PROJECT SUMMARY/ABSTRACT In contrast to other resistant bacteria, virtually no antibiotics are in the pipeline to deal with XDR A. baumannii. There is a critical need for new strategies to prevent and treat these infections. We spent the first grant period raising MAbs to A. baumannii capsule, and have now identified 4 anti-capsular MAbs (2 of which were used to generate a bi-specific MAb, leaving us with 3 MAb molecules) that collectively bind to 80-90% of US clinical isolates and protect mice from lethal infection. These 3 lead candidates are all highly potent, achieving 100% protection in bacteremia models at single doses of ≤ 50 µg. They are also protective in pneumonia models of infection, and synergize with antibacterials. Furthermore, the bi-specific MAb has increased potency compared to each of its individual MAbs, and retains binding for all target strains, and efficacy in vivo. This lead three- MAb therapeutic has begun translation into full GMP and toxicity, planning for a future Phase I clinical trial. Our goals for the renewal are to enhance feasibility of clinical development and deployment of the MAbs by closing any coverage gaps against international strains, defining surrogate efficacy markers, and validating key assays to support clinical trials and future clinical deployment. We have obtained a new global strain collection, and entered into key partnerships to further these aims, including experts at multi-valent MAb synthesis, clinical microbiology laboratory operations, and statistics. Our Aims are to: Specific Aim 1: Define and optimize strain coverage and surrogate efficacy markers for international clinical strains of A. baumannii. We have collected 50 strains each from Taiwan, Southeast Asia, China, Europe, and South America. We will survey our 3 MAbs against all acquired strains, assessing flow binding and macrophage uptake, and will assess efficacy in our IV bacteremia model for representative strains. We will raise news MAbs as needed to close international strain coverage gaps. Specific Aim 2: Validate bioassays to enable clinical trials of the MAbs, including potency and human surrogate efficacy markers. We will validate LC-MS/MS for the specific amino acid sequences of our variable regions to quantify our MAbs when spiked into human blood, distinct from background antibodies. We will adapt our well-established HL-60 assays to quantify opsonic activity of MAb in human plasma. Finally, we will use multiplex Luminex assays to quantify cytokine modulation of fresh human leukocytes. Specific Aim 3: Optimize a rapid in vitro binding assay as a “susceptibility testing”-equivalent to support clinical trials and deployment of the MAbs. We will validate rapid, high throughput flow binding assays to correlate with protection in mice as a “susceptibility-test equivalent”. Novel solutions for A. baumannii infections are a critical unmet need. We have developed a promising MAb regimen that improves outcomes during blood and lung...