IT IS MY PLEASURE TO INTRODUCE DR. MAUREEN GOODENOW, WHO IS THE DIRECTOR OF THE OFFICE OF AIDS RESEARCH, AND SHE OVERSEES ALL OF THE HIV/AIDS BUDGET TO THE NIH. SO IF I'M NOT MISTAKEN, THAT'S OVER $3 BILLION. SO IT'S A LOT OF MONEY. AND PERHAPS EVEN GREATER INTEREST, IT LOOKS LIKE THIS YEAR, IT LOOKS LIKE THERE MAY BE AN INCREASE, WHICH HASN'T HAPPENED IN MANY YEARS, AND THAT THOSE MONEYS, HOW THEY WILL BE ALLOCATED, WILL BASICALLY BE UP TO MAUREEN. SO BUT THEY'RE SAYING PREVENTION, SO VACCINES AND CURE. SO MAUREEN, THANK YOU VERY MUCH. WE LOOK FORWARD TO HEARING WHAT YOU HAVE TO SAY. THANKS, DIANA. GOOD MORNING. NICE TO SEE EVERYONE HERE AGAIN. SO, YES, THANK YOU FOR THAT INTRODUCTION. IT ALWAYS SOUNDS LIKE A LOT OF MONEY. I HAVE TO TELL YOU, I DO NOT HAVE A PERSONAL BANKING ACCOUNT AT THE NIH AND I'VE NEVER SEEN $3 BILLION. LIKE. AND JUST ONE CLARIFICATION, WE DID ACTUALLY HAVE AN INFUSION OF NEW MONEY IN 2019, $45 MILLION, WHICH WAS THE FIRST INCREASE IN ABOUT EIGHT YEARS OR MORE. SO THAT CAME OUT OF -- IT WAS THE FIRST YEAR THIS YEAR THAT ACTUALLY THE APPROPRIATION TO THE NIH HAD FLEXIBLE MONEY IN IT. THE LAST FEW YEARS WHERE THERE'S BEEN A BIG INCREASE IN THE NIH BUDGET, THERE WAS -- IT WAS VIRTUALLY ALL EARMARKED FOR SPECIFIC PROGRAMS, AND EVEN DR. COLLINS COULDN'T DO ANYTHING WITH IT. HE HAD TO SPEND IT, IT HAD TO BE SPENT THE WAY IT WAS DETERMINED BY CONGRESS. SO THIS WAS THE FIRST YEAR THERE WAS PART OF THE ALLOCATION WAS FLEXIBLE, NON-EARMARKED MONEY, SO $45 MILLION OF THAT WAS INVESTED IN THE HIV PROGRAM, SO WE'RE VERY EXCITED ABOUT THAT. SO FOR THOSE OF YOU WHO DON'T KNOW ME AND I KNOW WE HAVE COLLEAGUES HERE FROM OTHER FIELDS, JUST TO LET YOU KNOW THAT I HAVE BEEN IN YOUR POSITION MOTION OF MY MOST OF MY CAREER, IT'S ONLY BEEN THREE YEARS SINCE I'VE BEEN IN THIS POSITION, AND IT'S BEEN AN INTERESTING CHALLENGE TO MAKE THE CHANGE FROM ACADEMIA TO GOVERNMENT, AND IT'S REALLY -- IT'S BEEN REALLY EXCITING. SO OFFICE OF AIDS RESEARCH IS ACTUALLY PART OF DR. COLLINS' OFFICE, THE OFFICE OF THE DIRECTOR, AND IT IS AN OFFICE THAT'S ROLE IS TO, AS DIANA MENTIONED -- WE SET THE RESEARCH PRIORITIES WITH ADVICE, OF COURSE, IN CLOSE COLLABORATIONS WITH THE ICs, AND WE ALSO COORDINATE THE RESEARCH AGENDA AND FUNDING ACROSS THE NIH TO THE 20 OR SO INSTITUTES, CENTERS AND OTHER OFFICES THAT HAVE RESEARCH AGENDAS. SO THERE'S A BIG PORTFOLIO FOR THIS LARGE INVESTMENT AT THE NIH. WE ALSO ARE RESPONSIBLE FOR TRACKING AND REPORTING TO CONGRESS ON AN ANNUAL BASIS HOW THE MONEY IS SPENT. SO IN ORDER TO CARRY OUT ALL OF THESE ACTIVITIES, THE OFFICE OF AIDS RESEARCH HAS ABOUT 45 PEOPLE, WE HAVE A LARGE NUMBER OF SCIENTISTS, AND MANY OF YOU IN THE HIV AREA KNOW THE HSAs, THE SENIOR SCIENCE AT VISORS, BUT BEHIND THE SCENES, THE PEOPLE YOU DON'T SEE, ARE THE BUDGET TEAM AND THE TEAM WHO RUN THE OPERATIONS OF THE OFFICE AND DEVELOP ALL THE I.T. SYSTEMS THAT WE NEED TO DO ALL THE WORK THAT WE DO. THE OFFICE WAS ESTABLISHED VERY EARLY IN THE EPIDEMIC. IT WAS ESTABLISHED OFFICIALLY IN 1988, BY CONGRESSIONAL AUTHORIZATION, AND THAT WAS REALLY AFTER ALMOST FIVE YEARS OF NIH INVESTMENT IN HIV RIGHT FROM THE BEGINNING OF 1983 FROM THE EPIDEMIC, BUT IT WAS FORMALIZED IN 1988 BY CONGRESS, AND THE OFFICE HAS BEEN GIVEN, BY AUTHORIZATION, A NUMBER OF UNUSUAL, I WOULD SAY, ACTIVITIES THAT WE DO, AND I'M NOT GOING TO GO THROUGH ALL OF THEM, THERE ARE A COUPLE I JUST WANTED TO MENTION TO YOU, AND ONE IS THAT WE ARE AUTHORIZED TO HAVE AN ADVISORY COUNCIL AND THE AUTHORIZATION IS VERY SPECIFIC ABOUT THE TYPES OF EXPERTISE THAT WE'RE SUPPOSED TO HAVE, AND I'M HAPPY TO SEE LIZ KONIG HERE BECAUSE SHE HAS BEEN ON THE OAR ADVISORY COUNCIL FOR ABOUT FOUR YEARS, BEFORE I STARTED, BUT SHE JUST ROTATED OFF THIS YEAR AND WE'RE REALLY SORRY THAT SHE'S NOT THERE ANYMORE. SO THE ORAC IS AN IMPORTANT ACTIVITY. THE OTHER ACTIVITY WE HAVE AUTHORIZATION TO CARRY OUT IS THAT WE'RE ALLOWED TO TRANSFER, EACH YEAR, UP TO 3% OF THE HIV BUDGET, SO THAT'S $90 MILLION, AND WE'RE ALLOWED TO TRANSFER IT AROUND THE NIH, SO WE CAN MOVE MONEY. AND THIS IS A REALLY IMPORTANT ASPECT OF THE ACTIVITIES OF THE OFFICE BECAUSE IF YOU REMEMBER YESTERDAY, WHEN TONY WAS DESCRIBING THE HUGE INCREASE, SEVEN FOLD INCREASE IN THE INVESTMENT IN CURE OVER THE PAST SIX TO EIGHT YEARS DURING A PERIOD WHEN WE HAD VIRTUALLY NO INCREASE IN THE OVERALL MONEY, THE REASON WE WERE ABLE TO GO FROM A FEW MILLION TO OVER $250 MILLION WAS IN PART BECAUSE WE COULD MOVE MONEY AROUND. AND THIS IS HOW WE DO THE INVESTMENTS WHEN WE'RE -- PARTICULARLY UNDER A FLAT BUDGET, WHICH IS WHAT WE'VE BEEN UNDER. SO OUTSIDE THE NIH, THE OAR HAS OTHER ACTIVITIES AS A RESULT OF WHAT CONGRESS HAS AUTHORIZED US AND OUTLINED FOR US TO DO, AND ONE OF THE PLACES THAT I WANTED TO FEATURE IS THE ACTIVITIES THAT WE DO THROUGH THE INTERNATIONAL AIDS SOCIETY. ONE OF THE ACTIVITIES THAT IAS DOES IS HAVE A CURE INITIATIVE, AND THERE IS SCIENTIFIC ADVISORY BOARD FOR THAT, AND SO OAR HAS A SEAT ON THE BOARD AND DIANA FINZI FROM NIAID IS ON THAT BOARD. WHAT THAT ALLOWS US TO DO IS REALLY HAVE A NEUTRAL SPACE PROVIDED BY IAS FOR STAKE STAKEHOLDERS FROM AROUND THE WORLD TO CON SEEN WHO CONVENE WHO ARE WORKING IN THE CURE SPACE. IT'S NOT ONLY THE BASIC RESEARCHERS, THE CLINICAL RESEARCHERS, BUT IT'S ALSO PEOPLE WHO HAVE -- OTHER TYPES OF STAKEHOLDERS INCLUDING ADVOCACY AND INCLUDING TECHNOLOGY AND PHARMACEUTICAL COMPANIES. SO THIS IS A ROLE THAT WE PLAY ON BEHALF OF THE NIH INVESTIGATORS IN THE INTERNATIONAL ARENA. THEN ANOTHER ACTIVITY THAT IS PS ALSO GOING ON IN PART SUPPORTED BY IAS AND THE BILL AND MELINDA GATES FOUNDATION IS THE RETREAT THAT WAS HELD EARLIER THIS YEAR IN CALIFORNIA, AND THAT WAS ATTENDED BY A NUMBER OF STAKEHOLDERS AS WELL, AND THE PURPOSE OF THAT IS ONGOING BUT REALLY TO DEVELOP, WHAT DOES A CURE LOOK LIKE IN 10 OR 15 YEARS AND WHAT IS A TARGET PRODUCT PROFILE THAT THESE CURES, INITIATIVES OR APPROACHES, I'M ASSUMING THERE MIGHT BE MORE THAN ONE, WHAT WOULD THESE LOOK LIKE AND HOW ARE THEY GOING TO BE PARTNERED, BECAUSE THERE IS, SIMILAR TO THE VACCINE AREA, THERE ISN'T REALLY ANY ONE FUNDER THAT'S GOING TO BE ABLE TO DO THIS ALONE. SO THAT JUST GIVES YOU A LITTLE BIT OF OVERVIEW OF WHAT OAR DOES AND HOW WE INVEST THE TAXPAYERS' MONEY. I'M VERY HAPPY TO BE HERE AND I'LL BE AROUND THE REST OF THE MORNING AND UNTIL THE END OF THE SESSION SO IF YOU HAVE ANY QUESTIONS, LET ME KNOW. THANKS. [APPLAUSE] I'M UNO O'DOHERTY FROM THE UNIVERSITY OF PENNSYLVANIA. I WANT TO THANK THE ORGANIZERS FOR INVITING ME TO ATTEND, AS WELL AS CHAIR THIS SESSION. I HAVE THE PLEASURE OF INTRODUCING THREE UP AND COMING SCIENTISTS WHO HAVE ALREADY CONTRIBUTED VERY SIGNIFICANTLY TO HOW WE ANALYZE RESERVOIRS, AND THE FIRST SPEAKER, BRANDON KEELE, WHO ACTUALLY HAS MANY HATS AT FREDERICK BUT ALSO SIMULTANEOUSLY DOES REALLY AWESOME RESEARCH, PROBING SIV RESERVOIR. TODAY HE'S GOING TO TELL US ABOUT BARCODED SIV AND HOW WE CAN USE THIS TO CALCULATE OR ESTIMATE REACTIVATION RATES. SO BRANDON. OH, AND I WANT TO SAY ONE MORE THING. IT'S A REALLY AWESOME EXAMPLE OF AN EXPERIMENT THAT YOU CAN ONLY DO IN A PRIMATE. >> GREAT. THANK YOU. SO I DON'T KNOW WHAT $3 BILLION LOOKS LIKE EITHER, BUT WE MAKE 3 BILLION VIRUSES ALL THE TIME, SO THAT'S NOTHING. [LAUGHTER] SO ALL RIGHT, THIS IS -- I'M REALLY EXCITED TO BE HERE. THANKS TO THE ORGANIZERS. THIS IS AN INTERESTING MODEL FOR NON-HUMAN PRIMATES AND I HOPE IT'S HELPFUL. JUST BY WAY OF BACKGROUND, THERE'S TWO TYPES OF VIRUS POPULATIONS THAT YOU CAN USE TO INFECT ANIMALS WITH. ONE WOULD BE A SWARM WHERE IT'S AN ISOLATE WHERE IT HAS GENETIC DIVERSITY BUILT INTO IT OR A CLONAL ONE. AND OF COURSE YOU CAN MAKE THESE IN SIV OR IN SHIV, AND AS I GOT INTO THE NON-PRIMATE FIELD, IT BECAME CLEAR THAT THERE WAS SOME ROOM FOR THIS TO BE DEVELOPED FURTHER. PEOPLE HAD THEIR FAVORITE CLONES AND THEIR FAVORITE SWARMS, BUT WE REALLY FELT LIKE WE COULD ADD SOMETHING TO THE FIELD HERE. SO THERE'S PROS AND CONS TO ALL OF THESE MODELS, AND I JUST WANT TO TAKE A MOMENT JUST TO RECOGNIZE THAT AND PUT IT OUT THERE, AND IT DOESN'T NEGATE THE& CONS JUST TO HAVE -- I MEAN THE PROS JUST TO HAVE A CON IN YOUR MODEL. YOU JUST HAVE TO ACKNOWLEDGE IT AND THE LIMITATIONS AND USE TO ANSWER QUESTIONS THAT YOU CAN. SO SWARMS GENERALLY HAVE THE DIVERSITY OF A SINGLE HIV INFECTED PERSON. AND YOU CAN USE THAT DIVERSITY TO SEQUENCE AND GENETICALLY ANALYZE THE MONKEY THAT'S INFECTED WITH THAT SWARM. CLONAL STOCKS, OF COURSE, ARE MORE CONSISTENT BETWEEN ANIMALS, AND THAT GREATLY FACILITATES THE REPLICATION OF PUBLISHED STUDIES, HAVING DISTINCT SWARMS CAN SOMETIMES INTERFERE WITH INTERPRETATION OF STUDIES BETWEEN INDIVIDUAL GROUPS. SO FOR RESERVOIR RESEARCH, THE LIKELY MOST RELEVANT MODEL IS TO ESTABLISH THE DIVERSE POPULATION THAT LOOKS LIKE AN HIV INFECTED PERSON, CHRONIC INFECTION, ALTHOUGH MORE AND MORE, PATIENTS ARE BEING TREATED EARLIER AND EARLIER AND MIGHT HAVE LESS AND LESS DIVERSITY. SO OUR MODEL WAS TO COMBINE THE ADVANTAGES OF BOTH OF THESE SYSTEMS BY CREATING A SYNTHETIC SWARM, AND THIS IS -- THE VIRUS IS IDENTICAL OUTSIDE OF THE BARCODE THAT WE USE TO DISCRIMINATE LINEAGES. OUR GOAL WAS REALLY JUST TO GENETICALLY DEFINE THE REBOUND-COMPETENT VIRAL RESERVOIR. OF COURSE THIS IS THE RESERVOIR THAT MATTERS. ALL THE OTHER MEASURES ARE USEFUL AND MIGHT PREDICT THINGS -- THESE ARE THE ACTUAL VERIONS COMING OUT OF EITHER LATENTLY INFECTED CELLS OR ANY RESERVOIR AND PRODUCING VIREMIA IN THE ANIMAL. SO THIS IS WHAT WE'RE TRYING TO TRACK AND FOLLOW. SO THE WAY WE DID THIS IS VERY SIMPLE, WE BUILT UPON A MODEL WE USED FOR TRANSMISSION, AND IN THIS SYSTEM, WE SIMPLY INSERT USING RESTRICTION ENZYME THIS YELLOW PIECE OF GENOME SEQUENCE INSIDE THE VIRAL GENOME PLASMID BETWEEN THE VBX AND VPR GENES. THIS ALLOWS ALL THE VIRUS PRODUCED TO HAVE THE RANDOM BASES IN THE MIDDLE WHICH WE CAN THEN SEQUENCE AND OF COURSE THIS IS A TRICK THAT PEOPLE HAVE USED FOR PRIMARY D AND WE HEARD YESTERDAY IN SEQUENCING RNA. IN THIS CASE, THE VIRUS CARRIES THE BARCODE WITH IT AS IT REP TI LATES, AND AS IT CARRIES WITH IT ALL THE LINEAGE FROM THE ORIGINAL FOUNDER, HAS THE SAME BARCODING, YOU CAN TRACK THEN THAT LINEAGE OF VIRUS IN THE ANIMAL OVER TIME. SO IN OUR FIRST MODEL WHICH WE CALL -- WE FOUND THERE'S ABOUT 10,000 VIRUSES THAT CAN REPLICATE AND WERE FOUND IN THE POPULATION. SO NOW THE MODEL IS WE CAN SEE THE ANIMAL WITH DIFFERENT VIRUSES THROUGHOUT THE ANIMAL AND THEN TRACK THOSE INDIVIDUALLY. SO FAR WEES MADE FIVE VERSIONS WITH DIFFERENCES THAT I WON'T GO INTO TODAY AND WE HAVE SIX SHIV STOCKS THAT ARE AVAILABLE. THE PLACE WHERE I WORK, WE HAVE FROZEN DOWN 3,000 1ML ALIQUOTS IN HOPES THE FIELD WILL USE SOME OF THEM IN PRIMATE RESEARCH AND WE'RE HAPPY TO SHARE THOSE AS NEEDED. SO WE USE NEXT GENERATION SEQUENCING AND ANALYSIS PIPELINE TO ANALYZE OUR BARCODES AND WE'VE DEVELOPED THESE OVER THE YEARS. THE BARCODE, BASED ON WHERE IT SIT, WE SIMPLY ADD THE PRIMERS AND INCLUDED IN THE PRIMER IS THE INDEX AND ADAPTER FOR SEQUENCING ON BOTH ENDS. SO THIS DIRECTS FOR DIRECT PCR AMPLIFICATION AND DIRECT MISEQ SEQUENCING. AT THE BOTTOM ARE TWO ANIMALS THAT WERE INFECTED WITH BARCODED VIRUS, AND ON THE X AXIS, YOU CAN SEE THE MEAN LOG PROPORTION AND THEN THE Y AXIS IS THE 95% CONFIDENCE INTERVAL ON 10 REPLICATES ON THE ANALYSIS. AS YOU CAN SEE, THEY'RE VERY TIGHTLY CORRELATED AND WE ONLY SEE KIND OF VARIABILITY AT THE BOTTOM END OF THIS GRAPH. SO THE SEQUENCING IS ACCURATE AND REPRODUCIBLE, AND WE CAN DO THIS OVER AND OVER AND WE'VE MADE MILLIONS AND MILLIONS OF SEQUENCES OF BARCODED VIRUSES. THANKS TO SOME EARLY ADOPTERS, LEWIS PICKER AND OTHERS, WE WERE ABLE TO INFECT OVER 150 RHESUS OR -- MACAQUES SO FAR AT DIFFERENT DOSES. YOU CAN SEE ON LEFT THE TIME TO PEAK VIREMIA, THE GROWTH RATE OF THESE DIFFERENT DOSES OF INITIAL I.V. INOCC LUM AND THE NUMBER OF BARCODES OF COURSE CORRELATES TO THE DOSE, AND WHAT'S REALLY IMPORTANT IS THIS LARGEST BARCODE, AND AT LOWER INFECTIOUS DOSES YOU HAVE SOME OF THE BARCODES REPRESENT A LARGE PROPORTION OF THE TOTAL, BUT WHEN YOU GET DOWN HERE TO THESE LOWER ONES, THEY'RE VERY WELL DISTRIBUTED. SO IN THESE ANIMALS, WE WANTED TO MAKE SURE ALL THE BARCODES WERE REPLICATION-COMPETENT. SO IN THE RED IS JUST A HISTOGRAM OF THE BARCODES OF THE STOCK THAT WE SEQUENCED, AND THEN IF YOU LOOK IN THE BLUE IN THE MIDDLE PANEL, YOU CAN SEE THE NUMBER OF BARCODES ACTUALLY DETECTED IN AT LEAST ONE OF THOSE ANIMALS REPLICATING IN THE PLASMA. YOU CAN SEE THAT THE MAJORITY OF BARCODES ARE REPLICATION-COMPETENT IN VIVO AND DOING JUST FINE. SO WHEN WE THINK ABOUT -- I'M GLAD BOB USED THE LITTLE DOTS BECAUSE THAT'S WHAT THE RESERVOIR IS, IT'S LITTLE CELLS OF DOTS THAT ARE DIFFERENT FROM EACH OTHER. BUT WHEN WE LOOK AT THE INDIVIDUAL PROPORTION, WHAT WE'RE LOOKING AT HERE IS ACTUALLY -- ONCE -- IS RELEASED, WE'RE LOOKING AT INDIVIDUAL CELLS REACTIVATING AND EVENTUALLY LEADING TO VIREMIA. ALL THE PRIMARY EVENTS SURROUNDING THE ACTIVATION AND ITS REPLICATION BEFORE VIREMIA, WE ARE TRYING TO FIGURE OUT AND THAT'S VERY IMPORTANT. BUT TIME TO DETECT WILL REBOUND ONLY MEASURES THE FIRST REACTIVATION EVENT. THERE MAY OR MAY NOT BE MANY REACTIVATION EVENTS. SO IN FACT, IF THERE WERE A SECOND ONE, IT WOULD SHOW UP HERE AND A THIRD AND A FOURTH AND A FIFTH, AND THESE ARE ALL AVAILABLE TO SEE IF YOU'VE CAN DISCRIMINATE THE REACTIVATION EVENTS GENETICALLY. THAT'S WHAT THE BARCODE ALLOWS US TO DO, TO REPLACE TIME TO DETECTABLE REBOUND WITH A REACTIVATION RATE. THAT'S THE TIME BETWEEN WHEN THE CELLS -- HOW OFTEN THE CELLS ARE GOING TO REACTIVATE THAT LEADS TO PRODUCTIVE VIREMIA. THE SUM TOTAL, NOT JUST THE REACTIVATION EVENT BUT ITS PROGENY AND ITS LIFE UNTIL WE CAN DETECT IT. SO IT'S THAT WHOLE REACTIVATION RATE. OF COURSE THIS IS HAPPENING ON ART, AND THEY'LL HAPPEN AFTER WE STOP MEASURING IT BUT THIS IS JUST THE RATE AT WHICH THESE CELLS ARE PRODUCING. WE DON'T KNOW IF IT'S -- CONSISTENT OR VARIABLE, AND THESE ARE THE TYPES OF QUESTIONS WE CAN ANSWER WITH THE VIRUS. I THINK THEY'RE IMPORTANT. SO OUR FIRST STUDY TO ASSESS THE REACTIVATION RATE WAS IN A VERY SIMPLE MODEL, ACTUALLY THE SIMPLEST WE COULD DEVISE. WE TREATED VERY EARLY WITH ART, AND THEN WE LET THEM GO A YEAR AT VARIOUS TIMES AND YOU CAN SEE WE RELEASED THEM IN PAIRS AND SEQUENCED THE REBOUND VIRUS BARCODE TO SEE WHAT THEY LOOKED LIKE. WHAT YOU SEE IS EACH BAR REPRESENTS A DIFFERENT BARCODE AND THEY'RE NUMBERED HERE, JUST RANDOM BARCODE, AND THEIR RELATIVE PROPORTION IS SHOWN. THEN IF YOU LOOK AT THE VIRAL LOAD AND DARK BLACK, YOU CAN SEE YOU CAN JUST TRACE BACK THE THEORETICAL INITIATION OF THAT WHEN THAT CELL BECAME ACTIVATED AND STARTED TO PRODUCE VIRUS BASED ON VIRAL LOAD OF THAT ANIMAL. ABOVE EACH GRAPH, YOU CAN SEE THE REACTIVATION RATE, SO ONE EVENT EVERY TWO DAYS, ONE DAY OR THREE DAYS, AND ON AVERAGE ACROSS ALL SIX OF THESE ANIMALS, ONCE EVERY TWO DAYS, IT'S FAIRLY CONSISTENT BETWEEN 1.2 AND 3.2 DAYS. YOU CAN SEE THE NUMBER OF REBOUNDERS VARY FROM TWO TO LIKE SEVEN OR SOMETHING LIKE THAT. THOSE ARE THE CELLS THAT REACTIVATE IT AND THEY PRODUCE ENOUGH VIRUS TO BE DETECTABLE IN THE PLASMA DURING THIS WINDOW OF OPPORTUNITY HERE. SO WHEN YOU LOOK AT TIME TO REBOUND VERSUS REACTIVATION RATE, WE DON'T KNOW WHICH ONE IS A BETTER PREDICTER OF RESERVOIR SIZE, I'M JUST SHOWING YOU THE ACTUAL COMPARISON, YOU CAN SEE THEY DON'T REALLY CORRELATE VERY WELL. SO IN YELLOW, IT WAS THE LONGEST DELAY BEFORE DETECTABLE REBOUND AND YOU CAN SEE THAT THERE'S VARIABILITY IN THIS TIME TO DETECTION. AND OF COURSE THE REBOUND RATE IS NOT SENSITIVE TO WASHOUT TIME, SO IF THE WASHOUT TIMES ARE A LITTLE DIFFERENT BETWEEN ANIMALS, THE RATE WILL BE ACCURATE REGARDLESS OF THE WASHOUT TIME. WHEREAS THE TIME TO DETECTABLE WILL BE VARIABLE. WE DON'T KNOW IF THAT'S THE REASON FOR THIS, WE'RE STILL LOOKING INTO THAT. SO WHAT IF YOU TREAT ANIMALS LATER? ON THE RIGHT, WE TREATED ANIMALS AT DAY 27 WITH ART, THE SAME EXPERIMENTAL DESIGN. ANIMALS WERE RELEASED AT THESE VARIOUS TIMES AND WE FOUND THE REACTIVATION RATE WAS DOUBLE WHAT IT WAS WHEN ART WAS TREATED AT DAY 4. BUT INTERESTINGLY, AND SOMETHING WE DON'T YET UNDERSTAND, IS THE CELL ASSOCIATE DNA LEVELS THAT PERSISTED WERE 100 FOLD, TWO LOGS MORE THAN WHAT WE FOUND, SO THE REACTIVATION RATE WENT UP TWOFOLD AND THE RESERVOIR THAT'S DNA-RESISTANT VIRUS WENT UP 100 FOLD. SO NOT ALL OF THOSE ARE REAL RESERVOIR OR THE REACTIVATION RATE IS MEASURING SOMETHING OUTSIDE OF WHAT CAN REACTIVATE. SO TO ESTABLISH THIS IN DETAIL, WE WORKED WITH LEWIS AND WE SET UP AN EXPERIMENT WITH FIVE ANIMALS TREATED AT EACH OF THE DAYS REPRESENTED IN THE TOP LEFT. SO EACH OF THESE ANIMALS, FIVE ANIMALS IN EACH GROUP WERE TREATED AT THE DAYS INDICATED. WE MEASURED THE NUMBER OF BARCODES IN EACH ANIMAL, THEY HAD PLENTY OF BARCODES AND OF COURSE THE ANIMALS THAT WERE TREATED EARLY, THEY HAVE FEWER DETECTABLE, THAT'S JUST BECAUSE THE VIRAL LOAD IS VERY LOW, BUT WE SEQUENCED ALL OF THESE HERE. THEN OF COURSE THE CELL ASSOCIATED DNA LEVELS INCREASED IN THESE ANIMALS AFTER THEY'D BEEN TREATED BASED ON WHEN THEY STARTED ART. SO THIS IS A MIRRORED EXPERIMENT TO LEWIS' RECENT PUBL BAITION AND WHAT PUBLICATION AND WHAT WE ANTICIPATE IS THAT THE ANIMALS THAT STARTED ART EARLY WILL HAVE FEWER REBOUND VIRUSES THAN THOSE THAT STARTED LATER. THESE ANIMALS ARE GOING TO BE RELEASED IN THE NEXT WEEK, SO WE'RE REALLY EXCITED TO GET THESE DATA AND TO FIND THE ANSWER, TO FIND THE RELATIONSHIP BETWEEN ABSOLUTE RESERVOIR SIZE AND THE REACTIVATION RATE. SO SO REACTIVATION RATES CAN BE -- RESERVOIR REDUCING THERAPIES, SO WE CAN TREAT THESE ANIMALS. WORK DONE BY MYLES DAVENPORT SUGGESTS THAT EIGHT ANIMALS IN EACH GROUP TREATED FOR ONE YEAR, WE CAN CLEARLY DETECT A GREATER THAN -- A LESS THAN ONE LOG REDUCTION RESERVOIR SIZE. SO WE THINK THAT'S EXCITING. THERE'S ALSO SOME FUNDAMENTAL FEATURES OF THE RESERVOIR THAT WILL BE ABLE TO EXAMINE THE DECLINE OF THE VIRUS, DURING THE DECLINE, WE'LL BE ABLE TO SEE HOW THAT HAPPENS. THE MAINTENANCE PHASE INCLUDING MEASURING THE BLIPS AND WHAT CONSTITUTES VIRAL BLIPS, WE CAN LOOK AT THE EVOLUTION OF THE VIRUS OVER TIME, WE CAN LOOK AT CLONAL EXPANSION, AND THEN WHEN ANIMALS ARE RELEASED FROM THERAPY, WE CAN LOOK AT THE RESERVOIR, HOW IT'S RECEDED, HOW THE POPULATION CHANGES BASED ON THE REBOUND AND WHICH VARIANTS WERE REBOUNDING. WE ANTICIPATE THAT THE RESERVOIR WILL CHANGE, GIVEN THESE RECEDINGS, BUT WE NEED TO DEFINE THAT AND WE NEED TO DECIDE HOW LONG TO LET THE REBOUND OCCUR. THESE ARE ALL THINGS INHERENTLY DOABLE IN THE SYSTEM. SO AS I MENTIONED, WE'VE DEVELOPED ADDITIONAL SHIVs IN COLLABORATION WITH SEVERAL PEOPLE THAT I'LL MENTION AT THE END, AND THESE SHIVs NOW, WE'VE BARCODED ALL OF THESE, AND THE NUMBER OF DISTINCT BARCODES IN EACH STOCK IS INDICATED -- RANGING ANYWHERE FROM 3,000 TO 65,000 BARCODES IN EACH OF THESE STOCKS. THE BARCODE IN THE SHIVs ARE IN THE SAME POSITION AS THEY WERE IN THE SIV 239 OF COURSE THE ENVELOPES ARE DIFFERENT. SOME OF THESE ENVELOPES, YOU'LL RECOGNIZE, THERE'S # 8, THERE'S 1054, THERE'S SOME NEW SHIV Cs, SUB-TYPE Cs THAT WE'VE GENERATED. THESE ARE ALL AVAILABLE IF ANYONE WOULD LIKE TO TRY THESE OUT. IN ADDITION, WE REALIZED THAT THERE WAS THIS NEED FOR GREATER NUMBER OF BARCODES THAN JUST 10,000. FIRST WE MADE THIS VIRUS FOR TRANSMISSION HAD 10 VIRUSES IN IT, THEN WE MADE THE -- 10,000, WE THOUGHT THAT WAS GRAND, WHO NEEDED MORE THAN 10,000. BUT IT TURNS OUT IF YOU ACTUALLY WANT TO CAPTURE SINGLE FOUNDER VIRUSES OF EACH BARCODE, YOU HAVE TO HAVE A VERY LARGE POPULATION TO MAKE SURE THAT THEY'RE NOT DUPLICATED. THIS IS THE BIRTHDAY PHENOMENON, RIGHT? YOU'VE GOT A ROOM FULL OF PEOPLE, SOME OF THEM WILL SHARE A BIRTHDAY. BIRTH DATE. SO TO DO THAT, WE GENERATED THE SECOND GENERATION, SAME BARCODE, SAME APPROACH, BUT IN THIS CASE, WE HAVE 145,000 BARCODES IN THIS STOCK. DOWN HERE YOU CAN SEE A SIMULATION, IF YOU HAVE 500 FOUNDER VIRUSES IN THE NEW STOCK, ACTUALLY ONLY ON OF THOSE WILL BE LIKELY A DUPLICATE OF THE SAME BARCODE TWICE IN THE ANIMAL. EVERYTHING ELSE WILL BE UNIQUE. IF YOU GO OUT TO 2,000, ONLY 20 OF THEM WILL ACTUALLY BE DUPLICATED. SO YOU CAN AFFECT THE ANIMAL WITH 2,000 DIFFERENT THINGS, AND ONLY 20 OF THEM WILL BE DUPLICATED ANYWHERE. THIS REALLY ALLOWS US TO LOOK FOR FOUNDER EFFECTS IN THE ANIMAL ITSELF. SO ONE BIG PROBLEM WITH OUR MODEL WAS BOB'S DATA SUGGESTING A LOT OF THESE VIRUSES HAD LARGE DELETIONS. OF COURSE THEY DELETE RIGHT IN THE MIDDLE WHERE THE BARCODE IS, AND WE DIDN'T WANT THAT. OUR MODEL WAS SET UP TO ESTABLISH THE INFECTION QUICKLY OR DO SOME INTERVENTION. YOU DON'T WANT THE VIRUSES TO REPLICATE FOR A VERY LONG PERIOD OF TIME, BARCODES CAN GET LOST, THE BARCODES CAN MAKE POINT 3450U MUTATIONS TO FIND WHICH IS A REAL BARCODE. SO IN OUR MODEL SYSTEM, WE SIMPLY LOOKED AT OUR DAY 10 TREATED ANIMALS AND DAY 27 TREATED ANIMALS, AND WHAT WAS REMARKABLE, I'LL SHOW YOU ON THE DAY 101ST, WAS THAT WE FOUND THAT ABOUT 85% OF ALL OF THE GENOMES THAT WE MEASURED A YEAR AFTER TREATMENT WERE INTACT, AND MOST REMARKABLE WAS THAT 50% OF THOSE ACTUALLY HAD NO NUCLEOTIDE CHANGE IN THE ENTIRE GENOME. SO THIS EXACTLY WHAT WE SAW WITH TRANSMITTED FOUNDER VIRUSES YEARS AGO, THE ACCUMULATION OF SINGLE NUCLEOTIDE CHANGES OCCUR SLOWLY IN THE POPULATION, AND YOU CAN CAPTURE THESE EVENTS BEFORE THESE HAVE TAKEN OVER THE POPULATION. AT DAY 27, WE SEE ABOUT THE SAME DELETIONS, AND THEN ACCUMULATION OF MORE SINGLE NUCLEOTIDE CHANGES. BUT IF YOU GO OUT A YEAR, THEN WE BEGIN TO SEE WHAT WE SEE IN MACAQUES OR WHAT BOB JUST MEASURED, AND WHAT MOST HUMANS HAVE, SO THIS IS ONE YEAR BEFORE THERAPY AND ONE YEAR ON THERAPY, YOU SEE ONLY A FRACTION OF THE GENOMES ARE INTACT. AND WE SEE LARGE DELETIONS,ET CETERA. SO IN CONCLUSION, THE BARCODED VIRUSES ARE FUNCTIONAL, THE VAST MAJORITY ARE REPLICATION-EXE AT THE PRESENT TIME, THEY PROVIDE KEY INSIGHTS TO MEASURING THE REBOUND-COMPETENT VIRAL RESERVOIR. TIGHT CONTROL OVER NUMBER OF VARIANTS IS CRITICAL IN THIS MODEL, IT ALLOWS FOR ASSESSMENTS, STOCKS ARE AVAILABLE TO USE, AND FOR ART INITIATED LATE AND CHRONIC INFECTION, THE MODEL BECOMES LESS RELEVANT DUE TO THE LOSS OF BARCODES, THE INCREASE IN DOMINANT BARCODES, AND MORE FREQUENT DELETED GENOMES. BUT I THINK OVERALL, WE HAVE A GREAT MODEL FOR ASSESSING THE REBOUND COMPETENT RESERVOIR. THESE ARE THE PEOPLE THAT DID THE WORK, AND I JUST WANT TO ACKNOWLEDGE MYLES DAVENPORT, MY COLLABORATOR IN THIS, LEWIS PICKER AND AFM FOR BEING GREAT COLLABORATORS AND THESE GUYS DOWN HERE ALL DID THE SHIV WORK. THANK YOU VERY MUCH. [APPLAUSE] >> THANK YOU, BRANDON. OKAY. WE DON'T HAVE TIME FOR A LOT OF QUESTIONS. WE CAN GO A LITTLE OVER? OKAY, EVERYBODY, LET'S BE QUICK. I'LL LET LEWIS PICKER GO FIRST. >> I WANTED TO MAKE ONE COMMENT JUST TO GIVE ANOTHER EXAMPLE, [[INAUDIBLE] -- WITH BARCODED VIRUSES IN WHICH WE COMPARED REBOUND DIANA DYNAMICS IN ANIMALS THAT ARE CD8 -- DEPLETED OR CONTROL TREATED IN WHICH THAT DEPLETION OCCURRED BEFORE RELEASE [INAUDIBLE] THE END RESULT WAS ABOUT A 2 LOG DIFFERENCE IN SET POINT BETWEEN THESE ANIMALS BUT THERE WAS NO DIFFERENCE IN THE REBOUND RATE THAT SUGGESTS THAT THE CA T-CELLS DID NOT INTERCEPT [INAUDIBLE] IT SUGGESTS THE CAT CELLS DID NOT INTERCEPT THE REBOUNDING INFECTION UNTIL WELL INTO THE REBOUND, WHICH I THINK IS USUALLY AN IMPORTANT POINT BECAUSE OUR GOAL IS TO HAVE THE CAT CELLS INTERCEPT THE RESERVOIR, IF IT DOESN'T INTERSET EVEN THE FIRST WEEK AFTER REBOUND [INAUDIBLE]. THAT'S JUST ANOTHER EXAMPLE OF HOW [INAUDIBLE]. >> THAT'S REALLY BEAUTIFUL. QUESTION ABOUT THE REACTIVATION RATE. THAT'S A RATE OF CELLS PER DAY, IS THAT RIGHT? WHAT HAPPENS IF YOU NORMALIZE BY THE NUMBER OF CELLS WITH INTACT PRO VIRUS TO GET A RATE OF REACTIVATION OF CELLS THAT HAVE THE POTENTIAL TO REACT? >> SO THAT'S GOOD. SO WE DON'T HAVE ENOUGH RATES YET, BUT THAT'S A GOOD IDEA. WE'LL DO THAT ONCE WE HAVE THESE CHRONIC ANIMALS THAT SHOULD HAVE LESS INTACT MORE DEFECTIVE GENOME. RIGHT NOW THEY'RE SO INTACT THAT IT ONLY MAKES A SMALL FRACTION. >> RELATED QUESTION. I THINK AN INTEREST IS THE RATE OF REACTIVATION FROM LATENCY VERSUS THE RATE FROM CELLS THAT ARE ALREADY EXPRESSING RNA. DID YOU MEASURE CELL ASSOCIATED RNA IN THESE STUDIES, CELL ASSOCIATED DNA? >> WE DO. THEY CORRELATE ROUGHLY WITH THE DNA LEVELS. SO YOU'RE EXACTLY RIGHT, WHAT WE DON'T KNOW FUNDAMENTALLY IS HOW MANY CELLS REACTIVATE AND PRODUCE INFECTIOUS VIRUS THAT DON'T GO ANYWHERE, THAT ARE -- THEY'RE TERMINAL, RIGHT? THEY DON'T END UP IN THE PLASMA. AND THAT'S WHAT WE DON'T KNOW. SO THEY'RE REACTIVATING, THEY MAY OR MAY NOT MAKE INFECTIOUS VIRUS AND THEN THE QUESTION IS, DID THAT INFECTIOUS VIRUS MAKE AN -- LARGE ENOUGH TO CONTINUE PROPAGATING. >> ONE MORE QUESTION. >> REALLY A BEAUTIFUL SYSTEM. WHEN YOU LOOK AT BARCODE DIVERSITY, IS THERE ANY EVIDENCE OF BOTTLENECK IN THE MACAQUE? IN OTHER WORDS, HAVE YOU LOOKED ACROSS TISSUES FOR DIVERSITY MARKERS? >> I DON'T NORMALLY SHOW THAT. WE'VE LOOKED AT SO MANY TISSUES, IT'S ALL ABOUT TIMING. IT'S HOW MANY REPLICATION CYCLES THE VIRUS HAS GONE THROUGH. WHEN WE GO OUT, YOU KNOW, NINE MONTHS WITHOUT ANY ART, WE SEE A LOSS OF SOME OF THE BARCODES, IT JUST SHRINKS A LITTLE BIT, DELETION, POINT MUTATIONS. EVERYTHING YOU CAN PIECE BACK GENETICALLY TO THE FOUNDER, BUT IT LOSES ITS SPEED AND UTILITY BY LETTING IT GO SO LONG. BUT THE NORMAL ACCUMULATION OF CHANGES -- LIKE THE VIRUS WILL. >> THANK YOU. [APPLAUSE] >> THANK YOU. I'M SURE YOU'LL GET MORE QUESTIONS IN THE BREAK. THE NEXT SPEAKER IS EDWARD BROWNE, WHO TO MY KNOWLEDGE WAS THE FIRST TO REALLY USE TRANSCRIPTOME ANALYSIS OF LATENT CELLS. THIS IS A GREAT EXAMPLE, I THINK, OF HOW AN IN VITRO MODEL CAN BE STUDIED TO GREAT PURPOSE. THANKS. >> THANKS TO THE ORGANIZERS FOR THE OPPORTUNITY TO PRESENT HERE. THIS HAS BEEN A REALLY FASCINATING MEETING SO FAR. SO I'M GOING TO BE TELLING YOU TODAY ABOUT SOME WORK FROM MY LAB TO CHARACTERIZE A PRIMARY -- USING SINGLE CELL METHODS. SO THE CENTRAL QUESTION THAT I'M INTERESTED IN IS JUST THE QUESTION OF JUST HOW AND WHY DOES HIV GO INTO LATENCY, WHAT ARE THE MOLECULAR MECHANISMS THAT GO WITH THAT. WHEN IT INFECTS THE HOST CELL, MOST OF THE TIME WHAT YOU GET IS ACTIVE VIRAL TRANSCRIPTION AND PRODUCTIVE VIRAL INFECTION, LOTS OF LITTLE VIRUSES GETTING MADE. OTHER TIMES, FOR REASONS WE DON'T COMPLETELY UNDERSTAND, THE VIRUS EITHER SHUTS DOWN TRANSCRIPTION OR NEVER STARTS IN THE FIRST PLACE, WHAT YOU GET IS LATENCY. SO MOI THOUGHT WAS IF WE START WITH A MODEL SYSTEM, WE CAN SEE DIVERSE -- LEARN TO PREDICT WHICH LATENCY IS GOING TO OCCUR. SO IN ORDER TO DO THIS I HAD TO USE A PRIMARY MODEL OF CELL LATENCY. IT'S WORTH DESCRIBING IN SOME DETAIL, I THINK. SO WHAT WE START OFF WITH IS JUST TOTAL CD4 T-CELLS, WE ACTIVATE THEM AND THEN INFECT THEM WITH A GFP EXPRESSING VIRUS. WE THEM FLOW SORT THEM TO OBTAIN A PURE INFECTED POPULATION, AND THEN WE CULTURE THESE CELLS FOR A FAIRLY LONG PERIOD OF TIME, ABOUT SIX TO EIGHT WEEKS USING A -- CELL LINE AND WHAT WE OBSERVED IS AFTER THAT PERIOD OF CULTURE IS THAT WE SEE DIVERSE TRANSCRIPTIONAL FATES FOR THE PRO VIRUSES. SO SOME OF THE CELLS MAINTAIN VERY HIGH LEVELS OF VIRAL TRANSCRIPTION, SOME OF THEM HAVE THIS KIND OF -- INTERMEDIATE PHENOTYPE, OTHERS LOSE -- EXPRESSION AND APPARENTLY GO INTO LATENCY. SO A FEW OTHER POINTS ABOUT THESE CELLS, YOU CAN SORT THE GFP POSITIVE AND NEGATIVE CELLS. THEY MAINTAIN THEIR PHENOTYPES. THESE PHENOTYPES ARE REASONABLY STABLE AT LEAST FOR UP TO -- SO THE GFP NEGATIVE CELLS STAY NEGATIVE AND THE POSITIVE CELLS SAY POSITIVE. WHAT I THOUGHT WAS KIND OF INTERESTING, YOU CAN REACTIVATE THESE CELLS WITH VARIOUS LATENCY REVERSING AGENTS. IN THIS CASE WE'RE USING -- AND THEY TURN GREEN AGAIN, WHICH TELLS YOU THAT THEY'RE ACTUALLY LATENTLY INFECTED. THEY CAN RESPOND TO A UM IN BER OF CLINICAL LATENCY REVERSING AGENTS, SO THEY SEEM TO RECAPITULATE SOME OF THE MOLECULAR MECHANISMS THAT SEEM TO OPERATE IN THE CLINICAL RESERVOIR. WHAT I FOUND STRIKING WAS IF YOU TAKE THESE CELLS AND JUST WAIT A FEW NOR DAYS, MORE DAYS, THEY GO STRAIGHT BACK TO BEING GFP-NEGATIVE AGAIN. YOU CAN STIMULATE THEM OVER AND OVER AGAIN AND THEY ALWAYS GO BACK TO BEING GFP-NEGATIVE, SOMETHING QUITE STRIKING. I THINK IT'S SOMETHING THAT'S INTRINSIC TO A CELL THAT REMEMBERS LATENCY THROUGH DIFFERENT ROUNDS OF STIMULATION AND PULLS THE VIRUS BACK INTO LATENCY AGAIN. WHAT YOU'RE INTERESTED IN THIS HERE, WHY DO SOME CELLS GO INTO LATENCY AND OTHERS NOT AND YOU CAN USE THIS AS A MODEL SYSTEM TO LEVERAGE NATURAL DIVERSITY OF THIS SYSTEM TO TRY AND UNDERSTAND MOLECULAR CORRELATES OF LATENCY. AND SO WE'VE LOOKED AT A BUNCH OF THINGS. THE TWO THINGS WE LOOKED AT, HOST CELL GENE EXPRESSION USING RNA SEQ. MORE RECENTLY WE'VE BEEN LOOKING AT THE HOST CELL EPIGENOME USING CHP AND SINGLE CELL -- I'LL BE TALKING ABOUT BOTH THOSE THINGS TODAY. SO WE STARTED OFF WITH SINGLE CELL RNA SEQ. SO WE PROFILED SEVERAL THOUSAND INFECTED CELLS, SO ON THE LEFT HERE IS A -- PLOT, EACH DOT REPRESENTS AN INFECTED CELL. ON THE RIGHT HERE, YOU'VE GOT A HEAT MAP OF VIRAL GENE EXPRESSION SO THE MORE RED IT IS, THE HIGHER THE LEVEL OF GENE EXPRESSION. TWO TAKE HOME MESSAGES. ONE IS THAT YOU CAN SEE LATENTLY INFECTED CELLS WHICH ARE THE GRAY ONES IN ALL THE DIFFERENT CLUSTERS, SO CLEARLY LATENCY CAN OCCUR IN DIVERSE TRANSCRIPTIONAL ENVIRONMENTS. BUT HAVING SAID THAT, THERE IS A CLEAR INFLUENCE OF THE UNDERLYING TRANSCRIPT PHENOTIME. CLUSTER ONE IS HIGH LEVELS OF VIRAL TRANSCRIPTION, UNDERLYING INFLUENCE OF THE HOST CELL ENVIRONMENT ON WHETHER THE VIRUS GOES INTO LATENCY OR NOT. SO FROM THIS WE WERE ABLE TO PUT TOGETHER A LIST OF 42 DIFFERENT TRANSCRIPTS WHOSE EXPRESSION LEVEL WAS ELEVATED IN LATENTLY INFECTED CELLS, A LATENTLY ASSOCIATED SIGHT, IF YOU WILL. WE IMMEDIATELY STARTED LOOKING THROUGH THIS LIST OF GENES TO TRY AND GAIN SOME SORT OF BIOLOGICAL INSIGHT. ONE THING THAT WAS IMMEDIATELY APPARENT WAS THAT THE LATENTLY INFECTED CELLS WERE ENRICHED WITH EXPRESSION WITH MARKERS OF TCM MARKERS. SUGGESTING CELLS THAT HAVE GONE TOWARDS THAT KIND OF PHENOTYPE ARE MORE CONDUCIVE TO LATENCY ANOTHER INTERESTING OBSERVATION THAT CAME OUT OF THIS EXPERIMENT WAS WHEN WE SORTED CELLS BASED ON VIRAL GENE EXPRESSION, SO WE SOUGHT THE GFP POSITIVE AND NEGATIVE CELLS IN PARALLEL, ACTIVATE THEM AND EXPAND THEM, WHAT WE FOUND CONSISTENTLY THE LATENT CELLS EXPAND MORE THAN THE GFP POSITIVE CELLS, SUGGESTING THIS VIRUS IS GETTING PREFERENTIALLY -- PROLIFERATIVE POTENTIAL, CONSISTENT WITH THE NOTION OF JUST SILENCING REALLY TO UNDERLYING BIOLOGICAL PROPERTIES OF THE CELL, NOT BEING COMPLETELY RANDOM. SO RELATED TO THAT, WE WERE ALSO INTERESTED IN CLONAL EXPANSION, SO IF WE KNOW THE CLONAL EXPANSION IS AN IMPORTANT MECHANISM OF MENTIONING -- IN VIVO, I DON'T KNOW WHETHER MY CULTURE SYSTEM IS A GREAT MODEL FOR IN VIVO EXPANSION OF THESE CELLS BUT WE COULD LOOK AT LEAST TO SEE WHETHER WE COULD SEE CLINICAL EXPANSION GOING ON. AND SURE ENOUGH, WE CAN SEE EXPANDED CLONES IN OUR CULTURE, AND THE INFECTED CELL POPULATION ABOUT 7% OF THE CELLS ARE EXPANDED CLONES. SO IT'S DEFINITELY HAPPENING. CURIOUSLY, WE SAW A LOT MORE OLIGOCLONALLITY THAN WE DID -- THERE'S POTENTIALLY SOMETHING INTERESTING HAPPENING THERE, I THINK. AND FROM THIS, WHAT WE'RE HAVING TO FIGURE OUT ARE THE , OTHER QUESTIONS WE CAN ASK, IS CLONAL EXPANSION RELATED TO VIRAL GENE EXPRESSION? IS IT THE GFP NEGATIVE CELLS THAT ARE EXPANDING, POSITIVE, BOTH? THE THING WE CAN FIGURE OUT FROM THIS, IS THERE A CLONAL EXPANSION SIGNATURE. THE TCR SEQUENCE TO FIGURE OUT THE CLONALITY, BUT FIGURE OUT WHETHER EXPANDING CELLS HAVE A PARTICULAR SIGNATURE, FOR EXAMPLE, THAT TELLS YOU SOMETHING ABOUT THE BIOLOGY OF WHY THEY'RE EXPANDING. SO THIS IS KIND OF A SUMMARY OF THIS PART OF THE SLIDE. SO THE MESSAGE HERE IS THAT THE SCIENCING IS NOT RANDOM, INTRINSIC BIOLOGICAL FEATURES OF THE HOST CELL. 42 GENE SIGNATURE THAT TRACKS WITH LATENCY THAT SEEMS TO BE ELEVATED IN CELLS WITH A TCM PHENOTYPE AND CELLS WITH ELEVATED PROLIFERATIVE POTENTIAL BUT WHAT WE WANT OUT OF THIS IS NOT JUST KIND OF GENERAL AND BIOLOGICAL PATHWAYS BUT WE WANT SPECIFIC GENES THAT REGULATE LATENCY THAT WE CAN TARGET THEIR PEUTLY. WE WANT NEW TARGETS FOR LRA, FOR EXAMPLE. WE'VE HAD SOME SUCCESS, WE FOUND THIS TO BE A FAIRLY SLOW AND TEDIOUS PROCESS. I'LL BE TELLING YOU A STORY ABOUT THE TWO FACTORS WE'VE BEEN FOCUSING ON, ONE CALLED AES WHICH WE THINK IS A LATENCY PROMOTING FACTOR AND GATA3, A LATENCY COUNTERING FACTOR. SO AES CAME OUT OF OUR SINGLE RNA SEQ STREAM IN THE LATENTLY INFECTED CELLS. THIS IS AN INTERESTING PROTEIN. IT'S A MEMBER OF THIS FAMILY OF TRANSCRIPTION REPRESSORS BASED ON HOMOLOGY -- THESE FAMILY MEMBERS HAVE TWO MOLECULAR FUNCTIONS, ONE IS THEY ARE NATURAL INHIBITORS OF NA KB AND ALSO -- IT HITS ON TWO PATHWAYS THAT WE KNOW ARE IMPORTANT FOR LATENCIES SO THAT PIQUED OUR INTEREST. WE ALSO DID A KNOCK DOWN IN THE JURKAT MODEL AND KNOCKED OUT AES WHICH SUGGESTED IT IS A BONA FIDE LATENCY REGULATOR. SO GATA 3 I THINK WORKS THE OTHER WAY, GFP POSITIVE CELLS, WE MADE A LENTIVIRUS, TO DO THIS, WE TRANSDUCED CELLS WITH THIS GATA 3 EXPRESSING LENTIVIRUS AT THE SAME TIME AS HIV INFECTION SO WE GET THIS DOUBLE INFECTION POPULATION, SO WE SORT THOSE OUT AND WHAT WE FIND IS GATA 3 HAD A HARDER TIME GOING INTO LATENCY. WE ALSO KNOCKED OUT THE GATA 3 BINDING SITES IN THE LTR OF HIV, SO ONE HYPOTHESIS IS THIS IS DIRECT, JUST DRIVING HIV EXPRESSION, BUT THE KNOCKOUT VIRUS GOES INTO LATENCY FASTER TO A GREATER DEGREE THAN THE WILD TYPE VIRUS, SUGGESTING THAT AT LEAST PART OF THIS PHENOMENON IS THROUGH DIRECT TRANSCRIPTIONAL -- I'M LOSING MY VOICE. I'M GOING TO CHANGE GEARS NOW TO TALK A LITTLE ABOUT CHROMATIN. KIND OF A NEW AVENUE FOR US. CHROMATIN AS I'M SURE YOU'RE ALL AWARE IS IMPORTANT FOR TRANSCRIPTIONAL REGULATION AND ALSO FOR HIV TRANSCRIPTIONAL REGULATION, AND IT INVOLVES THIS SERIES OF COVALENT MODIFICATIONS TO LONG HISTONE TAILS HERE THAT TYPICALLY METHYLATION OR ACETYLATION CAN CREATE AN ENVIRONMENT THAT'S CONDUCIVE FOR INHIBITORY -- WE TRIED TO -- THAT CORRELATED WITH LATENCY IN OUR MODEL. AND THE RESULTS ARE HERE ON THE LEFT. WESTERN BLOTS WITH DIFFERENT HISTONE MODIFICATIONS. MOST THINGS DIDN'T REALLY CHANGE AT ALL, DEPENDING ON WHETHER YOU'RE IN LATENCY OR ACTIVE VIRAL TRANSCRIPTION. THERE WAS ONE THING CONSISTENTLY DIFFERENT AND THAT WAS THAT LATELY INFECTED CELLS HAVE DIFFERENT TOTAL LEVEL OF H3K27 METHYLATION. WE ALSO LOOKED AT THE HIV PROMOTER SO YOU CAN SEE THE NUC1 HAS ELEVA TED LEVELS OF H3K27 METHYLATION AND THE NEGATIVE CELLS COMPARED TO THE POSITIVE. SO WE HAVE A VERY LOCAL CHROMATIN MODIFICATION AT THE HIV PROMOTER, BUT IT CORRELATES WITH A GLOBAL PROCESS BY WHICH THESE CELLS HAVE JUST GLOBAL HIGH LEVELS OF H36789K273K27 METHYLATION. SO THIS IS A KNOWN INHIBITING CHROMATIN MARKET, IT SHUTS DOWN -- TRANSCRIPTION, IT'S INVOLVED IN FACULTATIVE OR DYNAMIC CHROMATIN. THERE ARE OTHER INHIBITORS OF THIS COMPLEX AVAILABLE. THERE'S AT LEAST TWO PAPERS OUT THERE SHOWING H3K27 SHUTS DOWN VIRAL TRANSCRIPTION AND THAT INHIBITORS OF -- LATENCY REVERSING AGENTS. SO I THINK AS THESE ACTIVATED CELLS, INFECTED CELLS GO BACK TO REST, I THINK WHAT HAPPENS IS SOME OF THEM HAVE HIGH LEVELS OF H3K27 TRY METHYLATION AND THOSE ARE THE ONES WHERE THE VIRUS IS GOING INTO LATENCY. I DON'T THINK THERE'S ANYTHING MAGICAL ABOUT IT, IT'S JUST BASIC T-CELL BIOLOGY WHEREAS CELLS GO BACK TO REST TO A MEMORY PHENOTYPE, THEY JUST SHUT DOWN LOTS OF GENES WITH EXTRA H3K27 27 METHYLATION AND THE VIRUS GETS CAUGHT UP IN THAT, I THINK. DIFFERENT QUESTIONS THAT CAN ARISE HERE, WHY DO THESE CELLS HAVE HIGH LEVELS OF H3K27 TRY METHYLATION, 27 TRI-METHYLATION, AND WHAT DOES THAT MEAN BUY BIOLOGICALLY. QUESTIONS THERE, I THINK. IN THE LAST FEW MINUTES, I THOUGHT I'D TALK ABOUT MORE RECENT WORK, LOOKING AT ATEC SEQ. BASICALLY THIS IS A METHOD TO PROVE CHROMATIN STRUCTURE. IT'S VERY SIMILAR IN CONCEPT TO A DNA HYPERSENSITIVITY ASSAY. YOU HAVE THIS TRANSPOSON THAT CAN -- GENERATE FRAGMENTS OF DNA THAT YOU CAN SEQUENCE BY NEXT GENERATION SEQUENCING AND YOU GET PEAKS BASICALLY THAT TELL YOU WHERE THE CHROMATIN IS OPENING AND CLOSING IN DIFFERENT POPULATIONS SO YOU CAN INFER WHAT GENES ARE CHANGING, YOU CAN ALSO LOOK IN THESE PEAKS FOR DIFFERENT INFECTION AFFECTED BINDING SITES, SO IT'S A COOL METHOD. FOR OUR EXPERIMENT, BECAUSE WE CAN'T INFER VIRAL GENE EXPRESSION -- WE HAD TO PRESORT OUR -- INTO GFP NEGATIVE AND POSITIVE. WE THEN PROCESSED THESE FOR SINGLE CELL ATTACK SEQ AND WHAT WE GET OUT OF THIS IS SORT OF PEAKS FOR EACH CELL AND WHICH WE CAN CALCULATE A TRANSCRIPTION FACTOR -- MATRIX TO SEE WHICH TRANSCRIPTION FACTORS ARE AFFECTED IN DIFFERENT CLUSTERS. FIRST WE LOOKED AT THE VIRAL GENOME, YOU CAN SEE WHERE THE PEAKS -- FOR HIV, YOU CAN SEE TWO MAIN PEAKS THAT CORRESPOND TO THE TRANSCRIPTION -- SITE AND YOU CAN SEE IN THE GFP NEGATIVE CELLS, THESE PEAKS ARE SIGNIFICANTLY SMALLER, THE VIRAL GENOME IS SIGNIFICANTLY MORE CLOSED, IT HAS REDUCED ACCESSIBILITY. THEN WE LOOKED AT THE HOST CELL AND THIS IS PRELIMINARY DATA I SHOULD MENTION, WE'VE DONE THIS ON -- YOU CAN SEE THESE DOTS CORRESPONDING TO THE TRANSCRIPTION FACTORS MATRIX AND COMPARING THE GFP POSITIVE TO NEGATIVE CELLS, TWO OBSERVATIONS, ONE IS THE POSITIVE CELLS ARE MORE DIVERSE, THE CELLS CAN FOLD INTO FOUR DIFFERENT CLUSTERS WHEREAS FOR THE NEGATIVE CELLS, ALL OF THEM ARE ON KIND OF ONE BIG CLUSTER SO THEY'RE ALL KIND OF THE SAME EPIGENOMIC TYPE. AND WE CAN IDENTIFY TRANSCRIPTION FACTORS THAT DEFINE EACH CLUSTER, CLUSTER ONE AND CLUSTER FOUR, YOU CAN SEE MEMBERS OF THE I FAMILY. AND FOR THE GFP NEGATIVE CELLS, HERE CLUSTER I, AT LEAST THE TOP 10 TRANSCRIPTION FACTORS. POTENTIALLY SOME INTERESTING THINGS TO LOOK AT HERE. THERE'S ONE THING THAT STOOD OUT, THOUGH, THIS WAS INTERESTING BECAUSE -- THE FIGURE GOT SCREWED UP HERE BUT NEVER MIND. THERE'S A TRANSCRIPTION FACTOR THAT'S A REPRESSOR, AND THERE'S A SEQUENCE ELEMENT INSIDE THE HIV GENOME JUST DOWNSTREAM OF THE TRANSCRIPTION SATURDAY SITE. AND THERE'S SOME WORK FROM A BUNCH OF LABS, FROM HENDERSON'S LAB SHOWING THIS TRAN SCRIP TORE FABBING TORE -- THERE'S THIS WORK SHOWING IT'S ASSOCIATED WITH LOWER HIV RESERVOIR AND TRANSCRIPTION LEVELS IN CENTRAL MEMORY T-CELLS AND ALSO THE WORK FROM MICHELLE'S LAB, THE SIGNATURE THAT THEY SAW FOR REACTIVATED LATENTLY AFFECTED CELLS FOR PATIENTS. A FEW INTERESTING TIDBITS FROM THE LITERATURE LINKING THAT TO HIV SCIENCING. THAT'S MY SUMMARY SLIDE. HERE'S WHAT I'M DOING NEXT. PARTICULARLY MY TECHNICIAN, OTHER COLLABORATORS HERE AT UNC. THANKS MY FUNDING, THANKS TO THE NIH, OUR R01 JUST GOT FUNDED SO THANKS, NIH. THAT'S ALL. [APPLAUSE] >> I THINK I SHOULD ALLOW A COUPLE QUESTIONS JUST TO BE FAIR TO EDWARD, BUT PLEASE KEEP THEM QUICK. YA- CHI, GO AHEAD. >> SO THAT WAS VERY COOL. BUT 1 EXPRESION WILL BE STRONGLY CORRELATED WITH HIGH C27 AND OTHER THINGS, SO TWO RELATED QUESTIONS. WHEN YOU LOOK AT THE SINGLE CELL RNA SEQ, DO YOU SEE PRDMI EXPRESSION IN THOSE LATENT CELLS? >> WE DIDN'T SEE ANY -- I'M NOT SURE WHAT TO MAKE OF THAT RESULT YET. LIKE I SAID, THIS IS A PRELIMINARY RESULT. IT'S JUST FROM ONE DONOR. >> WELL, ONE SUGGESTION. SO THE IDENTIFICATION OR THE PREDICTION OF TRANSCRIPTION FACTOR BINDING SITES BY ATAC SEQ IS JUST PREDICTION BASED ON WHAT SEQUENCE COMES DOWN IN A CHIP SEQ SPAIRMS OR EXPERIMENT SO IT JUST MEAN S THAT CAN BIND THERE. OTHER TRANSCRIPTION FACTORS CAN ALSO BIND THERE AND HERE IN PARTICULAR -- DO YOU KNOW IF THERE ARE OTHER ZINC FINGER TRANSCRIPTION FACTORS THAT MAY BIND OR BE RELEVANT IF PRD1 ISN'T THERE? THEY'RE PRETTY PERMISSIVE. SO IT MIGHT BE INTERESTING TO LOOK AT THE SINGLE CELL RNA SEQ DATA FOR OTHERS IN FINGER TRANSCRIPTION FACTORS THAT MIGHT BE PROMISCUOUS BINDERS TO THOSE KINDS OF SITES. >> I TOTALLY AGREE. >> ONE MORE QUESTION. BRAD? >> HI. REALLY NICE WORK. JUST A BIT OF A COMMENT. I THINK IT WOULD BE REALLY COOL TO SEE WHAT YOU GET IF YOU ADD SOME IMMUNE EFFECTORS TO THE MIX AS WELL BECAUSE IT MIGHT BE THE CELLS THAT GO ON TO FORMULATE RESERVOIRS ARE THE ONES THAT JUST HAVE MORE TIME TO DO SO BECAUSE THEY SURVIVE THAT INITIAL KILLING EFFECT. ONE SIGNAL I THINK YOU'LL SEE COME OUT MORE IS THE PROLIFERATION SIGNAL THAT YOU'VE ALREADY SEEN BECAUSE CELLS UNDERGOING CELL CYCLE -- I'M NOT SURE ABOUT NK CELLS. SO THAT MIGHT BE -- >> I COMPLETELY AGREE. ADDING IMMUNE EFFECTORS TO THIS SYSTEM -- WE JUST DIDN'T HAVE THE BANDWIDTH QUITE TO DO IT, FIGURING OUT WHAT TO DO WITH ALL THIS DATA, BUT TRYING TO FIGURE OUT -- MY DREAM EXPERIMENT IS WHERE WE CAN HAVE LIKE A MICRO WELL WHERE YOU CAN SEE LATENCY OCCURRING, SEE LATENCY BEING REVERSED, KILLING ALL IN THE SAME WELL AND THEN TRY TO FIGURE OUT WHAT HAPPENED IN THAT WELL, IF WE CAN REVERSE-ENGINEER THAT, IT WILL TELL US WHAT WE'RE AIMING FOR. >> THE REPORTER PRO VIRUS WAS MADE BY BOB SOME YEARS AGO. THAT ONE IS EFFECTED IN GAG, THAT WOULD BE HARDED TO INTERPRET. >> WE'VE GOT OTHER VIRUSES WITH THE SIMILAR SORT OF METHODOLOGY. >> THANK YOU. OBVIOUSLY WE CAN HAVE MORE QUESTIONS DURING THE DISCUSSION PERIOD. NOW I'D LIKE TO INVITE -- OH, I'M SORRY. [APPLAUSE] >> JUST TRYING TO STAY ON TIME. AND NOW I'D LIKE TO INVITE YA-CHI HO WHO REALLY LED THE FIELD BY BEING THE FIRST TO SEQUENCE LARGE NUMBERS OF PRO VIRUSES FROM HIV INFECTED INDIVIDUALS, AND NOW SHE'S BEING EVEN MORE AMBITIOUS AND TRYING TO SORT LATENTLY INFECTED CELLS AND THEN SEQUENCE. >> I'D LIKE TO THAT CAN THE THANK THE ORGANIZERS. IT ALL STARTED BY MY MENTOR BOB, THEN WE HEARD A TALK BSH -- THAT DIRECTED ME INTO THE SINGLE CELL FIELD SO THAT'S AN EXAMPLE OF HOW COMBINATION OF TWO FIELDS MAY GIVE US SOME NEW INSIGHT AND INTEREST FOR YOUNG INVESTIGATORS, I GUESS. SO WE CARE ABOUT THIS SPECIFIC THING CALLED CLONAL EXPANSION BECAUSE MORE THAN 50% -- ARE MAINTAINED THROUGH CLONAL EXPANSION. FROM WORK BY WAGNER AND OTHERS SHOWING THAT A LARGE PORTION OF THESE UNDERGOING CLONAL EXPANSION, THESE CLONAL EXPANDED CELLS CAN INCREASE OVER TIME. SO WHEN THEY LOOKED AT THE HIV INTEGRATION SITE IN THESE CLONAL EXPANDED CELLS THEY FOUND DURING IN VITRO INFECTION, THEY'RE GOING TO TAKE CELL LINES OR THROW A VIRUS IN THERE, YOUR INTEGRATION SITES ARE RELATIVELY SCATTERED THROUGHOUT THE ACTIVE TRANSCRIPTION SITES BUT IT'S ONLY IN CELLS COMING FROM HIV INFECTED INDIVIDUALS SUPPRESSING ART, THEY FELT THE INTEGRATION SITE ARE REALLY ENRICHED VERY SMALL, LIKE 10% OF THE REGION OF THIS WHOLE CHAIN, AND THEY'RE ALL IN THE SAME ORIENTATION, AND THIS SPECIFIC ENRICHMENT CAN ONLY BE SEEN IN VIVO BUT NOT IN VITRO, SO SOMETHING IS GOING ON, THESE CELLS UNDERGO CLONAL EXPANSION. SO THIS SUGGESTS THAT IN VIVO PRESSURE DRIVING -- EXPANSION IS STILL UNCLEAR TO US AND IT'S LIKELY RELATED TO INTEGRATION SITE. SO THIS IS INTERESTING FOR US, EVEN FOR THE CURE AGENDA, BECAUSE -- SO HIV INFECTED CELLS -- SHOULD DIE AFTER ACTIVATION BECAUSE OF -- FOR EXAMPLE, COHEN THAT PROMOTE THE SURVIVAL OF THESE CELLS UNDERGOING REACTIVATION. OR A SECOND THING IS THAT BECAUSE OF THE INTEGRATION SITE, THAT HIV CAN TRY DIFFERENT KINDS OF -- WITH THE GENE IN WHICH HIV SITS. SO WE WANT TO UNDERSTAND THIS, BUT YOU CAN TAKE A PIECE OF MOUSE BRAIN, SINGLE RNA SEQ THEM, WE DON'T KNOW WHAT TO SEQUENCE WITH BECAUSE OF THIS RARITY OF HIV INFECTED CELLS AND A LACK OF MARKERS WHICH CAN PULL THEM OUT TO DO VERY IN DEPTH ANALYSIS. SO TO THIS END, WE DEVELOPED A SORTSEQ TO DIVIDE HIV INFECTED CELLS FROM SUPPRESSED INDIVIDUALS. WE USE HIV RNA AS A SURROGATE THAT CELLS EXPRESSING RNA -- INDUCIBLE HIV SO WE WANT TO PULL THEM OUT, SO IN SHORT, WE TAKE -- FROM INFECTED INDIVIDUALS AND TREAT THEM WITH -- FOR 16 HOURS TO INDUCE -- EXPRESSION SO USING A FISH METHOD SO WE CAN STAIN THIS RNA WITH 96 PROBES TARGETING RNA SO WE CAN SEE IN FLOW FLOW CYTOMETRY -- INFECTED IN VITRO WITH THREE CLINICAL ICE LATS AND -- OTHER CULTURES AND WE CAN DETECT TO 1 MILLION CD4 T-CELLS. SUGGESTING THAT THEY DO HAVE AUTHENTIC AND SUPPLIESABLE HIV ELEMENTS, PUTATIVE INTACT, POTENTIALLY INTACT PRO VIRUSES. SO THE FIRST QUESTION WE WANT TO KNOW IS WHAT DOES HIV DO DURING AN EARLY REACTIVATION, WHAT DOES DO TO CELL AT THE INTEGRATION SITE. SO WE DO HIV HOST CHIMERIC RNA, SUPPLIES TO HIV -- HIV SPLICING INTO HOST AND BECAUSE OF THAT, WE CAPTURE THEM IN RNA SEQ SO THAT WE CAN LOOK INTO WHERE THEY SIT. WE'RE SEQUENCING MANY OF THESE -- ACTUALLY SEQUENCING ARTIFACTS SO -- VERY CLOSELY -- TO MAKE SURE ALL THAT WE REPORTED ARE AUTHENTIC. SO WE FOUND SEVERAL OF THEM -- WHAT WE FOUND IS AN INTERESTING EXAMPLE WE WANT TO SHOW YOU, ISOLATED FROM HIV INFECTED INDIVIDUAL USING SOURCE C. WE CAN QUEENS THAT THROUGH MAJOR SUPPLIES DONOR INSTEAD OF SPLICING TO HIV RNA, CANONICAL ACCEPTOR SITE. THIS MEANS NOW IT'S DRIVEN BY HIV, IT'S NOT CONTROLLED BY THE HOST. THIS HAS BEEN REPORTED IN THE PAPER IN CLONAL EXPANDED CELLS IN VIVO. SO TWO DIFFERENT METHODS, THE SAME INTEGRATION. WHEN WE LOOK AT THESE SINGLE CELLS, IN THIS GENE, CANCER-RELATED GENE, RELATED TO HIV REACTIVATION IN CELLS WHICH DO NOT HAVE HIV INTEGRATED HERE, THEN THE TRANSCRIPTION IS RELATIVELY INTACT, BUT IN THE CELL WHICH HAS HIV INTEGRATED RIGHT HERE, THE TRANSCRIPTION DOWNSTREAM OF HIV IS HIGHLY INCREASED BUT UPSTREAM REMAINS INTACT. SO THIS SUGGESTS THAT IT'S HIV PROMOTER THAT DOMINATES RIGHT HERE OVER THE HOST, SO IT'S HIV DRIVES THIS TRANSCRIPTION. TO KNOW WHAT THIS DOES TO THE CELL, WE NEED TO DO A CELL LINE MODEL TO UNDERSTAND WHAT HAPPENS BECAUSE THE ONLY CELLS WE HAD -- CAPTURE IT BACK FOR OTHER ANALYSES, HIV INCREASES -- TRANSCRIPTION DOWNSTREAM OF THE INTEGRATION SITE BUT NOT UPSTREAM, SUGGESTING -- IT'S A BIOLOGY, IT'S NOT JUST A RANDOM SEQUENCING ART ARTIFACT. SO THIS HIV INTEGRATION INTO THE CANCER RELATED GENE, THAT ONE CREATES A TRUNCATE OF THAT ONE PROTEIN AND THIS HAS BEEN REPORTED TO INCREASE ONCOGENESIS POTENTIAL AT THIS INTEGRATION. SO TO UNDERSTAND WHETHER IT IS REALLY HIV THAT CHANGES THE LANDSCAPE AND POST PROTEIN PRODUCTION AT THIS INTEGRATION SITE, WE CONSTRUCTED CRISPR-BASED HIV LTR-SPECIFIC ACTIVATION OR REPRESSION. WE CAN INCREASE THE TRANSLATION OF THIS TRUNCATED PROTEIN AND WE CAN USE CRISPR TO KNOCK IT DOWN, THEN WE SEE THIS AVERAGE TRANSCRIPTION IS GONE. SO IT IS HIV THAT DRIVES ABERRANT PROTEIN PRODUCTION. SECOND, THIS CAN BE POTENTIALLY TARGETED BY CRISPR BASED OR OTHER SMALL MOLECULAR BASED METHODS TARGETING HIV LTR. THE SECOND QUESTION WE WANT TO KNOW IS WHAT HAPPENS TO THE CELL WHEN HIV IS REACTIVATED. THESE CELLS -- ONLY FOR HIV INFECTED INDIVIDUALS SO THEY'RE JUST THIS BIG CROWD OF LIKE SCATTERED DISTRIBUTION IN THE CD4 POSITIVE CELLS IN THEM. SO WE FOUND SEVERAL THINGS. FIRST SORTSEQ POSITIVE AND NEGATIVE CELLS, THEY SHOW EARLY ACT VAIGHTS ACTIVATION MARKERS, CAPTURING THESE EARLY ACTIVATED CD4 T-CELLS. SECOND, WE FOUND THESE CELLS ARE MORE POLARIZED IN ONE PHENOTYPE. THIS IS NOT SURPRISING BECAUSE TH1 IS ONE OF THE MOST DOMINANT POPULATION IN THE BLOOD BUT IT SEEMS LIKE THOSE CELLS WITH HIV, SINCE THEY ARE PROBABLY MORE PRIME FOR THIS TH1 RESPONSE BECAUSE THEY WERE LIKELY -- LATENCIES. WE FOUND THERE ARE TWO GENES RELATED TO T-CELL SURVIVAL THAT ARE ENRICHED, IKBKB AND IL2. INTERESTINGLY -- CAN INTRODUCE THE -- SHOWING INCREASED IKBKB OR IL2 MIGHT BE SOMEWHAT -- BY HIV IN OTHER WAYS. SO BY LOOKING AT THIS TRANSCRIPTOME LANDSCAPE THAT WE CAN CAPTURE POSITIVE VERSUS NEGATIVE CELLS, SO WE CAPTURE THAT HERE, RNA BINDING, RNA PROCESSING AND VIRAL GENE TRANSCRIPTION, GENES THAT ARE RELATED TO THIS, FOR EXAMPLE, IN RNA PROCESSING, WE CAPTURE THIS GENE CALLED UPF2 WHICH IS ONE OF THE PLAYERS, SO -- WHEN HOST MECHANISM TO ORGANIZE THIS WEIRD -- WITHPREMATURE -- 3.0 -- GENES IF SOMETHING IS MADE WRONG, UPF2 OR UPF1 IS A GENE TO CAPTURE THEM, A HOST DEFENSE SYSTEM. ACTUALLY HTLV-1 CAN BE TARGETED AND HTLV-1 TAX IS COUNTERACTING THEM. THERE ARE REPORTS SAYING THESE MAY POTENTIALLY RESTRICT -- SO WE'RE INTERESTED IN HOW WE CAN USE THIS TO RESTRICT HIV UPON REACTIVATION. AND THE SECOND REGARDING VIRAL GENE TRANSCRIPTION, SO HOW CAN WE REACH A FULL HIV REACTIVATION OR WHAT DOES HIV NEED TO MAKE A FULL REACTIVATION INSTEAD OF MANY TRANSCRIPTIONAL BLOCKS SHOWN BEFORE. SO WE FOUND THESE TWO GENES, IMPDH1 AND JAK1. IT'S THE ONLY THING IT CAN USE TO MAKE -- WE FOUND THAT ENRICHMENT OF THESE TWO GENES -- THERE ARE TWO ONGOING CLINICAL TRIALS USING INDIVIDUAL INHIBITORS LIKE IMPDH1 OR JAK1 INHIBITOR TO TEST WHETHER SUPPRESSING THESE CAN CHANGE THE HIV LATENT RESERVOIR OR THE LANDSCAPE. WE'RE LOOKING FORWARD TO THOSE RESULTS. SO INDIVIDUALLY IN OUR OWN HANDS, WE TESTED THEM ON PATIENTS AND WE -- WE SHOW IT CAN SUPPRESS HIV TRANSCRIPTION. WHEN WE PUT THESE IN THE CELL LINE MODEL THAT WE HAVE, WE SEE THAT HIV INDUCES ABERRANT HOSTING TRANSCRIPTION. TO SUPPRESS THE HIV DRIVEN -- SO WE'RE SHOWING THAT WE SEE THIS PHENOMENON THAT CAN BE TARGETED BY A COMPOUND FOR SOMETHING SPECIFIC TO HIV. SO THE FIRST I'M GOING TO SHOW YOU TODAY IS THAT WE WANT TO KNOW WHAT HAPPENS TO HIV AND WHAT HAPPENS TO THE CELL, BUT PEOPLE COMMONLY ASK US THAT WE NEED TO ACTIVATE THESE CELLS, AND WE UNDERSTAND THAT WE WANT TO KNOW WHAT HAPPENS WHEN HIV IS REACTIVATED BUT WE ALSO WANT TO KNOW WHAT HAPPENS WHEN THEY'RE NOT ACTIVATED. IT'S BEEN VERY HARD FOR US. BUT LET'S JUST TRY. SO THIS IS PRELIMINARY RESULTS WE HAVE. WE GET HIV INFECTED INDIVIDUALS DURING VIREMIA AND ONE YEAR AFTER ART. THEY HAVE A VIRAL LOAD TESTED SIX MONTHS BEFORE THAT, SO THE SECOND TIME POINT THAT WE GOT, THEY'RE REALLY UNDER SUPPRESSIVE -- FOR SIX MONTHS. WE WORK WITH PETER -- TO GET THIS FORWARD. SO THIS IS ALL DONE BY MY STUDENT JACK WHO NOT ONLY DID THE BENCH WORK BUT ALSO DID ALL THE ANALYSIS. SO WE CAN LOOK AT ALL OF THIS TRANSCRIPTOME LANDSCAPE, BY PULLING TOGETHER THREE HIV INFECTED AND UNINFECTED INDIVIDUALS, THIS IS THE LANDSCAPE THAT WE SEE. IF WE COLOR CODE THEM AS HIV INFECTED AND NON-INFECTED, THE BLUE ONES ARE THE NON-INFECTED ONES, YOU CAN SEE THEY'RE -- AWAY FROM THOSE HIV INFECTED INDIVIDUALS TELLING US THAT HIV -- DOES SOMETHING DIFFERENTLY TO CD4 T-CELLS, BECAUSE WE HAVE THE SURFACE PROTEIN SO WE CAN TELL THEY LOOK DIFFERENTLY, CD45RA AND RO, THEY LOOK VERY DIFFERENT, SO RNA SEQ CULTURING, WHEN WE OVERLAY THE PROTEIN MARKERS ON THEM, YOU CAN SEE THEY ARE CLUSTERING IN A VERY DISTINCT WAY SO WE CAN DISTINGUISH THEM. SO USING TRANSCRIPTOME ANALYSIS, WE CAN -- IN THESE TWO CLUSTERS,& THEY'RE ENRICHED IN VIE REMIBG INDIVIDUALS IN VI REMIC STATE, ISG15, IFITM3 WHICH IS ACTUALLY REPORTED BY ALEX. SO WE KNOW THE T-CELL RECEPTOR SEQUENCING, WE CAN IDENTIFY IN THIS MAP WHERE ARE THE CLONES, SO WE CAN SEE WITH THIS CLONE TYPE IN THIS INDIVIDUAL, WE CAN SEE WHERE THE CLONE IS. WE CAN ALSO MAP WHERE HIV IS BY MAPPING THEIR HIV RNA SO WE CAN CAPTURE THESE. NOW BECAUSE WE KNOW THERE'S T-CELL SEQUENCING WE KNOW THE HIV RNA, WE CAN CAPTURE THESE CLONAL CHANGES LIKE OVER TIME DURING HIV INFECTION. WHAT HAPPENS TO CD4 T-CELLS. IN VERY FEW CLONES HAVING HIV, WHAT HAPPENED TO THEM. BECAUSE WE KNOW THEIR CORRESPONDING TRANSCRIPTION LANDSCAPE, SURFACE MARKERS, WE'RE IN THE PROCESS OF UNDERSTANDING HOW THIS WOULD TELL US REGARDING HOW HIV RESPONDS AFTER ART ADMINISTRATION DURING THIS EARLY STAGE OF THESE -- LATENCY. SO IN SUMMARY, I'VE SHOWN OUR APPROACH INTO HIV BIOLOGY BY USING HIV SORTSEQ. THIS, WE CAPTURE VERY, VERY FEW CELLS, THIS IS THE BIOLOGY OF -- I'M VERY SORRY I CANNOT BRING MORE TO YOU, ANALYZE LIKE 10 PATIENTS, BUT THIS GIVES US THE CAPABILITY TO LOOK AT TOTAL RNA, NOT JUST -- CAPTURE, SO THEN I CAN SHOW YOU THE HIV HOST CHIMERA. BUT WE WANT TO LOOK BROAD OVERVIEW INTO WHAT HAPPENED TO THE HOST CD4 T-CELLS, WE'RE TRYING TO UNDERSTAND WHAT DOES HIV -- CELLS DO IN THE CONTEXT OF CLONAL EXPANSION, IN THE CONTEXT OF IMMUNE EXHAUSTION THAT HIV INFECTION MAY BRING ABOUT. SO THIS IS PRELIMINARY, WE STILL NEED A LOT OF HELP, BUT WE LOOK FORWARD TO PEOPLE WHO CAN HELP US TO ANALYZE HOW WE CAN MODEL OR UNDERSTAND THE DYNAMICS OF THESE SINGLE CELL CHANGES AND THEN WE ARE INTERESTED IN WORKING ON TISSUES SO THE VIABILITY -- WE NEED REALLY JUST VIABLE CELLS TO FLOW INTO BOTH OF THESE ASSAYS, THE FROZEN CELLS -- FOR THESE THINGS. SO I'D LIKE TO THANK MY LAB WHO DID MOST OF THIS WORK AND ESPECIALLY -- SO IT'S GINNY WHO JIMMY -- JACK, KRISTEN DEVELOPED THE HIV SPECIFIC GUIDE RNAs. I'D LIKE TO THANK OUR COLLABORATORS AND I'D LIKE TO THANK THE COLLABORATORY THAT WE HAVE MANY ONGOING STUDIES WITH THEM, SUPPORTING MY R01 AND I'D LIKE TO THANK ANNE DUERR AND THE DIFFERENT FUNDING AGENCIES. THANK YOU. [APPLAUSE] >> WE HAVE TIME FOR SOME QUESTIONS. THANK YOU FOR BEING ON TIME, YA-CHI. ANY QUESTIONINGS? QUESTION? WELL, I HAVE A QUESTION. SO CAN YOU JUST EXPLAIN BETTER THE SITE SEQ? THE PART I MISSED WAS HOW YOU'RE SORTING THE CELLS THERE. >> SITE SEQ IS PUBLISHED, SO BASICALLY YOU TAKE CELLS, SORT THEM, JUST TAKE CELLS, WE ISOLATE CD4 T-CELLS AND YOU STAIN THEM WITH -- SO IT'S DNA BARCODED ANTIBODIES AND PUT THEM INTO DROPLET AND YOU GET RNA SEQ OUT OF THIS. SO WE'VE BEEN ASKING ABOUT THIS QUESTION THAT -- WHY DO YOU WORK ON CD4s, WHY DO YOU WORK ON THESE RARE POPULATIONS? WHY DON'T YOU WORK ON CD8s AND NK CELLS? IT'S A HARD QUESTION FOR ME BECAUSE FOR ME, A PERSON WHO'S BEEN INTERESTED IN LATENT RESERVOIR, THIS IS THE HARDEST POPULATION TO WORK ON, YOU CAN SELECT SPECIFIC ONES AND TRY TO RNA SEQ THEM OUT BUT THIS HAS BEEN VERY HARD FOR US, WE JUST WANT TO TRY, LET'S TRY TO SHOOT FOR THE HARDEST THING, FOR EXAMPLE, THE BLACK HOLES IN IT. SO HIV INFECTED CELLS ARE BLACK HOLES FOR ME BECAUSE THEY'RE SO HARD TO FIND, BUT WE'RE TRYING TO WORK ON THIS DIRECTION. IT'S STILL VERY HARD. >> I DO HAVE A FOLLOW-UP QUESTION. HOW DO YOU KNOW -- BECAUSE YOU SAID YOU DON'T HAVE TO REACTIVATE. I DO UNDERSTAND HOW YOU'RE DOING IT BUT HOW DO YOU GET THE NOT REACTIVATED CELL? >> THEY'RE ACTUALLY NOT QUIET, SO THEY'RE ACTUALLY ACTIVATED IN VIVO, WHICH MEANS I DO NOT ACTIVATE THEM IN VITRO, JUST TAKE THEM -- INTO THE MACHINE. SO I DID NOT ADD -- SO THEY ARE EXPRESSING RNA IN VIVO SO THERE COULD BE SOME THAT BRANDON WAS SHOWING, THEY ARE REACTIVATED, MAYBE NOT, BUT STILL SHOWING SOMETHING. SO AT THIS POINT WE DON'T KNOW HOW LATENT ONES ARE LATENT. WE'RE TRYING TO UNDERSTAND WHY THE REACTIVATING ONES ARE REACTIVATED BECAUSE WE WANT TO TARGET THOSE. >> CAN I JUST ASK ONE MORE FOLLOW-UP? SO YOU WERE LOOKING AT THE TRANSCRIPTIONALLY ACTIVE CELLS? >> IT'S FOR THOSE THAT WE CAPTURE HIV RNA, WE MAP OUT HIV RNA. SO FOR THOSE THAT HAVE HIV BUT ARE NOT EXPRESSING RNA WE DON'T KNOW SO WE'RE TRYING TO SEE IF SOMEONE CAN HELP US TO SEE IF YOU HAVE TWO TIME POINTS YOU CAN TRACE BACK, THOSE ARE ACTUALLY THE SAME CELLS AND YOU CAN, LIKE, DO THEM BACK TO WHICH CELL IT IS. >> THNK YOU. SO GO AHEAD, BOB. >> SO YA-CHI, WHEN YOU DEFINE THE CLONES BASED ON TCR SEQUENCE, RIGHT, CAN YOU TELL WHAT FRACTION OF A CLONE THAT'S SPECIFIC FOR A GIVEN ANTIGEN ACTUALLY HAS ACQUIRED HIV INFECTION? BECAUSE IT COULD BE THAT IF THE INFECTION IS EARLY ON IN SORT OF THE LIFETIME OF THE CLONE, ALL OF THE MEMBERS OF THAT CLONE WILL HAVE PRO VIRUS, BUT IT ALSO COULD BE ONLY A SMALL FRACTION IF THE INFECTION OCCUR AFTER THE CLONAL EXPANSION. >> SO THIS IS HARD FOR US, THAT'S WHY WE SAY WE NEED HELP BECAUSE IF YOU BASE ALL THE TRANSCRIPTIONAL LANDSCAPE AND ALL THE SITE SEQ DATA WE HAVE, WE STILL MAY NOT TRACE BACK THE RIGHT ONE BECAUSE EVEN -- IT DOES NOT TELL THIS IS THE SAME CELL. SO WE THOUGHT T-CELL RECEPTOR SEQUENCING -- ACTUALLY YOU'RE NOT. -- 10 CELLS, THEY'RE HAVING THE SAME SEQUENCE. ONE GOT HIV INFECTED. ONE YEAR LATER, IT COULD BE THIS ONE IS GONE OR 10 OTHERS SO EVEN WE TRACE ON THEIR PROTEIN MARKERS IN RNA SEQ, WE'RE STILL STRUGGLING HOW WE CAN TRACE BACK EVEN IF WE KNOW -- >> [INAUDIBLE] >> SEVERAL THOUSAND. SIX SAMPLES FROM THREE INDIVIDUALS, WE FOUND 73. 7,000 TIMES SIX. VERY EXPENSIVE. >> ONE MORE HERE. >> I'M JUST WONDERING WHAT LEVELS OF RNA YOU FOUND IN EACH OF THE CELLS. LIKE WHEN YOU LOOKED AND YOU MAPPED HIV RNA READS FROM EACH OF THE CELLS THAT WAS NOT ACTIVATED BUT MAKING RNA, WHAT FRACTION OF THE READS WERE COMING FROM HIV? >> VERY, VERY FEW. IT DEPENDS ON THE CELL, BUT SOME HAVE VERY FEW, SOME HAS A LOT. BECAUSE TARGETING 50,000 READS PER CELL, SO IT'S NOT DEEP. IT'S NOT AS DEEP AS THE SORTSEQ -- WE HANDLE THEM WITH DEPTH SO WE CAN SEE A WHOLE LOT OF SEQUENCE THAT YOU WANT. BUT THESE ARE LIKE 50,000 READS PER CELL, NO, SO WE'RE WORKING ON TARGETED AMPLIFICATION TO CAPTURE THAT ONE. OTHERWISE IT DOESN'T GET THE DEPTH THAT WE WANT. >> SO SINCE YOU HAVE THE WHOLE TRANSCRIPT SEQUENCE, NOT JUST THREE PRIME, HAVE YOU LOOKED FOR ANY SOMATIC MUTATIONS IN THESE CELLS THAT MIGHT BE ASSOCIATED WITH SURVIVAL? >> WE'RE NOT THAT FAR YET BECAUSE -- THE BREADTH, THEY HAVE SOME LIMITATIONS, BUT WE CAN TRY. >> ALL RIGHT. THAT ENDS THIS SESSION. I'M SURE THERE WILL BE LOTS OF DISCUSSION OUTSIDE. THANKS TO THE SPEAKERS. I JUST WANT TO START BY THANKING OUR ORGANIZERS HERE. MIKE AND DIANA. FOR BRINGING US ALL TOGETHER TO THIS MEETING TO THINK ABOUT THIS INCREDIBLE PROBLEM, SO THANK YOU VERY MUCH. [APPLAUSE] SO WHAT I'VE BEEN STAS BEINGED TASKED WIT H IS STARTING THIS DISCUSSION, AND WHAT'S BEEN EXCITING ABOUT THIS MEETING, REALLY, IS BRINGING TOGETHER GROUPS OF PEOPLE WHO HAVE BEEN WORKING ON THIS PROBLEM FOR A VERY LONG TIME, AND IT'S A TREMENDOUS PROBLEM FOR THOSE OF YOU THAT ARE NEW TO IT. PERSONALLY I'M NEW TO IT MYSELF, HAVING BECOME INVOLVED IN THIS REALLY JUST A COUPLE OF YEARS AGO. AND IT'S FANTASTIC. THERE'S SO MUCH THAT'S NOT CLEAR, YOU KNOW, THERE'S THE WHAT, WHERE, AND WHY, ALL OF IT IS REALLY A BIT NEBULOUS, AND JUST THE IDEA OF BRINGING THESE NEW TOOLS TO BEAR ON THIS GLOBALLY IMPORTANT PROBLEM IS JUST VERY EXCITING. SO AGAIN, MIKE AND DIANA, IT'S FANTASTIC THAT YOU'VE DONE THIS. SO WITH THAT, I JUST WANT TO TURN IT OVER TO THE PANEL FIRST AND THEN TO ALL OF YOU TO THINK ABOUT WHAT WE'VE LEARNED HERE, WHAT WE'VE LEARNED ELSEWHERE CAN BE BROUGHT TO BEAR TO HELP SOLVE THIS PROBLEM, THIS LATENCY PROBLEM. SO MAYBE WE'LL JUST GET STARTED. >> I HAD A COUPLE OF THOUGHTS LAST NIGHT ON WHAT WE WERE GOING TO TALK ABOUT AND THERE ARE A COUPLE OF POINTS I'D LIKE TO BRING UP FOR DISCUSSION. LOVE THE FOCUS ON FINDING AND CHARACTERIZING THE REBOUND COMPETENT RESERVOIR. I THINK THERE'S ALSO POTENTIALLY OPPORTUNITY TO KIND OF CHARACTERIZE THE NON-REPLICATION VIRAL RESERVOIR. WE NEED TO THINK ABOUT WHAT THAT CAN INVOLVE. SO VEER LOGICALLY, THE DEFECTIVE VIRUS IS UNIMPORTANT BUT THEY COULD BE IMMUNOLOGICALLY QUITE IMPORTANT, FOR EXAMPLE. THEY COULD BE SHAPING THE IMMUNE REPERTOIRE, THEY COULD BE CAUSING INFLAMMATION IN PEOPLE WHO ARE SICK, SO IT COULD BE THAT WE CLEAR OUT THE REPLICATION RESERVOIR AND THESE PEOPLE ARE STILL SICK. SO THAT'S SOMETHING TO THINK ABOUT. SO LEARNING MORE ABOUT HOW DEFECTIVE PRO VIRUSES INTERACT WITH THE IMMUNE SYSTEM I THINK SOMETHING WE COULD POTENTIALLY LEARN. SOMETHING THAT HASN'T REALLY BEEN TALKED ABOUT HERE A LOT AS WELL IS LATENCY REVERSAL. I KNOW THAT THE OPINION IS DIVIDED ON WHETHER WE NEED LATENCY REVERSAL. SOME PEOPLE THINK WE JUST NEED A FUNCTIONAL CURE, BUT I KIND OF WORRY ABOUT IF WE GO KIND OF CHARACTERIZED IN VIVO RESERVOIR WITH SINGLE FLOW METHODS, WE FIND OUT WHERE IT IS, WHAT IT LOOKS LIKE AND WE FIND OUT IT'S EVERYWHERE, WHAT DO WE DO THERN. THEN. THEY HAVE A PRO VIRUS IN THEM IN WHICH CASE YOU'RE GOING TO NEED ANTIGEN -- REVERSAL IN VIVO COULD BE A KEY INSIGHT THAT COULD COME FROM THIS KIND OF FIELD. SO AN EXPERIMENT I THINK WE NEED TO DO IS, WE NEED TO IDENTIFY REACTIVATING CELLS IN VIVO AFTER CLINICAL LR -- ADMINISTRATION FOR EXAMPLE, WHY DO THE OTHER CELLS NOT REACTIVATE, CAN WE OPT MATE THE ENVIRONMENT, WHAT'S IT SURROUNDED BY, SO WE NEED TO KNOW WHAT THE GOALPOSTS ARE, I THINK, IN TERMS OF WHAT WE'RE AIMING FOR TO ACHIEVE EFFECTIVE LATENCY REVERSAL. SO THAT'S A COUPLE OF THINGS I COULD THINK. >> SO I'LL COMMENT A BIT ABOUT SOME OF THE CHALLENGES I'VE BEEN THINKING ABOUT QUITE A BIT IN TERMS OF CHARACTERIZING THE IN VIVO HIV LATENT RESERVOIR IN PEOPLE AND WHAT ARE SOME OF THE WAYS WE'RE THINKING ABOUT TO OVERCOME THOSE CHALLENGES. SO ONE OF THE FIRST CHALLENGES I THOUGHT ABOUT A WHILE AGO IS THAT AS WE ALL KNOW, THE PRO VIRUS IS OFTEN TRANSLATIONALLY AND TRANSCRIPTIONALLY SILENT, SO OFTEN TIMES THOSE LATENT CELLS, WE HAVE TO STIMULATE THEM. AS I TALKED ABOUT YESTERDAY, ONE OF THE APPROACHES WE'RE TRYING TO GET AROUND FACT THAT WHEN YOU STIMULATE, YOU GREATLY CHANGE THE PHENOTYPE OF THE CELLS, IS BASICALLY TO USE BIOINFORMATICS-BASED APPROACHES TO TRY TO PREDICT THE ORIGINAL STATE BEFORE THE STIMULATION. WE'RE ALSO TRYING TO DEVELOP APPROACHES WHERE WE CAN TRY TO NOT STIMULATE AT ALL, SO THIS IS A COLLABORATION THAT I'M DOING WITH JOSH VAZQUEZ AT UCSF WHERE WE ARE APPLYING A SYSTEM CALLED PLAYER TO BE COMPATIBLE TO DETECT HIV RNA. SO IT'S A SYSTEM DEVELOPED BY GARRY NOLAN AND COLLEAGUES AND ESSENTIALLY IT CAN -- WE'RE ADAPTING TO DETECT SPECIFICALLY HIV RNA. AND THE WAY PLAYER WORKS IS IT USES A ROLLING CIRCLE AMPLIFICATION METHOD THAT IS SIMILAR TO THE SNAIL METHOD THAT XIAO MENTIONED YESTERDAY, SO THAT'S DIFFERENT FROM THE BRANCH TREE AMPLIFICATION METHOD THAT'S USED IN FLOW FISH AS WELL AS THE RNA SCOPE. BUT THE GENERAL IDEA IS THAT YOU GREATLY AMPLIFY YOUR HIV RNA SIGNAL AND THEN YOU COME IN WITH COMPLEMENTARY PROBES AND THOSE PROBES WILL BE ATTACHED TO SPECIFIC MASS ELEMENTS WHICH CAN BE -- YOU GET 40 PARAMETER SIGH TO HAVE DATA TOGETHER WITH QUANTITATIVE INFORMATION ABOUT HOW MUCH OF THE HIV RNA WAS THERE. SO WE'VE GOTTEN THE SYSTEM TO WORK NOW WHERE IN VITRO INFECTED PDMCs, WE CAN DO 40 PARAMETER CYTOFF PHENOTYPING AND GET INFO ON WHICH CELLS HAVE RNA AND HOW MUCH OF THE RNA IS THERE. IT'S WORKING VERY WELL, WHERE WE WANT TO MOVE THAT NOW IS INTO PATIENT SAMPLES WITH THE IDEA THAT MAYBE AN UNSTIMULATED CELLS, YOU MAY HAVE QUITE A BIT OF TRANSCRIPTIONAL INITIATION. THEY'VE SHOWED -- CAN ART PLAYER PROBES DETECT SOMETHING, THAT'S SOMETHING WE'RE VERY INTERESTED IN DOING. WE WOULD ALSO EVENTUALLY LOVE TO ADAPT THE PLAYER SYSTEM SO WE CAN DETECT HIV DNA AS WELL. OF COURSE WE NEED TO INCREASE THE SENSITIVITY FOR THAT, YOU ALSO DON'T NEED TO STIMULATE THE CELLS, YOU CAN DIRECTLY PHENOTYPE THEM. ANOTHER CHALLENGE, I THINK, IS THAT THESE CELLS, OF COURSE, ARE RARE. IT'S LESS OF AN ISSUE IN CYTOFF WHERE WE CAN TAKE IN MILLIONS AND MILLIONS OF CELLS AND ANALYZE THEM BUT FOR RNA SEQ ANALYSES, THE THRESHOLD IS LOW. WHEN YOU HAVE SUCH RARE EVENTS, THE SIGNAL TO NOISE RATIO GOES WAY DOWN AND SO YOU GET A LOT OF FALSE POSITIVES. SO WE AND OTHERS HAVE OVERCOME THIS ISSUE BY USING ANTIBODIES, MULTIPLE ANTIBODIES DIRECTED AGAINST MULTIPLE HIV PROTEINS SO THAT WE CAN BASE CLIN BASICALLY INCREASE THE SPECIFICITY OF WHAT WE'RE TESTING. IN THE DATA I SHOWED THREE TO FOUR ANTIBODIES DIRECTED AGAINST HIV GAG AS WELL AS -- THE POSITIVE FOFER ALL OF THESE THINGS, IT'S LIKELY TO BE A REAL EVENT. SO THE ISSUE ALSO IS THAT WHEN IT'S SO RARE, YOU HAVE TO ENRICH FOR THESE CELLS, AND THEY'VE DEVELOPED A NICE ASSAY WHERE YOU CAN ACTUALLY ENRICH FOR REAK SERRATED CELLS THROUGH ENRICHMENT AND THEN SUBSEQUENT -- I THINK THESE ARE ALL TECH NEEGS THAT WE NEED TO APLAY IN ORDER TO BE ABLE TO ANALYZE THESE CELLS BY SINGLE CELL RNA SEQ. AND ALSO ONE FINAL CHALLENGE THAT I'LL JUST COMMENT ON IS IN THE CASE OF CITING THE IN VIVO LATENT CELLS IN HUMANS, A LOT OF THOSE CELLS ARE PROBABLY IN TISSUES AND IN HUMANS, WE CAN ONLY TYPICALLY SAMPLE MAYBE FNAs, GUT SPECIMENS, GENERAL SPECIMENS, BUT THAT MAY NOT BE WHERE WE CAN CAPTURE A LOT OF THE LATENT CELLS. SO I THINK THAT'S WHERE THE LAST -- THAT SARAH AND -- ARE WRITING, THAT'S REALLY KEY WHERE YOU CAN ACTUALLY LOOK IN NATISH EU. ANY SCH TISSUE. >> SO I GUESS AS A REPRESENTATIVE OF THE NON-HIV NATIVE CROWD, I GUESS I CAN SPEAK MOSTLY TO JUST WHAT MY IMPRESSIONS ARE IN TERMS OF THE WAY IN WHICH TECHNOLOGY IS INCREASINGLY BROUGHT TO BEAR ON A LOT OF THE QUESTIONS OF WHAT A LOT OF PEOPLE ARE INTERESTED IN HERE. I THINK WHAT STRUCK ME ABOUT KIND OF ALL THE DATA THAT'S BEEN SHOWN IS THAT REALLY AS KIND OF THE QUANTITY OF THIS DATA IS KIND OF INCREASING, WE'RE KIND OF AT THE.WHERE THERE SEEMS TO BE KIND OF A CHANGE IN SORT OF THE QUALITATIVE NATURE ABOUT THE INFORMATION THAT YOU CAN ACTUALLY GET. AS WE GET MORE AND MORE INFORMATION ABOUT BOTH SORT OF TRANSCRIPTIONAL STATES AS WELL AS KIND OF THE SEQUENCE, SPECIFICALLY DATA ABOUT THE PRO VIRUSES AND WHATNOT, I THINK IT BECOMES INCREASINGLY PLAUSIBLE TO TRY TO CORRELATE INFORMATION ON SORT OF THE LINEAGE TREES OF LATENT CELLS AND TRANSCRIPTIONAL STATES THAT ACTUALLY CAUSED DIFFERENT CLAIFS OF THESE LINEAGE TREES PROLIFERATE MORE, PROLIFERATE LESS AND I THINK THAT KIND OF PROVIDES NEW POSSIBILITIES TO REALLY KIND OF SPECIFICALLY TEASE OUT INFORMATION ABOUT THE WAY IN WHICH TRANSCRIPTIONAL STATES MIGHT CONTROL THE -- DYNAMICS OF T-CELLS THAT HARBOR THE PRO VIRUS. ON THE OTHER HAND, I THINK FROM THE SPATIAL STANDPOINT, I THINK THERE'S ENORMOUS OPPORTUNITY TO TRY TO BETTER DEFINE THE ROLE OF SORT OF THE HIGHER ORDER DEFINITION OF CELL STATES, SO NOT ONLY WHAT THE CELL SEES WITHIN ITSELF, BUT ALSO KIND OF THE WAY IN WHICH CELLS NEARBY POTENTIALLY DEFINE KIND OF THE OUTCOME OF WHETHER OR NOT A T-CELL HARBORING THE PRO VIRUS STAYS LAY TEBT OR LATENT OR BECOMES ACTIVATED. I THINK BEING ABLE TO INCLUDE ALL OF THAT DATA COLLECTIVELY COULD PLAWSLY EXPAND THE RANGE OF HYPOTHESES THAT THIS COMMUNITY CAN TEST. AND BECAUSE OF THAT, I THINK SORT OF -- THERE'S A VERY, VERY STRONG ROLE POTENTIALLY IN THAT SPATIAL TRANSCRIPTOMICS ESPECIALLY CAN PLAY IN FURTHERERRING THESE TYPES OF INVESTIGATIONS. >> SO ALSO FROM THIS TWO-DAY MEETING, MY GENERAL IMPRESSION IS REGARDING TECHNOLOGY DEVELOPMENT, I FOUND THERE SEEMS LIKE TWO MORE IMPORTANT ISSUES. ONE IS THE SENSITIVITY. THE OTHER IS MULTIMODAL DETECTION NOT ONLY LIMITED TO RNA BUT FOR DNA AND RNA PROTEIN TO BE DETECTED IN THE SAME SAMPLE. SO REGARDING THE SENSITIVITY ISSUE, MOST OF THE PEOPLE HAVE MENTIONED THAT -- MANY PEOPLE HAVE MENTIONED THAT SINGLE CELL RNA SEQUENCING SOMETIMES DO NOT HAVE SUFFICIENT EFFICIENCY TO BE ABLE TO DETECT THIS LOW -- NUMBERS OF RNA VIRUS OR DNA INTEGRATION PIECE SO REGARDING THAT, I THINK IT'S ALSO -- IT'S A GENERAL NEED FOR BIOLOGICAL TOPICS SO IN THE FUTURE, I THINK I WILL TRY TO MAKE THE KNOW SURE OVERALL MORE SENSITIVE. IF YOU ARE THINKING ABOUT DETECTING LOW COPY NUMBER LIKE -- 1 OR 2, SO I THINK PROBABLY THE CURRENT MEASURE IS NOT GOOD ENOUGH. SO THIS IS ABOUT THE SENSITIVITY. ABOUT THE MULTIMODAL DETECTION, I THINK MOST OF THE HYBRIDIZATION OR IN SITU SEQUENCING, THEY ARE PERFECTLY FINE TO DETECT BOTH DNA AND RNA, THE OTHER PROBLEM IS HOW ABOUT PROTEIN, SO I THINK IN THE FUTURE, I WILL ALSO TRY TO SEE WHAT'S BEST WAY TO INTEGRATE PROTEIN DETECTION AS WELL. AND THE ONE ISSUE I THINK HAS BEEN DISCUSSED YESTERDAY IS THAT IT SEEMS THE ANTIBODIES NOT IDEAL ENOUGH NOW SO I DON'T KNOW IF PEOPLE HAVE EVER TRIED BEFORE OR NOT, SO IT'S POSSIBLE TO ALSO DEVELOP LIKE RNA BASED -- APPROACH BECAUSE IF YOU'RE THINKING ABOUT THOSE PROTEINS LIKE -- THEY'RE SUPPOSED TO WORK ON -- ACID SO PROBABLY WE CAN TRY TO DO SOME ACT MER DEVELOPMENT OR OR A COMBINATION OF ANTIBODY AND ACTIMER. SO I THINK THAT WILL BE ALSO USEFUL BECAUSE FOR ACTIMER FOR RNAs, IT'S ALSO GOOD FOR RNA DETECTION AS WELL. SO I THINK THAT'S ANOTHER DIRECTION WE CAN TRY TO WORK ON TO SEE HOW WE CAN DETECT PROTEIN RNA SIMULTANEOUSLY. SO THAT'S MY THOUGHTS. >> THANK YOU. I'D LIKE TO OPEN IT UP NOW TO EVERYONE, TO PLEASE COMMENT. PARTICULARLY WE'RE INTERESTED IN THINKING ABOUT HOW THESE NEW TECHNOLOGIES, THE SINGLE CELL TECHNOLOGIES, THE IMAGING TECHNOLOGIES, ALL OF THEM COULD CONTRIBUTE TO FURTHERING THIS PROBLEM. >> SO I DON'T HAVE ANYTHING TO SAY ABOUT THE TECHNOLOGIES BECAUSE I DON'T KNOW ANYTHING, ALTHOUGH I THINK THOSE ARE AWESOME IDEAS, I ESPECIALLY LIKE THAT APTIMER. I JUST WANT TO BACK UP SOMETHING THAT ED BROWNE SAID AND THAT I THINK WE SHOULDN'T ALWAYS IGNORE THE DEFECTIVE SEQUENCES FOR LOTS OF REASONS BECAUSE THEY ARE THE MAJORITY SEQUENCE. YOU KNOW, AND WE'RE ALREADY AT RARE CELL DETECTION. AND CERTAINLY THERE'S SOME THEORY THAT -- I THINK IT'S DANIEL REEVES, A MATH MODELER, HAS BEEN PROPOSING THAT MAYBE THEY'RE ALL CLONES AFTER A WHILE ON ART. IF THAT'S TRUE AND THERE IS NO VIRAL REPLICATION FOR THE MOST PART OR MAYBE AT ALL, THEN DEFECTIVES WOULD BE TEACHING YOU ABOUT SELECTION PRESSURES. SO I JUST THINK CERTAINLY WE WANT TO GET RID OF THE INTACT, NO QUESTION THAT'S THE MOST IMPORTANT. I DON'T MEAN TO GO THAT FAR. BUT I THINK IT IS ONE WAY TO GET INFORMATION AND INCREASE YOUR N. SO THAT WAS JUST A COMMENT. >> I DON'T SEE ANYBODY ELSE RAISING THEIR HAND SO I'M GOING TO PICK ON PEOPLE ACTUALLY. I'M JUST GOING TO PICK ON SCOTT FOR A MINUTE. BECAUSE YOU'RE REPRESENTING SOME OF THE IMAGING PEOPLE. JUST WONDERING WHAT YOUR THOUGHTS MAY BE. >> I THINK ONE OF THE CHALLENGES WITH MOST IMAGERS AS THEY WORK HARD TO MAKE HIGH RESOLUTION PICTURES OF THINGS AND NOT THINK ABOUT DETECTING COMBINATION OF THINGS. FOR EXAMPLE, IF WE WANT TO BE ABLE TO SAY IF IT'S A FULL LENGTH PRO VIRUS, IT'S NOT ENOUGH TO HAVE A LOW CAPTURE REAGENT THAT SEES SOME FRACTION OF IT, WHICH WOULD BE GOOD ENOUGH TO DO CELL SORTING ON IF YOU WERE JUST LOOKING FOR A PRETEEN THAT MIGHT ALMOST BE ON THE SURFACE, SO WE'VE GOT TO BE THINKING ABOUT HOW TO MAKE OUR TECHNOLOGIES ROBUST ENOUGH THAT THE ABSENCE OF A SIGNAL MEANS SOMETHING. I THINK MOST OF US HAVE BEEN PUSHING TOWARDS GETTING THE PRESENCE OF A SIGNAL, ANY SIGNAL, RATHER THAN THE ABSENCE OF A SIGNAL, AND YOUR COMMENT ABOUT LOOKING AT THE DEFECTIVE, BEING ABLE TO SEE THE PARTIAL BINDING EVENTS AND THINGS OF THAT SORT. I MYSELF AM SORT OF NERVOUS ABOUT THINGS LIKE APTIMERS BECAUSE EVEN THOUGH THEY'RE POWERFUL, THEY'RE ALSO VERY FLEXIBLE AND ABLE TO GIVE A HIGH BACKGROUND BINDING RESULT THAT OFTEN PEOPLE HAVE HAD PROBLEMS WITH. SO I THINK WHAT WE NEED TO BE THINKING ABOUT AND WHAT I AM HOPING THE PEOPLE IN THE MEETING HERE WILL HELP US THINK ABOUT IS WHAT ARE THE VALIDATIONS AND QUALITY CONTROLS WE NEED TO BE THINKING, REALLY TAKING INTO THE DESIGN RIGHT FROM THE START, HOW IMPORTANT IS A FALSE POSITIVE OR A FALSE NEGATIVE OR A FALSE COMBINATION OF RESULTS, AND I THINK EACH OF US WITH EACH OF OUR TECHNIQUES HAVE A DIFFERENT SPECTRUM OF CAPABILITIES IN THAT AREA, AND IT'S LIKELY THAT ONE LAB'S TRASH HEAP IS ANOTHER LAB'S REAL BENEFIT BECAUSE, YOU KNOW, IF IT'S SOMETHING THAT'S GOT A HIGH BACKGROUND RATE BUT YOU CAN MAKE SENSE OF IT BY, FOR EXAMPLE, LOOKING FOR MULTIPLE BINDINGS OF DIFFERENT ANTIBODIES TO A SITE TO SAY IT REALLY IS A POSITIVE. YOU KNOW, YOU COULD IMAGINE MAKING -- BASICALLY MAKING LEMONADE OUT OF THE LEMONS OR REALLY TREASURE OUT OF OTHER PEOPLE'S TRASH. IS THERE A CLEARINGHOUSE, IS THERE A WAY THAT WE COULD MAKE A CLEARINGHOUSE FOR THOSE OF US THAT HAVE SOME SUCCESSES AND SOME MISTAKES SO THAT OTHERS CAN HELP US MAKE IT INTO SOMETHING THAT COULD SOLVE PROBLEMS? AND UNFORTUNATELY, MY EXPERIENCE IS THAT NIH HAS NOT BEEN ESPECIALLY GOOD ABOUT FUNDING THINGS THAT HAVE A VERY GREAT ASPIRATIONAL GOAL THAT I DON'T ALREADY HAVE IN HAND. >> YOU'RE IN CHARGE. >> OKAY. SO -- >> [INAUDIBLE] >> THAT'S FANTASTIC, SCOTT. THANK YOU VERY MUCH. I WANT TO GO FROM THE TECHNOLOGY TO THE BIOLOGY, SO BOB, MAYBE IF I COULD CALL ON YOU TO SAY SOMETHING ABOUT HOW YOU THINK MAYBE THESE TECHNOLOGIES COULD IMPACT WHAT WE'RE ALL THINKING ABOUT. >> OKAY, THANKS, MICHELLE. [LAUGHTER] >> WE'LL FIGURE OUT A WAY TO RETURN THE FAVOR. >> SO I JUST WANTED TO SORT OF MENTION SOMETHING THAT ED SAID. THESE TECHNOLOGIES ARE PHENOMENAL, AND I THINK THE IDEA OF THIS MEETING WAS PHENOMENAL. BUT IF YOU LOOK AT THE TITLE, "FIND THE RESERVOIR," WHAT IF IT'S NOT IN ANY SPECIFIC TISSUE AND WHAT IF THE CELLS DON'T HAVE A UNIQUE TRANSCRIPTOME THAT'S DIFFERENT THAN UNINFECTED CELLS. THOSE TWO THINGS ARE ACTUALLY QUITE LIKELY. WOULD HE WE CAN TAKE BLOOD FROM ANY WITH HIV INFECTION AND DEMONSTRATE THE PRESENCE OF THESE CELLS. NOT SAYING THAT THE RESERVOIR IS IN THE BLOOD, BUT IT'S JUST A CONDUIT AS THESE CELLS RECIRCULATE AND PROBABLY ARE EVERYWHERE THAT CD4 CELLS ARE. IN TERMS OF THEIR UNIQUE CHARACTERISTICS WHILE IN THE LATENT STATE, ONE THING THAT ALWAYS WORRIES ME IS WE KNOW SOME OF THESE CELLS CAN REMAIN IN A LATENT STATE FOR YEARS. THE EXPERIENCE OF, FOR EXAMPLE, THE MISSISSIPPI BABY. SO IS THERE A PERMANENT CHANGE IN THE CELL PHENOTYPE THAT PERSISTS FOR YEARS THAT WE'RE GOING TO BE ABLE TO USE TO TARGET THE RESERVOIR. MY OWN WORRY IS THAT THERE'S NOT GOING TO BE SORT A MAGIC CD32-LIKE MARKER AND THAT WE HAVE TO GET BACK TO KIND OF THE REALLY HARD PROBLEM OF WHAT DO WE ACTUALLY DO ABOUT THIS, AND FOR EXAMPLE, THE WORK THAT DAVID MARGOLIS HAS DONE OF TRYING TO REACTIVATE THESE CELLS SO THAT NOW WE CAN DEFINITIVELY IDENTIFY THEM AND HAVE A WAY TO ELIMINATE THEM. SO IT'S JUST A CONCERN AND I HOPE THESE -- THESE APPROACHES ARE CERTAINLY VERY IMPORTANT BUT THERE'S STILL ANOTHER PIECE THAT NEEDS TO BE DONE IN TERMS OF HOW DO WE TURN THE VIRUS BACK OND AND ON AND GET RID OF IT, OR FIGURE OUT A WAY TO CUT OUT THE PRO VIRUS. >> I JUST WANTED TO MAKE A COMMENT BECAUSE THERE'S SO MANY PEOPLE HERE WHO ARE INTERESTED, UP WITH OF THE QUESTIONS THAT YOU REALLY NEED US TO ANSWER IS YOU HAVE A TECHNOLOGY, SO NEXT MONTH WE'LL BE GETTING THE GENE THERAPY FOLKS TOGETHER IN SEATTLE. ONE OF THE THINGS WE'RE REALLY FOCUSED ON, WHAT ARE THE WEAK POINTS OF THE VIRUS WHERE WE THINK WE CAN HIT IT. ONE OF THE PLACES WE'RE LOOKING IS AT REACTIVATION, AND I WOULD JUST POINT OUT THAT WOULD WE DON'T HAVE A VERY GOOD VIEW AT ALL -- BRANDON'S TALK ON THE BARCODES AND REACTIVATION RATE AND THEN REALLY FRIGHTENED FROM WHAT LEWIS SAID THAT THE T-CELLS CAN'T ACTUALLY GET THERE IN TIME TONESS NECESSARILY AFFECT THAT REACTIVATION THING. SO I THINK WE NEED TO HAVE A REALLY NICE VIEW ABOUT WHAT THOSE MEAN, IS IT A CAR-T CELL OR DO WE NEED A SOLUBLE PRODUCT -- >> CALLED AN ANTIBODY. >> YEAH, BUT THIS SORT OF VIEW OF THOSE EARLY ACTIVATION EVENTS CAN REALLY HELP US FOCUS ON WHAT WE NEED TO FOCUS ON AS AN INTERVENTION. SO I WOULD JUST ENCOURAGE FOLKS TO REALLY NOT FORGET THAT AT ALL. >> TOM. >> THIS MIGHT BE MORE OF AN ACTION ITEM, I GUESS, BUT I WAS TALKING EARLIER -- [INAUDIBLE] [INAUDIBLE] -- THE NEXT WEEK OR SO. [OFF MIC] >> SO RELATED SORT OF TO THE QUESTION THAT YOU'RE ASKING THEM, I WANTED TO JUST PUSH BACK A TINY BIT, BOB, WITH WHAT YOU SAY IN TERMS OF THE HIV DNA IS A TARGET, SO TO ME, IF YOU THINK OF, IN THE CANCER WORLD, WHERE THERE'S SO MUCH -- I THINK WE HAVE THE ADVANTAGE AT LEAST OF HAVING THAT DNA, SO OBVIOUSLY WHETHER IT'S EXPRESSED ON THE SURFACE AND THE WHOLE CD32 PROBLEM -- IT'S NOT -- CLEARLY NOT EASY, BUT SORT OF TO WHAT TOM WAS ASKING, AND WHAT WE'RE ASKING YOU, IF YOU DID HAVE A NUMBER OR A WAY TO LOOK AT THE INTEGRATED PRO VIRUS, AND THEN LOOK AT OTHER CHARACTERISTICS OF THE CELLS, I THINK TOM IS ASKING ALSO, RIGHT, HOW WOULD YOU DO THAT, OR WHAT WOULD YOU LOOK AT? >> SO WHAT ARE THE THINGS THAT YOUR METHODS CAN DO IN THE TISSUE, BECAUSE WE HAVE THIS PROBLEM OF DIFFERENT CELLS CAN BE EXTRACTED FROM THE TISSUE WITH DIFFERENT EFFICIENCIES, AND SO THAT'S THE TRUE VALUE OF THESE METHODS WHERE WE CAN -- YOU CAN WORK WITHIN THE TISSUE ITSELF, AND SO -- AND I REALIZE WE'RE INTERESTED IN THESE LATENT CELLS, OTHER CELLS. THE REASON WE ARE TAKING THIS APPROACH TO THE REBOUND SITE IS WE'RE ASSUMING NOT ONLY ARE THERE CELLS REBOUNDING THERE BUT OTHER CELLS YET TO REBOUND IN THE SAME NEIGHBORHOOD, SO YOU MIGHT BE ABLE TO SEE THOSE AT THE SAME TIME. SO AGAIN WHAT DO YOU THINK YOU COULD EXTRACT OR WHAT WOULD BE THE IDEAL SAMPLE TO TRY TO ASK THIS KIND OF A QUESTION. >> I'D LIKE TO PICK ON ALEX NEXT. AS ANOTHER REPRESENTATIVE OF TECHNOLOGY. OH, SORRY. OH, YOU WERE GOING TO ANSWER. GO AHEAD. >> SO I THINK FIRST ABOUT THE TISSUE PREPARATION, WE CAN EITHER WORK WITH FRESH FROZEN TISSUES OFF -- FIXED TISSUES AS LONG AS THEY ARE NOT OVERFIXED. SO I THINK THE EASIEST WAY IS JUST TO -- FRESH FROZEN -- WE -- WILL BE STABLE FOR SEVERAL MONTHS. REGARDING THE DETECTION FOR DNA AND RNA, WE JUST NEED TO DESIGN PROBES SO THOSE PROBES WILL WORK WITH POST DNA RNA -- HYBRIDIZATION PARAMETERS, SO WE HAVE SEEN LIKE SOMETIMES IF WE DESIGN -- WE INCREASE THE HYBRIDIZATION -- DNA AND RNA. SO -- AND I THINK DNA WILL HAVE NO PROBLEM, AND THE PROBLEM -- RNA IS LIKE -- FRESH FROZEN REALLY QUICKLY TO PREVENT ANY RNA DEGRADATION. >> I'D LIKE TO MAKE A FOLLOW-UP COMMENT TO DIANA AND BOB'S COMMENT. I ALSO DON'T THINK THERE WILL BE A UNIVERSAL CD32 BUT DO WE REALLY NEED TO CHARACTERIZE EVERY SINGLE LATENT CELL IN THAT INFECTED INDIVIDUAL? THEIR POST -- THE SAN FRANCISCO PATIENT THAT WAS PRESENTED YESTERDAY, IF WE CAN JUST UNDERSTAND IT ENOUGH, WHAT IS NECESSARY TO CONTROL IT, THERE COULD STILL BE LATENT CELLS THAT ARE EVEN CAPABLE REACTIVATING EX VIVO, BUT AS LONG AS WE CAN ACHIEVE THE FUNCTIONAL CURE, THEN THAT MIGHT BE SUFFICIENT FOR NOW. >> I AGREE WITH THAT. IF YOU COULD MAKE SOME COMMENTS, ALEX? >> ANYTHING SPECIFIC TO COMMENT ON? >> YES, HOW ARE WE GOING TO USE THESE TECHNOLOGIES TO SOLVE THESE PROBLEMS? >> I MEAN, I THINK THERE ARE MULTIPLE DIFFERENT WAYS OF APPLYING THESE TECHNOLOGIES. THE REAL QUESTION BECOMES WHAT PROBLEM DO WE WANT TO SOLVE. TO BOB'S POINT, THERE MAY NOT BE A UNIVERSAL MARKER FOR ALL INFECTED CELLS THAT WE CAN JUST GO SAY, WELL, LET'S QUANTIFY THIS, BUT WE MAY DISCOVER THINGS ABOUT REACTIVATION FROM THE FACT THAT WE CAN'T REACTIVATE ALL OF THE CELLS AND THAT MAY TELL US SOMETHING ABOUT WHAT MOLECULAR PATHWAYS WE NEED TO HIT IN ORDER TO DEVELOP BETTER SHOCK AND KILL STRATEGIES. I THINK XIAO'S POINT IS INCREDIBLY GOOD GIVEN WHAT TOM BROUGHT UP ABOUT THE ANIMAL AVAILABILITY THAT'S PRESSING. WHEN WE DO THINGS IN THE CANCER SPACE, WE THINK A LOT ABOUT HOW WE CAN PROCESS OUR TISSUES SO WE CAN USE THEM FOR MULTIPLE DIFFERENT ASSAYS, SO WE ROUTINELY TAKE PIECES AND DO MULTIPLE DIFFERENT THINGS AND SO I THINK THAT LINING OUT AHEAD OF TIME HOW WE MIGHT WANT TO PREP ALL THE SAMPLES SO THAT ANY TECHNOLOGY THAT MIGHT BE USEFUL& TO SORT OF TRY ON THESE DIFFERENT THINGS TO FIGURE OUT WHAT THE VALUE OF EACH ONE IS AND TO DO SOME OPTIMIZATION IS A GOOD THING TO HAVE A CONVERSATION ABOUT AT THIS POINT SO THAT WE CAN TAKE THE BEST ADVANTAGE OF THESE ANIMALS. I DO THINK TO A POINT THAT I BROUGHT UP YESTERDAY THAT IT'S GOING TO BE VERY HARD, IT WAS BROUGHT UP EARLIER ON THE PANEL, IT'S GOING TO BE VERY HARD TO FIGURE OUT WHAT'S HAPPENING IN ALL THE DIFFERENT TISSUES AROUND THE BODY GIVEN OUR AVAILABILITY IN HUMANS. I THINK WE HAVE TO THINK CAREFULLY ABOUT MODELS AND ABOUT THINGS THAT WE CAN MEASURE. IT MAY BE THAT SOME OF THESE DELTAS THAT COME THROUGH REACTIVATION ARE GOOD WAYS OF QUANTIFYING WHAT REMAINS EVEN IF WE CAN'T REACTIVATE EVERYTHING BUT LOOKING TO SEE WHEN THERE'S A CHANGE WHEN WE DO SOME SORT OF STIMULATION. BUT MY SENSE IS THAT THESE ARE GOING TO GIVE US A BETTER UNDERSTANDING OF THE BIOLOGY ASSOCIATED WITH THE RESERVOIR AND WITH ITS CHARACTERISTICS. BUT I'M NOT 100% SURE THAT THERE ARE GOING THEY'RE GOING TO BE DIAGNOSTICALLY RELEVANT, WE'RE GOING TO USE OTHER APPROACHES LIKE THE PET THAT CAME UP YESTERDAY AS A WAY OF REALLY LOOKING TO SEE WHERE THINGS ARE, AND MICHELLE, THE ONE QUESTION I WOULD HAVE FOR YOU IS I WAS THINKING A LOT WHEN I WAS LOOKING YESTERDAY AT THE ANIMALS ABOUT HOW YOU GET SIGNAL IN DIFFERENT TISSUES THROUGHOUT THE BODY, SO WHAT DO YOU THINK IS GOING TO BE THE BEST APPROACH, DO YOU THINK ANTIBODIES IS THE BEST WAY TO GO IN TERMS OF TRYING TO LABEL ALL THE DIFFERENT RESERVOIR SITES THROUGH THE BODY, HOW DO YOU THINK WE ACTUALLY GO BACK AND QUANTIFY IN HUMANS WHAT REMAINS OF A RESERVOIR, PARTICULARLY GIVEN THE REPLICATION COMPETENT/INCOMPETENT PIECE THAT CAME UP TODAY? >> HOW DO YOU QUANTITATE THE RESERVOIR. YEAH, SO, YOU KNOW, THERE ARE MULTIPLE METHODS, AND I THINK BOB GAVE A FANTASTIC LECTURE AT THE BEGINNING ABOUT WHERE WE STAND AS A FIELD IN TERMS OF THE QUANTITATION OF THE RESERVOIR, AND WHAT WE HAVE AVAILABLE, IS, AS HE SAID, IN SOME WAYS REPRESENTATIVE OF WHAT'S EVERYWHERE, ALTHOUGH THERE ARE CELLS IN TISSUES THAT LIVE IN TISSUES AND THAT THERE IS LESS ACCESS TO. SO ONE POSSIBILITY ABOUT THE RESERVOIR IS SO CALLED LEFT EARLOBE HYPOTHESIS. IT LIVES IN THE LEFT EARLOBE AND IT STAYS IN THE LEFT EARLOBE, AND YOU CAN'T GET IT BECAUSE IT'S IN THE LEFT EARLOBE. BUT THEN THERE'S THE OTHER IDEA, WHICH IS THAT IN FACT WHAT YOU GET IN CIRCULATION IS SOMEHOW REPRESENTATIVE OF WHAT'S OUT THERE. I THINK THE MACAQUE MODELS REALLY ARE PROBABLY THE RIGHT MODELS IN TERMS OF TRYING TO UNDERSTAND THIS THING IN DETAIL WITH CAVEATS, AND I THINK PEOPLE THAT UNDERSTAND THOSE CALF YA THES AND ARE HONEST ABOUT SAYING THIS IS AN INFECTION WHICH IS DIFFERENT FROM HIV IN THIS WAY, THIS WAY AND THAT WAY, I BELIEVE THAT'S WHERE WE'RE GOING TO LEARN THE MOST. SO I'VE DONE MY LITTLE BIT, BUT NOW I WANT TO CALL ON AN IMMUNOLOGIST, SO JOHN IS THERE AS A REPRESENTATIVE IMMUNOLOGIST. SOME OF HIS THOUGHTS ABOUT HOW THESE DIFFERENT TOOLS MIGHT BE BROUGHT TO BEAR ON THIS PROBLEM. WE HAVE TO MOVE THINGS AROUND A LITTLE BIT. >> THANKS, MICHELLE. YOU KNOW, THERE ARE A COUPLE THINGS THAT YOU'LL GET -- THERE ARE TWO THINGS THAT KIND OF OCCURRED TO ME. AGAIN, I APOLOGIZE IF SOME OF THIS SEEMS INCREDIBLY OBVIOUS, BUT I'M AS A BIT AN OUTSIDER SITTING HEERYLY UNCLEAR HERE REALLY UNCLE AR BY WHAT EVERYBODY MEANS BY THE TERM RESERVOIR. IT SEEMS TO ME THAT TERM MEANS DIFFERENT THINGS DEPENDING ON WHO'S TALKING AND WHAT THE FRAMEWORK IS. AND I THINK THERE ARE THE OBVIOUS THINGS ABOUT LATENT VIRUS THAT'S NOT TRANSCRIPTIONALLY ACTIVE AND TRANSCRIPTIONALLY ACTIVE VIRUS, THERE ARE THE OBVIOUS THINGS ABOUT VIRUS IN T-CELLS VERSUS VIRUS IN MYELOID CELLS OR THAT YOU CAN FIND IN CIRCULATION VERSUS IN TISSUES. BUT I THINK GETTING A BETTER HANDLE ON WHAT THAT WORD MEANS, BOTH BIOLOGICALLY AND ALSO AS IT RELATES TO CURE, AND THE TWO THINGS I THINK ARE QUITE DIFFERENT AND THERE'S FUNCTIONAL CURE AND WE TALKED A LITTLE BIT ABOUT THAT. BUT I THINK THE TOPIC THAT SEEMS KIND OF INTERESTING THAT WE DIDN'T REALLY TOUCH ON IS THE IDEA OF HOW THE INTEGRATION LOCATION IN THE GENOME MAY MATTER NOT JUST DEPENDING ON THE LOCATION BUT DEPENDING WHAT KIND OF CELL IT'S IN. SO I THINK IT'S NOW BECOMING CLEARER THAT T-CELLS MANAGE INTEGRITY IN VERY UNUSUAL WAYS, AND I THINK THE IN VITRO MODEL PRESENTED EARLIER IS REALLY INTERESTING FROM THE STANDPOINT OF VIRUSES OR CELLS THAT HAVE VIRUS THAT GOES LATENT, REACTIVATE AND GOES RIGHT BACK TO GOING LATENT. IT WOULD SEEM THERE'S A T-CELL BIOLOGY ASPECT THAT WE NEED TO KEEP VERY MUCH IN MIND AND MAKE SURE WE'RE MATCHING OUR UNDERSTANDING OF LATENCY OR RESERVOIR WITH THE BIOLOGY OF THE T-CELLS. THERE ARE TYPES OF T-CELLS THAT FUNDAMENTALLY DIFFERENTLY IN THE WAY THEY MANAGE GENOME INTEGRITY AND REPLICATION, QIE I SENSE, QUIESCENCE A ND THE WAY THEY TURN THOSE PROGRAMS ON AND OFF MUCH MORE READILY THAN OTHER CELLS. SO THERE'S A REALLY IMPORTANT ROLE FOR THIS IN DEEP T-CELL BIOLOGY, PERHAPS DEEP MYELOID CELL BIOLOGY AS WELL, THAT NEEDS TO BE PAIRED WITH HOW WE THINK WITH WHAT RESERVOIR MEANS. SO THOSE WOULD BE THE TWO TOPICS AS AN OUTSIDER, I SEE ONE THAT I DIDN'T UNDERSTAND WHAT WE MEAN BY RESERVOIR, AND TWO, THAT THE CELLULAR IMMUNOLOGY AND THE THINGS WE CAN DO NOW WITH THE GENOMIC APPROACHES TO REALLY DEEPLY INTERROGATE THE UNDERLYING PROGRAM OF THE IMMUNE CELLS WE'RE INTERESTED IN. SO WE'RE NOW LEARNING MUCH MORE FROM EPIGENETIC PROGRAMMING ABOUT CELLULAR I'DITY WITHIN THE IMMUNE SYSTEM AND HOW THAT IDENTITY REMAINS CONSTANT OVER TIME, HOW THAT RELATES TO WHERE VIRUS INTEGRATES, HOW IT MIGHT REACTIVATE FROM -- I THINK WE NEED TO MATCH WITH THE TYPE OF T-CELL AND T-CELL BEHAVIORS A EL WITH. AND THE SAME PROBABLY IS TREUL FOR MYELOID CELLS. >> I COMPLETELY AGREE WITH THAT. I THINK THAT IN ADDITION TO PULLING IN PEOPLE THAT ARE VERY INTERESTED IN TECHNOLOGIES, IN NEW TECHNOLOGIES, SINGLE CELL TECHNOLOGIES AND MICROSCOPY AND VISUALIZATION, THAT THE FIELD WOULD BENEFIT FROM BRINGING IN BASIC IMMUNOLOGISTS THAT KNOW A LOT ABOUT IT. CELL BIOLOGY AND A ABOUT IT. ABOUT IT. ABOUT T-CELL BIOLOGY AND MYELOID BIOLOGY. >> BASIC T-CELL BIOLOGY HAS EVERYTHING TO DO WITH LATENCY AND AS A VIROLOGIST BY TRAINING, I FIND MYSELF BEING DRAWN MORE TO RELEARNING BASIC T-CELL METHODOLOGY AS I GO THROUGH THIS PROJECT, SO BRINGING IMMUNOLOGISTS TOGETHER TO VIROLOGISTS TO WORK ON THIS IS ESSENTIAL. >> I THINK RELEARNING THE BASIC T-CELL BIOLOGY IS ONE THING BUT THE T-CELL BIOLOGISTS ARE STILL ARGUING ABOUT ALL OF THIS, I THINK VERY VIGOROUSLY. SO I WOULD SAY THIS IS SORT OF A MERGING OF TWO AREAS THAT STILL ARE QUITE CONTENTIOUS, TO BE CANDID, AND I DON'T THINK ALL T-CELL BIOLOGISTS WOULD AGREE ON WHAT A MEMORY T-CELL IS, FOR EXAMPLE. SO WE NEED TO BASICALLY -- >> AND WHETHER THAT'S A STABLE PHENOTYPE, YES. >> EXACTLY. >> LEWIS. WE'LL CALL ON YOU. >> I'VE LIVED LONG ENOUGH NOW TO WATCH THE EVOLUTION OF TECHNOLOGIES. I MEAN, AS A PATHOLOGIST, I STUDY THE HISTORY OF PATHOLOGY AND WATCH WHEN HNE WAS A NEW TECHNOLOGY, ALL WE LEARNED FROM THAT, FLOW CYTOMETRY, ALL THESE OBSERVATIONAL METHODS WERE GREAT, THEY ALLOW YOU TO CLASSIFY THINGS BUT THEY DON'T REALLY TELL YOU ABOUT THE DYNAMICS. 20 YEARS AGO, I LEFT HUMAN PATHOLOGY IMMUNOLOGY AND MOVED TO THE MONKEY FACILIY SO THAT I COULD ASK QUESTIONS AND I WOULD JUST REITERATE THAT ALL THESE TECHNOLOGIES BY THEMSELVES AREN'T GOING TO ANSWER THE QUESTION BECAUSE WE'RE NOT GOING TO KNOW WHAT THAT PARTICULAR CELL YOU IDENTIFY EVERY TBEEN FOR HAS THE POTENTIAL IN VIVO BECAUSE IT'S SITTING IN YOUR SEQUENCER. SO WHAT REALLY HASN'T BEEN TALKED ABOUT HERE IS ALL THIS HAS TO BE PAIRED WITH INTERVENTIONAL EXPERIMENTS. EVEN IN -- OBVIOUSLY I'M A MONKEY PERSON WHICH ALLOWS TO YOU DO THIS A LITTLE MORE READILY, BUT BOTH IN MONKEYS AND IN PEOPLE, AND VERY TECHNOLOGICALLY SIMPLE QUESTIONS, WOULD A MONKEY BE CURED IF YOU GOT RID OF EVERY CD4 T-CELL, WOULD HE BE CURED IF YOU GOT RID OF EVERY MEMORY CELL, EVERY FOLLICULAR HELPER T-CELL. OR IF IT'S NOT CURED, WHAT WOULD BE THE RESITD RESIDUAL POPULATION THAT WOULD KRNT TO REBOUND. SO I THINK AT THE SAME TIME, WE'RE GOING THROUGH WHAT ALL THESE FANTASTIC TECHNOLOGIES, WE REALLY HAVE TO THINK ABOUT TECHNOLOGY THAT WOULD ALLOW US TO INTERVENE IN VIVO IN INCREASINGLY SOPHISTICATED WAYS. WE'RE DEVELOPING CAR-T CELLS TO GET RID OF CD4 T-CELLS, GET RID OF B CELLS. GETTING RID OF B CELLS ACTUALLY HELPS, IT ACCELERATES THE INTERCEPT OF THE INFECTION BECAUSE THE FOLLICLE IS SANCTUARY. SO I JUST WANT TO REITERATE THAT IN ORDER TO MAKE PROGRESS, WE'RE GOING TO HAVE TO GO HAND IN HAND WITH CLEVER INTERVENTIONAL EXPERIMENTS THAT ARE GOING TO TOTALLY CHANGE THE PARADIGM, AND DICTATE HOW WE'RE GOING TO USE THIS TECHNOLOGY. >> LEWIS, I ACTUALLY WANT TO CALL KATIE FOR A MINUTE. AS A REPRESENTATIVE CLINICIAN. BECAUSE I THINK IN THE END, THIS IS A CLINICAL PROBLEM. >> I WANT TO SPEAK TO THE QUESTION OF DYNAMICS. I AGREE ENORMOUS MORE FOCUS SHOULD BE ON THE PER TEU BAYTIVE EFFECTS BUT I WOULD PUSH BACK A LITTLE BIT ON THE ISSUE THAT THERE HAS BEEN KIND OF A SHIFT WITH THESE MULTIMODAL MEASUREMENTS TO BEING ABLE TO ACTUALLY TRACK DYNAMICS IN A VERY REAL WAY FOR INDIVIDUAL CELLS. >> INFERRED DYNAMICS. [INAUDIBLE] >> I'D AGREE WITH THAT, ALTHOUGH AGAIN, YOU ARE MAKING RECORDINGS BUT -- RIGHT, THE DIFFERENCE HAS TO DO WITH THE STOW CASS TISSITY OF THE MEASUREMENTS, SO IN THE SAME WAY THAT OBVIOUSLY YOU CAN CERTAINLY TRACK -- YOU MIGHT BE ABLE TO TRACK THINGS MORE DETERMINISTICALLY BY LOOKING AT SOMETHING BUT OF COURSE YOU'RE STILL DEALING WITH KIND OF THE NOISE OF POPULATION WIDE MEASUREMENT. SO I THINK THE POINT I WOULD MAKE WOULD BE THAT WITH KIND OF THE ABILITY TO SEQUENCE, FOR EXAMPLE, THE CHANGES OF PRO VIRUSES OVER TIME, YOU KNOW, DUE TO MUTATION, DUE TO THE CUMULATION OF MUTATION, PAIR THOSE WITH TRANSCRIPTIONAL MEASUREMENTS. WE ARE, IN FACT, KIND OF AT THE CUSP OF BEING ABLE TO REALLY GET DYNAMIC INFORMATION ABOUT WHAT IS, IN FACT, CAUSING CHANGE IN PROLIFERATIVE DYNAMICS OVER TIME, WHAT ARE THE CAUSAL NATURES OF THAT IN A WAY THAT I DON'T THINK THAT FLOW CYTOMETRY AND OTHER TYPES OF METHODS COULD IN PRINCIPLE ACCESS. SO I WOULD JUST SAY THAT -- RIGHT. >> THAT'S NOT TRUE. IT'S JUST MORE SPHIS SOPHISTICATED. FLOW CYTOMETRY -- [OFF MIC] [INAUDIBLE] >> RIGHT. >> IT'S REALLY THE SAME CONCEPT, IT'S JUST MORE THE TECHNOLOGY. I'M NOT DISAGREEING WITH THE USEFULNESS OF THIS, I'M JUST SAYING ULTIMATELY SOMEBOD HAD TO GO IN AND SHOW HOW HEMATOPOIETIC -- WORKS IN VIVO. >> MY POINT IS THAT ALWAYS HAD TO BE A DESTRUCTIVE MEASUREMENT, WHEREAS KIND OF TERMINAL MEASUREMENT OF A CELL'S MUTATIONAL -- BASICALLY TELLS INFORMATION ABOUT THAT SPECIFIC CELL'S HISTORY, AND THAT IS A QUALITATIVE CHANGE IN THE NATURE OF THE TECHNOLOGY. >> SO I GUESS FROM MY VANTAGE POINT WHERE I ATTEMPT TO SIT IN THIS TRANSLATIONAL SPACE BETWEEN THE -- AND CLINICAL TRIALS WORLD, I THINK ONE OF THE MOST IMPORTANT MAYBE THINGS TO FOCUS ON IN THIS EVOLVING CURE WORLD IS REALLY CLOSE COMMUNICATION BETWEEN ALL THESE DIFFERENT AREAS. SO I TOTALLY SUPPORT THE IMPORTANCE OF THE BASIC SCIENCE INVESTIGATION OF THE RESERVOIR AND ANY SORT OF INTERVENTIONS WE CAN DO THERE, TRANSLATING THAT INTO HUMAN INTERVENTION STUDIES AND HUMAN PRIMATE MODEL WHICH I THINK CAN REALLY TEST A LOT OF THESE HYPOTHESES AND WE CAN MOVE THE FIELD FORD WITH BUSINESS DIG COVER RES, BUT I THINK WE NEED TO THINK OF PHASE 1 EXPERIMENTAL MEDICINE CLINICAL TRIALS BECAUSE ALL OF THESE FABULOUS DISCOVERIES THAT HAVE BEEN SHOWN IN NON-HUMAN PRIMATE MODELS OR HUMANIZED MOUSE MODELS, THEY'RE VERY EXCITING BUT THE NUANCES AND THE DIFFERENCES BETWEEN THE MODELS AND BETWEEN HUMAN SYSTEMS ARE REALLY IMPORTANT, AND WE NEED TO THINK ABOUT HOW TO USE OUR CLINICAL TRIALS ARCHITECTURE, THIS REALLY AMAZING GROUP OF ALTRUISTIC PARTICIPANTS IN OUR TRIALS, HIV-POSITIVE INDIVIDUALS WHO ARE HIGHLY MOTIVATED TO PARTICIPATE IN TRIALS THAT MAY NOT BENEFIT THEM PERSONALLY, BUT WILL MOVE THE FIELD FORWARD, AND TAKE THESE EXCITING CONCEPTS THAT ARE DISCOVERED BASIC NON-HUMAN PRIMATE HUMANIZED MICE WHATEVER MODELS AND TEST THEM IN SMALL GROUPS OF HUMANS WHERE WE CAN ASK DID WE SEE THE SAME SURROGATE MEASURES, DO WE SEE THE SAME REAL EFFECTS. WE'RE NOT GOING TO CURE INITIALLY BUT DO WE SEE THESE SAME THINGS IN A HUMAN SYSTEM. I THINK THAT COULD BE A VERY IMPORTANT WAY TO CONTINUE TO MOVE THE FIELD FORWARD THROUGH COLLABORATION OF ALL OF THESE DIFFERENT AREAS. I DO THINK THIS EXPERIMENTAL MEDICINE PHASE 1 TRIAL HAS A LITTLE BIT MORE TRACTION MAYBE IN THE VACCINE FIELD AND OTHER AREAS, AND WE SHOULD THINK ABOUT IT IN THIS ARENA AS WELL. >> SO I DON'T THINK WE HAD TROUBLE FINDING THE RESERVOIR. I THINK IT'S WIDESPREAD. IN TERMS OF CHARACTERIZING IT, EVERY TIME I SEE DATA FROM A GROUP OF PATIENTS, PARTICULARLY BOB'S RATIO OF IPDA TO TOTAL DNA, I'M STRUCK BY ONE THING. THE DIVERSITY OF THE MEASUREMENTS, THE RANGE OF VALUES. THERE'S SUCH VARIABILITY WITHIN AN INDIVIDUAL AND ACROSS AN INDIVIDUAL, I THINK IT COULD BE AN INVIE NIGHT PROBLEM TRYING TO CHARACTERIZE THE RESERVOIR. JUST AS IT IS TRYING TO CHARACTERIZE CANCER. AND THAT THE CHARACTERIZATION IS USEFUL FOR TARGET DISCOVERY LIKE ED WAS SUGGESTING, BUT TO COME UP WITH THE HOLY GRAIL OF THIS IS THE RESERVOIR, I'M GOING TO SAY IS PROBABLY FUTILE ACROSS ALL INDIVIDUALS, BECAUSE I DON'T THINK TWO INDIVIDUALS ARE ALIKE. I MEAN, THE PHILOGYNY OF THE RESERVOIR IS, YOU KNOW, ENDLESS, AND THE DIVERSITY OF CELLS THAT CAN BE INFECTED IS ENORMOUS. SO I THINK WHERE WE NEED TO FOCUS MORE ON IS WHETHER TO MAKE -- WHETHER WE CAN MAKE A DENT IN THE RESERVOIR. AND I THINK WE HAVE REASONABLE TOOLS, THEY'RE NOT THE BEST, BUT THEY'RE REASONABLE TOOLS TO ASSESS WHETHER WE'VE AFFECTED THE RESERVOIR OR WHETHER WE'VE AFFECTED IMMUNE CONTROL OF ART. THINK WE HAVE DECENT TOOLS TO DO THAT. AND WHERE THE CHARACTERIZATION IS GOING TO GO IS WHEN WE ACHIEVE REMISSION IN SOME INDIVIDUALS AND NOT OTHERS AND CHARACTERIZING THE RESERVOIR THAT CAUSES THE RELAPSE WILL TAKE A LOT OF ENERGY AND EFFORT. SO I AGREE WITH LEWIS THAT ALL THIS WORK NEEDS TO BE DOVETAILED WITH EXPERIMENTAL MEDICINE TO SEE IF ANYTHING WE'RE DOING IMPACTS THE RESERVOIR. WE COULD SPEND DECADES CHARACTERIZING THE RESERVOIR, BUT UNLESS WE TEST WHETHER ANY OF THE INTERVENTIONS THAT WE'RE TALKING ABOUT AFFECT THE RESERVOIR, I DON'T THINK WE'RE GOING TO MAKE PROGRESS. >> I WANT TO SEE IF BRAD WANTS TO SAY SOMETHING. >> SO MICHELLE OPENED THE SESSION BY ASKING HOW WE CAN USE THESE NEW TECHNOLOGIES TO ASK THE WHERE, WHAT AND THE WHY OF THE RESERVOIR. I THINK AGREE WITH JOHN THAT THE WHERE IS BASICALLY EVERYWHERE -- >> DON'T WHREEF IN BELIEVE IN THE LEFT EARLOBE? >> NO, HAVEN'T CHECKED. AND THE WHAT, IT'S VERY HETEROGENEOUS POPULATION OF CELLS BUT IF WE ASK OURSELVES WHY, I THINK WE ALL HAVE THE ONE KIND OF REASON IN MIND THAT THESE AFFECTED CELLS HIDE FROM THE IMMUNE SYSTEM. IS THAT THE ONLY WHY, AND CAN THESE TECHNOLOGIES GIVE US SOME OTHER REASONS WHY THESE CELLS MIGHT PERSIST AND I THINK IN YA-CHI'S DATA, AND LI CHTERFELD, THERE'S INTERESTING FINDINGS ASSOCIATED WITH SURVIVAL, SO MAYBE THAT'S ANOTHER WHY, THESE CELLS ARE INTRINSICALLY RESILIENT, INTRINSICALLY TOUGH, AND MAYBE THERE'S OTHER WHY'S, BUT THE WHY'S AREN'T QUITE AS OVERWHELMING AS THE WHERE'S AND THE WHAT'S. SO MY 2 CENTS. >> DOES ANYBODY ELSE WANT TO MAKE ANY COMMENTS? >> SORRY, I JUST HAVE A VERY NAIVE QUESTION. SO DOES ANY OF THE CRISPR SYSTEM BE ABLE TO, LIKE, DESTROY THE DNA OR RNA PIECE OF EACH HIGHWAY, AND THE FIRST NAIVE QUESTION. THE SECOND ONE, IS THERE ANY CELL TYPES THAT CANNOT BE AFFECTED BY HIV? I MEAN, BECAUSE IF IT IS EVERYWHERE, I'M JUST WONDERING IS THERE ANY PARTICULAR CELL TYPE THAT CANNOT BE AFFECTED BY DNA SPECIAL RECEPTOR OR GENE EXPRESSION PROGRAM AS A -- OF THIS HIV VIRUS? >> I THINK I'LL JUST TAKE A SHOT AT THAT. BEING A NOVICE AS WELL, BUT -- CRISPR SYSTEMS WORK, YES. THERE'S GOOD EVIDENCE FOR THAT. THERE IS A LIMITATION ON THE CELLS THAT ARE INFECTED. IT HAS TO DO WITH THE RECEPTORS THAT THE VIRUS USES TO GET INTO CELLS AND IT'S PRETTY MUCH IN THE HUMAN LIMITED THAT WAY. AND IT'S MYELOID CELLS AND T-CELLS. >> CAN I ALSO ADD TO THAT, SO IN TERMS OF THE RESERVOIR BEING EVERYWHERE, I SOMEWHAT AGREE BUT I ALSO SOMEWHAT DISAGREE, SO OUR DATA ACTUALLY SUGGESTS THAT NOT ALL RESTING MEMORY CD4 T-CELLS CAN LATENTLY INFECTED IN VIVO, AND WE DO SEPA SEE PATTERNS IN DIFFERENT INDIVIDUALS AND I THINK IT WOULD BE INTERESTING TO -- WHY SOME TEND TO BE LATENT AND OTHERS TEND TO BE PREFERENTIALLY LATENT. >> DOES ANYBODY ELSE WANT TO MAKE ANY COMMENTS? >> SO I THINK ONE OF THE THINGS THAT REALLY FASCINATED ME FROM VERY, VERY EARLY ON WAS THAT YOU CAPTURE THE VIRUS IN THIS MOMENT IN TIME, AND THE WHOLE IDEA OF IMMUNOLOGIC MEMORY, SO IT SORT OF GOES BACK TO WHAT YOU WERE SAYING AND HOW DEEPER UNDERSTANDING IS OF THE CELL. SO IN MY MIND, I GO BACK AND FORTH THINKING THEY MAY HAVE NO CHARACTERISTICS AS BOB SAYS, AND THEN COMPLETELY THE OTHER WAY, THERE MAY BE SOME BIOLOGICAL ROLES THAT THESE CELLS HAD A CERTAIN TIME TO DO CERTAIN THINGS THAT MIGHT BE HARD TO CAPTURE AS YEARS PASS, BUT THEY MAY HAVE HAD SOME COMMON MOMENT IN WHICH THEY SERVED A ROLE, AND SO GOING BACK TO WHAT YOU'RE SAYING ABOUT THE FACE RECOGNITION, IS THERE SOMETHING THERE WITH THOSE CELLS. >> AGAIN, IS THERE ANYBODY ELSE THAT WANTS TO SAY SOMETHING? PLEASE. >> SO I ALSO WAS TRAINED AS AN IMMUNOLOGIST, SO REALLY IN THE PROCESS OF STUDYING IMMUNE CONTROL MECHANISM OF THE CONTROLLERS, I CAME ACROSS WITH PRO VIRUS LANDSCAPE, THAT'S HOW I GOT INVOLVED IN THIS EXCITING WORKSHOP. I -- WHAT JOHN SAID, IT'S REALLY TRUE WHEN I TRIED TO ENTER THIS FIELD TO THINK ABOUT IT, I'M CONFUSED WITH THE RESERVOIR DEFINITION AS WELL. THAT'S WHY I LISTED A BUNCH OF THEM YESTERDAY TO SAY WHAT IS -- ARE YOU CONSIDERING ANY CELL AS A PRO VIRUS YOU CALL RESERVOIR, THAT'S VERY BROAD, OR ARE YOU REALLY TALKING ABOUT REBOUND COMPETENT INTACT PRO VIRUSES THAT ONLY THING THAT MATTERS. I THINK SOMEBODY IN THE FIELD LONG ENOUGH SHOULD REALLY USE THIS WORKSHOP TO GIVE A REAL MORE DEFINITIVE DEFINITION FOR THIS. THAT'S ONE THING TO UNDERSTAND. BUT ALSO FROM WHAT I UNDERSTAND, THE PROFILE OF THE RESERVOIR CHANGING REALLY OVER TIME DEPENDS ON -- TREATMENT, LIKE ONE SIMPLE FACTOR. EVEN AFTER ONE OR TWO YEARS OR UP TO THREE YEARS INFECTION, THERE'S STILL MANY -- BEING DETECTED. SO -- SURPRISING SEE A LOT OF TRANSCRIPTION OR TRANSLATIONAL ACTIVITIES GOING ON, BUT 10 YEARS AFTER TREATMENT, THE RESERVOIR COMPOSITION COULD BE QUITE DIFFERENT FROM WHAT YOU SEE IN THREE YEARS OF TREATMENT. SO OUR TARGET ON DIFFERENT PATIENTS OR DIFFERENT DURATION OF TREATMENT, MAYBE THERE WILL BE DIFFERENT STRATEGY, AFTER LONG TERM TREATMENT WOULD YOUR -- BUT IF YOU WANT TO TREAT SOMEBODY OR CURE SOMEBODY, ONLY THREE YEARS AFTER INFECTION, YOU HAVE PROBABLY TO CONSIDER A LOT MORE DEFECTIVE PRO VIRUSES THAT'S ALSO GAVE RISE TO TRANSCRIPTIONAL TRAN LAITIONAL ACTIVITY THAT ACTUALLY CAUSE A LOT OF CHRONIC IMMUNOACTIVATION, PROBABLY VERY SIGNIFICANT ISSUE NOT ONLY BY CURE, THE REBOUND COMPETENT VIRUSES. SO ANYBODY? >> BOB WANTS TO SAY SOMETHING. >> SO I GUESS TO ANSWER JOHN'S QUESTION, I THINK IT'S IMPORTANT WE BE PRECISE ABOUT WHAT WE MEAN. IN A PAPER WHICH AS FAR AS I CAN TELL NO ONE HAS EVER READ, WE TRIED TO PROPOSE -- >> I GOT LOTS OF THOSE TOO. EVERYBODY'S GOT THOSE. >> YEAH. WELL, I HAVE A LOT OF THEM. I THINK WE SORT OF NEED A PRACTICAL DEFINITION, AND IT REALLY RELATES TO THE CLINICAL REALITY, WE'RE GOING TO TRY TO CURE PEOPLE WHO HAVE BEEN ON LONG TERM ART. SO TO ME, THE ONLY THING THAT MATTERS IS CELLS IN WHICH A REPLICATION-COMPETENT FORM OF THE VIRUS CAN PERSIST IN A PATIENT ON OPTIMAL ART FOR A SIGNIFICANT AMOUNT OF TIME. NOW CLEARLY, THERE'S OTHER CELLS THAT HAVE HIV AND THEY MAY TURN OVER MORE QUICKLY AND SO FORTH, BUT CLINICALLY WHAT MATTERS, AND I MAYBE WOULD ASK JOHN IF HE AGREES, IS THE POPULATION THAT HARBORS HIV IN THE SETTING OF OPTIMAL TREATMENT FOR A LONG PERIOD OF TIME. THAT'S WHAT I WOULD SAY IS SORT OF A CLINICAL PRACTICAL DEFINITION OF A RESERVOIR. >> I SORT OF AGREE WITH THAT. >> I CAN'T REMEMBER WHO IT WAS, BUT SOMEBODY USED THE TERM REBOUND-COMPETENT RESERVOIR IN ONE OF THE EARLIER -- YOU'RE SHAKING YOUR HEAD, JOHN, THAT'S NOT A GOOD IDEA? I LIKE THAT IDEA. >> WELL, I DON'T, BECAUSE -- [INAUDIBLE] I LIKE IT IN THAT IT ADDS SOME CLAIREITY, BUT IN CLARITY, BUT IN MY MIND, THE RESERVOIR IS ANYTHING, ANY FORM OF THE VIRUS, ANY CELL THAT CAN LEAD TO REBOUND. AND THE RESERVOIR IS WHAT LEADS TO REBOUND. UNDER ANY CIRCUMSTANCE. >> THAT'S PRETTY MUCH, I THINK, WHAT DAVID WAS SAYING. >> OH, BUT YOU'RE NOT GOING TO HAVE REBOUND UNLESS IT'S REPLICATION-COMPETENT, SO I THINK IT'S REDUNDANT. >> [INAUDIBLE] >> NO, IT'S THE REBOUND THAT IS THE PHENOTYPE, THE CLINICAL PHENOTYPE. IT'S NOT WHETHER THE PRO VIRUS IS REPLICATION COMPETENT, BECAUSE A REPLICATION COMPETENT PRO VIRUS COULD SIT THERE FOR THE LIFETIME OF THE INDIVIDUAL IN HETEROCHROMATIN. >> [INAUDIBLE] >> NO, NO. YOU'RE SAYING REPLICATION-COMPETENT REBOUND. NO, NO, NO -- >> I THINK WE PRETTY MUCH AGREE -- >> I'M GOING TO USE THE TERM -- >> REBOUND-COMPETENT IS THE TERM THAT I LIKE. >> REBOUND-COMPETENT, FINE. [APPLAUSE] >> WE ACCOMPLISHED SOMETHING! >> WELL, WITH THAT, YES, PLEASE. LAST COMMENT. >> GIVEN THAT WE JUST SAW THAT GO FROM COMPLETE DISCORD TO COMPLETE AGREEMENT -- >> YES! >> I'M VERY CURIOUS HOW MANY OF THE OTHER THINGS THAT WE'VE TALKED ABOUT MIGHT BE THAT WAY IF WE WERE ABLE TO GET TOGETHER IN A ROOM AND HAVE MORE THAN ONE, 1 1/2 DAY LONG TIME. I THINK MOST OF THE PROBLEMS THAT HAVE BEEN PRESENTED, THERE ARE DIFFERENT PEOPLE IN THE ROOM THAT HAVE SOLUTIONS FOR. AND WE NEED A PROCESS TO MAKE THE MATCH MAKING. I HAVE NOT HEARD ONE QUESTION BROUGHT UP THAT I CAN'T MATCH SOMEBODY BUILDING TECHNOLOGIES COULDN'T SOLVE. IF THEY HAD THE GUIDANCE FROM THE RIGHT PEOPLE. I ALSO KNOW A LOT OF TECHNOLOGY DEVELOPERS THAT ARE MAKING TECHNOLOGIES THAT NOBODY IN THE WORLD WILL EVER WANT TO USE BECAUSE THEY DON'T SOLVE A PROBLEM. [LAUGHTER] SO IT'S A MOUSETRAP. ANYWAY, SO THE QUESTION IS, CAN WE IMAGINE MAKING A PROCESS THAT DOES THE SORT OF MATCH MAKING HE LOVE HERE, MAYBE LEADING TO HUGS AT THE END, BUT MAYBE INSTEAD OF HUGS AT THE END, IT LEADS TO DEFINING WHAT THE OPPORTUNITIES ARE AND THEN DOING THEM. AND THE FEW TIMES WE'VE DONE THAT BEFORE BY GETTING THE BUILDERS AND THE USERS TOGETHER, THE BUILDERS MADE THINGS THAT MATTERED MORE AND THE USERS GOT THE SOLUTIONS THEY NEED NEEDED. >> BRAVO, AND A WONDERFUL WAY TO END THE SESSION. I THINK THAT'S EXACTLY WHAT MIKE AND DIANA HAD IN MIND, AND WE'LL JUST SEE WHERE IT GOES FROM HERE. SO IT'S MIKE AND DIANA, IF YOU COULD GO AHEAD AND MAKE POTENTIAL OPPORTUNITIES, AS THE NAME OF THE SESSION. >> I'M NOT GOING TO TALK FROM THE BACK OF THE ROOM. THAT WAS REALLY MAGICAL, WHAT I JUST HEARD. [LAUGHTER] MICHELLE ASKED ME LAST NIGHT, WHAT AM I SUPPOSED TO DO ON THIS SESSION? DIANA, COME ON UP HERE. SO YEAH, THIS HAS REALLY BEEN AN INTERESTING MEETING AND I THANK ALL OF YOU FOR COME, I THANK THE SPEAKERS AND THE PANELISTS AND ALL OF YOU PARTICIPATING. I THANK YOU ESPECIALLY BECAUSE OF THE UNCERTAINTY THAT MANY OF YOU HAD WHEN YOU CAME. AS FRANCIS SAID, MANY DID WRITE TO SAY -- EVEN WHILE I WAS HERE, JOHN ASKED ME, WHY AM I HERE? IT WAS A BIT OF AN EXPERIMENT. AT DINNER LAST NIGHT, I WAS ASKED BY STEVE WHY WERE NOT THE EXPERIMENTAL GUIDELINES EXPLAINED FIRST. THAT WOULD HAVE BEEN PERTURBING IT. WHAT I WANT TO DO IS GIVE YOU NOW SOME BACKGROUND ABOUT WHY WE SET IT UP THIS WAY. AS I THINK MANY OF YOU KNOW, I'VE TALKED TO YOU OVER THE PAST YEAR IN A HALF, TO TRY TO MOVE THEM OFF THE DIME TO BRING CURATIVE INTERVENTIONS TO RESOURCE LIMITED PARTS OF THE WORLD. I NOT ONLY MEAN SUB-SAHARAN AFRICA, WHICH IS THEIR TARGET, BUT VIRGINIA, OTHER PLACES WHERE THE EPIDEMIC IS BOILING IN OUR COUNTRY. THAT HAS NOW CULMINATED IN A PROGRAM WHICH THEY'VE SANCTIONED AND IT'S MOVING FORWARD, AND IT'S BEING DONE IN LOCK STEP WITH THE NIH, AND THIS MEETING CULMINATED FROM THE EARLY DISCUSSIONS WHICH WE HAVE HAD AND WHICH STARTED ACTUALLY AS SOON AS I STARTED WORKING PART-TIME AT THE GATES FOUNDATION ABOUT THIS TIME LAST YEAR. IN WHICH WE'LL MOVE FORWARD. THIS IS AN ASPIRATIONAL GOAL. ANYTHING THAT'S GOING TO HAPPEN FOR HIV CURE AND RESEARCH, WE'RE TALKING 15 TO 25 YEARS. MY ORIGINAL HESITATION ABOUT WHETHER OR NOT IT WOULD BE ADOPTED BY THE FOUNDATION, EVEN THE ACADEMIC COMMUNITY REALLY IS NOT SET OUT TO DO THE KIND OF THING THAT WE'RE TALKING ABOUT. THERE'S GOT TO BE A MOTIVATION TO KEEP IT MOVING, INCENTIVES TO KEEP PEOPLE MOVING TOWARDS IT, AND THEN A PLAN THAT WILL ALLOW THAT TO HAPPEN AN THEN RESOURCES. THE FOUNDATION HAS PUT RESOURCES BEHIND IT, NIH WILL BE PUTTING RESOURCES BEHIND IT. WE HAVEN'T WORKED OUT ALL THE DETAILS OF HOW THIS WILL HAPPEN. THE GENERAL SCOPE OF REALLY WHAT HAS TO HAPPEN IS THIS. SO FIRST IF YOU IMAGINE GOING INTO RESOURCE POOR PARTS OF THE WORLD, ROUGHLY HALF ARE VIE REMIBG, THERE'S NO WAY THOSE 16 MILLION ARE GOING TO RECEIVE ANTIRETROVIRAL THERAPY, THE INFRASTRUCTURE JUST CAN'T BE BUILT. SO YOU HAVE TO HAVE AN INTERVENTION THAT WILL BE AMENABLE TO ADOPTION BY INDIVIDUALS WHO TEND NOT TO GO TO HEALTHCARE PROVIDERS EITHER BECAUSE -- OR BECAUSE THERE ARE NO DOCTORS. SO IT HAS TO BE SOMETHING THAT WORKS, IT HAS TO BE SOMETHING THAT'S SAFE, IT HAS TO BE SOMETHING WHICH I THINK NEEDS TO PREVENT REINFECTION BECAUSE I DON'T FORESEE A SITUATION IN WHICH INDIVIDUALS WILL NOT BE EXPOSED TO THE VIRUS IN THE FUTURE. IT HAS TO BE SOMETHING WHICH IS DURABLE. IT HAS TO BE SOMETHING WHICH PROBABLY CAN BE DELIVERED -- WELL, YOU KNOW, I THINK OF IT AS A SINGLE SHOT, THAT'S A EUPHEMISM IN A WAY, BUT SOMETHING THAT'S REALLY EASY. MAYBE IT'S TWO SHOTS, ONE IN EACH ARM. BUT A SINGLE ENCOUNTER, DONE AND OUT. A VACCINE. >> MAYBE NOT SO EASY. RIGHT NOW. >> SO BUT THE DREAM IS WE WOULD TAKE ADVANTAGE OF ALL THE TECHNOLOGY THAT HAS BEEN DEVELOPED OVER THE PAST SEVERAL DECADES, VECTORIZING VIRAL AND NON-VIRAL VECTORS, AN INTERVENTION THAT WOULD BE A PERCUTANEOUS INTERVENTION TO TAKE VECTORS THAT WOULD THEN BE DELIVERED TO THE RIGHT SPOTS, AND WHAT WOULD THOSE SPOTS BE FOR A DURABLE INTERVENTION, YOU KNOW, PROBABLY BE A HEMATOPOIETIC STEM CELL OR DIVIDING T STEM CENTRAL MEMORY CELL, WHAT WOULD YOU DELIVER, MAYBE YOU COULD KNOCK OUT VIRUS FROM ALL THE CELLS THAT ENG CYST IN THE BODY THAT HAVE VIRUS. WHO KNOWS. I MEAN, THIS IS ALL -- THIS IS NOT ROCKET SCIENCE NOW, PEOPLE ARE DOING THIS. MOST PEOPLE IN THE UNITED STATES ARE DOING IT EX VIVO, WELL, BECAUSE WE KNOW HOW TO DO IT AND THAT'S WHERE THE COMMERCIAL PLANS DO BEST. SINGLE SHOT IS NOT GOING TO BE ADOPTED BY MANY COMPANIES IN THE UNITED STATES BECAUSE THAT'S JUST NOT GOING TO MAKE MONEY. AND YET THE TECHNOLOGIES ARE THERE, THE CONFERENCE THAT KEITH MENTIONED IN AUGUST WILL SHOWCASE IN A SESSION, SO 12 DIFFERENT SPEAK ARES WHO ARE WORKING ABOUT HALF AND HALF ON VIRAL VERSUS NON-VIRAL VECTORS FOR IN VIVO DELIVERY. AND WHAT ARE MOST OF THOSE VECTORS NOW BEING DELIVERED TOWARDS? IT'S FOR TREATMENT OF MONOGENIC DISEASES. SICKLE CELL IS A GOOD EXAMPLE, RIGHT? WE KNOW PRECISELY WHAT TO DO WITH SICKLE. AND IT'SING DONE. IT'S BEING DONE. IS IT GOING TO BE ADAPTABLE THAT HAVE IT IN THE UNITED STATES? WELL, OUR DEBATES LAST NIGHT SUGGESTED TO ME PROBABLY NOT. IT'S JUST NOT GOING TO BE AFFORDABLE. IS IT GOING TO BE ADAPTABLE TO PEOPLE THAT ARE ACTUALLY IN SUB-SAHARAN AFRICA WHERE THE VAST MAJORITY, THE BURDEN OF DISEASE EXISTS? CERTAINLY NOT. BUT IF WE PUSH PEOPLE, I THINK IT MIGHT BE POSSIBLE TO DELIVER FOR SICKLE INTERVENTIONS BY IN VIVO. FOR HIV, IT'S A TOTALLY DIFFERENT MATTER. I HAVE WHAT I CALL MY BEST BETTS. YES, MICHELLE, INCLUDING AMONGST THEM AND WE GOT OUR CCR5 KNOCKOUTS, MAYBE WE CAN USE CRISPR TO KNOCK OUT EVERY GENOME IN THE BODY. THESE ARE THE BEST BETTS THAT WE HAVE NOW. I THINK THEY'RE WORTH PURSUING SO WE WILL, BUT DO I SLEEP WELL AT NIGHT WITH THOSE BEST BETTS? NO. ANYBODY THAT'S POSITIONED, ALL OF US, WE THINK ABOUT EXPERIMENTS, YOU THINK OF THE WORST-CASE SCENARIO, I THINK IN LIKE FIVE, 10 YEARS FROM NOW, IF WE FOLLOW THOSE BEST BETTS, WE'RE GOING TO FAIL. BECAUSE WE SIMILARLY DON'T UNDERSTAND THE BIOLOGY OF THIS BUG, HIV. SO THE OTHER PART OF THE PROGRAM THAT THE FOUNDATION HAS EMBRACED IS TO DO WHAT I CALL A DEEP DIVE ON THE RESERVOIR. IT COULD BE THAT WE'RE MEASURING, TRYING TO DEFINE IT. GOODNESS GRACIOUS, 30 YEARS AFTER THE EPIDEMIC, ARE WE HAVING APPLAUSE FINALLY? IT'S CRAZY. SO AS MANY OF YOU KNOW, WE'VE EMBARKED ON A PROCESS TO ENGAGE IN A NUMBER OF LABS, MANY LABS ACTUALLY INTO A CONSORTIUM, THIS IS NOT YET -- IT'S IN THE PROCESS OF BEING FORMED, TO DO THIS DEEP DIVE. IT WILL INVOLVE NON-HUMAN PRIMATES AS WELL AS HUMANS, HUMANS NOT JUST IN THE UNITED STATES BUT HUMANS WHO ARE LIVING IN SUB-SAHARAN AFRICA, ACKNOWLEDGING UP FRONT THAT THERE IS LIKELY -- WELL, TESTING THE POSSIBILITY THAT THERE MAY BE HETEROGENEITY IN THE RESERVOIR, WHATEVER IT IS, IN DIFFERENT PEOPLE OF DIFFERENT GENDERS OF DIFFERENT AGES IN DIFFERENT PARTS OF THE WORLD WHO HAVE DIFFERENT COINFECTIONS THAT COULD IMPACT UPON THE IMMUNE SYSTEM. THAT'S GOING TO BE A DURABLE EFFORT. IT'S GOING TO BE WELL -- NOT AS WELL RESOURCED AS SOME OF US WOULD LIKE BUT IT'S GOING TO BE WELL RESOURCED, AND IT'S GULF COAST TO MOVE GOING TO MOVE FORWARD. PART OF THE INTENT OF THIS MEETING WAS TO BRING TOGETHER PEOPLE WHO ARE THINKING ABOUT THE RESERVOIR AND THINKING ABOUT THE TECHNOLOGIES. I'M A FIRM BELIEVER IN THE FACT THAT TECHNOLOGIES DRIVE INTERACTIONS BETWEEN PEOPLE, PEOPLE THAT HAVE BIOLOGICAL QUESTIONS DRIVE THE AGENDA FORWARD. AS PART OF THIS, THIS IS A POTENTIAL OPPORTUNITY DISCUSSION. WHAT I WOULD DO IS CHALLENGE YOU OR BESEECH YOU TO USE THE OPPORTUNITY THAT YOU HAVE NOW TO THINK ABOUT HOW YOU MIGHT USE WHAT YOU'VE LEARNED IN THE PAST COUPLE DAYS OR WHAT YOU HAVE IN YOUR HEAD ALREADY TO DEVELOP EXPERIMENTS, SO THERE'S MANY DIFFERENT THINGS WE TALKED ABOUT, MANY DIFFERENT EXPERIMENTS THAT COULD BE DEVELOPED. TO ME, THE MOST IMPORTANT THING THAT WE NEED TO DO NOW IS TO HAVE A WAY TO QUANTITATE, REALLY, OR AT LEAST BE ABLE TO DETECT THE REBOUND-COMPETENT RESERVOIR. I'D LOVE TO BE ABLE TO DO IT IN THREE DIFFERENT WAYS. I ONLY -- ONE WAY, I'D LOVE TO BE ABLE, LIKE MY PATIENTS WHO HAVE CANCER, LUNG DISEASE, HEART DISEASE, OR ANY OTHER DISEASE, I'D LOVE TO BE ABLE TO QUANTITATE THE LEVEL OF DISEASE WHILE THEY'RE ON TREATMENT. SO QUANTITATE THE REBOUND COMPETENT RESERVOIR ON ART. IS THAT POSSIBLE? I DON'T KNOW. BUT I'VE ASKED -- I'VE CHALLENGED PEOPLE, IT'S REALLY INTERESTING SOMETIMES, WHEN YOU CHALLENGE PEOPLE TO DO IT, SOMETIMES THEY THROW IT OUT AND DISMISS IT. SOME PEOPLE WILL SAY OKAY, FINE, I'LL TRY. GO FOR IT, I'LL SAY. THE SECOND THING I'D LOVE TO DO IS HAVE A NON-VIRAL CIRCULATING MARKER OR SET OF BIOMARKERS THAT CAN TELL ME THAT SOMEBODY IS ABOUT TO REBOUND WITH VIRUS. WHY? FOR TWO REASONS. ONE IS, I'D LOVE TO LATER HAVE A DETECTION DEVICE THAT WILL SEE THAT SO THAT PEOPLE KNOW BEFORE THEY BECOME -- BEFORE THEY'RE THEN ABLE TO TRANSMIT. WE'VE ACTUALLY DONE MODELING, THERE'S A PAPER THAT JUST CAME OUT, A GROUP AT THE IMPERIAL COLLEGE, THAT IF WE CREATE AN INTERVENTION THAT DELIVERS A SO CALLED CURE INTO A HIGH INCIDENCE AREA, AND WE DON'T HAVE WAYS OF DETECTING FAILURE, THAT IT WILL LEAD TO AN INCREASE IN THE INCIDENCE OF HIV INFECTION IF WE CONTINUE THE WAY THAT WE ARE NOW WITH THE INFRASTRUCTURE THAT WE HAVE. I CAN SEND THIS OUT TO PEOPLE IF YOU'RE INTERESTED. IT'S ALL MODELING, TAKE IT FOR WHAT IT'S WORTH, BUT IT MAKES SENSE TO ME INTUITIVELY. ANY MODEL, MATH MATTEL MODEL THAT MAKES SENSE TO ME INTUITIVELY. THEY'VE GOT TO START. SO ANYWAY, I THINK IT'S IMPORTANT TO HAVE A MARKER THAT CAN TELL PEOPLE THAT THAT'S HAPPENING, A DETECTION DEVICE THAT CAN SEE IT. AND THEN FINALLY, I THINK IT'S HIGHLY LIKELY THAT WHEN WE TEST INTERVENTIONS AGAINST HIV, THAT AS IN THE CASE FOR EVERYOTHER INTERVENTION FOR EVERY OTHER DISEASE THAT I'VE EVER SEEN, WE'RE GOING TO HAVE TO HAVE INCLUSION AND EXCLUSION CRITERIA TO CAREFULLY STRATIFY GROUPS THAT ARE BEING TESTED. RIGHT NOW WE'RE THROWING EVERYBODY INTO THE SAME HOPPER. IF WE DID THAT IN THE EARLY STAGES OF THE EP DEIN IK, WE TOOK EPIDEMIC, WE CAME IN WITH EVERYBODY THAT HAD LATE STAGE DISEASE, AND SAID LET'S NOW PUT THEM INTO A CURE PROGRAM, EVERYTHING WOULD HAVE FAILED. IT'S HIGHLY LIKELY THAT WE'RE GOING TO HAVE DIFFERENT INTERVENTIONS AT DIFFERENT STAGES OF HIV DISEASE, AND WE HAVE TO HAVE A WAY TO QUANTITATE THEM. AND THE QUANTITATION MAY INCLUDE QUALIFIERS AS WELL. THERE MAY BE, YOU KNOW, DIFFERENT AMOUNTS OF VIRUS AND DIFFERENT TYPES OF CELLS IN THE RIGHT EARLOBE. I DON'T KNOW. WHAT I THINK IS CLEAR IS I FEEL VERY IGNORANT NOW ABOUT THE ASPECTS OF THE RESERVOIR THAT I'D LIKE TO SEE, AND I FEEL ACTUALLY QUITE FORTUNATE THAT WE HAVE NOW BACKING AND COLLABORATION TO TRY TO PUSH THIS DISCUSSION FORWARD, PRECISELY IN THE WAY IN WHICH YOU MENTIONED. I DON'T THINK THAT WE CAN DO THIS BY WORKING IN A UNIDISCIPLINARY VACUUM. THERE HAS TO BE DISCUSSION. SO ANYWAY, I WILL PUT OUT A WHITE PAPER TO DESCRIBE WHAT I'D LIKE -- I'M TALKING PRECISELY ABOUT -- THESE WOULD BE SHORT TERM HIGH RISK PILOT EXPERIMENTS THAT IF YOU HAVE TRAINING, YOU WOULD SAY GO FORWARD ON THIS PLANK AND ONLY WALK UNTIL THAT POINT, IF IT DOESN'T WORK, THEN STOP. THAT KIND OF EXPERIMENT. AND IT CAN BE -- IT WILL BE NESTED WITHIN, SUPPORTED BY RESOURCES FROM THE LARGER CONSORTIUM, SO I WOULD SEE THESE AS SUPPLEMENTARY GRANTS, IF YOU WILL, THAT WILL BE FLED IN THE LARGER CONSORTIUM, AND WE CAN TURN THESE AROUND QUICKLY. AUGUST 8TH, XIAO, YOU WANT TO GUESS THOUGH CELLS FROM TOM, I'M JUST JOKING, BUT WE CAN TURN THESE AROUND VERY QUICKLY. SO I'M GOING TO SEND THESE OUT, I INVITE YOU TO WRITE ME, TO CALL ME. THOSE OF YOU, IF ANYBODY IS OUT IN CYBERSPACE LISTENING, GET MY EMAIL, I'M VERY TOLERANT OF LISTENING TO IDEAS, BRING IT ON. SO THAT'S WHAT I'LL SAY. DIANA. SHE SAID SHE WOULD NOT WANT TO SAY MUCH, BUT -- >> RIGHT. MOST OF MY COLLEAGUES KNOW THAT I'M SPEAKING S NOT MY FAVORITE THING. SO I AM GOING TO ASK CARL TO COME UP AND GIVE CLOSING REMARKS BUT WHAT I WILL SAY IS THAT I'VE REALLY ENJOYED WORKING WITH YOU, AND I'M EXCITED BY THE OPTIMISM PARTICULARLY OF OUR PANEL PEOPLE HERE, ALEX AND THE PEOPLE FROM OUTSIDE THE FIELD, YOUR OPTIMISM IS WHAT WE NEED IN TERMS OF DOING THIS WORK, AND SO REALLY WANTED TO THANK YOU ALL. CARL? >> SO I GET THE PLEASURE OF TRYING TO CLOSE OUT A DAY AND A HALF MEETING WHICH I WILL ABSOLUTELY NOT TRY TO DO. I THINK THAT THE DISCUSSION SPOKE FOR THEMSELVES AND I THINK THIS LAST PANEL WAS ABSOLUTELY A PERFECT WAY TO END THE DISCUSSION WITH A LOT OF QUESTIONS. THE USUAL AMOUNT OF APPROPRIATE TENSION, I THINK ESSENTIAL FOR THE FIELD. I THINK THE KIND OF VISION THAT MIKE HAS IS ONE WHERE PEOPLE ARE COOPERATIVE, YET COMPETITIVE. BECAUSE I THINK WHAT DRIVES SCIENCE ULTIMATELY IS COMPETITION, BUT AT THE SAME TIME, THE COMMENT ABOUT CAN WE HAVE A CLEARINGHOUSE FOR DEAD DUMB IDEAS, I THINK IS A REALLY GOOD ONE. AT THE SAME TIME, LET'S TAKE A STEP BACK. WHERE ARE WE IN THE HIV EPIDEMIC IN 2019 HEADED INTO 2020? WE HAVE AMAZING MEDICATIONS THAT ARE ONE PILL ONCE A DAY, WITH ANY LUCK AT ALL WITHIN A COUPLE OF YEARS, WE WILL HAVE MEDICATIONS THAT ARE INJECTABLE OR IMPLANTABLE FOR TREATMENT AND PREVENTION THAT COULD LAST SIX MONTHS OR A YEAR. THEY CAN BE THE BASIS OF MIKE'S ONE-SHOT CONCEPT FOR THERAPY. YOU COULD HAVE DRUG IN ONE ARM AND CURE IN THE OTHER. ADDITIONALLY, WE NEED PREVENTION. WHEN IT ULTIMATELY IS DRUG BASED OR VACCINE, WE HAVE TO CONTINUE TO WORK ON IT. THOSE ARE OUR TWO MAJOR CHALLENGES. THEY'RE NOT NECESSARILY DISASSOCIATED. IMMUNITY IS AN ESSENTIAL COMPONENT OF VIRUS CONTROL, LAST TIME I CHECKED, AND I THINK WE NEED TO CONTINUE TO WORK IN PARALLEL WITH OUR COLLEAGUES ON THE PREVENTION VACCINE SIDE AS WELL AS THE PREVENTION DRUG SIDE. MOVING FORWARD, WHEN I THINK ABOUT THE SAMPLING QUESTION HERE TODAY, THAT'S OUR FUNDAMENTAL CHALLENGE, ULTIMATELY WE'RE GOING TO BE SAMPLING SOMETHING THAT IS AN ULTIMATELY INCREDIBLY RARE EVENT. SO AS MIKE SAID, WE'RE GOING TO NEED TO BE ABLE TO HAVE SURROGATE MARKERS FOR REBOUND. THAT'S A GIVEN. BUT AT THE END OF THE DAY, WE NEED TO -- AND WE TALK ABOUT CHARACTERIZATION. IT'S NOT JUST ENOUGH TO CHARACTERIZE BECAUSE WE CAN DEFINE LIKE LEWIS SAID, IT'S GREAT TO DO HNEs AND UNDERSTAND AND CAT CATEGORIZE DISEASE, BUT WHAT DOES THAT TELL US ABOUT MECHANISM? I THINK WE CANNOT LOSE SIGHT THAT THERE ARE FUNDAMENTAL MECHANISMS WE STILL DON'T UNDERSTAND, AND THAT HAS TO BE ALSO A FOCUS OF THIS RESEARCH ENDEAVOR, BECAUSE IT HAS TO BE AN ITERATIVE CYCLE BETWEEN BASIC TO MONKEYS TO UNDERSTAND AT A MECHANISTIC LEVEL BEFORE WE JUMP IN TO PEOPLE SO WE HAVE SOMETHING THAT WE ARE CONFIDENT WE CAN TEST. ULTIMATELY PHASE 1 EXPERIMENTAL MEDICINE TRIALS ARE ESSENTIAL, BUT WE ALSO HAVE TO HAVE A SENSE THAT THEY'RE GOING TO DO SOMETHING, AND SO FAR, WE HAVEN'T TESTED MUCH THAT CAN DO ANYTHING'S. WE HAVE YET TO BUDGE THE RESERVOIR, BUDGE THE NEEDLE. I THINK IF I CAN PICK ON JOHN A MINUTE, BACK TO THE EARLY 90s, WHEN PCR WAS COMING UP AND WE HAD NO WAY OF REALLY UNDERSTANDING WHAT WAS HAPPENING, AND THEN JEFF AND OTHERS PUBLISHED THE PAPER ABOUT CONSTANT VIRAL LOAD, WE KNEW THEN WE HAD AN ASSAY THAT COULD MAYBE GIVE US A DYNAMIC RANGE. MAYBE THE NEW ASSAY THAT HAS BEEN DEVELOPED BY THE SILL COULD KNOW LAB AND NOW IT SEEMS TO BE MOVING FORWARD WILL BE THE TOOL THAT WE NEED, BUT WE HAVE TO START AND TEST IT AND IF IT'S NOT THE ASSAY, MAKE A BETTER ONE. SO IN SUMMARY, I AM THRILLED BY HAVING THIS NEW PARTNERSHIP WITH THE GATE FOUNDATION. THE DIVISION OF AIDS HAS HAD MAJOR PROJECTS THAT HAVE TENDED TO BE VERY CLINICAL WITH THE GATES FOUNDATION. YOU THINK ABOUT THE BIG TRIALS THAT GATES AND NIAID HAVE GONE THROUGH OVER THE YEARS. THIS IS AN OPPORTUNITY TO DO SOMETHING NEW AND UNIQUE WITH THE FOUNDATION, AND IT'S GOING TO BE A LOT OF FUN AND SOME CHALLENGE, BUT MIKE STARTED TALKING TO US A YEAR AGO, AND IT TOOK US ABOUT A YEAR TO GET TO THIS POINT. SO I'M REALLY THRILLED TO HAVE BEEN ABLE TO STAY FOR THE WHOLE MEETING AND PARTICIPATE AND HEAR THIS, BUT AGAIN, ANOTHER COMMENT ABOUT FINDING PARTNERS. THE BEST MARRIAGES ARE MADE BY YOU. YOU DO NOT NECESSARILY WANT THE NIH TO BE SAYING HERE'S YOUR PARTNER, GO DANCE. IT DOESN'T WORK. SO I ENCOURAGE EVERYBODY TO TAKE THE LIST OF ATTENDEES, STAY IN TOUCH WITH EACH OTHER, AND TAKE MIKE UP ON HIS WORD BECAUSE HE IS A LOT MORE TOLERANT THAN I AM ABOUT EMAILS. JUST KIDDING. BUT IN THE MEANTIME, LET'S CONTINUE THE DIALOGS THAT WE STARTED HERE TODAY, KEEPING IN MIND THAT IT'S GOING TO TAKE SIGNIFICANT DISCOVERIES, BEING ABLE TO BE TRANSLATED AND UNDERSTOOD TO NON-HUMAN PRIMATES AND ULTIMATELY WE WILL NEED COMPANIES AND OTHERS TO COME TOGETHER TO HELP US BUILD THE MOLECULES THAT CAN BE TESTED, BECAUSE NONE OF US HAVE THE SKILL SET OR THE MONEY TO TAKE THINGS TO A SCALE TO IMPLEMENT AN HIV CURE. SO I'LL STOP THERE, AND OFFICIALLY CLOSE THE MEETING. I THINK A MINUTE OR TWO EARLY. EXACTLY? OR A MINUTE LATE. OH, WELL. WE'LL TAKE IT -- SO THANK YOU ALL FOR YOUR PARTICIPATION. I WOULD LIKE TO ALSO THANK DIANA AND MIKE AND IN THE BACK -- ANNE IN THE BACK, FOR HER TREMENDOUS EFFORTS. [APPLAUSE] THE PEOPLE IN THE BASIC SCIENCE PROGRAM AND THE DIVISION OF AIDS THAT ALSO CONTRIBUTED AND ALL OF YOU FOR JOINING US HERE AT THE CLOISTERS.