Identification of ongoing human immunodeficiency virus type 1 (HIV-1) replication in residual viremia during recombinant HIV-1 poxvirus immunizations in patients with clinically undetectable viral loads on durable suppressive highly active antiretroviral therapy

Abstract

In most human immunodeficiency virus type 1 (HIV-1)-infected individuals who achieve viral loads of <50 copies/ml during highly active antiretroviral therapy (HAART), low levels of plasma virus remain detectable for years by ultrasensitive methods. The relative contributions of ongoing virus replication and virus production from HIV-1 reservoirs to persistent low-level viremia during HAART remain controversial. HIV-1 vaccination of HAART-treated individuals provides a model for examining low-level viremia, as immunizations may facilitate virus replication and sequence evolution. In a phase 1 trial of modified vaccinia virus Ankara/fowlpox virus-based HIV-1 vaccines in 20 HIV-infected young adults receiving HAART, we assessed the prevalence of low-level viremia and sequence evolution, using ultrasensitive viral load (<6.5 copies/ml) and genotyping (five-copy sensitivity) assays. Viral evolution, consisting of new drug resistance mutations and novel amino acid changes within a relevant HLA-restricted allele (e.g., methionine, isoleucine, glutamine, or arginine for leucine at position 205 of RT), was found in 1 and 3 of 20 subjects, respectively. Sequence evolution was significantly correlated with levels of viremia of between 6.5 and <50 copies/ml (P = 0.03) and was more likely to occur within epitopes presented by relevant HLA alleles (P < 0.001). These findings suggest that ongoing virus replication contributes to low-level viremia in patients on HAART and that this ongoing replication is subject to CD8(+) T-cell selective pressures.

Trial registration: ClinicalTrials.gov NCT00107549.

FIG. 1.

Maximum likelihood phylogenetic tree of HIV-1 RT sequences as a function of time postvaccination. Representative prevaccine replication-competent viral isolates, subtype B, C, and D reference sequences for HIV-1 RT, and the vaccine sequence (vaccine) are shown. The plasma sequences amplified during episodes of rebound viremia postvaccination are shown with a strike through the symbols. Wild-type variants, drug-resistant variants, and variants with amino acid substitutions at position 205 of HIV-1 RT are indicated. Bootstrap values of >80% are shown.

FIG. 2.

Amino acid alignment, relative to the consensus region of HIV-1_HXB2 RT, showing nonsynonymous substitutions detected during low-level viremia postvaccination within relevant CD8⁺ T-cell epitopes. Homology to the consensus sequence is represented by dots. (a) Subject 8 developed the nonsynonymous substitutions V108I and G112S in an HLA-A*02 epitope (red box), and the I135T substitution within an HLA-A*02/B*51 epitope (blue box) later became fixed in the free plasma virus present at low levels. The selection of substitutions at position 205 occurred within an HLA-A*02 epitope (red box) for three subjects (subject 7 [b], subject 15 [d], and subject 17 [e]). For the one subject (subject 11) (c) who showed temporal clustering of low-level viremia genotypes during the trial and subsequently failed therapy while maintained on HAART, and who is HLA-A*6801 positive (which is also an HLA-A*03 supertype), the L205M position also lies outside the HLA-A*68 (green box) and HLA-A*03 (red box) epitopes.

FIG. 3.

Frequency of episodes of low-level viremia above 6.5 copies/ml for the subjects experiencing sequence evolution (n = 4) at viral loads of <50 copies/ml compared to the frequency for those who did not (no evolution) following HIV-1 recombinant poxvirus vaccinations. Sequence evolution was defined as either new drug resistance mutations (subject 8) or novel changes at amino acid position 205 of HIV-1 RT while still being suppressed on HAART (subjects 7, 11, and 17). The one subject (subject 15) who also developed substitutions at amino acid position 205 of RT at week 40 in rebounding virus was excluded from this analysis. Median values and IQR are indicated.

FIG. 4.

Changes from baseline in HIV-1-specific CD8⁺ IFN-γ ELISPOT responses to overlapping peptides spanning amino acids 1 to 556 of HIV-1 Pol at weeks 6 and 24 or 26 for the subjects experiencing sequence evolution in low-level viremia compared to those without detectable sequence evolution. The one subject (subject 15) who also developed substitutions at amino acid position 205 at week 40 in rebounding virus was excluded from this analysis. One subject (subject 8) did not receive any additional vaccine doses after week 4 and therefore did not have immune studies performed at week 24 or 26 of the study. The colored dots indicate individual subject data. The black symbols represent data from the one subject (subject 11) who developed rebound viremia during the trial while still on HAART.