Browsing by Author "Happel, Anna-Ursula"

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    Cervicovaginal Human Papillomavirus Genomes, Microbiota Composition and Cytokine Concentrations in South African Adolescents
    (Multidisciplinary Digital Publishing Institute, 2023-03-15) Happel, Anna-Ursula; Balle, Christina; Havyarimana, Enock; Brown, Bryan; Maust, Brandon S.; Feng, Colin; Yi, Byung H.; Gill, Katherine; Bekker, Linda-Gail; Passmore, Jo-Ann S.; Jaspan, Heather B.; Varsani, Arvind
    The interaction between cervicovaginal virome, bacteriome and genital inflammation has not been extensively investigated. We assessed the vaginal DNA virome from 33 South African adolescents (15–19 years old) using shotgun DNA sequencing of purified virions. We present analyses of eukaryote-infecting DNA viruses, with a focus on human papillomavirus (HPV) genomes and relate these to the vaginal bacterial microbiota (assessed by 16S rRNA gene sequencing) and cytokines (assessed by Luminex). The DNA virome included single-stranded (Anelloviridae, Genomoviridae) and double-stranded DNA viruses (Adenoviridae, Alloherpesviridae, Herpesviridae, Marseilleviridae, Mimiviridae, Polyomaviridae, Poxviridae). We identified 110 unique, complete HPV genomes within two genera (Alphapapillomavirus and Gammapapillomavirus) representing 40 HPV types and 12 species. Of the 40 HPV types identified, 35 showed positive co-infection patterns with at least one other type, mainly HPV-16. HPV-35, a high-risk genotype currently not targeted by available vaccines, was the most prevalent HPV type identified in this cohort. Bacterial taxa commonly associated with bacterial vaginosis also correlated with the presence of HPV. Bacterial vaginosis, rather than HPV, was associated with increased genital inflammation. This study lays the foundation for future work characterizing the vaginal virome and its role in women’s health.
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    Characterising vaginal Lactobacillus strains from young South African women with persistently optimal vaginal microbiota – Developing the framework for an African vaginal probiotic product development platform for reproductive health
    (2024) Chicken, Anika; Kullin, Brian; Passmore, Jo-Ann; Happel, Anna-Ursula
    Background: Bacterial vaginosis (BV) is a genital condition with a high prevalence in sub-Saharan Africa, which can cause adverse sexual and reproductive health outcomes. Treatment with antibiotics such as metronidazole results in only a brief resolution of the vaginal dysbiosis present in BV and the recurrence rates are high. Probiotics have been explored as a possible adjunctive treatment option, but most commercially available formulations do not contain vaginally-derived strains and there is limited evidence for their efficacy in local populations. Therefore, there is an urgent need for the development of vaginal probiotics tailored towards sub-Saharan African women containing strains with useful probiotic characteristics that have the potential to persistently colonise the female genital tract. Aim: This project aimed to isolate and characterise Lactobacillus strains from young South African women with a stable optimal vaginal microbiota for consideration as candidate strains in a vaginal probiotic product. Methods: Samples from a previously completed study cohort where hormonal contraceptive preferences were examined in adolescent and young women in Cape Town (UChoose) were used for this study. Samples were collected from healthy, HIV uninfected young women (aged 15-19) at three timepoints, 16 weeks apart. To identify women with a stable and optimal vaginal microbiota over time, vaginal microbial communities were classified using VALENCIA. Lactobacillus spp. were selectively isolated from participants with longitudinally stable Lactobacillus crispatus- dominant communities and assayed for their ability to inhibit Prevotella bivia, a BV-associated organism using a soft agar overlay method. Candidates with strong inhibition potential against P. bivia were characterised further fortheir inhibition potential against regionally relevant BV-associated organismsisolated from women within the same study cohort, as well astheir growth at physiologically relevant pH (pH 4, 4.5 and 5), their ability to produce lactic acid and finally their resistance to commonly used BV and urinary tract infection antibiotics. Additionally, whole genome sequencing (WGS) data were available for three strains and these were assessed to determine additional safety characteristics of the probiotic candidates. The strains were screened for antimicrobial resistance genes using several online databases (ResFinder, ARG-ANNOT, CARD, NCBI AMRFinderPlus and VFDB), the presence of integrated prophages using VirSorter and phage defence mechanisms using the DefenseFinder tool. Lastly, putative bacteriocins were identified using BAGEL4. Results: The most common community state type (CST) identified across all participants and visits was the highly diverse CST IV, which is dominated by a variety of anaerobes and shows a strong association with BV. In total 3 participants had an optimal, L. crispatus-dominant vaginal microbiota (CST I) across all three study visits and did not use any antibiotics during the study. Samples from these participants yielded a combined 337 isolates. Isolates displayed a wide variety in inhibition potential against P. bivia ATCC 29303T . The isolates selected for further characterisation were all identified as L. crispatus using 16S rRNA sequencing. More than half of the selected isolates inhibited local Prevotella strains more than they inhibited the P. bivia ATCC 29303T type strain. All L. crispatusisolates produced some of each lactic acid isomer, with the top three L-lactate producers all isolated from the same participant (UC101). No correlation was found between the amount of lactate produced and antimicrobial activity against Prevotella strains suggesting that the inhibition was not lactate dependent. All isolates were resistant to metronidazole, but susceptible to other BV and urinary tract infection treatment antibiotics indicating that co administration with metronidazole is a possibility for these candidate probiotic strains. Of the three genomes analysed, two of the isolates harboured plasmids. No previously characterised antimicrobial resistance determinants were identified. Each of the three strains harboured five putative bacteriocins, which may play a role in activity against competing bacterial strains. Additionally, 12 putative prophages were identified, with none found on the plasmid. Overall, seven different potential phage defence mechanisms were identified on the bacterial chromosomes. Conclusion: The proportion of women with longitudinally stable, optimal vaginal communities in our setting was relatively low. However, targeting samples provided by these women for the isolation of potential probiotic bacteria yielded a large number of isolates with inhibitory activity against a BV-associated pathogen. Phenotypic variation among isolates illustrates the importance of screening multiple strains of the same species per sample. The WGS screen did not identify any virulence or antimicrobial resistance genes suggesting that the probiotic candidates are safe to use. The putative bacteriocins found in the isolates could be targeted for future research. This pilot study provides proof-of-principle for a pipeline that will screen L. crispatus strains with probiotic potential from African women to treat BV and improve reproductive health in Africa.
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    Correlates of tuberculosis and non-tuberculosis morbidity and immunity in sub-Saharan African HIV-exposed, uninfected infants
    (2024) Iwase, Saori Christina; Jaspan, Heather; Happel, Anna-Ursula
    Background: Perinatal HIV transmission has been considerably reduced due to successful intervention programs. Consequently, there is a growing population of infants who are HIV-exposed but uninfected (iHEU), particularly in sub-Saharan Africa. These infants experience an increased risk of morbidity compared to infants who are HIV-unexposed and uninfected (iHUU), predominantly due to infectious diseases. Although the mechanisms underlying this increased vulnerability remain unclear, it may be associated with their altered immunity and/or gut microbiota. Bacillus Calmette-Guérin (BCG) vaccination is an effective intervention to prevent severe tuberculosis (TB) disease in children. BCG vaccination also enhances heterologous protective immunity against infections through epigenetic reprogramming of innate immune cells (known as “trained innate immunity”). However, whether iHEU receive comparable protection from BCG induced immunity against TB and non-TB infection as iHUU remains elusive. Gut microbiota plays a critical role in immune development during infancy. A close relationship between gut microbiota and vaccine responses has been reported in iHUU, including tetanus toxoid (TT) vaccination. However, a limited number of studies longitudinally investigated the effect of in utero HIV exposure on the gut microbiota, and results are often conflicting. In addition, not many studies have compared the trajectory of gut microbiota between iHEU and iHUU across multiple countries. While several studies have indicated reduced immune responses against TT vaccination in iHEU compared to iHUU, the interplay between HIV exposure, gut microbiota, and vaccine response is largely unexplored. Aims: In this dissertation, we examined three potential contributing factors that may underlie the higher risk of morbidity observed among iHEU in sub-Saharan Africa. The specific aims were to examine whether BCG affords the same protection against TB infection (TBI) and disease in iHEU (corresponds to Aim 1), effect of HIV exposure on longitudinal gut microbiota composition and its association with TT vaccine response (corresponds to Aim 2), and optimization of epigenetic assay protocol, intended for future investigation of BCG-induced histone modifications in iHEU (corresponds to Aim 3). Methods and results: To assess TBI prevalence among iHEU and iHUU, a total of 418 mother-infant pairs from South Africa and Botswana were included. All infants received BCG vaccination at birth as per standard of care. T-SPOT.TB (ELISpot-based interferon-gamma release assay) was performed using cryopreserved peripheral blood mononuclear cells (PBMCs) from infants aged 9-18 months. The prevalence of TBI did not differ by the infant HIV exposure status, with 10 cases (3.4%) among iHEU and four cases (3.2%) among iHUU, none with symptoms of active TB disease. This trend was the same across two different African countries where the burden of HIV and TB is high. However, because of the lower T-SPOT.TB positivity than initially anticipated, we were under powered to conclude the effect. To assess whether gut microbial succession alters immunity in iHEU, we profiled longitudinal gut microbiota composition and associated this with TT vaccine responses in 354 mother-infant pairs from South Africa and Nigeria. Stool samples were collected at 1 and 15 weeks of life, and 16S ribosomal ribonucleic acid (rRNA) gene sequencing was performed. Plasma IgG anti-tetanus antibody titers were measured by enzyme-linked immunosorbent assay (ELISA). The effect of HIV exposure on infant gut microbiota composition was relatively modest compared to the impact of age and geographical factors. However, HIV exposure and specific gut microbes were independently associated with the TT vaccine response at 15 weeks of age. Results for South Africa and Nigeria differed, possibly due to higher maternal anti-tetanus IgG titers and hence infant baseline titers in Nigeria. To optimize an epigenetic assay that can be applied to infant samples, monocytes and natural killer (NK) cells were isolated from cryopreserved PBMCs using fluorescence-activated cell sorting (FACS). Cleavage Under Targets and Tagmentation (CUT&Tag) was optimized for assessing the histone modifications, acetylation of histone H3 at lysine 27 (H3K27Ac), trimethylation of histone H3 at lysine 4 (H3K4me3), and trimethylation of histone H3 at lysine 27 (H3K27me3; also used as a positive control). The optimized protocol was then applied to a subset of infant samples (n = 14; aged between six and seven weeks). Optimal input cell number, polymerase chain reaction (PCR) cycles, and sequencing depth were carefully determined for the CUT&Tag assay. These adjustments were necessary to achieve the assay's feasibility and data quality. The optimized CUT&Tag protocol and fine-tuned data analysis strategy successfully exhibited its capability to analyze multiple histone modifications using only 5,000 infant monocytes or NK cells as an input sample. Conclusions: Prenatal HIV exposure and gut microbiota may independently influence infant TT vaccine response. This supports the existing notion that iHEU exhibit altered immunity. Although previous studies have indicated that iHEU experience a higher risk of infection than iHUU, our data suggested that BCG vaccination was equally protective against TBI, irrespective of HIV exposure status. The optimized CUT&Tag protocol will offer a useful tool for investigating histone modifications using ultra-low input samples. This will be employed in the future study to explore whether iHEU exhibit comparable epigenetic modifications induced by BCG vaccination as for iHUU, providing valuable insight into whether iHEU receive similar non-specific protection from BCG vaccination compared to iHUU.
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    Evaluation of probiotic and vaginal Lactobacillus species for the treatment of bacterial vaginosis and promotion of vaginal health in South African women
    (2018) Happel, Anna-Ursula; Passmore, Jo-Ann; Froissart, Rémy
    Background: Bacterial vaginosis (BV) increases women's risk for adverse reproductive outcomes and acquisition of sexually transmitted infections (STIs), including HIV. The etiology of BV is still unclear, and it has been hypothesized that lytic or temperate Lactobacillus bacteriophages may contribute. The current standard of care for BV are antibiotics like metronidazole or clindamycin, although these are not effective long-term, as rates of BV recurrence are high. There is thus an urgent need for durable treatment of BV to be developed. Probiotics administered adjunctively to antibiotics may improve efficacy and durability of BV treatment, but no randomized trial comparing antibiotic treatment to probiotics as an adjunct to antibiotics has been performed in South Africa, despite BV rates >50%. The South African Health Products Regulatory Authority (SAHPRA) regulates drugs and health supplements, into which probiotics are categorized locally. However, no probiotic product has yet been registered with SAHPRA. Further, there have been no recent surveys of the availability of probiotics for vaginal health in South Africa; neither has their suitability to be used in the treatment for BV been evaluated. Aims: The specific aims of this dissertation were (1) to survey the South African probiotic market and evaluate locally available products marketed explicitly for vaginal health in vitro, (2) to determine the efficacy of the most promising local overthe- counter (OTC) probiotic for vaginal health for BV treatment in South African women in a pilot randomized clinical trial that is approved by the regulatory authorities in South Africa, in order to explore the local regulatory landscape for future trials; (3) to screen and thoroughly characterize vaginal Lactobacillus strains isolated from healthy South African women in vitro for the development of a geographically-specific probiotic for vaginal health; and (4) to evaluate the role of bacteriophages in the etiology of BV and Lactobacillus spp. survival in vitro. Approach and results: To review probiotics available on the South African retail market, a cross-sectional survey using two-stage cluster sampling was conducted in Durban and Cape Town. Of the 104 unique probiotic products identified, only four were explicitly for vaginal health, although they contained bacterial species commonly found in the gastro-intestinal tract (GIT) and not lower female genital tract (FGT). The probiotics marketed for vaginal health were analysed for the bacterial contents, concentration per dose, growth kinetics, influence of bacterial growth on culture pH, adhesion to cervical cells, production of L-and D-lactic acid as well as H2O2, inhibitory activity against G. vaginalis and P. bivia and Group B Streptococcus (GBS), their susceptibility to antibiotics, and mucosal safety (using cytokine biomarkers). Batch testing revealed that they mainly contained the bacterial species and dose as claimed by the manufacturers. The contained bacterial isolates had promising probiotic characteristics, but there was some variability in the biological characteristics of isolates from different lot numbers of some of the products. A single-blind, randomised SAHPRA-approved trial enrolling BV positive, STI negative (including discharge-causing STIs; C. trachomatis, T. vaginalis, M. genitalium and A. vaginae) South African women was initiated to compare standard of care (SOC, MetroGelTM V, n=20) to a combination of metronidazole and a commercially-available probiotic (Vagiforte® PLUS Combo Pack, containing L. rhamnosus, L. acidophilus, B. longum and B. bifidum in oral capsules and vaginal spay, treatment duration 15 days) marketed for vaginal health, including treatment of BV and vaginal thrush, in South Africa (n=30, intervention group). The primary endpoint of the pilot study was BV cure one month after treatment completion. BV was assessed by Nugent scoring, vaginal pH was measured, IL-1α concentrations in cervicovaginal fluid were measured by ELISA as a biomarker of genital inflammation, and quantitative PCR (qPCR) was performed to measure the abundance of several vaginal Lactobacillus spp. (including L. crispatus, L. gasseri, L. jensenii, L. vaginalis, L. mucosae and L. iners), in addition to key BV-associated bacteria (including G. vaginalis, P. bivia, A. vaginae, BVAB2 and Megasphaera 1), the bacterial species contained in Vagiforte® (including L. acidophilus, L. rhamnosus, B. bifidum and B. longum) and Candida spp. (including C. albicans, C. dubliniensis, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, and C. tropicalis), as a common side effect of metronidazole treatment is vaginal thrush. An interim analysis of the first 24 participants who have completed the trial is included in this dissertation, as the trial is still ongoing. The probiotic was found to be well accepted and no product related adverse events were reported, although women commonly experienced vaginal Candida infections after topical metronidazole use. In the interim analysis, BV cure rates were similar between the SOC and intervention group, as was vaginal pH and the abundances of Lactobacillus spp. and most BV-associated bacteria. Women randomized to the intervention group had higher levels of B. bifidum and B. longum after treatment, which tended to go along with increased levels of Candida spp. and some BV-associated bacteria, while L. crispatus levels were lower in these women. This shows the urgent need to develop a vaginal probiotic containing Lactobacillus strains that are commensal to the FGT, to ensure achieving the desired effect of adjunctive probiotics in BV treatment. Thus, the characteristics of the commercially in South Africa available probiotic strains were compared to clinical vaginal Lactobacillus strains isolated from healthy South African women. A weighted scoring system was developed to select candidate strains for the development of a vaginal probiotic. Towards this aim, 57 Lactobacillus strains were isolated from healthy South African women (including 10 L. crispatus, 9 L. gasseri, 18 L. jensenii, 8 L. vaginalis, and 12 L. mucosae strains) which were distinct to the commercially available probiotic strains, and several isolates exhibited better probiotic characteristics in vitro than the commercially available probiotic bacterial strains (such as ability to lower pH and adherence to cervical cells), although this appeared to be highly strain- and not species specific. Based on weighted scores, two L. crispatus, two L. jensenii, and one L. vaginalis and one L. gasseri strain isolated from BV and STI negative South African women were selected for the development of a local probiotic for vaginal health. The presence of bacteriophages that target vaginal Lactobacillus spp. in cervicovaginal secretions of women with and without BV was evaluated using serial bacteriophage transfer and plaque assays. No lytic bacteriophages that targeted vaginal Lactobacillus spp. (including L. crispatus, L. gasseri, L. jensenii, L. vaginalis and L. mucosae) were isolated from FGT secretions, although CRISPR loci were common in publically available full Lactobacillus genome sequences. However, temperate bacteriophages were induced from the majority (71.8%) of the clinical Lactobacillus strains and 61.1% of the probiotic Lactobacillus strains screened using Mitomycin C, which was confirmed by transmission electron microscopy. Based on their morphology, these Lactobacillus bacteriophages belonged to the families of Sipho-, Myo- and Podoviridae. Conclusions: There are very few probiotics for vaginal health on the South African market, and the development of a probiotic containing commensals of the lower FGT should urgently be considered. Lytic bacteriophages targeting Lactobacillus spp. were not found in this study, although temperate bacteriophages were common and could influence Lactobacillus survival in vivo. Screening women with vaginal discharge for the SAHPRA-acknowledged pilot probiotic trial of Vagiforte PLUS® confirmed a high burden of STIs in Cape Town, South Africa, and that the symptom vaginal discharge is a very poor predictor for BV. The pilot trial showed that large doubleblind, randomized, placebo-controlled trials with adequate screening and enrolment algorithms and sample sizes, using a product containing vaginal Lactobacillus spp., are needed to determine the efficacy of adjunctive probiotics on BV cure and recurrence in South African women. Finally, while the products currently being marketed for vaginal health in South Africa and worldwide mostly do not contain Lactobacillus spp. commonly found in the lower FGT, several promising candidates from the FGTs of healthy, young, HIV- and BV- South African women were isolated and characterized that may prove more efficacious in treating BV. These have the potential to make a big impact on reproductive outcomes and HIV risk in young South African women.
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    In Silico Characterisation of Putative Prophages in Lactobacillaceae Used in Probiotics for Vaginal Health
    (2022-01-20) Happel, Anna-Ursula; Kullin, Brian R; Gamieldien, Hoyam; Jaspan, Heather B; Varsani, Arvind; Martin, Darren; Passmore, Jo-Ann S; Froissart, Rémy
    While live biotherapeutics offer a promising approach to optimizing vaginal microbiota, the presence of functional prophages within introduced Lactobacillaceae strains could impact their safety and efficacy. We evaluated the presence of prophages in 895 publicly available Lactobacillaceae genomes using Phaster, Phigaro, Phispy, Prophet and Virsorter. Prophages were identified according to stringent (detected by ≥4 methods) or lenient criteria (detected by ≥2 methods), both with >80% reciprocal sequence overlap. The stringent approach identified 448 prophages within 359 genomes, with 40.1% genomes harbouring at least one prophage, while the lenient approach identified 1671 prophages within 83.7% of the genomes. To confirm our in silico estimates in vitro, we tested for inducible prophages in 57 vaginally-derived and commercial Lactobacillaceae isolates and found inducible prophages in 61.4% of the isolates. We characterised the in silico predicted prophages based on weighted gene repertoire relatedness and found that most belonged to the Siphoviridae or Myoviridae families. ResFam and eggNOG identified four potential antimicrobial resistance genes within the predicted prophages. Our results suggest that while Lactobacillaceae prophages seldomly carry clinically concerning genes and thus unlikely a pose a direct risk to human vaginal microbiomes, their high prevalence warrants the characterisation of Lactobacillaceae prophages in live biotherapeutics.
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    Presence and Persistence of Putative Lytic and Temperate Bacteriophages in Vaginal Metagenomes from South African Adolescents
    (2021-11-23) Happel, Anna-Ursula; Balle, Christina; Maust, Brandon S; Konstantinus, Iyaloo N; Gill, Katherine; Bekker, Linda-Gail; Froissart, Rémy; Passmore, Jo-Ann; Karaoz, Ulas; Varsani, Arvind; Jaspan, Heather
    The interaction between gut bacterial and viral microbiota is thought to be important in human health. While fluctuations in female genital tract (FGT) bacterial microbiota similarly determine sexual health, little is known about the presence, persistence, and function of vaginal bacteriophages. We conducted shotgun metagenome sequencing of cervicovaginal samples from South African adolescents collected longitudinally, who received no antibiotics. We annotated viral reads and circular bacteriophages, identified CRISPR loci and putative prophages, and assessed their diversity, persistence, and associations with bacterial microbiota composition. Siphoviridae was the most prevalent bacteriophage family, followed by Myoviridae, Podoviridae, Herelleviridae, and Inoviridae. Full-length siphoviruses targeting bacterial vaginosis (BV)-associated bacteria were identified, suggesting their presence in vivo. CRISPR loci and prophage-like elements were common, and genomic analysis suggested higher diversity among Gardnerella than Lactobacillus prophages. We found that some prophages were highly persistent within participants, and identical prophages were present in cervicovaginal secretions of multiple participants, suggesting that prophages, and thus bacterial strains, are shared between adolescents. The number of CRISPR loci and prophages were associated with vaginal microbiota stability and absence of BV. Our analysis suggests that (pro)phages are common in the FGT and vaginal bacteria and (pro)phages may interact.
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    Probiotics for vaginal health in South Africa: what is on retailers’ shelves?
    (BioMed Central, 2017-01-19) Happel, Anna-Ursula; Jaumdally, Shameem Z; Pidwell, Tanya; Cornelius, Tracy; Jaspan, Heather B; Froissart, Remy; Barnabas, Shaun L; Passmore, Jo-Ann S
    Background: Probiotics are widely used to improve gastrointestinal (GI) health, but they may also be useful to prevent or treat gynaecological disorders, including bacterial vaginosis (BV) and candidiasis. BV prevalence is high in South Africa and is associated with increased HIV risk and pregnancy complications. We aimed to assess the availability of probiotics for vaginal health in retail stores (pharmacies, supermarkets and health stores) in two major cities in South Africa. Methods: A two-stage cluster sampling strategy was used in the Durban and Cape Town metropoles. Instructions for use, microbial composition, dose, storage and manufacturers’ details were recorded. Results: A total of 104 unique probiotics were identified in Cape Town and Durban (66.4% manufactured locally). Cape Town had more products than Durban (94 versus 59 probiotics), although 47% were common between cities (49/104). Only four products were explicitly for vaginal health. The remainder were for GI health in adults (51.0%) or infants (17.3%). The predominant species seen overall included Lactobacillus acidophilus (53.5%), L. rhamnosus (37.6%), Bifidobacterium longum ssp. longum (35.6%) and B. animalis ssp. lactis (33.7%). Products for vaginal health contained only common GI probiotic species, with a combination of L. acidophilus/B. longum ssp. longum/B. bifidum, L. rhamnosus/L. reuteri or L. rhamnosus alone, despite L. crispatus, L. gasseri, and L. jensenii being the most common commensals found in the lower female reproductive tract. Conclusion: This survey highlights the paucity of vaginal probiotics available in South Africa, where vaginal dysbiosis is common. Most vaginal products contained organisms other than female genital tract commensals