Browsing by Author "Ndung'u, Peter Thumbi."

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Analysis of viral inhibitory activity of cytotoxic T. Lymphocytes targeting identical epitopes restricted by different class 1 HLA alleles from the same HLA supertype.
(2015) Ogunshola, Funsho Japhet.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
Human leukocyte antigen (HLA) polymorphism and the genetic diversity of human immunodeficiency virus (HIV) are the major obstacles for designing an effective HIV Cytotoxic T Lymphocytes (CTLs) based vaccine. Interestingly, recent studies have demonstrated that multiple class I alleles can recognize common epitopes “supertopes” due to the homology of amino acids within the major binding pockets of the peptide binding cleft. The implications of this for vaccine design is that a vaccine containing a small number of highly promiscuous supertopes can confer protection against a wide range of HIV variants. This notion makes supertopes immunogen design an attractive option. However, it is not clear whether supertopes presented in the context of different class I HLA alleles would induce functional equivalent CTL responses. In this study, we investigated the inhibitory activity of CTLs targeting identical epitopes presented by class I HLA alleles from the same superfamily. The viral inhibitory activity was measured using a newly developed CEM-GFP reporter T-cell line (GXR-cell) as target cell. We first compared the inhibitory activity of CTLs from 8 subjects targeting TPQDLNTML (Gag p24 residue 180-188-TL9) epitope presented by HLA-B*81:01 or B*42:01 alleles. We then assessed the inhibitory activity of the 8 subjects’ CTLs when presented with in-vivo occurring mutant (Q182S)-TL9 epitope by HLA-B*81:01 or B*42:01 alleles. Furthermore, we compared the inhibitory activity of CTLs from 4 subjects targeting ISPRTLNAW (Gag p24 residue 147-155-IW9) epitope presented by HLA-B*57:03 or B*58:01 alleles. Comparative analysis of the inhibitory activity of the 8 subjects’ CTLs showed no statistical significant difference when TL9 epitope was presented by HLA-B*81:01 or B*42:01 alleles (1:1; p-value = 0.8785, paired t test), even at low target to effector ratio (1:8; p-value = 0.4418). No statistical significant difference was observed in the inhibitory activity of the 8 subjects’ CTLs when mutant (Q182S)-TL9 epitope was presented by HLA-B*81:01 or B*42:01 alleles (1:1; p-value = 0.8042), same result was observed at low target to effector ratio (1:8; p-value = 0.9396). Comparative analysis of the inhibitory activity of the 4 subjects’ CTLs targeting identical IW9 epitopes presented by HLA-B*57:03 or B*58:01 alleles showed a trend towards significance at target to effector ratio 1:1 (1:1; p-value = 0.0924), but at low target to effector ratio, no significance difference was observed (1:8; p-value = 0.1496). In conclusion, we have demonstrated that there is no observable significant difference in the inhibitory activity of CTLs targeting wildtype TL9 or mutant (Q182S)-TL9 epitopes presented in the context of HLA-B*81:01 or B*42:01 alleles. Thus, TL9 epitope could be immunogenic for individuals expressing HLA-B*81:01 or B*42:01 alleles. We have also shown that the inhibitory activity of CTLs targeting identical IW9 epitopes presented by HLAB* 57:03 or B*58:01 alleles is comparable. Indicating that IW9 epitope could be included in immunogen design for individuals expressing HLA-B*57:03 or B*58:01 alleles. These findings are relevant for HIV vaccine approach that seeks to identify immunogenic supertopes that can be cross-presented in a broadly cross-reactive T cell based vaccine design.
APOBEC3G expression is dysregulated in primary HIV-1 infection and polymorphic variants influence CD4R T-cell counts and plasma viral load.
(Lippincott Williams & Wilkins., 2008) Reddy, Kavidha.; Winkler, Cheryl Ann.; Werner, Lise.; Mlisana, Koleka Patience.; Abdool Karim, Salim Safurdeen.; Ndung'u, Peter Thumbi.
Objectives: In the absence of HIV-1 virion infectivity factor (Vif), cellular cytosine deaminases such as apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) inhibit the virus by inducing hypermutations on viral DNA, among other mechanisms of action. We investigated the association of APOBEC3G mRNA levels and genetic variants on HIV-1 susceptibility, and early disease pathogenesis using viral load and CD4+ T-cell counts as outcomes. Methods: Study participants were 250 South African women at high risk for HIV-1 subtype C infection.We used real-time PCR to measure the expression of APOBEC3G in HIV-negative and HIV-positive primary infection samples. APOBEC3G variants were identified by DNA re-sequencing and TaqMan genotyping. Results: We found no correlation between APOBEC3G expression levels and plasma viral loads (r=0.053, P=0.596) or CD4+ T-cell counts (r=0.030, P=0.762) in 32 seroconverters. APOBEC3G expression levels were higher in HIV-negative individuals as compared with HIV-positive individuals (P<0.0001), including matched pre and postinfection samples from the same individuals (n=13, P<0.0001). Twenty-four single nucleotide polymorphisms, including eight novel, were identified within APOBEC3G by re-sequencing and genotyping. The H186R mutation, a codon-changing variant in exon 4, and a 3' extragenic mutation (rs35228531) were associated with high viral loads (P=0.0097 and P<0.0001) and decreased CD4+ T-cell levels (P=0.0081 and P<0.0001), respectively. Conclusion: These data suggest that APOBEC3G transcription is rapidly downregulated upon HIV-1 infection. During primary infection, APOBEC3G expression levels in peripheral blood mononuclear cells do not correlate with viral loads or CD4+ T-cell counts. Genetic variation of APOBEC3G may significantly affect early HIV-1 pathogenesis, although the mechanism remains unclear and warrants further investigation.
Association of genetic polymorphisms in select HIV-1 replication cofactors with susceptibility to HIV-1 infection and disease progression.
(2011) Madlala, Paradise Zamokuhle.; Ndung'u, Peter Thumbi.; Kormuth, Emil.
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.
Association of polymorphisms in the LEDGF/p75 gene (PSIP1) with susceptibility to HIV-1 infection and disease progression.
(Lippincott Williams & Wilkins., 2011) Madlala, Paradise Zamokuhle.; Gijsbers, Rik.; Christ, Frauke.; Hombrouck, Anneleen.; Werner, Lise.; Mlisana, Koleka Patience.; An, Ping.; Abdool Karim, Salim Safurdeen.; Winkler, Cheryl Ann.; Debyser, Zeger.; Ndung'u, Peter Thumbi.
Objective: LEDGF/p75, encoded by the PSIP1 gene, interacts with HIV-1 integrase and targets HIV-1 integration into active genes. We investigated the influence of polymorphisms in PSIP1 on HIV-1 acquisition and disease progression in black South Africans. Methods: Integrase binding domain of LEDGF/p75 was sequenced in 126 participants. Four haplotype tagging SNPs rs2277191, rs1033056, rs12339417 and rs10283923 referred to as SNP1, SNP2, SNP3 and SNP4, respectively, and one exonic SNP rs61744944 (SNP5, Q472L) were genotyped in 195 HIV-1 seronegative, 52 primary and 403 chronically infected individuals using TaqMan assays. LEDGF/p75 expression was quantified by real-time RT-PCR. The impact of Q472L mutation on the interaction with HIV_1 IN was measured by AlphaScreen. Results: rs2277191 (SNP1) A was more frequent among seropositives (P=0.06, Fisher’s exact test). Among individuals followed longitudinally SNP1A trended towards association with higher likelihood of HIV-1 acquisition [relative hazard (RH)=2.21, P=0.08; Cox model] and it was also associated with rapid disease progression (RH=5.98, P=0.04; Cox model) in the recently infected (primary infection) cohort. rs12339417 (SNP3)C was associated with slower decline of CD4+ T cells (P=0.02) and lower messenger RNA (mRNA) levels of LEDGF/p75 (P<0.01). Seroconverters had higher preinfection mRNA levels of LEDGF/p75 (P<0.01) and these levels decreased after HIV-1 infection (P=0.02). Conclusions: Genetic variants of PSIP1 may affect HIV-1 outcomes. Further studies are needed to confirm the effect of genetic variation of PSIP1 on HIV-1 pathogenesis in different cohorts.
Association of polymorphisms in the regulatory region of the cyclophilin A gene (PPIA) with gene expression and HIV/AIDS disease progression.
(Wolters Kluwer Health., 2016) Madlala, Paradise Zamokuhle.; Singh, Ravesh.; An, Ping.; Werner, Lise.; Mlisana, Koleka Patience.; Abdool Karim, Salim Safurdeen.; Winkler, Cheryl Ann.; Ndung'u, Peter Thumbi.
Abstract available in PDF file.
Association of TRIM22 with the Type 1 Interferon Response and Viral Control during Primary HIV-1 Infection.
(American Society for Microbiology., 2010) Singh, Ravesh.; Gaiha, Gaurav.; Werner, Lise.; Mlisana, Koleka Patience.; Luban, Jeremy.; Walker, Bruce D.; Abdool Karim, Salim Safurdeen.; Ndung'u, Peter Thumbi.; Brass, Abraham.; McKim, Kevin.
Type 1 interferons (IFNs) induce the expression of the tripartite interaction motif (TRIM) family of E3 ligases, but the contribution of these antiviral factors to HIV pathogenesis is not completely understood. We hypothesized that the increased expression of select type 1 IFN and TRIM isoforms is associated with a significantly lower likelihood of HIV-1 acquisition and viral control during primary HIV-1 infection. We measured IFN-a, IFN-b, myxovirus resistance protein A (MxA), human TRIM5a (huTRIM5a), and TRIM22 mRNA levels in peripheral blood mononuclear cells (PBMCs) of high-risk, HIV-1-uninfected participants and HIV-1-positive study participants. Samples were available for 32 uninfected subjects and 28 infected persons, all within 1 year of infection. HIV-1-positive participants had higher levels of IFN-b(P=0.0005), MxA (P=0.007), and TRIM22 (P=0.01) and lower levels of huTRIM5a (P< 0.001) than did HIV-1-negative participants. TRIM22 but not huTRIM5a correlated positively with type 1 IFN (IFN-a, IFN-b, and MxA) (all P<0.0001). In a multivariate model, increased MxA expression showed a significant positive association with viral load (P=0.0418). Furthermore, TRIM22 but not huTRIM5a, IFN-a, IFN-b, or MxA showed a negative correlation with plasma viral load (P=0.0307) and a positive correlation with CD4 T-cell counts (P=0.0281). In vitro studies revealed that HIV infection induced TRIM22 expression in PBMCs obtained from HIV-negative donors. Stable TRIM22 knockdown resulted in increased HIV-1 particle release and replication in Jurkat reporter cells. Collectively, these data suggest concordance between type 1 IFN and TRIM22 but not huTRIM5a expression in PBMCs and that TRIM22 likely acts as an antiviral effector in vivo.
Case 15-2011: A 19-year-old South African woman with headache, fatigue, and vaginal discharge.
(Massachusetts Medical Society., 2011) Venter, W. D. Francois.; Ndung'u, Peter Thumbi.; Abdool Karim, Quarraisha.
No abstract available.
CD8+ T cell breadth and ex vivo virus inhibition capacity distinguish between viremic controllers with and without protective HLA class I alleles.
(American Society for Microbiology., 2016) Koofhethile, Catherine Kegakilwe.; Ndhlovu, Zaza Mtine.; Thobakgale, Christina Fanesa.; Prado, Julia G.; Ismail, Nasreen.; Mncube, Zenele.; Mkhize, Lungile.; Van der Stok, Mary Elizabeth.; Yende-Zuma, Fortunate Nonhlanhla.; Walker, Bruce D.; Goulder, Philip Jeremy Renshaw.; Ndung'u, Peter Thumbi.
Abstract available in PDF file.
Cellular immunity, immune activation and regulation in HIV-1 infected mother-child pairs : what are the determinants of protective immunity.
(2011) Moodley-Govender, Eshia S.; Ndung'u, Peter Thumbi.; Addo, Marylyn.; Goulder, Philip Jeremy Renshaw.
Background: Prevention of Mother-to-child transmission (PMTCT) of human immunodeficiency virus (HIV) remains a significant challenge in resource-poor settings despite the advances in antiretroviral (ARV) treatment. HIV-1 infected individuals are able to achieve viral control naturally, however the underlying mechanisms of immunological control in children remains poorly understood. This study was conducted from 2006 to 2010 to investigate correlates of immune control in HIV-1 clade C infected mother-child pairs in the absence of ARVs. Genotypic and phenotypic viral characteristics, cellular immune responses to HIV-1 and host genetics were characterized and correlated with clinical markers of disease progression. Materials and Methods: To achieve the objectives of the study, three cohorts of mother-child pairs were investigated. The first cohort included 60 untreated mother-child pairs and a further ten uninfected children as controls. The second cohort comprised of ARV treated pairs (n=60). The third cohort consisted of 374 mothers and 374 children (infected, exposed uninfected, HIV negative). Plasma viral loads and absolute CD4+ T cell counts were routinely performed in all three cohorts. HIV-specific CD8+ T cell responses were analyzed by interferon gamma (IFN-γ) enzyme linked immunosorbent spot (ELISpot) assays. Viral replicative fitness was assessed using a green fluorescent protein reporter cell line (GFP).Multi-parameter flowcytometry allowed for the investigation of T cell regulation, exhaustion and activation using CD127/CD25, TIM-3/PD-1 and HLA-DR/CD38 markers respectively. IL-10 promoter single nucleotide polymorphisms (SNPs) at positions -592 and -1082 were determined by TaqMan allelic discrimination assays. Plasma IL-10 levels were measured using a luminex assay. Results: To describe the CTL responses elicited to various regions of the HIV proteome in HIV-infected treatment naïve children. Sixty children under one year of age in the untreated cohort were analyzed for CTL responses spanning the HIV genome, for which only 30 had detectable responses. There was no significant difference in viral load between respondersand non-responders (p=0.2799). The responders predominantly targeted Nef (49%), Gag (17%) and Env (14%) regions. Markers of T cell exhaustion and regulation and theirrelationship to markers of disease progression, were next investigated as these parameters may explain the inability of T cells to effectively control HIV infection. T cell phenotyping compared treated, untreated and uninfected subgroups. In infected children, CD8+ T cells were significantly higher for both the inhibitory marker TIM-3 (p=0.001) and exhaustion marker PD-1 (p=0.0001) compared to uninfected children. Median expression of TIM-3 was higher on CD8+ T cells (46%) compared to CD4+ T cells (20%). TIM-3 and PD-1 expression on T cells were maintained at high levels over time. The frequency of absolute Tregs (p=0.0225) were found to be significantly higher in untreated compared to treated children. HLA-DR+CD38+ on CD8+ T cells were significantly up-regulated in untreated children compared to treated (p=0.002) and uninfected children (p=0.0177). HLA-DR+CD38+ was also significantly higher in children less than 6 months compared to older children on CD4+ (p=0.0437) and CD8+ T cells (p=0.00276). Interestingly, we observed a significant negative correlation between magnitude of CTL response and CD25+CD127- (p=0.0202; r=-0.7333) as well as HLA-DR+CD38+ (p=0.0408; r=-0.5516) on CD8+ T cells. IL-10 is an important immunoregulatory cytokine that has been shown to affect the outcome of chronic viral infections. IL-10 polymorphisms have previously been associated with IL-10 levels and HIV-1 outcomes in adults. Polymorphisms associated with different levels of IL-10 production and their relationship with transmission, markers of disease progression and immune responses were next investigated in this mother-child HIV transmission setting. Genetic analysis of IL-10 in cohort three revealed that HIV-1 acquisition was not associated with either IL10 -592 (AA/CA vs CC) or IL10 -1082 (AA/AG vs GG) single nucleotide polymorphisms (SNPSs). There was a significant association between IL10 -1082 and HIV-1 transmission (p=0.0012). No correlation was observed between IL10 -592 (p=0.4279) or IL10 -1082 SNPs (p=0.6361) and mortality rates in children. IL10 -592C was associated with an elevated magnitude of IFN-γ CD8+ T cell response compared to IL10 -529A (p=0.0071). We found a significant positive correlation between IL-10 plasma levels and viral loads (p=0.0068; r=0.4759) and the ages of the children (p=0.0312; r=0.1737). Conclusion: CD8+ T cell responses and viral fitness did not explain differences in disease progression in selected HIV-1 untreated clade C transmission pairs. T cell activation and regulatory markers influence CTL immune responses resulting in poor clinical outcome. IL10 -1082 polymorphisms may be used as a predictor of HIV-1 transmission. The association between increased IL-10 plasma levels and high viral loads suggest that IL-10 contributes to immune dysfunction in paediatric HIV-1 infection. This study has extended our understanding of immunological and genetic correlates of mother-to-child transmission and disease outcome in ARV naïve (naturally controlling) and HIV treated infected children.
Changes in natural killer cell activation and function during primary HIV-1 infection.
(Plos., 2012) Naranbhai, Vivek.; Altfeld, Marcus.; Abdool Karim, Salim Safurdeen.; Ndung'u, Peter Thumbi.; Abdool Karim, Quarraisha.; Carr, William Henry.
Background. Recent reports suggest that Natural Killer (NK) cells may modulate pathogenesis of primary HIV-1 infection. However, HIV dysregulates NK-cell responses. We dissected this bi-directional relationship to understand how HIV impacts NK-cell responses during primary HIV-1 infection. Methodology/Principal Findings. Paired samples from 41 high-risk, initially HIV-uninfected CAPRISA004 participants were analysed prior to HIV acquisition, and during viraemic primary HIV-1 infection. At the time of sampling post-infection five women were seronegative, 11 women were serodiscordant, and 25 women were seropositive by HIV-1 rapid immunoassay. Flow cytometry was used to measure NK and T-cell activation, NK-cell receptor expression, cytotoxic and cytokine-secretory functions, and trafficking marker expression (CCR7, α4β7). Non-parametric statistical tests were used. Both NK cells and T-cells were significantly activated following HIV acquisition (p = 0.03 and p<0.0001, respectively), but correlation between NK-cell and T-cell activation was uncoupled following infection (pre-infection r = 0.68;p<0.0001; post-infection, during primary infection r = 0.074;p = 0.09). Nonetheless, during primary infection NK-cell and T-cell activation correlated with HIV viral load (r = 0.32'p = 0.04 and r = 0.35;p = 0.02, respectively). The frequency of Killer Immunoglobulin-like Receptor-expressing (KIRpos) NK cells increased following HIV acquisition (p = 0.006), and KIRpos NK cells were less activated than KIRneg NK cells amongst individuals sampled while seronegative or serodiscordant (p = 0.001;p<0.0001 respectively). During HIV-1 infection, cytotoxic NK cell responses evaluated after IL-2 stimulation alone, or after co-culture with 721 cells, were impaired (p = 0.006 and p = 0.002, respectively). However, NK-cell IFN-y secretory function was not significantly altered. The frequency of CCR7+ NK cells was elevated during primary infection, particularly at early time-points (p<0.0001). Conclusions/Significance. Analyses of immune cells before and after HIV infection revealed an increase in both NK-cell activation and KIR expression, but reduced cytotoxicity during acute infection. The increase in frequency of NK cells able to traffic to lymph nodes following HIV infection suggests that these cells may play a role in events in secondary lymphoid tissue.
Characterization of anti-HIV-1 neutralizing and binding antibodies in chronic HIV-1 subtype C infection.
(Elsevier., 2012) Archary, Derseree.; Rong, Rong.; Gordon, Michelle Lucille.; Boliar, Saikat.; Madiga, Maphuti C.; Gray, Elin Solomonovna.; Dugast, Anne-Sophie.; Hermanus, Tandile.; Goulder, Philip Jeremy Renshaw.; Coovadia, Hoosen Mahomed.; Werner, Lise.; Morris, Lynn.; Alter, Galit.; Derdeyn, Cynthia A.; Ndung'u, Peter Thumbi.
Neutralizing (nAbs) and high affinity binding antibodies may be critical for an efficacious HIV-1 vaccine. We characterized virus-specific nAbs and binding antibody responses over 21 months in eight HIV-1 subtype C chronically infected individuals with heterogeneous rates of disease progression. Autologous nAb titers of study exit plasma against study entry viruses were significantly higher than contemporaneous responses at study entry (p=0.002) and exit (p=0.01). NAb breadth and potencies against subtype C viruses were significantly higher than for subtype A (p=0.03 and p=0.01) or B viruses (p=0.03; p=0.05) respectively. Gp41-IgG binding affinity was higher than gp120-IgG (p=0.0002). IgG–FcγR1 affinity was significantly higher than FcγRIIIa (p<0.005) at study entry and FcγRIIb (p<0.05) or FcγRIIIa (p<0.005) at study exit. Evolving IgG binding suggests alteration of immune function mediated by binding antibodies. Evolution of nAbs was a potential marker of HIV-1 disease progression.
Characterization of CD4+ and CD8+ T cell responses in HIV-1 C-Clade infection.
(2011) Ramduth, Dhanwanthie.; Kiepiela, Photini.; Ndung'u, Peter Thumbi.; Walker, Bruce D.
HIV-1 specific CD4+ T cell activity in clade C infected subjects has not been studied. CD4+ T cells play a vital role in controlling infectious diseases and there is a need to augment our knowledge of HIV immunology to aid vaccine design. We therefore embarked on a study to characterize HIV-1 specific CD4+ T cell activity in both adults and infants; assess the relationship between CD4+ and CD8+ immune responses; and the relationship between CD4+ T cell activity and markers of disease progression (viral loads and CD4 counts). Our study revealed that the magnitude of CD8+ T cell responses correlated significantly with CD4+ T cell responses, but that the percentage of CD8+ T cells directed against HIV-1 was always greater than that of CD4+ T cells. Gag was the frequently targeted HIV-1 protein by CD4+ T cells and had the highest density of epitopes targeted by CD4+ T cells. Patients with either a dominant CD4 or CD8 T cell response against Gag had significantly lower viral loads than patients in whom non-Gag proteins were the main target (p< 0.0001 for CD4 activity and p= 0.007 for CD8 responses). Single IFN- producing CD4+ T cells were present in significantly higher numbers than cells producing both IFN- and IL-2 simultaneously (p=0.009). Gag also dominated the CD4+ T cell response in acutely infected infants with IFN- production detected more frequently than IL-2 or TNF- . Longitudinal analysis of infants receiving early ARV treatment and then ceasing after 12 months revealed that early treatment conferred no protection against increasing viremia and disease progression. CD4+ T cell responses were detected sporadically in untreated infants indicating a dysfunctional immune response in the face of constant exposure to high levels of viremia. Taken together, the data reveal that a vaccine inducing Gag specific CD4+ T cell responses has the potential to confer some degree of protection, but other immunological parameters need to be investigated especially in infants.
Characterization of nucleoside reverse transcriptase inhibitor-associated mutations in the RNAse H region of HIV-1 subtype c infected individuals.
(Viruses., 2017) Ngcapu, Sinaye.; Theys, Kristof.; Libin, Pieter.; Marconi, Vincent C.; Sunpath, Henry.; Ndung'u, Peter Thumbi.; Gordon, Michelle Lucille.
Abstract available in pdf.
Characterizing the role of CD4+ T cell immunoregulatory networks in peripheral blood and lymphoid tissue during HIV-1 clade C infection.
(2018) Laher, Faatima.; Ndhlovu, Zaza Mtine.; Ndung'u, Peter Thumbi.
HIV eradication efforts have been unsuccessful due to virus persistence in cellular and tissue reservoirs. Recent evidence suggests that germinal centers (GCs) within lymph nodes (LN) contain a novel subset of regulatory T cells (TREGs), termed follicular regulatory T (TFR) cells. These cells control the magnitude and specificity of the GC response and like TREGs are essential for the maintenance of self-tolerance and immune homeostasis. However, the exact role of TFR cells in HIV infection and their contribution to viral control is not completely understood, possibly due to their low frequency, heterogeneity and more so, the difficulty in accessing human lymphoid tissue samples to fully study them. Thus, we set out to comprehensively investigate TFR cells in LN and peripheral blood (PB) samples, using a multifaceted approach including flow cytometry, MHC class II tetramers, immunofluorescence microscopy (IF), ELISA, digital droplet PCR and singlecell RNA sequencing (SeqWell), in HIV-1 clade C infection. Furthermore, we aimed to determine the effect of very early treatment on the frequency and function of this cell subset. Overall, our studies contributed various notable findings to the field. Firstly, we were able to develop MHC class II tetramers, specific in our HIV-1 clade C setting, as a more sensitive method of identifying very low cell frequency antigen-specific CD4+ T cells without relying on function. Tetramers eliminate the bias associated with in vitro stimulation required for functional assays and the limitation associated with only detecting subsets of cells capable of secreting a cytokine. Notably, we used class II tetramers to demonstrate that HIV-specific CD4+ T cell responses restricted to DRB1*11-Gag41 are associated with immune control of HIV-1 infection. We next focused on understanding the role of CD4+ regulatory cells during HIV-1 infection. Firstly, we showed that TFR cell frequencies were significantly higher in LN compared to PB samples. Secondly, TFR are a phenotypically and transcriptionally distinct subset compared to regulatory T cells (TREGs) and T Follicular Helper cells (TFH). Thirdly, we were able to detect HIV-specific TFR using our newly synthesized MHC class II tetramers, and showed higher frequencies observed in LNs during untreated HIV infection. Fourthly, as measured by both flow cytometry and IF, most of TFR localized outside of the GC, with very early ART initiators displaying larger proportions of TFR within the GC. Lastly, TFR cells exhibited a potential suppressive functional capacity as they produced IL-10, which is a canonical suppressive cytokine and they were also positively associated with gp41 IgG antibodies titers. Overall, the data presented in this thesis highlights the advantage of MHC class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. More so, the results give important insights into regulatory cells within lymph nodes; their biology, function and their role in the setting of very ART initiation.
Clinical and mycological predictors of cryptococcosis-associated immune reconstitution inflammatory syndrome.
(Wolters Kluwer Health., 2013) Chang, Christina C.; Dorasamy, Afton A.; Gosnell, Bernadett I.; Elliott, Julian H.; Spelman, Tim.; Omarjee, Saleha.; Naranbhai, Vivek.; Ndung'u, Peter Thumbi.; Moosa, Mahomed Yunus Suleman.; Lewin, Sharon R.; French, Martyn A.; Coovadia, Yacoob Mahomed.
Abstract available in PDF file.
Compartmentalisation of innate immune responses in the central nervous system during cryptococcal meningitis/HIV co-infection.
(Wolters Kluwer Health., 2014) Naranbhai, Vivek.; Chang, Christina C.; Durgiah, Raveshni.; Omarjee, Saleha.; Lim, Andrew.; Moosa, Mahomed Yunus Suleman.; Elliott, Julian H.; Ndung'u, Peter Thumbi.; Lewin, Sharon R.; French, Martyn A.; Carr, William Henry.
Abstract available in PDF file.
Coreceptor utilization and primary cell tropism by HIV-1 subtype C strains.
(2010) Singh, Ashika.; Ndung'u, Peter Thumbi.
Human immunodeficiency virus type 1 (HIV-1) isolates can be differentiated based on their ability to use particular coreceptors – R5 viruses use CCR5, X4 viruses use CXCR4 and R5X4 (dual tropic) viruses use both CCR5 and CXCR4. It is widely reported that HIV-1 subtype C (HIV-1C) has a unique viral coreceptor evolution pattern in that a complete switch from the predominant CCR5 (R5) to CXCR4 (X4) phenotype is less common for this subtype compared to other subtypes. However, dual tropic HIV-1C isolates have occasionally been described. Furthermore, it has been reported that certain highly active antiretroviral drugs (HAART) may select for X4 viral variants. Therefore, this thesis study was undertaken to better understand the functional and genotypic characteristics of dual tropic HIV-1C isolates, and to characterize drug resistance and coreceptor usage patterns in HAART-naïve versus HAART-failing HIV-1C infected patients. Thirty-five functional HIV-1 env clones derived from seven dual tropic HIV-1C strains were generated and their coreceptor usage characterized in transformed cell lines. All 35 env clones efficiently infected transformed cells expressing CXCR4. Twenty of 35 clones (57%) also utilized the CCR5 receptor. No R5-only clones were detected. Functional coreceptor usage data was correlated to env gene sequence data. The ability of the HIV-1C env clones to facilitate infection of primary lymphocytes and monocyte-derived macrophages was next investigated. The majority of clones characterized as X4 or R5X4 on cell lines used either CXCR4 alone or CXCR4 and CCR5, respectively, in primary cells. A few viruses displayed comparable CCR5 and CXCR4 usage and clones from one virus preferred CCR5 usage in macrophages. Thus in a few cases coreceptor phenotyping in transformed cell lines does not predict usage in primary cells. Genetic determinants for coreceptor usage in primary cells require further investigation. Finally the patterns of drug resistance mutations were studied and coreceptor usage among 45 HAART-naïve and 45 HAART-failing HIV-1C infected patients analyzed. Ninety-five percent of HAART-failing patients had viruses with at least one drug resistance mutation. Thymidine analog resistance mutations (TAMs) were present in 55% of patients. HAART-failing patients had significantly higher prevalence (59%) of X4/R5X4-utilizing viruses compared to HAART-naïve patients (30%) (p<0.02) using the Trofile Co-receptor Tropism Assay while 41% of HAART-failing patients used CCR5 and 70% of HAART-naïve patients used CCR5. Functional results correlated with predictive algorithm methods. This study enhances our understanding of HIV-1 pathogenesis and the results have important implications for the use of coreceptor antagonists for the clinical management of HIV-1C infection.
Development and optimization of real-time PCR assays to detect anti-microbial immune factors and their response to type I and II interferons.
(2016) Kieswetter, Nathan Scott.; Ndung'u, Peter Thumbi.; Wong, Emily.
Abstract available in PDF file.
Drug resistance and viral tropism in HIV-1 subtype C-infected patients in KwaZulu-Natal, South Africa : implications for future treatment options.
(Lippincott Williams & Wilkins., 2011) Singh, Ashika.; Sunpath, Henry.; Green, Taryn N.; Padayachi, Nagavelli.; Hiramen, Keshni.; Lie, Yolanda.; Anton, Elizabeth D.; Murphy, Richard.; Reeves, Jacqueline D.; Kuritzkes, Daniel R.; Ndung'u, Peter Thumbi.
Background: Drug resistance poses a significant challenge for the successful application of highly active antiretroviral therapy (HAART) globally. Furthermore, emergence of HIV-1 isolates that preferentially use CXCR4 as a coreceptor for cell entry, either as a consequence of natural viral evolution or HAART use, may compromise the efficacy of CCR5 antagonists as alternative antiviral therapy. Methods: We sequenced the pol gene of viruses from 45 individuals failing at least 6 months of HAART in Durban, South Africa, to determine the prevalence and patterns of drug-resistance mutations. Coreceptor use profiles of these viruses and those from 45 HAART-naive individuals were analyzed using phenotypic and genotypic approaches. Results: Ninety-five percent of HAART-failing patients had at least one drug-resistant mutation. Thymidine analog mutations (TAMs) were present in 55% of patients with 9% of individuals possessing mutations indicative of the TAM1 pathway, 44% had TAM2, whereas 7% had mutations common to both pathways. Sixty percent of HAART-failing subjects had X4/dual//mixed-tropic viruses compared with 30% of HAART-naïve subjects (P < 0.02). Genetic coreceptor use prediction algorithms correlated with phenotypic results with 60% of samples from HAART-failing subjects predicted to possess CXCR4-using (X4/dual/mixed viruses) versus 15% of HAART-naïve patients. Conclusions: The high proportion of TAMs and X4/dual/mixed HIV-1 viruses among patients failing therapy highlight the need for intensified monitoring of patients taking HAART and the problem of diminished drug options (including CCR5 antagonists) for patients failing therapy in resource-poor settings.
Duffy-Null–Associated Low Neutrophil Counts Influence HIV-1 Susceptibility in High-Risk South African Black Women.
(Oxford University Press., 2010) Ramsuran, Veron.; Kulkarni, Hemant.; He, Weijing.; Mlisana, Koleka Patience.; Wright, Edwina J.; Werner, Lise.; Castiblanco, John.; Dhanda, Rahul.; Le, Tuan.; Dolan, Matthew J.; Guan, Weihua.; Weiss, Robin A.; Clark, Robert A.; Abdool Karim, Salim Safurdeen.; Ahuja, Sunil K.; Ndung'u, Peter Thumbi.
Background. The Duffy-null trait and ethnic netropenia are both highly prevalent in Africa. The influence of pre-seroconversion levels of peripheral blood cell counts (PBCs) on the risk of acquiring human immunodeficiency virus (HIV)–1 infection among Africans is unknown. Methods. The triangular relationship among pre-seroconversion PBC counts, host genotypes, and risk of HIV acquisition was determined in a prospective cohort of black South African high-risk female sex workers. Twenty seven women had seroconversion during follow-up, and 115 remained HIV negative for 2 years, despite engaging in high-risk activity. Results. Pre-seroconversion neutrophil counts in women who subsequently had seroconversion were significantly lower, whereas platelet counts were higher, compared with those who remained HIV negative. Comprising 27% of the cohort, subjects with pre-seroconversion neutrophil counts of <2500 cells/mm3 had a ~3-fold greater risk of acquiring HIV infection. In a genome-wide association analyses, an African-specific polymorphism (rs2814778) in the promoter of Duffy Antigen Receptor for Chemokines (DARC -46T>C) was significantly associated with neutrophil counts (P = 7.9 x10-11). DARC -46C/C results in loss of DARC expression on erthyrocytes (Duffy-null) and resistance to Plasmodium vivax malaria, and in our cohort, only subjects with this genotype had pre-seroconversion neutrophil counts of <2500 cells/mm3. The risk of acquiring HIV infection was ~3-fold greater in those with the trait of Duffy-null–associated low neutrophil counts, compared with all other study participants. Conclusions. Pre-seroconversion neutrophil and platelet counts influence risk of HIV infection. The trait of Duffy-null–associated low neutrophil counts influences HIV susceptibility. Because of the high prevalence of this trait among persons of African ancestry, it may contribute to the dynamics of the HIV epidemic in Africa.