An in vitro-transcribed-mRNA polyepitope construct encoding 32 distinct HLA class I-restricted epitopes from CMV, EBV, and Influenza for use as a functional control in human immune monitoring studies.

Interest and activity in the areas of clinical immunotherapy and therapeutic vaccines are growing dramatically, thus there is a pressing need to develop robust tools for assessment of vaccine-induced immunity. CD8+ T cell immunity against specific antigens is normally measured by either flow cytometry using MHC tetramer reagents or via biological assays such as intracellular cytokine staining or ELISPOT after stimulation with specific peptide epitopes. However, these methodologies depend on precise knowledge of HLA-restricted epitopes combined with HLA typing of subjects. As an alternative approach, electroporation of antigen presenting cells (APC) with in vitro-transcribed mRNA (IVT-mRNA) encoding the antigen of interest bypasses the requirements for HLA typing and knowledge of specific epitopes. A current limitation of the IVT-mRNA technique is the lack of robust positive control RNAs to verify the efficacy of electroporation and to ensure that the electroporated APC retain the ability to stimulate T cells. Herein we describe an IVT-mRNA construct wherein all 32 HLA class I-restricted epitopes of the widely used CEF (Cytomegalovirus, Epstein-Barr Virus and Influenza Virus) positive control peptide pool have been genetically spliced together to generate a single polyepitope construct. Each epitope is flanked by three amino- and three carboxy-terminal amino acids from the original parent protein to facilitate proteolytic processing by the proteasome. Using cells obtained from a panel of normal healthy donors and cancer patients we report that dendritic cells, CD40-activated B cells, PHA blasts, and even tumor cells can be transfected with CEF polyepitope IVT-mRNA and can elicit robust CEF-specific responses from autologous T cells, as measured by IFN-gamma ELISPOT. Moreover, the response elicited by CEF IVT-mRNA-transfected APC was similar in magnitude to the response elicited by the complete pool of CEF minimal peptide epitopes, implying that the polyepitope parent protein encoded by the CEF mRNA was efficiently processed into individual epitopes by the proteolytic machinery of the APC. In summary, the CEF polyepitope IVT-mRNA described herein comprises a robust positive control for immunomonitoring studies requiring IVT-mRNA transfection and potentially provides a unique tool for assessing MHC class I processing regardless of HLA haplotype.