Currently, there is no licensed vaccine to protect humans against urinary tract infection (UTI) in the U.S. Indeed, UTI is the second most common infection in humans after those of the respiratory tract. This high frequency of infection results not only in huge annual economic costs, but in decreased workforce productivity and high patient morbidity. At least 80% of these infections are caused by uropathogenic Escherichia coli (UPEC). Antibiotic treatment is generally effective for eradication of the infecting strain. However, increasing antibiotic resistance, allergic reaction to certain pharmaceuticals, alteration of normal gut flora, and failure to prevent recurrent infections, represent significant barriers to treatment. Our long-term research goal is to develop approaches to prevent UTI by vaccination, which represents a gap that must be addressed. Our objective is to strengthen antigen combinations to include siderophores and novel adjuvants for inclusion in an effective intranasal UTI vaccine. In the current funding period, we determined that intranasal immunization with two UPEC antigens, Hma and IutA, using dmLT [double mutant of E. coli heat-labile toxin: (R192G/L211A)] as adjuvant, reduces bladder bacterial load following challenge with UPEC. Our central hypothesis states that a multi-subunit vaccine elicits an immune response that protects against experimental challenge with UPEC strains. Working toward a more effective vaccine, we have identified additional protective antigens, siderophore receptor FyuA and two siderophores, yersiniabactin and aerobactin that also target UPEC. LTA1 has also been identified as an effective novel adjuvant. Although many women experience recurrent UTI and UPEC heterogeneity complicates vaccine design, data from our animal model and human studies offer encouragement for successful UPEC vaccine development. The rationale for the proposed work is to protect women from the development of recurrent UTI by simple administration of a vaccine. We will test our central hypothesis and complete our objectives by carrying out two specific aims: 1) Combine protective antigens Hma and IutA with FyuA and siderophores and novel adjuvants to establish an effective intranasal multi-subunit vaccine for prevention of recurrent urinary tract infection; and 2) Identify the mechanism of protection for the optimized vaccine against uropathogenic E. coli in urinary tract infection. For specific aim 1, we will build on our determination that an intranasal vaccine containing iron acquisition proteins Hma and IutA can protect mice from UTI by UPEC when delivered intranasally with a mucosal adjuvant. For specific aim 2, we will conduct specific immunological and physiological experiments to determine how the host is protected from UPEC infection by vaccination. The proposed research is innovative because we propose to combine discovery of two protective antigens with siderophores and novel adjuvant to protect against UTI by UPEC. T...