Clonal sequences recovered from plasma from patients with residual HIV-1 viremia and on intensified antiretroviral therapy are identical to replicating viral RNAs recovered from circulating resting CD4+ T cells

Abstract

Despite successful antiretroviral therapy (ART), low-level viremia (LLV) may be intermittently detected in most HIV-infected patients. Longitudinal blood plasma and resting CD4(+) T cells were obtained from two patients on suppressive ART to investigate the source of LLV. Single-genome sequencing of HIV-1 env from LLV plasma was performed, and the sequences were compared to sequences recovered from limiting-dilution outgrowth assays of resting CD4(+) T cells. The circulating LLV virus clone was identical to virus recovered from outgrowth assays from pools of millions of resting CD4(+) T cells. Understanding the sources of LLV requires evaluation of all possible reservoirs of persistent HIV infection.

Fig. 1.

Longitudinal viral load analyses of patient 15 (A) and patient 41 (B). Patient 15 was diagnosed with established HIV-1 infection in December 2003, and ART was promptly initiated. Patient 41 was diagnosed with acute HIV-1 infection in June 2004 and initiated ART in December 2004. HIV-1 RNA viral loads determined by Roche Amplicor assay are shown in black, and those determined by single-copy assay (SCA) are shown in red (24). Asterisks represent time points when leukophoresis was performed to obtain virus for resting cell outgrowth. Daggers represent time points when blood plasma was assayed by single genome amplification.

Fig. 2.

Phylogenetic analyses of patients 15 and 41. DNA sequences were aligned using CLUSTAL W (32). Phylogenetic trees were constructed using the neighbor-joining method (27) implemented in CLUSTAL W with Kimura's correction (17) by using Mega 4.0 (30). Evolutionary distances were computed using the maximum-composite-likelihood method (31). (A) Neighbor-joining tree of 37 SGA-derived full-length env sequences from resting CD4⁺ T lymphocytes in patient 15. Black circles represent SGA-derived env sequences. (B) Neighbor-joining tree of viruses derived from resting CD4⁺ T cells and blood plasma in patient 15. Closed red circles, full-length SGA-derived env sequences from plasma; open red circles, hypervariable V1-V2 SGA-derived sequences from plasma; closed black circles, full-length SGA-derived env sequences derived from cell supernatants from first leukophoresis; open black circles, hypervariable V1 to V5 bulk PCR sequences from cell supernatants from first leukophoresis; open black triangles, hypervariable V1-V5 bulk PCR sequences from all supernatants at second leukophoresis. (C) Highlighter analysis of viruses derived from resting CD4⁺ T cells and blood plasma from patient 41. The consensus sequence is included as the master. Sequence names are indicated on the right. Each vertical tick represents a mismatch from the master as indicated in the figure. (D) Neighbor-joining tree of V1 to V5 amplicons derived from resting CD4⁺ T cells (black circles) in August 2009, blood plasma in January 2009 (open red circles), and blood plasma in March 2010 (filled red circles). In each neighbor-joining tree, the percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (values of >60% with 500 replicates) are shown next to the branches. The trees are drawn to scale, and the horizontal scale bar represents genetic distance (nucleotide subsititutions per site). All positions containing gaps and missing data were eliminated from the data set. Strain HXB2 is included in the trees as an outgroup.