Browsing by Author "Mann, Jaclyn Kelly."

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Functional and clinical consequences of immune-driven sequence variation of gag-protease in HIV-1 subtypes A, C, D and recombinants.
(2016) Kiguoya, Marion Wangui.; Ndung'u, Peter Thumbi.; Mann, Jaclyn Kelly.
Abstract available in PDF file.
Gag-protease driven viral replication capacity among HIV-1 subtypes: Implications for disease progression, epidemic spread and vaccine design.
(2020) Farinre, Omotayo Oluwaremilekun.; Ndung'u, Peter Thumbi.; Mann, Jaclyn Kelly.
The HIV-1 epidemic in sub-Saharan Africa is heterogeneous with diverse unevenly distributed subtypes and regional differences in prevalence. Subtype-specific differences in disease progression rate and transmission efficiency have been reported, but the underlying biological mechanisms have not been fully characterized. In this study, I tested the hypothesis that the subtypes prevalent in the East African epidemic, where adult prevalence rate is higher, have lower viral replication capacity (VRC) than their West African counterparts where adult prevalence rates are lower. Materials and methods: Gag-protease sequencing was performed on plasma samples from 213 and 160 antiretroviralnaïve participants from West and East Africa, respectively. Online bioinformatic tools were used to infer HIV-1 subtypes and recombination patterns. Replication capacities of patientderived gag-protease chimeric viruses from West (n=178) and East (n=114) Africa were determined using a green fluorescent protein reporter-based cell assay. Subtype and regional differences in viral replication capacity and amino acid variants impacting replication capacity were identified using appropriate statistical methods. Results: Subtypes identified in West Africa were CRF02_AG (65%, n=139), G (7%, n=15), A3 (5%, n=10), other CRFs (12%, n=26), various pure subtypes (9%, n=19) and A1G recombinants (2%, n=4). Subtypes A1 (64%, n=103), D (22%, n=35), AD (11%, n=17) and AC (3%, n=5) were identified in East Africa. Chimeric viruses from West Africa had significantly higher VRC compared to those from East Africa (p < 0.0001), with subtype-specific differences found among strains within West and East Africa (p < 0.0001). Recombination patterns showed a preference for subtypes D, G or J rather than subtype A in the p6 region of gag, with evidence that subtype-specific differences in this region impact viral replication capacity. Furthermore, the Gag A83V polymorphism was associated with reduced viral replication capacity in CRF02_AG (median < 0.86). HLA-A*23:01 (p = 0.0014) and HLA-C*07:01 (p = 0.002) were associated with significantly lower viral replication capacity in subtype A infected individuals from East Africa. Conclusion: Overall, the data showed that viruses from West Africa displayed higher replication capacity than those from East Africa, which is consistent with the hypothesis that lower viral replication capacity is associated with higher population prevalence.
In vitro testing of the predicted viral fitness landscape for the HIV-1 Nef protein.
(2015) Rajkoomar, Erasha.; Mann, Jaclyn Kelly.; Ndung'u, Peter Thumbi.
Abstract available in PDF file.
Intersubtype differences in the effect of a rare p24 Gag mutation on HIV-1 replicative fitness.
(American Society for Microbiology., 2012) Chopera, Denis Rutendo.; Cotton, Laura A.; Zawaira, Alexander.; Mann, Jaclyn Kelly.; Ngandu, Nobubelo K.; Ntale, Roman.; Carlson, Jonathan M.; Mlisana, Koleka Patience.; Woodman, Zenda.; de Assis Rosa, Debra.; Martin, Eric.; Miura, Toshiyuki.; Pereyra, Florencia.; Walker, Bruce D.; Gray, Clive M.; Martin, Darren Patrick.; Ndung'u, Peter Thumbi.; Brockman, Mark A.; Abdool Karim, Salim Safurdeen.; Brumme, Zabrina L.; Williamson, Carolyn.
Certain immune-driven mutations in HIV-1, such as those arising in p24Gag, decrease viral replicative capacity. However, the intersubtype differences in the replicative consequences of such mutations have not been explored. In HIV-1 subtype B, the p24Gag M250I mutation is a rare variant (0.6%) that is enriched among elite controllers (7.2%) (P 0.0005) and appears to be a rare escape variant selected by HLA-B58 supertype alleles (P<0.01). In contrast, in subtype C, it is a relatively common minor polymorphic variant (10 to 15%) whose appearance is not associated with a particular HLA allele. Using site-directed mutant viruses, we demonstrate that M250I reduces in vitro viral replicative capacity in both subtype B and subtype C sequences. However, whereas in subtype C downstream compensatory mutations at p24Gag codons 252 and 260 reduce the adverse effects of M250I, fitness costs in subtype B appear difficult to restore. Indeed, patient-derived subtype B sequences harboring M250I exhibited in vitro replicative defects, while those from subtype C did not. The structural implications of M250I were predicted by protein modeling to be greater in subtype B versus C, providing a potential explanation for its lower frequency and enhanced replicative defects in subtype B. In addition to accounting for genetic differences between HIV-1 subtypes, the design of cytotoxic-T-lymphocyte-based vaccines may need to account for differential effects of host-driven viral evolution on viral fitness.
Nef-mediated down-regulation of CD4 and HLA class I in HIV-1 subtype C infection: association with disease progression and influence of immune pressure.
(Elsevier., 2014) Mann, Jaclyn Kelly.; Chopera, Denis Rutendo.; Omarjee, Saleha.; Kuang, Xiaomei T.; Le, Anh Q.; Anmole, Gursev.; Danroth, Ryan.; Mwimanzi, Philip.; Reddy, Tarylee.; Carlson, Jonathan M.; Radebe, Mopo.; Goulder, Philip Jeremy Renshaw.; Walker, Bruce D.; Abdool Karim, Salim Safurdeen.; Novitsky, Vladimir.; Williamson, Carolyn.; Brockman, Mark A.; Brumme, Zabrina L.; Ndung'u, Peter Thumbi.
Abstract available in pdf.
No evidence for selection of HIV-1 with enhanced gag-protease or nef function among breakthrough infections in the CAPRISA 004 tenofovir microbicide trial.
(2013) Chopera, Denis Rutendo.; Mann, Jaclyn Kelly.; Mwimanzi, Philip.; Omarjee, Saleha.; Kuang, Xiaomei T.; Ndabambi, Nonkululeko.; Goodier, Sarah A.; Martin, Eric.; Naranbhai, Vivek.; Abdool Karim, Salim Safurdeen.; Abdool Karim, Quarraisha.; Brumme, Zabrina L.; Ndung'u, Peter Thumbi.; Williamson, Carolyn.; Brockman, Mark A.
Background: Use of antiretroviral-based microbicides for HIV-1 prophylaxis could introduce a transmission barrier that inadvertently facilitates the selection of fitter viral variants among incident infections. To investigate this, we assessed the in vitro function of gag-protease and nef sequences from participants who acquired HIV-1 during the CAPRISA 004 1% tenofovir microbicide gel trial. Methods and Results: We isolated the earliest available gag-protease and nef gene sequences from 83 individuals and examined their in vitro function using recombinant viral replication capacity assays and surface protein down regulation assays, respectively. No major phylogenetic clustering and no significant differences in gag-protease or nef function were observed in participants who received tenofovir gel versus placebo gel prophylaxis. Conclusion: Results indicate that the partial protective effects of 1% tenofovir gel use in the CAPRISA 004 trial were not offset by selection of transmitted/early HIV-1 variants with enhanced Gag-Protease or Nef fitness.
Prevention of HIV-1 acquisition and determinants of disease progression.
(2021) Moyano De Las Muelas, Ana.; Mann, Jaclyn Kelly.; Sigal, Alexander.; Ndung'u, Peter Thumbi.
Introduction HIV-1 infection can be managed using multiple strategies, including preventative approaches and therapeutic approaches. Current preventative and treatment strategies are suboptimal and there is a need to develop an effective prophylactic or therapeutic vaccine and to improve the public health approaches against the virus. This requires more detailed understanding of the infection, from prevention to natural disease progression. We performed several studies that cover a range of infection attributes, from understanding the mechanism of action of pre-exposure prophylaxis (PrEP) and determining the effectiveness of different compounds in blocking initial infection, to gaining further insight into potential mechanisms of natural control of HIV-1 disease progression in viraemic controllers (VC) with (VC+) and without (VC-) protective class I human leukocyte antigen (HLA-I) alleles. In order to cover this range of infection attributes we investigated two hypotheses: (i) initial low dose infection can be cleared with suboptimal drug inhibition, which allows ongoing viral replication, as long as the drug mechanism acts before the first cell is infected; and (ii) individuals without protective HLA-I alleles have CD8+ T cell-independent mechanisms of control. Methods To understand the mechanism of action of PrEP, the probability of extinction of new infections in the presence of two drug mechanisms (interference of initial infection with tenofovir (TFV), or reduction of burst size with atazanavir (ATV)), or with no drug, was modelled as a function of initial infected cells and viral replication ratio. The fraction of extinguished infections was experimentally determined with low viral input in the presence of either drug, or with no drug, in an in vitro model of PrEP. To gain insight into potential mechanisms of control, we studied immune cells in 12 VC+ and 9 VC- and, compared these 21 controllers with 5 rapid progressors (RP). Measurements included the magnitude and breadth of CTL responses using the ELISpot assay, as well as flow cytometry-based characterization of NK cell and T cell populations, which included the measurement of surface markers for activation, maturation, and exhaustion on these populations. Further, NK cell function was measured by intracellular cytokine staining following stimulation of these cells. Results Our study showed that TFV dramatically increased clearance while ATV did not, both for our mathematical model and our experimental study. We observed that both VC, in particular VC-, had a higher contribution of Gag CTL responses to the total CTL response than RP (p=0.04), however there was no significant difference in the magnitude and breadth of CTL responses between VC+ and VC-. In addition, VC- NK cells had higher levels of the activation markers HLA-DR (p=0.007) and co-expression of CD38 and HLA-DR (p=0.03) when compared to VC+ and uninfected individuals (UI), and lower cytokine expression (MIP-1β and TNF-α) than VC+ NK cells (p=0.05 and p=0.04, respectively). We found a negative correlation between the expression of MIP-1β and the co-expression of CD38 and HLA-DR (r =-0.45, p=0.05). Furthermore, VC- T cells had higher levels of CD38 and HLA-DR co-expression (p=0.05), and a trend of higher HLA-DR (p=0.07) as well as CD57 expression (p=0.09) when compared to VC+. Conclusions The ability of drugs to clear initial but not established infection depends only on the ability to target initial infection. This implies that in diseases which involve transmission of low pathogen numbers upon exposure, but have robust replication when established, such as HIV-1, a possibility to clear infection should exist even with relatively weak inhibition as long as the drug has the mechanism of targeting the initial infection. This finding is particularly relevant in scenarios of variable adherence that result in sub-optimal drug levels or possible future PrEP strategies with drugs that have long half-lives yet do not completely suppress viral replication. VC have a more Gag focused CTL response than RP, however this feature did not distinguish VC+ from VC-. NK and T cell profiles differ between VC+ and VC-. VC- have a more activated NK cell profile with lower cytokine expression, and a more active and terminally differentiated T cell profile than VC+. A possible explanation for our results is that the increased CD38+HLA-DR+ NK cells in VC- may represent NK cells acting as antigen presenting cells (APCs), which may then directly interact with a more activated and terminally differentiated population of T cells observed in VC-. Further work to test this hypothesis is necessary to better understand the mechanisms underlying control in these two groups of VC patients. It is also suggested that transcriptomic studies may contribute further to understanding the distinct NK and T cell profiles observed between VC+ and VC- and how these may result in differing mechanisms of control.
The role of Nef-mediated SERINC5 down-regulation on HIV-1 disease progression.
(2021) Naicker, Marshlin Delon.; Mann, Jaclyn Kelly.
HIV-1 Nef is a small accessory protein that plays a vital role in enhancing HIV-1 pathogenesis, evidenced by a strongly attenuated disease course following infection with a virus with gross Nef defects. Nef has multiple cellular effects, which enhance HIV-1 replication and immune evasion. Major activities of Nef include CD4 down-regulation, HLA-I down-regulation, and CD4-independent enhancement of virion infectivity. Recent studies have uncovered Nefmediated down-regulation of the host restriction factor SERINC5 as an important mechanism by which Nef enhances virion infectivity. However, there is a lack of studies defining the role of this function in HIV-1 pathogenesis. Previous studies indicated that Nef-mediated CD4 down-regulation and enhancement of infectivity are likely the major contributors to Nef’s effect of enhancing pathogenicity; the relative significance of each Nef function for HIV-1 disease progression remains incompletely understood. Given the key role of Nef-mediated SERINC5 down-regulation in enhancing virion infectivity, the primary aim of the present study was to determine if this Nef activity contributes significantly to disease progression in individuals infected with HIV-1 subtype C, which is the dominant HIV-1 subtype worldwide. To investigate this, SERINC5 down-regulation activity of 106 Nef clones derived from patients with early HIV-1 subtype C infection were evaluated in a CD4+ T cell line using a flow cytometry-based assay and subsequently related to viral load set point and to the rate of CD4+ T cell decline using linear regression analysis. The second aim of this study was to assess the overall contribution of SERINC5 down-regulation to Nef function, using linear regression analysis with E values as a proxy for overall Nef function in vivo. The third aim of the study was to identify amino acid variants that significantly alter Nefmediated SERINC5 down-regulation using a codon-by-codon sequence-function analysis tool available online. No significant relationship was found between each Nef function and viral set point (SERINC5 down-regulation, p=0.28) or rate of CD4+ T cell decline (SERINC5 down-regulation, p=0.48). CD4 down-regulation (p=0.02) and SERINC5 down-regulation (p=0.003) were significant determinants of the E value in univariate analyses, and SERINC5 down-regulation remained significant in the multivariate analysis (p=0.003). We found several amino acids that were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. In conclusion, none of the Nef functions in our study, including SERINC5 down-regulation, were found to be significant individual contributors to disease progression. However, interestingly we found CD4 down-regulation and SERINC5 down-regulation to be the largest contributors, of the Nef functions considered here, to overall Nef function and that the contribution of SERINC5 down-regulation was the most significant. Taken together, this could be explained by multiple Nef functions acting together to facilitate the enhancement of viral spread and immune evasion in vivo that ultimately enhance disease progression. We found several amino acid variants that either increased or decreased Nef’s ability to down-regulate SERINC5; however, further studies in the form of site-directed mutagenesis are warranted to further understand their effect on SERINC5 down-regulation activity. In summary, the results suggest that SERINC5 down-regulation is a strong contributor to overall Nef function and identifies potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.
Roles of single nucleotide polymorphisms in the promoter regions of tumour necrosis factor-α and interleukine-1o genes in Schistosoma haematobium infection susceptibility.
(2020) Marume, Amos.; Mduluza, Takafira.; Mann, Jaclyn Kelly.
Background: Schistosomiasis remains a public health threat in sub-Saharan Africa which carries 85% of the global burden. With effective vaccines a distant future away, and no one allround intervention, research is still required to ensure that only effective programmes are introduced and implemented as well as evaluated and/or monitored. It is therefore key for researchers, policy makers and implementers to understand the epidemiology, immunology, immunopathology, immunogenetics, chemotherapy, management and control of Schistosoma haematobium for optimal elimination strategies to be implemented. A research study was therefore instituted to determine the prevalence, risk factors and host immunogenetic factors in S. haematobium infections among pre- and school going children living in endemic regions of Manicaland and Mashonaland central provinces in rural Zimbabwe. The relationship between single nucleotide polymorphisms (SNPs) of the promoter regions of tumor necrosis factor alpha (TNF-α or rs1800629) and interleukin- 10 (IL-10 or rs1800871) and susceptibility to Schistosoma haematobium was investigated. In addition, the relationship between these SNPs and cytokine levels, as well as the relationship between actual cytokine levels and susceptibility to Schistosoma haematobium was assessed. Methods: In this cross-sectional immune-epidemiological study Schistosoma haematobium was diagnosed by the microscopic examination of urine specimens for the presence of parasite eggs using the urine filtration technique. DNA for the genotyping was extracted from approximately 300μl whole blood using the QiagenFlexiGene DNA extraction kit, following the manufacturer’s protocol. IL-10 and TNF-α promoter region single nucleotide polymorphisms were genotyped using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR).The allele frequencies and genotype distribution of S. haematobium infected and uninfected participants were then analysed using the chi-square test. All analyses were performed using SPSS (version 21) and p-values <0.05 were considered statistically significant. The levels of the cytokines (IL-10 and TNF-α) were measured by indirect enzyme linked immunosorbent assay (ELISA) using MABTECH, 3510-1H-6 kits, according to the manufacturer’s instruction. Results: The overall prevalence of S. haematobium among children in endemic rural and farming communities of the two provinces of Zimbabwe assessed was 17.1% (158/924). Gender specific prevalences were similar (17.5% in girls and 16.7% in boys; p = 0.735). Age and location were significant risk factors for schistosomiasis in children living in endemic regions surveyed. The older the child the higher the risk of getting infected by S. haematobium xvii (10.5% in 0-5 year olds; 24.0% in 6-10 year olds and 30.7% in 11-15 year olds; p < 0.001). IL-10 -1082 G/A, IL-10 -819 C/T and TNF-α -308 G/A single nucleotide polymorphisms were not significantly associated with susceptibility to S. haematobium infection. TNF- α genotypes AA, GA and GG were associated with high, moderate to high and low production of TNF-α respectively. IL-10 TT, CT and CC genotypes were associated with low, moderate and high IL-10 plasma levels respectively. Higher TNF-α and lower IL-10 serum levels were negatively associated with schistosomiasis infection. Praziquantel treatment reduced prevalence among the study participants as reinfections were only recorded in 6 of the 59 (10.2%) who were infected at baseline of children. Discussion and Conclusion: The determined schistosomiasis prevalence puts the regions of Zimbabwe studied within the moderate range as described by the World Health Organisation (10 – 49% prevalence), hence more concerted efforts are required to fight schistosomiasis. Although cytokine genotypes were associated as expected with cytokine levels, genotypes did not directly correlate with schistosomiasis infection while cytokine levels did. This indicates that circulating TNF-α and IL-10 levels are a result of many factors apart from genotypes. Taken together with previous work, this study suggests that high TNF-α and low IL-10 serum levels confer protection against schistosomiasis infection. Since schistosomiasis prevalence was similar between boys and girls and prevalence was high in all age groups (increasing with age), all programmes aimed at eliminating schistosomiasis should include both genders and children of all age groups. Specific locations could be targeted in resource limited settings as location was a significant risk factor for infection. Praziquantel is effective, with few reinfections observed, and therefore remains central in schistosomiasis management. To clearly understand the role host genetic factors in infection and to inform effective control, elimination and eradication programmes, more research on risk factors and host immunogenetics is necessary