Philosophiae Doctor - PhD / Heterosexual transmission of HIV-1 via the female genital tract is the leading route of HIV infection in sub-Saharan Africa. Viruses then traffic between the cervical compartment and blood ensuring pervasive infection. Previous studies have however reported the existence of genetically diverse viral populations in various tissue types, each evolving under separate selective pressures within a single individual, though it is still unclear how compartmentalization dynamics change over acute and chronic infection in the absence of ARVs. To better characterize intrahost evolution and the movement of viruses between different anatomical tissue types, statistical and phylogenetic methods were used to reconstruct temporal dynamics between blood plasma and cervico-vaginal lavage (CVL) derived HIV-1 subtype C gp120 envelope sequences. A total of 206 cervical and 253 blood plasma sequences obtained from four treatment naïve women enrolled in the CAPRISA Acute Infection study cohort in South Africa were evaluated for evidence of genotypic and phenotypic differences between viral populations from each tissue type up to 3.6 years post-infection. Evidence for tissue-specific differences in genetic diversity, V-loop length variation, codon-based selection, co-receptor usage, hypermutation, recombination and potential N-linked glycosylation (PNLG) site accumulation were investigated. Of the four participants studied, two anonymously identified as CAP270 and CAP217 showed evidence of infection with a single HIV-1 variant, whereas CAP177 and CAP261 showed evidence of infection by more than one variant. As a result, genetic diversity, PNLGs accumulation and the number of detectable recombination events along the gp120 env region were lowest in the former patients and highest in the latter. Overall, genetic diversity increased over the course of infection in all participants and correlated significantly with viral load measurements from the blood plasma in one of the four participants tested (i.e. CAP177). Employing a structured coalescent model approach, rates of viral migration between anatomical tissue types on time-measured genealogies were also estimated. No persistent evidence for the existence of separate viral populations in the cervix and blood plasma was found in any of the participants and instead, sequences generally clustered together by time point on Bayesian Maximum Clade Credibility (MCC) trees. Clades that were monophyletic by tissue type comprised mostly of low diversity or monotypic sequences from the same time point, consistent with bursts of viral replication. Tissue-specific monophyletic clades also generally contained few sequences and were interspersed among sequences from both tissue-types. Tree and distance-based statistical tests were employed to further evaluate the degree to which cervical and blood plasma viruses clustered together on Bayesian MCC trees using the Slatkin-Maddison (S-M), Simmonds Association index (AI), Monophyletic Clade (MC), Wright’s measure of population subdivision (FST) and Hudson’s Nearest Neighbour (Snn) statistics, in the presence and absence of monotypic and low diversity sequences. Statistical evidence for the presence of tissue-specific population structure disappeared or was greatly reduced after the removal of monotypic and low diversity sequences, except in CAP177 and CAP217, in 3/5 of longitudinal tree and distance-based tests. Analysis of phenotypic differences between viral populations from the blood plasma and cervix revealed inconsistent tissue-specific patterns in genetic diversity, codon-based selection, co-receptor usage, hypermutation, recombination, V-loop length variation and PNLG site accumulation during acute and chronic infection among all participants. There is therefore no evidence to support the existence of distinct viral populations within the blood plasma and cervical compartments longitudinally, however slightly constrained populations may exist within the female genital tract at isolated time points, based on the statistical findings presented in this study.

Dendritic cells (DCs) are specialized members of the innate immune system that are responsible for the initiation of primary adaptive immune responses whose purpose is to resolve infection and inflammation. During most viral infections, mature dendritic cells present critical viral antigens to naïve T-cells within secondary lymphoid organs, resulting in the generation of an antigen-specific adaptive immune response and clearance of the virus. During infection with HIV-1 however, the virus is not cleared and a chronic systemic infection develops characterized by immune dysfunction, CD4+ T-cell depletion, systemic inflammation, and opportunistic infections. A growing body of evidence indicates that HIV-1 subversion of DCs contributes to both HIV-1 pathologies and viral dissemination. A number of similar effects by accessory HIV-1 peptides on DC physiology have also been reported. In vitro studies demonstrate that HIV-1 inhibits DC maturation and function. Ex vivo studies on the other hand describe partially mature, dysfunctional DCs collecting in secondary lymphoid organs. In vitro studies examining the effects of HIV-1-Tat and HIV-1-Vpr have described opposing effects on DC maturation. Therefore we undertook experiments to comprehensively describe the effects of HIV-1 and the Tat and Vpr accessory peptides on DC maturation and function. To understand the contributions of individual viral proteins to DC dysfunction we infected DCs with a dual tropic HIV-1 and examined phenotypic and functional changes after maturation with inflammatory cytokines. Following this we examined the influence of exogenous and endogenous HIV-1-Tat and HIV-1-Vpr on MDDC maturation and function using recombinant proteins and deletion mutant lab adapted HIV-1 strains. Live dual tropic HIV-1 was found to selectively inhibit aspects of phenotypic maturation as well as antigen capture and presentation functions. MDDC MAPK responsiveness to bacterial LPS remained intact however. Exogenous accessory HIV-1 Tat and Vpr did not affect MDDC phenotype but inhibited dextran endocytosis and viral peptide presentation. HIV-1-gp120 increased iMDDC maturation while blunting cytokine induced decreases in MDDC antigen capture abilities. The deletion of HIV-1-Tat did not affect MDDC phenotype, but was found to affect antigen capture decreases by R5 tropic HIV-1BaL. Deletion of HIV-1-Vpr likewise did not affect MDDC phenotype, however it was found to be influential in HIV-1 induced decreases in MDDC antigen presentation to autologous T-cells. These accumulated results indicate that HIV-1 subverts DC maturation and function through whole virus effects and individual accessory peptide influences. Understanding the mechanisms of DC dysfunction in HIV infection may provide some insight into infection prevention strategies and therapies leading to adaptive immune system activation and viral clearance.

Impaired cell mediated immunity is the clinical hallmark of HIV infection yet the manner in which CD8 T-cells are disabled is not yet fully understood. IL-7 signalling is essential for normal CD8 T-cell development and function. Our lab has previously shown decreased expression of the IL-7 receptor a-chain (CD127) on circulating CD8 T-cells in HIV+ patients is mediated by the HIV Tat protein which results in poor CD8 T-cell function. Soluble Tat protein is secreted by infected CD4 T-cells and taken up by neighbouring uninfected CD8 T-cells through endocytosis. Once in the cytoplasm, Tat translocates to the inner leaflet of the cell membrane where it binds directly to the cytoplasmic tail of CD127 inducing receptor aggregation, internalization, and degradation by the proteasome. By removing CD127 from the cell surface, the HIV Tat protein is able to reduce IL-7 signaling and impair CD8 T-cell proliferation and function. To determine which domain(s) in the cytoplamic tail of CD127 are required for interaction with Tat, a series of plasmids encoding for CD127 deletion mutants were successfully created. These series of mutant CD127 coding sequences were transfected into a eukaryotic expression system, the Jurakt cell line, where CD127 mutants were successfully expressed. Before determine which region on CD127 is required for Tat binding, an optimized Ni-NTA column system was used to successfully isolate histidine-tagged HIV-1 Tat at a high yield and purity from E. coli. This HIV Tat protein was used to treat the lysates of the Jurakt cells transfected with the panel of CD127 mutants. CD127 was then immunoprecipitated, followed by Western analysis of the immune complexes to detect Tat protein. Tat was immunoprecipitated with all CD127 mutants suggests neither tyrosine 449, box 1, the acidic region, serine region nor C-tail are specifically required for Tat binding to CD127.

PhD (Microbiology) / Department of Microbiology

Includes bibliographical references (leaves 33-42). / Aim: Neurological involvement related to HIV-1 infection is well described in the paediatric population and causes significant morbidity and mortality. This study aimed to describe specific neurological and neurobehavioural complications in this population. Method: Children infected with HIV-1 attending infectious diseases clinics were recruited for general and neurological assessments, developmental history screening and categorization of behavioural phenotype using the Aberrant Behaviour Checklist (ABC). Results: Eighty patients were assessed (males - 44/80: females - 36/80) (median age 5 years 1 month; range: 3 months - 12 yrs). Eighteen patients (23%) were not on antiretroviral (ARV) therapy at the time of testing. The Centre for Disease Control (CDC) immune categories of the patients at the time of assessment were: Category 1- n=6/80, Category 2- n=15/80 and Category 3- n=59/80. Thirty-three percent had a history of chronic lung disease, 10% had a history of an opportunistic central nervous system infection and 12.5% had epilepsy. 5 5 Anthropometric measurements identified that 19% of the patients were microcephalic, 17% of the patients were < 60% of their expected weight, 49% were 60-80% of expected weight and 45% were stunted. On neurological assessment 41% of the patients had global pyramidal tract signs, 7% had a hemiparesis, 5% had peripheral neuropathy, 16% had visual impairment, and 6% were hearing impaired. Of those who were screened for developmental deficits (patients < 6years of age) 66% had gross motor delay, 75% had fine motor delay, 70% had language delay and 73% had cognitive delay. Forty one percent had HIV Encephalopathy, 81% of whom a CD4 count < 15% and 48% were < 1year old. On the aberrant behaviour checklist (ABC) scale 24/80 patients had features of hyperactivity and 22/80 patients scored in the mild-moderate range on the lethargy / social withdrawal sub-scale reflecting a correlation with the affective and adjustment disorders. Conclusion: Diverse neurological and neurobehavioural deficits are common in children with HIV-1 infection especially those with CD4 < 15%, not on ARVs, with growth impairment and < 1yr of age. This study demonstrated the extent and spectrum of neurobehavioural and neurological complications in a defined HIV population. It stresses the need for early initiation of ARVs in the planning for future regimens and guidelines.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg 2014 / Human receptor CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes where it plays a key role in the activation of immunostimulatory T cells. This function is diverted by the Human Immunodeficiency Virus (HIV) envelope glycoprotein (gp120), which uses CD4 as its primary receptor for cell entry. The requirement of CD4 for viral entry has rationalised the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. While growing evidence suggests that redox exchange reactions involving CD4 disulphides (potentially catalysed by cell surface-secreted oxidoreductases) play an essential role in regulating the activity of CD4, their mechanism(s), biological utility and structural consequences that may be applicable to the designs of novel antiviral therapies and vaccines remain incompletely understood. Herein, a novel recombinant CD4 protein designed to bind gp120 through a targeted disulphide-exchange mechanism is described. This molecule contains a conservative Ser60 to Cys mutation on the CD4 domain 1 α-helix which, according to theoretical crystal structure modelling, positions a thiol in close proximity of the gp120 V1/V2 loop disulphide (126Cys–Cys196) resulting in the formation of an interchain disulphide bond. Experimental evidence for this effect is provided by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). This 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulphide exchange and this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. To gain more insights into the importance of redox activity in the mechanism of HIV entry, a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, were used to show that Thioredoxin (Trx), an oxidoreductase found on the cell surface, reduces 2dCD4 highly efficiently, catalysing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulphide-linked 2dCD4 dimer. HIV-1 gp120 was shown to be incapable of binding a fully oxidised, monomeric 2dCD4 in which both domain 1 and 2 disulphides are intact, but binds robustly to reduced equivalents that are the products of Trx-mediated isomerisation. This Trx-driven dimerisation of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is shown to be impaired when CD4 is bound to gp120. Finally, preliminary, low-resolution structural analysis of individual CD4 domains 1 and 2 are suggestive of intrinsic metastability in domain 2, and reduction of its resident allosteric disulphide bond likely underpins the structural rearrangements in CD4 that are required for efficient interaction with gp120. Overall, these findings emphasise the fundamental importance of redox pathways in the biochemical mechanism of HIV entry, and illustrate the feasibility of exploiting these for the development of novel antiviral ligands.

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