Association of genetic polymorphisms in select HIV-1 replication cofactors with susceptibility to HIV-1 infection and disease progression.

Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.Objective.Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1) infection and disease progression. This heterogeneity is attributed to the interplay between the environment, viral diversity, immune response and host genetics. This study focused on host genetics. We studied the association of single nucleotide polymorphisms (SNPs) in peptidyl prolyl isomerase A (PPIA), transportin 3 (TNPO3) and PC4 or SFRS1 interacting protein 1 (PSIP1) genes with HIV-1 infection and disease progression. These genes code for Cyclophilin A (CypA), Transportin-SR2 (TRN-SR2) and Lens epithelium derived growth factor/p75 (LEDGF/p75) proteins respectively, which are all validated HIV replication cofactors in vitro. Methods. One SNP A1650G in the PPIA gene was genotyped in 168 HIV-1 negative and 47 acutely infected individuals using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 6 intronic and 2 exonic haplotype tagging (ht) SNPs (rs13242262; rs2305325; rs11768572; rs1154330; rs35060568; rs8043; rs6957529; rs10229001) in the TNPO3 gene, 4 intronic ht SNPs (rs2277191, rs1033056, rs12339417 and rs10283923) and 1 exonic SNP (rs61744944, Q472L) in the PSIP1 gene were genotyped in 195 HIV-1 negative and 52 acutely infected individuals using TaqMan assays. The rs1154330, rs2277191, rs12339417 and rs61744944 were further genotyped in 403 chronically infected individuals. CypA and LEDGF/p75 messenger RNA (mRNA) expression levels in peripheral blood mononuclear cells (PBMCs) were quantified by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The impact of the Q472L mutation on the interaction of LEDGF/p75 with HIV-1 integrase (IN) was measured by AlphaScreen. Results. The minor allele (G) of SNP A1650G (1650G) in the promoter region of PPIA was significantly associated with higher viral load (p<0.01), lower CD4+ T cell counts (p<0.01) and showed a possible association with rapid CD4+ T cell decline (p=0.05). The 1650G was further associated with higher CypA expression post HIV-1 infection. The minor allele (G) of rs1154330 in the intron region of TNPO3 was associated with faster HIV-1 acquisition (p<0.01), lower CD4+ T cell counts, higher viral load during primary infection (p<0.05) and rapid CD4+ T cells decline (p<0.01). The minor allele (A) of rs2277191 (rs2277191A) in the intron region of PSIP1 was more frequent among seropositives (p=0.06). Among individuals followed longitudinally, rs2277191A was associated with higher likelihood of HIV-1 acquisition (p=0.08) and rapid CD4+T cell decline (p=0.04) in the recently infected (primary infection) cohort. In contrast, the minor allele (C) of rs12339417 (rs12339417C) also in the intron region of PSIP1 was associated with higher CD4+ T cell counts during primary infection. The rs12339417C was also associated with slower rate of CD4+ T cell decline (p=0.02) and lower mRNA levels of LEDGF/p75 (p<0.01). Seroconverters had higher preinfection mRNA levels of LEDGF/p75 compared to nonseroconverters (p<0.01) and these levels decreased after HIV-1 infection (p=0.02). The Q472L mutation showed approximately 2-fold decrease in the association constant (Kd), suggesting stronger binding to HIV-1 integrase. Our findings demonstrate, for the first time, that genetic polymorphisms in the TNPO3 and PSIP1 genes may be associated with susceptibility to HIV-1 infection and the disease progression. These data provide in vivo evidence that TRN-SR2 and LEDGF/p75 are important host cofactors for HIV-1 replication. This is also the first study to show the association of genetic polymorphisms in the PPIA gene with disease outcome in a population (South African) with high burden of HIV-1 infection. Conclusions. Genetic variation in HIV-1 replication cofactors may be associated with disease outcome in a South African population. These data strongly support the role of these HIV replication cofactors in disease pathogenesis in vivo and suggest that these factors are possible targets for therapeutic interventions. However, these data will need to be replicated in larger cohorts to confirm the effect of these genetic variants. Further studies on how to target these factors in antiviral strategies are needed

Young and Early Career Investigators: Report from a Global HIV Vaccine Enterprise Working Group

The scientific challenges facing HIV-1 vaccine development are unprecedented in the history of vaccinology. As a result, investigators, funders, and other stakeholders generally agree that &#x201c;game-changing&#x201d; ideas are required. While innovation can certainly arise from investigators at all career stages, young and early-career investigators, defined as those under 40 years of age or within 10 years of their final degree or clinical training, are especially key contributors of novel and transformative ideas. Young and early-career investigators bring energy, enthusiasm, and fresh perspectives that are unbiased by prevailing dogma and that are essential to scientific progress

Epidemiological studies on camel trypanosomosis (surra) and its control and economic impact in Somaliland

Surra is a disease caused by the pathogenic trypanosome, Trypanosoma evansi, and is distributed throughout Africa, Asia and South America. The study outlined in this thesis was conducted to determine the epidemiology of trypanosomosis in camels, its economic impact on camels raised under a traditional pastoral production system and potential vectors that could transmit the disease between camels in Somaliland. Prior to this study there was limited information on the distribution and impact of surra in camels in Somaliland, although field reports indicated significant losses of production and consequently impact on the livelihood of pastoralists. In this study 2,575 camels were sampled from 144 herds and tested with the CATT/T. evansi. The animal level test seroprevalence observed was 26.4% (95% CI 24.8, 28.2) (real prevalence after adjusting for test sensitivity and specificity was 38.2%; 95% CI: 36.0, 40.0). The seroprevalence varied significantly between regions (p < 0.05) with a higher level (37.2%) in Sahil than in Awdal (19.3%) or Waqoyi Galbed (17.4%). A susceptible-infectious-subclinical (SIC) model was constructed in order to determine criteria for successful disease control by mass and targeted chemotherapy. This was used to simulate and estimate the economic benefits of four different control options against surra in camels. Adopting biannual treatment of all camels, monthly targeted treatment of clinically sick camels or biannual targeted treatment of seropositive camels was estimated to result in a benefit of US$141,431, 170,577 and 114,625 per village (80 camels) for a five year period in the study area, respectively. The prevalence after five years of control was predicted to be 7.4, 6.4 and 6.7$404,630 (20159 camels studied) if no treatment was administered. The greatest loss was associated with decreased milk yield (US$314,630). As part of this research Nzi and biconical traps were set to trap tabanids responsible for transmitting trypanosomosis. Three genera of tabanids were trapped (Philoliche, Tabanus and Haematopota) and these flies were recognised as potential vectors of trypanosomosis in camels. Philoliche species were the most widely distributed and abundant biting flies in the area. The activity of the biting flies differed throughout the day, with the highest activity observed in the middle of the day and the lowest in the afternoon. There was a significant difference between the alighting sites of biting flies on camels, with the lower body and belly of camels being the preferred sites compared to the upper body (head, neck and hump). In this study on average 0.87 ± 0.34 flies of the Philoliche genus alighted on the lower body and belly of camels in the middle of the day. The results of this study strongly support the need for implementation of surveillance and control programs for trypanosomosis in camel herds in Somaliland

Screening and phytochemical characterization of a South African herbal concoction for anti-HIV-1 activity

A dissertation submitted to the Faculty of Science under the School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master in Science. Johannesburg, June 2017.In South Africa, the anti-HIV-1 activity of various indigenous plants has not been studied extensively. Most of the phytochemical screening work has focused on anti-cancer activity with less attention given to infectious diseases. A large proportion of South Africans (70-80%) still rely on traditional medicines for treatment of various ailments. And, therefore, there is a need to evaluate and validate the effectiveness of the traditional medicines. The aim of this study was to identify, screen, phytochemically characterize and isolate bioactive compounds from a South African herbal extract that exhibit the best anti-HIV-1 activity. Three extracts were prepared: an ethanol extract, a dereplicated ethanol extract and an aqueous extract from a herbal concoction comprised of a mixture of six plants. These herbal concoctions were investigated for anti-HIV-1 subtype C activity. Phytochemical profiling of the ethanol- and dereplicated ethanol- extracts from the herbal concoctions showed the presence of intermediate polar compounds (flavonoids, alkaloids, sugars and terpenes) for both extracts, while the aqueous extract contained predominantly highly polar compounds. Anti-HIV-1 screening of the three extracts showed that the ethanol and dereplicated ethanol herbal- extracts had the best anti-reverse transcriptase activity. The ethanol extract had mean IC50 values of 56.53, 53.96 and 55.39 μg/ml against MJ4, Du179 and CM9 HIV-1 subtypes C isolates, respectively. The dereplicated ethanol extract had mean IC50 values of 51.87, 47.56 and 52.81 μg/ml against MJ4, Du179 and CM9 HIV-1 isolates, respectively. The aqueous extract was inactive against HIV-1 activity. Moreover, both the ethanol- and dereplicated ethanol- extracts showed activity against HIV neutralization. The ethanol- and dereplicated ethanol- extracts had mean IC50 values of 36.33 and 32.06 μg/ml, respectively. Furthermore, they also potently neutralized Vesicular stomatitis virus (VSV) yielding mean IC50 values of 24.91 and 20.82 μg/ml for ethanol- and dereplicated ethanol- extracts, respectively. All extracts were inactive against Murine leukemia virus (MLV). The isolation and phytochemical characterization of the bioactive compound(s) was done by utilizing various chromatographic and spectroscopic methods. Four homoisoflavanoids were isolated and tested for anti-HIV-1 subtype C activity. Three compounds (1, 3a and 3b) were inactive while compound 2 was found to be bioactive against HIV-1 reverse transcriptase (RT) and yielded mean IC50 values of 7.23 ± 1.88, 12.83 ± 0.41 & 12.81 ± 0.10 μg/ml for MJ4, CM9 and Du179 HIV-1 subtype C isolates, respectively. Compound 2 had a mean CC50 value of 23.08 ± 0.1981 μg/ml against HEK293T cells. Overall, the data suggested that ethanol- and dereplicated ethanol- herbal extracts possess direct and indirect anti-HIV-1 activity. They possess a cocktail of phytochemicals that can inhibit HIV-1 RT, HIV-1 entry. Furthermore, these extracts possess phytochemicals that can lower the activation of inflammatory responses during an infection and, hence, reduction in the number new cells infected during the course of HIV-1 infection. Moreover, they possess phytochemicals that have antioxidant activity which, in relation to HIV infection, results in a boosted immune system response in order to ward off the virus.MT 201

Protective HLA class I alleles : investigation of viral control and lack of control in chronic HIV-1 subtype C infection.

Doctor of Philosophy in Immunology. University of KwaZulu-Natal, Medical School 2015.Abstract available in PDF file

Prevalence and risk factors associated with Trypanosomosis in Cattle Herds in Kilwa District of Lindi Region of Tanzania

Thesis(MSc)-University of Zambia,2015African Animal trypanosomosis (AAT) or Nagana and Human African Trypanosomosis (HAT) or Sleeping Sickness are complex chronic, debilitating, emaciating and often fatal diseases of animals and humans, respectively. This cross-sectional study was conducted to determine the prevalence and risk factors associated with bovine trypanosomosis in tsetse-infested Kilwa district, Lindi region, Southern Tanzania. Blood samples were collected from 420 cattle randomly selected from 86 herds from ten villages. A maximum of ten herds per village and at maximum six animals from each herd were selected for sampling. At the same time a questionnaire was administered. Individual animal samples were analysed using microscopy and pooled sample at herd level were analysed by loop mediated isothermal amplification (LAMP). A herd was considered positive if at least one animal in the herd was positive for AAT. A herd prevalence of 9.3 % (95% CI: 2.9-14.9) was recorded for AAT by microscopy, mainly caused by Trypanosoma congolense 5.8% (95% CI = 0.9-10.7), Trypanosoma brucei species 5.8% (95%, CI = 0.9-10.7) and Trypanosoma vivax 3.5% (95% CI = 0-7.4). Loop mediated isothermal amplification (LAMP) recorded a heard prevalence of 41.9% (95% CI: 30.0-51.4%), mainly caused by T. congolense 30.2% (95% CI: 20.5-39.9), T. brucei species 25.6% (95% CI: 16.4-34.8) and T. vivax 20.9% (95% CI: 12.3-29.7). Most of the cattle herds had mixed infections of these parasites. According to LAMP, Miteja and Matandu villages both had the highest AAT prevalence of 57% (95% CI: 20.3-93.7) while Mavuji had the lowest prevalence of 14% (95% CI: 0-39.7). Data from the present study suggest that district of origin, grazing in Game Reserve, season of increased vector, form of watering point, and affordability of the anti-trypanosomal drugs are risk factors associated with AAT in Kilwa district, southern Tanzania. Use of tsetse trapes and targets, continuous surveillance and monitoring of AAT using more sensitive and specific molecular tests, discouraging settlement and graing in the game reserve are recommended

Variation in Hiv-1 Nef Function Within and Among Viral Subtypes Reveals Genetically Separable Antagonism of Serinc3 and Serinc5

HIV Nef counteracts cellular host restriction factors SERINC3 and SERINC5, but our understanding of how naturally occurring global Nef sequence diversity impacts these activities is limited. Here, we quantify SERINC3 and SERINC5 internalization function for 339 Nef clones, representing the major pandemic HIV-1 group M subtypes A, B, C and D. We describe distinct subtype-associated hierarchies for Nef-mediated internalization of SERINC5, for which subtype B clones display the highest activities on average, and of SERINC3, for which subtype B clones display the lowest activities on average. We further identify Nef polymorphisms that modulate its ability to counteract SERINC proteins, including substitutions in the N-terminal domain that selectively impair SERINC3 internalization. Our findings demonstrate that the SERINC antagonism activities of HIV Nef differ markedly among major viral subtypes and between individual isolates within a subtype, suggesting that variation in these functions may contribute to global differences in viral pathogenesis

Relative Resistance of MHC-B to Nef-Mediated Downregulation is Conserved Among Primate lentiviruses and Influences Antiviral T cell Responses in HIV-1-Infected Individuals

Patient-derived HIV-1 subtype B Nef clones downregulate HLA-A more efficiently than HLA-B. However, it remains unknown whether this property is common to Nef proteins across primate lentiviruses, and how antiviral immune responses may be affected. We examined 263 Nef clones from diverse primate lentiviruses including different pandemic HIV-1 group M subtypes for their ability to downregulate MHC-A and MHC-B from the cell surface. Though lentiviral Nef proteins differed markedly in their absolute MHC-A and MHC-B downregulation abilities, all lentiviral Nef lineages downregulated MHC-A on average 11-32% more efficiently than MHC-B. Nef genotype/phenotype analyses in a cohort of HIV-1 subtype C-infected patients (N=168), together with site-directed mutagenesis, revealed Nef position 9 as a subtype-specific determinant of differential HLA-A vs. HLA-B downregulation activity. Nef clones harboring non-consensus variants at codon 9 downregulated HLA-B (though not HLA-A) significantly better than those harboring consensus at this site, resulting in reduced recognition of infected target cells by HIV-1-specific CD8+ effector cells in vitro. Among persons expressing protective HLA class I alleles, carriage of Nef codon 9 variants was also associated with reduced ex vivo HIV-specific T-cell responses. Our results demonstrate that Nef\u27s inferior ability to downregulate MHC-B compared to MHC-A is conserved across primate lentiviruses, and suggest that this property influences antiviral cellular immune responses