Replication fidelity in the mircroevolution of mycobacteria

This thesis aimed to elucidate the structure-function relationships determining the differential fidelities of the dnaE1- and dnaE2-encoded mycobacterial PolIIIα subunits under conditions of genotoxic stress. To this end, the role in DnaE1 intrinsic fidelity of highly conserved PHP domain residues was explored by site-directed replacement of targeted amino acids, resulting in a panel of Mycobacterium smegmatis mutants carrying selected dnaE1 alleles. A complementary approach investigated the contribution of the mycobacterial proofreading DnaQ subunit homolog to the maintenance of DnaE1-dependent replicative fidelity by generating a targeted dnaQ knockout mutant. The third component of this study focused on the inferred role of a highly-conserved N-terminal extension and C-terminal pentapeptide motif in the function of the alternative, error-prone DNA PolIIIα subunit, DnaE2

Effect of natural colonization by Streptococcus pneumoniae on the systemic immune responses to common pneumococcal protein antigens with immune protective potential

MSc., Faculty of Science, University of the Witwatersrand, 2011Background: Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), surface proteins of Streptococcus pneumoniae are being investigated for their role as potential vaccine candidates. There are limited data on natural antibody kinetics against pneumococcal surface proteins arising through exposure to pneumococcal nasopharyngeal (NP) colonization in African populations. Objectives: To characterize the natural antibody kinetics and sero-prevalence to 15 pneumococcal proteins with respect to age, PCV vaccination and HIV status as well as to explore the association between antibody titers and pneumococcal nasopharyngeal colonization in infants, older children and adults. Methods: We established a 15-plex Luminex assay for the following proteins: PspA, PspC, LytB, IgA1-proteinase, SP 0082, PdB, PcsB, PsaA, SP 0609, SP 0749, PpmA, SlrA, StkP, SP 2027 and SP 2194, and also validated the Luminex assay comparing it to a standard ELISA method for PspA, PspC, PsaA and PdB. We used the Luminex method to characterize the prevalence and dynamics of serum IgG antibodies against the pneumococcal proteins. The study involved 2 166 human subjects which included: i. A longitudinal cohort of children less than 2 years of age, who were vaccinated with the seven-valent pneumococcal conjugate vaccine (PCV-7) and were either a) HIV-exposed infected, b) HIV-exposed uninfected or c) HIV-unexposed uninfected. ii. A longitudinal cohort of PCV-7 unvaccinated children less than 2 years of age who were either: a) HIV-unexposed uninfected or b) HIV-exposed uninfected. The PCV-7 vaccinated and unvaccinated children were followed up from approximately 4 to 24 months of age. In addition, samples were also analyzed from HIV-uninfected and HIV-infected children Project ID: Pneumococcal protein antigens Student: Zanele Ditse Date: 04 October 2011 - 5 - aged between 4 to 7 years who received either a primary series of PCV-9 or placebo during infancy. Lastly, we analyzed cross-sectional samples from HIV-uninfected and HIV-infected women. Results: The multiplex Luminex assay correlated well with singleplex ELISAs for all four analyzed proteins with correlation coefficients of 0.86, 0.90, 0.87 and 0.96 for PspA, PspC, PdB and PsaA respectively. Antibody titers to PspC, PdB, LytB, SP 0082, PcsB and StkP showed increases in titer with respect to increasing age. Prevailing nasopharyngeal pneumococcal colonization in young children was associated with higher antibody titers to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB and StkP. Conversely higher antibody titers to PspC, PdB, LytB, SP 0082, PcsB and StkP were associated with lower prevalence of pneumococcal colonization in older children and adults. In children under two years of age, PCV vaccination was associated with lower antibody titers to PspA, PspC, LytB, PdB, IgA1-proteinase, PcsB and StkP as well as higher antibody titers against SP 0082 and PpmA at multiple time-points. In PCV-vaccinated children under two years of age, those who were HIV-unexposed , -uninfected had higher antibody titers to PspA, PspC, SP 0082, IgA1-proteinase, PpmA and StkP compared to HIV-exposed, uninfected children. Conclusion: There was an age-related increase in antibody titers to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB, and StkP in children under two years of age. PCV immunization was, however, associated with lower antibody titers to PspA, PspC, LytB, PdB, IgA1-proteinase, PcsB and StkP in young children which was not attributed to differences in the prevalence of nasopharyngeal colonization. Furthermore, HIV-infection status in young children was associated with higher antibody responses to PspA, PspC, PdB, SP 0082, LytB, IgA1-proteinase, PpmA, PcsB and StkP proteins in HIV-unexposed uninfected children compared to HIV-exposed uninfected and HIV-exposed infected children. Higher antibody concentrations to Project ID: Pneumococcal protein antigens Student: Zanele Ditse Date: 04 October 2011 - 6 - PspC, PdB, LytB, SP 0082, PcsB and StkP was negatively associated with nasopharyngeal pneumococcal colonization in older children and adults; indicating a protective role against colonization and a potential role as vaccine candidates

HIV-1 Subtype C-Infected Children with Exceptional Neutralization Breadth Exhibit Polyclonal Responses Targeting Known Epitopes

We have previously shown that HIV-1-infected children develop broader and more potent neutralizing antibody responses than adults. This study aimed to determine the antibody specificities in 16 HIV-1 subtype C-infected children who displayed exceptional neutralization breadth on a 22-multisubtype virus panel. All children were antiretroviral treatment (ART) naive with normal CD4 counts despite being infected for a median of 10.1 years with high viral loads. The specificity of broadly neutralizing antibodies (bNAbs) was determined using epitope-ablating mutants, chimeric constructs, and depletion or inhibition of activity with peptides and glycoproteins. We found that bNAbs in children largely targeted previously defined epitopes, including the V2-glycan, V3-glycan, CD4bs, and gp120-gp41 interface. Remarkably, 63% of children had antibodies targeting 2 or 3 and, in one case, 4 of these bNAb epitopes. Longitudinal analysis of plasma from a mother-child pair over 9 years showed that while they both had similar neutralization profiles, the antibody specificities differed. The mother developed antibodies targeting the V2-glycan and CD4bs, whereas bNAb specificities in the child could not be mapped until 6 years, when a minor V2-glycan response appeared. The child also developed high-titer membrane-proximal external region (MPER) binding antibodies not seen in the mother, although these were not a major bNAb specificity. Overall, exceptional neutralization breadth in this group of children may be the result of extended exposure to high antigenic load in the context of an intact immune system, which allowed for the activation of multiple B cell lineages and the generation of polyclonal responses targeting several bNAb epitopes. IMPORTANCE An HIV vaccine is likely to require bNAbs, which have been shown to prevent HIV acquisition in nonhuman primates. Recent evidence suggests that HIV-infected children are inherently better at generating bNAbs than adults. Here, we show that exceptional neutralization breadth in a group of viremic HIV-1 subtype C-infected children was due to the presence of polyclonal bNAb responses. These bNAbs targeted multiple epitopes on the HIV envelope glycoprotein previously defined in adult infection, suggesting that the immature immune system recognizes HIV antigens similarly. Since elicitation of a polyclonal bNAb response is the basis of next-generation HIV envelope vaccines, further studies of how bNAb lineages are stimulated in children is warranted. Furthermore, our findings suggest that children may respond particularly well to vaccines designed to elicit antibodies to multiple bNAb epitopes

Bridging the skills gap in the financial industry : uncovering the skills that banks require in the future world of work

Automation and digital transformation are replacing tasks that are performed by humans, therefore changing the skills that organisations are looking for to cope with the changing environment. The banking industry has also been undergoing this unprecedented change and to meet the challenges that come with the technological advances, they need to improve their skills. This can be realised through education. Qualitative, exploratory research methods were adopted. Twenty semi-structured, in-depth interviews were conducted with executives and graduates employed by the South African banking industry. The findings demonstrated that poverty, inequality, poor access to quality education, a lack of soft skills and practical experience, poor curriculum alignment with industry needs as well as how the skills are taught contribute to the skills gap in the South African banking industry. Programming, machine learning, robotics, coding, analytical and quantitative skills, collaboration, communication, problem-solving, social intelligence and agility were identified as critical hard and soft skills of the future. Experiential learning or learning through simulations were identified as some of the teaching methods that could enhance and equip graduates with practical experience and relevant skillsets. Recommendations on the role that industry and institutions of higher learning should play to bridge the skills gap included collaboration between both parties to ensure alignment in academic curriculum with industry needs, having industry experts in academia, vocational training and internal training provided by employers to upskill or reskill their current workforce. These findings suggest that in order to address the issue of skills gap in South Africa, engagement and collaboration between industry, academia and government will be required. The findings validate the human capital theory which highlights the importance of knowledge in skills development and the contribution of knowledge to the productivity and economic development of the country.Mini Dissertation (MBA)--University of Pretoria, 2020.pt2021Gordon Institute of Business Science (GIBS)MBAUnrestricte

Reduced neutralization of SARS-CoV-2 B.1.617 by vaccine and convalescent serum

SARS-CoV-2 has undergone progressive change with variants conferring advantage rapidly becoming dominant lineages e.g. B.1.617. With apparent increased transmissibility variant B.1.617.2 has contributed to the current wave of infection ravaging the Indian subcontinent and has been designated a variant of concern in the UK. Here we study the ability of monoclonal antibodies, convalescent and vaccine sera to neutralize B.1.617.1 and B.1.617.2 and complement this with structural analyses of Fab/RBD complexes and map the antigenic space of current variants. Neutralization of both viruses is reduced when compared with ancestral Wuhan related strains but there is no evidence of widespread antibody escape as seen with B.1.351. However, B.1.351 and P.1 sera showed markedly more reduction in neutralization of B.1.617.2 suggesting that individuals previously infected by these variants may be more susceptible to reinfection by B.1.617.2. This observation provides important new insight for immunisation policy with future variant vaccines in non-immune populations

Omicron-B.1.1.529 leads to widespread escape from neutralizing antibody responses.

On the 24 th November 2021 the sequence of a new SARS CoV-2 viral isolate spreading rapidly in Southern Africa was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses