Browsing by Author "Woodman, Zenda"

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    Can one size fit all? Approach to bacterial vaginosis in sub-Saharan Africa
    (BioMed Central, 2016-03-11) Woodman, Zenda
    Bacterial vaginosis (BV) is the most common vaginal disorder affecting women of reproductive age and is associated with increased risk of sexually transmitted infections such as human immunodeficiency syndrome (HIV-1). Sub-Saharan Africa has the highest BV and HIV-1 burden and yet very few studies have focused on understanding the aetiology of BV and its association with HIV in this region. It has been suggested that we need to accurately diagnose and treat BV to lower the risk of HIV infection globally. However, effective diagnosis requires knowledge of what constitutes a “healthy” cervicovaginal microbiome and current studies indicate that Lactobacillus crispatus might not be the only commensal protective against BV: healthy women from different countries and ethnicities harbour alternative commensals. Microbiotas associated with BV have also shown global variation, further complicating effective diagnosis via culture-based assays as some species are difficult to grow. Antibiotics and probiotics have been suggested to be key in controlling BV infection, but the efficacy of this treatment might rely on reconstituting endogenous commensals while targeting a specific species of BV-associated bacteria (BVAB). Alternatively, therapy could inhibit essential BV bacterial growth factors e.g. sialidases or provide anti-microbial compounds e.g. lactic acid associated with a healthy cervicovaginal microbiome. But without global investigation into the mechanism of BV pathogenesis and its association with HIV, selection of such compounds could be limited to Caucasian women from certain regions. To confirm this suggestion and guide future therapy we require standardised diagnostic assays and research methodologies. This review will focus on research papers that describe the global variation of BV aetiology and how this influences the identification of determinants of BV pathogenesis and potential probiotic and antimicrobial therapy
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    Characterisation of HIV-1 subtype C envelope functional determinants of dual infected individuals
    (2018) Omar, Shatha Sultan Ahmed; Woodman, Zenda
    Identification of HIV-1 Envelope (Env) fitness determinants could provide functionally constrained, accessible regions that could be included in subunit vaccines to induce broadly neutralising antibodies (bnAb). We hypothesised that Env fitness determinants are common to circulating variants but that the plasticity of Env structure limits identification. Rapid evolution; however, could select for sequence changes within the determinants coincident with alterations in function, making identification easier. Dual infection with two phylogenetically distinct HIV-1 variants under the same selective pressures might result in rapid functional evolution, facilitating identification of Env fitness determinants. It has been shown that the Env plays a significant role in viral adaptation to the host environment, which then increases disease progression. Therefore, this study used dual infections as a model system to characterise Env function, its role in in vivo viral outgrowth of variants and disease progression and to identify fitness determinants for future vaccine design. Single-genome amplification (SGA)-derived env sequences of four dual infected individuals sampled at enrolment (0 months), 3, 6, and 12 months post infection (mpi) were analysed using Highlighter plots, RIP, DNA pairwise distance and Neighbourjoining trees to determine the in vivo evolution of infecting viral populations and their relative frequency over time within each participant. Representative amplicons were cloned at each time point and compared using a pseudovirus (PSV) entry efficiency assay. 2 characterised by Affinofile system, T-20 IC50 and Western blotting to identify whether tropism, Env expression/cleavage, incorporation into viral particles and fusogenicity were most likely responsible for the variation in Env entry efficiency. All variants were R5- and T-tropic and only Env fusion capacity correlated significantly with Env entry efficiency data (p = 0.02, r = 0.59), suggesting that variants infecting dual infected participants evolved towards higher fusion capacity. Changes in Env fusogenicity indicated that gp41 might be a fitness determinant of PSV entry efficiency and analysis of SGA sequences indicated that recombination within gp41 was common to 3/4 participants. Env chimeras were generated where gp41 was swapped between clones that either had the same (CAP84) or different (CAP267) PSV entry efficiency. For both participants, and (CAP137) gp41 was identified as a potential determinant of Env fitness. Moreover, two potential N-glycan sites (PNG) at position N332 and N339, previously reported to be involved in neutralising antibody escape, were also identified. While N332 enhanced Env entry efficiency in one participant, N339 attenuated Env entry efficiency in another, potentially due to the escape mutation carrying a fitness cost. However, neither PNG seemed to affect Env expression/cleavage, incorporation into viral particles and fusogenicity. As Env phenotypic characterisation focussed on PSV assays, we wanted to determine whether viral replication was also similarly affected. Infectious molecular clones (IMCs) were generated from two participants using a recombination yeast assay and replication capacity (RC) in peripheral blood mononuclear cells (PBMCs) was assessed using parallel replication. A significant correlation between RC of viruses in PBMCs and Env entry efficiency in TZM-bl and fusion capacity (p = 0.03, r = 0.7; p = 0.04, r = 0.7, respectively) was determined. IMC RC was also associated with in vivo outgrowth of viral populations at 12 mpi although this relationship did not always coincide with the frequency of individual variants. Changes in the RC of the Env chimeras and mutants was not associated with phenotypic changes, suggesting that Env entry efficiency determinants did not play the same role in IMC RC as it did in PSV entry. Lastly, there was a significant negative association (p = 0.046, r = -0.59) between Env entry efficiency and CD4+ T decline, a marker of disease progression, supporting the previous finding that Env entry efficiency could be the driving agent of disease progression. This was also corroborated by the trend in association between RC of IMCs and faster CD4+ T decline. 3 Our findings suggest that despite different host pressures, viral competition in most dual infected individuals selected for rapid recombination within gp41 that enhanced fusion capacity. Enhanced gp41 fusogenicity of the dominant viral population at 12 mpi increased PSV entry efficiency and replicative fitness enabling viral outgrowth. Therefore, vaccines that target gp41 might prevent HIV infection or at least attenuate viral fitness and slow disease progression. On the other hand, we showed that targeting the PNG at position N339 of gp120 might influence viral fitness and increase viral load and/or decrease CD4 T cell count. This is in keeping with the association between CD4 T cell decline and PSV entry efficiency and IMC RC, suggesting that Env fitness plays a role in HIV pathogenicity.
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    Characterisation of the ectodomain shedding of angiotensin-converting enzyme
    (2003) Woodman, Zenda; Sturrock, Edward D
    Bibliography: leaves 240-266.
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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 genotypic and phenotypic properties of transmitted Human Immunodeficiency virus type 1 variants circulating in Mbeya Tanzania
    (2013) Nofemela, Andile; Williamson, Carolyn; Woodman, Zenda
    Includes abstract. Includes bibliographical references.
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    Extensive purifying selection acting on synonymous sites in HIV-1 Group M sequences
    (BioMed Central Ltd, 2008) Ngandu, Nobubelo; Scheffler, Konrad; Moore, Penny; Woodman, Zenda; Martin, Darren; Seoighe, Cathal
    BACKGROUND: Positive selection pressure acting on protein-coding sequences is usually inferred when the rate of nonsynonymous substitution is greater than the synonymous rate. However, purifying selection acting directly on the nucleotide sequence can lower the synonymous substitution rate. This could result in false inference of positive selection because when synonymous changes at some sites are under purifying selection, the average synonymous rate is an underestimate of the neutral rate of evolution. Even though HIV-1 coding sequences contain a number of regions that function at the nucleotide level, and are thus likely to be affected by purifying selection, studies of positive selection assume that synonymous substitutions can be used to estimate the neutral rate of evolution. RESULTS: We modelled site-to-site variation in the synonymous substitution rate across coding regions of the HIV-1 genome. Synonymous substitution rates were found to vary significantly within and between genes. Surprisingly, regions of the genome that encode proteins in more than one frame had significantly higher synonymous substitution rates than regions coding in a single frame. We found evidence of strong purifying selection pressure affecting synonymous mutations in fourteen regions with known functions. These included an exonic splicing enhancer, the rev-responsive element, the poly-purine tract and a transcription factor binding site. A further five highly conserved regions were located within known functional domains. We also found four conserved regions located in env and vpu which have not been characterized previously. CONCLUSION: We provide the coordinates of genomic regions with markedly lower synonymous substitution rates, which are putatively under the influence of strong purifying selection pressure at the nucleotide level as well as regions encoding proteins in more than one frame. These regions should be excluded from studies of positive selection acting on HIV-1 coding regions.
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    HIV-1 subtype C Envelope function becomes less sensitive to N-glycosylation deletion during disease progression
    (2019-06-17) Lumngwena, Evelyn N; Shuping, Liliwe; Bernitz, Netanya; Woodman, Zenda
    Objective As part of a larger study to understand how Envelope N-glycosylation influences HIV-1 pathogenesis, we selected a participant infected with a single Subtype C variant and determined whether deletion of specific potential N-glycan sites (PNGs) impacted Envelope function longitudinally. Results We deleted five PNGs previously linked to HIV-1 transmission of two matched Envelope clones representing variants at 5 and 173 weeks post-infection. The transmitted founder (TF) had significantly better pseudovirus entry efficiency than the chronic infection (CI) variant. Deletion of all PNGs significantly reduced TF entry efficiency, binding to dendritic cell-specific intracellular adhesion molecule 3 grabbing non-integrin (DC-SIGN) receptor and trans-infection. However, mutational analysis did not affect the phenotype of the CI Envelope to the same extent. Notably, deletion of the PNGs at N241 and N448 had no effect on CI Envelope function, suggesting that some PNGs might only be important during acute infection. Therefore, vaccines that elicit antibodies against N-glycans important for TF Envelope function could drive the loss of PNGs during immune escape, abrogating viral replication. Conversely, changes in N-glycosylation might have no effect on some variants, reducing vaccine efficacy. This finding highlights the need for further investigation into the role of Envelope N-glycosylation in HIV-1 pathogenesis.
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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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    The impact of HIV-1 subtype C Envelope N-glycosylation on DC-SIGN meditated modulation of DC function to facilitate transmission or enhance viral pathogenesis
    (2017) Lumngwena, Evelyn Ngwa; Woodman, Zenda
    N-glycosylation plays an important role in Envelope (Env) function and may be involved in the modulation of the immune response to HIV-1 infection. In this study, we hypothesized that Env N-glycosylation may affect viral pathogenesis by influencing Env structure and function. Furthermore, we also postulated that differences in Env glycosylation could affect interactions between Env and DC-SIGN of dendritic cells (DCs), activating alternative signalling pathways which stimulate the release of different immune modulators. We generated pseudovirus of eighteen Env clones (PSVs) with variable number and position of potential N-glycan sites (PNGs) and compared their ability to infect TZM-bl cells, bind to Raji+ DC-SIGN cells, trans-infect TZM-bl cells when captured by either Raji-DC-SIGN cells or monocyte-derived dendritic cells (MDDCs) and modulate MDDC signaling by investigating the release of Interleukin-10 (IL-10) and other immune modulatory cytokines and MAPK activation. Entry efficiency, DC-SIGN binding and trans-infection varied widely across all clones. The level of IL-10 secreted by MDDCs in response to PSV stimulation varied 32-fold. The induction of IL-10 secretion by purified gp140 confirmed that Env was the viral component that stimulated the secretion of IL-10 via interaction with DC-SIGN and potentially other undefined receptors. PSV and purified gp140 stimulated MDDC signaling via ERK and JNK phosphorylation, while p38 was not activated. The addition of recombinant DC-SIGN lowered the levels of secreted IL-10 and ERK /JNK phosphorylation, suggesting that DC-SIGN plays a role in these responses. As Env mannosylation correlated with DC-SIGN binding, five highly conserved Env PNGs (241, 262, 386, 392, and 448) previously identified to carry high mannose type N-glycans and hence thought to be involved in DC-SIGN binding were deleted in two Env clones by site-directed mutagenesis to confirm their importance in Env function. The potential role of these PNGs in Env entry efficiency, DC-SIGN binding, trans-infection, induction of MDDC IL-10 secretion and activation of MAPK phosphorylation was determined. Deletion of these sites significantly affected the entry efficiency, DC-SIGN binding, trans-infection and MDDC IL-10 secretion, with one Env clone proving to be more sensitive to mutation than the other. This suggests that PNGs influence Env function in a clone-specific manner. As deletion of highly conserved PNGs abrogated Env function we used sequence analysis to identify PNGs involved in binding DC-SIGN and inducing MDDC IL-10 secretion. We grouped PSVs based on the presence or absence of specific PNGs in Env sequences and compared entry efficiency, DC-SIGN binding, trans-infection, stimulation of MDDC IL-10 secretion and induction of MAPK phosphorylation. Three Env PNGs were significantly associated with entry efficiency (N356, N392, and N674), and three sites (N289, N356 and 674) were significantly associated with trans-infection while N674 also influenced DCSIGN binding. The majority of MDDC donors secreted higher levels of IL-10 when stimulated with PSVs that carried PNGS at N130 (p = 0.0016) and N332 (p = 0.0039) and lacked N674 (p = 0.033). When Envs were graded on whether they had 0, 1, 2 or 3 of the PNGs (e.g. -130, -332, +674; -130, +332 and +674, etc.) those that carried either one of the PNGs or the entire induction motif (N130+ N332+ N674-) significantly stimulated MDDCs to secrete higher levels of IL-10 than those that completely lacked the motif (p = 0.0335 and p = 0.0304, respectively). As the presence of N674 was linked to reduction in all functions of Env, it is likely that the presence of an N-glycan at this site affected Env structure and could skew the analysis. Excluding N674 indicated that the presence of PNGs at position 130 and 332 was sufficient to induce significantly higher IL-10 release than those that had either none or one of these sites (p = 0.0053). When we determined whether N130 and N332 were enriched in subtype C acute infection Envs, these sequences were not enriched with PNGs at either N130 or N332 compared to chronic infection viruses. However, when IL-10 levels were compared between MDDC donors stimulated with PSV of either acute or chronic infection clones, those from early infection significantly enhanced MDDC secretion of IL-10 (p = 0.0039). This suggests that even though PNGs at 130 and N332 could be involved in inducing MDDC IL-10 secretion, it is not the only requirement for enhanced stimulation. Although Env differentially activated ERK and JNK phosphorylation, ERK phosphorylation did not correlate with IL-10 secretion, suggesting that this MAPK signaling pathway was not solely responsible for triggering the release of MDDC IL-10 and other regulatory cytokines. PSVs also stimulated the release of TNFα, IL-1β, IL-6, IL-8, MIP-1a, and MIP-1b while having no effect on IL-12 levels. This suggests that HIV-1 binding to DCs in the genital tract could change the dynamics of DC immune responses, deregulating their cytokines secretion and destabilising the Th0 cell differentiation to facilitate viral survival and thus productive clinical infection. We therefore conclude that HIV-1 variants differentially stimulate MDDCs to release immunosuppressive IL-10 and that transmitted founders could be better at modulating immune responses in the genital tract compared to chronic infection variants.
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    Neutralizing antibody responses in HIV-1 dual infection : lessons for vaccine design
    (2013) Sheward, Daniel James; Williamson, Carolyn; Woodman, Zenda
    Includes abstract. Includes bibliographical references.
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    Screening for novel compounds produced by Actinobacteria and Gardnerella vaginalis that impact HIV-1 infection
    (2023) Cohen, Arad; Woodman, Zenda
    HIV-1 infection continues to impact the lives of millions, with the most vulnerable being women living in Sub-Saharan Africa. Variation in the outcome of antiretroviral (ARV) and preexposure prophylaxis (PrEP) clinical trials is most likely multifactorial which could include Bacterial Vaginosis (BV), a condition associated with increased HIV acquisition and decreased tenofovir (TVF) efficacy. It is therefore important to not only understand how BV impacts HIV infection but also to identify alternative, novel ARVs. Natural products (NPs) have been harnessed and used as antibiotics, antifungals, and anti-virals and may represent a source of novel HIV drugs. This study determined whether NPs produced by selected filamentous actinobacteria and the BV-associated bacterium (BVAB) Gardnerella vaginalis (GV) could impact HIV infection. NPs from 23 actinobacteria strains were extracted and tested on pseudovirus (PSV) infection. Ten strains showed a range of anti-viral activity from 50 µg/mL to 25 µg/mL, and the identity of the NPs was determined through mass spectrometry and the Global Natural Products Social Molecular Networking (GNPS) database. These strains produced known antimicrobials: Actinomycin D, Valinomycin, Antimycin, Puromycin and Anisomycin. As some commercial antibiotics have been shown to have antiviral activity, we tested the ability of the antimicrobials to inhibit PSV infection. However, we were unable to determine accurate IC50 values due to the cytotoxicity of all the compounds. Further analysis is required to identify antimicrobial concentrations that are not cytotoxic but are still able to inhibit PSV infection. Surprisingly, preliminary data indicated that GV, the most common bacterial species associated with BV, inhibited HIV infection (Unterpertinger and Abrahams, unpublished data). When PSV infection was measured in the presence of GV supernatant, PSV infection was significantly inhibited, suggesting that either the bacteria released a compound that impacted HIV infection or the culture medium, BHI was reducing infection. Ammonium sulphate (AS) precipitation was performed to isolate compounds from the supernatant of GV but, none of the fractions showed significant anti-viral activity. Subsequently, to confirm whether GV produced organic compounds that may be inhibitory, NPs were extracted from the abiotic culture medium using liquid-liquid extraction (LLE) and solid-phase extraction (SPE). A midpolar fraction was identified that displayed anti-viral properties without reducing cell viability. After the fraction was analyzed by mass spectrometry and screening of the GNPS database, Vitamin B6 was identified as the possible inhibitor. Therefore, this study has identified compounds produced by filamentous actinobacteria and GV that might be novel inhibitors of HIV infection and further study is required to confirm their efficacy.
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    The role of bacterial vaginosis-associated sialidase on HIV-1 infection
    (2022) Traviss, Riley James; Woodman, Zenda
    Studies suggest that women with Bacterial Vaginosis (BV) are at a higher risk of being infected by HIV-1. One possible reason is that bacterial vaginosis-associated bacteria (BVAB) release sialidases which alter either the virus and host cells directly or the viscosity of the female vaginal tract (FGT) mucosal barrier, increasing the likelihood of infection. This study aimed to determine the impact of bacterial sialidase on HIV-1 infection focussing specifically on the BVAB, G. vaginalis. Pseudovirus was produced by transfection of HEK293T cells and infection of TZM-bl cells was measured by luminescence. In the presence of purified commercial sialidase, pseudovirus infection increased significantly (2-fold) suggesting that sialidase enhanced HIV infection. The sialidase appeared to act on both the virus and the host cell, although the former interaction appeared to play a more important role in enhancing pseudovirus infection. Two G. vaginalis strains, 3H6 and 2HI, isolated from the FGT were cultured and tested for production of sialidase. Sialidase was purified from the culture medium of these strains by anion-exchange and gel filtration. Sialidase was successfully purified, as confirmed with a sialidase activity assay as well as SDS-PAGE. Analysis by SDS-PAGE showed two distinct bands, with approximate molecular weights of 40 – 45 kDA. Inclusion of the purified sialidase in a pseudovirus entry assay resulted in a 1.5-fold increase in HIV entry and LC/MS mass spectrometry confirmed the presence of sialidase. As not all G. vaginalis strains express sialidase, we tested whether the ATCC 14018 strain, which does not produce sialidase, could also impact HIV infection. Pseudovirus entry of TZM-bl cells was measured in the presence of the growth medium used to culture G. vaginalis. However, instead of an increase in pseudovirus infection, the culture medium inhibited virus entry. We then determined whether G. vaginalis growth altered the pH of the culture medium and/or produced an inhibitor that reduced HIV entry. When the culture medium was heated and the volume reduced, pseudovirus infection was restored albeit to a level lower than the virus only control. Increased culture medium pH did enhance infectivity but was less effective compared to heating. Overall, we concluded that BHI medium components reduced cell viability above 5ul in the absence of G. vaginalis growth and that ATCC 14018 secreted a heat labile factor that inhibited HIV infection. Therefore, it seems as though the expression of sialidase might be a very important determinant of how G. vaginalis affects HIV infection.
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    The role of HIV-1 subtype B Envelope transmission motifs in subtype C variant infectivity
    (2018) Meyer, Bahiah; Woodman, Zenda
    Transmitted founders (TF) might carry motifs that provide a phenotypic advantage that enables human immunodeficiency virus type-1 (HIV-1) to overcome immune barriers within the female genital tract. One study compared over 5000 subtype B TF and mismatched chronic infection envelope (env) sequences and identified two putative transmission motifs: Histidine at position 12 of the signal peptide (His12) and a potential N-glycan site (PNG) at position 413-415. Although, His12 was shown to be important for subtype B Env expression and viral infectivity, in our own sequence analysis subtype C variants did not carry the transmission motifs and the aim of this study was to determine whether His12 and PNG413 was important for subtype C Env expression, processing, function and viral replication. Mutagenesis of a subtype C Env clone indicated that His12 decreased pseudovirion (PSV) entry efficiency without influencing Env expression, secretion and cleavage with no changes in the N-glycosylation profile. This suggested that His12 had a fitness cost and was thus selected against. However, His12 significantly enhanced the entry efficiency of infectious molecular clones (IMCs), suggesting that it might be beneficial for in vivo replication. The variation between the PSV and IMC entry of TZM-bl cells could be due to differences in assay conditions. On the other hand deletion of PNG413 enhanced Env expression, secretion, cleavage and PSV and IMC entry efficiency of TZM-bl cells. This would suggest that subtype C TFs carrying a PNG at 413-413 would have lower viral replicative capacity due to poor expression and processing of Env. The benefit of this phenotype on HIV-1 subtype C transmission needs to be further investigated. Unfortunately, PSV and IMC entry of TZM-bl cells could not be confirmed by IMC replication in peripheral blood monocytes because the clones could not replicate to measurable levels in these cells over the culture period. Overall, this study has shown that amino acid residues at positions 12 and 415 do play a role in modulating Env processing and function however the actual mechanism by which these polymorphisms impact viral fitness most likely differ to that of subtype B, explaining why His12 is absent and PNG413 is present in subtype C TFs.
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    To compare the expression, processing, incorporation and function of pseudoviruses and infectious molecular clones using different cell types and HIV backbones
    (2019) Abrahams, Bianca; Woodman, Zenda
    Understanding HIV transmission mechanisms is essential for the design and development of an efficacious, broadly acting vaccine that targets features common to transmitted viruses. However, there is a lack of consensus amongst current HIV studies characterising transmitted founders (TFs). When investigating the methods employed across studies, it becomes clear that methodologies are highly variable and thus, could be impacting research outcomes. This study therefore aimed to determine whether Envelope (Env) expression and processing affects function and whether cell type and/or expression system were responsible for these differences. Our data suggest that even though we did not observe differential expression of recombinant Env clones across cell types, when pseudovirus and infectious molecular clone (IMC) backbones were introduced, expression of Env decreased. We also found differences in processing in the form of cleavage, N-glycosylation and incorporation of Env across cell types. We conclude from this that methods used to study Env characteristics are highly sensitive to cell type and HIV backbone which suggests that a more standardised system is required to make meaningful comparisons between studies. The results of our functional Env analysis revealed high variation depending on the methodology used. We found that entry of TZM-bl cells by pseudovirus (PSV) is dependent on the cell line used to produce the viral particles. Unfortunately, due to low IMC titre, we had to expand the virus in PBMCs, negating the effect that cell type might have had on IMC expression. We could thus not directly compare PSVs to IMCs. However, PSV and IMC entry as well as IMC replication in PBMCs suggested that CHO cells were not suitable for robust viral production and better suited for recombinant Env expression. Overall, the findings in this project support previous findings that PSVs and IMCs are not directly comparable due to multiple factors that influence Env expression and virus production. We suggest that researchers who focus on HIV functional analysis, particularly Env, with the end-point of informing vaccine design, need regulated methods across laboratories, similar to the way that neutralisation assays were standardised.
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    Transmission of HIV-1 CTL escape variants provides HLA-mismatched recipients with a survival advantage
    (Public Library of Science, 2008) Chopera, Denis R; Woodman, Zenda; Mlisana, Koleka; Mlotshwa, Mandla; Martin, Darren P; Seoighe, Cathal; Treurnicht, Florette; Rosa, Debra Assis de; Hide, Winston; Karim, Salim Abdool
    Author Summary Following infection with HIV, it is well established that a person's genetic makeup is a major determinant of how quickly they will progress to AIDS. Particularly important is the class I Human leukocyte antigen (HLA) gene that is responsible for alerting the immune system to HIV's presence. One of the reasons our immune systems are unable to beat HIV is that the virus can mutate to forms that our HLA genes no longer recognise. However, some people have versions of the HLA gene (for example HLA-B*57 and HLA-B*5801) that are known to force HIV to tolerate mutations that damage its ability to reproduce. Slower HIV reproduction is thought to be one reason that HLA-B*57 and HLA-B*5801 positive people progress to AIDS more slowly than most other HIV infected persons. We report here on a study of HLA-B*57 and HLA-B*5801 negative women in which better control of disease tended to be associated with their being infected with viruses carrying mutations that have been previously shown to reduce replication. These mutations characterise viruses found infecting HLA-B*57 and HLA-B*5801 positive people. This indicates for the first time that HLA-B*57 or HLA-B*5801 negative people that are infected by such reproductively compromised viruses may also experience better survival prospects.