Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood

Abstract

Plasma viral load and CD4 counts are effective for clinical monitoring, but they do not give a full representation of HIV-1 quasispecies in cellular reservoirs, the major repository of replication-competent HIV-1 in infected individuals. We sought to develop a diagnostic system that might stimulate the replication-competent HIV-1 reservoirs for enhanced clinical monitoring, including selection of antiretroviral regimens. Whole-blood samples from 45 HIV-infected individuals were collected into 1 ViraStim HIV-1 activation tube and 1 EDTA tube. Samples were tested for viral load and cell type-specific HIV-1 replication. Further, 7 matched activated/nonactivated samples were sequenced using the Trugene HIV-1 genotyping kit. The percentage of patients with replication-competent virus in peripheral blood mononuclear cells (PBMCs) varied, depending on the baseline plasma viral load in the EDTA tubes. Six out of 24 patients with a starting plasma viral load of <20 copies/ml (cp/ml), 6 out of 8 patients with starting viral loads of >20 and <1,000 cp/ml, and 8 out of 13 patients with starting viral loads of >1,000 all showed increases in viral replication of >5-fold. These increases came from cellular reservoirs in blood as determined by simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI). When resistance genotypes in plasma from activation tubes were compared to those from EDTA tubes for 7 patients, all patients showed additional mutations in the activation tube, while 3 patients demonstrated additional genotypic resistance determinants. We show that HIV-1 viral replication can be stimulated directly from infected whole blood. The sequencing results showed that 3 of 7 cases demonstrated additional drug resistance following stimulation.

FIG 1

Plasma viral load (VL) comparison between EDTA tubes and ViraStim tubes. Six out of 24 patients with starting plasma viral loads of <20 copies/ml (cp/ml) (A), 6 out of 8 patients with starting viral loads of >20 and <1,000 cp/ml (B), and 8 out of 13 patients (C) with starting viral loads of >1,000 demonstrated increases of >0.5 log in the ViraStim tubes compared to the EDTA tubes.

FIG 2

(A) Flow cytometric histograms demonstrating HIV-1 replication in CD3⁺ and CD4⁺ T lymphocytes using SUSHI. PBMCs from ViraStim tubes showed increased replication relative to that of PBMCs from EDTA tubes. (B) Histogram overlay of the HIV-1 hybridization controls using PMA-stimulated ACH-2 cells (red) that express high levels of HIV-1 mRNA as a positive control and unstimulated ACH-2 cells (black) that express little if any HIV-1 mRNA as a negative control.

FIG 2

(A) Flow cytometric histograms demonstrating HIV-1 replication in CD3⁺ and CD4⁺ T lymphocytes using SUSHI. PBMCs from ViraStim tubes showed increased replication relative to that of PBMCs from EDTA tubes. (B) Histogram overlay of the HIV-1 hybridization controls using PMA-stimulated ACH-2 cells (red) that express high levels of HIV-1 mRNA as a positive control and unstimulated ACH-2 cells (black) that express little if any HIV-1 mRNA as a negative control.

FIG 3

Fold changes in HIV-1 replication in the CD3⁺ CD4⁺ reservoir and in the plasma viral load using the formula ViraStim tube/EDTA tube. Included in the subset of patients were samples with adequate cells to determine the replication in cellular reservoirs. Increases in viral replication in cells (x axis) correlated (r² = 0.7, P = 0.006) with increases of virus in plasma (y axis).