Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM197 conjugate vaccine co-localizes with MHC II on the antigen processing cell surface

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI) antigens and do not induce immunological memory or antibodies in infants. Conjugation of PnPS to the carrier protein CRM197 induces PS-specific antibody in infants, and memory similar to T-dependent (Td) antigens. Conjugates have improved immunogenicity via antigen processing and presentation of carrier protein with MHC II and recruitment of T cell help, but the fate of the PS attached to the carrier is unknown. To determine the location of the PS component of PnPS-CRM197 in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM197 and 19F-CRM197 was specifically labeled by Alexa Fluor® 594 hydrazide (red). The CRM197 was separately labeled red in a reaction that did not label PS. Labeled antigens were incubated with APC which were fixed, permeabilized and incubated with anti-MHC II antibody labeled green by Alexa Fluor® 488, followed by confocal microscopy. Labeled CRM197 was presented on APC surface and co-localized with MHC II (yellow). Labeled unconjugated 14 or 19F PS did not go to the APC surface, but PS labeled 14-CRM197 and 19F-CRM197 was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM197. Brefeldin A and chloroquine blocked both CRM197 and PS labeled 14-CRM197 and 19F-CRM197 from co-localizing with MHC II. These data suggest that the PS component of the CRM197 glycoconjugate enters the endosome, travels with CRM197 peptides to the APC surface and co-localizes with MHC II.