Synthesis and Biological Evaluation of Peptide-Adjuvant Conjugate Vaccines with Increasing Antigen Content

Abstract

Synthetic vaccines that induce T cell responses to peptide epitopes are a promising immunotherapy for both communicable and noncommunicable diseases. Stimulating strong and sustained T cell responses requires antigen delivery to appropriately activated antigen presenting cells (APCs). One way this can be accomplished is by chemically conjugating immunogenic peptide epitopes with α-galactosylceramide (α-GalCer), a glycolipid that acts as an immune adjuvant by inducing stimulatory interactions between APCs and type I natural killer T (NKT) cells. Here we investigate whether increasing the ratio of antigen:adjuvant improves antigen-specific T cell responses. A series of conjugate vaccines was prepared in which one, two, four, or eight copies of an immunogenic peptide were covalently attached to a modified form of α-GalCer via the poly­(ethoxyethylglycinamide) dendron scaffold. Initial attempts to synthesize these multivalent conjugate vaccines involved attaching the bicyclo[6.1.0]­non-4-yne (BCN) group to the adjuvant-dendron structure followed by strain-promoted azide–alkyne cycloaddition of the peptide. Although this approach was successful for preparing vaccines with either one or two peptide copies, the synthesis of vaccines requiring attachment of four or eight BCN groups suffered from low yields due to cyclooctyne degradation. Instead, conjugate vaccines containing up to eight peptide copies were readily achieved through oxime ligation with adjuvant-dendron constructs decorated with the 8-oxo-nonanoyl group. When evaluating T cell responses to vaccination in mice, we confirmed a significant advantage to conjugation over admixes of peptide and α-GalCer, regardless of the peptide to adjuvant ratio, but there was no advantage to increasing the number of peptides attached. However, it was notable that the higher ratio conjugate vaccines required lower levels of NKT cell activation to be effective, which could be a safety advantage for future vaccine candidates.

A Glycolipid-Peptide-Hapten Tricomponent Conjugate Vaccine Generates Durable Antihapten Antibody Responses in Mice

“…Next, 6″-azido-6″-deoxy-αGalCer ( 17 ) was reduced to its corresponding amine ( 18 ) and then reacted with carbonate 16 to give the core αGalCer-bidentate unit 19 . Unexpectedly, the basic conditions used to couple 16 and 18 resulted in a small amount (<10%) of a contaminating product with a mass 16 Da more than that calculated for 19 , which was presumed to correspond to a cyclooctenone species analogous to that previously reported . While not isolated and formally identified, this postulated byproduct would be unreactive in the subsequent SPAAC reaction enabling it to be purged in the next step.…”