Viral diversification has been attributed to either increased viral replication or immunologic pressure. A strong immunologic response would be expected to restrict viral replication, so that viral diversification might not be evident but, if partially effective, it might result in viral escape, as demonstrated by an evolution of quasispecies. Since our patients were unable to restrict viral replication to <1,000 copies, we expected to see such evolution. Our observation that viral diversification was the greatest in subjects with the best viral suppression (i.e., a reduction of >0.7 log units), is not consistent with higher viral replication driving viral diversification. We had expected that a change in HIV envelope diversity would correlate with the vigor or the Env-specific CD8 response. Although we could not demonstrate a correlation between increased envelope diversity, as measured by the heteroduplex tracking assay (HTA) assay, and enhanced Env-specific IFN-γ responses to a heterologous Env vector, the sum of the responses to all four vectors (i.e., Env, RT, Gag, and Nef) suggested such a correlation, particularly in the 3 subjects with the largest change in viral load. The considerable differences that exist in the envelope sequence presented by our vaccinia vector from sequences expressed by autologous viruses may explain the observed lack of correlation. The frequency of responses to Env for 5 of the 8 subjects was very low. Also, studies comparing CD8 responses to other variable viral epitopes show enhanced responses to autologous peptides relative to heterologous peptides (1
). Ideally, vaccinia vectors that expressed autologous Env proteins or the use of short peptides that reflected autologous Env antigens would have been useful to better assess CD8 responses to autologous Env viruses. However, this approach was limited by the financial constraints of the study. Alternatively, the selection may be due to other immune pressures, such as escape from neutralizing antibody in the envelope region of HIV (15
). Neutralizing antibody to autologous virus, a potential cause of viral diversification, could not be tested in our study. This approach was attempted via collaboration with Monogram Biosciences, but many of the viral isolates were unable to be amplified for insertion into their vector.
Lack of correlation of immune pressure with one aspect of cellular immune response (i.e., IFN-γ production) and quasispecies selection is not entirely surprising. Lytic CD8 T cells may be required (13
), a response that may be dissociated from IFN-γ release (9
). Lu et al. studied vaccination with zinc finger-inactivated autologous virus presented by matured dendritic cells to stimulate HIV-specific immune responses and demonstrated an 80% decrease in viremia, with suppression persisting for as long as a year (11
). The response that correlated best with viral suppression was Gag-specific CD8 perforin release. However, the correlation between cellular responses to other HIV antigens (i.e., RT and Gag) with less variation in sequences from autologous virus suggests that cellular immune pressure may have played a role in some of the individuals. Other studies attempting to correlate virologic control in chronically infected individuals with IFN-γ responses to Env antigens have also not found an association, despite the fact that responses to Gag antigens were associated with control (2
). Virologic escape may also correlate with responses to other antigens (4
). This can explain increased HTA changes seen in some of our subjects with the greatest responses to the 4 antigens. Decreased viremia in those with the largest diversity may also reflect restrained viremia as a consequence of this escape (12
). Further studies, involving a larger cohort of individuals, elucidating the relationship between quasispecies' evolution and the pressure exerted on the virus by the various immune responses, may contribute to a better selection of vaccine candidates.