Fitness Cost of Escape Mutations in p24 Gag in Association with Control of Human Immunodeficiency Virus Type 1

Abstract

Mutational escape by human immunodeficiency virus (HIV) from cytotoxic T-lymphocyte (CTL) recognitionis a major challenge for vaccine design. However, recent studies suggest that CTL escape may carry a sufficient cost to viral replicative capacity to facilitate subsequent immune control of a now attenuated virus. In order to examine how limitations can be imposed on viral escape, the epitope TSTLQEQIGW (TW10 [Gag residues 240 to 249]), presented by two HLA alleles associated with effective control of HIV, HLA-B*57 and -B*5801, was investigated. The in vitro experiments described here demonstrate that the dominant TW10 escape mutation, T242N, reduces viral replicative capacity. Structural analysis reveals that T242 plays a critical role in defining the start point and in stabilizing helix 6 within p24 Gag, ensuring that escape occurs at a significant cost. A very similar role is played by Thr-180, which is also an escape residue, but within a second p24 Gag epitope associated with immune control. Analysis of HIV type 1 gag in 206 B*57/5801-positive subjects reveals three principle alternative TW10-associated variants, and each is strongly linked to concomitant additional variants within p24 Gag, suggesting that functional constraints operate against their occurrence alone. The extreme conservation of p24 Gag and the predictable nature of escape variation resulting from these tight functional constraints indicate that p24 Gag may be a critical immunogen in vaccine design and suggest novel vaccination strategies to limit viral escape options from such epitopes.

Availability of a Diversely Avid CD8+ T Cell Repertoire Specific for the Subdominant HLA-A2-Restricted HIV-1 Gag p2419–27 Epitope

“…In particular, the extreme conservation and intolerance of escape variations in Gag p24 indicate that it may be a critical target for vaccine design (35,36). There is only one well-defined HLA-A2-restricted epitope in p24, abbreviated as TV9 (TLNAWVKVV, Gag p24 19 -27 ).…”

Clinical Significance of Human Immunodeficiency Virus Type 1 Replication Fitness

“…The hypothesis that CTL escape mutants have reduced fitness is supported by studies of simian immunodeficiency virus in which cloned CTL escape mutants obtained from a macaque showed slowed growth in cell culture and reversion to the CTL-susceptible wild-type sequence after inoculation into macaques lacking the major histocompatibility class I determinants necessary for the recognition of the mutant epitopes (62). Studies of escape mutants in the Gag p24 protein demonstrated that these mutations do confer substantial reductions in replication fitness, as measured in a multiplecycle cell culture assay (117,134). It is interesting that these escape mutants, when they occur, are seen primarily in patients with HLA types that are associated with the successful control of HIV viremia.…”

“…Some examples of measuring virus directly include quantitation of a viral protein, such as p24, by enzyme-linked immunosorbent assay (106,156,163); measurement of reverse transcriptase activity (164, 175); and quantitation of proviral DNA (116). In addition, in growth competition assays, the relative amounts of test and reference strains can be quantified by sequence analysis of proviral DNA or viral RNA (65,69,78,86,117,154,155,169,174), heteroduplex tracking assays (HTAs) (140), or allele-specific real-time PCR (33,142,176). It should be noted that the latter methods do not provide information on the extent of viral replication.…”

“…Sequence analysis has also been used to quantify the relative amounts of the two variants in a growth competition assay either by direct sequence analysis of the bulk PCR product (65,69,78,86,117,154,155,174) or by analysis of individual clones derived from the PCR amplicon (119). One disadvantage of bulk sequencing is that differences in the surrounding sequence may influence the assay's sensitivity for detecting a minority variant at a given codon.…”

“…An assay used by several laboratories is a multiple-cycle recombinant-virus growth competition assay in which the relative proportion of test and reference strains is measured using either bulk or clonal sequence analysis (4,65,69,78,86,117,119,154,155,174). This type of assay is labor-intensive and difficult to apply to large numbers of clinical samples but has been commonly used to characterize the relative fitness of site-directed drug-resistant mutants of HIV-1 in protease and reverse transcriptase.…”