The population of genetically diverse viruses that develops in each infected individual confers on HIV-1 the capacity to rapidly and relentlessly adapt to host immune responses. Such immune adaptation contributes to ineffective immune control of viral replication [14
], and experiments in the SIV/macaque model underline that CTL escape will pose a significant threat to the durability and efficacy of vaccine-induced CD8+ T cell responses [16
]. To properly understand immune adaptation of HIV-1, it is critical to characterize the dynamics of viral evolution during acute infection when numerous adaptive changes take place. Recent studies examining near full-length genomes [12
] showed that at peak viremia the virus population remains virtually homogeneous with significant evolution occurring by week 6 of infection, including a dramatic increase in CTL immune-adaptive mutations, particularly in highly variable proteins such as Env and Nef [12
]. Interestingly, multiple mutations in targeted CTL epitopes were observed in both studies. Herbeck et al. [12
] noted that such mutations were often mutually exclusive, which suggests that the virus population can explore multiple adaptive pathways but that there are limits to the plasticity of individual viruses, even in highly variable proteins. Although such intensive longitudinal analyses of viral evolution during acute/early infection have been limited to a small number of individuals, next-generation sequencing technologies promise to scale up these efforts and to increase our understanding of early HIV-1 adaptations to host selection pressures. Chip-based sequencing technologies such as pyrosequencing (“454”) [19
] and four-color cyclic reversible termination sequencing (CRT) (“Illumina”) [20
] offer rapid and cost-effective production of up to 10,000-fold sequencing read coverage of the HIV-1 genome. This represents a substantial improvement over the costly and time-consuming amplification and sequencing of individual amplicons for the analysis of viral genetic variation. Indeed, the depth of coverage provided by these approaches allows the quantification of genetic variants at frequencies that are undetectable by standard sequencing approaches, and the increased sensitivity provides an unprecedented view of the earliest events in the adaptation of HIV-1 to frontline immune responses mounted during acute infection. The relatively short read lengths may, however, limit the utility of next-generation sequencing technologies for establishing linkage of mutations except over short regions of the genomes.
To date, next-generation sequencing of HIV-1 has predominantly been used to identify and quantify antiretroviral drug resistance mutations at frequencies below the limit of detection of commercial drug resistance genotyping assays (to approximately a 1% level), in order to understand the impact of minor variant resistance mutations on treatment outcomes [21
]. Recent efforts have been applied to HIV-1 evolution during the acute/early phase of infection. For example, by comparing chip-based pyrosequencing to conventional sequencing for the analysis of the evolution of specific CTL epitopes during acute/early infection in longitudinal samples from three patients, Fischer et al. [25
] showed that next-generation sequencing captured a greater degree of genetic diversity and detected CTL escape variants earlier than conventional approaches. The detection by “deep” sequencing of viral CTL escape variants earlier in infection than previously appreciated, e.g., as early as 17 days post-infection, was also recently reported in the SIV/macaque system [26
]. Next-generation sequencing also allows the characterization of CD8+ T cell epitopes that “shatter” during viral escape, i.e., exhibit multiple, highly variable, low frequency escape mutations, before coalescing on a single escape pathway, versus epitopes that are limited to escape at a single residue [27
]. As such, deep sequencing approaches are now capable of distinguishing between the early pathways of viral escape within different CTL epitopes, revealing critical information about the ease with which HIV-1 can evade these responses.
The recent development of novel assembly, alignment, and analysis tools has enabled high-throughput, next-generation sequencing of near full-length viral genomes (Henn and Allen, unpublished; Deng and Mullins, unpublished). This approach revealed that the earliest escaping epitopes were in more variable proteins of the virus (Vif, Env and Nef) [27
], and corresponded to highly immunodominant responses (Henn and Allen, unpublished). These data suggest that viral escape may be tied more closely to the kinetics and specificity of the immune response, than the functionality of the response. These advances will allow future studies to extend beyond analyses of the evolution of specific epitopes in a small number of samples to full-scale, unbiased screening of HIV-1 evolution in large, cross-sectional and longitudinal cohorts. Although individual-based studies are critical for providing fine detail of the interactions between specific viruses and specific host immune responses, population-based studies are necessary to identify common patterns of HIV-1 escape from the earliest immune responses, and more specifically, to define attributes associated with effective control of HIV-1.