The pathogenesis of persistent low-level viremia in patients on HAART is unclear, but it is important to understand to improve control of viral replication in patients on long-term HAART. Evidence exists for both ongoing virus expression from latent cellular reservoirs (or drug sanctuary sites) (2
) and low-level virus replication during HAART (6
). While these two mechanisms may not be mutually exclusive, it is likely that interpatient differences in the potency of suppression of virus replication below 50 copies/ml shift the contributions of virus release from long-lived infected cells and complete cycles of replication to sustaining residual viremia in the context of HIV-1 immunizations.
In this first study to examine serially the effects of immune activation with recombinant MVA/FPV-based HIV-1 vaccinations on low-level viremia in HAART-treated patients, we found evidence for both complete cycles of virus replication, with sequence evolution, in a subset of patients receiving durable successful HAART (current clinical criterion of a plasma viral load of <50 copies/ml) and virus expression from a latent reservoir. In particular, sequence evolution in free plasma virus was significantly more likely to occur within relevant CD8+
T-cell epitopes of HIV-1 RT and consisted of novel amino acid changes at a non-drug-resistance site, position 205 of HIV-1 RT, as well as new relevant drug resistance mutations. Furthermore, we found that sequence evolution following HIV-1 vaccinations was significantly associated with more episodes of quantifiable low-level viremia, suggesting ongoing cycles of virus replication that may be amenable to CD8+
T-cell selective pressures. At baseline, the subjects were heterogeneous with respect to the predicted potency of their HAART regimens (41
) and the presence of drug resistance mutations, but their respective HAART regimens had controlled plasma viremia to clinically undetectable levels for years before study entry. Furthermore, plasma viral loads remained low postvaccination even in the presence of relevant new drug resistance mutations or evidence of sequence evolution at non-drug-resistance sites during low-level viremia, except for in one subject. Importantly, for most subjects in this study, administration of MVA/FPV HIV-1 therapeutic vaccines was not associated with temporal shifts or sequence changes within persistent wild-type or drug-resistant plasma viral sequences in very low viremia or with new drug resistance mutations for up to 18 months postvaccination. The 40% of study participants without amplifiable variants prevaccination also did not have amplifiable genotypes postvaccination and thus did not provide evidence of either enhanced virus production or replication following HIV-1 vaccination. Virus replication was even controlled for the individuals for whom NNRTI-resistant variants were detected postvaccination, despite their remaining on the same nevirapine-based HAART regimen as that at study entry. While they are important for the safety profile of these vaccines (18
), these observations also support the clinical significance of HAART to limit HIV-1 replication in most patients. In particular, the current clinical criterion of <50 copies/ml indicates that HAART has significantly reduced the effective virus population size, thereby slowing virus replication and sequence evolution in most individuals (39
Because recent studies of treatment intensification have shown variable effects on low-level viremia (6
), study-to-study variation in the baseline level of viremia must be considered. In one study, baseline viremia was quite low, at 1 to 2 copies/ml, most likely reflecting the potency of the regimen, and no evidence of additional suppression was generated by the addition of raltegravir (24
). In contrast, abacavir intensification of an efavirenz and indinavir two-drug regimen in subjects with a baseline viral load of 10 copies/ml produced a lowering of the viral load to <2.5 copies/ml (22
). Our data demonstrate discernible sequence evolution in HAART-suppressed subjects experiencing low-level viremia of >6.5 copies/ml. Together, these findings suggest that a threshold level of viremia below 50 copies/ml may exist, above which productive infection may be a greater contributor to sustained viremia than low-level virus release from long-lived cellular reservoirs. Under this model, the sequence evolution we observed during therapeutic HIV-1 poxvirus vaccination, in or near epitopes recognizable based on HLA serotypes, would be facilitated by steady-state, low-level, productive infection. To test this will require further study of patients receiving newer, more potent HAART regimens than those used by the subjects in our study. Nevertheless, the data suggest that the current therapeutic target of control of viremia to <50 copies/ml, while clinically meaningful, is insufficient for distinguishing contributions of low-level virus expression from those of productive infection. A new therapeutic target for viremia may be necessary for clinical trials assessing HIV-1 treatment approaches involving newer drug combinations, therapeutic vaccinations, or treatment intensification and deintensification strategies.
Among nonimmunized individuals, substitution at position 205 of HIV-1 RT is exceedingly rare, appearing in only a single HIV-1 subtype C isolate (L205S) among 914 HIV-1 sequences in the Los Alamos HIV Sequence Database (http://www.hiv.lanl.gov
). Amino acid position 205 of HIV-1 RT lies within an HLA-A*02 cytotoxic T-lymphocyte epitope, KIEELRQHL (48
), and three of four subjects with L205 changes were HLA-A*02 positive (subjects 7, 15, and 17) in our study. While the fourth (subject 11) was HLA-A*6801 and -A*3001 positive, and not HLA-A*02 positive, the L205M substitution was also flanked by HLA-A*68 (FTTPDKKHQK) (9
) and HLA-A*03 (DLEIGQHRTK) (48
) epitopes. It is notable that HLA-A*6801 could share binding to these regions as an A*03 supertype (Fig. ). Additionally, in the one subject (subject 8) who developed new drug resistance mutations in low-level viremia postvaccination, the first NNRTI resistance mutation detected (V108I) lay within an HLA-A*02-restricted epitope (VLDVGDAYFSV) (47
) that was linked to an isoleucine-to-threonine substitution at amino acid position 135 of HIV-1 RT (I135T), known to confer decreased susceptibility to NNRTI (5
). This substitution also represents an amino acid change within relevant HLA-A*02 and -B*51 epitopes (KYTAFTIPSI and TAFTIPSI, respectively) (40
) for this individual, who was indeed HLA-A*02 and -B*51 positive (Fig. ). While the NNRTI resistance mutations were transient, the I135T substitution became fixed and persisted through week 72 of the trial, suggesting selection due to either immune or drug pressure. Moreover, in the one subject who developed rebound viremia while on HAART, decreasing sequence diversity in low-level viremia was associated with sustained CD8+
T-cell responses to HIV-1 Pol postvaccination, also suggesting specific HIV-1 immune selective pressure on residual replication during HAART. This idea is supported by reports of selection of immunity-escaping HIV-1 variants at clinically undetectable viral loads in long-term nonprogressors (4
Together, these findings imply that viral populations replicating at low levels may be targeted by HIV-1-specific immune responses in HAART-treated patients. We were unable to demonstrate that CD8+
T-cell responses to the peptide containing position 205 of HIV-1 RT were responsible for sequence evolution, though a finely detailed analysis of CD8+
T-cell recognition with overlapping peptides was not performed. Finer mapping of immunological responses, including interleukin-2-mediated responses (3
), to this region of HIV-1 RT may help to elucidate the role of CD8+
T-cell selective pressures in L205 substitutions.
This study has some limitations. These include restriction of genetic analysis to the HIV-1 RT region, the sensitivities of the low-level viremia genotyping and viral load assays, the lack of a placebo group to fully assess sequence evolution at viremia levels of <50 copies/ml within HLA-restricted CD8+
T-cell epitopes, and the small study population. Nevertheless, we identified in vivo selection and convergent sequence evolution associated with novel mutations within HLA epitopes in residual viremia among a subset of individuals receiving recombinant HIV-1 MVA/FPV vaccinations who had viral loads of <50 copies/ml on durable successful HAART. Moreover, this selection occurred in association with higher frequencies of episodes of low-level viremia above 6.5 copies/ml. The data support contributions of both low-level virus release (2
) and productive infection (6
), which suggests that a low-level threshold may exist where the balance shifts for these two contributors. Although reinforcing the benefits of HAART suppression of viremia levels of <50 copies/ml, this potential model suggests that a new therapeutic target for viremia may be necessary for clinical trials assessing newer treatment approaches for HIV-1, including vaccines. Furthermore, the potential for virus replication and sequence evolution within epitopes presented by relevant HLA sites observed in this study has implications for therapeutic HIV-1 vaccines. Both of these implications warrant further study.