Abstract Osteomyelitis is the bane of orthopaedic surgery, of which the majority is caused by Staphylococcus aureus. Prior to this P50 Center of Research Translation on the Osteoimmunology of Bone Infection (CoRTOBI), it was known that these rare infections following elective surgery (~1%), and our inability to control them (~40% reinfection rate), are due to specific host-pathogen interactions. While co-morbidities associated with infection were also known, the roles of adaptive immunity and S. aureus strain on clinical outcome had yet to be studied. Thus, Project 2 of CoRTOBI tested this using custom multiplex immunoassays, whole genome sequencing (WGS), animal models, and the AO-CPP Bone Infection Registry of 297 patients with confirmed S. aureus infection. The results formally established anti-Gmd antibodies as protective and anti-IsdB antibodies as susceptible determinants of the immune proteome, and that virtually all people have a susceptible immune proteome. Thus, we now hypothesize that: 1) there is a defect in anti-Gmd vs. anti-IsdB antibody secreting B cells, plasmablasts and/or long-lived plasma cells (LLPCs) that is responsible for the native susceptible immune proteome. 2) S. aureus strain specific factors contribute to susceptible immune proteome genesis. And, 3) multivalent nanoparticle mRNA vaccines can reverse the susceptible immune proteome. Three Specific Aims are proposed for Project 2 in this CoRTOBI renewal. In Aim 1 we will elucidate the mechanism of susceptible anti-S. aureus immune proteome genesis by quantifying anti-Gmd vs. anti-IsdB specific B cells and plasmablasts in blood, and LLPCs in bone marrow, in experimental mice and patients with S. aureus bone infections versus uninfected healthy controls. In Aim 2 we will complete WGS of the 297 S. aureus strains in the AO-CPP registry, and identify unique genes in the strains associated with the worst clinical outcomes. And in Aim 3 we will demonstrate the efficacy of multivalent mRNA nanoparticle vaccines to overcome the susceptible immune proteome against S. aureus, and protect mice from implant-associated osteomyelitis. In addition to Gmd, we have also demonstrated that anti-Amd, Hla, CHIPS & SCIN antibodies are significantly associated with infection control in osteomyelitis patients. Given the historic success of mRNA nanoparticle vaccines during the COVID-19 pandemic, we will use this technology to develop candidate vaccines for the five antigens. Dose response studies will be performed for monovalent vaccines to determine the minimal mRNA concentration that can induce antibodies >10ng/ml in sera. Then the efficacy of the mono and multivalent vaccines will be tested against the most virulent S. aureus strains in the AO-CPP registry using our prophylactic and therapeutic murine models of implant-associate osteomyelitis. Overall Impact: We will identify the mechanism responsible for the susceptible immune proteome, establish candidate mRNA vaccines with potential to o...