Modification of the Inhibitory Amino Acid for Epitope Peptide Binding onto Major Histocompatibility Complex Class II Molecules Enhances Immunogenicity of the Antigen

Abstract

Previously, the arginine at hen egg-white lysozyme 61 (HEL 61) was characterized as inhibiting T-lymphocyte stimulation due to the inefficient binding of the arginine-containing epitope peptide to the corresponding major histocompatibility complex class II molecules in C57BL/6 mice. In this study, we produced recombinant HEL, with arginine or alanine at HEL 61, and compared its ability to induce immune responses in mice to see whether modification of an inhibitory amino acid could enhance the immunogenicity of an inefficient antigen. Immunization of the mice with modified HEL induced strong antibody and T-cell immune responses against the native antigen. The enhanced T-cell immune response was due to a more specific elevation of the T-cell responses to the HEL 46-61 epitope region than to other epitope regions, although recognition of the other epitope peptides of HEL was generally increased. Mass spectrometric analyses of the epitope peptides generated by splenic antigen-presenting cells indicated that production of the epitope peptides encompassing HEL 46-61 was efficient using the modified antigen. These results suggest that modification of the critical amino acid residue(s) involved in hampering induction of an efficient immune response is an effective method to improve the immunogenicity of an inefficient antigen.

The Unveiling of Hidden T‐Cell Determinants of a Native Antigen by Defined Mediators of Inflammation: Implications for the Pathogenesis of Autoimmunity

“…We have previously shown that the crypticity of this epitope is attributable in part to the hindering amino acid residue (arginine, R) at position 61 (R61) [25]. This hindrance can be overcome by the deletion of R61 from peptide 46‐61 [25], by the substitution of R61 within HEL with an alanine residue [45] or by the use of phosphorylated HEL as the immunogen [46]. We suggest that the observed reversal of crypticity of 46‐61 in this study might involve one or more of the following: (1) fine processing of the epitope 46‐61 such that R61 is efficiently removed from the naturally processed epitope; (2) cleavage of the determinant region at a site slightly distal from R61 such that the extended C‐terminal residues could circumvent the hindrance caused by R61; and (3) the immune enhancing effects of cytokines on the APC (on Ii chain, co‐stimulatory molecules, etc.).…”

Induction of autoimmunity by immunization with hapten‐modified hen egg lysozyme in hen egg lysozyme‐transgenic mice

“…The epitope peptides were initially separated using high‐performance liquid chromatography (HPLC; Agilent Technologies, Boeblingen, Germany), and the peptide content and fragmentation of each peak were analysed using Agilent 1100 series LC/MSD Trap ion‐trap mass spectrometer coupled to an Agilent 1100 capillary LC system (Agilent Technologies). The LC system was operated with the electrospray ionization source in positive mode as previously described 16 …”

“…To analyse the peptides generated after antigen processing, splenocytes (5 · 10 8 ) from B6 mice were incubated with 250 lg of HEL or PC-HEL in 500 ll of medium for 8 hr at 37°to allow antigen processing as described previously. 16 The splenocytes were initially washed with PBS and then surface-bound processed peptides were eluted with 0Á75 M NaCl for 10 min at room temperature without cell lysis. The supernatant was then concentrated using a YM-3 Amicon concentrator (Millipore Co., Bedford, MA).…”

“…The LC system was operated with the electrospray ionization source in positive mode as previously described. 16…”