Figure 6 Kinetics of neutralizing antibodies to EV71 following immunization. Neutralizing antibodies in the sera of immunized mice to EV71 were measured by in vitro microneutralization assay. The neutralizing antibody titer was defined as the highest serum dilution that prevented the occurrence

of cytopathic effects. Each bar represents the mean reciprocal log2 endpoint titers and standard error. Neonatal mice as a model to verify in vitro neutralizing ability of chimeric VLP-immunized sera EV71 BrCr-TR strain was used for viral infection because of its high virulence in neonatal mice. Groups of one-day-old BALB/c suckling mice (n =10 Trichostatin A per group) were inoculated intraperitoneally (i.p.) with the virus-sera mixtures that had been incubated overnight at 37°C. After 7 days, control mice receiving EV71 with either PBS or anti- HBcAg VLPs sera started to show symptoms, such as reduced mobility, limb weakness, limb paralysis, and death (Figure 7A and B). The survival rates were 20% Lazertinib manufacturer and 40% for the PBS and anti-HBcAg VLPs sera recipient groups, respectively, at 16 day post-inoculation (Figure 7C). In contrast, 90% of mice treated with mixture of anti- chimeric VLPs sera remained healthy and survived throughout the course. These observations confirmed previous experiments using RD cells, that immune sera elicited by chimeric particles neutralized EV71 infection. Figure 7 Neonatal mice as a model to assess in-vitro neutralizing

effects of anti-sera. Groups of one-day-old BALB/c suckling mice were inoculated intraperitoneally (i.p.) with the virus-sera and virus-PBS mixture. (A) Mice with different antiserum

treatment at 11 days post-infection with EV71. The mouse on the left side received anti-chimeric VLPs sera and the one on the right side received anti-HBcAg VLPs sera. The appearance of limb paralysis in mouse is indicated by arrows. (B) Two representative GBA3 mice in the PBS-treated group die at 7 days post-infection with EV71. (C) Survival rates were recorded daily after infection for 16 days. 10 mice were used for each group. Identification of “core sequence” by S3I-201 solubility dmso epitope mapping VP4N20 peptide can elicit neutralizing antibody and conferred cross-protection against EV71 strains belonging to different genotypes in vitro. We further investigated the most immunologically essential sequence of the peptide by epitope mapping experiments to find out the minimal peptide sequence showing the highest efficiency for inducing the production of neutralizing antibody. A panel of peptides corresponding to the N- and C-terminal truncations of VP4N20 peptide was used for epitope mapping. As shown in Figure 8, the polyclonal antibodies raised against the VP4N20 peptide were very sensitive to truncation of either end of the peptide. Once six (N-terminal) or ten (C-terminal) residues were clipped from either end of the inoculation peptide, the polyclonal antibodies were no longer able to bind.