Understanding of the viral and host factors that determine time for progression to acquired immunodeficiency syndrome (AIDS) in individuals infected with human immunodeficiency virus type 1 (HIV-1) could aid in the design of an effective HIV-1 vaccine. Human leukocyte antigen (HLA) class I profile is strongly and consistently associated with differential rates of HIV-1 disease progression, however the mechanisms explaining this are not well understood. It has been hypothesised that “protective” HLA alleles select escape mutations in functionally important epitopes in the conserved group specific antigen (Gag) protein resulting in HIV-1 attenuation, which may result in slower disease progression. Many of the studies investigating the fitness cost of Gag escape mutations have concentrated on a few pre-selected mutations and have not assessed fitness consequences in the natural sequence background. Furthermore, the majority of studies have focussed on HIV-1 subtype B, while HIV-1 subtype C is the most prevalent subtype worldwide. Therefore, in the present study, a large population-based approach and clinically-derived Gag-protease sequences were used to comprehensively investigate the relationship between immunedriven sequence variation in Gag, viral replication capacity and markers of disease progression in HIV-1 subtype C chronic infection. The influence of Gag function on HIV-1 disease progression was further investigated in early HIV-1 subtype C infection. It was also hypothesised that Gag may contribute significantly to overall HIV-1 fitness and towards fitness differences between HIV-1 subtypes. Materials and Methods Recombinant viruses encoding Gag-protease, derived from antiretroviral naïve HIV-1 subtype C chronically (n=406) and recently (n=60) infected patients as well as a small subset of HIV-1 subtype B chronically infected patients (n=25), were generated by electroporation of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positively of an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positivelyof an HIV-inducible green fluorescent protein (GFP)-reporter T cell line with plasmaderived gag-protease PCR products and linearised gag-protease-deleted NL4-3 plasmid. The replication capacities of recombinant viruses, as well as intact HIV-1 isolates from peripheral blood mononuclear cells of patients chronically infected with HIV-1 subtype C (n=16), were assayed in the GFP-reporter T cell line by flow cytometry. Replication capacity was defined as the slope of increase in percentage infected cells from days 3-6 following infection, normalised to the growth of a wild-type NL4-3 control. Replication capacities were related to patient HLA alleles and markers of disease progression (viral load, CD4+ T cell count, and rate of CD4+ T cell decline in chronically infected patients, and viral set point and rate of CD4+ T cell decline in recently infected patients). Replication capacities were compared between isolates and recombinant viruses encoding Gag-protease from the same isolates, as well as between HIV-1 subtype B and C recombinant viruses matched for viral load and CD4+ T cell count. Bulk sequencing of patient -derived gagprotease amplicons was performed and mutations were identified that were significantly associated with altered viral replication capacity. The fitness effect of some of these mutations was directly tested by site-directed mutagenesis followed by assay of the mutant viruses. Results In HIV-1 subtype C chronic infection, protective HLA-B alleles, most notably HLA-B*81 (p<0.0001), were associated with lower replication capacities. HLA-associated mutations at low entropy sites (i.e. conserved sites) in or adjacent to Gag epitopes were associated with lower replication capacities (p=0.02), especially the HLA-B*81-associated 186S mutation in the TL9 epitope (p=0.0001). The fitness cost of this mutation was confirmed in site-directed mutagenesis experiments (p<0.001), and the co-varying mutations tested did not significantly compensate for this fitness cost. Replication capacity also correlated positively with baseline viral load (p<0.0001) and negatively with baseline CD4+ T cell count (p=0.0004), but not with subsequent rate of CD4+ T cell decline (p=0.73). In HIV-1 subtype C recent infection, replication capacities of the early viruses did not correlate with subsequent viral set points (p=0.37) but were significantly lower in individuals with below median viral set points (p=0.03), and there was a trend of correlation between lower replication capacities and slower rates of CD4+ T cell decline (p=0.09). Overall, the proportion of host HLA-specific Gag polymorphisms in or adjacent to epitopes was negatively associated with replication capacities (p=0.04) but host HLA-B-specific polymorphisms were associated with higher viral set points (p=0.01), suggesting a balance between effective Gag CD8+ T cell responses and viral replication capacity in influencing viral set point. A moderate statistically significant correlation was found between the replication capacities of whole isolates and their corresponding Gag-protease recombinant viruses (p=0.04) and the replication capacities of the subtype C recombinant viruses were significantly lower than that of the subtype B recombinant viruses (p<0.0001). The subtype-specific difference in the consensus amino acids at Gag codons 483 and 484 was found in site-directed mutagenesis experiments to largely contribute to the fitness difference between subtypes, possibly by influencing budding efficiency. Discussion The data support that protective HLA alleles, in particular HLA-B*81, attenuate HIV-1 through HLA-restricted CD8+ T cell-mediated selection pressure on Gag. Results suggest that viral replication capacity determined by sequence variability in Gag-protease has an impact on HIV-1 disease progression, but also indicate that a balance between HLA-driven fitness costs and maintenance of effective CD8+ T cell responses is important in determining clinical outcome. Gag-protease was observed to significantly contribute to overall HIV-1 replication capacity and variability in this region between HIV-1 subtypes B and C is suggested to partly explain the difference in viral fitness between these subtypes. Specific mutations in Gag-protease associated with viral attenuation were identified and it was also observed that mutations in conserved Gag regions carried the greatest cost to HIV-1 replication capacity. Overall, the data support the concept of, and may assist in the rational design of, an HIV-1 vaccine in which immune responses are directed towards several conserved epitopes, particularly in Gag, with the aim to constrain immune escape (thereby maintaining effective CD8+ T cell responses) and attenuate HIV-1 (in the event of partial escape), resulting in slower disease course and reduced HIV-1 transmission at the population level. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2011.

As South Africa begins its National HIV-1 treatment program, it is urgent that we collect data that will help define the phylogenetic relationships, transmissibility and drug responsiveness of C viruses. In this thesis, data is presented on the genetic diversity of locally circulating drug naive subtype C strains, as an indication of their natural susceptibility to antiretroviral drugs, prior to the national roll-out of antiretroviral therapy. At the time this thesis was initiated, antiretroviral therapy was only available in South Africa in a few clinical trials and in the private sector, and it was therefore difficult to obtain large numbers of samples from treatment-experienced patients. Nevertheless, valuable information on the prevalence and patterns of resistance mutations in subtype C infected patients was obtained from small studies on patients receiving HAART, concomitant HAART and TB treatment, HAART and treatment for Kaposi Sarcoma, and single dose nevirapine for the prevention of mother-to-child transmission of HIV-1 infection. The results show that the general antiretroviral drug naive population do not harbour any major resistance-associated mutations to the currently available protease and reverse transcriptase inhibitors, with no differences in genetic variation between the different ethnic groups infected with subtype C. Phenotyping of some of these isolates showed that they were susceptible to the available protease and reverse transcriptase inhibitors, and hyper-susceptible to the protease inhibitor, Lopinavir. Phylogenetic analysis of recent and retrospective subtype C isolates showed that there are multiple lineages of subtype C viruses circulating in South Africa, indicative of multiple introductions of subtype C across its many borders. Polymorphisms in the protease, reverse transcriptase and C2-V5 region of envelope in these drug naive samples lead to significant variation in the number, type and location of potential phosphorylation sites. There was also variation in the cleavage sites controlling the initiation and rate of Gag and Gag-Pol processing (p2/NC) and the activation of protease (TFP/p6gag) suggesting that there may be important differences in the way that B and C viruses regulate polyprocessing and virion assembly. Similar to studies on subtype B, 10 to 18% of the patients on HAART developed drug resistance. However, those on concomitant HAART and TB treatment developed resistance as early as one month after starting treatment. Generally, the resistance mutations that were seen were consistent with those seen in treatment experienced subtype B isolates. Of note was the high level of resistance to the entire class of NNRTIs. This could be reflective of the predominant use of NNRTI-based regimens, as well as the low genetic barrier in this class of drugs. The NNRTI mutations included the V106M mutation that is considered a signature mutation of EFV experienced subtype C isolates. Resistance was high (40%) in mothers and infants 6 weeks after each received a single dose of NVP. K103N was most common mutation in the mothers, while Y181C was most common in the infants. Of note were the changes in functional properties caused by these mutations, by the introduction or alteration of putative myristoylation and phosphorylation sites in the RT. Taken together, these data suggests that the pattern of resistance in African patients will be similar to that observed for the treatment of subtype B infection. However, patients should be closely monitored for viral rebound very early on in treatment. Also, given the high rate of resistance in mothers and infants after single dose NVP, the search for safer regimens to prevent MTCT should be intensified. Although the mechanisms are unknown, our results indicate that several of the phosphorylation-related substitutions in the pol and env genes of KZN and other C viruses are highly conserved and positively selected. It will be important to determine whether these sites play an important role in the replicative capacity and proteolytic processing of C viruses, and in viral entry. These data provide important benefits for public health policy and planning and for future patient treatment management. / Thesis (Ph.D.)-University of KwaZulu-Natal, Durban, 2004.

"February, 2002" / Includes bibliographical references (leaves 156-182) / xiv, 182, [26] leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Molecular Biosciences, 2002

The development and optimization of anti-viral screening methods are essential to develop newer more effective, treatments against HIV. The XTT method is a widely described method for antiviral screening. Both continuous HIVinfected cells and experimentally infected T-cells have been used in the XTT assay. We compared these methods to screen several plant-derived extracts for cytotoxicity. Several considerations were taken into account when performing these tests (effect of media, solvents and plant enymes). Experiments were performed to investigate these effects. In addition, p24 and viral load quantification were compared as antiviral screening methods. The study showed that several modifications were necessary when performing the XTT assay on plant extracts, due to the effect of media, solvents and plant enymes. The XTT assays and p24 assays performed using experimentally infected cells are far more specific than those using chronically infected cells. The use of viral loads as an antiviral screening method consistently demonstrated the expected efficacy of AZT. / Thesis(MMed.)-University of KwaZulu-Natal, 2009.