Characterization of the Opsonic and Protective Activity against <i>Staphylococcus aureus</i> of Fully Human Monoclonal Antibodies Specific for the Bacterial Surface Polysaccharide Poly- <i>N</i> -Acetylglucosamine

TLDR

Carbohydrate antigens are key targets of the immune system, yet the protective roles of distinct carbohydrate epitopes remain incompletely understood. The study aimed to determine how acetylation‑dependent and –independent epitopes on the staphylococcal polysaccharide PNAG influence antibody‑mediated protection. Fully human IgG2 monoclonal antibodies were generated by hybridoma, cloned to IgG1, and characterized for binding, complement activation, and opsonophagocytic activity in vitro. A backbone‑specific MAb (F598) induced stronger complement deposition, opsonophagocytosis, and mouse protection than acetyl‑specific MAbs, highlighting epitope specificity and suggesting deacetylated PNAG antibodies as potential therapeutics.

Abstract

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 (&gt;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.