Human immunodeficiency virus replication and genotypic resistance in blood and lymph nodes after a year of potent antiretroviral therapy

Abstract

Potent antiretroviral therapy can reduce human immunodeficiency virus (HIV) in plasma to levels below the limit of detection for up to 2 years, but the extent to which viral replication is suppressed is unknown. To search for ongoing viral replication in 10 patients on combination antiretroviral therapy for up to 1 year, the emergence of genotypic drug resistance across different compartments was studied and correlated with plasma viral RNA levels. In addition, lymph node (LN) mononuclear cells were assayed for the presence of multiply spliced RNA. Population sequencing of HIV-1 pol was done on plasma RNA, peripheral blood mononuclear cell (PBMC) RNA, PBMC DNA, LN RNA, LN DNA, and RNA from virus isolated from PBMCs or LNs. A special effort was made to obtain sequences from patients with undetectable plasma RNA, emphasizing the rapidly emerging lamivudine-associated M184V mutation. Furthermore, concordance of drug resistance mutations across compartments was investigated. No evidence for viral replication was found in patients with plasma HIV RNA levels of <20 copies/ml. In contrast, evolving genotypic drug resistance or the presence of multiply spliced RNA provided evidence for low-level replication in subjects with plasma HIV RNA levels between 20 and 400 copies/ml. All patients failing therapy showed multiple drug resistance mutations in different compartments, and multiply spliced RNA was present upon examination. Concordance of nucleotide sequences from different tissue compartments obtained concurrently from individual patients was high: 98% in the protease and 94% in the reverse transcriptase regions. These findings argue that HIV replication differs significantly between patients on potent antiretroviral therapy with low but detectable viral loads and those with undetectable viral loads.

FIG. 1

Amino acid residues associated with resistance to indinavir in different compartments from patient A at baseline and after 1 year. Amino acids in this figure and subsequent figures are given in the International Union of Pure and Applied Chemistry code. Dashes represent amino acids which are unchanged from the reference sequence. Numbers in the top row indicate protease codons. ?, codon not determined.

FIG. 2

Amino acid residues associated with resistance to AZT or 3TC in different compartments from patient A at baseline and after 1 year. Dashes represent amino acids which are unchanged from the reference sequence. na, not done.

FIG. 3

Amino acid residues associated with resistance to indinavir in different compartments from patient E at baseline and after 1 year. Dashes represent amino acids which are unchanged from the reference sequence. Numbers in the top row indicate protease codons. V, viral; ref., reference; ?, codon not determined; LN C RNA, RNA extracted from LN tissue sample C; LN D RNA, RNA extracted from a different piece, D, of the same LN.

FIG. 4

Amino acid residues associated with resistance to AZT or 3TC in different compartments from patient E at baseline and after 1 year. Dashes represent amino acids which are unchanged from the reference sequence. ?, codon not determined. Lymph node C RNA, RNA extracted from LN tissue sample C; Lymph node D RNA, RNA extracted from a different piece D of the same LN.

FIG. 5

Amino acid residues associated with resistance to indinavir in different compartments from patient F at baseline and after 1 year. Dashes represent amino acids which are unchanged from the reference sequence. Numbers in the top row indicate protease codons. V, viral; ref., reference; aa, amino acid; ?, codon not determined. LN C RNA, RNA extracted from LN tissue sample C; LN D RNA, RNA extracted from a different piece, D, of the same LN.

FIG. 6

Amino acid residues associated with resistance to AZT or 3TC in different compartments from patient F at baseline and after 1 year. Dashes represent amino acids which are unchanged from the reference sequence. Lymph node C RNA, RNA extracted from LN tissue sample C; Lymph node D RNA, RNA extracted from a different piece D of the same LN; na, not done.