Fig. 9: Opsonophagocytic killing activities of mouse antisera on three species of bacteria following immunization with the dPNAG-CRM197 conjugates as vaccine candidates.

Mouse antisera obtained 14 days after the final immunization with dPNAG-CRM197 conjugates were diluted 1:10 to 1:640. The red dashed lines indicate an OPK activity of 40%. Comparison of the OPK activities of sera obtained from mice immunized with non-acetylated dPNAG 4, 8, 12, and 18mer-NH₂-CRM197 conjugates against (A) S. aureus serotype Newman, (B) S. pneumonia serotype 19 A, and (C) A. baumannii strain ATCC 17978; comparison of the OPK activities of sera obtained from mice immunized with partially N-acetylated dPNAG 4, 8, 12, and 18mer-NHAc-CRM197 conjugates against (D) S aureus serotype Newman, (E) S. pneumonia serotype 19 A, and (F) A. baumannii strain ATCC 17978. The experiment was performed in biological replicates, and error bars represent the standard deviation from the mean of the data point (with n = 6 for S. pneumonia serotype 19 A and S. aureus serotype Newman, and n = 5 for S aureus serotype Newman). Statistical analysis was performed with one-way ANOVA followed by Tukey’s multiple comparison test using GraphPad Prism version 10.4.0 for macOS. Statistical comparisons of the killing at the same serum dilution, and with a P < 0.05, were defined as “a” for PNAG-8mer-NH₂ vs PNAG-4mer-NH₂, “b” for PNAG-8mer-NH₂ vs PNAG-12mer-NH₂, “c” for PNAG-8mer-NH₂ vs PNAG-18mer-NH₂, “d” for PNAG-8mer-40%Ac vs PNAG-4mer-45%Ac, “e” for PNAG-8mer-40%Ac vs PNAG-12mer-45%Ac, “f” for PNAG-8mer-40%Ac vs PNAG-12mer-60%Ac, and “g” for PNAG-8mer-40%Ac vs PNAG-18mer-40%Ac. The red asterisk indicates a significant difference between the negative control (PBS) with all other antisera tested at the same serum dilution in the assay. Multiple comparisons and P values are shown in Tables S4–S6.