town. METHODS: Vaccine-specific T-cell proliferation (Ki67 expression) and intracellular expression of four cytokines [interferon-gamma interleukin (IL)-2 IL-13 and IL-17] were measured after whole blood stimulation with antigens at 6 and 14 weeks of age. RESULTS: HEU infants demonstrated elevated BCG-specific CD4 and CD8 T-cell proliferative responses at 14 weeks (P = 0.041 and 0.002 respectively). These responses were significantly increased even after adjusting for birth weight feeding mode and gestational age. Similar to BCG increased CD4 and CD8 T-cell proliferation was evident in response to SEB stimulation (P = 0.004 and 0.002 respectively) although pertussis-specific T cells proliferated comparably between the two groups. Within HEU infants maternal CD4 cell count and length of antenatal antiretroviral exposure had no effect on T-cell proliferation to BCG or SEB. HIV exposure significantly diminished measurable cytokine polyfunctionality in response to BCG Bordetella pertussis and SEB stimulation. CONCLUSION: These data show for the first time when adjusting for confounders that exposure to HIV in utero is associated with significant alterations to CD4 and CD8T-cell immune responses in infants to vaccines and nonspecific antigens.

Problem How does menstrual cup (MC) compare to other genital sampling methods for HIV RNA recovery? Method of study We compared HIV RNA levels between MC, endocervical swab (ECS), and ECS-enriched cervicovaginal lavage (eCVL) specimens in 51 HIV-positive, antiretroviral therapy-naive women at enrollment, 3 and 6 months, with order rotated by visit. Paired comparisons were analyzed with McNemar's exact tests, signed-rank tests, and an extension of Somer's D for pooled analyses across visits. Results MC specimens had the highest proportion of quantifiable HIV VL at enrollment and month 3, but more MC specimens (n=12.8%) were insufficient for testing, compared with ECS (2%, P=0.006) and eCVL (0%, P

Vaccination with Mycobacterium bovis bacille Calmette-Guerin (BCG) has variable efficacy in preventing tuberculosis. Both BCG strain and route of administration have been implicated in determining efficacy; however, these variables are not considered in current clinical recommendations for vaccine choice. We evaluated antigen-specific immunity after percutaneous or intradermal administration of Japanese BCG or intradermal administration of Danish BCG. Ten weeks after vaccination of neonates, percutaneous Japanese BCG had induced significantly higher frequencies of BCG-specific interferon- gamma -producing CD4(+) and CD8(+) T cells in BCG-stimulated whole blood than did intradermal Danish BCG. Similarly, percutaneous vaccination with Japanese BCG resulted in significantly greater secretion of the T helper 1-type cytokines interferon- gamma, tumor necrosis factor- alpha , and interleukin-2; significantly lower secretion of the T helper 2-type cytokine interleukin-4; and greater CD4(+) and CD8(+) T cell proliferation. Thus, BCG strain and route of neonatal vaccination confer different levels of immune activation, which may affect the efficacy of the vaccine.

Objectives Sexually transmitted infections (STI) and bacterial vaginosis (BV) cause female genital tract inflammation. This inflammation, which is often present in the absence of symptoms, is associated with increased susceptibility to HIV infection. We aimed to evaluate genital cytokine profiles and the degree of inflammation associated with common STIs and BV. Methods HIV-uninfected women (n=227) were screened for BV, Chlamydia trachomatis , Neisseria gonorrhoeae , Herpes simplex virus type 2 (HSV-2), and Trichomonas vaginalis . Concentrations of 42 cytokines in cervicovaginal lavages and 13 cytokines in plasma were measured using Luminex. Changes in cytokine profiles were evaluated using Mann–Whitney U test, logistic regression and factor analysis. p Values were adjusted for multiple comparisons using a false discovery rate step-down procedure. Results Women with chlamydia or gonorrhoea had the highest genital cytokine concentrations, with 17/42 and 14/42 cytokines upregulated compared with women with no infection, respectively. BV was associated with elevated proinflammatory cytokine concentrations, but lower chemokine and haematopoietic cytokine concentrations. HSV-2 reactivation was associated with lower levels of inflammation, while trichomoniasis did not cause significant differences in genital cytokine concentrations. Genital infections did not influence plasma cytokine concentrations. Although certain STIs, in particular chlamydia and gonorrhoea, were associated with high genital cytokine concentrations, only 19% of women with an STI/BV had clinical signs. Conclusions Chlamydia was associated with the highest genital cytokine levels, followed by gonorrhoea, HSV-2, trichomoniasis, and BV. In regions where HIV is prevalent and STIs are managed syndromically, better STI/BV screening is urgently needed, as certain infections were found to be highly inflammatory.

Background. BCG vaccination prevents disseminated tuberculosis in children, but it is contraindicated for persons with human immunodeficiency virus (HIV) infection because it can result in severe disease in this population. In tuberculosis-endemic regions, BCG vaccine is administered soon after birth, before in utero and peripartum HIV infection is excluded. We therefore assessed the immunogenicity of BCG vaccine in HIV-exposed infants who received BCG at birth or at 8 weeks of age. Methods. HIV-exposed, uninfected infants were randomly assigned to receive BCG vaccination at birth (the early vaccination arm) or 8 weeks of age (the delayed vaccination arm). BCG-specific proliferative and intracellular cytokine responses were assessed in 28 infants per arm at 6, 8, and 14 weeks of life. Results. There was no difference in BCG-specific T-cell proliferation between the study arms 6 weeks after vaccination. However, at 14 weeks of age, the frequency of interferon γ–expressing CD4 + T cells and multifunctional BCG-specific responses in the delayed vaccinated arm were significantly higher than those in the early vaccination arm (P= .021 and P= .011, respectively). Conclusions. The immunogenicity of BCG vaccination in HIV-exposed, uninfected infants is not compromised when delayed until 8 weeks of age and results in robust BCG-specific T-cell responses at 14 weeks of age. These findings support further evaluation of this modified BCG vaccination strategy for HIV-exposed infants. Clinical Trials Registration. NCT02062580.

Immunogenicity of new tuberculosis (TB) vaccines is commonly assessed by measuring the frequency and cytokine expression profile of T cells.We tested whether this outcome correlates with protection against childhood TB disease after newborn vaccination with bacillus Calmette-Guérin (BCG).Whole blood from 10-week-old infants, routinely vaccinated with BCG at birth, was incubated with BCG for 12 hours, followed by cryopreservation for intracellular cytokine analysis. Infants were followed for 2 years to identify those who developed culture-positive TB-these infants were regarded as not protected against TB. Infants who did not develop TB disease despite exposure to TB in the household, and another group of randomly selected infants who were never evaluated for TB, were also identified-these groups were regarded as protected against TB. Cells from these groups were thawed, and CD4, CD8, and γδ T cell-specific expression of IFN-γ, TNF-α, IL-2, and IL-17 measured by flow cytometry.A total of 5,662 infants were enrolled; 29 unprotected and two groups of 55 protected infants were identified. There was no difference in frequencies of BCG-specific CD4, CD8, and γδ T cells between the three groups of infants. Although BCG induced complex patterns of intracellular cytokine expression, there were no differences between protected and unprotected infants.The frequency and cytokine profile of mycobacteria-specific T cells did not correlate with protection against TB. Critical components of immunity against Mycobacterium tuberculosis, such as CD4 T cell IFN-γ production, may not necessarily translate into immune correlates of protection against TB disease.

Introduction Untreated sexually transmitted infections (STIs) and bacterial vaginosis (BV) cause genital inflammation and increased risk of HIV infection. WHO-recommended syndromic STI and BV management is limited as large numbers of women with asymptomatic infections go untreated. The purpose of this study was to evaluate genital cytokine profiles as a biomarker to identify women with asymptomatic, treatable infections. Methods Luminex was used to measure the concentrations of 42 cytokines in cervicovaginal lavages (CVL) from 227 HIV-uninfected women from Durban, South Africa, and nine cytokines in endocervical swabs from 264 women from Bondo, Kenya and Pretoria, South Africa. Women were screened for BV and treatable STIs ( Chlamydia trachomatis , Neisseria gonorrhoeae , Trichomonas vaginalis , Mycoplasma genitalium ) using microscopy and molecular assays. Nonparametric receiver operating characteristic curves and logistic regression were used to identify cytokine profiles associated with STIs/BV. Results In women from Durban, concomitant increased IL-1α and IL-1β and decreased IP-10 concentrations in CVLs predicted the presence of a treatable genital condition, correctly classifying 76% of women (sensitivity 72%, specificity 81%, PPV 86% and NPV 64%). In a separate validation cohort of women from Bondo and Pretoria, IL-1α, IL-1β and IP-10 concentrations in endocervical swabs correctly classified 72% of the participants according to STI/BV status. This approach performed substantially better than clinical signs in both cohorts from Durban (sensitivity 19%, specificity 92%, PPV 79% and NPV 40%) and Pretoria and Bondo (sensitivity 29%, specificity 78%, PPV 68%, NPV 39%). Conclusion Across two cohorts of women residing in different regions in sub-Saharan Africa, genital IL-1α, IL-1β and IP-10 together was the best immunological predictor of the presence of an STI or BV. Supplementing syndromic management with point-of-care assessment of biomarkers of genital inflammation may improve STI/BV management for women, enabling more effective treatment of asymptomatic infections and potentially reducing their risk of HIV infection. Disclosure of interest statement This work was supported by a Strategic Health Innovation Partnerships (SHIP) grant from the South African Medical Research Council and grants from the Poliomyelitis Research Foundation (PRF) of South Africa and European and Developing Countries Clinical Trials Partnership (EDCTP). The cohorts were supported by grants from the Comprehensive International Program of Research on AIDS (CIPRA) of the Division of AIDS (DAIDS); National Institute of Allergy and infectious Disease (NIAID); National Institutes of Health (NIH) and US Department of Health and Human Services (DHHS) [grant number U19 AI51794]. FEM-PrEP was conducted under two grants funded by the United States Agency for International Development (USAID): the Contraceptive and Reproductive Health Technologies and Research Utilisation Program (GPO-A-00–05–00022–00), and the Preventive Technologies Agreement (GHO-A-00–09–00016–00). Early support was also provided by the Bill and Melinda Gates Foundation. Gilead Sciences, Inc. donated Truvada® and placebo. LM was supported by the PRF; South African Medical Research Council (MRC); the Carnegie Corporation; the National Research Foundation (NRF) of South Africa and the UCT Clinical Infectious Diseases Research Initiative/Wellcome Trust. No pharmaceutical grants were received in the development of this study.

Introduction The biological mechanisms underlying HIV risk in younger women is unclear. HIV is primarily transmitted across the genital mucosa and preferentially infects CD4 + T-cells. We investigated the influence of asymptomatic sexually transmitted infections (STIs) and bacterial vaginosis (BV) on CD4 + T-cell activation and inflammation in the genital tracts of adolescents from South Africa. Methods Cervical cytobrush mononuclear cells were isolated from 148 adolescents (16–22 years) from Cape Town, and expression of T-cell activation and proliferation markers (CD38, HLA-DR, Ki67, CCR5) was measured by FACs. Adolescents were screened for BV (Nugent) and STIs ( C. trachomatis , N. gonnorhoea, T. vaginalis , M. genitalium , HSV-2) by PCR. For comparison, 11 HIV-negative adult women were included. Concentrations of 48 cytokines, chemokines and growth factors were measured in matching menstrual cups by Luminex. Results Adolescents (median 18 years; IQR 17–20) had significantly higher frequencies of activated CD4 + T-cells (CD38 + , HLADR + , CD38 + HLADR + : each p C. trachomatis positive. Adolescents with an STI, despite these being asymptomatic, had higher frequencies of activated and proliferating T-cells compared to those with no STI/BV (CD4 + CD38 + HLADR + : p = 0.047; CD4 + Ki67 + : p = 0.020). Women positive for chlamydia had significantly higher frequencies of CD4 + CD38 + T-cells (p = 0.006). Women with both STIs and BV had the most pronounced increase in CD4 + T-cell activation (CD38 + : p = 0.002; CD38 + HLADR + : p = 0.001; Ki67+: p = 0.002). Higher cervical T-cell activation marker expression was directly associated with increased genital cytokine profiles. Conclusion Heightened levels of genital immune activation and inflammation found in South African adolescent females, partly due to the presence of asymptomatic STIs and BV could increase their risk for HIV infection.

Introduction South African adolescent females are at high risk of HIV acquisition, disproportionate to their sexual behaviour. We hypothesised that biological changes associated with puberty may influence this susceptibility. Methods This study was conducted in two South African sites, the Desmond Tutu Youth Centre, Masiphumele, Cape Town and the Perinatal HIV Research Centre, Soweto, Johannesburg. Cytokines were measured by Luminex. Sexual risk behaviour, contraceptive use and the prevalence of sexually transmitted infections (STIs) [ C. trachomatis (CT), N. gonorrhoeae (NG), T. vaginalis , M. genitalium , HSV-2, syphilis], bacterial vaginosis (BV) and candida were assessed in each women. Results The prevalence of STIs or BV was 71% in Cape Town and 54% in Johannesburg, with 47% of Cape Town and 42% of Johannesburg women having BV. The CT prevalence in Cape Town [62/148 (42%)] was substantially higher than Johannesburg [26/149 (17%); p Conclusion An alarmingly high STI and BV prevalence was found in these at risk populations, indicating a need for improved preventative strategies. In young women, BV caused a greater degree of inflammation than STIs and its effective management requires further investigation. This work was supported by a Strategic Health Innovation Partnerships (SHIP) grant from the South African Medical Research Council and European and Developing Countries Clinical Trials Partnership (EDCTP). The cohort was supported by grants from the EDCTP. SLB was supported by the HIV Vaccine Trials Network, the Fogarty Foundation and the South African Medical Research Council (MRC). No pharmaceutical grants were received in the development of this study.

We optimized a whole blood intracellular cytokine assay to quantitate the frequency of specific CD4+ and CD8+ T cells in small volumes of whole blood from infants from developing countries. The assay is performed in two steps. First, whole blood is stimulated in the presence of specific antigens for 6-18 h, ending with cryopreservation of fixed white cells. These stimulation steps were specifically adapted to be practical and reliable in a rural, developing country field setting. Later, in a more resourceful setting, interferon-gamma producing CD4+ or CD8+ T cells are detected by flow cytometry. The assay proved sensitive and specific for detecting mycobacteria-specific immunity 10 weeks after Bacillus Calmette-Guerin (BCG) vaccination of newborns from a rural field site.

Cervical cytobrushing, biopsy, or lavages have previously been used to collect mononuclear cells from the female genital tract. Compared with blood, obtaining cells from the female genital tract is more invasive and generally yields few cells for subsequent immune studies. Because of the value of including mucosal sampling in HIV vaccine trials, standardisation of methods for collection, processing, and analysis of immunity from cells derived from the female genital tract is important. The aim of this study was to assess the effect of transport conditions on the viability, recovery and antigenic responsiveness of cervical T cells. This was investigated in cervical cytobrush specimens collected from 215 chronically HIV-infected women. Cytobrushes were either processed immediately, after cryopreservation, or after 24h at 37°C, 4°C or room temperature. CD3+ T cell numbers were quantified using Guava automated cell counting. Viability was assessed using Trypan and Annexin/PI staining. Intracellular cytokine staining was used to evaluate IFN-γ responses to PMA, PHA and CEF peptides in cytobrush-derived T cells ex vivo and after delayed processing. In vitro polyclonal expansion of thawed cervical lymphocytes was conducted for 14days in the presence of anti-CD3 and IL-2. We found that CD3+ T cell recovery and viability was similar in cytobrushes processed immediately or after 24h irrespective of the conditions at which they were maintained. Fifty percent of the CD3+ T cells could be recovered after cryopreservation of cytobrushes and these could be polyclonally expanded in half of the cryopreserved samples. IFN-γ production following mitogenic stimulation was similar in ex vivo and delayed processing cytobrushes. Maintaining cytobrushes at 37°C prior to processing significantly improved the detection of CEF-specific T cell responses compared to ex vivo. We conclude that cervical cytobrush-derived T cells are robust and can preserve their viability, phenotype and function over 24h of mock transport.