Seattle Children's Hospital

IMMUNE MECHANISMS OF PROTECTION AGAINST MYCOBACTERIUM TUBERCULOSIS CENTER (IMPAC-TB)

THIS STATEMENT OF WORK (SOW) OUTLINES THE ACTIVITIES THAT UK HEALTH SECURITY AGENCY (UKHSA), PREVIOUSLY KNOWN AS PUBLIC HEALTH ENGLAND (PHE), WILL BE PERFORMING. NOTE THAT THE TASK NUMBERS BELOW REFLECT THE NUMBERING OF THE OVERALL ACTIVITIES IN THE PRIME CONTRACT. THROUGH THE EXECUTION OF THIS CONTRACT, WE WILL EXAMINE BASIC INDUCTION OF INNATE AND ADAPTIVE IMMUNITY FROM VACCINATION, INCLUDING STUDIES THAT ADDRESS NONTUBERCULOUS MYCOBACTERIA (NTM) INTERFERENCE, RNA DELIVERY, PRIME-BOOST REGIMENS (INCLUDING BCG PRE-EXPOSURE), TRANSCRIPTOMIC HOST SIGNATURES, SYSTEMS BIOLOGY AND DURABLE IMMUNE MEMORY FOR THE FURTHER ADVANCEMENT OF THE EFFORT TO DESIGN AND EVALUATE AN EFFICACIOUS VACCINE AGAINST MYCOBACTERIUM TUBERCULOSIS (M.TB). THE WORK UNDER THIS CONTRACT MAY INCLUDE THE POSSIBLE DISTRIBUTION OF DE-IDENTIFIED HUMAN SUBJECT PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC), SERUM, AND OTHER CLINICAL SAMPLES TO SCRI AND THE PHOENIX PARTNERS. NHP SAMPLES AND/OR DOWNSTREAM DATA MAY BE SHARED WITH THE PARTNERS ON THIS CONTRACT. OPTION 3.M72 ACTIVITIES: THE ANIMALS USED IN THESE STUDIES WILL BE RHESUS MACAQUES OF INDIAN GENOTYPE OBTAINED FROM ESTABLISHED UK BREEDING COLONIES AND WILL BE BETWEEN 3 AND 6 YEARS OF AGE (TYPICALLY BETWEEN 4 – 12 KG). ABSENCE OF PREVIOUS EXPOSURE TO MYCOBACTERIAL ANTIGENS AND/OR NTM RECOGNITION WILL BE CONFIRMED BY EX VIVO IFN-Γ ELISPOT TO MEASURE RESPONSES TO PURIFIED PROTEIN DERIVATIVE (PPD) FROM M.TB, POOLED 15-MER PEPTIDES OF ESAT-6 AND CFP-10 AND NTM MEGA PEPTIDE POOLS FOR SCREENING TIMEPOINTS AND MTB300 FOR POST VACCINATION AND M.TB CHALLENGE TIMEPOINTS AS AVAILABLE. THE PROJECT LICENSE ENABLING THESE STUDIES WILL BE APPROVED BY THE ANIMAL WELFARE AND ETHICAL REVIEW BODY AT UKHSA PORTON AND GRANTED BY THE HOME OFFICE, UK. ANIMALS WILL BE HOUSED IN SOCIALLY COMPATIBLE GROUPS AND ACCORDING TO THE CODE OF PRACTICE FOR THE HOUSING AND CARE OF ANIMALS BRED, SUPPLIED OR USED FOR SCIENTIFIC PURPOSES, DECEMBER 2014, THE NC3RS, AND THE GUIDELINES ON PRIMATE ACCOMMODATION, CARE AND USE, AUGUST 2006 (NC3RS, 2006). ANIMALS WILL BE MONITORED DAILY FOR BEHAVIORAL AND CLINICAL CHANGES. BEHAVIOR WILL BE EVALUATED FOR CONTRA-INDICATORS INCLUDING DEPRESSION, WITHDRAWAL FROM THE GROUP, AGGRESSION, CHANGES IN FEEDING PATTERNS, BREATHING PATTERN, RESPIRATION RATE AND COUGH. BLOOD SAMPLE COLLECTION (PAXGENE) FOR TRANSCRIPTOMICS WILL BE COLLECTED TWO DAYS AFTER EACH IMMUNIZATION OR CHALLENGE. BLOOD SAMPLE COLLECTION FOR M.TB ANTIGEN-SPECIFIC IMMUNE RESPONSES WILL BE EVALUATED AT REGULAR INTERVALS THROUGHOUT THE STUDY (HIGH PRIORITY TIMEPOINTS TO INCLUDE: TWO WEEKS POST PRIME AND BOOST IMMUNIZATIONS, ONE WEEK PRE-CHALLENGE, ONE, TWO, FOUR AND EIGHT WEEKS POST CHALLENGE), AEROSOL CHALLENGE AND EUTHANASIA; ANIMALS WILL BE WEIGHED, EXAMINED FOR GROSS ABNORMALITIES AND BODY TEMPERATURE MEASURED. RED BLOOD CELL (RBC) HEMOGLOBIN LEVELS WILL BE MEASURED TO IDENTIFY THE PRESENCE OF ANEMIA, AND ERYTHROCYTE SEDIMENTATION RATES (ESR) WILL BE MEASURED TO DETECT AND MONITOR INFLAMMATION INDUCED BY INFECTION WITH M.TB. TASK 3.M72: USE ULDA MODEL TO EVALUATE THE EFFICACY OF NEW TB VACCINE CANDIDATES NOTE: AFTER REVIEW OF ALL UPCOMING EXPERIMENTS, THE NIH, SCRI AND UKHSA AGREED THAT THE FOLLOWING EXPERIMENT COMBINING BOTH ID93 AND M72 VACCINE GROUPS IS PREFERRED GIVEN IT IS BOTH COST SAVING (SHARED CONTROLS) AND SCIENTIFICALLY MORE ROBUST (DIRECT HEAD TO HEAD) THAN THE EXPERIMENTAL DESIGNS AS SEPARATE TASKS. THEREFORE, THIS WORK IS DESCRIBED IN OPTION 3 SOW AS WELL AS OPTION M72.3 SOW. GROUPS BOLDED BELOW WILL BE SPECIFICALLY PAID FOR WITH 3.M72 FUNDING, WITH CONTROLS AS A SHARED COST WITH OPTION 3.3. TABLE 1: PRECLINICAL DESIGN FOR TASK M72.3 GROUP PRIME FIRST BOOST SECOND BOOST1 CHALLENGE2 1 SALINE SALINE SALINE CLINICAL M.TB ISOLATE HN878 - ULDA 2 BCG SALINE SALINE 3 BCG M72+GLA-LSQ M72+GLA-LSQ 4 BCG ID93+GLA-LSQ ID93+GLA-LSQ 5 BCG M72+GLA-SE M72+GLA-SE 6 BCG M72+UM-1098 M72+UM-1098 1 SECOND BOOST DELIVERED I.M. 56 DAYS POST FIR

AFRICA HEALTH RESEARCH INSTITUTE (AHRI) WILL CONDUCT GLOBAL MOLECULAR MEASUREMENTS ON TB CASES AND CONTROLS IN THE VUKUZAZI COHORT TO DEFINE CORRELATES OF PROTECTION AND TEST HYPOTHESIZED MECHANISMS OF PROTECTION ARISING FROM WITHIN THE CENTER OR OTHER PROGRAMS (YEARS 2-7). IN YEAR 4, AHRI WILL SPECIFICALLY: ACTIVITY 1: IDENTIFY EARLY TB CASES AND MTB-INFECTED UNDISEASED CONTROLS FROM PARTICIPANTS IN THE VUKUZAZI COHORT. ACTIVITY 2: IDENTIFY LATE TB CASES AND MTB-INFECTED UNDISEASED CONTROLS FROM PARTICIPANTS IN THE VUKUZAZI COHORT. ACTIVITY 3: CONDUCT TRANSCRIPTIONAL ANALYSIS ON WHOLE-BLOOD FROM TB CASES AND MATCHED CONTROLS IN THE VUKUZAZI STUDY. €ƒ

UNIVERSITY OF WASHINGTON PROTEIN PRODUCTION GROUP (UW-PPG), UNDER THE DIRECTION OF WESLEY C. VAN VOORHIS, M.D., PH.D., WILL PERFORM THE FOLLOWING WORK TO SUPPORT THE BASIC PROJECT REQUIREMENT FROM SEPT 1, 2025 THROUGH AUG 31, 2026. UW-PPG WILL GENERATE TARGET CLONES, PERFORM SMALL- AND LARGE-SCALE SCREENING, UPSCALE EXPRESSION AND PURIFICATION OF THE CLONES. UW-PPG WILL ALSO LEAD ONE STRUCTURE-BASED VACCINE AND THERAPEUTIC DEVELOPMENT PROJECT AND SUPPORT OTHER SPECIAL STUDIES AS NEEDED. A. CLONING, EXPRESSION TESTING, UPSCALE AND PURIFICATION OF SSGCID TARGETS: UW-PPG WILL PERFORM CLONING MAINLY USING HTP TECHNOLOGIES AND PCR-BASED LIGATION INDEPENDENT CLONING INTO E. COLI EXPRESSION VECTORS OR DESIGN AND ORDER SYNTHETIC CONSTRUCTS. UW-PPG WILL SCREEN THEIR EXPRESSION AND SOLUBILITY PROPERTIES USING HTP TECHNOLOGIES AND PROTOCOLS, INCLUDING, AS NEEDED, MULTIPLE EXPRESSION SYSTEMS AND CLEAVAGE SYSTEMS. UW-PPG WILL GENERATE LARGE SCALE EXPRESSIONS OF TARGETS FOR HIGHLY PURIFIED TARGET PROTEINS OR PROTEIN FRAGMENTS AND PRODUCE THEM IN SUFFICIENT QUANTITY AND QUALITY FOR STRUCTURE DETERMINATION THROUGH STANDARDIZATION AND AUTOMATION OF PROTOCOLS. UW-PPG WILL EVALUATE THE PURITY, CONCENTRATION AND BIOPHYSICAL PROPERTIES (E.G. PROTEIN FOLDING, AGGREGATION, AND/OR STABILITY) OF THE PURIFIED SAMPLES TO ENHANCE THE EFFECTIVENESS OF THE STRUCTURE DETERMINATION EFFORTS. UW-PPG WILL LEAD AND SUPPORT SEVERAL HIGH VALUE TARGETS (HVTS), COMMUNITY REQUEST PROJECTS (CRP), HIGHEST IMPACT PROJECTS (HIP) AND OTHER SPECIAL STUDIES WITHIN THE SSGCID PIPELINE, AS DECIDED BY THE SSGCID SCIENTIFIC LEADERSHIP TEAM (SLT). B. LEAD ONE STRUCTURE-BASED VACCINE AND THERAPEUTIC DEVELOPMENT PROJECT. UW-PPG MEMBERS WILL LEAD ONE SVTD PROJECT ENTITLED “STUDIES ON CHEMICAL-GENETICALLY VALIDATED PLASMODIUM FALCIPARUM PROTEIN KINASES (TOUSLED-LIKE KINASE 1[TLK1] AND AURORA KINASE 2 [ARK2]).” C. QUALITY CONTROL, DATA COLLECTION AND REPORTING: UW-PPG WILL ESTABLISH AND CONDUCT QUALITY CONTROL PROCEDURES AS NECESSARY TO ENSURE THE PRODUCTION OF HIGH-QUALITY DATA. UW-PPG WILL ENSURE THAT THE STATUS OF ALL WORK PERFORMED FOR SSGCID IS UPDATED IN A TIMELY FASHION, THROUGH THE CENTRAL TARGET TRACKING DATABASE (CTTDB), TRACKING SPREADSHEETS AND/OR ANY OTHER TRACKING SYSTEM REQUIRED BY THE PROJECT. KEY UW-PPG STAFF WILL PARTICIPATE IN SSGCID PROJECT MEETINGS AND NIAID REQUIRED MEETINGS TO ENSURE THE SUCCESS OF THE PROJECT. UW-PPG WILL CONTRIBUTE TO QUARTERLY AND SEMI-ANNUAL PROGRESS REPORTS AND ANY OTHER NIAID REQUIRED REPORTS AS NEEDED AND IN A TIMELY FASHION. UW-PPG WILL CONDUCT THEIR WORK WITH THE GOAL TO PUBLISH THEIR RESULTS IN HIGH IMPACT PUBLICATIONS

THE UNIVERSITY OF KANSAS (KU), UNDER THE DIRECTION OF SCOTT LOVELL, PH.D., WILL PERFORM THE FOLLOWING WORK TO SUPPORT THE BASIC PROJECT REQUIREMENT (9/1/2025-8/31/2026). KU WILL ACCEPT PROTEIN AND OTHER SAMPLES PRIMARILY GENERATED BY OTHER SSGCID SITES AND PREPARE THEM FOR X-RAY CRYSTALLOGRAPHY STRUCTURE DETERMINATION STUDIES, CONDUCT CRYSTALLIZATION EXPERIMENTS, COLLECT DIFFRACTION DATA, AND PERFORM STRUCTURE SOLUTION. KU WILL ALSO SUPPORT VARIOUS SPECIAL PROJECT STUDIES AS NEEDED AND LEAD ONE FUNCTIONAL STUDY. STRUCTURE DETERMINATION OF PROTEINS OR PROTEIN COMPLEXES BY X-RAY CRYSTALLOGRAPHY: KU WILL ACCEPT PROTEIN SAMPLES PRIMARILY RECEIVED FROM THE OTHER TEAM SITES FOR X-RAY CRYSTALLOGRAPHY STRUCTURE DETERMINATION STUDIES AND CONDUCT HIGH THROUGHPUT CRYSTALLIZATION EXPERIMENTS, INCLUDING CRYSTALLIZATION TRIALS, OPTIMIZATION, DATA COLLECTION AND INTERPRETATION. WHEN NECESSARY, KU WILL CONDUCT CO-CRYSTALLIZATION SCREENS OF THE TARGET IN COMPLEX WITH SMALL MOLECULES AND LIGANDS. KU WILL IDENTIFY AND HARVEST CRYSTALS THAT ARE SUITABLE FOR DIFFRACTION SCREENING AND CONDUCT LARGE SCALE CRYSTAL DIFFRACTION SCREENS, COLLECT DIFFRACTION DATA AND MONITOR QUALITY METRICS OF THE SUITABLE CRYSTALS. WHERE APPROPRIATE, KU WILL ALSO LIAISE WITH KUMC’S CRYOEM CENTER TO HELP SOLVE STRUCTURES BY CRYOEM. KU WILL PERFORM STRUCTURE SOLUTION, MODEL REFINEMENT AND VALIDATION FOR X-RAY CRYSTALLOGRAPHY. A STRUCTURE DETERMINATION PROJECT WILL BE CONSIDERED COMPLETE WHEN THE REFINEMENT AND VALIDATION OF THE FINAL STRUCTURE MODELS DO NOT PROVIDE ANY CLEAR SIGNAL OF WHERE THE MODEL CAN BE IMPROVED OR WHEN 3D STRUCTURE QUALITY PARAMETERS SATISFY THE MINIMUM QUALITY REQUIREMENTS ESTABLISHED AND AGREED UPON IN COORDINATION WITH THE CENTER FOR STRUCTURAL BIOLOGY OF INFECTIOUS DISEASES. KU WILL SUPPORT HIGH VALUE TARGETS (HVTS), COMMUNITY REQUEST PROJECTS (CRP), HIGHEST IMPACT PROJECTS (HIP) AND OTHER SPECIAL STUDIES WITHIN THE SSGCID PIPELINE, AS DECIDED BY THE SSGCID SCIENTIFIC LEADERSHIP TEAM (SLT). FUNCTIONAL STUDIES KU WILL LEAD ONE FUNCTION STUDY, ENTITLED “STRUCTURE AND FUNCTIONAL STUDIES OF THE TRANSCRIPTIONAL REGULATOR, GNTR FROM ENTEROCOCCUS FAECALIS.” QUALITY CONTROL, DATA COLLECTION AND REPORTING: KU WILL ESTABLISH AND CONDUCT QUALITY CONTROL PROCEDURES AS NECESSARY TO ENSURE THE PRODUCTION OF HIGH-QUALITY DATA. KU WILL ENSURE THAT THE STATUS OF ALL WORK PERFORMED FOR SSGCID IS UPDATED IN A TIMELY FASHION, THROUGH THE CENTRAL TARGET TRACKING DATABASE (CTTDB), TRACKING SPREADSHEETS AND/OR ANY OTHER TRACKING SYSTEM REQUIRED BY THE PROJECT. KEY STAFF MEMBERS FROM KU WILL PARTICIPATE IN SSGCID PROJECT MEETINGS AND NIAID-REQUIRED MEETINGS TO ENSURE THE SUCCESS OF THE PROJECT. KU STAFF WILL CONTRIBUTE TO QUARTERLY AND SEMI-ANNUAL PROGRESS REPORTS AND ANY OTHER NIAID REQUIRED REPORTS AS NEEDED AND IN A TIMELY FASHION AND WILL CONDUCT THE PROJECT WORK WITH THE AIM OF PRODUCING IMPACTFUL PUBLICATIONS. IT IS EXPECTED THAT SEVENTY-FIVE STRUCTURES WILL BE SOLVED THROUGH THIS WORK

THROUGH THE EXECUTION OF THIS CONTRACT, WE WILL EXAMINE BASIC INDUCTION OF INNATE AND ADAPTIVE IMMUNITY FROM VACCINATION, INCLUDING STUDIES THAT ADDRESS NTM INTERFERENCE, RNA DELIVERY, PRIME-BOOST REGIMENS (INCLUDING BCG PRE-EXPOSURE), TRANSCRIPTOMIC HOST SIGNATURES, SYSTEMS BIOLOGY AND DURABLE IMMUNE MEMORY FOR THE FURTHER ADVANCEMENT OF THE EFFORT TO DESIGN AND EVALUATE AN EFFICACIOUS VACCINE AGAINST MYCOBACTERIUM TUBERCULOSIS (M.TB). THE WORK UNDER THIS CONTRACT MAY INCLUDE THE POSSIBLE DISTRIBUTION OF DE-IDENTIFIED HUMAN SUBJECT PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC), SERUM, AND OTHER CLINICAL SAMPLES TO SCRI AND THE PHOENIX PARTNERS. NON-HUMAN PRIMATE (NHP) SAMPLES AND/OR DOWNSTREAM DATA MAY BE SHARED WITH THE PARTNERS ON THIS CONTRACT. OPTION 3 ACTIVITIES: EVALUATE OPTIMIZED M.TB VACCINE CANDIDATES FOR SAFETY, IMMUNOGENICITY AND EFFICACY IN THE NHP MODEL OF TB SCRI HAS WORKED WITH DR. RYAN MCNAMARA, WHO HAS PREVIOUSLY BEEN EMPLOYED AT THE RAGON INSTITUTE, TO DEVELOP A WORKPLAN FOR THE SYSTEM SEROLOGY ENDPOINTS FOR BOTH CLINICAL AND PRECLINICAL EVALUATIONS. WE HAVE CONFIRMED THAT A LIST OF 207 M.TB ANTIGENS THAT WERE RECENTLY PUBLISHED ARE NOT PROPRIETARY TO RAGON AND ARE AN APPROPRIATE SET FOR OUR INVESTIGATIONS. TASK 3.7 COMPARE IMMUNE PROFILES IN NHP, SMALL ANIMAL MODELS AND HUMANS TO IDENTIFY CORRELATES OF PROTECTION. SUBTASK 3.7.1 EVALUATE HUMORAL IMMUNITY AS A VACCINE-INDUCED CORRELATE OF PROTECTION USING SYSTEMS SEROLOGY IN THE NHP MODEL. SEVERAL EFFECTOR FUNCTIONS BASED ON THE FC PORTION OF THE ANTIBODY CAN CONTRIBUTE TO THE CLEARANCE OF ORGANISMS, INCLUDING COMPLEMENT ACTIVITY, ANTIBODY-DEPENDENT CELLULAR PHAGOCYTOSIS (ADCP), ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC), ANTIBODY-DEPENDENT CYTOKINE AND CHEMOKINE SECRETION. SYSTEMS SEROLOGY ANALYSIS WILL BE USED TO EVALUATE ANTIBODY EFFECTOR FUNCTIONS INCLUDING NK CELL ACTIVATION, ADCP, AND ADCC. THESE ASSAYS CAPTURE OVER 100 BIOPHYSICAL AND FUNCTIONAL CHARACTERISTICS OF POLYCLONAL ABS, INCLUDING CYTOLYTIC ACTIVITY, PHAGOCYTIC TARGET CLEARANCE, CYTOKINE SECRETION, AND IMMUNOMODULATOR RELEASE. THIS SUITE OF FUNCTIONAL ASSAYS WILL BE UTILIZED TO BROADLY DEFINE THE FC PROFILES OF ID93- AND BCG-SPECIFIC ABS. FOR ALL FUNCTIONAL ASSAYS, THE FUNCTIONAL ACTIVITY OF ABS WILL BE ASSESSED AGAINST ID93, PPD, INFLUENZA HEMAGGLUTININ (CONTROL), EBOLA VIRUS GLYCOPROTEIN (NEGATIVE CONTROL). ALL ANTIGENS WILL BE BIOTINYLATED AND ATTACHED TO STREPTAVIDIN-FLUORESCENT BEADS (THERMO FISHER). SIGMA INTRAVENOUS IG WILL BE USED AS A POSITIVE CONTROL. SAMPLES RECEIVED FROM COLLABORATORS ON THIS PROJECT (MOUSE, GUINEA PIG, NHP AND HUMAN) WILL BE USED TO CARRY OUT THE SYSTEMS SEROLOGY APPROACH DETAILED BELOW. SYSTEMS SEROLOGY CAPTURES LARGE NUMBERS OF DISTINCT ANTIGEN-SPECIFIC SERUM-BASED ANTIBODY PROFILE FEATURES THAT COMPREHENSIVELY AND OBJECTIVELY PROBES THE ANTIGEN-SPECIFIC FC-ANTIBODY-OME AT UNPRECEDENTED DEPTH. DISPARATE 53 DATA POINTS WILL BE INTEGRATED AND A SPECTRUM OF COMPUTATIONAL METHODS FOR CONSTRUCTING MULTI-VARIATE MODELS AND THEN APPLIED TO ASCERTAIN PREDICTIVE RELATIONSHIPS BETWEEN COMBINATIONS OF SYSTEM VARIABLES (STRUCTURE:FUNCTION, TRANSFER, ETC.) TO DEFINE NOVEL AND OFTEN UNEXPECTED INSIGHTS INTO THE ANTIBODY CHARACTERISTICS THAT DRIVE UNIQUE AND DISTINCT BIOLOGICAL OUTCOMES. THESE ANALYTICAL METHODS INCLUDE NON-NEGATIVE MATRIX FACTORIZATION, SELF-ORGANIZING MAPS, MULTI-LINEAR REGRESSION, PARTIAL LEAST-SQUARES DISCRIMINATION, DECISION TREES, AND RANDOM FORESTS AMONG OTHERS, INCLUDING FEATURE REDUCTION WHEN NECESSARY FOR CONSTRAINING THE NUMBER OF REGULATORY VARIABLES TO HELP PREVENT MODEL OVER-FITTING. TWO SPECIFIC APPROACHES WILL BE UTILIZED TO DEFINE IMMUNE RESPONSE AND IMMUNE CORRELATES OF PROTECTION ASSOCIATED CHANGES USING BOTH “FLAT” AND “HIERARCHICAL” FRAMEWORKS. IN THE “FLAT” FRAMEWORK, A SINGLE-LEVEL MODEL WILL BE UTILIZED – EXPLOITED EACH DATA POINT AS AN INDEPENDENT VARIABLE. THIS AGNOSTIC APPROACH ENABLES THE UNBIASED IDENTIFICATION OF INDIVIDU

THE DU PLESSIS/WALZL LABORATORY AT STELLENBOSCH UNIVERSITY (SU) WILL BE RESPONSIBLE FOR THE CLINICAL AND IMMUNOLOGICAL WORK (INCLUDING ON-SITE DATA MANAGEMENT) OF THE PROJECT. SU WILL COORDINATE THE STUDY DESIGN, PARTICIPANT SCREENING AND ENROLLMENT FOR THE CASCADE HOUSEHOLD CONTACT STUDY, PERFORM BRONCHOSCOPIES AND MANAGE SAMPLE SHARING TO PARTICIPATING SITES. SU WILL PROVIDE INPUT IN THE DESIGN, EXECUTION AND DATA ANALYSIS OF IMMUNOLOGICAL STUDIES AND PARTICIPATE IN THE LEADERSHIP TEAM OF €œCASCADE IMPAC-TB€Ͽ½ CENTER. SU WILL INTERACT AND COORDINATE ALL PROJECT ACTIVITIES WITH THE PI OF THE PROJECT. DR. DU PLESSIS AND DR. WALZL, THE SU CO-PI€„¢S WILL REVIEW ALL RAW DATA GENERATED BY THE SU TEAM, AND SUPERVISE THE LABORATORY WORK, AND THE RESEARCH TECHNICIANS. SPECIFIC DELIVERABLES FOR YEAR 6 OF THE CONTRACT AT SU INCLUDE THE FOLLOWING: A. RECRUITMENT: THE ANTICIPATED SCREENING AND RECRUITMENT GOALS HAVE BEEN ACHIEVED AND EXCEEDED. WHILE THE INITIAL RECRUITMENT TARGET WAS SET AT 20 PARTICIPANTS FOR EACH OF THE 8 CLINICAL GROUPS (N=160 TOTAL), WE EXCEEDED THIS TO ACHIEVE AND ENROLL A TOTAL NUMBER OF 185 STUDY PARTICIPANTS ACROSS ALL GROUPS FOR THE MAIN STUDY. THE COVID-19 PANDEMIC HAS RESULTED IN A REQUEST FOR A SOMEWHAT ALTERED CLASSIFICATION AND ENROLLMENT TARGET OF CLINICAL GROUPS, WHICH WAS APPROVED BY THE NIH IN 2021. THE SU HHC STUDY NOW CONSISTS OF GROUPS STRATIFIED NOT ONLY BY IGRA RESULTS OR PET/CT READOUT, BUT ALSO BY SARS-COV-2 SEROLOGY STATUS. THE BUDGET FOR PERIOD 6 WILL INCLUDE LIMITED PARTICIPANT RECRUITMENT AND BRONCHOSCOPIES, AS ORIGINALLY PROPOSED, WHICH WILL INCLUDE ONLY STUDY PARTICIPANTS FROM SELECTED €œBIOLOGICALLY INTERESTING€Ͽ½ CLINICAL PHENOTYPES FOR FURTHER TARGETED FUNCTIONAL ANALYSES THROUGH GRANULOMA MODEL MANIPULATION. SU HAS BUDGETED FOR N=12 BRONCHOSCOPIES. B. END-ASSAYS: IMMUNOPHENOTYPING EXPERIMENTS €€Œ ONE OF THE DELIVERABLES AT SU IS TO CONDUCT DEEP PHENOTYPING OF PAIRED BRONCHOALVEOLAR LAVAGE (BAL) AND PBMC FROM THE VARIOUS CASCADE HOUSEHOLD CONTACT STUDY CLINICAL GROUPS. SU HAS EVALUATED THE ASSAY PRIORITY LIST, AS WELL AS THE BAL AND PBMC SAMPLES COLLECTED FROM PARTICIPANTS ENROLLED IN THE CASCADE HOUSEHOLD CONTACT STUDY, TO ENUMERATE THE NUMBER OF STORED SAMPLES AVAILABLE FOR THE OBJECTIVES RELATED TO THE PROPOSED PLANS FOR THIS END-ASSAY. OF ALL MAIN STUDY PARTICIPANTS FROM WHICH PAIRED BAL AND PBMC HAVE BEEN COLLECTED (N=190), SUFFICIENT CELL NUMBERS WERE OBTAINED FROM 88% OF PARTICIPANTS, WHICH INCLUDES MAKING USE OF RESIDUALLY STORED BAL CELLS. THIS ALSO INCLUDES REPLACEMENT OR REPEATING OF BRONCHOSCOPIES FOR N=5 OF THE PARTICIPANTS WITH LOW CELL YIELDS. WHILE THE BAL CELL AUTOFLUORESCENCE IS LIKELY TO RESULT IN A SMALLER NUMBER OF AVAILABLE FLUORESCENT CHANNELS FOR OUR BAL MYELOID FLOW CYTOMETRY IMMUNE PHENOTYPING PANEL, THE OPTIMIZATION OF THE 24 MARKER MYELOID PANEL FOR PBMC HAS BEEN COMPLETED WHILE OPTIMIZATION OF THE 27 MARKER LYMPHOID PANEL FOR PBMC WILL BE FINALIZED IN OCTOBER 2024. SU EXPECTS TO COMPLETE IMMUNE PHENOTYPING OF ALL SU HHC PBMC SAMPLES BY JUNE 2025. IMMUNE PHENOTYPING OF ALL SU HHC BAL SAMPLES USING THE LYMPHOID FLOW CYTOMETRY PANEL WILL BE COMPLETED BY DECEMBER 2025. GRANULOMA MODEL/KILLING ASSAYS €€Œ SU WILL MAKE USE OF THE IN VITRO GRANULOMA MODEL ALONG WITH PARALLEL ASSESSMENTS IN MONOLAYER CULTURE AND UNFRACTIONATED PBMC AND BAL, TO ASSESS MTB KILLING AMONGST THE VARIOUS CLINICAL GROUPS. INTERESTING FINDINGS RELATED TO MTB CONTROL WILL BE TESTED IN FUTURE IN GRANULOMA MODEL EXPERIMENTS. BY USING CLINICAL SAMPLES FROM PARTICIPANTS NOT ELIGIBLE ACCORDING TO THE CASCADE HHC STUDY CRITERIA, SU HAS OPTIMIZED THE MODEL AND ALSO IMPLEMENTED CHANGES TO STREAMLINE BACTERIAL LOAD ASSESSMENT THROUGH MGIT CULTURE. SU EXPECTS TO COMPLETE THE KILLING ASSAY EXPERIMENTS FOR AT LEAST A QUARTER OF ALL SU HHC SAMPLES BY THE END OF PERIOD 6. THE RESULTS FROM VARIOUS CASCADE OMICS READOUTS (HUMAN AND LAB ANIMAL RESULTS) WILL INFORM