ENZYMATICALLY MEDIATED, GLYCOSIDIC CONJUGATION OF IMMUNOGLOBULINS WITH VIRAL EPITOPES

We developed a novel enzymatic procedure to couple a peptide to the su gar moieties of immunoglobulins (Igs). The synthesis of the conjugates consists in galactose (Gal) oxidation of desialylated Igs followed by covalent attachment of the peptides with concurrent stabilization of the Schiff bases upon mild reduction. The peptide used in this study, corresponds to the amino acid residues 110-120 of hemagglutinin (HA) o f PR8 A virus and is recognized by CD4 T helper cells in association w ith I-E(d) class II major histocompatibility complex (MHC). The degree of coupling as determined by competitive inhibition of radioimmunoass ay (IRIA) using FPLC purified conjugates was estimated at 11.4 peptide s per IgG molecule. Coupling of HA110-120 peptide to the sugar moiety of various mouse and human Igs was confirmed by Western blot analysis developed with anti-HA110-120 antibodies. Complete detachment of the p eptide from the conjugates by N-deglycosylation with PGNase F indicate d a defined specificity of coupling HA peptide to the N-linked oligosa ccharides of Igs. To facilitate quick release of the peptides from the conjugates into the lysosomal compartment of the antigen processing c ells (APC) we introduced at the cu amino terminus of the peptide (HAc1 10-120), a cleavage site for cathepsins (AAAL). The immunoglobulin-gal actose-HAc110-120 conjugates (IGP) were able to activate HA110-120 spe cific T hybridoma cells as efficient as influenza PR8 A virus and 40-1 00-fold higher than the synthetic peptide itself.