Antigen Processing of pneumococcal-CRM197 glycoconjugate vaccine: The polysaccharide component co-localizes with the carrier protein and MHC II on the APC surface (36.8)

Abstract

Pneumococcal (Pn) polysaccharides (PS) are T-independent (TI), induce no immunological memory and low Ab titers in infants. Conjugation of the Pn PS to the protein carrier CRM197 induces PS-specfic Ab in infants and memory similar to T dependent (Td) antigens. Glycoconjugates induce a Td PS immune response via antigen processing and presentation of carrier with MHC II, but the fate of the PS attached to the carrier is unclear. To determine the location of the PS of PnPS-CRM197 in the APC, we separately labeled PS and protein and tracked their location in APC. The PS of 19F-CRM197 was labeled by Alexa Fluor ® 594 hydrazide (labels PS red). The CRM197 was separately labeled in a reaction that did not label PS. Labeled 19F PS, 19F-CRM197 and CRM197 were incubated with 1x106 APC for 3–12 hours. APC were fixed and incubated with anti- HLA-DR Ab labeled by Alexa Fluor® 488 (green), followed by confocal microscopy. Labeled CRM197 was taken up and presented on the APC surface co-localized with MHC II (yellow; 42% positive cells). Labeled 19F PS was not internalized or presented on the surface. PS labeled 19F-CRM197 was internalized and co-localized on the surface with MHC II (23% positive). Brefeldin A and chloroquine blocked both CRM197 and 19F-CRM197 from co-localizing on the surface. These data suggest that PS of the 19F-CRM197 glycoconjugate enters the endosome, travels with CRM197 peptides to the APC surface and co-localizes with MHC II.