I'M MICHAEL GOTTESMAN DIRECTOR FOR THE INTRAMURAL RESEARCH. JOHN SCHILLER FROM THE NCI AND ROB TYCKO FROM NIDDK. REPRESENTATIVES FROM THEIR INSTITUTES WILL INTRODUCE THEM BUT I WANTED TO TAKE A MINUTE TO INTRODUCE THIS REMARKABLE CONCEPT OF A YEARLY MINI-SYMPOSIUM. IT WAS ESTABLISHED BY THE U.S. CONGRESS IN 1863. THIS WAS ABE LINCOLN'S IDEA. MEMBERSHIP IN THE NIS IS CONSIDERED TO BE ONE OF THE HIGHEST HONORS BESTOWED ON A SCIENTIST. THE ACADEMY MEMBERS NOMINATE THOSE THEY FEEL REPRESENT THE VERY BEST IN THEIR SCIENTIFIC FIELD. THEY MUST COME HAVE INSTITUTIONS OTHER THAN THEIR OWN. THESE NOMINEES ARE EXTENSIVELY VETTED AND IF THEY PASS MUSTER THEN CAST THE FINAL BALLOT AT A FINAL MEETING IN APRIL. A MAXIMUM OF 100 SUCHLTH U.S. MEMBERS ARE CHOSEN ANNUALLY. AND EVERY YEAR A PRINCIPAL INVESTIGATOR HAS BEEN ELECTED. THIS IS REMARKABLE FOR TWO REASONS. THERE'S ONLY ABOUT 1,000 PRINCIPAL INVESTIGATORS HERE AND UNIVERSITIES OFTEN HAVE MANY MORE AND THE NIH IS PREDOMINANTLY A BIO MEDICAL ENTERPRISE AND NIS ENCOMPASSES ZOOLOGIST AND AS -- AS STRONG MISTS. MORE THAN 50 OTHER NIHERS NOW RETIRED OR DECEASED HAVE BEEN HONORED WITH NIS MEMBERSHIP. THAT ADDS UP TO MORE THAN 100. AS THEY SAY IN LATE NIGHT INFO INFOMERCIALS THERE'S MORE. WE HAVE MORE THAN 60 ACTIVE P.I.s IN THE NATIONAL ACADEMY OF MEDICINE AND THIS YEAR PETER BASSER A SENIOR INVESTIGATOR IN NICHD WAS ELECTED TO THE BIO ACADEMY OF ENGINEERING. THE TAKE-HOME MESSAGE IS YOU'RE WORKING AMONG SOME OF THE WORLD'S GREATEST SCIENTISTS AND WE THINK THIS IS AN OPPORTUNITY TO CELEBRATE THEIR ACCOMPLISHMENTS AND TO LEARN FROM THEM. THAT'S WHY WE STARTED THE NIS MINI SYMPOSIUM 2014. NOW, LET ME HAND THE MIC OVER TO DOUG LOWY TO INTRODUCE OUR FIRST SPEAKER, JOHN SCHILLER. DOUG. >> GOOD MORNING, EVERYONE. THANKS VERY MUCH. I WANT TO APPLAUD MICHAEL FOR ORGANIZING THIS SYMPOSIUM AND THE PLAN TO HAVE IT BE AN ANNUAL EVENT. BEFORE INTRODUCING JOHN, I WANT TO HAVE ONE HOUSEKEEPING POINT, IF YOU HAVE QUESTIONS DURING EITHER TALK, PLEASE SUBMIT THEM VIA THE VIDEO WEB SITE AND LOOK FOR AND CLICK ON THE WORDS LIVE FEEDBACK FORUM. THIS IS AT THEBOTTOM OF THE WEB PAGE YOU'RE WATCHING AND WE'LL RELAY THE QUESTIONS TO THE SPEAKERS AT THE END OF THEIR LECTURES. NOW TO JOHN. HE RECEIVED HIS BACHELOR'S DEGREE FROM THE UNIVERSITY OF WISCONSIN AND Ph.D. FROM THE UNIVERSITY OF WASHINGTON. HE HAS WORKED AT THE NIH SINCE THE EARLY 1980s AND HE AND I HAVE WORKED TOGETHER FOR MORE THAN 30 YEARS. THE VAST MAJORITY OF OUR RESEARCH HAS BASKETBALL WITH HUMAN PAPILLOMA VIRUS AND BEST KNOWN FOR THE DEVELOPMENT OF THE TECHNOLOGY UNDERLYING THE HPV VACCINE. I WANT TO TELL YOU HOWEVER, THAT JOHN HAS GONE FAR BEYOND THAT RESEARCH AND YOU WILL HEAR BOTH ABOUT THE RESEARCH WITH THE VACCINE AS WELL AS OTHER RESEARCH ACCOMPLISHMENTS. I SHOULD ALSO NOTE IT IS MORE DIFFICULT FOR THE SECOND PERSON IN A DUO TO BE ELECTED TO THE NATIONAL ACADEMY OF SCIENCES AND IT IS A TESTIMONY TO JOHN'S STELLAR CREATIVITY AND ACCOMPLISHMENTS THAT ENABLES HIM TO BE ELECTED. BEFORE FINISHING, I JUST WANT TO SAY THAT JOHN HAS BEEN THE RECIPIENT OF MANY AWARDS. THIS IS JUST A SUBSET. THE NATIONAL MEDAL OF TECHNOLOGY AND INNOVATION FROM PRESIDENT OBAMA THE PROGRESS IN CANCER RESEARCH AND THE CLINICAL MEDICAL RESEARCH AWARD. SO WITHOUT FURTHER ADO I WANT TO PRESENT TO YOU JOHN BECAUSE YOU REALLY CAME HERE TO HEAR HIS PRESENTATION AND ROB TYCKO'S BECAUSE THEY'RE THE PEOPLE OF THE HOUR. >> THANKS. JUST TO LET YOU KNOW DOUG GOT ALMOST ALL THE SAME AWARDS AS WELL SO YOU CAN CONGRATULATE HIM AS WELL. THE TITLE OF MY TALK TODAY SEEMS PRETTY BROAD BASIC VIROLOGY TO BRING MOLECULAR MEDICINE TO UNDER SERVED POPULATIONS. ACTUALLY, WHAT I WILL BE CONCENTRATING ON IS REALLY ILLUSTRATING THE CONCEPT WITH ABOUT THREE AND A HALF INTERRELATED AND AS YET UNFINISHED STORIES. THE DEVELOPMENT OF THESE DIFFERENT PORTIONS REALLY SPAN A BROAD SPECTRUM FROM INTERVENTIONS THAT ARE CURRENTLY BEING DELIVERED IN HUNDREDS OF BILLIONS OF DOSES ALL OVER THE WORLD TO THOSE THAT ARE STILL IN PRE-CLINICAL AND UNPUBLISHED STUDIES. WHEN I FIRST CAME TO THE NIH AT THE EARLY '80s I CAME TO STUDY HOW HPVs CAUSE CANCER. THEY CAUSE 5% OF ALL CANCERS AND IMPORTANT FOR THIS LECTURE ABOUT 85% OF THESE CANCERS OCCUR IN LOW AND MIDDLE INCOME COUNTRIES. ALSO IMPORTANT THOUGH THERE ARE ABOUT 12 ONCOGENIC TYPES 20% OF CERVICAL CANCERS ARE CAUSED BY HPV, 16% OR 18% AND OTHER CANCERS ARE 16% AND 18% AND CERVICAL CANCER DOMINATES THE CANCER SPECTRUM WORLDWIDE. WE'VE FOCUSSED MORE ON VIRO LOGIC ASPECTS OF PHVs. THIS IS BASED ON TWO BROADLY ENABLING TECHNOLOGIES THAT HAVE BOTH BASIC APPLICATIONS AND APPLIED APPLICATIONS AND THE LAB HAS PRETTY MUCH HAD AN EQUAL PROPORTION OF STUDIES IN BOTH OF THESE. THESE ARE THE L1 VIRUS LIKE PARTICLES AND THEN AN EFFICIENT WAY TO MAKE HPV GENE TRANSFER BACKWARDS WHICH WE CALL PSEUDOVIRUSES AND THIS WAS WORKED ON PRIMARILY BY OTHERS. WHAT I'LL BE TALKING ABOUT FIRST IS AN APPLIED APPLICATION WHERE WE USE THE TECHNOLOGY TO DEVELOP THE CURRENT COMMERCIAL HPV VACCINES AND THEN STUDIES HOW THE VACCINES WORKED AND THE IMMUNE RESPONSE TO THEM LED US TO DEVELOP VIRUS-LIKE PARTICLE BASED VACCINES AGAINST CHRONIC DISEASES THAT HAVE WORLDWIDE APPLICABILITY, WE BELIEVE. WE ALSO DID BASIC STUDIES WITH WE USED THE PARTICLES TO LOOK AT HOW THE VIRUS INTERACTS WITH CELL SURFACES AND THIS LED TO SURPRISING RESULTS THAT GAVE US INSIGHTS INTO BROAD SPECTRUM CANCER THERAPIES WHICH I'LL ALSO TALK ABOUT. FOR THE PROPHYLACTIC VACCINE IT WORK BEGAN WHEN A DERMATOLOGIST CAME TO OUR LAB AS A POST-DOC. WHAT HE DID WAS HE INSERTED L1 EXPRESSION SYSTEM PRODUCED IN INSECT CELLS AND LO AND BEHOLD THIS FORMED COPIES A VLP THAT LOOK LIKE THE OUTER SHELL OF THE VIRUS AND NOT ONLY LOOK LIKE THE OUTER SHELL OF THE VIRUS BUT INDUCE HIGH TIDERS OF ANTIBODIES THAT PROTECTS MUCH LIKE THE REAL VIRUS. OF COURSE THIS WAS NOT INFECTIOUS BECAUSE IT'S A SINGLE PROTEIN AND NOT ONCOGENIC WITHOUT THE ONCOGENE AND BECAME THE BASIS OF THE CURRENT VACCINES. THIS WENT SWIMMINGLY FROM THE BEGINNING EXCEPT FOR ONE THING WHEN WE TRIED TO MAKE THE VLPs FOR THE TYPE MOST ASSOCIATED WITH CANCER. THEY DIDN'T ASSEMBLE TO VLPs HARDLY AT ALL. THEN WE FIGURED OUT THE PROTOTYPE EVERYBODY WAS USING AT THE TIME HAD A MUTANT IN THE L1 SEQUENCES PRESUMABLY BECAUSE IT ISOLATED FROM THE CANCER AND HAD AN ACID IN OTHER SCREENS THEY WERE ISOLATE FROM PRODUCTIVE LESIONS AND THESE WERE ABLE TO DEVELOP AND GENERATE HIGH TIDERS OF ANTIBODIES BECAUSE THEY FORMED VLPs. ONE QUESTION IS WHY DID WE NEED VLPs FOR THE PROPHYLACTIC HPV VACCINES. THERE WAS NO WAY TO MAKE REAL VIRUSES IN CULTURE CELLS AND THE ONCA GENES WILL PRECLUDE THESE AS AN ATTENUATED OR KILL VIRUS VACCINES AND THEY MIMIC THE SURFACE OF REAL VLPs SO THEY CREATE ANTIBODIES AND ACTIVATE THEM. IT WAS KNOWN AT THE TIME THE PEPTIDES DIDN'T INDUCE PROTECTIVE ANTIBODIES BECAUSE ALL NEUTRALIZING EPITOPES ARE CONFIRMATI CONFIRMATION DEP ENT AND WE LEARNED THE PARTICLES ARE AMAZING AND EXCEPTIONAL IN THE ABILITY TO PRODUCE STRONG AND DURABLE ANTIBODY RESPONSES. I'LL TALK ABOUT THAT IN A MINUTE. THIS LED TO THE DEVELOPMENT OF FOUR COMMERCIAL VACCINES. IN THE UNITED STATES WE ONLY HAVE GAURDOSIL 9 . THE FIRST TWO VACCINES WERE LICENSED STARTING IN 2006. THE NEWEST VACCINE ON THE BLOCK IS MADE IN CHINA BY INOVAX AND LICENSED ONLY IN CHINA. THE VACCINES ARE DELIVERED BY INTRAMUSCULAR VACCINATIONS IN SIX MONTHS. THEY'VE BEEN APPROVED FOR JUST VACCINATION IN PEOPLE UNDER 15 YEARS OLD BECAUSE OF IMMUNOLOGICAL EQUIVALENCIES. THEY HAVE THE ABILITY TO PROTECT AGAINST DISEASE. IF YOU LOOK AT DISEASE BY THE VACCINE TARGETED TYPES, IN GIRLS THAT DIDN'T HAVE THOSE PARTICULAR TYPE INFECTIONS AT ENTRY, CERVICAL, VAGINAL INFECTIONS YOU CAN SEE FOR PRE-CANCER ALL THREE VACCINES GAVE 100% PROTECTION AND GENITAL WART PROTECTION BY GAURDISIL IS 96% AS WELL. AND THESE HAVE BEEN APPROVED IN COUNTRIES WHICH IS A 20% INCREASE SINCE 2018. WE CONTINUE TO GET THESE VACCINES IN MORE AND MORE COUNTRIES. THIS IS RECENT META-ANALYSIS THAT LOOK AT THE CHANGES IN INCIDENT OF PRE-CANCER IN SCREENED WOMEN WHERE THE VACCINE COVERAGE RATES ARE LESS THAN 50%. IN THE YOUNGER AGE POPULATIONS, 15 TO 19, THE RATE OF PRECANCER REGARDLESS OF TYPE IS DROPPING DRAMATICALLY AS IN THE SLIGHTLY OLDER WOMEN BUT NOT IN THE OLDER WOMEN BECAUSE THEY'RE NOT PART OF THE VACCINATION PROGRAM OR HAVE BEEN INFECTED BEFORE VACCINATION. SO THE BIG ISSUE NOW IS COVERAGE. WE HAVE TO GET IT TO MORE PEOPLE. FOR GIRLS WHO HAD THE POTENTIAL OF BEING VACCINATED IN THE FIRST TWO YEARS SINCE INTRODUCED IN 2006, WITHOUT VACCINE, THERE WOULD BE EXPECTED TO BE 19 MILLION CASES OF CERVICAL CANCER AND 10 MILLION DEATHS IN THE NEXT 65 YEARS. THE QUESTION HOW MANY OF THOSE DEATHS HAVE WE PREVENTED. YOU CAN SEE WE HAVE A LONG WAY TO GO IT'S ONLY PROJECTED WE PREVENTED 365 CASES AND 155,000 DEATHS AND WHEN LUKE -- YOU LOOK WHERE THEY'VE BEEN PREVENTED IT'S UP AND MIDDLE INCOME COUNTRIES THOUGH THE BURDEN OF THE DISEASE IS IN LOWER INCOME DISEASES BECAUSE WE'RE NOT GETTING THE VACCINE TO THE WOMEN. HOW CAN WE BEST INCREASE VACCINE COVERAGE UPTAKE? IN LOW-RESOURCE SETTINGS? THERE'S A NUMBER OF THINGS BEING DONE BUT WE THINK THE ONE THING THAT COULD DO THE MOST TO INCREASE UPTAKE IS PROVE YOU ONLY NEED A SINGLE DOSE OF THE VACCINE IN ORDER TO GET PROTECTION. THIS SEEMS VERY UNLIKELY BECAUSE THERE'S NO SUBUNIT, NON-INFECTIOUS VACCINE THAT HAS EVER BEEN SHOWN TO BE SUCCESSFUL AS A SINGLE DOSE. WHY DO WE THINK THIS COULD OCCUR FOR THE HPV VACCINES? BCAUSE THE NCI ADMINISTRATION IN THEIR INFINITE WISDOM ALLOWED US TO DO A PUBLIC SECTOR TRIAL OF ONE OF THE VACCINES PRIOR TO LICENSURE. THIS IS A VACCINE THAT WAS HEADED HERE AT THE NCI THAT COMPARED AND IT WAS A CONTROL VERSUS THE OTHER THAT WENT ON FOR FOUR YEARS AND THERE WAS A LONG-TERM FOLLOW-UP. UNLIKE THE COMPANIES WE ASKED THE QUESTION, WHAT HAPPENED TO THE YOUNG LADIES WHO DID NOT RECEIVE THE FILL COURSE OF VACCINATION AND DID NOT RECEIVE FULL DOSES. 20% OF THE INDIVIDUALS IN THE TRIAL RECEIVED LESS THAN THREE DOSES. TO MAKE A LONG STORY SHORT, NOW AFTER 11 YEARS SINCE INITIAL VACCINATION, THERE'S NO DIFFERENCE IN THE NUMBER OF INFECTIONS IN WOMEN WHO GOD THREE DOSES OR -- GOT THREE DOSES OR ONE DOSE. THIS IS SAME FOR THE TARGETED 16 OF 18 AND HPV, 31, 33 AND 35 WHERE THERE'S SUBSTANTIAL PARTIAL PROTECTION. WHY IS THE VACCINE WORKING SO WELL EVEN BY SINGLE DOSE? IT'S REMARKABLE IN PRODUCING CONSISTENT HIGH TIDER LEVELS OF ANTIBODIES. THIS SHOWS THE DATA FROM COSTA RICA AND FOR ANTIBODY LEVELS AND IN BETWEEN YEAR TWO AND YEAR 11 THERE'S NO CHANGE IN ANTIBODY TIDERS AFTER A SINGLE DOSE. THE DIFFERENCE BETWEEN THREE AND FOUR DOSES IS ALMOST FOURFOLD. TO PROVIDE THE DATA CHANGE FOR ADOPTION OF ONE-DOSE TRIALS THE NCI IN COLLABORATION WITH COSTA RICA COLLABORATORS IS LOOKING TO EVALUATE OF A SINGLE VERSUS TWO DOSE IN THE PREVENTION OF CERVICAL TYPE HPV16 INFECTION THAT PERSISTS SIX MONTHS IN A FORMAL RANDOMIZED CONTROL TRIALS OF GIRLS 12 TO 16-YEAR-OLDS WITH 5,000 GIRLS PER ARM THAT ARE FOLLOWED FOR FOUR YEARS. WILL ALSO BE EVALUATING ONE DOSE OF THE VACCINE IN COMPARISON TO ZERO DOSES BY DOING AN EPIDEMIOLOGICAL SURVEY WHERE WE TAKE WOMEN AT THE END OF THE SURVEY AT THE BEGINNING AND END AND DETERMINE THE HPV VACCINATION STATUS TO GET PEUDOEFFICACY VERSUS NO DOSE. THE RESULTS WILL PROBABLY BE AVAILABLE IN 2020. THE REASON WE THINK THE VACCINE WORKS SO SURPRISINGLY WELL IS BECAUSE OF THE STRUCTURE OF THE ANTIGEN. WE THINK A LOT OF IT HAS TO DO WITH THE INTERACTION WITH THE CELL RECEPTORS -- B CELL RECEPTORS WHERE A SIMPLE SOLUBLE ANTIGEN WHEN THEY INTERACT WITH THE B CELL RECEPTORS LEAD TO WEAK ACTIVATION SIM BOWLS AND RELATIVELY -- SYMBOLS AND RELATIVELY LOW ANTIBODY LEVELS AND DON'T LAST LONG. WHEN THE REPETITIVE ELEMENTS ENGAGE THE B CELL RECEPTORS THEY INTERACTION WITH KINASE THAT SEND STRONG SURVIVAL AND PROLIFERATION SIGNALS THAT SENT ANTIBODIES WITH PLASMA CELLS THAT CONTINUE TO PUMP OUT ANTIBODIES THE REST OF YOUR LIFE IN THE ABSENCE OF ANTIGEN STIMULATION. AND WHAT'S INTERESTING IS THIS IDEA THAT THESE TYPES OF STRUCTURES ARE PARTICULARLY VIEWED AS DANGEROUS TO THE IMMUNE SYSTEM CAME OUT ABOUT THE SAME TIME IN 1993 IN A PAPER AND WHAT THEY PROPOSE IS THE CELLS LOOK AT EPITOPES FACING THEM AND USUALLY FOUND IN THE MICROBIAL SOURCES AS SHOWN IN THE PAPILLOMA VIRUSES AND THE COMPLEX OF THIS FACING RARELY OCCURS TO INVERTEBRATE ANIMALS. I LIKE TO THINK OF THE B CELL RECEPTOR AS ANTIGEN SPECIFIC RECOGNITION RECEPTORS. AND SO ONE OF THE THINGS WE'VE DONE IS TRY TO EXPLOIT THIS KNOWLEDGE OF THE HIGH IMMUNOGENICITY OF THE PARTICLES TO BRING THIS TO UNDER SERVED POPULATIONS. THE PROJECT STARTED WHEN A POST-DOC CAME TO THE LAB AND ADDRESSED A SIMPLE BUT PROFOUND QUESTION OF WHAT'S IMMUNODOMINANT. B CELL TO CELL ANTIGENS OR THE ACTIVATION OF THE B CELLS IN RESPONSE TO THE REPETITIVE DISPLAY OF PARTICULATE VIRAL ANTIGENS. THE STUDY THEY DID IS HE LINKED VLPs AND LINKED A CELL PROTEIN, TNF TO THE VLPs THROUGH A BRIDGE AND THIS AFFORDED THE OPPORTUNITY TO COMPARE THE INDUCTION OF ANTIBODIES TO FOREIGN EPITOPES IN CELLS IN EXACTLY THE SAME CONCEPT AND PUT THEM OUT AT HIGH DENSITY DISPLAY AND WHAT HE FOUND IN THE DATA WAS CLEAR. B CELL IMMUNITY TO VIRUS SURFACE STRUCTURE IS DOMINANT OVER DOMINANCE TO SELF. THIS IS AN EXAMPLE OF THE DATA. IF YOU TAKE THIS AND DON'T USE AN ADJUVANT YOU DON'T HAVE THE TIDERS TO FORM AND IF YOU LINK IT TO A STRONG USE IN HUMAN YOU GET A GOOD RESPONSE TO THE FOREIGN EPITOPES IN GREEN BUT SAY WEAK RESPONSE TO THE TNF AND THERE'S A HUNDRED FOLD DIFFERENCE IN THE ANTIBODIES TOO. IF YOU LINK THE SAME AMOUNT OF TNF FUSION PROTEIN TO THE VLPs, FOR FOREIGN YOU DO AS WELL AS USING AN ADJUVANT TOO STRONG TO USE IN PEOPLE AND GREATLY INCREASEAND THE RESPONSE TO CELL IN RELATIVE TO FOREIGN AND IF YOU ADD AN ADJUVANT PLUS YOU COMPLETELY BREAK THE ABILITY OF THE ANIMAL TO DISTINGUISH BETWEEN FOREIGN AND SELF. AND THE DENSITY SEEMS TO BE THE KEY. IF YOU TAKE THE HUGE AMOUNT OF THE FUSION PROTEIN AND DISPLAY IT EITHER AT HIGH DENSITY OR AT LOWER DENSITY ACROSS A LARGER NUMBER OF VLPs, WHAT YOU CAN SEE IS WHEN YOU DECREASE THE DENSITY YOU DECREASE THE ABILITY TO BREAK B CELL TOLERANCE LODGE RHYTHMICALLY AND THIS IS DISPLAYED. SO THEY WERE RIGHT IN THEIR HYPOTHESIS. SO WHY WOULD ONE WANT TO BREAK B CELL TOLERANCE THE ANSWER IS TO ACTIVATE CRITICAL MEDIATORS OF CHRONIC DISEASE. THERE'S SEVERAL IMPORTANT ADVANTAGES OF THIS APPROACH. FIRST A SLOWER DECREASE IN THE THERAPEUTIC MONOCLONAL THERAPEUTIC ANTIBODY LEVELS AND MAYBE IT WILL BE MORE EFFECTIVE BECAUSE IT COULD BE A POLY CLONAL RESPONSE AND THERE WOULD BE NO DRUG ANTIBODY INDUCTION BECAUSE THESE WOULD BE OUR OWN ANTIBODIES BUT MOST IMPORTANTLY FOR THE DISCUSSION TODAY, IT COULD BE DONE AT MUCH LOWER COST WHICH WOULD MAKE IT WORLDWIDE APPLICABLE. IF THERE'S ANYTHING WE'VE BEEN ABLE TO DELIVER WORLDWIDE IS VACCINES. BUT WE MUST ACKNOWLEDGE THAT THERE ARE SOME DISTINCT DISADVANTAGES FIRST THERE'S VARIABILITY TO RESPONSE TO VACCINES AND AN INDIVIDUAL CAN HAVE LOW ANTIBODY LEVELS AND THERE ARE SAFETY ISSUES WITH THIS APPROACH BECAUSE YOU CAN'T TURN IT OFF. YOU INDUCE ANTIBODIES AND THERE'S A PROBLEM IT WOULD BE AN ISSUE THAT WOULD BE DIFFICULT TO DEAL WITH. SO THEREFORE WE'RE THINKING ABOUT WHAT COULD BE THE IDEAL TARGET FOR AN AUTO ANTIBODY VACCINE? WE THINK THEY SHOULD MEET THESE FOUR CRITERIA. ONE, A CRITICAL MEDIATOR OF AN IMPORTANT CHRONIC DISEASE AND ACTIVE IN THE CIRCULATION IN LOW CONCENTRATIONS SO IT'S EASIER TO HAVE ANTIBODY EXCESSES. MONOCLONAL ANTIBODY SHOULD BE PROVEN TO BE SAFE AND EFFECTIVE AGAINST THE SAME TARGET PROOF OF CONCEPT AND IN ADDITIONAL IMPORTANT TO DEMONSTRATE INDIVIDUALS WITH NO MUTATIONS IN THEIR TARGET ARE PERFECTLY HEALTHY. pTHAT MEETS ALL THESE CRITERIA.T IT'S CALLED PCSK9 WHICH FOR A LONG TIME I HAD NO IDEA WHAT IT IS. SO WHAT IT IS IS A MOLECULE THAT INHIBITS THE LOW-DENSITY LIPO PROTEIN CHOLESTEROL METABOLISM. THE LDL CHOLESTEROL IS USUALLY TAKEN UP IN THE LIVER AND LDL IS DEGRADED BUT THE RECEPTOR IS RECYCLED BUT IN THE PRESENCE OF THIS SOLUBLE FACTOR IS TAKEN IN AND THE RECEPTOR DEGRADED AND THE SO IN ADDITION IT CAN LEAD TO AN INCREASE IN LDLC UPTAKE. SO THE RATIONALE FOR TARGETING PCSK9 BIO VACCINE TO PREVENT CARDIOVASCULAR DISEASE IS QUITE COMPELLING. SO THERE'S HUMAN GENETIC DATA WHERE NATIONALLY OCCURRING AND MUTATIONS ARE ASSOCIATED WITH REDUCED LDLC LEVELS AND TRIGLYCERIDE LEVELS AND DECREASED RISK OF CARDIOVASCULAR DISEASE FROM ATHEROSCLEROSIS AND SEEMS TO BE SAFE BECAUSE MUTATIONS IN THIS ARE NOT ASSOCIATED WITH ANY PATHOLOGY WHICH SUPPORTS THE CONCEPT THAT TARGETING THESE MOLECULES WILL BE SAFE. IN ADDITION, ANTI-PKSN ANTIBODIES ARE EFFECTIVE AT LOWERING CHOLESTEROL LEVELS BY GREATER THAN 60% IN COMBINATION WITH STATINS. ALSO, THEN PREVENTING CARDIOVASCULAR DISEASE OUTCOMES. THE BIGGI ISSUE IS COST. IT'S BEEN ESTIMATED IF EVERYBODY WHO COULD BENEFIT FROM THIS TREATMENT GOT IT IT WOULD ASTOUNDINGLY COST $500 BILLION FOR THIS TREATMENT IN THE UNITED STATES ALONE. I THINK WE COULD DELIVER A VACCINE FOR CHEAPER THAN THAT. SO BASED UPON THIS WE DEVELOPED A CONSORTIUM TO TRY TO DEVELOP A PCSK9 VACCINE. THIS CONSORTIUM IS LED FROM NHLBI AND A PROFESSOR AT UNIVERSITY OF NEW MEXICO AND THEN OUR GROUP. AND INCIDENTALLY, THIS CONSORTIUM WAS FORMED WHEN WE WERE WORKING OUT TOGETHER AT THE NIH GYM AND GOT TO TALKING ABOUT WHAT WE WERE DOING. SO FOR THE VACCINE WE'RE NOT USING -- AND THE PRODUCTION OF THEE VLPs IS WELL ESTABLISHED. YOU CAN GET A TON OF VLPs CHEAPLY FROM E. CHOLI AND PEPTIDES HAVE DEMONSTRATED TO BE SHAVE AND HAVE THE ABILITY TO DELIVER ANTIGEN INCLUDING SELF-PROTEIN ANTIGENS. BASED ON THE CRYSTAL GRAPHIC STRUCTURE, WE LOOKED AT THE INTERFACE BETWEEN LDR RECEPTOR AND PCSK9 AND LINKED THEM TO THE BETA PARTICLES AND LINKING THE P PEPTIDE LED TO CONSISTENT REDUCTION IN CHOLESTEROL WHEN USED AS A VACCINE IN MICE. WE WENT ON WITH A LARGER GROUP TO SHOW IT CONSISTENTLY INDUCED HIGH TITERS OF ANTIBODIES TO DELIVER CHOLESTEROL IN TRIGLYCERIDES IN THE VACCINATED ANIMALS. YOU CAN ALSO DO THIS IN MONKEYS IN A SMALL MACAQUE STUDIES WE WERE ABLE TO INDUCE GOOD ANTIBODY RESPONSES. IN THE ANIMALS THAT GOT THE PCSK9 VACCINE WE'RE NOW MOVING FORWARD AND HOPEFULLY NOW THAT COVID IS A LITTLE BIT UNDER CONTROL, BUT NOT REALLY, TO GO TO ANOTHER STUDY WHERE A LARGER STUDY WE WOULD VACCINE 24 MACAQUES WITH THE PARENTAL OR PCSK9 AND FOLLOW THEM FOR CHOLESTEROL AND LIPID LEVELS AND TREAT THEM WITH A STATIN AND SEE IF THEY CAN COLLABORATE WITH STATIN AND HOPEFULLY WITH THIS DATA WE COULD GENERATE MORE INSTITUTIONAL AND INDUSTRY ENTHUSIASM IN MOVING THIS FORWARD TO A CLINICAL TRIAL. NOW LET'S TURN TO CANCER. I MUST SAY AS I LOOK AT THE LANDSCAPE I REALLY THINK THAT ALMOST NOBODY IS WORKING ON BIO LOGICAL THERAPIES OF CANCER BETTER THAT ARE GEARED TO LOW RESOURCE SETTINGS. I THINK THERE'S A REAL OPPORTUNITY BECAUSE PEOPLE AREN'T THINKING ABOUT THIS. WHAT WOULD BE THE PRODUCT PROFILE FOR A BIOLOGICAL THERAPY FOR CANCER FOR APPLICATION IN LOW AND MIDDLE INCOME COUNTRIES? I MUST SAY THIS IS ASPIRATIONAL AND QUITE POSSIBLE WE WON'T BE ABLE TO MEET ALL THESE CHRIST BUT WE'D LIKE A BROAD RANGE OF CANCER TYPES WITH A BROAD ARRAY OF CANCER AND NO NEED FOR HISTOLOGICAL PROFILING SO THE OPPOSITE OF INDIVIDUALIZED MEDICINE WHICH SUN LIKELY TO HAVE SUBSTANTIAL IMPACT OF CANCER DEATHS. IT WON'T BEND THE CURVE AND THE COMPOSITION WOULD PREFERABLY NOT REQUIRE A COLD CHAIN AND SERIOUS ADVERSE EVENTS WOULD HAVE TO BE QUITE RARE BECAUSE OF THE LIMITED RESOURCES FOR TREATMENT OF ADVERSE EVENTS IN THE SETTINGS AND YOU'D HAVE TO HAVE AN INVESTMENT CASE FOR HIGH RESOURCE SETTINGS BECAUSE IF THE PRODUCT WOULD NOT BE APPLICABLE TO HIGH-INCOME COUNTRIES YOU WOULDN'T BE ABLE TO TRACK INDUSTRY DEVELOPMENT IN PRODUCTION TO MAKE THIS A REALITY. THE STORY STARTS WITH BASIC VIROLOGY. IT STARTS WHEN WE DECIDED TO FIGURE OUT HOW THE VIRUS INFECTS NORMAL TISSUE OF THE CERVICAL VAGINAL TRACT AND HOW ANTIBODIES AFFECT THAT AND THE VERIONS CAN'T FIND THE SURFACE OF NORMAL EPITHELIA BUT MUST FIRST FIND THE BASEMENT MEMBRANE IN UNDERGOING A SERIES OF CHANGES BEFORE THEY CAN BIND TO THE BASAL CYTES AND THE FACTOR ARE THE PROTEO GLYCAN THAT IS UNIQUE MODIFICATIONS THAT AREN'T FOUND ON THE SURFACE OF CELLS. THIS IS AN EXAMPLE WHERE IF YOU LOOK IN THE VAGINAL TRACT AND PUT IN PARTICLES THAT TRANSMIT A RED FLUORESCENT PROTEIN THERE'S NO BINDING TO THE SQUAMOUS EM -- EPITHELIA BUT WHEN YOU DISRUPT THE EPITHELIA IT LIGHTS UP. WHAT'S THIS HAVE TO DO WITH CANCER? IT TURNS OUT REMARKABLY THAT MOST TUMOR CELLS HAVE THE SAME TYPE OF MODIFICATIONS ON THEIR CELL SURFACES. WE DON'T KNOW KNOW WHAT THE MODIFICATIONS ARE YET BUT WE'RE LOOKING AT IT. SUFFICE TO SAY THEY BIND THE PARTICLES STRONGLY AND INSECTED WITHOUT HAVING TO INTERACT WITH THE BASEMENT MEMBRANE FIRST AND THIS IS TRUE OF A WIDE VARIETY OF TYPE AND HERE YOU SEE STRONG BIND INNI BINDING IN RED THAT CAN BE BLOCKED BY HEPARIN A STRONG FORM OF HSPGs. THIS OPENED UP THE DOOR FOR THIS TO BE USED AS GUIDED MISSILES FOR IMAGING DIAGNOSIS OR IMAGING OF CANCER. THINK MULTIPLE WAYS TO DO THIS FOR INSTANCE YOU COULD ATTACH OR ENCAPSULATE DRUGS, CYTOTOXINS OR SUICIDE GENES OR LABELS OR INFRARED DYE. I'LL TALK ABOUT AN APPLICATION WHERE WE PUT ON AN INFRARED DYE. IT'S A STRONG AND LONG-LASTING COLLABORATION WITH ORA BIO SCIENCE AND I WANT TO CALL OUT THE CEO AND A FORMER POST-DOC IN THE LAB. SO -- AURA BIO SCIENCE. THESE CONJUGATES ARE NICE BECAUSE THEY HAVE DUAL SPECIFICITY FOR CANCER AND THEY BIND WHEN ILLUMINATED WITH LIGHT. THIS CAUSES RELEASE OF THE CINGULATE OXYGEN WHICH DISRUPT THE MEMBRANE INTEGRITY WHICH BASICALLY PUNCHES HOLES IN IT CAUSING CELL DEATH BY NECROSIS AND THE CELLS SHRIVEL UP AND DIE AND THE CELLS TRIGGER T CELL RESPONSES AND WE THINK THERE'S DUAL ACTIVITY IN DIRECT KILLING AND INDUFG -- INDUCING IMMUNO RESPONSE AND I WANT TO GIVE A SHOUT OUT TO TWO PEOPLE TO DISCOVER THE ACTIVITY OF THE IR700 DYE WHEN ADJACENT TIE CELL SURFACE AND WHEN THEY BOUND IT TO EGF RECEPTOR MONOCLONAL ANTIBODIES. WE STRUGGLED TO DECIDE WHAT FIRST APPLICATION TO USE AND WE DECIDED ON AN MELANOMA. IT'S AN ORPHAN DISEASE BUT OFTEN DEADLY DUE TO LIVER CANCER METASTASES AND IT LEADS TO LONG-TERM RETINAL DAMAGE AND VISION LOSS AND IT'S ASSOCIATED WITH MORBIDITY. THIS PERMITS INTRAVITREOUS DELIVERY OF THE DRUG AND THERE WAS A TRANSPLANTATION OF CANCER CELLS AND IN THE ANIMAL MODEL YOU TREAT THE ANIMAL THAT DON'T REJECT THE HUMAN TUMORS YOU CAN DO INTRACHOROID IMPLANTATION AND THEN DO THE INJECTION OF THE DYE COUPLE PARTICLES AND DO THE LIGHT TREATMENT IF YOU DO THESE TWO WEEKS A PART AND LOOK TWO DAYS LATER YOU SEE MASSIVE NECROSIS OCCURRING IN A DOSE-DEPENDENT FASHION. MORE STRIKING, IF YOU WAIT EIGHT OR NINE DAYS AFTER THE SECOND TREATMENT, BASICALLY THE TUMORS HAVE DISAPPEARED AND MOST THE ANIMALS AT THE HIGHER DOSE AND THE HISTOLOGISTS TELL US THERE'S NO DAMAGE TO THE RETINA WHICH IS A CLEAR ISSUE WHICH IS JUXTA POSE TO THE CHOROIDAL PHASE. THIS LED TO A PHASE 1 AND PHASE 2 TRIAL AURA SPONSORED AND BASED ON INTELLECTUAL PROPERTY THEY LICENSE FROM US. IN A RELATIVELY COMPLICATED TRIAL, WE'VE BEEN ABLE TO VACCINE ABOUT 52 SUBJECTS SO FAR AND THE RESULTS HAVE BEEN VERY ENCOURAGING. THIS INVOLVES INTRAVITREOUS INJECTION OF PATIENTS WITH SMALL MELANOM MELANOMAS .1 TO .2 MILLIMETERS THICK AND VISUAL ACUTE HAS BASKETBALL -- HAS BEEN OBTAINED. AND ADVERSE EVENTS HAVE BEEN LIMITED TO MILD INFLAMMATION AND INCREASED PRESSURE WHICH HAVE BEEN TREATED WITH STANDARD STEROIDS IN TOPICAL HYPERTENSION DRUGS. IT LOOKS LIKE THEY'RE DOING A GOOD JOB AT TUMOR CONTROL AS WELL. IF YOU LOOK AT THE TUMORS AND THE RATE IN WHICH THEY'RE GROWING PRIOR TO TREATMENT AND EXTRAPOLATE THEY WOULD HAVE GOTTEN LARGER BUT WE SEE THERE'S BASICALLY THE TUMORS STOPPED GROWING. WE DON'T KNOW EXACTLY WHAT THIS MEANS BUT IN THE ONE TUMOR WE WERE ABLE TO BIOPSY, BIOPSIES ARE DIFFICULT GOING THROUGH THE RETINA IT WAS A LYMPHOCYTES WITH NO EVIDENCE OF MELANOCYTES AND WE LOOKED AT DISEASE PROGRESSION TO NORMAL STANDARD OF CARE AND FOR MAINTENANCE OF VISUAL ACUITY AND WE LOOKED AT AN OFF-THE-SHELF CANCER THERAPY AND WE LOOKED AT BLADDER CANCER BECAUSE IT'S ACCEPTABLE TO THE DRUG AND THE LASER AND THE ONLY TREATMENT WHO FAIL IS REMOVAL OF THE BLADDER AND WE'RE LOOKING TO IMPROVE THE PROFILE FOR LOW-INCOME SETTINGS AND WE'RE EXCITED ABOUT USING THE DYE COUPLE PARTICLES. THIS OVERCOMES TWO LIMITATIONS OF THE TECHNOLOGY. THE LIMITED PENETRATION OF INFRARED LIGHT AND THE LIMITED AVAILABILITY OF THE APPROPRIATE LASER. THIS WILL BE A COLLABORATION WITH A BIO ENGINEER AT OXFORD UNIVERSITY. WE THINK THE APPROACH MAY BE FEASIBLE TECHNICALLY BECAUSE THERE IS SOME EVIDENCE THAT YOU CAN USE ULTRASOUND TO ACTIVATE THESE TYPES OF DYE EVEN FOR TYPES OF THERAPY INVOLVING TUMORS WITH PRE-DYE. WE THINK THIS APPROACH MAY BE FEASIBLE PROGRAMMATICALLY BECAUSE THEY'RE, USED MORE AND MORE IN LOW-RESOURCE SETTINGS. THIS IS AN ARTICLE FROM THE NEW YORK TIMES WHERE IT SAYS A HANDHELD DEVICE BRINGS TECHNOLOGY TO REMOTE COMMUNITIES FOR THE FIRST TIME. THE VISION IS THE ULTRASOUND DEVICE WOULD BE USED TO GUIDE INJECTION OF THE DRUG INTO THE TOMB TUMOR, ACTIVATE THE DRUG IN THE TUMOR AND FOLLOW THE COURSE OF TREATMENT WITH THE SAME HANDHELD DEVICE WHICH CURRENTLY COSTS $1,000 AND THE PRICE IS COMING DOWN. IN MY LAST MINUTES I WANT TO TALK ABOUT HARVESTING IMMUNITY TO TREAT CANCER. TREATING IT AGAINST CANCER ANTIGENS RELIES UPON EX VIVO AMPLIFICATION AND IT'S DIFFICULT TO ACTIVATE THE DE NOVO RESPONSES. HOWEVER, MOST PEOPLE HAVE PRE-EXISTING FUNCTIONING T CELLS AGAINST VIRAL ANTIGENS. THE RESPONSES CAN BE RECRUITED TO TUMORS TO KILL THE CANCER CELLS AND INTRODUCE ANTIGEN SPREADING TO IMMUNOANTIGENS. WE'RE TRYING TO RECRUIT T CELL IMMUNITY AND IT'S GREAT FOR THIS. FIRST OF ALL, INFECTION IS HIGHLY PREVALENT IN MOST POPULATIONS. THERE'S REMARKABLY HIGH MAGNITUDE OF RESPONSES THAT REMAIN FUNCTIONAL AND INCREASE WITH AGE IN A PROCESS CALLED MEMORY INFLATION. AND WITH AN OLD GUY LIKE ME, 10% OF ALL MY FUNCTIONAL T CELLS BOTH CD8 AND CD4 ARE DIRECTED TOWARDS A LIMITED NUMBER OF CMV EPITOPES. CMV CELLS REMAIN FUNCTIONAL IN CANCERS. AND THEY HAVE BEEN SHOWN TO INFILTRATE TUMORS, EVEN TUMORS NEGATIVE FOR CMV INFECTION. THERE'S A MOUSE MODEL THAT'S GOOD FOR HPV INFECTION WHERE WE CAN DO PROOF OF CONCEPT. THE QUESTION IS HOW DO WE RECRUIT THE TUMORS? THE WAY WE THOUGHT ABOUT IT INITIALLY IS TO INFECT THEM WITH THE PSEUDOVIRUS EXPRESSING CMV ANTIGENS WITH SPECIFIC DELIVERY ONLY TO THE TUMORS IN KILLING ONLY THE TUMOR CELLS. THE THING WE'RE CONCENTRATING MORE RECENTLY IS TO INJECT MINIMAL EPITOPES IN THE TUMOR BECAUSE THIS IS A SIMILAR PRODUCTION SCHEME AND LIKELY TO HAVE MORE EFFECTS ON THE TUMOR ENVIRONMENT AND HAVE MORE OFF TARGET AFFECTS. SO BASICALLY THE EXPERIMENTAL DESIGN IS TO INFECT THEM WITH CMV AS YOUNG ANIMALS AND WAIT SIX MONTHS AND TUMOR THEM SUB CUTANEOUSLY AND THEN DO INTERTUMORAL INJECTIONS AND WATCH THE GROWTH AND THIS HAS BEEN DONE WITH HELP FROM OTHERS. AND SO WE INJECT IN VARIOUS COMBINATIONS CLASS 1 PEPTIDES, CLASS 2 PEPTIDES WITH AND WITHOUT A TLR POLY AGONIST AND I DON'T HAVE TIME TO SHOW YOU THE DATA BUT WHAT WE LEARNED SO FAR ENTIRELY IN THE DATA IS QUITE INTERESTING. SO IF WE INJECT THE CD8 PEP SIDES WE GET RAPID PROFOUND TUMOR REGRESSION SOMETIMES TO WHERE WE SEE TOXICITY IN THE ANIMAL. MANY OF THESE TUMORS EVENTUALLY GROW BACK. WITH THE CD4 PEPTIDES IF WE ADD IT BUT IF WE DON'T THE CHANGES IN THE TUMOR ENVIRONMENT WE SURPRISINGLY GET 50% OF THE ANIMALS WITH DURABLE PROGRESSION IN THE NON-TOXIC FASHION. THE COMBINATION OF ALL THREE GENERATES THE HIGHEST REGRESSION UP TO 30% IN SOME CASES AND IT'S IMPORTANT TO NOTE THIS DURABLE REGRESSION DOES NOT REQUIRE THE INJECTION OF AN IMMUNE CHECKPOINT INHIBITOR SO IT'S CRITICAL TO SEE HOW THE RESPONSE CAN GET OVER THE NEED FOR IMMUNE CHECKPOINT INHIBITORS. AND THE T CELLS THIS ONES THAT DON'T INCREASE WITH CELLS ARE MORE POTENT THAN THE INFLATIONARY ONES WE THINK BECAUSE THEY EXPAND MORE AFTER INJECTION OF THE PEPTIDE. THE PRODUCT PROFILE FOR HARNESSING THE ANTIVIRAL IMMUNITY IN LOW-INCOME SETTINGS WE CAN BE AN AGNOSTIC IMMUNITY FOR TUMORS AND WOULD CONSISTENT OF THE COMBINATION OF ALL IMMUNODOMINANT CD4 AND CD8 PEPTIDES THAT SPAN IN CLASS 1 ALLELES AND CLASS 2 PLUS POLY IC. IN THE LAST MINUTE TO GIVE YOU AN IDEA OF THE LESSONS I'VE LEARNED IN 35 YEARS AT THE NIH, FIRST TRY TO DO GOOD SCIENCE AND DEVELOP ENABLING TECHNOLOGIES AND LOOK FOR TECHNOLOGIES TO TRANSLATE INTO MEDICAL INTERVENTIONS. THINK BOLD, ATTACK BIG ISSUES AND ALSO BE REALISTIC. IS THIS INTERVENTION I'M DOING PRACTICAL? DO I NEED TO DO -- WHAT DO I NEED TO DO BEFORE A COMPANY WILL SPEND THEIR $500 MILLION TO SEE IF I'M RIGHT AND THEN TAKE THAT EXTRA STEP AND THINK HOW YOUR INTERVENTION MAY BE TAILORED TO UNDER SERVED POPULATION LIKELY TO BE THE MOST IN NEED OF IT AND LASTLY I'D LIKE TO LEAVE YOU WITH THE IDEA OF THIS APPROACH CAN WORK BY HAPPENSTANCE THEN BASICALLY WE DIDN'T HAVE THIS VISION AT THE BEGINNING AND BASICALLY FOLLOWED SERENDIPITY AND TOOK ADVANTAGE WHERE WE COULD BUT YOU CAN IMAGINE WHAT WOULD HAPPEN IF A P.I. OR INSTITUTE COULD ACCOMPLISH IF THIS WAS ACTUALLY THE CLEAR VISION FOR THAT INDIVIDUAL OR INSTITUTION FROM THE START? I THINK WE COULD DO A LOT TO ADDRESS ISSUES IN LOW-RESOURCE SETTINGS. SO THANK YOU. I'D LIKE TO ACKNOWLEDGE MY KEY COLLABORATORS ESPECIALLY DOUG LOWY WHO HAS BEEN MY PARTNER IN CRIME FOR THE LAST FEW YEARS SO THANK YOU FOR YOUR ATTENTION AND SORRY IF I WENT OVER TIME A LITTLE. >> JOHN, THANKS A LOT. I'M SURE PEOPLE UNDERSTAND THERE'S BOTH STUFF IN PEOPLE AND MAKING A LOT PROGRESS BUT ALSO A VISION FOR HOW TO DO THINGS IN A LOT OF DIFFERENT DIMENSIONS. I THINK WE HAVE TIME FOR ONE QESTION AND A QUESTION THAT CAME IN WAS WHAT PREVENTS A VACCINE FROM REACHING OTHER COUNTRIES MORE RAPIDLY? WHAT ARE THE CURRENT BARRIERS? >> THE MAIN BARRIERS ARE THE COST OF A VACCINE. $5 A DOSE IS MORE THAN THE SERIES AND SETTING UP IMPLEMENTATION PROGRAMS IN ADOLESCENTS. MOST ARE GEARED TOWARDS YOUNG CHILDREN. THE INFRASTRUCTURE OF SETTING UP A PROGRAM FOR ADOLESCENCE IS EXPENSE IN ITSELF. THAT'S WHY WE THINK GOING TO A SINGLE DOSE WILL DECREASE THE COST OF THE PRODUCT AND THE COST OF HAVING TO DELIVER THE VACCINE AS WELL. >> THANKS. IN THE INTEREST OF TIME, I THINK WE SHOULD PROBABLY GO ON. I BELIEVE THAT DR. GRIF RODGERS THE DIRECTOR OF NIDDK WILL INTRODUCE ROB TYCKO. >> THANK YOU, DOUG. I'M GRIFFEN RODGERS THE DIRECTOR OF THE NATIONAL INSTITUTE OF DIABETES, DIGESTIVE AND KIDNEY DISEASES. THANK YOU FOR JOINING US TODAY IN A VIRTUAL MINI-SYMPOSIUM RECOGNIZING DR. ROBERT TYCKO SELECTION TO THE NATIONAL ACADEMY OF SCIENCES. MEMBERSHIP IN THE NATIONAL ACADEMY IS CONSIDERED ONE OF THE HIGHEST HONORS BESTOWED ON U.S. SCIENTISTS AND I'M HONORED TO BE HERE TODAY TO CELEBRATE ROB'S ACHIEVEMENTS. I'LL ECHO DR. GOTTESMAN'S SENTIMENT THAT IT'S A PITY WE CAN'T BE MEETING IN PERSON AS WE DO EACH YEAR FOR THESE LECTURES HONORING NEW MEMBERSHIPS BUT HOW REMARKABLE TO THINK WE CAN DO THIS YEARLY. EACH YEAR WE HAVE NEW MEMBERS OF THE NATIONAL ACADEMY AT NIH ELECTED AMONG A VERY LIMITED NUMBER AND WE HAVE THEM HERE AT NIH TO CELEBRATE AND HAVE THIS LECTURE. SO TO MOVE FORWARD LET ME JUST TELL YOU A LITTLE BIT ABOUT ROB TYCKO. ROB IS A SENIOR INVESTIGATOR AND DEPUTY LABORATORY CHIEF OF NIDDK'S LABORATORY OF CHEMICAL PHYSICS. HE RECEIVED HIS UNDERGRADUATE DEGREE FROM PRINCETON UNIVERSITY IN 1980. HAS BEEN A Ph.D. FROM CHEMISTRY CALIFORNIA BERKELEY IN 1984. AFTER DOING POST-DOCTORAL RESEARCH TRAINING AT THE UNIVERSITY OF PENNSYLVANIA, HE JOINED THE AT&T BELL LABORATORY AS A MEMBER OF HIS TECHNOLOGY STAFF IN 1986 AND EIGHT YEARS LATER HE MOVED TO NIDDK WHERE I'M HAPPY TO SAY HE HAS STAYED EVERY SINCE. ROB'S RESEARCH INCLUDES CONTRIBUTIONS TO MAGNETIC RESONANCE, CONDENSED PHYSICS AND STRUCTURAL BIOLOGY. THIS PLACES HIM AT THE INTERSECTION OF PHYSICS, CHEMISTRY AND BIOLOGY. HE'S BEST KNOWN RECENTLY FOR THE APPLICATION OF SOLID STATE NUCLEAR MAGNETIC RESONANCE AND STUDY OF AMYLOID FIBRALES. HIS EXPERIMENTS HAVE FOUND NEW INSIGHT TO THE STRUCTURAL BEHAVIOR OF PROTEINS ASSOCIATED WITH ALZHEIMER'S DISEASE AND CRIONS AND AIDS AND TYPE II DIABETES. ROB HAS BROAD UNDERSTANDING OF THE PHYSICAL PROPERTIES OF FULLER NEEDS A TYPE OF CARBON STRUCTURE THAT INCLUDES [INDISCERNIBLE] AND A TYPE OF FIELD CONFIGURATION THAT CAN MODEL ENERGY PROPERTIES OF THE NUKE -- NUCLEA. HONORS STOWED AMONG ROB ARE FROM THE ALZHEIMER'S RESEARCH FROM THE ALZHEIMER'S ASSOCIATION. THE CHRISTIAN ANTHENSON AWARD PROTEIN SOCIETY. THE HILLIBRANT SOCIETY OF WASHINGTON AND NIH DIRECTOR'S AWARD AMONG OTHERS. BEFORE I HAND THE MIC OVER TO QUESTION DURING THE TALK, PLEASE SUBMIT IT VIA THE VIDEOCAST WEBSITE. LOOK FOR AND CLICK ON THE WORD LIVE FEEDBACK FORUM OR GO TO THE CHAT LINE. THIS IS AT THE VERY BOTTOM OF THIS WEB PAGE IF YOU'RE WATCHING THE LECTURE AND WE'LL RELAY THEM TO ROB AND ROB WILL SELECT THE QUESTIONS AT THE END OF HIS LECTURE. NOW, I'D LIKE TO TURN IT OVER TO ROB. THE TITLE OF HIS TALK TODAY IS MOLECULAR STRUCTURES AND SIGNIFICANCE OF AMYLOID FIBRILES. ALL YOURS, ROB. >> THANK YOU. I APPRECIATE THE GRACIOUS AND COMPREHENSIVE INTRODUCTION. AND I'D APPRECIATE OF THE AWARD AND TO WORK WITH NIDDK FOR THE PAST 26 YEARS. I BELIEVE THIS IS THE BEST JOB A SCIENTIST CAN HAVE IN THE U.S. BECAUSE OF THE CONSISTENT REPORT TO PURSUE THE RESEARCH. I APPRECIATE THAT VERY MUCH. SO I HOPE CAN SEE THAT AND MY CURSER. THIS SAY RECENT PHOTOGRAPH FROM A ZOOM ONLINE MEETING AND CURRENT MEMBERS OF MY LAB. AS DR. RODGERS SAID, MY LAB SPECIALIZES IN SOLID STATE NUCLEAR MAGNETIC RESONANCE A FORM OF NUCLEAR MAGNETIC RESONANCE, NMR. IT'S THE TEAK -- TECHNIQUE FOR STRUCTURES AND MOTIONS OF MOLECULES AND INTERACTIONS MOLECULE ARE SOLUBLE THAT LY DIFFUSE AND TUMBLE FREELY WITHIN SIMPLE SOLUTIONS, LIQUID STATES. SOLID STATE NMR MY LAB IS RELATED BUT SOMEWHAT DIFFERENT IN TERMS OF THE DETAILS OF THE METHODS AND THE EQUIPMENT WE USE AND WE SPENT A LOT OF TIME WORKING ON THOSE METHODS AND IN SOME CASE CONSTRUCTING HOMEMADE EQUIPMENT TO FACILITATE VARIOUS MEASUREMENTS. AND TECHNIQUES ARE TECHNIQUES THAT COULD BE APPLIED TO SOLID-LIKE MATERIALS. HALL -- MOLECULE NOT FREELY DIFFUSING AND COULD BE MEMBRANES AND MOLECULES AND PRODUCTS ASSOCIATED WITH MEMBRANES. IN SOME EXPERIMENTS INVOLVED FROZEN SOLUTIONS OF PROTEINS OR OTHER BIOLOGICAL MOLECULES AND NNR COULD BE APPLIED TO PROTEIN ASSEMBLIES AND CALLED AMYLOID FIBRILS IN THEIR STRUCTURE IN THE PAST 22 YEARS WHICH SAY -- IS A LONG TIME. THESE ARE SOME OF THE AMYLOID FIBRILS WE STUDIED IN MY LAB NEGATIVELY STATED MICROSCOPE IMAGES INCLUDING FIBRILS FORMED BY THE AMYLOID BETA AND THE PEPTIDE ASSOCIATE WITH ALZHEIMER'S DISEASE AND MOST MY TALK WILL BE DEVOTED TO THE ABETA FIBRILS AND WE STUDIED THE AMYLOID POLY PEPTIDE FIBRILS ASSOCIATED WITH TYPE II DIABETES. THE MAMMALIAN PROTEIN, YEAST PROTEINS IN COLLABORATION WITH NIDDK AND THE FIBRILS FORMED BY A YEAST PROTEIN AND RECENTLY FIBRILS FORMED BY LOW COMPLEXITY TI SEQUENCES AND THE RNA BINDING PROTEIN WITH A VARIETY OF FUNCTIONS AND INVOLVEMENTS IN VARIOUS DISEASES INCLUDING CANCER AND DEGENERATIVE DISEASES. SO ALL THESE PROTEINS AND PEPTIDES FORM AMYLOID FIBRILS AND LOOK SIMILAR AT THE LEVEL OF THE MICROSCOPE IMAGE AND LONG, HUNDREDS OF NANO METERS IN LENGTH AND STRAIGHT OVER THE LENGTH SCALES AND ON THE ORDER OF 10 NANO METERS ABOUT THE LIKES OF THE POLY PEPTIDE CHAINS ARE DIFFERENT AND THAT'S DIFFERENT FROM THE BIO PHYSICAL PERSPECTIVE. SEEMS TO BE A STRUCTURAL STATE THAT'S QUITE TOLERANT OF VARIATIONS IN IMMUNOACID SEQUENCE NEARLY GENERIC STRUCTURAL STATE OF POLYPEPTIDE STRAINS AND I SHOWED THE POLYPEPTIDES WITH THE COLOR CODING YOU'LL SEE WHERE GREEN REPRESENTS AMINO ACIDS AND MAGENTA ARE FROM DIFFERENT AND GREEN AND RED ARE POSITIVELY AND NEGATIVELY CHARGED CHAINS AND YOU'LL SEE THE AMYLOID BETA CHAINS HAVE SECTION IN THEM AND SEQUENCES LIKE THE LOW COMPLEX SEQUENCE HAVE NO HYDROPHOBIC SEQUENCES. WHEN WE GOT STARTED ON THIS WORK AROUND 1998, IT WAS UNCLEAR HOW THIS COULD HAPPEN. HOW CAN YOU HAVE A STRUCTURAL STATE WITH PROTEINS NEARLY INDEPENDENT OF AMINO ACID SEQUENCE. ALSO, YOU'LL SEE TWO OF THESE MICROSCOPE IMAGES ARE FIBRILS FORMED BY THE SAME PEPTIDE BUT THE FIBRILS LOOK QUITE DIFFERENT IN THE MICROSCOPE IMAGES. THESE ARE POLYMORPHIC FIBRIL STRUCTURES WHERE THERE'S MULTIPLE MORPHOLOGIES FORMED BY THE SAME AMINO ACIDS. HOW CAN WE DEBT THE POLYMORPHISM. SO BACK IN 1998 WE GOT STARTED WE KNEW THEY'RE MORPH LOGICALLY SIMILAR AND WHAT DOES THAT MEAN THE MOLECULAR STRUCTURE IT'S ALSO SIMILAR TO AN EXTENT AND HOW CAN THEY LEAD TO SIMILAR MOL EC LEAR STRUCTURES. -- MOLECULAR STRUCTURES. AND TO WHAT EXTENT DO MOLECULES WITHIN THE FIBRILS ARE NOT SPECIFIC CONFIRMATIONS AND HAVE SPECIFIC INTERACTIONS RATHER THAN BEING MORE RANDOMLY PACKED TOGETHER SOMEHOW. HOW DOES IT GIVE RISE TO SINGLE MORPHOLOGIES AND HOW DOES THE POLYMORPHISM ARISE AND IS WHAT IS THE BIOLOGICAL SIGNIFICANCE? WE STARTED WITH THE STRUCTURES IN 1998 AND WHAT WAS KNOWN THE AMYLOID FIBRILS CONTAIN BETA SHEETS WHERE YOU HAVE EXTENDED SEGMENTS ACTING THROUGH HYDROGEN BONDS THE GREEN BARS AND YOU HAVE BETA STRANDS COMING TOGETHER TO FORM A BETA SHEET AND THE CROSS-BETA STRUCTURE AND THE BETA SHEET IS ORIENTED IN A PARTICULAR WAY RELATIVE TO THE LONG ACCESS OF THE FIBRIL GROWTH DIRECTION AND THEY RUN PERPENDICULAR TO THE GROWTH AXIS. THE EVIDENCE CAME FROM X-RAY DATA FROM THE LAB AT BOSTON COLLEGE IN 1998 AND THE PEPTIDE SEQUENCE AND WHAT YOU SEE THE PATTERN OR PEAKS IN THE X-RAY SCATTERING INTENSITY AND THE SPACING ALONG THE FIBRIL GROWTH AC SIS -- AXIS AND IT'S THE SPACING BETWEEN BETA STRANDS AND SHEETS. THE OBSERVATION OF A PEAK THAT PROVIDES THE EVIDENCE FOR THE CROSS-BETA STRUCTURAL MOTIVE AND THE RECOGNITION FIRST CAME FROM THE PREDECESSOR OF NIDDK AS I UNDERSTAND IT THE NATIONAL INSTITUTE OF ARTHRITIS AND METABOLIC DISEASES IN 1968, 30 YEARS BEFORE THIS EXPERIMENTS IN THE LAB PROVIDED THE FIRST EVIDENCE THAT AMYLOID FIBRILS WERE CROSSBETA STRUCTURE AND WE'RE CONTINUING THAT WORK BUT STARTING 30 YEARS LATER AND TRYING TO LEARN MORE ABOUT THE STRUCTURE OF AMYLOID FIBRILS AND NOT MUCH KNOWN WAS THAT TIME BECAUSE THEY'RE NON-SOLUBLE PROTEINS AND NOT AMENABLE TO THE MOST WIDELY USED METHODS FOR STRUCTURE DETERMINATION. THAT IS X-RAY CRYSTALLOGRAPHY OR STANDARD LIQUID STATE SOLUTION. THOSE CAN'T BE APPLIED DIRECTLY TO NON-CRYSTAL AMYLOID FIBRILS AND IT'S A GOOD TARGET FOR THE METHODS MY LAB KNOWS HOW TO IMPLEMENT AND METHODS WE DEVELOP OURSELVES. WE DIDN'T DO THE FIRST MEASUREMENTS ON AMYLOID FIBRILS. A COUPLE OTHER LABS DID MEASUREMENTS BEFORE WE DID IN PARTICULAR WHAT GOT US INTERESTED IN THE FIELD WAS A PAPER PUBLISHED IN 1998 USING LOOKING AT THE RESIDUE PIECE OF THE PEPTIDE USING SOLID STATE NMR TO PROVIDE EVIDENCE THE CROSS-BAY TRA STRUCTURES WERE -- CROSS-BAY TRA -- CROSS-BETA STRUCTURES AND ONE IS WHERE THEY RUN IN THE SAME DIRECTION AND ANTI-PARALLEL WERE THE ALTERNATING LEFT TO RIGHT AND RIGHT TO LEFT AND LEFT TO RIGHT. IN THE LITERATURE EVEN DATING BACK TO THIS EARLY PAPER THERE WAS AN ASSUMPTION THEY WERE ANTIPARALLEL STRUCTURES AND FROM THE COLLEAGUES AT THE UNIVERSITY OF CHICAGO PROVIDED EVIDENCE THEY COULD BE PARALLEL BETA SHEETS AND THAT WAS CONTROVERSIAL. OUR ORIGINAL WORK WAS TO CHECK IF THEY GOT IT RIGHT AND LOOK AT THE PEPTIDE SEQUENCE. THIS WORK WAS STARTED BY A VISITOR WHO CAME TO MY LAB IN 1998, 1999 AN ENERGETIC INDIVIDUAL WHO CREATE THE FIRST MEASUREMENT ON MY LAB AND GOT THE PROJECT STARTED. I WANT TO GIVE HIM CREDIT FOR DOING THAT AND CONTINUE TO INTERACT WITH HIM FOR YEARS AFTERWARDS AND IN THE EXPERIMENT O OLEG DID WAS INTRODUCE CARBON 13 ISO TOPIC LEVELS WITH METHYL CARBON LEVELS AND MEASURE THE COUPLINGS WEN -- BETWEEN THE NUCLEI WE INTRODUCED AND THEY'RE DISTANCE DEPENDENT. AND WHERE R IS THE DISTANCE BETWEEN THE CARBON 13 LABELS. AND SO IN A PARALLEL BETA SHEET YOU'LL HAVE A RELATIVELY SHORT DISTANCE AROUND .8 ANGSTROMS AND WE COULD DISTINGUISH BETWEEN THE TWO POSSIBILITIES AND IN FACT THE MEASUREMENT WE DID AT THE TIME WAS SOMEWHAT MORE COMPLICATED TYPE CALLED QUANTIBLE NMR AND WE COULD DISTINGUISH A STRUCTURE WHERE YOU JUST HAVE PAIRS OF PARALLEL CHAINS THEN ALTERNATING RELATIVE TO ONE ANOTHER ON THE GROWTH AXIS FROM STRUCTURES WHERE YOU HAVE THREE OR FOUR OR MORE PARALLEL CHAINS IN A ROW AND YOU HAVE AT LEAST FOUR IN A ROW. SO IT'S A PARALLEL BETA SHEET STRUCTURE RATHER THAN A TRIMERIC STRUCTURE. WE THEN WENT ON TO DO SIMILAR MEASUREMENTS ON OTHER SYSTEMS IN SOME CASES SIM PARTICULAR MEASUREMENTS TO MEASURE THE COUPLINGS. WE FOUND THIS PARALLEL BETA SHEET STRUCTURE IS WHAT WE FIND AND THE AMYLOID PEPTIDES AND THE PROTEINS IN MAMMALIAN PROTEINS IN THE LOW COMPLEXITY SEQUENCE PROTEINS WE'VE STUDIED RECENTLY AND IN OTHER LABS HAVE SHOWN THIS FOR OFFICE KNEW -- NUKE LEE -- AND IN LOOKING AT TAO. IF YOU LOOK AT THE SEQUENCE WITH THE COLOR CODING DIAGRAM AS SHOWN HERE FOR THE STRUCTURE AND MEANING THE CHAINS LINE UP EXACTLY IN PARALLEL WITH ONE ANOTHER VERSUS AN ANTI-PARALLEL ARRANGEMENT IN THE REGISTERED PARALLEL CROSS-BETA STRUCTURE HAVE YOU PATCHES OF HYDROPHOBIC RESIDUES AGAINST ONE ANOTHER TO HAVE FAVORABLE HYDROPHOBIC INTERACTIONS WITHIN A SINGLE CROSS-BETA SHEET. YOU CAN ALSO HAVE FAVORABLE INTERACTIONS BETWEEN BETA SHEETS IF THEY'RE STACKED ON TOP OF ONE ANOTHER AND YOU DON'T GET THAT IN AN ANTI-PARALLEL BETA STRUCTURE AND HAVE OTHER FAVORABLE INTERACTIONS FOR EXAMPLE, GLUTAMINE RESIDUES LINED UP AND CAN HAVE HYDROGEN BONDING BETWEEN CHAINS OF GLUTAMINE WHICH CAN BE STABLIZING INTERACTIONS. BY HAVING THE PARALLEL ARRANGEMENT CAN MAXIMIZE THE FAVORABLE HYDROPHOBIC INTERACTIONS AND FAVORABLE INTERACTIONS SUCH AS THE INTERACTIONS. THE ONE THING THAT WOULD BE BAD WITH A PARALLEL ARRANGEMENT IS YOU BRING LIGHT CHARGES UP AGAINST ONE ANOTHER. IF YOU HAVE GLUTAMATE NEGATIVELY CHARGED CHAINS THEY WOULD TEND TO DESTABILIZE THE STRUCTURE. IT MEANS YOU CAN'T HAVE TOO MANY CHARGED AMINO ACID WITHIN THE CORE AND THE DETAILS OF THE STRUCTU STRUCTURE HAVE TO BE ARRANGED TO MINIMIZE THE REPULSION OF THE SIDE CHAINS. WE WANTED TO GO BEYOND THAT SIMPLE CHARACTERIZATION AND IN OUR EARLY WORK WE WERE INTERACTING AND COLLABORATING WITH THE SCIENTIFIC DIRECTOR AND RICHARD CONTRIBUTED A LOT AND MANY IMPORTANT DISCUSSIONS IN EXPLICIT COLLABORATIONS WITH RICHARD. ONE MEASUREMENT HE SUGGESTED WE DO HE INTRODUCED US TO THE ELECTROMICROSCOPY WAS A MEASUREMENT OF THE INDIVIDUAL FIBRILS. AT THE TIME WE KNEW THEY WERE CROSS-BETA STRUCTURES AND BELIEVED THERE WAS SOME STACKING OF MOTIFS ON TOP OF ONE ANOTHER AND HOW MANY WOULD YOU ACTUALLY HAVE WITHIN ONE FIBRIL? HE THOUGHT HE COULD QUANTIFY THE INDIVIDUAL AMYLOID FIBRILS USING AN ELECTRON MICROSCOPE WHICH HE HAD IN HIS LAB. THE IDEA IS IF YOU HAVE A SINGLE CROSS-BETA MOTIF ON THE GROWTH DIRECTION OF THE FIBRIL WILL BE THE MOLECULAR WEIGHT OF THE INDIVIDUAL POLY PEPTIDE CHAIN AND PROTEIN DIVIDED BY THE SPACING BETWEEN MOLECULES AND IN THE CROSS-BETA STRUCTURE IS .4 NANOMETERS FOR THE FIBRILS WE LOOKED AT THE WEIGHT AND WE COULD JUST QUANTIFY THAT IF YOU HAVE A SINGLE CROSS-BETA STRUCTURAL MOTIF YOU GET NINE PER NANOMETER IF YOU HAVE MULTIPLE MOTIFS STACKED OR BUNDLED YOU EXPERIMENTALLY DETERMINE A MULTIPLE OF THAT AND THEY DID THE MEASUREMENTS PREPARED AND THE FIRST TIME HE GOT A HISTO GRAM PEAKED AT 27 IT MEANT THE FIBRILS HAD THREE CROSS BETA MOTIFS AND THE SECOND TIME HE GOT 18 SO HOW WAS THAT POSSIBLE? WHY SIT ONE DAY YOU GET THREE AND ANOTHER DAY TWO. THIS IS CONFUSING AND I KEPT TELLING RICHARD YOU MUST HAVE MADE A MISTAKE. THERE'S A SINGLE STRUCTURE. HOW CAN YOU GET TWO DIFFERENCE ANSWERS ON TWO -- DIFFERENT ANSWERS ON TWO DIFFERENT DAYS. WE FIGURED OUT THERE WERE TWO TYPES OF FIBRILS AND TWO POLYMORPHS THAT GIVE RISE TO A VALUE OF THIS HAVE A TWISTED PAIR APPEARANCE IN ELECTRON MICROSCOPE IMAGES OF THREE SUBUNITS PUT TOGETHER AND THE FIBRILS WITH A MINIMUM OF 18 ARE WHAT I CALL RIBBONS THESE FLAT RIBBON LIKE FIBRILS COMPRISED OF FI FILAMENTS AND THEY HAVE DIFFERENT VALUES. THAT'S AN INDICATION THE STRUCTURE FOR POLYMORPHS ARE DIFFERENT FROM ONE ANOTHER. A POST-DOC IN MY LAB IN THE EARLY 2000s CARRIED OUT CHARACTERIZATION OF GROWTH AND ELECTRON MICROSCOPE AND DISCOVERED YOU COULD CONTROL THE PREDOMINANT MORPHOLOGY IN THE TEST TUBE IN VITRO BY SUBTLE VARIATIONS IN THE GROWTH CONDITIONS AND TAKING THE SAME PEPTIDE SOLUTION AND ALLOWING THE FIBRILS TO GROW OR GENTLY AGITATING THE SOLUTION AND SLOSHING THE SOLUTION BACK AND FORTH TO GET FIBRILS WITH DIFFERENT MORPHOLOGY A TWISTED PAIR APPEARANCE OR RIBBON APPEARANCE AND PROPAGATE THAT BY SEEDED FIBRIL GROWTH. THAT WILL BE IMPORTANT AS YOU'LL SEE IN LATER WORK AND THE IDEA THE MORPHOLOGY MEANS YOU PREPARE FIBRILS UNDER SOME CONDITIONS YOU BREAK THE FIBRILS INTO SHORT PRAGMENT. WE CALL THEM -- FRAGMENTS WE CALL SEED AND ADD THEM TO A FRESH SOLUTION AND THE FIBRILS GROW TO PRODUCE THE NEXT GENERATION AND REPEAT THAT MULTIPLE TIMES AND THE MORPHOLOGY PROPAGATES FROM ONE GENERATION TO THE NEXT. AND THE FIRST EXAMPLE THAT I'M SHOWING YOU THIS IS A SPECTRUM WE DON'T HAVE TIME TO EXPLAIN WHAT THIS MEANS OR HOW WE DO IT BUT YOU CAN THINK OF THIS AS SOME PATTERN OF SIGNAL INTENSITY THAT IS SENSITIVE TO THE DETAILS OF THE MOLECULAR STRUCTURE. IT'S A STRUCTURAL FINGERPRINT FOR THE PURPOSE OF THIS TALK. YOU SEE THE FINGERPRINTS FOR THE TWO FIBRILS THAT ARE GROWN UNDER AGITATED CONDITIONS INITIALLY. THESE ARE THE FIBRILS GROWN UNDER QUIESCENT CONDITIONS AND THESE IN THE CIRCLED REGIONS ARE DIFFERENT AND THEY PROPAGATE FROM ONE GENERATION TO THE NEXT. I SHOULD SAY THAT IT'S ONLY THE PARENT THE ORIGINAL GENERATION GROWN UNDER DIFFERENT GROWTH CONDITIONS AFTER THAT THE SAME GROWTH CONDITIONS ARE USED IN THE GROWTH OF THE DAUGHTER AND GRANDDAUGHTER. THEN IT BECOMES INDEPENDENT OF GROWTH CONDITIONS. IT'S JUST A MATTER OF WHAT THE SEEDS ARE. THIS SHOWS YOU CAN HAVE MOLECULAR STRUCTURAL DIFFERENCES AND THEY CAN PROPAGATE FROM ONE GENERATION TO THE NEXT. SO THIS WAS A CLEAR DEMONSTRATION OF MOLECULAR STRUCTURES AND THE AMYLOID STRUCTURES ARE NOT DEFINED BY THE AMINO ACIDS IN THE PAPER WE COLLABORATED WITH THE NATIONAL INSTITUTE OF AGEING TO TEST THE TOXICITY OF THE TWO FIBRIL TYPES, TWO DIFFERENT MORPHOLOGIES AND POLYMORPHS. IN PRIMARY NEUONNAL CELL DIFFERENCES THERE WAS A FACTOR OF TWO IN THE CELL CULTURE TOXICITY OF THE TWO MORPHOLOGIES. AND STATISTICALLY SIGNIFICANT. THIS WAS SOME OF THE FIRST INDICATION ALSO THAT THERE MIGHT BE BIO MEDICAL SIGNIFICANCE OF THE AMYLOID POLYMORPHISM. WE THEN WENT ON TO CARRY OUT MEASUREMENTS ON MANY SYMBOLS THAT WERE ISOTOPICALLY DIFFERENT AND COMPLETE STRUCTURAL MODELS. FOR THE TWO MORPHOLOGIES FOR THE T ST STRIATED RIBBON MORPHOLOGY THESE HAVE THEM GOING OUT OF THE SCREEN TOWARDS YOU THE TWO SUBUNITS. EACH SUBUNIT THERE ARE TWO EXTENDED SEGMENTS, TWO BETA STRAND SEGMENTS CONNECTED BY A LOOP. THE TWO BETA STRAND SEGMENTS FORM PARALLEL BETA SHEETS. EACH MOLECULE PARTICIPATES IN THE PARALLEL SHEET STRUCTURE AND FOR THE OTHER MORPHOLOGY A POST-DOC IN MY LAB NOW AT PROFESSOR AT GEORGIA TECH UNIVERSITY DETERMINED THE STRUCTURAL MODEL FOR THE TWISTED PAIR OF MORPHOLOGIES. THEY'RE NOT REALLY TWISTED PAIRS BUT HOW THEY LOOK IN THE MICROSCOPE IMAGES AND THEY HAVE A TRIANGULAR SUBSECTION. THE SUBUNITS ARE SIMILAR. SOME ARE SPECIFIC DIFFERENCES IN MOLECULAR CONFIRMATION BUT THE OVERALL POP TOLL GY IS SIM -- TOPOLOGY IS SIMILAR AND IN THIS CASE THREE COME TOGETHER. WE ALSO STUDIED A PROTO FIBRIL. THE INTERMEDIATE THAT CONFORMS IN THE COURSE OF FIBRIL GROWTH WHERE WE GIVE FIBRIL LIFE ASSEMBLIES GENERALLY SHORTER AND MORE HIGHLY CURVED. THESE ARE CALLED PROTO FIBRILS IN THE LITERATURE AND ANOTHER POST-DOC IN MY LAB AT BINGHAMTON UNIVERSITY CHARACTERIZED THE MOLECULAR STRUCTURE FORMED BY THE DISEASE ASSOCIATED FAMILIAL EARLY ON SET NEURAL DEGENERATION REPLACED BY THESE MODELS AND BETA SHEETS AND THE U-SHAPED CONFIGURATIONS STACK ALONG THE FIBRIL GROWTH DIRECTION IN ALTERNATING FASHIONS AND YOU HAVE AN ANTI-PARALLEL STRUCTURE THAT WE SEE IN THE MATURE FIBRILS. THESE STRUCTURES CAN EXIST. THIS WAS A SURPRISE TO ME WHEN WE FIRST REALIZED THIS WAS HAPPENING BUT THEY'RE LESS STABLE. THE PROTO FIBRILS WILL CONVERGE TO FIBRILS WITH A PARALLEL CROSS-BETA STRUCTURE. THOSE ARE SOME OF THE THINGS WE LEARNED ABOUT FIBRILS WE PREPARED AND PEOPLE ARE INTERESTED IN THE AMYLOID BETA PEPTIDE AND FIBRILS BECAUSE OF THEIR ROLE IN NEURO GEE DEGENERATION AND CLEAVAGES PRODUCED BY THE PROTEIN AND ABROGATES THE SPACE INTO A VARIETY OF STRUCTURES INCLUDING AMYLOID FIBRILS AND LEADS TO NEURO DEGENERATION. BASED ON WHAT WE HAD LEARNED ON FIBRILS PREPARED IN IN VITRO WE STARTED TO WONDER IF FIBRILS THAT GROW IN HUMAN BRAIN TISSUE ARE SIMILAR OR POLYMORPHIC AND IS IT SIMILAR TO WHAT WE SAW IN IN VITRO EXPERIMENTS COULD THEY CORRELATE TO THE CHARACTERISTICS OF THE DISEASE LIKE SEVERITY OF NEURO DEGENERATION AROUND OTHER CHARACTERISTICS. WE WANTED TO DO SIMILAR STUDIES THAT DEVELOP IN BRAIN TISSUE. WE CAN GET AMYLOID FROM AUTOPSIES OF ALZHEIMER'S DISEASE PATIENTS BUT CAN'T CHARACTERIZE THE STRUCTURE FOR TWO REASONS. FIRST, THERE'S NOT ENOUGH MATERIAL THERE AND IT'S NOT ISOTOPICALLY LABELLED AND IT DEPENDS ON INTRODUCING CARBON 13 AND NITROGEN 15 LABELS AS SPECIFIC SITES OR THROUGH THE PEPTIDE SERIES AND WE HAD SHOWN YOU CAN LOOK AT THE STRUCTURAL GROWTH. BY USING FIBRIL GROWTH WE CAN START WITH AN AMYLOID EXTRACTED FROM HUMAN BRAIN TISSUE FROM AUTOPSY TISSUE FROM ALZHEIMER'S DISEASE PATIENTS. THESE ARE GENTLE EXTRACTION PROCEDURE TO PREPARE AN EX TRACT THAT STILL CONTAINS FRAGMENTS OF THE TISSUE AND USE IT AS A SEED AND ADD THEM TO A SOLUTIN THAT CONTAI CONTAINS SYNTHETIC OR KEY COMBINANT AND GO FROM MY GROW -- MICRO GRAMS TO MILLIGRAMS AND EXPERIMENTS OF THIS TYPE WERE FIRST DONE BY A POSTDOC NOW IN CHINA. AND SHE DID THE FIRST EXPERIMENTS ON A PATIENT WE CALLED PATIENT 1. WE HAD A LOT OF CORTICAL BRAIN TISSUE FROM THE UNIVERSITY OF CHICAGO. WE GENERATED LARGE QUANTITIES OF FIBRILS AND EVERYTHING WORKED WELL. WE GOT THESE BEAUTIFUL SOLID STATE WE EVER OBTAINED IN FACT THAT WE GOT SHARP SIGNALS THAT WERE REPRODUCIBLE IN INDEPENDENT PREPARATIONS FROM DIFFERENT REGIONS OF THE BRAIN. AND IT WAS DIFFERENT FROM DIFFERENT SAMPLES. THIS WAS A DIFFERENT MOLECULAR STRUCTURE AND THE SAME IN MULTIPLE INDEPENDENT SAMPLES AND NOT POLYMORPHIC. THIS WAS A SURPRISE. THEN AND THEY WENT ON TO CARRY ON A LARGE SET OF MEASUREMENTS WHICH I DON'T HAVE TIME TO DESCRIBE AND EVENTUALLY WE SHOW IT IN THE CROSS SECTION HERE AND AGAIN I DON'T HAVE TIME TO DESCRIBE THE DETAILS. IT RESEMBLES STRUCTURES WE HAD SEEN FROM IN VITRO FIBRILS BUT DIFFERENT IN CERTAIN IMPORTANT RESPECTS. THE QUESTION WAS IS THIS THE ONLY STRUCTURE THAT DEVELOPS IN THE BRAIN STRUCTURE OF OTHER ALZHEIMER'S PATIENTS. IN THE SAME PAPER IN 2013 WE LOOKED AT A PATIENT CALLED PATIENT 2 DID THE SAME THIN AND GOT DIFFERENT RESULTS. THEY HAD A DIFFERENT STRUCTURE DEVELOPING IN HER BRAIN COMPARED TO PATIENT. -- PATIENT 1. WE THEN WENT BACK AND LOOKED AT THE AUTOPSY REPORTS AND BASED ON THE SKETCHY INFORMATION AVAILABLE PATIENT 1 HAD ATYPICAL AND HAD AN INITIAL DIAGNOSIS OF LEWY BODY AND PATIENT 2 HAD TYPICAL A.D. SYMPTOMS AND THE INITIAL DIAGNOSIS WAS CONFIRMED BY THE AUTOPSY. THIS SUGGESTED THERE MAY BE A CONNECTION BETWEEN AMYLOID STRUCTURES THAT DEVELOP IN THE BRAIN TISSUE IN THE COURSE OF THE DISEASE. THAT'S ONLY TWO PATIENTS. THEN WE STUDIED A LARGER SET OF TISSUE SAMPLES PROVIDED BY THE COLLABORATOR OF COLLEGE OF LONDON INTERESTED IN DISEASES AND OTHER NEURO DEGENERATIVE DISEASES IN PROTEIN-BASED STRAINS. THIS LAB PROVIDED TISSUE SAMPLES FROM THREE TYPES OF PATIENTS. TYPICAL LONG DURATION ALZHEIMER'S DISEASE PATIENTS, PATIENTS WITH A VARIANT CALLED POSTERIOR ATROPHY NOT OBSERVED IN TYPICAL ALZHEIMER'S AND IN RAPIDLY PROGRESSING ALZHEIMER'S DISEASE AFTER THE DIAGNOSIS. USUALLY IT'S A SLOW PROGRESSING DISEASE. FROM THIS LARGE SET OF TISSUE SAMPLES WE PREPARED FIBRILS USING THIS GROWTH PROTOCOL USING THE 40 AND 42 RESIDUE PEPTIDE SEQUENCE. THERE'S TWO VARIETIES. THE 40 RESIDUE SEQUENCE IS THE ONE THAT PRODUCED THE GREATEST ABUNDANCE IN HUMANS AND THERE'S A 42 RESIDUE VERSION SLIGHTLY A MORE HYDROPHOBIC THAT REPRESENTS A PERCENTAGE PRODUCED AND BELIEVED BY SOME THE ABETA 42 MAY BE THE SERIES. WE RECORDED IT AND IT TOOK A LONG TIME. MORE THAN A YEAR OF JUST DATA ANALYSIS AND THE BOTTOM LINE IF YOU COMPARE THE NMR SPECTRA YOU FIND THE SPECTRA OF THE A BETA PEPTIDE FROM THE CORTICAL ATROPHY PATIENTS ARE REPRESENTED BY THE HEAT MAP OF THE DEVIATION BETWEEN SIGNAL INTENSITIES AND I APOLOGIZE I DON'T HAVE TIME TO EXPLAIN IN MUCH DETAIL BUT THE BLUE MEANS THE STRUCTURES ARE SIMILAR AND WHAT WE FOUND IN THE PCM PATIENTS WHERE THERE WAS DIFFERENT SPECTRA RAPTED BY -- REPRESENTED BY THE RED SHADES. MOREOVER WE FOUND THERE'S A SINGLE STRUCTURE WE IDENTIFIED BY SOLID STATE AMR UP MOST SAMPLES. IT'S THE SAME AS THE PATIENT 2 STRUCTURE FROM OUR 2013 PAPER. IN CONTRAST THE FIBRILS FROM THE RAPIDLY PROGRESSING SAMPLES TENDED TO BE MORE POLYMORPHIC. IT CONTAINED SOME PREDOMINANT STRUCTURE AND ADDITIONAL STRUCTURES. ONE POSSIBILITY IS THEY MAY IN SOME WAY BE NEURO TOXIC LEADING TO THE MORE RAPID PROGRESSION. THAT HYPOTHESIS REMAINS TO BE VERIFIED. IN THE CASE OF THE BETA 42 FIBRILS WE DIDN'T SEE SIGNIFICANTLY SIGNIFICANT MORPHISMS BUT WE IDENTIFIED TWO FIBRIL STRUCTURES IN MOST TISSUE SAMPLES. THIS KIND OF WORK WAS PUBLISHED IN AND THE RESULTS ARE STILL SUGGESTIVE THERE'S STILL AN ASSOCIATION BETWEEN POLYMORPHISM AND THE COURSE OF THE DISEASE. WE HAVEN'T SHOWN THAT DEFINITIVELY BUT IT'S AN AN ACTIVE AREA OF RESEARCH IN MY LAB AND OTHER LABS AROUND THE CORRELATIONS BETWEEN DETAILED MOLECULAR STRUCTURES OF AMYLOID FIBRILS AND THE NEURO DEGENERATIVE DISEASES THEY'RE ASSOCIATED WITH IN TAO AND IN THE PROTEIN DISEASES WHERE STRUCTURALLY BASED STRAINS AND THERE'S POSSIBILITY THERE'S NEURO DEGENERATIVE DISEASES IS AN ACTIVE AREA OF RESEARCH STIMULATED BY RESULTS FROM MY LAB. I'M OUT OF TIME. I JUST WANT TO QUICKLY IN A COUPLE MINUTES SHOW YOU A COUPLE THINGS WE'RE WORKING ON. ONE IS TO TRY TO IDENTIFY THE PREDOMINANT STRUCTURES WE IDENTIFIED FROM OUR STUDIES OF BRAIN TISSUES AND FOR THE PREDOMINANT POLYMORPH WE'VE SEEN IN MOST CASES WE'RE NOW USING MICROSCOPY TO DEVELOP STRUCTURAL MODEL. THROUGH THIS WORK SOLID STATE NMR IS THE ONLY WAY TO IDENTIFY THE FIBRILS. AND OVER THE LAST FEW YEARS IT'S IMPROVED IN TERMS OF THE TECHNOLOGY THE MICROSCOPES AND CAMERAS AND THE SOFTWARE. WE'RE NOW TAKING ADVANTAGE OF THAT AND THIS IS A MODEL FOR THE PREDOMINANT POLYMORPH FROM ALZHEIMER'S DISEASE BRAIN. THIS IS WORK WE DID TO IMPROVE THE IMAGE TO DEVELOP A DENSITY MAP. HIGH ENOUGH RESOLUTION TO UNAMBIGUOUSLY FIT THE SEQUENCE IN. WE'RE ALSO WORKING ON A DIFFERENT TYPE OF FIBRILS FORMED BY SO-CALLED LOW-COMPLEXITY SEQUENCE LIKE THE RNA BINDING SEQUENCE SHOWN HERE. IT'S A NON-HYDROPHOBIC SEQUENCE DIFFERENT FROM THE ABETA SEQUENCE OR OTHER SEQUENCES MOST WIDELY STUDIED AND RESIDUES. YOU MIGHT THINK SEQUENCES LIKE THIS BECAUSE THEY'RE NOT HYDROPHOBIC WOULD BE HIGHLY SOLUBLE BUT THEY'RE RATHER INSOLUBLE AND FORM BEAUTIFUL AMYLOID FIBRILS. YOU CAN'T SEE THE MICROSCOPE IMAGE. I APOLOGIZE FOR THAT. ONE THING WE EXPECTED IS THE FIBRILS WOULD BE HIGHLY POLYMORPHIC BECAUSE THE AMINO ACID COMPOSITION IS UNIFORM OVER THE 240 AMINO ACID SEQUENCE. YOU'D EXPECT THERE WOULD BE MANY WAYS OF FORMING A FIBRIL CORE USING DIFFERENT SEGMENTS AND THE POSSIBLE STRUCTURES WOULD HAVE CERTAIN ABILITY TO COEXIST AND YOU END UP WITH A HIGHLY POLYMORPHIC SITUATION AND THESE ARE NOT POLYMORPHIC AND WE ALWAYS GET THE SAME FIBRIL CORE STRUCTURE DETERMINED BY MY POST-DOC AND IT'S ALWAYS FORMED BY THE SAME SEQUENCE RESIDUES 39 TO 95. NOW WE'RE TRYING TO UNDERSTAND WHY IS THAT? WHAT'S SO SPECIAL ABOUT THIS SEGMENT OR IS THERE ANYTHING SPECIAL ABOUT THIS SEGMENT THAT LEADS TO A NON-POLYMORPHIC STRUCTURED AMYLOID FIBRIL CORE. AND SOME ARE INCAPABLE OF FORMING FIBRILS AND THAT TURNED UT NOT TO BE TRUE. A POTT DOCK HAS BEEN STUDIED THE SEQUENCE. AND DEVELOPED A STRUCTURE MODEL. THE SECOND HALF OF THE SEQUENCE IS CAPABLE OF FORMING QUIT STABLE STRUCTURED AMYLOID FIBRILS. SO WE'RE STILL TRYING TO UNDERSTAND WHAT DETERMINES WHAT THE CORE FORMING SEGMENT IS IN A REPETITIVE AMINO ACID SEQUENCE USING THE STRUCTURE. WE CAN NOW DO A BEAUTIFUL MOLECULAR DYNAMIC SIMULATION. IT'S A GOOD WAY TO END THE TALK BY SHOWING A MOVIE TO ILLUSTRATE HOW THE MOLECULES MAY BE MOVING AROUND WITHIN AN AMYLOID FIBRIL AND THIS ALLOWS US TO IDENTIFY SOME OF THE IMPORTANT STABLIZING INTERACTIONS WITHIN THE CORE AND WE'RE LOOKING AT NON HYDROPHOBIC FIBRILS. I WANT TO THANK YOU FOR YOUR ATTENTION AND THANK EVERYBODY WHO'S CONTRIBUTED TO THIS WORK AND AGAIN THANK THE INTRAMURAL RESEARCH PROGRAM AT NIDDK AND MY COLLABORATORS AT THE NIH FOR THEIR MANY CONTRIBUTIONS TO THIS WORK. IF THAT'S ANY TIME FOR QUESTIONS I'LL TRY TO ANSWER THEM. >> DO WE HAVE TIME FOR QUESTIONS? >> WE'LL HAVE TO SAVE THE QUESTIONS FOR OFFLINE. I WANTED TO THANK BOTH YOU AND JOHN FOR REALLY SPECTACULAR EXAMPLES OF HOW THE MOST BASIC SCIENCE CAN HAVE PROFOUND CLINICAL IMPLICATIONS BOTH IN TREATMENT AND DIAGNOSIS OF DISEASE AND THANK BOTH OF YOU FOR OUTSTANDING TALKS AND I LOOK FORWARD TO NEXT YEAR'S MINI-SYMPOSIUM FROM OUR NEW NATIONAL ACADEMY MEMBERS. THANK YOU VERY MUCH AND GOOD-BYE TO EVERYBODY.