Yekaterina Timofeyeva

Staphylococcus aureus Manganese Transport Protein C Is a Highly Conserved Cell Surface Protein That Elicits Protective Immunity Against S. aureus and Staphylococcus epidermidis

Staphylococcus aureus and other staphylococci cause severe human disease, and there are currently no vaccines available. We evaluated whether manganese transport protein C (MntC), which is conserved across the staphylococcal species group, could confer protection against S. aureus and Staphylococcus epidermidis. In vivo analysis of S. aureus MntC expression revealed that expression occurs very early during the infectious cycle. Active immunization with MntC was effective at reducing the bacterial load associated with S. aureus and S. epidermidis infection in an acute murine bacteremia model. Anti-MntC monoclonal antibodies have been identified that can bind S. aureus and S. epidermidis cells and are protective in an infant rat passive protection model and induce neutrophil respiratory burst activity. This is the first description of a protein that has the potential to provide protection across the staphylococcal species group.

Heterogeneous in vivo expression of clumping factor A and capsular polysaccharide by Staphylococcus aureus: Implications for vaccine design

There is a clear unmet medical need for a vaccine that would prevent infections from Staphylococcus aureus (S. aureus). To validate antigens as potential vaccine targets it has to be demonstrated that the antigens are expressed in vivo. Using murine bacteremia and wound infection models, we demonstrate that the expression of clumping factor A (ClfA) and capsular polysaccharide antigens are heterogeneous and dependent on the challenge strains examined and the in vivo microenvironment. We also demonstrate opsonophagocitic activity mediated by either antigen is not impeded by the presence of the other antigen. The data presented in this report support a multiantigen approach for the development of a prophylactic S. aureus vaccine to ensure broad coverage against this versatile pathogen.

Expression of Staphylococcus epidermidis SdrG Increases following Exposure to an In Vivo Environment

ABSTRACT SdrG is a surface-associated fibrinogen binding protein present in most strains of Staphylococcus epidermidis. Surface expression of SdrG was not detected by flow cytometry or immunofluorescence microscopy on S. epidermidis 0-47 grown in nutrient broth or in the presence of human serum. sdrG transcript levels increased 1 hour following a shift from growth in nutrient broth to growth in the bloodstream of a mouse and resulted in a concomitant increase in protein levels as detected by immunofluorescence microscopy. The environmental signal(s) resulting in the increase in expression is elusive, as growth under conditions known to mimic in vivo conditions (elevated CO2, iron limitation, human serum, and citrated human blood) did not affect expression of SdrG. Immunizing mice with either the N1N2N3 (amino acids 50 to 597) or N2N3 (amino acids 273 to 597) subdomain of the N-terminal A domain of recombinant SdrG (rSdrG) elicited a robust antibody response; however, only mice vaccinated with rSdrGN23 exhibited a significant reduction in 0-47 recovered after experimental infection. Since SdrG is expressed early during infection in response to specific host environmental cues present in the bloodstream and since antibodies to it are effective in reducing bacteremia, SdrG possesses attributes of a vaccine component effective against the pathogenic form of the ubiquitous human commensal S. epidermidis.

Demonstration of the preclinical correlate of protection for Staphylococcus aureus clumping factor A in a murine model of infection

The Staphylococcus aureus virulence factor clumping factor A (ClfA) is a component of an investigational S. aureus prophylactic vaccine. ClfA enables S. aureus to bind to fibrinogen and platelets during the initial stages of invasive disease. Here we demonstrate that ectopic expression of ClfA is sufficient to render nonpathogenic Lactococcus lactis lethal in a murine model of systemic infection. In contrast, L. lactis expressing ClfAY338A, which cannot bind fibrinogen, did not cause death in the mice. Pathogenicity was also prevented by immunization with ClfA. This model was then used to define a preclinical correlate of protection by measuring functional antibody in a S. aureus fibrinogen binding inhibition assay (FBI) and correlating that titer with protective outcomes. Although many humans have pre-existing antibodies that bind to ClfA, only sera with a threshold functional titer in the FBI were protective in this preclinical model. This confirms that fibrinogen binding is critical for ClfA-mediated pathogenesis and demonstrates that functional antibodies against ClfA are sufficient to protect against ClfA-mediated pathogenesis in vivo, enabling the definition of a preclinical correlate of protection for ClfA-containing vaccines based on FBI titer.

Immunofluorescence Microscopy for the Detection of Surface Antigens in Methicillin-Resistant Staphylococcus aureus (MRSA)

Immunofluorescence microscopy is a widely used laboratory method which allows detection and visualization of specific antigens. The method employs the specificity of antibodies to deliver fluorophore to a specific target and then visualize it with a microscope. The power of the technique is that it requires relatively little manipulation and relatively few bacterial cells, enabling the detection of antigen expression where other methods cannot, such as during an actual infection in an animal. Here, we apply the method to follow antigen expression on the surface of MRSA cells over time in in vivo infection models.

O-Acetylation is essential for functional antibody generation against Staphylococcus aureus capsular polysaccharide

ABSTRACT Staphylococcus aureus produces an antiphagocytic polysaccharide capsule to evade neutrophil-mediated killing. Many vaccines against encapsulated bacterial pathogens require generation of functional anti-capsular antibodies to mediate protection against infection and disease. Here it is shown that the generation of such antibody responses to S. aureus in vivo and in vitro requires the presence of O-acetyl modifications on the capsular polysaccharides. O-acetylation of S. aureus capsular polysaccharide therefore should be monitored carefully during vaccine development and production. This finding may provide additional insight into the previous failure of a S. aureus capsular polysaccharide conjugate vaccine.