Comparative opsonic and protective activities of Staphylococcus aureus conjugate vaccines containing native or deacetylated Staphylococcal Poly-N-acetyl-beta-(1-6)-glucosamine

Abstract

Staphylococcus aureus and Staphylococcus epidermidis both synthesize the surface polysaccharide poly-N-acetyl-beta-(1-6)-glucosamine (PNAG), which is produced in vitro with a high level (>90%) of the amino groups substituted by acetate. Here, we examined the role of the acetate substituents of PNAG in generating opsonic and protective antibodies. PNAG and a deacetylated form of the antigen (dPNAG; 15% acetylation) were conjugated to the carrier protein diphtheria toxoid (DT) and used to immunize animals. Mice responded in a dose-dependent fashion to both conjugate vaccines, with maximum antibody titers observed at the highest dose and 4 weeks after the last of three weekly immunizations. PNAG-DT and dPNAG-DT vaccines were also very immunogenic in rabbits. Antibodies raised to the conjugate vaccines in rabbits mediated the opsonic killing of various staphylococcal strains, but the specificity of the opsonic killing was primarily to dPNAG, as this antigen inhibited the killing of S. aureus strains by both PNAG- and dPNAG-specific antibodies. Passive immunization of mice with anti-dPNAG-DT rabbit sera showed significant levels of clearance of S. aureus from the blood (54 to 91%) compared to control mice immunized with normal rabbit sera, whereas PNAG-specific antibodies were ineffective at clearing S. aureus. Passive immunization of mice with a goat antiserum raised to the dPNAG-DT vaccine protected against a lethal dose of three different S. aureus strains. Overall, these data show that immunization of animals with a conjugate vaccine of dPNAG elicit antibodies that mediated opsonic killing and protected against S. aureus infection, including capsular polysaccharide types 5 and 8 and an untypable strain.

FIG.1.

Gel filtration elution profiles of PNAG-DT (A) and dPNAG-DT conjugate vaccines (B) on a Superose 6 prep-grade column. The presence of polysaccharide (open circles) in fractions was monitored by the hexosamine assay (OD₆₅₀) and the presence of protein (closed circles) in the fractions was monitored by the Bradford assay (OD₅₉₅). Double-headed arrows indicate the fractions containing clearly conjugated protein and polysaccharide, as well as where the unconjugated polysaccharide (PNAG or dPNAG) and protein (DT) eluted.

FIG. 2.

Mean titers of IgG antibodies in sera pooled from 10 mice immunized three times at weekly intervals with the indicated dose of PNAG-DT (A) or dPNAG (B) conjugate vaccines. Sera were collected weekly for 4 weeks starting 1 week after the last dose of conjugate vaccine and were tested by ELISA with the homologous immunizing antigen, either PNAG (A) or dPNAG (B) as a coating antigen. Bars represent means and error bars indicate standard deviations. All preimmune titers were <25.

FIG. 3.

IgG subclass distributions in mouse sera obtained 4 weeks after the third booster immunizations with PNAG-DT and dPNAG-DT conjugate vaccines.

FIG. 4.

Opsonophagocytic killing of various Staphylococcal strains indicated in each panel by rabbit sera raised to PNAG-DT conjugate vaccine (open symbols) or dPNAG-DT conjugate vaccines (solid symbols). Points represent mean of quadruplicate determinations, two for each of the two rabbits immunized with each vaccine, and error bars indicate standard deviations. Opsonic killing of >30% is considered biologically significant and is statistically significant at P < 0.01; analysis of variance (ANOVA) and Fisher probable least squares difference (PLSD) posthoc, pairwise analysis.

FIG. 5.

Opsonic killing of native and Δica S. aureus strains MN8 and Newman by 1:10 dilutions of either preimmune serum or antiserum raised to native PNAG-DT or dPNAG DT in the presence of 5% final concentration of complement. Bars represent mean percentages of killing and error bars indicate the standard deviation. Killing of wild-type strains by antisera to either native PNAG or dPNAG was significant at P values of <0.05, compared to killing by NRS.

FIG. 6.

Inhibitory capacity of PNAG and dPNAG in the opsonophagocytic assay. Inhibition of opsonic killing of the staphylococcal strain indicated in each panel by rabbit serum raised to PNAG-DT conjugate mixed with purified PNAG (solid bars) or dPNAG (open bars) antigens at the indicated concentration. Bars represent means of quadruplicate determinations and error bars indicate the standard deviations. Inhibition of >40% is considered biologically significant and is statistically significant at P values of <0.01; ANOVA and Fisher PLSD posthoc, pairwise analysis.

FIG. 7.

Inhibitory capacity of PNAG and dPNAG in the opsonophagocytic assay against antisera raised to dPNAG-DT. Inhibition of opsonic killing of the staphylococcal strain indicated in each panel by rabbit serum raised to dPNAG-DT conjugate mixed with purified PNAG (solid bars) or dPNAG (open bars) antigens at the indicated concentration. Bars represent means of quadruplicate determinations, and error bars indicate the standard deviations. Inhibition of >40% is considered biologically significant and is statistically significant at P values of <0.01; ANOVA and Fisher PLSD posthoc, pairwise analysis.