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

Harvard Medical School, 181 Longwood Ave., Boston, MA 02115, USA.
Infection and Immunity (Impact Factor: 4.16). 06/2006; 74(5):2742-50. DOI: 10.1128/IAI.74.5.2742-2750.2006
Source: PubMed

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.

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    • "In addition to the wide studies pertaining to groups of S. aureus surface epitopes, much research has been done on specific staphylococcal antigens, i.e., peptidogylcan, the cell WTA (a glycopolymer covalently linked to peptidoglycan), polymeric-N-acetylglucosamine (PNAG) (29, 30), N-acetylglucosamine (GlcNac) (31), iron-regulated surface protein B (IsdB) (32, 33), and ClfA (34, 35). As an example, interesting studies have been published on the opsonizing capacity of peptidoglycan. "
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    ABSTRACT: Initial elimination of invading Staphylococcus aureus from the body is mediated by professional phagocytes. The neutrophil is the major phagocyte of the innate immunity and plays a key role in the host defense against staphylococcal infections. Opsonization of the bacteria with immunoglobulins and complement factors enables efficient recognition by the neutrophil that subsequently leads to intracellular compartmentalization and killing. Here, we provide a review of the key processes evolved in neutrophil-mediated phagocytosis of S. aureus and briefly describe killing. As S. aureus is not helpless against the professional phagocytes, we will also highlight its immune evasion arsenal related to phagocytosis.
    Frontiers in Immunology 09/2014; 5:467. DOI:10.3389/fimmu.2014.00467
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    • "Notably, most humans (>95%) have high titers of natural antibody directed to the acetylated epitopes of native PNAG, and this antibody is poorly opsonic and not protective in animal models. Some, but not all, human infections with S. aureus induce opsonic antibodies to dPNAG [14], [15], and ∼3% of normal humans have natural dPNAG-specific opsonic antibody (unpublished finding). The validity of raising antibody to the deacetylated glycoform of PNAG to produce protective antibody was strongly validated in work that used a synthetic oligosaccharide composed of nine b-1-6-linked monomers of glucosamine (9GlcNH2) conjugated to tetanus toxoid (TT) as a vaccine. "
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    ABSTRACT: Staphylococcus aureus is a major cause of nosocomial and community-acquired infections for which a vaccine is greatly desired. Antigens found on the S. aureus outer surface include the capsular polysaccharides (CP) of serotype 5 (CP5) or 8 (CP8) and/or a second antigen, a β-(1→6)-polymer of N-acetyl-D-glucosamine (PNAG). Antibodies specific for either CP or PNAG antigens have excellent in vitro opsonic killing activity (OPKA), but when mixed together have potent interference in OPKA and murine protection. To ascertain if this interference could be abrogated by using a synthetic non-acetylated oligosaccharide fragment of PNAG, 9GlcNH(2), in place of chemically partially deacetylated PNAG, three conjugate vaccines consisting of 9GlcNH(2) conjugated to a non-toxic mutant of alpha-hemolysin (Hla H35L), CP5 conjugated to clumping factor B (ClfB), or CP8 conjugated to iron-surface determinant B (IsdB) were used separately to immunize rabbits. Opsonic antibodies mediating killing of multiple S. aureus strains were elicited for all three vaccines and showed carbohydrate antigen-specific reductions in the tissue bacterial burdens in animal models of S. aureus skin abscesses, pneumonia, and nasal colonization. Carrier-protein specific immunity was also shown to be effective in reducing bacterial levels in infected lungs and in nasal colonization. However, use of synthetic 9GlcNH(2) to induce antibody to PNAG did not overcome the interference in OPKA engendered when these were combined with antibody to either CP5 or CP8. Whereas each individual vaccine showed efficacy, combining antisera to CP antigens and PNAG still abrogated individual OPKA activities, indicating difficulty in achieving a multi-valent vaccine targeting both the CP and PNAG antigens.
    PLoS ONE 10/2012; 7(10):e46648. DOI:10.1371/journal.pone.0046648 · 3.23 Impact Factor
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    • "This reduction in susceptibility of the ClfA-negative strain DU5852 in comparison to wild type S. aureus Newman is most likely attributable to the variation in PNAG retention on the bacterial cell surface when other factors are changed, as retention is often due to post-synthetic effects such as that of the extracellular IcaB deacetylase [8], [9]. Based on our previous experience in testing mAbs and polyclonal sera to dPNAG in the OPKA we found that opsonic killing levels >30% identify antisera with in vivo protective activity [4], [10]. "
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    ABSTRACT: The increasing frequency, severity and antimicrobial resistance of Staphylococcus aureus infections has made the development of immunotherapies against this pathogen more urgent than ever. Previous immunization attempts using monovalent antigens resulted in at best partial levels of protection against S. aureus infection. We therefore reasoned that synthesizing a bivalent conjugate vaccine composed of two widely expressed antigens of S. aureus would result in additive/synergetic activities by antibodies to each vaccine component and/or in increased strain coverage. For this we used reductive amination, to covalently link the S. aureus antigens clumping factor A (ClfA) and deacetylated poly-N-β-(1-6)-acetyl-glucosamine (dPNAG). Mice immunized with 1, 5 or 10 µg of the dPNAG-ClfA conjugate responded in a dose-dependent manner with IgG to dPNAG and ClfA, whereas mice immunized with a mixture of ClfA and dPNAG developed significantly lower antibody titers to ClfA and no antibodies to PNAG. The dPNAG-ClfA vaccine was also highly immunogenic in rabbits, rhesus monkeys and a goat. Moreover, affinity-purified, antibodies to ClfA from dPNAG-ClfA immune serum blocked the binding of three S. aureus strains to immobilized fibrinogen. In an opsonophagocytic assay (OPKA) goat antibodies to dPNAG-ClfA vaccine, in the presence of complement and polymorphonuclear cells, killed S. aureus Newman and, to a lower extent, S. aureus Newman ΔclfA. A PNAG-negative isogenic mutant was not killed. Moreover, PNAG antigen fully inhibited the killing of S. aureus Newman by antisera to dPNAG-ClfA vaccine. Finally, mice passively vaccinated with goat antisera to dPNAG-ClfA or dPNAG-diphtheria toxoid conjugate had comparable levels of reductions of bacteria in the blood 2 h after infection with three different S. aureus strains as compared to mice given normal goat serum. In conclusion, ClfA is an immunogenic carrier protein that elicited anti-adhesive antibodies that fail to augment the OPK and protective activities of antibodies to the PNAG cell surface polysaccharide.
    PLoS ONE 09/2012; 7(9):e43813. DOI:10.1371/journal.pone.0043813 · 3.23 Impact Factor
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