The HIV-1 reservoir in eight patients on long-term suppressive antiretroviral therapy is stable with few genetic changes over time

Abstract

The source and dynamics of persistent HIV-1 during long-term combinational antiretroviral therapy (cART) are critical to understanding the barriers to curing HIV-1 infection. To address this issue, we isolated and genetically characterized HIV-1 DNA from naïve and memory T cells from peripheral blood and gut-associated lymphoid tissue (GALT) from eight patients after 4-12 y of suppressive cART. Our detailed analysis of these eight patients indicates that persistent HIV-1 in peripheral blood and GALT is found primarily in memory CD4(+) T cells [CD45RO(+)/CD27((+/-))]. The HIV-1 infection frequency of CD4(+) T cells from peripheral blood and GALT was higher in patients who initiated treatment during chronic compared with acute/early infection, indicating that early initiation of therapy results in lower HIV-1 reservoir size in blood and gut. Phylogenetic analysis revealed an HIV-1 genetic change between RNA sequences isolated before initiation of cART and intracellular HIV-1 sequences from the T-cell subsets after 4-12 y of suppressive cART in four of the eight patients. However, evolutionary rate analyses estimated no greater than three nucleotide substitutions per gene region analyzed during all of the 4-12 y of suppressive therapy. We also identified a clearly replication-incompetent viral sequence in multiple memory T cells in one patient, strongly supporting asynchronous cell replication of a cell containing integrated HIV-1 DNA as the source. This study indicates that persistence of a remarkably stable population of infected memory cells will be the primary barrier to a cure, and, with little evidence of viral replication, this population could be maintained by homeostatic cell proliferation or other processes.

Fig. 1.

Phylogenetic analysis of viral sequences from plasma isolated before the initiation of cART and cells isolated from peripheral blood and GALT after several years of suppressive cART. ML trees of sequences from pretherapy plasma (gray filled circles), on-therapy plasma (red filled circles), peripheral blood [all memory (blue filled triangles), central/transitional memory (blue filled squares), effector memory (blue open squares), and naïve (blue open circles) T cells], and GALT [central/transitional memory (green filled squares), effector memory (green open squares), and naïve (green open circles) T cells] from a subset of patients initiating antiviral therapy during acute/early HIV-1 infection (patients 3 and 5) (A and B), and during chronic HIV-1 infection (patients 7 and 8) (C and D: red circles indicate identical sequences isolated from different cells). Patient 7 initiated therapy in chronic infection and had identical clonal HIV-1 sequences containing a large 380-bp deletion (C). The trees representing the sequences isolated from patients 1, 2, 4, and 6 are in Fig. S3.