Peptide impurities in commercial synthetic peptides and the implications for vaccine trial assessment

The US Military HIV Research Program, Rockville, MD. USA; JPT Peptide Technologies, Berlin, Germany; National Institute for Communicable Diseases, Sandringham, Johannesburg; Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; Mbeya Medical Research Programme, Mbeya, Tanzania; Department of Infectious Diseases and Tropical Medicine, University of Munich, Munich, Germany

^* To whom correspondence should be addressed. Email: jcurrier{at}hivresearch.org.

	Abstract

The advent of T cell assay methodologies amenable to high throughput coupled with the availability of large libraries of overlapping peptides has 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-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 1% 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, which can affect cellular immune-based assay outcome and interpretation.