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Exploiting innate immune evasion strategies of Staphylococcus aureus for the design of novel antibody-based therapeutic agents for the treatment of bacteremia

Final Report Summary - COMPSTAPH (Exploiting innate immune evasion strategies of Staphylococcus aureus for the design of novel antibody-based therapeutic agents for the treatment of bacteremia)

Staphylococcus aureus is a human pathogen that has an increasingly negative impact on human health. It is a leading cause of hospital and community-acquired infections of human skin or soft tissue abscesses. Contraction of S. aureus can lead to potentially fatal conditions (endocarditis, septicemia), as well as infections associated with implanted medical devices (intravascular catheters, pacemakers). The massive consumption of antibiotics has led to the selection of drug-resistant strains, designated MRSA for methicillin–resistant S. aureus. An effective vaccine that can protect from S. aureus infections is not yet available by the scientific community. The emerging multidrug resistance of S. aureus, the poor response of hospital-endemic strains to broad-spectrum antibiotics and the diverse repertoire of strategies that the pathogen uses to evade the immune response urge for the development of novel therapeutic platforms exploiting more targeted approaches to neutralize virulence-promoting factors. In view of the adverse effects and therapeutic limitations of IVIG treatment, combinatorial monoclonal antibody (mAb)-based therapies are now gaining momentum as promising means for combating S. aureus infections through passive immunization.
Addressing these emerging priorities in antibacterial therapeutics and S. aureus vaccine design, through FP7-COMPSTAPH we sought to generate and characterize novel, highly selective human antibodies that can display neutralizing properties over key pathogenic determinants and virulence factors of S. aureus. Through this project we have generated and characterized human monoclonal mini-antibodies (miniAbs) that selectively recognize and neutralize the function of Efb, a key immune evasion and virulence-promoting protein of S. aureus. These miniAbs have also shown therapeutic efficacy and virulence-neutralizing profiles in in vitro and in vivo models of bacterial infection and inflammation.
In vitro testing of the complement modulatory protein Efb-C, during the first period of the program, demonstrated its ability to inhibit the alternative pathway (AP) of complement. To evaluate the consequences of this inhibition, we established an ex vivo whole blood model of bacteremia which recapitulates the clinical hallmarks of the human condition. Addition of Efb-C to S. aureus-exposed whole blood resulted in a markedly increased survival of the pathogen, confirming previous studies that inhibition of AP activation results in decreased surface opsonization of the pathogen and consequently to decreased phagocytosis.
Human antibodies (miniAbs) selectively recognizing Efb-C were generated by screening a HuCal® human mini-antibody library. To characterize the specificity and binding kinetics of 12 miniAbs to their target protein Efb-C, we employed a combination of fine biochemical (SPR-based protein interaction analysis) and immunochemical (ELISA, immunoblot) approaches. Based on their distinct binding affinities, miniAbs were grouped in high, intermediate and low binders. Immunoblot analysis revealed that miniAbs recognize mostly linear epitopes on Efb-C. Efb-C was tested both as a recombinant protein and as its naturally secreted form in S. aureus culture supernatants. Both forms of the protein were recognized by the miniAbs suggesting that miniAbs can also recognize the protein in blood-borne infection settings.
The main aim of the study was to generate miniAbs which would block the interaction of Efb-C with its binding partner C3d, a fragment of the complement protein C3. To test the miniAbs for their possible blocking activity, a competition ELISA assay was set up. MiniAbs A1-A4 appeared to block significantly the binding of Efb-C to C3d/C3. MiniAb A2 which was found by SPR analysis to bind to Ehp, did not inhibit the interaction of C3d/Ehp, probably because Ehp has two binding sites on C3d and the miniAb A2 reacts with only one of them, thus being unable to inhibit the binding of the two partners. The binding/selectivity profiles of miniAbs A1-A2 render them interesting tools for further studies. To gain insight into the structural basis of the blocking activity of the selected miniAbs, non-active Efb-C mutant proteins were tested by SPR for their binding to these miniAbs. The non-active mutants did not bind to the blocking miniAbs indicating that their neutralizing capacity arises from their ability to disrupt contacts essential for the Efb-C/C3d complex formation. Evaluation of the blocking miniAbs in the whole blood model of bacteremia, revealed interception of Efb-C activity and significant reduction of the survival of S. aureus. The expression and secretion of Efb by S. aureus was also confirmed in various clinical isolates both in culture and in whole blood assays. Our findings in the whole blood model have significant implications for vaccine design since this ex vivo model of bacterial inflammation closely resembles the pathological landscape of human bacteremia.
Evaluation of the impact of the blocking miniAbs on the release of the inflammation markers C5a and IL-6 in the whole blood model of bacteremia revealed increase in C5a generation and decrease in IL-6 levels. Increase of C5a leads to recruitment of phagocytic cells and increased bacterial killing. The reduced bacterial burden is reflected by the lower IL-6 levels. Importantly, these latter findings corroborate our previous data on the mechanism of function of the miniAbs (Figure A).

Figure A (see attachment): Schematic overview of the proposed mode of action of the anti-EfbC mini-antibodies generated through the COMPSTAPH project

The testing of the miniAbs in an in vivo model of kidney colonization was a prerequisite for the evaluation of their therapeutic potential as part of a multivalent vaccine. Treatment of mice with a single dose of blocking miniAbs resulted in a significantly lower bacterial burden in infected kidneys. Histological analysis of these kidneys revealed absence of bacterial foci and less signs of inflammation compared to the control animals. Finally, in vitro testing of the half-life of the blocking miniAbs in mouse plasma showed that they are stable for at least 36 hr. Overall, these in vivo data are considered promising and further testing of these blocking miniAbs in different preclinical models of S. aureus-mediated infectious disease is warranted.

Project significance and impact: The generation of miniAbs against S. aureus is anticipated to lead to a multivalent vaccine which will afford protection against the devastating symptoms of an S. aureus infection through passive immunization. Currently no anti-S. aureus vaccine has been approved or licensed by FDA or EMA. Therefore the development of such therapeutic antibodies will open new avenues for vaccine design in the field of infectious diseases and particularly in treating MRSA patients. Such vaccines are anticipated to provide significant relief from the profound socioeconomic burden imposed by hospitalization costs of S. aureus-infected patients worldwide. The long-term goal of our research is the development of novel Ab–based therapeutics for the treatment of infectious diseases caused by the emerging pathogen S. aureus.