Characterization of the opsonic and protective activity against Staphylococcus aureus of fully human monoclonal antibodies specific for the bacterial surface polysaccharide poly-N-acetylglucosamine

Abstract

Carbohydrate antigens are important targets of the immune system in clearing bacterial pathogens. Although the immune system almost exclusively uses antibodies in response to foreign carbohydrates, there is still much to learn about the role of different epitopes on the carbohydrate as targets of protective immunity. We examined the role of acetyl group-dependent and -independent epitopes on the staphylococcal surface of polysaccharide poly-N-acetylated glucosamine (PNAG) by use of human monoclonal antibodies (MAbs) specific for such epitopes. We utilized hybridoma technology to produce fully human immunoglobulin G2 (IgG2) MAbs from B cells of an individual post-Staphylococcus aureus infection and cloned the antibody variable regions to produce an IgG1 form of each original MAb. Specificity and functionality of the purified MAbs were tested in vitro using enzyme-linked immunosorbent assays, complement deposition, and opsonophagocytic assays. We found that a MAb (MAb F598) that bound the best to nonacetylated or backbone epitopes on PNAG had superior complement deposition and opsonophagocytic activity compared to two MAbs that bound optimally to PNAG that was expressed with a native level (>90%) of N-acetyl groups (MAbs F628 and F630). Protection of mice against lethality due to S. aureus strains Mn8 and Reynolds further showed that the backbone-specific MAb had optimal protective efficacy compared with the acetate-specific MAbs. These results provide evidence for the importance of epitope specificity in inducing the optimal protective antibody response to PNAG and indicate that MAbs to the deacetylated form of PNAG could be immunotherapeutic agents for preventing or treating staphylococcal infections.

FIG. 1.

ELISA reactivities of IgG1 (A and B) and IgG2 (C and D) MAbs to PNAG (A and C) and dPNAG (B and D) antigens. (A) Binding of isotype-switched IgG1 MAbs F598, F628, and F630 to native PNAG. (B) Binding of IgG1 MAbs to dPNAG antigen. (C) Binding of IgG2 MAbs to PNAG antigen. (D) Binding of IgG2 MAbs to dPNAG antigen. The irrelevant antibody is a human IgG1 antibody specific for P. aeruginosa alginate.

FIG. 2.

Immunofluorescence study of binding of MAbs F598 and F628 to wild-type (WT) and Δica S. aureus strain Mn8, showing specificity of binding to the PNAG-producing parental strain.

FIG. 3.

Relative deposition onto purified PNAG of the third component of complement (C3) by IgG1 (filled symbols) and IgG2 (open symbols) MAbs. C3 deposition was measured by ELISA with a goat anti-human C3. The MAb to alginate is an irrelevant human IgG1 antibody specific for the P. aeruginosa alginate capsular antigen.

FIG. 4.

Opsonophagocytic activity of IgG1 and IgG2 MAbs. Target strain is S. aureus Mn8. Bars represent means; error bars represent the standard errors of the means for three to eight replicates. Percent reduction in CFU was calculated compared to controls containing complement, PMNs, bacteria, and IgG control antibodies.

FIG. 5.

Opsonophagocytic killing of several staphylococcal strains with MAb F598 at 12 μg/ml. Bars represent means; error bars represent the standard errors of the means. S. aureus strains Mn8, Newman, 10833, and Reynolds and S. epidermidis strain M187 represent methicillin-sensitive clinical and laboratory isolates. Strain 123 (also designated MW2 or USA400) is an MRSA strain carrying the PVL genes whose genome has been sequenced. Strains 192 and 193 represent MRSA, PVL-positive isolates from community-acquired infections. Strain 157 is a methicillin-sensitive, PVL-positive S. aureus isolate from a patient with lethal necrotizing pneumonia.

FIG. 6.

Protection against S. aureus strain Reynolds with IgG1 MAbs (12 mice per group). (A) A 600-μg volume of each MAb was administered 4 h before bacterial challenge. (B) A 300-μg volume of each MAb was administered 4 h before bacterial challenge. Challenge dose, 2.5 × 10⁸ CFU/mouse; *, P value less than 0.05 compared to IgG1 control MAb.

FIG. 7.

Protection against S. aureus MN8 lethal infection with IgG1 MAbs to PNAG (eight mice per group). (A) A 400-μg volume of each MAb was administered 4 h before challenge. Strain Mn8 challenge dose, 5 × 10⁸. (B) A 200-μg volume of each MAb was administered 4 h before challenge. Strain Mn8 challenge dose, 9 × 10⁸; *, P value less than 0.05 compared to IgG1 control MAb.