Contribution of conformational stability of hen lysozyme to
induction of type 2 T‐helper immune responses

Abstract

SUMMARYIt is important to identify characteristics that confer on proteins the potential to induce allergenic sensitization and allergenic disease. Protein allergens carry T-cell epitopes that are capable of inducing a type 2 T helper (Th2) cell response. There is limited information regarding factors that govern the allergenicity of proteins. We previously reported that a decrease in the conformational stability of hen-egg lysozyme (HEL) enhanced its capacity to activate HEL-speci®c T cells owing to the increased susceptibility to intracellular antigen processing. To determine whether the conformational stability of HEL makes for a critical contribution to allergenic sensitization in vivo, we immunized BALB/c mice with HEL derivatives of different conformational stability, but which retained a similar three-dimensional structure. The magnitude of in vivo T-cell responses, evaluated by ex vivo proliferative responses of lymph node T cells from mice primed with various HEL derivatives, was inversely correlated with conformational stability, as was interferon-c (IFN-c) and interleukin-4 (IL-4) production by splenic T cells in response to HEL. Immunization of the least stable derivative led to a potent IL-4 response and to immunoglobulin E (IgE) antibody production. We propose that the intrinsic allergenicity of proteins can be determined by the degree of conformational stability.

A Protein’s Conformational Stability Is an Immunologically Dominant Factor: Evidence That Free-Energy Barriers for Protein Unfolding Limit the Immunogenicity of Foreign Proteins

“…Because protein unfolding may be a crucial step in intracellular Ag processing, the conformational stability of the protein Ag is important in determining the immune response. In a previous study using modified hen egg white lysozyme (HEL), we found that the T cell-triggering response and, therefore, the immune response, were governed by the degree of conformational stability of the protein Ags (18,19). This finding was supported by a study using derivatives of snake toxin with different stabilities, which found that Ag presentation was controlled by the stability of the toxin (20).…”

“…The immunization experiments using lysozyme were performed according the methods of So et al (19), with slight modification. Five mice were injected i.p.…”

“…with 50 mg wild-type Phl p 7 or M5C mutant Phl p 7 (50 mg/mouse) adsorbed to 2 mg aluminum hydroxide on days 0, 7, 14, 21, 28, 35, and 42, and blood samples were drawn from the orbital sinus into capillary tubes every week. Serum IgG levels to each protein Ag were determined by ELISA, according to previously described methods (19).…”

“…We used the Freund's adjuvant to evaluate the difference in IgG response between immunizing proteins, according to a previous study (19). The reactivities of these antisera to the respective HEL derivatives or 3mut-ML mutants were analyzed using ELISA.…”

Immunogenicity of Antibody Drug Conjugates: Bioanalytical Methods and Monitoring Strategy for a Novel Therapeutic Modality

“…As a result, self-protein sequences (including human antibody germ line sequences) have the potential to be recognized as foreign, as a consequence of occurring in a conjugated protein entity alone. Observations related to this possibility have been made in murine models, where modifications in protein conformational stability (53) or susceptibility to lysosomal proteolysis (54) have been shown to contribute to immunogenicity. Another example is the c o n j u g a t i o n o f a h e n e g g l y s o z y m e ( H E L ) t o phosphorylcholine.…”