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

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