Multivalent human antibody-centyrin fusion protein to prevent and treat Staphylococcus aureus infections.

Treating and preventing infections by antimicrobial-resistant bacterial pathogens is a worldwide problem. Pathogens such as Staphylococcus aureus produce an array of virulence determinants, making it difficult to identify single targets for the development of vaccines or monoclonal therapies. We described a human-derived anti-S. aureus monoclonal antibody (mAb)-centyrin fusion protein ("mAbtyrin") that simultaneously targets multiple bacterial adhesins, resists proteolysis by bacterial protease GluV8, avoids Fc engagement by S. aureus IgG-binding proteins SpA and Sbi, and neutralizes pore-forming leukocidins via fusion with anti-toxin centyrins, while maintaining Fc- and complement-mediated functions. Compared with the parental mAb, mAbtyrin protected human phagocytes and boosted phagocyte-mediated killing. The mAbtyrin also reduced pathology, reduced bacterial burden, and protected from different types of infections in preclinical animal models. Finally, mAbtyrin synergized with vancomycin, enhancing pathogen clearance in an animal model of bacteremia. Altogether, these data establish the potential of multivalent mAbs for treating and preventing S. aureus diseases.

Characterization of the mechanism of protection mediated by CS-D7, a monoclonal antibody to Staphylococcus aureus iron regulated surface determinant B (IsdB).

We previously reported the development of a human monoclonal antibody (CS-D7, IgG(1)) with specificity and affinity for the iron regulated surface determinant B (IsdB) of Staphylococcus aureus. CS-D7 mediates opsonophagocytic killing in vitro and protection in a murine sepsis model. In light of recent data indicating that IsdB specific T cells (CD4+, Th17), not Ab, mediate protection after vaccination with IsdB, it is important to investigate the mechanism of protection mediated by CS-D7. The mAb was examined to determine if it blocked heme binding to IsdB in vitro. The mAb was not found to have heme blocking activity, nor did it prevent bacterial growth under in vivo conditions, in an implanted growth chamber. To assess the role of the mAb Fc a point mutation was introduced at aa 297 (CS-D7·N297A). This point mutation removes Fc effector functions. In vitro analysis of the mutein confirmed that it lacked measurable binding to FcγR, and that it did not fix complement. The mutein had dramatically reduced in vitro opsonic OP activity compared to CS-D7. Nonetheless, the mutein conferred protection equivalent to the wild type mAb in the murine sepsis model. Both wild type and mutein mAbs were efficacious in FcγR deletion mice (including both FcγRII(-/-) mice and FcγRIII(-/-) mice), indicating that these receptors were not essential for mAb mediated protection in vivo. Protection mediated by CS-D7 was lost in Balb/c mice depleted of C3 with cobra venom factor (CFV), was lost in mice depleted of superoxide dismutase (SOD) in P47phox deletion mice, and as previously reported, was absent in SCID mice (Joshi et al., 2012). Enhanced clearance of S. aureus in the liver of CS-D7 treated mice and enhanced production of IFN-γ, but not of IL17, may play a role in the mechanism of protection mediated by the mAb. CS-D7 apparently mediates survival in challenged mice through a mechanism involving complement, phagocytes, and lymphocytes, but which does not depend on interaction with FcγR, or on blocking heme uptake.

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.

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.

A novel Staphylococcus aureus vaccine: iron surface determinant B induces rapid antibody responses in rhesus macaques and specific increased survival in a murine S. aureus sepsis model.

Staphylococcus aureus is a major cause of nosocomial infections worldwide, and the rate of resistance to clinically relevant antibiotics, such as methicillin, is increasing; furthermore, there has been an increase in the number of methicillin-resistant S. aureus community-acquired infections. Effective treatment and prevention strategies are urgently needed. We investigated the potential of the S. aureus surface protein iron surface determinant B (IsdB) as a prophylactic vaccine against S. aureus infection. IsdB is an iron-sequestering protein that is conserved in diverse S. aureus clinical isolates, both methicillin resistant and methicillin sensitive, and it is expressed on the surface of all isolates tested. The vaccine was highly immunogenic in mice when it was formulated with amorphous aluminum hydroxyphosphate sulfate adjuvant, and the resulting antibody responses were associated with reproducible and significant protection in animal models of infection. The specificity of the protective immune responses in mice was demonstrated by using an S. aureus strain deficient for IsdB and HarA, a protein with a high level of identity to IsdB. We also demonstrated that IsdB is highly immunogenic in rhesus macaques, inducing a more-than-fivefold increase in antibody titers after a single immunization. Based on the data presented here, IsdB has excellent prospects for use as a vaccine against S. aureus disease in humans.

A novel actinomycete strain de-replication approach based on the diversity of polyketide synthase and nonribosomal peptide synthetase biosynthetic pathways.

The actinomycetes traditionally represent one of the most important sources for the discovery of new metabolites with biological activity; and many of these are described as being produced by polyketide synthases (PKS) and nonribosomal peptide synthetases (NRPS). We present a strain characterization system based on the metabolic potential of microbial strains by targeting these biosynthetic genes. After an initial evaluation of the existing bias derived from the PCR detection in a well defined biosynthetic systems, we developed a new fingerprinting approach based on the restriction analysis of these PKS and NRPS amplified sequences. This method was applied to study the distribution of PKS and NRPS biosynthetic systems in a collection of wild-type actinomycetes isolated from tropical soil samples that were evaluated for the production of antimicrobial activities. We discuss the application of this tool as an alternative characterization approach for actinomycetes and we comment on the relationship observed between the presence of PKS-I, PKS-II and NRPS sequences and the antimicrobial activities observed in some of the microbial groups tested.