In the last decade, multi-drug resistant organisms (MDROs) have become endemic in healthcare systems throughout the world. Of highest priority for new treatment strategies are infections caused by extreme drug resistant (XDR) Gram negative bacilli (GNB), which are resistant to carbapenems and all other antibiotics except for colistin or tigecycline. For example, infections caused by XDR, KPC-expressing Klebsiella pneumoniae have spread across the US and are associated with a 50% mortality rate. Like KPC, the NDM-1 resistance mechanism is spread by transmissible plasmids, and has been found worldwide, including in, Asia, Western Europe and the US. New ways to prevent or treat infections caused by these XDR bacteria are critically needed, especially given the dearth of antibiotics in the pipeline to treat them. A novel potential means to deal with pathogens expressing KPC and NDM-1 carbapenemases is to neutralize the activity of the enzymes and/or target bacteria expressing them for destruction. Polyclonal anti- KPC-1 and NDM-1 antibodies were raised by immunization with recombinant KPC-1 (rKPC1) or NDM-1 (rNDM1). When purified IgG was covalently linked to an iron siderophore to enhance bacterial uptake, 2.5 ?g/ml of the complexed immune but not control IgG-siderophore killed NDM-1-expressing K. pneumoniae. Siderophore conjugation to immune but not control IgG Fabc fragments, formed by reducing the disulfide bonds at the IgG heavy chain hinge region, enabled killing of both NDM-1- and KPC-expressing strains. The mechanisms by which the IgG kill the bacteria is unclear. It is hypothesized that conjugation to the siderophore increases cidal activity either by increasing antibody uptake into the periplasmic space, or by antibody-mediated obstruction of the siderophore uptake channel, thereby blocking iron uptake needed for growth. Thus, the specific aims are to 1) Define the role of antibody uptake and iron in in vitro killing of siderophore-conjugated polyclonal IgG and Fabc raised against NDM-1 and KPC-1 and 2) Optimize efficacy by engineering monoclonal antibodies (MAbs) targeting consensus KPC and NDM enzymes and conjugating them to iron siderophores or not. The proposed investigations will define the mechanisms of an innovative, promising technology to attack highly resistant GNB. Enhanced MAb uptake via siderophores is particularly promising because many antibiotic resistance targets exist in the periplasmic space, which is also the immediate location siderophores deposit iron in GNB. Hence, results from the current proposal have fundamental implications for enhancing antibody-based therapeutics against GNB in general, and have the potential to enable an entirely new approach to attacking XDR pathogens. The current results will support a future R01 and/or SBIR/STTR application to humanize the MAbs, define their potential to treat multiple strains and species of XDR GNB in vivo, and define biochemical mechanisms of killing.
KPC and NDM1 are special enzymes that make bacteria resistant to almost all antibiotics. Infections caused by such bacteria kill half of the people the infect because there are no good antibiotics available to treat them. The purpose of the current grant is to determine if antibodies made by the immune system can block the enzymes and kill the bacteria that make them.
|Spellberg, Brad; Doi, Yohei (2015) The Rise of Fluoroquinolone-Resistant Escherichia coli in the Community: Scarier Than We Thought. J Infect Dis 212:1853-5|
|Bruhn, Kevin W; Spellberg, Brad (2015) Transferrin-mediated iron sequestration as a novel therapy for bacterial and fungal infections. Curr Opin Microbiol 27:57-61|
|Spellberg, Brad; Bartlett, John; Wunderink, Rich et al. (2015) Novel approaches are needed to develop tomorrow's antibacterial therapies. Am J Respir Crit Care Med 191:135-40|
|Lin, Lin; Pantapalangkoor, Paul; Tan, Brandon et al. (2014) Transferrin iron starvation therapy for lethal bacterial and fungal infections. J Infect Dis 210:254-64|
|Spellberg, Brad (2014) Antibiotic judo: working gently with prescriber psychology to overcome inappropriate use. JAMA Intern Med 174:432-3|
|Spellberg, Brad; Gilbert, David N (2014) The future of antibiotics and resistance: a tribute to a career of leadership by John Bartlett. Clin Infect Dis 59 Suppl 2:S71-5|
|Spellberg, Brad; Rex, John H (2013) The value of single-pathogen antibacterial agents. Nat Rev Drug Discov 12:963|