No abstract

The advent of T-cell assay methodologies that are amenable to high throughput coupled with the availability of large libraries of overlapping peptides have revolutionized the fields of vaccine efficacy testing and cellular immune response assessment. Since T-cell assay performance is critically dependent upon the quality and specificity of the stimulating peptides, assurance of high-quality and reliable input peptides is an important aspect of assay validation. Herein, we demonstrate that individual peptides from large human immunodeficiency virus (HIV)-based peptide library sets obtained directly from two independent custom peptide suppliers contained contaminating peptides capable of giving false-positive results, which were consistent with nominal antigen-specific CD8 ؉ T-cell responses. In-depth investigation of the cellular response in terms of responding CD8 ؉ T-cell frequency and human leukocyte antigen (HLA) restriction led to the conclusion that one set of HIV type 1 (HIV-1)-derived peptides was contaminated with a peptide from human cytomegalovirus (HCMV), which is commonly used in cellular immunology research applications. Analytical characterization of the original stock of the suspect HIV-1 peptide confirmed the presence of ϳ1% by weight of the HCMV peptide. These observations have critical implications for quality assurance (QA) and quality control (QC) of peptides used in clinical trials where cellular immune-based assays are important end-point determinants. We propose a simple schema of biological QA/QC protocols to augment the standard biochemical QA/QC analyses as a means to circumvent this and other problems that can affect cellular immune-based assay outcome and interpretation.The identification and characterization of immunogenic Tcell epitope-containing regions of the proteomes of infectious agents and candidate cancer-and tumor-specific proteins are an essential phase in the rational development of prophylactic vaccines and immunotherapeutic strategies. 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. Synthetic peptides are usually specific to the particular application of the assay, d...