Designing CD8 T cell messenger RNA vaccines targeting the viral large T antigen to prevent BK polyomavirus disease...

BK polyomavirus (BKPyV) continues to threaten kidney transplantation outcomes by directly or indirectly causing premature allograft failure. Insufficient BKPyV-specific immunity underlies the onset and duration of BKPyV-DNAemia and nephropathy. BKPyV-specific T cells have been correlated with protection and include cytotoxic CD8 T cells targeting immunodominant 9-mer epitopes encoded in the viral large tumor antigen (LTag). To develop vaccines increasing LTag-specific T cells, we reduced the 695 amino acid-long LTag to 143 residues or less, comprising 4 clusters of immunodominant 9-mers presented by at least 54 human leukocyte antigen alleles. Prediction algorithms assisted in improved global coverage, alanine-alanine-tyrosine-linker placement, proteasomal processing, and solubility. Using a human cell culture vaccination model, transfection of adherent monocytes with large T antigen-coding nucleotide sequence 1-messenger RNA (LTAG1-mRNA) and LTAG2-mRNA demonstrated the expected 13.9 and 15.2 kD immunogens, respectively. LTAG2-mRNA induced higher CD8 T cell responses than LTAG1-mRNA, with an interferon-γ-positive CD62L-CD45RA-effector memory phenotype and cytotoxicity for BKPyV-replicating primary human renal tubular epithelial cells. We tested 3 additional candidates (LTAG3-mRNA, LTAG4-mRNA, and LTAG5-mRNA) by changing alanine-alanine-tyrosine linkers and extending immunogenic regions. The encoded immunogens were expressed and increased upon proteasome inhibitor treatment. LTag5-expanded T cells showed BKPyV-specific interferon-γ expression and cytotoxicity. Our results demonstrated novel approaches to designing modular mRNA-based vaccine candidates for clinical development of nonsecreted antigens using BKPyV as a paradigm of nonenveloped DNA viruses.