Analysis of distinct variants of immunoglobulin G binding protein EAG on humoral immunity and bacterial clearance of Streptococcus equi subspecies equi.

Abstract

OBJECTIVE: Streptococcus equi subspecies equi (S. equi) causes strangles, one of the most prevalent and highly contagious equine infectious diseases with significant welfare and economic impacts. Alpha 2-macroglobulin and immunoglobulin G binding protein (EAG) has been identified as a key antigen and plays a crucial role in the immune evasion of S. equi. In this study, we aimed to investigate the genetic polymorphism of EAG and to determine the effects of genetic variation on its function and immunogenicity. METHODS: Phylogenetic analysis indicated these EAG mutants belong to two clades, respectively. Three recombinant EAG mutants-EAG5012 (K123E and 155-169), EAG823 (A73V), and EAGHT1112 (T196I)-were expressed and purified. A mouse model and equine were then used to evaluate the immunogenicity and protection efficacy of these mutants. Antibody levels, phagocytosis inhibition, and opsonophagocytosis were assayed. RESULTS: Our results showed that the EAG5012 mutant elicited the highest levels of specific immunoglobulin G (IgG), IgG1, and IgG2a, protecting immunized mice against intraperitoneal challenge of three different S. equi strains, with a protection rate of 80 %-86.7 %. Additionally, the EAG5012 mutant exhibited significantly enhanced phagocytic inhibition, and its antisera showed increased opsonophagocytic activity. Conversely, the EAG823 mutant displayed the lowest IgG levels, opsonophagocytic capability, and protection rate (66.7 %-73 %). The results suggest that the variations located at K123E and 155-169 in the EAG5012 mutant are significantly affected the opsonophagocytic activity of PMNs. CONCLUSION: The conformational epitope altered by these variations significantly impacts IgG immune responses and antibody-mediated opsonophagocytic activity. Our findings may be useful for the future development of effective multivalent subunit vaccines against strangles.