The glycolipid α-galactoslyceramide (α-GalCer) acts as an adjuvant for peptide-specific T cell responses by activating natural killer T (NKT) cells. This mechanism relies on NKT/ dendritic cell (DC) interactions that result in DC activation. Critically, in a manner similar to conventional CD4 T cell help, the peptide and α-GalCer must be presented on the same DC. We have developed a series of conjugate vaccines comprising antigenic peptides covalently linked to an α-GalCer prodrug via a cathepsin-cleavable linker, and have shown that these enhance peptide-specific T cell responses in mice. Here we aimed to determine whether these glycolipid-peptide conjugate vaccines are active in human cells, to inform their development for clinical applications.

We synthesized a conjugate vaccine comprising an α-GalCer prodrug linked to the immunodominant HLA-A*02-restricted cytomegalovirus (CMV) peptide NLVPMVATV (‘NLV’). Conjugate activity was assessed using PBMCs from healthy human donors.Cultures containing the conjugate led to NKT cell activation and proliferation, with cytokines IFN-γ, GM-CSF and IL-12p70 all produced in a CD1d-dependent manner. The conjugate enhanced expression of co-stimulatory molecules on DCs in the presence of NKT cells, and processed NLV peptide was detected on the DC surface. Across multiple donors, cultures containing the conjugate led to higher numbers of activated NLV-specific CD8⁺ T cells than either peptide alone or an admix of unconjugated peptide and α-GalCer. These T cells also produced IFN-γ and expressed the cytotoxicity marker CD107a. Finally, a similar conjugate vaccine containing an influenza matrix protein epitope also induced peptide-specific responses. We conclude that glycolipid-peptide conjugate vaccines activate NKT cells and enhance peptide-specific CD8⁺ T cell responses in human cells in vitro. This approach has potential clinical application for the immunotherapy of infectious diseases and cancer.