Antibodies Against Ebola and Lassa: A Global Collaboration

Antibodies Against Ebola and Lassa: A Global Collaboration


>>GOOD AFTERNOON, LADIES AND GENTLEMEN, WELCOME TO THE NOW 2017 JOSEPH J. KINOUN MEMORIAL LECTURE. MAY I HAVE HAVE NEXT SLIDE. SINCE 1979 WE AT NIAID HAVE HOSTED THIS LECTURE SERIES IN HONOR OF DR. KINYOUN PIONEER OF MODERN BIOMEDICAL RESEARCH. HE FOUNDED THE HYGIENIC LABORATORY IN 1887 IN NEW YORK CITY WHICH PAVED THE WAY FOR A NEW ERA OF COMBATING INFECTIOUS DISEASES THROUGH RIGOROUS SCIENTIFIC RESEARCH. SINCE THEN HIS ONE ROOM LABORATORY IN THE ATTIC OF THE STATEN ISLAND HAS GIVEN RISE TO THE 27 INSTITUTES AND CENTERS OF TODAY’S NIH AND HIS VISION CONTINUES TO INSPUE OUR APPROACH TO IMPROVING HUMAN HEALTH IN AMERICA AND AROUND THE GLOBE. MAY I HAVE THE NEXT SLIDE. TODAY’S SPEAKER, DR. ERICA OLLMANN SAPHIRE IS DIRECTOR OF THE VIRAL HEMORRHAGIC FEVER IMMUNOTHERAPEUTIC CONSORTIUM REFERRED TO AS VIC AND GLOBALLY RENOWN RESEARHER OF SMALL GENOME VIRUSES. AT THE VIC DR. OLLMANN SAPHIRE OVERSEES COLLABORATIVE NETWORK OF SCIENTISTS ON FIVE CONTINENTS WORKING TOGETHER TO INVESTIGATE THE STRUCTURES, FUNCTIONS AND WEAKNESSES OF SOME OF THE WORLD’S DEADLIEST VIRUSES INCLUDING LASSA AND EBOLA. BY FACILITATING LABS TO WORK TOGETHER THE VIC HELPED ACCELERATE DISCOVERIES THAT MIGHT OTHERWISE HAVE TAKEN MUCH MORE TIME, EFFORT AND FUNDING TO COMPLETE. FOR EXAMPLE, AFTER THE VIC’S FORMATIONNA 2013 THE CONSORTIUM CONNECTED RESEARCHERS FROM LABORATORIES ACROSS THE GLOBE IN AN EFFORT TO SYSTEMATICALLY INVESTIGATE PROMISING ANTIBODIES AGAINST EBOLA VIRUS. AS MEMBERS OF THE VIC, THESE RESEARCHERS ORCHESTRATED AND PERFORMED LARGE SCALE EXPERIMENTS ON THEIR SHARED COLLECTIONS OF ANTIBODIES ALLOWING THEM TO HONE IN ON THE MOST PROMISING CANDIDATES FOR EBOLA TREATMENTS FAR FASTER THAN ANY SINGLE LABORATORY COULD HAVE DONE ALONE. TODAY ONGOING WORK ON EBOLA, LASSA MARBERG AND OTHER VIRUSES AND EXPANDING OUR KNOWLEDGE AND PROVIDING INSIGHT HOW THEY CAUSE DISEASE. DR. OLLMANN SAPHIRE EARNED HER Ph.D. IN 2000 FROM THE SCRIPPS RESEARCH INSTITUTE IN LA JOLLA WHERE SHE’S PROFESSOR OF IMMUNOLOGY AND MICROBIAL SCIENCE WITH A JOINT APPOINTMENT AS PROFESSOR OF INTEGRATIVE STRUCTURAL AND COMPUTATIONAL SCIENCE. SHE ALSO SERVES AS A MEMBER OF SCRIPPS INSTITUTIONAL STEERING COMMITTEE AND THE SKAGGS INSTITUTE FOR CHEMICAL BIOLOGY. DURING THE PAST 17 YEARS HER RESEARCH PROVIDED REMARKABLE INSIGHTS INTO VIRUSES WITH EXTREMELY COMPACT GENOMES. OFTEN CONTAINING ONLY FOUR TO SEVEN GENES. SHE HAS INVESTIGATED ANTIBODY RESPONSES IN PATIENTS SUFFERING FROM EBOLA VIRUS DISEASE AND LASSA FEVER WHICH IS YIELDED VALUABLE INSIGHTS TO VIRAL STRUCTURE AND FUNCTION. ADDITIONALLY, HER RESEARCH TEAM WAS RESPONSIBLE FOR THE GROUND-BREAKING DISCOVERY THAT CERTAIN VIRAL PROTEINS CAN CHANGE THEIR STRUCTURES TO PLAY SEVERAL ROLES OVER TIME ALLOWING MORE INFORMATION AND FUNCTIONING TO BE ENCODED IN EXTREMELY CONCISE VIRAL GENOMES. TODAY SHE WILL ELABORATE ON RECENT DISCOVERIES RELATED TO THE EBOLA AND LASSA VIRUSES AND DESCRIBE HOW COLLABORATIVE RESEARCH CAN HELP FILL GAPS IN KNOWLEDGE AND LEAD TO INNOVATIVE NEW THERAPIES AN PREVENTION STRATEGIES. SO PLEASE JOIN ME IN WELCOMING DR. ERICA OLLMANN SAPHIRE. [APPLAUSE]>>THANK YOU VERY MUCH FOR HAVING ME HERE TODAY. IT’S A REAL HONOR FOR ME TO BE KINOUN AND HIS CONTRIBUTIONS, IT’S A PLEASURE TO SEE YOU AND TO GET TO HEAR ABOUT SOME OF THE SCIENCE HAPPENING HERE TODAY. SO I’M GOING TO TELL YOU TODAY ABOUT TWO VILLAGES. A FIGURATIVE VILLAGE, THIS ONE AND A LITERAL VILLAGE THAT HELPED OUR WORK AGAINST EBOLA AND LASSA. HERE IS THE LITERAL VILLAGE. MY LABORATORY FOCUSES ON TWO CLASSES OF VIRUSES THE FILA VIRUSES AND RAY RENA VIRUS. YOU HAVE SEEN THESE ON THE NEWS LONG FILAMENTOUS PARTICLES AND THERE WAS ONE 28,000 PERSON OUTBREAK. THE ARENA VIRUSES ARE SMALLER AND EXIT ALL OVER THE WORLD AND MUCH LARGER PUBLIC HEALTH PROBLEMS WITH HUNDREDS OF THOUSANDS OF CASES EVERY YEAR. THAT’S WHAT WE UNDERSTOOD ABOUT EBOLA VIRUS WHEN DISCOVERED IN 1976. THIS IS MORE WHAT WE UNDERSTAND ABOUT IT NOW. FROM THE WORK OF MY LABORATORY AND OTHERS. IT’S A LONG FILL MEANT US PARTICLE WITH AN OLD TELEPHONE CARD NUCLEAR CAPSID AT CENTER AND SINGLE GLYCO PROTEIN ON THE SURFACE. THE ONLY MOLECULE IT HAS TO ATTACH TOLL AND ENTER A NEW HOST CELL. SO GP IS THE MAJOR COMPONENT OF VACCINES BECAUSE IT’S THE MAJOR TARGET OF THE ANTIBODY RESPONSE AGAINST THE VIRUS. ONE OF THE MAJOR QUESTIONS THE CONSORTIUM I WILL TELL YOU ABOUT SET OUT TO ANSWER, WHAT ARE THE RIGHT KINDS OF ANTIBODIES AGAINST EBOLA VIRUS AND WHY AND HOW TO FIND THEM RAPIDLY AND WHAT MAKES THEM EFFECTIVE? SO LET ME INTRODUCE YOU TO THE MOLECULE ON THE SURFACE OF THE VIRUS, THE GLYCO PROTEIN GP. THIS IS DAVID STEWART’S STRUCTURE. TWO SUBUNITS, ONE IN BLUE IS GP 1, THE RECEPTOR BINDING SITE THAT ATTACHES TO THE CELL. AND GRAY IS SUBUNIT CALLED GP 2, THIS IS THE FUSION, THIS IS DRIVES FUSION OF THE VIRUS IN HOST MEMBRANES. IN THE RECEPTOR BINDING SUBUNIT THERE’S RECEPTOR BINDING CORE, A REGION CALLED THE GLYCAN CAP, NOT DRAWING SUGARS HERE BUT FOUR END LINK GLYCOSYLATION SITES IN THE CLUSTER AND FURTHER UPWARD THERE’S A 75-KILODALTON USE THAT HAS 20 OTHER SUGARS FLEXIBLE IN DISORDER, WE DON’T HAVE A HIGH RESOLUTION STRUCTURE, WE HAVE ENVELOPE FROM EM AND TOMOGRAPHY. THE FUSION SUBUNIT THERE’S SECOND HEAD TO HEAD PEAK WHICH MAKES THE STALK AT THE BOTTOM, THE FIRST PEP TOYED REPEAT WHICH WRAPS THE RECEPTOR BINDING CORE AND ALSO THE HYDROPHOBIC FUSION LOOP THAT IS SLAP ON THE OUTSIDE OF THE TRIMER LIKE A FLY SWATTER IN INFECTION THIS FUSION LOOP UNWRAPS FROM TRY MARE REACH UP AND PENETRATE THE TARGET CELL COLLAPSING THE SECOND DEBT REPEAT INTO THE FIRST ONE TO MAKE A SIX HELIX BUNDLE, IT THAT OPENS THE PORE IN THE ENDOSOME ALLOWING THE VIRAL MATERIAL TO PASS AND THE CELL BE INFECTED SO THIS IS THE MACHINE THAT DOES THE GEL GETTING EBOLA INTO THE CELLS. TRYING TO FIND THE RIGHT KIND OF ANTIBODIES AGAINST EBOLA GLYCO PROTEIN THERE’S TWO COMPLICATIONS. FIRST IS THIS, THAT’S NOT ACTUALLY THE MAJOR PRODUCT OF THE GLYCO PROTEIN GENE. THIS IS. IT’S A SECRETED DIMER CALLED SGP FOR SECRETED OR SOLUBLE. THE SURFACE GLYCO PROTEIN TRIMER THAT THE VIRUS NEEDS TO PROPAGATE IS ONLY A 20% OF THE TRANSCRIPTS OF THAT GENE. CAN ONLY BE ACHIEVED THROUGH SOME TRANSCRIPTIONAL EDITING EVENT. INSTEAD THE PRIMARY TRANSCRIPT MAKES AND SHIFTS THIS DIMER ABUNDANTLY TO INFECTED PERSON’S SEE RA. THIS DIMER SGP IS LIKE TWO GP 1 CORES STAPLED TOGETHER WITH BOND. SO IT ABUNDANTLY MAKE THIS IS PROTEIN HERE. THERE ARE ANTIBODIES SPECIFIC FOR THE VIRAL SURFACE GP RECOGNIZED CONTAINING GB 2, RECOGNIZED, THERE ARE OTHER ANTIBODIES THAT CROSS REACT BETWEEN TWO PROTEIN IT IS BECAUSE THEY SHARE SO MUCH SEQUENCE. AND SO THOSE KINDS OF ANTIBODIES MIGHT BE DISTRACTED BY THAT SHAPED DIMER IN INFECTION. ANOTHER COMPLICATION IS THAT THAT GLYCO PROTEIN IS A MOVING TARGET. THAT’S WHAT IT LOOK LIKE IN SURFACE OF THE VIRUS, SURFACE OF INFECTED CELLS. BUT AFTER THE VIRUS ENTERS CELLS SO IT ENTERS BY MACRO PIANO CYTOSIS, THE VIRUS IS SWALLOWED HOLE. ONCE INSIDE THE CELL, IN THE ENDOSOME, AWAY FROM IMMUNE SURVEILLANCE, IT HIGH JACKETS HUMAN ENZYMES CALLED CAP SAY SIN S TO STRIP THE MUCIN LIKE DOMAINS AND CAPS LEAVING BEHIND THIS RECEPTOR BINDING CORE, SO THE RESTORE BINDING SITE WHICH RECOGNIZED NPC 1 IS UNSHEATHED IN THE ENDOSOME AWAY FROM THE IMMUNE SUFFER VALENCE SO FINDS A INTRACELLULAR RECEPTOR, EIGHTH TAS BY ANY ONE OF A NUMBER OF LECTINS AROUND THE GLYCO PROTEIN OR BY INTERACTIONS WITH THE TOES FILL SERINE IN THE MEMBRANE. SO IT DROPS THE GP 1 ON ITS WAY IN TO THE ENDOSOME. THAT’S IMPORTANT FOR ANTIBODIES BECAUSE YOU CAN ELICIT A LOT OF THEM I AGAINST THE OUTER DOMAINS BUT ONCE INSIDE THE CELL IT DOESN’T CARE. IT USES CAP SAY SIN TO DROP THEM OFF LIVING BEHIND THE FUNCTIONAL RECEPTOR BINDING CORE. THOSE ARE COMPLICATION, HOW DO WE RAIDS AND EVALUATE THEM? WE RAISE A PANEL OF ANTIBODIES, WE TYPICALLY EVALUATE THEM FIRST BY CELL CULTURE NEUTRALIZATION, THE ABILITY TO BROCK INFECTION. THOSE THAT NEUTRALIZE GO INTO MICE FIRST, THEY GO INTO PROTECT THE GUINEA PIGS THEY GO TO NON-HUMAN PRIMATES THEN PROTECT NON-HUMAN PRIMATE THEY’RE A CANDID THERAPY FOR EMERGENCY USE. WE HAVE ALMOST NEVER DONE IS GO TO PRIMATES WITHOUT FIRST FILTERING AND FUNNELING OTHER SYSTEMS TO VET. WE DO THIS BECAUSE IT WORKS AND BECAUSE WE DON’T WANT TO USE PRIMATES UNLESS WE VETTED THINGS. BUT WE HAVE TO CONSIDER EACH PROCESS WE DOWN SELECT ANTIBODIES IS APPLYING SELECTION CRITERIA. WE MAY NOT ALWAYS UNDERSTAND WHAT IT IS THAT WE ARE SELECTING ON. WE HAVE DONE NEUTRALIZATION AS PRIMARY PASS. BECAUSE IT IS EASY CHEAP AND FAST AND IN OUR EXPERIENCE WE HAVE THOUGHT NEUTRALIZATION WOULD FORECAST PROTECTION IN VIVO IN THESE LIVING ANIMAL MODELS. THE TROUBLE WITH THAT IS IT’S NOT ALWAYS TRUE AND THERE ARE OUTLIERS IN THE LITERATURE. THERE’S THIS ANTIBODY, CALLED KC 52 FROM HUMAN SURVIVOR WHICH NEUTRALIZES IN CELL CHURL BUT NOT THE NON-HUMAN PRIMATES. THEY DIED. THERE’S COMBINATION OF THREE ANTIBODIES AGAINST THE MUCIN DOMAIN GLYCAN CAP, NEUTRALIZE POORLY IN CELL CULTURE BUT PUT THEM TOGETHER THE NON-HUMAN PRIMATES LIVE. SO WE HAVE ONE NEUTRALIZATION PROTECTION AND ONE HAS PROTECTION IN ABSENCE OF NEUTRALIZATION. THE OTHER ONE THIS ANTIBODY BOUND THE CORE THAT STAYS THE SAME THROUGH THE PROCESS AND THEY FOUND THE PARTS THAT GET CUT OFF. SO WHEN THAT PAIR OF CONFLICTING RESULTS CAME OUT IN THE LITERATURE WE THOUGHT WE DON’T REALLY UNDERSTAND AS MUCH AS WE THINK ABOUT THE SYSTEM. WHEN WE HAVE DONE NEUTRALIZATION, HAVE WE DONE IT THE RIGHT WAY, DIFFERENT WAYS TO GIVE DIFFERENT RESULTS, CERTAIN WILL I DIFFERENT PEOPLE IN THE FIELD HAD ANSWERS WHETHER SOMETHING WAS OR WAS NOT NEUTRALIZING ANTIBODY. IS NEUTRALIZATION THE RIGHT OR ONLY ASSAY TO DO? DO WE UNDERSTAND WHAT IT IS COULD CONFER PROTECTION AND HOW TO FIND I. IN THIS PROCESS WHAT IS IT WE HAVE BEEN FILTERING FOR, WHAT FALLS THROUGH THE TUNNELS AND WHAT COULD WE HAVE BEEN MISSING THE WHOLE TIME. WHEN WE THINK ANTIBODIES WE MIGHT THINK FOR TWO USES FIRST POST EXPOSURE IMMUNOTHERAPEUTIC, A PERSON IS VERY SICK YOU CAN DELIVER IT AND KNOCK DOWN THE LOAD AND GIVE THE PERSON A CHANCE TO RECOVER. THAT’S ONE TO THREE WELL CHARACTERIZED MONOCLONAL ANTIBODIES DELIVERED AFTER INFECTION. THAT’S HARD TO DELIVER ON A PUBLIC HEALTH SCALE. WHAT MIGHT ULTIMATELY DRAW A RING AROUND OUTBREAK SOMETHING TO DELIVER TO ABUNDANCE OF PEOPLE WOULD BE A VACCINE VACCINE ELICIT AS POLYCLONAL RESPONSE, THE VARIETY OF DIFFERENT TYPES OF ANTIBODIES WITH DIFFERENT FEATURES AND TYPES OF PROTECTION. SO IT WOULD BEHOOVE US TO DO A BETTER JOB UNDERSTANDING THE VARIETY OF ANTIBODIES THE LEAD TO PROTECTION SO WE CAN BETTER UNDERSTAND HOW TO MAKE THESE TWO DESIRABLE THINGS FOR HUMAN HEALTH. IN IGG AS YOU KNOW, HAS FAB ARMS, HYPERVARIABLE, WHAT SPECIFICALLY ANCHOR TO THE ANTIGEN. AND IT HAS AN FC DOMAIN, THAT IS WHAT LINKS TO THE IMMUNE SYSTEM. SO WHERE IS THE FAB, BINDS ON THE FOREIGN ANTIGEN AND DO THE JOB OF MECHANICAL NEUTRALIZATION SO PHYSICALLY BLOCK RECEPTOR BINDING CHANGES IN INFECTION ALL THE MAGIC OF INTERACTING WITH AND RECRUITING IMMUNE CELL KILLING OF INFECTED CELLS AND CLEARANCE OF PATHOGENS, HAPPENS THROUGH THAT FC. NOW, ONE MIGHT AFTER THAT WHEN WE APPLY THESE FUNNELS, WHAT DOES SUCCESSFULLY FALL THROUGH CONSISTENTLY MIGHT BE MECHANICAL NEUTRALIZATION. IF BINDING OF THAT FAB FRAGMENT PHYSICALLY BLOCKS RECEPTOR BINDING OR ANCHORS THE GLYCO PROTEIN TOGETHER SO IT CAN’T UNDERGO CONFIRMATIONAL CHANGE OR FUSE, THAT WILL HAPPEN IN A DISH IN RODENT AND PRIMATE AND HUMAN PROBABLY EQUALLY WELL. BUT ANYTHING INVOLVING SOMETHING FANCY LIKE RECRUITING PROPER IMMUNOEFFECTOR FUNCTIONS DIFFER. FC FUNCTIONS ARE DIFFERED IN RODENTS AND PRIMATES, THEY MAY NOT HAVE BEEN PRESENT IN THAT DISH. SO WE MIGHT HAVE BEEN MISSING ANTIBODIES LIKE THAT ALL ALONG. SO TO TRY TO UNDERSTAND WHAT IT IS THAT WORKS AND WHY, I HAVE THE PROBABLE THAT WE HAVE HAD, WHEN WE DESIGNED THE STUDY IN 2013 BEFORE THE OUTBREAK WE HAD A SMALL SAMPLE POOL, N OF 9 LOOKENING LITERATURE, MAYBE DIDN’T HAVE ENOUGH ANTIBODIES TO COME UP WITH STATISTICALLY SIGNIFICANT CONCLUSIONS. WE NEED AD BIGGER POOL. WHILE LOOKING AT A BIGGER POOL WE’RE COGNIZANT OF THE FACT ANTIBODIES HAVE LOTS OF VARIABLES IN STRUCTURES AN FUNCTIONS AND IN ORDER TO UNDERSTAND THAT WE NEED TO LOOK AT A LOT OF THEM. IF WE WANT TO SEE WHAT WE MIGHT HAVE MISSED WE CERTAINLY ARE GOING TO NEED TO LOOK FOR THOSE OUTLIERS. NOT ANTIBODIES THAT ONLY CONFORM TO OUR PREVIOUS SELECTION PROCESS BUT OTHERS THAT MAY HAVE FALLEN THROUGH. SO THIS IS WHAT WE FORM, THE VIRAL HEMORRHAGIC FEVER IMMUNOTHERAPEUTIC CONSORTIUM, VIC FOR SHORT. @WE HAVE GATHERED 200 MONOCLONAL ANTIBODIES AGAINST EBOLA GP FROM 43 LABS FIVE CONTINENTS. WE LET THE INVESTIGATORS DECIDE WHAT THEY WANT TO SEND US WHY, SOME SENT TWO OR THREE FAVORITE ANTIBODIES SOME OPENED FREEZERS AND SAID EVERYTHING. YOU’LL SEE BOTH ANTIBODIES AS I HOE YOU THE DATA. WHEN THEY COME INTO THE HEAD QUARTER LAB, THEY ARE ALL BLIND. THEY’RE GIVEN CODE NAMES. VIC 1,, 2, 3, THE GOAL IS TO MAKE IT FAIR. NOBODY KNOWS WHICH ANTIBODIES, JUST ANTIBODIES. YOU CAN ARGUE WHAT VIC 42 WAS WITHOUT INTERFERING WITH THE ABILITY OF THE INVESTIGATOR TO DISCOVER THE ANTIBODY SO THEY COME IN STANDARDIZED, BLINDED AND IDENTICAL BLOCK SETS AND SEND THEM PEOPLE IN THE FIELD TO DO WHATEVER THEY DO TO EVALUATE, THEY’RE GOING TO AT THIS TIME ON THE SAME SET. AND 3508 THE RESULTS WHEN WE GET TOGETHER AND THE GOAL IS TO NOT ONLY EVALUATE THIS PANEL OF ANTIBODIES AN SEE WHAT’S GOOD AND WHY, BUT AT THE SAME TIME EVALUATE THE ASSAYS TO FIGURE OUT WHAT EACH ONE FINDS AND WHY. SO E THIS IS WHAT WE DID. A LOT OF LABS WORKING TOGETHER OVER THREE AND A HALF YEARS, FROM THAT PANEL OF ANTIBODIES WE DID EVERYONE OF THESE STUDIES AND EVERYONE OF THOSE ANTIBODIES. STRUCTURE CROSS REACTIVITY, UNACCESSED BY DIFFERENT CONFIRMATION, IMMUNE FUNCTION, GLYCAN STRUCTURE, ABILITY TO PROTECT. MOUSE MODEL. SO HERE AGAIN IS GLYCO PROTEIN COLORED BY DIFFERENT PLACES ANTIBODY BINDS, WE WEDNESDAY INTO EPITOPE GROUPS SO WE HAVE 43 AGAINST THE FLY CAN CAP, 14 BIND AGAINST THE BASE. IF THERE ARE DEEP SHADE HUMAN ANTIBODY, A LIGHT SHADE MOUSE ANTIBODY SO THIS IS THE POOL WE HAVE, WE HAVE NUMBERS AND GROUPS OF DIFFERENT EPITOPES THIS IS WHAT WE LOOK LIKE. SO IN THE 8 ANTIBODIES AGAINST FUSION LOOP IN PURPLE SINGLE PARTICLE, THEY HIT ROUGHLY THE SAME PLACE. THEY COME AT IT FROM DIFFERENT ANGLES, MAYBE THAT’S IMPORTANT BUT WE CAN SAY THIS CLASS OF ANTIBODIES BINDS RELATIVELY OVERLAPPING FOOTPRINT AND THEY PROBABLY FUNCTION THE SAME WAY. WE CAN LOOK AT DIFFERENCES AMONG THEM. SO THOSE ARE ANTIBODIES AGAINST THE HEAD, GLYCAN CAP, THE MUCIN POP AND BASIC BOTTOM AND THE STOCK AT THE BOTTOM. IN SOME WE HAVE ANTIBODIES AGAINST ALMOST EVERY PLACE ORDERED ON THE GLYCO PROTEIN SO WE HAVE NEARLY COMPLETE COVERAGE OF THE MOLECULE WHICH IS KIND OF INTERESTING. WE TOOK THE ANTI-BOY BODIES SORTED TO EPITOPE GROUPS. AND WE DID THREE KINDS OF NEUTRALIZATION ASSAYS, WE LOOK AT ABILITY TO NEUTRALIZE A BSL 2 MODEL, THE GLYCO PROTEIN PSEUDOTYPE VESICULAR VIRUS, WE LOOK AT ABILITY OF ANTIBODY TO NEUTRALIZE A PARTICLE MISSING A GENE, OPERATE AT BSL 2, 3, ANOTHER MODEL AND LOOK AT THE ABILITY TO NEUTRALIZE A EBOLA VIRUS IN BSL 4, THESE ARE ALL VIRAL CELLS. WE LOOK AT THE FRACTION LEFT BEHIND WITH HIGHERS CONCENTRATION OF ANTIBODIES SO CONFIRMATION OR POPULATION OF GP NEVER ACCESSED. WE CAN RANK THEM BY CAPACITY NEUTRALIZE POTENTLY NOT AT ALL OR IN THE MIDDLE. THERE WAS ALSO LIKE THIS. SO WE CAN LINE UP ALL THIS WAY, WE ASSUME NEUTRALIZE IN EVERY ASSAY, WE HAVE MANY THAT NEVER NEUTRALIZE IN ANY ASSAY AND EVERYTHING ELSE IN BETWEEN. THERE’S THIS GROUP WHICH OFTEN NEUTRALIZES AND PARTICULARLY AT LOW — AT A LOW CONCENTRATION, THEY DON’T NEED MUCH. THERE’S THIS GROUP WHICH SOMETIMES NEUTRALIZES BUT NEED A HIGHER CONCENTRATION TO DO SO. THIS INTERESTING GROUP WHICH ONLY NEUTRALIZE EBOLA VIRUS AND NOTHING ELSE SO BACK TO THAT LATER. WE LOOK IN THE MIDDLE AND SAY WHAT IS SOURCE OF VARIABILITY. THE ANTIBODIES THAT COME UP TYPICALLY HAVE ONE OR BOTH TWO THINGS IN COMMON. FIRST IS THEY MIGHT HITTING T OR NEAR THRESHOLD CONCENTRATION. IF YOU NEED BIT MORE OR LESS HOVERING BY THE LINE, MIGHT REGISTER AS HIT OR NOT. THE SECOND THING THAT’S TRUE, THEY TEND TO RECOGNIZE EPITOPES THAT WE CAN’T FULLY CHARACTERIZE BY EM. SO SOMETHING MOBILE, SOMETHING HARD TO NAIL DOWN. úCOMMON THAT THEY HAVE ARE IN SIGNIFICANT PERCENTAGE OF VIRIONS THEY CAN’T NEUTRALIZE EVEN AT HIGHEST ANTIBODY CONCENTRATION. WHEN THERE’S A GLYCO PROTEIN MISSED THAT COULD BE FLY CAN STRUCTURE PRESENT OR ABSENT SO MEANING THAT COPY OF GLYCO PROTEIN CAN OR CANNOT BE IN ANTIBODY OR EXISTING IN DIFFERENT CONFIRMATIONS. ONE IS ACCESSIBLE, ONE IS NOT. THAT IS WHY THEY’RE DISCORDANT IN THE MIDDLE. SO WE RAN THROUGH SEVEN MEASURES OF IMMUNE EFFECTIVE FUNCTIONS WE LOOK AT THESE ARE HIGH THROUGH PUT IN VITRO ASSAYS ARRAY DON, WE LOOK FOR ABILITY TO RECRUIT PHAGOCYTOSIS AND MONOCYTES NEUTROPHILS AND MOUSE AND HUMAN CELLS AND ABILITY TO RECRUIT THREE DIFFERENT FUNCTIONS, NATURAL KILLER CELLS. YOU CAN PLOT RESULTS LIKE THIS. THIS IS THE FIRST 171 ANTIBODIES ALONG THIS AXIS, EVERY COLORED SPOT IS DIFFERENT IMMUNE ASSAY AND THE HEIGHT IS THE STRENGTH OF THAT FUNCTION. YOU CAN SUM UP AND GET A MEASURE OF POLYFUNCTIONALTY, ABILITY TO DO LOTS OF STUFF WELL. WE HAVE SOME ANTIBODIES THAT ARE HIGHLY FUNCTIONAL, WE HAVE SOME THAT ARE POORLY FUNCTIONAL AND WE HAVE EVERYTHING ELSE IN BETWEEN. THEN WE CAN MAKE EACH THE ARRAY OF FUNCTIONS AT THAT TIME PIE CHART SAY WHETHER STRONG MODERATE OR WEAK. AND GET IMMUNE EFFECTOR FOOTED PRINT OF EVERY ANTIBODY IN THERE, RELEASE GLYCANS TO FUNCTIONS, AND THEN EVALUATE FOR PROTECTION. SO WE PUT EVERYONE OF THE ANTIBODIES INTO THE MOUSE MODEL, EBOLA VIRUS LOOKING AT TEN MICE PER ANTIBODY, 100 MICROGRAM DOSE TWO DAYS POST EXPOSURE, MORE STRINGENT THAN THE LITERATURE STARTED AN ABILITY TO SURVIVE. WE HAVE 30 HIGHEST LEVEL PROTECTION, 103 DISAPPOINTING, 17 INTERMEDIATE GROUPS. SO AT THE END OF THREE AND A HALF YEARS HERE ARE THE ANTIBODIES, AND HERE ARE ALL THE FEATURES WE HAVE MEASURED IN EVERY ANTIBODY. WE DID MACHINE LEARNING. TO TEASE APART AND SEE WHAT MAKES SENSE. FIRST QUESTION YOU MIGHT ASK IS THERE A RELATIONSHIP BETWEEN EPITOPE AND NEUTRALIZATION, IS WHERE THE ANTIBODY BIND TELL YOU WHETHER OR NOT IT WILL NEUTRALIZE IN CELL CULTURE. IT MEANS ABSOLUTELY YES. HERE IS A GLYCO PROTEIN DOMAIN, A COLORED SPOT FOR EPITOPE GROUPS AND PERCENTAGE OF ANTIBODIES THAT FALL INTO THAT GROUP THAT NEUTRALIZE. ALL STOCK ANTIBODIES ANNUAL TRAILIZE, 63% POTENTLY, MOST FUSION ANTIBODIES, NOT SO MUCH, GET CUT AUTISM NEUTRALIZATION CONCENTRATES IS IN THIS ENVELOPE, THIS SHOULD LOOK FAMILIAR. THIS IS THE SHAPE OF GPCL, THAT’S THE CLEAVED FORM OF GLYCO PROTEIN THAT’S LEFT BEHIND THE ENDOSOME AFTER THE PROCESSING. SO IF THE ANTIBODY CAN ANCHOR WITH GOOD AFFINITY, ON THAT RECEPTOR BINDING CORE. IT WILL NEUTRALIZE IN CELL CULTURE. BY BLACKING RECEPTOR BINDING AND CONFIRMATIONAL CHANGES. IS THERE A RELATIONSHIP BETWEEN NEUTRALIZATION AND CELL CULTURE? AND PROTECTION IN A LIVING THING? YES. OKAY. INSTEAD OF COLORED SPOT FOR ALL OF THEM, I’M SHOWING YOU A SPOT FOR EVERY INDIVIDUAL ANTIBODY THAT GIVES DECENT LEVEL PROTECTION, ACCORDING TO ITS SET SO TEN AGAINST THE BASE, FIVE AGAINST THE STOCK, THESE ARE THE ONES THAT PROTECT NEUTRALIZATION STRONGLY CORRELATES PROTECTION, IN THAT ENVELOPE. ON GPCL SO IF THE ANTIBODY ANCHORS TO THAT RECEPTOR BINDING CORE, STAYS BOUND IN ENDOSOME AND NEUTRALIZES IT WILL PROBABLY PROTECT. THAT IS THE STRONGEST CORRELATION IN STUDY, 31 ANTIBODIES THAT FALL INTO THAT CATEGORY. IF YOU NEED TO FIND IMIMMUNOTHERAPEUTIC FAST FOR NEW VIRUS, DO THAT. IF YOU DO JUST THAT ONE THING, YOU WILL GET SOMETHING THAT IS USEFUL. OKAY. HERE IS THIS, IF ANCHORS WELL IN GPCL NEUTRALIZE AND PROTECT. HOWEVER, WE DID SOMETHING DIFFERENT HERE. WE DID NOT — WE FOUND OUTLIERS. BECAUSE WHICH DID BIG AGNOSTIC STUDY. WE DIDN’T DO THIS, WE STARTD WITH THE ASSUMPTION WE KNEW NOTHING T ALL AND GOING TO RUN EVERY ANTIBODY FOR EVERY EXPERIMENT AND SEE WHAT SHOOK OUT AT THE END. HERE AGAIN ARE ALL THE ANTIBODIES THAT PROTECT. HERE ARE THREE THAT DON’T NEUTRALIZE AT ALL IN ANY ASSAY WE HAVE, YET CONFER PROTECTION. THOSE ARE MODERATE, 60 TO 70%. LOOK AT THAT AGAINST MUCIN LIKE DOMAIN THAT PROTECT IT IS MICE, IN ABSENCE OF ANY MEASURABLE NEUTRALIZATION ANY ASSAY WE HAVE. LET’S — WORTH PAYING ATTENTION TO. HERE ARE SIX ANTIBODIES THAT ONLY NEUTRALIZE AUTHENTIC EBOLA VIRUS AND NOTHING ELSE AND REALLY BAD BUT NOT NEUTRALIZE AS WELL, IT REGISTERS MODERATE, HITS THERE AND NOTHING ELSE. THEY CAN CONFER 60 TO 90% PROTECTION IN MOUSE MODEL. HERE ARE FOUR MORE THAT DO REGISTER AS NEUTRALIZERS BUT VERY WEAK. YOU WOULDN’T WRITE HOME ABOUT THE NEUTRALIZATION, NEED HIGH CONCENTRATION TO DO SO, LEAVE A BIT BEHIND YET STILL CONFER 70 TO COMPLETE PROTECTION IN THE MOUSE MODEL, IN THE PRESENCE OF TRULY CRU MANY,MY NEUTRALIZATION SO 13 ANTIBODIES FALL INTO THE CATEGORY OF PUNCHING ABOVE APPARENT WEIGHT MEASURED IN NEUTRALIZATION, THEY DON’T DO IT AT ALL OR DON’T DO IT WELL. IF U YOU’RE SELECT AGNEW TRAILIZATION ALONE YOU WOULDN’T PICK THESE. BUT THEY PROTECT. AND THE OTHER THING THEY HAVE IN COMMON THEY BIND UPPER AND OUTER DOMAINS. WHERE WE CAN MAP THEM IN THE UPPER AND OUTER REGIONS. WELL, IF THEY’RE NOT PROTECTING BY PHYSICALLY INACTIVATING THE GLYCO PROTEIN, WHAT ARE THEY DOING? ONE POSSIBILITY IS USING FC TO TO RECRUIT EFFECTOR FUNCTIONS MAYBE BINDING AND TAGGING INFECTED CELLS FOR DESTRUCTION OR TAGGING FOR CLEARANCE. HERE IS A GLYCO PROTEIN, EVERY EPITOPE SPOT LOCATION. DIVIDE INTO PHYSICAL TIERS. TIER 1 ANTIBODIES RECOGNIZE THE TOP, TIER TWO MIDDLE, TIER THREE AT THE STALK. THOSE ARE LIKE THE THREE TIERS FOR THE GLYCO PROTEIN. IN THAT UPPER OUTER TIER, ARE WHAT WE CALL THE HEAD, THE GLYCAN CAP AND MUCIN LIKE DOMAIN. THAT’S TIER 1. TIER 1 SEEMS TO BE SPECIAL IN RECRUITING IMMUNE EFFECTOR FUNCTION. HERE PLOTTING POLYFUNCTIONALTY OF ALL ANTIBODIES IN THE STUDY WHERE THEY BIND ON GP, ABILITY TO RECRUIT SUM OF AS MANY FUNCTIONS AS POSSIBLE AS WELL AS POSSIBLE. TIER 1 ARE STRONGER THAN TIERS 2 AND 3. THAT WE THINK COMES FROM THE PARTICULAR MEASURES OF PHAGOCYTOSIS THAT ARE PART OF THE POLYFUNCTIONALTY. AND EVERY MEASURE OF PHAGOCYTOSIS TIER 1 IS A BIT STRONGER THAN TIERS 2 AND 3. SO LOOKS LIKE FURTHER UP AND OUT YOU GO BETTER ABLE YOU ARE TO RECRUIT PHAGOCYTIC DESTRUCTION OF CELLS AND OTHER IMMUNE EFFECTOR FUNCTIONS. THAT MAKES SENSE. IF YOU’RE UP OUT THERE WAYING IN THE BREEZE INSTEAD OF HID BELOW THE MEMBRANE YOU MIGHT HAVE BETTER PHYSICAL ACCESS TO THE IMMUNE CELLS. ALSO THOSE DOMAINS ARE FLEXIBLE, MOBILE, DISORDERED. WE HAVE NEVER GOTTEN A HIGH RESOLUTION STRUCTURE WITH MUCIN LIKE DOMAIN, MAYBE THAT FLEXIBILITY ALSO HELPS COUPLING IN SOME WAY OR HELPS AGGREGATION OF THE TWO FCs YOU MIGHT NEED TOGETHER TO GET CELL KILLING FUNCTIONS. SO WE CAN FIND TWO MAPS OF INFORMATION. NEUTRALIZATION HAPPENS IN THAT SHAPE. IF IT BINDS GPCL NEUTRAL’S CELL CULTURE AND PROTECT BIASIBILITY AND CONNER THAT MACHINE. IF IT ANCHORS TO THAT TIER IT HAS AN ADVANTAGE IN POLYFUNCTIONALTY, IN PARTICULAR PHAGOCYTOSIS. SO THOSE ARE THE KINDS OF ANTIBODIES WE FOUND TO ACHIEVE SOME PROTECTION IN THE ABSENCE OF MECHANICAL NEUTRALIZATION. IF WE ALSO KNOW THERE’S SOMETHING INTERESTING HERE THE GREEN SPOT, THE HEAD EPITOPE, THAT’S THE SWEET SPOT THAT HITS BOTH. THOSE ARE ANTIBODIES LIKE 114 THAT BIBBED THE HEAD AS MONOTHERAPY, MAYBE ABLE TO ACHIEVE PHYSICAL MECHANICAL NEUTRALIZATION AND RECRUIT EFFECTOR FUNCTION AS WELL SO A SPECIAL CLASS OF ANTIBODY. HERE IS THE OTHER THING. BECAUSE WE DID A BIG AGNOSTIC STUDY WE CAN QUANTITATE WHAT WE FINE. 18% SAMPLE POOL ARE ANTIBODIES THAT NEUTRALIZE WELL AND PROTECT BY THAT MECHANISM. 8% OF SAMPLE POOL GOES TO PROTECT IN ABSENCE OF GOOD NEUTRALIZATION, AND RECRUIT IMMUNE EFFECTOR. WE KNOW WE WOULD FIND THESE BECAUSE WE SOUGHT THEM ALL ALONG BUT IT WAS A SURPRISE HOW MANY OF THOSE WE FOUND. THOSE CAME FROM PEOPLE THAT SAID HERE IS A BOX WHATEVER IN MY FREEZER GO NUTS. WHEN SOMEBODY SAID TWO OR THREE OFTEN ONES FOCUSED ON OTHER SELECTION CRITERIA WHICH MAKES ME WONDER IF THERE WOULD BE MORE LIKE THIS ALL ALONG BUT HADN’T THOUGHT TO GO LOOK FOR THEM, MAYBE IN THE CUTTING ROOM FLOOR. NOW THAT WE HAVE 13 THAT FALL CERTAIN BIN AND HAVE CERTAIN FEATURES, WE CAN STUDY IT. WE CAN FIGURE OUT WHAT HAVE THEY GOT, THAT IS HELPFUL, AND CAN WE USE TO ENGINEER NEUTRALIZING ANTIBODIES AND MAKE THEM BETTER. SO I THINK THAT’S KIND OF A NEW FRONTIER. WE UNDERSTAND KNEW THAT WILLIZATION, YOU CAN SEE IT WITH STRUCTURAL BIOLOGY, WE KNOW HOW TO ACCESS IT. WE DON’T HAVE AS GOOD HANDLE ON HOW TO MEDIATE ALL THOSE IMMUNE CELL FUNCTIONS THAT ARE VERY IMPORTANT, ALSO PATHOGEN CLEARANCE AND SURVIVAL. DO WE HAVE ASSAYS WE NEED TO UNDERSTAND THE — DO WE KNOW HOW TO HARNESS AND ENGINEER ON TO IMMUNE THERAPEUTICS WE HAVE. DO WE KNOW WHAT QUESTION WE LEARN FROM THOSE 13 ENGINEER ON TO THE ONES WE MIGHT PUT IN THERAPY. BECAUSE POST EXPOSURE THERAPY 10 TO THE 9 VIRUSES PER ML YOU WANT THE MOST POTENT NEUTRALIZER YOU CAN FIND, IF YOU CAN ADD CELL KILLING MIGHT BE BETTER. IF A VACCINE IS A POLYCLONAL RESPONSE, THOSE ANTIBODIES ARE IN THERE. WHAT CAN WE LEARN FROM STUDYING MONOCLONAL TO HELP BETTER EVALUATE AND DESIGN OTHER VACCINES. WE ARE NOT THE ONLY ONES THINKING THIS WAY. MY DATA FOR EBOLA BUT NUMBERS OF GROUPS WITH OTHER VIRUSES LOOK AT CONTRIBUTION OF EFFECTOR FUNCTION ON TOP OF OR IN ABSENCE OF NEUTRALIZE RESPONSE ESPECIALLY WHEN IT’S VERY DIFFICULT TO GET A NEUTRALIZING RESPONSE IN THE CASE OF HIV VACCINE. SO WE HAVE GONE AND MADE CORRELATION NETWORKS FORE ALL VARIABLES MEASURED, PUT ON THE WEB THIS IS A SLIDER TO CLICK ANY FEATURE YOU WANT, SUGAR, EPITOPE AND LOOK AT ALL THE POSITIVE CORRELATES OF PROTECTION AND NEGATIVE CORRELATES OF PROTECTION FOR EVERY FEATURE AND HOW THEY’RE LINKED TOGETHER, HOW SPECIES LINKS TO SUGAR WHICH MAY OR MAY NOT LEAD TO CERTAIN EFFECTOR FUNCTIONS SO YOU CAN PLAY WITH THAT ON YOUR DESK. SO THAT WAS EBOLA VIRUS WHICH HAD ONE QUITE SIGNIFICANT OUTBREAK. I SAID THE ARENA VIRUSES ARE GENUINE PUBLIC HEALTH PROBLEM, EVERY SINGLE YEAR, HUNDREDS OF THOUSANDS OF CASES. THEY EXIST ALL OVER THE THE WORLD. THERE ARE A NUMBER OF DISEASE CAUSING ARENA VIRUSES IN THE NEW WORLD, THAT CAUSE A LETHAL HEMORRHAGIC FEVER. THERE ARE OTHERS IN THE OLD WORLD, ANOTHER ONE IS LCMV, A LABORATORY MODEL FOR US BUT ALSO CAUSES BIRTH DEFECTS IN FETUS, 2 TO 5% IS SERO POSITIVE. ARENA VIRUSES ARE THE SMALL ROUND PARTICLES LIKE EBOLA PUT ONE GLYCO PROTEIN ON THEIR SURFACE, ALL THEY HAVE TO MEDIATE ATTACHMENT AND ENTRY OF TARGET CELL. LIKE EBOLA AND HIV AND FLU AND EVERYTHING ELSE WE LOVE THEY HAVE A RECEPTOR BINDING SUBUNIT, A FUSION SUBUNIT, A TRANSMEMBRANE DOMAIN. AND THEY FORM A TRIMER ON THE VIRAL SURFACE, ONCE THE VIRUS IS INTO THE ENDOSOME RESPONSE TO RECEPTOR BINDING AND LOW PH RECEPTOR BINDABLE SUGGESTION UNIT IS RELEASED AND GP 2 REARRANGES TO SIX HELIX BUNDLE LIKE HIV FLU AND EVERYTHING ELSE. OVER THE LAST TEN YEARS, A NUMBER OF STRUCTURES HAVE EMERGED FOR KEY PARTS OF THIS FAMILY, GLYCO PROTEINS. HERE ARE STRUCTURES OF THE SIX HELIX BUNDLE FOR DIFFERENT POST FUSION ARENA VIRUS, THERE’S A STRUCTURE OF THE C TERMINAL CYTOPLASMIC DOMAIN, THERE’S A COUPLE OF STRUCTURES OF THE MONOMERIC RECEPTOR BINDING SUBUNIT BY EVENT AND FROM OXFORD ANGSTROM TOE MOW GRAPHIC RECONSTRUCTION OF GP TRIMER IN THE SURFACE OF THE VIRUS. BUT WHAT WE NEVER HAD WAS HIGH RESOLUTION STRUCTURE OF THE ENTIRE GLYCO PROTEIN ASSEMBLY, THE TRIMER ON THE VIRAL SURFACE. THAT’S WHAT WE WANTED IN ORDER TO UNDERSTAND HOW TO MAKE A VACCINE FOR LASSA, OTHER VIRUSES OR HOW TO INTERPRET ANTIBODY THERAPEUTIC. THAT WAS THE PROJECT THAT KATE HASTEY SET OUT TO SOLVE IN MY LAB IN 2007 TO START HER Ph.D.. SHE TOLD HER COMMITTEE FOR MY Ph.D. I WANT TO SOLVE ARENA VIRUS PRO FUSION TRIMER THEY LOOKED AND SAID GOOD LUCK, LADY. TEN YEARS LATER SHE GOT IT. SHE STAND IN MY LAB FOR Ph.D. POST-DOC AND SHE GOT OTHERS ALONG THE WAY, GOT HER DEGREE BUT IT’S HER PERSEVERANCE AND CREATIVITY THAT MADE THIS HAPPEN. THE ONE SHE GOT WAS FROM LASSA VIRUS WE GOT IT BECAUSE WE WENT TO AFRICA TO COLLABORATE WITH THE SCIENTISTS THERE TO DO THE WORK. SO WE WORK IN H SIERRE LEONE, FRY INTO FREE TOWN, DRIVE TO KINEMA, A COUPLE OF HOURS EAST, IN THERE IS A GOVERNMENT HOSPITAL. THIS IS THE LARGEST HOSPITAL FOR MILES AROUND. IN THE HOSPITAL THERE IS THE LOSS OF WORD, OLE WORD NEW WARD IS NOW UNDER CONSTRUCTION. THE LASSA WARD. UNTIL THE EBOLA VIRUS OUTBREAK THIS WAS THE ONLY WARD IN THE WORLD SOLELY DEDICATED TO TREATING VIRAL HEMORRHAGIC PATIENTS. HOLLOWED GROUND FOR TRAINING. ATTACHED TO THAT IS THE LAB TOY. BSL 3 WE SHY ACCESS TO PATIENT AND RODENT SERA. THERE ARE PEOPLE WORKING THERE DOING DIAGNOSTICS GENETICS STRUCTURAL BIOLOGY, IMMUNOTHERAPEUTICS AND EVALUATING VACCINES AND ALL KINDS OF WORK. THIS IS SOMETHING TULANE REBUILT AFTER THE CIVIL WAR, LOOKING INTO THE OLD LASSA WARD AND VIEW INTO THE LASSA LAB. SO PART OF WHAT THE GROUP DUD IS WE GO OUT ON THE ROADS INTO THE VILLAGES AND IN THE VILLAGES, THIS IS LENA MOSES, ONE OF THE PUBLIC HEALTH SCIENTISTS, SHE’S FEEDING THE CHIEF AND ONE OF HIS WIVES IN A VILLAGE. THEY GO IN WITH A TRAINED SIERRE LEONE ECOLOGISTS LOOKING FOR EVIDENCE OF RODENT INFESTATION, THEY’RE SPREAD BY UBIQUITOUS HOSTS, THIS IS A TYPICAL MUD AND STEEK CONSTRUCTION. YOU CAN SO THE LIT HOLES IN THE BOTTOM. THOSE ARE THE RODENT BUREAUS SO LOOKING FOR EVIDENCE OF RODENT INFESTATION WHERE THERE’S BEEN LASSA AND THEY TRACK AND SURVEY THE RODENTS IN THE HOUSE, THEY CONSENT AND SURVEY THE PEOPLE THEN AS NIGHT FALLS, ANOTHER GROUP THE PUBLIC HEALTH OUTREACH TEAM, AND THEY BRING IN A BED SHEET AND LAB TOP AND GENERATOR AND AS NIGHT FALLS THEY SHOWED MOVIES THEN START RAPPING. THROUGH RAP MOVIES AND SONGS TELL THEM ABOUT LASSA VARIETY, HOW TO STORE FOOD IN WELL SEALED BUCKETS. IF THEY HAVE SYMPTOMS THEY CAN GO TO A HEALTH POST WITH A CELL PHONE TO CALL THE LAND ROVER TO GET THEM AND BE TREATED FOR FREE. SO IT’S A WONDERFUL PUBLIC HEALTH OUTREACH SCIENTIFIC RESEARCH WRAPPED UP IN ONE SO WE WORKED IN THAT ENVIRONMENT FOR TEN YEARS. IN THIS COLLABORATIVE CIRCLE WHERE IN CALIFORNIA WE ENGINEERED GLYCO PROTEIN, WE USE THAT TO USE FOR HUMAN ANTIBODIES FOR PATIENTS SURVIVORS EMPLOYEES, THEY GOT THEIR JOBS BECAUSE THEY’RE SURVIVORS AND IMMUNE THEN ANALYZED IN TULANE, FOR TEN YEARS WEENING NEARED GLYCO PROTEINS TO PLUG ANTIBODIES TO IMPROVE THE GLYCO PROTEINS TO ANALYZE THE ANTIBODIES. AND FROM THE PATIENTS AND STAFF WE RAISED A PANEL OF ANTIBODIES. THIS IS WHAT THEY FOUND. THE BEST ANTIBODIES FROM SPECIFIC FOR THE TRIMER. THEY DON’T BIND, THERE’S THE SIMPLE PARTS. THOSE KNOWN BODIES ALSO GAVE US THE FIRST STRUCTURE OF THE PREFUSION ARENA VIRUS TRIMER. LET ME SEE IF THIS WILL START UP. SO THE HUMAN EYE HAS EVOLVED TO SENSE MOVEMENT. SO YOU CAN SEE PREDATORS IN THE FOREST SO YOU CAN APPRECIATE A STRUCTURE BETTER IF YOU CAN SEE IT MOVE. HERE IS THE LASSA — THIS IS OUR FIRST LOOK AT THE PREFUSION TRIMER FOR ARENA VIRUS GLYCO PROTEIN. WE WORKED WITH TULANE, COLORED IN MARDI GRAS COLORS, THE GP 1 SUBUNIT IS IN THE LIGHT SHADE AT TOP, GP 2 FUSION SUBUNIT IN DARK FADED BOTTOM. GP 1 IS ROUND LIKE A SCOOP OF ICE CREAM, GP 2, HELICES MAKE A CONE THAT THE GP 1 SCOOP SITS IN. THEN HERE THERE’S A LONG MELTING DRIP OF GP 1 THAT COMES DOWN TO MAKE A BETA SHEET WITH GP 2, THAT LOOKS LIKE FLU TO ME. SO THAT’S THE ARRANGEMENT OF THE PRE-FUSION TRIMER. NOW IF YOU ROLE IT AROUND LOOK DOWN THE CENTER YOU CAN SEE WHERE THE GLYCANS ARE, THERE’S 11 PREDICTED GLASS OSCILLATION SITES ON EACH MONOMER, 33 IN THE TRIMER EVERYONE IN THE CRYSTAL STRUCTURE. SO THE WHOLE THING IS COVERED WITH FLY CAN. I SHOW YOU THE AMOUNT OF SUGAR IN CRYSTALS BUT YOU KNOW THERE’S MORE DISORDER. THIS SUGAR IS PROBABLY THIS LONG SO THERE’S A GLYCAN CODE, LOOK AT PETER CONGRESS’S WORK, IT WILL LOOK LIKE THAT, PROBABLY NOT AS HAIRY. THERE’S A COUPLE OF PLACES ON THIS GLYCO PROTEIN NOT MASS SPEC CARBOHYDRATE. HERE IS ONE T. GET BETTER LASER. AT THE BOTTOM AND THE BASE. THAT IS EXPOSED. ANOTHER SITE IS HERE ON ON THE SIDE, ANOTHER ONE THE TOP NEAR WHERE THE NEW VIRUS S BIND DIFFERENT RECEPTOR, THOSE ARE THE SPOTS ANTIBODY RECOGNIZED. SO THIS IS THE FIRST ONE THAT CRYSTALLIZE, ONE POTENT NEUTRALIZER. CALLED 37.7H, WHAT IT’S DONE IS FOUND BARE SPOT AT THE BOTTOM. HERE IS THE OTHER COOL THING ABOUT IT. NOW YOU SEE WHY SPECIFIC FOR THE TRIMER. IT DOESN’T BIND POST FUSION GP 2 OR 1, ANTIBODY SIMULTANEOUSLY INTERACTS WITH DIFFERENT COPIES OF THE TRIMER AT THE SAME TIME. WE PUT ENGINEERING INTO THAT PROTEIN TO MAKE IT STAY STABLE IN PREFUSION CONFIRMATION INSTEAD OF DOING WHAT IT WANTS TO DO, SPRING TO POST FUSION SIX HELIX BUNDLE, BUT WE GOT IT RIGHT BECAUSE IT FITS THE ENVELOPE OF THE TOPO GRAPHIC RECONSTRUCTION ON A FENTY CLASS OF VIRUSES, SUGARS DISORDERED. SO NEUTRALIZING ANTIBODIES, THIS IS THE VAST MAJORITY OF NEUTRALIZING MONOCLONAL HIT THAT SPOT. THAT’S THE MAJOR SITE OF NEUTRALIZATION. THAT’S THE FUSION MACHINERY AT THE BOTTOM. IT’S SPECIFIC FOR THE TRIMER, IT RECOGNIZES THE QUATERNARY EPITOPE BECAUSE EVERY GLYCO PROTEIN SIMULTANEOUSLY INTERACTS WITH TWO ANTIBODIES AND EVERY ANTIBODY BINDS TWO GLYCO PROTEINS AT THE SAME TIME. I’M SHOWING YOU ONE OF THOSE GLYCO PROTEIN IT IS HOW IT’S BOUND BY TWO. I WILL RECOLOR THE FUSION SUBUNIT BY COMPONENT PARTS, PEPTIDE REPEAT 1 H LITTLE BITS, PEPTIDE 2 IN GREEN, THIS IS THE FIRST TIME WE HAVE SEEN THE FUSION MACHINERY FOR THE ARENA VIRUSES, HAS THE FUSION LOOP AND FUSION PEPTIDE. IF WE EXTRACT THAT PREFUSION GP 2 AND COMPARE T TO THE MONOMER YOU CAN SEE CONFIRMATIONAL CHANGES THAT HAPPEN IN FUSION. ALL THOSE BROKEN YELLOW HELICES REASSEMBLE TO THE ONE ROD, THAT GREEN PEPTIDE REPEAT 2 FLIPS FROM BOTTOM TO TOP, AS THE FUSION MACHINERY GOES UP AND T LOOP CHANGES FROM A STRAND TO HELIX IN LOOP SO THAT’S CONFIRMATIONAL CHANGE IN FUSION SUBUNIT FROM THE META STABLE TRIAL TORE THE SUPER STABLE SIX HELIX BUNDLE INFUSION. NOW WE EXPECTED THAT T. AS VIROLOGISTS WE HAVE GROWN UP ON MOTHER’S KNEE LEARNING THAT CLASS 1 GLYCO PROTEINS SPRING INTO SIX HELIX BUNDLE. WHAT WE DIDN’T EXPECT WAS CONFIRMATIONAL CHANGE AND RECEPTOR SUBUNIT AS WELL. HE SAW THE STRUCTURE OF LASSA GP 1 AS MONOMER LOW PH 5 ENDOSOME. COMPARE HIS STRUCTURE TO OURS AT THE TRIMER AT PH 8 THERE’S A NUMBER OF DIFFERENCES. SEE THEM BETTER IF I MORPH ONE THAT ARE COORDINATE INTO THE OTHER. AS WE GO FROM GP 1, THE GP 1 RELEASE IN ENDOSOME YOU CAN SEE THE BETA SHEATHE AT CENTER STAYS THE SAME BUT EVERYTHING ELSE REARRANGES, ALL THE HELICES AND LOOPS. GP 1 ONCE OFF ITS TRIMER IS THE WRONG SHAPE. OR DIFFERENT SHAPE THAN GP 1 IN TRIMER. ANTIBODIES RAISED AGAINST RELEASE GP 1 MIGHT NOT RECOGNIZE TRIMER IN VIRAL SURFACE. WE LEARN BOTH SUBUNITS ARE DIFFERENT SHAPES OUT OF THE TRIMER THAT — IN THAT TRIMER. WE LEARN THAT THE MAJORITY OF THE NEUTRALIZING ANTIBODY RESPONSE FOR LASSA NEEDS THAT TRIMER TO BE PREFUSION, AND NEEDS TO BE A TRIMER. THOSE ANTIBODIES ARE HIGHLY EFFECTIVE PASSIVE IMMUNOTHERAPY THAT’S A THE NEWSPAPER FROM TOM GUISEMAN’S LAB THE ANTI-BOY DOS BY THEMSELVES CONFER PROTECTION AND PRIMATE MODELS PRETTY LATE DISEASE COURSE. SO ANTIBODIES AGAINST LASSA CAN BE GOOD, IT CAN BE PROTECTIVE. BUT YOU HAVE TO SHOW THEM THE RIGHT THING TO GET THEM. WE LEARNED THE BEST ANTIBODIES SPECIFIC FOR THE ASSEMBLED TRIMER THEY DON’T BIND DISASSEMBLING OR DISASSEMBLE PARTS. THAT’S IMPORTANT BECAUSE WHY WOULD TYPE GLYCO PROTEIN, WE SPENT TEN YEARS ENGINEERING THAT. THE WILD TYPE GLYCO PROTEIN MAKES ALL OF THOSE SO IF YOU PUT THE WILD TYPE GLYCO PROTEIN ON A VACCINE, YOU WILL GET ALL THOSE DEPENDING HOW YOU TREAT IT, THIS IS THE STORY YOU MIGHT GET MORE THAN YOU WANTED. THEN IF YOU READ THE LASSA VACCINE LITERATURE, THIS IS WHAT YOU READ, THE T-CELL RESPONSE IS A CLEAR CORRELATE OF PROTECTION ANTIBODY HAS NOT YET BEEN FOUND TO BE. ANTIBODIES DO NOT CORRELATE PROTECTION. HERE IS OUR NEW IDEA. CAN WE USE THIS HARD ONE STRUCTURE AS BLUEPRINT? WE HAVE ENGINEERED TO REMAIN IN THE SAME STRUCTURE, IT FAITHFULLY PRESENT IT IS NEW NEUTRALIZING EPITOPES IF WE PUT THAT ON VACCINE PARTICLE CAN WE IMPROVE NEUTRALIZING ANTI-BE DIRESPONSE. MAYBE HAVING BOTH ANTIBODIES AND T-CELLS TOGETHER WOULD BE BETTER THAN T-CELLS ALONE, SO THERE’S A NUMBER OF GROUPS USING THIS GLYCO PROTEIN, TRYING TO MAKE A BETTER VACCINE FOR LASSA VIRUS, USING THIS TEMPLATE TO TRY TO UNDERSTAND THE DIFFERENT ARENA VIRUSES THAT CAUSE PROBLEMS FOR HUMAN HEALTH WORLDWIDE. SO THIS IS THE TEAM FOR LASSA VIRUS. I ESPECIALLY WANT TO THANK KATE WHO HUNG IT UP FOR TEN YEARS AND TRIED EVERYTHING TO MAKE THE STRUCTURE WORK AND HER TECHNICIAN WITH MICHELLE, THAT’S KATE IN AFRICA AND IN CALIFORNIA. LEWIS BARANCO WHO WORKED WITH US LINEAGE SPECIFICITY PANEL OF ANTIBODIES AND HERE IS LEWIS IN AFRICA AND MARYLAND. AUGUSTINE GOTA IN THE LAB AND DONALD GRANT MD MANY THE LASSA WARD. THIS IS MY GROUP AT SCRIPPS. THEY’RE ALL INSIDE PACKING CRATE NUMBER 18 OF 18 OF NEW ELECTRON MICROSCOPE AND I WANT TO THANK ALL THESE PEOPLE THAT DONATE AD LOT OF TIME AND A LOT OF WORK. STUDY AND SKILL WOULD ONLY HAVE BEEN POSSIBLE FOR PEOPLE WILLING TO DO A LOT OF WORK. OTHER PEOPLE SAMPLE TO SEE WHAT WE COULD FIND. THANKS TO THE TWO PROGRAM MANAGERS FOR TREMENDOUS AM OF WORK ORGANIZING THIS AND KEEPING TRACK OF THIS AND SHIPPING AN COORDINATING THE DATA. I WOULD LIKE THE THANK THE PEOPLE WITH THE HEAVY LIFTING, BSL 4. JOHN DYE AND GARY CARPENTER, I WOULD LIKE TO THANK NIH TO THEIR SUPPORT. EVERY DOLLAR IS SUPPORTED BY EXTRAMURAL FUNDS AND WE ARE TREMENDOUSLY GRATEFUL AND COULDN’T HAVE DONE IT WITHOUT THEM AND THANKS TO THE PROGRAM OFFICER WHOSE OVER THE YEAR SHEPHERDED THE PROGRAMS FROM RECEPTION TO THERAPEUTIC. I WOULD LIKE TO THANK YOU FOR YOUR ATTENTION, THAT’S THE FOUNDING PRINCIPLE OF THE LAB AND I WOULD BE HAPPY TO ANSWER ANY QUESTIONS. [APPLAUSE]>>THAT WAS ABSOLUTELY SPECTACULAR. QUESTIONS, COMMENTS? WHILE THEY’RE THINKING, SO I COULDN’T BE STRUCK BY YOUR LONG TERM FOCUS IS ON MONOCLONAL ANTIBODIES THAT COULD BE PASSIVELY TRANSFERRED. ARE YOU DOING ANY WORKER DO YOU SEE ANY LIGHT TO LOOK AT THE EPITOPES THAT ARE PROTECTIVE BUT NOT NECESSARILY NEUTRALIZING AND CAN YOU INDUCE THEM OR ARE THEY IMMUNODOMINANT OR ARE THEY NOT IMMUNODOMINANT? IF YOU VACCINATE WILL YOU GET THOSE OR WON’T YOU? ANY IDEA? THAT’S WHAT WE’RE DEALING WITH.>>THAT’S WHAT WE OTHER DOING NOW, COUPLE OF THINGS. WE ARE LOOKING AT THOSE ANTIBODIES THAT POTENTLY NEUTRALIZE ALL THE DIFFERENT — WHAT DO YOU RECOGNIZE AND HOW DO WE MAKE A MOLECULE THAT BETTER DISPLAYS THAT. MAYBE IT WORK, MAYBE IT WON’T, MAYBE IT WILL ALSO WORK JUST TO DISPLAY THEM ONE BY ONE IN WILD TYPE. SO LOOKING AT THAT, INTERMARRY GP TO DISPLACE THOSE THINGS I’M REALLY INTERESTED IN THAT QUESTION OF WHAT THE VARIETY OF THINGS ELICITED BY VACCINATION, HOW DO THEY TOGETHER CONTRIBUTE TO PROTECTION. WHICH LOOK AT ANTIBODIES INDIVIDUALLY BUT WHEN YOU DELIVER TWO OR THREE TOGETHER OR POLYCLONAL RESPONSE THEY INTERACT. WE DO SEE PATTERNS WHEN ANTIBODIES MINE SOME EPITOPES THEY ENHANCE EXPOSURE OF OTHER EPITOPES. IS THAT IMPORTANT AND CAN WE HARNESS BY TETHERING OR CO-DELIVERING THEM. TO WHAT EXTENT ARE THOSE EFFECTOR FUNCTIONS CRITICAL FOR CLEARANCE? HOW DO WE STEER THAT? IT’S A BALANCE BETWEEN YOU’RE NERVOUS ABOUT THE EPITOPES BECAUSE THINK OOHER MORE VARIABLE IN SEQUENCE, THE MACHINERY IS CONSTANT SO IF THE VIRUS MUTATES AS THEY DO, YOU MIGHT LOSE THEM BUT THEY SEEM TO BRING SOMETHING INTERESTING AND IMPORTANT. (OFF MIC)>>WHAT DO WE KNOW ABOUT EBOLA, NOT AS MUCH AS WE SHALL. WE DO KNOW IF THE A ANTIBODY FALLS OFF, IT WILL BE — ONES REMAIN ANCHORED THAT’S A GOOD CRITERIA TO LOOK FOR. WHY THE ABSOLUTE REQUIREMENT FOR LOW PH OR IN DIRECT OPERATING WITH SOMETHING ELSE OR JUST THERE, WE HAVEN’T FULLY TEASED APART, SOMETHING WE DON’T FULLY UNDERSTAND EVERYTHING REQUIRED TO SPRING GP OR JUST MISCELLANEOUS DESTRUCTION BY VARIETY OF PROTEASES. REMAINING BOUND WILL BE CRITICAL FOR KNEW THAT WILLIZATION FOR ENDOSOME. THE ONES THAT TAG FOR DESTRUCTION LESS IMPORTANT. WE SURE DON’T UNDERSTAND EVERYTHING WE NEED TO.>>THANK YOU. THAT WAS REALLY FUN FOR ME TO WATCH YOU DO THAT. I HAVE A LOT OF QUESTIONS. SO IF SOMEBODY ELSE HAS ONE YOU CAN INTERRUPT. YOU SHOWED MOST ANTIBODIES WERE TO THE STEM. LOOKED LIKE A STEM OR MAYBE THE PLACE BETWEEN THE STEM AND THE HEAD. THOSE ARE FUSION INHIBITING. DO YOU HAVE ANY ANTIBODIES THAT BLOCK ATTACHMENT?>>I DO. MY LAPTOP FAILED SPECTACULARLY LAST NIGHT. I DON’T HAVE THOSE PICTURES. WE HAVE SHOWN YOU THE ONES THAT WE HAVE BOUND — WE GOT STRUCTURE FIRST THAT ANCHOR — FOCUS ON THAT BECAUSE COULDN’T NEUTRALIZE THE ANTIBODIES BIND IN SAME PLACE SO WE HAVE BUNCH OF STRUCTURES TO FIGURE WHAT’S THE BETTER ONE, THE WORSE ONE, HIGHER AFFINITY, MORE CONTACTS THE BETTER IT IS, HOW IT IS WITH MACHINERY. THOSE ONES THAT WE THINK WORK BY BLOCKING FUSION ALSO INHIBIT BINDING THE LAMP RECEPTOR, BUT WE THINK IT’S NOT DIRECT BECAUSE LAMP IS HERE AND FABS ARE THERE WHAT THEY DO IS PREVENT — LOOK AT THE OXFORD TO MAG PHI OF THE GLYCO PROTEIN HERE AND LOW PH IT OPENS UP LIKE ONION AT APPLEBEE’S. IT OPENS LIKE THIS. AND WE THINK THAT WHEN THE ANTIBODIES ANCHOR TO THE BOTTOM IT CAN’T QUITE OPEN. WE HAVE OTHERS THAT DO NOT NEUTRALIZE AS POTENTLY BUT DO ANCHOR THE SIDE AND FIDSICALLY BLOCK SO WE HAVE THOSE AS WOMEN — AS WELL AND OTHERS THAT ANCHOR STEM MACHINERY WE’RE TRYING TO TEASE APART.>>PART OF THAT QUESTION WAS TO UNDERSTAND WHAT DO YOU THINK THE FIRST TRIGGER IS FOR STARTING THE REARRANGE PROCESS? TRIGGERED BY RECEPTOR BINDING OR IS IT TRIGGERED BY THE STEMS PULLING APART FROM EACH OTHER? WHAT IS IT THAT TRIGGERS THE REARRANGEMENT?>>CHICKEN OR EGG? LAMPER LOW Ph.D. BECAUSE ONE IS REQUIRED FOR THE OTHER. I DON’T REALLY KNOW. I KNOW THAT ONE CELL SURFACE ATTACHMENT REQUIRES INTACT TRIMER THOSE POINT MUTATIONS THAT CHANGE ARMSTRONG CHANGE THE AFFINITY, KEEP IT FROM MAINTAINING THIS, SO IT HAS TO BE THIS TO ANCHOR IN THE CELL SURFACE, ONCE INTO THE ENDOSOME MY GUESS IS LOW PH WILL START OPERATING ON A HISTIDINE TRIAD AND TRIGGERING THAT REASSEMBLING GP 1 THAT RELEASES FROM DP 2, EXPENT TO WHICH HATCH DRIVES THAT ITSELF OR LOCATION, I DON’T FULLY KNOW. I’M GOING TO GUESS LOW PH GETS IT GOING. MAYBE SOMEBODY ELSE HAS NEWER BETTER DATA.>>ACTIVE MORE CLINICAL QUESTION IF — KAY.>>EXTENT STRUCTURAL BIOLOGIST CAN ANSWER IT.>>THERE MAYBE A STRUCTURAL ANSWER TO THIS PROBLEM. BECAUSE IMMUNE VIRUSES THAT CAUSE ARGENTINIAN HEMORRHAGIC FEVER IS ARENA VIRUS. CLASSICALLY ARENA VIRUSES, THAT’S WHAT WE ALL HAVE LEARNED T-CELL. ARENA VIRUSES YOU THINK T-CELLS BUT VIRUS YOU CAN TREAT WITH ANTIBODY SO TAKE PUBLIC ANTIBODY AND TREAT IT. THE THING I WANT TO KNOW IF YOU UNDERSTAND IS 10% PEOPLE WHO ARE TREATED AND SURVIVE LEAD VIRUS INFECTION END UP WITH CEREBRAL DISORDER THAT EVENTUALLY KILLS AND PROBABLY HAS SOME VIRUS TO THE BRAIN ANTI-BE DITREATMENT, THAT’S NOT VERY UNDERSTOOD SO ANTIBODIES ALONE GIVEN PASSIVELY FAIL IN SOME PERCENTAGE OF THOSE EVEN POLYCLONAL ANTIBODIES FAIL. SO THE QUESTION IS, HAVE YOU BEEN ABLE TO RECAPITULATE ANYTHING LIKE THAT EITHER ANNOUNCING — WHO YOU WORK WITH OR LASSA OR OTHER THINGS, WHEN YOU JUST GIVE PASSIVELY YOU END UP WITH PROBLEMS IN THE BRAIN.>>I HAVEN’T, ONLY ANTIBODY TREATED PEOPLE.>>WHO DOESN’T EVER HAVE SURVIVING BY REGULAR INFECTION. ONLY HAPPENS WHEN YOU’RE TREATED WITH (INAUDIBLE).>>IS IT PASSIVE HUMAN ANTIBODY? THAT’S INTERESTING. (OFF MIC)>>I DON’T KNOW. INTERESTING QUESTION.>>IF THERE ARE NO OTHER QUESTIONS, THANK YOU VERY MUCH.

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About the Author: Oren Garnes

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  1. In Korea up 12 miles south of the dmz.We had 10 guys tha got Hemorrhagic fever.9 died within 3 days after noticing the first sympoms. The only reason one guy survived was he received dialysis quick enough.Scary shit since it can lie dormant 45 days.

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