Molecular characterization of HIV-1 Subtype C strains from KwaZulu-Natal, South Africa, with a special emphasis on viral fitness and drug resistance.
Gordon, Michelle Lucille.
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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.