Project Summary Our goal is to develop a novel L. lactis probiotic-based therapeutic for the treatment of rheumatoid arthritis (RA). RA is a chronic, systemic inflammatory disorder affecting as much as 0.24% worldwide, and as much as 1% of the US population. The exact frequencies depend on geographical origin and gender, women being three times more affected than men. While many organs are affected, the main targets of RA are synovial joints, and approximately half of the affected patients become disabled over the progression of the disease. Treatment of RA remains a significant unmet medical need. Since TNF-? is a major facilitator of disease progression, TNF-? antagonists can effectively diminish inflammation and attenuate destruction of cartilage and bone. However, a large number of patients either fail to respond to, or relapse with, anti-TNF therapy. In addition, prolonged treatment with TNF-? antagonists can have deleterious side effects, making patients more susceptible to opportunistic infections. Immunotherapy for autoimmune disorders is also especially attractive for correcting inflammatory diseases without having to resort to immunosuppressive drug therapies. The two main goals of such an approach are to 1) ?switch off? the immune response against the host?s own tissues and 2) maximally balance the relationships between effector cells of different lineages to prevent relapses of chronic inflammation. Originally conceived as a diarrheal vaccine for humans when using Salmonella vaccine vectors, we found colonization factor antigen I (CFA/I) from human enterotoxigenic E. coli (ETEC) is potently effective in preventing and treating experimental models for arthritis and multiple sclerosis. In fact, purified CFA/I fimbriae, when given orally or nasally, and administered in lieu of live Salmonella-CFA/I as a source of fimbriae, can effectively attenuate inflammation without the associated side-effects from Salmonella. To avoid efforts and costs associated with producing sufficient quantities of recombinant CFA/I fimbriae, and to increase the PK/PD properties of CFA/I fimbriae following oral administration, we have successfully engineered a Lactococcus lactis strain to express CFA/I fimbriae (L. lactis-CFA/I). Similar to purified CFA/I fimbriae, this L. lactis-CFA/I is effective in preventing and treating experimental arthritis. The next step in the development process is to produce a food-grade L. lactis-CFA/I strain (called VTC-CFA) for clinic development. The specific aims of this proposal are to: 1) generate L. lactis clones expressing CFA/I from a genome-integrated operon; 2) select a VTC-CFA clinical candidate based on growth rate, CFA/I expression and stability, and 3) demonstrate CD4+ regulatory T cell expansion with oral treatment of VTC-CFA in normal mice. Successful commercialization of VTC-CFA will ultimately provide a profound front-line medical advancement in the treatment of RA.