Background HIV-1 has been shown to possess mechanisms to evade its host's immune system, especially pressure from cytotoxic T-lymphocytes (CTL) (1,2,3,4). One mechanism of such viral escape is epitope mutation, which may hamper CTL function by way of non-recognition or antagonism (1). The presence or absence of escape mutations is determined by the net interaction of immune pressure and fitness costs for each potential epitope escape mutation (5). For example, virions that are recognized as “fit” in the absence of immune pressure may not be allowed to replicate when confronted by CTLs specific for its epitopes. On the other hand, mutants that are poorly recognized by CTLs but suffer severe fitness loss also fail to replicate. To carry an overall fitness advantage, a potential epitope mutation must gain an advantage in evading CTLs that outweighs the loss in fitness sustained by the change. Fitness costs are probably determined by the direct impact of mutations on the ability of HIV-1 to replicate or the indirect effects on viral functions important for persistence in vivo (5). Despite high rates of viral replication and mutation, which ensures HIV-1 diversity in vivo, such mutations have been inconsistently observed (2). The aims of this study are to: (1) verify that CTLs exert immune pressure on HIV-1 in vivo, resulting in escape mutations (2) to create conditions for the virus to replicate in the absence of immune pressure in vitro; and (3) to demonstrate viral reversion in vitro to its “fittest” state in the absence of such immune pressure.
Methods We obtained blood from HIV-positive individuals, isolated Peripheral Blood Mononuclear Cells (PBMC), and cultured them with interleukin-2 (IL-2) for 2 weeks. After harvesting these cells, we isolated HIV-1 virus from the supernatant and used it to infect CD4+ T-cells from an uninfected individual in vitro. We cultured these cells with interleukin-2 (IL-2) for 2 weeks, and harvested both the supernatant and cells. At this stage (1) virus was isolated for another round of infection, and (2) genomic DNA was isolated for sequencing. Selected fragments, corresponding to previously determined epitopes, were amplified using PCR and ligated into plasmid vectors for bacterial transformation. After obtaining multiple bacterial colonies, we used the plasmid inserts for sequencing analysis. This procedure was repeated at weeks 2, 4, 6, 8, and 10 for 3 different patients.
Data In one patient, multiple amino acid reversions in 2 epitopes over a 10 week period. Further data is currently being analyzed.
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