Background/Rationale: Human lymphocyte antigen (HLA)-restricted cytotoxic T lymphocytes (CTL) target HIV infected cells expressing cognate HlV-1 epitopes. Numerous studies have examined the dynamics of CTL response and escape mutations following acute infection and disease progression. CTL escape mutations are typically associated with a fitness """"""""cost"""""""", inferred by the reversion of these mutations in absence of CTL selection pressure. It is important to note that multiple factors may contribute to in vivo fitness but fitness """"""""cost"""""""" is typically defined as a decrease in replicative capacity within target cells. Thus, this """"""""cost"""""""" is often ascribed to (DTL escape mutations due to their absence in the CTL-susceptible or """"""""wild type"""""""" sequence but few studies have actually empirically examined replicative fitness of HIV-1 escape variants. During the current grant period we accessed the costs of CTL escape mutations on replicative fitness of the infecting HIV-1 isolate. In general, we observed that in a subject's HIV-1 population, CTL escape mutations impart different fitness costs. CTL escape mutations in Gag emerged later in infection and were associated with higher fitness costs than the CTL escape mutations selected in Env (go to article). These pilot studies involved HlV-1 sequence and IFN-y ELISpot analyses to first identify 19 escape and compensatory mutations that emerged in two epitopes encoded by gag and five in env during infection of SEAPIP subject 1362 (PIC1362). Each of the 19 mutations was then individually introduced into the autologous HlV-1 gag and env genes from the founder virus. Fitness analyses were performed using direct competitions between the chimeric viruses, which harbored the subject gag or env genes with or without the CTL escape mutations. This is the most comprehensive analysis to date that examined the interplay between CTL response, selection of escape variants, and the associated fitness costs. The findings were quite striking in that 8/9 CTL escape mutations in env were either neutral or resulted in a fitness increase, rather than any loss in replicative capacity. Based on our initial findings, we have structured this renewal proposal to first compare the cost of CTL escape mutations in more conserved versus more heterogeneous HIV-1 coding regions throughout the genome (AIM 1). We propose that low genetic diversity and limited functional plasticity within HlV-1 coding regions will slow the appearance of CTL escape mutations due to higher fitness costs. Even in the same epitope under the same HLA-restriction, divergent HIV-1 isolates often take distinct evolutionary pathways towards different CTL escape mutations. Thus, In AIM 2, we will explore how specific CTL escape mutations in an HLA A3, A25, B27, and B57-restricted p24 epitopes affect replicative fitness of a virus derived from the autologous capsid (CA) p24 sequence, heterologous p24 sequence (but from a subject sharing the same restricting HLA allele), and of a laboratory strain. Again, preliminary data would suggest a minimal fitness impact in the autologous versus heterologous background and emphasize the need to study these CTL escape mutations in the native context. Given the protective effects of B57 and B27 HLA alleles, we will also assessed whether CTL escape mutations confer higher fitness costs in unique B57- or B27-specific epitopes. Our preliminary studies on drug resistant and CTL escape mutations suggest that the entire HIV-1 genome evolves to compensate for any fitness loss. For this reason, fitness costs are often over-emphasized when analyzed in a heterologous HIV-1 background.
In AIM 3, we examine how a loss in replicative fitness conferred by CTL escape mutations will be compensated by mutations within the targeted gene and throughout the genome. Specifically, we have found that the HIV-1 env gene and the efficiency of host cell entry is dominant over all other HIV-1 genes and replication steps in determining fitness. In fact, we found that many drug resistant HIV-1 isolates maintain high replicative fitness by increasing the efficiency of host cell entry to compensate for a loss in protease (PR) or reverse transcriptase (RT) activity. The same scenario may exist with CTL escape mutations.
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