Continued production of drug-sensitive human immunodeficiency virus type 1 in children on combination antiretroviral therapy who have undetectable viral loads

Abstract

Highly active antiretroviral therapy (HAART) can suppress plasma human immunodeficiency virus type 1 (HIV-1) levels to below the detection limit of ultrasensitive clinical assays. However, HIV-1 persists in cellular reservoirs, and in adults, persistent low-level viremia is detected with more sensitive assays. The nature of this viremia is poorly understood, and it is unclear whether viremia persists in children on HAART, particularly those who start therapy shortly after birth. We therefore developed a reverse transcriptase PCR (RT-PCR) assay that allows genotyping of HIV-1 protease even when viremia is present at levels as low as 5 copies of HIV-1 RNA/ml. We demonstrated that viremia persists in children with plasma virus levels below the limit of detection of clinical assays. Viremia was detected even in children who began HAART in early infancy and maintained such strong suppression of viremia that HIV-1-specific antibody responses were absent or minimal. The low-level plasma virus lacked protease inhibitor resistance mutations despite the frequent use of nelfinavir, which has a low mutational barrier to resistance. Protease sequences resembled those of viruses in the latent reservoir in resting CD4(+) T cells. Thus, in most children on HAART with clinically undetectable viremia, there is continued virus production without evolution of resistance in the protease gene.

FIG. 1.

Nested RT-PCR assay used for genotyping the HIV-1 protease gene from the plasma in patients in whom plasma HIV-1 RNA levels are below 50 copies/ml. (A) The sensitivity of the assay was established using reaction mixtures containing serial dilutions of in vitro-transcribed HIV-1 NL43 RNA in the presence (RT+) or absence (RT−) of RT. Control PCRs for the first (1st) RT-PCR and the second (2nd) nested PCR are also shown. (B) Representative amplifications of protease from 4 ml of plasma from two subjects (subject C11, top panel; subject C40, bottom panel) whose viral loads were <50 copies/ml at the time of analysis.

FIG. 2.

Viral load data from study subjects who were monitored longitudinally. Plasma HIV-1 RNA levels are indicated by closed symbols. Open symbols indicate that the level of viremia was below the limit of detection of the assay used (400, 200, or 50 copies/ml); the plotted values serve to indicate the limit of detection. Solid arrows indicate times of sampling. Dashed arrows indicated previously reported time points (20).

FIG. 3.

Maximum-likelihood phylogenetic analysis of HIV-1 protease gene sequences from plasma (red circles) of study subjects treated with HAART either later during childhood (A) or from early infancy (B). Sequence information obtained from latently infected CD4⁺ T cells from the early-treated group (black circles) are included in panel B to confirm the patient-specific nature of the protease sequences amplified at low levels and to emphasize the homogeneity of HIV-1 protease sequences in these individuals. Previously published plasma virus sequences are indicated in open circles (A) and are included to provide a complete picture of the evolutionary profile of HIV-1 in plasma at low levels during prolonged suppressive HAART. Reference sequences from clades A and C are labeled as follows: A*, A.SE.SE8131; A1, A.UG.U455; A2, A2.CD.CDKFE4; C*, C.IN.IN21068; C**, C.ET.ETH2220. Strain B.F.R.HXB2R was used as a reference sequence for clade B.

FIG. 3.

Maximum-likelihood phylogenetic analysis of HIV-1 protease gene sequences from plasma (red circles) of study subjects treated with HAART either later during childhood (A) or from early infancy (B). Sequence information obtained from latently infected CD4⁺ T cells from the early-treated group (black circles) are included in panel B to confirm the patient-specific nature of the protease sequences amplified at low levels and to emphasize the homogeneity of HIV-1 protease sequences in these individuals. Previously published plasma virus sequences are indicated in open circles (A) and are included to provide a complete picture of the evolutionary profile of HIV-1 in plasma at low levels during prolonged suppressive HAART. Reference sequences from clades A and C are labeled as follows: A*, A.SE.SE8131; A1, A.UG.U455; A2, A2.CD.CDKFE4; C*, C.IN.IN21068; C**, C.ET.ETH2220. Strain B.F.R.HXB2R was used as a reference sequence for clade B.

FIG. 4.

Maximum-likelihood phylogenetic analysis of HIV-1 protease gene sequences from individual study subjects. (A) Phylogenetic trees showing sequences obtained from plasma virus (diamonds) and from latently infected resting CD4⁺ T cells (circles). Time of sampling is indicated by the color scale. The red bracket used with the patient C11 data indicates sequences bearing drug resistance mutations. (B) Plots of genetic distance versus time on HAART for subjects who provided multiple specimens during HAART. An MRCA sequence was reconstructed for each subject, and distances from the MRCA to each sequence were calculated using the HKY85+G model. Sequences obtained from plasma (diamonds) and latently infected CD4⁺ T cells (circles) are indicated.

FIG. 5.

Representative HIV-1 Western blots of samples from children who were treated with HAART from early infancy (C101, C103, and C108) or who initiated HAART at later times (C10). Patient C102 had no viremia detectable using our RT-PCR assay. Ages at the time of HIV-1 antibody testing are indicated in parentheses. y, year.