Comparative Opsonic and Protective Activities of Staphylococcus aureus Conjugate Vaccines Containing Native or Deacetylated Staphylococcal Poly-N-Acetyl- -(1-6)-Glucosamine

Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA.
Infection and Immunity (Impact Factor: 3.73). 11/2005; 73(10):6752-62. DOI: 10.1128/IAI.73.10.6752-6762.2005
Source: PubMed


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.

Download full-text


Available from: Donald A Goldmann, May 03, 2015
  • Source
    • "More recently, both PNAG and N-deacetylated (dPNAG) have been studied as potential vaccine targets in therapeutic approaches against a range of human microbial pathogens [8] [9]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Staphylococcus aureus and Staphylococcus epidermidis cause dangerous and difficult to treat medical device-related infections through their ability to form biofilms. Extracellular poly-N-acetylglucosamine (PNAG) facilitates biofilm formation and is a vaccination target, yet details of its biosynthesis by the icaADBC gene products is limited. IcaC is the proposed transporter for PNAG export, however a comparison of the Ica proteins to homologous exo-polysaccharide synthases suggests that the common IcaAD protein components both synthesise and transport the PNAG. The limited distribution of icaC to the Staphylococcaceae and its membership of a family of membrane-bound acyltransferases, leads us to suggest that IcaC is responsible for the known O-succinylation of PNAG that occurs in staphylococci, identifying a potentially new therapeutic target specific for these bacteria.
    FEBS letters 04/2014; 588(10). DOI:10.1016/j.febslet.2014.04.002 · 3.17 Impact Factor
  • Source
    • "S. aureus isolates used were CP8 strain MN8 [23], CP5 strain Newman [24], and nontypable (NT) USA 300 MRSA strain LAC [25]. Isogenic variants lacking cap, ica, clfA, clfB, and isdB have been previously described [10], [26], [27], [28], [29], [30], [31], [32]. Mutants were constructed in spontaneous streptomycin-resistant (Smr) isolates of wild-type S. aureus strains Newman [33] that were selected on tryptic soy agar (TSA) plates containing 0.5 mg/ml streptomycin as described before [16]. "
    [Show abstract] [Hide abstract]
    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
  • Source
    • "Polymorphonuclear cells (PMNs) were prepared from fresh human blood collected from healthy adult volunteers as described earlier [4] under a protocol approved by the Institutional Review Board of Partner's Healthcare System and the concentration adjusted to 2.5×107/ml in modified minimal essential medium (MEM)+1% BSA. "
    [Show abstract] [Hide abstract]
    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
Show more