title: Antigen processing of glycoconjugate vaccines; the polysaccharide portion of the pneumococcal CRM(197) conjugate vaccine co-localizes with MHC II on the antigen processing cell surface.
abstract: 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 CRM(197) 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-CRM(197) in the APC, we separately labeled PS and protein and tracked their location. The PS of types 14-CRM(197) and 19F-CRM(197) was specifically labeled by Alexa Fluor 594 hydrazide (red). The CRM(197) 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 CRM(197) 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-CRM(197) and 19F-CRM(197) was internalized and co-localized with MHC II. Monoclonal antibody to type 14 PS bound to intracellular type 14 PS and PS-CRM(197). Brefeldin A and chloroquine blocked both CRM(197) and PS labeled 14-CRM(197) and 19F-CRM(197) from co-localizing with MHC II. These data suggest that the PS component of the CRM(197) glycoconjugate enters the endosome, travels with CRM(197) peptides to the APC surface and co-localizes with MHC II.
authors.affiliation: Department of Pediatrics, Tufts University School of Medicine, Boston, MA 02111, USA.
authors.forename: Zengzu
authors.initials: Z
authors.lastname: Lai
authors.forename: John R
authors.initials: JR
authors.lastname: Schreiber
mesh_terms: D000818:Animals; D017951:Antigen Presentation; D001426:Bacterial Proteins / Q000276:immunology*; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000032:analysis*; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D022242:Pneumococcal Vaccines / Q000276:immunology*; D011134:Polysaccharides / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology; D018074:Vaccines, Conjugate / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D000949:Histocompatibility Antigens Class II; D022242:Pneumococcal Vaccines; D011134:Polysaccharides; D018074:Vaccines, Conjugate; C028581:CRM197 (non-toxic variant of diphtheria toxin)