Project Grant R01AI180451

INFERRING THE RULES OF ANTIBODY EVOLUTION USING REPLICATED GERMINAL CENTERS - PROJECT SUMMARY GERMINAL CENTERS (GCS) ARE THE SITE OF AFFINITY MATURATION, A PROTOTYPICAL DARWINIAN PROCESS THAT IS REQUIRED TO GENERATE THE POTENT ANTIBODIES THAT PROTECT AGAINST INFECTIOUS DISEASE. OVER THE PAST DECADES, WORK BY SEVERAL GROUPS, INCLUDING OUR OWN, HAS LED TO A COMPREHENSIVE GENERAL UNDERSTANDING OF THE CELLULAR AND MOLECULAR MECHANISMS THAT DRIVE EVOLUTION IN THE GC. HOWEVER, THIS MECHANISTIC UNDERSTANDING HAS YET TO REACH SUFFICIENT GRANULARITY TO ALLOW FOR ACCURATE PREDICTION OF THE OUTCOMES OF GC EVOLUTION AND EFFICIENT GUIDANCE OF GC B CELLS ALONG PREDETERMINED MUTATIONAL TRAJECTORIES. SEVERAL POPULATION-LEVEL PHENOMENA OBSERVED IN GCS REMAIN POORLY UNDERSTOOD, INCLUDING THE APPARENT STOCHASTICITY OF "CLONAL BURSTS," PROLIFERATIVE SPREES IN WHICH THE ENTIRE GC IS TAKEN OVER BY THE DESCENDANTS OF A SINGLE CELL IN A MATTER OF A FEW DAYS, THE CONTINUOUS PRESENCE OF B CELLS WITH LOW AFFINITIES IN GCS EVEN AT LATE STAGES OF THE REACTION, AND THE APPARENT INABILITY OF GCS TO FIND CERTAIN SOMATIC MUTATIONS DESPITE THEIR BENEFIT IN TERMS OF AFFINITY. GREATER UNDERSTANDING OF THESE APPARENTLY ABERRANT EVOLUTIONARY PATHWAYS MAY IMPROVE OUR ABILITY TO PREDICT AND POTENTIALLY CONTROL THE OUTPUT OF THE GC REACTION. IN THIS APPLICATION, WE DEVELOP A FULL TOOLSET CONSISTING OF A MOUSE MONOCLONAL FORGENES ENCODING AN ANTIBODY TO A CLASSIC PROTEIN ANTIGEN, A DEEP MUTATIONAL SCAN (DMS) EXPERIMENT IN WHICH THE EFFECTS ON AFFINITY OF EVERY POSSIBLE MUTATION IN BOTH CHAINS OF THISARE MEASURED EXPERIMENTALLY, AND A COMPUTATIONAL FRAMEWORK THAT ALLOWS US TO ASSIGN AFFINITIES TO ANYSEQUENCE THAT WE RECOVER FROM THESE MONOCLONAL GC B CELLS. WE WILL USE THESE TOOLS TO CONDUCT REPLICATED EVOLUTION EXPERIMENTS ON HUNDREDS OF GCS, RECONSTRUCTING THE EVOLUTIONARY PATHS AND AFFINITIES OF THOUSANDS OF B CELLS IN THE COURSE OF GC MATURATION. WE PROPOSE TO USE THIS APPROACH TO GAIN INSIGHT INTO HOW B CELL EVOLUTION PLAYS OUT WITHIN GCS IN VIVO, FOCUSING ON THE INTERPLAY BETWEEN REPRODUCIBILITY AND STOCHASTICITY IN EVOLUTION. UNDERSTANDING SUCH EVOLUTIONARY ASPECTS WILL BE IMPORTANT FOR THE FIELD'S EFFORTS TO GUIDE B CELL CLONES THROUGH DEFINED AFFINITY MATURATION TRAJECTORIES THROUGH VACCINATION.