**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** ANIMAL HEALTH IS OF GREAT IMPORTANCE, IN AGRICULTURAL, FOOD SECURITY, GENERAL ECONOMIC AND PUBLIC HEALTH TERMS. THE DISEASES THAT THIS GRANT PROPOSES TO INVESTIGATE (TUBERCULOSIS, LEPTOSPIROSIS, JOHNE'S DISEASE, AND PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS (PRRSV)) CAUSE BILLIONS OF DOLLARS IN LOSSES TO U.S. AGRICULTURAL PRODUCERS. IN ADDITION, TUBERCULOSIS, LEPTOSPIROSIS, AND JOHNE'S DISEASE ARE ZOONOTIC DISEASES, IN WHICH ANIMALS CAN SERVE AS RESERVOIRS AND VECTORS OF DISEASE FOR HUMANS. FOR EXAMPLE, MYCOBACTERIUM TUBERCULOSIS AND MYCOBACTERIUM BOVIS, THE BACTERIA THAT CAUSE TUBERCULOSIS, INFECT ONE THIRD OF THE WORLD'S HUMAN POPULATION, AND ARE RESPONSIBLE FOR 1.5 MILLION HUMAN DEATHS WORLDWIDE PER YEAR. WHILE MYCOBACTERIUM BOVIS INFECTION OF LIVESTOCK AND HUMANS HAS BEEN NEARLY ELIMINATED IN THE U.S. BY DECADES OF CULLING INFECTED ANIMALS AND PASTEURIZING MILK, IMPORTATION OF INFECTED LIVESTOCK AND DAIRY PRODUCTS FROM MEXICO AND INFECTED DEER RESERVOIRS HAVERESULTED IN CATTLE AND HUMAN INFECTION AND DEATHS IN THE U.S. THE RISE IN CONSUMER DEMAND FOR RAW UNPASTEURIZED MILKALSO INCREASES THE DANGER OF A MYCOBACTERIUM BOVIS OUTBREAK.HISTORY HAS SHOWN THAT THE BEST WAY TO TREAT DISEASE IS TO PREVENT IT THROUGH VACCINATION, WITH THE LONG-TERM GOAL OF DRIVING IT INTO EXTINCTION. SMALLPOX AND RINDERPEST VIRUS ARE TWO EXAMPLES OF PATHOGENS THAT ARE NOW EXTINCT BECAUSE OF VACCINATION. THIS IS ESPECIALLY TRUE IN AGRICULTURE, IN WHICH DISEASES MAY NOT BE FATAL TO ALL ANIMALS, BUT SEVERELY IMPACT PRODUCERS BECAUSE ANIMALS FAIL TO REACH MARKET WEIGHT IN A TIMELY FASHION, INCUR VETERINARY MEDICAL BILLS, AND FAIL TO DELIVER VIABLE YOUNG. CHRONICALLY INFECTED ANIMALS CAN INFECT THE WHOLE HERD OR FLOCK. THUS, IT IS CRITICAL TO DEVELOP EFFECTIVE VACCINES TO PREVENT ANIMAL PATHOGENS FROM GAINING A FOOTHOLD. MOST CLASSIC VACCINES RELY ON ACTIVATING A WHITE BLOOD CELL CALLED A B CELL, WHICH PRODUCE ANTIBODY PROTEINS THAT BIND SPECIFICALLY TO THE PATHOGENAND LEAD TO ITS ELIMINATION. THESE B CELLS PRODUCE ONLY ONE TYPE OF ANTIBODY PER CELL AND DISPLAY A MEMORY RESPONSE IN THAT THEY ACT FASTER THE SECOND TIME THEY ENCOUNTER THE PATHOGEN AND ARE THUS ABLE TO PREVENT INFECTION. HOWEVER, MANY PATHOGENS ARE ABLE TO EVADE THESE ANTIBODY PROTEINS THROUGH MUTATION BECAUSE THE ANTIBODIES ARE SO SPECIFIC. FOR THESE PATHOGENS, IT HAS PROVEN BENEFICIAL TO DESIGN VACCINES THAT RECRUIT A SECOND TYPE OF IMMUNE CELL CAPABLE OF A MEMORY RESPONSE, THE T CELL. THERE ARE TWO TYPES OF T CELLS: ALPHA BETA T CELLS AND GAMMA DELTA T CELLS. MUCH MORE IS KNOWN ABOUT ALPHA BETA THAN GAMMA DELTA T CELLS, BUT WE DO KNOW THAT GAMMA DELTA T CELLS RESPOND MUCH MORE QUICKLY THAN B CELLS OR ALPHA BETA T CELLS, THAT THEY CAN RECOGNIZE PATHOGEN MOLECULES THAT THE PATHOGENS CAN'T MUTATE BECAUSE THEY ARE INTEGRAL TO PATHOGEN SURVIVAL, AND THAT, UNLIKE B CELLS, THEY MAKE THE POTENT PROTECTIVE CHEMICAL INTERFERON-GAMMA.WE HAVE SHOWN THAT THE EFFICACY OF A VACCINE TO THE BACTERIA LEPTOSPIRA IN CATTLE IS CORRELATED WITH THE DEVELOPMENT OF AN EARLY-ACTING GAMMA DELTA T CELL MEMORY RESPONSE, AND THAT A GAMMA DELTA T CELL PROTEIN CALLED WC1-3, WHICH BINDS DIRECTLY TO LEPTOSPIRA, IS INVOLVED. WC1-3 IS ONLY ONE OF 13 WC1 GENES IN CATTLE THAT HAVE BEEN CONSERVED OVER MILLIONS OF YEARS OF EVOLUTION. BECAUSE THE NORMAL TENDENCY OF NUCLEOTIDE SEQUENCES IN GENES THAT FULFILL NO FUNCTION IS TO ACCUMULATE MUTATIONS THAT RENDER THEM NON-FUNCTIONAL PSEUDOGENES, IT IS LIKELY THAT EACH WC1 GENE HAS BEEN PRESERVED THROUGH RECOGNITION OF A DIFFERENT CLASS OF PATHOGENS. CONSISTENT WITH THIS HYPOTHESIS, WE HAVE FOUND THAT GAMMA DELTA T CELLS RESPOND TO MYCOBACTERIUM, AND THAT TWO OTHER WC1 PROTEINS BIND TO MYCOBACTERIUM. IN THIS GRANT PROPOSAL, WE PROPOSE TO GENERATE RESEARCH AND POSSIBLY THERAPEUTIC TOOLS RECOGNIZING WC1 CALLED NANOBODIES, AND TO INVESTIGATE THE MECHANISM OF WC1 PROTEINS INTERACTION WITH, AND ACTIVATION BY, PATHOGENS. UNDERSTANDING HOW WC1 ACTIVATES GAMMA DELTA T CELLS WILL ALLOW US TO CREATE MORE EFFECTIVE DESIGNER VACCINES THAT RECRUIT MULTIPLE ARMS OF THE IMMUNE RESPONSE, INCLUDING THE EARLY AND POTENT GAMMA DELTA T CELL RESPONSE.