Project Grant R41AI177227

FACTOR H FC FUSIONS AS NOVEL THERAPEUTICS FOR BURKHOLDERIA PSEUDOMALLEI INFECTIONS - BURKHOLDERIA PSEUDOMALLEI (BP) IS THE CAUSATIVE AGENT OF MELIOIDOSIS, A MAJOR CAUSE OF MORBIDITY AND SEPTIC DEATH IN TROPICAL/SUBTROPICAL REGIONS WORLDWIDE. IT IS PARTICULARLY VIRULENT IN DIABETIC PATIENTS AND IS ALSO CONSIDERED A TIER 1 SELECT AGENT DUE TO ITS POTENTIAL USE AS A BIOWEAPON. THESE BACTERIA ARE NATURALLY RESISTANT TO MANY ANTIBIOTICS AND THERE ARE CURRENTLY NO VACCINES AVAILABLE. THUS, THERE IS A GREAT INTEREST IN IDENTIFYING TARGETS FOR CURATIVE/PREVENTATIVE THERAPIES. LIKE MANY HUMAN PATHOGENS, BP IS ADEPT AT EVADING KILLING BY COMPLEMENT, A CRITICAL ARM OF INNATE IMMUNITY. PRELIMINARY DATA GENERATED BY A CO-INVESTIGATOR ON THIS GRANT SHOWED THAT VIRULENT BP ARE VERY EFFICIENT AT BINDING HOST FACTOR H (FH), A REGULATORY PROTEIN THAT INHIBITS C3 DEPOSITION, THUS PREVENTING COMPLEMENT ACTIVATION ON HOST CELLS. FH COMPRISES 20 DOMAINS CALLED SHORT CONSENSUS REPEATS (SCR), BUT ONLY THE FOUR N-TERMINAL SCR (SCR 1-4) POSSESS COMPLEMENT INHIBITING ACTIVITY. THE ABILITY TO BIND HOST FH, EITHER VIA SCR6-7 OR SCR18-20, IS A COMMON MECHANISM USED BY SEVERAL PATHOGENS (NOW INCLUDING BP) TO ESCAPE COMPLEMENT-MEDIATED KILLING. PLANET BIOTECHNOLOGY INC (PBI) IS DEVELOPING THERAPEUTIC RECOMBINANT PROTEINS CONTAINING SPECIFIC FH SCRS (THOSE THAT BIND TO PATHOGENS) FUSED TO IGG FC. IN PRELIMINARY EXPERIMENTS WE HAVE SHOWN THAT A CONSTRUCT WITH IGG3 FC N-TERMINAL TO SCR18-20 (FC3/SCR(18-20)), PROMOTES COMPLEMENT DEPOSITION AND SHOW POTENTIAL FOR MEDIATING KILLING OF BP. THE GOAL OF THIS PROPOSAL IS TO FURTHER MODIFY FC3/SCR(18-20) TO BECOME MORE EFFECTIVE AT PROMOTING DIRECT KILLING, PHAGOCYTE UPTAKE AND KILLING OF BP, AND POTENTIALLY ACTIVATING HOST CELL CYTOPLASMIC KILLING PATHWAYS. WE WILL START BY PRODUCING VARIANTS OF FC3/SCR(19-20) WITH BOTH SHORT AND LONG HINGES, INTENDED TO OPTIMIZE EITHER DIRECT COMPLEMENT-MEDIATED KILLING OR OPSONOPHAGOCYTOSIS, RESPECTIVELY. WE WILL ALSO PRODUCE A CONSTRUCT WITH FC MODIFICATIONS INTENDED TO IMPROVE BINDING TO THE INTRACELLULAR FC RECEPTOR, TRIM21, AND THUS ENHANCE ANTIBODY-DEPENDENT INTRACELLULAR NEUTRALIZATION (ADIN). WE WILL PURIFY AND CHARACTERIZE SUFFICIENT AMOUNTS OF THESE NEW FH-FC VARIANTS TO BE TESTED IN VITRO AND IN VIVO AGAINST VIRULENT STRAINS OF BP. WE WILL TEST THE ABILITY OF THE NEW FC3/SCR(19-20) VARIANTS TO: 1) BIND TO BP, 2) PROMOTE C3 DEPOSITION AND MEMBRANE ATTACK COMPLEXES (MAC) FORMATION ON BP, 3) PROMOTE DIRECT KILLING IN THE PRESENCE OF SERUM, 4) PROMOTE UPTAKE AND INTRACELLULAR KILLING BY MACROPHAGES AND NEUTROPHILS, AND 5) PROMOTE KILLING OF BP IN MICE IF ADMINISTERED EITHER BEFORE OR AFTER INFECTION, SO AS TO SIGNIFICANTLY DECREASE THE DEVELOPMENT OF MELIOIDOSIS. TO EVALUATE POTENTIAL ACTIVITY OF FC3/SCR(19-20) AGAINST THE "NORMAL" INTRANASAL FLORA, WE WILL OBTAIN CULTURES REPRESENTATIVE OF HUMAN NASAL COMMENSAL (NON-PATHOGENIC) SPECIES FROM ATCC AND EVALUATE THEIR SENSITIVITY TO KILLING BY COMPLEMENT IN THE PRESENCE AND ABSENCE OF FC-SCR(19-20). WE WILL ALSO EVALUATE THE ABILITY OF FC-SCR(19-20) TO ENHANCE COMPLEMENT DEPOSITION ON NORMAL HUMAN ERYTHROCYTES.