Browsing by Department "Division of Medical Virology"

Now showing 1 - 13 of 13
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    Open Access
    A Molecular Epidemiological Study of Human Parainfluenza 4 in the Western Cape, South Africa
    (2021) Parsons, Jane; Hardie, Diana; Smuts, Heidi
    Background Human parainfluenza 4 (HPIV 4) is a recognised cause of acute respiratory infection (ARI). However, there is no published data on the epidemiology of this virus in South Africa. This thesis describes the molecular epidemiology of HPIV 4 over a 4-year period (2014-2017). Respiratory samples from infants, children and adults presenting with respiratory illness in the Western Cape, South Africa were studied. Method A retrospective 4-year study using routine diagnostic samples from patients with ARI was conducted in Western Cape, South Africa. A database search of positive HPIV 4 samples detected by the Seegene Anyplex RV 16 diagnostic assay was extracted. Epidemiological information was recorded to determine age, gender, hospital ward (used as a proxy for disease severity), specimen type (upper or lower respiratory tract) and collection date (to indicate seasonality). To determine genetic evolution, novel primers targeting the haemagglutinin-neuraminidase (HN) in both HPIV 4 subtypes were designed to amplify a 733 bp and 738 bp sequence for HPIV 4A and HPIV 4B respectively. This product was then sequenced and aligned with known reference sequences from GenBank, using BioEdit. These aligned sequences were analysed using the phylogenetic analysis tool, MEGA 6, and Highlighter plots to determine sequence divergence events and evolution. A real-time PCR assay, targeting the phosphoprotein, was developed to rapidly distinguish subtype A and B viruses. Results HPIVs were the 6th most common respiratory viruses detected in diagnostic samples. In all, there were 312/7456 (4.2 %) HPIV 4 positive samples in patients with a median age of 12 months. Males had a higher infection rate. HPIV 4 was the most prevalent of the HPIVs accounting for 47% of all HPIVs. Respiratory infections due to HPIV 4 were seasonal, peaking in autumn and mid-winter (March to August). The overall prevalence of HPIV 4 increased over the study period. Of the HPIV 4-positive samples that were subtyped, 59 were subtype A and 26 subtype B. Both subtypes co-circulated during each season. 71 % of patients who were positive for HPIV 4 were co-infected with one or more additional respiratory virus with Adenovirus (27 %), Human Rhinovirus (23 %) and Bocavirus (19 %) as the most common. HPIV 1 and HPIV 3 were both able to co-infect patients with HPIV 4, but no co-infections with HPIV 2 were detected. Phylogenetic trees constructed using neighbour joining (NJ) method showed that most of the South African HPIV 4 subtypes did not group with the closest significant reference sequences from GenBank. The phylogenetic tree for HPIV 4A revealed 4 genetic groupings. There were many nucleotide changes increasing with time as well as a non-synonymous change in HPIV 4A, at location N161D. HPIV 4B had an amino acid change in location G198R in the HN protein sequenced. Conclusion HPIV 4 with an overall prevalence of 4 % over the study period was identified as a significant cause of ARI in the Western Cape, South Africa. Mono-infection with HPIV4 was associated with severe disease. In hospitalized infants who were HPIV 4 positive, between ¼ to 1/3 were from patients in ICU. Of these almost half (46 %) had HPIV 4 as a single infection. Further studies are needed to fully understand the molecular epidemiology of this infection.
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    An investigation into the specific function of the vaccinia virus :13.8 kDa protein encoded by the N1L gene
    (2005) Abrahams, Melissa-Rose Hilda; Kotwal, Girish J
    Vaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies.
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    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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    Characterization of Mycobacterium tuberculosis-specific Th22 cells in HIV-TB co-infection
    (2020) Makatsa, Mohau Steven; Burgers, Wendy A; Riou, Catherine
    Tuberculosis (TB) remains the infectious disease causing the greatest global mortality, with an estimated 10 million incident cases of TB and 1.45 million deaths in 2018. Although there is a cure for TB, the success of the treatment is hampered by multidrug resistant TB and HIV infection. There is an urgent need for an effective TB vaccine to prevent ongoing transmission. The development of a new and efficacious TB vaccine will likely be dependent on our understanding of protective immunity to TB. Although it is well established that Th1 cells are crucial in the response against Mycobacterium tuberculosis (Mtb), Th1 cytokines may not be sufficient to control Mtb infection. A major focus of this thesis is the contribution of an understudied Th subset in Mtb immunity, namely Th22 cells, producing the cytokine IL-22. IL-22 functions to preserve mucosal barriers and induce antimicrobial peptides, contributing to protective immunity to a range of extracellular and intracellular bacteria. A recent study in IL-22-deficient mice described a protective role for IL-22 during the development of TB. In humans, soluble IL-22 has been detected at sites of extra-pulmonary tuberculosis (TB), and a polymorphism in the IL-22 promoter has been linked to TB susceptibility. However, much remains to be understood about Th22 cells and their role in protective immunity to Mtb. In this study, we investigated the contribution of Th22 cells to TB immune responses by providing a detailed characterisation of Mycobacterium tuberculosis-specific Th22 cells in latent TB infection (LTBI), TB disease and HIV co-infection, using flow cytometric techniques. In Chapter 2, we optimised detection of IL-22 and determined the factors that contribute to Mtb-specific IL-22 production by CD4+ T cells, as well as characterising some aspects of Th22 cell biology. In Chapter 3, we examined the impact of TB disease and HIV infection on Th22 cells, compared to Th1 and Th17 cells. Finally, in Chapter 4, we explored Mtb-specific cytokine production by CD8+ T cells and CD4+ T cells following Mtb peptide stimulation, and the effect of TB disease and HIV infection. We detected significant IL-22 production from CD4+ T cells in healthy individuals following whole blood stimulation with Mtb whole cell lysate (MtbL). However, IL-22 responses were poorly detectable when peripheral blood mononuclear cells (PBMC) were stimulated with MtbL. Therefore, we sought to investigate conditions that influence IL-22 detection in whole blood and PBMC, and characterise Th22 cells further. We found that PBMC are able to produce IL-22 in response to Mtb but appear to lack the physiological environment for optimal induction of IL-22. We also discovered that TCR blocking inhibited Mtb-specific IL-22 production, suggesting that responses are stimulated through recognition of Mtb antigen by the TCR, rather than through bystander activation. IL-22 is produced by CD4+ T cells that appear to be conventional, rather than MAIT, γδ or iNKT cells. Indeed, analysis of the TCR clonality using vβ repertoire typing revealed similar repertoire usage between IL-22, IFN-γ-producing CD4+ T cells, and total CD4+ T cells. Overall, these data shed more light on the biology of IL-22-producing CD4+ T cells. Next, we examined the effects of HIV infection and TB disease on the magnitude, memory profile and activation phenotype of Mtb-specific Th22 cells, compared them to Th1 and Th17 cells. Blood samples were collected from 72 individuals classified into four groups based on their HIV-1 and TB status, namely HIV-/LTBI, HIV+/LTBI HIV-/active TB and HIV+/active TB. Blood was stimulated with MtbL and analysed for cytokine production using multiparameter flow cytometry. We observed similar frequencies of IL-22 to IFN-γ-producing CD4+ T cells in LTBI. Mtb-specific Th22 cells were reduced to a greater extent than Th1 cells by a combination of HIV infection and TB disease. Th22 cells demonstrated differences in their memory and activation phenotype compared to Th1 and Th17 cells. In the context of active TB, Th1 cells were characterised by a high expression of the activation marker HLA-DR. In contrast, Th22 cells did not demonstrate activation using this marker during TB disease. Similarly, Th1 cells were more differentiated in TB disease irrespective of HIV status, while there was no difference in the memory phenotype of Th22 cells during different disease states. Finally, we characterised Mtb peptide-specific CD4+ and CD8+ T cell responses in LTBI, active TB and HIV infection. CD4+ T cells did not produce detectable IL-22 when blood was stimulated with Mtb peptides, and there was also no IL-22 response from CD8+ T cells. Th1 cytokines IFN-γ and TNF-α were detectable from CD4+ and CD8+ T cells in response to Mtb peptides. Consistent with previous studies, there was a higher proportion of individuals with detectable CD8+ responses during active TB and HIV co-infection compared to HIV-infected LTBI individuals, but no difference is the magnitude of response was observed. Interestingly, HIV infection and TB disease induced similar levels of activation in Mtb-specific CD8+ compared to CD4+ T cells. Moreover, active TB and HIV co-infection impaired memory differentiation of Mtb-specific CD8+ T cells towards a less differentiated profile, compared to LTBI. These results confirm that both CD4+ and CD8+ T cells contribute to TB immune responses. In summary, we confirm that Th22 cells constitutes a substantially portion of CD4+ T cell response to Mtb . IL-22 appears to be produced by conventional CD4+ T cells but may require specific antigen presentation requirements to optimally induce its production. Interestingly, HIV infection during TB disease led to a near absence of Th22 cells in blood. Our results warrant further study of the role of Th22 cells in TB immunity, which may lead to insights that could assist the development of an effective vaccine against TB.
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    Characterizing the genotypic and phenotypic diversity of Gardnerella vaginalis from vaginal clinical samples
    (2018) Masete, Kopano Valerie; Froissart, Rémy; Passmore, Jo-Ann
    Bacterial vaginosis (BV) is a common vaginal condition affecting reproductive-age women, especially in sub-Saharan Africa. With poor treatment outcomes, BV has been associated with pregnancy complications, pelvic inflammatory disease as well as acquisition and transmission of sexually transmitted diseases. While the etiology of BV is not well characterized, it is understood that Gardnerella vaginalis plays a critical role in BV by initiating the formation of the polymicrobial biofilm that characterizes BV and by degrading protective vaginal mucus through the release of sialidase. Recent evidence suggests that the G. vaginalis species is more heterogeneous that initially thought and that not all G. vaginalis may be involved BV. The aim of this study was thus to characterize the genotypic and phenotypic diversity of G. vaginalis isolates. This was achieved in vitro, using 109 G. vaginalis isolates that were previously purified from vaginal samples of 109 French women who were BV-positive (n = 75), BV-intermediate (n = 20) or BV-negative (n = 14), as diagnosed by Nugent scoring. To determine the genotypic diversity of G. vaginalis isolates, 90 isolates were successfully genotyped using their chaperonin-60 (cpn60) sequences, revealing the presence of four phylogenetic clades (subgroups A-D) made up of 13 subgroup A, 17 subgroup B, 58 subgroup C and 2 subgroup D isolates. To determine the phenotypic diversity of G. vaginalis isolates, sialidase activity, biofilm formation and susceptibility to antibiotics used to treat BV were measured. Sialidase activity was not detected in subgroup A and D isolates but was detected, at similar levels, in subgroup B and C isolates. Isolates from all subgroups of G. vaginalis could form similar amounts of biofilm. G. vaginalis isolates (n = 45) were largely resistant to metronidazole (71%), but sensitive to clindamycin (100%), moxifloxacin (91%) and augmentin (100%). The presence of prophages in G. vaginalis isolates was also investigated, revealing the presence of bacteriophage (phage)-like particles that could not be classified into any known phage families, whose phage status remains to be confirmed. In conclusion, G. vaginalis subgroup B and C isolates were the only ones that formed biofilm as well as had detectable sialidase activity suggesting that G. vaginalis subgroups B and C are most likely to be involved in BV. These results contribute to our knowledge of BV and could be useful in future studies that aim to design better treatment strategies for BV.
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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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    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 Viral Load Testing in the South African Public Health Setting in the Context of Evolving ART Guidelines and Advances in Technology, 2013 - 2022
    (Multidisciplinary Digital Publishing Institute, 2023-08-22) Hans, Lucia; Cassim, Naseem; Sarang, Somayya; Hardie, Diana; Ndlovu, Silence; Venter, W.D. Francois; Da Silva, Pedro; Stevens, Wendy
    HIV viral load (VL) testing plays a key role in the clinical management of HIV as a marker of adherence and antiretroviral efficacy. To date, national and international antiretroviral treatment recommendations have evolved to endorse routine VL testing. South Africa (SA) has recommended routine VL testing since 2004. Progressively, the centralised HIV VL program managed by its National Health Laboratory Service (NHLS) has undergone expansive growth. Retrospective de-identified VL data from 2013 to 2022 were evaluated to review program performance. Test volumes increased from 1,961,720 performed in 2013 to 45,334,864 in 2022. The median total in-laboratory turnaround time (TAT) ranged from 94 h (2015) to 51 h (2022). Implementation of two new assays improved median TATs in all laboratories. Samples of VL greater than 1000 copies/mL declined steadily. Despite initial increases, samples of fewer than 50 copies/mL stagnated at about 70% from 2019 and declined to 68% in 2022. Some variations between assays were observed. Overall, the SA VL program is successful. The scale of the VL program, the largest of its kind in the world by some margin, provides lessons for future public health programs dependent on laboratories for patient outcome and program performance monitoring.
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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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    Poxvirus Protein N1L Targets the I-κB Kinase Complex, Inhibits Signaling to NF-κB by the Tumor Necrosis Factor Superfamily of Receptors, and Inhibits NF-κB and IRF3 Signaling by Toll-like Receptors
    (2004) DiPerna, Gary; Stack, Julianne; Bowie, Andrew G; Boyd, Annemarie; Kotwal, Girish; Zhang, Zhouning; Arvikar, Sheila; Latz, Eicke; Fitzgerald, Katherine A; Marshall, William L
    Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways.
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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.
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    Tanned gelatin: some biophysical and biochemical properties of a new gel exclusion agent and its application to the chromatography of proteins and viruses
    (1970) Katz, Woolf
    The principal aim of this study was to develop a new bed material suitable for gel exclusion chromatography, which would embrace all the desirable characteristics of available materials used for this purpose and if possible improve on some of them. The two most important limitations of existing gel media concern their exclusion limits and rigidity. Agar and agarose have been found satisfactory for the separation of larger molecules including some viruses, but difficulties may arise at low gel concentrations due to the low mechanical strength of the granules. The dextran and polyacrylamide gels have in general a rigidity equal to that of agar and agarose, but cannot be prepared with such large pore sizes and are therefore limited to the separation of smaller molecules.
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    Wesselsbron virus : a biophysical, biochemical and serological study
    (1966) Parker, Joan Rae
    The application of variety of virological techniques to the study of Wesselsbron virus hos resulted in the compiling of much worthwhile information on the biophysical, biochemical and serological properties of this virus. Wesselsbron virus , like most arboviruses, showed marked sensitivity to the action of bother ether and sodium deoxycholate.