Project Grant F32AI191475

EVALUATING CORRELATES OF ANTIBODY REPERTOIRE AND VIRAL TOLERANCE OF RESERVOIR SPECIES VIA COMPLEMENTARY PROTEOMICS APPROACHES - PROJECT SUMMARY/ABSTRACT PATHOGENS CAUSING SEVERE DISEASE IN HUMANS APPEAR TO BE TOLERATED BY CERTAIN BAT SPECIES. STUDIES OF BAT IMMUNITY POINT TO POTENTIAL DIFFERENCES IN CONSTITUTIVE INTERFERON EXPRESSION AND PATHWAYS LEADING TO INFLAMMASOME ACTIVATION, YET MUCH REMAINS UNEXPLORED REGARDING BAT ADAPTIVE IMMUNITY. THE CIRCULATING ANTIBODY REPERTOIRE PROVIDES AN INDIVIDUAL SIGNATURE OF PATHOGEN EXPOSURE ARISING FROM THE INTERACTION BETWEEN AN INDIVIDUAL'S GENETICALLY ENCODED REPERTOIRE AND THEIR ENVIRONMENT. REPERTOIRE ANALYSIS AT THE PROTEIN LEVEL MAY PROVIDE MORE ACCURATE INFORMATION REGARDING CLONE ABUNDANCE AND ACTIVE EXPOSURE RESPONSES COMPARED TO B- AND T-CELL REPERTOIRE SEQUENCING. BOTTOM-UP APPROACHES, IN WHICH PROTEINS ARE DIGESTED INTO SMALL PEPTIDES, ARE ROUTINELY APPLIED TO STUDY PROTEINS IN BIOLOGICAL FLUIDS. HOWEVER, SUCH STRATEGIES ARE NOT WELL SUITED FOR ANTIBODY ANALYSIS, SINCE MUTATIONS OF THE GERMLINE SEQUENCE COMPLICATE THE ALREADY CHALLENGING PROBLEM OF PIECING TOGETHER PROTEIN SEQUENCES FROM SMALL PEPTIDES. DR. LUCA FORNELLI HAS PIONEERED TECHNIQUES FOR INTACT PROTEIN ANALYSIS USING MASS SPECTROMETRY (MS) THAT HAVE IMPROVED SEQUENCING COVERAGE AND STRUCTURAL CHARACTERIZATION OF ANTIBODIES. HIS COLLEAGUE AT THE UNIVERSITY OF OKLAHOMA, DR. DANIEL BECKER, HAS LED KEY STUDIES ADVANCING UNDERSTANDING OF HOST-PATHOGEN INTERACTIONS AND FACTORS ASSOCIATED WITH SPILLOVER EVENTS. DR. JOAN MERRILL OF THE OKLAHOMA MEDICAL RESEARCH FOUNDATION HAS EXTENSIVE EXPERTISE IN RIGOROUS CLINICAL TRIAL DESIGN AND DATA INTERPRETATION FOR STUDIES OF ANTIBODY PROFILES IN AUTOIMMUNE DISEASE. THEIR EXPERTISE AND RESOURCES AVAILABLE IN THE FORNELLI AND BECKER LABS PROVIDE A UNIQUE OPPORTUNITY TO LEVERAGE ADVANCES IN MS ANALYSIS TO CHARACTERIZE THE ANTIBODY REPERTOIRE OF BATS. INSPIRED BY WORK TO PROFILE THE HUMAN IMMUNOGLOBULIN G REPERTOIRE AND MS TECHNIQUES DEVELOPED BY DR. FORNELLI, ANTIBODIES ISOLATED FROM BAT PLASMA WILL BE SUBJECTED TO LIMITED DIGESTION TO YIELD THE FAB, THE SMALLEST ANTIBODY FRAGMENT THAT INCLUDES ALL ANTIGEN BINDING REGIONS. A MULTI-TIER MS PLATFORM WILL COMBINE SEQUENCE INFORMATION FROM THE FAB AND ITS SUBUNITS AND PROTEOLYTIC PEPTIDES. SEQUENCE TAGS DETERMINED FROM THE MS DATA WILL BE USED TO CORRECT FAB SEQUENCE TEMPLATES BASED ON IMMUNOGLOBULIN GENES PREDICTED FROM BAT GENOMES. SEQUENCES OF ABUNDANT FAB SUBUNITS WILL BE DETERMINED FOR TWO GROUPS OF BAT PLASMA SAMPLES, CORONAVIRUS POSITIVE VERSUS NEGATIVE VIA PCR. REPERTOIRE FEATURES WILL BE DEFINED BASED ON FAB SEQUENCE PATTERNS, GENE USAGE, AND REPERTOIRE COMPLEXITY AND EVALUATED FOR THEIR IMPORTANCE IN DISTINGUISHING CORONAVIRUS POSITIVE VERSUS NEGATIVE INDIVIDUALS. THE OVERALL OBJECTIVE IS TO IDENTIFY FEATURES IMPORTANT FOR SIGNALING VIRUS EXPOSURE. THE PROPOSED RESEARCH WILL ESTABLISH A NOVEL STRATEGY FOR BIOSURVEILLANCE IN SPECIES TOLERANT OF VIRUSES THAT POSE SIGNIFICANT THREATS TO HUMAN HEALTH. TECHNICAL SKILLS AS WELL AS MENTORSHIP AND PROJECT MANAGEMENT EXPERIENCE DEVELOPED AS PART OF THE TRAINING WILL PROVIDE THE NECESSARY FOUNDATION FOR LEADING COLLABORATIVE RESEARCH EFFORTS TO MINE ANTIBODY REPERTOIRES FOR INSIGHTS RELEVANT TO EXPOSURE AND INFECTION MONITORING AND EVEN DISEASE DIAGNOSIS.