Intracellular Staining for HIV-Specific IFN-γ Production: Statistical Analyses Establish Reproducibility and Criteria for Distinguishing Positive Responses

Abstract

The cellular immune response plays a pivotal role in controlling the spread of HIV-1 infection by lysing virally infected cells and producing potent antiviral cytokines, such as interferon-gamma (IFN-gamma). Flow cytometric methods have been established to evaluate the contribution of both CD4 and CD8 subsets of T lymphocytes to the immune response to HIV by measuring their production of intracellular IFN-gamma following brief antigenic stimulation. We present a statistical treatment of intracellular cytokine staining (ICS) data that is aimed at establishing the reproducibility and robustness of this assay for use in HIV clinical trials. Comparisons of responses from HIV-seronegative and seropositive individuals were used to establish a 2-fold criterion for distinguishing positive responses with a low probability of false positives (<1%). Additional comparisons established that the reproducibility of the assay is between 1.4 and 2.0-fold depending on the magnitude of the response. Little variability was demonstrated between multiple operators for both the execution and analysis components of these experiments (<10% difference with 95% confidence). We conclude that the statistical criteria established by these analyses allow for the accurate detection and comparison of positive responses. Using these statistical criteria, the ICS assay is sufficiently robust for use in HIV-specific vaccine trials.

Comparative Immunogenicity of Human Immunodeficiency Virus Particles and Corresponding Polypeptides in a DNA Vaccine

“…4A, left panel). We have typically seen lower signals in CD4 ϩ cells than in CD8 ϩ cells (15), consistent with previous observations that CD4 ϩ responses are lower than CD8 ϩ responses by the intracellular cytokine staining assay (6,17,21), possibly because relevant posttranslational modifications are lacking in the peptides, because ␥-IFN and TNF-␣ are less efficiently synthesized in these CD4 ϩ cells than in CD8 ϩ cells, or because antigenpresenting cells are limiting. In contrast, the CD8 ϩ responses to Env were detectable and showed no statistical difference between separate Gag and Env and pVLP groups (P ϭ 0.69) (Fig.…”

Peptide Impurities in Commercial Synthetic Peptides and Their Implications for Vaccine Trial Assessment

“…Custom peptide manufacturers and suppliers have responded to this demand by increasing their supply capacity to meet the growing demand in both research and therapeutics markets (15). Since peptide libraries used for screening cellular immune responses may contain hundreds or even thousands of individual peptides, and each peptide is synthesized and purified independently, QA/QC of peptide libraries used in both basic research and clinical research protocols has become a significant and daunting task (4,16,24,27,28). Stringent QA/QC is an absolute requirement for clinical trials where biological assay outcome may be the end-point determinant for advancement or nonadvancement of a vaccine or therapeutic product (10,25).…”

“…Recent methodological advances, particularly enzyme-linked immunospot (ELISPOT) assays and cytokine-based flow cytometry (CFC) assays coupled with overlapping pooled peptide technology, give the opportunity for detailed and precise analyses of specific cellular immune responses (1,3,11,19,22,29). As cellular immune response assays proceed from being used primarily as research tools to be being used as tools for clinical evaluation and assessment of end points in different phases of vaccine development, the required standards of quality assurance (QA)/quality control (QC) for these assays rise dramatically (4,10,16,17,21,27,28). While assay techniques and equipment can be standardized by employing standard operating procedures and assay reagents and can be standardized by use of manufacturer-certified assay kits, a critical component of the assay that is not typically subject to standardized QA/QC is the synthetic peptides used for stimulating the T cells.…”

Influence of GB virus C on IFN-γ and IL-2 production and CD38 expression in T lymphocytes from chronically HIV-infected and HIV-HCV-co-infected patients

“…Normalised samples in which = 1% of cells produced cytokines in response to antigen specific stimulation were considered responsive (Trigona et al 2003, Chen et al 2005. Of the 135 patients challenged with Nef, 66 (48.8%) presented antigen-specific responses in at least one of the three cellular populations, with a total of 119 responses when the three cell populations and the two cytokines were considered individually.…”