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.
References
[1]
Lowy FD (1998) Staphylococcus aureus infections. N Engl J Med 339: 520–532.
[2]
Lowy F (2005) Staphylococcal infections. Harrisons Principles of Internal Medicine.
[3]
Proctor RA (2012) Challenges for a universal Staphylococcus aureus vaccine. Clin Infect Dis 54: 1179–1186.
[4]
Maira-Litran T, Kropec A, Goldmann DA, Pier GB (2005) Comparative opsonic and protective activities of Staphylococcus aureus conjugate vaccines containing native or deacetylated Staphylococcal Poly-N-acetyl-β-(1–6)-glucosamine. Infect Immun 73: 6752–6762.
[5]
Smith RL, Gilkerson E (1979) Quantitation of glycosaminoglycan hexosamine using 3-methyl-2-benzothiazolone hydrazone hydrochloride. Anal Biochem 98: 478–480.
[6]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
[7]
Maira-Litran T, Kropec A, Abeygunawardana C, Joyce J, Mark G, et al. (2002) Immunochemical properties of the staphylococcal poly-N-acetylglucosamine surface polysaccharide. Infect Immun 70: 4433–4440.
[8]
Cerca N, Jefferson KK, Maira-Litran T, Pier DB, Kelly-Quintos C, et al. (2007) Molecular basis for preferential protective efficacy of antibodies directed to the poorly acetylated form of staphylococcal poly-N-acetyl-β-(1–6)-glucosamine. Infect Immun 75: 3406–3413.
[9]
Vuong C, Kocianova S, Voyich JM, Yao Y, Fischer ER, et al. (2004) A crucial role for exopolysaccharide modification in bacterial biofilm formation, immune evasion, and virulence. J Biol Chem 279: 54881–54886.
[10]
Kelly-Quintos C, Cavacini LA, Posner MR, Goldmann D, Pier GB (2006) Characterization of the opsonic and protective activity against Staphylococcus aureus of fully human monoclonal antibodies specific for the bacterial surface polysaccharide poly-N-acetylglucosamine. Infect Immun 74: 2742–2750.
[11]
Hall AE, Domanski PJ, Patel PR, Vernachio JH, Syribeys PJ, et al. (2003) Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A. Infect Immun 71: 6864–6870.
[12]
Lee J-C, Park JS, Shepherd SE, Carey V, Fattom A (1997) Protective efficacy of antibodies to the Staphylococcus aureus type 5 capsular polysaccharide in a modified model of endocarditis in rats. Infect Immun 65: 4146–4151.
[13]
Fattom AI, Sarwar J, Ortiz A, Naso R (1996) A Staphylococcus aureus capsular polysaccharide (CP) vaccine and CP-specific antibodies protect mice against bacterial challenge. Infect Immun 64: 1659–1665.
[14]
Josefsson E, Hartford O, O'Brien L, Patti J, Foster T (2001) Protection against experimental Staphylococcus aureus arthritis by vaccination with clumping factor A, a novel virulence determinant. J Infect Dis 184: 1572–1580.
[15]
Risley AL, Loughman A, Cywes-Bentley C, Foster TJ, Lee JC (2007) Capsular polysaccharide masks clumping factor A-mediated adherence of Staphylococcus aureus to fibrinogen and platelets. J Infect Dis 196: 919–927.
[16]
Domanski PJ, Patel PR, Bayer AS, Zhang L, Hall AE, et al. (2005) Characterization of a humanized monoclonal antibody recognizing clumping factor A expressed by Staphylococcus aureus. Infect Immun 73: 5229–5232.
[17]
Vernachio JH, Bayer AS, Ames B, Bryant D, Prater BD, et al. (2006) Human immunoglobulin G recognizing fibrinogen-binding surface proteins is protective against both Staphylococcus aureus and Staphylococcus epidermidis infections in vivo. Antimicrob Agents Ch 50: 511–518.
[18]
McAdow M, Kim HK, Dedent AC, Hendrickx APA, Schneewind O, et al. (2011) Preventing Staphylococcus aureus sepsis through the inhibition of its agglutination in blood. PLoS Pathog
[19]
Duthie ES, Lorenz LL (1952) Staphylococcal coagulase; mode of action and antigenicity. J Gen Microbiol 6: 95–107.
[20]
Kropec A, Maira-Litran T, Jefferson KK, Grout M, Cramton SE, et al. (2005) Poly-N-acetylglucosamine production in Staphylococcus aureus is essential for virulence in murine models of systemic infection. Infect Immun 73: 6868–6876.
[21]
McDevitt D, Francois P, Vaudaux P, Foster TJ (1994) Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol Microbiol 11: 237–248.
[22]
Kreiswirth BN, L？fdahl S, Betley MJ, O'Reilly M, Schlievert PM, et al. (1983) The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature 305: 709–712.
[23]
Holden MTG, Feil EJ, Lindsay JA, Peacock SJ, Day NPJ, et al. (2004) Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc Natl Acad Sci USA 101: 9786–9791.
[24]
O'Connell DP, Nanavaty T, McDevitt D, Gurusiddappa S, Hook M, et al. (1998) The fibrinogen-binding MSCRAMM (clumping factor) of Staphylococcus aureus has a Ca2+-dependent inhibitory site. J Biol Chem 273: 6821–6829.
