6″-Modifed α-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 -derived peptide conjugated to a rearranged form of α-galactosylceramide (α-GalCer) could stimulate liver-resident memory T (T) cells that were effective killers of liver-stage ANKA (Pba)-infected cells. To investigate if similar or even superior T responses can be induced by modifying the α-GalCer adjuvant, we created new conjugate vaccine cadidates by attaching an immunogenic -derived peptide antigen to 6″-substituted α-GalCer analogues. Vaccine synthesis involved developing an efficient route to α-galactosylphytosphingosine (α-GalPhs), from which the prototypical iNKT cell agonist, α-GalCer, and its 6″-deoxy-6″-thio and -amino analogues were derived. Attaching a cathepsin B-cleavable linker to the 6″-modified α-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 oxime ligation to create new glycolipid-peptide (GLP) conjugate vaccines. A single dose of the vaccine candidates induced acute NKT and -specific CD8 T cell responses that generated potent hepatic T responses in mice. Our findings demonstrate that attaching antigenic peptides to 6″-modifed α-GalCer generates powerful self-adjuvanting conjugate vaccine candidates that could potentially control hepatotropic infections such as liver-stage malaria.