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Comparison of the two lumpy skin disease virus vaccines, Neethling and Herbivac, and construction of a recombinant Herbivac-Rift Valley fever virus vaccineOmar, Ruzaiq January 2015 (has links)
There are two broad aims to this project. The first aim is to compare and characterise two lumpy skin disease virus (LSDV) vaccines namely the vaccine based on attenuated Neethling LSDV (nLSDV) and Herbivac®LS (Herbivac). The second aim is to construct a recombinant LSDV expressing Rift Valley fever virus (RVFV) genes. An LSDV vaccine is critical for sustainable control of lumpy skin disease (LSD). There are four commercially available live attenuated vaccines for LSDV, nLSDV, Herbivac, Lumpyvax and the Kenyan strain sheeppox virus (KS-1). In this study Herbivac was characterised by comparing it to its parent, nLSDV. Growth curves of the two viral strains were conducted in cell culture as well as in embryonated hens’ eggs. No notable difference in the growth rate of the two strains could be detected when the viruses were grown in cell culture, however a notable difference was detected when the viruses were grown on the chick allantoic membranes (CAMs) of embryonated hens’ eggs. When grown on CAMs a faster growth rate was observed for nLSDV compared to Herbivac. nLSDV also killed the embryos at 4 d.p.i where Herbivac did not. The two strains were then further characterised through histological analysis of CAMs after infection with each of the viruses. Overall, higher levels of hyperplasia and hypertrophy were observed in CAMs infected with either nLSDV or Herbivac compared to uninfected CAMs. Herbivac-infected CAMs resulted in thicker chorionic membranes and larger pocks compared to nLSDV. RVFV and LSDV both contribute to the disease burden among cattle in Africa and the Arabian Peninsula. The main aim of this study was to construct a recombinant Herbivac which expresses immunogenic proteins of Rift Valley fever virus (Herbivac-RVFV). Herbivac-RVFV was designed to express specific RVFV genes selected for their antigenic properties. The genes selected are also representative of the genes from recent viral outbreaks in the horn of Africa. The selection of outbreak relevant RVFV genes involved phylogenetic analysis of all full length M-segment and NC gene sequences available on Genbank. Phylogenetic trees were constructed for M-segments and NC genes and groups identified which were highly representative of sequences from recent outbreaks of the virus. Consensus sequences were derived from these groups and included in the transfer vector. The phylogenetic analysis also revealed that the sequences of current RVFV vaccines are phylogenetically distant from viruses isolated from current outbreaks, although high levels of sequence conservation was maintained across all viral strains. This is the first study in which the RVFV genes coding for proteins that will induce a protective immune response (Gn and Gc, as well as the nucleocapsid (NC) gene) were selected so as to be representative of current outbreak strains of the virus. These genes were inserted between LSDV ORFs 49 and 50, a novel insertion site. The transfer vector also contained an eGFP marker gene and an ECO-GPT selection gene, located outside of the LSDV flanking sequences. This meant a two-step isolation procedure, first to isolate the recombinant containing the entire transfer vector with eGFP and ECO-GPT, and then to isolate a recombinant with only the RVFV genes and not eGFP and ECO-GPT. Transient expression of RVFV proteins in cells infected with Herbivac and then transfected with the transfer vector was confirmed via western blotting and immunofluorescence. Here the proteins Gn, Gc and NC were shown to be expressed. In the present study, a single crossover Herbivac-RVFV recombinant was isolated through multiple passaging of cell lysates, originally obtained from Herbivac-infected FBT cells transfected with the transfer vector, in the presence of mycophenolic-acid selection medium.
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Characterisation of HIV-1 Envelope features of breakthrough infections from the CAPRISA 004 Microbicide TrialIsmail, Sherazaan Dineo January 2016 (has links)
The CAPRISA 004 trial demonstrated the safety and a 39% efficacy of a 1% tenofovir (TFV) gel for the prevention of HIV-1 acquisition in young African women. It was subsequently shown that women assigned to the TFV arm who became infected had higher viral loads, slower anti-HIV-1 antibody avidity maturation, and higher Gag-specific IFN-γ+ CD4+ T cell responses; although replication capacity, as measured by Gag-Pro recombinant viruses, did not differ between arms. We thus aimed to investigate if there were differences in Envelope function, or TFV susceptibility, which may be selected for during transmission in those who became infected despite being assigned to the TFV arm. Viruses from 39 out of 48 recently HIV-1 infected individuals from the trial (matched on time post-infection and the presence of protective HLAs) were isolated. Isolate env genes were sequenced using a single genome amplification approach and were compared to plasma sequences from the same time-point. To evaluate phenotypic characteristics of env, inhibition assays were performed using the following inhibitors: tenofovir, maraviroc, T20, PSC-RANTES and anti-CD4 antibody clone SK3. In addition, envs for 19 participants were cloned and used to generate pseudoviruses which were evaluated for entry efficiency. Viral isolates were identical or very similar to viruses in circulation in vivo; however had a lower diversity, indicating that they were representative of in vivo virus but did not reflect the entire quasispecies in plasma. The TFV arm viruses were not more resistant to TFV than those in the placebo arm. A comparison of variable loop characteristics, distance to a consensus representative of viruses circulating in the region, and sensitivity to inhibitors or entry efficiencies of the viruses, also found no difference in genotypic nor phenotypic properties between study arms. When assessing the impact of viral phenotype on markers of disease progression, it was found that sensitivity to inhibitors did not contribute to VL or CD4+ count in this cohort. To evaluate envelope in isolation of the rest of the genome, pseudoviruses were generated from 11 participants. We found that PSV entry efficiency did not correlate with VL at isolation, 3 months post-infection and set-point, or with CD4+ counts at set-point. However, pseudovirus inhibitor sensitivities were significantly different to those of isolates for the inhibitors T20, anti-CD4 antibody SK3 and PSC-RANTES. Overall, the isolate env genotypic and phenotypic characteristics investigated in this study did not differ between trial arms. Interestingly, pseudoviruses showed significant differences in their sensitivity to entry inhibitors when compared to their corresponding isolate, highlighting the importance of caution when interpreting data from in vitro studies, and motivates for further evaluation of in vitro models.
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Hepatitis A seroprevalence in South Africa: Are we in epidemiological transition?Enoch, Annabel January 2018 (has links)
Hepatitis A virus (HAV) is the most common cause of viral hepatitis worldwide. Infection with HAV is vaccine preventable, however, a vaccine against HAV is not included in the Expanded Programme on Immunization in South Africa (SA). South Africa was considered to be a high endemic country for hepatitis A in the past, hence there was no need for routine immunization against the virus. Our hypothesis is that SA is changing from high to intermediate endemic setting for hepatitis A. To test our hypothesis, we conducted a cross-sectional seroprevalence study in the 1-7 year age group in the Western Cape Province. Our samples for this study were from specimens, collected between August and October 2015, sent for routine diagnosis to referral hospitals in the Western Cape Province. We tested remaining serum of 482 samples sent for routine tests. A Siemens enzyme immunoassay was used to test for hepatitis A antibodies. We also analysed hepatitis A test results from the National Health Laboratory Services (NHLS) Disa*Lab database at Groote Schuur hospital from 2009-2014, as well as hepatitis A surveillance data from the National Institute for Communicable Diseases (NICD) from 2009-2014, to look at the past hepatitis A prevalence trend. Our cross-sectional study showed the seroprevalence to be 44.1% in the 1-7 year age group. The NHLS data showed a seroprevalence of <90% up to age 10 years, indicating intermediate endemicity. The NICD data showed that a substantial number of symptomatic hepatitis A infections occurred in the 7-40 year age group, suggesting an increasing proportion of a susceptible population to HAV infection. Taken together, these results indicate the need for further studies designed to aid the development of vaccination policies against HAV infection in South Africa.
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Analysis of HIV early infant diagnosis and linkage to care in the Western Cape: a laboratory perspectiveHsiao, Nei-Yuan January 2012 (has links)
Includes abstract. / Includes bibliographical references. / Prevention of mother-to-child transmission (PMTCT) of HIV is the cornerstones of HIV prevention programs. The principle of using antiretrovirals (ARV) to reduce the risk of transmission from mother to child is well established as a range of PMTCT regimens with varying efficacies have been widely studied and reviewed1. In South Africa and other Sub-Saharan countries, single dose Nevirapine, amongst other cost- effective regimens, have been adopted as part of the national HIV prevention program2 since 2003.
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The Influence of HIV-1 Subtype C LTR Genotype on Latency PotentialDoolabh, Deelan Sudhir 18 February 2019 (has links)
The persistence of latent viral reservoirs, that are insensitive to antiretroviral therapy (ART), remains the greatest barrier to HIV-1 eradication. The role that viral factors play in HIV-1 latency establishment and maintenance is poorly understood, and characterisation of these factors is imperative for the development of curative strategies or interventions that could lead to HIV-1 remission in infected individuals. Subtype level genotypic variation within regulatory elements of the HIV-1 promoter, the long terminal repeat (LTR), has been shown to influence latency establishment in in vitro models. We investigated the influence of inter-participant subtype C LTR genotypic variation on the establishment of latency in a dual reporter HIV-1 plasmid model and evaluated potential correlates of this latency potential. Long terminal repeats from 11 ART-naïve, acutely subtype-C infected women in the CAPRISA 004 cohort from Durban, South Africa were cloned into an HIV-1-expressing vector (pRGH) used to generate pseudovirions following HEK293T transfection. Pseudoviruses harboured a gag-eGFP gene under the control of the participant LTR, allowing measurement of active replication, and an mCherry gene under the control of a constitutive CMV promoter allowing measurement of viral integration. Latency potential was expressed as the ratio of mCherry only (latent) to eGFP and mCherry (active replication), as measured by flow cytometry after infection of Jurkat E6-1 and CEM.NKR CCR5+ cell lines before and after T cell activation with PMA/Ionomycin. A panel of LTRs cloned into a pGL4.10 luciferase expression vector were used to measure basal LTR expression and Tat-induced LTR expression. All LTR sequences were classified as subtype C, with an average inter-participant pairwise DNA distance of 7.6%. The median basal LTR activity was approximately two times higher than that of the BaL isolate (interquartile range: 1.38-2.14), and Tatinduced activity approximately nine times higher than that of BaL (interquartile range: 6.16-10.33). We observed consistently greater proportions of latently infected cells than actively infected cells. In Jurkat E6-1 cells, the median latent:active infection ratio was 1.97 (range 0.86-2.83; three experiments). Latency was reversible in a proportion of cells as the median latent:active infection ratio decreased to 0.55 (range 0.46-0.78). The latent:active ratio was unchanged, post-stimulation, in CEM.NKR CCR5+ cells and was therefore found not to be a suitable cell-line for the model. Latency potential did not correlate with basal or Tat-induced activity (Spearman correlation tests, basal p=0.25, r=-0.38, Tat-induced p=0.42, r=-0.27). The DNA distance in characterised functional sites from consensus did not correlate with latency potential (Spearman correlation test p=0.67, r=0.14). Our data suggest that HIV-1 LTRs have intrinsic properties which influence latency potential and the proportion of latently infected cells early post-infection. However, since differences were independent of basal and Tat-induced LTR activity, other factors such as regulatory element interaction and the efficiency of recruitment of molecules responsible for establishing latency, such as histone modifiers, may play a role.
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Investigating cytokine responses to different strains of Mycobacterium tuberculosisBooi, Zandile 31 January 2022 (has links)
Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), is one of the world's most prevalent fatal pathogens, infecting one third of the global population. The highly specialised pathogen that infects humans today evolved from a group of bacteria, known as the Mycobacterium tuberculosis complex (MTBC). It is divided into different lineages, based on phylogeography and genetic structure. It is well established that infection with different strains can lead to different types of innate immune responses. Recent studies indicate differences in the adaptive immune response (IL-22 and IL-17) induced by various Mtb strains, however these differences have not been clearly defined. The aim of this study was to investigate CD4+ T cell adaptive cytokine responses (IFN-γ, IL-22 and IL-17) in blood from human volunteers with prior exposure to Mtb, when stimulated with different strains within the MTBC, using a whole blood assay, flow cytometry and enzyme-linked immunosorbent assay (ELISA). My results indicate that the frequency of IFN-γ and IL-22-producing CD4+ T cells was similar for the two strains, as no significant differences were observed. In contrast, IL-17 responses for HN878 was almost 2-fold higher than H37Rv (p=0.04), consistent with published observations. Moreover, three additional Mtb strains (Indo-oceanic, East African-Indian and the Euro-American outbreak strain CDC1551) were tested for their ability to detect cytokine responses. While all five strains enabled the detection of Mtb-specific CD4+ T cell IFN-γ, IL22 and IL-17, no significant differences were observed when comparing the responses between strains. The highest responses were observed for IFN-γ, followed byIL-22 and IL17. In addition to flow cytometry, soluble cytokine concentrations of IFN-γ and IL-22 were examined in plasma in whole blood stimulated with Mtb lysate derived from H37Rv and HN878, using an ELISA. Responses were tested at both 24 and 48 hours of stimulation, and while median responses were higher at the later time point, they were not significantly different. IFN-γ showed no difference between H37Rv and HN878 at 24 hours in the ELISA. In contrast, HN878 IFN-γ responses were lower than H37Rv at 48 hours (p=0.04). The amount of secreted IL-22 did not differ significantly between the twos trains at either time point. Further work is required to confirm these findings in a larger cohort to understand differences in adaptive responses to different Mtb strains. In conclusion, understanding Mtb strain modulation of host immune function is of major importance, as it may provide better insights into human TB immunity and could assist in the development of an effective TB vaccine.
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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 AfricaBarnabas, Shaun Lawrence 14 July 2022 (has links) (PDF)
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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Evaluation of probiotic and vaginal Lactobacillus species for the treatment of bacterial vaginosis and promotion of vaginal health in South African womenHappel, Anna-Ursula 20 October 2022 (has links) (PDF)
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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The design, development and characterization of a self-replicating DNA expression technologyde Moor, Warren Ralph Josephus 19 October 2022 (has links) (PDF)
High quality T-cell immunogenicity can be an elusive type of immunity to generate and one that is often sought after by virologists, immunologists and cancer researchers alike. When T-cell immunity is generated using current methodologies the quality and magnitude of the immunological response achieved is often weak and unable to create protective immunity. Among current methods, DNA vaccines, generate highly specific T-cell immunity towards targeted antigens, and do not suffer from issues like misdirected vector targeted immunity, like viral based vectors. DNA vaccines, however, face a variety of their own weaknesses. These include, inefficient delivery, high biological loss inside the body, and the inability to counteract or avoid immediate innate cellular defence mechanisms, which limit their ability to persist inside a host cell. For these reasons, DNA vaccines are usually combined with more conventional viral vaccines in what is known as a DNA prime and viral boost regiment strategy. Combining them works well and results in improved immunity towards targeted antigens that is superior to what is obtained when either DNA or recombinant vaccines are used alone. To address many of the core issues faced by DNA vaccines, I report here on the design, development and characterization of a self-replication DNA gene expression technology. This novel DNA expression system employs a form of DNA replication (known as rolling circle replication) to generate a self-replicating DNA amplicon that can amplify its own copy number and the relative localised levels of antigen expression inside transfected mammalian cells in tissue culture and within Balb/cJ mice. These capabilities help effectively mitigate many of the core issues faced by DNA vaccines. The technology developed was shown to significantly increase gene expression for eGFP and Luciferase reporter genes, with an overall average increase in expression of approximately two-fold by 48 h post transfection in HeLa S3 cells. More specifically, an increase of at least two-fold in the absolute maximum level of the gene of interest per cell was also observed. Such localised doubling in antigen expression, at the cellular level, is believed to enhance innate immune activation and improve the overall immune response. Experimental results indicated that gene expression levels by this technology is non static in nature and appears to increase in magnitude within affected cells over time as was hypothesised. This provided strong evidence that the replication technology appears to be functioning as was expected and was able to demonstrate the ability to elevate antigen expression over time, potentially starting from extremely low and otherwise ineffective starting concentrations. This ability has potential to effectively mitigate many of the issues associated DNA vaccines such as low and ineffective delivery. This capability was observed in tissue culture as a steady increase in reporter gene expression levels across the entire range of DNA transfection levels. Furthermore, the increases in gene expression were observed to continue to amplify over time, eliminating the presence of weakly fluorescing cells in tissue culture. By 11 days post transfection, every observable cell transfected with the replication expression system, was observed to have extremely high levels of fluorescence. With recorded fluorescence levels being as bright or brighter than the highest levels obtained under normal transfections with no replicative plasmids (~48-72 h). Unique cellular responses to the presence of the replicating gene expression technology were also observed. These included an apparent slowdown in cellular metabolic activity and growth among cells transfected with replicating vectors. This was observed as a decrease in cellular division and total cell number by ~50%, by 48 h post transfection. This was accompanied by significant increases in cell size, internal cellular granularity, and gene-of-interest expression per cell. These changes were observed among all cells regardless of their relative DNA transfection level. This was demonstrated by assessment of the change in the range, mean, median, skewness and standard deviation of the cellular distribution curves for eGFP expression, cell size and internal cellular granularity. These observations provided further evidence of the dynamically changing and active nature of this technology. This also provided evidence that the replicating gene expression technology has a definitively different kind of cellular impact and effect on transfected cells compared to non-replicating DNA expression systems. Pilot studies to test the technology in Balb/cJ mice indicated, the technology appears to be functional within this animal model and was able to increase gene of interest (eGFP) expression levels compared to an equivalent non-replicating DNA expression vector control. Furthermore, these animal experiments also demonstrated significant increases in the maximum possible level of expression achieved within localised ‘hot spots' of muscle fibre bundles. This effect appeared to increase following transient addition of additional replication associated protein (Rep), giving further evidence this technology appears to be functional within the Balb/cJ animal model. Suggesting that the rate at which the replication amplification process occurs, may also be manipulated by adjusting Rep concentration. Finally, an antiviral response gene array was run to look for evidence that the replicating gene expression technology could increases antiviral response gene activation, to possibly improve T-cell activation and immunity. The array provided evidence improved antiviral response gene activation was occurring however the data was inconclusive in nature and further investigation is needed to verify these preliminary findings. The array also showed significant evidence of Rep induced Caspase 10 (CASP10), gene suppression. This suggests that Rep may play a role in the survival and virulence ofBFDV by acting as a suppressor of cellular apoptosis in a concentration-dependant manner and is worth investigating further.
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Development and Characterization of a Modified Vaccinia Ankara Vaccine Candidate Expressing the SARS-CoV-2 Spike GlycoproteinKhumalo, Fezokuhle Ncedile 15 March 2023 (has links) (PDF)
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2, SARS-CoV-2. Given the ongoing COVID-19 pandemic and the continued evolution of the virus to escape host immunity, new vaccines and refinement of first-generation vaccines to improve protection against SARS-CoV-2 variants of concern is vital. In Africa, the cost of vaccine manufacturing as well as the scarcity in resources for storage and distribution have all contributed to the inequitable access to vaccines and heavy reliance on donations. Modified Vaccinia Virus Ankara (MVA) is a low-cost production vector platform which is suitable in this context. This project falls into a bigger study where our group compared different vector platforms, including MVA. The project serves as a proof of concept that this platform can be used to produce vaccines encompassing different variants of SARS-CoV-2 as they emerge. The most recent variant, Omicron, has proven to be highly immune evasive and demonstrates this need well. As the virus mutates, the variants of concern each present with differing characteristics and subsequently differ in immunogenicity and pathogenicity. Sub-Saharan Africa has been ransacked by the pandemic, resulting in loss of lives and livelihoods; the effects of which will undoubtedly be felt for decades to come. This study had two aims: 1. The development of a candidate vaccine, MVA-SARS-CoV2-S∆TM by using the widely used MVA platform and poxvirus recombinant vaccine strategies used in our research group, 2. The testing of this vaccine's immunogenicity in mice. The MVA-based vaccine was constructed by infection of BHK21 cells with wildtype MVA, and transfection with transfer vector pMVA-FNK2. The transfer vector contains a truncated form of the SARS-CoV-2 spike glycoprotein gene, the vaccinia virus host-range gene K1L and reporter gene eGFP, flanked by gene sequences to allow homologous recombination into MVA. The presence of the K1L gene in the recombinant virus allowed for selection by passaging in RK13 cells, which were not permissive for the parent MVA. Following the potentially successful isolation, the recombinant MVA-SARS-CoV2-S∆TM was validated and characterized by PCR, Sanger sequencing, Western blot analysis and immunofluorescence, which all confirmed the presence and expression of the spike protein. Large scale propagation of the vaccine was done in RK13 cells, and the stock was titrated to yield a titre of 3.9 x 106 ffu/ml. Balb/c mice were inoculated three times with MVA-SARS-CoV2-S∆TM at a dose of 3 x 105 to assess immunogenicity of the vaccine. Results from the immunogenicity assessments demonstrated that the vaccine induced T-cell responses shown by an enzyme-linked immunosorbent spot (ELISpot) assay. An enzyme-linked immunosorbent assay (ELISA) confirmed the ability of MVA-SARSCoV2-S∆TM to induce increasing titres of binding antibodies in mice over a period of 56 days. However, the vaccine did not induce neutralizing antibodies against a matched SARS-CoV-2 pseudovirus, highlighting the need for further refinement of the vaccine. In conclusion, recombinant MVA expressing a truncated SARS-CoV-2 spike protein was successfully constructed and tested for immunogenicity in mice. The candidate vaccine induced good T-cell responses and binding antibodies, but not neutralizing antibodies. This study provides additional evidence that MVA can be used as a platform for a SARS-CoV-2 vaccine, as has been previously demonstrated by others, and this could potentially be adapted for emerging variants of concern. This work also allows for the direct comparison of the MVA platform to other platforms employed by our group (DNA and plant-based subunit) as SARS-CoV-2 vaccines.
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