WE ARE HERE IN REMEMBRANCE OF A FRIEND AND FORMER COLLEAGUE IN DR. DAVID DERSE, THOSE OF YOU WHO HAD THE PRIVILEGE OF KNOWING DAVE AND WORKING WITH HIM, THOSE WHO WERE HIS FRIENDS AND COLLEAGUES, HIS FAMILY WILL KNOW THAT I CANNOT DO HIM JUSTICE AS A SCIENTIST OR AS A PERSON IN A FEW BRIEF MINUTES. DAVE WAS BOTH A GOOD FRIEND AND A VALUED COLLEAGUE BUT HE DIED TOO SOON OF CANCER, SENDS THE CLEAREST POSSIBLE MESSAGE THAT WE NEED BETTER DIAGNOSIS AND BETTER TREATMENT. WE SHOULD BE BASED ON AND DERIVED FROM MORE AND BETTER SCIENTIFIC RESEARCH. HIS DEATH IS OUR LOSS, IT REMINDS THOSE OF US, PART OF THE SCIENTIFIC COMMUNITY THAT UNDERLYING CHALLENGES POSED BY EXPERIMENTS AS YET UNDONE AND PROBLEMS AS YET UNSOLVED ARE IN FACT THE PREMATURE DEATHS OF WONDERFUL PEOPLE LIKE DAVE DERSE. IT WAS MY GOOD FORTUNE TO HAVE DAVE NOT ONLY AS A FRIEND AT WORK AS HE IS DEPICTED HERE BUT ALSO AS A FRIEND AWAY FROM WORK. WE BOTH LOVE TO FISH AND I HAD THE GOOD FORTUNE OF TAKING THAT PICTURE ON A TRIP TO CHESAPEAKE BAY, A SPRING DAY SEVERAL YEARS AGO. AND THIS PICTURE WAS TAKEN ON TOWN CREEK AND THAT'S A PLACE THAT'S REALLY SPECIAL TO BOTH DAVE AND ME. AND NO ONE IS IN THIS PICTURE BECAUSE SADLY DAVE IS NO LONGER WITH US. THAT MEANS WHEN I GO TO CHESAPEAKE OR TO TOWN CREEK I WILL NOT HAVE THE PLEASURE OF HIS COMPANY. NOR CAN WE GET FURTHER BENEFITS FROM HIS WISDOM. WE CAN HOWEVER REMEMBER AND HOLD THE WARMTH OF HIS SPIRIT IN OUR HEARTS AND KEEP THESE IDEAS IN OUR CONTRIBUTIONS IN OUR MEMORIES, IN SUCH A GOOD MAN SHOULD NEVER BE FORGOTTEN. AND IN THAT SENSE WE SHOULD BE HERE IN CELEBRATION, CELEBRATION OF A LIFE WELL LIVED, OF A GOOD FRIEND WHO LOVED RESEARCH AND WHOSE WORK HAS DONE SO MUCH TO ADVANCE OUR KNOWLEDGE OF VIROLOGY. WE'VE MADE PART OF THAT CELEBRATION A SEMINAR THAT WE THINK DAVE WOULD HAVE ENJOYED. NOT ONLY ONE THAT TAKES US TO NEW PLACES INTELLECTUALLY BUT ONE THAT WILL AS HAS BEEN OUR TRADITION, BE GIVEN BY SOMEONE WHO HAS BOTH PERSONAL AND PROFESSION CONNECTIONS TO DAVE AND THAT PERSON FOR THIS LECTURE IS DR. WALTHER MOTHES, AND WE ARE HONORED TO HAVE HIM AS THE DAVID DERSE LECTURER. THIS AWARD IS SPONSORED BY THE HIV DRUG RESISTANCE PROGRAM, WITH SUPPORT FROM DR. KATE DERSE, THE FOUNDATION FOR NIH AND THE FRIENDS AND COLLEAGUES WHO CONTRIBUTED TO THE DAVE DERSE MEMORIAL FUND. HUMANS ARE VISUAL LEARNERS, FOR US AS THE TALK SO CLEARLY STATES, SEEING IS BELIEVING AND TODAY WE WILL GET TO SEE SOMETHING THAT HUMANS DON'T OFTEN SEE. WE GET TO SEE HIV INFECT CELLS. PLEASE JOIN ME IN WELCOMING DR. WALTHER MOTHES. [ APPLAUSE ] AND WE'RE DELIGHTED TO HAVE YOU HERE AND IN ADDITION TO GIVING THE TALK, THERE IS TO GO WITH IT, KATE, WOULD YOU JOIN US PLEASE? THIS IS AS MUCH FOR YOU AS FOR EVERYONE, PLEASE, I INSIST. YES. [ APPLAUSE ] [ APPLAUSE ] >> YEAH, MANY THANKS, STEVE ANDICATE AND ALL OF YOU, IT'S AN HONOR TO BE HERE WITH YOU TODAY AND REMEMBER DAVE I MET DAVE VERY LATE IN HIS LIFE AND I MET HIM FIRST THROUGH HIS EXCELLENT PEOPLE. I WAS LOOKING AT [INDISCERNIBLE]--AND ALSO BIOSTATTISTICAL INFORMATION SO WE SHARED THE INTEREST IN BIOSYNTHESIS INFORMATION AND DAVE WAS INCREDIBLY SUPPORTIVE AND FOR THE CONSTRUCTS THAT THEY GENERATED AT THE TIME THEY WERE ABLE TO QUANTIFY THE VIRUS AND THEN WITH HIV TRANSMISSION QUANTITATIVELY AND STUDY A CERTAIN ASPECTS AND VERY GRATEFUL FOR THIS INCREDIBLE SUPPORT. AND THIS PAPER CAME OUT AFTER DAVID LOST THE BATTLE AGAINST CANCER AND IN THE NEXT YEAR. AND WITH THIS [INDISCERNIBLE] THE CONSTRUCTS WITH THE LUCIFERASE IN THIS CASE, IT'S EXPRESSION AND THE PRODUCERS ARE SUPPRESSED BECAUSE OF THESE CARRY INTERIM SOOOSE ONLY EXPRESSED AFTER IT INFECTS THE NEXT CELL AND THE NEXT CELL ALLOWED US TO QUANTSIFY THE TRANSMISSION AND WE HAVE A COUPLE OF JOINED PAPERS [INDISCERNIBLE] AND THEN THIS IS OUR FIRST WORK ON HIV AND OF COURSE THIS WAS PUSHED INTO [INDISCERNIBLE] BUT I'M VERY PROUD OF THIS WORK, WORKED VERY HARD, ACTUALLY. SO BECAUSE OF THIS HISTORY, I WOULD LIKE TO START WITH SOME DATA THAT WERE REPORTED EARLIER FOR THE SECOND GENERATION AND WITH THE TRANSMISSION BY DIFFERENT MODES OR TRANSMISSION AND DIRECTLY CONTACT THE TARGETS AND THOSE TRANSMISSIONS HAVE ADVANTAGES AND I BELIEVE THAT BIAS LIKE HIV THAT GOES TO SPREAD AND WITH OTHER SPREAD DISASTERS WHERE OUR GOAL HAS THE ABILITY TO TAKE ADVANTAGE OF BOTH MODES OF TRANSMISSIONS AND CERTAIN CIRCUMSTANCES. SO THEN THERE ARE CERTAINLY ADVANTAGE TO THE VIRUS, DESPITE INVITRO, THERE'S ANY EVIDENCE THAT HIV [INDISCERNIBLE] FOR CELL TO CELL TRANSMISSION IN TISSUE CULTURE AND VERY EFFICIENTLY, LOTS MORE EFFICIENTLY THAN [INDISCERNIBLE]. AND A KEY FEATURE MAY BE THE DEFINING FEATURE OF THIS MODE OF TRANSMISSION IS THAT THERE CAN BE HIGH LOCAL PARTICLES THAT OFTEN RESULTS IN THE HIGHER NUMBER OF INTEGRATION SITES THAT I MENTIONED. AND SIMULTANEOUSLY LEARNING MORE CORRECTLY REALIZE [INDISCERNIBLE] THAT HIGH LOW TO MRI, MEANS YOU NEED HIGHER CONCENTRATION OF THE INHIBITORS TO INHIBIT CELL TO CELL TRANSMISSION. AND HE SHOWED THAT TAMIFIVIR INHIBITS AND SPREAD IN TISSUE CULTURE. SO THEY ALSO CONCLUDED THIS IS THE MECHANISM FOR ONGOING REPLICATION DESPITE ANTIRETROVIRAL THERAPY WHICH IS LIKELY QUESTIONABLE BECAUSE PARTICULARLY HERE AT THE NCI, THEY'VE SHOWN LITTLE EVIDENCE FOR ONGOING REPLICATION SO THESE VARY BECAUSE MANY PEOPLE KNEW THE DATA FOR THIS APPLICATION BECAUSE CLINICIANS EXPERIENCE EVERY DAY THAT AIDS PATIENTS CAN LIVE A LONG LIFE BECAUSE OF ANTIRETROVIRAL THERAPY WORKS. THE FIELD CONCLUDED THAT HIV MUST SPREAD IN PEOPLE BY [INDISCERNIBLE] BECAUSE IT SHOULD SHOWN THAT IT DOESN'T WORK IN CELL TO CELL TRANSMISSION SINCE THE ANTIRETROVIRAL THERAPIES WORK AND MANY CONCLUDED THAT HIV SPEDS BY CELL RATE. NOW THIS WAS A CONCLUSION THAT WE VERY MUCH DOUBTED AND LUIS AGOSTO WORKED ON THE ANTIRETROVIRAL THERAPY AND CELL TO CELL TRANSMISSION AND THE EXTERNAL SYSTEM THAT HE USED WAS BASED ON THE CONCEPTS [INDISCERNIBLE] IN THE LABORATORY WHERE COMPARED FOR THE FREE CELL BIAS IN CELL TO CELL TRANSMISSION AND THIS WAS THE [INDISCERNIBLE]. THEY MADE A DRUG CELL LINE THAT STABLY EXPRESSES THE INTERREGULATED [INDISCERNIBLE] SO THIS WAS MADE BY TRANSSECTION, NOT BY INSECTION. BY TRANSSECTION SO IT CARRIED THE LUCIFERASE AND THEN THE INFECT WITH HIV AND HIV WAS REPLICATE AND THE PACKAGE THIS REPORTER AND THEN [INDISCERNIBLE] THESE CELLS OF PRIMARY HUMAN CD4 T-CELLS TO MEASURE THE GENERATION THE SECRETION OF THE LUCIFERASE. AND THEN WE TAKE THE INFECTED CELLS AFTER THIS [INDISCERNIBLE] AND THE FACTS OUT THE P24 POSITIVE CELLS AND THEN WE FIND THAT THE POPULATION TO PERFORM PC R IN A POPULATION THAT THERE'S A SIX FOLD HIGHER BIOCONTENT AND THE CONDITIONS OF CELL TO CELL SPREAD COMPARED TO CELL FREE TRANSMISSION. SO LUIS USES THE SYSTEMATICALLY TEST THE ANTIRETINAL LOCATION ARE VIRAL INHIBITORS AND IN COMBINATION, AND TO SHOW YOU HERE, THIS IS AN EXPERIMENT HERE, WHERE THEY PRODUCE THE ORIGINAL BALTIMORE OBSERVATION SO THIS IS ENCASING THE TAMOFOVIR, AND YOU SEE THIS WITH THE CELL FREE AND CLOSE TO THE [INDISCERNIBLE] SO TURNOVER THE AREA IN SUPPRESSING [INDISCERNIBLE] HIV BUT THE TRIAL S&P A MUCH HIGHER DOSE TO SUPPRESS HIV OF CELL TO CELL TRANSMISSION AND A THOUSAND FOLD HIGHER AND THIS IS IMPORTANT BECAUSE OF THE MEASUREMENT IN THE PATIENTS FOR THE ANTIRETROVIRAL THERAPY. THIS CANNOT SUPPRESS CELL TO CELL TRANSMISSION, SO WE SEE SIMILAR PHENOTYPE FOR AZT. THIS IS HIGHER TRANSMISSION FOR AZT TO SUPPRESS CELL TO CELL TRANSMISSION. SO OTHER RTIs THAT THE CURSE MOVE CLOSER AND THE MOVEMENT WE TEST RTIs AND INHIBITORS, PROTEASE INHIBITORS THAT ARE ON TOP OF EACH OTHER. WE FOUND THAT THEY FAILED TO INHIBIT CELL TO CELL TRANSMISSION AND THEN THE PROTEASE INHIBITORS ARE ALL EFFECTIVE, NO MATTER IRRESPECTIVE OF THE MODE OF TRANSMISSION. THIS IS QUANTIFIED IN THE GRAPH, THERE YOU SEE IN THE DIFFERENT CLASSES OF INHIBITORS AND THIS IS FAILURE FOR DIFFERENCE WITH THE CELL TO CELL TRANSMISSION AND YOU SEE THAT HERE, [INDISCERNIBLE] AZT ARE THE ONES THAT FAIR BUT THE OTHERS ARE FAIRLY EFFECTIVE AND IN FACT, THIS IS [INDISCERNIBLE], AND JUST MOVING THROUGH [INDISCERNIBLE] VIRUS, THE EFFECT IS ACTUALLY MUCH SMALLER. THERE'S 20-34 CONCENTRATION IN CELL TO CELL TRANSMISSION SO IT'S ALSO DEPENDENTOT HIV ISOLATE. SO NOW, WE WANTED TO NEXT ASK, SO WE CAN CERTAINLY WE KNOW THAT TAMOFIVIR IS AN ANTIRETROVIRAL THERAPY AND IT'S EFFECTERRIVE AND IT'S USING INDEED COMBINATIONS. SO WE WANTS TO LOOK AT COMBINATIONS AND WE WANT TO ASK WHAT HAPPENS IF WE COMBINE TAMOFIVIR, AND INDIVIDUALLY OR IF WE COME BINE THEM. SO IN RED WE SEE AZT ALONE WHICH YOU WILL SEE FROM THE PREVIOUS AZT ALONE THIS IS DATA AND THIS IS TAMOFIVIR AND BLACK YOU WILL SEE THE CAUSE FOR THE COMBINATION, YOU WILL SEE THAT THE COMBINATION OF THE TWO DRUGS THAT FAIL INDIVIDUALLY ARE EFFICIENT IN SUPPRESSING CELL TO CELL TRANSMISSION AS WELL AS [INDISCERNIBLE] SO THIS LIKELY EXPLAINS WHY NRTI THAT ARE FAIR TO INHIBIT HIV SPREAD BY CELL TO CELL CONTACT AS INDIVIDUAL DRUGS, THEY WORK IN COMBINATIONS. SO HERE YOU WILL SEE OTHER COMBINATIONS AND AGAIN COMPARED TO THE INDIVIDUAL, YOU WILL SEE THE TWO CURVES IN BLACK AND GRAY ON TOP OF EACH OTHER. SO THEN WE ASK WHAT DOES THE MECHANISM OF THE SYNERGISTIC EFFECT AND WE CANNOT ANSWER THIS ALL INHIBITORS THAT THIS VARIOUS MECHANISMS HERE TAKE BUT WE NOTICE THAT IT HAS BEEN DESCRIBED WITH SYNERGY AND RTI, FOR THE INHIBITION OF THE [INDISCERNIBLE] SO WHEN YOU REDUCE AND INCORPORATE ENVELOPE, YOU CAN SOMETIMES FANATICALLY FOR AZT AS DESCRIBED IS VERY [INDISCERNIBLE] AND STILL A CONFLICT SO IT SLIDES BACK AND THEN IT CAN BE EXERCISED AND THEN ALL THE NUCLEOTIDE PLACED THERE. AND THIS HAS BEEN DESCRIBED TO OCCUR IN COMBINATION THERAPY AND TO USE THIS, IT TESTED THIS MUTANT. THIS IS A TEAM MUTATION THAT MAKES IT HYPER SENSITIVE TO AZT, BECAUSE AZT CAN NO LONGER EXCISE. AND NOW COMPARED TO WILD-TYPE IN RED, YOU SEE NOW THAT IN MUTE ANT, THE CURSE THE CELL ON TOP OF EACH OTHER SO THE MOMENT YOU HAVE NUCLEOTIDE EXCISION REMOVED, THE DRUG IS EFFECTIVE AND SUPPRESSING CELL TO CELL TRANSMISSION. SO WE THEN WANTED TO ASK IF AGAIN COMING BACK, IT APPEARS TO BE THE DEFINING FEATURE OF CELL TO CELL TRANSMISSION AND ASK IF THIS IS REALLY TRUE, THEN IS WE HAVE ABOUT SIX POOLED VIRUSES IN OUR CONDITIONS AND WE SHOULD BASICALLY MIMIC THE CELL TO CELL TRANSMISSION BY INCREASING MRI OF THE CELLS, THIS IS THE CASE, SO HERE ARE THE DRUGS THAT FAIL TO INHIBIT CELL TO CELL TRANSMISSION AND THEN INCREASE FROM ONE TO FIVE TO 25. YOU SEE THAT WE NEED CORRESPONDINGLY MORE INHIBITOR TO INHIBIT THE SPREAD OF THIS VIRUS AND IN CONTRAST, THE COMBINATIONS AND THE MMRTI, BECAUSE THEY ARE MRI DEPENDENT, WITH ALL THOSE IRRESPECTIVE OF HOW MANY PARTICLES YOU HAVE, SO WHAT THIS SHOWS YOU IS THAT CHARACTERISTIC OF EFFECTIVE ANTIRETROVIRAL THERAPY IS THAT THEY ARE MOI DEPENDENT AND IT'S DIFFICULT TO PERFORM THE TRANSMISSION--AND WE LEARNED FROM LUIS WORK IT WORKS AND CYTOSOL INFORMATION BECAUSE EFFECTIVE DRUGS OF COMBINATION THERAPY IS WORK AGAINST HIGHER CONCENTRATION OF PARTICLES. SO NOW WE MOVE THE ARGUMENT OF HOW THE FIELD RESPOND TO BALTIMORE PAPER SAYING THAT ALL SPREAD BECAUSE OF THE ANTIRETROVIRAL THERAPY IS FAIL IN CELL TO CELL IS SIMPLY NOT TRUE BECAUSE IT'S ONLY OBSERVED FOR TWO OR THREE INHIBITORS AND FOR ALL THE OTHER RETROVIRAL INHIBITORS AND COMBINATIONS, IT'S ACTUALLY EFFECTIVE AGAINST HIV CELL TO CELL TRANSMISSION. OF COURSE, THIS DOESN'T ANSWER THE [INDISCERNIBLE] OF HOW HIV IS SPREAD IN VIVO, IT ADJUSTS AND MOVES THIS ARGUMENT AND I WOULD LIKE TO SAY, INTERESTED TO CONTRIBUTE TO THIS QUESTION AND WHAT WE ARE TRYING TO DO IS DIRECTLY VISUALIZE VALUABLE SPREAD IN LIVING MICE AND I WOULD LIKE TO REMIND YOU THERE ARE TWO CONCEPTS THAT HAVE BEEN DEVELOPED INVITRO OF HOW VIRUS IT SPREAD, ONE IS AROUND THE VIROLOGICAL SYNAPSE, WHERE TRAY EXPRESS THE GLYCOPROTEIN AND THEY IT FUNCTIONS AS PROTEIN AND IT'S CELL THERE AND THEN, RETROVIRAL CAPSULE IS REDIRECTED TO THE INTERFACE AND BIOPARTICLES AT THE CYTOSOL AS CONTACT FOR EFFICIENT TRANSMISSION. SO IT'S INDEPENDENT PROCESS AND IT CAPTURES AND PARTICLES ACCUMULATE IN THE IRPT FACE. THE SECOND CONCEPT IS OF TRANSINFECTION, IT WAS ORIGINALLY CALLED INFECTIOUS SYNAPSE AND IT IS TO CAPTURE HIV AND TRANSFER TO A T-CELL WITHOUT BECOMING INFECTED. SO I WILL SHOW YOU EVIDENCE FOR BOTH PROCESSES [INDISCERNIBLE] AND SPREAD IN VIVO THAT'S WHAT WE'RE TRYING TO DO. AND THIS WAS ESTABLISHED IN OUR LABORATORY BY XAVER SEWALD, AND THE FIRST QUESTION WE WANT TO ASK IS DO I HAVE LODGEICAL SYNAPSES WITH THE INFECTED AND UNINFECTED CAN THEY EXIST IN VIVO? AND IN ORDER TO DO THIS BY PERFORMING A SCREEN, THEN WE IDENTIFY WHERE THEY PREPARE THE B-CELLS MACROPHAGES AND THE MODELS SO THEY HAVE THE LYMPHOCYTES AND THEN THE INFECT ANT AND GFP EXPRESSING VIRUS, AND THAT IS INFECTED MICE, CELLS BACK INTO THE MOUSE AND VISUALIZE THE BEHAVIOR IN THE LYMPHNODE OF THE LIVING MOUSE. AND WE USE VARIOUS AS A MODEL VIRUS BECAUSE WE HAD DEVELOPED THE [INDISCERNIBLE] AND IT'S ALSO A VERY GOOD MODEL TO STUDY CELL TO CELL SPREAD BECAUSE [INDISCERNIBLE] IS AN ENDOGENOUS VIRUS IN MICE. WE ARE CURRENTLY TRYING TO DO THIS EXPERIMENT FOR HIV IN THE MOUSE BUT IT'S NOT A GOOD PERFECT MODEL BECAUSE IT'S A HYBRID SYSTEM AND THAT'S ALL INTERACTIONS BETWEEN HUMAN LYMPHOCYTES IN THE MOUSE TISSUES ARE COMPLEMENT, IN CONTRAST, THE MMV MUST STUDY THE INFECTION IN ITS COURSE, SO THE DESIGN SECTION IS PRETTY MUCH THE BEST IT CAN DO. SO NOW, IF YOU CAN LOWER THE LIGHT TO SEE--WE WILL SEE WHAT--I HAVE A COUPLE OF MOVIES. SO THE SCREEN--THE ABILITY OF PRIMARY CELLS WHEN THEY BECOME INFECTED AND BE ASHES DATA PROTECTIONIVELY TRANSFERRED AND BACK INTO THE MOUSE, WE ASK IS THERE ANY CELL TYPE THAT CAN FORM LONG LASTER BIOLOGICAL SYNAPSES. AND THE--SO WHAT WE DO, SO WHAT THE--BUT THE IDENTIFIED, THE IDENTIFIED B-CELLS--IT'S OKAY. SO WE ARE--WE ARE HERE DIRECTLY--GREAT, THANK YOU. THAT'S PERFECT, YEAH. SO WE HAVE THE SCREEN, AND THE STANDARD ARE THE BETAS SO WHAT YOU SEE IS THE SIDE OF A LYMPHNODE OF A LIVING MOUSE AND BLUE IT IS A COLLAGEN IN RED, YOU WILL SEE THE B-CELLS THAT PREPARED FROM AN RFP MODEL AND THEY CONCENTRATE IN THE AREAS THAT B-CELL FOLLICLES AND AGREE IN THE CELLS, BECAUSE THESE ARE THE B-CELLS THAT ARE INFECTED BY MMV THAT EXPRESSES GFP. AND THEN WE TRACT THE INFECTED CELLS COMPARED WITH THE UNINFECTED CELLS, WE FOUND THAT MANY OF THE INFECTED CELLS ACTUALLY IMMOBILE AND THEN THE [INDISCERNIBLE] INTO THIS [INDISCERNIBLE] BUT WE FOUND WAS THAT THE INFECTED CELLS THAT GAG POLARIZES--GAG-GFP POLARIZES TO ONE SIDE OF THE CELL AND WE EXPECT THE CELL TO BE IN CONTACT WITH ANOTHER LYMPHOCYTE AND IN FACT YOU CAN SEE THE EXCHANGE OF MATERIAL IN THIS AREA. AND THEN PROBABLY, WE WOULD ONLY OBSERVE THIS POLARIZATION OF GAG FOR THE CELL IF THIS WAS INFECTED BY THE WILD-TYPE VIRUS. IF THE INFECT S&P THE CONSTRUCT BUT THE ENVELOPE IS NOT EXPRESSED THEN YOU DON'T OBSERVE THE BIOLOGICALS. THIS POLARIZATION, SO AS I MENTION IN THE BEGINNING, THE ENVELOPE INDEPENDENT POLARIZATION, GFP, SO, REALLY THE HALLMARK OF THE BIOLOGICAL SIGN APSE AND WE ARE VERY EXCITED BECAUSE IT'S PARTICULARLY--PARTICULARLY TO ASK THIS SUCH AS [INDISCERNIBLE] VIROLOGICAL SYNAPSES AND IT'S IN THE LYMPHNODE. WE THEN QUANTIFIED THE GFP MEMBER YOU SEE HERE, WE SEE THE POLARIZATION TO ONE SIDE WHICH IS NOT OBSERVED IN AND THE CELL IS INFECTED IN VIRUS IN THE NEXT ENVELOPE AND IMPORTANTLY JUST THE POINT MUTATION IN THE ENVELOPE THAT USES THE AFFINITY FOR THE END CAP AS THE SAME PHENOTYPE SO THIS POLARIZATION OF THE GAG IS THE CONCENTRATE OF THE AFFINITY OF THE GLYCOPROTEIN FOR THE NCAT AND WE'RE REALLY LOOKING AT BIOLOGICAL SYNAPSES. SO WE THEN USED IDENTIFIED THE CELLS THAT THESE INFECTED B-CELLS CONTACT AND INVITRO WOULD BE INCUBATED FOR THESE INFECTED B-CELLS TOTAL LYMPHSITES FROM THE LYMPHNODE, FROM THE GREEN MOUSE AND YOU SEE THE BIOLOGICAL SYNAPSES AND DETERMINE THAT THE B-CELLS ARE CONTACTED WITH THE CD4 T-CELLS AND UNINFECTED B-CELLS. OF COURSE, THE--THEY THEN WANT TO ASK IF YOU HAD PERFORMED THE VICIOUS SCREEN AND WE WANTED TO GO BACK AND ASK WHAT HAPPENS WHEN THE MOUSE IS INFECTED WITH THE VIRUS AND INFECT THE MOUSE AT THE LEG BECAUSE THE IMAGED IN THE POPLITEAL LYMPHNODE SO WE WANT TO STUDY THE NATURAL INFECTION OF THE MOUSE GOING TO THE SAME ROUTE, THIS MODE OF TRANSMISSION CAN BE OBSERVED AND ALSO WITH THE TRANSMISSION IN MICE SO TWO DAYS AFTER INFECTION, WE DO FIND INFECTED CELLS IN THE POPLITEAL LYMPHNODE AND NOT IN SECONDARY LYMPHNODE SO THE INFECTION IS CONTAINED TO THE DRAINING LYMPHNODE. AND THE INFECTED CELLS ARE THE CD4 T-CELLS AND B-CELLS. SO IMPORTANTLY AS SHOWN HERE IF WE INFECT THE MICE, THE INFECTION IS MUCH REDUCED, SO THIS WAS THE FIRST STEP IN OUR [INDISCERNIBLE] OF BIOLOGICAL SYNAPSES CAN EXIST IN VIVO AND PERFORMED DIVISION SCREEN AND IDENTIFIED B-CELLS, AND BASHING-CELLS ARE AMONG THE TYPES AND FOR THE ESTABLISHMENT OF AN INFECTION IN MICE. WHICH SUGGESTED THEY COULD PLAY IN A TRANSMISSION. BUT THIS, I THINK THE WEAKNESS OF OUR FIRST REPORT AND THIS WAS THAT WE DIDN'T STUDY THE WHOLE PROCESS DURING INNATE INFECTION, SO THIS IS REALLY WHAT WE WANT TO DO NEXT, STUDY ALL THE STEPS OF AN INFECTION OF A MOUSE, THIS FLUORESCENT PARTICLES, RATHER THAN ADOPTIVE TRANSFERRING IT IN CELLS OF THE INFECTED EXVIVO. AND WHAT WE REALIZED IS WE HAD DETECTED BIOLOGICAL SYNAPSE IN THE--IN THE [INDISCERNIBLE] OF THE LYMPHNODE, BUT IF WE WATCH THE LYMPHNODE FROM THE PERIPHERY AND THERE'S NO CHANCE AT THAT TIME [INDISCERNIBLE] CAN OF THE SEVEN KILODALTON SIZE EXCLUSION. SO IT WAS CLEAR THAT IT HAD TO BE AN INFECTION EVENT SO IT HAD TO BE ANTIGEN PRESENTING FIRST THAT CAPTURED THE BIAS AND TRANSFER THE BIAS TO THE [INDISCERNIBLE] TO INFECT THEM AND WE WANTED TO VISUALIZE THIS ENTIRE SYSTEM BY INFECTING A MOUSE THAT WAS FLUORESCENTLY LABELED PARTICLES AND WATCHING THE ARRIVAL OF THE PARTICLES AT THE LYMPHNODE. SO IN THE CASE OF THE MICRO SCOPE OF THE LYMPHNODE AREA, ARRIVING AT THE LYMPHNODE SEE WE THE COLLAGEN AND THE VIRUS ACCUMULATES IN THE SINUS OF THE LYMPHNODE. AND THIS SUBCAPSULAR SINUS IS FORMED BY THE MACROPHAGE. SO THIS IS THIS PICTURE COMES FROM TO EXPLAIN THIS IS NOW AN ENTIRE LYMPHNODE, HISTOLOGY FOR INN TIRE LYMPHNODE IN BLUE, YOU SEE THE COLLAGEN FOR THE LYMPHNODE CAPSULE AND IN GREEN IS THE VIRUS, SO THE VIRUS IS CAPTURED OF THIS MACROPHAGE THAT ARE DISCLOSED TO THE B-CELL FOLLICLES USUALLY BECAUSE THE CELL TYPE REQUIRES--REQUIRES CYTOKINES THAT ARE RELEASED ON B-CELLS SO THEY CANNOT EVEN CAPTURE [INDISCERNIBLE] IT CAN ONLY STUDY THIS IN VIVO. AND THEN WHEN YOU COMPARE THE SYSTEM FOR THE CD169 POSITIVE MACROPHAGES, YOU SEE THAT THEY LOCALIZE IN THIS AREA THAT THE VIRUS IS CAPTURED, SO WE REQUIRE THIS MACROPHAGE BECAUSE IT'S REQUIRED FOR THE CAPTURE OF MLV, BECAUSE WE CAN SPECIFICALLY DEPLETE THE CELL LAYER USING CHLODROINATE AND WE CAN SEE THE DEPLETION AND THEN WE CAN CAPTURE THE MATERIAL BY THE MACROPHAGES. ACTUALLY IT'S IN HIGHER LYMPHNODE SO IT'S A METHOD WHERE IT CANNOT CAPTURE IT. NEXT WE WILL DETERMINE THE MECHANISM, THE MECHANISMS HAVE BEEN DESCRIBED OF HOW THE VIRUS IS CAPTURED AND ONE IS THE GLYCOPROTEIN IS RECOGNIZED FOR C-TYPE LECTIN SUCH AS IT DESIGN. AND ANOTHER MORE RECENTLY THE BANGLIO SITES IN THE LIPIDS, AND THE LIPID BY-LAYER OF THE VIRUS, AND THESE THROUGH THE SAME PROTEIN. SO TO THESE MODELS, YOU CAN PREPARE IT AND CAPTURE THE WILD-TYPE BUT THEN YOU GENERATE [INDISCERNIBLE] OF THOSE BIOSYNTHESIS AND YOU CAN SEE THAT THE G-ONE STAINING AND THE NUMBER OF SIDES IN THE BIAS AND THEN THE CAPTURE IS LOST. SO THE GANGLIO SIGHTS IN THE BIOMEMBRANE ARE RECOGNIZED BY THIS MACROPHAGES WHICH IS--WHAT? >> [INDISCERNIBLE]. [INDISCERNIBLE] >> BUT THE [INDISCERNIBLE] WE HAVE COMPARED TO THE DIFFERENT SPECIES AND THEY ALL HAVE [INDISCERNIBLE]. SO WITH THE NEXT SLIDE CD169 INJECTS ANTIBODIES OF 169 BEFORE WE CAPTURE AND THEN INJECT THEM WITH THE ANTIBODIES AGAINST THE [INDISCERNIBLE] OF THE OTHERS THEY CAPTURE LIKE WILD-TYPE AND THEN YOU CAN DO MICE THAT ARE NOT [INDISCERNIBLE] FOR CD61 AND YOU SEE A MORPHOLOGY HERE [INDISCERNIBLE] AND YOU CAN BARELY, BARELY DETECT THE PARTICLES. SO WE MONITOR WHAT IS THE ROLE OF CD169. THIS IS THE EFFECTOR OF [INDISCERNIBLE] TO INFECT [INDISCERNIBLE] OR PART OF THE IMMUNE DEFENSE SYSTEM THAT THE MOUSE IS CLEAVE--I HAVE CD169 TO CAPTURE VIRUSES AND PRESENT THE B-CELLS AND YOU GENERATE AN IMMUNE RESPONSE AND DETECT--PROTECT THE ANIMAL FROM THE INFECTION AND IT TURNS OUT THAT THAT THE INFECTION LEVELS IN THE KNOCK OUT MOUSE ARE VERY VIRAL SO IT'S A POOR VIRAL EFFECTOR AND THEY'RE EVOLVED TO USE THIS MOLECULE TO BE TRANSINFECTED AND CAPTURED BY THE MACROPHAGE AND THEN [INDISCERNIBLE]. THIS IS [INDISCERNIBLE] AND THE ANTIBODY RESPONSE IN THE CD169 KNOCK OUT MOUSE IS PRO-VIRAL AND HOW THEY'RE [INDISCERNIBLE] AND THEY MAY BE OF CD169 TO BE CAPTURED BY [INDISCERNIBLE]. SO AS I SHOW YOU THAT THE EXCEPTION OF THE CELL CAPTURE THE LYMPHNODE AND WE PREDICTED THAT SOMEHOW B-CELLS NOW MUST COME TO IMPACT THE CELL LAYER TO PICK UP THE VIRUS AND WHAT WE DID TO ADDRESS THIS IS WE GENERATED B-CELLS FROM MOUSE TO MICE AND ADOPTED TRANSFER THEM INTO THE MOUSE INTO THE RGFP VIRUS AND TO OUR BIG SURPRISE AND THAT THOSE ARE ANSWERED INTO THE MOUSE BECAME GREEN. SO THAT THAT WAS PUZZLING TO US BECAUSE IF WE TAKE THE LYMPHNODE AND PERFORM TASKS, THE VIRUS INFECTS B-CELLS BUT IT CANNOT INFECT NAIVE B-CELLS SO IT INFECTS SUBPOPULATION OF CELLS AND WHEN YOU PREPARE NAIVE B-CELLS, IT MUST BE A SUBSET OF ACTIVATED B-CELLS. SO--SO IT TURNS OUT USING VARIOUS MARKERS THAT THE SUBSET MAY BE IN EFFECT OF LCDFIVE, CDNINE AND 43 AND NIGD LOW AND HAS ALL THE CHARACTERISTICS OF B-1-AFIRST, SO NORMAL B-CELLS WILL BE THERE LOWER BTWO, AND THESE ARE B-1, AND IF YOU WILL NOW, INTRODUCE OR PREPARE THE MICE, ADAPTIVELY TRANSFER THEM INTO THE MOUSE AND NOT INFECT AGAIN THEN THE BIAS THERE WILL INFECT THESE RED CELLS OF COURSE THEY ALSO INFECT THE ENDOGENOUS PONE CELLS THAT ARE AVAILABLE. SO THIS IS HERE QUANTIFIED BY FACTS, IF THE--IF WE INFECT, THE NAIVE B-CELLS, THE VIRUS CANNOT INFECT OF THESE CELLS AND THE VIRUS CANNOT EFFECT THE B-CELL POPULATION, SO WHAT IS SO SPECIAL ABOUT W-ONE ACELLS IS THAT FOR THE PURPOSE OF OUR TALK IS THAT THEY'RE RENEWING AND THAT MEANS THEY'RE RECYCLING AND THE REENTER DEPENDS ON CELL CYCLING. THE MAJORITY OF THIS IS IN THE PERO TON EEL CAVITY AND THIS EXPLAINS RETINAL LOCATION ARE SPECTIVELY WHY THE MLV RESEARCHERS LIKE TO INJECT IT ENTASES THE MAIN TARGET IS THE PERO TONE EEL AND IT'S VERY LOW IN THE PERIPHERY BUT DESPITE THESE IT TARGETS THIS POPULATION IN THE LYMPHNODE. SO NOW WE WANTED OF COURSE, SPECIALIZE THIS DIRECTLY AND B-CELL NOW IN THE LYMPHNODE, THESE ARE B-ONE A CELLS SO WE CAN IMAGE THE BEHAVIOR AND WE NOW ADD FLUORESCENT PARTICLES AND WE SEE HOW THIS B-ONE A CELLS AND THEY'RE IN THE LYMPHOCYTES AND--AND B-ONEA CELLS ARE THE ONLY ONES THAT INTERACT FROM LONG LASTING COMPLEXES, THIS IS THE MACROPHAGES THAT CAPTURED FLUORESCENT PARTICLES. SO THESE ARE WE BELIEVE TRANSINFECTION EVENTS, INFECTIOUS SYNAPSES WHERE THE VIRUS IS TRANSFERRED BECAUSE TWO DAYS LATER THESE B-CELLS ARE INFECTED. WE CAN TRACK THE BEHAVIOR OF THESE CELLS, FOR THE INFECTION AND IF--IF--BEFORE INFECTION THEY MIGRATE QUICKLY AND THEY'RE IN INVERSE AND INVERSE PYRAMID, BUT THEN WE HAD THE VIRUS, NOW HE SAYS THIS TIME, MIGRATE MORE SLOWLY IN THE SUBPOPULATION THAT BECOMES ARRESTED BECAUSE THEY FORM BIOLOGICAL SYNAPSE, SO EVEN AT THE POPULATION IF YOU TRACK ALL SORTS, WE CAN SEE THE DIFFERENCE FOR THE INFECTED LYMPHNODES AND THE UNINFECTED LYMPHNODES WHERE THERE BE ONE POPULATION THAT BECOMES IMMOBILIZED IN THE SYNAPSIS ONE OR TWO HOURS AFTER ADDITION OF THE BIAS. OKAY. SO, B-ONEA SAYS MACROPHAGE IN TO PICK UP THE VIRUS AND TWO DAYS LATER WE FIND THEM IN THE FOLLICULAR SPACE AND SHOW THE--WE SUSPECT THAT IT IS A BIOLOGICAL SIGN UPS, SOMETHING WE WANT TO SPECIALIZE, SO AGAIN, HERE THIS IS THE INFECTED CELLS, SO THESE ARE THE INTERFOLLICULAR SPACE AND THE B-ONEA SAYS, THE LOCALIZATION EXCEPT FOR THE WAY, MIGRATE TO THE PERIPHERY TO PICK UP THE VIRUS ONCE THEY'RE INFECTED THEY RETURN TO THE PLACE WHERE THEY'RE USUALLY LOCATED. AND NOW WHEN THEY INFECT THESE MICE NOW, FROM THE VIRUS INFECTS SOME OF THE TRANSFERRED B-ONEA CELLS, IT ALSO INFECTS ENDOGENOUS B-ONEA CELLS AND THEY MOVE INTO THESE AREAS, THEY ACTUALLY OBSERVE--WE OBSERVE THE FORMATION OF AGAIN, WE SEE THE [INDISCERNIBLE] OF GFP ON ONE SIDE OF THE CELL WHICH IS AN INDEPENDENT POLARIZATION. SO THIS IS INNATE INFECTION WHERE THE MLV INFECTS THE B-ONEA CELLS AND THEY FORM BIOLOGICAL SYNAPSES LOCALIZED ON ONE SIDE. OKAY, I WILL SHOW YOU SOME MORE EXAMPLES OF THIS. BUT THESE ARE B-ONEA CELLS FROM THE WHITE MOUSE AND YOU WILL SEE THESE, JUST SHOWED YOU THESE MOVIES AND THEN WE HAVE THESE EVENTS WHERE THEY GET GFP AND FORM--BECAUSE IN SOME SYNAPSE THEY FORM THE T-CELLS AS WELL AS UNINFECTED B-ONE CELLS, AND IF FOR EXAMPLE WELL YOU SEE THE BIOLOGICAL SYNAPSE WHERE THEY HAVE THE FORMER CELL, AND THE TARGET CELL POST LABOR, SO HE HAS INFECTED B-ONE AND YOU SEE ACCUMULATION OF GFP, THIS IS QUANTIFIED HERE. WE HOPE TO CAPTURE GFP POLARIZES TO THE ENDOPHASE, HERE WE CAN TRACK THE DONOR TARGET CELL AND MIGRATE QUICKLY UNTIL THEY INTERACT AND THEN IMMOBILIZE. SO THEY MIGRATE FREELYY AND THEN THEY FORM BIOLOGICAL SYNAPSE IS THEN THEY SLOW DOWN AND THEN THEY IMMOBILIZE TO INTACT EACH OTHER, IT TAKES A FEW MOMENTS TO CONTACT THE AND A SIMILAR EVENT IS SHOWN ON THIS MOVIE WHERE THE TARGET SAYS IT FREELYY INTERACT AND FORM A STABLE POLAR BIOLOGICAL SYNAPSE. OKAY SO WHAT I'VE SHOWN YOU IS SPECIALIZED THE ARRIVAL OF THE VIRUS OF THE LYMPHNODE, WHERE THE PART CAPTURED [INDISCERNIBLE] AND MACROPHAGES AND THEN B-ONEA SAYS UPINTO THIS LAYER TO PICK UP THE VIRUS AND THEN FORM BIOLOGICAL INAPSEIS IN THE LYMPHNODE. SO THIS IS FIRST TIME THAT WE BELIEVE IT CAN IMAGE, THERE'S A LOT MORE TO DO, WE'RE TRYING TO DO BUT WE BELIEVE IT CAN GET THE FIRST HINTS OF HOW IN THE SECTION AND BIOLOGICAL SYNAPSE EVENT VS BEEN IN VIVO DURING THE EARLY INFECTION EVENTS OF A MOUSE BY THE VIRUS. THE MOD HILIGHTS POTENTIAL FROM IN VIVO STUDIES CAN CAN TEST CONCEPTS LIKE I'VE SHOWN, YOU TEST THESE SECTIONS AND THE BIOLOGICAL SYNAPSE TO IN VIVO, WE CAN CHARACTERIZE THE SPECIFIC SUBSETS OF CELLS THAT PLAY A ROLE IN THIS PROCESS, IT'S NOT A DENDRITIC CELL THAT CAPTURES THE VIRUS ESSENTIALLY IS A SPECIFIC SUBSET OF MACROPHAGES. AND MAYBE IT DOESN'T INFECT ALL THE [INDISCERNIBLE] TARGETS AVAILABLE SPECIFICALLY, THE B-CELL SUBSET, B-1-A, AND THEN USING KNOCKOUT MICE, YOU CAN ALSO ADDRESS MOLECULAR MECHANISM OF TRANSMISSION. SO NOW WE WANT TO VISUALIZE THE MICE. SO THIS IS--YEAH? >> [INDISCERNIBLE] >> SO THIS IS AT LEAST A HALF AN HOUR. >> YEAH WE ARE TRYING TO IMAGE MOVIES JUST AN HOUR AND THE MAXIMUM WE CAN IMAGE IS JUST ABOUT FOUR HOURS BUT I AGREEN CELLS IT WOULD BE INCITEFUL TO REALLY HAVE THE INTIRE LIFE CYCLE OF THE B-ONEA, WE HAVE DONE IT IN VIVO, JANE HAS DONE THIS IN VITRO AND WE HAVE DONE THE [INDISCERNIBLE] ASKING TO BE INSPIRED [INDISCERNIBLE] SHOW HOW MANY MINUTES IT TAKE FOR THE CONTACT AND THEN SEPARATED. >> I AGREE WE WOULD LIKE TO KNOW THAT IN VIVO TOO. >> [INDISCERNIBLE]. >> YES THIS ACTUALLY USES THE CD69 MACROPHAGES SO IT'S AMAZING THAT THIS MOLECULE THAT WAS IDENTIFIED AS A MARKER FOR THIS MACROPHAGE S&P A MOLECULE THAT CAPTURES THE VIRUS,. >> THESE ARE IN THE EXPRESSION OR IS THAT PRETTY CONSISTENT? >> WE FIRST TESTED IS IT WOULD BE POSSIBLE TO THE 169, TOO BECAUSE IT COULD HAVE BEEN A DOUBLE--IN SECTIONS AND SCENARIO AND THAT'S NOT THE CASE, DON'T EXPRESS CD169 AND WE BELIEVE THAT THE TRANSFER FROM [INDISCERNIBLE] MACROPHAGE IS A B-CELL INDEPENDENT FORCES AND WE BELIEVED HERE, SYNAPSIS AND INDEPENDENT PROCESS WHERE THE BI AS DID INFECT THE CELL BUT [INDISCERNIBLE] AND THE ENVELOPE NCAT ACTION BUT THIS IS AN EXPERIMENT WE ARE CURRENTLY DOING. >> I'M SAYING THE NEED TO REPEAT AND WE MAY IGNORE IT BECAUSE BECAUSE IT MAY BE SOMETHING AND IT MAY MOT BE SOMETHING IT MAY DEEMPHASIZE THIS BECAUSE THIS IS FOR INTERFERON LIKE THIS WAS. ALL RIGHT, SO FOR YEARS WE STUDIED SINGLE PARTICLE IMAGING TO STUDY THE [INDISCERNIBLE] OF VIRUSES AND THEY'RE DOCUMENTED TO TRANSMISSION OF SIGNAL PARTICLES AND [INDISCERNIBLE] YOU WILL DOING TO MOVE THE SYSTEM INTO LIVING ANIMALS. AND THE SECOND MENTION THAT I WOULD LIKE TO ADD WHAT I HAVE BEEN DONE BEFORE AND TO ALSO MOVE IT, SO THIS IS MOVING FROM TISSUE TO MOVE INTO ANIMALS AND HE WILL MOVE INTO THE NANNY SCALE AND VISUALIZING MOLECULES AND THIS WAS ESTABLISHED BY [INDISCERNIBLE] WHO BIOPHYSICIST AND THE IDEA HERE IS RATHER THAN IMAGING THE LABELING THE ENTIRE VIRUS, THEY INTRODUCE JUST TWO MOLECULES AND INTROTHOSE THIS A SINGLE ENVELOPE AND SINGLE MOLECULE AND BY INTRODUCING TWO GUYS WE CAN ACTUALLY MONITOR BECAUSE [INDISCERNIBLE] IT DEPENDS ON THE [INDISCERNIBLE] WE CAN MONITOR CONFIRMATION OF CHANGES AND THE SINGLE POLARIZED KIEWL AND IN THIS COLLABORATION, WE ARE SURE THAT IN THE COURSE OF THE VIRUS LIKE PARTICLES IN THE COURSE OF TRANSITION FROM A CONTEXT TO AN EXTENDED STRUCTURE AND HERE WE WANTED TO DO THE SAME FOR THE ENVELOPE SO WE'RE INTERESTED IN CONFIRMATION EVENTS DURING HIV ENTRY. AND SO THEY'RE INTERESTED IN LARGE SCALE DYNAMICS THESE MOTIONS ARE DETECT INDEED THE MILLI SECONDS RANGE. AND ALL TIMES THIS IS AGENTS WHICH ARE MUCH FASTER IN THE GLOBAL REARRANGEMENTS AND THIS IS ONE METHOD THAT GIVES YOU ACCESS TO THIS CHANGES AND IT'S--IT MEASURES THE ERNEERGY TRANSFER FROM [INDISCERNIBLE] TO AN ACCEPTOR AND IT'S DEPENDENT ON THE ENERGY TRANSFER EFFICIENCY IS DEPENDENTOT DISTANCE. SO THESE TWO DYES ARE VERY CLOSE AND THE IMAGE IS HIGH AND THE ABILITY TO DETECT THE [INDISCERNIBLE] FROM THE GLOBAL FLORA FLOR, TO SET THOSE UP FOR HIV, AT THE TIME [INDISCERNIBLE] PUBLISHED AND INDICATED THAT THE TRIMER IS CLOSE AND INVOLVES PLAYING A ROLE AT THE APEX OF THE MOLECULE AND NONE OF THE RESPONSE TO CD4 WOULD OPEN UP SO IT WAS VERY CLEAR THAT THIS IS OPENING UP THE TRIMER IS SOMETHING WE WANTED TO SEE. SO IT WAS CLEAR THAT ONE OF [INDISCERNIBLE] HAD TO BE IN THE KD--SALLY WAAND THEN WE NEEDED THE SECOND FLOOR FOR TO REPORTOT RELATIVE OPENING OF THIS AND THEY FOUND THIS IN THE BFOUR AND B-FIVE GROUPS AND WE FOUND THIS TOO FOR THIS ON THE SURFACE OF COMPLETE VIRAL PARTICLE SO THE CHALLENGE WAS TO INCORPORATE A SINGLE MOLECULE AND THE OTHER TWO MOLECULES USING THE TRIMER, ALL OTHER TRIMERS ARE ON THE BOTTOM, ONLY ONE MOLECULE CARRIED THE TWO DIMERS. AND TO INTRODUCE THE [INDISCERNIBLE] WE USE ENZYMES SO THOSE ARE ALL ENZYMES THAT RECOGNIZE CERTAIN PEPTIDES, SIX, EIGHT, 24, PEPTIDES, AND THEY ANSWER SUBSTRATES TO THESE PEPTIDES AND BECAUSE THE DICE TO THE SUBSTRATES, THESE ENZYMES WERE VERY INCORPORATE AT THESE POSITIONS IN THE PEPTIDES. AND WHAT JAMIS TO IDENTIFY TO USE VARIOUS METHODS TO HAVE EVEN MORE AND WE IDENTIFIED POSITIONS OF THE PEPTIDE SEQUENCE ARE TOLERATE INDEED A VALUABLE LOOP OF THE GLYCOPROTEIN, YOU SEE MANY [INDISCERNIBLE] AND ONLY SOME PEPTIDES IN CERTAIN ENVIRONMENTS ARE TOLERATED SO IT'S REALLY--IT REALLY HELPS TO HAVE A VARIETY OF DIFFERENT CHEMISTRIES AVAILABLE TO IDENTIFY SOME THAT ARE TOLERATED AND THEN YOU COMBINE THE TOLERATED ONES TO GENERATE AND LABELED--IT REMAINED INFECTIOUS AND INCORPORATE INTO BIAS AND THEN ASK THEM TO NEUTRALIZING ANTIBODIES AS [INDISCERNIBLE] SO THEY DIDN'T CHANGE THE [INDISCERNIBLE] SO THESE ANTIBODIES ARE RECOGNIZED THE INTACT TRIMER IN THESE PEPTIDES DIDN'T CHANGE THE STRUCTURE OF THE TRIMER, AND THE END OF UP WITH THREE VIRUSES, TWO FOR [INDISCERNIBLE] AND AND WE WANTED TO DO THIS STUDY FOR NEUTRALIZATION SENSITIVE METHOD SO LIKE FOR THREE AND CONTRAST THE UTILIZATION OF THE CLINICAL ISOLATE SUCH AS JSL, SO THEN WE COULD DO THAT SECOND AND THEN THE PREPARED VIRUS SO THAT ONLY A SINGLE MOLECULE SYSTEM COUPLED INTO [INDISCERNIBLE], YOU HAVE THE LINKS PLUS THE PEPTIDES IN THE VARIABLES AND THE ENVELOPE, AND THEN WE GO WITH THE 44 AXIS OF WILD-TYPE, SO THAT STATISTICALLY, BECAUSE THEY HAVE SEVEN-15 TRIMERSOT VIRUS, THIS IS STATISTICALLY ONLY ABOUT EVERY SECOND VIRAL PARTICLE, THEY CARRY A SINGLE MOLECULE WE THEN CALL TO PEPTIDES AND EVERYTHING ELSE IS WILD-TYPE AND THEN OACCORDING TO THE STATISTICS, THEY HAVE LABELING EFFICIENCY OF 50%, 25% OF THE PARTICLES OF [INDISCERNIBLE] FLORA FORALSO CARRY THE ACCEPTOR FLORA TOR, NOW THIS CORRELATES BIOSIS, THE ACCEPTOR MISSION AND THE FILTER [INDISCERNIBLE] AND YOU CAN SEE THE DONOR FLUORESCENCE IN THESE EVENTS AND YOU SEE THE DONOR COLLAPSES AND YOU SEE THE [INDISCERNIBLE] WHICH ARE INDICATIVE OF THREAT, THIS IS RESIDING THREAT EFFICIENCY. SO CAN YOU SEE IMMEDIATELY FROM THE STRATEGIC PLANUS, YOU CAN SEE THAT THE UNLIGANDED HIV ENVELOPE IN THE ABSENCE OF ANY LIGANDS ON THE SURFACE OF THE VIRUS IS CONFIRMATION OF EPIDEMIC, IT CONSTANTLY CHANGES ITS SHAPE. AND YOU CAN QUANTIFY THE VARIOUS TRACES IN THE HISTOGRAM AND JULE SEE THAT THE ACTUAL ENVELOPE IS THE SAME CONFIRMATIONS THAT HAVE THE LOW THREAT AND IMMEDIATE THREAT AND THEN HIGH THREAT. SO THE YOU ANALYZE THE SERVER AND ASK HOW ARE THESE THREE CONFIRMATIONS LINKED TO EACH OTHER AND PERHAPS A LOOK AT THIS DATA TO REMIND YOU THAT THE MOST POPULATED STATE IS BY DEFINITION THE STATE OF THE UNLIGANDED [INDISCERNIBLE] ENVELOPE OF THE TRIMER. SO I'M FOLLOWING THE MODELING, JAMES WAS ABLE TO PLOT ALL TRANSITION IN THE TRANSITION DENSITY PLOT AND KD--SALLY IN A WAY THIS IS THE INITIAL FOR THE [INDISCERNIBLE] FOR FRET, IF THE MOLECULE IS IN THE LOW FRET, TO BEGIN WITH HERE, IT HAS THE HIGHEST PROBABILITY OF THE ENTIRE DENSITY FOR THE LOW FRET STATE IS TRANSITIONING TO THE HIGH FRET AND THIS IS WHAT YOU SEE HERE, SO THIS IS LOW FRET INTO THE HIGH FRET. THEN WHEN IT'S IN THE HIGH FRET IT HAS THE HIGHEST ABILITY TO GO BACK TO LOW FRET BUT YOU SEE THESE ARE HIGH FRET AND BACK TO LOW FRET. BUT IN ADDITION YOU SEE FROM THE HIGH FRET, IT CAN POSITION TO THE IMMEDIATE FRET THIS, IS WHAT YOU SEE HERE FOR THE INTERMEDIATE FRET AND THEN FROM THE IMMEDIATE FRET, IT GOES BACK TO THE HIGH FRET. SO WE HAVE TWO SYMMETRICAL TRANSITIONS, LOWER AND HIGH, AND HIGH AND INTERMEDIATE. THAT ARE SYMMETRICAL BECAUSE THEY ARE--BECAUSE [INDISCERNIBLE]. NOW, THE TRANSITION THAT IT'S MISSING IF THE TRANSITION FROM THE [INDISCERNIBLE] DIRECTLY INTO THE INTERMEDIATE THREAT STATE SO THIS TRANSITION IS [INDISCERNIBLE] HIGH ENERGY BARRIER. SO NOW WE COMPARE [INDISCERNIBLE] AND THESE ARE THE DATA RESENTED TO YOU, THIS [INDISCERNIBLE] THAT ARE TWO OR THREE ISOLATE. AND FIRST OF ALL ALL ENVELOPES HAVE THREE CONSERVATIONAL STATES AND THEY'RE FUNDAMENTALLY THE SAME SAME MOLECULAR MACHINES AND YOU LOOK AT THE PLOT, REMEMBER FOR NLFOUR-THREE, IT OPENS UP TO THE CONSTRUCT, SO JR-FL IS MOSTLY CLOSED, IT'S RARELY OPENS. WHEN IT OPENS IT'S TRANSITIONS BACK AND FORTH WITH HIGH FRET AND IMMEDIATE FRET. YOU WILL SEE IT WITH THE PLOT. THE CONSEQUENCE IS THAT IT'S MUCH LESS POPULATED AND THIS YOU WILL SEE AS MEANINGFUL. SO NOW WE ASK HOW THE [INDISCERNIBLE] LANDSCAPE CHANGES IN RESPONSE TO LIGANDS, VERSES THE UNLIGAND EVER AND THE CD4, AND THE HIGH FRET STABILIZED AND THEN CD4 AND 17 B IN THE IMMEDIATE STATE IS STABILIZED AND 17 B, BINDS TO THE BINDING SITE FOR ANTIBODY AND PROBABLY THE HIGH FRET AND IMMEDIATE FRET IS TO LIGANDS ALREADY EXIST SO THE UNLIGAND OF THE ENVELOPE HAS THE ABILITY TO SPONTANEOUSLY SAMPLE THE CONFIRMATIONS THAT ARE ACCESS AND STABILIZED BY CD4 IN THE CORE RECEPTOR OF ANTIBODY 17 B. SO THIS IS REALLY IMPORTANT THE ENVELOPE HAS SPONTANEOUS ACCESS TO THE CONFIRMATIONS OF THE STABILIZED VIRAL RECEPTOR SO COMPARED TO THE [INDISCERNIBLE] RESPONSE MORE RELUCTANT TO LIGANDS IN GENERAL, SO THEN WE HAVE CD4 AND INCREASED AND THEN INCREASED HIGH FRET AS INCREASED AND THEN WE HAVE IMMEDIATE FRET. THE 17 P WE DO SEE STABILIZATION OF 17 B, BUT IN GENERAL THIS ENVELOPE IS JUST RESPONSIVE TO LIGAND EVEN WHEN WE HAVE A 55 HIGHER NEUTRALIZATION CONCENTRATION OF LIGAND OF 54 VASCULARIZATION SO BELIEVE THAT THIS REFLECTS CONFIRMATIONAL MASKING. >> [INDISCERNIBLE] >> YES? >> IS THE FINDING SIMILAR BETWEEN [INDISCERNIBLE] >> IT IS, YES, 17 B, YES. >> SO YOU REMEMBER TO NOW LOOK AT 17 B BECAUSE THE [INDISCERNIBLE] VIRUS IS THAT IT'S FREQUENTLY OPENS UP THAT'S [INDISCERNIBLE] AND SURE ENOUGH WHEN WE ADD 17 B, 17 B ALONE CAN STABILIZE THE MUSCLE INDEEDLY IN THE ENTIRE STATE. SO THIS IS THE KORESEPTORSOR CONFIRMATION AND CD4 EPITOPE BUT IN THE [INDISCERNIBLE], THE ISOLATE CAN OPEN UP. SO IN CONTRAST JFL, CLINICAL DOESN'T RESPOND TO 17 B, IT DOES IF YOU ADD CD4. BUT IN THE ABSENCE OF CD4, THERE'S NO RESPONSE, ONLY AT 90 MINUTES YOU SEE THE SLIDE AND THE INTERMEDIATE THREAT SPACE. SO THE HIGHER OBJECT [INDISCERNIBLE] OF THE THREAT STATE HERE, REALLY MAKE THIS IS ENVELOPE WIDER TO CAPTURE THE CONFIRMATION FROM THE CAPTURE AND HERE WE SEE THE CONFIRMATION OF THE MOUSE. SO HERE WE HAVE THE [INDISCERNIBLE] TO MEASURE NEUTRALIZING ANTIBODIES AND PARTICULAR INTERESTED IN BROADLY NEUTRALIZING ANTIBODIES TO PROTECT [INDISCERNIBLE] ANIMALS FROM HIV AND SIV, VERY IMPORTANT FOR NEUTRALIZING ANTIBODIES, AND DEVELOP BY FEWER PATIENTS AND WHAT WE FOUND TO OUR SURPRISE AND THIS IS AGAIN THE UNLIGAND WE FOUND THAT ALL THESE BROADLY NEUTRALIZING ANTIBODIES STABILIZE SO THIS IS THE OPEN CONFIRMATIONS. SO SUCH AS [INDISCERNIBLE] THE CHAMPION HERE IS PGT 122, WITH THE STATE SO THEY ALL STABILIZE THE STATE AND THAT MEANS FIRST OF ALL THE STATE, SO BROADLY NEUTRALIZING ABET BODIES RECOGNIZING CLOSE CONFIRMATION AND SECOND STABILIZE THE TRIMER [INDISCERNIBLE] STATE SO THEY HAVE FEATURES THAT PREVENTS THE ACTIVATION OF THE PROTEIN AFFUSION. SO THE EPITOPE OF THIS HAS A SIMILAR FEATURE. OKAY, NOW, THE ACTIVATION OF PG122 NOW IN RETROSPECT EXPLAINS WHY [INDISCERNIBLE] AND THEY CRYSTALLIZE, THEY HAD THE CONFIRMATION OF A DYNAMIC TRIMER IN THE CONFIRMATION IN CONPLEX WITH THE ANTIBODY. IT'S ALSO SOLUBLE TRIMER TO GENERATE THESE AND LOOK AT THIS STRUCTURE. AND THE FIRST SUCH AS ONLY ABOUT FIVE BY DIVISION IN THAT LABORATORY NOT HIGH ENOUGH TO [INDISCERNIBLE] GP31, BUT THE SOURCE RECENTLY WAS [INDISCERNIBLE] LABORATORY AT 3.5-ANGSTROMS NOW RESOLVE THE GP41 AND IT'S AN INTERESTING SECTION. BECAUSE THE M& C OF THE GPONE AND THE INTERIOR OF THE GP41. SO THE WAY IT IS SYNTHESIZED THE END TERMINUS OF THE GP160 DELAYED. SO THE FUNCTIONS LIKE AN ANCHOR WHICH TIES THE [INDISCERNIBLE] TO THE MEMBRANE AND THEN GP31 COMES OUT, IT FOLDS--IT WRAPS AROUND THE GP120 AND THEN THIS IS AN AMAZING STRUCTURE AND YOU CAN IMAGINE THEN CD4 AT THE GP120, THAT THE WHOLE THING EXPLODES. AND GP41 CAN SUSPEND THE MACHINE [INDISCERNIBLE] SO THEY'RE--THIS IS [INDISCERNIBLE] THIS IS THE GP41 HRONE REGION AND YOU SEE THE LONG HELIX IS A TWO HEADED HEAT EXCITATORY CONNECTED BY A LOOP SO WHAT HAPPENS DURING FUSION IS THAT THE TWO HELIXES EXTEND AND DO THE FUSION PEPTIDE INTO THE TARGET REMEMBER, THAT'S HYPOTHETICAL BUT THIS IS AWAITING CONFIRMATION BUT ALSO TWO THE PEPTIDES HELIX TO DRIVE THE [INDISCERNIBLE] REMEMBRANCE TOGETHER FOR FUSION, AND THIS STRUCTURE OF HOW GP 41 IS FOLDED IN THE PREFUSION AND POST FUSION IS IDENTICAL WHEN ALL THESE DIFFERENT TYPE ONE, CLASS ONE FUSION ISSUES. OKAY, NOW THE STRUCTURE, AND ALSO STRUCTURE FOR THE ENVELOPE TRIMER IN COMPLEX OF PGAC129. AND 122. SO THIS IS REALLY--AND OUR WORK SHOWS THAT THE BROADLY NEUTRALIZING ANTIBODIES RECOGNIZED AND BIND TO THE GROUND STATE AND IT'S NEUTRALIZING ANTIBODIES SO ANY VACCINE SHOULD PRESENT THIS CLOSE CONFIRMATION TO THE IMMUNE SYSTEM TO ILLICKITY THE BROADLY NEUTRALIZING ANTIBODIES AND MANY [INDISCERNIBLE] AND MANY--OOPS, WHAT HAPPENED HERE? OKAY, GOOD. SO WE KNOW EXACTLY WHAT THE EPITOPES ARE IN THE TRIMER AND THIS IS WHY WE BELIEVE THAT THE [INDISCERNIBLE] OR THE SENSATION ARE OF THE STATURE AND THE DYNAMICS OF THE TRIMER HOPEFULLY THIS BREAKS IN THE DESIGN OF THE TOUR AND ILLICIT THE NEUTRALIZING ANTIBODIES AND WITH THIS I LIKE TO THANK THE PEOPLE IN MY LAB [INDISCERNIBLE], PERFORM THE WORK AND THE EFFICIENCY OF THE RETROVIRAL THERAPY IN CELL TO CELL TRANSMISSION AND [INDISCERNIBLE] INITIATED THE ENTIRE WORK ON THE IN VIVO IMAGING AND THAT'S NOW THREE PEOPLE HAVE JOINED AND PEOPLE HAVE--USED TO WORK ON [INDISCERNIBLE] JOINED IN VIVO IMAGING TEAM AND FINALLY JAMES INITIATE THE MOLECULE WORK IN OUR LAB [INDISCERNIBLE] AND POINT TO THIS MOVE THEY--THE IS OUR INTERPRETATION OF WHAT IS HAPPENING AND YOU WILL SEE--[LAUGHTER] NTHIS IS THE EFFECTOR RACE WHERE I EXPLAINED OPEN STATE AND THE TWO OPEN CONFIRMATIONS AND YOU CAN SEE THE ONLY HAVE THE STRUCTURALLY ARE OF THE CLOSE CONFIRMATION. SO YOU HAVE TO MAKE THE HUMAN MOVE TOW EXPLAIN WHAT'S GOING ON. WE WOULD ALSO LIKE TO THANK OUR COLLABORATORS AND FUNDING AND AS I MENTIONED SHE'S SLIDES ARE [INDISCERNIBLE] AND THE TRANSMISSION AND IN VIVO IMAGING AND THE [INDISCERNIBLE] SUPPORT AND CRITICAL AMOUNT HERE AND MGH, SIGNAL MOLECULE IMAGING, THIS WORK, [INDISCERNIBLE] AND PETER QUONG, AND THE MACULAR BMS, THANK YOU FOR YOUR ATTENTION. [ APPLAUSE ] >> [INDISCERNIBLE] WHY DO YOU GET [INDISCERNIBLE]? >> I WOULD ALWAYS ASK THE WITH ONE DRUG LIKE AZT, BUT THEN YOU GET A PRETTY GOOD DECLINE RIGHT AWAY, AND AT LEAST THAT WELL OVER REPLICATION FOR VIRUSES AND BLOOD INSTITUTE INCOLLIDE THE VIRUS, YOU ARE SEEING THERE S&P PROBABLY IS NOT THE CONSEQUENCE OF CELL TO CELL TRANSMISSION. >> MAYBE, I MEAN WE DON'T KNOW. >> FAILURE OF THOSE DRUGS OF COURSE IS RESISTANCE IF NOT--[INDISCERNIBLE] >> SO ONE THING IN OUR DATA THAT IS INTERESTING THAT'S RESIST CANT IF YOU COMBINE TWO THAT DON'T WORK YOU WILL GET COMPLETE LACK OF TRANSMISSION, BUT WHEN YOU'RE--YOU PICK UP THE RESISTANCE AGAIN ONE OF THE TWO DRUGS, IT BEGINS TO REPLICATE. SO THEN OUR MODEL WE CAN SHOW THEM THEY HAVE--DRUG RESISTANCE COMBINATION, THEY HAVE THE PHENOTYPE OF THE DIRECTED [INDISCERNIBLE]. SO THAT IS A POSSIBILITY THAT THE DRUG IS PERSISTENT, VIRUS AMPLIFY LOCALLY IN THE TISSUE BECAUSE THERE'S SPREAD MORE EFFICIENTLY BY CELL TO CELL. BUT WE DON'T HAVE ANY YET. ERIC IN. >> HAVE YOU LOOKED AT MINIATURE [INDISCERNIBLE] VERSES MINIATURE [INDISCERNIBLE] AND WHAT HAPPENS FOR EXAMPLE [INDISCERNIBLE]? >> YEAH, YEAH, WE ARE DOING THE CONCEPT ALREADY, WORKING ON THAT IMAGE SOURCE, YEAH. >> YEAH. >> GOING BACK TO JOHN'S POINT, HOW MUCH DO YOU THINK MIGHT BE CELL TYPES THAT SOME OF IT IS THE ABILITY OF THE CELL TO TAKE UP THE DRUG AND THEN HAVING TO PHOSPHORYLATE IT, MAYBE THOSE RATES ARE JUST NOT [INDISCERNIBLE]? >> YEAH, ABSOLUTELY, SO WHAT WE HAVE DONE IS PREINCUBATED ALL OF OUR CELLS FOR OVERNIGHT AT LEAST OR GAVE ALL KNOWLEDGE FROM THE DRUGS, ALL LOWERED THE DRUGS BUT THE [INDISCERNIBLE] THE TARGET IS THE PRIMARY PBMCs SO WE DIDN'T--DIDN'T TEST VERY CLEAR THE DIFFERENCES IN THE METABOLISM, YEAH. >> DO YOU RECALL IF MALCOM DID THOSE EXPERIMENTs AS WELL [INDISCERNIBLE] IN CO CULTIVATING THE DISEASE OR INFECTED EACH [INDISCERNIBLE]? >> THANK YOU. [ APPLAUSE ] >> THANK YOU VERY MUCH. [ APPLAUSE ]