6 ''-Modifed alpha-GalCer-peptide conjugate vaccine candidates protect against liver-stage malaria

Self-adjuvanting vaccines consisting of peptide epitopes conjugated to immune adjuvants are a powerful way of generating antigen-specific immune responses. We previously showed that a Plasmodium-derived peptide conjugated to a rearranged form of alpha-galactosylceramide (alpha-GalCer) could stimulate liver-resident memory T (T-RM) cells that were effective killers of liver-stage Plasmodium berghei ANKA (Pba)-infected cells. To investigate if similar or even superior T-RM responses can be induced by modifying the alpha-GalCer adjuvant, we created new conjugate vaccine cadidates by attaching an immunogenic Plasmodium-derived peptide antigen to 6 ''-substituted alpha-GalCer analogues. Vaccine synthesis involved developing an efficient route to alpha-galactosylphytosphingosine (alpha-GalPhs), from which the prototypical iNKT cell agonist, alpha-GalCer, and its 6 ''-deoxy-6 ''-thio and -amino analogues were derived. Attaching a cathepsin B-cleavable linker to the 6 ''-modified alpha-GalCer created pro-adjuvants bearing a pendant ketone group available for peptide conjugation. Optimized reaction conditions were developed that allow for the efficient conjugation of peptide antigens to the pro-adjuvants via oxime ligation to create new glycolipid-peptide (GLP) conjugate vaccines. A single dose of the vaccine candidates induced acute NKT and Plasmodium-specific CD8(+) T cell responses that generated potent hepatic T-RM responses in mice. Our findings demonstrate that attaching antigenic peptides to 6 ''-modifed alpha-GalCer generates powerful self-adjuvanting conjugate vaccine candidates that could potentially control hepatotropic infections such as liver-stage malaria.