Vaccination with Clumping Factor A and Fibronectin Binding Protein A to Prevent Staphylococcus aureus Infection of an Aortic Patch in Mice

Carlos Arrecubieta Iwao Matsunaga 1 Tomohiro Asai 1 Yoshifumi Naka 1 Mario C. Deng 0 Franklin D. Lowy 0 Cardiology, Department of Medicine, and Departments of 1 Surgery, College of Physicians and Surgeons, Columbia University , New York , New York Staphylococcus aureus is a leading cause of ventricular assist device-related infections. This study evaluated the protective effect against S. aureus infection of active and passive immunization that targeted 3 proteins involved in bacterial attachment to a murine intra-aortic polyurethane patch. Active immunization of mice with a combination of the A domains of clumping factor A (ClfA), fibronectin-binding protein A (FnBPA) and fibronectin-binding protein B or passive immunization with monoclonal antibodies against ClfA and FnBPA resulted in a higher level of protection than that obtained by vaccination with either protein or antibody alone. The combination of antibodies or protein antigens appears to provide enhanced protection against prosthetic-device infection. - Ventricular assist devices (VADs) are an important form of therapy for patients with end-stage congestive heart failure. However, a high incidence of VAD-related infections, often caused by Staphylococcus aureus, has been a serious limitation to the longterm use of VADs [1]. Eradication of infection usually requires removal of the device, posing a major threat to survival [1, 2]. The VAD surface undergoes a number of changes over time as host components adhere and form a dynamic neointimal layer [3]. The first step in the pathogenesis of VAD-related infections involves bacterial adhesion to the prosthetic neointimal surface. For this process, S. aureus possesses an array of surface proteins that adhere to both cellular and extracellular host components [4]. Via their fibrinogen-binding domains, 3 adhesins clumping factor A (ClfA), fibronectin-binding protein A (FnBPA), and fibronectin-binding protein B (FnBPB)mediate in vitro adherence of S. aureus to explanted VAD membranes, as well as in vivo binding to an implanted aortic patch in mice [5]. In this study, we investigated the potential role of the A domains of ClfA, FnBPA, and FnBPB (rAClfA, rAFnBPA, and rAFnBPB, respectively) as protective antigens against S. aureus infection in an aortic patch murine model by both active and passive immunization. Materials and methods. S. aureus strain LS-1 [6] was grown at 37C in tryptic soy broth or mannitol salt agar (BD Biosciences). Escherichia coli strain XL-1 Blue (Stratagene) was grown at 37C in Luria Bertani broth (BD Biosciences), with or without 1% agar, supplemented with ampicillin (100 g/mL). Oligonucleotides used to generate the appropriate DNA fragments that code for the A domains of ClfA, FnBPA, and FnBPB were described elsewhere [7, 8]. DNA fragments were amplified from S. aureus strain LS-1 using Platinum PCR Supermix High Fidelity (Invitrogen). The fragments were digested, ligated to pQE-30 (Qiagen) by use of the appropriate restriction enzymes (New England Biolabs), and introduced into E. coli strain XL1-Blue. Recombinant polyhistidine-tagged proteins were purified using HisTrap HP columns (GE Healthcare) in accordance with the manufacturers instructions and dialyzed extensively against phosphate-buffered saline (PBS). Protein concentrations were determined using the Bio-Rad Protein Assay. Monoclonal antibody (MAb) 12-9 (anti-rAClfA) was raised against rAClfA from S. aureus strain Newman and has been described elsewhere [5]. MAb 43-677.2 (anti-rAFnBPA) recognizes the peptide-spanning repeats N2 and N3 of FnBPA from S. aureus strain 8325-4 and inhibits rAFnBPA binding to fibrinogen (kindly provided by John Vernachio [Inhibitex]). Between 3 and 6 h after implantation of the aortic patch, C57BL/6J mice were immunized by intraperitoneal injection with 1 mg of the corresponding antibodies unless otherwise stated. Murine IgG1 (Sigma) was used as an isotypic control antibody. For active immunization experiments, C57BL/6J mice were immunized using a previously described protocol [9], and each mouse received 30 g of protein per injection. Bovine serum albumin (BSA [Sigma]) was used as the antigen control. The first subcutaneous immunization was performed on day 21, followed by a booster injection on day 10. Aortic patch implantation was performed on day 0. Blood samples were obtained by retro-orbital bleeding on day 0 for measurement of antibody concentrations. Implantation of polyurethane patches and infection of mice were performed using our previously described model with minor modifications [5]. Briefly, S. aureus strain LS-1 was cultured overnight to stationary phase, diluted into fresh medium and allowed to reach logarithmic growth phase. Cells were harvested and resuspended in PBS to an OD600 of 0.15. Sterile polyurethane patches (size, 1 4 mm [Thoratec Corporation]) were implanted in the abdominal aorta. Twenty-four hours later, all mice were challenged by tail vein injection with an identical bacterial inoculum (7 10610 106 colony-forming units [CFU]). This inoculum had been previously shown to achieve an infection rate of 100%. A blood sample was obtained 30 min after inoculation to confirm bacteremia. Twenty-four hours after inoculation, animals were anesthetized, and polyurethane patches were excised and processed as previously described [5]. Bacterial loads were calculated by serial dilution of homogenized samples, plating onto tryptic soy agar, and incubation for 24 h at 37C. Serum antibody concentrations from individual mice were determined by enzyme-linked immunosorbent assay (ELISA). Triplicate microtiter wells were coated with 0.25 g of the A domains of ClfA, FnBPA, or FnBPB. Wells were blocked with 2% skim milk in PBS and incubated with sera from immunized mice (dilution, 1:1000 in PBS), followed by incubation with anti-mouse IgG horseradish peroxidase conjugated antibody (Sigma). Peroxidase activity was measured using 1-Step UltraTMB-ELISA (Pierce), according to the manufacturers instructions. Statistical evaluation was done by analysis of variance. Whole-cell dot immunoblot was performed as follows: serial dilutions of a bacterial suspension with an OD600 of 50 were prepared in PBS, and 1- L drops were adsorbed to nitrocellulose filters, air dried, blocked with 5% skim milk in PBS supplemented with 0.05% Tween-20 (PBS-T), and incubated with anti-rAFnBPA (dilution, 1:5000 in PBS-T), followed by antimouse IgG horseradish peroxidase conjugated antibody (dilution, 1:5000 in PBS-T) and Immobilon Western substrate (Millipore), according to the manufacturers instructions. The Institutional Animal Care and Use Committee of Columbia University approved this study. Results. Patches from mice actively immunized with rAClfA had significantly lower levels of S. aureus than patches explanted from control mice immunized with BSA (P .01), suggesting that an immune response against rAClfA was able to partially protect against S. aureus colonizatio (...truncated)