Development of a rat central venous catheter model for evaluation of vaccines to prevent Staphylococcus epidermidis and Staphylococcus aureus early biofilms.

Indwelling central venous catheters are a common and important source of nosocomial Staphylococcus epidermidis and S. aureus infections, causing increased morbidity and mortality during hospitalization. A model was developed to reflect the clinical situation of catheter colonization by transient hematogeneously spread staphylococci, in order to investigate potential vaccine candidates. Rats were cannulated in the right jugular vein, followed by challenge through the tail vein with either S. epidermidis RP62a, or S. aureus Becker. At 24 hr post challenge, colonizing bacteria were found to be present on the catheter in an early biofilm, as evidenced by the presence of polysaccharide intercellular adhesin (PIA). For vaccination studies, rats were first immunized, surgically cannulated, and then challenged via the tail vein. At 24 hr post challenge, the catheters were harvested and cultured on mannitol salt agar plates. The catheters were scored as positive if there was outgrowth of bacterial colonies, and negative if no colonies were observed. A S. epidermidis antigen (SERP0630, MenD), and a S. aureus antigen (SACOL1138, iron regulated surface determinant B, IsdB) were found to have significant protective activity in this model, compared to mock immunized controls. Using SERP0630 as the test immunogen, it was also determined that a single vaccination of rats after cannulation was sufficient for significant catheter protection. This model may be used to evaluate antigens for protective activity against transient hematogenous spread of staphylococci resulting in catheter colonization and early biofilm formation.

Staphylococcus aureus capsule type 8 antibodies provide inconsistent efficacy in murine models of staphylococcal infection.

Staphylococcus aureus is a clinically important capsule-forming bacterium. The capsule polysaccharide (CPs) occurs as different chemical structures depending on the serotype of the organism, but one form, capsular polysaccharide type 8 (CPs8) found in clinical isolates, is largely unstudied. The potential of CPs8 as a vaccine target was evaluated using two approaches. The first approach used a conjugate vaccine, made by chemically linking purified CPs8 to the outer membrane protein complex of N. meningitidis serotype B (OMPC). In efficacy studies, the CPs8-OMPC conjugate vaccine was immunogenic in Balb/c mice, however the immune response gave no protection from death after a lethal intravenous (IV) challenge with S. aureus Becker. In the second approach, two monoclonal antibodies were produced against CPs8 (mAbs 8E8 and 1C10). These were found to have functional activity in an opsonophagocytic killing assay (OPA), and provided protection from a lethal challenge when bacteria were pre-opsonized ex vivo before intra-peritoneal (IP) challenge. However, mAb 8E8 was not efficacious in the lethal challenge model, in which antibodies were passively transferred to the peritoneum and the animals were infected via the tail vein 18-24 h later. Additionally, the monoclonal antibodies did not opsonize capsule-expressing S. aureus Becker obtained from in vivo growth conditions. These results indicated that functional capsule antibodies may not be sufficient for protection from S. aureus under all in vivo conditions.

Immunization with Staphylococcus aureus iron regulated surface determinant B (IsdB) confers protection via Th17/IL17 pathway in a murine sepsis model.

We have previously shown that IsdB, a conserved protein expressed by Staphylococcus aureus, induces a robust antibody response which correlates with protection in a murine challenge model. Here we investigate the role of cellular immunity in IsdB mediated protection using lymphocyte deficient SCID mice. As opposed to WT CB-17 mice the CB-17 SCID mice were not protected against a lethal challenge of S. aureus after active and passive immunizations with IsdB. Adoptive transfer of in vitro isolated lymphocyte subsets revealed that reconstituting mice with IsdB specific CD3+ or CD4+ T-cells conferred antigen specific protection while CD8(+) T-cells, CD19(+) B-cells and plasma cells (CD138(high)B220(int)CD19(lo)) alone were not protective. A combination of CD3(+) T-cells plus CD19(+) B-cells conferred protection in CB-17 SCID mice, whereas bovine serum albumin (BSA) immune lymphocytes did not confer protection. Active immunization experiments indicated that IsdB immunized Jh mice (B-cell deficient) were protected against lethal challenge, while nude (T-cell deficient) mice were not. In vitro assays indicated that isolated IsdB specific splenocytes from immunized mice produced abundant IL-17A, much less IFN-γ and no detectable IL-4. IL-23 deficient mice were not protected from a lethal challenge by IsdB vaccination, pointing to a critical role for CD4(+) Th17 in IsdB-mediated vaccination. Neutralizing IL-17A, but not IL-22 in vivo significantly increased mortality in IsdB immunized mice; whereas, neutralizing IFN-γ did not alter IsdB-mediated protection. These findings suggest that IL-17A producing Th17 cells play an essential role in IsdB vaccine-mediated defense against invasive S. aureus infection in mice.

A fully human monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB) with functional activity in vitro and in vivo.

A fully human monoclonal antibody (CS-D7, IgG1) specific for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus was isolated from the Cambridge Antibody Technology (CAT) scFv antibody library. As compared to previously described IsdB specific murine monoclonals, CS-D7 has a unique, non-overlapping binding site on IsdB, and exhibits increased in vivo activity. The antibody recognizes a conformational epitope spanning amino acids 50 to 285 and has a binding affinity of 340 (± 75) pM for IsdB. CS-D7 bound to a wide variety of S. aureus strains, but not to an isdB deletion mutant. The antibody mediated opsonophagocytic (OP) killing in vitro and mediated significant protection in vivo. In a murine lethal sepsis model, the antibody conferred protection from death when dosed prior to challenge, but not when dosed after challenge. Importantly, in a central venous catheter (CVC) model in rats, the antibody reduced bacteremia and prevented colonization of indwelling catheters. Protection was observed when rats were dosed with CS-D7 prior to challenge as well as post challenge. IsdB is currently being investigated for clinical efficacy against S. aureus infection, and the activity of this human IsdB specific antibody supplements the growing body of evidence to support targeting this antigen for vaccine development.

Selection and characterization of murine monoclonal antibodies to Staphylococcus aureus iron-regulated surface determinant B with functional activity in vitro and in vivo.

In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (> or =50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.