Rapid quantification of the latent reservoir for HIV-1 using a viral outgrowth assay

Abstract

HIV-1 persists in infected individuals in a stable pool of resting CD4(+) T cells as a latent but replication-competent provirus. This latent reservoir is the major barrier to the eradication of HIV-1. Clinical trials are currently underway investigating the effects of latency-disrupting compounds on the persistence of the latent reservoir in infected individuals. To accurately assess the effects of such compounds, accurate assays to measure the frequency of latently infected cells are essential. The development of a simpler assay for the latent reservoir has been identified as a major AIDS research priority. We report here the development and validation of a rapid viral outgrowth assay that quantifies the frequency of cells that can release replication-competent virus following cellular activation. This new assay utilizes bead and column-based purification of resting CD4(+) T cells from the peripheral blood of HIV-1 infected patients rather than cell sorting to obtain comparable resting CD4(+) T cell purity. This new assay also utilizes the MOLT-4/CCR5 cell line for viral expansion, producing statistically comparable measurements of the frequency of latent HIV-1 infection. Finally, this new assay employs a novel quantitative RT-PCR specific for polyadenylated HIV-1 RNA for virus detection, which we demonstrate is a more sensitive and cost-effective method to detect HIV-1 replication than expensive commercial ELISA detection methods. The reductions in both labor and cost make this assay suitable for quantifying the frequency of latently infected cells in clinical trials of HIV-1 eradication strategies.

Figure 1. Two-step bead depletion procedure yields highly purified resting CD4⁺ T cells from HIV-1 infected patients.

A two-step negative selection strategy to purify resting CD4⁺ T cells from patient PBMC. (A) Representative FSC/SSC plot indicating live cell population after the two-step bead depletion procedure. (B) Representative dot plot indicating purity of resting CD4⁺ T cells. Purified cells were stained with antibodies to CD4 and HLA-DR.

Figure 2. The standard and the MOLT-4/CCR5 viral outgrowth assays.

The frequency of HIV-1 latent infection of resting CD4⁺ T cells can be measured using a viral outgrowth assay. PBMC are collected from HIV-1 infected individuals and resting CD4⁺ T cells (CD25⁻, CD69⁻, HLA-DR⁻) are purified. Resting T cells are plated in 5-fold serial dilutions in duplicate, such that the input number of patient cells ranges from 1,000,000 to 320 cells per well. To reverse latency in the cells that harbor a replication-competent HIV-1 provirus, patient cells are activated with PHA and a 10-fold excess of irradiated PBMC from healthy donors. The next day, target cells for HIV-1 infection are added to allow outgrowth of replication-competent HIV-1 released from infected cells in which latency has been reversed. In the standard viral outgrowth assay, CD4⁺ lymphoblasts from healthy donors are added on days 2 and 7 of the assay. In the MOLT-4/CCR5 viral outgrowth assay, MOLT-4/CCR5 cells are added on day 2 only. For the standard assay, HIV-1 p24 antigen ELISA is used to identify wells positive for HIV-1 outgrowth at 14 days. For the MOLT-4/CCR5 assay, RT-PCR is used to identify wells positive for outgrowth at 7 days. The frequency of latently infected cells can be determined using limiting dilution statistics based on the input number of patient cells in the wells positive for outgrowth. This frequency is reported in infectious units per million (IUPM).

Figure 3. The standard and the MOLT-4/CCR5 viral outgrowth assays yield comparable frequencies of latent infection.

(A) The frequency of latently infected resting CD4⁺ T cells was measured in 3 viremic patients and 14 HAART-suppressed patients using both the standard and the MOLT-4/CCR5 viral outgrowth assays, with HIV-1 p24 antigen ELISA used as the endpoint assay of viral outgrowth at day 14. (B) Statistical comparison of the IUPM values measured using the standard viral outgrowth assay and the MOLT-4/CCR5 viral outgrowth assay by Wilcoxon rank sum test. (C) The correlation of IUPM values measured using the standard viral outgrowth assay and the MOLT-4/CCR5 viral outgrowth assay (Pearson's correlation coefficient, r).

Figure 4. Kinetics of HIV-1 outgrowth from latently infected CD4⁺ T cells measured by HIV-1 p24 antigen ELISA and HIV-1 specific RT-PCR.

Resting CD4⁺ T cells were isolated from HAART patient S15, whose latent reservoir was previously measured to be 3.25 IUPM. Twenty-nine replicate wells were plated in which 200,000 resting cells were activated with PHA and irradiated PBMC from a healthy donor and subsequently cultured with MOLT-4/CCR5 cells. Outgrowth of reactivated HIV-1 was measured in positive wells over 14 days using both (A) HIV-1 p24 antigen ELISA and (B) HIV-1 specific RT-PCR. (C) The difference between the day on which a particular well becomes positive by RT-PCR versus p24 ELISA. (D) The day of detection of HIV-1 outgrowth from the latent reservoir is shown for HIV-1 p24 antigen ELISA and HIV-1 specific RT-PCR.

Figure 5. Accurate measurement of IUPM at day 7 using HIV-1 specific RT-PCR.

(A) Using the rapid MOLT-4/CCR5 viral outgrowth assay, the frequency of latently infected cells was measured for HAART patients S1–S14 and viremic patients V1–V3 at day 7 with the HIV-1 specific RT-PCR assay and at both days 7 and 14 with HIV-1 p24 antigen ELISA. Statistical significance of the differences in IUPM values was assessed by Wilcoxon rank sum test. (B) Correlation of the IUPM measured at day 7 using HIV-1 p24 antigen ELISA with the IUPM measured at day 14 using HIV-1 p24 antigen ELISA (Pearson's correlation coefficient, r). (C) Correlation of the IUPM measured at day 7 using HIV-1 specific RT-PCR with the IUPM measured at day 14 using HIV-1 p24 antigen ELISA (Pearson's correlation coefficient, r). (D) Correlation of the IUPM measured at day 7 using the rapid MOLT-4/CCR5 outgrowth assay with the IUPM measured at day 14 using the standard outgrowth assay (Pearson's correlation coefficient, r).