La Jolla Institute For Immunology

CONSORTIUM FOR IMMUNOTHERAPEUTICS AGAINST EMERGING VIRAL THREATS

HUMAN IMMUNE SIGNATURES OF DENGUE VIRUS AND MYCOBACTERIUM TUBERCULOSIS EXPOSURE IN INFECTION, DISEASE AND VACCINATION

STRATEGIC HERPESVIRUS IMMUNE EVASION AND LATENCY DEFENSE (SHIELD)

FUNCTIONAL AND DYSFUNCTIONAL HUMAN CD4 T CELL AND B CELL RESPONSES TO BACTERIA AND VIRUSES

HIPC DATA COORDINATING CENTER - PROJECT SUMMARY/ABSTRACT THE HUMAN IMMUNOLOGY PROJECT CONSORTIUM (HIPC) WAS FOUNDED IN 2010 TO CREATE A NETWORK OF INDEPENDENT CENTERS FOCUSED ON MEASURING HUMAN IMMUNE RESPONSES WITH HIGH-THROUGHPUT SYSTEMS IMMUNOLOGY APPROACHES COUPLED WITH DETAILED CLINICAL PHENOTYPING. WE PROPOSE TO DEVELOP A HIPC COORDINATING CENTER (HCC) THAT WILL PROVIDE DATA MORE RAPIDLY AND EFFECTIVELY TO THE BROADER SCIENTIFIC COMMUNITY, AND ALSO SERVE THE ENTIRE HIPC BY INCREASING THE VALUE OF THE RESEARCH PERFORMED AT HIPC CENTERS. IN PARALLEL, THE HCC WILL SERVE AS A PROMOTER OF CROSS-HIPC COLLABORATIONS THROUGH THE ORGANIZATION OF MULTI-CENTER ANALYSIS PROJECTS AND A CENTRALIZED PORTAL TO PROVIDE A SPACE FOR THE EXCHANGE OF IDEAS. SPECIFICALLY, THE HCC WILL CONTINUE THE DEVELOPMENT OF SHARED DATA STANDARDS, PROVIDE A CENTRAL KNOWLEDGEBASE OF STUDY RESULTS, ESTABLISH TOOLS TO VISUALIZE AND ANALYZE DATA, LEAD SYNERGISTIC CROSS-CENTER ANALYSIS EFFORTS, PROVIDE A PORTAL TO MAKE THE TOOLS AND DATA BROADLY ACCESSIBLE, AND PROVIDE ADMINISTRATIVE SUPPORT FOR THE ACTIVITIES OF HIPC SUBCOMMITTEES AND MANAGEMENT OF THE INFRASTRUCTURE AND OPPORTUNITY FUND (IOF) PROGRAM. OUR TEAM HAS PROVEN EXPERIENCE IN RUNNING PROGRAMS OF SIMILAR SCALE AND COMPLEXITY. WE ALSO HAVE THE NECESSARY FAMILIARITY WITH THE HIPC NETWORK, WITH OUR TEAM INCLUDING CURRENT PIS OF THE DATA STANDARDS IOF PROJECT, THE SIGNATURES IOF PROJECT, CLINICAL INFORMATION CAPTURE, AND THE EXISTING IMMUNESPACE DATA PORTAL. THIS PROPOSAL LEVERAGES THIS COMBINED EXPERIENCE AND INFRASTRUCTURE WHILE, IN PARALLEL, IMPLEMENTING VARIOUS SIGNIFICANT AND NECESSARY IMPROVEMENTS FACILITATED BY THE TIGHTER INTEGRATION AND THE 5-YEAR TIME HORIZON OF THIS DEDICATED HCC GRANT.

GLUCOSE, INSULIN IN DIABETIC VASCULAR DISEASE

NEW PLAYERS IN IMMUNE FUNCTION: IDENTIFICATION THROUGH RNAI AND MICRO-RNA SCREENS

RESEARCH RESOURCES: EPIGENOMIC AND TRANSCRIPTOMIC PROFILES OF HUMAN IMMUNE CELLS

GENETIC SCREENS FOR KEY TRANSCRIPTIONAL REGULATORS OF ANTIVIRAL T CELL IMMUNITY

INDUCTION AND MAINTENANCE OF REGULATORY T CELLS

SIGNAL TRANSDUCTION AND GENE INDUCTION IN T LYMPHOCYTES

TET ENZYMES AS GUARDIANS OF GENOME STABILITY

BZIP PROTEINS, NFAT, AND LYMPHOCYTE GENE INDUCTION

EPIGENOMICS OF T CELLS AND INNATE IMMUNE CELLS IN HUMAN ASTHMA

LIAI EPITOPE VALIDATION CENTER: CHARACTERIZATION OF ALLERGEN SPECIFIC T CELLS

THERAPEUTIC FULLY HUMAN MONOCLONAL ANTIBODIES AGAINST VACCINA VIRUS AND SMALLPOX

DEVELOPING COMPUTATIONAL MODELS TO PREDICT THE IMMUNE RESPONSE TO B. PERTUSSIS BOOSTER VACCINATION

LJI EPITOPE VALIDATION CENTER: CHARACTERIZATION OF EPITOPE-SPECIFIC T CELLS RESPONDING TO FOOD, FUNGAL AND INNER CITY ALLERGENS

MAXIMIZING GERMINAL CENTERS AND SOMATIC HYPERMUTATION TO HIV ENV IMMUNOGENS - ABSTRACT WE PROPOSE THAT THE FEATURES OF GERMINAL CENTER BIOLOGY IMPORTANT FOR DEVELOPING HIGH AFFINITY B CELLS TO A VERY DIFFICULT EPITOPE, SUCH AS A TIER 2 NAB EPITOPE ON HIV ENV TRIMER, ARE VERY DIFFERENT THAN THE WELL CHARACTERIZED FEATURES OF GC BIOLOGY FOR CONVENTIONAL ANTIGENS, SUCH AS HAPTENS. WE HAVE DEMONSTRATED THAT SLOW DELIVERY IMMUNIZATION CHANGES FUNDAMENTAL ASPECTS OF THE IMMUNE RESPONSE, WHICH CAN RESULT IN DRAMATIC IMPROVEMENTS IN NAB RESPONSES (CELL 2019). CONVENTIONAL IMMUNIZATION STRATEGIES WILL LIKELY BE INSUFFICIENT FOR THE DEVELOPMENT OF A BNAB VACCINE TO HIV OR OTHER DIFFICULT PATHOGENS DUE TO THE IMMUNOLOGICAL HURDLES POSED, INCLUDING B CELL IMMUNODOMINANCE AND GC QUANTITY AND QUALITY. WE FOUND THAT TWO INDEPENDENT METHODS OF SLOW DELIVERY IMMUNIZATION OF RMS RESULTED IN MORE ROBUST TFH CELLS AND MORE GC B CELLS WITH ENV-BINDING, TRACKED BY LONGITUDINAL LYMPH NODE (LN) FINE NEEDLE ASPIRATES (FNA) 1. IMPROVED GCS CORRELATED WITH THE DEVELOPMENT OF > 20-FOLD HIGHER TITERS OF AUTOLOGOUS TIER 2 NEUTRALIZING ABS (NABS). BCR SEQUENCING AND AB MAPPING DEMONSTRATED TARGETING OF IMMUNODOMINANT NON-NEUTRALIZING (NNAB) EPITOPES BY CONVENTIONAL BOLUS IMMUNIZED ANIMALS, WHILE SLOW DELIVERY IMMUNIZED ANIMALS TARGETED A MORE DIVERSE SET OF EPITOPES, INCLUDING MULTIPLE TIER 2 NAB EPITOPES. WE WILL CONTINUE THESE GROUNDBREAKING STUDIES TO USE NOVEL SLOW RELEASE TECHNOLOGIES TO PROBE THE BIOLOGY OF GERMINAL CENTERS RELEVANT TO AFFINITY MATURATION AGAINST A DIFFICULT HIV TRIMER IMMUNOGEN IN NON-HUMAN PRIMATES (NHP, RHESUS MACAQUES).

EPIGENETIC CONTROL OF FOXP3 EXPRESSION IN INDUCED T REGULATORY CELLS

REGULATION OF STORE-OPERATED CA2+ ENTRY: STIM AND ORAI

THE ROLE OF TRAIL IN IMMUNE CONTROL OF CYTOMEGALOVIRUS

GENERATION OF LONG-TERM B CELL MEMORY AND ANTIBODY RESPONSES TO VIRAL INFECTIONS

STIMULATING NEO-ANTIGEN SPECIFIC T CELL RESPONSES IN HEAD AND NECK CANCERS

THE CANCER EPITOPE DATABASE AND ANALYSIS RESOURCE - ABSTRACT RECENT YEARS HAVE WITNESSED A DRAMATIC RISE IN INTEREST TOWARDS CANCER EPITOPES IN GENERAL, AND NEOEPITOPES THAT ENCOMPASS MUTATIONS ARISING IN A GIVEN TUMOR IN PARTICULAR. CURRENT LINES OF RESEARCH EXAMINE HOW THE EPITOPE LOAD IN A GIVEN TUMOR RELATES TO THE SUCCESS OF CHECKPOINT BLOCKADE TREATMENTS, AND HOW TO UTILIZE EPITOPE-BASED VACCINES AND ADOPTIVE TRANSFER OF EPITOPE-SPECIFIC T CELLS FOR PERSONALIZED THERAPIES. FOR THESE PURPOSES, NEOEPITOPES THAT ARE RECURRENTLY RECOGNIZED IN DIFFERENT INDIVIDUALS ARE OF PARTICULAR INTEREST, WHICH HAS ALSO RE-IGNITED INTEREST IN EPITOPES IDENTIFIED IN CLASSIC TUMOR-ASSOCIATED ANTIGENS. ALONG WITH THE INTEREST IN CANCER EPITOPES, THERE IS ALSO INTEREST IN THE TCRS AND BCRS SPECIFICALLY RECOGNIZING THEM, AS THESE HAVE THE POTENTIAL TO BE USED IN THERAPEUTIC APPROACHES, AND THEY CAN AID IN BASIC STUDIES TO INFER THE SPECIFICITY OF T CELLS OR B CELLS CHARACTERIZED IN SINGLE CELL SEQUENCING DATA. THIS RESURGENCE OF INTEREST IN EPITOPES HAS CREATED A NEED TO CATALOG AND MAKE ACCESSIBLE TO THE SCIENTIFIC COMMUNITY ALL EPITOPE DATA, ALSO LINKED TO THE BIOLOGICAL, IMMUNOLOGICAL, AND CLINICAL CONTEXTS. THE ULTIMATE GOAL IS TO COME “FULL CIRCLE” AND LINK EPITOPE RECOGNITION AND IMMUNOLOGICAL READOUTS TO CLINICAL OUTCOMES AND TREATMENT STRATEGIES ALIKE. IN PARALLEL, THERE IS AN URGENT NEED TO DEVELOP RESOURCES FOR EPITOPE PREDICTION AND ANALYSIS TOOLS THAT PROVIDE ACCESS TO PREDICTIVE STRATEGIES AND PROVIDE OBJECTIVE EVALUATIONS OF THEIR PERFORMANCE IN THE RELEVANT BIOLOGICAL, IMMUNOLOGICAL, AND CLINICAL CONTEXTS. RECENT YEARS HAVE ALSO WITNESSED THE PUBLICATION OF MULTIPLE ORIGINAL METHODOLOGIES THAT REPORTED SOMETIMES IMPRESSIVE GAINS IN THE PREDICTIONS OF CANCER EPITOPES. HOWEVER, SEVERAL OF THESE STUDIES WERE DIFFICULT TO EVALUATE, BECAUSE THE METHODOLOGIES AND/OR DATASETS WERE NOT FULLY AVAILABLE IN A FORMAT THAT WAS READILY EXECUTABLE. AS A RESULT, THEIR PERFORMANCE COULD NOT BE PROPERLY BENCHMARKED ON INDEPENDENT DATASETS. THIS IS ALSO BECAUSE EFFECTIVE BENCHMARKING ON INDEPENDENT DATASETS REQUIRES THE ASSEMBLY OF NOVEL DATASETS OF SUFFICIENT SIZE AND DIVERSITY. TO OVERCOME ALL OF THESE INFORMATION TECHNOLOGY CHALLENGES, WE PROPOSE TO DESIGN AND IMPLEMENT THE CANCER EPITOPE DATABASE AND ANALYSIS RESOURCE (CEDAR), WHICH WILL PROVIDE A FREELY ACCESSIBLE, COMPREHENSIVE COLLECTION OF CANCER EPITOPE AND RECEPTOR DATA CURATED FROM THE LITERATURE, AND PROVIDE EASILY ACCESSIBLE EPITOPE AND TCR/BCR TARGET PREDICTION AND ANALYSIS TOOLS. AS THE CANCER EPITOPE DATA ARE CURATED, THEY WILL BE USED AS A TRANSPARENT BENCHMARK OF HOW WELL PREDICTION TOOLS PERFORM, AND ALSO TO DEVELOP NEW PREDICTION TOOLS FOR THE ANALYSIS RESOURCE COMPONENT OF CEDAR. CEDAR WILL LEVERAGE OUR EXPERTISE FROM DEVELOPING THE IMMUNE EPITOPE DATABASE AND ANALYSIS RESOURCE (IEDB), WHICH IS FULLY OPERATIONAL AND WIDELY USED BY RESEARCHERS GLOBALLY. CEDAR WILL DIRECTLY COMPLEMENT OTHER PROJECTS CURRENTLY FUNDED THROUGH THE NIH ITCR PROGRAM THAT PROVIDE RESOURCES AND TOOLS RELATED TO CANCER OMICS DATA. FINALLY, WE WILL ENGAGE IN OUTREACH ACTIVITIES TO IMPROVE FUNCTIONS, USER INTERFACES, AND INTEROPERABILITY WITH OTHER ITCR TOOLS AND PROMOTE THE USE OF CEDAR IN CANCER RESEARCH.

MAXIMIZING GERMINAL CENTERS AND SOMATIC HYPERMUTATION TO HIV ENV IMMUNOGENS

IDENTIFYING MECHANISMS AND CORRELATES OF LONG-LASTING VACCINE-INDUCED IMMUNITY TO WHOOPING COUGH

DEFINING INTERACTION QUANTITATIVE TRAIT LOCI (IQTLS) IN THE HUMAN GENOME

BIOMARKERS OF INFLAMMATION AND VASO-OCCLUSION IN SICKLE CELL DISEASE

DEVELOPMENT AND FUNCTION OF NKT CELLS

EPIGENOME-WIDE ASSOCIATION STUDY OF CHILDHOOD ASTHMA

REGULATED ACCESSIBILITY OF A CYTOKINE GENE LOCUS

IMMUNE REGULATION BY SLAT, A NOVEL TH2-EXPRESSED PROTEIN

SARS-COV-2-REACTIVE TISSUE-RESIDENT MEMORY T CELLS IN HEALTHY AND CANCER SUBJECTS

DEVELOPMENT OF A REPLICON RNA-BASED VACCINE AGAINST DENGUE AND ZIKA - ABSTRACT THE LONG-TERM GOAL OF THIS PROJECT IS TO DEVELOP A DENGUE-ZIKA VACCINE THAT PROVIDES PROTECTION AGAINST THE FOUR SEROTYPES OF DENGUE (DENV1-4) AND ZIKA (ZIKV) VIRUSES WITH MAXIMAL SAFETY AND EFFICACY. TO DATE, FLAVIVIRUS VACCINE DEVELOPMENT HAS FOCUSED ON THE INDUCTION OF NEUTRALIZING ANTIBODIES (NABS), AS THEY HAVE BEEN ASSUMED TO BE THE KEY MECHANISM FOR PROTECTION AGAINST NATURAL INFECTION. HOWEVER, DENV AND PERHAPS ZIKV ARE UNUSUAL IN THAT WEAK AB RESPONSES TO VACCINATION OR PRIOR INFECTION CAN INDUCE ANTIBODY-DEPENDENT ENHANCEMENT (ADE) OF INFECTION AND PATHOGENESIS DURING SUBSEQUENT REINFECTIONS. IN FACT, DENV DISEASE WITH SEVERE SEQUALAE HAS BEEN DOCUMENTED IN CHILDREN GIVEN THE ONLY CURRENTLY LICENSED DENV VACCINE. THUS, THE PRIMARY OBJECTIVE OF THIS APPLICATION IS TO DEVELOP AN EFFECTIVE VACCINE AGAINST DENV AND ZIKV THAT CANNOT MEDIATE ADE. WE HYPOTHESIZE THAT THIS VACCINE WILL NEED TO ELICIT BOTH STRONG NAB RESPONSES AND STRONG T CELL EFFECTOR RESPONSES THAT WILL COUNTERBALANCE THE PRESENCE OF ANY ADE-MEDIATING ABS, BASED ON OUR WORK INVESTIGATING THE INTERPLAY BETWEEN AB AND T CELL RESPONSES TO DENV AND ZIKV. IN PARTICULAR, WE HAVE SHOWN THAT CD8 T CELLS MEDIATE CROSS- PROTECTION AGAINST HETEROTYPIC DENV AND ZIKV INFECTIONS, AND THAT DENV VACCINE-ELICITED CD8 T CELLS CAN PREVENT ADE. IN ADDITION, OUR PRELIMINARY DATA SHOW THAT AN RNA REPLICON-BASED VACCINE EXPRESSING ZIKV NONSTRUCTURAL PROTEIN 3 ELICITS ONLY T CELL BUT NOT NAB RESPONSES AND CONFERS PROTECTION AGAINST ZIKV CHALLENGE IN MICE. THUS, WE HYPOTHESIZE THAT OUR COMBINATORIAL DENV-ZIKV VACCINE EXPRESSING BOTH AB- AND T CELL-TARGETING PROTEINS OF DENV1-4 AND ZIKV WILL PRODUCE HUMORAL AND CELLULAR IMMUNE RESPONSES THAT PROVIDE ROBUST, LONG-TERM PROTECTION AGAINST ALL FIVE VIRUSES. WE WILL TEST THIS HYPOTHESIS BY ACHIEVING THE FOLLOWING SPECIFIC AIMS: 1) TO EVALUATE IMMUNOGENICITY AND EFFICACY OF A DENV-ZIKV VACCINE. 2) TO DETERMINE THE DURABILITY AND MECHANISTIC UNDERPINNINGS OF DENV-ZIKV VACCINE-INDUCED PROTECTIVE IMMUNITY.

BIOACTIVE METABOLITES MODULATE IMMUNE-RELATED ADVERSE EVENTS IN CANCER IMMUNOTHERAPY - PROJECT SUMMARY IMMUNE CHECKPOINT BLOCKADE (ICB) THERAPY HAS DEMONSTRATED SIGNIFICANT CLINICAL BENEFIT IN LATE-STAGE PATIENTS WITH MELANOMA, RENAL CELL CARCINOMA, HEAD AND NECK CANCER, HODGKIN LYMPHOMA, BLADDER CANCER, NON-SMALL CELL LUNG CANCER, GASTRIC CANCER, LIVER CANCER, CERVICAL CANCER, MERKEL CELL CARCINOMA, AND FOR ALL MICROSATELLITE- UNSTABLE TUMORS. A MAJOR LIMITATION OF ICB THERAPIES TARGETING THE CTLA4 OR PD1 IMMUNE CHECKPOINTS IS THAT A SIGNIFICANT PORTION OF PATIENTS WILL EXPERIENCE IMMUNE-RELATED ADVERSE EVENTS (IRAES), WHICH CAN RESULT IN PERMANENT OR EVEN FATAL TOXICITY AND DISCONTINUATION OF LIFE-SAVING IMMUNOTHERAPY. COMPOUNDING THIS PROBLEM IS THE FACT THAT THERE CURRENTLY EXIST NO MOLECULAR MODULATORS FOR ICB-DRIVEN IRAES. THIS R01 APPLICATION EXAMINES CIRCULATING LPC 18:2 A NOVEL SMALL MOLECULE MODULATOR AND THERAPEUTIC FOR IRAES BY STUDYING HUMAN CANCER PATIENTS AND RELEVANT PRECLINICAL MODELS OF ICB-DRIVEN IRAES AND TUMOR REGRESSION. THE PROPOSED AIMS WILL SYSTEMATICALLY I) EXAMINE ASSOCIATION BETWEEN LPC 18:2 AND IRAES ACROSS MULTIPLE HUMAN CANCER COHORTS (E.G. MELANOMA, NON SMALL LUNG CANCER, HEAD AND NECK SQUAMOUS CELL CARCINOMA) AND ICB THERAPIES (E.G. ANTI- CTLA4 IPILIMUMAB, ANTI-PD1 PEMBROLIZUMAB AND COMBINATION THERAPIES); II) EXAMINE RELATIONSHIP BETWEEN PLASMA LPC 18:2 LEVELS AND ICB-DRIVEN TUMOR REGRESSION OR NATURAL AUTOIMMUNE DISEASE; III) STUDY THE IMMUNOLOGICAL MECHANISMS BY WHICH LPC 18:2 RESTRAINS ICB-DRIVEN IRAES AND AUTOIMMUNE COLITIS; IV) MECHANISTICALLY PROBE NOVEL EFFECTS OF LPC 18:2 AND LPC-G2A SIGNALING ON DEVELOPMENT AND FUNCTION OF INFLAMMATORY NEUTROPHILS. THIS STUDY USES A HIGHLY INNOVATIVE APPROACH LEVERAGING CANCER PATIENT BIO-SAMPLING ACROSS MULTIPLE INDEPENDENT CLINICAL TRIALS WITH STATE-OF-THE-ART RAPID MASS SPECTROMETRY PROFILING OF SMALL MOLECULE METABOLITES AND MECHANISTIC STUDIES. THIS IS A COLLABORATIVE STUDY BETWEEN A CANCER IMMUNOLOGIST AND BASIC SCIENTIST AT LA JOLLA INSTITUTE FOR IMMUNOLOGY AND UCSD MOORES CANCER CENTER, AN ANALYTICAL CHEMIST AT UCSD, A STATISTICAL EPIDEMIOLOGIST AT CEDARS-SINAI MEDICAL CENTER, CLINICAL IMMUNE-ONCOLOGY COLLABORATORS AND EXPERTS ON FUNDAMENTAL IMMUNOLOGY AND NEUTROPHILS. VALIDATING LPC 18:2 AS A THERAPEUTIC MOLECULE FOR IRAE TOXICITIES ADDRESSES AN URGENT NEED AT THE CLINICAL LEVEL TO DEVELOP THE VERY FIRST THERAPIES THAT CAN MINIMIZE RISK, MAXIMIZE BENEFIT, AND MORE ACCURATELY PERSONALIZE ICB THERAPIES FOR THOSE PATIENTS WHO STAND TO BENEFIT FROM CANCER IMMUNOTHERAPY.

INTEGRATED FUNCTIONAL HISTOPATHOLOGY OF THE DIABETIC HUMAN PANCREAS

ITCH IN T CELL ACTIVATION AND TOLERANCE

THE ROLE OF CYTOPLASMIC AND NUCLEAR THEMIS IN IMMATURE AND MATURE T CELLS

CD4 T CELL CONTROL OF CYTOMEGALOVIRUS

MYELOID CELL INTERACTIONS WITH T CELLS IN ATHEROSCLEROSIS

MATERNAL ANTIBODY-MEDIATED ENHANCEMENT OF DENGUE PATHOGENESIS - ABSTRACT DENGUE VIRUS (DENV) REPRESENTS A MAJOR THREAT TO GLOBAL HEALTH. HOWEVER, THE PRECISE ROLE OF THE IMMUNE SYSTEM IN PROTECTING AGAINST AND PATHOGENESIS OF THE FOUR DENV SEROTYPES, WHICH SHARE ANTIGENIC SIMILARITIES AND GEOGRAPHIC RANGES WITH EACH OTHER AND OTHER CLOSELY RELATED FLAVIVIRUSES ARE POORLY UNDERSTOOD. IN PARTICULAR, ANTIBODIES CAN CONTRIBUTE TO DENV PATHOGENESIS BY MEDIATING ANTIBODY (AB)-DEPENDENT ENHANCEMENT OF INFECTION (ADE). THIS PROJECT FOCUSES ON DEFINING THE FEATURES OF THE ANTI-FLAVIVIRUS AB RESPONSE THAT CONTRIBUTES TO ADE VS PROTECTION USING EPIDEMIOLOGICALLY RELEVANT MOUSE MODELS IN WHICH DENV INFECTION OF MOUSE PUPS IS ENHANCED BY MATERNALLY ACQUIRED FLAVIVIRUS ABS. OUR PUBLISHED AND NEW DATA DEMONSTRATE THAT FLAVIVIRUS VACCINATION-INFECTION COMBINATIONS CAN PROMOTE EITHER PATHOGENESIS OR PROTECTION. OUR PRELIMINARY DATA ALSO SHOW THAT MICE LACKING T FOLLICULAR HELPER (TFH) CELL RESPONSES ARE UNABLE TO INDUCE DENV IGG RESPONSE, AND THAT MICE TREATED WITH AN AGONISTIC AB THAT STIMULATES OX40, A T CELL COSTIMULATORY MOLECULE BELONGING TO THE TNF RECEPTOR SUPERFAMILY, EXHIBIT A BOOSTED DENV IGG RESPONSE, SUGGESTING THAT THE MAGNITUDE OF THE TFH RESPONSE CORRELATES WITH THE LEVEL OF AB RESPONSE TO DENV. THEREFORE, WE HYPOTHESIZE THAT PROMOTING TFH RESPONSES WILL INCREASE THE PRODUCTION OF BROADLY-NEUTRALIZING AB (BNAB) RESPONSES THAT MEDIATE PROTECTION AND MINIMIZE ADE DURING DENV INFECTION. WE WILL TEST THIS HYPOTHESIS BY USING AN RNA REPLICON-BASED VACCINE PLATFORM THAT INDUCES ROBUST T CELL AND AB RESPONSES IN THE FOLLOWING SPECIFIC AIMS: (1) DETERMINE HOW VACCINATION WITH DIFFERENT FLAVIVIRUS ANTIGENS AFFECTS TFH CELL AND AB RESPONSES IN MATERNAL MICE AND SUSCEPTIBILITY TO DENV ADE IN THEIR OFFSPRING. (2) TEST WHETHER MANIPULATION OF IMMUNIZATION VARIABLES AND CANDIDATE T CELL COSTIMULATORY PATHWAYS BOOSTS MATERNAL TFH CELL AND AB RESPONSES AND INDUCES A PROTECTIVE RESPONSE TO DENV2 INFECTION IN OFFSPRING. THESE STUDIES WILL PROVIDE CRITICAL INSIGHTS INTO THE FACTORS AND MECHANISMS THAT REGULATE ADE VS PROTECTIVE IMMUNITY TO DENV2 INFECTION. THIS KNOWLEDGE IS URGENTLY NEEDED TO INFORM DEVELOPMENT OF DENV VACCINES THAT PROTECT INFANTS AND YOUNG CHILDREN, THE HIGHLY VULNERABLE POPULATIONS, AND OTHER FLAVIVIRAL VACCINES THAT ARE SAFE AND EFFECTIVE WORLDWIDE, INCLUDING IN COUNTRIES WITH CO-CIRCULATION OF 2 OR MORE FLAVIVIRUSES. THE PROPOSED WORK IS BASED ON OUR STRONG TRACK RECORD IN INVESTIGATING HUMORAL AND CELLULAR IMMUNE MECHANISMS DURING FLAVIVIRUS INFECTIONS USING STATE-OF-THE-ART MOUSE MODELS. THE PROJECT WILL ALSO BENEFIT FROM OUR COLLABORATORS’ EXPERTISE IN TFH CELLS, T CELL COSTIMULATORY MOLECULES, FLAVIVIRAL AB RESPONSE IN HUMANS, DEVELOPMENT OF NOVEL VACCINE PLATFORMS, AND GENOMICS ASSAYS.

SELECTIVE INSTRUCTIONS FOR MEMORY PRECURSOR T CELLS

HUMORAL IMMUNITY TO VACCINIA VIRUS

MUCOSAL IMMUNE REGULATION BY CD8AA+ CELLS

HOST NUTRIENTS PERMIT IMMUNE EVASION OF NKT CELL ANTI-BACTERIAL RESPONSES

TRAINING IN IMMUNOLOGICAL MECHANISMS

BZIP PROTEINS AND LYMPHOCYTE GENE INDUCTION

ABCG1 FUNCTION IN DIABETES

UNCOVERING THE MISSING LINK THAT DETERMINES SUSCEPTIBILITY TO AUTOIMMUNITY

T REGULATORY LYMPHOCYTES, HDL FUNCTION, AND ATHEROSCLEROSIS

DIFFERENTIATION OF SUBSETS OF NKT CELLS

CONTROL OF THE FUNCTIONAL FATE OF CD4 T CELLS BY LNCRNA-SWITCH

HVEM: A TNF FAMILY RECEPTOR THAT INFLUENCES MUCOSAL IMMUNITY AND THE MICROBIOME

EXPLORING THE POTENTIAL OF TET INHIBITION IN CANCER IMMUNOTHERAPY

THE ROLE OF LIGHT IN COLITIS PATHOGENESIS

A NOVEL STRATEGY FOR VACCINE-INDUCED PROTECTION AGAINST MATERNAL-TO-FETAL TRANSMISSION OF ZIKA VIRUS - PROJECT SUMMARY THE LONG-TERM GOAL OF THIS PROJECT IS TO DEVELOP A VACCINE THAT CONFERS ROBUST AND DURABLE PROTECTION AGAINST TRANSPLACENTAL TRANSMISSION OF ZIKA VIRUS (ZIKV). ACCOMPLISHING THIS GOAL MAY BE CHALLENGING IN THAT THE VACCINE MAY NEED TO INDUCE BOTH ANTIBODY AND T CELL RESPONSES TO CONFER HIGHLY EFFECTIVE PROTECTION AGAINST ZIKV AT THE MATERNAL-FETAL INTERFACE (MFI). STUDIES WITH PREGNANT WOMEN IN BRAZIL HAVE SUGGESTED THAT ANTIBODY RESPONSES MAY CONTRIBUTE TO PATHOGENESIS OF CONGENITAL ZIKA SYNDROME (CZS) AND NEUTRALIZING ANTIBODY RESPONSES MAY NOT CORRELATE WITH PROTECTION AGAINST CZS. RECENT HUMAN STUDIES ALSO SUGGEST THAT THE ANTI-ZIKV ANTIBODY RESPONSE MAY BE LESS DURABLE THAN T CELL RESPONSE. HOWEVER, ONGOING ZIKV VACCINE DEVELOPMENT EFFORTS ARE FOCUSED ON ELICITING MAINLY ANTIBODY RESPONSES. BASED ON OUR PUBLISHED DATA DEMONSTRATING A CRITICAL ROLE FOR CD8 T CELLS IN PROTECTING AGAINST ZIKV INFECTION IN MULTIPLE MOUSE MODELS, WE WILL TEST OUR CENTRAL HYPOTHESIS THAT A ROBUST ZIKV VACCINE-INDUCED CD8 T CELL RESPONSE IN MOTHERS IS REQUIRED TO PROVIDE STRONG AND DURABLE PROTECTION AGAINST TRANSPLACENTAL TRANSMISSION OF ZIKV. OUR REPLICON RNA VACCINE EXPRESSING ZIKV PREMEMBRANE (PRM) AND ENVELOPE (E) OR NONSTRUCTURAL PROTEIN 3 (NS3) INDUCES ROBUST PROTECTION AGAINST ZIKV INFECTION IN PREGNANT MICE BUT ONLY PARTIAL PROTECTION IN THEIR FETUSES. THEREFORE, WE WILL USE THESE REPLICON RNA VACCINES AND MOUSE MODELS TO ACHIEVE THE FOLLOWING SPECIFIC AIMS: 1) IMPROVE VACCINE-INDUCED PROTECTION AGAINST ZIKV INFECTION DURING PREGNANCY, AND IDENTIFY THE MATERNAL IMMUNE RESPONSES ASSOCIATED WITH THE MOST PROTECTIVE AND DURABLE VACCINES. 2) TEST THE ROLE OF CD8 T CELLS IN VACCINE-INDUCED PROTECTION, AND DETERMINE PRECISE FEATURES OF MFI CD8 T CELLS ELICITED BY THE MOST PROTECTIVE AND DURABLE VACCINE. WE HAVE EXPERTISE IN EXAMINING FLAVIVIRAL PATHOGENESIS AND IMMUNITY USING MOUSE MODELS. WE ALSO HAVE A LONGSTANDING COLLABORATION WITH COLLEAGUES AT OUR INSTITUTE AND UC SAN DIEGO TO INVESTIGATE VIRUS-HOST INTERACTIONS USING GENOMICS AND HISTOPATHOLOGY-INFORMED APPROACHES.

HIV MRNA VACCINE STRATEGIES FOR EFFICIENT PRIMING, DIVERSITY AND DURABILITY OF IMMUNE RESPONSES - SUMMARY CONVENTIONAL VACCINE STRATEGIES HAVE NOT INDUCED SUCCESSFUL PROTECTION TO DIVERSE HIV STRAINS. MRNA VACCINE PLATFORMS ENABLE NOVEL MODALITIES OF ANTIGEN DELIVERY FOR INNOVATIVE VACCINE STRATEGIES. THE USE OF MRNA TECHNOLOGY FOR HIV VACCINES HOLDS PROMISE TO CONQUER MAJOR BARRIERS TO INDUCE PROTECTIVE RESPONSES VIA BROADLY NEUTRALIZING ANTIBODIES (BNABS). HOWEVER, MRNA VACCINES TO EXPRESS HIV ANTIGENS CAPABLE OF EFFECTIVELY CONFERRING PROTECTIVE IMMUNITY HAVE NOT BEEN FULLY EXPLORED. GERMINAL CENTER (GC) RESPONSES ARE PARAMOUNT FOR PROPHYLACTIC VACCINES AS THEY PROVIDE A HIGHLY SPECIALIZED ENVIRONMENT TO AFFINITY MATURE ANTIBODIES TO DIFFICULT EPITOPES ON HIV. WE HAVE DEMONSTRATED THAT EFFICIENT PRIMING AND LONGER DURATION OF GC RESPONSES ARE LIKELY FAVORABLE FOR BROADER NEUTRALIZING RESPONSES AGAINST HIV (NATURE 2022). MRNA VACCINES AGAINST SARS-COV-2 HAVE BEEN SHOWN TO INDUCE LONG-LASTING GC RESPONSES AFTER 2 DOSES, BUT THE BIOLOGY AND EFFECTIVE MANIPULATION OF THIS PROCESS REMAINS ELUSIVE. PARTICULARLY, A BIG KNOWLEDGE GAP IS HOW MRNA VACCINE-INDUCED GCS DIFFER FROM PROTEIN VACCINES. A SECOND KNOWLEDGE GAP IS WHAT MECHANISMS DRIVE LONG- LASTING GCS? A THIRD KNOWLEDGE GAP IS WHAT IMMUNOLOGICAL SIGNALS REGULATE THE OUTPUT OF DURABLE IMMUNE MEMORY? HIV ENVELOPE (ENV) TRIMER IS THE SOLE ANTIGENIC TARGET FOR NEUTRALIZING ANTIBODIES. MANY EXPERIMENTAL HIV VACCINES USE SOLUBLE FORMS OF ENV THAT EXPOSE THE IMMUNODOMINANT BASE OF THE TRIMER ELICITING NON- NEUTRALIZING ANTIBODIES. MRNA VACCINES ALLOW THE REALIZATION OF MEMBRANE-BOUND ENV EXPRESSION, WHICH CAN PRESENT ANTIGEN IN A MORE NATIVE FORM AVOIDING EXPOSURE OF THE BASE. MEMBRANE-BOUND ENV TRIMERS CAN ALSO BE EXPRESSED ON NANOPARTICLES SUCH AS VIRUS-LIKE PARTICLES (VLPS) VIA MRNA DELIVERY RESEMBLING HIGHER DENSITY EXPRESSION ON NATIVE VIRIONS. GIVEN THE POTENTIAL OF LONG-LASTING GC RESPONSES AND THE NEW MODES OF ANTIGEN DELIVERY BY MEMBRANE-BOUND ENV OR MULTIMERIC ENV NANOPARTICLES, WE SEEK TO INVESTIGATE: ARE HIV MRNA VACCINES CAPABLE OF INDUCING LONG-LASTING GC RESPONSES? HOW CAN HIV MRNA VACCINES OPTIMALLY PRIME LONG- LASTING GC RESPONSES? WHAT SEQUENTIAL IMMUNIZATION STRATEGIES WORK BEST WITH HIV MRNA VACCINES TO INDUCE DIVERSE AND DURABLE MEMORY RESPONSES? NON-HUMAN PRIMATES (NHPS) ARE AN INVALUABLE MODEL FOR STUDYING THIS BIOLOGY AND IMMUNOLOGY, BECAUSE OF THEIR RELATEDNESS TO HUMANS.

A TREG CELL-INTRINSIC CTLA4-PKC-ETA SIGNALING PATHWAY MEDIATING CONTACT-DEPENDENT SUPPRESSION OF TUMOR IMMUNITY: A NOVEL TARGET FOR CANCER IMMUNOTHERAPY

HISTAMINE-RELEASING FACTOR OLIGOMERS IN FOOD ALLERGY

TRANSCRIPTIONAL CONTROL OF MONOCYTE DEVELOPMENT

TOWARDS A PREDICTIVE UNDERSTANDING OF INFLUENZA IMMUNITY THROUGH EXPERIMENTAL DATA INTEGRATION, ITERATIVE MODEL DEVELOPMENT, AND RIGOROUS ASSESSMENT OF MODEL QUALITY - PROJECT SUMMARY/ABSTRACT: OUR CENTRAL HYPOTHESIS IS THAT COMPUTATIONAL MODELS CAN ACCURATELY PREDICT INFLUENZA VACCINE BREADTH AND DURABILITY BASED ON UNDERLYING IMMUNOLOGICAL FACTORS, INCLUDING AN ASSESSMENT OF INNATE RESPONSES TRIGGERED BY THE VACCINE, ANTIGEN-SPECIFIC B CELLS AND T CELLS, THE IMMUNE EXPOSURE HISTORY OF THE HOST, AND GENETIC FACTORS INTRINSIC TO THE HOST. WE PROPOSE TO CAPTURE THESE VARIABLES AND THEIR RELATIONSHIP TO VACCINE OUTCOMES FROM PUBLIC DATASETS AND NEWLY GENERATED EXPERIMENTAL DATA. WE WILL BUILD COMPUTATIONAL MODELS WITH COMPLEMENTARY APPROACHES THAT USE THESE DATA TO PREDICT VACCINE OUTCOMES, AND WE WILL HOST AN ANNUAL PREDICTION CONTEST THAT IS OPEN TO THE PUBLIC TO EVALUATE THE PREDICTIVE PERFORMANCE OF THESE MODELS ON UNSEEN DATA. OUR AIMS ARE TO: 1) CAPTURE EXISTING INFLUENZA VACCINE DATA AND GENERATE NEW EXPERIMENTAL DATA THAT CONNECT IMMUNOLOGICAL VARIABLES WITH VACCINE BREADTH AND DURABILITY. WE WILL: 1.1) COLLECT AND STANDARDIZE DATA FROM EXISTING STUDIES THAT PROFILE THE BREADTH AND/OR DURABILITY OF INFLUENZA VACCINE RESPONSES USING ANY OF THE KEY IMMUNE VARIABLES WE WILL ASSESS (GENETIC FACTORS OR INNATE, B CELL, OR T CELL IMMUNITY). THIS WILL RESULT IN A COMPREHENSIVE TRAINING SET FOR OUR COMPUTATIONAL MODELS. 1.2) GENERATE NEW EXPERIMENTAL DATASETS THAT MEASURE ALL OF THESE VARIABLES, ALONG WITH VACCINE BREADTH AND DURABILITY, TO CREATE AN INDEPENDENT TESTING SET FOR OUR ANNUAL COMPETITION. 2) GENERATE COMPUTATIONAL MODELS PREDICTING THE BREADTH AND DURABILITY OF VACCINE RESPONSES AND THE CASCADE OF IMMUNE EVENTS LEADING UP TO IT. WE WILL: 2.1) ADAPT AND REFINE COMPUTATIONAL MODELS DEVELOPED BY OUR CENTER INVESTIGATORS TO PREDICT VACCINE BREADTH, DURABILITY, AND KEY IMMUNE EVENTS CONNECTED TO THESE VACCINE OUTCOMES. 2.2) IMPLEMENT MODELS PUBLISHED IN THE LITERATURE TO SERVE AS A COMPARISON. 2.3) COMBINE THE BEST- PERFORMING MODELS TO DEVELOP AN INTEGRATED UNDERSTANDING OF INFLUENZA VACCINE RESPONSES. 3) RIGOROUSLY EVALUATE MODEL PREDICTION PERFORMANCE ON UNSEEN DATA IN AN OPEN COMPETITION. WE WILL 3.1) PERFORM AN ANNUAL COMPETITION USING DATA FROM AIM 1.2) THAT WAS HELD BACK FROM THE PUBLIC TO EVALUATE MODEL PERFORMANCE. 3.2) ENGAGE THE BROADER SCIENTIFIC COMMUNITY TO PARTICIPATE IN THE PREDICTIVE MODELING COMPETITION AND THEREBY MAXIMIZE THE DIVERSITY OF INDEPENDENTLY ASSESSED COMPUTATIONAL MODELING APPROACHES. OVERALL, THIS OPEN, TRANSPARENT, AND QUANTITATIVE PROCESS TO BUILD AND EVALUATE COMPUTATIONAL MODELS OF INFLUENZA VACCINATION-INDUCED IMMUNITY WILL TEST OUR CENTRAL HYPOTHESIS AND QUANTIFY HOW WELL COMPUTATIONAL MODELS PREDICT VACCINE BREADTH AND DURABILITY.

ELUCIDATING THE SIGNALING AND PROTEIN INTERACTION NETWORKS OF THE O-GLCNAC TRANSFERASE DURING EMBRYONIC STEM CELL STATE TRANSITIONS - PROJECT SUMMARY/ABSTRACT O-GLCNAC IS A SINGLE N-ACETYLGLUCOSAMINE COUPLED TO SERINE AND THREONINE RESIDUES OF NUCLEAR AND CYTOPLASMIC PROTEINS. ANALOGOUS TO PHOSPHORYLATION, O-GLCNAC SIGNALING IS DYNAMIC, RAPIDLY ADDED AND REMOVED FROM PROTEINS IN A SITE-SPECIFIC MANNER IN RESPONSE TO CELLULAR PERTURBATIONS AND EXTRACELLULAR CUES. BECAUSE BOTH MODIFICATIONS OCCUR ON THE SAME RESIDUES IT IS HYPOTHESIZED THAT THERE IS A FUNCTIONAL CROSSTALK BETWEEN O- GLCNAC AND PHOSPHORYLATION, WHERE ONE MAY AFFECT DEPOSITION OR REMOVAL THE OTHER. UNLIKE PHOSPHORYLATION, HOWEVER, WHICH IS CATALYZED BY OVER 500 KINASES AND ROUGHLY 300 PHOSPHATASES, THE MAMMALIAN GENOME ONLY ENCODES A SINGLE O-GLCNAC TRANSFERASE (OGT) AND A SINGLE HYDROLASE (OGA). WHILE MANY KINASES RECOGNIZE SPECIFIC AMINO ACID SEQUENCES IN THEIR SUBSTRATES, THE DETERMINANTS GUIDING OGT ARE UNCLEAR AND LIKELY MANIFOLD. THIS INTRACELLULAR GLYCOSYLATION IS IMPLICATED IN NEARLY EVERY CELLULAR PROCESS FROM GENE EXPRESSION AND SIGNAL TRANSDUCTION TO CELL DIVISION AND DIFFERENTIATION. DESPITE THE UBIQUITOUS NATURE OF THIS POST-TRANSLATIONAL MODIFICATION IN HEALTH AND DISEASE, THE SPECIFIC FUNCTIONS OF OGT AND THE BASIC PRINCIPLES OF O-GLCNAC SIGNALING REMAIN ALMOST ENTIRELY ELUSIVE. THIS GAP IN OUR KNOWLEDGE HAS BEEN LARGELY DUE TO THE MAJOR LACK OF TOOLS AND TECHNOLOGIES AVAILABLE TO STUDY O-GLCNAC SIGNALING OR PERTURB THE ESSENTIAL OGT. HERE, WE AIM TO UNCOVER THE BASIC PRINCIPLES OF OGT AND O-GLCNAC SIGNALING, AND THEIR ROLE IN TRANSCRIPTIONAL REGULATION OF CELLULAR DIFFERENTIATION. WE HAVE RECENTLY DEVELOPED A HIGHLY SENSITIVE AND SPECIFIC ENRICHMENT REAGENT TO ANALYZE O-GLCNAC-MODIFIED PEPTIDES FROM CELLS AND TISSUES BY MASS SPECTROMETRY. USING THESE NEW ANTI-O-GLCNAC ANTIBODIES WE WILL ELUCIDATE THE GLOBAL, SITE-SPECIFIC TEMPORAL DYNAMICS OF O-GLCNAC SIGNALING DURING THE TRANSITION FROM TOTIPOTENCY TO NAÏVE AND PRIMED PLURIPOTENCY. COMBINED WITH PHOSPHOPROTEMIC PROFILING OF THE SAME SAMPLES, WE WILL MONITOR FOR CROSSTALK BETWEEN THESE TWO POST-TRANSLATIONAL MODIFICATIONS. TO GAIN INSIGHT INTO HOW OGT TARGETS ITS DIVERSE ARRAY OF SUBSTRATES, WE WILL DECONVOLUTE THE EXTENSIVE OGT INTERACTOME EMPLOYING CHEMICAL CROSSLINKING AND BIOCHEMICAL FRACTIONATION, FOLLOWED BY MASS SPECTROMETRIC ANALYSIS. TO EXPLORE HOW OGT USES ADAPTOR PROTEINS TO TARGETS SUBSTRATES, WE WILL USE AN INNOVATIVE APPROACH, DEGRADING SPECIFIC OGT INTERACTING PROTEINS AND ASSESSING CHANGES IN DOWNSTREAM O-GLCNAC SIGNALING USING OUR NEW QUANTITATIVE GLYCOPROTEOMIC APPROACH. INTEGRATING THESE TWO RESEARCH PROGRAMS, WE WILL CREATE A HOLISTIC, HIGH- RESOLUTION UNDERSTANDING OF THE PRINCIPLES OF O-GLCNAC SIGNALING. THE MIRA MECHANISM WILL NOT ONLY ENABLE THE INVESTIGATION OF BASIC O-GLCNAC BIOLOGY, BUT WILL PROVIDE THE FLEXIBILITY TO CONDUCT DATA-DRIVEN FOLLOW-UP, FUNCTIONAL ANALYSES OF DYNAMIC O-GLCNAC/CROSSTALK SITES AND DISTINCT OGT COMPLEXES, AND THEIR ROLE IN TRANSCRIPTIONAL REGULATION OF SOME OF THE EARLIEST DEVELOPMENT DECISIONS.

RELATIONSHIPS BETWEEN TFH CELLS, SOMATIC HYPERMUTATION, AND THE DEVELOPMENT OF BROADLY NEUTRALIZING ANTIBODIES

LYMPHOCYTE ACTIVATION IN SICKLE CELL LUNG DISEASE

REGULATION OF CD45 ALTERNATIVE SPLICING BY HNRPLL

CD4 T CELL, GERMINAL CENTER, AND ANTIBODY RESPONSE DEFECTS IN RECURRENT TONSILLITIS

FUNCTIONS OF NONCLASSICAL MONOCYTES IN THE VASCULATURE

LIPID HOMEOSTASIS, IMMUNITY AND ATHEROSCLEROSIS

REGULATION OF INNATE LYMPHOID CELLS IN INFLAMMATION

DIFFERENTIATION AND ANTI-VIRAL PROTECTIVE AND PATHOGENIC ROLES OF CD4 CTL

THE ROLE OF NATURAL KILLER T CELLS IN THE INNATE RESPONSE TO LUNG INFECTION

GLOBAL PROTEIN PALMITOYLATION AND DHHC PROTEINS IN T CELL ACTIVATION AND ANERGY

CELLULAR AND MOLECULAR REGULATION OF CD8+ T CELL MEMORY

CONTROL OF AIRWAY TOLERANCE

NUCLEAR RECEPTORS IN MYELOID DEVELOPMENT, INFLAMMATION, AND ATHEROSCLEROSIS

ASSESSMENT OF CYTOKINES IN HUMAN ISLETS FROM PATIENTS WITH DIABETES

THE MOLECULAR BASIS OF OX40 ON CD4 T CELLS

G2A RECEPTOR IN ATHEROSCLEROSIS

FUNCTION AND ASSEMBLY OF THE EBOLA VIRUS NUCLEOCAPSID

TARGETING TUMOR ENDOTHELIUM FOR CANCER SURVEILLANCE AND IMMUNOTHERAPY

MECHANISMS OF PROTEIN UBIQUITINATION IN REGULATING AIRWAY INFLAMMATION

MITOCHONDRIA, APOPTOSIS AND THE BCL-2 FAMILY

PROGRAMMING OF CD8+ T CELL TOLERANCE BY B CELL APC

TWEAK AND SKIN INFLAMMATION

DYSREGULATION OF TET DIOXYGENASE FUNCTION AS A SOURCE OF ABERRANT TRANSPOSABLE ELEMENT EXPRESSION DURING HUMAN AGING - ABSTRACT IT IS WELL ESTABLISHED THAT AGING IS ACCOMPANIED BY CHRONIC LOW-GRADE INFLAMMATION THAT CORRELATES WELL WITH MORTALITY AND MORBIDITY, BUT THE ASSOCIATION BETWEEN AGING AND INFLAMMATION IS NOT WELL UNDERSTOOD. SIMILARLY, CHANGES IN DNA CYTOSINE METHYLATION OCCUR REPRODUCIBLY WITH AGE, SUGGESTING THAT DNA METHYLATION CONTRIBUTES TO THE AGING PROCESS, BUT THE BIOLOGY REMAINS UNCLEAR. CERTAIN FAMILIES OF TRANSPOSABLE ELEMENTS (TES) ARE REPRESSED IN SOMATIC CELLS VIA DNA METHYLATION; MOREOVER, TES ARE ACTIVATED IN AGING MICE AND IN CULTURED HUMAN CELLS UNDERGOING REPLICATIVE SENESCENCE, AND INCREASED TE EXPRESSION LEADS TO DNA DAMAGE, MUTATIONS AND INFLAMMATION. THESE CONSEQUENCES HAVE ALL BEEN OBSERVED IN CELLS FROM AGED INDIVIDUALS, BUT THE UNDERLYING MECHANISMS ARE OBSCURE. UNDERSTANDING THE MECHANISTIC BASIS FOR THE ASSOCIATION OF AGING WITH INFLAMMATION AND CHANGES IN DNA METHYLATION WILL BE ESSENTIAL TO DESIGN RATIONAL INTERVENTIONS FOR AGE-ASSOCIATED DISORDERS. HERE WE TACKLE THE QUESTION OF HOW CHANGES IN DNA METHYLATION RELATE TO INFLAMMATION DURING AGING. DNA METHYLATION IS REGULATED BY DNA METHYLTRANSFERASES (DNMTS) AND TET METHYLCYTOSINE OXIDASES, WHICH CONTROL DNA METHYLATION AND DEMETHYLATION RESPECTIVELY. DNMT DEFICIENCY PREDICTABLY RESULTS IN DECREASED DNA METHYLATION, BUT TET DEFICIENCY IS PARADOXICALLY ALSO ASSOCIATED WITH A STRIKING LOSS OF DNA METHYLATION IN HETERO- CHROMATIN. MOREOVER, TET AND/OR DNMT-DEFICIENT CELLS SHOW INCREASED TE EXPRESSION AND CELL-INTRINSIC INFLAMMATION, SIMILAR TO THAT OBSERVED IN CELLS FROM AGED HUMANS AND SENESCENT CELLS IN CULTURE. WE WILL TEST THE HYPOTHESIS THAT AGING INVOLVES A PROGRESSIVE DECREASE IN TET AND/OR DNMT ACTIVITY WITH AGE, LEADING TO SELECTIVE LOSSES OF DNA METHYLATION IN HETEROCHROMATIN WHERE REPRESSED TES RESIDE, AND HENCE IN INCREASED TE EXPRESSION AND CELL-INTRINSIC (“STERILE”) INFLAMMATION. IN AIM 1, WE WILL DEFINE THE DNA MODIFICATION (5MC, 5HMC) STATUS OF TRANSPOSABLE ELEMENTS IN IMMUNE CELLS (CD4+ T LYMPHOCYTES AND MONOCYTES) OF YOUNG, MIDDLE-AGED AND OLD HEALTHY SUBJECTS FROM THE BLSA AND GESTALT COHORTS AT NIA, AND RELATE THEM TO THE OBSERVED INCREASE IN EXPRESSION OF TES AS A FUNCTION OF AGE. THESE STUDIES PROBE THE HYPOTHESIS THAT DECLINE OF TET AND/OR DNMT ACTIVITY WITH AGE DYSREGULATES TE EXPRESSION. IN AIM 2, WE WILL CORRELATE INCREASED TE EXPRESSION IN IMMUNE CELLS FROM THE BLSA AND GESTALT COHORTS WITH SIGNATURES OF INFLAMMATION AND SERUM LEVELS OF PRO-INFLAMMATORY CYTOKINES DURING AGING. IN AIM 3, WE WILL PERFORM COMPLEMENTARY STUDIES IN GENETICALLY TRACTABLE MOUSE MODELS OF CLONAL HEMATOPOIESIS, A MALADY OF BOTH AGING AND INFLAMMATION. USING MICE DEFICIENT IN TET2 AND/OR DNMT3A, WE WILL DEFINE THE DEVELOPMENT OF HETEROCHROMATIC DNA HYPOMETHYLATION AND INFLAMMATION IN IMMUNE CELLS OF THESE MICE WITH AGE, AND DETERMINE THE RELATION TO INCREASED TE EXPRESSION. OUR STUDIES WILL TEST THE NOVEL HYPOTHESIS OF A LINK BETWEEN TET/DNMT DEFICIENCY, LOSS OF HETEROCHROMATIC DNA METHYLATION AND INCREASED TE EXPRESSION WITH AGE, AND DEEPEN OUR UNDERSTANDING OF THE ELUSIVE MECHANISMS THAT CONNECT HETEROCHROMATIN DYSFUNCTION WITH INFLAMMATION, AGING AND ONCOGENESIS.

MAST CELL STAT5-REGULATORY PATHWAY IN ATOPIC DERMATITIS

INTERACTION OF HISTAMINE-RELEASING FACTOR WITH IMMUNOGLOBULINS IN ASTHMA

IRF-3/5/7-INDEPENDENT ANTIVIRAL IMMUNITY

ROLE OF MICRO-RNAS IN T CELLS AND OTHER IMMUNE/HAEMATOPOIETIC CELLS

ROLE OF TET PROTEINS IN MYELOID MALIGNANCIES

UNCOVERING THE MISSING LINK THAT DETERMINES SUSCEPTIBILITY TO AUTOIMMUNITY

ACHIEVING THERAPEUTIC ANTIGENT-SPECIFIC TOLERANCE IN TYPE 1 DIABETES

STRUCTURAL IMMUNOLOGY OF CD1 AND CD1-TCR COMPLEXES

MUCOSAL VACCINATION AGAINST DENGUE VIRUS INFECTION

ROLE OF TET PROTEINS IN ES CELL PLURIPOTENCY AND FUNCTION

VACCINATION WITH MHC-II RESTRICTED APOB100 PEPTIDES TO PREVENT ATHEROSCLEROSIS

PROTEOME-WIDE BASE EDITOR SCREENS TO ASSESS PHOSPHORYLATION SITE FUNCTIONALITY IN IMMUNOSENESCENCE - ABSTRACT WE OFTEN DEPICT SIGNALING PATHWAYS BY A DOZEN OR SO LANDMARK PHOSPHORYLATION EVENTS THAT CULMINATE IN THE TRANSCRIPTION OF A GENE OR INDUCTION OF A PHENOTYPE. IN REALITY, A CASCADE OF HUNDREDS TO THOUSANDS OF PHOSPHORYLATION SITES CREATE DYNAMIC BIOCHEMICAL NETWORKS THAT ORCHESTRATE ESSENTIALLY EVERY CELLULAR PROCESS FROM EXPRESSION OF TRANSCRIPTIONAL PROGRAMS AND CELL PROLIFERATION TO MIGRATION AND CYTOTOXIC EFFECTOR FUNCTIONS. T CELL SIGNALING IS AN IMPORTANT EXAMPLE. SIGNAL TRANSDUCTION THROUGH THE T CELL RECEPTOR AND CO-STIMULATORY MOLECULES IS INCREDIBLY COMPLEX AND LEADS TO IMPORTANT BUT DISTINCT DOWNSTREAM EFFECTS NECESSARY FOR PROPER IMMUNE RESPONSES TO PATHOGENS, CANCER, AND VACCINES. AS HUMANS AGE, T CELL SIGNALING BECOMES DETRIMENTALLY ALTERED, LEADING TO “IMMUNOSENESCENCE” AND LESS EFFICIENT PROTECTION FROM MALIGNANCIES. MASS SPECTROMETRY HAS ENABLED THE PROFILING OF TENS OF THOUSANDS OF PHOSPHORYLATION SITES FROM A DIVERSE RANGE OF CELLS AND TISSUES. UNFORTUNATELY, FUNCTIONALLY CHARACTERIZING THE TENS OF THOUSANDS OF POST-TRANSLATIONAL MODIFICATIONS CELLS USE TO COORDINATE ESSENTIALLY ALL CELLULAR AND ORGANISMAL PROCESSES REMAINS A FUNDAMENTAL CHALLENGE IN BIOLOGY. HERE, WE PROPOSE AN INNOVATIVE TECHNOLOGY THAT WILL ENABLE THE FUNCTIONAL ASSESSMENT OF THOUSANDS OF PHOSPHORYLATION SITES IN HIGH THROUGHPUT. WE WILL EMPLOY CRISPR-MEDIATED BASE EDITORS, WHICH MUTATE CODONS IN GENOMIC DNA, COUPLED TO PHENOTYPIC SCREENS TO PROBE THE SIGNALING EVENTS THAT LEAD TO SPECIFIC T CELL STIMULATION-SPECIFIC GENE EXPRESSION PROGRAMS OR PROLIFERATION RESPONSES. PRELIMINARY DATA FROM OUR LABORATORY STRONGLY SUGGESTS THAT WE CAN FUNCTIONALLY SCREEN TENS OF THOUSANDS OF PHOSPHORYLATION SITES FOR THEIR CONTRIBUTION TO NFAT OR NFKB SIGNALING IN STIMULATED T CELLS. IN THIS PROPOSAL, WE WILL FURTHER OPTIMIZE THIS TECHNOLOGY TO CREATE A ROBUST, RELIABLE SCREENING PLATFORM. TO VALIDATE OUR SCREENING TECHNOLOGY, WE WILL CHARACTERIZE A SUBSET OF PHOSPHORYLATION SITE MUTANTS USING CONVENTIONAL GENOMIC ENGINEERING METHODS TO UNDERSTAND HOW OUR SCREENING PLATFORM COMPARES TO CLASSIC APPROACHES TO STUDY PHOSPHORYLATION SITE FUNCTION, I.E. ONE MUTATION AT A TIME. WE WILL ALSO EXTEND THIS APPROACH TO PRIMARY HUMAN CD8+ T CELLS, TO SHOW THAT WE CAN UNCOUPLE THE SIGNALING EVENTS THAT LEAD TO CELL PROLIFERATION OR EXPRESSION OF THE ACTIVATION/EXHAUSTION MARKER PD1. HIGH THROUGHPUT FUNCTIONAL SCREENING OF PHOSPHORYLATION SITES IN PRIMARY IMMUNE CELLS WILL REVOLUTIONIZE THE WAY WE STUDY SIGNALING PATHWAYS AND CELLULAR DECISION MAKING, AND THE WAY WE APPROACH DRUG DEVELOPMENT OR ADOPTIVE CELL THERAPIES TO TREAT CANCER AND A VARIETY OF HUMAN AILMENTS ASSOCIATED WITH AGING.

NEXT GENERATION CRISPR READY, HLA CLASS I AND II TRANSGENIC MOUSE PLATFORM - ABSTRACT T CELL RESPONSES ARE CRITICAL IN THE PATHOGENESIS, PREVENTION, AND TREATMENT OF A BROAD RANGE OF DISEASES FROM INFECTION TO CANCER, AUTOIMMUNE DISEASE, AND TRANSPLANT REJECTION. HLA TRANSGENIC MICE HAVE BEEN PROVEN TO PREDICT HUMAN T CELL RESPONSES; YET CURRENTLY AVAILABLE HLA TRANSGENIC MICE ARE SPECIFIC TO INDIVIDUAL HLA ALLELES AND BASED ON GENETIC ENGINEERING TECHNOLOGIES WHICH DATE TO BEFORE THE TURN OF THE CENTURY. FURTHER, AVAILABLE HLA TRANSGENIC MICE ARE INCOMPATIBLE FOR BREEDING TO PRODUCE COMBINED HLA CLASS I AND CLASS II MODELS. THIS PROJECT WILL CREATE AND VALIDATE A MODULAR CAS9-EXPRESSING HLA TRANSGENIC MOUSE PLATFORM STRAIN FROM WHICH ANY HLA CLASS I AND OR CLASS II TRANSGENIC MOUSE CAN BE GENERATED WITH NOW BROADLY AVAILABLE CRISPR METHODS. OUR PUBLISHED DATA DEMONSTRATE OUR TRACK RECORD OF WORKING WITH HLA TRANSGENIC MICE AND OF DESIGNING, EVALUATING, AND VALIDATING NOVEL MOUSE MODELS. FURTHER, OUR ESTABLISHED RELATIONSHIP WITH TRANSVIRAGEN INC., ESTABLISHED GENETIC ENGINEERING EXPERTS, ASSURES THAT THE MICE WILL BE ENGINEERED AS PRECISELY AND EFFICIENTLY AS POSSIBLE. AS PRELIMINARY DATA WE INCLUDE INFORMATION FROM OUR COLLABORATION WITH SYNBAL INC. WHICH IN LESS THAN 2 YEARS’ TIME GENERATED TRIPLE-HUMANIZED MICE ON BOTH C57BL/6 AND BALB/C BACKGROUNDS WHICH ARE DISTRIBUTED BY THE THE JACKSON LABORATORY. WE WILL PRODUCE A CAS9-EXPRESSING PLATFORM MOUSE STRAIN WITH HUMANIZED Β2M, CD8 AND CD4, AND ABSENCE OF MOUSE MHC II. THE THREE KEY ADVANTAGES OF THIS STRAIN WILL BE: (A) INVESTIGATORS STUDYING A BROAD RANGE OF DISEASES WILL BE ONE CRISPR STEP OR TRANSGENE INJECTION AWAY FROM A MOUSE MODEL WITH HLA CLASS I OR CLASS II ALLELE OF THEIR CHOICE, (B) CROSSES OF ALLELE-SPECIFIC STRAINS WILL ALLOW INVESTIGATORS TO MODEL VIRTUALLY ANY COMBINATION OF HLA CLASS I AND II ALLELES BEGINNING AT F1, AND (C) ANY PLATFORM-DERIVED MOUSE FOR SOMATIC MUTAGENESIS EXPERIMENTS IN THE CONTEXT OF HUMANIZED CD4, CD8 AND Β2M. THE SPECIFIC AIMS ARE (1) PRODUCE A CRISPR-READY PLATFORM STRAIN AND TRANSGENIC MOUSE STRAINS THAT ARE HLA ALLELE-SPECIFIC FOR AUTOIMMUNE DISEASES AND THE TOP HLA CLASS I AND II ALLELE FREQUENCIES OF US ETHNIC GROUPS, (2) TEST AND VALIDATE T CELL REACTIVITY IN HLA CLASS I AND HLA CLASS II MICE DERIVED FROM PLATFORM STRAIN. MICE WILL BE DEPOSITED IN THE JAX REPOSITORY, AND VALIDATION DATA WILL BE COMPILED AND PUBLISHED IN A PUBLICLY VIEWABLE ONLINE DATABASE. THIS HLA TRANSGENIC PLATFORM STRAIN WILL PROVIDE A BROAD RANGE OF INVESTIGATORS WITH A MODULAR SMALL ANIMAL IN VIVO MODEL OF HUMAN RELEVANT T CELL RESPONSES FOR THE NEXT DECADE AND BEYOND.

INDIRECT RECOGNITION OF MICROBES BY NKT CELLS

THE IMMUNOGENETICS OF MACAQUES USED IN BIODEFENSE RESEARCH

MITOCHRONDRIAL REGULATION OF APOPTOSIS

VIRUSES AND AUTOIMMUNITY POI

GENETIC STUDIES LINKING LSP1 FUNCTION IN T CELLS TO INFLAMMATORY BOWEL DISEASE - PROJECT SUMMARY/ABSTRACT INFLAMMATORY BOWEL DISEASES (IBD) CAUSE SUBSTANTIAL MORTALITY AND MORBIDITY. CURRENT TREATMENTS THAT BLOCK PATHOLOGICAL INFLAMMATORY RESPONSES HAVE IMPROVED CLINICAL OUTCOMES IN SOME PATIENTS BUT THEY ARE NOT HIGHLY EFFECTIVE IN MODIFYING DISEASE PROGRESSION OR PREVENTING RELAPSES. HENCE, THERE IS A LARGE UNMET NEED TO DEVELOP NOVEL THERAPEUTIC TARGETS. GENOME-WIDE ASSOCIATION STUDIES (GWAS) OFFER AN UNBIASED APPROACH TO IDENTIFY THERAPEUTIC TARGETS IN RELEVANT IMMUNE CELL TYPES SUCH AS CD4+ T CELLS THAT PLAY KEY ROLES IN IBD PATHOGENESIS. BECAUSE T CELLS ARE QUIESCENT IN THE ABSENCE OF EXTRINSIC STIMULATION, IT IS NOT POSSIBLE TO FULLY EXAMINE THE EFFECTS OF DISEASE-RISK VARIANTS ON FUNCTIONALLY RELEVANT EFFECTOR GENES UNDER RESTING CONDITIONS. TO IDENTIFY IBD-RISK GENES IN ACTIVATED CD4+ T CELLS, WE PERFORMED THE FIRST LARGE-SCALE SINGLE-CELL EQTL STUDY ON ACTIVATED CD4+ T CELLS. WE FOUND THAT REDUCED EXPRESSION OF LEUKOCYTE-SPECIFIC PROTEIN 1 (LSP1), SPECIFICALLY IN ACTIVATED CD4+ T-CELL SUBSETS SUCH AS TH1 AND TH17 CELLS, WAS ASSOCIATED WITH THE RISK OF IBD. IN THIS R01 PROPOSAL, WE WILL INVESTIGATE HOW REDUCED LEVELS OF LSP1 INFLUENCES THE DIFFERENTIATION AND FUNCTION OF CD4+ T CELLS TO DRIVE DISEASE PATHOGENESIS, AND WILL TEST THE HYPOTHESIS THAT LSP1 PLAYS A KEY ROLE IN RESTRAINING THE RE-PROGRAMMING OF CD4+ T CELLS INTO A MORE PATHOGENIC CELL STATE IN IBD PATIENTS. IN AIM 1, WE WILL DETERMINE THE FUNCTIONAL IBD-RISK VARIANTS THAT REDUCE LSP1 EXPRESSION IN CD4+ T CELLS. WE WILL EMPLOY LUCIFERASE REPORTER ASSAYS TO DETERMINE FUNCTIONAL VARIANTS IN THE ENHANCERS AND PROMOTER OF LSP1, PERFORM CRISPR-MEDIATED EDITING OF PRIORITIZED IBD-RISK EQTLS TO DEFINE CAUSAL VARIANTS, AND PERFORM CRISPRI AND CHIP ASSAYS TO DETERMINE FUNCTIONAL ENHANCERS, RELEVANT UP-STREAM REGULATORS AND WHETHER THE FUNCTIONAL LSP1 EQTLS DIRECTLY PERTURB THE BINDING OF KEY TRANSCRIPTION FACTORS THAT MODULATE LSP1 EXPRESSION. IN AIM 2, WE WILL DETERMINE THE ROLE OF LSP1 IN REPROGRAMMING OF CD4+ T CELLS INTO THE PATHOGENIC STATE OBSERVED IN IBD. TO DETERMINE WHETHER LSP1 INFLUENCES THE DIFFERENTIATION AND PATHOGENIC FUNCTION OF CD4+ T CELLS FROM HEALTHY AND IBD DONORS, WE WILL REDUCE AND INCREASE LSP1 LEVELS AND ASSESS THE EFFECTS ON CD4+ T-CELL ACTIVATION, APOPTOSIS, PROLIFERATION AND PROINFLAMMATORY CYTOKINE PRODUCTION. IN AN ADOPTIVE T CELL TRANSFER MODEL OF COLITIS, WE WILL COMPARE THE ABILITY OF LSP1- SUFFICIENT (WILD-TYPE) AND LSP1-DEFICIENT CD4+ T CELLS IN DRIVING COLONIC INFLAMMATION AND PATHOGENIC TH1 AND TH17 DIFFERENTIATION. WE WILL DETERMINE WHETHER REDUCING LSP1 EXPRESSION IN CD4+ T CELLS ENHANCES THEIR PATHOGENICITY IN MOUSE MODELS OF COLITIS, THUS IMPLICATING AN IMPORTANT T CELL-INTRINSIC ROLE FOR LSP1 IN IBD PATHOGENESIS. OVERALL, THIS STUDY, EXAMINING THE FUNCTION, EXPRESSION, ACTIVATION AND REGULATION OF LSP1 IN CD4+ T CELLS, WILL PROVIDE IMPORTANT MECHANISTIC INSIGHTS INTO THE GENETIC BASIS OF RISK FOR INFLAMMATORY BOWEL DISEASE.

SIGNAL TRANSDUCTION IN T LYMPHOCYTE ACTIVATION

FLUOROGENIC ASSAYS FOR CELL-BASED HTS OF TYROSINE PHOSPHATASE INHIBITORS

HOW IL-10R BLOCKADE CAN RESOLVE PERSISTENT VIRAL INFECTIONS

SERVICES TO SUPPORT THE OBO FOUNDRY STANDARDS

NANOSCALE REGULATION OF STORE-OPERATED CALCIUM ENTRY THROUGH STIM-ORAI SIGNALLING

PROTECTIVE ROLE OF NONCLASSICAL MONOCYTES IN IMMUNOTHERAPIES FOR SOLID CANCERS

TNFR MEMBERS IN T CELL IMMUNITY TO VACCINIA

IMMUNE REGULATION BY DEUBIQUITINATION

REGULATION OF T CELL TOLERANCE BY OX40

RECOGNITION OF BACTERIAL ANTIGENS BY NKT CELLS

THE ROLE OF 4-IBB IN T CELL AND APC INTERACTIONS

MODULATING LYMPHOTOXINS FOR VIRAL DEFENSES

UNDERSTANDING LYMPHOID TISSUE IMMUNOLOGY IN HUMAN SLE AND THE IMPACT OF THERAPEUTIC INTERVENTIONS - PROJECT SUMMARY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS A COMPLEX AUTOIMMUNE DISEASE CHARACTERIZED BY SELF-REACTIVE ANTIBODIES (AUTOANTIBODIES), AND THIS CHRONIC DISEASE RESULTS IN SIGNIFICANT LOSS OF QUALITY OF LIFE AND IRREVERSIBLE TISSUE DAMAGE THAT CAN RESULT IN ORGAN FAILURE AND DEATH. WITH SLE BEING A DISEASE THAT AFFECTS MANY ORGANS WITH A RANGE OF SEVERITY, NEWER TARGETED THERAPIES ARE NEEDED. DESPITE ADVANCES IN UNDERSTANDING SLE, MOST HUMAN STUDIES HAVE RELIED ON PERIPHERAL BLOOD, LIMITING INSIGHTS INTO THE LYMPHOID TISSUE COMPARTMENTS WHICH ARE LIKELY TO BE THE PRIMARY SITES WHERE AUTOREACTIVE T AND B CELLS ARE ACTIVATE. AS A RESULT, MAJOR GAPS REMAIN IN OUR UNDERSTANDING OF TISSUE-SPECIFIC IMMUNE MECHANISMS IN SLE PATHOGENESIS AND TREATMENT RESPONSE. TO ADDRESS THESE KNOWLEDGE GAPS, WE PROPOSE A NOVEL APPROACH USING MINIMALLY INVASIVE NASAL SWAB LYMPHOID TISSUE SAMPLING COMBINED WITH HIGH-DIMENSIONAL IMMUNOLOGIC ANALYSIS IN SLE. UNLIKE BLOOD-BASED TESTING, THIS METHOD ENABLES DIRECT STUDY OF IMMUNE CELLS IN A LYMPHOID TISSUE. OUR TEAM HAS DEVELOPED AND VALIDATED A SAFE AND REPRODUCIBLE NASAL SWAB PROTOCOL, PAIRED WITH FLOW CYTOMETRY AND TRANSCRIPTOMIC PROFILING, TO MEASURE LYMPHOID TISSUE IMMUNE CELL POPULATIONS WITH HIGH RESOLUTION. THIS METHOD ALLOWS DIRECT INTERROGATION OF IMMUNE RESPONSES IN LYMPHOID TISSUE OVER TIME, WHICH WILL BE A KEY ADVANCE FOR SLE RESEARCH. IMPORTANTLY, SUCH MEASUREMENTS CAN BE DONE REPEATEDLY, LONGITUDINALLY OVER TIME, ALLOWING FOR NOVEL KINETIC INSIGHTS INTO SLE PATHOPHYSIOLOGY. THIS APPROACH, COMBINED WITH OUR HUMAN IMMUNOLOGY EXPERTISE, CAN POTENTIALLY PROVIDE UNPRECEDENTED INSIGHTS INTO SLE IMMUNE EVENTS. WE WILL (1) CHARACTERIZE IMMUNE CELL POPULATIONS IN LYMPHOID TISSUE FROM SLE PATIENTS VS. HEALTHY CONTROLS; (2) IDENTIFY FEATURES THAT CORRELATE WITH DISEASE ACTIVITY OVER TIME; AND (3) ASSESS PRE- AND POST-TREATMENT IMMUNE DYNAMICS IN PATIENTS RECEIVING COMMON AND NEW SLE THERAPIES. OUR CENTRAL HYPOTHESIS IS THAT LONGITUDINAL ANALYSIS OF LYMPHOID TISSUE IMMUNE SIGNATURES WILL UNCOVER CRITICAL DRIVERS OF SLE PATHOGENESIS AND TREATMENT RESPONSE. ADDITIONALLY, WE AIM TO IDENTIFY KEY FEATURES OF THESE T AND B CELLS THAT COULD SERVE AS TARGETS FOR NOVEL THERAPEUTIC INTERVENTIONS AND/OR BIOMARKERS OF DISEASE ACTIVITY AND RESPONSES TO TREATMENT, THUS POTENTIALLY IDENTIFY NOVEL SLE THERAPEUTIC INTERVENTIONS AND BIOMARKERS. THE PROJECT IS ENTIRELY FOCUSED ON HUMAN IMMUNOLOGY IN SLE PATIENTS, DIRECTLY EXAMINING LYMPHOID TISSUE. FURTHERMORE, WE WILL, FOR THE FIRST TIME, COLLECT DETAILED LONGITUDINAL DATA FROM LYMPHOID TISSUES FROM SLE PATIENTS, INCLUDING PATIENTS RECEIVING THERAPIES. BY ADVANCING UNDERSTANDING OF HUMAN ADAPTIVE IMMUNITY IN LUPUS, THIS WORK COULD SIGNIFICANTLY IMPROVE PATIENT STRATIFICATION AND TREATMENT OUTCOMES IN SLE.

DEVELOPMENT OF A THERAPEUTIC ANTIBODY COCKTAIL AGAINST NEW WORLD ARENAVIRUSES - PROJECT SUMMARY/ABSTRACT THE MAMMARENAVIRUS GENUS OF THE ARENAVIRIDAE FAMILY, ENCOMPASSES SEVERAL SEVERE HUMAN PATHOGENS, INCLUDING OLD WORLD ARENAVIRUSES (OWAS) LIKE LASSA VIRUS AND NEW WORLD ARENAVIRUSES (NWAS) SUCH AS MACHUPO AND JUNÍN VIRUSES, EACH OF WHICH CAUSE HEMORRHAGIC FEVER. CURRENTLY, THE ONLY AVAILABLE VACCINE, THE LIVE-ATTENUATED CANDID #1 FOR JUNV, IS NOT FDA-APPROVED AND PROVIDES LIMITED NEUTRALIZATION BREADTH, LEAVING POPULATIONS VULNERABLE TO DIVERSE ARENAVIRUSES. MONOCLONAL ANTIBODY (MAB) THERAPIES REPRESENT ONE OF THE MOST IMPORTANT TREATMENT OPPORTUNITIES IN MODERN MEDICINE. THEY ARE BEING USED TO TREAT AND CURE NUMEROUS DISEASES, INCLUDING DIFFERENT TYPES OF CANCER AND AUTOIMMUNE DISEASES. SINCE THE 1980S, MABS LIKE SYNAGIS AGAINST RSV, AND EBANGA™ AND INMAZEB™ AGAINST EBOLA VIRUS DISEASE HAVE BEEN USED TO TREAT VIRAL HEALTH THREATS. HOWEVER, THERE REMAINS NO LICENSED THERAPIES FOR TREATMENT OF ANY ARENAVIRUS INFECTION. AS SUCH, DEVELOPMENT, ESPECIALLY OF BROADLY APPLICABLE THERAPEUTICS, IS AN URGENT NEED. PIONEERING DISCOVERIES FROM OUR GROUP ILLUMINATED THE “ACHILLES HEELS” ON THE LASSA VIRUS SURFACE GLYCOPROTEIN AND LED TO A FIRST-IN-CLASS PRE- CLINICAL THERAPEUTIC FOR LASSA VIRUS INFECTION. HERE, WE EXTEND THIS CUTTING-EDGE WORK TO NEW WORLD ARENAVIRUSES. EMPLOYING A NOVEL PANEL OF PREFUSION-STABILIZED NWA GPCS, WE WILL ELUCIDATE THE STRUCTURAL AND FUNCTIONAL PROPERTIES OF THESE PROTEINS, PROVIDING ESSENTIAL TEMPLATES TO INTERPRET ANTIBODY RESPONSES. OUR APPROACH INCLUDES ADVANCED METHODS SUCH AS HIGH-RESOLUTION CRYO-ELECTRON MICROSCOPY TO VISUALIZE NAB INTERACTIONS AT A MOLECULAR LEVEL. WE WILL ISOLATE A DIVERSE ARRAY OF BROADLY PROTECTIVE NABS FROM IMMUNIZED ANIMAL MODELS AND HUMANS VACCINATED WITH CANDID #1, ASSESSING THEIR BREADTH AND POTENCY TO IDENTIFY CANDIDATES FOR THERAPEUTIC DEVELOPMENT. FURTHERMORE, WE WILL CONDUCT COMPREHENSIVE STRUCTURAL AND BIOCHEMICAL ANALYSES TO DELINEATE THE ANTIGENIC LANDSCAPE OF NWA GPCS AND ESTABLISH MECHANISTIC RULES UNDERLYING THEIR NEUTRALIZATION. THIS MULTIFACETED INVESTIGATION PROMISES TO YIELD GROUNDBREAKING INSIGHTS INTO THE IMMUNE RESPONSE TO NWAS, PAVING THE WAY FOR THE DEVELOPMENT OF EFFECTIVE VACCINES AND IMMUNOTHERAPEUTICS TO COMBAT THESE UNPREDICTABLE AND OFTEN FATAL INFECTIONS.

ILLUMINATING IMMUNOREGULATORY MECHANISMS OF INTERLEUKIN-1 RECEPTOR 8 AND NOVEL THERAPEUTIC STRATEGIES - PROJECT SUMMARY AS A KEY IMMUNE RESPONSE, INFLAMMATION UNITES INNATE AND ADAPTIVE IMMUNITY TO FIGHT AGAINST INFECTIONS, INJURIES AND OTHER DANGER SIGNALS. HOWEVER, CHRONIC INFLAMMATION CAN CAUSE AND ACCELERATE AUTOIMMUNE DISEASES, ORGAN DISORDERS AND CANCERS. AT A MOLECULAR LEVEL, IMMUNE BALANCE IS ORCHESTRATED BY PRO- AND ANTI- INFLAMMATORY SIGNALS THAT ARE INITIATED AND MEDIATED BY CYTOKINE/RECEPTOR INTERACTIONS. INTERLEUKIN-1 RECEPTOR 8 (IL-1R8) IS A POTENT IMMUNOREGULATORY FACTOR THAT NOT ONLY BLOCKS INFLAMMATORY SIGNALS BY ACTING AS A DECOY RECEPTOR FOR NUMEROUS PRO-INFLAMMATORY TOLL-LIKE RECEPTORS (TLRS) OR IL-1RS, BUT CAN ALSO TRANSDUCE IL-37- INDUCED ANTI-INFLAMMATORY SIGNALING AS A CYTOKINE CO-RECEPTOR. IL-1R8 IS WIDELY EXPRESSED AND DAMPENS PRO- INFLAMMATORY SIGNALS IN MANY TISSUES, AND THE DOWNREGULATION OR DYSFUNCTION OF IL-1R8 IS ASSOCIATED WITH MANY INFLAMMATION-PROMOTED DISEASES. ON THE OTHER HAND, SILENCING OF IL-1R8 CAN UNLEASH NK OR CD8+ T CELL- MEDIATED CYTOTOXICITY AGAINST INFECTIONS AND CANCERS. THESE MULTI-FACETED FEATURES MAKE IL-1R8 A PROMISING TARGET WITH SUPERIOR DRUGGABILITY. HOWEVER, THE STRUCTURAL BASES OF IL-1R8-MEDIATED ANTI-INFLAMMATORY MECHANISMS ARE STILL UNKNOWN, WHICH HINDERS THE DEVELOPMENT OF THERAPEUTICS THAT TARGET IL-1R8. IN THIS PROJECT, WE WILL FULLY INTERPRET THE ANTI-INFLAMMATORY MECHANISMS OF IL-1R8 AT AN ATOMIC LEVEL BY SOLVING THE HIGH-RESOLUTION STRUCTURES OF IL-1R8 IN COMPLEX WITH PRO-INFLAMMATORY TLR4 OR IL-1R1, AS WELL AS THE TRIPARTITE COMPLEX OF IL- 1R8/IL-18RΑ/IL-37, WHICH MEDIATES NOVEL ANTI-INFLAMMATORY SIGNALING. WE WILL ALSO DISCOVER, CHARACTERIZE AND OPTIMIZE IL-1R8-TARGETING MONOCLONAL ANTIBODIES (MABS) FROM MICE IMMUNIZED WITH AN ENGINEERED IL-1R8 IMMUNOGEN. TAKING THE STRUCTURAL INFORMATION AS A BLUEPRINT AND MABS AS CRITICAL BUILDING BLOCKS, WE WILL DESIGN AND EVALUATE A SERIES OF NOVEL BIOLOGICAL DRUG CANDIDATES WITH A HIGH POTENTIAL FOR PRECISELY HARNESSING THE ANTI- INFLAMMATORY AND ANTI-TUMOR ACTIVITIES OF IL-1R8 IN VARIOUS PATHOLOGICAL CONDITIONS.

NOVASEQ5000 HIGH THROUGHPUT SEQUENCER

MONOCYTE ADHESION TO ATHEROMA IN GENE TARGETED MICE

CYTOF MASS CYTOMETER

HOUSE DUST-MITE ALLERGEN-SPECIFIC T CELL SUBSETS IN ALLERGY AND ASTHMA - PROJECT SUMMARY ONE THIRD OF THE POPULATION SUFFER FROM ALLERGIC DISEASES INCLUDING ASTHMA, RHINITIS, ATOPIC DERMATITIS AND FOOD ALLERGY. HOUSE-DUST MITE (HDM) IS THE MOST COMMON ALLERGEN WITH NEARLY UBIQUITOUS PRESENCE IN US HOMES AND ~30% SENSITIZATION. IT IS AN ENIGMA WHY SOME PEOPLE DEVELOP ALLERGY TO A UBIQUITOUS ALLERGEN SUCH AS HDM WHILE OTHERS DON’T. HDM IS ALSO THE MOST IMPORTANT ALLERGEN DRIVING SENSITIZATION AND RISK OF ALLERGIC DISEASES LIKE ASTHMA. BUT, AGAIN, ONLY SOME PEOPLE WITH HDM SENSITIZATION DEVELOP ASTHMA AND/OR RHINITIS, FOR UNKNOWN REASONS. IN THIS PROPOSAL, WE WILL EXPLORE THE MECHANISMS AND CELL TYPES THAT DRIVE EITHER NATURAL TOLERANCE OR ALLERGEN SENSITIZATION AND THE DEVELOPMENT OF ASTHMA. WE FOCUS ON CD4+ HELPER T CELLS (TH) BECAUSE OF THEIR IMPORTANCE IN ALLERGY AND ASTHMA. TO DETERMINE THE DIVERSITY OF THESE TH CELL SUBSETS IN ALLERGY AND ASTHMA, WE PERFORMED ONE OF THE FIRST SINGLE-CELL TRANSCRIPTOMIC STUDIES OF HDM ALLERGEN-SPECIFIC TH CELLS. WE IDENTIFIED OF A NOVEL SUBSET OF HDM-SPECIFIC TH CELLS CHARACTERIZED BY AN INTERFERON RESPONSE (IFNR) GENE SIGNATURE (THIFNR CELLS), WHICH WAS NEGATIVELY ASSOCIATED WITH HDM SENSITIZATION. IN ADDITION, HDM- SPECIFIC TH2 CELLS WITH INCREASED EXPRESSION OF IL9 WERE INCREASED IN HDM-SENSITIZED SUBJECTS WITH ASTHMA COMPARED TO THOSE WITHOUT ASTHMA. BASED ON THESE FINDINGS, WE HYPOTHESIZE THAT HDM-SPECIFIC THIFNR CELLS PROTECT AGAINST HDM-SENSITIZATION, AND IL9-EXPRESSING TH2 CELLS IN THE AIRWAYS, SPECIFICALLY TISSUE-RESIDENT MEMORY T CELLS (TRM CELLS) PROMOTE THE DEVELOPMENT OF HDM-ALLERGIC ASTHMA. IN AIM 1, WE WILL DETERMINE THE ASSOCIATION BETWEEN HDM-SPECIFIC THIFNR CELLS AND PROTECTION AGAINST HDM SENSITIZATION. WE WILL ASSESS SUBJECTS ENROLLED IN THE ISLE OF WIGHT WHOLE POPULATION BIRTH COHORT (IOWBC; N=1456). WE WILL IDENTIFY SUBGROUPS OF PARTICIPANTS (N=140) WITH: (I) PERSISTENT HDM-SENSITIZATION SINCE CHILDHOOD (II) NEVER HDM- SENSITIZATION SINCE CHILDHOOD, (III) ADULT-ONSET HDM-SENSITIZATION, AND (IV) ADULT-ONSET HDM TOLERANCE. USING LONGITUDINALLY-COLLECTED PERIPHERAL BLOOD MONONUCLEAR CELLS, WE WILL ISOLATE HDM-SPECIFIC T CELLS AND PERFORM SINGLE-CELL TRANSCRIPTOME AND TCR-SEQ ANALYSIS TO ENUMERATE THE FREQUENCY, PROPERTIES, PERSISTENCE AND CLONALITY OF HDM-SPECIFIC T CELL SUBSETS INCLUDING THE NOVEL THIFNR CELL SUBPOPULATION, AND DETERMINE THEIR ASSOCIATION WITH PROTECTION AGAINST DEVELOPMENT OF HDM-SENSITIZATION AT DIFFERENT AGES AND WITH ADULT-ONSET TOLERANCE. IN AIM 2, WE WILL IDENTIFY HDM-SPECIFIC T CELL SUBSETS IN THE BLOOD AND AIRWAYS ASSOCIATED WITH THE DEVELOPMENT OF ASTHMA IN SUBJECTS WITH HDM-SENSITIZATION. WE WILL ENROLL 125 SUBJECTS WITH HDM-ALLERGY FROM THE IOWBC (N=55 WITH ASTHMA, N=70 WITHOUT ASTHMA), OBTAIN BLOOD AND AIRWAY SAMPLES LONGITUDINALLY COLLECTED, AND IN A SUBGROUP FOLLOWING HDM-ALLERGEN BRONCHIAL CHALLENGE. WE WILL PERFORM SINGLE-CELL TRANSCRIPTOME AND TCR-SEQ ANALYSIS TO DETERMINE THE FREQUENCY, PROPERTIES, PERSISTENCE AND CLONALITY OF HDM- SPECIFIC TH CELL AND TRM SUBSETS IN THE BLOOD AND AIRWAYS, AND THEIR ASSOCIATION WITH ASTHMA DEVELOPMENT.

DEVELOPMENT OF A REPLICON RNA-BASED UNIVERSAL VACCINE AGAINST DENGUE AND ZIKA

ALLOSTERIC ENHANCEMENT OF ADENOSINE RECEPTORS

PURIFICATION & CHARACTERIZATION OF ADENOSINE RECEPTORS

MITOCHONDRIAL PATHWAYS IN APOPTOSIS

ILLUMINA HISEQ 2500 SEQUENCING SYSTEM

TRAIL-MEDIATED REGULATION OF T HELP FOR CTL

DEORPHANIZING A NOVEL GPCR EXPRESSED BY TISSUE RESIDENT MEMORY CELLS - PROJECT SUMMARY LUNG INFECTIONS REMAIN A SIGNIFICANT SOURCE OF MORBIDITY AND MORTALITY ACROSS ALL AGE GROUPS. THE RECENT SARS- COV-2 PANDEMIC UNDERSCORES THE CRITICAL NEED FOR THE SCIENTIFIC COMMUNITY TO URGENTLY DEVELOP STRATEGIES THAT ENHANCE MUCOSAL IMMUNITY AGAINST A WIDE RANGE OF PATHOGENS. WHILE VACCINES OFFER PROTECTION AGAINST SEVERE DISEASE, THEIR EFFECTIVENESS ARE NOT OFTEN DURABLE AND FOR MANY INFECTIONS, SUCH AS TUBERCULOSIS, THEY ARE NOT EFFECTIVE. THEREFORE, GAINING A DEEPER UNDERSTANDING OF THE MECHANISMS SUPPORTING LONG-TERM PROTECTIVE IMMUNITY AT BARRIER SITES, LIKE THE RESPIRATORY TRACT, IS VITAL FOR DEVELOPING THERAPIES THAT BOOST MUCOSAL IMMUNE RESPONSES. TISSUE-RESIDENT MEMORY T CELLS (TRM CELLS) HAVE EMERGED AS THE GUARDIANS OF BARRIER IMMUNITY. TRM CELLS ARE A DISTINCT POPULATION OF MEMORY T CELLS THAT RESIDE WITHIN TISSUES AND RESPOND IMMEDIATELY AGAINST PATHOGENS INVADING BARRIER TISSUES, THUS REPRESENTING THE FIRST-LINE OF DEFENSE. DR. VIJAYANAND’S TEAM (LJI PI) HAS LONG-STANDING EXPERTISE IN HUMAN LUNG TRM CELLS HAVING SHOWN THEIR IMPORTANCE IN DRIVING NATURAL AND ANTI-PD1 THERAPY-INDUCED T CELL RESPONSES IN LUNG CANCER. GIVEN THE PROFOUND IMPORTANCE OF TRM CELLS IN PROTECTIVE IMMUNITY, THE GENERATION AND MAINTENANCE OF ROBUST TRM RESPONSES IS CONSIDERED IMPORTANT FOR THE SUCCESS OF VACCINES AIMED AT PREVENTING SEVERE LUNG INFECTIONS. HOWEVER, CURRENTLY, THERE ARE NO THERAPEUTIC OPTIONS FOR BOOSTING OR MAINTAINING NATURAL AND VACCINE-INDUCED TRM RESPONSES IN THE LUNGS. OUR TRANSCRIPTOMIC STUDIES HAVE IDENTIFIED A NOVEL ORPHAN G-PROTEIN COUPLED RECEPTOR (GPCR) THAT IS EXPRESSED AT HIGH LEVELS IN LUNG TRM CELLS. USING GENETIC KNOCK-OUT MODELS, WE FOUND THAT THIS GPCR PLAYS A KEY ROLE IN THE DEVELOPMENT OF LUNG TRM CELLS. IN THIS PROPOSAL, OUR OBJECTIVE IS TO DE-ORPHANIZE THIS GPCR. WE HAVE ALREADY DETECTED LIGAND ACTIVITY IN MURINE THYMIC EXTRACTS USING THE B-ARRESTIN RECRUITMENT ASSAY IN HEK293 CELLS. IN AIM 1, DR. CHANGLU LIU’S TEAM (PI) AT SBP, WILL IDENTIFY AND PURIFY THE LIGAND FROM THYMIC EXTRACTS AND THYMIC-DERIVED CELL TYPES, AS DESCRIBED IN THEIR PREVIOUS WORK TO DEORPHANIZE OTHER GPCR LIKE GPR183 AND GPCR135. WE WILL PURIFY THE LIGAND FROM THYMIC TISSUE THROUGH ORGANIC EXTRACTIONS FOLLOWED BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC). FRACTIONS WITH LIGAND ACTIVITY WILL BE ANALYZED BY MASS SPECTROMETRY TO IDENTIFY THE NATURE AND SEQUENCE OF THE LIGAND. THE PUTATIVE LIGAND WILL THEN BE TESTED IN A DOSE-DEPENDENT MANNER TO DETERMINE AFFINITY AND SPECIFICITY OF THE LIGAND TO THE ORPHAN GPCR. IN AIM 2, WE WILL TEST FUNCTIONAL ACTIVITY OF THE PUTATIVE GPCR LIGAND IN PRIMARY T CELLS. WE INITIALLY TEST IN VITRO THE FUNCTIONAL ACTIVITIES OF THYMIC TISSUE/CELLULAR EXTRACTS IN T CELLS. THEN, WE WILL PERFORM SIMILAR STUDIES USING THE PURIFIED LIGAND AND DETERMINE ITS ACTIVITY ON T CELL SIGNALING, ACTIVATION, PROLIFERATION, CYTOKINE PRODUCTION AND TRM CELL GENERATION IN VIVO. STUDIES IN THIS AIM WILL IDENTIFY THE PHYSIOLOGICAL LIGAND THAT CAN ENHANCE THE ACTIVITY OF AN IMPORTANT TARGET (GPCR) IN TRM CELLS. THIS LIGAND OR ITS ANALOGUES HAVE THE POTENTIAL TO ENHANCE THE GENERATION OF LUNG TRM CELLS, THEREBY BOOSTING PROTECTIVE MUCOSAL IMMUNE RESPONSES IN HUMANS.

SUPER-RESOLUTION CONFOCAL MICROSCOPE

GENETIC CONTRIBUTIONS TO T CELL MEDIATED IMMUNOPATHOLOGY AFTER VIRAL INFECTION

ACTIVATION OF NKT CELLS BY CYTOMEGALOVIRUS

PRE-FUSION ARENAVIRUS GP TRIMERS: STRUCTURAL TEMPLATE FOR FUNCTIONAL ANALYSIS

A NOVEL CD4+ T CELL SUBSET ASSOCIATED WITH ALLERGY PROTECTION - PROJECT SUMMARY DISEASE ASSOCIATED WITH ALLERGIES SUCH AS ASTHMA ARE A RISING HEALTH PROBLEM WITH NO CURRENT CURATIVE SOLUTIONS. CD4+ HELPER T CELLS (TH) THAT RESPOND TO COMMON ALLERGENS PLAY AN IMPORTANT ROLE IN DRIVING AIRWAY INFLAMMATION IN ASTHMA. TO BETTER UNDERSTAND THE DIVERSITY OF T CELL SUBSETS IN ALLERGY AND ASTHMA, WE ANALYZED THE SINGLE-CELL TRANSCRIPTOME OF ~50,000 HOUSE DUST MITE (HDM) ALLERGEN-REACTIVE TH CELLS FROM ASTHMATICS AND NON-ASTHMATICS, WITH AND WITHOUT HDM ALLERGY. FROM OUR ANALYSIS, BESIDES CANONICAL CLUSTERS OF CELLS SUCH AS TH2, TH17, AND TH1, WE IDENTIFIED A NOVEL SUBSET OF ALLERGEN-REACTIVE TH CELLS CHARACTERIZED BY AN IFN RESPONSIVE GENE SIGNATURE THAT WE CALLED THIFNR CELLS (SEUMOIS ET AL. SCIENCE IMMUNOLOGY, 2020). PROPORTIONS OF THIFNR CELLS WERE SIGNIFICANTLY INCREASED IN NONALLERGIC INDIVIDUALS COMPARED TO ALLERGIC PATIENTS, SUGGESTING AN ALLERGEN-SPECIFIC HOST SPECIFIC RESPONSE EVEN IN NON-ALLERGIC INDIVIDUALS. MOREOVER, THE EXCLUSIVE PRESENCE OF THE ALLERGEN-REACTIVE TH2 CELLS IN THE ALLERGIC PATIENTS SUGGESTS A PROTECTIVE ROLE (ANTI-TH2 RESPONSE) OF THE THIFNR CELLS IN THE NON- ALLERGIC PATIENTS WITH EXPOSURE TO ALLERGEN. THIS POTENTIAL PROTECTIVE ROLE WAS REINFORCED BY OUR IN VITRO STUDIES SHOWING THAT TNF-RELATED APOPTOSIS-INDUCING LIGAND (TRAIL) PRODUCED BY THIFNR CELLS DIRECTLY INHIBITS T CELL ACTIVATION TRIGGERED BY TCR ENGAGEMENT. IN FOLLOW-UP STUDIES, WE FOUND THIFNR CELLS AMONG VIRAL-REACTIVE TH CELLS DIRECTED TOWARDS FLU OR SARS-COV2, SUGGESTING A BROADER ROLE OF THOSE CELLS IN IMMUNE RESPONSES. ALSO, WE FOUND THIFNR CELLS AS A STABLE TH SUBSET IN A LARGE COHORT OF HEALTHY INDIVIDUALS. BECAUSE OF THE RECENT DISCOVERY OF THIFNR CELLS, VERY LITTLE IS KNOWN ABOUT THEIR ORIGINS, DIFFERENTIATION, PHENOTYPE, AND FUNCTION. WE HYPOTHESIZE THAT THESE THIFNR HDM-REACTIVE T CELLS COULD PLAY A ROLE THROUGH TRAIL ENGAGEMENT IN DAMPENING TH2 INFLAMMATION IN ALLERGY AND ASTHMA. IN AIM 1, WE WILL DEFINE THE FUNCTIONAL PROPERTIES OF THIFNR CELLS. WE WILL PERFORM CO-CULTURE EXPERIMENTS TO TEST THE ABILITY OF THIFNR CELLS TO INHIBIT PROLIFERATION AND FUNCTION OF TH2 CELLS IN A TRAIL-DEPENDENT MANNER. IN AIM 2 WE WILL DETERMINE OPTIMAL CONDITIONS TO INDUCE DIFFERENTIATION OF THIFNR CELLS IN VITRO AND COMPARE THEIR FUNCTIONAL PROPERTIES WITH THOSE GENERATED IN VIVO. FINALLY, IN AIM 3, WE WILL DETERMINE IF HDM-REACTIVE THIFNR CELLS SHOW PERSISTENCE AND PLASTICITY IN VIVO. WE WILL PERFORM SINGLE-CELL TCR SEQUENCING OF HDM-REACTIVE THIFNR CELLS AND OTHER MEMORY CD4+ T CELL SUBSETS ISOLATED FROM LONGITUDINALLY COLLECTED BLOOD SAMPLES OF HDM ALLERGIC AND NON-ALLERGIC SUBJECTS. BY COMPARING TCR REPERTOIRE IN LONGITUDINAL SAMPLES, WE WILL ASSESS PERSISTENCE OF THIFNR CELLS IN VIVO. OVERALL, FUNCTIONAL STUDIES IN THIS R21 PROGRAM WILL PROVIDE IMPORTANT INSIGHTS INTO THIS NOVEL CD4+ T CELL SUBSET; AND FORM THE BASIS OF A FUTURE R01 APPLICATION TO CONDUCT STUDIES TRANSLATING FINDINGS TO THE CLINIC.

STRUCTURE OF THE SARS-COV-2 NUCLEOCAPSID: BUILDING BLOCK TO VIRAL CAPSID - SARS-COV-2, THE CAUSATIVE AGENT OF AN UNPRECEDENTED GLOBAL PANDEMIC, HAS JUST FOUR STRUCTURAL PROTEINS. OF THESE, THE NUCLEOCAPSID N IS THE MOST ABUNDANT PROTEIN IN THE VIRION, AND PLAYS ESSENTIAL ROLES IN GENOME ENCAPSIDATION AND VIRAL ASSEMBLY. HOWEVER, N HAS THUS FAR DEFIED STRUCTURE DETERMINATION OF ITS FULL-LENGTH MOLECULE. INDEED, THERE ARE CURRENTLY NO HIGH-RESOLUTION STRUCTURES OF FULL-LENGTH N FOR ANY CORONAVIRUS, ALTHOUGH MULTIPLE STRUCTURES EXIST FOR INDIVIDUAL DOMAINS WITHIN N. THE LACK OF STRUCTURAL INFORMATION ON N, ITS ASSEMBLY AND ITS INTERACTIONS AND ENCAPSIDATION OF THE GENOME STEM FROM THE INHERENT FLEXIBILITY CONTRIBUTED BY THREE INTRINSICALLY DISORDERED REGIONS. IN PREVIOUS WORK, N, IN THE ABSENCE OF RNA OR IN THE PRESENCE OF RANDOM BACTERIAL RNA DERIVED FROM THE EXPRESSION SYSTEM, WAS TOO FLEXIBLE TO ALLOW DETERMINATION OF A HIGH-RESOLUTION STRUCTURE. THE ASSEMBLED CAPSID IN THE VIRION IS ALSO TOO HETEROGENEOUS IN ITS FLEXIBILITY, POSITIONS AND CONFORMATIONS TO AFFORD HIGH-RESOLUTION INFORMATION. THROUGH CAREFUL ANALYSIS USING ELECTROMOBILITY SHIFT ASSAYS, SIZE-EXCLUSION AND SCREENING BY ELECTRON MICROSCOPY, WE HAVE NOW IDENTIFIED PORTIONS OF THE SARS-COV-2 GENOME THAT YIELD STRUCTURALLY HOMOGENEOUS, PURIFIED N DIMERS, OCTAMERS, AND 16-MERS THAT ARE AMENABLE TO HIGH-RESOLUTION STRUCTURAL ANALYSIS, AND WHICH REPRESENT THE BASIC BUILDING BLOCK AND LIKELY ASSEMBLY INTERMEDIATES OF THE FULL CAPSID. WE HAVE FURTHER PRODUCED A POLYMERIZED FULL-LENGTH CAPSID IN VITRO THAT IS ALSO AMENABLE TO STRUCTURAL STUDY. HERE WE PROPOSE CRYOEM OF THE DIMER, ASSEMBLY INTERMEDIATES AND FULL LENGTH IN VITRO CAPSID, COMPLEMENTED BY INNOVATIVE NATIVE MASS SPECTROMETRY AND STRAIGHTFORWARD SPECIFIC ANTIBODY-MEDIATED DOMAIN IDENTIFICATION. THIS WORK WILL ILLUMINATE (I) THE STRUCTURE AND ASSEMBLY OF THE CORONAVIRUS CAPSID; (II) HOW THE RNA GENOME INTERACTS WITH MULTIPLE DOMAINS OF THE FULL- LENGTH N AND CONNECTS ALONG POLYMERIZED COPIES OF N; (III) CONFORMATIONAL ADJUSTMENTS THAT OCCUR IN ASSEMBLY, PROTEIN-PROTEIN AND PROTEIN-RNA INTERACTION SITES; AND (IV) SITES THAT MAY BE AMENABLE TARGETS FOR ANTIVIRAL DEVELOPMENT.

AUTOMATED MULTISCALE SUPER-RESOLUTION CONFOCAL MICROSCOPE - ABSTRACT THE LA JOLLA INSTITUTE FOR IMMUNOLOGY (LJI) IS A PREMIER IMMUNOLOGY RESEARCH INSTITUTE IN THE US WHOSE 20 PRINCIPAL INVESTIGATORS ARE SUPPORTED BY ~$39 MILLION IN NIH GRANTS AND CONTRACTS. AT LJI, WE STUDY THE BEHAVIOR OF IMMUNE CELLS IN DISEASES PROMINENT IN THE US AND AROUND THE WORLD, INCLUDING VIRAL BIOLOGY, TYPE 1 DIABETES, ATHEROSCLEROSIS, CANCER, AND INFLAMMATION. NEARLY ALL OF OUR SCIENTISTS USE IMAGING TO VISUALIZE CELLULAR INTERACTIONS, SIGNALING, AND FUNCTION WITH RESULTS PUBLISHED IN NATURE, SCIENCE, AND OTHER PROMINENT JOURNALS. HOWEVER, AS OUR RESEARCH AIMS EVOLVE, AND TO PROGRESS FURTHER IN THE RESEARCH PROJECTS LISTED IN THIS APPLICATION, WE NEED A RELIABLE AND VERSATILE SUPER-RESOLUTION CONFOCAL MICROSCOPE WITH MULTISCALE IMAGING CAPABILITY. THE LJI MICROSCOPY AND HISTOLOGY CORE FACILITY IS A DYNAMIC, SCIENTIFICALLY DRIVEN, MULTI-USER RESOURCE WITH A REMARKABLE POTENTIAL FOR GROWTH. THE INSTRUMENTATION IN THE LJI MICROSCOPY CORE FACILITY INCLUDES AN AGING ZEISS LSM 780 THAT IS USED BY MANY NIH FUNDED PROJECTS, WITH AIMS RANGING FROM THE INVESTIGATION OF MATRIX PROTEIN ASSEMBLY IN SARS-COV-2, NOVEL CELL TYPES IN ATHEROSCLEROSIS, MOLECULAR PATHWAYS LEADING TO TYPE 1 DIABETES, AND MANY OTHERS. THIS SYSTEM IS NEARING THE END OF ITS USEFUL LIFETIME AND A REPLACEMENT MICROSCOPE IS NEEDED. HERE, WE SEEK SUPPORT FOR PURCHASING AN AUTOMATED, STATE-OF-THE-ART, SUPER-RESOLUTION MICROSCOPE THAT ALLOWS FOR MULTISCALE, HIGH-THROUGHPUT INVESTIGATION OF MOUSE AND HUMAN TISSUES. DYNAMIC BIOLOGICAL PROCESSES CAN BE PROBED THANKS TO INNOVATIVE SOFTWARE, HIGHLY SENSITIVE DETECTORS, AND UNPARALLELED ACQUISITION SPEED. AFTER COMPARING INSTRUMENTS ON THE MARKET FROM ALL MAJOR COMPANIES, IT IS CLEAR THAT THE ZEISS LSM 980 WITH AIRYSCAN 2 SYSTEM IS THE BEST SUITED FOR THE NEEDS OF LJI INVESTIGATORS AND COLLABORATORS, AND IF FUNDED, IT WILL UNDOUBTEDLY HAVE A SIGNIFICANT IMPACT ON NIH-SUPPORTED SCIENTIFIC PROGRESS.

FACSARIA II CELL SORTER

LINKING SPECIFIC PHOSPHORYLATION EVENTS TO DISTINCT TRANSCRIPTIONAL STATES - ABSTRACT: LINKING SPECIFIC PHOSPHORYLATION EVENTS TO DISTINCT TRANSCRIPTIONAL STATES THE CELLULAR BEHAVIORS THAT UNDERLIE HEALTH AND DISEASE ARE DIRECTED BY SIGNAL TRANSDUCTION PATHWAYS THAT DRIVE GENE EXPRESSION. LINKING SIGNALING TO SPECIFIC GENE EXPRESSION IS A FUNDAMENTAL GOAL OF BIOLOGY. OUR ABILITY TO IDENTIFY CAUSAL LINKS BETWEEN SPECIFIC PHOSPHORYLATION EVENTS AND THE EXPRESSION OF CERTAIN GENES WOULD NOT ONLY BETTER OUR COMPREHENSION OF CELLULAR DECISION-MAKING, BUT WOULD REVEAL A BREADTH OF NOVEL POINTS OF THERAPEUTIC INTERVENTION FOR US TO MODULATE CELL FUNCTIONS AND BEHAVIORS. WE PROPOSE AN INNOVATIVE DATA-DRIVEN TECHNOLOGY SOLUTION THROUGH INTEGRATING GLOBAL, TEMPORALLY-RESOLVED TRANSCRIPTOMIC AND PHOSPHOPROTEOMIC DATA WITH STATISTICAL MODELING TO SYSTEMATICALLY IDENTIFY OF SIGNALING EVENTS PREDICTIVE OF CONTEXT-SPECIFIC GENE SETS. WE WILL VALIDATE THE PREDICTED LINKS BETWEEN PHOSPHOSITES AND SPECIFIC GENE EXPRESSION PROGRAMS EMPLOYING NOVEL CRIPSR-MEDIATED BASE EDITING TECHNOLOGY TO MUTANT INDIVIDUAL PHOSPHORYLATION SITES AT ENDOGENOUS LOCI TO ASSESS THEIR IMPACT ON GENE EXPRESSION AT AN UNPRECEDENTED SCALE, AS WELL AS CORROBORATE USING CONVENTIONAL METHODS. AS A MODEL SYSTEM, WE WILL FOCUS ON CYTOKINE INDUCED SIGNALING AND GENE EXPRESSION IN MACROPHAGES, WHICH ARE CRITICAL MODULATORS OF INFLAMMATION. SPECIFICALLY, WE WILL INTERROGATE THE CYTOKINES IL-6 AND IL-10, WHICH SIGNAL THROUGH COMMON JANUS KINASES (JAKS) AND SIGNAL TRANSDUCERS AND ACTIVATORS OF TRANSCRIPTION (STAT) TRANSCRIPTION FACTORS TO PROMOTE CONTRASTING PRO- AND ANTI-INFLAMMATORY FUNCTIONS, RESPECTIVELY. WE WILL EXPLORE THE HYPOTHESIS THAT SIGNALING EVENTS OCCURRING OUTSIDE OF THE JAK/STAT AXIS ENCODE CYTOKINE-INDUCED TRANSCRIPTIONAL DIVERSITY AND CAN BE TARGETED TO MODULATE CONTEXT-SPECIFIC CYTOKINE INDUCED GENES. SUCCESSFUL COMPLETION OF THIS PROPOSAL WILL PROVIDE A ROBUST EXPERIMENTAL AND COMPUTATIONAL FRAMEWORK TO IDENTIFY AND PERTURB KEY PHOSPHORYLATION EVENTS, AND MEASURE THE SPECIFICITY OF SIGNALING-TO-TRANSCRIPTION LINKS THAT SHAPE THE DIVERSITY OF CELLULAR RESPONSES. THE WORKFLOW DEVELOPED HERE WILL BE BROADLY APPLIED ACROSS A VARIETY OF STIMULUS, CELLULAR, AND DISEASE CONTEXTS, IMPROVING EFFICIENCY OF THE THERAPEUTIC DEVELOPMENT PIPELINE.

FILOVIRUS NUCLEOCAPSID ASSEMBLY, CAUGHT IN THE ACT, BY IN SITU STRUCTURAL BIOLOGY - FILOVIRUSES EMERGE NEARLY ANNUALLY AND CAUSE HEMORRHAGIC FEVER WITH HIGH MORTALITY, AND FREQUENT UNPREDICTABLE OUTBREAKS. DEVELOPMENT OF BROAD-SPECTRUM ANTIVIRAL AGENTS IS NEEDED AND WILL BE GUIDED BY A CLEAR UNDERSTANDING OF THE STRUCTURES AND ASSEMBLIES OF ESSENTIAL VIRAL MACHINERY. THE FILOVIRUS NUCLEOCAPSID FORMS THE CORE OF THE VIRION AND IS ESSENTIAL FOR REPLICATION AND TRANSCRIPTION OF THE VIRAL GENOME AND HOST IMMUNE SUPPRESSION. THE VIRAL REPLICATION AND NUCLEOCAPSID ASSEMBLY OCCUR IN VIRAL REPLICATION FACTORIES IN HOST CYTOPLASM. ASSEMBLY OF THESE NUCLEOCAPSIDS IN REPLICATION FACTORIES AND THEIR SUBSEQUENT TRANSPORT TO THE CELL SURFACE, AND PACKAGING INTO VIRIONS ARE ALL CRITICAL STEPS IN THE FILOVIRUS LIFE CYCLE. MUCH ABOUT THESE ESSENTIAL PROCESSES, HOWEVER, REMAINS UNKNOWN, BECAUSE THE ASSEMBLY PROCESSES HAPPEN INSIDE CELLS, AND TRADITIONAL STRUCTURAL BIOLOGY APPROACHES INVOLVED USE OF PURIFIED, OFTEN RECOMBINANT, SAMPLES OUTSIDE CELLS. THUS, WE DO NOT YET HAVE STRUCTURAL UNDERSTANDINGS OF WHAT FILOVIRUS ASSEMBLY, TRANSPORT, MATRIX-INTERACTION AND VIRION-RECRUITMENT PROCESSES LOOK LIKE. FOR EXAMPLE, STRUCTURES ARE AVAILABLE FOR THE RECOMBINANT N-TERMINAL HALF OF THE EBOV NUCLEOPROTEIN (NP) COILED WITH CELLULAR RNA, BUT WE DON’T YET UNDERSTAND THE STRUCTURE OF THE C-TERMINAL HALF OF NP THAT RECRUITS THE OTHER NC PROTEINS, NOR DO WE UNDERSTAND HOW OTHER VIRAL PROTEINS ASSEMBLE TO FORM COMPLETE NUCLEOCAPSID STRUCTURE OR HOW THE NUCLEOCAPSID INTERACTS WITH THE VIRUS MATRIX. A NEW TECHNOLOGY, IN SITU CRYO-ELECTRON TOMOGRAPHY, NOW ALLOWS US TO CARRY OUT STRUCTURAL BIOLOGY ANALYSES INSIDE CELLS AND ALLOWS US TO COLLECT PREVIOUSLY INACCESSIBLE INFORMATION ABOUT THE STRUCTURE OF THE ASSEMBLING NUCLEOCAPSID INSIDE INFECTED CELLS. THE GOAL OF THIS PROJECT IS TO APPLY CRYO-FOCUSED ION BEAM (FIB) MILLING-ENABLED IN SITU CRYO-ELECTRON TOMOGRAPHY (CRYO-FIB-ET) TO UNDERSTAND HOW THE EBOLA VIRUS NUCLEOCAPSID IS ASSEMBLED INSIDE CELLS AND HOW THE STRUCTURES CHANGE BEFORE AND AFTER VIRION INCORPORATION. STUDY OF THESE COMPLEXES INSIDE CELLS, FROM BOTH TRANSFECTED AND INFECTED CELLS, WILL PROVIDE US OUR FIRST INFORMATION ABOUT THE ASSEMBLY PROCESS OF VIRUS REPLICATION FACTORIES INSIDE CELLS AND COMPLETE STRUCTURAL MODEL OF FILOVIRUS NUCLEOCAPSID STRUCTURE. THE FUNDAMENTAL STRUCTURAL ORGANIZATION AND CRITICAL PROTEIN INTERFACES REVEALED IN THIS STUDY ARE LIKELY CONSERVED AMONG ALL FILOVIRUSES. HENCE, THE PROPOSED WORK WILL PROVIDE INSIGHT INTO THE FUNDAMENTAL STRUCTURAL PRINCIPLES OF FILOVIRUS ASSEMBLY. THE KNOWLEDGE AND EXPERTISE GAINED HERE MAY ALSO BE RELEVANT FOR UNDERSTANDING THE STRUCTURES OF OTHER NEGATIVE-STRAND RNA VIRUSES THAT HAVE A SIMILAR PROTEIN STRUCTURAL ORGANIZATION.

EXPERIMENTAL IDENTIFICATION OF FUNCTIONAL GWAS VARIANTS LINKED TO COVID-19 SEVERITY IN IMMUNE CELLS - PROJECT SUMMARY/ABSTRACT THE CLINICAL PRESENTATION OF SARS-COV-2 INFECTION IN HUMANS CAN RANGE FROM VERY MILD OR NO SYMPTOMS TO SEVERE RESPIRATORY FAILURE. ALTHOUGH HYPERACTIVATION OF VARIOUS CELLULAR COMPONENTS OF THE IMMUNE SYSTEM HAVE BEEN OBSERVED IN PATIENTS WITH SEVERE COVID-19 ILLNESS, THE HOST GENETIC FACTORS THAT DETERMINE SUSCEPTIBILITY TO SEVERE COVID-19 ILLNESS ARE NOT WELL UNDERSTOOD. GWAS STUDIES HAVE REPORTED SEVERAL GENETIC LOCI THAT ARE SIGNIFICANTLY ASSOCIATED WITH SEVERE COVID-19 AND DEFINED SEVERAL TARGET GENES BASED ON THEIR PROXIMITY TO THE RISK LOCI, ALTHOUGH THIS APPROACH DOES NOT ACCURATELY PRIORITIZE CAUSAL GENES. WE CONDUCTED ONE OF THE FIRST TRANSCRIPTOME-WIDE ASSOCIATION STUDY (TWAS) IN PRIMARY IMMUNE CELLS TO DEFINE GENES THAT ARE ASSOCIATED WITH COVID-19 SEVERITY. IN THIS R21 PROPOSAL, WE WILL FOCUS ON DEFINING FUNCTIONAL COVID-19 RISK VARIANTS LINKED TO TOP TWO CANDIDATE GENES (OAS1 AND IL10RB) FROM OUR GENETIC ANALYSES. IN AIM 1, WE WILL DETERMINE THE FUNCTIONAL COVID-19 RISK EQTLS ASSOCIATED WITH OAS1 EXPRESSION IN NON- CLASSICAL MONOCYTES (NCM). OUR TWAS STUDY SHOWED SIGNIFICANT ASSOCIATION OF COVID-19 SEVERITY WITH REDUCED EXPRESSION OF INTERFERON-INDUCIBLE GENE (OAS1) SPECIFICALLY IN NCM. OAS1 ENCODES FOR OLIGOADENYLATE SYNTHASE FAMILY OF PROTEINS THAT DEGRADE VIRAL RNA AND ACTIVATE ANTI-VIRAL RESPONSES, THUS VARIANTS ALTERING ITS EXPRESSION LEVELS, ESPECIALLY IN NCM, A CELL TYPE WHICH HAS BEEN IMPLICATED IN COVID-19 PATHOGENESIS, ARE LIKELY TO HAVE AN IMPORTANT ROLE IN HOST IMMUNE RESPONSES. HERE, WE WILL UNDERTAKE EXPERIMENTAL STUDIES TO DEFINE THE FUNCTIONAL VARIANT(S) IN THE DENSE OAS1 NEANDERTHAL HAPLOBLOCK HARBORING >100 SNPS. BRIEFLY, WE WILL PERFORM (I) CRISPRI ASSAYS TO DETERMINE FUNCTIONAL ENHANCERS THAT OVERLAP COVID-19 RISK SNPS, (II) LUCIFERASE REPORTER ASSAYS TO DETERMINE THE FUNCTION VARIANTS IN SUCH ENHANCERS, (III) CHIP TO DETERMINE ALLELE-SPECIFIC BINDING OF THE TRANSCRIPTION FACTOR RXRA THE BINDING OF WHICH IS PREDICTED TO BE DISRUPTED BY COVID-19-RISK VARIANTS, AND (IV) HDR-MEDIATED EDITING OF PRIORITIZED COVID-19-RISK EQTLS TO DEFINITIVELY ESTABLISH FUNCTIONALITY. IN AIM 2, WE WILL DETERMINE THE FUNCTION COVID-19 RISK EQTLS ASSOCIATED WITH IL10RB EXPRESSION IN NK CELLS AND T CELLS. OUR TWAS SHOWED THAT INCREASED EXPRESSION OF IL10RB IN NK CELLS WAS SIGNIFICANTLY ASSOCIATED WITH COVID-19 SEVERITY. IL10RB ENCODES FOR IL-10 RECEPTOR BETA, AND GIVEN THE IMMUNOMODULATORY ROLE OF IL- 10, IT IS LIKELY THAT THE HIGHER EXPRESSION ON THE IL10RB IN NK CELLS AND T CELLS MAY ENHANCE THEIR RESPONSIVENESS TO IL-10. HERE, WE WILL PERFORM EXPERIMENTAL STUDIES AS DESCRIBED IN AIM 1 TO DEFINE THE FUNCTIONAL COVID-19- RISK VARIANTS THAT ARE PREDICTED TO PERTURB THE BINDING OF THE TRANSCRIPTION FACTORS TCF12 AND GATA-3 AND THEIR ROLE IN THE MODULATION OF IL10RB EXPRESSION IN NK CELLS IN T CELLS. OVERALL, FUNCTIONAL STUDIES IN THIS R21 PROGRAM WILL PROVIDE IMPORTANT MECHANISTIC INSIGHTS INTO THE GENETIC BASIS OF COVID-19 SEVERITY.

CROSSTALK BETWEEN FCERI AND MAVS SIGNALING PATHWAYS IN MAST CELLS

REGULATION OF T CELL ANERGY BY PALMITOYL ACYL TRANSFERASES

MHC RESTRICTION AND ANTIGEN CHARACTERIZATION OF MUCOSAL CD8AA TCRAB IEL

ILLUMINATING LUJO VIRUS GLYCOPROTEIN STRUCTURE, RECEPTOR ENGAGEMENT AND NEUTRALIZING ANTIBODY EPITOPES - PROJECT SUMMARY THE MAMMARENAVIRUS GENUS OF THE ARENAVIRUS FAMILY CONTAINS MULTIPLE ZOONOTIC PATHOGENS WITH THE POTENTIAL TO CAUSE HEMORRHAGIC FEVER. THESE INCLUDE THE SOUTH AMERICAN VIRUSES JUNIN VIRUS (JUNV; ARGENTINIAN HEMORRHAGIC FEVER) AND MACHUPO VIRUS (MACV; BOLIVIAN HEMORRHAGIC FEVER), AND LASSA VIRUS (LASV), WHICH CAUSES THOUSANDS OF CASES OF LASSA FEVER IN WEST AFRICA EACH YEAR. THE CASE FATALITY RATE FOR THESE VIRUSES IS 20-70%. LUJO VIRUS (LUJV) IS THE MOST RECENTLY IDENTIFIED AFRICAN ARENAVIRUS. THIS VIRUS WAS RESPONSIBLE FOR FIVE INFECTIONS, OF WHICH FOUR WERE FATAL. NOTABLY, THIS OUTBREAK WAS CHARACTERIZED BY HUMAN-TO-HUMAN TRANSMISSION RATHER THAN TRANSMISSION BETWEEN RODENT AND HUMAN, AS IS MOST COMMON FOR OTHER ARENAVIRUSES. ARENAVIRUSES ARE GENETICALLY AND GEOGRAPHICALLY DIVIDED INTO NEW WORLD (E.G. JUNV AND MACV) AND OLD WORLD (E.G. LASV) GROUPS. ITS AFRICAN LOCATION PLACED LUJV INTO THE OW GROUP. HOWEVER, LUJV IS GENETICALLY DIVERGENT FROM OTHER AFRICAN ARENAVIRUSES AND IS PHYLOGENETICALLY EQUIDISTANT BETWEEN THE NW AND OW GROUPS. FURTHER, ITS GLYCOPROTEIN GPC RECOGNIZES A DIFFERENT RECEPTOR AND IS ANTIGENICALLY DISTINCT FROM THE OTHER ARENAVIRUSES. AS THE ONLY PROTEIN EXPRESSED ON THE VIRAL SURFACE, GPC IS RESPONSIBLE FOR RECEPTOR ENGAGEMENT, CELL TROPISM AND ENTRY, AND IS THE PRIMARY TARGET OF ANTIBODIES. UNDERSTANDING THE UNIQUE STRUCTURE AND SURFACE CHEMISTRY OF LUJV GPC IN ITS NATIVE CONFORMATION IS KEY TO UNDERSTANDING RECEPTOR RECOGNITION IN THE NATIVE TRIMER CONTEXT, WHAT ANTIBODY TARGETS MIGHT BE ON THIS DIVERGENT VIRUS, AND HOW WE MIGHT DESIGN VACCINES AND THERAPEUTICS SHOULD THE VIRUS RE-EMERGE. THE PREMISE OF THIS PROPOSAL IS THAT STRUCTURES OF THE MEDICALLY RELEVANT LUJV GP ALONE AND IN COMPLEX WITH ITS CELL SURFACE RECEPTOR WILL REVEAL REASONS FOR ITS UNIQUE CELL ENTRY REQUIREMENTS AND ANY DIFFERENCES IN ITS EPITOPE LANDSCAPE. WE WILL USE STATE-OF-THE-ART BIOPHYSICAL TECHNIQUES SUCH AS CRYO ELECTRON MICROSCOPY, SURFACE PLASMON RESONANCE AND COMPOSITION-GRADIENT MULTIANGLE LIGHT SCATTERING, TO CHARACTERIZE THE INTERACTION OF THE PREFUSION-STABILIZED LUJV GP TRIMER (PFGP-TD) WITH ITS RECEPTOR NRP2. IN AIM 2, WE WILL IDENTIFY ANTIBODIES FROM MICE IMMUNIZED WITH LUJV PFGP-TD USING THE BERKELEY LIGHTS BEACON PLATFORM. RESULTS FROM THE INNOVATIVE RESEARCH PROPOSED HERE WILL LAUNCH MULTIPLE LINES OF INQUIRY FOR FUTURE STUDIES AND WILL HELP GUIDE DEVELOPMENT OF VACCINES AGAINST A DIVERSE RANGE OF ARENAVIRUSES.

UNDERSTANDING PH-DEPENDENT RECEPTOR UTILIZATION OF LASSA VIRUS - THE OLD-WORLD ARENAVIRUS LASSA (LASV) CAN CAUSE SEVERE HEMORRHAGIC LASSA FEVER (LF), WHICH IS ASSOCIATED WITH SIGNIFICANT MORBIDITY AND MORTALITY IN WEST AFRICA EVERY YEAR. AS THE MOST EXPORTED HEMORRHAGIC FEVER, LASV ALSO POSES A SIGNIFICANT GLOBAL HEALTH RISK. LF IS RECOGNIZED AS A “PRIORITY DISEASE” BY THE WORLD HEALTH ORGANIZATION (WHO) AND THE COALITION FOR EPIDEMIC PREPAREDNESS INNOVATIONS (CEPI). THE TRIMERIC GLYCOPROTEIN COMPLEX (GPC) IS THE ONLY VIRAL PROTEIN EXPRESSED ON THE VIRION SURFACE AND RESPONSIBLE FOR LASV CELL ENTRY. EACH MONOMER WITHIN THE TRIMER IS COMPOSED OF THE NON-COVALENTLY ASSOCIATED STABLE SIGNAL PEPTIDE, THE RECEPTOR-BINDING SUBUNIT, GP1, AND THE FUSION MACHINERY, GP2. LASV SEQUENTIALLY UTILIZES TWO HOST RECEPTORS FOR CELL ENTRY, THE MATRIGLYCOSYLATED ALPHA-DYSTROGLYCAN (MATRI-Α-DG) AT THE CELL SURFACE AND THE LYSOSOMAL-ASSOCIATED MEMBRANE PROTEIN 1 (LAMP1) IN THE ENDOSOME DURING ENDOCYTOSIS OF THE VIRION. THE RECEPTOR SWITCH REQUIRES AN ACIDIC PH-INDUCED CONFORMATIONAL REARRANGEMENT OF THE GPC TRIMER, CALLED GPC PRIMING. STRUCTURAL BIOLOGY HAS ONLY CAPTURED ATOMIC SNAPSHOTS: PREFUSION GPC IN COMPLEX WITH MATRIGLYCAN, AND THEN INDIVIDUAL SUBUNITS: POST-FUSION GP2, AND A LOW-PH INCUBATED GP1 TRUNCATION PRESUMED TO BE IN THE PRIMED STATE. THE INHERENT METASTABILITY OF GPC HAS THWARTED EFFORTS TO OBTAIN HIGH-RESOLUTION STRUCTURES OF TRIMERIC GPC IN PRIMING STATES AND THE GPC:LAMP1 COMPLEX. IN THIS PROJECT, WE WILL FILL IN THESE IMPORTANT MISSING STRUCTURES AND ILLUMINATE PH-DRIVEN CHANGES IN THE GPC TRIMER AND MECHANISMS FOR RECEPTOR SWITCHING. THE PROPOSED STRUCTURAL WORK FOR THIS PROGRAM IS MADE FEASIBLE BY A NOVEL LASV GPC CONSTRUCT ENGINEERED BY THE PI THAT MAINTAINS NATIVE CONFORMATION AT NEUTRAL PH AND IS CAPABLE OF PRIMING AND LAMP1 BINDING AT ACIDIC PH, WITHOUT DISSOCIATION OF THE GP1 AND GP2 SUBUNITS. IMPORTANTLY, INSIGHTS FROM THESE STUDIES CAN BE APPLIED TO OTHER MEDICALLY RELEVANT, PH-DEPENDENT RECEPTOR-SWITCHING ARENAVIRUSES, SUCH AS THE UBIQUITOUS LYMPHOCYTIC CHORIOMENINGITIS VIRUS AND THE RARE, BUT POTENTIALLY MORE FATAL, LUJO VIRUS.

WHAT IS THE HUMAN ANTIBODY RESPONSE TO MEASLES VIRUS VACCINATION? - PROJECT SUMMARY/ABSTRACT VACCINES ARE THE GREATEST BANG FOR OUR PUBLIC HEALTH BUCK, YET VACCINE DEVELOPMENT IS STILL SOMETIMES HIT OR MISS. RECENT EFFORTS WITH SARS-COV-2 DEMONSTRATED THAT ACCELERATION OF VACCINE PROGRAMS IS POSSIBLE, ESPECIALLY WHEN SUPPORTED BY PRIOR STUDIES CHARACTERIZING SUCCESSFUL IMMUNE RESPONSES AGAINST RELATED PROTOTYPE PATHOGENS. THE MEASLES VIRUS VACCINATION CAMPAIGN IS A GLOBAL SUCCESS STORY; THE LIVE-ATTENUATED VACCINE DEVELOPED IN THE 1960’S STILL ELICITS HIGHLY PROTECTIVE ANTIBODIES TO STRAINS CIRCULATING TODAY. YET MEASLES OUTBREAKS PERSIST DUE TO OBSTACLES IN VACCINATION PROGRAMS SUCH AS GLOBAL HEALTH INEQUITIES, VACCINE HESITANCY, EXTREME WEATHER EVENTS RELATED TO CLIMATE CHANGE, AND THE SARS-COV-2 PANDEMIC. FURTHERMORE, A GROWING POPULATION WITH COMPROMISED IMMUNE SYSTEMS CANNOT RECEIVE LIVE-ATTENUATED VIRAL VACCINES. THUS, THE THREAT OF MEASLES REMAINS (THERE ARE STILL APPROXIMATELY 9-10 MILLION CASES ANNUALLY), AND INNOVATIVE SOLUTIONS WILL BE NEEDED TO ACHIEVE GLOBAL MEASLES ERADICATION. HOWEVER, VERY LITTLE IS KNOWN ABOUT THE HUMAN ANTIBODY RESPONSE TO MEASLES INFECTION OR VACCINATION BEYOND NEUTRALIZING ANTIBODY TITERS. IMPORTANTLY, THERE ARE NO STRUCTURES OF ANY ANTIBODIES IN COMPLEX WITH ANY MEASLES VIRUS ANTIGENS. AS A RESULT, WE DO NOT KNOW WHICH SITES ON MEASLES VIRUS ARE IMMUNODOMINANT AND PROTECTIVE. MUCH OF THIS FUNDAMENTAL KNOWLEDGE IS LACKING LARGELY BECAUSE MEASLES WAS EFFECTIVELY ERADICATED FROM DEVELOPED NATIONS BEFORE NEEDED SCIENTIFIC TOOLS HAD BEEN DEVELOPED. IN THIS WORK, WE WILL USE STATE-OF-THE-ART TOOLS TO DIRECTLY VISUALIZE THE STRUCTURE OF HUMAN ANTIBODIES FROM VACCINEE POLYCLONAL SERA BOUND TO MEASLES SURFACE GLYCOPROTEINS TO UNDERSTAND WHICH ANTIGENIC SITES DOMINATE IN THE HUMAN VACCINEE RESPONSE. WE WILL ALSO USE MICROFLUIDICS AND RAPID MICROSCALE MULTIPLEXED COMPETITION ANALYSES TO RAPIDLY DISCOVER AND ANALYZE INDIVIDUAL HUMAN MONOCLONAL ANTIBODIES AGAINST MEASLES VIRUS FOR THE FIRST TIME. THIS WORK WILL REVEAL WHAT COMPONENTS OF THE HUMAN IMMUNE RESPONSE TO THIS PROTOTYPE PARAMYXOVIRUS LEAD TO VACCINE-MEDIATED PROTECTION, AND WILL HELP GUIDE DEVELOPMENT OF MODERN VACCINES FOR THE IMMUNOCOMPROMISED AND OTHER VACCINES AGAINST PARAMYXOVIRUSES YET TO EMERGE.

VAGINAL ADAPTIVE IMMUNITY AGAINST ZIKA VIRUS

DEVELOPMENT OF A PIPELINE TOOL TO PREDICT IMMUNOGENICITY OF CANCER NEO-EPITOPES

DESIGN AND EVALUATION OF HLA-A, -B, AND -C BINDING PEPTIDES THAT DISRUPT INHIBITORY KIR/MHC INTERACTION AND ACTIVATE NK CELLS

TARGETING ANGIOGENESIS PATHWAYS FOR THERAPEUTIC PROTECTION AGAINST DENGUE

NECROPTOSIS AND ADAPTIVE IMMUNITY TO VIRUS INFECTION

MECHANISM OF IMMUNE REGULATION BY CD11B+ MYELOID CELLS IN THE INTESTINE

NOVEL PTPRD INTERACTION CONTROLLING T CELL RESPONSIVENESS

DEUBIQUITINATION IN IMMUNE REGULATION

STRUCTURAL BASIS OF UL141 MEDIATED NK CELL INHIBITION BY HCMV

MODULATION OF TEFF/TREG CELLS BY NOVEL PKC-THETA ALLOSTERIC INHIBITORY STRATEGIES

ANAPHYLAXIS AND IGE HETEROGENEITY

DEFINING FUNCTION AND REGULATION OF THE NOVEL SLE-SUSCEPTIBILITY GENE ILRUN IN T CELLS - PROJECT SUMMARY/ABSTRACT SYSTEMIC LUPUS ERYTHEMATOUS (SLE) CAUSES SUBSTANTIAL MORTALITY AND MORBIDITY. CURRENT TREATMENTS ARE NOT HIGHLY EFFECTIVE IN MODIFYING DISEASE PROGRESSION, ESPECIALLY IN SEVERELY AFFECTED PATIENTS WITH RENAL INVOLVEMENT. HENCE, THERE IS A LARGE UNMET NEED TO DEVELOP NOVEL THERAPEUTIC TARGETS. GENOME-WIDE ASSOCIATION STUDIES (GWAS) OFFER AN UNBIASED APPROACH TO IDENTIFY GENES THAT PLAY AN IMPORTANT ROLE IN DRIVING DISEASE PATHOGENESIS. TO IDENTIFY RISK GENES IN IMMUNE CELL TYPES RELEVANT TO SLE PATHOGENESIS, WE PERFORMED THE FIRST LARGE-SCALE SINGLE-CELL EQTL STUDY ON ACTIVATED CD4+ T CELLS AND FOUND THAT SLE-RISK VARIANTS WERE STRONGLY ASSOCIATED WITH INCREASED EXPRESSION OF THE POORLY UNDERSTOOD GENE ILRUN. OUR STUDY PROVIDES THE FIRST DIRECT EVIDENCE THAT ACTIVATED CD4+ T CELLS ARE THE KEY CELL TYPES IN WHICH SLE-RISK VARIANTS INCREASE THE EXPRESSION OF ILRUN. IN AIM 1, WE WILL IDENTIFY THE FUNCTIONAL SLE-RISK VARIANTS ASSOCIATED WITH ILRUN EXPRESSION IN ACTIVATED CD4+ T CELLS. WE WILL EMPLOY CRISPRI ASSAYS TO DETERMINE FUNCTIONAL ENHANCERS THAT OVERLAP ILRUN EQTLS, PERFORM LUCIFERASE REPORTER ASSAYS TO DETERMINE FUNCTIONAL VARIANTS IN ILRUN PROMOTER AND ENHANCERS, AND PERFORM CHIP ASSAYS TO IDENTIFY THE FUNCTIONAL ILRUN EQTLS THAT DIRECTLY PERTURB THE BINDING OF KEY TRANSCRIPTION FACTORS AND MODULATE ILRUN EXPRESSION. IN AIM 2, WE WILL ASSESS THE FUNCTIONAL ROLE OF ILRUN IN CD4+ T CELLS IN THE CONTEXT OF SLE AND DETERMINE WHETHER ILRUN INFLUENCES THE ACTIVATION, APOPTOSIS, PROLIFERATION, DIFFERENTIATION AND CYTOKINE PRODUCTION OF CD4+ T CELLS. OVERALL, THE STUDIES EXAMINING THE EXPRESSION, REGULATION AND FUNCTION OF ILRUN IN CD4+ T CELLS WILL PROVIDE IMPORTANT MECHANISTIC INSIGHTS INTO THE GENETIC BASIS OF SLE.

CELL-BASED FUNCTIONAL GENETICS OF AUTOIMMUNITY THROUGH TARGETED GENE INTEGRATION

STRUCTURE AND FUNCTION OF PEPTIDE PRESENTATION BY CD1D

TNF FAMILY MEMBERS AND CONTROL OF FIBROTIC DISEASE

THYMIC DEVELOPMENT AND MHC RESTRICTION OF CD8AA TCRAB INTRAEPITHELIAL LYMPHOCYTES

PROTEIN DEUBIQUITINATION IN LYMPHOCYTE SIGNALING

REGULATION OF LUNG INFLAMMATION BY PROTEIN UBIQUITINATION

PATHOGENIC ROLE OF ANTIBODIES TO DENGUE VIRUS

THE ROLE OF TRAIL IN IMMUNE CONTROL OF CYTOMEGALOVIRUS

(PQ 5) ROLES OF MITOCHONDRIAL HETEROGENEITY IN CELL SURVIVAL AND ONCOGENESIS

CMV UL144 TARGETS THE HVEM-BTLA COSIGNALING SYSTEM

LOCAL CONTROL OF ANTI-TUMOR/ANTI-SELF REACTIVITY IN LOW-AFFINITY ACT

UBIQUITINATION IN IMMUNE TOLERANCE

STRUCTURAL IMMUNOLOGY OF CD1 AND CD1-TCR COMPLEXES

DYNAMIC ANALYSIS OF BETA CELL ATTACKS IN RESPONSE TO VIRUS INFECTION IN HUMAN PANCREATIC SLICES - PROJECT SUMMARY TYPE 1 DIABETES (T1D) IS A CHRONIC AUTOIMMUNE DISEASE OF UNKNOWN ETIOLOGY LEADING TO THE DESTRUCTION OF PANCREATIC BETA CELLS, WHICH SECRETE INSULIN AND REGULATE GLUCOSE HOMEOSTASIS. THE INCIDENCE OF T1D HAS BEEN INCREASING OVER THE YEARS, A TREND INEXPLICABLE BY ONLY GENETIC PREDISPOSITION, SUGGESTING THAT ENVIRONMENTAL FACTORS SUCH AS VIRUSES PLAY AN IMPORTANT ROLE IN TRIGGERING THE DISEASE. DECADES OF RESEARCH HAVE PROVIDED EPIDEMIOLOGICAL EVIDENCE FOR AN ASSOCIATION BETWEEN T1D AND VIRUSES, PARTICULARLY COXSACKIEVIRUSES OF THE ENTEROVIRUS GENUS. PATIENTS WITH T1D HAD HIGHER RATES OF ANTIBODIES ANTI-CVB, VIRAL RNA AND VIRAL CAPSID PROTEIN COMPARED TO NON-DIABETIC. STUDIES HAVE ALSO SHOWN THAT CHILDREN WHO HAD DEVELOPED ISLET AUTOANTIBODIES HAD MORE CVB INFECTIONS. VIRAL INFECTION CAN IMPACT BETA CELLS THROUGH A DIRECT CYTOLYTIC EFFECT OR BY BYSTANDER ACTIVATION OF THE IMMUNE SYSTEM LEADING TO GRADUAL DESTRUCTION. HOWEVER, THERE IS STILL LITTLE DIRECT CAUSAL EVIDENCE FROM HUMAN PANCREATA THAT WOULD ESTABLISH A LINK BETWEEN VIRAL INFECTIONS AND T1D DEVELOPMENT. ALTHOUGH MOUSE STUDIES HAVE BEEN VERY USEFUL IN UNDERSTANDING SOME OF THE MECHANISMS OF THE DISEASE, THERE REMAIN SUBSTANTIAL GAPS AND DIFFERENCES TO HUMAN PATHOLOGY. THEREFORE, STUDIES ON HUMAN PANCREATIC TISSUE SAMPLES BECOME ESSENTIAL. PANCREAS TISSUE SLICES ARE 150-200 ΜM THICK SECTIONS OF LIVING HUMAN DONOR PANCREATIC TISSUE. THEY ENABLE FUNCTIONAL ANALYSIS OF INTACT ISLETS IN THEIR ENDOGENOUS ENVIRONMENT AND 3D MORPHOLOGICAL ASSESSMENT OF THE ENDOCRINE AND EXOCRINE PANCREAS AS WELL AS THE BLOOD VASCULATURE, NEURONS AND TISSUE RESIDENT IMMUNE CELLS. IN PRELIMINARY STUDIES, WE HAVE BEEN ABLE TO CULTURE AND INFECT HUMAN PANCREATIC SLICES USING COXSACKIEVIRUS B3 (CVB3)-GFP AND IDENTIFY THE BETA CELLS AND TISSUE-RESIDENT MACROPHAGES AS TARGETS. THUS, WE HYPOTHESIZE THAT VIRUS INFECTIONS INDUCE CHANGES IN TISSUE-RESIDENT MACROPHAGES WHICH THEN INDUCE T CELLS TO ATTACK AND DESTROY BETA CELLS. AIM 1 WILL EVALUATE THE IMPACT OF CVB3-GFP INFECTION ON BETA CELLS FUNCTION AND VIABILITY. AIM 2 WILL CHARACTERIZE TISSUE-RESIDENT MACROPHAGES DURING CVB3 INFECTION BY SINGLE CELL RNA-SEQ. AIM 3 WILL EXAMINATE IF THE INFECTION COULD LEAD TO AN IMMUNE DESTRUCTION OF BETA CELLS WHEN COCULTURED WITH HLA MATCHED OR AUTOLOGOUS PBMCS. AIM 4 WILL CHARACTERIZE THE IMPACT OF CVB3 INFECTION ON AUTOPHAGY IN INFECTED HUMAN PANCREATIC SLICES. OVERALL, THIS EFFORT WILL GENERATE A BETTER UNDERSTANDING OF THE CONSEQUENCES OF VIRAL INFECTIONS IN THE PANCREAS AND ITS ROLE IN THE ONSET OF T1D.

USING COMMON FUND DATASETS FOR PRIORITIZATION OF DISEASE-ASSOCIATED GENETIC VARIANTS - ABSTRACT GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE HIGHLIGHTED THAT DISEASE-ASSOCIATED HUMAN GENETIC VARIANTS ARE PREVALENT IN NONCODING REGIONS AND FOR MOST OF THEM THE BIOLOGICAL FUNCTION OR GENE TARGET REMAIN UNCHARACTERIZED. TO BETTER ANNOTATE SUCH DISEASE VARIANTS, NIH-FUNDED CONSORTIA CREATED COMPREHENSIVE MAPS OF PUTATIVE REGULATORY ELEMENTS AND IDENTIFIED SNPS ASSOCIATED WITH GENE EXPRESSION (EQTLS) FOR DIFFERENT TISSUES AND PRIMARY CELL TYPES. IN PARALLEL, BREAKTHROUGHS IN CAPTURING THE 3D GENOME STRUCTURE HAVE DEMONSTRATED THE IMPORTANCE OF CELL-TYPE-SPECIFIC PHYSICAL PROXIMITY BETWEEN GENES AND THEIR REGULATORY ELEMENTS. THIS 3D VIEW PROVIDED A NEW WAY THROUGH WHICH DISEASE-ASSOCIATIONS OF CERTAIN VARIANTS CAN BE EXPLAINED. THERE IS AN INCREASING INTEREST IN UTILIZATION OF CHROMATIN LOOPS FOR GWAS VARIANT ANNOTATION, HOWEVER, TO THE BEST OF OUR KNOWLEDGE, THERE IS NO COMPREHENSIVE STUDY INCORPORATING EQTL DATA AND HIGH-RESOLUTION CHROMATIN LOOPING INFORMATION ACROSS MANY DIFFERENT MATCHED/RELATED CELL TYPES AND TISSUES TO INTERPRET GWAS VARIANTS IDENTIFIED FOR A LARGE SET OF DISEASES. TO GOAL OF THIS PROPOSAL IS TO UTILIZE NIH COMMON FUND DATASETS (GTEX AND 4D NUCLEOME) AS WELL AS OTHER PUBLISHED CHROMATIN LOOP AND EQTL DATA TO CARRY OUT DIFFERENT INTEGRATIVE APPROACHES FOR BETTER ANNOTATION OF DISEASE-ASSOCIATED GENETIC VARIANTS. THIS WILL LEAD TO THE DEVELOPMENT OF A FRAMEWORK AND BEST PRACTICES FOR INTEGRATIVE ANALYSIS OF LOOPS, EQTLS AND GWAS SIGNALS. THE DEVELOPED FRAMEWORK WILL BE TESTED ON A LARGE NUMBER OF DISEASES AND DISEASE-RELEVANT CELL TYPES TO CREATE A SUBSTANTIAL ONLINE RESOURCE FOR RESEARCHERS. FOR A SUBSET OF THE STUDIED DISEASES, FOR WHICH WE HAVE ONGOING RESEARCH INTERESTS, WE WILL FURTHER ANALYZE THE IDENTIFIED NOVEL GENES, GENETIC VARIANTS AND OVERLAPPING REGULATORY ELEMENTS TO DETERMINE POTENTIAL TARGETS THAT WARRANT FURTHER INVESTIGATION.

TNFR MEMBERS IN T CELL IMMUNITY TO VACCINIA

CXCR3 CHEMOKINES IN TYPE I DIABETES

GIF REGULATION OF B CELLS AND CD4 CELLS

MAPPING THE HUMORAL IMMUNE RESPONSE TO THE SURFACE GLYCOPROTEINS OF THE MUMPS VIRUS - ABSTRACT MUMPS VIRUS IS ONE COMPONENT OF THE MMR VACCINE, WHICH HAS BEEN SUCCESSFUL IN THE GLOBAL EFFORT AGAINST MEASLES, MUMPS AND RUBELLA. HOWEVER, OF THE THREE VIRUSES, PROTECTION AGAINST MUMPS IS THE LEAST EFFECTIVE. CYCLES OF MUMPS VIRUS OUTBREAKS CONTINUE TO OCCUR, ESPECIALLY AMONG YOUNG ADULTS AND EVEN IN THE VACCINATED. 10% OF PATIENTS IN A RECENT SAN DIEGO COUNTY OUTBREAK WERE HOSPITALIZED. BREAKTHROUGH INFECTIONS COULD BE LINKED TO SEQUENCE DIFFERENCES BETWEEN THE VACCINE STRAIN AND THE NEWLY EMERGED MUMPS GENOTYPE G. SURPRISINGLY, ALTHOUGH WE HAVE HAD THIS VACCINE SINCE 1971, MEDICAL SCIENCE STILL DOES NOT KNOW WHAT THE HUMAN ANTIBODY RESPONSE IS AGAINST MUMPS VIRUS, FROM EITHER VACCINATION OR NATURAL INFECTION. NO HUMAN ANTIBODIES AGAINST MUMPS HAVE BEEN YET DESCRIBED, AND NO STRUCTURES OF ANY ANTIBODIES AGAINST MUMPS HAVE YET BEEN DETERMINED. THUS, WE ARE LACKING UNDERSTANDING OF WHICH ANTIBODIES AND WHICH EPITOPE TARGET SITES ON THE SURFACE PROTEINS HN AND F ARE MOST EFFECTIVE, AND WE ARE LACKING THE ANTIBODIES THEMSELVES WHICH MIGHT BE INCORPORATED INTO POST-EXPOSURE TREATMENTS. IN THIS PROGRAM, WE WILL DEVELOP PANELS OF HUMAN MONOCLONAL ANTIBODIES AGAINST MUMPS HN AND F FROM BOTH VACCINATED AND CONVALESCENT INDIVIDUALS. RESULTS OBTAINED FROM THIS PROGRAM WILL ILLUMINATE WHAT HUMANS TARGET IN THEIR IMMUNE RESPONSES, AND WHICH TARGET SITES, OR EPITOPES, LEAD TO BROADEST VIRAL GENOTYPE RECOGNITION, ESPECIALLY BETWEEN VACCINE-DERIVED GENOTYPE A AND CURRENTLY CIRCULATING GENOTYPE G OF THE VIRUS. THE RESULTS OF THIS PROGRAM WILL ALSO PROVIDE THE ESSENTIAL MABS THEMSELVES FROM WHICH WE MAY DEVELOP POST-EXPOSURE TREATMENTS AND GUIDE DEVELOPMENT OF BOOSTERS.

MAPPING ANTI-FILOVIRUS HUMORAL IMMUNE RESPONSES IN BROADLY SEROREACTIVE INDIVIDUALS - PROJECT SUMMARY/ABSTRACT: FILOVIRUSES ENCOMPASS A BROAD GROUP OF HIGHLY LETHAL PATHOGENS, INCLUDING ORTHOEBOLAVIRUSES (EBOLA (EBOV), BUNDIBUGYO, SUDAN, AND TAÏ FOREST VIRUSES) AND ORTHOMARBURGVIRUSES (MARBURG (MARV) AND RAVN VIRUSES). THESE VIRUSES CAUSE SPORADIC DISEASE OUTBREAKS, BUT WHICH VIRUS WILL EMERGE AND WHERE CANNOT BE PREDICTED. AS SUCH, RESIDENTS OF AFFECTED AREAS, AS WELL AS MILITARY, AID WORKERS AND TRAVELERS IN CENTRAL AND WEST AFRICA CONTINUE TO BE AT RISK OF INFECTION. FURTHER, CURRENTLY APPROVED MONOCLONAL ANTIBODY (MAB) TREATMENTS AND VACCINES ARE AVAILABLE ONLY FOR EBOV. A FEW KNOWN MABS CAN CROSS-REACT AMONG ORTHOEBOLAVIRUSES, BUT NONE ARE YET KNOWN TO OFFER BROAD FILOVIRUS NEUTRALIZATION. IT IS POSSIBLE THAT OUR ABILITY TO UNCOVER HUMAN MABS OF BROAD NEUTRALIZATION WAS LIMITED BY THE NATURE OF THE HUMAN SAMPLES USED FOR DISCOVERY. PRIOR ANTIBODY DISCOVERY CAMPAIGNS TO DATE ISOLATED ANTIBODIES FROM INDIVIDUALS WHO HAD BEEN INFECTED ONCE WITH JUST ONE OF THESE VIRUSES (USUALLY EBOV, ONE WITH MARV, ETC.), AND MANY STUDIES WERE PERFORMED ON RETURNING AMERICANS WHO WOULD NOT HAVE HAD MORE THAN ONE FILOVIRUS EXPOSURE. THIS PROPOSAL, HOWEVER, UTILIZES A DIFFERENT COHORT. SEROLOGY STUDIES IN THE DEMOCRATIC REPUBLIC OF THE CONGO, WHERE MULTIPLE FILOVIRUSES ARE ENDEMIC, REVEALED 23 INDIVIDUALS WITH EXTRAORDINARILY BROAD AND STRONG ANTIBODY RESPONSES AGAINST A BROAD RANGE OF FILOVIRUS SURFACE GLYCOPROTEINS (GP). SOME OF THESE INDIVIDUALS HAVE STRONG REACTIVITY TO ALL OF THEM: ALL FOUR PATHOGENIC ORTHOEBOLAVIRUSES AND BOTH ORTHOMARBURGVIRUSES. THESE INDIVIDUALS HAVE LIVED ALL OR MOST OF THEIR LIVES IN REMOTE VILLAGES THAT HAVE HAD KNOWN OUTBREAKS AND HAVE OCCUPATIONS THAT LIKELY LED TO MULTIPLE FILOVIRUS EXPOSURES OVER TIME. IN THIS PROJECT, WE WILL ANALYZE, IN DETAIL, THE ANTIBODY REPERTOIRES OF THESE MULTIPLY EXPOSED INDIVIDUALS. WE WILL ANALYZE REACTIVITY AT THE POLYCLONAL AND MONOCLONAL LEVELS ALIKE. WE EXPECT THAT MABS IDENTIFIED IN THESE INDIVIDUALS WILL HAVE GREATER BREADTH AND BROADER FILOVIRUS ACTIVITY THAN HAVE BEEN DISCOVERED BEFORE. THE RESULTS FROM THIS STUDY COULD LEAD TO THE DISCOVERY OF A NOVEL, BROAD-SPECTRUM FILOVIRUS MAB THERAPEUTIC, GUIDE THE DEVELOPMENT OF MORE BROADLY APPLICABLE VACCINES, AND WILL DEEPEN OUR UNDERSTANDING OF HOW A LIFETIME OF EXPOSURE SHAPES THE HUMORAL IMMUNE RESPONSE TO THESE VIRUSES.

DEUBIQUITINATION IN IMMUNE REGULATION

TUMOR SUPPRESSIVE FUNCTIONS OF TET PROTEINS IN B CELL MALIGNANCIES: ROLE OF G-QUADRUPLEX STRUCTURES

MAPPING THE GERMINAL CENTER AND MEMORY B CELL LANDSCAPE TO COMPLEX ANTIGENS

TOLEROGENIC INTERACTIONS OF 4-1BB

T CELL RESPONSE TO DENGUE VIRUS

NKT CELL RECOGNITION OF STREPTOCOCCUS PNEUMONIAE

MITOCHONDRIAL INJURY IN METHAMPHETAMINE-INDUCED NEURODEGENERATION

NFAT, BZIP PROTEINS, AND TRANSCRIPTIONAL PROGRAMS IN LYMPHOCYTES

STRUCTURAL CHARACTERIZATION OF 4-1BB-GALECTIN-9 COMPLEXES

THE ROLE OF ALTERNARIA FUNGAL SENSITIZATION IN ASTHMA PATHOGENESIS - PROJECT SUMMARY ALTERNARIA ALTERNATA (ALT) IS THE MOST COMMON OUTDOOR AIRBORNE FUNGUS FOUND WORLDWIDE. THE PREVALENCE OF ALT SENSITIZATION RANGES FROM 2% TO 23.8% IN THE GENERAL POPULATION AND MUCH HIGHER AMONG ASTHMATICS. ALT SENSITIZATION HAS BEEN ASSOCIATED WITH AIRWAY HYPER-RESPONSIVENESS, ASTHMA AND ADVERSE ASTHMA OUTCOMES. IT IS AN ENIGMA WHY SOME PEOPLE DEVELOP SENSITIZATION TO A COMMON FUNGAL ALLERGEN (ALT) WHILE OTHERS DON’T. MOREOVER, THE REASON WHY ALT SENSITIZATION IS ASSOCIATED WITH INCREASED RISK OF DEVELOPING ASTHMA IS NOT KNOWN. IN THIS PROPOSAL, WE WILL EXPLORE THE MECHANISMS AND CELL TYPES THAT DRIVE ALT SENSITIZATION AND THE DEVELOPMENT OF ASTHMA. WE RECENTLY IDENTIFIED A NOVEL SUBSET OF HOUSE DUST MITE (HDM)-SPECIFIC T CELLS CHARACTERIZED BY AN INTERFERON RESPONSE (IFNR) GENE SIGNATURE (THIFNR CELLS), WHICH WAS NEGATIVELY ASSOCIATED WITH HDM SENSITIZATION. IN A PILOT STUDY, FOCUSSED IN ALT ALLERGEN, WE FOUND THAT THE PROPORTION OF ALT-SPECIFIC THIFNR CELLS WAS INCREASED IN HEALTHY SUBJECTS WITHOUT ALT SENSITIZATION WHEN COMPARED TO ASTHMATIC SUBJECTS SUGGESTING THAT IT MAY ALSO PLAY A PROTECTIVE ROLE IN ALT-SENSITIZATION AND ASTHMA. WE ALSO FOUND THAT ALT-SPECIFIC TH2 CELLS FROM ASTHMATICS DISPLAYED PATHOGENIC FEATURES THAT WERE DISTINCT FROM HDM- SPECIFIC TH2 CELLS. BASED ON THESE FINDINGS, WE HYPOTHESIZE THAT ALT-SPECIFIC THIFNR CELLS PROTECT AGAINST ALT-SENSITIZATION IN HEALTHY SUBJECTS, AND A NOVEL PATHOGENIC TH2 SUBSET IN THE AIRWAYS, SPECIFICALLY TISSUE- RESIDENT MEMORY T CELLS (TRM CELLS) PROMOTE THE DEVELOPMENT OF ASTHMA IN ALT SENSITIZED SUBJECTS. IN AIM 1, WE WILL DETERMINE THE ASSOCIATION BETWEEN ALT-SPECIFIC THIFNR CELLS AND PROTECTION AGAINST ALT SENSITIZATION. WE WILL ASSESS SUBJECTS ENROLLED IN THE ISLE OF WIGHT WHOLE POPULATION BIRTH COHORT. WE WILL IDENTIFY SUBGROUPS OF PARTICIPANTS (N=100) WITH: (I) PERSISTENT ALT-SENSITIZATION SINCE CHILDHOOD (N=25), (II) ADULT-ONSET ALT- SENSITIZATION (N=25), (III) HDM-SENSITIZED WITHOUT ALT-SENSITIZATION SINCE CHILDHOOD (N=25), AND (IV) NON- ATOPIC SINCE CHILDHOOD (N=25). WE WILL ISOLATE ALT-SPECIFIC T CELLS FROM BLOOD AND PERFORM SINGLE-CELL RNA-SEQ AND TCR-SEQ TO ENUMERATE THE FREQUENCY, PROPERTIES, PERSISTENCE AND CLONALITY OF ALT-SPECIFIC T CELL SUBSETS, INCLUDING THE NOVEL THIFNR CELL SUBPOPULATION, AND DETERMINE THEIR ASSOCIATION WITH RISK AND PROTECTION AGAINST DEVELOPMENT OF ALT-SENSITIZATION AT DIFFERENT AGES. IN AIM 2, WE WILL IDENTIFY ALT-SPECIFIC T CELL SUBSETS IN THE BLOOD AND AIRWAYS ASSOCIATED WITH THE DEVELOPMENT OF ASTHMA IN SUBJECTS WITH ALT-SENSITIZATION. WE WILL ENROLL 50 SUBJECTS WITH ALT-SENSITIZATION FROM THE IOWBC (N=25 WITH ASTHMA, N=25 WITHOUT ASTHMA), AND AS CONTROLS, 50 ASTHMATIC SUBJECTS WITHOUT ALT-SENSITIZATION (N=25 WITH HDM- SENSITIZATION, AND N=25 WITH NO SENSITIZATION TO COMMON ALLERGENS), OBTAIN BLOOD AND AIRWAY SAMPLES (SPUTUM). WE WILL PERFORM SINGLE-CELL TRANSCRIPTOME AND TCR-SEQ ANALYSIS TO DETERMINE THE FREQUENCY, PROPERTIES, PERSISTENCE AND CLONALITY OF ALT-SPECIFIC TH CELL AND TRM SUBSETS IN THE BLOOD AND AIRWAYS, AND THEIR ASSOCIATION WITH ASTHMA.

HUMAN UPPER AIRWAY IMMUNE MEMORY KINETICS AND DURABILITY AGAINST RESPIRATORY PATHOGENS - PROJECT SUMMARY PERIPHERAL BLOOD IS THE PRIMARY SAMPLE TYPE COLLECTED TO EVALUATE A VAST RANGE OF MEDICAL CONDITIONS. HOWEVER, PERIPHERAL BLOOD TESTING MAY NOT REFLECT PROCESSES OCCURRING IN TISSUE, LIKE THE UPPER AIRWAY. THE SARS- COV-2 (SARS2) PANDEMIC HIGHLIGHTED THE IMPORTANCE OF BEING ABLE TO SAMPLE THE UPPER AIRWAY FOR PURPOSES INCLUDING EVALUATION OF VIRAL INFECTION AND CLEARANCE. ALTHOUGH THE UPPER AIRWAY REPRESENTS THE PRIMARY SITE OF INFECTION FOR MANY HUMAN PATHOGENS, CRITICAL QUESTIONS REMAIN REGARDING UPPER AIRWAY IMMUNITY AND PROTECTION. NASAL CAVITY SWAB SAMPLING CAN BE USED TO BRIDGE KNOWLEDGE GAPS REGARDING UPPER AIRWAY IMMUNITY TO IMPORTANT RESPIRATORY PATHOGENS LIKE SARS2 AND TO ADDRESS IMPORTANT IMMUNOLOGIC QUESTIONS WHERE PERIPHERAL BLOOD SAMPLING IS INHERENTLY INSUFFICIENT. I ESTABLISHED NOVEL METHODS FOR REPRODUCIBLE, LONGITUDINAL SAMPLING OF UPPER AIRWAY IMMUNE CELL POPULATIONS USING SWABS AND DEMONSTRATED THAT SUFFICIENT NUMBERS OF VIABLE IMMUNE CELLS COULD BE COLLECTED TO ALLOW FOR HIGH RESOLUTION DOWNSTREAM ANALYSES, INCLUDING MULTIPARAMETRIC FLOW CYTOMETRY AND SINGLE CELL RNA SEQUENCING. UPPER AIRWAY RESIDENT MEMORY B AND T CELL POPULATIONS, INCLUDING SARS2- SPECIFIC MEMORY B AND T CELLS WERE CHARACTERIZED USING THESE METHODS. THERE IS GREAT INTEREST IN DEVELOPING NEXT GENERATION VACCINES, INCLUDING VACCINES THAT CAN ELICIT ROBUST MUCOSAL IMMUNE RESPONSES. THIS REQUIRES KNOWLEDGE OF THE IMMUNOLOGIC CONTEXTS IN WHICH MUCOSAL IMMUNITY IS GENERATED AND THE REQUIREMENTS FOR MAINTENANCE OF UPPER AIRWAY IMMUNE MEMORY. I HYPOTHESIZE THAT LOCAL ANTIGEN EXPOSURE IS REQUIRED TO DEVELOP DURABLE UPPER AIRWAY IMMUNE MEMORY, AND INTRAMUSCULAR IMMUNIZATION ALONE MAY FAIL TO ELICIT UPPER AIRWAY IMMUNITY DESPITE GENERATING CIRCULATING IMMUNITY. I WILL TEST THIS HYPOTHESIS BY STUDYING UPPER AIRWAY MEMORY B AND T CELL FREQUENCIES, DIVERSITY, KINETICS AND DURABILITY IN DISTINCT IMMUNOLOGIC CONTEXTS INCLUDING SARS2 BREAKTHROUGH INFECTION (BTI) AND COVID-19 BOOSTER VACCINATION USING THIS AWARD. OTHER RESPIRATORY PATHOGENS OF PUBLIC HEALTH IMPORTANCE WILL ALSO BE EXAMINED. IF FUNDED, THIS K08 WILL ASSIST WITH MY TRANSITION TO BECOMING A SUCCESSFUL INDEPENDENT PHYSICIAN SCIENTIST INVESTIGATOR IN HUMAN IMMUNOLOGY AND INFECTIOUS DISEASES. THE LA JOLLA INSTITUTE FOR IMMUNOLOGY (LJI) IS A SUPERB TRAINING ENVIRONMENT AND LOCATED ON THE CAMPUS OF ANOTHER EXCELLENT ACADEMIC RESEARCH AND MEDICAL INSTITUTION, THE UNIVERSITY OF CALIFORNIA, SAN DIEGO (UCSD). I WILL RECEIVE ONGOING MENTORSHIP FROM AN ESTABLISHED INVESTIGATOR WITH A TRACK RECORD FOR PRODUCING SUCCESSFUL MENTEES, PROFESSOR SHANE CROTTY, PHD, A WORLD- RENOWNED EXPERT IN THE FIELD OF IMMUNOLOGY. LJI AND UCSD ARE CLOSELY AFFILIATED, AND DR. CROTTY HAS AN ADJUNCT APPOINTMENT AT UCSD. I WILL HAVE ACCESS TO RESOURCES AT BOTH LJI AND UCSD FOR THIS CAREER DEVELOPMENT AWARD. AS AN ASSOCIATE PHYSICIAN IN UCSD’S DIVISION OF INFECTIOUS DISEASES I WILL RECEIVE ADDITIONAL MENTORSHIP IN ACADEMIC CAREER DEVELOPMENT, AND MAINTAIN MY CLINICAL ACUMEN BY PROVIDING PART-TIME MEDICAL CARE AND CONSULTATION SERVICES FOR MEDICALLY COMPLEX PATIENTS.

MECHANICS OF LCR ACTION ON PERFORIN TO ESTABLISH CYTOTOXICITY IN NK CELLS AND CTL

INVARIANT NKT CELL RESPONSES TO VIRAL INFECTIONS AND TUMORS

ROLE OF ABCA7 IN INKT DEVELOPMENT AND FUNCTION AND ITS IMPACT ON ATHEROSCLEROSIS

THE ROLE OF ABCG1 IN ATHEROSCLEROSIS PROGRESSION AND TH17 DIFFERENTIATION

THE ROLE OF HVEM IN INFLAMMATION AND T CELL FUNCTION

RABIES VIRUS GLYCOPROTEIN STRUCTURE DETERMINATION AND RECEPTOR BINDING INTERACTIONS

PKCTHETA-CD28 INTERACTION: A NOVEL DRUG TARGET FOR AUTOIMMUNITY AND INFLAMMATION

IN VIVO INVESTIGATION OF NON-CLASSICAL MONOCYTE PATROLLING MECHANISM

GENETIC ANALYSIS OF THE NKT CELL RESPONSE TO STREPTOCOCCUS PNEUMONIAE

THE ROLE OF INTESTINAL EPITHELIAL CELLS IN THE GENERATION OF PROTECTIVE CD4 CYTOTOXIC T LYMPHOCYTES