Surface plasmon resonance analysis of antipolysaccharide antibody specificity: responses to meningococcal group C conjugate vaccines and bacteria

Abstract

Antibody (Ab) responses to polysaccharides (PS), such as Neisseria meningitidis group C PS (MCPS), are characterized as being thymus independent and are restricted with regard to clonotype and isotype expression. PS conjugated to proteins, e.g., MCPS coupled with tetanus toxoid or the diphtheria toxin derivative CRM197, elicit thymus-dependent responses. The present study developed a surface plasmon resonance approach to evaluate Ab responses to MCPS conjugate vaccines, including either O-acetylated (OAc+) or de-O-acetylated (OAc-) forms of the PS. The results were generally consistent with those obtained by enzyme-linked immunosorbent assay and showed that sera from mice immunized with conjugate vaccines contain Abs that bind more effectively to OAc+ and OAc- MCPS than sera from mice immunized with fixed bacteria. The data suggest a critical shared or overlapping epitope recognized by all the conjugate vaccine immune sera and strategies for assessing polyclonal Ab avidity.

FIG. 1.

Total IgM and IgG end point titers, determined by FELISA, of anti-meningococcal serogroup C Abs in the pooled sera from groups of 10 mice immunized with different conjugate vaccines or fixed bacteria. PBS was used as a control. The titers are expressed as the log reciprocal dilution. The data represent the averages of three experiments.

FIG. 2.

SPR sensorgrams of MCPS binding with four murine anti-MCPS MAbs, C2/1076.10, C2/655.7, 2016.3, and 2055.5, with different FELISA specificities. (A) Binding to OAc⁺-BSA conjugate (243.7 RU immobilized). (B) Binding to OAc⁻-BSA conjugate (242.8 RU immobilized).

FIG. 3.

Concentration-dependent MCPS binding of four murine anti-MCPS MAbs with different specificities. The markers represent the averages of near-equilibrium binding, and the error bars (within the symbols) represent the range of two determinations. (A) C2/1076.10 and 2016.3 binding to OAc⁻ and OAc⁺ surfaces. (B) C2/655.7 and 2055.5 binding to OAc⁻ and OAc⁺ surfaces. The OAc⁺ and OAc⁻ MCPSs conjugated to BSA were used as the coating Ags.

FIG. 4.

Concentration-dependent MCPS binding of murine serum Abs induced by immunization. The markers represent the averages of near-equilibrium binding, and the error bars represent the ranges of two determinations. (A) Sera from hosts immunized with MCC conjugate vaccines by Vac1, Vac2, and Vac3 binding to OAc⁻ and OAc⁺ surfaces. (B) Sera from hosts immunized with PBS, OAc⁺ fixed bacteria, and OAc⁻ fixed bacteria binding to OAc⁻ and OAc⁺ surfaces.

FIG. 5.

Apparent dissociation half-lives of anti-MCPS Abs. The apparent dissociation half-lives were calculated by exponential decay modeling as described in Materials and Methods. (A) Averages of four determinations after injection of Abs C2/1076.10, 2016.3, and C2/655.7 at a concentration of 3.125 or 6.25 μg/ml are shown for OAc⁻ and OAc⁺ surfaces. The error bars represent the standard deviations, and the numbers in parentheses are the numerical averages. For Ab 2055.5, the bars and averages reflect two determinations and the error bars reflect the range. (B) Averages of four determinations using polyclonal sera after injection of 1/20 or 1/40 dilutions; determinations are shown for OAc⁻ and OAc⁺ surfaces. The error bars represent the standard deviations, and the numbers in parentheses are the numerical averages. (C) Examples of SPR sensorgrams of Vac2 immune sera binding to OAc⁻-BSA conjugate (242.8 RU immobilized) at concentrations of 1/10, 1/20, and 1/40.

FIG. 6.

Inhibition of binding by MAbs with different specificities. The average of duplicates for a 1/20 dilution of PBS immune serum, Vac1 immune serum, Vac2 immune serum, and Vac3 immune serum are shown. The blocking MAbs were added at 100 μg/ml, or buffer was added as a control. (A) OAc⁺-specific MAb 2055.5. (B) OAc⁻ ≫ OAc⁺-specific MAb 2016.3. (C) OAc⁺ ≈ OAc⁻ >>> K92-specific MAb C2/1076.10.