Alanine scanning mutagenesis of SP70 epitope in characterizing species-specific antibodies induced by enterovirus 71-based antigens

Abstract

The enterovirus 71 (EV71) SP70 epitope, derived from amino acids 208-222 of VP1, is a neutralizing epitope. The present study aimed to assess the inter-species differences of the antibodies induced by EV71-based antigens in responses to SP70 mutant peptides. BALB/c mice and Lou/C rats were immunized with EV71 SP70. Monoclonal antibodies (Mabs) were produced by hybridoma clones. Serum polyclonal antibodies (Pabs) were produced from BALB/c mice and New Zealand white rabbits immunized with recombinant EV71 VP1 (rEV71-VP1) protein or inactivated EV71. Micro-neutralization and immunofluorescence assays were used to evaluate the capacity of the antibodies to bind to EV71. Reactivity of Mabs and Pabs to mutated SP70 were determined by alanine scanning mutagenesis. Furthermore, serums from EV71-infected patients were collected to examine the affinity of SP70 antibody in the serum to mutated SP70, using competitive ELISA. The binding affinity of mouse Mabs to the SP70 epitope was increased by alanine substitution at sites of 210, 212, 213, 214, and 221. The binding affinity of rat Mabs to the SP70 epitope was increased by alanine substitution at sites 210, 217, 219, and 221. Mouse serum Pabs elicited by inactivated EV71 bound wild-type SP70, but lost affinity for mutated peptides. Conversely, rabbit serum Pabs elicited by inactivated EV71 robustly recognized SP70 mutants. Mouse serum Pabs elicited by rEV71-VP1 presented the same trend as mouse Mabs. Mutations at sites 214, 215, and 217 led to loss of recognition by rabbit Pabs elicited by rEV71-VP1, while most mutations did not influence antibody binding. Compared with the wild-type, mutations at the sites 209, 219 and 221 of SP70 lead to increased affinity with the serum antibodies produced by the EV71-infected patients. Antibody responses triggered by inactivated EV71, rEV71-VP1 and EV71 SP70 differed among species in neutralizing capacity and affinity for SP70 mutant peptides.