Browsing by Author "Masson, Lindi"

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    Open Access
    Characterisation of the HIV inhibitory activity of vaginal lactobacilli isolates from young South African women at high risk of HIV acquisition
    (2020) Manhanzva, Monalisa Tatenda; Masson, Lindi; Passmore, Jo-Ann; Woodman, Zenda
    Bacterial vaginosis (BV) is an important predisposing factor for the acquisition of human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs) in South African women. However, the microbial causes and the immunomodulatory effects of BV are not yet fully understood, and effective treatment strategies do not exist. BV is associated with upregulated inflammatory cytokine levels in the female genital tract (FGT), which in turn may increase HIV infection risk by recruiting and activating HIV target cells, reducing epithelial barrier function and directly promoting HIV replication. Lactobacillus species on the other hand are thought to protect against HIV by competitive exclusion, producing virucidal hydrogen peroxide (H2O2), maintaining an acidic pH by producing lactic acid and regulating immune responses in the FGT. This dissertation aimed to characterise the relative HIV inhibitory properties of clinical Lactobacillus isolates, to evaluate the immunoregulatory properties of lactobacilli, and determine the mechanisms underlying these relationships. Vaginal Lactobacillus isolates (n=103), including L. crispatus, L. jensenii, L. johnsonii, L. mucosae, L. plantarum, L. ruminis, L. salivarius and L. vaginalis, were isolated from young South African women who participated in the Women's Initiative in Sexual Health (WISH) study. The production of pro-inflammatory cytokines (IL-6, IL-1α, IL-1β), chemokines (IL-8, IP-10, MIP-3α, MIP-1α, MIP-1β) and regulatory IL-1RA by vaginal epithelial cells in response to lactobacilli in the presence or absence of Gardnerella vaginalis ATCC 14018 and Prevotella bivia ATCC 29303, was measured using Luminex. Growth rates, bacterial sizes, adhesion to cervical (Ca Ski) and vaginal epithelial cells (VK2), culture pH changes and D/L-lactate production by the lactobacilli were also measured in vitro. The properties of vaginal Lactobacillus isolates were also compared to those of commercial probiotics and ATCC reference strains. In order to evaluate differences between lactobacilli isolates that induced low (termed “non-inflammatory”) versus high (termed “inflammatory”) levels of inflammatory cytokine production, the proteomic profiles of 22 inflammatory and 22 non-inflammatory Lactobacillus isolates were analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the underlying mechanisms leading to the different inflammatory profiles. Lastly, the influence of Lactobacillus culture supernatants (n=16) on HIV infectivity was evaluated using a Luciferase Reporter Gene Assay in TZM-BL cells. Lactobacilli isolated from women with non-optimal microbiota produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1a, IL-1b, MIP-1a and MIP-1b production significantly elevated. Proteomics analysis showed that 164 proteins were differentially abundant between inflammatory lactobacilli and non-inflammatory lactobacilli. Functional analysis revealed that isolates inducing low levels of inflammatory cytokine production had a significantly higher relative abundance of membrane-associated cellular components, metabolic biological processes and enzymatic molecular functions compared to isolates that induced higher levels of inflammation. A subset of sixteen lactobacilli significantly suppressed IL-6 (adjusted p<0.001) and IL-8 (adjusted p=0.0170) responses to G. vaginalis while L. crispatus isolates suppressed inflammatory cytokines responses to P. bivia. Culture supernatants from the same 16 isolates significantly suppressed HIV infectivity in TZM-BL cells (p=0.0078). Lactobacilli adhesion to VK2 cells correlated negatively with IL-6, IL-8, MIP-1a and IL-1RA production. Lactobacillus beneficial characteristics were highly strainspecific and vaginal isolates out-performed commercial probiotics and ATCC strains. Lactobacillus growth rates, bacterial sizes and adhesion to VK2 cells did not differ significantly between isolates from women with non-optimal microbiota versus those from women with optimal microbiota. These findings show that, while cervicovaginal lactobacilli suppressed overall inflammatory responses to G. vaginalis and P. bivia, isolates from women with non-optimal microbiota were more inflammatory, had lower relative protein abundance and produced less antimicrobial lactic acid than isolates from women with optimal microbiota. Additionally, vaginal Lactobacillus isolates performed better than existing commercial probiotics, suggesting room for improvement of current probiotic formulations available on the South African market to improve BV treatment outcomes and reduce inflammation in the FGT.
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    Diversity of vaginal microbiota associated with bacterial vaginosis and the impact on pregnancy outcomes in HIV-infected and uninfected women.
    (2019) Mugabe, Muchaneta; Jaspan, Heather Beryl; Mavenyengwa, Rooyen T.; Masson, Lindi
    The composition of vaginal microbiota in pregnancy is important as it may influence susceptibility to adverse pregnancy outcomes. Dysbiosis of the vaginal microbiota caused by the replacement of protective resident microorganisms such as Lactobacillus spp. by anaerobic bacteria is known as bacterial vaginosis (BV). BV is prevalent in women of African descent and may be a cause of higher rates of adverse birth outcomes in these women. Adverse birth outcomes have been reported to be higher in HIV-infected women both on treatment or treatment naïve compared to HIV-uninfected women. The relationship between the vaginal microbiome, HIV and pregnancy outcomes has not yet been established in Zimbabwean women, nor reported in sub Saharan Africa, where the burden of disease is high. Identification of specific organisms and immune factors associated with adverse pregnancy outcomes could lead to interventions or diagnostic algorithms to prevent and predict these outcomes in this population. We recruited 420 pregnant Zimbabwean women [48 (11.4%) HIV infected, 372 (88.6%) HIV uninfected], between 13-35 weeks of gestation with a median gestational age of 30 weeks. Vaginal swabs were collected at enrolment and women were followed until pregnancy outcome was determined. Bacterial DNA was extracted from the vaginal swabs using an optimized Phenol chloroform extraction method with an addition of an enzymatic cocktail step. Library preparation was done using the Universal primers (515F/806R) for the hypervariable V4 region of the 16S rRNA gene. For the first PCR KAPA Hotstart + primers (Roche Lifescience, UK), were used for amplification. After amplification AMPure XP beads (Beckman Coulter, Brea, CA, USA) were used for cleaning of the PCR products at all stages. For the second PCR Nextera XT, Index Kit (Illumina) was used adding unique sequencing adapters to the amplicons. Quantitative PCR was used for library quantification and pooled libraries were the sequenced using Illumina MiSeq. Upstream analysis of sequencing data was done using QIIME and UPARSE and downstream analysis using custom R scripts on samples with good quality reads (>5000). Concentrations of 27 cytokines were measured in vaginal swab samples using a Bio-Plex Pro Human Cytokine 27- plex Assay (Bio-Rad Laboratories Inc., USA). Assay plates were read using a Bio-Plex Suspension Array Reader (Bio-Rad Laboratories Inc., USA). Data were analyzed using Bio-Plex manager software (version 4). Cytokine levels that were below the lower limit of detection of the assay were reported as the mid-point between zero and the lowest detectable level measured for that given cytokine. Pregnant women in this cohort had vaginal microbiota that clustered into 3 main community state types (CST): 109/356 (31%) CST1 - Lactobacillus iners dominant, 102/356 (29%) CST2 - Lactobacillus crispatus dominant and 145/356 (41%) CST3 - Gardnerella vaginalis dominant. There was a high prevalence of dysbiotic vaginal communities and L. iners was the predominant taxa found in > 90% of the women. When exploring whether the vaginal microbiota is associated with pregnancy outcomes, L. iners was highly associated low birth weight (LBW) and small for gestational age deliveries (SGA) while in contrast G. vaginalis was associated with normal birth outcome. There were no strong relationships identified between preterm birth (PTB) and vaginal microbiota. There was a strong correlation between vaginal microbiota and proinflammatory and adaptive cytokines. BV associated organisms (Gardnerella, Aerococcus, Prevotella Coriobacteriaceae and Dialister) were highly inflammatory while Lactobacillus spp were associated with low inflammation. Low levels of IL-13 and PDGF-BB cytokines were associated with LBW and PTB. We found that HIV status was highly associated with high vaginal microbial diversity. HIV-infected women had significantly higher alpha diversity in their vaginal microbiota, and they were more likely to have CST3. Surprisingly, having a diverse community type was not associated with high levels of vaginal inflammation. However low levels of IL13 and IL-I7 while high levels of TNF-α were associated with HIV status. In conclusion this study found that a highly dysbiotic vaginal environment is characteristics of Zimbabwean pregnant women and that vaginal microbiota is weakly associated with some poor pregnancy outcomes. The high prevalence of L. iners and its association with poor pregnancy outcomes suggests that vaginal microbiota may partly explain the high incidence of adverse pregnancy outcomes in African women. Low level of Th2 cytokines and growth factors may lead to adverse birth outcomes. Longitudinal studies of vaginal microbiota and soluble factors are needed in African women.
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    Effect of progestin-based hormonal contraceptives on genital inflammation and Th17 cell activation in adolescents at high risk for HIV infection
    (2019) Konstantinus, Iyaloo; Passmore, Jo-Ann; Jaspan, Heather; Masson, Lindi
    Background: Adolescent girls and young women (AGYW) are at high risk for HIV infection, particularly in Southern Africa. In addition, some hormonal contraceptives (HC), such as progestin only-injectable contraceptives DMPA and NET-EN, have been associated with significantly increased risk for HIV infection. These HC together with sexual immaturity may increase activation of CD4+ T cells in the female reproductive tract (FRT), which are target cells for HIV infection. NuvaRing, also a long-acting progestin-containing contraceptive albeit topical, has recently been introduced to South Africa, and may offer an improved safety profile over NET-EN and DMPA in terms of HIV risk for young women. Recently, Th17 cells have been found to be disproportionally susceptible to HIV infection compared to the other T helper subsets although the impact of HC use on Th17 cell frequency and activation has not been investigated. Here, the impact of NuvaRing, NET-EN and combined oral contraceptive pills (COCPs) on the vaginal microenvironment of the FRT in AGYW was investigated as this relates to HIV risk, with particular focus on cervical Th17 cells and their related cytokines (Th17- related cytokines). Methods: One hundred and thirty HIV-negative adolescent girls between the age of 15 and 19 years were enrolled into a randomized, controlled crossover study comparing NuvaRing (n=45), NET-EN (n=45), and COCPs (n=40) for 16 weeks (~4 menstrual cycles). At crossover (16 weeks), the AGYW changed to another method for the following 16 weeks: 23 of those who used NuvaRing changed to NET-EN while 8 changed to COCPs; 23 of those who used NET-EN and 24 of those who used COCPs changed to NuvaRing. The protocol included three study visits in total (screening, crossover, study completion visits). Of the 130 adolescents enrolled, 107/130 reached the crossover visit and 92/130 reached the study exit visit. All adolescents were screened for STIs (multiplex PCR for Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium), BV (by Nugent scoring), and yeast (visible hyphae on gram stain) at all study visits. Data on relative abundance of vaginal microbial community types (CTs) from ectocervical swabs was available for this study, determined by 16S rRNA sequencing of the V4 region. Several genital samples were collected, including Digene cervical cytobrushes (for flow cytometry of cervical T cells) and menstrual cups at each study visit (for measurement of genital cytokine concentrations). Multiparameter flow cytometry was performed on cytobrushes to determine the frequency and activation status (CD38 and HLA-DR) of Th17 cells (defined by expression of CCR6+CCR10-), total CD3+CD4+ T cells and CD3+CD8+ including Tc17 cells (CD8+ CCR6+). A panel of fifteen Th17-related cytokines (IL-17A, IL-17F, IL-21, IL-22, IL-6, IL-1β, IL-23, IL-33, TNF-α, IL-4, IL-10, IL-25, IL-31, IFN-γ and sCD40L) were measured in genital secretions by Luminex. Results have been presented as an intention to treat (ITT) and per protocol (PP; which accounted for early switching of HC products prior to the crossover visit). Unless otherwise stated, all statistical testing was non-parametric, and P≤0.05 were considered significant. The BenjaminiHochberg method was used to adjust for multiple comparisons. Results: In the FRT of adolescents at baseline (before randomization), Th17 cells (CCR6+CCR10-) were found to be the major CD4+ T cell subset in cytobrushes (median 54.4%, IQR 43.7% - 64.3%) compared to CCR6-CCR10- (median 42.2%, IQR 33.5% - 52.6%), CCR6+CCR10+ (median 1.2%, IQR 0.4% - 2.8%) and CCR6-CCR10+ (median 0.8%, IQR 0.2% - 1.9%). Higher frequencies of Th17 cells expressed CCR5 compared to CCR6-CCR10- CD4+ T cells (median 68.0% vs 56.2% respectively, p< 0.0001). However, Th17 cell frequencies did not correlate with genital tract Th17-related cytokines at baseline. The presence of BV or STIs did not appear to influence either the frequencies or activation status of cervical Th17 cells. Although BV (Nugent 7-10) and having a nonLactobacillus-dominated vaginal microbiome (C1) was associated with increased concentrations of all Th17-related cytokines (IL-17A, IL-17F, IL-21, IL-22, IL-6, IL-1β, IL-23, IL-33, TNF-α, IL-4, IL-10, IL-25, IL-31, IFN-γ and sCD40L) compared to those without BV (Nugent 0-3) or C2/3, while adolescents with any STI had increased concentrations of IL-1β and IL-17A compared to those without an STI. After being randomized on to HC for 16 weeks, cervical cytobrush-derived immune cells were analysed within individuals in each arm (intra-individual) and between individuals in the three contraceptive arms, using both ITT and PP approaches. Although the frequency and activation status of cervical Th17 cells was similar across the three HC arms, adolescents using NuvaRing and NET-EN had significantly increased activation (CD38+HLA-DR+) on Th17 cells compared to their respective baselines (p=0.02 and p=0.03, respectively), which was not evident in those using COCPs. Furthermore, adolescents using NuvaRing had reduced frequencies of Th17 cells compared to baseline (p=0.001), which was not evident in those using NET-EN or COCPs. Although it was hypothesized that NuvaRing would offer some safety advantage over NET-EN in terms of mucosal HIV target cell activation, intra-individual analysis showed a significant increase in the frequency of highly activated cervical Th17 cells in those adolescents who started using the ring. A significant increase in genital tract concentrations of several Th17-related cytokine concentrations (including IL-21, IL-1β, IL-33, TNF-α, IL-4, IFN-γ and sCD40L) was noted in adolescents assigned to NuvaRing after 16 weeks of use, suggesting that the presence of the vaginal ring likely increased genital cytokine responses. Although the frequency and activation of CD8+ T cells was similar across HC arm, intra-individual analysis showed changes in the frequency of activation markers on CD8+ T cells in all HC arms. Moreover, frequencies of Tc17 cells were significantly reduced after 4 months of contraceptive use in each HC arm compared to baseline frequencies. Conclusion: In summary, CCR6+CCR10- Th17 cells were confirmed to be the major CD4+ T cell subset in the FRT of young adolescents. The use of NuvaRing led to decreased frequencies of Th17 cells which were highly activated, and was also associated with an increase in Th17-related cytokines compared to NET-EN and COCPs. All HC altered activation of cervical CD8+ T cells and reduced the frequencies of Tc17 cells. The dramatic alterations observed in cervical immune cells associated with the use of NuvaRing compared to NET-EN and COCPs warrant further investigations.
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    Gene expression patterns of the female genital tract and immunomodulation by Lactobacillus species
    (2020) Abrahams, Andrea Gillian; Masson, Lindi; Alisoltani-Dehkordi, Arghavan; Jaspan, Heather
    Inflammation in the female genital tract (FGT) is associated with increased HIV-1 viral replication, HIV-1 transmission and HIV-1 acquisition. The optimal commensal Lactobacillus bacterial species is associated with reduced inflammation in the FGT and dampened immune responses to non-optimal bacteria in vitro. Using a transcriptomics approach, this research aimed to investigate gene expression patterns in the FGT of HIV-infected women compared to peripheral blood. Furthermore, transcriptomics was used to investigate interactions between different vaginal Lactobacillus species and the host to elucidate its immunomodulatory mechanisms. Cervical cytobrushes and blood samples were collected from chronically HIV-infected South African women. Cervical and peripheral blood mononuclear cells (CMCs and PBMCs) were isolated and mRNA was extracted for microarray analysis using the Illumina HumanHT-12 v3 Expression BeadChip system. Eight Lactobacillus isolates, two of each L. jensenii, L. mucosae, L. crispatus and L. vaginalis species were included in this study. The effects of these lactobacilli on cytokine production by vaginal epithelial (VK2) cells stimulated with Gardnerella vaginalis (ATCC 14018) were tested in vitro, RNA was extracted and used for Affymetrix Genechip whole transcript microarray analysis. This study found that significantly over-expressed genes in CMCs compared to PBMCs were mapped to proinflammatory signaling pathways (including Nuclear factor kappa B (NFκB), Tumor necrosis factor (TNF), Toll-like receptor (TLR) and Nucleotide-binding and oligomerization domain (NOD)-like receptor). Concurrently, a signature of reduced potential for adaptive immunity was observed in CMCs compared to PBMCs, as evidenced by underrepresentation of the T cell receptor signaling and natural killer cell mediated cytotoxicity pathways. G. vaginalis induced a potent proinflammatory cytokine response by VK2 cells in vitro. Over-expressed genes in G. vaginalis-stimulated VK2 cells compared to unstimulated VK2 cells were mapped to inflammatory signalling pathways. In contrast, 3/8 Lactobacillus isolates, including two L. mucosae and one L. vaginalis species, reduced inflammatory cytokine production by VK2 cells in response to G. vaginalis and were thus termed “cytokinesuppressive”. Several genes, 7/8 of which are involved in inflammation, were downregulated in VK2 cells co-cultured with lactobacilli and G. vaginalis in combination compared to coculture with G. vaginalis only. Futhermore, when gene expression changes were investigated in cells cultured with cytokine-suppresive lactobacilli versus non-cytokine-suppressive lactobacilli, it was found that SAMD9L, DDX58, IFIT1 gene expression was downregulated exclusively in VK2 cells co-cultured with cytokine-suppressive lactobacilli and G. vaginalis compared to co-culture with G. vaginalis only. The findings of this study have identified distinct gene expression patterns in the FGT compared to peripheral blood. Furthermore, key genes that may play a critical role in the immunomodulatory effects of vaginal lactobacilli were identified, motivating for further confirmatory research.
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    HIV-1 subtype C transmitted founders modulate dendritic cell inflammatory responses
    (2020-07-02) Lumngwena, Evelyn N; Metenou, Simon; Masson, Lindi; Cicala, Claudia; Arthos, James; Woodman, Zenda
    Background Heterosexual transmission remains the main route of HIV-1 transmission and female genital tract (FGT) inflammation increases the risk of infection. However, the mechanism(s) by which inflammation facilitates infection is not fully understood. In rhesus macaques challenged with simian immunodeficiency virus, dendritic cell (DC) mediated recruitment of CD4+ T cells to the FGT was critical for infection. The aim of this study was to delineate the mechanisms underlying DC-mediated HIV infection by comparing chemokine and pro-inflammatory cytokine production in response to transmitted founder (TF) and chronic infection (CI) Envelope (Env) pseudotyped viruses (PSV). Results Monocyte-derived DCs (MDDCs) were stimulated with PSV and recombinant gp140 representing matched TF and CI pairs of four individuals and cytokine secretion measured by multiplex immuno-assay. We found that 4/9 Env induced robust MDDC inflammatory responses and of those, three were cloned from TFs. Overall, TF Env induced MDDCs from healthy donors to secrete higher concentrations of inflammatory cytokines and chemokines than those from CI, suggesting TF Env were better inducers of inflammation. Assessing the signalling pathway associated with inflammatory cytokines, we found that PSV of matched TF and CI variants and a gp140 clone activated ERK and JNK to similar levels. Recombinant soluble DC-SIGN inhibited cytokine release and activation of ERK by PSV, suggesting that Env-DC-SIGN binding was partly involved in MDDC stimulation. Therefore, Env clones might differentially stimulate MDDC immune responses via alternative, yet unidentified signalling pathways. Conclusion Overall, this could suggest that the genetics of the virus itself influences inflammatory responses during HIV infection. In the absence of pre-existing infections, induction of greater inflammatory response by TFs might favour virus survival within the healthy FGT by driving an influx of target cells to sites of infection while suppressing immune responses via IL-10.
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    Impact of long-acting contraceptives on female genital tract cytokine profiles in a randomised controlled trial
    (2020) Radzey, Nina; Masson, Lindi
    The Evidence for Contraceptive Options and HIV Outcomes (ECHO) trial found no substantial difference in HIV acquisition risk between women randomised to injectable depot medroxyprogesterone acetate (DMPA-IM), copper intrauterine device (IUD) or the levonorgestrel (LNG) implant. However, it remains unknown whether these contraceptives increase HIV risk relative to other forms of contraception or no contraception. This study investigated the impact of DMPA-IM, copper IUD and LNG implant on cervicovaginal inflammatory profiles previously associated with HIV acquisition, among a sub-cohort of ECHO participants. This study included 167 ECHO participants at the Setshaba Research Centre in Pretoria and MatCH Research Unit in Durban, South Africa. Eleven cytokines and antimicrobial peptides were measured in lateral vaginal wall swabs in duplicate using Luminex. Differences in baseline cytokine profiles were assessed using demographic data, including site, age, body mass index (BMI) and sexually transmitted infection (STI) status. Changes in cytokine concentrations were assessed using Wilcoxon signed ank test. Fold changes in cytokine concentrations were compared between arms using Mann-Whitney U test. P-values were adjusted for multiple comparisons using a false discovery rate procedure. Overall cytokine profiles were compared using principal components analysis and unsupervised hierarchical clustering. Mixed effects linear regression was used for longitudinal analysis and multinomial logistic regression was used to adjust for potential confounders that were assessed at baseline. Concentrations of IL-6 and MIP-3α were significantly higher in women enrolled at the Setshaba Research Centre compared to the MatCH Research Unit. Several immune mediators were elevated in younger women and this trend was significant for the pro-inflammatory IL1β and the chemokines IL-8 and MIP-1α. Women that were seropositive for herpes simplex virus type 2 (HSV-2) had significantly lowerconcentrations of MIP-1α. The copper IUD and LNG implant were associated with rapid increases in inflammatory markers following contraceptive initiation. Pro-inflammatory IL-1β and IL-6 and chemotactic IL-8, IP-10, MIP1⍺ and MIP-1β were significantly elevated one month following copper IUD insertion. No changes were evident at one-month post LNG implant insertion, however at three months, TNF-⍺, IP-10, MIP-3⍺ and SLPI were significantly raised relative to baseline. No significant changes in immune mediator concentrations were detected following DMPA-IM initiation but the trend was towards a decrease, particularly for SLPI. After adjusting for potential confounders, including site, age and infection status with chlamydia, gonorrhoea and HSV-2, IL-6 and IP-10 were significantly elevated in the copper IUD compared to the DMPA-IM arm at months 1 and 3, while IP-10 and SLPI were higher in the LNG implant arm at month 3 compared to the DMPA-IM arm. The copper IUD and the LNG implant are associated with increased cervicovaginal inflammatory markers that have been linked to HIV infection risk and the chemokine IP-10 appears to play a central role. Recent studies have demonstrated the importance of the interplay between inflammation, the microbiome, contraception and HIV risk. Continued research to understand these effects are critical for safe contraceptive use and to inform novel contraceptive development.
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    The impact of sexually transmitted infections and inflammation in the female genital tract and blood on susceptibility to HIV-1 infection and disease progression
    (2011) Masson, Lindi; Passmore, Jo-Ann; Williamson, Carolyn; Little, Francesca
    Background. In sub-Saharan Africa, which has the highest prevalence of HIV-1 worldwide, most newHIV-1 infections occur by sexual transmission to women. Recent studies in non-human primates have demonstrated that pro-inflammatory cytokine production in the genital tract is necessary for immune cell recruitment and establishment of simian immunodeficiency virus (SIV) infection following vaginal inoculation. The aims of this study were to evaluate the relationships between inflammation in the female genital tract and (i) susceptibility to HIV-1 infection and (ii) subsequent disease progression in women who became infected. Additionally, genital inflammation was investigated as a mechanism for breakthrough HIV-1 infections in women who became infected even though they were using 1% tenofovir (TFV) microbicide. In the systemic compartment, the level of T cell activation and soluble markers of immune activation during HIV-1 infection are associated with disease outcome. Therefore, the relationships between plasma cytokine concentrations during early HIV-1 infection and disease progression were evaluated Methods. The participants of this study included 230 HIV-uninfected women from the CAPRISA 002cohort who were followed longitudinally for HIV-1 infection, 49 women who were enrolled during acuteHIV-1 infection and followed until 12 months post-infection and 166 HIV-uninfected women who were enrolled in the CAPRISA 004 1% TFV microbicide trial (62 of whom later became HIV-1-infected).Cytokine concentrations were measured in cervicovaginal lavage (CVL) and plasma samples from these women using Luminex and ELISA.
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    Impact of the mycobiome on the health of the female genital tract
    (2022) Gangiah, Tamlyn Kirstey; Mulder, Nicola; Masson, Lindi
    The female genital tract microbiome comprises a community of microorganisms. Imbalances in the microbiome are associated with vaginal diseases such as bacterial vaginosis (BV) and sexually transmitted infections (STIs). These diseases greatly burden South Africa, and young women in this region are at an increased risk of contracting vaginal diseases. Consequently, it is vital to investigate the factors that influence FGT health. The fungal constituent of the microbiome (the mycobiome) has been demonstrated to play a role in regulating mucosal health, especially when the bacterial component is disturbed. However, we have a limited understanding of the vaginal mycobiome since many microbiome studies have focused on bacterial communities and have neglected low abundance taxonomic groups, such as fungi. To reduce this knowledge deficit, we present the first large-scale metaproteomic study to define the taxonomic composition and potential functional processes of the vaginal mycobiome in South African women. We examined vaginal fungal communities present in optimal and nonoptimal states (BV, STIs, and genital inflammation) by collecting lateral vaginal wall swabs from 123 women for liquid chromatography-tandem mass spectrometry. Taxonomic analysis requires representative sequence databases; however, since mycobiome research is relatively new, fungal databases are still in their infancy. As a result, metaproteomic methods are not optimized for fungal research. Therefore, we optimized a metaproteomic approach to increase fungal protein group assignments. With this, 50 fungal proteins belonging to 39 different genera were identified post quality-control and analysed for taxonomic and functional distributions. Taxonomic analysis revealed that the vaginal mycobiome had a high relative abundance of Candida across optimal and non-optimal states. We observed changes in differential abundance at the genus and biological process level between optimal and non-optimal states for BV and Mycoplasma genitalium. In the BV positive state, most fungal proteins were significantly underabundant (p< 0.05) compared to the BV negative state, with the exception of Malassezia and Condiobolus. Correspondingly, Nugent score was negatively associated with total fungal protein intensity, implying that the microenvironment during BV is less suitable for fungal growth. Furthermore, we assessed which clinical variables were associated with driving fungal community composition; results indicated that Nugent score, pro-inflammatory cytokines, chemokines, vaginal pH, Chlamydia trachomatis, and the presence of clue cells were involved. Lastly, we used publicly available vaginal proteome data to confirm our fungal identifications and suspect Candida, Debaryomyces, Kluyveromyces, Malassezia, Penicillium, Yarrowia, Aspergillus, Cryptococcus, Wallemia, Trichosporon, and Saccharomyces are likely true vaginal inhabitants. Thus, this study sets the groundwork for understanding the vaginal mycobiome and its association with prevalent vaginal diseases.
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    Sexually transmitted infections, bacterial vaginosis and genital inflammation as risk factors of HIV acquisition in adolescent girls and young women in South Africa
    (2019) Barnabas, Shaun Lawrence; Passmore, Jo-Ann; Bekker, Linda-Gail; Masson, Lindi
    Background: In South Africa, young women are at increased risk of HIV infection, predominantly through heterosexual contact. Socio-behavioural and biological factors most likely play an important role at increasing risk. BV, STIs and genital yeast infections are known to influence HIV risk, although few studies have been conducted in African adolescent girls and young women (AGYW). Understanding the role of behavioural and biological risk factors in this key population is essential to inform on new prevention strategies. Aims: (1) To define the prevalence of sexual risk behaviour, bacterial vaginosis (BV), sexually transmitted infections (STIs), and genital fungal infections in South African AGYW; (2) to assess the relationship between symptoms and an etiological STI diagnosis; (3) to evaluate the influence of electronic and paper-based data collection methods on reported demographics and sexual risk behavior; (4) to examine the impact of non-infectious and infectious causes on genital inflammatory cytokine profiles; and (5) to investigate the use of cytokine biomarkers to identify young women with asymptomatic vaginal dysbiosis and STIs. Approach: To address these aims, the multi-center Women's Initiative in Sexual Health (WISH) study was conducted as a longitudinal observational trial that included HIV-negative, South African AGYW between the ages of 16-22 (EDCTP Strategic Primer 2013-2015), of which 149 from Masiphumelele, Cape Town were enrolled longitudinally for three visits and the 149 from Soweto, Johannesburg were seen cross-sectionally. Genital samples were collected to test for STIs (including Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, Mycoplasma genitalium, herpes simplex virus (HSV)-1 and -2, Haemophilus ducreyi, Treponema pallidum and Lymphogranuloma venerum), BV (Nugent scoring) and yeast infections, to assess vaginal pH, and to measure prostate specific antigen (PSA) as a marker for the presence of semen. Multi-locus sequence typing (MLST) was performed on samples positive for C. trachomatis. In addition, 44 inflammatory, adaptive, regulatory cytokines, chemokines and growth factors were measured in genital secretions byLuminexR. Concentrations of sex hormones (estrogen, progesterone and luteinizing hormone), HSV-serology and cotinine (smoking) were measured in blood plasma. A HIV test was done at screening and each visit. Only HIV negative women were enrolled in the study Results: The prevalence of BV (Nugent 7-10) in South African AGYW were similarly high in Masiphumelele and Soweto (48% and 45%, respectively), as was intermediate microbiota (12% and 16%, respectively). In addition, 40% of South African AGYW were infected with any STI. C. trachomatis and N. gonorrhoeae were more prevalent in Masiphumelele compared to Soweto (42% vs. 18% for C. trachomatis and 11% vs. 5% for N. gonorrhoeae, respectively), while the prevalence of the other STIs tested for, including T. vaginalis, M. genitalium and HSV-2, was similar at both sites. Two-thirds (67%) of AGYW had HPV infection while 10% had a fungal infection. The indicators of risky behavior (including partner's HIV status being unknown [57%], having unprotected vaginal sex in the last three months [40%], and being in HIV discordant relationships [20%]) were high in this population, and tended to be more prevalent in Soweto than Masiphumelele. It was striking that only 24% of women with a diagnosed STI or BV were symptomatic, underlining the inadequacy of syndromic management in this population. The most sensitive symptom in this cohort was dysuria, which had 50% sensitivity in diagnosing a STI, and the least sensitive was abnormal vaginal discharge with a sensitivity of 22%. Having multiple, concurrent conditions (multiple STIs or a STI and BV) did not increase the symptom frequency or severity. In Chapter 3, two methods were compared to obtain demographic and sexual risk behaviour data – an electronic tablet device and a paper-based questionnaire. This was done to explore the presence of reporting bias and to improve the frequency of it in the cohort. There were no obvious differences seen in the reporting of risk between the two arms. It was striking that geographical differences in risk reporting were observed, with AGYW from Soweto reporting a higher frequency of HIV discordant relationships and intergenerational relationships than women from Masiphumelele, independently of the data collection method used. As genital inflammatory cytokines are known to influence HIV risk, physiological and pathological factors that may influence the genital cytokine profiles of AGYW were examined in Chapter 4 and 5. A change of vaginal pH appeared to play a crucial role, as it was associated with a significant increase in half (22/44) of the cytokines measured in AGYW with no STIs, no yeast infections and a Nugent score ≤3. Further, in these adolescents that one would consider healthy, a pronounced effect was seen with hormonal contraception choice, with Net-En significantly increasing the median concentrations of 31/44 cytokines, and DMPA upregulating 27/44 cytokine concentrations. Recent sex (being PSA positive) did not influence genital inflammation. Next, the impact of BV, viral STIs (HPV and HSV-2) and yeast infections on genital inflammation in adolescents was investigated. BV was the most inflammatory condition seen with 34/44 cytokines being upregulated. Interestingly, BV was also associated with downregulation of chemokines at both sites, including GRO-α, IP-10 and MIG. BV alone was more inflammatory than coinfection of BV with C. trachomatis, BV with another STI, or BV, C. trachomatis and another STI. In this cohort, HPV and HSV-2 shedding did not influence genital inflammation. In Chapter 5, the influence of bacterial and parasitic STIs (including C. trachomatis, N. gonorrhoeae, T. vaginalis and M. genitalium) on the genital inflammatory profile was explored. C. trachomatis and T. vaginalis were equally inflammatory, with 23/44 cytokines being elevated. M. genitalium infections had a very little impact with only GM-CSF being significantly downregulated in Masiphumelele, while N. gonorrhoea infection did not influence the genital inflammatory milieu. Geographical variation in genital cytokine responses were observed in adolescents infected with T. vaginalis, which was moderately inflammatory in AGYW from Masiphumelele but not in AGYW from Soweto. With C. trachomatis infection, however, there was a more pronounced inflammatory response seen in adolescents from Soweto (where 23/44 genital cytokines were significantly increased) than those from Masiphumelele (no cytokines were changed), compared to adolescents with no STIs, no yeast infections and a Nugent score ≤3. Using MLST, further differences were associated with C. trachomatis infection: sequence type (ST) 3 was the most inflammatory C. trachomatis ST detected with 14/44 cytokines upregulated; ST137 caused no significant changes in cytokine markers, and ST100b was the least inflammatory with four cytokines being down regulated. Exploring the relationship between years of sexual experience and C. trachomatis infection showed low levels of inflammation in women with only with <2 year of sexual experience (only MIG was upregulated), while C. trachomatis infection in AGYW with ≥2 years sexual experience was associated with an increase in 19/44 cytokines. Finally, in Chapter 6 the cytokine biomarkers IL-1α, IL-1β and IP-10 were evaluated in the classification of asymptomatic STIs, BV and intermediate microbiota. These biomarkers correctly classified 76% of women using their SoftcupR samples, 76% of women using lateral vaginal wall swabs and 73% using vulvovaginal swab. The addition of pH to the biomarkers model improved the classification (82%) and sensitivity (87%) of results, but reduced specificity (64%). The validation of these biomarkers could lead to the development of a point-of-care test that could be used to triage women into risk categories independently of symptoms. Conclusion: The results show a high prevalence of risky behavior, STIs and BV in this vulnerable population, with poor correlation between clinical symptoms and diagnosis of STIs or BV, with most cases in adolescents being asymptomatic. Injectable hormonal contraceptive use led to increased genital inflammation in adolescents. Despite the lack of symptoms, most cases of BV and STIs were associated with significantly elevated concentrations of genital cytokines in the genital mucosa of these at-risk adolescents compared to their uninfected counterparts, with variation in levels of inflammation seen by geographic location, type of infection, and years of sexual experience. These results supported the use of biomarkers as a potential method to identify at risk women. Overall, these findings highlight the urgent need to include adolescents in the design and testing of new HIV prevention strategies and the importance of active identification and management of any infection or dysbiosis in this key at risk population.