Comparison of immunologic assays for detecting immune responses in HIV immunotherapeutic studies: AIDS Clinical Trials Group Trial A5181

Abstract

This study was designed to evaluate which of several T-cell-specific, immune response assays are the most relevant in measuring the key characteristics of an effective immune response to HIV-1. Using 5 HIV-1 antigens as stimulants, we assessed lymphocyte proliferation, supernatant gamma interferon (IFN-gamma) cytokine production (CP), single-cell IFN-gamma production by enzyme-linked immunospot (ELISPOT) assay, with and without Epstein-Barr virus-transformed B-lymphoblastoid cell lines (B-LCLs), and intracellular cytokine production (ICC) for IFN-gamma and interleukin 2 (IL-2) by flow cytometry. We used these to compare specimens from HIV-1-infected subjects who were virally suppressed with a stable antiretroviral therapy (ART) regimen (group A) with specimens from subjects not on ART but with HIV-1 viremia of <3,000 copies/ml (group B). The lymphocyte proliferation assay (LPA) did not significantly differentiate between the two groups. Using fresh peripheral blood mononuclear cells (PBMCs), the CP and ELISPOT assays for IFN-gamma detected the greatest differences between the two groups, specific for three of the five HIV-1 antigens, whereas significant differences were seen only in response to one antigen when cryopreserved cells were used. The strongest correlations were seen between the CP and ELISPOT assays. The ELISPOT B-LCL assay showed a cell concentration-dependent increase in IFN-gamma production compared to that shown by the standard ELISPOT assay but did not differentiate between the groups. In the ICC assay, greater numbers of IFN-gamma-producing T cells were seen in group B, and little or no detectable IL-2 production was seen in both groups. These studies highlight complexities of immunologic monitoring of T-cell responses in multisite clinical trials in HIV infection and outline considerations for optimizing these efforts.

FIG. 1.

T-cell responses assessed by the LP, CP, and ELISPOT assays in group A versus group B using fresh PBMC specimens (a) and using cryopreserved specimens (b). The CP and ELISPOT assays were used to detect IFN-γ. For each assay, data were averaged over 1 to 3 separate time points for each individual and each antigen, and differences between the two arms were tested by the Wilcoxon rank sum tests. Comparisons marked with asterisks reached clinical significance.

FIG. 2.

Median ELISPOT B-LCL and standard ELISPOT responses in week 12 cryopreserved PBMC samples (n = 7; n = 6 in all 3 × 10⁵-cell concentrations). ELISPOT B-LCL points with asterisks indicate statistically significant differences compared to results from the standard ELISPOT assay.

FIG. 3.

Correlation between the ICC-flow assay for IFN-γ (sum of IFN-γ-producing CD4⁺ and CD8⁺ T cells) versus the ELISPOT and CP assays using fresh PBMCs (rank-based Spearman correlation).

FIG. 4.

Correlations between fresh and cryopreserved PBMCs in the ELISPOT assay (rank-based Spearman correlation).