>> ALL RIGHT. GOOD MORNING,
EVERYBODY. ON BEHALF OF THE
ENTIRE NIH DYNAMICS AND
REPLICATION I WOULD LIKE TO
WELCOME YOU FOR VIRUSES AND
RESEARCH AND MEDICINE. BEFORE
WE JUMP INTO THE PROGRAM I WOULD
LIKE TO BRIEFLY MENTION A FEW
UPCOMING EVENTS TO THIS
AUDIENCE. NOVEMBER 2ND WE WILL
HOLD THE 12TH DAVID DURSY AWARD
AND SPEAKER IS FROM UNIVERSITY
OF MINNESOTA -- THROUGH
COMPARATIVE ANALYSIS NOVEMBER
11TH UNIVERSITY OF MARYLAND
COLLEGE PARK PROGRAM WILL HOLD
THE FALL RESTRATES AND VERY
DISTINGUISHED GROUP OF SPEAKERS
INCLUDING DANY AND OUR OWN ALEX
COMPTON. IF YOU WOULD LIKE TO
ATTEND AND HAVEN'T ALREADY
CONTACTED ME, REACH OUT AND WE
WILL PUT YOU ON THE LIST.
DECEMBER EIGHTH, NIH WILL HOLD
THE 25TH SYMPOSIUM IN AWARDS AND
BASIC AND CLINICAL VIROLOGY.
YOU CAN CHECK OUT THE WEBSITE
FOR MORE INFORMATION. DECEMBER
EIGHTH, THE NCI CRYPTER FOR
EXCELLENCE AND HIV CANCER
VIROLOGY WILL HOLD THE FALL
THINK TANK AT NATURAL CONFERENCE
CENTER IN BETHESDA AND CHAIRS
FOR THIS CORRECTOR OF
EXCELLENCE, YOU CAN CONTACT THEM
AT U MASS FOR ADDITIONAL
INFORMATION AND FINALLY WOULD
LIKE TO MAKE A PLUG FOR THE
FIFTH INSTALLMENT OF THE
INTERNATIONAL VIRUSES CONFERENCE
SPONSORED BY VIRUSES, VIRUSES
2024 WORLD OF VIRUSES HOLD IN
BARCELONA SPAIN FEBRUARY 14TH OR
16TH AND GREAT GROUP OF SPEAKERS
IS CHARLIE AND LOUIS AND LORDES
AND CHECK OUT THE WEBSITE AND
REGISTER AND THERE IS A PROGRAM
FOR TALKS AND POSTERS DRIVEN BY
ABSTRACTS.
OKAY. BACK TO THE FOCUS OF
TODAY.
AS MEMBERS OF SOCIETY AND
SCIENTISTS, WE ARE USED TO
THINKING OF VIRUSES AS ENEMIES
OR AT LEAST CYBER SERIES.
WE HAVE SEEN A WAVE AFTER WAVE
OF EPIDEMICS AND PANDEMICS
EMERGING AND RE-EMERGING AND
SARS V COVID 2 AND MERS AND
RESPIRATORY AND [INDISCERNIBLE]
ZIKA EBOLA AND INFLUENZA POX
REMAINING CONSTANT THREAT.
VIRUSES HAVE A POSITIVE SIDE
APART FROM ALL THIS.
VIRUSES SERVE AS PLATFORMS FOR
VIRUS BASED VACCINES
IMMUNOTHERAPY AND VIRAL FACTORS
AND FAMOUSLY PROBABLY CC1
INVOLVED IN PRIMARY PLACENTA
DEVELOPMENT AND USED IN
RESTRICTION FACTORS TOWARDS THE
END OF THE DAY AND THOUGHT IT
WOULD BE FUN TO EXPLORE TOPICS
IN THIS YEAR'S CONTENTS AND
ASSEMBLING AN ALL-STAR CAST TO
TELL US ABOUT THEIR WORK IN THIS
AREA.
FROM DUKE, WE HAVE JEN FROM NCI
AND SARAH WOOTON FROM ONTARIO
VETERAN COLLEGE UP IN CANADA AND
RECRUITING FROM BOSTON
UNIVERSITY AND RECRUIT BACK TO
HARVARD AND FROM OREGON HEALTH
AND SCIENCES UNIVERSITY AND CHIP
FROM UCSD AND DAVID FROM
UNIVERSITY OF MINNESOTA AND FIN
WILL I JOHN COUGHIN FROM TUFTS.
WITHOUT FURTHER ADO, I WOULD
LIKE TO INTRODUCE THE FIRST
SPEAKER MATAIS FROM UNIVERSITY
OF SDERM -- DISCONTINUED TO THE
PRESENT AND THIS TALK IS
ENTITLED CANCER AND NEW THERAPY
WITH COMMON POLIO VIRUS.
MATTIAS.
>> AUDIENCE: [APPLAUSE].
>> THANK YOU VERY MUCH FOR THE
INTRODUCTION.
THANK YOU VERY MUCH FOR HAVING
US FOR THIS IN-PERSON EVENT. IT
IS KIND OF EMOTIONAL TO ME THE
VIRUS I WILL TALK YOU TO ABOUT
WAS MANUFACTURED ACROSS THE
STREET. THE CLINIC IS GREAT.
SO, I'M GOING TO UPDATE YOU
WHERE WE ARE WITH CANCER
IMMUNOTHERAPY AND BRAIN TUMORS
COMBINO POLIO VIRUSES AND IN MY
VIEW SCIENCE OF WHAT WE HAVE
LEARNED IN THE PAST TWO YEARS.
I'M CONFLICTED AND USED TO GET
PAID BY A COMPANY AND HOLD
EQUITY IN A COMPANY THAT
DEVELOPS THIS THERAPY.
SO, YOU ALL HAVE SEEN THIS.
THIS IS HOW EVERYBODY, MORE OR
LESS, AGREES ENTERTUMOR THERAPY
WORKS AND WE HAVE DENDRITIC
CELLS AND CDC ONES AND
CROSS-[INDISCERNIBLE] 2
ANTROEJENS AND T-CELL IMMUNITY
AND HOPEFULLY T-CELL WILL ENTER
THE TUMOR KILLING CELLS.
THOSE PATIENTS WHERE THIS IS
SOMEWHAT WORKING HAVE BENEFIT TO
RESPONDING TO IMMUNOCHECKPOINT
BLOCKADE THAT WORKS HERE IN
THESE STAGES AND VAST MAJORITY
OF CANCER PATIENTS DON'T HAVE
THIS WORKING. THIS IS TRUE FOR
BRAIN TUMORS AND CHECKPOINT
COMPLETELY FAILED IN BRAIN TUMOR
PATIENTS THAT WE CAN SAFELY
ASSUME THAT VIRTUALLY NO BRAIN
TUMOR PATIENTS HAVE INTERTUMOR
IMMUNITY.
WHAT IS THE PROBLEM.
I AGREE WITH WHAT IS STATED BY
IRA MELMAN AND MANY COLLEAGUES.
THE PROBLEM IS HERE AND IT IS
TUMOR ANTROEJEN TRAFFICKING
CROSS-[INDISCERNIBLE] T-CELL
PRIMING AND WAY TO FIX THIS IS I
AGREE WITH MOST LEADING EXPERTS
IS ENDOGENOUS SUSTAINED TYPE 1
INTERFERON CDCS AND WILL TRY TO
CONVINCE YOU THIS IS VERY
DIFFICULT TO ACHIEVE. WE GOT IT
WITHOUT TRYING.
THAT IS WHAT I WILL TELL YOU.
THE VIRUS I WORK WITH IS PDS AND
BRIEFLY EXPLAINED WHAT IT IS.
WILD TYPE POLIO VIRUS MAHONEY
AND LAB STRAIN USED AND TYPE 1
VACCINE THAT IS A VACCINE THAT
EVERYONE IN THIS ROOM GOT AND
LIFE ATTENDED VACCINE IS A
TISSUE CULTURE ADAPTIN OF THIS.
WE TOOK THE TYPE 1 SALINE
VACCINE AND REPLACED IT AND I
WILL EXPLAIN BRIEFLY WHAT IT IS
A LITTLE BIT LATER WITH
CORRESPONDING ELEMENTS FROM
HUMOON VIRUS TYPE 2.
SO, I MADE THIS VIRUS MYSELF IN
1996.
IT IS EMBARRASSING. WE FIGURED
OUT NOW WHAT MAKES THIS
INTERESTING. IT IS WAY MORE
INTERESTING THAN WE THOUGHT AND
TOOK AN ETERNITY TO FIGURE THIS
OUT. I WILL TELL YOU.
WHAT THIS VIRUS IS, WE HAVE
THREE TYPES OF CELLS NATURALLY
TARGETED BY POLIO VIRUS.
THESE CELLS ARE KILLED
EFFICIENTLY AND NEUR OWNIAL
CELLS AND MYELOID CELLS AND
CERTAIN EPITHELIAL CELLS SUBSET
IN GI TRACT AND LYMPH NODES AND
HIGHLY CRYTOPATHOGENIC IN CELLS
AND NEUROLINEAGE PHENOTYPE AND
NOT MUCH IN OTHER TYPE NONNEURAL
CELLS AND SOMEWHAT OF A MYSTERY
WHY IT WAS SAFE IN USE OF
VACCINE AND VIRUS WE WORK WITH
IS COMPLETELY DIFFERENT AND
CRYTOPATHOGENIC AND WON'T EFFECT
OR KILL THEM AND ONCHOLYTIC
VIRUS THAT WORD IS NOT ALLOWED
IN MY LAB NOT SURE IF CAN KILL
MALIGNANT CELLS CAN KILL CELL
LINES BUT IS A TISSUE ARTIFACT.
REPORTING IN 2018 SUCCESSFUL
CLINICAL TRIAL [INDISCERNIBLE]
BLASTOMA COMMON BRAIN TUMOR THAT
HAS NO ADEQUATE THERAPY --
CHEMOAND RADIATION THAT THEY
ALMOST INVARIABLY DO AND FOR
THIS CONDITION IS NO APPROVED
THERAPY.
WE TREATED PATIENTS WITH A
SINGLE INTERTUMOR INOCULATION OF
PDS RIPO AND TUMOR WE INJECTED
HERE AND SAW THE CLASSIC
IMMUNOTHERAPY TAIL.
YOU HAVE A SUBGROUP OF PATIENTS
DOING EXTREMELY WELL AND
BECOMING APPARENT LATE AFTER
THERAPY AND PATIENT TAIL LOOKS
LIKE THIS. INJECT A TUMOR AND
OVER A VERY LONG PERIOD,
SOMETIMES CAN TAKE FIVE YEARS,
THE TUMOR SLOW LY DISAPPEARS AS
SEEN IN MRIS AND WHEN WE PUBLISH
THIS WORK, WE GOT INVITED BY
DIRECTOR OF SOOEBERITY OF PETER
MARKS TO ASK FDA FOR ACCELERATED
APPROVAL.
THAT DID NOT GO OVER TOO WELL
FOR SEVERAL REASONS AND POINT
OUT MOST IMPORTANT ONE. WE HAVE
80% OR SO OF PATIENTS DERIVING
ABSOLUTELY NO BENEFIT OF THIS.
THAT IS THE PROBLEM.
I WILL TELL YOU TODAY WHAT WE
WILL DO TO FIX THAT AND STARTED
A NEW TRIAL THAT IS COMPLETELY
DIFFERENT AND REVOLUTIONARY FOR
BRAIN TUMORS AND WILL EXPLAIN
WHY WE DO THIS.
THE MOST IMPORTANT EXPERIMENT IN
MY CAREER IS THIS.
SO, WHAT WE TRY TO DO HERE IS TO
UNDERSTAND WHAT THE VIRUS DOES
IN BRAIN TUMORS AND USE EX-VIVO
TOMBO CITES ASSAY. SUPER
INTERESTING IF THINKING ABOUT
DOING IT, DO IT. WE TAKE TUMOR
TISSUES FROM THE OR FRESH TUMOR
TISSUES NOT PROCESSED BUT WE
DISSECT IT AND [INDISCERNIBLE]
THAT IS AN AGONIST FOR MDA5
PATTERN RECEPTOR. C GAS ALSO.
WE PICK THESE AGONISTS BECAUSE
THEY ARE THE CLASSIC TYPE 1
INTERFERON GENERATING PATHWAYS
IN THE INNATE IMMUNE SYSTEM. WE
TREAT THE SLICES AND MEASURE
CYTOKINE RESPONSES AND SEE IN
BLUE THE CYTOKINE RESPONSE TO
PDSRIPO THAT STICKS OUT BECAUSE
OF SOMETHING WE CALL TYPE 1
INTERFERON DOMINANT INFLAMMATION
POLO CANE TB1 SIGNATURE THAT IS
STATE OF POINT HERE IS
INTERFERON ALPHA THAT I WILL
EXPLAIN IS VERY IMPORTANT
EXPLAINING WHY THIS IS HAPPENING
INCAPABLE OF INTERFERON RELEASE
WITH INNATE AGONIST
IMMUNOTHERAPY TESTED
RIBOSTAND-OUT SIGNATURES
SUSTAINED INTERFERON RELEASE AND
SEE THAT TLRS PRODUCE SOMETHING
COMPLETELY DIFFERENT AGONIST.
IT IS MORE A CUP OF B CYTOKINE
SIGNATURE TNF10 AND IT IS DEVOID
OF CYTOKINE RELEASE SYNDROME
CYTOKINES AND THIS INTERFERON
RESPONSE STEMS FROM MACROPHAGES.
SO, I THETA IS ISG AND SEE
SIGNATURE IN MACROPHAGES AND
TREATING TUMOR WITH PDS RIPO YOU
DON'T KILL ANYTHING, NO
ONCOLSIS. YOU SEE INFECTION OF
MYELOID CELLS AND SEE AN
INTERFERON SIGNATURE.
SO, I HAVE TO EXPLAIN TO YOU THE
BRILLIANT STRATEGY THAT POLIO
VIRUS USES THAT IS COMPLETELY
DIFFERENT FROM RNA VIRUSES
COMING TO THE INNATE RESPONSE
AND IN CONTRAST TO MESSENGER RNA
A 5 PRIME METHO GUAN ADEAN CAP
USED FOR TRANSLATION AND EF
TRANSLATION FACTOR TO THE CAP
USED TO RECRUIT ERG FOR THE
RIBOSOME AND RNA VIRUSES HAVE A
PROBLEM. THEY DON'T ACQUIRE A
CAP STRUCTURE LIKE MESSENGER
RNAS DO IN THE N NUCLEUS.
THEY ACQUIRE CAP SOMEHOW ON
THEIR OWN.
POLIO VIRUS. I USE THE WORD
POLIO BUT MEAN ENTROE VIRUSES
USE SOMETHING ENTIRELY
DIFFERENT. THEY HAVE A PROTEIN
COVALENTLY LINKED TO THE 5 PRIME
END AND POLIO VIRUS CAN'T U
USE-DEPENDENT TRANSLATION LIKE
MOST RNA VIRUSES DO. IT HAS TO
USE A METHOD OF TRANSLATION WITH
INTERNAL ENTRY SITE THAT IS
DISCOVERED NECKERITIES LAB AND
ENTRY WORKS ABOUT I RECRUITING
EFRG DIRECTLY TO THE VIRAL RNA
THROUGH THE VIRUS WITHOUT THE
NEED FOR E.
WHAT POLIO VIRUS DOES THEN IS
CLEAVES THE EFR G-PROTEIN SUCH
THAT THE HOST CAN NO LONGER
DIRECT PROTEIN SYNTHESIS LACKING
PORTION OF EFRG THAT RECRUITS
THE RIBOSOME BUT VIRUS CAN
CONTINUE TRANSLATING BECAUSE C
TERMINAL PORTION OF EFRG IS
SUFFICIENT TO RECRUIT RIBOSOMES
TO THE VIRAL GENOME.
THIS STRATEGY HAS HUGE
IMPLICATIONS FORINATE IN
CONTRAST TO RNA VIRUSS THAT IS
PREDOMINANTLY SENSED IN
CYTOPLASM REGULARI POLIO VIRUS
IS EXCLUSIVELY SENSED BY MDA5
THAT IS VERY IMPORTANT FOR WHAT
WE ARE DOING.
YOU CAN SEE THIS PLAY OUT HERE.
I WANT TO EXPLAIN WHAT RIPO IS
AND WHY I'M SAYING IT IS NOT
CRYTOPATHOGENIC AND NOT ALLOWED
TO USE WILD-TYPE POLIO ANYMORE
AND CLOSE RELATIVE TO JUST
IGNORE THIS.
IT IS WHAT WILD TYPE POLIO LOOKS
LIKE AND PAIRING FOR EFRG
CLEAVAGE THAT IS VERY RAPIDLY
CLEAVED BY WILD TYPE INTRO
VIRUSES TO CAP 21 AND SEEING
CLEAVAGE PRODUCT HERE THAT IS
VIRAL PROTEIN.
AS A RESULT OF THIS CLEAVAGE,
THE HOST CELL IS INCAPABLE OF
MOUNTING INNATE INTERVIRAL
RESPONSE.
SO, WE REPRODUCE THE CLASSIC
TYPE 1 INTERFERON RESPONSE THAT
IS DESCRIBED BY [INDISCERNIBLE]
ET AL 2000 AND EXPLICITLY
EXPRESSED IRF3 AND SEE
INTERFERON RELEASE AND IR3
ACTIVATION AND PHOSPHOLATION 3
DOES OCCUR AND DOESN'T RESULT IN
A PRODUCTIVE INTERFERON RESPONSE
WITH WILD TYPE VIRUS BECAUSE WE
DON'T SEE PHOSPHO STAT 1.
ENTROE VIRUSES FAMOUSLY AND OLD
VIROLOGISTS AND MANY DECADES AGO
KNEW THIS THEY DON'T MAKE
INTERFERON RESPONSES.
THIS RIPO VIRUS BECAUSE OF IRIS
IS INCAPABLE OF CLEAVING EFRGS
IN-TACT EFRG PREVAILS AND
SURPRISINGLY IS VIRAL ARCTIC
TRANSLATION IN THE CELLS AND
LOOK AT UNOPPOSED TYPE 1
INTERFERON RESPONSE.
PHOSPHORYLATION 3 AND MASSIVE
INTERFERON RELEASE AND INDUCTION
OF STAT 1. HERE IS INTERESTING
PARTS OF THIS. IT IS IRF7 AND
THIS IS AN INTERFERON
TRANSCRIPTION FACTOR THAT DRIVES
TRANSCRIPTION OF INTERFERON
ALPHA ISOFORMS.
VERY LATE MD5 INDUCTION THAT
HASN'T REACHED ITS PEAK BY 60
HOURS.
SO, WHY IS THIS SO SIGNIFICANT.
THE LATE INTERFERON RESPONSE AND
INTERFERON AUTHORED RELEASE AND
PRIMATE EVOLUTION WE ACQUIRE
APPLICATION OF INTERFERON ALPHA
GENES OVER TIME AND 12 DIFFERENT
INTERFERON ALPHA PROTEINS AND
MOST PEOPLE AGREE IT IS UNKNOWN
WHY WE HAVE THESE AND WHAT THE
EVOLUTIONARY BENEFIT IS. MOST
PEOPLE AGREE WE HAVE THESE
ISOFORMS TO BE ABLE TO EXTEND
INTERFERON RESPONSES TIME-WISE.
YOU CAN ACHIEVE DIFFERENT
PATTERNS BY HAVING MULTIPLE
TYPES OF INTERFERON A--LE FA
GENES.
THAT IS WHAT YOU SEE HERE.
THIS IS SUPER LATE SUSTAINED
INTERFERON ALPHA RESPONSE DUE TO
UNOPPOSED MDA-5 ORCHESTRATED IN
SENSING OF PDSRIPO THAT CAN'T
OPPOSE THE INTERFERON RESPONSE.
IT CAN'T CLEAVE THE EFRG.
THIS IS REALLY INTERESTING IN
THE NATURAL TARGET FOR FOLIO
VIRUS THAT WE SEE IN THE SLIDES
I WOULD SAY AND THOSE ARE
MYELOID CELLS.
THESE ARE MDMS.
AGAIN WHAT I SHOWED YOU BEFORE
IS IN THE CANCER CELL LINE AND
MDM AND MONOCITE MACROPHAGES NOT
INFECTED WITH PDS RIPO OR
INFECTED WITH CLEAVAGE AND LOW
VIRAL TRANSLATION AND MASSIVE
TYPE 1 INTERFERON DEFENSE AND
PHOSPHORYLATION ARE 3 AND FOR
FORILATION OF 7 INDUCTION AND
ESIG EXPRESSION AND THESE CELLS
RESPOND BETTER THAN ANY OTHER
TYPE OF CELL BECAUSE OF HIGHER
LEVELS OF MDA5 AT BASELINE.
AGAIN, HERE AT 48 HOURS IN MDMS,
WE SEE INTERFERON THAT IS A
DOMINANT RESPONSE WITH TYPE 13
INTERFERONS LACKING
[INDISCERNIBLE] CYTOKINES
CONTRASTING TO TLR AGONISTS AND
REALLY INTRIGUING.
WHEN WE LET CELLS SIT IN THE
INCUBATOR WE DON'T FEED THEM OR
ANYTHING JUST LET THEM GO AND
MDMS MIGHT NOT SURVIVE LONGER
THAN FIVE DAYS. THESE CELLS
WILL LIVE AT LEAST A MONTH.
TAKING THEM OUT AFTER 2 WEEKS
RUNNING CRATO KIND ESSAYS SEE
PDS RIPO MACROPHAGES SECRETE
INTERFERON ALPHA AND NOTHING
ELSE AND THESE MOCK INFECTED
CELLS ARE DEAD.
THIS IS WHAT WEIN DODGE NOWS
SUSTAINED TYPE 1 INTERFERON
BELIEFS MYELOID CELLS HOLY GRAIL
IF YOU WANT IN CANCER
IMMUNOTHERAPY AND PDRIPO THAT IS
INTRIGUING AND A SUSTAINED
SIGNATURE.
WHY IS THIS HAPPENING?
I WON'T GO INTO LOTS OF DETAILS
AND DURATION OF THE RESPONSE
THAT IS PATTERNS THAT DICTATE IT
BY VIRAL RNA REPLICATION AND IF
YOU SEE HERE FORGETTING THE
TREATMENT THAT IS NOT IMPORTANT
HERE AND SEE INNATE RESPONSE
THAT IS INTERFERON SIGNALING
CORRELATES WITH ABUNDANCE OF
VIRAL RNA AND TRANSLATION IS
ALMOST GONE BY 48 HOURS AND
VIRAL RNA REPLICATION STARTS TO
TAKE OFF AND VIRUS INCAPABLE OF
DRIVING VIRAL TRANSLATION
EFFICIENTLY AND CLEAVING FRG IS
CAPABLE ENOUGH TO DIRECT VIRAL
RNA REPLICATION THAT FUELS AN
INTERFERON RESPONSE.
SO, WE HAVE SHOWN THIS
QUALITATIVELY SUPERIOR INNATE
PATTERN THAT IS IMPORTANT IN
CANCER IMMUNOTHERAPY.
WHEN YOU TREAT B-16 MELANOMAS
WITH POLY-RIPOCLS YOU SEE
TRANSLATION -- YOU SEE WAY MORE
CYTOKINES AND T-CELLS COMING IN
WITH AGONISTS ARE STUPID T-CELLS
AND ARE CONFUSED AND DON'T KNOW
WHAT THEY ARE DOING.
LOOK AT RIPO T-CELLS THAT ARE
[INDISCERNIBLE] POSITIVE AND
INTERFERON GAMMA POSITIVE AND
QUALITATIVELY ARE BETTER T-CELLS
THAT YOU CAN ACTUALLY SEE THIS
IN THIS IN THE PICTURE YOU CAN
SEE THEM AND IS QUITE
IMPRESSIVE. HUGE RIPO AND
BIGGER THAN [INDISCERNIBLE] AND
WE HAVE QUALITATIVELY BETTER
INNATE PATTERN WITH PBSRIPO
GENERATED T-CELLS ARE CAPABLE TO
[INDISCERNIBLE] TUMOR AFTER A
TRANSFER.
OKAY. MOVING, I EXPLAINED TO
YOU INNATE PATTERN THAT WE ARE
EXCITED ABOUT.
NOW, WHAT ABOUT THE SPATIAL PART
OF IT?
WHICH CELLS ARE TARGETED?
THIS IS THE MOST IMPORTANT SLIDE
IN THE TALK.
POLIO VIRUS WAS MISUNDERSTOOD
FOR ALMOST A CENTURY.
WHAT DO I MEAN BY THAT?
IN THE 50S, THERE WAS INTRIGUING
WORK IN CHIMPANZEES BY MULTIPLE
PEOPLE AND THIS DAVID'S WORK AND
IS EASY TO DETERMINE POLIO VIRUS
DISTRIBUTION CAN TAKE UP ORGANS
AND SAY THAT IS WHAT THEY DID.
THEY SAW VIRUS IN LYMPHATIC
STRUCTURES AND SO IT IS NOWHERE
ELSE AND DON'T UNDERSTAND WHY IT
HAPPENS AND MAIN SITE OF VIRUS
REPLICATION IS THE LYMPHATIC
SYSTEM AND INTERESTING STUDY IN
PRIMATES PUBLISHED IN 2017 AND
NIH A CO-SPONSOR OF THIS. I SAW
THIS AND ALMOST FELL OUT OF MY
CHAIR. WE HAD NEVER KNOWN --
SO, THIS LYMPHATIC PRESENCE OF
VIRUS WAS NEVER UNDERSTOOD BUT
ACTUALLY MISINTERPRETED IN THE
50S.
PEOPLE DIDN'T KNOW WHAT TO MAKE
OF IT.
IN THE STUDY WE BEGAN TO
UNDERSTAND WHAT IT IS ABOUT.
THEY IDENTIFIED THE KEY TARGET
FOR POLIO VIRUSES, WILD TYPE
POLIO IN PRIMATE SYSTEM ARE CFRG
MACROPHAGES AND LYMPHOCYOCYTES
SPECULATE POLIO VIRUS IS LYMPHO
TROPIC VIRUS TARGETING CDCS
USING THEM TO HITCHHIKE A RIDE
TO DRAGGING LYMPH NODES WHERE IT
REPLICATES AND WE TESTED THIS
WITH PBS RIPO AND IN THE BRAIN
WE HAVE INTRIGUING LYMPHATIC
DRAINAGE SYSTEMS IN 2015 SYSTEM
OF LYMPHATIC DRAINAGE AND TRYING
TO DRAIN DEEP CERVICAL LYMPH
NODES AND INJECTING PEOPLE IN
MOUSE GLEOMAS IN HEMISPHERES AND
INJECTED HEMISPHERES WE SEE A
STEADY DROP OF VIRUSES IN
INJECTED HEMISPHERES AND NO
INCREASE OF [INDISCERNIBLE] AND
THIS VIRUS DOESN'T KILL ANYT
ANYTHING.
WE SAW IMPRESSIVE INCREASE OF
VIRUS IN DEEP CERVICAL LYMPH
NODES GOING ON SIX DAYS DROPPING
PRECIPITOUSLY AND THIS IS TAKING
OF INTERVIRION IMMUNE RESPONSE
AND MICE TRANSGENIC POLIO
RECEPTOR IN THE STUDIES AND
REPEATING THE EXPERIMENT LOOKING
AT DAY 5 AND COMPARING
TRANSGENIC MICE WITH WILD TYPE
MICE THIS ONLY HAPPENS IN MYOCIN
TRANSGENIC AND WE -- THIS IS A
WORK IN PROGRESS AND SHOWING
THAT CELLS DO TRANSPORT VIRUS TO
A CERVICAL LYMPH NODES ARE
INDEED CDC1 POSITIVE CDC ONES
AND INJECTING POLIO VIRUS IN
TUMOR TARGETS CDCS AND LYMPH
NODES CROSS-PRESENTING TUMOR
[INDISCERNIBLE] AND LOTS HAVE
NOT BEEN PROVEN YET AND LARGER
SENSE BECOMING MORE AND MORE
INTERESTED IN INNATE IMMUNE
COMPARTMENTS IN CDS AND IN
THERAPY AND SHOWING CHEMISTRY
FOR CDC45 IMMUNE CELLS AND MOUSE
GLEOMA AND SEE TUMORS ARE
INTERLACED WITH MYELOID CELLS
ALL THROUGHOUT.
AND THIS IS KS67.
PROLIFERATION STRAINS ARE
PROLIFERATING TUMOR CELLS THAT
WE GET A DOSE OF PDS RIPO. THIS
VIRUS DOESN'T KILL ANYTHING
EFFECTING MYELOID CELLS AND
MICROGLIA AND SEE THIS MYELOID
EXPLOSION THAT IS A PROFOUND
REACTION ENGULFING THE CNS AND
MYELOID RESPONSE AND AS
CONSEQUENCE SEE TUMOR REMAINING
AND KS67 SIGNAL WE SEE HERE
ACTUALLY PROLIFERATING
MICROGLIA.
I HAD A STUDENT IN MY LAB WHO
GENERATED THESE WHO TREATED MICE
WITH PDS RIPO AND SENT SLICES
FROM BRAINS IN A BLINDED FASHION
TO OUR CLINICAL BOARD CERTIFIED
NEUROPATHOLOGIST.
NOBODY IN MY FIELD DOES THAT.
IT IS A DANGEROUS CAREER THAT HE
DIDN'T ASK ME.
I HAVE A CALL FROM A PATHOLOGIST
AND NEVER SPOKE TO HER BEFORE.
HE SAID I HAVE NEVER SEEN
ANYTHING LIKE IT BEFORE. HE IS
AN HIV NEUROPATHOLOGY PERSON.
HE SAID WHAT YOU HAVE IS A
MASSIVE AND VERY UNUSUAL AND
VERY WIDESPREAD MICROGLIA
RESPONSE AND MAIN OUTCOME FOR
ANALYSIS AND WAY THIS LOOKS LIKE
IS SEEING EXTENSIVE PROFUSE
MICROGLIA PROLIFERATION AND
MICROGLIA ACTIVATION AND
INTERESTING IT OCCURS IN THE
ENTIRE CNS. THIS IS NOT DRIVEN
BY VIRAL DAMAGE TO NEURONAL OR
GLIAL CELLS THAT IS DOGMA IN
COLITIS BUT IS DRIVEN BY
INFECTION OR DIRECT INFECTION OF
MICROGLIA CELLS.
SO, MICROGLIA ASSUMED PROFOUND
MORPHOLOGICAL CHANGES SEEN HERE
THAT IB1 POSITIVE AND TMM119
CATEGORICAL MITOGLEEA MARKER AND
CD11B PULLDOWNS AND MICROGLIA IN
FACT ARE INFECTED BY PDS RIPO
THAT IS A WORK IN PROGRESS AND
WE DON'T QUITE HAVE A FULL GRASP
ON THIS.
WE BELIEVE THAT THIS DRAMATIC
RESPONSE IS DUE TO INFECTION.
SO, NOW, WHAT ARE WE GOING TO DO
WITH ALL THIS IN A CLINIC?
MY INTEREST HAS SHIFTED AWAY
FROM THE TUMOR.
I ACTUALLY HAVE ZERO CONFIDENCE
THAT WE WILL EVER GET ANYTHING
INTERESTING OR HELPFUL FROM THE
TUMOR ITSELF AND EVEN DEPRESSED
ABOUT MYELOID COMPARTMENTS IN
TUMORS AND MYELOID COMPARTMENT
IS ABUNDANT AND VICIOUS OF ALL
CANCERS AND TUMORS ARE 80% OF A
MACROPHAGE AND TUMOR ASSOCIATED
MICROGLIA OR MACROPHAGES TO DO
ANYTHING HELPFUL IS SINKING BY
THE DAY AND IS ABCERTIFIED TO
BELIEVE YOU CAN EVER GET
IMMUNITY IN THE BRAIN WITHOUT
ENGAGING TISSUE RESIDENT
DEPARTMENT AND GLIM FATTIC
SYSTEM AND THIS IS HOW WE
APPROACH OUR CLINICAL STRATEGY.
SO, THE NEW PROTOCOL THAT HAS
BEEN FDA APPROVED -- REAL TRIAL
WON'T BEGIN UNTIL NEXT YEAR AND
RECURRENT GBM AND RECURRENT
SECTION AND WE TAKE OUT ALL THIS
JUNK THAT IS IMPORTANT BECAUSE
WE WANT TO GET RID OF
IMMUNOSUPPRESSIVE EFFECT OF
HAVING A TUMOR IN THERE AND MOST
TUMORS HAVE HYPOXY AND NECROTIC
AND WORST KIND OF MACROPHAGE YOU
CAN THINK OF AND THINK IT IS
BETTER TO GET RID OF IT RATHER
THAN TO HOPE TO ENGAGE THIS
STUFF AND WE INTERJECT PATIENTS
AND GIVE TWO TANDEM INOCULATIONS
AND FIVE DAYS APART AND INNATE
PRIME
PRIMING PHILOSOPHY AND TWO
INJECTIONS INSTEAD OF ONE AND
DON'T GIVE INSIDE OF THE TUMOR
BUT FLAIR THAT IS A TYPE AND
BRAIN SURROUNDING TUMOR THAT IS
INFILL TRAYED IN IT AND
COMPROMISED TUMOR ITSELF AND
INJECT NEXT TO THE TUMOR SEEING
PROFOUND CNS INFLAMMATORY
CHANGES IN THE MOUSE AND TWO
INJECTION GIVEN BACK-TO-BACK
INTO FLAIR NOT TUMOR AND BACK IT
UP WITH EVERY THREE WEEKS
[INDISCERNIBLE] INOCULATION OF
PDS RIPO AND INJECT PDS RIPO IN
THE AREA OF THE SECOND FROM THE
TOP DEEP CERVICAL LYMPH NODE.
SO, A LOT OF POLIO VIRUS
INJECTED ALL OVER THE PLACE IN
THE BRAIN MULTIPLE TIMES AND FDA
DID THIS WITHOUT COMMENT AND WAS
SURPRISED. THIS IS A RANDOMIZED
TRIAL THAT WE HAD TO DO THIS AND
WE DON'T LIKE IT.
BUT, THIS IS THE TYPE OF
SETTING THAT WE HOPE WILL GET US
MORE RESPONSES, BETTER RESPONSES
AND MORE DURABLE RESPONSES AND
IN MORE PATIENTS AND ARE VERY
EXCITED ABOUT KICKING THIS OFF.
SO, THAT IS MY LAST SLIDE.
AND I AM ACTUALLY DOING GOOD ON
TIME AND WILL HAVE TIME FOR
QUESTIONS IF YOU HAVE ANY.
>> AUDIENCE: [APPLAUSE].
>> QUESTIONS FROM THE
AUDITORIUM?
>> I WONDER WHAT YOU WILL HAVE.
>> IN VITRO AS I HAVE SHOWN WE
USE MDMS DENDRITIC MOUSE CELLS.
AS LONG AS WE CAN KEEP CELLS
ALIVE, THEY HAVE THIS SIGNATURE.
MY FORMER COLLABORATOR ALWAYS
TELLS ME DON'T SAY THAT. COULD
BE A BAD THING; RIGHT?
WE DON'T THINK OF RONIC CYTOKINE
RESPONSES NECESSARILY AS A GOOD
THING.
IN VIVO, WE HAVE NO GOOD DATA
FROM PATIENTS AND BRAIN TUMOR
PATIENTS, YOU CAN'T DO A
RESEARCH BY OPSY COMING FROM
RISK OF DEATH THAT IS AN
OPERATION AND YOU CAN'T JUST
TAKE OUT TUMOR TO CHECK; RIGHT?
WE DON'T KNOW IF DURABLE
INTERFERON RESPONSE WE SEE IN
VITRO ACTUALLY HAPPENS IN VIVO.
CELLS THAT DO MOUNT THE
PARTICULAR RESPONSE DON'T
EXPRESS VIRAL ANTIGEN AND VIRAL
TRANSLATION STOPS EARLY AND THEY
DON'T REPLICATE VIRUS AND ARE
NOT VISIBLE AS BEING INFECTED
AND COULD GO ON FOR A WHILE AND
WE JUST DON'T KNOW.
>> FASCINATING TALK.
I GUESS I'M WONDERING, YOU KNOW,
HOW MUCH OF THE ANTITUMOR RISK
EFFECT THAT YOU ARE SEEING WHEN
IT WORKS IN PATIENTS AND ALSO
MOUSE MODELS IS EFFECTIVE
ADAPTIVE IMMUNE RESPONSE VERSUS
ASPECTS OF INNATE IMMUNE CELLS
SOMEHOW, YOU KNOW, DAMAGING
TUMOR CELLS KILLING THEM.
>> THE T-CELL RESPONSE.
>> YEAH.
>> YEAH, WE THINK THAT.
>> WHAT IS THE EVIDENCE IN THE
MICE?
>> NONE. OUR MOUSE WORK
SUGGESTS THAT AND WHAT WE THINK
AND THIS IS VERY DIFFICULT TO
TEST. WE DON'T HAVE TUMORS, AS
I SAID; RIGHT?
WE CAN'T JUST -- AS YOU SEE IN
THE TRIAL. SO, WE HAD A BY OPSY
SOMEWHERE IN HERE THAT IS
[INDISCERNIBLE] FOR OUR
SURGEONS.
SO, WE HAVE TUMOR MATERIALS AND
TCS SEQUENCE MATERIALS AND IS A
CHALLENGE THAT WE BELIEVE IS THE
CASE.
>> ARE YOU PLANNING ON
[INDISCERNIBLE]?
>> WE HAVE. SO, WE HAVE A, I
THINK, INTERESTING PAPER THAT I
WILL IN 2021 COMMUNICATIONS
STUDYING THIS VERY CLOSELY AND
WE FOUND -- SO, ACTUALLY IT IS
COUNTERINTUITIVE.
SO, THESE PATIENTS HERE HAVE
EXTREMELY LOW TUMOR MUTATIONAL
BURDEN AND PUBLISHING OUR PAPER
THERE IS MORE STUDIES IN BRAIN
TUMOR PATIENTS AND CHECKPOINTS
THAT CONFIRM THAT AND IN
HYPOTHESIS, THERE IS ACTUALLY
DATA THAT LOW TUMOR MUTATIONAL
BURDEN TUMORS ARE MORE IMMUNE
COMPETENT AND MORE IN-TACT
IMMUNE ENVIRONMENT AND LOW TUMOR
MUTATION IS SIGN OF ACTIVE
IMMUNE EDITING AND IS ACTIVE
PROCESS OF IMMUNOLOGIC REACTION
IN TUMOR AND EVERYONE SEES THIS.
IT PREDICTS IMMUNOTHERAPY IS TOO
SIMPLISTIC; RIGHT?
WE SEE THE OPPOSITE HERE.
>> SO, THAT IS AN AMAZING TRIAL
AT THE END.
WHAT IS THE OUTCOME MEASURE?
HOW ARE YOU FOLLOWING THE
PATIENTS?
>> YEAH. GENOMES ARE ALWAYS
SURVIVAL. WE HAVE SOME
BIOLOGICAL RESEARCH ENDPOINTS
AND HAVE SOME VERY INTERESTING
BLOOD MONITORING THAT WE ARE
ALREADY DOING AND PARALYMPHATIC
INJECTIONS WITH REPEAT EXPOSURES
AND ARE VERY EXCITED ABOUT IT.
IT WILL BE SURVIVAL.
>> AND WITH RESPECT TO THE
MARKERS, YOU WOULD EXPECT HOW WE
THINK THE CD-14 MIGHT BE
RELEASED FROM ACTIVATED
MACROPHAGES.
COULD YOU FOLLOW THAT MARKER?
>> NO. SO, I'M NOT AN
IMMUNOLOGIST.
I HAVE TO SAY THAT.
WE MONITOR IN BLOOD WE GET
REALLY EXCITING RESULTS AND
CAN'T MONITOR THE BRAIN. WE
DON'T HAVE TISSUE.
IN BLOOD, WE SEE A DECREASE IN
T-CELL EXHAUSTION MARKERS AND
INCREASE IN CDA NUMBERS AND CDA
INTERFERON GAMMA AND WE SEE
INCREASE IN CDC CIRCULATING IN
CDC ONES IN BLOOD AND THINGS OF
THAT NATURE AND EXCITED ABOUT
THAT WE GET CEREAL BLOOD SAMPLES
8 OR 9 NOW FROM THE FIRST TWO
SUBJECTS WE ENROLLED AND MARKERS
HAVE AN OUTWARD TREND OVERTIME
AND HAVE A MORE SUSTAINED IMMUNE
-- AT LEAST AS WE CAN TELL
PERIFERL ENGAGEMENT AND
PARALYMPHATIC INJECTIONS HE WE
HAVE.
>> HOW ABOUT IMAGING? YOU
INJECT THE FLAIRS.
>> YEAH.
>> HOW DOES THAT LOOK OVER TIME
AND DO THE LYMPHATICS LIGHT UP
WHEN THEY ARE IN PLAY IN THE
VIRUS OR NOT ACTIVATED?
>> IMAGING IS A HUGE PROBLEM;
RIGHT?
PEOPLE -- THIS IS NOT WHAT YOU
THINK IT IS THIS IS A TUMOR
GOING AWAY AND NOBODY KNOWS WHAT
IT SHOWS BUT IT IS SIMPLY A
MEASURE OF CONTRAST BEING ABLE
TO ACCUMULATE SOMETHING NOT IN A
NORMAL BRAIN AND WE HAVE THE
PATIENT THAT IS A CR FOR SEVEN
YEARS AND THINK THAT YOU WILL BE
A CR FOREVER HOPEFULLY AND THIS
PATIENT NEVER HAD ANYTHING.
TUMOR WENT AWAY AND NO SIGNS ON
MRI THAT ARE INDICATIVE OF ANY
TROUBLE. HE HAD NO CLINICAL --
WE HAVE A LOT OF PROBLEMS WITH
PERITUMOR INFLAMMATION AND EDEMA
SEIZURES IN PATIENTS WITH
INTERTUMORAL INOCULATIONS AND
THIS PATIENT WITH A BIG HOLE
HERE VIRUS DIDN'T END UP
CONTRAST VIRUS END INOCULATION
AND KNOW THAT INOCULATION DIDN'T
PROPERLY TARGET THE TUMOR AND
VIRUS IS VERY MUCH IN WAY WE
INTEND NAIL AND VIRUS PROBABLY
TARGETED AREAS AROUND THE TUMOR.
THIS IS WHAT WE KIND OF HOPED
FOR; RIGHT?
NO BAD INFLAMMATION IN THE BRAIN
WAS NOT A GOOD THING; RIGHT?
WE CAN'T TOLERATE A LOT AND IN
RESPONDERS, WE TYPICALLY SEE
NOTHING.
>> SCAN?
>> WE HAVE DONE THAT. IT IS
VERY INTERESTING.
WE HAVE DONE THIS ONCE.
WE LOVE IT BUT INSURANCE WON'T
PAY FOR IT FOR THESE PATIENTS SO
--
>> ONE LAST QUESTION.
>> USING MD POLIO VIRUS IN HERE?
>> OH, YEAH. VERY MUCH. THESE
ARE PATIENTS THAT ARE ALL
IMMUNE, OF COURSE.
WE BOOST THEM. THEY GET A
BOOSTER WITH A PEDIATRIC VACCINE
ON PURPOSE AND SUPER EXCITED
ABOUT RECALL AND PUBLISHED A
PAPER ON GYPSY ABOUT THIS THAT
IS VERY COMPLICATED AND CAN'T
PUT INTO MY TALK AND PART OF
THIS AND LOVE IT AND IF DIDN'T
HAVE VACCINE WOULDN'T BE ALLOWED
TO DO THIS AND PATIENTS WITH
PRE-EXISTING [INDISCERNIBLE] THE
BETTER AND IN MOUSE MODELS WE DO
BETTER IN VACCINATED MICE
COMPARED TO UNVACCINATED MICE.
WE ARE VERY EXCITED ABOUT
RECALL.
>> THANK YOU VERY MUCH.
FANTASTIC.
>> I WONDER IF I CAN GET A
VOLUNTEER TO GO ON TO THE ZOOM
LINK LOOKING FOR QUESTIONS IN
THE CHAT AND WOULD LIKE TO
ENGAGE THE VIRTUAL AUDIENCE THAT
IS SEVERAL TIMES LARGER THAN
IN-PERSON AUDIENCE AND RAISING
HAND AND READING QUESTIONS
SHOWING UP IN THE CHAT AND NOT
SURE PEOPLE CAN VIRTUALLY UNMUTE
THEM SFELVES ASKING QUESTIONS
DIRECTLY AND WILL FIND OUT AND
MOVING ALONG NEXT SPEAKER IS JEN
RECEIVING DEGREE FROM EMORY
UNIVERSITY SCHOOL OF MEDICINE
AND DID FELLOWSHIP TRAINING IN
MEDI
MEDICAL ONCOLOGIST -- RESEARCH
GROUP OF JAMES IN THE NCI
ASSOCIATE RESEARCH POSITION.
AND JEN USES CAR-T-CELL THERAPY
TO INTRODUCE CANCERS IN THE
BLOOD.
>> [APPLAUSE].
>> ALL RIGHT. THANKS SO MUCH
FOR HAVING ME.
I GAVE A SIMILAR TALK AT
WASHINGTON HOSPITAL CENTER FOR
GRAND ROUNDS.
I FEEL LUCKY TO HAVE WORKED AT
BETHESDA CAMPUS AND NCI AND
STEVE ROSENBERG AND I WILL TRY
TO HIGHLIGHT THAT NOW IF YOU CAN
INCLUDE MY TRAINED FELLOWSHIP 10
YEARS OF CAR T-CELL THERAPY
WORK.
SO, DISCAN CLOSURE I'M ON
SCIENTIFIC ADVISORY COMMITTEE A
SMALL PHARMACEUTICAL COMPANY AND
FEDERAL EMPLOYEE SO THAT IS AN
UNPAID POSITION.
ALL RIGHT. I HAVE TWO CASE
PRESENTATIONS TO TRY TO CENTER
THE RESEARCH AROUND.
MR. A, A GENTLEMAN I KNEW FOR A
LONG TIME. 66-YEAR-OLD
GENTLEMAN WITH NON-GERM NAL CELL
TYPE LYMPHOMA AND MANY PRIOR
RESIDENTS AND THEME PATIENTS
COME TO US HAVING HAD MANY PRIOR
[INDISCERNIBLE] MALIGNANCIES AND
HAD VERY STANDARD UP-FRONT
INTERCYCLING-BASED REGIMENT.
COMBINING THAT WITH THE SMALL
MOLECULE ORAL -- HE KEPT HAVING
A LITTLE REMISSION BUT HAVING
RESIDUAL LYMPHOMA AND THAT IS A
VERY BAD INDICATOR. HE HAD
RADIATION THERAPY TO BIG
ABDOMINAL MASS THAT BER AGED HIM
AND STEM CELL TRANSPLANT MEANT
TO BE CONSOLIDATIVE AND HAD
COMPLETE REMISSION AND RELAPSED
4 MONTHS LATER. AGGRESSIVE
B-CELL LYMPHOMA NOT RESPONSIVE
TO TREATMENT AND 66 YEAR OLD
VERY FIT HAD WELL CONTROLLED
DEPRESSION GLAUCOMA AND
ARTHRITIS AND BRINGING US TO
OBLIGATORY BACKGROUND AND WHAT
IS A CAR?
HERE ARE COMPONENTS.
HERE IS THE -- I SHOULD SAY THE
[INDISCERNIBLE] FOR ANNE WHO
PUBLISHED THIS NOW 10 YEARS AGO
IN NATURE REVIEW OF CLINICAL
ONCOLOGY AND STILL WORKS AND
GREAT FIGURE AND THESE ARE THE
ACTIVA
ACTIVATION JUST AS YOU WOULD
HAVE IN NORMAL ENDOGENOUS T-CELL
AND COMPONENTS OF CHIMERA WHICH
IS LIGHT AND HEAVY VARIABLE
CHAINS THAT ARE CONNECTED BY A
LINKER TYPICALLY AND THIS IS
WHAT STANDARD CONSTRUCTS LOOK
LIKE AND PROTEINS ARE CONNECTED
BY HINGE AND TRANSMEMBRANE
REGION AND WILL TALK LITTLE
ABOUT OUR VIRAL VECTOR.
I'M SORRY IT IS VERY, VERY
IMPORTANT AND CAR GENES ARE
INCERTIFIED IN VECTOR AND GAMMA
RETROVIRAL VECTOR AND RESULT OF
THIS, GENES OF T-CELL AND NOW
CAR EXPRESSING T-CELL ARE
PERMANENTLY GENETICALLY MODIFIED
AND IT EXPRESSES CAR T-CELL
PROTEIN ON SURFACE AND GROSSLY
SIGNIFIED THERE ARE THOUSANDS OF
RECEPTORS ON T-CELL SURFACE AND
CLASSIC VERSION 1.0 IS TREATING
MALIGNANCY CDC CAR T-CELL --
MALIGNANT CANCER CELLS THAT ARE
DERIVED FROM B-CELLS AND SO NOW
THERE ARE VARIATIONS ON THIS
THEME AND DIFFERENT ANDROEJEN
TARGETS AND CARS I WILL TALK
ABOUT LATER AND IDEA IS I TOLD
THEM ONCOLOGY FELLOWS ARE
FINGERS COMING AND SENSING THE
ANTIGEN AND RESULT T-CELL IS
ACTIVATED INTERCYTOPLASMIC
[INDISCERNIBLE] AND IT IS A
FIGURE THAT WORKS AND GIVING
THIS I WILL SAY AGAIN THIS IS
STANDARD VERSION OF EVENTS AND
CLINICAL TRIALS ARE VARIATIONS
OF THIS I CAN TALK ABOUT LATER
IF DESIRED AND ESSENTIALLY
T-CELLS PDMC IS COLLECTED FROM
THE PATIENT AND TELL THEM THIS
IS A PROCESS AS IF DONATING
BLOOD THAT IS SIMPLE AND DONE
IN-HOUSE IN OUR CAN CELL
PROCESSING CENTER AND ARE TAKEN
FOR EX-VIVO PROCESSING AND
T-CELLS ACTIVATED AND TRANCE
DEUCED WITH THE GAMMA VIRAL
VECTOR -- CAN ALSO BE SUCCESSFUL
AND USUALLY A VIRAL VECTOR.
THE T-CELLS PROLIFERATE EX-VIVO
AND IN THE MEANTIME PATIENT GETS
CHEMOTHERAPY AND PATIENTS ASK ME
IF THIS IS CHEMOTHERAPY TO
DECREASE BURDEN OF MALIGNANCY
AND GREAT WHEN IT HAPPENS AND
FREQUENTLY HAPPENS AND THESE
FACTORS ARE RETREATING AND
PRIMARILY WE ARE DEPLETING WITH
THE SITES CALLED CONDITIONING
CHEMOTHERAPY OR LYMPHO DEPLETING
CHEMOTHERAPY AND TWO AGENTS
TOGETHER ARE TYPICAL REGIMENT
ENDOGENOUS LYMPHOOCYTES AND
LYMPHOCYTES ARE SYNC FOR
CYTOKINES THAT EFFECTIVELY MAKE
IT SO CYTOKINE BOILS IN BLOOD
AND IT PROMOTES CAR T-CELL
ACTIVITY IN VIVO WHEN THEY ARE
INFUSED INTO THE PATIENT THAT IS
A STANDARD THING WE DO TO TREAT
PATIENTS WITH CAR T-CELL THERAPY
AND MORE OFTEN THAN NOT --
INFUSION CENTER TO GET
LYMPHOCYTE DEPLETING
CHEMOTHERAPY AND CAR T-CELLS
IN-PATIENT AND OUT-PATIENT --
PHASE 1 TRIALS CURRENTLY IN OUR
GROUP WE ARE EXCLUSIVELY IN MANY
PATIENTS TO IN-PATIENT SERVICE
FOR NEEDS TO TOXICITY MONITORING
AND WHERE WERE WE JULY 1ST 2013
AND STARTED INTERNSHIPS AND
STARTING MEDICAL ONCOLOGY
HEMOTOLOGY FELLOWSHIP AND IN A
DIFFERENT PLACE THAN TODAY AND
NO APPROVED CAR-T CELL PRODUCTS
FOR MALIGNANCY AND SOME ANTI-C19
CAR T-CELL CLINICAL TRIALS AND
CAN SEE THERE ARE SOME REPORTS
HERE.
SO, THE -- MY MENTOR ARE FIRST 2
PUBLISH IN BLOOD AS BRIEF REPORT
ACTIVITY OF GIVING ANTI-C9
[INDISCERNIBLE] WHO IS NOW MORE
THAN A DECADE IN REMISSION AND
STARTED THAT STORY THIS IS A
REPORT IN THE JOURNAL FROM GROUP
OF UNIVERSITY OF PENNSYLVANIA
DR. PORTER KARL JUNE TALKING
ABOUT EARLY EXPERIENCE GIVING
CAR-T CELLS TO PATIENTS WITH
[INDISCERNIBLE] WITH REALLY
IMPRESSIVE REMISSIONS AND
IMPRESSIVE TOXICITIES CITTOKIND
RELEASE SYNDROME AND MEMORIAL
KETERRING BACK IN 2013 WITH
SERIES OF INDIVIDUALS WITH ACUTE
LYMPHO BLASTIC LEUKEMIA AND
IMPRESSIVE REMISSIONS INCLUDING
MRD NEGATIVE AND NEGATIVE
REMISSIONS AND ALSO I STARTED
FELLOWSHIP THAT ARE NO
ANTI-B-CELL MADE CAR-T CELL
PRODUCTS AND [INDISCERNIBLE]
ANTIGEN THAT IS WELL-EXPRESSED
ON PLASMA CELLS AND DIFFERENT
SITUATION THAN WHERE WE ARE NOW.
WHERE ARE WE NOW?
WE HAVE SIX FDA APPROVED CAR
T-CELL PRODUCTS FOR FOUR
HEMOLOGIC MALIGNANCIES AND WE
HAVE [INDISCERNIBLE] AND YOU
NOTICE WE HAVE CUTE LITTLE
NICKNAMES FOR THE PRODUCTS WITH
LONG AND CUMBERSOME NAMES
DIFFICULT TO ANNOUNCE AND THAT
WAS THE PRODUCT DEVELOPED BY MY
[INDISCERNIBLE] FOR STEVE
ROSENBERG STARTING BACK IN 2007
AND PRETTY GOOD TIMELINE; RIGHT?
FDA APPROVAL IN 2017 AND NOW
SECOND LINE AS WELL THAT IS
GREAT AND PATIENTS CAN GET
FIRST-LINE THERAPY AND IF
DOESN'T WORK CAN GET CAR-T CELL
THERAPY THAT IS EXCITING AND
APPROVED FOR LARGE B-CELL
LYMPHOMA AND ONE FOR MAN TELL
CELL LYMPHOMA AND ALL, IT IS THE
VERY FAMOUS UNIVERSITY OF
PENNSYLVANIA PRODUCT.
THIS IS THE FIRST FDA APPROVED
CAR-T CELL BACKED IN 27 TAENFOR
PEDIATRIC B-CELL ALL AND WE HAVE
ADULT B-CELL ALL PRODUCT AND
APPROVED AND TWO MULTIPLE
MYELOMA PRODUCTS TARGETED
AGAINST [INDISCERNIBLE] AND I
MENTIONED EARLIER.
THESE ARE EXCITING THERAPIES
THAT WORK WHEN CHEMOTHERAPY
DOESN'T.
IT IS A VERY -- THERE ARE NARROW
RANGE OF THERAPIES THAT CAN DO
THAT IN PATIENTS IN SOME CASES
AND INCURABLE -- LARGE B-CELL
LYMPHOMA AND WE ESTABLISHED AT
THIS POINT WE CAN CURE THOSE
PATIENTS AND LIKELY SUBSET OF
PATIENTS WITH INCURABLE B-CELL
MALIGNANCIES LYMPHOMAS AND
LEUKEMIAS WE CAN CURE. BARRIER
I WILL TALK ABOUT IS PRICE
CELLULAR PRODUCTS COST HUNDREDS
OF THOUSANDS OF DOLLARS FOR
PRODUCT ITSELF AND NEEDS PATIENT
MIGHT NEED TO BE ADMITTED OR
POTENTIALLY HAVE LONG-TERM
EFFECTS IN TERMS OF CITTOPENIAS
AND B-CELL AMRASHIA THAT IS NOT
TRIVIAL AND COST BENEFIT
ANALYSIS ACTUALLY HAVE SHOWN IF
YOU ARE A PERSON WITH CURABLE
MALIGNANCY WITH ALL B-CELL
LYMPHOMA GETTING LOTS OF
THERAPIES AND DYING BECAUSE THEY
DON'T WORK IT IS PROBABLY COST
EFFECTIVE TO GET T-CELL THERAPY
AND HOPE IN FIELD IS VERSION 3.0
AND 5.0 WILL BE MUCH MORE
EFFECTIVE.
TALKING A LITTLE ABOUT THE STORY
OF WORKING WITH MENTOR STEVE
ROSENBERG WORKING ON 19 CAR --
AND TALKING ABOUT FUTURE
WORKABILITY ON THAT. PATIENTS
WITH LARGE B-CELL LYMPHOMA HAVE
POOR OUTCOMES AND TRY TO BUILD
THIS STORY WITH ANECDOTE OF MR.
A THAT WILL COME BACK TO YOU IN
A LITTLE BIT.
PATIENTS WITH LARGE LYMPHOMA
GROUP CHEMOTHERAPY REFRACTORY
LASTING 12 MONTHS AFTER IN STEM
CELL TRANSPLANT THEY HAVE
PROGNOSIS AND PATIENTS ARE ALIVE
LONG-TERM AND IS VERY DIFFERENT
THAN IN THE FRONTLINE AND 60 OR
70% OF PATIENTS MIGHT BE CURED
WITH LARGE CELL LYMPHOMA WITH
CHEMOTHERAPY AND UP-FRONT IS NOT
CURATIVE AND LONG-TERM OUTLOOK
HISTORICALLY IS POOR AND STEVE
ROSENBERG WORKS ON CONSTRUCTING
ANTI-C9 CAR AND IT IS THE NAME
OF THE [INDISCERNIBLE] AND
REMEMBER THAT ANTIBODY DERIVED
RECOGNITION AND FINGERS EXTRA
CELLULARLY AND DOMAIN AND THIS
CONSTRUCT IS WHAT THEY DEVELOPED
AND STARTING BACK IN 2007 AND
RAN THE FIRST PHASE 1 CLINICAL
TRIAL LOOKING AT CAR-T CELL
THERAPY FOR NON-HODGE KINES
LYMPHOMA AND REPORTS IN JOURNAL
OF CLINICAL ONCOLOGY IN 2017
REPORTING EARLY PHASE 1 PATIENTS
AND OVERALL RESPONSE RATE LOVELY
73% WITH CR COMPLETION RATE OF
[INDISCERNIBLE] OCCURRING IN
HALF PATIENTS THAT IS PER
NCICTCA SYSTEM AND NEUROLOGIC
TOXICITY THEY ARE NOT FUNCTIONAL
AND DIVERSE TOXICITIES AND HAND
TREMORS APHASIAS AND MOTOR
DEFECTS SOMETIMES AND ATAXIA AND
APHASIA IS SOMETIMES VERY DENSE
AND PATIENT CAN'T SPEAK AND
INCREASED SAL MAN ILLA AND
PATIENT WANTS TO NAP A LOT AND
BE ON A VENTILATOR AND BE
INTUBATED FOR AIRWAY SUPPORT AND
GREAT BEGINNING WE ARE GIVING
INTERLEUKIN TWO INFUSIONS TO
CAR-T CELLS TO PROMOTE ACTIVITY
IN VIVO AND BECAUSE OF THAT
BECOMING CLEAR VERY SOON THIS IS
NOT IL2 EFFECT.
IT IS A DIFFERENT FLAVOR OF
NEUROLOGIC TOXICITY ON THE
SEVERE END WAS VERY COMMON.
CYTOTOXICITY AND RESULTS OF
EFFICACY IS EXCITING AND TAKEN
TO PHASE 2 MULTICENTERED TRIAL
AND CONSTRUCT USED IN EXTRA
CELLULAR PRODUCT AND AXY CELL
FOR SHORT AND DEDICATED TRIAL
FOR LARGE B-CELL LYMPHOMA AND
THEY ARE UP TO [INDISCERNIBLE]
23 AND HAVE CUTE AND GILLAD
TRIALS HAVE SHORT NAMES AND THIS
IS A TRIAL UNDER ONE PATIENTS
RETREATED GETTING FAIRLY
STANDARD CONDITIONED REGIMENT
WITH HIGH RESPONSE RATES YOU CAN
SEE HERE WITH OVERALL RESPONSE
RATE OF 0% AND 0 TO 54% AND
DURABLE IN MANY PATIENTS AND NOW
HAVE LONG-TERM FOLLOW UP DATA
PUBLISHED THIS YEAR IN BLOOD AND
30% OF PATIENTS ARE CURED
FOREVER FROM REFRACTORY LARGE
B-CELL LYMPHOMA AND HIGH GRADE
NEUROLOGIC TOXICITIES AND
DIVERSITIES CYTOKINE RELEASED
SYNDROME AND TOXICITIES ARE
MAJOR BARRIER TO USE IN PRODUCTS
TODAY AND KATIE WHO AT THE TIME
WAS A FELLOW WORKING WITH OUR
GROUP AND WENT INTO PRACTICE AND
CAME BACK AND IS WORKING IN THE
LAB AND WE ARE EXCITED ABOUT
THAT AND WENT BACK AND LOOKED
LONG-TERM AT PATIENTS TREATED
WITH AXY SILICON CONSTRUCT AND
PATIENTS GAINING 43 PATIENTS
GAINING 46 CELL INFUSIONS THAT
IS A MIX OF LARGE B-CELL
LYMPHOMA AND LOW GRADE NON-HODGE
KINES B LYMPHOMAS AND CLL AND
NICE DURABILITY YOU CAN SEE HERE
IN THIS CAP LYNNE MEYER PLOT
WITH HALF PATIENTS HAVING FAIRLY
DURABLE REMISSIONS OUT THREE
YEARS AND BEYOND.
I WANT TO TALK ABOUT THE
[INDISCERNIBLE] FOR LAB AND
CLINICAL RESEARCH TEAMS
EXPERIENCE TRYING TO DEVELOP
ESSENTIALLY VERSION 2.0 OF THE
AXY CELL CONSTRUCT FOR B-CELL
NON-HODGE KINES LYMPHOMA AND IS
WORK BY LEAH WITH LAB PUBLISHED
BACK IN 207 TAEN/* -- 2017 AND
T-CELL ACTIVATION COSTIMULATORY
DOMAIN TRANSMEMBRANE REGION AND
SINGLE CHAIN OF SINGLE CHAIN
VARIABLE FRAGMENTOR REGION HEAVY
CHAINS CONNECTED BY A LINKER AND
ON FIGURE AND TRANSMEMBRANE
REGIONS LOOK INACTIVE SKIPPING
THEM GIVING INITIAL CAR SCHEMA
AND HOW IT WORKS AND RECOGNIZED
EARLY ON BY LEAH FROM COLLEAGUES
THAT MEMBRANE DOMAINS OF CARS
CAN IMPACT FUNCTION AND
COMPARING CARS AND REGION DERIVE
FROM [INDISCERNIBLE] CONSTRUCTS
WITH DIFFERENT TRANSMEMBRANE
REGION DERIVED FROM CD8 AND
RATHER THAN CD28 AND CELLS ARE
DIFFERENT HINGE AND
TRANSMEMBRANE REGION DERIVED
FROM CD8 HAVE LOWER LEVELS OF
CYTOKINE [INDISCERNIBLE] STILL
WORKS AND STILL HAS CITTOLYTIC
PROPERTIES.
SO, THIS WAS VERY INTERESTING.
WE KNOW AND I REVIEW THIS BACK
IN 2016.
WE KNOW MOST CAR-T CELL
TOXICITIES ARE DUE TO CYTOKINES
BOTH PRODUCED BY T-CELLS
THEMSELVES AND DOWNSTREAM CAR-T
CELLS AND CAN WE IMPROVE INITIAL
CONSTRUCT DEVELOPED IN NCI WITH
INITIAL PUBLICATIONS BACK IN
2015?
LEAH AND OTHERS IN THE LAB FOR
NORRIS AS WELL DEVELOPED A NEW
CONSTRUCT THAT HAS A FULLY HUMAN
SINGLE CHAIN OF V AND CD8
MEMBRANE REGION AND WHAT ARE
POTENTIAL ADVANTAGES HERE?
HOPE WAS THAT THE FULLY HUMAN
AND ALL HUMAN PROTEINS AND
IMMUNOGENIC AND CAR CONSTRUCT
WITH CD8 HINGING TRANSMEMBRANE
REGION WOULD BE LESS TOXIC WITH
LOW CYTOKINE MODEL IN MOUSE
MODELS AND USING CONSTRUCT 28Z
CONSTRUCT IN A CLINICAL TRIAL
FOR PATIENTS THIS IS PHASE 1
FAIRLY STANDARD 3 + 3 AND
STARTING FAIRLY LOW DOSE CELL
DOSE LEVEL WORKING TO HIGH DOSE
LEVELS BASED ON TOXICITY PROFILE
AND 20 PATIENTS LARGE B-CELL
LYMPHOMA IS MOST KIND AND B-CELL
NON-HODGE KIN LYMPHOMA AND
POPULATIONS ARE ENRICHED IN
PATIENTS AND WE HAVE OTHER
LYMPHOMAS YOU CAN SEE HERE AND
OVERALL RESPONSE AND CR RATE IS
SIMILAR TO WHAT WE SEE IN OUR
CENTER AND IN AXY CELL PRODUCT
AND NOW WITH COMMERCIAL USE.
WE HAD -- VERY NICE RESPONSE
RATES AND FIVE PATIENTS WITH
COMPLETE REMISSION OVER FIVE
YEARS AND ABOUT PROBABLY ABOUT A
QUARTER OF PATIENTS THAT WE ARE
CURING.
ALSO, IT ENDS UP BEING FAIRLY
COMPARABLE TO CELL FOLLOW UP.
YOU SEE HERE FAIRLY SMALL DOSE.
WE HAD RESPONSES AND TOXICITIES
AND DURABLE RESPONSE LEVELS AND
MIDDLE LEVEL IS THE LOSER HERE
AND PATIENTS WITH LOW CAR-T CELL
LEVEL DID VERY WELL AND BACK TO
MR. A AND CASE I TALKED ABOUT
EARLIER AND HE RETRIEVED A
REMISSION AFTER FIVE YEARS OF
CAR T INFUSION AND FAIRLY LOW
DOSE AND PET SCAN AND MASSIVE WE
WILL SEE IN LIVER AND RETRO
PERITY ON EEL LESIONS AND VIDEO
ON PET SCAN AND WE CAN'T SEE
THIS IN REAL-TIME AND IT WASN'T
AN AREA THAT WAS AMENABLE AND
SEE A THEME THAT WE DON'T KNOW
WHAT IS GOING ON AND IS COMMON
IN OUR PATIENTS WE HAVE RESIDUAL
ACTIVITY ON A PET SCAN ABOUT A
MONTH AFTER TREATMENT AND
GRADUALLY ALL GOES AWAY AND
BECOMES CLEAR TO PATIENTS IN
COMPLETE REMISSION AND WHAT WE
SAW HERE AND HOW WE DO IN TERMS
OF TOXICITY PROFILE AMEEZED WIT
PROFILE AND PRIOR SINGLE CELL
EXPERIENCE USING AXY CELL
CONSTRUCT AND HIGH-GRADE
NEUROLOGIC TOXICITIES AND ALL
SIGNIFICANTLY LESS WITH LOW
NUMBER OF PATIENTS SIGNIFICANTLY
LESS WITH PHASE 1 TRIAL PRODUCT
THAT WAS GREAT AND EASY TO USE
PRODUCT AND CYTOKINE RELEASE
SYNDROME THAT IS MANAGEABLE AND
LOW RATES OF NEUROLOGIC TOXICITY
AND WHY IS THIS?
EXACTLY WHAT WE AS MOUSE MODELS
SHOWED AND CYTOKINES AND
PROTEINS IMPLICATED IN
NEUROLOGIC TOXICITY ARE LOWER IN
CAR-T CELLS THAN STANDARD AXY
CELL CONSTRUCT CAR-T CELLS
INCLUDING AIL2 AND PLAYERS
INTERFERON GAMMA KNOWN TO BE
NEUROLOGIC TOXICITY.
I WANT TO TALK BRIEFLY ABOUT SAM
A FELLOW WORKING WITH OUR TEAM
AND GOING BACK LOOKING AT OUR
EXPERIENCE TREATING BESHG ET
LYMPHOMA A RARE NON-HODGE KINES
LYMPHOMA WITH RAPID PROGRESSIVE
DISEASE AND REPRESENTED IN
CLINICAL TRIALS ANTI-C9 CAR-T
CELLS BECAUSE THEY PROGRESS SO
QUICKLY THEY CAN'T HANDLE THE
WAIT TIME FOR CELL PROCESSING IN
LARGE MULTI-CENTERED TRIALS WITH
CENTRAL CELL PROCESSING AND WE
HAD ON CAMPUS CELL PROCESSING
THAT IS RELATIVELY SHORT WE WERE
ABLE TO GET THESE PATIENTS IN
QUICKLY TREATING THEM WITH CAR-T
CELL THERAPY AND PATIENTS ARE
CURED IN COMPLETE REMISSION MORE
THAN FIVE YEARS AND ONE WITH
RISK OF STANDARD CELL MC63 CAR
CONSTRUCT AND ONE WITH OUR HUME
19 CONSTRUCT.
I WANT TO PIVOT A LITTLE BIT.
HOW ARE WE DOING ON TIME? WE
CAN COVER IT TALKING ABOUT
EXPERIENCE AT NCI DEVELOPING
CAR-T CELLS FOR MULTIMYELOMA.
I WON'T GO THROUGH IT IN DETAIL.
HE HAD EVERY PRIOR REGIMENT THAT
WAS AVAILABLE FOR PATIENTS WITH
MULTIPLE MYELOMA AT THE TIME HE
ENTERED THE STUDY WE KNEW HE HAD
TO MANAGE THOSE GOING INTO THE
TRIAL.
WHY IS IT A GOOD TARGET? STANDS
FOR B-CELL MATURATION ANTIGEN
AND IN LOW LEVELS MAJORITY OF
CASES MULTIPLE MYELOMA GREAT
CAR-T CELL ANTIGEN MILOMA AND
B-CELLS ARE EXPENDABLE AND CAR-T
CELLS DO A GREAT JOB ERADICATING
THE CELL IN THE BODY YOU WILL BE
OKAY YOU ARE IMMUNOCOMPROMISED
BUT PATIENTS CAN LIVE WITH THAT
LEVEL OF IMMUNOCOMPROMISED. NOT
TISSUES THAT WE VITALLY NEED AND
MULTIPLE MYELOMA IS A TERRIBLE
MALIGNANCY CAUSING LESIONS IN
THE BONE CREATING BONY PAIN FOR
PATIENTS CAUSING ANEMIA AND
RENAL FAILURE AND ALL IS
RECOGNIZED FAIRLY LATE IN THE
DISEASE COURSE AND PATIENTS NO
ONE KNOWS WHY THEY ARE ANEMIC
AND GIVE IRON AND YEARS LATER
FIND OUT THEY HAVE MULTIPLE
MYELOMA THAT IS INCURABLE.
SO, THE LAB DEVELOPED THE FIRST
ANTI-B-CELL CAR CLINICAL RESULTS
PUBLISHING CANCER RESEARCH IN
2013 AND AS I ARRIVED AT NCI FOR
FELLOWSHIP AND MI VERY GOOD
COLLEAGUE A COUPLE YEARS AHEAD
OF ME IN FELLOWSHIP STARTED ON
THE GROUND WITH FIRST PHASE 1
TRIAL EVER OF CAR-T CELLS
MULTIPLE MYELOMA STARTING IN
2014 AND HE AND GYM AND GROUP
PUBLISHED FIRST REPORT OF FIRST
TWO PATIENTS WITH IMPRESSIVE
REMISSIONS IN BLOOD AND 24
PATIENTS WORK HEAVILY AND EVERY
LINE OF PRIOR THERAPY THAT WAS
AVAILABLE AND PATIENTS HAD
REALLY IMPRESSIVE MINIMAL
DISEASE NEGATIVE REMISSIONS AND
I FOLLOWED PATIENTS CREATING
MORE AT EFFECTIVE DOSE LEVEL AND
FINDING OVERALL RESPONSE RATE OF
63% BEING GOOD PARTIAL AND
COMPLETE REMISSION AND BONE
MARROW WAS CLEAR IN MULTIPLE
MYELOMA AND MARKERS GO DOWN AND
LIKE EVERY MYELOMA THERAPY
EVERYONE RELAPSED AND EVENT FOR
SURVIVAL IS 31 WEEKS SEVERAL
MONTHS OFF THERAPY THAT PATIENTS
HAD AMAZING QUALITY OF LIFE
AFTER SINGLE INFUSION OF CAR-T
CELLS AND LEARNING CURVE
MANAGING TOXICITY AND MR. B
RECEIVED ICU CARE AND DELIRIUM
THAT IS LOW-GRADE NEUROLOGIC
TOXICITY AND IL-6 RECEPTOR
ANTAGONIST AND STRESS REDUCED
STEROIDS AND MINIMAL RESIDUALS
OF NEGATIVE REMISSION LASTING 66
WEEKS AND AT RELAPSE HAD
[INDISCERNIBLE] A BC NEGATIVE
POPULATION AND YOU SEE FLOW AND
PRE-TREATMENT VERY HIGH BCMA
LEVELS OF MAZ MA CELL POPULATION
AND DEFINITELY PROPORTION OF
CELLS ARE NEGATIVE AND RELAPSE.
SO, I WILL GO THROUGH THIS
BRIEFLY SO WE HAVE TIME FOR
QUESTIONS AND FIRST COMMERCIALLY
AVAILABLE MADE B-CELL CAR-T CELL
THERAPY IS SDERM AND NICE SHORT
NICKNAME AND CONSTRUCT IS
DEVELOPED COLLABORATION WITH
BLUE BIOAND FIRST CENTER TO
ENROLL PATIENTS TREATED A
HANDFUL OF PATIENTS ON TRIAL
THAT IS EXCITING AND THEME THAT
IS HEAVILY PRETREATED PATIENTS
AND NICE OVERALL RESPONSERATE OF
88 TO 5% AND LESS THAN A YEAR IN
TERMS OF MEDIAN PROGRESSION FREE
SURVIVAL AND THERAPY AND DISEASE
THAT PASHIENTS ARE KNOWN TO
BEING TREATED AND GETTING
TREATMENT AND VERY FEW BREAKS
AND HAVING A YEAR OFF THERAPY
WAS AMAZING FOR PATIENTS SEEING
DAUGHTER GET MARRIED AND
GRANDCHILDREN BORN AND THIS
PRODUCT HAD A MANAGEABLE
TOXICITY PROFILE WITH 6% MORE
SEVERE CYTOKINE RELEASE
SYNDROME.
SO, DIFFERENT LAB WENT BACK AND
WHAT IS VERSION 2.0 OF THIS CELL
AND MAKING ANTI-BCMA CAR-T CELLS
BETTER AND WORKING HARD ON THIS
CONSTRUCT THAT YOU CAN SEE HERE
IS HEAVY SHADE ONLY.
NO LINKER MAKING POTENTIAL
IMMUNE RESPONSES AND SINGLE
CHAIN FV ARE DECREASED AND ONLY
AND FULLY HUMAN THAT IS VERY
SMALL AND HELPFUL. IN THE
FUTURE OF CAR-T CELL WORLD WE
THINK PERHAPS WE CAN PACK MORE
CARS TO A SINGLE VECTOR AND
LIMITED ROOM OF COURSE AND
SMALLER COULD BE BETTER FOR
COMBINED THERAPY IN THE FUTURE
AND UNDERSTAND IT IS FULLY HUMAN
WITH IDEA WE COULD ELIMINATE
MASS IMMUNE RESPONSES AND FELLOW
WITH OUR GROUP THAT IS NOW
FACULTY AT STANFORD AND HITTING
UP PHASE 1 TRIAL AND BEAUTIFUL
OVERALL RESPONSE RATE OF 92%
WITH NICE MAJORITY OF PATIENTS
WITH CR AND VGPR AND 65 WEEKS
AND GETTING PATIENTS HERE BEYOND
A YEAR AND CS AND NEUROLOGIC
TOXICITY AND NUMBERS ARE BIG AND
IMPRESSED HOW MANAGEABLE AND
EASY PATIENTS ARE AND SHORT ICU
STAYS --
WE DID NOT SEE ANY OF THAT ON
THE TRIAL.
IT WAS A RELIEF AND VERY
REASSURING AND SAW
IMMUNORESPONSES THAT IS
SOMETHING THAT LAB IS LOOKING
INTO AND HOW WE CAN MAKE THIS
LESS IMMUNOGENIC THAT IS UP FOR
GRABS AT THIS POINT.
WHAT IS NEXT? TARGETING THINGS
WE ARE DOING ON LONG NEXT LIST
AND TRIAL FOR SPECIFIC --
EXPRESSED ON B-CELLS AND I'M
HEARTENED BY NUMBER OF NOVEL
ANTIGENS WORKING AND KATIE THAT
I TOLD YOU EARLIER IS WORKING ON
THE LAB FOR T-CELL THERAPY AND
GAINING EXCITING RESULTS AND I
WON'T GET INTO THIS AND NOT
READY FOR PRIME TIME AND HAVE
POTENTIAL BENEFITS AND UPCOMING
TRIAL AND CONSTRUCT 19 CAR
T-CELLS CULTURE PROCESS THAT IS
DEVELOPING LVPI IN THE SPRING
AND JIM FOR AMAZING MENTORSHIP
AND SURGERY BRANCH AMAZING PLACE
TO WORK AND PROCESSING
DEPARTMENT TRANSFUSION OF
MEDICINE COULDN'T BE BETTER AND
HAVE AMAZING PATHOLOGISTS AS
WELL WORKING WITH FLOW CYTOMETRY
AND [INDISCERNIBLE].
THANK YOU FOR YOUR TIME. I
REALLY APPRECIATE IT.
>> SO YOU TALKED ABOUT
[INDISCERNIBLE]. ARE THEY
SUSTAINED OR BRIEF AFTER.
>> YEAH. THEY ARE CONSIDERED
REVERSIBLE. THEY ARE ALMOST
ALWAYS REVERSIBLE IF YOU DON'T
HAVE ACUTE MORTALITY THAT IS NOW
REPORTED COMMERCIAL PRODUCTS
VARY INFREQUENT AND GETTING
PATIENT LAW IS REVERSIBLE IN
MOST CASES AND REPORTS HERE AND
THERE OF PATIENTS THAT ARE WEEKS
IN AND HAVING MAYBE PROLONGED
COGNITIVE DEFECTS AND DIE FROM
MALIGNANCY AND YOU ARE NOT
HEARING SO MUCH OF PATIENTS THAT
ARE CURED AND NOT KNOWN TO WHAT
EXTENT THERE IS A VERY, VERY
SMALL MINORITY OF PATIENTS WITH
LONG-TERM TOXICITIES AND
ANTI-BCMA CAR-T CELLS AND
DELAYED AFTER WEEKS OF CAR-T
CELL INFUSION.
>> HOW IS THIS ESCAPING?
>> YEAH. LOTS OF PEOPLE NOT SO
MUCH OUR LAB BUT LOTS OF PEOPLE
PUBLISHED ON THIS AND MULTIPLE
MOLECULAR MECHANISMS BY WHICH
CAR ITSELF COULD BE
DOWNREGULATED OR I SHOULD SAY 20
[INDISCERNIBLE] ITSELF AND FOR
SURE AND TARGETS COULD BE
DOWNREGULATED AND ARE MULTIPLE
ESPECIALLY FOR C19 PRETTY WELL
DESCRIBED AND MULTIPLE GENETIC
CHANGES TO THE RECEPTOR TO
ANTIGEN TARGET I WOULD SAY THAT
STILL EXIST AND CAN'T COME IN
AND RECOGNIZE IT ESSENTIALLY.
>> SECOND CAR CD19.
>> EXACTLY. RIGHT.
>> CHALLENGES TO THAT OR
STRAIGHTFORWARD?
>> GREAT QUESTION.
MULTIPLE MODELS AND SAYING MY
PATIENT ESCAPED AND WILL TARGET
AND EXPRESSING CD20 AND WILL
GIVE THEM THAT CAR AND GROUPS IN
CHINA HAVE DONE THAT A LOT AND
GOOD RESULTS I WILL SAY AND WHAT
SEEMS TO BE THE THEME IS WHEN
YOU COME IN AND CD19 IS
SUCCESSFUL AND VERY
SERENDIPITOUS AND ANTIGEN AND
GREAT TARGET ANTIGEN AND CASE OF
ALL AND CD20 AND PATIENTS
REMISSIONS DOESN'T SEEM LIKE A
TAIL OF CURE IS QUITE AS GOOD.
I THINK THAT IS NOT VERY WELL
UNDERSTOOD.
JIM THINKS THAT WELL YOU WEREN'T
HERE WITH CAR-T CELLS MALIGNANCY
IS WITH CAR-T CELLS ANTIGEN IS
BAD NECESSARILY AND HARD TO BE A
WAY FOR MALIGNANCY TO CURE YOU
AND EXPENSIVE FOR MULTIPLE
VECTORS AND PRODUCTS IN A KIND
OF INDUSTRY LEVEL AND WHY SO
MUCH INTEREST AND CAN WE PACK A
COUPLE OR THREE CARS INTO A
VECTOR AND HAVE A CELL EXPRESS
THOUSANDS OF ANTI-CD20 AND CD19
AND WILL IT BE EFFECTIVE AND
RESULTS ARE EARLY AND PATIENTS
CAN GO INTO REMISSION AND WILL
IT BE DURABLE AND BETTER OR LESS
TOXIC?
WE STILL DON'T KNOW ALL THAT.
>> OKAY.
>> YEAH. SORRY.
>> HI.
>> GREAT TALK. WE UNDERSTAND
CONSTRUCT OF THE CAR IS CRUCIAL
FOR SUCCESS OF THE GENE THERAPY
AND WHAT ABOUT THE VECTORS AND
DO THE VECTORS HAVE DOWNSTREAM
EFFECTS ON IT AND DO THEY CAUSE
INSERTION OF MUTOGENESIS?
>> YEAH. SEEMS VERY, VERY
LITTLE THAT IT IS EXTREMELY,
EXTREMELY, EXTREMELY RARE.
>> CASE REPORTS AND RECENT ONE
USING TRANSPOISON SYSTEM AND
UNRELATED T-CELL LYMPHOMA THAT
IS A LITTLE MYSTERIOUS AND
DIFFERENT SYSTEM.
VECTORS SEEM TO BE VERY, VERY
SAFE AND TO THE EXTENT THAT
THERE IS A CASE REPORT BY PENN
ACTUALLY INSERTABLE MUTOGENESIS
IN TAP 2 GENE AND MIGHT BE BACK
IN 2018 AND CAR T-CELLS
DEFINITELY CHANGED CAR-T CELLS
AND ENDED UP BEING VERY
EFFECTIVE AND KILLING OFF THE
MALIGNANCY AND EVERYTHING THAT
IS WONDERFUL AND SHOWS THAT KIND
OF THING IS POSSIBLE, YES.
>> OKAY.
>> HOW ABOUT THE SUSTAINABILITY
OF THE EXPRESSION OF THE CAR?
DO YOU LOOK LONGITUDINALLY IN
PATIENTS SEEING IF LEVEL OF
EXPRESSION IS MAINTAINED?
>> SOMETIMES IT GOES DOWN, THE
CAR EXPRESSION IS.
>> DOES IT HAVE TO BE RELATED TO
INTEGRATION SITE?
>> I DON'T.
>> IS THAT STUDIED?
>> I DON'T KNOW. IT DOESN'T
MEAN IT HASN'T BEEN STUDIED.
I HAVE TO LOOK FOR YOU AND THE
QUESTION.
>> INTEGRATE.
>> RIGHT. RIGHT.
>> YEAH.
>> I DON'T KNOW. I HAVE TO LOOK
IT UP HERE.
>> DELETIONS WITHIN --
>> I SEE WHAT YOU ARE SAYING.
>> HASN'T BEEN DONE, I DON'T
KNOW.
>> MIGHT HAVE BEEN. I HAVE TO
LOOK. YEAH.
>> HAVE YOU LOOKED AT
DISTRIBUTION AND BEFORE YOU PUT
IT INTO THE PATIENT AND TIMES
DOWN THE LINE AND CD ONES ARE
EXPANDING AND.
>> WE HAVE IT. PEOPLE HAVE,
YEAH.
PEOPLE HAVE. I JUST HAVE TO
LOOK AND I CAN'T HAVE A GREAT --
GO AHEAD.
>> HOW LOW OF AN ANTIGEN LEVEL
ON THE SURFACE DO YOU THINK?
CD19 AND HIGHER AND YOU KNOW YOU
HAVE NEGATIVES.
WHAT DO YOU THINK IS THE LIMIT?
>> VERY GOOD QUESTION AND
PRODUCT AND CONSTRUCT DEPENDENT
AND CONSTRUCTS WITH DIFFERENT
THRESHOLDS AND BC MAY CELLS FOR
INSTANCE AND PRODUCTS WE
DEVELOPED AND PART OF IT
HONESTLY IS DETECTION METHODS
AND YOU HAVE TO GET USED TO
CHEMISTRY AND FLOW CYTOMETRY AND
PCR IF YOU SET IT UP THAT WAY
AND BCMA EXPRESSION COULD BE
QUITE LOW.
IF UNIFORM, SOME CONSTRUCTS ARE
GOOD AND T-CELLS CAN GO AFTER
AND T-CELLS ARE RELATIVELY -- IN
THE LITERATURE, I SEE THERE IS
VARIATION IN WHAT YOUR CAR
CONSTRUCT CAN PICK UP AND WHAT
ANTIGEN DENSITY.
>> YES?
>> OKAY. SO, FOR PATIENTS
PREVIOUSLY [INDISCERNIBLE].
COULD YOU USE [INDISCERNIBLE]?
>> GREAT QUESTION. YEAH. THIS
IS MOSTLY -- IT IS MOSTLY IN THE
LEUKEMIA LITERATURE THEY ARE
LOOKING AND THEY HAVE
[INDISCERNIBLE] WHICH IS -- AND
NOW THERE ARE SOME ANTI-C19
ANTIBODY THERAPIES IN B-CELL
NON-HODGE KINES LYMPHOMA AS
WELL.
SHORT ANSWER IS, YES. IT CAN
SOMETIMES WORK.
THERE HAS BEEN A -- I WILL SAY
THIS IS A LITTLE VARIETY OF THIS
IN THE LITERATURE AND ARE
REPORTS PATIENTS WITH ALL AND
[INDISCERNIBLE] AND THEY HAVE
MORE OF THE -- SORRY.
FEEDBACK.
THESE CD19S THAT ARE LOW
EXPRESSING AND CD19 CLONES
WITHIN THE MALIGNANCY THAT COULD
MEAN YOUR PATIENT FAIRS POORLY
TO CAR-T CELLS SOME LEUKEMIA
PHYSICIANS WANT TO
[INDISCERNIBLE] AND GET CELLS
MANUFACTURED TO DO THAT FIRST
BEFORE THERE HAS FSH BEEN ANY CD
THERAPY AND THERE IS A LOT TO
LEARN THERE.
>> ANYBODY ONLINE?
ACTUALLY, ONLINE CAN UNMUTE AND
ASK THE QUESTION DIRECTLY. CAN
THEY HEAR ME?
>> OKAY. THANK YOU VERY MUCH.
>> THANK YOU.
>> [APPLAUSE].
>> NEXT SPEAKER IS SARAH WHO
RECEIVED PHD IN VIROLOGY UP IN
CANADA AND DID POST DOCTORAL
TRAINING WITH DUFTY MILLER AND
RECRUITED TO ONTARIO VEGETARIAN
COLLEGE AND IS A PROFESSOR AND
RESEARCH FOCUSES ON GENE THERAPY
AND VIRAL VECTOR VACCINES AND
VIRAL PATHOGENESIS TITLE OF THE
TALK AND MONO CLONAL ANTIBODIES
IS .
>> THANK YOU. CAN EVERYONE HEAR
ME?
PROTECTIONS AGAINST INFECTIOUS
DISEASES AND POINTING OUT THIS
IS A COLLABORATIVE BODY OF WORK
BETWEEN MY LAB AT UNIVERSITY OF
GUELTH.
WE HAVE A LAB IN CANADA AND
STARTING WITH A BRIEF OVERVIEW
WHAT WE DO IN OUR LAB WE ARE
MOLECULAR WITH FOCUS ON MYOGENIC
DISEASES INFECTIOUS DISEASES AND
CANCER AND -- WHICH WILL BE
FOCUS OF MY TALK TODAY AND HAVE
A PROGRAM FOR LUNG GENE DELIVERY
FOCUS ON DELIVERING THERAPEUTIC
GENES AND CYSTIC FIBROSIS AND
USE A PEROMIXO VIRUS AND VIRAL
VECTOR VACCINE AND ONCO LYTIC
VIRUS AND WE HAVE ONE OR TWO
STUDENTS WORKING ON A POX VIRUS
SHEEP VIRUS.
OKAY.
SO IN CASE MANY ARE FAMILIAR
WITH AV AND THOUGHT I WOULD GIVE
A BRIEF INTRODUCTION AND AV IS A
SMALL AND NAKED DNA VIRUS.
IT IS IN THE FAMILY PIROVIRADAY.
IT IS REPLICATION DEFECTIVE
VIRUS AND DEPENDS ON IT TO
COMPLETE A LIFE CRYINGLE AND
OVER 200 -- AND OF COURSE THERE
ARE A NUMBER OF AVS RECEIVING
APPROVAL AND FDA WITH RECENT
BEING [INDISCERNIBLE] AND
MUSCULAR DISTROPHY AND JEN
MENTIONED THEY ARE MORE
EXPENSIVE AND DELIVERING MORE
VECTOR TO THE PATIENTS IN THIS
CASE AND NUMEROUS GENE THERAPIES
WAITING REGULATORY APPROVAL AND
40% ARE AD AND AD HAS A STRONG
SAFETY RECORD AND EFFICACY
RECORD IN CLINICAL TRIALS AND
TODAY I WILL TALK ABOUT THE
IMMUNOTHERAPY FOR INFECTIOUS
DISEASES AND KNOW THAT
MONOCLONAL ANTIBODY THERAPIES
ARE AN IMPORTANT TOOL IN OUR
[INDISCERNIBLE] TO PREVENT
INFECTIOUS DISEASES AND
ESPECIALLY FOR VIRUSES WE LACK
VACCINES AND EFFECTIVE THERAPIES
AND ISSUES WITH RECOMBINANT
THERAPY IS IT IS SHORT-LIVED
REQUIRING REPEAT DOSING AND
CIRCUMSTANCES ANTIBODIES HAVE TO
BE DELIVERED SYSTEMICALLY
PROBLEMATIC IN CASES AND MAPPING
COCKTAIL DELIVERED INTRAVENOUSLY
DURING A WEST AFRICA OUTBREAK
AND FRAGILE AND REQUIRE
[INDISCERNIBLE] AND DIFFER FROM
HOST DEPENDING HOW THEY ARE
PRODUCED SEEING WITH WEST AFRICA
OUTBREAK DIFFICULT TO SEE THESE
SCALE OUT AN OUTBREAK AND WE AND
OTHERS IN THE LAB HAVE EXPLORED
THIS ALTERNATIVE THAT IS VEKOR
EXPRESSION ANTIBODIES IN VIVO.
SO, PETER -- BALTIMORE AND BLAKE
COINED THE TERM AND AIVVP THAT I
WILL SAVE FROM HERE ON IN AND
USE OF GENE DELIVERY VECTOR THAT
IS AV VECTOR TO EXPRESS BROADLY
NEUTRALIZING MONOCLONAL -- SO,
IN A SENSE YOU TURN YOUR HOST
INTO THE VIRAL REACTOR.
THIS IN MOST CASES REQUIRES A
SINGLE INTRAMUSCULAR INJECTION
AND STUDIES IN LAB SUGGEST
SPRAYING DOSE OVER MULTIPLE
DOSES ENHANCES MONOCLONAL
ANTIBODY PRODUCTION AND
GLYCOSYLATION PATTERNS MATCH THE
HOST AND THIS CAN BE USED IN
IMMUNOCOMPROMISED INDIVIDUALS
AND WE AND OTHERS HAVE SHOWN
THIS LEADS TO VERY LONG AND
SUSTAINED ANTIBODY EXPRESSION IN
SOME SPECIES FOR THE LIFETIME OF
THE ANIMAL AND THIS APPROACH WAS
FIRST INVESTIGATED FOR TREATING
AND PREVENTING HIV AND ARE TWO
CL
CLINICAL TRIALS FOR PREVENTING
HIV INFECTIONS IN HUMANS AND
SHORT TIME I HAVE I WILL TRY TO
GO THROUGH WORK WE HAVE DONE FOR
VARIETY OF INFECTIOUS DISEASES
AND RECENTLY WORKING ON
BACTERIAL INFECTIONS AND GIVING
A LITTLE INTRODUCTION TO EBOLA
VIRUS AND NEGATIVE SENSE RNA
VIRUS IN FAMILY FILO VER ADAY
AND FOUR CCS OF EBOLA VIRUS AND
[INDISCERNIBLE].
THIS IS A ZOONOTIC INFECTION
TRANSMITTED FROM ANIMALS TO
HUMANS FROM BATS TO MONKEYS TO
HUMANS OR BATS DIRECTLY TO
HUMANS VIRUS ENTERS BODY TO
EXPOSURE TO CONTAMINATED BLOOD.
IT HAS A HIGH FATALITY RATE
BETWEEN 40 TO 90% AND AS MANY
WILL REMEMBER, WE HAVE LARGEST
OUTBREAK OF EBOLA VIRUS IN WEST
AFRICA BETWEEN 2014 AND 2016 AND
OVER 20,000 CONFIRMED CASES AND
11,000 DEATHS AND FATALITY RATE
AND DURING THE OUTBREAK A NUMBER
OF VACCINES AND THERAPIES WERE
TESTED AND WE HAVE A LICENSED
VACCINE RECOMBINANT VIRUS AND
VACCINE FOR EBOLA THAT ENCODES
PROTEIN ON THE SURFACE AND
ANTIBODY THERAPY THAT IS
PARTIALLY DEVELOPED HERE IS
APPROVED AND THEN IN SUBSEQUENT
OUTBREAKS ADDITIONAL MONOCLONAL
ANTIBODIES RECEIVED APPROVAL.
WHEN I FIRST STARTED TO WORK ON
THE VECTOR IMMUNOPROPHYLAXIS IS
AT THE BEGINNING OF THE WEST
AFRICA OUTBREAK AND GOING TO LAB
TO WORK ON ANTIBODIES THAT ARE
ISOLATED AND ARE MOUSE
ANTIBODIES STARTING WITH 2G4
THAT IS COMPONENT OF SAID MAP A
NEUTRALIZING ANTIBODY EVALUATING
5D2.
THESE WERE DERIVED FROM MICE
THAT WERE VACCINATED WITH THE
VACCINE.
SO, WE WERE NOT SURE THAT MICE
WERE MOUNTED IMMUNE RESPONSE TO
HUMAN CONSTANT DOMAIN AND
ENGINEERED AS IG2A ANTIBODIES
AND USED SAME CONSTRUCT DESIGN
AS ALEX BLAZE WITH A STRONG
MUSCLE SPECIFIC MOTOR AND
CLEAVAGE AND MICHAEL IS CRINGING
AT RIGHT NOW AND INCLUDING DP AS
WELL AND ANTIBODIES COULDN'T
QUANTIFY MOUSE OF AMOUNT OF
ANTIBODY PRODUCED IN MICE AND
USING EBOLA PROTILLIZES AND SEE
IN 7 DAYS WE HAD ANTIBODIES IN
THE BLOOD AND THEY INCREASE AND
PLATEAU AND THESE WERE JUST DATA
TO 125 DAYS AND LONG-TERM GENE
EXPRESSION AS WELL WE HAVE.
OKAY. WE KNEW WE COULD EXPRESS
ANTIBODIES IN MICE AND MOVE TO
CHALLENGE STUDIES AND TYPICAL
REGIMENT IS TO ADMINISTER
GENOMES OF AV TO THE MUSCLE AND
28 DAYS LATER WE KNOW WE HAVE
HIGH LEVELS OF ANTIBODY
EXPRESSION AND CHALLENGE MICE
AND CHALLENGED WITH MOUSE
ADAPTED EBOLA VIRUS PERITONEAL
CHALLENGE AND THREE ANTIBODIES
VECTORIZED LED TO CASES COMPLETE
PROTECTION FROM CHALLENGE AND IN
CASE 2G4 WE LOST ONE OF THE
ANIMALS AND GENERALLY HIGH LEVEL
RATE OF SURVIVAL AND EVALUATING
THERAPEUTIC WINDOW AND I MEAN IT
IS 28 DAYS THAT IS IDEAL FROM
POINT OF VIEW FROM OUR
EXPERIMENTS THAT WE WANTED TO
SEE IF WE COULD SHORTEN THE
THERAPEUTIC WINDOW AND SAME DOSE
OF 1E11 AND CHALLENGED WITH
EBOLA VIRUS 2 WEEKS 7 DAYS 1 DAY
OR CHALLENGED THEM BEFORE WE
GAVE AV AND SEEING AT 7 DAYS
AFTER AV WAS ADMINISTERED 100%
OF MICE WERE PROTECTED REDUCING
TO THREE DAYS WE HAD 20%
SURVIVAL RATE AND SOMEWHERE
BETWEEN 3 AND 7 DAYS FOR
PARTICULAR ANTIBODIES WOULD BE
THE THERAPEUTIC WINDOW.
EVALUATED LONGEVITY OF
EXPRESSION AND ABILITY FOR
ANTIBODIES TO PROTECT LONG-TERM
AND IN CASE MICE RECEIVED TWO
DIFFERENT AVMABS ONE IN EITHER
LEG AND LET SIT IN ANIMAL
FACILITY FOR FIVE MONTHS AND
SHIPPED TO WINE PEG THAT WERE
CHALLENGED AND IN TERMS OF LONG
TERM MONITORING DELIVERING AV
INTRAMUSCULARLY SHOWN HERE OR
INTRANASALLY SUSTAINED ANTIBODY
EXPRESSION AND IN RECENT STUDIES
EXPRESSED HUMAN ANTIBODIES TO
QUANTIFY AMOUNT OF ANTIBODY IN
THE BLOOD AND SEE THAT
ANTIBODIES WERE EXPRESSED IN
LIFETIME OF ANIMAL AND NATURAL
AEBD POINT THEY HAVE 200
MICROMILLIGRAMS OF ANTIBODY AND
BLOOD AND QUESTION THAT IS ASKED
IF YOU HAVE SO MUCH ANTIBODY AND
CIRCULATION WILL IT IMPEDE
ABILITY OF ANIMAL TO RESPOND TO
ANALOGOUS CHALLENGE AND IN THIS
CASE DESIGNING AN EXPERIMENT
ADMINISTERING AVMABS AND TWO
WEEKS LATER VACCINATED AGAINST
INFLUENZA AND MICE WERE BOOSTED
70 DAYS LATER AND EVALUATED
ANTI-INFLUENZA ANTIBODY RESPONSE
AND MICE RECEIVING AV MABS ARE
EXPRESSING EBOLA-SPECIFIC
ANTIBODIES AND HERE ARE ANTI-HA
TIRES AND MICE GIVEN AV HAD HA
TIGHTERS DISTINGUISHABLE FROM
MICE GIVEN PDS AND HIGH
CONCENTRATION OF ANTIBODIES
CIRCULATING IN SIERRA MIST
DOESN'T IMPEDE ANIMAL FOR
RESPONDING TO THIS CHALLENGE AND
ANTIBODY CLONING THERAPIES AND
STRATEGIES OF DRAMATICALLY
IMPROVED OVER THE YEARS AND WE
HAVE A NUMBER OF VERY HIGHLY
POTENT HUMAN ANTIBODIES CLONED
FROM HUMAN SURVIVORS AND TWO
INCLUDED MAB100 AND 114 AND
ISOLATED FROM SURVIVOR OF EBOLA
VIRUS AND B-CELLS FROM N1995
OUTBREAK AND MOVED TO EXPRESSING
ANTIBODIES HUMAN IGG1 ANTIBODIES
WE CAN DETECT NOW IN THE SERUM
AND USING SAME CONSTRUCT
DESIGNED AND STUDIES MOST
EDAPTED TO EBOLA AND SEE AT
DOSES OF EBOLA DOSES 5 TIMES TO
THE 9 WE HAVE HIGH SURVIVAL AND
MAB114 WE START WITH LOWER DOSE
AND DOSE TO THE 10 WE HAVE HIGH
SURVIVAL IN ANIMALS LD50 DOSE
AND HIGH DOSE OF EBOLA WE HAVE.
AND SO WE STARTED TO, YOU KNOW,
WE HAVE A VACCINE FOR EBOLA.
AND WE DECIDED TO THEN FOCUS ON
A PATHOGEN WE DON'T HAVE A
VACCINE OR ANTIVIRALS AND
FOCUSED ON THE VIRUS AND VIRUSS
ARE SIMILAR TO EBOLA VIRUS AND
SIMILAR CASE FATALITY RATE AND
SIMILAR CLINICAL MANIFESTATION
AND WHO HIGH PRIORITY PATHOGEN
WITH EPIDEMIC POTENTIAL AND
CONTINUE TO BE OUTBREAKS OVER
THE YEARS AND RECENT OUTBREAKS
WERE IN FEBRUARY OF 2023 IN
NEWGUINEA AND REPORTS OF
OUTBREAKS STILL HAPPENING IN
JUNE AND NO LICENSE VACCINES OR
THERAPEUTICS AVAILABLE.
MAKING VERY GOOD ANTIBODIES
ISOLATED FROM HUMAN SURVIVOR
THAT IS MR191 AND NICE
EXPRESSION KINETICS DOSE OF 1
TIMES 1011 AND PEAK LEVELS OF
300 MICROGRAMS PER ML IN THE
BLOOD AND 1 x 10 TO THE 11 OR 10
TIMES 10 GENOMES 100% PROTECTION
FROM MOUSE ADAPTED VIRAL
CHALLENGE AND INTERESTED IN
MOVING AVV PLATFORM INTO HIGHER
ORDER SPECIES WE MOVE TO THE
GUINEA PIG MODEL AND ADAPTED
VIRUS AND IN CASE ANIMALS ARE
LARGER HIGH DOSES OF AV RANGING
FROM 5 x 12 TO THE VECTOR
GENOMES AND 5 x 11 AS A LOW DOSE
AND YOU CAN SEE AT MID AND HIGH
DOSE WE HAVE HIGH LEVELS OF
PROTECTION AND AGAIN 1,000 LD50
GUINEA PIG VIRUS CHALLENGE.
WE ARE SEEING EXPRESSION LEVELS
OF BETWEEN 50 TO 100 MICROGRAMS
PER MILL AND HUMAN ANTIBODIES IN
A GUINEA PIG AND GOAL IS TO
EVALUATE AVVIP IN NON-HUMAN
PRIMARY MODEL AND BEFORE MOVING
TO THE MODEL WE WANT TO EVALUATE
HOW THE VECTOR PERFORMED IN A
LARGE ANIMAL SPECIES AND I HAVE
ACCESS TO PRODUCTION ALMOST AND
ELECTED TO USE SHEEP AND
OBTAINED THREE LANDS TWO WEEKS
OF AGE ADMINISTERED AV
EXPRESSING ANTIBODY THAT IS A
HUMAN ANTIBODY DOSED 5 x 10 TO
THE 12 -- WE DID ADMINISTER IN
TWO DIFFERENT DOSES.
HERE ARE THE -- WE ARE ABLE TO
MONITOR SHEEP FOR THREE YEARS
AND ANTIBODY EXPRESSION LEVELS
OVER TIME WE HAVE AND SHEEP HAD
TO LEAVE THE STUDY. HE WAS
PARTICULARLY ORNERY.
WE HAVE -- HE WAS NOT A NICE
ANIMAL.
WE WERE ABLE TO KEEP THE
REMAINING TWO FEMALES OUT TO
ALMOST THREE YEARS AND YOU CAN
SEE THERE IS A GRADUAL INCREASE
IN THE ANTIBODY EXPRESSION
LEVELS THAT STARTS TO SLOWLY
WAYNE AND AT AN ENDPOINT THEY
HAVE BETWEEN 20 TO 40 MICROGRAMS
PER ML OF HUMAN ANTIBODY IN THE
BLOOD AND 5 KILOGRAMS GROWING TO
80 KILOGRAMS AND LOTS OF CELL
TURNOVER AND MUSCLE MASS GREW
OBVIOUSLY AND ULTIMATELY DIDN'T
LEAD TO DILUTION OF VECTOR AND
AV INDUCED CELLS TO -- THIS IS
FIRST TIME WE DECIDED TO LOOK AT
RESPONSES THAT WE DID SEE
ANTI-CAPSIDATED VIRUS RESPONSES
THAT ARE UNEXPECTED.
PR
THIS ASSAY ONLY EVALUATED
RESPONSES THAT WE DIDN'T LOOK AT
RESPONSES AGAINST VARIABLE LIGHT
CHANGE ON NAMES AND TYPE OF
ANTIBODIES TO ACTUALLY QUANTIFY
THIS AND BOTTOM LINE LITTLE TO
NO DRUG ANTIBODY RESPONSE THAT
IS WHY WE WERE ABLE TO SEE
LONG-TERM ANTIBODY EXPRESSION.
WE ARE NOT SURE WHY THIS IS THE
CASE.
AGAIN WITH NON-HUMAN PRIMATE
STUDIES THAT IS NOT WHAT PEOPLE
SEE. WE HAVE A NUMBER OF
EXPERIMENTS TO EVALUATE WHAT IS
GOING ON AND COULD BE BECAUSE OF
NATURE OF ANTIBODY AND NEXT
STEPS FOR AVPIP IS TO WRITE A
MONKEY STUDY THAT IS SCHEDULED
FOR THE SUMMER OF 2024 AND
REGARDING THE GENOME WE HAVE
MICCAC DOMAINS AND WE HAVE W2A
AND PRA AND NEED TO REVISE THE
CONSTRUCT DESIGN AND SO FAR WE
HAVE SEEN VERY GOOD EXPRESSION
OF THIS ANTIBODY IN MICE.
THE PLAN IS TO DOSE 5 x 10 TO
THE 12 VECTOR GENOMES PER KG AND
CHALLENGE 28 DAYS LATER WITH
WILD TYPE AUTHENTIC -- BIOLOGY
LAB IN CANADA AND WORKING ON
RESPECT TO AVIP FILO VIRUSES TO
DEVELOP A VIRUS AVVIP AND
PROTEINS DIFFER SIGNIFICANTLY
AND RANGE BETWEEN 55 AND 66 AM
EVENO ACID IDENTITY AND PROTEINS
IS MUCH LOWER AND 31% IMMUNOACID
IDENTITY AND WE DON'T HAVE PAN
FILO VIRUS ANTIBODIES AND WE
[INDISCERNIBLE] BISPECIFIC AN
ANTIBODY AND HEAVY CHAINS AND
INSERTED THE SCFV FOR A MODERN
VIRUS ANTIBODY MR1 AND WE HAVE
BEEN ABLE TO DETECT ANTIBODY
EXPRESSION IN VIVO.
THIS IS A LARGER CONSTRUCT THAT
IS A LITTLE OVERSIZED DIDN'T
HAVE WERPV BUT SEE EXPRESSION
STILL THAT IS VERY EXCITING AND
HAVE A POSTER TO TALK ABOUT
CHALLENGE RESULTS FROM THE
STUDY.
OKAY. I STILL HAVE TIME AND WE
WILL MOVE ON TO RESPIRATORY
INFECTIONS.
OBVIOUSLY, YOU KNOW, FILO
VIRUSES ARE IMPORTANT AND
INTERESTING.
THIS IS NOT REALLY SOMETHING
THAT THE CAN AIDIAN GOVERNMENT
IS WORRIED ABOUT AND MOVING TO
WORKING ON RESPIRATORY PATHOGENS
AND ANYWAY WE DECIDED TO
EVALUATE WHETHER A PLATFORM
WOULD WORK FOR RESPIRATORY
INFECTIONS AND VIRUS AND
INFLUENZA VIRUS.
AS YOU KNOW RSVS ARE PROBLEMATIC
IN INFANTS AND ELDERLY
IMMUNOCOMPROMISED.
THIS REQUIRES MONTHLY
INTRAMUSCULAR INJECTIONS NOT TO
QUALIFY AT LEAST IN CANADA THERE
IS STRICT CRITERIA AND BABIES
ENTITLE TODAY HAVE ANTIBODY IN
FIRST YEAR OF LIFE AND WE KNOW
RESPONDING POORLY TO INFLUENCE
OF VACCINATION EVALUATING AVVIP
FOR THESE TWO RESPIRATORY
PATHOGENS AND DEALING WITH THEM
WE WANT TO INVESTIGATE WHETHER
OR NOT ANTIBODIES WOULD MAKE WAY
TO MUCOSAL SURFACES VERY GOOD
ANTIBODY EXPRESSION LEVELS THAT
WE USED ANTIBODY FROM JAMES
RSV90 AND ACTIVATED
[INDISCERNIBLE] AND EUTHANIZED
MICE WE COULD DETECT ANTIBODIES
IN LUNG AND INTESTINES AND
VAGINA AND ARE CONFIDENT THAT
ANTIBODIES ARE MAKING WAY TO
MUCOSAL SURFACES.
FIRST AGAIN IS STANDARD 1 x 1011
-- IN 28 DAYS LATER THEY WERE
CHALLENGED WITH RSVA2 STRAIN
INTERNASALLY DOSE OF 1 x 6PFU
AND DON'T DEVELOP CLINICAL SIGNS
VIRUS INFECTION AND CAN SEE
EVIDENCE OF VIRUS REPLICATION
AND FIVE DAYS POST CHALLENGE
QUANTIFYING AMOUNT OF VIRUS IN
THE LUNGS AND SEE IN MICE WE ARE
TREATED WITH VECTORIZED
[INDISCERNIBLE] AND NO EVIDENCE
OF REPLICATING VIRUS IN THE
LUNGS AND CONTROLLED AV AGAINST
EBOLA VIRUS THAT YOU CAN SEE
THERE IS A VIRUS REPLICATION IN
THE LUNGS AND PROBLEM FOR
INFANTS WANTING TO INVESTIGATE
IF THESE ANTIBODIES COULD BE
TRANSMITTED TO OFFSPRING.
28 DAYS LATER THEY WERE ALLOWED
TO AND DID IT WITH MICE AND
PREGNANT AND MOVED TO DIFFERENT
CAGES AND MONITORED ANTIBODY
EXPRESSION IN THEIR OFFSPRING.
YOU CAN SEE AT THE TIME WHEN THE
PUPS WERE BORN WE EUTHANIZED
PUPS AND EXTRACTED BLOOD AND
HIGH LEVELS OF ANTIBODY
EXPRESSION IN THE BLOOD AND
WANED OVER TIME AND BECAME
DETECTIBLE THE ANTIBODIES COULD
TRANSFER TO THE OFFSPRING AND
OFFER PROTECTION AND QUESTION WE
ARE ASKED IS CAN YOU TURN OFF
ANTIBODY EXPRESSION AND P SITES
ON EITHER SIDE OF CDNA FOR
ANTIBODY AND ADMINISTERED TO
MICE 28 DAYS LATER ADMINISTERED
VECTOR A STRONG MUSCLE SPECIFIC
CAPSID AND PROTEINS THAT IS
FUSED TO THE HIV TAP PROTEIN
THAT IS SELF-PENETRATING PEPTIDE
AND RESULTS THAT USE ANTIBODY
EXPRESSION DAY 28 AND
ADMINISTERED [INDISCERNIBLE] OR
RECOMBINANT TAT PROTEIN AT THIS
INITIAL CONCENTRATION DIDN'T
INTERFERE OR OBLATE ANTIBODY
EXPRESSION IN RED AND LOOKING AT
BLUE LINE AV DELIVERED WE SAW A
BLENDING OF ANTIBODY EXPRESSION
NOT ABLATION OF EXPRESSION BUT
HIGHER DOSES MIGHT HAVE BEEN
ABLE TO OBLATE IT AND
ADMINISTERED HIGHER DOSE CREATED
AT A LATER TIME POINT AND SAW A
REDUCTION AND POTENTIALLY HOPE
FOR ADMINISTERING RECOMBINATION
PROTEIN IF THIS WERE EVER TO
BECOME AN ISSUE.
MOVING ON TO INFLUENZA VIRUS WE
VECTORIZED ANOTHER ANTIBODY AND
THIS IS AN ANTIBODY EXPRESSING
ONE IN VIVO AGAIN 100 OR 150
MICROGRAMS PER ML AND STANDARD
VIRAL ANTIBODIES AND SAW FAIRLY
GOOD PROTECTION WITH MALE MICE
AND FEMALE MICE DIDN'T CONFER A
SIGNIFICANT AMOUNT OF
PROTECTION.
REVISING ANTIBODY AND SEQUENCE
TO INCLUDE A MICRO-RNA SCAFFOLD
DOWNSTREAM WITH CODING SEQUENCE
FOR THE ANTIBODY AND THIS
SCAFFOLD INCLUDES THREE SRNAS
AGAINST THREE GENE SEGMENTS.
WITHOUT -- THIS IS A BUSY GRAPH
THAT I WANTED TO POINT OUT BY
INCLUDING MICRO-RNA WE INCREASE
EFFICACY OF THIS PLATFORM AND
THIS IS THE ANTIBODY ON THE
[INDISCERNIBLE] AND HERE IS ANTI
BODY PLUS MICRON RNA AND HERE
THIS IS AGAIN MICRON RNA
EXPRESSING VECTOR AND IS A WAY
TO POTENTIALLY INCREASE THE
POTENCY OF VIP PLATFORM AND NOT
NECESSARILY INCREASING GHOSTS
AND NOW I WILL MOVE TO USE OF
AVVIP FOR BACTERIAL INFECTIONS
AND FOCUSING ON [INDISCERNIBLE]
AND HAVE INTEREST IN CYSTIC
FIBROSIS A CAUSE OF CHRONIC LUNG
INFECTION AND PEOPLE WITH CYSTIC
FIBROSIS AND RESPONSIBLE FOR 10
TO 15% OF INFECTIONS INHERENT
WORLDWIDE AND DRUG RESISTANT AND
NO VACCINES AVAILABLE AND TWO
PROMISING CANDIDATES THAT IS
POLYSACK ARIDE APSEUDOMOWNIS AND
COMPONENT OF TYPE 3 SECRETION
SYSTEM AND ALSO DEVELOPED
BISPECIFIC ANTIBODY COMBINING
CSFE FOR ANTIBODIES AND
VECTORIZE THE ANTIBODIES
EVALUATING EXPRESSION OF
KINETICS IN MICE AND SEE 100 TO
400 AND 8 MICROGRAMS PER ML WE
EVALUATED AND CONCENTRATIONS AT
11 VECTOR DOSE.
YOU KNOW, WE ARE GETTING ONE TO
2 UPWARDS OF THREE MLES AND
COULDN'T TELL YOU WHY IT
EXPRESSED 10 TIMES -- EVERYTHING
ELSE WAS THE SAME.
WE ALSO EXPRESSED BISPECIFIC
ANTIBODY AND WPRE WE THOUGHT
WOULD BE PROBLEMATIC AND HAD
REASONABLE EXPRESSION AND
EXPRESSION WANED FASTER THAT
CONVENTIONAL MONOCLONAL
ANTIBODIES AND PERFORMED ACUTE
CHALLENGE WITH APSEUDOMOWNIS
MODEL AND MICE WERE ADMINISTERED
AND ONE OF THREE VECTORS
INTRAMUSCULARLY AND 28 DAYS
LATER STANDARD PROTOCOL AND MICE
CHALLENGED WITH LETHAL DOSE OF
PA1 AND PA14 AND BISPECIFIC
ANTIBODY CONFERRED HIGH LEVELS
OF PROTECTION AGAINST A LETHAL
BACTERIAL CHALLENGE 18 TO 20
HOURS POSTED ADMINISTRATION.
WE WANTED TO INVESTIGATE IMPACT
OF BIODISTRIBUTION IN THE MODEL
AND MICE WERE CHALLENGED 28 DAYS
AFTER RECEIVING THE AV.
18 HOURS POST CHALLENGED MICE
WERE EUTHANIZED AND QUANTIFIED
AMOUNT OF COLOFTENIES IN VARIOUS
TISSUES DIRECTING GROUPS HERE
AND LIKE IN INFLUENZA WE SAW
DIFFERENCES AND SEX DIFFERENCES
IN TERMS OF THE CHALLENGE MODEL
AND YOU CAN SEE HERE BISPECIFIC
OR DOSE OF BOTH ANTI-PCRV AND
ANTI-PCRL YOU REDUCE BURDEN IN
TISSUES WE EXAMINED IN BOTH
CASES AND THESE ARE CONTROLS WE
WERE REPLACING THAT AND IN TERMS
OF FUTURE DIRECTIONS FOR THE
PROJECT WE HAVE SHOWN WE CAN
PREVENT AND USE PROPHYLACTICALLY
AND WOULD LOVE TO SEE IF WE CAN
USE THIS THERAPEUTICALLY AND WE
WILL EVALUATE COLONIZATION AND
BIOFILL FORMATION AND AVVP CAN
BE USED TO TREAT COLONIZATION
AND PREVENT IT AND HAVE INTEREST
IN CYSTIC FIBROSIS AND USING
CFTR CORRECTED MICE AND THESE
MICE WILL BE NATURALLY COLONIZED
PSEUDOMOWNIS THAT YOU CAN ADD TO
WATER AND NASAL CAVITIES CAN
BECOME THEM AND PE FORM NASAL
SWABS TO QUANTIFY THE NUMBER AND
ALSO INCIDENTALLY IN MODELS
EXPRESSING CFTR THIS PREVENTS
COLONIZATION AND NICE MODEL AND
EVALUATING WHETHER AVVIP CAN BE
USED TO TREAT COLONIZATION
THERAPEUTICALLY.
SUMMARY AND THIS IS ACTUALLY THE
CAP THAT WE ENGINEERED IN OUR
LAB NEGATES RAPID AND PROLONGED
SERUM ANTIBODY EXPRESSION AND WE
HAVE SHOWN THIS IS A GOOD
PLATFORM FOR PROPHYL ACTACTIVE
EFFICACY.
IF YOU ADMINISTER EXPRESSING
THESE -- THESE CAN BE
TRANSFERRED MATERNALLY AND
INCREASE POTENCY OF PLATFORM BY
INCLUDING CASSETTE -- OF COURSE
IN THIS SITUATION DELIVER THE AV
TO TARGET ORGAN AND COMBATING
INFLUENZA VIRUS INFECTIONS TO BE
DELIVERED TO THE NASAL CAVITY OR
LUNG.
WE BELIEVE AVVIP HOLES GREAT
POTENTIAL.
ALSO, FOR INDIVIDUALS THAT ARE
IMMUNODIFISHIENT AND COULD BE
USED IN OUTBREAK SCENARIOS FOR
PATHOGENS THAT DON'T HAVE
VACCINES BUT THERAPIES.
I WOULD LIKE TO THANK MEMBERS OF
MY LAB CONTRIBUTING TO WORK
INCLUDING ANNE WONG AND JORDAN
AND COLLABORATORS IN WINNIPEG.
I INITIALLY STARTED TO WORK ON
AVVIP WHEN I WENT TO WORK WITH
GARY AT UTMB AND COLLABORATORS.
I WOULD LIKE TO THANK THE
FUNDING AND THANKS FOR YOUR
ATTENT
ATTENTION.
>> YOU TOUCHED ON SEX-SPECIFIC
DIFFERENCES. WHAT DO YOU THINK
IS GOING ON THERE?
>> PARTIALLY FEMALE MICE WEIGH 7
OR 8 GRABS LESS THAN MALE MICE
AND THOUGHT ABOUT IT AFTER THE
FACT AND DIDN'T TAILOR DOSE TO
INFLUENZA IN MALE AND FEMALE
MICE AND POTENTIALLY SHOULD DOSE
BASED ON WEIGHT AND WORKING WITH
INFLUENZA MODEL DOSING BASED ON
WEIGHT AND EASY TO DOSE AND WE
WILL GIVE A HIGH DOSE OF
INFLUENZA.
>> THANK YOU.
>> SO, DO YOU IN ANY OF THE
SITUATIONS, DO YOU THINK THAT IN
PEOPLE ANTIBODIES COULD BE
GENERATED? IF SO ANY WAY TO
PREDICT WHICH MIGHT AND WHICH
MIGHT NOT GIVE IT?
>> VERY GOOD QUESTION AND
PRIMATE STUDIES EVALUATING AVVIP
AND MY UNDERSTANDING STUDIES
BROADLY INVOLVE [INDISCERNIBLE]
EXCEPTION OF WORK WHICH IS
SLIGHTLY DIFFERENT TYPE OF
ANTIBODY.
IT IS DIFFERENT. YES.
IF YOU HAVE A BROADLY
NEUTRALIZING ANTIBODY DERIVED
AGAINST HIV THEY INDUCE
ANTIVIRAL DRUG RESPONSES AND
EXCITED TO DO NON-HUMAN PRIMATE
STUDY WITH THESE ANTIBODIES IS
THIS IS AN ANTIBODY HARVESTED
FROM B-CELLS IN PATIENT AFTER
ACUTE INFECTION AND NOT SOMATIC
HYPERMUTATION AND DIDN'T SEE
ANTIDRUG ANTIBODY RESPONSES IN
THE SHEEP BODES WELL FOR
NON-HUMAN PRIMATES AND ANSWERING
QUESTION ISSUE RELEVANT WITH HIV
ANTIBODY WITH THOSE DERIVED WITH
RSV ANTIBODY FOR EXAMPLE AND
INFECTIONS.
>> THAT IS THE PLATFORM COMPARED
TO MRNA PLATFORM TO EXPRESS
ANTIBODIES, YOU KNOW?
>> I HAVE SEEN PAPERS PEOPLE
EVALUATED DNA AND DAVID'S GROUP
IS SEEING MAYBE NOT THE SAME
LEVEL OF ANTIBODY EXPRESSION AND
DURATION OF THE EXPRESSION SEEMS
QUITE SIMILAR AND I HAVE NOT
FOLLOWED MRNA LITERATURE AND
IMPRESSION IS YOU WON'T HAVE
ANYWHERE NEAR SUSTAINED
EXPRESSION. NOT SURE IF ANYONE
TRIED THAT.
>> I HAVE A FEELING IT WON'T
WORK FOR THIS PURPOSE.
>> MRNA DOESN'T ACHIEVE THE
LEVELS OF VECTOR OR ANTIBODY.
DURABLE IS FINE AND GETTING TO
THERAPEUTIC LEVELS.
>> INTERESTING.
>> [INDISCERNIBLE].
>> GO AHEAD.
>> INTERESTING EXPECTING THE
EXACT OPPOSITE THAT IS
INTERESTING.
>> YES.
>> A COUPLE OF NEGATIVES IN
THERE THAT IS GREAT.
BUT ONE QUESTION, YOU ARE
INTERPRETING THE SHEEP AS HAVING
CONFID
CONFIDENCE.
THEY WERE TALKING ABOUT EARLIER
SUCCESSFUL SUPPRESSION STUDY AND
YOU STARTED WITH LACK.
>> I DID, YES.
>> THAT MIGHT BE A KEY VARIABLE
THERE. YOU ARE RIGHT. NATURE
OF ANTIBODIES AND OTHER QUESTION
IS TARGETING THE VIRUS THAT IS
THE VIRUS AND CELLS AND HOW IS
THAT WORKING?
>> TECHNIQUE THAT GETS AV TO THE
LUNG.
I SHOULD -- I MEANT TO HIGHLIGHT
THAT AND FORGOT AND VECTOR WE
WILL USE IS A POTTIENT
TRANSDUCER OF LUNG AND MODEL AND
LUNG AND OTHER SPECIES AS WELL.
YEAH.
>> [INDISCERNIBLE].
>> ABOUT TO QUANTIFY ANTIBODY IN
THE LUNGS WITH A SORT OF RECENT
STUDY AND VECTORS WITH JUST
MICRO-RNA ON THEIR OWN
EXPRESSING GFP AND DIDN'T SEE A
LOT OF PROTECTION FROM THOSE.
HOWEVER, I THINK WE NEED TO
INCREASE THE DOSE AND HAVE TO DO
OPTIMIZATION AND HAVE DONE ONE
STUDY WITH MICRO-RNAS.
YOU HAVE TO HAVE ANTIBODY AND
EXPRESSED IN CELL TYPE VIRUS
WILL INFECT AND BE USEFUL
PACKAGED INTO EXOSOMES AND THE
OTHER THING.
>> TALKING ABOUT ENGINEERING AND
KILL SWITCH.
>> YES.
>> POTENTIAL DOWNSIDES OF
LONG-TERM ANTIBODY EXPRESSION,
WHY WOULD YOU --
>> [INDISCERNIBLE]. I MEAN I
THINK FOR STUDIES IN LARGE
ANIMALS LEVELS OF ANTIBODY
EXPRESSIONS ARE DESIRABLES AND
200 MICROGRAMS PER MILL IS A
NICE LEVEL OF ANTIBODY
EXPRESSION IS A THERAPEUTIC AND
NOT SEEING IN ONE STUDY GETTING
MGS PER MIL AND IF YOU USE AV TO
EXPRESS ANTIBODIES FOR
INDICATIONS LIKE CROHN'S DISEASE
OR LONG-TERM A ANTIBODY
EXPRESSION FOR A DISEASE WOULD
BE AN ISSUE AND FEEL THAT HAVING
ANTIBODIES AROUND ME PROTECT
AGAINST INFECTIONS.
IT IS GOOD.
>> YES, MA'AM.
>> VECTORS I COULD UNDERSTAND
HOW THEY WOULD BE EFFECTIVE FOR
RESPIRATORY VIRUSES MENTIONING
HIV FOR A FEW TIMES AND CAN YOU
MEASURE ANTIBODIES AND TISSUES
IN THE BODY AND HOW ABOUT
THROUGH THE BRAIN?
HOW IS THAT?
>> I HAVE NOT DONE WORK AND
STAYED AWAY FROM HIV AND MICHAEL
DID WORK AND PUT YOU ON THE HOT
SEAT AND QUANTIFIED.
>> RATE IS MUCH, MUCH LOWER AND
GOING ONE-TO-1,000.
>> HOW ABOUT IN OTHER TISSUES?
>> LESS IS ENOUGH AND POPULATED
AND WE SELL IT.
>> SO, FOR NON-HIV ANTIBODY WE
COULD SEE MUCOSAL SURFACES IN
THE INTESTINE.
>> DO YOU KNOW HOW LONG IS THE
EXPRESSION OR SUSTAINED IS IT?
>> MICE.
>> RIGHT.
>> SHEEP WE HAVE GONE OUT AS FAR
AS THREE YEARS FOR NON-HUMAN
PRIMATE STUDIES IS THE MONKEY
AND 10 YEARS NOW.
>> PROVIDING DRUG CELLS AND
SUSPECT THEY ARE VERY LONG AND
ESPECIALLY MUSCLES LONG-LIVED
AND PEOPLE ARE TARGETING MUSCLE
FOR GENE THERAPIES AND SUSPECT
QUITE LONG AND MADE
ADMINISTRATION STUDIES AND GOING
TO THE MUSCLE AND DIDN'T HAVE
TIME TO TALK ABOUT THAT.
>> WITH RESPECT TO THE QUESTION
FEMALE MICE WITH PR8 IS A
WIDESPREAD DISTRIBUTION AND WHAT
DID MICE DIE OF? PULMONARY
DISEASE?
>> BOTH NUMBER OF CRITERIA
MEETING FOR ENDPOINT AND WAS
WEIGHT LOSS AND LOOKING AT
LUNGS, THEY WERE DECIMATED. THE
LUNG.
>> LUNG AND SPLEEN AND WILL GO.
THAT ALSO?
>> LUNGS AND SECTIONS THAT LUNGS
WERE CONSOLIDATED AND HARD TO --
I'M SURPRISED THEY COULD
BREATHE, YEAH.
>> GOOD PLACE TO LOOK FOR
ANTIBODY LEVELS.
>> YEAH.
>> THAT IS THE NEXT THING THAT
WE ARE DOING.
THANKS.
>> ALL RIGHT. THANK YOU VERY
MUCH.
>> THANK YOU.
>> AUDIENCE: [APPLAUSE].
>> LAST TALK BEFORE LUNCH IS
NANCY SULLIVAN WHO DID PHD AT
HARVARD AND POST DOC WITH GARY
AT RESEARCH CENTER IN BETHESDA
AND TENURED AT BRC AND HEAD OF
DEFENSE RESEARCH SECTION AND
GROUNDWORK ON EBOLA VIRUS
VACCINE DEVELOPMENT AND HEARD
ABOUT IN PREVIOUS TALK AND
RECRUITED TO SERVE AS DIRECTOR
NATIONAL EMERGING INFECTIOUS
DISEASE LAB AT BOSTON UNIVE
UNIVERSITY.
>> THANK YOU, ERIC.
IT HAS BEEN A MINUTE SINCE I
HAVE BEEN TOGETHER WITH ALL MY
OLD HIV COLLEAGUES THAT I REALLY
APPRECIATE BEING HERE AND
STRIKES ME FRONT ROW LOOKS SAME
AS IT DID AT MEETINGS 30 YEARS
AGO.
OKAY.
I WILL TALK A LITTLE -- NOT SO
MUCH ABOUT VACCINES AND EFFICACY
AND USING EBOLA MODEL TO REALLY
UNDERSTAND HOW VACCINE VIRAL
VECTORS CAN WORK AND WHAT
ADVANTAGES ARE AND DISADVANTAGES
OVER OTHER PLATFORMS.
GOING BACK TO THE VERY BEGINNING
WHEN I FIRST STARTED TO WORK ON
EBOLA, THERE HAD BEEN MANY
VACCINES ELICITING STRONG
NEUTRALIZING ANTIBODY RESPONSES
KILLING VACCINES AND IN 90S
PROTEIN VACCINES BECOMING AN
ATTAINABLE APPROACH AND
NEUTRALIZING TIGHTERS WERE SUPER
HIGH AND ANIMALS DIED AND IN
SOME CASES GAVE ENHANCED DISEASE
LIKE IN GUINEA PIG MODEL AND GOT
ME THINKING DECIDING TO WORK ON
VACCINES EBOLA VIRUS THINKING
ABOUT WHAT WOULD BE NEEDED FOR
EFFECTIVE IMMUNE RESPONSE AND
EBOLA VIRUS CELLS AND STABLE
[INDISCERNIBLE] AND HIV AND
GLYCOE PROTEIN COVERS THE WHOLE
THING AND IS DENSE AND GIVEN
SIGN OF VARRION IT IS
[INDISCERNIBLE] AND
ACCESSIBILITY OF -- NEUTRALIZING
ANTIBODIES NEED A LITTLE HELP.
IT HAS A BROAD TROPHISM.
SO, WHEN YOU THINK ABOUT
ANTIBODIES THAT ARE MOSTLY
CIRCULATING AND HAVE A VIRUS
GETTING INTO EVERY TISSUE EXCEPT
T LYMPHOCYTES YOU NEED ANTIBODY
AND NEED IT EVERYWHERE.
OTHER THING THAT IS SO
SUCCESSFULLY THE SELECTED TO DO
IS RAPIDLY IS INTERNALIZED INTO
CELLS AND THE RECEPTOR BINDING
DOMAIN IS NOT ACCESSIBLE UNTIL
AFTER THE VIRUS IS
INTRACELLULAR.
I THOUGHT ABOUT WHAT WOULD BE A
BETTER APPROACH THAN PROTEIN AND
LIVE VIRUS THAT ARE ATTENUATED
VIRUS WOULD BE SOMETHING TO GIVE
US A POTENT T-CELL IMMUNE
RESPONSE TO AUGMENT ANTIBODIES.
I STARTED TO THINK ABOUT VIRAL
VECTORS BECAUSE MANY CAN
DIRECTLY TARGET DEN DRIDIC CELLS
THAT YOU GET BOTH
CROSS-PRESENTATION AND DIRECT
PRESENTATION RAMPING UP T-CELL
RESPONSE AND FIRST THING STARTED
WITH GOING BACK MANY YEARS AGO
WHEN PEOPLE JUST STARTED TO WORK
WITH PRIME BOOST APPROACHES THAT
HAVE VIRAL VECTORS.
SO, JUST TO DESCRIBE THE MODEL,
IT ALWAYS IS MACAQUES THAT ARE
SMALL STUDIES BECAUSE OF
LETHALITY AND HIGH CONTAINMENT
LEVEL NEEDED THAT IS HIGH
CHALLENGE DOSE AND UNIFORMLY IN
MACAQUE MODEL AND TARGETS
NUCLEOPROTEIN THOUGHT WOULD GIVE
BETTER T-CELL RESPONSES BECAUSE
OF HIGH AND ROWEN EXPRESSION AND
TRIED A HETEROL GAUS APPROACH
PRIMING WITH DNA AND WAS A LONG
REGIMENT AND FOUND WE GOT
UNIFORM PROTECTION IN MONKEYS
THAT FOR US WAS A BREAKTHROUGH
IN THINKING ABOUT HOW WE MIGHT
DEVELOP THIS FURTHER FOR
VACCINES AND MANY YEARS WE
MANAGED TO BREAK THIS DOWN TO A
SINGLE DOSE ADENO VIRUS
PROTECTING THAT GAVE ANTIBODY
AND T-CELL RESPONSES AND SEE
HERE A SINGLE SHOT PROTECTION
THAT IS GOOD AS THE PRIME BOOST
AND WE WANTED TO UNDERSTAND A
LITTLE MORE ABOUT WHAT VECTORS
WERE DOING.
AND WHAT WAS THE BALANCE OF
IMMUNE RESPONSES?
SO, TO GET AT THAT, WE DOSED
DOWN THE VACCINE TO A POINT WE
HAVE BREAKTHROUGH IN PROTECTION
TO START ASSOCIATING IMMUNE
RESPONSES AT THIS BREAKTHROUGH
DOSE BETWEEN 10 TO THE 9TH AND
10 TO THE 11TH AND LOOKING AT
T-CELLS WE DO BY INTRACELLULAR
CYTOKINE STAINING AND PBCS FROM
THE ANIMALS AND YOU CAN SEE WHAT
MIGHT BE A LITTLE BIT OF A TREND
FOR HIGHER CD8 T-CELL RESPONSES
IN SURVIVORS THAN FATALITIES AND
WHAT STOOD OUT WAS IN THE
BREAKTHROUGH DOSE WE HAD A CLEAR
DISTINCTION BETWEEN SURVIVORS
AND MORTALITIES AND WHOLE
PREMISE HAD BEEN THAT YOU NEEDED
MORE THAN AN ANTIBODY RESPONSE.
THIS WAS A LITTLE PERPLEXING WE
ARE USING VECTORS AND LOOKS LIKE
AN ANTIBODY RESPONSE DOING THE
WORK AND IS A SERIES OF STUDIES
TRYING TO OPTIMIZE THE ANTIGEN
AND NOTICE THAT A COUPLE
REGIMENTS AGAIN GAVE US
BREAKTHROUGH THAT WASN'T A DOSE
DIFFERENCE BUT CHANGE IN INSERT
AND FIRST TIME WE SAW NOT
ANTIBODIES BUT T-CELLS NOW THAT
WERE SEGREGATING WITH THE
ANIMALS THAT WERE PROTECTED IN
THE VACCINE REGIMENT.
WE WANTED TO TEST THIS MORE
DIRECTLY.
SO, WE LOOKED AT AFTER
VACCINATION, IN -- SORRY.
IN VACCINATING A CONTROL GROUP
AND HAVING ANOTHER GROUP WASN'T
VACCINATED WE TOOK
VACCINE-INDUCED ANTIBODIES FROM
A COHORT OF ANIMALS INFUSING
ANTIBODIES PRIOR TO INFECTIOUS
CHALLENGES AND DETERMINED A
CUTOFF FOR ANTIBODIES TO BE 1 IN
3,000.
WE SAW THE VACCINATED GROUP HAD
A CUTOFF FOR PROTECTION AND
INFUSED ANTIBODY WAS ORDERS OF
MAGNITUDE WE NEED FOR PROTECTION
AND UNVACCINATED ANIMALS DIDN'T
HAVE ANYTHING AND TO SURPRISE
HAD LITTLE PROTECTION OF ANIMALS
MASSIVE [INDISCERNIBLE] TO
ANTIBODIES AND POINTING OUT
T-CELL RESPONSE IS IMPORTANT
THAT IS NOW VERY CHALLENGING TO
TRY TO INFUSE T-CELLS AND WE
FLIPPED THE EXPERIMENT AROUND
AND VACCINATED AND DEPLETED THE
T-CELLS PRIOR TO INFECTION.
AND WHAT WE SAW HERE NOW WAS
THAT WE REALLY ELIMINATED
PROTECTION ON WHAT ANIMALS
DIDN'T HAVE A CD8 T-CELL
RESPONSE AND SUGGESTED THAT
OBVIOUSLY ANTIBODIES AND T-CELLS
ARE DOING SOMETHING AND
ANTIBODIES ARE NOT SUFFICIENT
AND CD8 T-CELLS ARE REQUIRED
TELLING US MORE ABOUT WHAT
VECTORS ARE DOG AND WE HAD
ANOTHER INDIRECT WAY OF SEEING
THIS THAT IS BY CHANCE.
SO, WE HAD VACCINATED A COHORT
OF ANIMALS WITH DNA WHEN WE
THOUGHT THAT WE PROABLE WILL I
WOULD END UP WITH
[INDISCERNIBLE] AND ABOUT THE
TIME THAT VIRUS WAS DISCOVERED
IN 2007.
WE THOUGHT COULD WE POSSIBLY GET
CROSS-PROTECTION IF WE HAVE A
GOOD CD8 RESPONSE AND
CROSS-PROTECTION AGAINST A
DIFFERENT SPECIES OF EBOLA
VIRUS?
ABOUT A YEAR AFTER ANIMALS WERE
PRIMED WITH DNA BOOSTED THEM
WITH SINGLE SHOT OF ADENOVIRUS
AND NOT SURPRISINGLY T-CELL
EPITOPES ARE MORE EASIER TO GET
CROSS -- ACROSS DIFFERENT
SPECIES.
THEY ARE SMALLER AND GOOD CD4
RESPONSE AND ALL ANIMALS AGAINST
[INDISCERNIBLE] OR CD4 AND 8.
WHAT WAS ENCOURAGING IS WE HAD A
NICE CD4 RESPONSE AND CD8
RESPONSES AND ANIMAL THAT WERE
FOR EBOLA EARLIER AND LOOKING AT
ANTIBODIES HAD NO
CROSS-REACTIVITY.
THAT IS BECAUSE FILO VIRUSS ARE
DIFFERENT AT GLYCOPROTEIN THREE
DIMENSIONAL LEVELS.
WE HAVE 100% SURVIVAL THAT SAYS
REALLY WHAT IS IMPORTANT HERE IS
T-CELL RESPONSE AND ONE ANIMAL
WITH VIREMIA LIVED AND TURNED
OUT TO BE THIS ANIMAL WITH
LOWEST CD8 RESPONSE AND AGAIN
POINTING IN A DIFFERENT WAY TO
THE IMPORTANCE OF THE ABILITY OF
THE VIRAL VECTORS TO GENERATE
T-CELL RESPONSES.
NOW, WE HAD BEEN BASICALLY OUR
PLATFORM ON AD5 BACK BEFORE HIV
STEP CROWD RESULT SHOWING IF
PEOPLE ARE AD5 IMMUNE BECAUSE OF
[INDISCERNIBLE] WITH ACTUAL
VIRUS THEY DON'T RESPOND TO
VACCINES AND IN CASES MIGHT BE
WORSE.
WE DECIDED TO LOOK AT THIS IN
MONKEYS AND HAD NAÏVE MONKEYS
AND ADD FIVE IMMUNE MONKEYS WE
VACCINATED WITH AD5 VECTOR AND
INTERESTING THAT WE WERE ABLE TO
GENERATE ANTIBODIES WITH AD5 IN
AD5 IMMUNE MONKEYS AND COULDN'T
GENERATE T-CELL RESPONSES AND IN
THE END WE COULD NOT PROTECT IN
THOSE ANIMALS LOSING CD8 T-CELL
RESPONSE.
INDEPENDENT EVIDENCE THAT T-CELL
RESPONSE IS IMPORTANT AND NICE
FOR UNDERSTANDING MECHANISMS AND
NOT SO NICE FOR VACCINE THAT WE
ARE TRYING TO DEVELOP THAT IS
BASED ON AD5.
WE LOOKED AT AND ARE WORKING
WITH CRUCEL AT THE TIME TO
MANUFACTURE AD5 AND AD26 AND
AD35 AND LOOKING AT ZERO
PREVALENCE RED IS HIGH AND BLUE
IS LOW AND ADD 26 AND ADD 35 AND
IN US HAD ALMOST ZERO PREVALENCE
IN AFRICA 35 AND OTHER PARTS OF
AFRICA AND WE DECIDED TO MOVE
FORWARD WITH AD26 AND AD35 AND
SET UP SAME EXPERIMENTS AND
MONKEYS IN SAME DOSE RANGE I'M
SHOWING HERE 10 TO THE 10TH AND
10 TO THE ELEVENTH AND REMEMBER
IS A NONREPLICATING VECTOR AND
DOSES SEEM HIGH BUT NOT WHEN YOU
CONSIDER IT IS NOT
NONREPLICATING AND SURPRISING AT
LOW DOSE DIDN'T GET GOOD
PROTECTION AND HIGH DOSE WE GET
NO PROTECTION AND WAS SO
STRANGE. WE ARE THINKING ADENO
VIRUS IS AN ADENO VIRUS AND WHY
COULD THIS BE AND LOOKED AT AD26
AND FOUND SAME THING NOT GIVING
SAME RESULT THAT AD5 GAVE US AND
THOUGHT MAYBE T-CELLS ARE BETTER
AND LOOKING AT CD4 AND CD8 AND
STUNNING NO DIFFERENCES IN CD4
AND CD8 RESPONSES YET WE WERE
NOT GETTING PROTECTION AND
ANTIBODY RESPONSES WERE SIMILAR
AND T-CELL RESPONSES WERE
SIMILAR BUT NOT GETTING
PROTECTION AND AT TIME MARIO AT
VRC HAD DONE AN AWFUL LOT TO
DEFINE T-CELL PHENOTYPES BASED
ON INTRACELLULAR CYTOKINE
STAINING AND WAY WE DO THIS IS
IF YOU MEASURE IBT FEARON GAMMA
IN CRM OR CELLS IBT FEARON GAMMA
IS NOT COMING FROM SITE THAT
MAKES CYTOKINE COMING FROM GAMMA
WITH -- 3 AND 4 AND 5 CYTOKINES
AND TURNS OUT CHOOSING THREE
COMBINATIONS OF CYTOKINES YOU
CAN REALLY DISTINGUISH BETWEEN
TERMINAL EFFECTORS, EFFECTORS
AND MEMORY T-CELLS AND DECIDING
TO TAKE A LOOK AT THIS IN OUR
EXPERIMENT.
EACH OF THE SUBSETS IS
FUNCTIONALLY DISTINCT.
COULD WE SEE A DIFFERENCE?
FIRST THING THAT STRUCK US IS
PROTECTIVE VACCINE IS GENERATING
MORE CELLS PRODUCING TWO
CYTOKINES SIMULTANEOUSLY.
AND THAT IS NOT MISSING BUT MUCH
LOWER IN THE NONPROTECTIVE
VACCINE AND WHAT WAS MORE
STRIKING IS THAT WHICH
CATEGORIES THIS FELL INTO.
THE PROTECTIVE VACCINE WAS
GIVING INTERFERON GAMMA TNF
ALPHA DUAL SECRETING CELLS
BETTER ASSOCIATED WITH EFFECTOR
MECHANISMS AND HAVE SOME MEMORY
AND VACCINE THAT WASN'T
PROTECTIVE WASN'T GIVING US
THOSE BUT MORE OF A SINGLE
CYTOKINE IL2 THAT IS ASSOCIATED
WITH MEMORY AND NOT INITIAL
EFFECTOR RESPONSE THAT YOU WOULD
NEED FOR AN EBOLA INFECTION THAT
IS PRETTY AGGRESSIVE AND
SCRATCHING HEADS AGAIN ABOUT WHY
THIS IS. TURNS OUT IT IS A
SIMPLE EXPLANATION AND DIFFERENT
ADENO VIRUS VECTORS TARGET
DIFFERENT DENDRITIC CELLS AND
I'M SURE OTHER CELLS ARE
DIFFERENT AND SEEN EVEN WHEN
DOING TISSUE BIOPSIES YOU CAN
SEE INFLUX OF IMMUNE CELLS TO
SITE OF INFECTION IS SPECIFIC TO
THE VECTOR AND EXPLAINED IT FOR
US AND DIDN'T SOLVE OUR PROBLEM
THAT IS NOT HAVING A VIRAL
VECTOR AS A VACCINE AND NEEDED A
SINGLE SHOT THAT WOULD ACT VERY
QUICKLY AND TURNED TO NON-HUMAN
DERIVED ADENOVIRAL VECTOR OR
VECTORS BEING DISCOVERED BY A
TINY COMPANY IN ITALY HAD DONE
THESE VIRUSES AND PICKED ONE IN
AD5 FAMILY THAT IS WORKING
THROUGH SAME RECEPTOR AND
ANOTHER FROM THE GROUP E.
WE WANTED A SECOND VACCINE AND
LOOKING AT T-CELL RESPONSES IS
SHOWING YOU HOW SIMILAR AD26 AND
AD35 ARE FOR PROFILES AND
LOOKING AT 5 AND CHIMP AD3 AND
POST DOC ACTUALLY THAT DID THIS
I ASKED HIM TO GO BACK AND REDO
IT.
I COULDN'T BELIEVE THAT CHIMP
AD3 LOOKED SIMILAR TO AD5 AND
QUALITIES BY INTRACELLULAR
CYTOKINE CAN BE TRICKY AND I
REALLY DIDN'T BELIEVE IT AT
FIRST.
TURNED OUT TO BE TRUE IT WAS
REALLY REPLICATING WHAT WE SAW
FOR AD5 THAT WAS SPECIFICALLY
TNF INTERFERON GAMMA A NICE
BALANCE BETWEEN EFFECTOR AND
MEMORY AND GIVES A REALLY GOOD
RESPONSE AND THEY DON'T BURN OUT
QUICKLY.
WE TESTED THESE IN MONKEYS AND
SHOWED IT WAS INTERESTING THAT
CHIMP AD PERFORMED WELL IN TERMS
OF ANTIBODY RESPONSES AND NEVER
PURSUED THAT TURNED OUT TO BE A
GREAT VECTOR ANYWAY AND CD4 AND
CD8 AND COMPARED TO 5 CHIMP AD5
VECTOR GAVE BETTER RESPONSES
THAN WHAT WE HAVE SEEN ON
AVERAGE AND FOR THE AD5 RESULT
AND WANTED TO ASK ABOUT
PROTECTION AND LOOKING AT ACUTE
PROTECTION THAT WE ARE DOING
MOST OF THE TIME FOR THE STUDIES
IS WE WANT A FAST-ACTING VACCINE
AND CHIMP AD3 GAVE SAME
PROTECTION AS CHIMP AD5 AND 63
IS PREDICTED FROM LOWER
RESPONSES ANTIBODY AND T-CELLS
DIDN'T AFFORD PROTECTION.
WHAT HAD NOT BEEN DONE TO THIS
POINT I THINK IS NOT STILL BEING
DONE FOR VACCINE PLATFORMS IS
TRYING TO UNDERSTAND WHAT IS
REQUIRED FOR DURABLE PROTECTION
AND WHEN YOU MAKE A VACCINE TO
GIVE YOU FAST PROTECTION YOU ARE
ASKING IT TO DO TWO DIFFERENT
THINGS, FAST AND LONG AND CAME
UP WHEN TALKING ABOUT MRNA.
YOU EXPECT TO PEAK AND CRASH
FAST AND LOOKING AT SINGLE SHOT
VACCINE FOR PROTECTION IN CHIMP
AD3 AND GAVE 50% PROTECTION AT A
YEAR THAT IS GOOD AND CHIMP AD63
DID NOT AND WAS DEPENDENT GOING
UP A LITTLE ON THE DOSE AND GOT
US THINKING ABOUT, YOU KNOW, HOW
CAN WE USE VIRAL VECTORS AND
SEEN WE CAN TUNE THEM NOW WITH
DNA AND ADENOVIRUS AND TUNE
IMMUNE RESPONSES AND CAN WE DO
THIS MORE AND EXTEND MEMORY AND
MBA IS KNOWN TO GENERATE VERY
POTENT CD4 RESPONSES THAT YOU
NEED FOR MEMORY AND GOOD CD8
RESPONSES AND GAVE YOU LONG-TERM
MEMORY AND DECIDED TO TRY PRIDE
BOOSTING WITH CHIMP 3 FOR FAST
RESPONSE AND MVA TO EXTEND
DURABILITY AND WILL SHOW YOU
IMMUNE RESPONSES IN BOOST THAT
WE WILL CALL PEAK RESPONSES AND
MEMORY RESPONSE AT TIME OF THE
CHALLENGE AND SO WHEN WE LOOK AT
ANTIBODY, THE CHIMP AD 3 BOOSTED
BY MVA GIVES MEMORY RESPONSES
THAT ARE HIGHER THAN THE PEAK
SINGLE SHOT SO THAT MVA IS
BOOSTING AND IS DURABLE WHEN WE
LOOK OUT AT A LONG TIME AND
MAGNITUDE AT PEAK PRIME BOOST IS
FOR -- LIKEWISE FOR CD4, MEMORY
RESPONSE AT A YEAR BOOSTED BY
MVA WAS AS HIGH AS PEAK RESPONSE
AFTER A SINGLE SHOT.
THAT WAS NOT THE CASE FOR CD8.
THIS WAS SOMEWHAT EXPECTED.
WE KNOW MVA IS GOOD AT BOOSTING
CD4 RESPONSES THAT TELLS YOU NOW
THAT WITH A DIFFERENT PRIMING
AND BOOSTING YOU CAN DIRECT
IMMUNE RESPONSE TO GO IN
WHATEVER DIRECTION YOU WANT AND
SO WHEN WE COMPARE I WANT TO GO
A LITTLE ABOUT THE PRIMING
MATTERS.
SO, WE ALSO BOOSTED CHIMP AD 63
AND SEE PEAK RESPONSES AFTER MVA
ARE LOWER AND MEMORY RESPONSES
ARE LOWER AND NOT BOOST THAT IS
DETERMINING WHAT YOU GET BUT IS
PRIME FOR MAGNITUDE AND TESTING
DURABLE PROTECTION WITH MVA
BOOST WE CAN EXTEND IT AND 50%
PROTECTION WE GET 100%
PROTECTION AND CD4 RESPONSE IS
GIVING LONGER TERM MEMORY AND
WANT TO GO IN A LITTLE MORE TO
THIS IDEA TUNE IMMUNE RESPONSE
AND GOING TO DNA PRIMED ANIMALS
WE HAD. WE WANTED TO UNDERSTAND
HOW DOES THE NUMBER OF PRIMES
INFLUENCE WHAT YOU GET AFTER A
BOOST.
WE HAVE ANIMALS PRIMED 3 TIMES
OR 2 TIMES OR ONCE WITH DNA AND
RECEIVING ADENO VIRUS AND IN
CASE OF THE BOOST IT IS SAME
INTERVAL BETWEEN THE LAST PRIME
AND BOOST.
AND THIS WAS -- THIS IS ACTUALLY
INTERESTING. SOME THAT WORK
WITH DNA VECTORS MIGHT NOT BE
SURPRISED.
YOU CAN CONSIDER THIS A PRIME
AND THIS A BOOST AND DON'T GET
MUCH BOOSTING OF ANTIBODIES WITH
DNA.
DNA IS NOT GOOD AT INDUCING
ANTIBODIES AND CD4 RESPONSE IS
GOOD AND YOU CAN SEE AFTER THREE
DNA INJECTIONS THAT ANTIBODY
RESPONSE IS MEASLY.
WHAT YOU CAN SEE NOW IS THAT THE
NUMBER OF PRIMES CAN INCREASE
THE EFFICACY OF THE BOOST.
SO, WITH MORE DNA PRIMES AND DNA
ITSELF IS NOT DOING IT WE ARE
GETTING BOOST WITH ADENO VIRUS
TUNING THAT IMMUNE RESPONSE AND
LOOKING AT CD4 AND CD8 RESPONSES
THIS IS QUITE INTERESTING
BECAUSE PREPONDERANCE OF CD4
CELLS INCREASED WITH DNA PRIMES.
YOU ARE REVERSING BIAS TOWARDS
CD4 OR CD8 DEPENDING HOW YOU
PRIME FOR CD8 AND QUALITY TO SEE
TRIPLE POSSIBILITY QUALITY THAT
IS LONG-TERM MEMORY THREE TIMES
PRIMING WITH DNA AND PHENOTYPES
STAYED THE SAME AND IS SELECTIVE
WITH WHAT IS BEING BOOSTED AND
HERE WITH CD8 WE SEE 1 TIMES
PRIME DOING GIVING MORE
DESIRABLE RESPONSE.
ONE TIME PRIME FOR T-CELL
QUALITY LOOKS LIKE ADENO VIRUS.
OKAY. NOW WHEN WE LOOK AT
MEMORY AND LOOK THREE YEARS
LATER AT IMMUNE RESPONSES AND
GIVE ONE DNA BOOSTED BY AD5 OR
THREE DNAS BOOSTED BY AD5 WE ARE
NOT SEEING ONE DAY LOOK LIKE
ADENO VIRUS BUT DNA AND STRONG
CD4 RESPONSES CD8 RESPONSES
SUFFER AND CD8 RESPONSES ARE NOT
DURABLE AND NEED CD4 FOR THAT
AND SUSPECT IF WE BOOST ANIMALS
HERE WE GET GOOD CD8 RESPONSES
THAT TELLS YOU, YOU CAN SET UP
THE IMMUNE RESPONSE TO DO WHAT
YOU WANT WITH A DIAL DEPENDING
HOW YOU USE THESE VECTORS.
LIKEWISE DOWN HERE YOU EXPECT
MORE IS BETTER FOR ANTIBODY
RESPONSE AND IN FACT THAT IS NOT
THE CASE.
LOOKING AT INTERVAL WE HAVE A
GROUP WHERE WE PRIME AND BOOSTED
14 WEEKS LATER VERSUS PRIMING
AND BOOSTING A YEAR LATER.
WHAT WE SEE IS THAT WHEN WE
BOOST 8 WEEKS POST DNA PRIME, WE
ARE GETTING A SOLID CD4 RESPONSE
NOT SO MUCH CD8 RESPONSE AND
BOOSTING A YEAR AFTER THE PRIME
IT LOOKS LIKE ADENOVIRUS AND
WHAT HAPPENED IS AD RESPONSE IS
CONTRACTED AND DON'T HAVE CD4
THERE THAT YOU DO WITH DNA AND
IS BOOSTING WHAT IS SEEN
RECENTLY AND IS BOOSTING WHAT IS
THERE AND TUNING IT TOWARDS THE
VECTOR SEEMS MOST RECENTLY AND
THINK I WILL STOP THERE TO LEAVE
TIME FOR QUESTIONS.
HOW DOES THAT WORK?
BOTTOM LINE IS DIFFERENT VIRAL
VECTORS WORK IN DIFFERENT WAYS
AND VSV I DIDN'T TALK ABOUT IS
COMPLETELY DIFFERENT.
THAT VACCINE DESIGN IS SUCH THAT
IT IS DIRECTING THE VECTOR TO
EVERY TISSUE YOU WOULD SEE EBOLA
AND INTRAMUSCULAR RESPONSES GO
EVERYWHERE AND COMPLETELY
DIFFERENT STORY AND DIDN'T TALK
ABOUT INNATE RESPONSES WHICH IS
DIFFERENT AND IS USING VIRAL
VECTORS TO TUNE ADAPTIVE
IMMUNITY TO SEE CHOICE OF ADENO
VIRUS VECTOR TO DIRECT NEW ONE
DIRECTION OR ANOTHER.
SEQUENCE OF PRIME BOOSTING WHAT
COMES FIRST OR SECOND AND HOW
MANY PRIMES YOU HAVE BEFORE THE
BOOST AND INTERVAL BETWEEN PRIME
AND BOOST ARE TOOLS TO USE WITH
VECTORS TO TUNE IMMUNE RESPONSE
TO WHAT YOU KNOW IS THE
MECHANISM OF IMMUNITY IN MOST
CASES HOPEFULLY BEFORE YOU START
STUDIES.
I WILL STOP WITH A SLIDE AND
LOTS OF YEARS AND PEOPLE.
THIS IS NOT ALL HANDS OF COURSE
AND APPRECIATE WHAT THEY DONE
AND APPRECIATE YOU FOR YOUR
ATTENTION.
THANK YOU.
>> AUDIENCE: [APPLAUSE].
>> I WILL ASK A STUPID QUESTION.
[INDISCERNIBLE].
>> SO, DEPENDS ON WHEN YOU ARE
LOOKING; RIGHT?
IF YOU LOOK SHORT TERM THREE
TIMES IS ACTUALLY BETTER FOR
ANTIBODY RESPONSES.
DURABLE RESPONSE.
I THINK WHAT HAPPENS WHEN
LOOKING AT CONTRACTED RESPONSE
WHAT YOU BOOST WITH WILL TAKE
OVER PHENOTYPE YOU SEE AND IS
IMMUNOLOGICALLY BASED.
YOU COULD THINK OF SCENARIOS TO
PRIME TOO MUCH AND GET FEEDBACK
THAT REALLY -- THAT HAPPENS. I
KNOW WITH THE VSV VACCINE YOU
GET A WHOPPING RESPONSE IN THE
BEGINNING.
BOTHINATE AND ADAPTIVE THAT IS
SHUT DOWN PRETTY QUICKLY AND
DURABILITY OF VACCINE IS NOT
GREAT AND MIGHT HAPPEN WITH MRNA
AND DON'T KNOW IF IT IS AN
EXPRESSION ISSUE OR FEEDBACK
FROM IMMUNOGENIC NATURE OF MRNA
AND CAN'T BE TOO MUCH IN THE
BEGINNING THAT SHUTS DOWN
DIFFERENT PATHWAYS.
>> WITH PRIMES, WHAT IS THE
LENGTH OF TIME BETWEEN FIRST AND
SECOND?
>> I DID TWO EXPERIMENTS.
FIRST IN THE SECOND PRIMES.
YEAH. THOSE ARE ALL FOUR WEEKS.
YEAH. YEAH.
>> CAN YOU TELL ME THE
[INDISCERNIBLE] BETWEEN
DIFFERENT ADENO TYPES
[INDISCERNIBLE] 25 TO SORT OF
SEE IF WE CAN GENETICALLY
PREDICT THE TARGET CELLS?
>> YEAH. SO, DAN VARUKE 2R50ID
MAKING A CHIM ERIC VECTOR TO DO
JUST THAT.
IT WAS ADD 48 AND ADD 5.
IT DIDN'T WORK.
I THINK IT IS A LITTLE
COMPLICATED AND HEXON AND FIBER
ARE REALLY DICTATING WHAT
HAPPENS AT THE SURFACE OF THE
CELL. WE ARE NOT QUITE THERE
YET WHERE YOU CAN PREDICT THE
RECEPTOR FROM THE SEQUENCE.
>> THANK YOU.
>> SOMETHING ABOUT THIS. SO
INTERESTING.
AT THE VERY BEGINNING, I WAS
LOOKING AT 21 THOUSAND
DIGITALIZATION TIGHTERS AS DOING
[INDISCERNIBLE] AND THEN I
THOUGHT THAT IT IS EBOLA AND HOW
MUCH OF THAT IS NEUTRALIZED?
>> NEUTRALIZING RESPONSES, WELL,
I HAVE TO PARSE THIS NOW.
YOU CAN DO THE VIRUS
NEUTRALIZATION AND DO VSV
NEUTRALIZATION AND CAN DO WHOLE
VIRUS NEUTRALIZATION.
WHEN YOU LOOK IN INFECTED
MONKEYS, POST INFECTION
NEUTRALIZING RESPONSES BY PLAQUE
REDUCTION IS IMPRESSIVE AND MORE
TO DO WITH ASSAY AND DON'T THINK
IS NEUTRALIZATION, PER-SE, MIKE.
I DON'T KNOW. YOU PROBABLY
REMEMBER THAT PAPER BY ERICA
SAPPHIRE WHERE SHE TESTED 50 OR
100 MONOCLONALS IN MICE THAT
GAVE BEAUTIFUL NEUTRALIZING
ACTIVITY NO MATTER HOW YOU
LOOKED THEY DIDN'T PROTECT.
[INDISCERNIBLE] THAT WE
DEVELOPED WAS DONE AND WASN'T
LOOKING FOR ANY ANTIBODIES. I
WAS LOOKING FOR AN ANTIBODY THAT
WOULD BIND TO THE SURFACE
GLYCOPROTEIN AND BIND TO THE
CLEAVED PROTEIN ONCE IT IS
INSIDE OF THE CELL. THAT WAS
THE ONLY ANTIBODY I COULD FIND
THAT DID THAT.
SAM DID ALL THAT WORK. IT WAS
BEAUTI.
IT IS NOT NEUTRALIZATION HOW WE
THINK ABOUT IT AND FC AND IN
TERMS OF ANTIGEN PRESENTATION
FOR CD8 RESPONSES, FC WILL BE
HELPFUL AND ALL EXPERIMENTS
MUTATING FC SHOWING ROLE FOR FC
IN ANTIBODY RESPONSES WHEN YOU
MUTATE THE IF. C YOU ARE DOING
A WHOLE ARRAY OF THINGS TO THE
IMMUNE RESPONSE. IT IS NOT JUST
KNOCKING OUT THE FC EFFECTOR
FUNCTION THAT WE THINK ABOUT.
>> OTHER QUESTIONS HERE? HOW
ABOUT ONLINE? YOU HAVE BEEN
QUIET THIS MORNING.
OKAY. IF NOT, THANK YOU VERY
MUCH.
>> THANK YOU!
>> AUDIENCE: [APPLAUSE].
>> WE ARE ON TIME AND WILL BREAK
FOR LUNCH UNTIL 1:20.
THERE IS A CAFETERIA JUST TO THE
LEFT AS YOU LEAVE THE AUDITORIUM
AND WILL BE A POSTER SESSION.
PLEASE COME BACK AFTER LUNCH AND
JOIN US.
IT IS ABOUT TIME WE GET STARTED.
I AM NOT SURE PEOPLE OUT IN THE
LOBBY CAN HEAR ME.
TIME TO GET STARTED.
>>.
HI. WELL, LET'S GET STARTED.
I'M HOPING MORE PEOPLE WILL JOIN
US FROM THE LOBBY AND THERE IS A
CHANGE IN PROGRAM AND SWITCHING
ORDERS AND TRULY WILL SWITCH
PLACES AND NEXT SPEAKER IS CHIP
WHO RECEIVED MD FROM JOHNS
HOPKINS UNIVERSITY DOING
INFECTIOUS DISEASE SCHOLARSHIPS
AT ANYAD MASS GENERAL JOINING IN
HARVARD MEDICAL SKIEN IN 1981
AND -- IN 1990 CHIP WAS
RECRUITED TO UNIVERSITY OF
COLORADO INSIDE OF THE DIVISION
OF INFECTIOUS DISEASES AND
SHARED NIH CLINICAL TRIALS GROUP
FROM 1985 TO 2002 TO REALLY
EXPAND THE SCOPE OF THAT GROUP.
2000 FIVE JOINED THE PATH OF
UCSD AND OTHER APPOINTMENTS AND
APPLICATIONS AND THERAPEUTICS
AND VERY ACTIVE IN HIV RESEARCH
AND IS A PIONEER IN FIELD OF AGE
THERAPY THAT IS TOPIC OF HIS
TALK TODAY.
>>.
>> THANK YOU. PLEASURE TO BE
BACK HERE FREDERICK AND SEE JOHN
AND ALL OF STEVE AND ALL 12 FEET
OF HIM.
IT IS GREAT TO BE BACK HERE AND
I WILL TALK TODAY ABOUT
SOMETHING I STUMBLED INTO A
NUMBER OF YEARS AGO.
IT IS AN INFORMAL TALK IN THE
AFTERNOON BUT ONE THAT I PROMISE
IS IN A FIELD THAT IS MOVING
RELATIVELY RAPIDLY. IF YOU
INDULGE ME IN A LITTLE BIT OF A
CULTURE SHOCK AND TALK ABOUT
WHERE THERAPEUTICS MIGHT GO IN
THE NEXT FEW YEARS AND THESE ARE
UNPAID DISCLOSURES AND SOMETHING
THAT IS KNOWN TO ALL OF YOU HERE
AND INFECTIONS BECOMING
INCREASING PROBLEM AROUND THE
WORLD AND IS A SLEEPER AND DON'T
REALIZE HOW MANY PEOPLE DIED
FROM THE INFECTIONS AND ONE AT A
TIME HERE AND THERE AND OPPOSED
TO OBVIOUS WAVES OF CORONAVIRUS
AND CUMULATIVE DEATH RATE AND
WHERE THEY ARE HEADED THIS IS
MAJOR INFECTIOUS DISEASE THREATS
OF THE ERA AND WE THOUGHT THAT
[INDISCERNIBLE] WITH ANSWER AND
AT TOP OF THE LINE YOU CAN SEE
THE DISCOVERY LINE OF
ANTI-BIOTICSES GOING BACK TO THE
EARLY PART OF THE LAST CENTURY
1930S AND STEADY SERIES OF NEW
ANTI-BIOTICS CLASSES DISCOVERED
IN THE NEXT 30 OR 40 YEARS AND
SEE SHORTLY AFTER EACH
ANTI-BIOTICS WAS INTRODUCED TO
THE CLINIC [INDISCERNIBLE] THERE
IS LONG DRY SPELL FROM 1970 TO
2000 WITH NO NEW ANTI-BIOTICS
DISCOVERED AND SENSE THEN YOU
HAVE TO CALL THEM SINGLES IF A
BASEBALL PLAYER AND TOP ITSELF
IS QUITE DRY AND BACTERIAL
PATHOGENS CONTINUE TO GO ALONG.
THIS IS WHERE THE STORY STARTS.
TOM PATTERSON A COLLEAGUE OF
MINE AT FACULTY OF UNIVERSITY OF
SAN DIEGO WAS IN EGYPT OVER THE
THANKSGIVING HOLIDAY AND HIPAA
POLICE ARE HERE THEY WROTE A
BOOK ABOUT THIS.
TOM IS A CO-AUTHOR AND THIS IS
PUBLIC DOMAIN AND THE BOOK GOES
INTO MORE DETAIL THAN I WILL
TODAY.
IT APPARENT WILLY HEADED FOR HO
WOD AND -- STEPHANIE TEXTED ME
AND THOUGHT HE DEVELOPED FOOD
POISONING AND TALKED TO HIM
MORE. HE WAS DRINKING WATER
CONTINUOUSLY AND WAS FEBRUARY
RILE AND HAD LOTS OF ABDOMINAL
PAIN AND NO DIARRHEA AND
VOMITING AND WORRIED ABOUT
PANCREATITIS AND DIABETIC THAT
DRINKS A BIT AND TOOK HIM TO A
HOSPITAL AND FOUND A BIG
PSEUDOCYST AND GOT EVACUATED TO
FRANKFORT WHERE THE PSEUDOCYST
WASTURED -- WAS PUNCTURED IN AN
ORGANISM THAT IS LARGE IN THAT
PART OF THE WORLD AND UC SAN
DIEGO DID EFFORT TO DRAIN THE
ABSCESS AND THE ABSCESS BEGAN TO
METASTASIZE AND TRYING TO GET
SURGEONS INVOLVED AND IS TOO
SICK TO OPERATE ON ONE OR TWO
DRAINING AND SAID HE IS GETTING
BETTER AND DON'T WANT TO OPERATE
ON HIM NOW AND WENT ON FOUR
MONTHS AND HE WAS OBTUNDED IN
SCU RENAL FAILING AND TALKING
ABOUT COMFORT CARE AND HIS WIFE
STEPHANIE IS A BULLDOG WHO RAN
ACROSS A PAPER FROM GEORGIA NOT
OURS BUT THEIR GEORGIA ABOUT
BACTERIOPHAGES THAT WERE FOUND
AND KILLED [INDISCERNIBLE] IN
THE LABORATORY AND GOT IN TOUCH
WITH ME AND ASKED IF WE COULD
USE THESE TO TREAT HER
HUSBANDED. AUDIENCE KNOWS WELL
WHAT BACTERIOPHAGES ARE BACTERIA
OF VIRUSES AND FOUND IN 8 HOLES
IN [INDISCERNIBLE] OF BACTERIA
AND WERE EATING BACTERIA HENCE
BACTERIOPHAGES AND FOR A WHILE
WERE THOUGHT IT BE THE ANSWER TO
BACTERIAL INFECTIONS AND WERE
SOLD OVER THE COUNTER IN
MULTIPLE PARTS OF THE WORLD
MAINLY SYNDROMICALLY FOR EXAMPLE
FOR SPHERICALS OR DIARRHEA AND
HEALTHY BACTERIOLOGIC DECISION
AND WERE PARTICULARLY VIRUSS
WITH VERY NARROW HOST RANGE AND
WERE SOLD AS COCKTAILS
HOLEARIZED COCKTAILS THAT
SOMETIMES WORKED AND SOMETIMES
DIDN'T AND NOBODY KNEW WHY.
BEFORE LONG THEY FELL OUT OF
FAVOR IN THE WESTERN WORLD
PRIMARILY BECAUSE ANTI-BIOTICSES
CAME ALONG AND WERE EASY TO
MAKE.
WHEN YOU MADE THEM YOU COULD
SELL THEM TO KILL ALL KINDS OF
BACTERIA THAT WERE
STRAIGHTFORWARD AND ENTERED THE
LEXICON IN THE WESTERN WORLD AND
SUPPLANTED BACTERIOPHAGES AND
SOON BACTERIOPHAGES WERE
PRIMARILY USED IN SOVIET UNION
AND IN THE SOCIALIST REPUBLICS
PRIMARILY BECAUSE THEY WERE
CHEAP AND ANTI-BIOTICSES WEREN'T
AND WHEN BACTERIOPHAGES WERE
USED TOPICALLY ON WOUNDS AND
BURNS AND SOME GIVEN ORALLY FOR
DIE REAL DISEASE AND WERE NOT
GIVEN PERENNIAL AND PATIENT THAT
DISSEMINATED AND ASKED TO VECTOR
VIRAL ON INFECTION AND WASN'T
CLEAR TO FIND LYTICPHAGES FOR
INFECTION AND GETTING HIM TO
SITE OF INFECTION. I WASN'T
CONFIDENT IT WOULD WORK AND FELT
IT WAS WORTH THE TRY GIVEN WE
HAD NOT MADE PROGRESS WITH
TRADITIONAL APPROACHES IN THE
LAST FEW MONTHS GETTING FDA
APPROVAL TO DO THIS TURNED OUT
TO BE EASIEST THING AROUND AND
WAS AN EASY PATHWAY FOR PATIENTS
WITH LIFE THREATENING INFECTIONS
WITH LICENSED PRODUCT EMERGENCY
IND PATHWAY REACH ON PHONE AND
TELL WHAT YOU WANT TO DO AND
DOESN'T SOUND TOO CRAZY CAN
GENERALLY GIVE APPROVAL THAT
DAY.
I WOULD EXPECT TO RUN INTO
PROBLEMS AND REGULATOR THERE
ANSWERED THE PHONE AND KNOW WHAT
I DO ALL DAY?
>> NO.
>> I DO FECAL TRANCE PLANTS AND
HATE IT. THIS SOUNDS
INTERESTING. WE SHOULD DO THIS.
ARE YOU FROM THE GOVERNMENT
TRYING TO HELP ME? I ACTUALLY
AM AND KNOW WHERE YOU CAN GET
THEPHAGES AND DRD AND WHERE WE
GET BACK TO FREDERICK AND SHE
REFERRED ME TO PEOPLE WORKING IN
BIOMEDICAL DEFENSE RESEARCH
THEREIN HAMILTON AND
[INDISCERNIBLE] WHO HAD BEEN
WORKING ON [INDISCERNIBLE] FOR
PEOPLE COMING BACK FROM IRAQ AND
AFGHANISTAN WITH INJURIES AND
INJURIES FROM IMPROVISED
EXPLOSIVE DEVICES AND WORKING
WITH [INDISCERNIBLE] AND WITHIN
ABOUT 10 DAYS WE HAD FOURPHAGES
FROM EACH SOUR ACTIVE AGAINST
THE ORGANISM THAT IS SHOWN HERE.
THESE ARE NAVY PHAGES SHOWING
SUPPRESSION OF BACTERIAL
RESIDENT PRATION IN THIS LIQUID
ASSAY THAT CAN BE DONE IN 96
WELL PLATE AND SEE BLACK LINE IS
RESID
RESPIRATION UNIT AND CAUSING
SUPPRESSION OF THAT BACTERIA
OVER A 20-HOUR PERIOD. WITHOUT
GOING INTO ALL OF THE DETAILS
TOM WAS LUCKY HE DID THIS WHEN
HE DID.
THIS IS ABOUT THE TIME IT IS
POSSIBLE TO CLEAR SUFFICIENTLY
TO BE ABLE TO GIVE THEM
PERENNIALLY AND USING CHLORA
KREN TROE FUGUATION AT THE NAVY
AND EXTRACTION PROGRAM AT SAN
DIEGO STATE PREPARATIONS WERE
CLEAN ENOUGH TO GIVE THEM
INITIALLY INTO HIS ABSCESSED
CAVITIES ON THE LEFT AS ANDREW
PICEL AND STUDYING PHAGES TO
DRAIN FROM ABSCESSED CAVITIES
FROM TEXAYNIMPHAGES AND MORE
CONSULTATION AND IN THINKING
MORE ABOUT IT TOM WAS AT THAT
POINT ON TWO PRESSERS AND
KIDNEYS WERE FAILING AND LIVER
WAS BEGINNING TO FAIL.
WE DECIDED WE NEEDED TO TRY TO
GO A LITTLE MORE SYSTEMICALLY
AND HE HAD ORGANISMS IN PERITY
ON EEL CAVITY AND DECIDED TO
TREAT THEM INTRAVENOUSLY AND
FELLOWS THAT ARE GIVING FIRST
DOSE OF FAITHS INTRAVENOUSLY AND
48 HOURS LATER HE WOKE UP AND
RECOGNIZED HIS DAUGHTER AND
FIRST TIME HE WAS AWAKE IN ABOUT
THREE MONTHS AND OVER THE NEXT
TWO OR THREE MONTHS MANAGED TO
GET OUT OF THE HOSPITAL.
I WILL SHOW YOU MORE ABOUT
REASONS WE THINK WE HAD IN VIVO
ACTIVITY IN A FEW MINUTES AND
FACULTY VISITING HIM IN 2018
BACK ON THE FACULTY AND WHEN
THEY CAME TO SAN DIEGO AND IN
THIS CASE, STEPHANIE, HER -- HIS
WIFE AND I, SHE IS AN INFECTIOUS
DISEASE EPIDEMIOLOGISTS DECIDED
TO DO TWO THINGS, ONE, PUT
TOGETHER A CENTER ALLOWING US TO
TAKE ADVANTAGE OF EXPERIENCE
WITH TOM AND MAKING AVAILABLE
FOR PEOPLE PROVIDING ADVICE
ANDPHAGES AND TO DEVELOP A BENCH
TO BENCH SIDE OR PHAGE RESEARCH
PROGRAM TO UNDERSTAND A MORE
RIGOROUS WAY HOW BEST TO
USEPHAGES IN THE CONTEXT OF
ANTI-MICROBIAL INFECTIONS AND
TREATMENT PROGRAM IS
STRAIGHTFORWARD.
WE HAVE A WEBSITE.
PEOPLE CONTACT US ABOUT PEOPLE
THAT MIGHT NEEDPHAGE THERAPY,
PHYSICIANS AND FAMILY MEMBERS
AND SO FORTH AND LAST FOUR YEARS
NOT QUITE 2,000 REQUESTS BUT
MANY TURN OUT TO BE PATIENTS
THAT ARE SENSITIVE TO
ANTI-BIOTICSES AND TALK TO
PHYSICIANS AND HELP THEM WITH
APPROACHES AND SOME HAVE BEEN --
I AM TRYING NOT TO BE BONAFIDE
CASES AND INITIATED HENCE ABOUT
3/4 OF THOSE.
FOR A WHILE WE HAD A LOT OF
TROUBLE FINDING PHAGES AND GOT
BETTER OVER TIME AND GMP ACCESS
TO PHAGES TO BEGIN PERENNIALLY
AND PROJECT WE ARE WORKING ON
AND ORGANISMS YOU MIGHT EXPECT
AND MULTIDRUG RESISTANT AND ESB
CHOLI.
AND STAFF IS THERE AND NOT --
HELLO JIM HOXY.
STAFF IS NOT A MULTIDRIVER
ORGANISM BUT ONE THAT HANGS OUT
ON AND PLANTED DEVICES BECAUSE
OF BIOFILMS.
PHAGES WILL BREAK UP BIOFILMS.
THEY ARE -- WE USEPHAGES TO
TREAT PEOPLE WITH INFECTED
CROSS-BCS AND CARDIAC DEVICES
WITH THOSE THAT PREEF YO USUALLY
FAILED ANTI-BIOTICSES AND
REQUESTS COME IN GETTING IN
TOUCH WITH NETWORK OF PEOPLE
THAT WORK IN DIFFERENT PLACES
AND WITH DIFFERENT ORGANISMS AND
GARY WORKS ON PSEUDOMOWNIS AND
AND SO FORTH.
AND WHEN WE HAVE DECIDED THAT A
PATIENT IS A GOOD CANDIDATE AND
PATIENT PHYSICIAN WANT TO
PROCEED WE GET THEM TO THE
LABORATORY AND THEY LOOK FOR
APHAGE AND WORK ON GETTING THEM
SENT AND OPPORTUNITY IN 2023 I
WILL TALK ABOUT BOTH OF THEM IN
RAPID FIRE AND PERHAPS WE WILL
HAVE A CHANCE TO TALK ABOUT SOME
OF THEM.
CHALLENGE IS RESISTANCE
DEVELOPED TOPHAGES AS THEY DO
ANTI-BIOTICSES WITH A NARROW
HOST RAGE AND CHALLENGING
SOMETIMES TO FIND ACTIVE PHAGE
OF AN ORGANISM AND LYSOGENIC
LIFESTYLE FOR NONPHAGEOLOGISTS
THAT INTEGRATE INTO DNA OF
BACTERIUM THEY ARE TRYING TO
TREAT AND JUMP AROUND AND CARRY
ANTI-BIOTICS RESISTANT GENES
WITH LYSOGENIZE. YOU DON'T WANT
TO GIVE LYSOGENICPHAGES TO A
THERAPEUTIC SETTING.
WHAT ABOUT RESISTANCE? BACK TO
PATIENT TOM PATTERSON.
UPPER RIGHT YOU HAVE SEEN
ALREADY, THIS IS THE NAVIPHAGES
SHOWING ACTIVITY AGAINST HIS
ORGANISM BEFORE TREATMENT AND
RIGHT PANEL YOU SEE IS FIXED
ANDPHAGES WERE NOT SUPPRESSING
HIS ORGANISM OVER THE SAME
TIMEFR
TIMEFRAME.
DAY 10 IS LITTLE EVIDENCE OF
PHAGE ACTIVITY AND SAME WITH ON
THE LEFT AND TEXAS AND A & M AND
NOT SURPRISING ANDPHAGES
INTERACTING WITH BACTERIA 300
MILLION YEARS AND TWO HAVE BEEN
ON A DANCE FOR A LONG TIME OF
EVOLUTION WITH PHAGES GOING ON
AND DEVELOPING SO ON AND SO
FORTH.
THERE IS DIVERSE INTERESTING
MECHANISMS FOR BACTERIA. I'M
SORRY THEY HAVE DEVELOPED VERY
INTERESTING MECHANISMS TO AVOID
PHAGE PREDATION AND ENZYMES NOT
DEVELOPED FOR MOLECULAR OLOG
BIOLOGIST AND FOR DATA AND WERE
THINGS COMING OUT OF BATTLE
BETWEEN BACK TIERIANPHAGES AND
ADAPTING THINGS LIKE METHYLATING
GENOMES TO AVOID RESTRICTION
ENZYMES AND WAR WENT ON AND
THING THAT HAPPENS MOST OFTEN IN
VITRO STARTING WITH A PARTICULAR
ORGANISM AND IN A PARTICULAR
PHAGE IS EVOLUTION OF SURFACE
RECEPTOR AND INTRINSIC
MECHANISMS RESTRICTION ENZYMES
SO ON AND SO FORTH DON'T INVOLVE
A TREATMENT AND OFTEN ORGANISMS
WILL EVOLVE IN WAY THAT EFFECTS
ABILITY OF THE PHAGE TO BIND TO
THE SURFACE YOU CAN GET ORGANISM
AND LOOK ATPHAGE AND IN FACT BRD
AND TOM'S ORGANISM, THIS IS A --
THESE ARE NEWPHAGES THAT I WILL
NOW SUPPRESS AND YOU CAN SEE
BELOW THE TP3 AND ORGANISM THAT
IS RESISTANT TO ALL OTHER PHAGES
AT DAY 10 AND GO BACK AND TREAT
PATIENT WITH NEWPHAGES AND WHAT
WE DID WITH THIS PATIENT AND
ANOTHER THING TO DO TO PREVENT
PHAGE RESISTANCE IS DO
COMBINATION THERAPY LIKE WE HAVE
DONE WITH ANTI-BIOTICSES AND
VIRAL DRUGS AND WHAT WE LEARNED
IN THIS CASE IS WE STARTED WITH
FOUR PHAGES FROM AND IN THIS
CASE ON NAVY ON THE RIGHT AND
SEE THEY ARE MYOFAIPHAGES AND A
TIME NOT ABLE TO SEQUENCEPHAGES
USING COMMERCIAL KITS THAT WERE
HEAVILY MODERATED AND WERE
SIMILAR IN TERMS OF ESSENTIALLY
BEING ONEPHAGE WITH MINIMALED
GENETIC DIFFERENCES ALONG THEM
AND NEWPHAGE I SHOWED YOU A
MINUTE AGO WAS A DIFFERENT CLASS
OF PODOPHAGE THINKING ABOUT HOW
TO COMBINE THESE DIFFERENT
RECEPTORS AND RESISTANCE
PATHWAYS AND BEING PURSUED AND
OTHER THING TO BE DONE IS TO TRY
TO USE THEM IN CONCERT WITH
PAUL TURNER AT YALE HAS DONE
SOME VERY INTERESTING WORK USING
PHAGES THAT CAUSE BACTERIA TO
DOWNREGULATE THE B FLUX PUMP AND
BASICALLY CHOKE ON ANTIBIOTICS
TO WHICH THEY WERE RESISTANT
BEFORE, NO LONGER ABLE TO PUMP
THEM OUT.
THE OTHER THING THAT HAPPENS
WHEN THE PHAGE MODIFIES ITS
OUTER MEMBRANE PROTEINS IS THEY
CAN END UP LOSING THEIR CAPSULE.
IN TOM'S CASE THAT IS EXACTLY
WHAT HAPPENED.
PRE-TREATMENT, DAY 6, DAY 10,
YOU CAN SEE THAT BIG JUICY
CASSELL AT PRE-TREATMENT ON THE
LEFT, AND THE CAPSULE IS HARD TO
SEE THE THIRD TIME AROUND.
AT TEXAS A & M, THE BACTERIA
WERE SEQUENCED AND AGAIN TO MAKE
A LONG STORY SHORT, THE PRIMARY
DIFFERENCES WERE REALLY AT THE
SITE WHERE THE OUTER MEMBRANE
PROTEIN IS REGULATED.
IN THIS CASE AND OTHERS THAT
HAVE BEEN REPORTED SINCE THEN,
WHEN THIS HAPPENS, THE BACTERIUM
IS NO LONGER AS INVASIVE AND
IT'S NO LONGER AS RESILIENT, SO
WHAT IT GIVES UP TO THE PHAGE
MAKES IT MORE SUSCEPTIBLE TO
HOST IMMUNE RESPONSES, AND
AGAIN, MAYBE ONE OF THE
MECHANISMS BY WHICH PHAGE
THERAPEUTICS IT WORK DESPITE
RESISTANCE.
THE OTHER CHALLENGE IS -- AND
LIFESTYLE, I'VE ALREADY KIND OF
TALKED ABOUT WHY HOST RANGE --
WHY LIFESTYLE IS A PROBLEM, AND
HOST RANGE BEING VERY NARROW, IT
WOULD BE NICE TO BE ABLE TO
BROADEN THE HOST RANGE OR TO
MAKE PHAGES MORE RESILIENT TO
EVOLUTION OF THE CELL SURFACE.
PHAGE ENGINEERING CAN BE DONE.
THIS IS ANOTHER KIND OF ANECDOTE
WITH -- WORK WITH GRAHAM HATHEL,
WHO IS A MYCOBACTERIAL PHAGE
RESEARCHER AT THE UNIVERSITY OF
PITTSBURGH.
THIS IS A PATIENT HE AND I SAW
ABOUT SIX MONTHS AFTER TOM
PATTERSON, WHO WAS A YOUNG WOMAN
WHO HAD MYCOBACTERIUM INFECTION
STARTING WHEN SHE WAS 7 YEARS
OLD THAT PROGRESSED IN A CONTEXT
OF HER CYSTIC FIBROSIS,
TRANSPLANTATION WHEN SHE GOT TO
BE ABOUT 15.
SHE WAS INVOLVED FOR SEVERAL
WEEKS POST TRANSPLANT AND THE
INFECTION CAME BACK.
YOU CAN SEE THE NODULES ON HER
FOREARM ON THE LEFT, THE STERNAL
WOWPPEDWOUND ON THE RIGHT, PET S
SIGNS OF INFECTION ON THE LEFT
SIDE HERE.
NOW, THE PROBLEM WITH THE
MYCOBACTERIAL PHAGES THAT GRAHAM
HAD, HE HAS ABOUT 15,000 IN HIS
FREEZER FROM A VERY LARGE
NSF-SUPPORTED PROGRAM THAT HAS
UNDERGRADUATES LOOKING FOR
PHAGES ACTIVE AS PART OF A
COLLEGE COURSE, SOME OF THEM
TURNED OUT TO BE ACTIVE AGAINST
MYCOBACTERIAL INFECTION,
INCLUDING -- THE PROBLEM IS THEY
ALL ARE LYSOGENIC.
HOW DO YOU DO THAT?
GET RIT OF INTEGRATE STREAM OR
THE REPRESSOR GENE.
AND GRAHAM HAS A WAY TO DO THAT,
WHICH IS BASICALLY AN APPROACH
IN WHICH -- IS USED INTO A HOST
STRAIN OF NON-T -- JUST ELECTRO-
WHAT YOU WANT TO TAKE OUT WITH
STICKY ENDS AND SELECTING CLONES
THAT HAD -- THAT LACK THAT --
SHORTER, ESSENTIALLY, IN TERMS
OF DNA AND YOU END UP WITH
PLAQUES THAT YOU CAN THEN PUR
PURIFY, THEY'RE NOW JUST LYTIC
AND NO LONGER CAPABLE OF
INTEGRATING.
GOING BACK TO THE PATIENT, HE
WAS ABLE TO DO THAT AND WE WERE
ABLE TO US A SOME PHAGES THAT
WERE ACTIVE AGAINST YOUR
ORGANISM.
YOU CAN SEE ON THE LEFT, THE
SKIN LESIONS MELTING AWAY -- I'M
SORRY, ON THE RIGHT.
YOU CAN ALSO SEE THE PET SCAN
BEGINNING TO LOSE THE INTENSITY
SHE HAD AT THE ACUTE PHASE,
OBVIOUSLY YOU CAN STILL SEE HER
BLADDER, BUT SHE SHOWED BOTH
CLINICAL IMPROVEMENT AND
RADIOLOGIC IMPROVEMENT, FROM
WHAT WAS ESSENTIALLY PRE-HOSPICE
CARE, YOU CAN SEE HER PFTs
THRANS PLANT IMPROVE AND WITH
PHAGE THERAPY, IMPROVED FURTHER.
WE HAD A LITTLE BIT OF TROUBLE
WITH THIS.
THIS IS THE FIRST PATIENT
TREATED WITH GENETICALLY
MODIFIED PHAGE BECAUSE WE WERE
TRYING TO FIGURE IN THE U.K.
WHERE THEY DON'T EVEN LIKE
GENETICALLY MODIFIED CHEERIOS,
AND WE HAD TO ARGUE WITH THE
REGULATORS ABOUT THIS.
THEY SAID, WELL, YOU KNOW, THIS
IS A GMO AND WE SAID, WELL,
THERE ARE PERIODICALLY --
MUTATIONS IN LYSOGENIC PHAGES,
WHICH THEY THEN BECOME LYTIC,
AND THE PROBLEM IS THEY CAN
MUTATE -- THEY CAN -- MUTATE.
WE JUST DON'T -- THEY REALLY
ARE -- SOMETHING BETTER THAN
NATURE, WE DIDN'T ADD ANYTHING.
THEY BOUGHT THAT, SO GRAHAM WAS
ABLE TO USE THESE NOW IN ABOUT
40 PATIENTS.
WE REPORTED 20 OF THEM IN
CLINICAL -- DISEASES ABOUT A
YEAR AGO, AND TO MAKE A LONG
STORY SHORT, THE SUCCESS RATE IS
ABOUT 55%.
WE'VE LEARNED A LOT ABOUT
EVOLUTION OF ANTIBODIES TO
PHAGES IN THESE PATIENTS BECAUSE
THESE ARE PATIENTS THAT HAVE
BEEN TREATED FOR A LONG TIME
BECAUSE OF THEIR IMMUNE
SUPPRESSIVE CONDITION AND WE
LEARNED QUITE A BIT ABOUT PHAGE
RESISTANCE.
THESE PATIENTS GENERALLY DON'T
DEVELOP RESISTANCE, MYCOBACTERIA
ARE SLOW TO GROW AND HE HAS SOME
PHAGES THAT ARE QUITE ACTIVE
AGAINST A PRETTY IR VARIANT
SURFACE PROTEIN.
SO LET'S TALK ABOUT
OPPORTUNITIES.
WE'VE OBVIOUSLY TALKED ABOUT
INDIVIDUAL PATIENTS.
AGAIN I WON'T GO INTO ALL THE
DETAIL.
WE HAVE A SERIES WE PUBLISH FROM
UCSD, CASE REPORTS IN THE
LITERATURE KIND OF EVER SINCE,
AND WHEN YOU LOOK AT WHAT THESE
CASE REPORTS ARE ABOUT, THEY'RE
PRIMARILY PATIENTS WHO HAVE
IMPLANTED DEVICES AND PULMONARY
INFECTIONS.
NOW YOU SAY WELL, THIS IS -- OF
COURSE IT IS.
THAT'S WHAT CASE REPORTS ARE.
BUT IT ALSO TELLS YOU WHAT
PEOPLE THINK ARE WORKING.
AND IT GIVES YOU SOMETHING TO
THINK ABOUT IN TERMS OF HOW YOU
PRIORITIZE YOUR MORE STRUCTURED
RESEARCH STUDIES.
SO IN TERMS OF AREAS THAT PEOPLE
THINK MIGHT BE AMENABLE TO PHAGE
THERAPEUTICS, LIKE NOW THERE'S A
LOT OF -- INFECTIONS ASSOCIATED
WITH MEDICAL DEVICES.
THERE IS INTEREST IN -- THERE'S
A COMPANY IN DENMARK THAT IS
WORKING ON PHAGES THAT ARE
ACTIVE AGAINST ENTERIC
GRAM-NEGATIVE ORGANISMS TO TRY
TO TREAT PEOPLE WHO ARE ABOUT TO
UNDERGO CHEMOTHERAPY TO TRY TO
REDUCE MULTIDRUG RESISTANT
SEPSIS IN THE CONTEXT OF KNEW
NUTRAPENA.
THERE ARE THEORIES ABOUT MICRO
BIOMIBG DRIVERS OF INFLAMMATORY
BOWEL DISEASE AND SO FORTH.
SO THERE ARE A LOT OF POTENTIAL
MEDICAL -- AND STOP ACTING LIKE
THEY'RE SOME MAGICAL THING FROM
GEORGIA OR RUSSIA.
THEY REALLY ARE ANTIBIOTICS BUT
THEY'RE ANTIBIOTICS THAT HAPPEN
TO BE ALIVE.
SO OUR GOAL WITH THEM IS TO DO
THE SAME THING WE WOULD WITH A
SMALL MOLECULE, KILL ORGANISMS,
BUT THE MEANS BY WHICH YOU DO IT
IS A BIT DIFFERENT BECAUSE
UNLIKE ANTIBIOTICS, THEY'RE
REALLY QUITE LIMITED.
YOU GIVE THEM INTRAVENOUSLY AND
NEXT THING YOU KNOW, THEY'RE
BEING ELIMINATED BY THE KIDNEY,
ELIMINATED BY THE LIVER.
YOU GET A PHAGE, IT FINDS THE
ORGANISM AND AMPLIFIES ITSELF AT
THE SITE OF THE ORGANISM.
IT'S A MUCH SMARTER ANTIBUY
KROAB YAANTIMICROBIAL.
THE GOOD NEWS IS THERE ARE A LOT
OF CLINICAL TRIALS THAT HAVE
PICKED UP SINCE THAT TIME.
THEY'VE BEEN PICKING UP
INCREASINGLY SINCE THE LATE --
THE LAST HALF OF THE LAST
DECADE.
THE BLUE BARS HERE, THESE ARE
THERAPEUTIC INDICATIONS.
THE ORANGE ONES ARE PEOPLE
TRYING TO ASSESS -- USING PHAGES
AS A TOOL BUT THERAPEUTIC
RESEARCH IN BLUE BARS HAS BEEN
PICKING UP.
THE BAD NEWS ABOUT THESE PHAGE
TRIALS ARE MOST ARE BEING
UNDERTAKEN BY THE BIOTECH
INDUSTRY, TRYING TO FIND A QUICK
EDUCATION FOR SPECIFIC INFECTION
THAT CAN BE TAKEN TO THE FDA.
AND THEY'RE OFTEN DONE WITHOUT
DUE DILIGENCE BEFORE THEY'RE
STARTED IN TERMS OF WHAT DOSE,
WHAT FREQUENCY, WHAT ROUTE OF
ADMINISTRATION, AND NEEDLESS TO
SAY, MANY OF THEM HAVE NOT GONE
VERY WELL.
WOULD NEVER DEVELOP AN
ANTIBIOTIC THE SAME WAY.
WHAT WE I THINK IN MY VIEW NEED
TO BE DOING IS TAKING ADVANTAGE
OF WHAT WE LEARNED FROM HIV,
WHICH IS TO TAKE AN INTEGRATED
APPROACH AND ESSENTIALLY ITERATE
BETWEEN INNOVATION IN THE
LABORATORY WITH ENGINEERED
PHAGES, PHAGES OF DIFFERENT
TYPES THAT WE HAVE DISCOVERED,
COMBINED THEM WITH OTHER NOVEL
ANTIMICROBIAL APPROACHES, TAKE
THEM INTO CLINICAL TRIALS THAT
ARE WELL THOUGHT OUT TO DO
CLINICAL TRIALS BUT DON'T JUST
HAVE CLINICAL END POINTS BUT
WELL BACKED UP SO WHEN THINGS
WORK WE KNOW WHY, WHEN THEY
DON'T WORK, WE KNOW WHY, WE CAN
THEN TAKE THEM BACK AFTER DOING
SOME MATHEMATICAL MODELING AND
ITERATE, DO A BIT BETTER THE
NEXT TIME.
THIS IS THE TYPE OF RESEARCH
THAT NEEDS TO BE DONE.
UNFORTUNATELY THE BIOTECH
INDUSTRY IS NOT CAPITALIZED
ENOUGH TO DO THIS, AND THEY
CONTINUE TO KIND OF STAND UP TO
THE PLATE AND TRY TO HIT AN
INDICATION THAT WILL LET THEM
MOVE ON TO THE NEXT INVESTMENT.
NOW SOME STUDIES ARE BEING DONE
THAT ARE ACTUALLY QUITE
INTELLIGENT.
THIS IS A STUDY REPORTED WEEK
BEFORE LAST.
THIS ONE DONE BY A COMPANY
CALLED BIOMIX FROM ISRAEL.
THIS IS -- IN WHICH PEOPLE WHO
HAVE CYSTIC FIBROSIS AND ARE
SHEDDING PSEUDOMONAS AND DON'T
NEED TO BE TREATED ACUTELY
RECEIVE AI AI AEROSOLIZED -- ONN
THE LEFT, THE ACTIVE TREATMENT,
PATIENTS GENERALLY HAVE A
DECLINE IN THEIR PSEUDOMONAS
SHEDDING THERAPY LASTED A WEEK,
YOU CAN SEE THIS IS A WEEK POST
THERAPY.
COMPARED TO A COUPLE OF CONTROLS
ON THE RIGHT.
THEY HAVE ANOTHER 30 PATIENTS
THAT WILL BE UNBLINDED SOMETIME
IN THE NEXT MONTH OR SO, SO
WE'LL HAVE MORE NUMBERS.
BUT THE BOTTOM PANEL HERE, YOU
CAN SEE THEY DETECT PHAGES IN
THE SPUTUM AND CAN STILL DETECT
IT IN 2 OF THE 6 ACTIVE PATIENTS
A WEEK LATER.
THEY'RE GOING TO BE DOING SOME
MODELING TO TRY TO UNDERSTAND
THE RELATIONSHIPS BETWEEN
POPULATIONS HERE OF THE SORTS OF
THINGS THAT PEOPLE LIKE JOHN
KAUFMAN AND AL DID WITH HIV.
TRULY COME BACK AT THIS WITH A
MORE INTELLIGENT APPROACH NEXT
TIME AROUND.
ANOTHER OPPORTUNITY I'LL CLOSE
WITH IS THINKING ABOUT USING
PHAGES FOR OUTBREAK
INTERVENTIONS.
LAST FIVE OR 10 YEARS ARE
OUTBREAKS OF NDR INFECTIONS
AROUND THE WORLD, MOST RECENTLY
AN OUTBREAK OF HIGHLY DRUG
RESISTANT PSEUDOMONAS AERGENOSA
OF PRESIEVETIVE-FREE EYE DROPS.
PEOPLE WHO HAVE DRY EYE -- YOU
HAVE TO GIVE THEM FREQUENTLY FOR
DRY EYE, IT CAUSES
CONJUNCTIVITIS, CAN MAKE THINGS
WORSE.
PRESERVED DRUGS CAN GET
CONTAMINATED, DOESN'T TAKE MUCH
FOR PSEUDOMONAS TO GROW, AND TWO
PLANTS IN INDIA HAVE
CONTAMINATED THEIR EYE DROPS AND
CAUSED A LARGE OUTBREAK OF
ORGANISMS THAT YOU DON'T WANT TO
HAVE AROUND YOUR HOSPITAL.
THIS SHOWS YOU TH HOW THEY'RE DG
RESISTANT TO MOST OF THE
ANTIBIOTICS WE HAVE AVAILABLE TO
TREAT PATIENTS WITH.
AS OF MARCH, THERE HAVE BEEN 80
CASES IN 18 STATES IN THE U.S.,
14 PEOPLE HAVE LOST VISION,
THERE HAVE BEEN SOME
ENUCLEATIONS AND SOME DEATHS.
FROM THE CASE REPORTS, THIS KIND
OF SHOWS YOU WHAT THIS DOES TO
AN EYE.
THESE ARE VERY INVASIVE
INFECTIONS.
EYE IS NOT WELL VASCULARIZED,
AND ONCE THEY GET STARTED WITH
AN ABRADED CORNEA, THEY CAN BE
DIFFICULT TO ERADICATE.
NOW WE WERE ABLE TO GET THE
ORGANISMS FROM THE CDC AND THEN
DAVID PRIDE IN OUR GROUP FOUND
SOME PHAGES THAT HE'D BEEN
WORKING WITH ALREADY, SOME JUMBO
PHAGES ON A -- WITH SOME
COLLEAGUES AT UCSD WHO ARE
WORKING ON -- WITH THESE PHAGES
FOUND THREE PHAGES.
YOU CAN SEE DOWN THE Y AXIS,
THESE ARE VIJ PHA INDIVIDUAL PH,
THOSE ARE PSEUDOMONAS ISOLATES.
ONE IS FROM THE OUTBREAK.
YOU CAN SEE PHAGES THAT HAVE
ACTIVITY AGAINST EACH OF THESE
THREE ORGANISMS.
THE NICE THING ABOUT OUT DK
BREAK SITUATIONS, WHEN SOMEBODY
SHOWS UP IN A DIFFERENT TOWN
FROM THE SAME OUTBREAK, THOSE
PHAGES ARE VERY LIKELY TO BE
ACTIVE AS WELL.
SO WE'RE WORKING WITH THE CDC ON
TRYING TO DEVELOP A PROGRAM BY
WHICH THEY IDENTIFY IC ISOLATESF
CONCERN, WE FIND PHAGES THEN WE
START TRYING TO GROW THEM UP TO
BE USED IN OUTBREAK CONTROL.
SO I'LL FINISH -- PHAGES ARE
NARROW, ANTIBIOTICS ARE HOW THE
MICROBIOME DISRUPTING, PHAGES
ARE GENERALLY NOT.
YOU CAN DEVELOP A BACTERIOPHAGE
OR ISOLATE ONE RELATIVELY
QUICKLY.
THE COST OF PRODUCTION REALLY OF
AN INDIVIDUAL PHAGE IS QUITE
TRIFTRIVIAL.
THESE ARE ACTUALLY QUITE EASY TO
WORK WITH WITH THE SAME SORTS OF
APPROACHES USED FOR BIOLOGI
BIOLOGICAL -- THE SAME KIND OF A
PLANT COULD EASILY MAKE PHAGES
FOR THERAPY.
I TALKED TODAY PRIMARILY ABOUT
HUMAN MEDICINE BUT THERE ARE A
NUMBER OF OTHERS INCLUDING
BIODEFENSE, FOOD SAFETY.
THERE'S ALREADY A PHAGE APPROVED
FOR LISTERIA PREVENTION ON MEAT.
PEOPLE HAVE TALKED ABOUT
USING -- OR REPLACING
TETRACYCLINE IN CHICKENS WITH
PHAGES.
THERE'S SOME NICE WORK BEING
DONE ON THAT IN THE U.K. AND YOU
CAN THINK ABOUT OTHER
APPLICATIONS WITH PHAGES USED AS
VECTORS WITH SOME -- WE HEARD
ABOUT THIS MORNING.
SO I'LL STOP THERE AND THEN JUST
WANT TO DEDICATE THIS TO
CAPTAIN -- WHO IS A --
UNFORTUNATELY PASSED AWAY TWO
WEEKS AGO.
HE WAS THE COMMANDER OF THE --
HERE AT FORT DIETRICH UNTIL HE
RETIRED EARLIER THIS YEAR.
HE MA PLAYED A MAJOR ROLE FOR
LETTING TOM PATTERSON BE
TREATED.
THANKS VERY MUCH.
[APPLAUSE]
>> WE'RE GOING TO GO HOME AND
THROW OUR EYE DROPS AWAY.
QUESTIONS.
>> YOU BRIEFLY ALLUDED TO THE
HOST IMMUNE RESPONSE OF A PHAGE,
AND -- THAT WOULD SEEM TO ME TO
BE A SIGNIFICANT CONSIDERATION.
COULD YOU -- A LITTLE BIT?
>> SURE.
SO PHAGES WERE USED FOR A LONG
TIME AS A WAY TO STUDY HUMAN
IMMUNE RESPONSES IN -- CAUSING
ANTIBODIES.
YOU CAN INDUCE -- ANTIBODIES BY
THE -- ADMINISTRATION OF PHAGES.
WHAT ISN'T CLEAR IS WHAT IMPACT
THAT HAS ON THEIR EFFICACY FROM
TWO PERSPECTIVES.
FIRST, YOU'RE TREATING ACUTE
INFECTION, MOST OF WHAT YOU DO
WITH AN ANTIBIOTIC OR PHAGE IS
DONE IN THE FIRST WEEK.
SOME WITH CHRONIC INFECTIONS
LIKE PATIENTS WHO HAVE ORGAN
TRANSPLANTS AND -- ONLY ABOUT
HALF OF THEM DEVELOP ANTIBODIES,
AND WHEN GRAHAM AND -- HAVE
LOOKED AT THOSE PATIENTS, ONLY
ABOUT HALF OF THOSE SEEM TO HAVE
A CHANGE IN THE IMPACT OF THE
PHAGES WHEN THOSE ANTIBODIES
DEVELOP.
NOW WHY MIGHT THAT BE?
IT MIGHT BE WHEN YOU THINK
ABOUT -- WHEN YOU GIVE PHAGES
INTRAVENOUSLY, THEY'RE CLEARED
RAPIDLY FROM THE BLOODSTREAM,
AND THE GOAL IS TO BOLUS THE
PHAGES AND HOPE THAT THEY GET TO
THE SITE OF INFECTION BEFORE
THEY'RE CLEARED.
WHEN YOU HAVE ANTIBODIES, YOU
INCREASE THE RATE OF CLEARANCE
BUT IF THEY GET TO A SITE OF
INFECTION LIKE IN THE LUNG, THEY
CAN REPLICATE IN THE INFECTING
ORGANISM AND SPREAD LOCALLY.
TO JUST AS HIV CAN PERSIST IN
THE PRESENCE OF NEUTRALIZING
ANTIBODY, IT'S POSSIBLE THAT
PHASES ARE PERSISTING IN THEIR
PRESENCE OF NEUTRALIZING
ANTIBODIES BECAUSE OF THE
CONTINUITY OF THEIR TARGETS.
WAYS AROUND THAT INCLUDE USING
PHAGES OF DIFFERENT CLASSES.
BIOPHAGES AND -- VERY MUCH LIKE
THE APPROACH TO TRY TO PREVENT
PHAGE DEVELOPMENT COCKTAILS.
ANIMAL MODEL STUDIES HAVE LOOKED
AT THIS TOO AND LIKE MANY ANIMAL
MODEL STUDIES, YOU CAN SET THEM
OUT TO SHOW WHATEVER YOU WANT
TO.
WITH ABOLI APOLOGIES TO UNIVERSY
OF -- I'M JUST KIDDING.
BUT THERE IS AT LEAST ONE MODEL
IN WHICH THE PEOPLE AT WALTER
REED TOOK A -- IT WAS A -- MODEL
OF A WOUND INFECTION, AND I
IMMUNIZED HALF THE ANIMALS WITH
THE PHAGE WE WERE GOING TO USE,
THEN ESTABLISHED THE INFECTION,
AND TREATED A PARALLEL GROUP
THAT HADN'T BEEN IMMUNIZED AND
THERAPEUTIC RESPONSE WERE THE
SAME.
WE HAD OTHER ANIMAL MODELS WHICH
ANTIBODIES DO CAUSE A PROBLEM.
SO THE SHORT ANSWER IS, IT'S
WORK IN PROGRESS.
ONE OF THE THINGS THAT NEEDS TO
BE LOOKED AT IN SOME OF THE
CLINICAL TRIALS MOVING FORWARD,
SO WE UNDERSTAND WHAT ROLE THEY
ACTUALLY DO PLAY, RATHER THAN
HAVING SOMETHING NOT WORK AND
NOT REALIZE IT'S AN ANTIBODY
RESPONSE.
>> TWO QUICK QUESTIONS
ASSOCIATED WITH -- SO DO THEY --
TO THE PHAGE THAT OVERLAP --
ALSO WITH TUBERCULOSIS, AND THE
SECOND QUESTION IS, YOU MUST
HAVE THOUGHT OF THIS HARD ABOUT
SOME OF THE CHALLENGES WITH
TB -- EVERYTHING ELSE, IS THAT
REALLY A -- PROBLEM, A -- TAR
TARGET?
>> SO THE SHORT ANSWER IS THAT
SOME OF THE PHAGES THAT ARE
ACTIVE THAT ARE FROM THIS
COLLECTION ARE ACTIVE AGAINST
TB.
GRAHAM AND SOME IN VITRO
EXPERIMENTS THINKS HE HAS PHAGES
THAT ARE ACTIVE AGAINST ABOUT
85% OF TB THAT YOU ENCOUNTER
CLINICALLY.
NOT ALL OF THEM ARE ACTIVE.
THE PHAGES HAVE NOT BEEN USED IN
PEOPLE WITH TUBERCULOSIS YET.
THERE WAS A PATIENT WITH
ACTUALLY RTB IN GERMANY THAT WAS
BEING TEED UP TO BE TREATED,
UNFORTUNATELY DIED BEFORE THE
PHAGE COULD BE GOTTEN TO HIM.
THE CHALLENGE THAT PEOPLE KEEP
TALKING ABOUT IS, WELL, IT'S AN
INTRACELLULAR INFECTION, WILL
THE PHAGES GET THERE.
AND I -- SORRY?
>> ALSO CYST FORMATION.
>> YOU'VE GOT GRANULOMAS AND ALL
THIS.
MY VIEW HAS BEEN, YOU CAN SPEND
A LOT OF TIME TRYING TO FIGURE
THAT OUT IN MICE AND RATS, WHY
NOT GIVE TO HUMANS, JUST LIKE
YOU WOULD AN ANTITUBERCULOSE
DRUG, LOOK AT COLONIES IN THE
SPUTUM, YOU CAN SEE DECLINE IN
COLONY FORMATION WITHIN A WEEK
WHEN YOU DO THAT.
AND THEN PEOPLE -- THEN CAN YOU
DEVELOP CLINICALLY IN PEOPLE AND
MICE CAN FIGURE THAT OUT.
THIS KIND OF CYCLE OF WE HAVE TO
SEE IF IT GETS INTO THEM BEFORE
YOU GIVE TO PEOPLE.
I DON'T THINK THE ANIMAL MODELS
ARE THAT GOOD FOR HUMAN TB
ANYWAY.
SO IT'S A GREAT QUESTION, I'D
LOVE TO GET A LITTLE MORE
IMPATIENT WITH IT AND PUSH IT
FORWARD, AND I THINK SO WOULD
GRAHAM.
YES.
>> YOU MENTIONED THE -- AND
DIFFICULTY OF TARGETING SPECIFIC
BACTERIA.
MY QUESTION IS HOW OFTEN DO YOU
COME ACROSS A TARGET WHERE YOU
DON'T FIND A SUITABLE LYTIC
PHAGE, AND CAN YOU DO ANYTHING
BY ENGINEERING THE PHAGE TO
EXPAND ITS HOST RANGE?
>> THE ANSWER TO THE FIRST
QUESTION IS IT DEPENDS ON THE
ORGANISM YOU'RE TREATING.
SOME ORGANISMS LIKE STAPH AUREUS
HAVE ORGANISMS ACTIVE AGAINST A
PRETTY WIDE SWATH OF STAPH.
YOU CAN COME UP WITH A FIVE-PAGE
COCKTAIL THAT WOULD KNOCK OFF 8E
CLINICALLY.
-- TO HAVE THAT SAME COVERAGE,
YOU'D HAVE TO HAVE A LAY BREAR F
2- OR 300 PHASES.
-- TRYING TO USE THINGS IN THE
CLINIC.
YOU COULD HAVE A FIXED COCKTAIL
FOR STAPH, PROBABLY PSEUDOMONAS
AND PERHAPS FOR ENDO BACTERACIA
USED LIKE AN ANTIBIOTIC WHILE
YOU DO TESTING BUT YOU'D NEVER
DO THAT -- YOU'D HAVE TO HAVE A
LIBRARY OF CURATED PHAGES READY
TO GO, SCREEN AND THEN CHOOSE
THE ONES YOU WANT TO USE.
PEOPLE HAVE CHANGED HOST RANGE
BY CO-EVOLUTION OF PHAGES WITH
TARGETS.
PEOPLE HAVE ALSO -- THE -- BIOME
THAT HAS A CRISPR-BASED APPROACH
AND IS USING THAT APPROACH FOR
TRYING TO DECOLONIZE THE G.I.
TRACT AS MODIFIED -- HAS
MODIFIED SOME OF THE TAIL FIBERS
TO BE MORE INTERACTIVE WITH A
BROADER RANGE OF ORGANISMS.
I THINK IT'S ONE OF THE THINGS
THAT MAY MAKE IT POSSIBLE TO
EVEN GET AFTER -- BACTER A
LITTLE BIT BETTER.
YOU HAD A QUESTION.
SAME QUESTION?
OKAY, GOOD.
THINKING THE SAME WAY.
>> SO FOR THE -- PHAGE, THERE
MUST BE A HUGE NUMBER OF PHAGES.
HAS THERE BEEN ANY ML DONE, LIKE
WHAT ARE WE DOING -- ALL OF THE
PHAGES AND WHAT THEIR ACTIVITY
IS?
>> NO, THEY'RE 10 TO 31ST
PHAGES -- UNIQUE PHAGES, SO
THERE'S A LOT OF WORK TO DO
HERE.
THE PERSON WHO HAS DONE ALL
THIS, HE'S LOOKED AT THEM IN SEA
WATER, THE ESTIMATE IS THERE ARE
ABOUT 10 PHAGES FOR ANY
BACTERIUM IN THE WORLD RIGHT
NOW.
SO THE DIVERSITY IS REMARKABLE.
AND THAT'S ONE OF THE REASONS
WHY AT LEAST IT'S FEASIBLE TO
FIND A PHAGE FOR ANY ORGANISM
SOMEBODY MIGHT GET INFECTED
WITH.
>> IT SEEMS LIKE HIGH-THROUGHPUT
SCREENING, IT'S NOT A REACH,
RIGHT?
>> NO, IT'S NOT A REACH AT ALL.
IF YOU'RE LOOKING FOR PHAGES
ACTIVE AGAINST E. COLI, YOU GO
TO THE SEWAGE TREATMENT PLANT.
IF YOU LOOK AT THOSE ACTIVE
AGAINST STAPH, YOU TAKE KIND OF
A SOUPY PIECE OF GAUZE OFF A
WOUND THAT'S INFECT AND YOU
SCREEN THAT.
SO -- WATERING HOLE.
JOHN.
>> IS THERE ANY PROSPECT FOR
FINDING I WOULDN'T CALL THEM
PHAGES BUT VIRUSES ACTIVE
AGAINST PARASITIC ORGANISMS AND
FUNGI?
>> THERE ARE -- PEOPLE ARE
BEGINNING TO LOOK AT THAT.
THE MYCOBACTERIUM THAT PEOPLE
WOULD BE -- I MEAN THE FUNGUS --
WHICH IS GOING TO BE A BIGGER
AND BIGGER PROBLEM WITH DEVICES
AROUND THE WORLD, I WAS HAVING
DINNER WITH SOME COLLEAGUES FROM
HONG KONG LAST WEEK AND THEY
TOLD ME THAT KYU NHUN HAS SOME.
I SAID HAVE THEM CALL ME BECAUSE
WE'D LOVE TO LOOK AT THOSE.
PEOPLE ARE BEGINNING TO LOOK IN
THAT AREA AS WELL.
BUT GREAT QUESTION.
>> I WAS WONDERING, WHAT IS THE
SAFETY -- LOOK LIKE FOR PHAGE
THERAPIES?
DOES THAT CHANGE BASED ON THE --
OR DISEASE STATE OR --
>> WERE YOU ASKING WHAT THE
SUCCESS RATE IS?
>> SAFETY AND --
>> THE SAFETY RATE, OKAY.
SAFETY HAS ACTUALLY BEEN
SOMETHING THAT HAS BEEN WORKED
OUT OVER THE LAST FEW HUNDRED
THOUSAND YEARS.
THERE ARE MORE PHAGES IN US THAN
THERE ARE BACTERIA.
AND SO WE'RE JUST KIND OF --
WE'RE KIND OF A CORAL REEF ON
WHICH BACTERIA AND PHAGE LIVE.
WHEN YOU TALK TO THE FDA, THEIR
CONCERN IS NOT SAFETY.
THE FACT THAT THEY'VE SAID TO ME
AND OTHERS, WE DON'T WANT ANY
MORE STUDIES ABOUT SAFETY, WE
JUST WANT TO KNOW IF THEY WORK.
SO THEY DON'T ASK YOU TO DO ANY
TOXICITY STUDIES, THEY DON'T ASK
FOR STUDIES ON ANIMALS.
THEY JUST WANT YOU TO SHOW THEM
THE CMC DATA ABOUT HOW YOU GOT
RID OF THE ENDO TOXIN AND
PREAPED THEM, BUT SAFETY HAS NOT
BEEN AN ISSUE WITH THEM.
OBVIOUSLY I THINK WE'VE GOT TO
KEEP WATCHING, BUT RIGHT NOW
JUST GOT TO FIGURE OUT IF THEY
WORK AND, IF SO, HOW TO MOST
RIGOROUSLY USE THEM.
SO APPRECIATE YOUR PATIENCE FOR
A FREE ASSOCIATION TALK.
THANK YOU.
[APPLAUSE]
>> WE'RE GOING TO NOW MOVE ON TO
CLASS CREW.
POSTDOCTORAL WORK IN LA HOI
WHAT.
IN 1994, HE JOINED -- RESEARCH
PROGRAM THERE.
IN 2000 HE JOINED THE OREGON
HEALTH AND SCIENCES UNIVERSITY
VACCINE GENE THERAPY INSTITUTE.
RESEARCH FOCUS ON THE MOLECULAR
REACTION BETWEEN BIOPATHOGENS
AND THEIR HOST.
HIS TALK HAS PIONEERED THE
DEVELOPMENT OF DB VACCINES.
TIE IS UNIQUE BIOLOGY OF --
BASED VACCINES.
[APPLAUSE]
>> THANK YOU.
THANK YOU FOR THE INTRODUCTION
AND THANK YOU FOR A GREAT
MEETING.
SO WHILE WE WORK ON -- IT'S OF
COURSE A VIRUS THAT INFECTS A
LOT OF US, AND MOST OF US ARE
CHRONICALLY INFECTED AND THE
REASON IS THAT CME IS QUITE
UNIQUE IN ITS ABILITY TO ELICIT
AND MAINTAIN VERY STRONG EFFECT
ON MEMORY T-CELL RESPONSES.
THIS IS FROM A PAPER FROM
LEWIS -- FROM 2005 SHOWING
THAT -- CHRONICALLY INFECTED
WITH CME, 10% -- CD4 T-CELL
RESPONSES -- LEWIS HAD THIS IDEA
WHAT IF WE COULD USE THIS AND
REDIRECT THESE EFFECT ON MEMORY
T CELLS TO OTHER PATHOGENS.
WHAT THAT WOULD MEAN IS THAT YOU
HAVE A CONSTANT IMMUNE FIELD
AGAINST THESE PATHOGENS AND YOU
DON'T NEED THE RECALL RESPONSE
OR IN CASE OF PRIMARY INFECTION
FIRST TIME RESPONSE WHICH TAKES
TWO OR THREE WEEKS TO DEVELOP.
SO IN THE CASE OF SIV, FOR
INSTANCE, IN MONKEYS, YOU WILL
SEE DATA THAT WITHIN A WEEK, YOU
GO FROM VIRTUALLY NOTHING WITH
FAIRLY LOW DOSE INFECTION UP TO
10 TO THE 6TH, 10 TO THE 7TH
VIRUSES PER MILLILITER OF BLOOD.
SO BY TEAM EVEN IN THE
VACCINATED ANIMAL, YOUR IMMUNE
SYSTEM KICKS IN, YOUR T-CELL
RESPONSE KICKS IN, IT HAS TO
PLAY CATCH-UP FOR THE REST OF
THE TIME IN CONTROLLING THIS
INFECTION.
SO THE FIRST QUESTION IS OF
COURSE CAN WE USE CMV TO ELICIT
IMMUNE RESPONSES TO OTHER
ANTIGENS, DO THOSE HAVE THE SAME
PHENOTYPE.
THE ANSWER TO THAT IS YES.
IN THE MEANTIME, -- PROBABLY 50
OR EVEN MORE DIFFERENT ANTIGENS
INTO A RHESUS CME VECTOR AND
POINT OUT THAT THE WHOLE
STUDIES -- ARE NON-HUMAN
PRIMATES.
AND YOU ALWAYS GET THESE IMMUNE
RESPONSES SAME AS THE IMMUNE
RESPONSES TO THE CME ANTIGENS
AND THIS IS A VERY TYPICAL GRAPH
HERE, IF YOU VACCINATE AGAINST
SIV USING RHESUS -- OR --
ADENOVECTORS, THE T-CELL
RESPONSE IN THE PERIPHERY WILL
DISAPPEAR WITH THE OTHER VECTORS
BECAUSE THEY'RE CENTRAL MEMORY
RESPONSES AND THEY NEED TO BE
RECALLED.
WHEREAS IN THE CMV CASE, THESE
STAY AT A CERTAIN LEVEL IN THESE
ANIMALS FOR THE REST OF THE LIFE
OF THESE ANIMALS.
THEIR EFFECT ON MEMORY BIAS IS
SHOWN HERE BUT THE SURFACE MARK
STAINING, AND THEY ARE NOT JUST
IN THE BLOOD BUT ALSO IN THE
TISSUES, AND SOME OF THOSE
RESPONSES ARE REALLY HIGH, THIS
IS AN ANIMAL I THINK SEVEN YEARS
AFTER ORIGINAL INNOCULATION, 10,
20% OF THE T CELLS IN THE LIVER
AND SPLEEN ARE AGAINST THE
INSERTED ANTIGEN.
MOST IMPORTANTLY, THEY WORK.
SO USING THE SIV MODEL, LEWIS IN
SEVERAL STUDIES NOW HAS SHOWN
THAT WHEN YOU CHALLENGE ANIMALS
WITH SIV AND THIS IS A LOW DOSE
INTRARECTAL -- HIGHLY VIRULENT
IN RHESUS MACAQUES, ALL THE
ANIMALS IN THE CONTROL GROUP ARE
DEVELOP AIDS, THIS VERY
EXPLOSIVE INFECTION EARLY ON.
WHEN YOU VACCINATE WITH
ADENOVECTORS, WHAT YOU SEE IS
YOU REDUCE THE P VIRAL LOAD BUT
ULTIMATELY THESE VECTORS DON'T
PROTECT.
IN THE CASE OF CMV, WE SEE THIS
VERY INTERESTING -- ABOUT HALF
OF THE ANIMALS, THEY ARE
INFECTED AND THEN CONTROL THE
VIRUS, AND THE OTHER HAVE NO
PROTECTION, THERE'S NO
IMMUNOLOGICAL DIFFERENCE.
THE VACCINE WORKS IN BOTH
ANIMALS THE SAME WAY.
THE REASON WHY THIS IS A SUBJECT
OF A DIFFERENT TALK BUT WE HAVE
SOME INDICATION THAT IT'S THE
INNATE SET POINT OF THESE
ANIMALS THAT DICTATES THAT.
WHEN YOU MIX THE TWO, ADENOVIRUS
DOES THIS THING AND CMV DOES ITS
THING.
SO WE'VE SHOWN ALSO THAT SIV
IS -- CLEAR FROM THESE ANIMALS,
EARLY ON IN INFECTION YOU CAN
TRANSFER BONE MARROW FROM
CONTROL ANIMALS BUT YOU STILL
CAN TRANSFER SIV TO RECIPIENT
ANIMALS.
WHEN YOU DO THIS LATER, THERE'S
NO VIRUS IN THE RECIPIENT
ANIMALS, AND THIS IS VERY
DIFFERENT FROM ANIMALS THAT
ARE -- END UP ON ANTIRETROVIRAL
THERAPY, YOU CAN ALWAYS FIND
VIRUS AND ALWAYS TRANSFER VIRUS.
SO BASICALLY THEY'RE CURED OF
SIV BECAUSE OF THIS INTERSECTION
THAT HAPPENS VERY EARLY ON, AND
BY THE WAY, THIS IS VERY SIMILAR
TO WHAT YOU SEE IN ANIMALS THAT
ARE TREATED VERY EARLY ON WITH
ANTIRETROVIRAL THERAPY SO THERE
IS POSSIBILITY FOR A CURE EARLY
IN -- WE TRIED THERAPEUTIC
VACCINATION IN THE SIV MODEL
WITH -- AND THAT -- THIS REALLY
IS THE MODEL OF THIS EARLY
INTERCEPT.
SO IN ADDITION TO SIV, SHOWN IN
OTHER PATHOGEN MODELS, THERE WAS
A PAPER FROM -- AGAINST' BOW LA
VIRUS, THERE IS -- WE'VE SHOWN
IT PROTECTS AGAINST TB AND THEN
THERE'S SOME INDICATION IT MIGHT
WORK AGAINST MALARIA ALSO IN
THAT IT REDUCES THE LIVER STAGE
OF MALARIA PARASITES AND WORKS
AGAINST VIRUSES, BACTERIA AND
PARASITES.
SO I STARTED TO WORK WITH LEWIS
BECAUSE OF THESE DATA.
SO I WAS INTRIGUED BY THE FACT
THAT HE WAS USING RHESUS CMV IN
ANIMALS THAT ARE ALREADY CME
POSITIVE AND FOR ADENOVECTORS
AND SO ON, THIS IS A HUGE
PROBLEM WITH ASSOCIATED VECTORS.
WE CAN GO INTO -- AT ANY DOSE
AND WITH RESPECT TO THE T-CELL
RESPONSE, THERE'S NO DIFFERENCE.
EVEN AT VERY LOW DOSES IN CME
POSITIVE ANIMALS WE GET THE SAME
LEVEL OF RESPONSE T-CELL
RESPONSES AS WE DO WITH HIGHER
DOSE.
WE CAN USE THE SAME VECTORS OVER
AND OVER AGAIN IN INDUCING
IMMUNE RESPONSE TO NEW ANTIGENS
THAT WE INSERTED INTO THESE
ANIMALS.
SO LEWIS AND I TEAMED UP AND
WHAT WE'VE SHOWN IS THAT IT'S
THE ABILITY OF CMV TO
DOWNREGULATE CLASS ONE MOLECULES
AND BASICALLY HIDE FROM THE T
CELLS THAT USES -- THAT ENABLES
TO BE INDUCED, TO BE USED IN CMV
POSITIVE ANIMALS.
IF YOU KNOCK OUT THOSE DOWN
REGULATING GENES FROM CMV IT NO
LONGER ABLE TO DO THAT.
SO -- BOTH IN USING BUT ALSO
RELATING THE T-CELL RESPONSES IT
THEN USES.
SO THIS WORK THEN LED US TO
SOME -- WE FOUND THAT THE
EPITOPE SPECIFICITY WAS
DIFFERENT IN THESE ANIMALS, DOWN
DIAGNOSE REGULATING GENES, SO WE
STARTED MAPPING THE EPITOPES AND
C MOLECULES THAT REPRESENT THESE
EPITOPES AND THAT -- INTO A VERY
UNEXPECTED AND VERY INTERESTING
STORY.
BECAUSE WHAT WE FIND IS THAT THE
CD8 T-CELL RESPONSES INDUCED BY
THE RHESUS CMV VECTORS, THEY'RE
NOT RESTRICTED BY MHC CLASS 1.
SO THE PEPTIDES PRESENTED TO DCE
THAT WE'VE EVER HEARD UP,
THEY'RE NOT RECOGNIZED, DO NOT
RECOGNIZE THE PEPTIDES IN THE
CONTEXT OF MHC CLASS 1.
INSTEAD IN THE CONTEXT OF MHC IT
OR IN THE CONMHC2 OR A -- THE WW
THIS IS THESE ARE EACH
INDIVIDUAL ANIMALS.
EACH OF THESE SQUARES INDICATES
PEPTIDES THAT GIVE BACKGROUND
RESPONSE IN THESE ANIMALS, THEN
YOU CAN BLOCK THOSE T-CELL
RESPONSES WHICH ARE -- STAINING,
USING ANTIBODIES OR IN THE CASE
OF -- A PEPTIDE THAT BINDS TO
HMCE AND SHOWS THIS SPECIFICALLY
BLOCKS THOSE RESPONSES.
SO VERY SURPRISING FINDING.
SOME OF THE PEPTIDES, THEY GIVE
RESPONSES IN EVERY SINGLE ANIMAL
THAT WE EVER TESTED.
THAT'S NOT SO SURPRISING FOR
HMC.
BECAUSE IT'S A HIGHLY CONSERVED
MOLECULE, BUT MHC2, THESE ARE
ANIMALS THAT HAVE DIFFERENT MHCT
QUITE UNDERSTAND THE
BIOMECHANISMS FOR THIS, WE CAN
EASILY TEST ONE OF THESE --
WHETHER THESE RESPONSES ARE
THERE OR NOT LOOKING
SPECIFICALLY FOR THESE
SUPERTOPES.
SO SURPRISINGLY -- SO WHY HAS
THIS NEVER BEEN SEEN BEFORE IN
PEOPLE CME POSITIVE, THE T CELLS
RESPONSES ARE MHC1 RESTRICT.
THE SAME IS TRUE FOR MONKEYS --
INFECTED.
THE IMMUNE RESPONSE IN MONKEYS
NATURALLY INFECTED WITH CMV
AGAINST CMV PROTEINS, THEY ARE
CLASSICALLY RESTRICTED AS SHOWN
HERE IN RED.
IT'S ONLY THE ONES THAT GOT OUR
VECTORS THAT WERE -- RESTRICTED
EVEN TO THE CME PROTEINS AND
WHEN YOU GO INTO ANIMALS THAT
ARE CME POSITIVE WE HAVE ALL
THREE KINDS OF RESTRICTIONS.
SO WHAT'S GOING ON HERE?
THE SOLUTION WAS THAT WE USE
JUST BY SHEER COINCIDENCE THE
ONLY REI REI RE -- IT WAS ISOLAN
1968, 68-1, AND THAT VECTOR
LACKS TWO GENE REGIONS THAT ARE
PRESENT IN THE -- CMV, YOU HAVE
TO THIS FLIFOVER OF THIS GENE
REGION AND DELETION OF THESE TWO
GENE REGIONS AND IT TURNS OUT
BOTH OF THOSE REGIONS NEED TO BE
DELETED AND WE GET THESE
UNCONVENTIONAL T-CELL RESPONSES,
IF EITHER ONE OF THEM IS
REPAIRED, WE GET CONVENTIONAL
MHC CLASS 1 RESTRICTED CD8 T
CELLS.
SO THESE THREE REGIONS CONTAIN
CHEMOKINES, TO BE HONEST WE
DON'T KNOW HOW THEY PREVENT THE
CONDUCTION OF THESE
UNCONVENTIONAL RESPONSE BUT
THAT'S WHAT THEY DO.
THE OTHER TWO GENES ARE
HOMOLOGUES AND THEY ARE VERY
WELL-KNOWN ALTHOUGH THE
CHEMOKINE HOMOLOGUES, THEIR
FUNCTION IS TO BE PART OF A
RECEPTOR OF A PENT -- OMERE
COMPLEX.
SO -- CONTAIN THESE CHEMOKINE
GENES THAT PREVENT THE
UNCONVENTIONAL RESPONSES FROM
HAPPENING.
SO RHESUS AND HUMAN CMV HAS
HOMOLOGUES, SO THESE VIRUSES GO
AT GREAT LENGTHS TO PREVENT
THOSE RESPONSES FROM HAPPENING.
WE JUST ST STUMBLED ACROSS THEM.
THIS IS ALSO VERY
SPECIES-SPECIFIC.
WE JUST PUBLISHED A STUDY USING
PSEUDOMONAS MONKEYS AND WE
SHOWED SU PSEUDOMONAS MONKEY CMS
ALSO PROTECTED IN -- MACAQUE BUT
IT'S HIGHLY SPECIES-SPECIFIC.
SO IF WE HAVE RHESUS CMV AND
RHESUS -- WE GET THE
UNCONVENTIONAL RESPONSES.
IF WE PUT RHESUS CMV IN
PSEUDOMONAS MONKEYS, WE DON'T
GET THOSE UNCONVENTIONAL
RESPONSES.
-- DELETE THOSE TWO GENE
REGIONS, WE GET THE UNCONGRESS
UNCONVENTIONAL RESPONSES, IF YOU
PUT IT IN RHESUS, WE DON'T GET
IT, SO WE'RE CURRENTLY TRYING TO
FIGURE OUT IF THERE ARE --
SPECIES SPECIFICITY.
I'M THINKING ABOUT HUMAN CMV,
THAT ALSO MEANS WE CAN'T USE
RHESUS CMV, WE HAVE TO USE HUMAN
CMV TO GET THOSE UNCONVENTIONAL
RESPONSES.
YOU ALREADY KNOW IF WE PUT HUMAN
CMV IN MONKEYS, THEY ARE
CONVENTIONAL CD8 T-CELL
RESPONSES.
SO -- HAS HOMOLOGUES AND EACH
ONE OF THEM WHEN WE PUT THAT
INTO RHESUS CMV, IT ALSO ALSO
PREVENTS THE INDUCTION OF THESE
RESPONSES.
-- NOT TO SAME VIRUS AND CMV
HAS 250 GENES AS SOME OF THEM
ARE HOMOLOGOUS, SOME ARE NOT.
SO WE WERE WORRIED THAT THERE
ARE ADDITIONAL INHIBITORS IN
HUMAN CMV THAT ARE NOT CONSERVED
IN THE RHESUS CMV.
SO WHAT WE DID THEN IS WE
BASICALLY CLONED OUT -- WE
INSERTED ALL THE NON-C NON-CONSD
INTO RHESUS -- AND THEN TESTED
WHETHER WE GOT THOSE SUPERTOPE
RESPONSES.
INDEED THERE WAS ONE GENE CALLED
UL18 WHICH INHIBITED BOTH THE
MHC CLASS 1 -- AND THE -- SUP
SUPERTOPE RESPONSES.
IT'S BEEN STUDIED, CRYSTALLIZED
BECAUSE IT'S AN MHC CLASS
1 HOMOLOGUE THAT HUMAN CMV HAS
BUT THE RHESUS CMV DOES NOT HAVE
IT.
AND IT ASSOCIATES WITH -- AND IT
EVEN ASSOCIATES WITH PEPTIDE.
BUT IT DOESN'T PRESENT PEPTIDE
TO T CELLS BECAUSE IT'S HIGHLY
GLYCOSYLATED ON TOP OF THE
BINDING GROOVE, BUT UL18 IS,
IT'S A HIGHLY -- INHIBITOR
RECEPTOR AND IT BINDS TO -- WITH
A MUCH HIGHER AFFINITY THAN HOST
MHC MOLECULES SO IT'S REALLY
SENDING AN INHIBITORY SIGNAL --
EXPRESSED -- BUT ALSO ON T CEL
CELLS.
ZOUH1 EXPRESSES UL18 BECAUSE WE
GET CONVENTIONAL -- RESTRICTED
T-CELL RESPONSES AND ALSO SHOW
THIS DEPENDS ON THIS INTERACTION
WITH LEA1.
INTERESTINGLY, IT'S HIGHLY
EXPRESSED IN CME-SPECIFIC
T-CELLS THAT'S BEEN SHOWN IN
HUMANS AS WELL.
SO THE WAY YOU SHOWED THIS
BASICALLY POINT MUTATION NO
LONGER BINDS TO UL1 AND LOSE
THIS ABILITY TO INHIBIT THESE
RESPONSES.
SO THESE STUDIES DEMONSTRATE WE
HAVE A WHOLE RANGE OF GENES THAT
PREVENT INDUCTION OF THESE
UNCONVENTIONAL CD8 T-CELL
RESPONSES.
BUT WHY ARE THEY IN THE FIRST
PLACE, WHAT IS THE MECHANISM
THAT ENABLES THEM TO INDUCE
THESE RESPONSES AND NO OTHER
VECTOR HAS BEEN FOUND TO BE ABLE
TO DO THAT.
SO START WITH MHCE.
MHCE IS A CLASS 1 MOLECULE THAT
BINDS TO A PEPTIDE THAT IS IN
THE SINGLE PEPTIDE OF OTHER, OF
THE POLYMORPHIC CLASS
1 MOLECULES, AND OF COURSE THE
SINGLE PEPTIDE IS CLIPPED OFF IN
TRANSLOCATION AND LOADED ON TO
MHCE OR U -- TRANSPORTED TO THE
CELL SURFACE AND THIS PEPTIDE
THAT IS PRESENT IN ALL THE OTHER
CLASS 1 MOLECULES TOGETHER WITH
HLE BASICALLY INHIBITORY SIGNAL
FOR INHIBITORY AND K CELL
RECEPTOR CAN ALSO ACTIVATE IN
HUMANS WHO ARE -- POSITIVE OR
WELL-KNOWN TO HAVE AN EXPANSION
OF NK CELLS ACTIVATING -- AND
THIS IS PROBABLY ONE OF THE
REASONS WHY.
SO INTERESTINGLY, I TOLD YOU
THAT CMV IS VERY GOOD AT
ELIMINATING CLASS 1 MOLECULES SO
IT ELIMINATES THE NATURAL LIGAND
OF HLAE, AND THAT WOULD CREATE A
PROBLEM BECAUSE NO LONGER YOU
ARE INHIBITING THE NK CELLS.
BUT THOSE -- AND RHESUS CMV
ENCODE A GENE THAT HAS A DEPLOY
LOADED ON TO HLE BECAUSE IT ALSO
BLOCKS TAP AND THAT SERVES AS AN
INHIBITORY SIGNAL TO NK CELLS.
SO THE QUESTION WAS IS THIS IN
ANY WAY INVOLVED IN THE
INDUCTION OF THESE -- RESTRICTED
T CELLS AND THE ANSWER WAS YES.
POINT MUTATION -- SEQUENCE OF
THE VIRUS, WHEN WE GO INTO
MONKEYS NOW WITH THIS MODIFIED
VECTORS, WE LOSE ALL THE MHCE
RESPONSES AND NOW THE T-CELL
RESPONSES, THE CD8 T-CELL ROW
SPONSES ARE ALL RESTRICTED BY
MHT CLASS 2.
SO NOW WE HAVE A VECTOR THAT
MAKES EXCLUSIVELY MHC TO
RESTRICTED T CELLS, BOTH THE
CD4s AND THE CD8s.
THE CD4s ARE NOT AFFECTED AT
ALL BY ALL OF THE -- BY ALL OF
THIS T-CELL PROGRAMMING.
AND -- CAN REPLACE THE RHESUS --
BECAUSE IT HAS AN INTACT -- --
RESTRICTED T-CELL RESPONSES
BACK.
SO THAT WAS SOMEWHAT SURPRISING
BECAUSE THE WAY WE SHOW THAT OUR
IMMUNE RESPONSES ARE H LAE
RESTRICTED IS TO ADD THE
PEPTIDE -- CLASS.
SO WHY WOULD YOU NEED THE --
LINE PEPTIDE TO GET THE
RESPONSES TO NON- -- ALIGNED
SEQUENCES.
SO WHAT WE THINK IS HAPPENING IS
THAT THE VIRUS LOADS THIS VL9 ON
TO HLAE AND THAT DRIVES TO THE
CELL SURFACE BUT EVENTUALLY THIS
HAS TO BE EXCHANGED WITH OTHER
PEPTIDES WHICH ARE THEN
RECOGNIZED BY THE T CELLS.
BUT AT THIS POINT, SPECULATION,
WE'RE WORKING ON IT BUT WE DON'T
HAVE ACTUALLY SHOWN THIS.
THE KIND OF PEPTIDE EXCHANGE.
SO THEN WE ALSO LOOKED AT DOING
A BROAD SCREEN OF ANY OTHER
VIRAL GENE PRODUCTS THAT WE NEED
TO GET THOSE UNCONVENTIONAL
T-CELL RESPONSES.
SO WE DELETED LARGE CHUNKS OF
THE GENOME THAT ARE
NON-ESSENTIAL FOR GROWTH IN
VITRO, AND WE DID FIND ONE GENE
REGION WHICH IS A HOMOLOGUE OF
THE VIRAL T -- RESP RECEPTOR CAD
US28.
SO RHESUS -- IS FIVE HOMOLOGUES
AND TWO OF THOSE ARE REQUIRED TO
GET THE IMAGE -- RESTRICTED T
CELLS, SO THE PHENOTYPE OF THOSE
VIRUSES LOOKS EXACTLY LIKE THE
ONE I JUST SHOWED YOU, WITH ONLY
TWO RESPONSES.
WHAT WE HAVEN'T FOUND YET IS ANY
GENE PRODUCT THAT IS REQUIRED TO
GET THE MHC2 RESTRICTED
CD8 T-CELL RESPONSES SO WE DON'T
KNOW HOW AND WHY THOSE ARE BEING
INDUCED BY RHESUS CME.
BUT WHAT IT DOES INDICATE IS THE
FACT THAT YOU NEED THE
EXPRESSION OF VIRAL GENES TO GET
THOSE RESPONSES, THESE RESPONSES
HAVE TO BE PRIMED BY INFECTED
CELLS.
SO THEN THE NEXT QUESTION WAS
WHAT TYPES OF CELLS NEED TO BE
INFECTED TO GET THOSE TYPES OF
UNCOUNCONVENTIONAL T-CELL RESPO.
THE MHC1 RESPONSES THAT YOU
USUALLY GET AT ALL WAS CMV
POSITIVE MAKE, THEY'RE USUALLY
PRIMED NOT BY DIRECT
PRESENTATION BUT INDIRECT BY
CROSS PRESENTATION.
SO TO TEST THAT QUESTION, THEY
CAME UP WITH THIS CLEVER
STRATEGY THAT INTRODUCED
TARGETING SITE OF TISSUE OR
CELL-SPECIFIC MICRORNAs INTO
ESSENTIAL GENES OF EITHER -- OF
TRANSCRIPTION FACTORS, WE NEED
TWO OF THEM -- AND THE OTHER FOR
THE LATE GENE TRANSCRIPTION, AND
THEN IN CELLS THAT EXPRESS THOSE
MICRORNAs, THIS VIRUS CANNOT
REPLICATE.
BUT IN OTHER CELLS DON'T EXPRESS
THESE MICRORNAs, WHICH TARGET
THOSE ESSENTIAL GENES, THERE IS
REPLICATION, THIS IS SHOWING FOR
ENDOTHELIAL CELLS, NO GROWTH IN
ENDOTHELIAL CELLS BUT NORMAL
GROWTH IN MY B FIBROBLASTS.
IT TURNS OUT THIS WAS ACTUALLY
QUITE STRIKING IN THAT IF WE
TARGET -- IF WE INSERT
MICRORNAs THAT ARE
SPECIFICALLY EXPRESSED IN
MYELOID CELLS -- 142, WE LOSE
ALL THE T-CELL RESPONSES TO ALL
THE RESPONSES TO -- RESTRICTED,
WHICH MEANS TO GET THE IMAGE
TO -- RESPONSES WE NEED
INFECTION OF -- AND THIS, BY THE
WAY, MIGHT BE THE REASON WHY WE
NEED THESE GCPRs BECAUSE THEY
ARE IMPORTANT FOR LATENCY AND
REACTIVATION IN MYELOID CELLS.
WHAT WAS FASCINATING WAS THE
FLIP SIDE.
WHEN WE INTRODUCED ENDOTHELIAL
CELL MICRORNA TARGETING SITES WE
LOST ALL THE MHC2 RESPONSES AND
WE HAVE -- SO MHC2 RESPONSES
MUST BE PRIMED BY ENDOTHELIAL
CELLS INFECTED BY CMV,
ENDOTHELIAL CELLS ARE USUALLY
NOT EXPECTED TO INDUCE
MHC2 RESTRICTED RESPONSE BUT
KNOWN TO REGULATE UPON INFECTION
QUITE WELL ACTUALLY.
AND AS WE ALL RECEIVED -- SO IN
THIS CASE WHEN WE BLOCK
REPLICATION IN BOTH THE MYELOID
LINEAGE AND ENDOTHELIAL LINEAGE,
NOW WE'RE REVERTING BACK TO
MHC1A RESTRICT RESPONSES, WE
NEVER HAVE A VECTOR THAT DOESN'T
INDUCE CD8 T CELLS, SO THIS IS
ALWAYS ONE OF THE DEFAULT
PATHWAY AND THAT HAPPENS BY
CROSS PRESENTATION.
WHAT WE DON'T UNDERSTAND IS WHY
DID WE LOSE THESE RESPONSES WHEN
WE GET THE OTHER ONES?
WHY ARE THEY NOT ALWAYS THERE?
SO THERE MUST BE A MECHANISM FOR
DIRECT PRIMING.
IN A WAY WE CAN NOW -- WE HAVE
THIS WHOLE ZOO OF VECTORS, WE
CAN PRIME ALL OF THESE DIFFERENT
KINDS OF CD8 T CELLS, US11, IF
WE DELETE THAT GENE, YOU CAN
ALWAYS ADD MHC1A RESPONSES TO
IMMUNODOMINANT PEPTIDES TO ANY
OTHER VECTOR BACKBONE.
AND THIS ALLOWS US NOW TO
TEST -- THIS IS SO COOL IN
IMMUNOLOGY, ANY OF THOSE
PATHOGEN CHALLENGE EXPERIMENTS.
TO MAKE A VERY LONG STORY SHORT
IS FOR SIV, WE FIND THAT WE
COMPLETELY LOSE PROTECTION
AGAINST -- 9 IF WE DON'T HAVE
MHC-RESTRICTED T CELLS.
EITHER WE HAVE MHC-ONLY
VECTORS -- AND IN EACH CASE WE
HAVE PROTECTION IN ANY OF THE
CASES WHERE WE DON'T HAVE THE
MHC RESPONSES, WE DON'T HAVE
PROTECTION.
AND THE PROTECTION IS ALWAYS
THIS SORT OF BINARY PROTECTION,
HALF OF THE ANIMALS ARE
PROTECTED, THE OTHER HALF --
FROM EXPERIMENT TO EXPERIMENT
BUT NONE OF THESE VECTORS WE HAD
HIGH HOPES FOR THEIR MHC ONLY
THAT WILL PROTECT BETTER BUT IT
DOESN'T.
SO THAT IS A DIFFERENT REASON.
SO WHAT IT MEANS IS THAT THE
MHCE RESPONSES ARE QUIET BUT NOT
SUFFICIENT FOR PROTECTION IN
THESE CELLS.
BUT THEY HELP TO CONTROL SIV
INFECTION.
SO THE LAST COUPLE OF MINUTES,
SINCE THIS IS NCI, I THOUGHT I'D
TALK A LITTLE BIT ABOUT CANCER.
SO MHCE OR HLAE IN HUMANS IS
ACTUALLY A REALLY INTERESTING
TARGET FOR CANCER.
AND IT IS ALREADY USED AS A
CHECKPOINT BECAUSE MANY --
NORMAL CELLS DON'T EXPRESS A LOT
OF MHCE BUT IN CANCER CELLS YOU
OFTEN SEE IT BEING UPREGULATED.
IT TURNS OUT -- T CELLS OFTEN
EXPRESS A LOT OF -- CURRENTLY IN
CLINICAL TRIALS TO TRY TO BLOCK
THIS PATHWAY, THESE ARE
ANTIBODIES THAT TARGET -- AND
THAT'S BEEN NICE -- DATA SHOWING
ENHANCED EFFICACY OF VACCINES.
THEY PROBABLY DON'T WORK BY
THEMSELVES LIKE OTHER CHECKPOINT
INHIBITORS BUT THEY MAKE T CELLS
MORE EFFECTIVE -- T CELLS.
SO WE ASKED THE QUESTION, CAN WE
ELICIT -- AND THEN -- RESTRICTED
T CELLS TO RECOGNIZE TUMOR
CELLS.
AND THE ANSWER IS YES, WE GET
THE SAME TYPE OF UNCONVENTIONAL
T-CELL RESPONSE IF WE USE --
VECTORS TO VERY WELL DESCRIBE
TUMOR ANTIGENS LIKE -- OR -- AND
THEY HAVE THE SAME PHENOTYPE,
SAME FREQUENCY, LONGEVITY, ET
CETERA.
THE ONE DIFFERENCE WAS THESE
CANONICAL T CELLS THAT WE CANNOT
INDUCE THEM AGAINST CANCER
ANTIGENS BECAUSE THEY ARE
SUBJECT TO CENTRAL TOLERANCE.
SO WE GET A VERY NICE IMMUNE
RESPONSE, AND THEY DO RECOGNIZE
CANCER CELLS, SO THIS IS JUST
ONE PIECE OF DATA, THIS IS A
COLLABORATION WITH MOUNT SINAI
WHERE WE BASICALLY TOOK PROSTATE
CANCER CELLS, PUT THEM IN TISSUE
CULTURE, EXPOSED THEM TO MONKEYS
IMMUNIZED AGAINST PROSTATE
CANCER ANTIGEN AND SHOW THOSE T
CELLS GET STIMULATED BY THESE
PROSTATE CANCER CELLS AND THIS
IS MHCE RESTRICTED BECAUSE WE
CAN BLOCK IT WITH THE
VL9 PEPTIDE.
CONCLUSIONS AND QUESTIONS.
CME VECTORS IS REALLY THE FIRST
VACCINE MODALITY THAT CAN ELICIT
THESE UNCONVENTIONAL T-CELL
RESPONSES AND THEY'RE VERY
DISTINCT DEPENDING ON THE VECTOR
PHENOTYPE.
WE IDENTIFIED SOME OF THE VIRAL
GENOME PRODUCTS THAT ARE
RESPONSIBLE FOR THIS, BOTH
POSITIVE AND NEGATIVE REGULA
REGULATORS, AND MOST OF THEM ARE
CONSERVED BETWEEN HUMAN AND
RHESUS CMV BUT WE STILL DON'T
FULLY UNDERSTAND WHY CMV ARE
USING THOSE RESPONSES.
IT'S ALSO NOT CLEAR WHY SIV IS
SO SUSCEPTIBLE TO THESE
RESPONSES.
WE DINES NORMAL T-CELL
RESPONSES, WE HAVE NO
PROTECTION.
SO MAYBE IT'S SIV HASN'T LEARNED
RESPONSES AS WELL AS OTHER
RESPONSES BUT WE SIMPLY DON'T
KNOW SO WE'RE WORKING ON IT.
SO THIS STRICT DEPENDENCE ON
MHC-RESTRICTED T CELLS WEEFN
ONLY SEEN SO FAR FOR SIV.
SO IN TB, FOR INSTANCE, IT WORKS
BOTH WITH -- AND RESTRICTED T
CELLS, SAME FOR FLU.
SO WHAT WILL THESE STUDIES SHOW,
IT SEEMS MHCE, IN ADDITION TO
PRESENTING THIS PL9 PEPTIDE,
THERE'S ALWAYS BACKGROUND OF
OTHER PEPTIDES BEING PRESENTED
BY IMAGE -- THIS IS PROBABLY NOT
A DEDICATED MECHANISM, IT'S JUST
THE VL9 PEPTIDE GETS LOST AT
SOME POINT AND UNLIKE OTHER
CLASS 1 MOLECULES, MHCE IS QUITE
STABLE WITHOUT PEPTIDE AND
PROBABLY THAT'S WHY IT CAN PICK
UP SOME OTHER PEPTIDES EVEN LOW
AFFINITY ONES AND THEY CAN BE
TARGETED BY T CELLS BUT THEY'RE
NOT GOOD ENOUGH IN TERMS OF
BINDING AND HIGH AFFINITY AND
SURFACE FREQUENCY TO INDUCE
THOSE T CELLS.
SO THERE ARE SOME PARTICULAR
BACTERIAL PATHOGENS -- BUT
GENERALLY THEY'RE NOT INDUCED.
SO WHAT IT ALSO SUGGESTS IS TO
USE AN HCMV-BASED HIV VACCINE,
THE GOAL HAS TO BE TO ELICIT
HLE-RESTRICTED T-CELL RESPONSES
AND WE JUST STARTED THE CLINICAL
TRIAL WITH A VECTOR THAT LACKS
ALL THE GENES WE'VE IDENTIFIED
AS INHIBITORS INCLUDING UL18
AND -- LIF FUSION PROTEIN THAT'S
OPTIMIZED FOR T-CELL EPITOPE
COVERAGE.
THIS IS DONE BY THE COMPANY WE
STARTED A COUPLE YEARS AGO
FOR -- TECHNOLOGY, AND FOUNDED
BY THE -- FUNDED BY THE BILL AND
MELINDA GATES FOUNDATION AS
WELL -- WITH -- CLINICAL TRIALS.
SO THIS IS, OF COURSE, WORK OF
MANY PEOPLE OVER MANY YEARS, I
JUST WANT TO POINT OUT -- WHO'S
BEEN MAKING ALL THESE HUNDREDS,
THOUSANDS, ACTUALLY, OF
DIFFERENT VECTOR CONSTRUCTS,
SCOTT HANSEN IS RUNNING ALL THE
I MU NOLG EXPERIMENTS IN MONKEYS
AND MANY COLLABORATORS WHO HELP
US WITH THIS AND LOTS OF OUTSIDE
COLLABORATORS AS I MENTIONED,
STARTING WITH MOUNT SINAI, LEW'S
WORK WAS DONE WITH A GROUP AT
PITTSBURGH, AND THEN ALSO
WORKING, OF COURSE, HERE WITH
JEFF LITZEN, A LONG-TIME
COLLABORATOR, WE HAVE A GRANT TO
ACTUALLY GET TO THE QUESTION WHY
HLE RESPONSES ARE SO GOOD AT
CONTROLLING AND -- SO THANK YOU
VERY MUCH.
[APPLAUSE]
>> THANKS VERY MUCH, CLAUSE.
>> SO YOU RAISED THE ISSUE, I
REMEMBER FROM THE FIRST
PRESENTATION OF THIS, I DON'T
KNOW WHEN IT WAS 15 YEARS AGO OR
SOMETHING, AND THAT IS WHY --
THE ANIMALS.
ONE MIGHT IMAGINE THERE'S SOME
GENETIC DIFFERENCE, FOR EXAMPLE,
TO TRACK DOWN THE GENES.
THOUGHTS?
>> SO LOUIS AND MICHAEL --
TEAMED UP AND THEY DID BLOOD
TRANSCRIPTOMICS ON THESE
ANIMALS, AND IT SEEMS WITHIN THE
VERY FIRST COUPLE OF DAYS, THE
RESPONSE TO THE VACCINE IS
DIFFERENT.
IT'S NOT THE T-CELL RESPONSE,
NOT THE RESPONSES I TALKED
ABOUT, BUT IT'S THE INNATE
RESPONSE, PARTICULARLY IN IL15
SIGNATURE THAT IS SUSTAINED AND
HIGH IN THE ANIMALS THAT BOTH
WILL BE PROTECTED IN THE END
WHEREAS THE ONES THAT ARE NOT
PROTECTED, YOU DO NOT HAVE THIS
RESPONSE, AND IT SEEMS TO BE
PREDETERMINED EVEN BEFORE YOU
INJECT THE VACCINE, IT'S NOT THE
MAGNITUDE OF THE RESPONSE, IT'S
THE DELTA.
SO THE MONKEYS THAT HAVE LESS
IL15 RESPONSE AND THEN MAKE A
LOT OF IT, THOSE ARE THE ONES
THAT ARE PROTECTED.
SO WE'RE TRYING TO FIGURE OUT,
IL15 IS A VERY -- CYTOKINE
FOR -- AND K CELLS AND SO ON AND
TRY TO FIGURE OUT WHAT THAT
MEANS, BUT THAT CORRELATED QUITE
WELL WITH WHICH ANIMALS WILL BE
PROTECTED AND WHICH ANIMALS WILL
NOT BE PROTECTED.
>> SO HAVE THEY GONE DIGGING
THROUGH -- RESPONSE GENES AND --
INTERFERONS --
>> NOT YET.
ONE THING WE DID TR TRY IS WE DD
PUT IL15 INTO -- AND ACTUALLY WE
GOT PRETTY GOOD PROTECTION WITH
THOSE BUT IT WAS NOT 100%.
THERE WAS A LITTLE BIT OF A -- I
DON'T KNOW IF IT WAS
STATISTICALLY SIGNIFICANT.
>> I WANT TO JUST EXPRESS
CONFUSION ABOUT THE EDUCATION
OF -- RESPONSIVE T CELLS.
HOW IS THAT WORKING POSITIVE OR
NEGATIVE, OR AM I THINKING IN
THE RIGHT FRAMEWORK?
>> THESE ARE VERY WEIRD T CELLS.
SO ONE EXPERIMENT LOUIS DID
IS -- MONKEYS AND HE STILL GETS
THOSE RESPONSES.
WE ALSO HAVE IDENTIFIED SOME
T-CELL RECEPTORS THAT
CROSS-REACT WITH IMMUNODOMINANT
CLASSICALLY RESTRICTED MH
MHC1 PEPTIDES FROM RHESUS CMV.
SO WHAT WE THINK IS HAPPENING IS
THESE ARE NOT TWO PRIME EVENT,
IT'S NOT THAT YOU HAVE NAIVE T
CELLS THAT THEN GET PRIMED, BUT
IT'S A REPROGRAMMING.
ALSO A DE-PROGRAMMING OF THE
PRE-EXISTING MEMORY IN THE GIVEN
ANIMAL.
AND I'M SAYING DE-PROGRAMMING
BECAUSE WE ALSO FIND THAT WE GET
THESE VERY BROAD RESPONSES.
YOU NOTICE THERE'S HALF OF THE
PIP TIPEPTIDES SHOW UP POSITIVET
WE DON'T HAVE THE EQUIVALENT
BREADTH OF THE TCR USAGE.
SO FEW CLONAL TYPES GIVE YOU
THESE -- RESPONSES WHICH MEANS
EACH TCR IS HIGHLY REACTIVE EVEN
TO PEPTIDES THAT ARE NOT VERY
RELATED.
SO THAT'S WHY DE-PROGRAMMING, SO
I THINK -- AND WE DON'T KNOW HOW
IT WORKS.
SOMEHOW CMV ARE INDUCED IN THESE
T CELLS IN THE ABILITY TO
RESPOND TO THESE LOW AFFINITY
PEPTIDES PRESENTED BY MHCE AND
VERY TRANSIENTLY BECAUSE THEY
HAVE VERY LOW AFFINITY.
IT MIGHT EVEN BE -- IN THE
MOLECULE ITSELF THAN A TYPICAL
PEPTIDE MHC RECOGNITION.
SO THE TCRs ARE KRE CROSS
REACTIVE, THE T CELLS ARE VERY
CROSS REACTIVE, SO CMV IS THE
LOWEST THRESHOLD OF THESE T
CELLS TO BE ABLE TO RESPOND TO
THIS.
BUT AGAIN, IT'S ONE OF THOSE
AREAS WHERE WE'RE ACTIVELY
WORKING ON IT BUT IT'S BEEN VERY
DIFFICULT.
ALSO THAT'S WHAT WE'RE WORKING
ON WITH JEFF HERE.
IT'S BEEN REALLY HARD TO CLONE
ALL THESE TCRs AND THEN GET
THEM TO FIRE.
WHEN YOU PET THEM INTO JUST ANY
RANDOM T-CELL.
SO THAT ALSO TELLS US THERE IS
SOMETHING IN THE EPIGENETIC
LANDSCAPE OF THESE T CELLS THAT
CHANGES WHEN THEY BECOME
HLE-RESTRICTED.
>> GREAT.
>> I HAVE A VERY -- QUESTION.
I KNOW -- THE -- WILL ACTUALLY
DICTATE A NEW RESPONSE.
-- SO I JUST WONDER WHAT --
WHETHER YOU CONSIDER ALL OF
THESE IN THE NEXT WORK
ESPECIALLY THE -- OF THE
SUPERTOPE IN THAT NETWORK AND
HOW IT WILL CORRESPOND TO THE --
>> I'M NOT SURE I QUITE
UNDERSTAND YOUR QUESTION.
THE NETWORK IN THE HIV WORLD
MEANS THAT YOU HAVE THESE HIGHLY
CONSERVED EPITOPES OR THESE --
SOME ARE MORE IMPORTANT FOR THE
VIRUS THAN OTHERS THAT ARE LESS
IMPORTANT.
IF THAT'S WHAT YOU'RE GETTING
AT.
WHAT I CAN SAY IS, WHAT I JUST
SAID TO MIKE, EVERY TCR WE
IDENTIFIED SO FAR RESPONDS TO A
SUPERTOPE.
AND THEN TO SOME UNIQUE OTHER
EPITOPES THAT ONLY THIS TCR
RECOGNIZES IN A WAY.
BUT THE SUPERTOPES FOR SOME
REASON SEEM TO BE THE -- OF
THESE IMMUNE RESPONSES.
I DON'T KNOW IF THAT'S A NETWORK
IN THE CLASSICAL SENSE.
I THINK IT'S MORE OF A --
REACTIVITY VERY SENSITIVE TO A
GIVEN TCR.
AGAIN, WHY THOSE ARE SUPERTOPES,
IS IT THEY'RE MOST ABUNDANTLY
MADE, HIGH AFFINITY, WE DON'T
KNOW AT THIS POINT.
THERE'S NO OBVIOUS REASON.
ALSO THERE IS NO HOMOLOGY IN
MOST CASES BETWEEN ANY OF THOSE
PEPTIDES AND THE EL9 SEQUENCE
WHICH IS EVENTUALLY WHICH HAS
EVOLVED TO BIND.
SO THESE BIND SOME OF THEM WITH
AFFINITY TO MHC.
I DON'T KNOW IF THIS ANSWERS
YOUR QUESTION.
OTHERWISE WE'LL TALK LATER.
THANK YOU.
[APPLAUSE]
>> SO WE'RE GOING TO GO BACK IN
THE PROGRAM TO MICHAEL FARZAN.
MICHAEL DOES PH.D. RESEARCH
IMMUNOLOGY AT HARVARD MEDICAL
SCHOOL, JUST LIKE NANCY DID.
HE STAYED ON AT HARVARD,
EVENTUALLY SERVING AS PROFESSOR
IN THE DEPARTMENT OF
MICROBIOLOGY AND IMMUNOLOGY.
HE THEN SPENT A DECADE AS
CO-CHAIR AND THEN CHAIR OF THE
DEPARTMENT OF IMMUNOLOGY AND
MICROBIOLOGY AT SCRIPPS,
FLORIDA.
HE RETURNED TO HARVARD MEDICAL
SCHOOL EARLIER THIS YEAR, AS
DIRECTOR OF VIROLOGY RESEARCH AT
BOSTON CHILDREN'S.
HE'S WORKED EXTENSIVELY ON HIV
AND MORE RECENTLY ON SA SARS-CO.
[APPLAUSE]
>> SO THIS IS GOING TO BE --
THIS IS NEW WORK AND RELATIVELY
SIMPLER WORK SO EVERYBODY CAN
SORT OF RELAX A LITTLE BIT.
EVERYTHING I'M GOING TO EXPLAIN
IS ACTUALLY REASONABLY
UNDERSTANDABLE, I HOPE.
MAYBE EXCEPT FOR THE LAST PART,
BUT LET'S GO THROUGH THE EASY
PART 1ST OF ALL.
SO WHAT WE'RE DOING IS, WE'RE
EDITING PRIMARILY MURINE CELLS,
EDITING THEM IN SUCH A WAY,
VARIABLE CHANGE, THEY'RE NOW
TYPICALLY A HUMAN ANTIBODY,
BROADLY NEUTRALIZING ANTIBODY
AGAINST EITHER HIV OR SARS.
AND SO THE STRUCTURE OF THIS
TALK IS WHAT WE DO, WHY WE DO
IT, HOW WE DO IT, HARD WORK AND
WHAT'S NEXT.
I SHOULD MENTION THAT SOME OF
THESE SLIDES WERE A LITTLE
GEARED TOWARDS GRADUATE
STUDENTS, SO I'LL GO BY THEM
QUICKLY BUT I DON'T NEED TO
CONSULT ANYBODY, THERE'S A LOT
OF THINGS I'M SURE YOU
UNDERSTAND.
BUT THE BASIC GOAL HERE IS THAT
WE MIGHT FIND A B CELL RECEPTOR
AND WE DO SO BY EDITING IT WITH
CRISPR.
AGAIN YOU PUT IN THE HEAVY LIGHT
CHAIN -- WE GRAFT THOSE CELLS
BACK INTO MICE, AND THEN WE
MEASURE A BUNCH OF THINGS.
THE ACTIVITY THAT -- REPERTOIRE
BY DEEP SEQUENCING -- TOLERANCE,
BLAH, BLAH, BLAH.
AND SO WE DO THAT FOR A NUMBER
OF REASONS, BUT BEFORE I GET
THERE, I WANT TO SNEAK IN TWO
SORT OF TECHNICAL POINTS.
THE FIRST IS WHEN WE DO THIS IN
MICE, WE CAN DISTINGUISH BETWEEN
THE CELLS THAT WE EDITED AND THE
HOST BECAUSE THEY'RE MARKED
WITH -- OR TWO MARKERS, SO THE
ORIGIN MICE ARE CD4 -- .1 AND --
.2.
SO THAT SORT OF PINKISH BOX IN
THE CORNER IS REALLY OUR SECRET
SAUCE.
SO I'M GOING TO SAY WE DO THIS A
LITTLE BETTER THAN MOST PEOPLE
IN THIS FIELD BECAUSE WE HAVE A
COUPLE OF EFFICIENCY BONUSES.
ONE BONUS IS WE'RE NOT USING
CONVENTIONAL CAS9, WE'RE USING
CAS12A.
-- THAT WE DON'T OWN BUT SORT
OF DEVELOPED ITS USE.
AND THEN THE SECOND THING WE'RE
USING IS AAV, AND THIS IS KIND
OF THE HOOK INTO THE MEASURE OF
THESE -- WHICH IS WHEN ARE
VIRUSES USEFUL.
A, THESE EXTREMELY -- FOR A
NUMBER OF REASONS, BUT ONE OF
ITS BEST USES, IT SERVES AS A
WONDERFUL WAY TO DELIVER A
HOMOLOGY -- REPAIR TEMPLATE.
THAT'S THE ONE PLACE WHERE THE
VIRUS CAN BE ESPECIALLY USEFUL.
IN THIS TALK.
OKAY.
SO WHY ARE WE DOING THIS IN THE
FIRST PLACE?
WELL, THERE'S FOUR REASONS.
THE FIRST THING I'LL SPEND A
LITTLE TIME IS, WE'RE HARNESSING
A MAMMALIAN LYMPH NODE TO
DEVELOP ANTIBODIES, TO AFFINITY
MATURE ANTIBODIES.
OUR PREMISE IS THAT THE
EVOLUTION SELECTS MUCH BETTER
THAN PHAGE DISPLAY OR OTHER
TECHNIQUES FOR IMPROVING
ANTIBODIES IN VITRO AND SHOWS
SOME EVIDENCE FOR THAT.
IT'S MORE THEORY THAN IT IS
PROOF, BUT I SHOW SOMETHING IN
SUPPORT OF THAT.
THE SECOND THING IS WE WANT TO
CREATE MORE RELEVANT VACCINE
MODELS.
THIS IS ESPECIALLY IMPORTANT FOR
CLASS OF VACCINE -- THE HIV
FIELD WHICH RELIES ON A UNIQUE
PROPERTY OF THE KI DIVERSITY CHS
OF THE HUMAN REPERTOIRE, NOT
FOUND IN MONKEYS, NOT FOUND IN
MICE.
SO IN ORDER TO REPLICATE THAT
AND TO MAKE A MOUSE MODEL OF
THAT, WE HAVE TO ENGINEER THE B
CELLS TO THOSE -- CHAINS.
SOME DAY WE CAN THINK ABOUT CAR
B CELLS IN THE SAME MANNER WE
THINK OF CAR-T-CELLS, ALTHOUGH I
CHALLENGE YOU TO THINK OF THE
USE CASES IN CAR B CELLS.
THE ONE I CAN THINK OF
IMMEDIATELY IS SUPPRESSING THE
ESTABLISHED HIV INFECTION WITH
ANTIBODIES.
SO I THINK THIS IS THE POINT
WHERE I'M KIND OF INTRODUCING
EVERYBODY TO THINGS THEY KNOW,
JUST TO REMIND YOU AN ANTIBODY
IS AN SC REGION, VARIABLE
REGIONS.
WE'RE ONLY INTERESTED IN
MODIFYING THOSE VARIABLE
REGIONS -- THE ONES THAT CONTACT
ANTIGEN, OF COURSE, THEY HAVE
CDLs 1, 2 AND 3s.
THEY CAN BE SHORT OR LONG, AND
ESPECIALLY GENERALLY PROVIDES A
LOT OF BINDING AND -- MOST
DIVERSE ELEMENT OF ANTIBODIES.
SO WE'RE VERY FOCUSED ON THAT.
YOU CAN SEE IN PART WHY IT IS,
BECAUSE IT'S AT THE JUNCTION OF
THE V, D AND J REGIONS, AND ALL
THE -- DIVERSITY AND ALL THOSE
GOOD THINGS YOU LEARN IN YOUR
IMMUNOLOGY TEXT, THAT'S WHERE
MOST OF THAT HAPPENS OR A HUGE
AMOUNT OF DIVERSITY.
AND IT CAN THEY CAN BE SHORT AND
THEY CAN BE LONG.
THERE'S AN EXAMPLE OF THE LONG
OVER THERE.
IT'S AN APEX -- HIV NEUTRALIZING
ANTIBODIES AND THAT LONG -- VERY
IMPORTANT TO TALK ABOUT THE
SECOND PART OF THE TALK.
BASICALLY I WANTED TO REMIND YOU
WHAT OUR TROUBLE IS -- PRIMARY B
CELLS.
BIG PROBLEM IS IT'S A RELATIVELY
RANDOM PROCESS WHERE -- V, D AND
J RECOMBINES, WE COMBINE IN A
RANDOM WAY, CREATE -- THERE'S
OTHER FORMS OF RANDOMIZATION AND
DIVERSIFICATION, BUT WE DON'T
KNOW WHAT'S THERE.
SO WE HAVE TO TRY TO SOLVE THE
PROBLEM OF TRYING TO ADD
SOMETHING, WHICH WE DON'T KNOW
WHAT IT IS REALLY.
SO WHAT DOES THAT REALLY COME
DOWN TO?
THE HOMOLOGY ARM, SO HOMOLOGY
DIRECTED REPAIR TEMPLATE.
SOMETHING THAT GUIDES THE DNA
REPAIR PROCESS, IT'S WHAT'S
NORMALLY GOING ON IN YOUR BODY,
IS THAT WHEN THERE'S SAY A
DOUBLE STRANDED BREAK ON ONE
CHROMOSOME BUT LOOKS OVER TO THE
OTHER CHROMOSOME AND FIXES IT.
WE FOOL THE SYSTEM, FIX IT WITH
SOME DOUBLE STRANDED DNA THAT WE
OURSELVES PROVIDE AND AS I'LL
MENTION AAV IS A VERY GOOD WAY
TO DO THAT.
OUR PROBLEM IS THAT WE REQUIRE
SOMETHING THAT'S THERE AND AGAIN
WE DON'T KNOW WHAT'S THERE
ESPECIALLY ON THE -- PRIME END.
LET ME PAUSE FOR A SECOND AND
TELL YOU A SPECULATIVE REASON
WHY AAV IS PROBABLY A VERY GOOD
WAY TO EDIT THINGS, AND ALSO WHY
IT'S A LITTLE BIT OF A PROBLEM
WHEN WE THINK ABOUT AAV USING AS
PAIRP IN PARTICULARLY IN CELLS
THAT ARE NOT DIFFERENTIATED.
THAT REALLY SOOMS IN ZOOMS IN E
STRANDED BREAK.
A SPECULATIVE HYPOTHESIS I TELL
PEOPLE SO THEY REMEMBER IS THAT
THE AAV REPLICATION CYCLE ALSO
REQUIRES THE SAME REPAIR ENZYMES
TO GO THROUGH ITS PROCESS OF
SECOND STRAND SYNTHESIS,
CIRCLIZATION, ALL OF THESE
REQUIRE -- USED BY THE CELL, THE
PRESUMPTION IS THAT THE
COLOCALIZATION -- WHAT'S
BRINGING THE AAV EPSOME TO THE
DOUBLE STRANDED BREAK.
NOW YOU REMEMBER THAT AAV LIKES
TO GO TO A DOUBLE STRANDED BRE
BREAK.
WEAVES IN EXTENSIVELY, PART OF
THE SECRET SAUCE THAT GIVES US
EFFICIENCY TO ACCOMPLISH WHAT
WE'RE DOING.
ANYWAY, THAT WAS AN ASIDE STORY
BECAUSE OF THE NATURE OF THESE
TALKS, BUT THE MAIN STORY IS,
HOW DO WE SOLVE THE PROBLEM OF
NOT KNOWING WHAT'S THERE?
SO THE FIELD DOES IT BY IGNORING
THE DIVERSE REGION AND HITTING
WHERE THOSE BLUE ARROWS ARE
GOING, WHICH IS AN INTRON
BETWEEN THE LAST J REGION AND
THE FIRST REGION -- WHICH IT'S
TYPICALLY SPLICED INTO.
THAT COMES WITH PROBLEMS AS I
SHOW YOU.
DIFFERENT HARDER APPROACH IN
WHICH WE EDIT -- WE MAKE A
SINGLE CUT AND THEN REACH TO A
VERY DISTANT 5 PRIME REGION,
VARIABLE REGION, AND BRIDGE
THOSE TWO AND SURPRISINGLY IT
WORKS.
ONE THING TO SAY IS THAT WE
DON'T NEED A TON OF EFFICIENCY
BECAUSE WHAT ARE WE GOING TO DO?
WE'RE GOING TO PUT THE CELL BACK
INTO AN ANIMAL, YOU DON'T NEED
80% EFFICIENCY, YOU DON'T NEED
EVEN 10% EFFICIENCY.
WE THINK YOU JUST NEED SOMETHING
ON THE ORDER OF .8% EFFICIENCY.
THAT'S 15,000 CELLS -- SO
SOMETHING ON THAT ORDER.
SO THERE ARE SEVERAL WAYS TO
EDIT THE B CELL RECEPTOR.
THIS IS THE WAY THAT IT'S
COMMONLY DONE.
THIS IS SORT OF OUR EXAMPLE OF
HOW WE SHOULDN'T DO THAT.
I HAVE TO GIVE PROPS TO THE
PEOPLE THAT DEVELOPED IT.
I'M USING ESPECIALLY MICHELLE'S
MODEL HERE, BRILLIANT WORK, ET
CETERA, BUT I'M GOING TO USE IT
AS SORT OF A COUNTER EXAMPLE.
WE ARE GOING TO DO IT IN FAVOR
OF THE REPLACING THE GENES
DIRECTLY OVERRIDING THE ORIGINAL
MURINE GENES WITH THE HUMAN
VERSION OF THOSE GENES.
AND NOT MESSING WITH THE
REGULATORY MECHANISM THAT YOU DO
WHEN YOU USE THAT CONSTRUCT OF
THERE, AND I WON'T COUNT THROUGH
ALL THE WAYS THAT IS A NATURAL
CONSTRUCT THAT LEADS TO
DYSREGULATION OF THE B CELL, I
WILL JUST SHOW YOU THAT IT
DISREGULATES THE B CELL.
THERE'S A THIRD TRICK WE DO
MOSTLY FOR VACCINE, WHERE WE
OVERWRITE JUST THE CDR, AND WE
DO SO BY RELYING ON SMALL
CONSENSUS REGIONS IN THE
VARIABLE REGION AS WELL AS
CUTTING THE SAME J REGION WE
ALWAYS CUT.
WHAT WE'RE TRYING TO DO IS
REPLACE HIV NEUTRALIZING
ANTIBODIES -- THE VARIABLE GENES
OF MURINE B CELLS WITH THE
VARIABLE DOSE OF HIV
NEUTRALIZING AND BODIES.
ON THE LEFT, OVER HERE, YOU CAN
SEE WHAT WE'RE DOING IS WE'RE
REPLACING WHAT'S KNOWN AS
CAPTIVE 56 OR VR62625 APEX
ANTIBODY, AND THEN OVER HERE,
101074.
THE DIFFERENCE IN EFFICIENCIES
ARE PROBABLY TRACEABLE TO THE
FOLLOWING FACT.
VRC2625 ONLY REQUIRES HEAVY
CHAIN.
-- WE'RE GETTING ESSENTIALLY
HALF OF THE EFFICIENCY OF
101074.
IT'S JUST IMPORTANT TO REMEMBER
THAT A SUBSET OF THE VRC2625
CELLS ARE CARRYING THE MURINE
LIGHT CHAIN.
IT DOESN'T REALLY MATTER, WHEN
YOU ENGRAFT THOSE CELLS BACK IN
TO ANIMALS AND THEN -- ONE, TWO
AND THREE TIMES, YOU GET BETTER
AND BETTER NEUTRALIZING --
PASSING A BENCHMARK HERE OF
2-MICROGRAM PER ML, PROTECTIVE
IN A MONKEY MODEL.
SIMILAR, 101074, ONE
VACCINATION, TWO VACCINATION AND
THESE THREE MUST KIND OF SPLIT
UP TOURING THAT SECOND
VACCINATION.
THIS WAS A 20-MICROGRAM PER ML
DOPED IN TO THE SAME NAIVE SERA
SERVING AS OUR BENCHMARK HERE.
WHAT'S INTERESTING I THINK AND
WORTH NOTING IS THAT MICE --
IT'S KIND OF WELL-KNOWN, MOST
MICE DO NOT RAISE NEUTRALIZING
ANTIBODIES IF THERE ARE NO -- IF
WE DON'T ADD THESE MODIFIED B
CELLS INTO THEM.
SO THOSE ARE THE CONTROLS.
SAME NUMBER OF VACCINATIONS BUT
NO ENGRAFTED B CELLS.
THIS IS JUST TO MAKE THE SIMPLE
POINT THAT IF YOU DO IT THE
OTHER WAY, WHAT WE CALL THE
INTRON EDITED WAY, YOU PUT HEAVY
AND LIGHT CHAIN BOTH IN THAT
INTRON, YOU DISRUPT THE
MECHANISM -- THE UNDERLYING
MECHANISM AS INDICATED HERE IN
GREEN LINES BY THE FACT THAT THE
NEUTRALIZING RESPONSES ARE MUCH
LOWER WHEN YOU DO SO.
SAME NUMBER OF -- CELLS GOING
INTO THE MICE, MUCH LOWER
NEUTRALIZING SERA.
ANOTHER WAY TO LOOK AT THIS IS
LOOK AT THE RATES OF SOMATIC
HYPERMUTATION IN THESE MICE.
THE BLUE ARE A COUPLE MICE THAT
HAVE BEEN EDITED IN THIS OTHER
WAY, BY INTRODUCING THE INTRON.
SO WE'RE KIND OF KICKING A DEAD
HORSE HERE.
YOU ABSOLUTELY WANT TO HAVE YOUR
HEAVY LIGHT CHAIN PROPERLY
PLACED WHERE THE MURINE OR THE
OTHER ANIMALS HEAVY LIGHT CHAIN
ARE, DON'T DO IT THE OTHER WAY.
BUT YOU CAN SEE ONE OF THE
REASONS WHY IT'S NOT VERY
GOOD -- I THINK IT'S MORE OF AN
EFFECT THAN ANYTHING ELSE.
BUT RATES OF SOMATIC
HYPERMUTATION ARE POOR, AND AS A
CONSEQUENCE OF THAT, YOU DON'T
GET WITH INTRON -- BUT YOU DO
GET UNIQUELY WITH NATIVE --
AFFINITY MATURATION OF THESE B
CELLS.
SO WE LIKE THIS.
THIS IS A BIG MILESTONE FOR US.
AND THE WAY WE DID THIS, WE
PULLED OUT A BUNCH OF ACTUALLY
ONLY HEAVY CHAINS HERE.
MORE OR LESS RANDOM, AND THOSE
ARE SHOWN IN PINK AND RED, THE
ONES THAT WE PICKED OUT OF THESE
THREE MICE THAT ARE ENGRAFTED.
THESE ARE JUST INDIVIDUAL
SEQUENCES, AND THEN WE
CHARACTERIZED THEM AGAINST THE
WILD TYPE ANTIBODY.
THESE ARE THE CHAINS AGAINST THE
WILD TYPE HEAVY CHAIN ALL IN THE
CONTEXT OF THE WILD TYPE LIGHT
CHAIN, AND YOU CAN SEE THAT ALL
OF THESE OUTPERFORMED THE WILD
TYPE ANTIBODY, FOUR OF THESE
VERY CONSISTENTLY ACROSS ALL
ASSAYS WE USED.
THESE FOUR ALWAYS WORKED OUT AND
THOSE FOUR, WHEN IT CAME RIGHT
DOWN TO IT, THEY SHARED A COMMON
ELEMENT, TWO MUTATION IN THE
CDR3 AS IT TURNED OUT, AND THESE
WERE BASICALLY DOING ALL THE
WORK TO IMPROVE THE POTENCY OF
THESE ANTIBODIES.
I DON'T HAVE THE GLOBAL PANEL
BUT THERE'S ANOTHER FIGURE IN
THE PUBLISHED PAPER THAT SHOWS
THE GLOBAL PANEL.
THE REASON WE IN PART WANT TO DO
THIS IS WE KNOW VERY WELL THAT
WHEN YOU TRY TO IMPROVE AN
ANTIBODY USING A CELL CULTURE
SYSTEM, YOU USE MAMMALIAN -- OR
INTELLIGENT DESIGN OF SOME SORT,
YOU ALWAYS GET BETTER AFFINITY.
THAT'S ALL YOU GO.
AND ALMOST INVARIABLY, YOU
UNDERMINE OTHER ELEMENTS,
IMPORTANT ELEMENTS OF THESE
ANTIBODIES, INCLUDING ESPECIALLY
THE HALF-LIFE AND THE
BIOAVAILABILITY.
AND WE'VE KNOWN THAT, I REMEMBER
BEAUTIFUL WORK BY PUTTING THE W
TRYPTOPHAN RIGHT IN THE MIDDLE
OF THAT.
AND THAT JUST CRASHED AND BURNED
ON BIOAVAILABILITY.
THIS ANTIBODY HERE, THIS IS
ANOTHER EXAMPLE OF SOMEBODY
INTELLIGENTLY COMBINING 101074
WITH ANOTHER ANTIBODY -- WITH
ELEMENTS OF ANOTHER ANTIBODY
MAKING IT MORE POTENT BUT IT
TURNS OUT TO BE A WORSE
ANTIBODY.
SO 101074 WAS KIND OF A GOLD
STANDARD.
IT'S A LOVELY, BEAUTIFUL, BEST
BEHAVED ANTIBODY THAT I'VE EVER
WORKED WITH, AND IT'S KIND OF A
GO-TO, IT'S A GOLD STANDARD FOR
EVERYBODY WORKING WITH THESE
ANTIANTIBODIES.
WE DID A COUPLE OF ASSAYS.
ONE POLYREACTIVITY.
AS YOU CAN SEE WE DIDN'T HARM
THE POLYREACTIVITY BY
INTRODUCING THE CHANGES THAT WE
DID, WHEREAS THE INTELLIGENTLY
DESIGNED FORM OF THE SAME
ANTIBODY DID INDEED IN THIS
PARTICULAR CASE HARM THE
POLYREACTIVITY.
BY THE WAY, THIS IS BJORKMAN'S
ANTIBODY AND SORT OF ANOTHER BAD
ACTOR IN THE HIV FIELD.
SIMILARLY, WHAT HAPPENS WHEN YOU
SEE THIS MAJOR POLL
REREACTIVITY, WE DID MANAGE TO
SQUEEZE SOME -- I WON'T MAKE TOO
MUCH OF IT BUT BASICALLY NO HARM
DONE TO THE BIOAVAILABILITY WHEN
YOU'RE IMPROVING THE POTENCY OF
THE ANTIBODY.
SAME SOURCE.
SO WHAT WE DID WAS WE STARTED
WITH SOME ANTIBODIES, WUHAN
STRAIN, ANCESTRAL STRAIN AND DID
NOT WORK AGAINST A COUPLE OF
OMICRON, SO WE PUT THEM INTO
MICE, DROVE THEM WITH VARIOUS
OMICRON ISOLATES AND TRIED TO
IMPROVE THEM IN VARIOUS WAYS.
INTERESTING FACT IS OF COURSE
MICE DO MAKE GOOD ANTIBODIES
AGAINST SARS.
ONE OR THE OTHER.
SO OUR ABILITY TO SEE A
DIFFERENCE BETWEEN THE WILD TYPE
AND THE B CELL ENGRAFTED MICE
WAS NOT VERY STRONG.
IT DIDN'T MATTER.
WE PRAY PLAYED THE SAME GAME AG.
THIS IS JUST STRAIGHT OUT
AFFINITY OUT OF -- WE WERE IN A
HURRY SO WE JUST CHARACTERIZED
THAT GUY THERE, WHICH IS OUR
BEST BINDER OF THE SET OF
VARIANTS THAT WE TRIED.
WE GOT A 61 FOLD, THIS IS OUR
BEST -- INCREASE IN AFFINITY
AGAINST THE -- BUT IT ALSO
EXTENDED TO SOME OF THE OTHER
OMICRONS AS WELL.
WE GOT THIS INTERESTING ON ONE
OF THE OMICRONS, WE GOT A GAIN
OF NEUTRALIZATION, AND THEN BACK
TO THE ANCESTRAL, WHICH IS --
BA.5 IMPROVEMENT.
THAT COLLATED WITH AFFINITY, AND
THIS IS WHAT THAT CHANGE LOOKED
LIKE ACROSS A BUNCH OF THESE
DIFFERENT VIRUSES, AND IN
CONTRAST, THIS GUY OVER HERE,
AND IT DID GREAT AGAINST THE --
BUT OF COURSE IT'S NOT A
GUARANTEE HERE AND WE DIDN'T GET
THE SAME KIND OF BREADTH WE SAW
THE FIRST TIME.
VERY QUICKLY SAME STORY WITH
BIOLOGICS.
DON'T PUT IN AN ANTIBODY, PUT IN
A BIOLOGIC, WE HAD BEEN
INTERESTED IN THE CD4, WORKING
WITH A MOLECULE CALLED EDC4,
THIS IS A COMPANY I'M CO-FOUNDER
OF THAT DEVELOPS CD4.
AT THIS POINT WE'RE MAKING A
BETTER CD4 OUT OF THEIR CD4,
PLAYING THE EXACT SAME GAME WE
DID BEFORE, ONLY WITH AN
UNGAMELY CONSTRUCT, IT HAS TO DO
WITH THE PRESENCE OF THE
C1 BINDER OF THE LIGHT CHAIN.
AGAIN, THE AFFINITY MATURATION,
WE CHARACTERIZE BOTH SILENT AND
NON-SILENT MUTATION.
TWO VERY ROBUST MUTATIONS CAME
OUT AND ALL 10 OF THE MICE WE
INOCULATED, THIS WAS VERY
CONSISTENT, EVERY ONE OF THESE
MICE SHOWED UP, SHOWED UP
SOMEWHERE BETWEEN 30 AND 90% OF
ALL THE CLONES WE SEQUENCED, AND
THEN SOME OF THESE OTHER
MUTATIONS AS WELL AS -- THE
INTERESTING THING IS THE SILENT
MUTATIONS SHOWED UP IN WHAT ARE
CALLED AID HOT SPOTS, SO --
SITES OF MUTATION WHERE IT'S THE
NON-CODING MUTATIONS DID NOT
SHOW UP ON NID HOT SPOTS, IN
PARTICULAR, THESE TWO.
SO SOMEHOW OR ANOTHER, THE
IMMUNE SYSTEM HAD TO WORK HARDER
TO GET THERE.
LONG STORY SHORT, WE
CHARACTERIZE THESE MUTATIONS,
MANY SHOWED UP DIRECTLY IN THE
BINDING SITE.
WE DID DEVELOP A PANEL AND THIS
IS CONFUSING YOU HERE PAUSE THIS
OVER HERE IS ORIGINAL CD4,
THAT'S NOT WHAT WE STARTED WITH.
THIS IS THE CD4 WE STARTED WITH.
THIS IS AN IMPROVED CD4 WE'VE
BEEN WORKING WITH FOR SOME TIME,
AND THIS IS THE CONSEQUENCES OF
OUR VARIOUS CONSTRUCTS THAT HAD
THESE VARIOUS MUTATIONS AND
COMBINATIONS.
AGAIN, THIS MUTATION AND THIS
MUTATION ARE DOING ALL THE
LIFTING, REALLY.
AND THERE'S AN INTERESTING STORY
ABOUT THE FIRST ONE.
AGAIN SAME GAME, NO REAL CHANGE
IN POLYREACTIVITY.
TWO OUT OF THREE THAT WE
CHARACTERIZED IN VITRO RETAINED
THE HALF-LIFE OF OUR HALF-LIFE
IMPROVED CD4.
ONE DID NOT, AND THE ORIGINAL
CD4, WE KNEW TO BE A DISASTER.
SO THAT'S WHAT WE'VE DONE WITH
BIOLOGICS.
I'M GOING TO DO THIS VERY
QUICKLY NOW FOR VACCINES.
THOFS MORE DEVELOPED WORK.
THERE WILL BE SOME SPECULATION
HERE AND SOME PREACHING I THINK
TO SOME EXTENT.
SO THIS IS A GOOD PICTURE
PROVIDED BY DENNIS BURTON, WHO
GENERATED A LOT OF THE DATA
UNDERLYING THIS PICTURE, WHICH
IS THE MAIN NEUTRALIZING
EPITOPES, I WOULD PROBABLY ARGUE
THAT THE THREE MOST IMPORTANT
WERE THE CD4 BINDING SITE.
SURELY THE V3 GLYCAN, KIND OF A
SUBSTITUTE FOR THE CORECEPTOR IN
THE UNCD4 BOUND STATE AND THEN
THE APEX, AND WE CAN ARGUE ABOUT
WHETHER THESE ARE USEFUL OR
NON-USEFUL EPITOPES.
BUT WHAT I'M GOING TO TELL YOU
IS, WE'RE GOING TO FOCUS ON THE
APEX AND THE REASON WE'RE GOING
TO FOCUS ON THE APEX, THIS IS
PREACHING, THIS IS THE SHORTETH
PATH BETWEEN THE IMMUNE REP
REPERTOIRE, THE ANTIBODY
REPERTOIRE THAT'S IN EACH OF US,
AND REASONABLY GOOD PROTECTION
AGAINST THE VIRUS.
THAT'S PREACHING, THAT'S NOT
SCIENTIFICALLY STATING THAT.
BUT LET ME TELL YOU ABOUT THESE
ANTIBODIES AND TELL YOU WHY I
THINK THAT.
FIRST THING TO SAY IS THEY BIND
THE APEX REGION.
YOU ONLY NEED ONE TO BIND.
THEY BIND -- THEY INTERFACE AT
THE TOP.
LONG TYROSINE SULFATED H
CDR3s.
THEY'RE VERY POTENT, IN TIGHT
BECAUSE THEY NEUTRALIZE THE
ENTIRE SPIKE WITH ONE ANTIBODY
RATHER THAN MAYBE TWO OR THREE
FOR THE OTHER CLASSES OF
ANTIBODIES.
VERY LITTLE SOMATOHYPERMUTATION
IS NEEDED.
THAT'S IN STRONG CONTRAST, WHERE
A GOOD DEAL OF HEROIC CHANGES
ARE NECESSARY TO MAKE THESE
ANTIBODIES.
THESE ANTIBODIES ARE VERY
SIMILAR TO WHAT'S CIRCULATING IN
YOUR BODY RIGHT NOW.
SO LET ME -- THEY'RE THERE NOT
FOR THE RETHE EPITOPE ISRARELY D
INDIVIDUALS IN ITS WHOLE --
FORM.
SO THE GAIN IS TO FOCUS THE
IMMUNE SYSTEM IN ON THAT CLASS
OF ANTIBODIES.
SO NOW KNOWING THAT I'M SHORT OF
TIME, I'M GOING TO TELL YOU MY
PUNCH LINE FIRST.
THE SORT OF ORGANIZING
HYPOTHESIS THAT IS DRIVING OUR
CURRENT VACCINE WORK.
THE FIRST THING IS THAT WE
PUBLISHED RECENTLY THAT THE CDRS
SUFFICIENT TO DEVELOP A
PROTECTIVE RESPONSE.
SO THESE ANTIBODIES AND OF THEIR
PROGENITORS, THEIR PRESUMED
UNMUTATED PRECURSORS.
THIS MEANS TO ME THAT THE
PROBLEM WITH AN HIV VACCINE CAN
BE DISTILLED DOWN TO ELICITING
THESE 25 TO 30 AMINO ACID
SEQUENCES.
WE KNOW VERY MUCH ABOUT WHAT
THESE 25 TO 30 AMINO ACID
SEQUENCES SHOULD LOOK LIKE.
WE KNOW WHICH DIVERSITY CHAINS
THEY SHOULD COME FROM, THEY KNOW
HOW TO BE POSITIONED, WE KNOW
EXACTLY AND WE KNOW THEIR
FREQUENCY.
SO THIS IS GOOD, BECAUSE THESE
PRECURSORS AS WE DEFINED THEM UP
TO THIS POINT ARE VERY COMMON IN
US.
SECOND GOOD POINT.
CURRENT SOSIP TRANSAMERICA TRANC
VACCINES ARE CLOSE TO ELICITING
THESE.
THIRD, WE CAN BRING THE SOSIP
CLOSER TO OUR CURRENT REPERTOIRE
AND THEN OF COURSE MOVE THEM
WITH A MUCH SMALLER STEP THAN
FOR EXAMPLE THE CD4 BINDING SITE
TO WHERE THEY NEED TO BE TO MAKE
A NEUTRALIZING ANTIBODY.
OKAY.
LET'S SEE.
OKAY.
THIS IS BASICALLY TALKING ABOUT
HOW WE DEVELOPED OUR SYSTEM.
AGAIN WE'RE JUST CHANGING THE
CDR3s IN AN OTHERWISE KIE
VERSE BACKGROUND OF MURINE
VARIABLE CHAIN.
SO AGAIN, HUMAN CDR3s, MURINE
B CELLS, MURINE B CELL RECEPTOR,
MURINE B CELLS.
SO THE ONLY THING WE'RE
INTERDEUTING NOW IS JUST A CDR3.
THE CDR3 IS THERE.
AND THIS IS AN EXAMPLE.
SO NOW WE PUT IN THE
VRC2625CDR3.
IT'S A BIG LONG CDR3 --
SULFATES, INTO THE BACKGROUND OF
MURINE B CELLS AND THEN WE
IMMUNIZE.
THIS IS IMMUNIZATION WITH
PROTEIN.
AGAIN WHAT YOU CAN SEE IS WE DO
GET NEUTRALIZATION HERE.
AGAIN -- AND THIS POSITIVE
CONTROL IS 2-MICROGRAM PER ML
LEVEL HERE.
SO JUST FOR THE CVR3, WE CAN
DRIVE THAT TO A 2-MICROGRAM PER
ML LEVEL OF THIS VERY POTENT
ANTIBODY.
AS YOU WOULD EXPECT, IT'S A GOOD
DEAL OF HYPERSOMATIC MUTATION
WITHIN THE CDR3, AND WE KNEW
THIS WAS ACTUALLY A MUTATION
THAT EMERGED AND CHANGES THE
PROPERTY OF THE ANTIBODY SO IT'S
ACTUALLY -- THAN THE ORIGINAL
ANTIBODY.
SO SOME OF THESE CHANGES, WE'RE
AGAIN ABLE TO PLAY THE GAME OF
IMPROVING THE ANTIBODY, WHICH
DERIVES TO IN VIVO SELECTION.
BUT WHAT WAS IMPORTANT TO US, I
THINK, WAS THE IDEA THAT THE
UNDERLYING BACKGROUND OF
VARIABLE CHAINS THAT ARE BURYING
THE CDR3, THEY ARE VERY DIVERSE.
SO AFTER THIRD IMMUNIZATION, WE
GOT SOME THAT POPPED UP MORE
FREQUENTLY, BUT WE WERE ABLE
TO -- IN BOTH OF THE GERMINAL
CENTERS AND THE FULL PBMC, SEE A
LOT OF VARIABLE CHAINS PRESENTED
IN THE CDR3.
SO -- IT IS THE CDR3 AS IS
WELL-KNOWN FOR -- WE PLAY THIS
SAME GAME WITH OTHER CDR3s,
OKAY?
SO THESE ARE THE CDR3s OF TWO
WELL-KNOWN AND RELATED ANTIBODY,
PG9 AND PG16 AGAIN AT THIS
APACKS CLASS, AGAIN LONG,
HYDROGEN SULFATED.
THIS IS THE SAME ORIGINAL
ANTIBODY, BUT IT'S UNMUTATED
COMMON ANCESTOR.
THE WAY IT WAS CALCULATED TO
EMERGE DURING THE -- BE ABLE TO
DRIVE UP ANTIBODY RESPONSES
THERE.
BY THE WAY, THESE CONTROLS ARE
COMING UP BECAUSE THAT'S AN MRNA
VACCINE, NOT A PROTEIN VACCINE.
BUT ANYWAY, BOTTOM LINE IS,
WE'RE VERY INTERESTED IN A
SUBSET OF APEX ANTIBODIES, IN
PARTICULAR THIS DOUBLE TYROSINE
YYDF MOTIF.
EVERYTHING WE'VE BEEN ABLE TO DO
THAT BINDS THE SOSIP THAT WE'VE
BEEN WORKING WITH.
AND THIS IS JUST TO SHOW YOU
THAT.
AND THEN THE ORIGIN OF THAT.
WHICH IS THIS PARTICULAR
DIVERSITY CHAIN, 1 IN 24 THAT
WE'RE CARRYING IN OUR BODY,
WE'VE ALL GOT THIS DIVERSITY
CHAIN, READ IT IN DOM NAPT
FRAMEDOMINANTFRAME, THIS IS IT,S
WHAT WE THINK WE NEED TO ELICIT
WITH OUR NEW ANTIGENS.
ONE QUESTION COULD BE -- AND
YOUR FINAL SLIDE, WHAT IS THE
FREQUENCY OF THESE ANTIBODIES IN
UNAFFECTED PATIENTS.
REPERTOIRES OF TIME -- BOTTOM
LINE, THE YYDF MOTIF SHOWS UP IN
ABOUT 1 OF 2,000 OF YOUR
ANTIBODIES, AND THEN IF YOU
BROADEN THAT A LITTLE BIT, YOU
CAN ADD SOME MORE IN THERE, BUT
THAT'S THE BOTTOM LINE, AND SO
WHAT'S NEXT FOR US?
WE WANT TO KNOW CAN THESE -- CAN
MODIFIED SOSIPs, THE ONES
WE'RE SELECTING FOR, ENRICH FOR
BCRs FROM PROPERTIES FROM
PBMC.
CAN I JUST SORT OUT THE
ANTIBODIES THAT I CARED ABOUT
USING THE SOSIPs THAT I'M
MAKING.
NOW THE SECOND THING IS, CAN I
MAKE A NEUTRALIZING ANTIBODY
FROM USING THE SYSTEM I JUST
SHOWED YOU, WHERE I PUT IN THE
HEAVY AND THE LIGHT CHAIN.
CAN I TURN AN ANTIBODY THAT I
PULL OUT OF A REGULAR HIV
NEGATIVE PERSON AND TURN IT INTO
A NEUTRALIZING ANTIBODY.
AND CAN WE DO SOMETHING
ANALOGOUS IN A NON-HUMAN PRIMATE
WITH A RELATED DIVERSITY CHAIN.
SO THOSE ARE -- AND THEN SIMPLY
TO ACKNOWLEDGE THE PEOPLE WHO
HAVE BEEN INVOLVED IN THIS.
BASICALLY INVENTED -- EDITING.
THERE SHE IS THERE.
AND THEN OUR VACCINE WORK IS
BEING LED BY THIS GREAT TEAM AT
THE BROAD, COMPOSED OF -- WITH
HELP FROM SOME OF THESE OTHER
PEOPLE TOO.
THANKS.
[APPLAUSE]
>> QUESTIONS?
OKAY.
WE'RE GOING TO TAKE A BREAK
UNTIL 3:50.
POSTERS ARE OUT THERE.
SEE THE WORK, AND PLEASE COME
BACK.
>> ALL RIGHT, LET'S GET STARTED
AGAIN.
WE'RE COMING IN THE HOME STRETCH
HERE, TWO MORE TALKS.
SO OUR NEXT SPEAKER IS DAVID,
WHO RECEIVED HIS PH.D. IN
MOLECULAR BIOLOGY AT UW-MADISON.
DAVE AND I WERE GRADUATE
STUDENTS TOGETHER IN THAT LAB.
HOW MANY DECADES AGO.
LOST TRACK.
DAVE LOOKS A LOT YOUNGER THAN I
DO, BUT ANYWAY.
HE THEN CAME TO THE NCI
FREDERICK TO DO A POSTDOC WITH
NANCY JENKINS AND NEIL COPELAND.
IN 1996, HE WAS RECRUITED TO THE
FACULTY OF UNIVERSITY OF
MINNESOTA.
AMONG MANY OTHER THINGS, DAVE IS
WORKING TO EXPLORE MUTAGENESIS
TO IDENTIFY AND UNDERSTAND GENES
AND PATHWAYS THAT GOVERN CANCER
CELL BEHAVIOR.
HIS GROUP PIONEERED THE USE OF
AN ACTIVE SYSTEM KNOWN AS
SLEEPING BEAUTY FOR --
MUTAGENESIS.
HIS TALK IS ENTITLED THE ENEMY
WITHIN, TURNING TRANSPOSONS FROM
FOES TO FRIENDS.
THANK YOU.
[APPLAUSE]
>> THANK YOU.
IT'S GREAT TO BE BACK FOR
FREDERICK, WHICH I CAN CALL HOME
FOR FIVE YEARS AS I WAS HERE AS
A POSTDOC TORE FELLOW WITH NEIL
AND NANCY.
SO THESE ARE MY DISCLOSURES.
SOME OF THE WORK I'LL DISCUSS IS
FUNDED BY GENENTECH.
I'M GOING TO TALK ABOUT
TRANSPOSONS IN HUMAN BIOLOGY, IN
PARTICULAR HOW WE USE THEM AS A
TOOL IN CANCER AND IF THERE'S
TIME, I'LL TALK ABOUT IT. CELL
PHENOTYPE, WHICH IS SOMETHING
NEW WE'RE DOING WITH SLEEPING
BEAUTY TRANSPOSON MUTAGENESIS.
SO EVERYONE HAS BEEN TALKING
ABOUT VIRUSES.
I'M GOING TO TALK ABOUT
TRANSPOSON, BUT WHAT WE'RE DOING
WITH TRANSPOSONS HAS REALLY BEEN
INSPIRED BY PRIOR WORK WITH
VIRUS, I WOULD SAY.
SO TRANSPOSONS ARE TRANS --
TRANS POE SAL ELEMENTS ARE
SEQUENCES OF DNA THAT CAN JUMP
FROM ONE PLACE TO ANOTHER WITHIN
A CELL, SO THEY'RE CALLED
JUMPING GENES OR MOBILE GENETIC
ELEMENTS.
BASICALLY THERE'S TWO TYPES.
CLASS 1 THAT MOVES TO AN --
INTERMEDIATE, AND CLASS II,
PHYSICALLY MOVED FROM ONE
LOCATION TO ANOTHER IN THE
TRANSPOSITION PROCESS.
USUALLY THEY'LL EXPRESS THE
PROTEINS NECESSARY FOR
MOBILIZATION.
SO FOR EXAMPLE, TRANSPOSONS WERE
DEVELOPED IN 1952, WHICH IS
BEFORE THE STRUCTURE OF DNA WAS
KNOWN, SO HOW DID SHE DO THAT?
SHE SHOWED USING GENETIC MEANS
THAT A GENE SHE HAD DEFINED
WHICH TURNED OUT TO BE A
TRANSPOSON COULD CHANGE POSITION
WITHIN A SINGLE GENERATION, AND
SHE RECEIVED THE NOBEL PRIZE FOR
THAT IN 1983.
BUT TRANSPOSONS ACTUALLY GO BACK
TO THE BEGINNING OF MODERN
GENETICS.
-- NOW KNOWN TO BE CAUSED BY A
TRANSPOSON INSERTION, ENCODING
STARCH -- ENZYME.
TRANSPOSONS ARE EVERYWHERE, SO
THERE'S AN UNCOUNTED NUMBER OF
THEM.
THEY'RE FOUND IN NEARLY ALL
GENOMES.
THEY HAVE INTERESTING DIVERSE
MODES OF REPLICATION, AND
THEY'RE A SOURCE OF USEFUL
TOOLS.
THE HUMAN GENOME ISN'T ANY
DIFFERENT.
IT'S ACTUALLY ABOUT 45% COMPOSED
OF TRANSPOSABLE ELEMENTS.
THE VAST MAJORITY OF THESE ARE
INACTIVE FOSSIL REMNANTS
ALTHOUGH IN ALL PEOPLE, THEY'RE
STILL UNDERGOING TRANSPOSITION.
TRANSPOSONS HAVE HAD A MASSIVE
INFLUENCE ON OUR EVOLUTIONARY
HISTORY, SO MANY SEQUENCES HAVE
BEEN DOMESTICATED FOR USE BY
THEIR HOST SPECIES.
ONE FAMOUS COMPACT IS THE RAD
1 AND 2 ENVIEMS INVOLVED IN
RECOMBINATION, ACTUALLY DERIVED
FROM ANCIENT TRANSPOSES.
-- CAN ALSO BE DOMESTICATED FOR
USE OF REGULATORY ELEMENTS.
IN
GENE EXPRESSION.
BUT THEY CAN ALSO CAUSE DISEASE.
SO GERMLINE AND SOMATIC CELERY
ARRANGEMENTS, DELETIONS AND
INSERTIONS, AND IT'S NOW KNOWN
THAT CRYPTIC PROMOTERS BECOME
ACTIVATED.
THESE CRYPTIC PROMOTERS BECOME
ACTIVATED DURING CANCER
DEVELOPMENT AND DRIVE EXPRESSION
OF NOVEL TRANSCRIPTS ENCODING
NEW PROTEIN ISOFORMS THAT CAN
DRIVE CANCER.
AND IT WAS OBSERVATIONS LIKE
THIS THAT MOTIVATED ME TO LOOK
TO TRANSPOSONS AS TOOLS FOR
MAMMALIAN GENETICS, AND SINCE I
WAS IN NEIL AND NANCY'S LAB HERE
IN FREDERICK, I WAS ALWAYS
INTERESTED IN THE IDEA OF DOING
FORWARD GENETICS IN THE MOUSE
MODEL.
SO REVERSE -- THIS IS IN
CONTRAST TO REVERSE GENETICS,
WHERE YOU START WITH A GENE AND
YOU ALTER IT AND SEE WHAT
PHENOTYPE YOU'LL GET, IN FORWARD
GENETICS, YOU USE RANDOM
MUTAGENESIS TO ISOLATE CELLS OR
ORGANISMS THAT HAVE A PHENOTYPE
YOU'RE INTERESTED IN AND THEN
YOU FIGURE OUT WHAT GENE GOT
ALTERED IN THE PROCESS.
AND SO THAT WAS REALLY THE
MOTIVATION FOR DEVELOPING
TRANSPOSON MUTAGENESIS IN MICE.
MORE SO ALSO WAS THE STUDY OF
SLOW TRANSFORMING RETROVIRUSES.
WHEN I WAS IN NEIL AND NANCY'S
LAB, THAT'S WHAT I WAS WORKING
ON.
I WANTED, WHEN STA -- THAT WOULE
SIMILAR TO HUMAN TE TUMORS BUT
ALTERED DURING TUMOR
DEVELOPMENT.
THE EXPERIENCE I HAD WORKING
WITH NEIL AND NANCY WAS GREAT,
BECAUSE WORKING WITH OTHERS, THE
THREE OF US FOUND SOME REALLY
THINGS THAT I THOUGHT WERE
REALLY COOL, IN TERMS OF ACUTE
MYELOID LOU KEEM LEUKEMIA INDUCA
RETROVIRUS, WE FOUND THAT HOXA
GENES -- IF HUMAN HOXA-9 GENE
WAS ACTIVATED BY FUSIONS TO A
NUCLEAR PORIN PROTEIN GENE TO
CAUSE LO LEUKEMIA.
WE THOUGHT WOULDN'T IT BE
AWESOME IF WE COULD DO THE SAME
THING BUT FOR OTHER TISSUES?
THE PROBLEM WITH RETROVIRUS IS
WE HAVE STUDIES -- FOR MAMMARY
TUMORS, BUT THESE VIRUSES DON'T
REPLICATE WELL AND INFECT POORLY
REPLICATING TISSUES VERY WELL
THAT ARE HOSTING CELL TYPE
RESTRICTIONS AND THEY ALSO HAVE
VERY BIASED INSERTION
PREFERENCE.
SO THIS IS WHY I STARTED TO
THINK ABOUT OTHER OPTIONS, AND
IN PARTICULAR, TRANSPOSONS.
NOW SOMETIMES IN SCIENCE YOU
JUST GET LUCKY, SO I RETURNED TO
MINNESOTA WHERE I'M FROM, AND I
STARTED MY LAB, AND I WAS GOING
FOR A WALK WITH ANOTHER
PROFESSOR, PERIHACKETT, AND HE
TOLD ME ABOUT THE PAPER THAT HE
HAD UNDER REVIEW AT CELL THAT
DESCRIBED SLEEPING BEAUTY, WHICH
PAVED THE WAY FOR
TRANSPOSON-BASED TOOLS.
THEY CALL IT SLEEPING BEAUTY
BECAUSE HE AND THE PEOPLE IN HIS
LAB CLONED A BUNCH OF COPIES OF
A TC1 TRANSPOSEASE FROM
DIFFERENT SPECIES OF FISH.
THEY DOESN'T KOANT FIND ANY
ACTIVE VERSIONS BUT THEY LINED
UP ALL THE INACTIVE COPIES AND
THEY DEDUCED THE SEQUENCE, IT
MUST HAVE BEEN ACTIVE MILLIONS
OF YEARS AGO, IN ANCESTORS TO
THESE FISH AND THEY FIXED ALL
THE MUTATIONS THAT OCCURRED AND
THEY GOT, IN THE END, AN EB SIEM
THEY CALLED SB10 WHICH WOULD
CATALYZE THE CUT AND PASTE
REACTION.
SO IT WAS THE FIRST VER --
ALTHOUGH MANY HAVE BEEN FOUND
SINCE THEN OR CREATED.
I STARTED WORKING WITH PERIAS
WELL AS STEVE AND SCOTT OVER
THERE ON KIND OF IMPROVING THE
SLEEPING BEAUTY SYSTEM AND USING
IT FOR A VARIETY OF THINGS.
SO YOU CAN THINK OF IT AS A
TWO-PART SYSTEM, THE TRANS POE
SAYS AND TRANSPOSON VECTOR THAT
YOU PROVIDE TO A CELL, FLANKED
BY REPEATS THAT CAN BE BOUND BY
THE TRANSPOSE ACE, IT EXCISING
FROM THE DONOR LOCUS, THAT TA IS
DUPLICATED, EXCISION SITE IS
REPAIRED BY THE HOST SELL
MACHINERY.
SO WHAT CAN YOU DO WITH IT?
YOU CAN DELIVER -- TO CELLS OR
YOU CAN USE TO DAMAGE CELLS BY
INSERTIONAL MUTAGENESIS, SO TWO
VERY DIFFERENT THINGS.
YOU CAN ALSO IMAGINE SEPARATING
THOSE INTO -- SO FOR EXAMPLE, WE
SHOWED YOU IT COULD BE USED TO
ENHANCE THE WAY THE GERMLINE
TRANS GENESIS DRAMATICALLY.
CAN YOU ALSO DELIVER GENES TO
SOMATIC CELLS FOR LONG TERM GENE
TRANSFER AND EXPRESSION AND --
GENE THERAPY, FOR EXAMPLE, AND
SLEEPING BEAUTY HAS BEEN USED TO
DELIVER CHIMERIC ANTIGEN
RECEPTOR TRANS GENES TO HUMAN T
CELLS IN CLINICAL TRIALS, SO
THAT'S SOME THINGS YOU COULD DO
WITH IT.
WE WERE MORE INTERESTED IN USING
IT AS A GENETIC TOOL FOR FORWARD
GENETICS, SO WE SHOWED WE COULD
ACHIEVE GERMLINE MUTAGENESIS AND
CREATE MICE WITH HERITABLE
MUTATIONS, BUT MOST OF OUR WORK
HAS BEEN TO USE THE TRANSPOSON
TO -- BY MOBILIZING IT IN
SPECIFIC TISSUES OF TRANS GENG
MICE.
GENIC
MICE.
I'LL TALK ABOUT THESE
APPLICATIONS FOR CANCER.
I MENTIONED SLEEPING BEAUTY CAN
MEDIATE STABLE -- IN THE CONTEXT
OF GENETICALLY INJURED MOUSE
MODEL THMMODEL OF CANCER -- TO T
TISSUES OF MICE.
OFTENTIMES WE ALSO DELIVER
CRISPR REAGENTS TO KNOCK OUT
TUMOR SUPPRESSORS AND WE CAN
CREATE VARIOUS TYPES OF CANCER
BY DOING THAT.
THAT'S USEFUL BECAUSE US CAN
CREATE DIVERSE GENETICALLY
DISTINCT MODELS OF CANCER
WITHOUT HAVING TO MAKE GERM
LINES TRANSGENIC MICE.
SO FOR EXAMPLE, WE SHOWED WE CAN
INDUCE VARIOUS TYPES OF BRAIN
TEU NORS OVER THE YEARS IN MICE
BY DELIVERING SB TRANSPOSON
ENCODED ONCOGENES TO THE MICE
OF -- TO THE BRAINS OF NEONATAL
MICE TO MAKE THESE DIFFERENT
FORMS OF BRAIN TUMORS.
WE SHOWED THAT WE COULD USE
SLEEPING BEAUTY TO DLI DELIVER S
TO CELLS IN THE LIVER FOR
CHOLANGIOCARCINOMA.
THESE ARE USEFUL FOR LOTS OF
OTHER CLEVER PEOPLE WHO DID MORE
IMPORTANT WORK THAN WE DID TO
STUDY THE ROLE OF INNATE
ADAPTIVE IMMUNE SYSTEM IN GLIOMA
GENESIS, FIND GLIOMA
VULNERABILITIES AND TEST NEW
PRE-CLINICAL THERAPIES, AND LOTS
OF INTERESTING WORK HAS ALSO
BEEN DONE ON MODELS OF LIVER
TUMORS USING THIS BASIC
APPROACH.
THEY CREATED THE WORLD'S FIRST
PORCINE MODEL OF GLIOGLASS THOMA
WHERE THEY USE APPROACHES
SIMILAR TO WHAT I DESCRIBED TO
DELIVER TRANSPOSONS AS WELL AS
CRISPR AGENTS TO THE BRAINS OF
NEONATAL PIGS, AND THEY CAN
INDUCE CLASSICAL GDM IN THESE
PIGS.
AND HAVE DEVELOPED REPORTERS
THEY CAN USE IN SERUM AND HOPE
TO USE IT FOR TESTING NEW
THERAPIES FOR GVMs.
NOW I'M GOING TO TALK FOR THE
REST OF THE TIME ABOUT USING --
IT'S PRETTY SIMPLE.
WE MADE ONE LINE OF MICE THAT
CARRY THE TRANSPOSON SO PART 1
OF THE SB SYSTEM.
IN ALL THESE EXPERIMENTS IT'S
PRESENT AS A MULTICOPY
CONCATAMER IN THE MOUSE GENOME.
THE REASON FOR THAT, WE DIDN'T
THINK WE COULD INDUCE CANCER BY
MOBILIZING A SINGLE TRANSPOSON.
CANCER TAKES THE -- WITHIN A
CELL OR CLONE OF CELLS, SO
THAT'S WHY THERE'S MULTIPLE
COPIES OF THE TRANSPOSON.
THAT'S THE LAUNCH PAD FOR THESE
TRANS POE SO
TRANSPOSONS.
NOW YOU HAVE A MOUSE THAT HAS
BOTH PARTS OF THE SLEEPING
BEAUTY SYSTEM.
WE SHOWED MANY, MANY SOMATIC
MUTATION INSERTIONS THAT WERE
OCCURRING AND WE HOPE THAT THIS
COULD RESULT IN CANCER.
IN MY VERY FIRST GRADUATE
STUDENT WHO THEN WENT TO
POSTDOC, HE AND MY LAB DESIGNED
THIS TRANSPOSON, WE HOPE THAT
COULD INDUCE CANCER, CALLED IT
T2 ONC.
WE INCLUDED ELEMENTS THAT WERE
REALLY INFORMED BY PRIOR STUDIES
ON SLOW TRANSFORMING RETROVIRAL
AND HOW THOSE COULD CAUSE
CANCER.
IN FACT, THE VERY FIRST VERSION
OF THE T2 ONC MADE HAS A PORTION
FROM THE MARINE STEM REUR EEN S.
WE INCLUDED COUNCIL STREAM OF
THAT AN ARTIFICIAL EXON WITH A
SPLICED DONOR.
SUPPLIES ACCEPTORS IN BOTH
ORIENTATIONS AND A
BI-DIRECTIONAL POLYA WITH VIRUS
FROM SB40.
THE IDEA WAS THIS COULD CAUSE
GAIN OF FUNCTION -- OR LOST
MUTATIONS IN TUMOR SUPPRESSOR
GENES.
REMEMBER WE'RE MOBILIZING FROM A
DONOR CHROMOSOME SITE WHERE
THERE'S MULTIPLE TRANSPOSON
VECTORS.
PERRY HACKETT, HE SAID I'M
TRYING TO DO GENE THERAPY AND
YOU JUST MADE THE TRANSPOSON
FROM HELL.
ACTUALLY IT DID CONFUSE
REGULATORS IN WASHINGTON.
THEY'RE LIKE, YOU WANT TO USE
THIS THING THAT CAUSES CANCER
FOR GENE THERAPY?
BUT YOU KNOW, WE MADE LIKE THE
WORST CASE GENE THERAPY VECTOR.
IT'S GOT NO GENE AND ALL IT DOES
IS DAMAGE ENDOGENOUS GENES.
SO THE WHOLE THING WORKS, AND TO
CARRY OUT A SLEEPING BEAUTY
TRANSPOSON SCREEN, YOU CROSS THE
TRANSPOSON LINE TO A TRANS POE
SAYS -- YOU AGE THE MICE,
OFTENTIMES THESE MICE HAVE A
CANCER PRED PREDISPOSING MUTATIN
THE BACKGROUND.
YOU AGE THEM.
THE GRADUATE STUDENT OR POSTDOC
STARTS TO GET STRESH STRESSED
OUT AFTER ABOUT A YEAR.
MOST OF THESE EXPERIMENTS, WE
AGED THE MICE UP TO TWO YEARS,
COLLECTED THE TUMORS, THEN
SEQUENCED THE TRANSPOSON
INSERTION SITES.
SO HERE'S AN EXAMPLE WHERE WE
DID THIS IN LIVER, THE LIVER
WOULD HAVE MULTIPLE TUMORS IN
IT, YOU COLLECT THE TUMORS, PCR
TO AMPLIFY THE INTERGR INTEGRATE
SO A PIECE OF DNA INTEGRATED IN
ADJACENT GENOMIC DNA FROM A
CELL.
AND DO NEXT GEN SEQUENCING.
WE DID IT IN A BARCODED FASHION
SO YOU COULD HAVE A DIFFERENT
BARCODE FOR EACH TUMOR AND
SEQUENCE IN BULK.
THEN WORK WITH THE
BIOINFORMATICIANS, YOU RECOVERED
THOUSANDS PER TUMOR.
YOU MAKE THE GOOD STUFF, DISTILL
THAT DOWN, IT'S LIKE MAKING
WHISKEY.
WHAT WE'RE LOOKING FOR ARE THE
CANCER-CAUSING INSERTIONS, AND
TO DO THAT, WE CLONE A BUNCH OF
INSERTIONS FROM MULTIPLE
INDEPENDENT TE TUMOR, AND WE'RE
LOOKING FOR REGIONS CALLED
COMMON INSERTION SITES, WHICH IS
THE SAME TERMINOLOGY USED IN
SLOW TRANSFORMING PRE TROA VIRUS
RESEARCH.
AFFECTING THE SAME GENE IN THE
SAME WAY SO AS TO GIVE THAT CELL
A SELECTIVE ADVANTAGE.
AND WHEN WE DID THESE SCREENS,
IT BECAME CLEAR THAT THE WHOLE
THING WAS WORKING BECAUSE WE SAW
ACTIVATION OF KNOWN AND NOVEL --
OF MANY KNOWN CANCER GENES LIKE
NOTCH 1 OR BRAF, AS WELL AS LOSS
OF FUNCTION INSERTION IN KNOWN
TUMOR SUPPRESSOR GENES LIKE NF1,
NF2.
IN FACT IF WE HAD DONE THIS 20
OR 30 YEARS EARLIER, I WOULD
HAVE DISCOVERED ALL OF THESE
CANCER GENES.
BUT OF COURSE SLEEPING BEAUTY
DIDN'T EXIST THEN EITHER.
SO ANYWAY.
SO YOU CAN ACTIVATE ONCOGENES
WHERE AN INTERNALLY TRUNK ATED
ACTIVATED FORM OR C TERMINALLY
TRUNK ATED ACTIVATED FORMS, OR
YOU JUST GET KNOCKOUT OF THE
TUMOR SUPPRESSOR GENE.
IF IT'S A GENE THAT DOESN'T HAVE
A KNOWN ROLE IN CANCER, YOU
COULD GUESS WHETHER IT WAS A TEU
TUMOR SUPPRESSOR GENE OR
ONCOGENE BY LOOKING AT THE
PATTERN AND ORIENTATION
PREFERENCES OF THE INSERTION.
SO IF IT LOOKS LIKE THIS WHERE
IT SPREAD THROUGHOUT THE GENE
WITHOUT A STRONG ORIENTATION
BIAS, MAYBE THAT'S A TUMOR
SUPPRESSOR GENE.
BUT THEY'RE ALL LINED UP IN THE
SAME PLACE AND THE SAME
TRANSCRIPTIONAL ORIENTATION,
YOU'RE PROBABLY CREATING A
FUSION TRANSCRIPT THAT'S
OVEREXPRESSED.
SO WE FIRST REPORTED ON THIS
DOING MUTAGENESIS SCREENS IN
2005 AND SOME OF THAT WORK WAS
WITH NEIL AND NANCY.
AND BY 2009, WE WERE DOING THESE
SCREENS IN TISSUE-SPECIFIC
MANNER.
AND ADAM CAME HERE TO FREDERICK
TO WORK WITH NEIL, NANCY AND
LINO AND MADE THIS CRE ROCKS
REGULATED OF THE TRANS POE SAYS
TRANS GENE WHICH HAS BEEN VERY
USEFUL, SO YOU CAN JUST COMBINE
IT WITH DIFFERENT TISSUE
SPECIFIC CRES TO MODEL DIFFERENT
FORMS OF CANCER JUST LIKE WE
HOPED IN THE BEGINNING.
AT THIS POINT THERE'S OVER 80
PUBLICATIONS OR ANOTHER TRANS
POE SAL ELEMENT, SCREENS FOR
CARCINOMAS, SARCOMAS, LOTS OF
DIFFERENT TYPES OF CANCER.
AS I SAID OFTEN ON A
PREDISPOSING BACKGROUND OR
CANCER PROMOTING ENVIRONMENT.
AND NOW WE'RE TRYING TO USE THE
TECHNOLOGY TO ASK MORE
INTERESTING QUESTIONS.
WHAT ABOUT CANCER EVOLUTION,
EVOLUTION OF DRUG RESISTANCE OR
METASTASIS?
SO ONE THING I WANT TO EMPHASIZE
ABOUT THE NATURE OF THESE
GENETIC EVENTS INTRODUCED BY THE
TRANSPOSON IS THEY CREATE FUSION
TRANSCRIPTS THAT CAN BE
RECOVERED BY RNA SEQ.
SO WHEN A TRANSCRIPT INITIATES
WITHIN THE TRANSPOSON AND FUSES
TO DOWNSTREAM EXONS, YOU
COULD -- OR THE SUPPLIES
ACCEPTORS INTERRUPT
TRANSCRIPTION INITIATED AT THE
CELLULAR PROMOTOR, YOU SHOULD BE
ABLE TO SEE ALL THOSE EVENTS IN
RNA SEQUENCING DATA.
SO IF YOU DO THAT WITH SLEEPING
BEAUTY ACCELERATED TUMORS, YOU
CAN ALSO GET THE GENE EXPRESSION
PROFILE OF THAT TUMOR, AND
WORKING WITH AN INFORMATACIST
NAMED AARON SARVER, WE TRIED TO
SIMPLIFY THE GENE EXPRESSION
PROFILES BY LOOK FORKING FOR
CLUSTERS OF GENESES THAT ARE
CO-REGULATED.
YOU CAN TAKE THAT CLUSTER AND --
CLUSTER TO GIVE US A CERTAIN
SCORE AND AARON CALLS THOSE GENE
CLUSTER EXPRESSION SUMMARY
SCORES, OR GCESS.
WHAT'S BEEN INTERESTING ABOUT
THIS IS IF WE DO THIS RNA SEQ ON
TRANS POE ZONE INDUCED -- AND
THEN LOOK AT THE FUSION
TRANSCRIPTS TO GET THE GENOTYPE
AND MAKE GENOTYPE PHENOTYPE
CORRELATIONS, AND WE'VE DONE
THIS WITH A NUMBER OF DIFFERENT
MODEL SYSTEMS, AND I'LL TALK A
LITTLE BIT ABOUT THE MAMMARY
TUMORS ACCELERATED ON A
PI3 KINASE MUTANT BACKGROUND AND
OSTEOSARCOMAS ACCELERATED ON A
P53 MUTANT BACKGROUND.
SO WE USED -- TO ACCELERATE
OSTEOSARCOMA GENESIS ON WILD
PIPE OR P53 MUTANT BACKGROUND ON
MICE.
AND THIS FEELS -- VERSUS AGE IN
MONTHS, YOU CAN SEE HOW LONG
THESE EXPERIMENTS TAKE.
AND P53 MUTATION ALONE WAS KNOWN
TO CAUSE OSTEOSARCOMA.
OSTEOSARCOMA COULD BE INDUCED ON
A WILD TYPE BACKGROUND WITH
SLEEPING BEAUTY MUTAGENESIS, BUT
IF YOU COMBINE THE TWO, YOU GET
THE FASTEST DEVELOPING AS YOE
SARCOMAS, AND YOU ALSO TEND TO
SEE MORE PER MOUSE.
IF IT WAS SLEEPING BEAUTY EXCEL
BRAITED ON A P53 MUTANT
BACKGROUND.
SO THESE ARE THE RECURRENT
TRANSPOSON GENE FUSIONS THAT WE
SEE.
SO EACH OF THE COLUMNS HERE IS A
GIVEN AS Y OSTEOSARCOMA, YOU CAE
GENE FUSIONS.
IT GOES DOWN FROM THERE.
, FROM PTEN.
SO THE LIST IS COULD BE SPIC
WITH US FOR CAMPAIGNING MANY
KNOWN OR SUSPECTED -- SO THAT'S
ALL -- THAT'S GOOD.
WHAT ABOUT THE OTHER GENES?
WELL, MANY OF THEM DO SHOW
RECURRENT COPY NUMBER
ALTERATIONS IN OSTEOSARCOMA, BUT
BUT -- HAVE A LOT OF GENE COPY
NUMBER CHANGES SO MAYBE THESE
AREN'T IMPORTANT.
BUT ALSO MANY OF THESE GENES
SHOWED RECURRENT COPY
ALTERATIONS IN CANINE AS YOE
SARCOMA AS WELL, SO WE THINK A
LOT OF THEM ARE MEANINGFUL,
BECAUSE THE GENE COPY -- IN THE
SAME DIRECTION AS HUMAN
OSTEOSARCOMA.
BUT DO ANY OF THEM CORRELATE
WITH CLINICALLY RELEVANT
PHENOTYPES?
SO IT TURNS OUT THAT THE SB
ACCELERATED OSTEOSARCOMAS --
IMMUNE CELL INFILTRATION.
SO FOR EXAMPLE, THIS CLUSTER OF
COVARIANT GENES DOWN HERE IS --
OUR CELL CYCLE GENES.
IT'S REALLY -- THESE ARE
OSTEOSARCOMA CELL LINES WE
ISOLATED FROM THE TUMORS BUT IT
ALSO VARIES IN THE TUMORS.
SIMILAR FOR THE IMMUNE CELL
INFILL TAITION.
SOME OF THE SAMPLES ARE
IMMUNE-COLD AND OTHERS ARE MORE
IMMUNE-HOT.
SO WE CAN USE INFORMATION LIKE
THIS TO GENERATE GRAPHS LIKE
THIS ONE, WHERE YOU CAN LOOK AT
THE TOTAL -- THE GCESS SCHOR,
SCORE.EACH DOT IS A TUMOR, AND E
SIZE OF THE DOT REFLECTS THE
TOTAL NUMBER OF TRANSPOSON GENE
FUSIONS, IN THIS CASE D WHAT YOU
CAN SEE FROM THIS IS SOMETHING
THAT YOU ALL WOULD HAVE
PREDICTED, WHICH IS THAT MYC
LEVELS ARE HIGH IN SAMPLES WHERE
THERE'S A TRANSPOSON ACTIVATED
FUSION, AND THAT ALSO TENDS TO
BE THE SAMPLES WITH THE HIGHEST
GCESS SCORE, SO NICK IS DRIVING
A -- THERE'S A FEW CANDIDATE,
ONE OF WHICH IS ACTIVATION OF
THE COLONY STIMULATING FACTOR
1 RECEPTOR.
SO TUMORS WITH HIGH LEVELS OF
THIS CAUSED BY TRANSPOSON
ACTIVATION ARE LOW IN IMMUNE
CELL INFILTRATION.
SO WE WENT AND AHEAD AND -- ON A
P53 MUTANT BACKGROUND.
THIS IS THE CURVE RIGHT HERE AND
THESE MICE DEVELOP OSTEOSARCOMAS
FASTEST.
WHEN YOU LOOK AT THESE
OSTEOSARCOMAS, THEY HAVE LOWER
IMMUNE INFILTRATION THAN
OSTEOSARCOMAS WITH P53 ALONE.
SO THIS LEADS US TO BELIEVE THAT
CSF 1 RECEPTOR EXPRESSION CAN
APPROPRIATE AND/OR THAT SHEDS
CSF 1 RECEPTOR SOMEHOW IS
SEQUESTERING LIGANDS AND
PREVENTING IMMUNE CELL
INFILTRATION.
NOW, YOU MIGHT WONDER IF IT'S
EVEN IMPORTANT IN HUMAN
OSTEOSARCOMA.
IT TURNS OUT IT IS.
THIS IS THE SAME KIND OF YOU AAL
CYST WITH HUMAN OSTEOSAR COMA
LOOKING AT CELL CYCLE AND IMMUNE
ILL FILTRATION.
HIGH IMMUNE CELL INFILTRATION OF
THE PRIMARY TUMOR, A BETTER
OUTCOME.
SO WE SHOULD CARE ABOUT THESE
PHENOTYPES IN OSTEOSARCOMA.
NOW WE HAVE VERY SIMILAR DATA
WSH WE AK EL --ON A -- THIS KNOE
LOX REGULATED EXPRESSION OF THE
THIS ACTIVATED FORM.
THIS MUTATION IS COMMON IN HUMAN
MAMMARY TUMORS.
AND WE COMBINE THAT WITH -- THAT
EXPRESSED IN DIFFERENT
COMARTMENTS OF THE MAMMARY GLAND
EPITHELIAL TISSUES AND USED A
DIFFERENT TRANSPOSON THAT'S MORE
ACTIVE IN EPITHELIAL CELLS, AND
THEN THE SAME CONDITIONAL
SLEEPING BEAUTY TRANS TRANSPOSE
GENE.
I WON'T GO INTO THE DETAILS ON
THIS, BUT IT DID ACCELERATE
MAMMARY TUMORS ON A PICK
3 RELATED TO BACKGROUND WITH
BOTH LOCK OR MTB -- THE
ACCELERATED TUMORS WERE MOSTLY
ADENOCARCINOMAS, BUT
INTERESTINGLY, WE COULD GET SOME
THAT WERE ER POSITIVE AND SOME
THAT WERE ER NEGATIVE.
THAT WAS INFLUENCED BY THE
TARGET CELL TRANSFORMATION,
WHERE THEY TENDED TO BE NEGATIVE
IF WE USE THE THE GENE.
WE FOREMANNED WHOLE EXOME
SEQUENCING AND RECOVERY OF THE
TRANSPOSON INSERTION SITES FROM
TUMORS AND MATCHED NORMAL
EPITHELIAL TISSUES FROM THESE
MICE.
ON 164 TUMORS AND MATCHED NORMAL
TISSUE.
WE NOW RECOVER THESE BY A
HYBRIDDATION APPROACH, WHICH
GIVES US A BETTER REPRESENTATION
OF THE CLONALITY OF EVERY
INSERTION SITE.
THERE WERE ONLY 34 CODING
MUTATION FROM 24 OF THESE
TUMORS, AND THEY WERE IN SOME
INTERESTING GENES BUT IT DOES
SHOW YOU THAT THE MAJORITY OF
THE GENETIC DAMAGE BEING DONE IN
THESE MODEL SYSTEMS IS BY THE
TRANSPOSON.
AND THERE WERE MANY THOUSANDS OF
INSERTION SITES RECOVERED WITH
STRONG EVIDENCE FOR CLONAL
EXPANSION OF INSERTIONS WITHIN
TUMORS.
IT'S ALLOWED US TO DEFINE WHAT
ARE THE LIKELY INITIATORS AND
WHAT CAUSES PROGRESSION IN THIS
MODEL.
WE ALSO DID RNA SEQUENCING IN
THESE MAMMARY TUMORS COMPARED TO
HUMAN AND SOUGHT NEW TUMOR
PHENOTYPES AND DISCOVERED
DRIVERS.
AND THIS IS THE KIND OF DATA WE
HAVE FROM THIS MODEL WHERE EACH
DOT IS A TUMOR.
THESE ARE U MAP PROJECTIONS
SHOWING DIFFERENT CLUSTERS.
SO WE DON'T GET ALL ONE
PHENOTYPE.
WE GET DIFFERENT TYPES OF
MAMMARY TUMORS.
AND THEY'RE SIMILAR TO DIFFERENT
TYPES OF HUMAN BREAST CANCER.
THEY ALSO TEND TO HAVE DIFFERENT
MUTATIONS, SO THIS CLUSTER OVER
HERE IS THE ONE WITH THE P10
INSERTIONS.
THIS ONE IS WITH ACTIVATION OF A
GENE -- THEN WHEN YOU LOOK AT
THE GCESS CLUSTERS, YOU CAN SEE
INTERESTING THINGS LIKE THIS IS
THE TUMORS THAT ARE ER POSITIVE
AND HAVE HIGH LEVELS OF
EXPRESSION OF ER-DEPENDENT GENE
EXPRESSION.
THESE TUMORS ARE VERY I MAWN
COLD AND HIGH CELL CYCLE, SO YOU
CAN CORRELATE THESE MUTATIONS
WITH A GIVEN CANCER PHENOTYPE.
AND SO WE'VE DONE THAT TO
DEVELOP A BUNCH OF
GENOTYPE/PHENOTYPE CORRELATIONS.
ONE THAT INTERESTS US, BECAUSE
OF THE IMMUNE CELL -- PHENOTYPE
IS -- SO THERE'S A SUBSET OF
HYPOMETHYLATED JUP HIGH TUMORS,
AND AS I SAID, THIS JEAN ENCODES
PLACHYCOBAN, THEREFORE IT'S
CONFUSING WHETHER IT'S A TUMOR
SUPPRESSOR OR AN ONCOGENE.
IT CAN INCREASE 2D AND 3D GROWTH
IN MODEL SYSTEMS, INCREASES --
SIGNALING AND ENHANCED -- GENE
EXPRESSION.
IF YOU LOOK IN HUMAN BASAL
BREAST CANCERS, HIGH JUMP IS
ASSOCIATED WITH THE WORST OUT
COME, AND IMMUNE SELL -- AS
MEASURED BY T CELLS IN IHC
EXPERIMENTS IN HUMAN BREAST
CANCER.
SO I SHOWED HOW YOU COULD USE
RNA SEQ TO GENERATE GENOTYPE
PHENOTYPE CORRELATIONS AND THAT
ARE SHOWN HERE.
AND WE THINK THESE ARE
CLINICALLY RELEVANT ASSOCIATIONS
THAT NEED TO BE EXPLORED.
SO IT MAKES US THINK, IMMUNE
CELL INFILTRATION AND ACTIVITY
SYNERGIZE WITH NEW ADJUVANT
CHEMOTHERAPY TO ALLOW SOME
OSTEOSARCOMA PATIENTS TO BE
CURED AND MAY IMPROVE BREAST
CANCER OUTCOMES AS WELL.
SO MAYBE IF WE COULD MODULATE
THE PATHWAYS WE TALKED ABOUT
EARLIER IN THOSE DISEASES, BUT
IT ALSO MADE US WONDER WHAT
IMMUNE CELL AUTONOMOUS
MECHANISMS COULD BE MODULATED TO
IMPROVE T-CELL -- INTO THE TUMOR
AND ACTIVITY.
NOW INSPIRED BY WORK FROM
UNIVERSITY OF IOWA, WE'VE BEEN
DOING SCREENING FOR T-CELL
AUTONOMOUS GENES AND PATHWAY TO
REGULATE INFILTRATION INTO
TUMORS.
TO DO THIS, WE BUILT A NEW TYPE
OF TRANS GENE WHERE WE COULD
TURN SLEEPING BEAUTY, IN THIS
CASE AN IMPROVED VERSION, WE
COULD TURN IT ON AND OFF.
SO THIS IS BOTH PREREGULATED
AND -- AND DOX REGULATED.
THE IDEA IS WE CAN HAVE ENOUGH
TRANSPOSITION TO GET NEW CELL
PHENOTYPES BUT NOT SUCH
TRANSPOSITION THAT YOU INDUCE
CANCER.
SO WE DID THIS WITH CD2 CRE AND
CREATED MICE WITH DOXYCYCLINE
AND I DUED T-CELL MUTAGENESIS.
WE TOOK THOSE MICE AND OR THE
TOPICALLY INJECTED A CANCER, SO
IN THIS CASE, A MOUSE MODEL OF
PANCREATIC DUCTAL
ADENOCARCINOMA, CALLED KPC MAU,
IN COLLABORATION WITH UNIVERSITY
OF MINNESOTA.
WE LET TUMOR GROW FOR THREE
WEEKS OR SO, HARVESTED TUMOR AND
SPLEEN AND THEN LOOK AT THE --
IN THE SPLEEN AS A CONTROL OR
TUMOR.
A AND WHAT WE'RE LOOKING FOR ARE
MUTATIONS AND T-CELLS IN TUMORS
THAT ARE NOT PRESENT OR ENRICHED
GREATLY ABOVE WHAT YOU'D SEE IN
THE SPLEEN FROM THE SAME MOUSE.
SOME EVENTS OCCUR SPECIFICALLY
IN T CELLS THAT ARE PRESENT IN
TUMOR AND NOT SPLEEN.
THE NUMBER ONE CANDIDATE WE GOT
FROM THAT, ALTHOUGH WE GOT A
NUMBER OF CANDIDATES, IS CALLED
O27B.
THIS GENE IS ALREADY IMPLICATED
IN T-CELL BIOLOGY, WHERE IT --
IT'S A DEUBIQUITIN ASE, AND FUSE
THEM INTO KPC DUE MORE BEARING
MICE.
THE MICE WERE TREATED WITH
IL2 EVERY OTHER DAY.
AT INPUT -- BUT LATER, IN TUMOR,
YOU SEE DOUBLE THAT NUMBER OF
O27B POSITIVE CELLS, SO THEY'RE
ENRICHED IN THE TUMOR, NOT IN
THE LYMPH NODE.
NOW THEY'RE ALSO ENRICHED IN
SPLEEN AND BLOOD, AND THAT MIGHT
BE BECAUSE OF THE IL-2 WE'RE
GIVING. SO WE THINK THIS IS THE
TBINNING OF MAYBE A VALIDATION
OF THIS GENE AS A GENE WHO COULD
IMPROVE -- IN THE INTEREST OF
TIME I WON'T GO THROUGH THIS BUT
WE DID THE SAME KIND OF SCREEN
BUT FOR -- EXHAUSTION.
WE GOT -- SEEMS TO CONFER, DRK
INCLUDING SOME GENES THAT HAVE
ALREADY BEEN IMPLICATED IN
T-CELL EXHAUSTION.
SO I THINK THE IDEA HERE IS WE
HAVE CANDIDATE GENES THAT MIGHT
CHANGE T-CELL PHENOTYPE WITH
REGARD TO ANTITUMOR ACTIVITY.
WE'D LIKE TO TAKE THOSE
CANDIDATES, TEST THEM IN
TUMOR-REACTIVE T CELLS OR TILs
OR IN FUTURE, WE'RE THINKING
ABOUT BROADENING THIS IDEA OF
SCREENS TO OTHER IMMUNE CELL
TYPES OR OTHER CELL TYPES THAT
HAVE POTENTIAL FOR REGENERATIVE
REGENERAL --WE ALSO HOPE SINGLEL
INSERTION ANALYSIS WILL HELP US
IN OUR GOALS.
WHEN WE THINK ABOUT IT, CA CANCR
CELLS -- HAVE EXTENDED
LONGEVITY, AND A COMPETITIVE
ADVANTAGE OVER HOST CELLS SO
THAT YOU DON'T HAVE TO -- THEY
CAN MAKE THEIR OWN ROOM.
BUT WHAT YOU DON'T WANT THEM TO
DO IS PROLIFERATE WITHOUT NORMAL
CONTROL OR LOSE THEIR
DIFFERENTIATION POTENTIAL.
SO -- AND I THINK THIS SB SYSTEM
WILL HELP US FIND GENES THAT
COULD HELP SALES DO THIS AND TRY
TO AVOID THIS.
AND THAT'S REALLY WHAT -- KIND
OF WHAT WE'RE THINKING ABOUT
WITH THIS LAST BIT OF WORK.
SO THESE WERE THE PEOPLE THAT
WERE INVOLVED, AND SOME OF OUR
FUNDING, AND I'M HAPPY TO TAKE
ANY QUESTIONS YOU HAVE NOW.
[APPLAUSE]
>> SO IT'S A FASCINATING SYSTEM.
IS THERE ANY BIAS, ARE THERE HOT
SPOTS OR REFRACTIVE SEQUENCES OR
SITES?
>> I GLOSSED OVER ONE MAJOR
THING, SOURCE OF BIAS, WHICH IS
MANY OF THE INSERTIONS LAND
WITHIN -- THEY JUMP AND THEY
LAND LOCALLY.
SO WE TRY TO IGNORE THE LOCAL
HOPS AND DO EVERY SCREEN WITH
TWO DIFFERENT DONORS, ONE WHERE
IT'S ON ONE CHROMOSOME AND
WITHIN WHERE THE TRONS POE SONS
ON ANOTHER.
WE ALSO THINK THE RNA SEQ
ANALYSIS OF THESE POPULATIONS
HELPS CLARIFY WHAT'S BEING
SELECTED FOR, BY SEEING THE SAME
KIND OF FUSION TRANSCRIPTS.
>> SO YOU SHOULD BE ABLE TO SEEP
THAT WITH SOME BIOINFORMATICS.
>> YEAH.
WITH REGARD TO OTHER GENOMIC
FEATURES, IT'S RELATIVELY
NEUTRAL, SO SB TRANSPOSON
INSERTION IS AFFECTED A LOT LESS
BY GENOMIC CONTEXT THAN BY
VIRUSES OR OTHER TRANSPOSONS IN
USE.
>> SO YOU THINK ABOUT LOOKING AT
ALSO EPIGENETIC CHANGES COMPARED
TO GENETIC CHANGES?
>> THAT'S A GREAT QUESTION.
WE HAVE -- YOU KNOW, I THINK IF
WE CAN DO THE SINGLE CELL
ANALYSES, YOU KNOW, THAT I
ALLUDED TO AT THE END, WE COULD
SEE FOR ANY GIVEN INSERTION HOW
FAR IS THE EFFECT, YOU KNOW, ON
NEIGHBORING GENES.
A MORE CREATIVE THING, INSTEAD
OF MOBILIZING A PROMOTOR,
MOBILIZE SIS REGULATORY
SEQUENCES, LIKE BORDER ELEMENTS
AND SEE WHAT YOU COULD LEARN BY
PERTURBING THE GENOME IN THAT
WAY.
I WOULD SAY THE CANCER SCREENS
THAT WE DO, THEY DO REVEAL
RECURRENT MUTATIONS IN READERS
AND WRITERS FOR SURE, JUST LIKE
IN HUMAN CANCER.
>> OTHER QUESTIONS?
>> GREAT TALK.
IN THE TUMOR SUPPRESSOR GENE
KNOCKOUT, DO YOU NEED BOTH
COPIES TO BE INACTIVATED?
>> GOOD QUESTION.
WHEN WE FIRST DID THIS, WE
THOUGHT WE WON'T GET ANY TUMOR
SUPPRESSORS BECAUSE IT HAS TO
HIT BOTH COPIES AND THAT'S NOT
GOING TO HAPPEN LIKELY BY
CHANCE.
BUT INSTEAD WE RECOVER A LOT OF
TUMOR SUPPRESSOR GENE INSERTIONS
AND IN SOME TUMORS, IT'S ONE
INSERTION IN EACH ALLELE.
IN OTHER CASES, WE THINK WHAT'S
HAPPENED IS HAPLOINSUFFICIENCY,
SO IN THE RIGHT GENETIC CONTEXT,
YOU ONLY NEED TO LOSE ONE COPY
OF P10.
AND WE HAVE SOME EVIDENCE FOR
THAT IN TUMORS WHERE WE TOOK THE
TUMOR, WE MADE CLONAL CELL LINES
AND YOU CAN SEE THAT EXACT KIND
OF DIVERSITY WHERE SOME SELLS IN
THE TUMOR HAD THOSE INSERTION
AND THEN -- IN OTHERS THEY
RETAIN A WILD TYPE ALLELE.
>> ANYBODY ELSE?
ANYBODY ONLINE?
>> THE CSF R HAD EXPRESSION --
MEANS OF IMMUNE SILENCING.
YOU WEREN'T SURE WHETHER THAT
WAS WORKING THROUGH DECOY OR
SOME OTHER PROGRAM.
SO I GUESS I MIGHT HAVE THROWN
CSF ON TO THAT OR USED A DECOY
TO TRY TO RECAPITULATE IT OR
SOMETHING LIKE THAT?
>> THAT'S WHAT WE WANT TO DO
NOW.
SO WHEN WE LOOKED AT THE SERUM
OF THESE MICE, INDEED THEY HAD
HIGH LEVELS OF SHED CSF 1R,
COMING FROM THE TUMOR.
AND THEY HAD LOWER LEVELS IN
CIRCULATION, SO THERE IS A DECOY
EFFECT, BUT WHETHER THAT'S
RESPONSIBLE FOR THE IMMUNE COLD
ENVIRONMENT OR NOT, WE'RE NOT
SURE.
SO IT'S WEIRD BECAUSE -- BECAUSE
PEOPLE THINK -- SO THIS WOULD
SUGGEST WE SHOULD PUT CSF 1 IN
THE TUMOR AND GET THE IMMUNE
CELLS BACK, BUT GENERALLY CSF
1 EXPRESSION IN TUMORS IS
THOUGHT OF AS A BAD THING
BECAUSE IT RECRUITS
IMMUNOSUPPRESSIVE MACROPHAGE.
BUT I THINK THAT REMAINS TO BE
SEEN.
>> THANKS.
[APPLAUSE]
>> LAST BUT NOT LEAST, JOHN
KAFFIN.
ONE MORE UW-MADISON GRADUATE
HERE.
WORKING WITH HOWARD TENMAN.
HE DID A POSTDOC AND IN 1975 WAS
RECRUITED TO THE FACULTY AT
TUFTS.
MOST NOTABLY FOR SPHOD TODAY'S ,
HE SERVED AS DIRECTOR FROM 1997
UNTIL 2005.
IN ADDITION TO HIS FACULTY
APPOINTMENT AT TUFTS, JOHN HAS
MAINTAINED HIS CLOSE TIES TO THE
DRP AND THE NCI SERVING AS A
SPECIAL ADVISER TO THE DIRECTOR
OF NCI CENTER FOR CANCER
RESEARCH.
JOHN'S RESEARCH FOCUS ON GETTING
A BETTER UNDERSTANDING OF THE
REACTION OF RETROVIRUSES AND
HOST CELLS AND ORGANISMS, HE'S A
LEADING AUTHORITY IN THIS AREA.
THE TITLE OF JOHN'S TALK TODAY
IS VIRUS VERSUS VIRUS, HUMAN
ENDOGENOUS RETROVIRUSES AS
ANTIRETROVIRAL RESTRICTION
FACTORS.
[APPLAUSE]
>> THANK YOU, ERIC.
THIS HAS BEEN A REALLY FUN DAY
FOR ME.
I HAVE TO SAY, IT'S BEEN A
REALLY WONDERFUL SERIES OF TALKS
PRECEDING THIS.
ON ALL OF THE TALKS I NOTE IT
INVOLVED VERY ELEGANT GENETIC
ENGINEERING TO ACCOMPLISH GOALS
PARTICULARLY USING VIRUSES AND
FOR VARIOUS PURPOSES.
I WANT TO CHANGE THE SUBJECT A
LITTLE BIT.
THE THING -- ENGINEERING A VIRUS
TO DO THINGS THAT ARE USEFUL FOR
YOU, BUT WITHOUT A HUMAN TOUCH,
ENTIRELY BY NATURE OVER THE
COURSE OF THE LAST 10 MILLION
YEARS OR SO IS THE RANGE WE'RE
GOING TO SPAN IN THIS TALK.
SO I'LL FIRST SHOW YOU MY VIEW
OF SORT OF THE PROCESS OF HOST
RETROVIRUS EVOLUTION.
THE THING TO REMEMBER ABOUT
VECTOR VIRUSES, UNLIKE ALMOST
ALL OTHER VIRUSES WITH A VERY
FEW RARE EXCEPTIONS, THEY'VE
LEFT BEHIND A FOSSIL RECORD THAT
WE CAN LOOK AT.
THIS HAS BEEN ALLUDED TO BEFORE,
AND IT'S IN THIS FOSSIL RECORD
WE'RE USING TO TRY TO CARVE OUT
IMPORTANT FEATURES.
YOU CAN VISUALIZE, WITH MY
ELEGANT COMPUTER GRAPHICS, THE
PROCESS OF HOST-VIRUS EVOLUTION
IN VERY BROAD TERMS.
IN GENERAL, THE -- REVOLUTION OF
THE VIRUS AND HOST IS OFTEN THE
RELATIVELY BENIGN RELATIONSHIP.
SO LIKE COMMON COLD VIRUSES AND
OTHER VIRUSES THAT CIRCULATE
WITHOUT KILLING US, THE VIRUSES
DO OKAY, WE MAY GET A LITTLE
UNCOMFORTABLE BUT WE DO OKAY IN
GENERAL, THAT'S THE USUAL
CIRCUMSTANCE.
BUT WHEN WE GO TO ANOTHER
SPECIES, ALL OF THE CONTROLS
THAT ARE BUILT INTO THAT OR A
LOT OF THEM ON THE HOST SIDE
HAVE GONE AWAY, AND YOU GET A
SITUATION WHERE THE VIRUS CAN BE
QUITE LETHAL ACTUALLY, OR CAUSE
CONSIDERABLE MORTALITY.
THAT CAN'T GO ON.
THERE'S GOT TO BE ONE OR ANOTHER
END POINT OF THIS, ONE OF WHICH
IS THE HOST CAN'T RECOVER -- CAN
REBOUND, THOSE EXTINCT, THOSE --
THE VIRUS GOES EXTINCT, AND THE
OTHERS, THEY RE-ESTABLISH A NICE
WORKING RELATIONSHIP LIKE YOU
SEE IN THE BEGINNING.
WE CAN'T TELL WHAT HAPPENED
BECAUSE WE HAD NO TRACES LEFT OF
THIS PROCESS, EXCEPT IN THE CASE
OF RETROVIRUSES, WHERE THEY
OFTEN, NOT ALWAYS BUT OFTEN CAN
GET INTO THE GERMLINE AND LEAVE
BEHIND A PROVIRUS THAT CAN LAST
FOREVER ESSENTIALLY THROUGH MANY
MILLIONS OF YEARS OF SUBSEQUENT
EVOLUTION AND LEAVE US WITH A
FOSSIL RECORD OF THIS PROCESS.
IN ADDITION THE VIRUS CAN
SOMETIMES PARTICIPATE IN THE
EXTINCTION AND THAT'S WHAT I
WANT TO TALK ABOUT TODAY.
SO AS THE COURSE OF EVOLUTION
GOES ALONG, YOU EFFICIENTLY END
EEFNTEVENTUALLY ENDUP WITH HUMAS
RETROVIRUS, AFFECT NATTILY CAL
CALLED HRS, AND THE FOSSIL
RECORD WE STUDY.
THE BLUE SQUARES, SUBSPECIES,
ALL OF WHICH ARE VISIBLY RELATED
TO EXTANT EXOGENOUS VIRUSES,
INFECTIOUS VIRUSES AND OTHER
SPECIES, AND THEY TELL US THAT
THROUGHOUT OUR EVOLUTIONARY
HISTORY, LOOKING BACK MAYBE
20 MILLION YEARS OR SO, MAYBE
EVEN A LITTLE MORE, WE CAN SEE
EVIDENCE FOR AT LEAST 30
PREVIOUS PANDEMICS THAT HAVE
AFFECTED OUR DISTANT ANCESTORS.
AND I'M GOING TO FOCUS ON ONE
GROUP IN PARTICULAR, WHICH IT'S
CALLED HERV-K HML-2.
IT'S THE ONE THAT LOOKS MOST
LIKE THE MAMMARY TUMOR VIRUS OF
MICE, AND THAT SITS WAY IN THE
RETROVIRUS GENUS WAY OVER IN THE
CORNER THERE, AND THIS IS A MOST
RECENT VIRUS.
EVEN TODAY, I SAW THE ENDOGENOUS
RETROVIRUSES REFERRED TO AS --
RETRO TRANSPOSONS.
THESE ARE NOT TRANSPOSONS.
ONCE THEY'RE IN THE GERMLINE,
THERE'S NO EVIDENCE, ALTHOUGH
PEOPLE OFTEN SAY IT, THERE'S NO
EVIDENCE THAT THESE CAN
TRANSPOSE WITHIN THE SINGLE CE
CELL.
THEY CAN MOVE SOMEWHERE ELSE IN
THE BODY, BUT IT GETS INTO THE
GERMLINE -- THAT'S THE ONLY
MECHANISM WE KNOW FOR ALMOST ALL
OF THESE VIRUSES.
VERY OCCASIONALLY THEY WILL
EVOLVE INTO A TRANSPOSON.
MORE RECENT ONES HAVE DONE THIS.
SO I THINK I DARE TO THE GENOME
NOT AS THE BATTLEGROUND BUT AS
THE GRAVEYARD FOR THESE VIRUSES.
BUT THE REMAINS THAT ARE IN
THESE GRAVES, LOOKING AT THEM
CAN TELL US A LOT, AND ALSO, OF
COURSE, OCCASIONALLY THESE
VIRUSES ARE DOING SOMETHING
USEFUL FOR US.
SO IN BROAD TERMS, THEY CAN DO
THINGS LIKE INDUCING A NEW OR
VIRAL GENE THAT CAN BE COOPTED
LIKE SIN SIGH DIN WHICH WAS
MENTIONED ALREADY TODAY TO PLAY
AN IMPORTANT ROLE IN FETAL
DEVELOPMENT.
THEY CAN LEAD TO INSERTIONAL
INACTIVATION OR ACTIVATION OF
GENES.
INTRODUCTION OF THE
TRANSCRIPTIONAL MACHINE -- THEY
CAN MODIFY GENE EXPRESSION, BY
TURNING IT ON IN CELL TYPES
WHERE IT'S NOT ALWAYS PRESENT,
THEY CAN BECOME PARTS OF -- YOU
CAN STILL SEE THEM AS EXPRESSED
THAT WAY.
THEY CAN ALSO DO PATHOGENESIS,
BY REPLICATION OF PROGENY OF
INTACT PROVIRUS, THIS IS NOT
POSSIBLE IN HUMANS AS FAR AS WE
KNOW, THEY PROVIDE A FOSSIL
RECORD, BUT MOST IMPORTANT THING
THAT THESE CAN DO IS THEY CAN
PROVIDE PROTECTION FROM
INFECTION BY RELATED VIRUS,
WHICH IS ITSELF QUITE
PATHOGENIC.
AND I POSTULATE AND WE THINK ONE
OF THE IMPORTANT THINGS IF YOU
THINK BACK TO THAT TREE, ONE
CHARACTERISTIC THAT'S SHARED BY
ALL OF THE VIRUSES, THEY'RE ALL
EXTINCT.
ALL OF THE INFECTIOUS VIRUSES --
EXTINCT.
I THINK THIS MAY BE DUE IN PART
TO THIS PHENOMENON, SO THAT'S
WHAT I WANT TO TALK ABOUT.
THERE ARE SEVERAL MECHANISMS
THAT HAVE BEEN AROUND A LONG
TIME, PARTICULARLY IN ANIMAL
MODELS, IT'S EASY TO VISUALIZE
HOW AN EXOGENOUS PROTEIN CAN
PREVENT INFECTION OF THE SAME
VIRUS FROM OUTSIDE.
FOR EXAMPLE, IN THE TOP DIE
GRAND CANYONDIAGRAMTHERE, IT CAR
FOR THAT PARTICULAR VIRUS AND
THAT CAN OF COURSE BLOCK
INFECTION FROM OUTSIDE, AND THAT
COULD BE EITHER A FULL LENGTH --
GENE IN SOME CASES, THERE IS A
PROTEIN CA WHICH IS JUST A FRAGT
THAT CONTAINS RECEPTOR-BINDING
DOMAIN, AND THEY BOTH DO THE
SAME THING.
ANOTHER CASE, THIS IS WHAT WE'LL
GET TO THIS WITH THU THIS ONE, E
YOU HAVE A SELF ASSEMBLING
PROTEIN THAT CONSISTS OF -- A
VUK TOUR THAT CONSISTS OF MANY
COPIES OF THE SAME MOLECULE.
THAT OCCASIONALLY CAN BE.SOINED
BEPOISONED BY A MUTANT BUT THAT
CANNOT BE ADDED ON TO, OR OTHER
WAYS TO DISRUPT THE STRUCTURE
AND FUNCTION OF THAT STRUCTURE.
VERY STRONG DOMINANT FASHION.
SO YOU GET A TRANS DOMINANT GENE
THAT CAN BLOCK THAT KIND OF
ASSEMBLY.
ONE CASE THAT'S KNOWN, THE
INCOMING VIRUS BLOCKS INFECTION
AND ANOTHER NOAB CASE IS A CASE
OF A VIRUS ACTUALLY RELATED TO
THE ONES I'M GOING TO TALK ABO
ABOUT, THERE'S AN ENDOGENOUS
VIRUS THERE THAT CAUSES THE
ASSEMBLING GAG GENE TO GO TO THE
WRONG PLACE IN THE CELL AND GET
DEGRADED.
THE RETROVIRUSES OVERALL,
THERE'S SOMETHING LIKE EIGHT'
THOUSAND DIFFERENT BITS AND
PIECES OF -- 80,000 DIFFERENT
BITS AND PIECES OF DNA THAT
COMES FROM OUR GENOME FROM THE
PROCESS OF ENDOGENOUS PR
PROVIRUSES, NOT IN OUR DNA, FOR
THE MOST PART THEIR EXPRESSION
IS HIGHLY REGULATED BY BOTH
EPIGENETIC CELL-SPECIFIC
EFFECTS, TRANS EFFECTS, AND SOME
OF THAT PROBABLY REFLECTS TO
SOME EXTENT PRESERVATION, SO IF
YOU CAN LOOK AT THAT, CAN YOU
GET SOME INFORMATION FROM THAT,
ALTHOUGH I'M NOT GOING TO TALK
ABOUT THAT RIGHT NOW.
AT THE TIME OF INTEGRATION OF
THE GENOME, ALL BUT A FEW,
HOWEVER, WERE VIRUS, NOT
TRANSPOSONS.
SO WE ACTUALLY KNOW THAT FOR
SOME VIRUSES BECAUSE IT'S BEEN
SHOWN -- STRONG PURIFYING
SELECTION, MEANING IT'S NEEDED
TO OFFER ALL THE -- THE VIRUS
HAS BEEN GOING BACK AND FORTH
AROUND THE GENOME.
AS FAR AS WE KNOW, THE MOST
RECENT ACQUISITION OF ANY HERV
WAS BY -- OF HML-2s WAS OVER
300,000 YEARS AGO.
THIS IS THE ONLY ONE THAT'S
POLYMORPHIC IN THE GENOME OF
HUMANS.
A LOT OF THE PROVIRUSES STILL
ARE CAPABLE OF BEING EXPRESSED
AND EVEN EXPRESSING PROTEINS AND
EVEN EXPRESSING DEFECTIVE
VIRULENCE.
AND THAT'S OFTEN SEEN IN CANCER
AND VARIOUS OTHER DISEASES.
SOME CANCERS, FOR EXAMPLE, ARE
CHARACTERIZED BY THE COPIOUS
PRODUCTION.
-- ONE SPECIFIC PROVIRUS THAT
HAPPENS TO HAVE A GOOD GAG GENE.
IT'S NOT LIKE ALL OF THEM ARE
BEING EXPRESSED IN ONE DIFFERENT
WAY.
ONE OF THEM HAPPENS TO BE
EXPRESSED BECAUSE OF ITS
LOCATION OR ITS LTR COMPOSITION
TO BE EXPRESSED IN THIS KIND OF
CANCER CELL, AND MAKE COPIOUS
PARTICLES.
THE NOMENCLATURE IS VERY MESSY.
WE REFER TO THEM BY THEIR
CHROMOSOMAL -- ROUGHLY
CHROMOSOMAL LOCATION, I.E., THE
CHROMOSOME BAND THAT THEY'RE IN.
SO THE ONE I WANT TO TALK ABOUT
PARTICULARLY IS ONE AT 38Q12.3,
CHROMOSOME 3 -- 12.3, AND THAT
IS ONE THAT'S REALLY GOING TO BE
THE FOCUS OF THE REST OF THE
TALK.
THIS CHART WAS PUT TOGETHER IN
MY LAB SOME YEARS AGO.
HE WENT THROUGH THE WHOLE HUMAN
GENOME AS A SEQUENCE OF WHAT WAS
KNOWN AT THAT TIME.
AND PULLED OUT ALL THE
PROVIRUSES OF THIS GROUP.
AND THEN LOOKED AT THEM, YOU SEE
A LITTLE BIT OF MARKS ON THE
PROVIRUSES.
THAT'S A MUTATION THAT CAUSES
DEFECTIVENESS OF A PARTICULAR
GENE.
YOU CAN SEE ALMOST ALL OF THESE
ARE AFFECTED ONE WAY OR THE
OTHER.
WITH VERY FEW EXCEPTIONS.
ONE POWERFUL THING HERE IS THAT
WHEN YOU KNOW THIS, YOU KNOW THE
CODING CAPACITY, YOU JUST LOOK
AT THE DATA UNDERLYING IT AND
SEE WHAT THE CODING CAPACITY OF
THIS IS, THEN YOU LOOK AT THE
TUMOR OR SOMETHING, PULL OUT
JUST DO RNA SEQ AND PULL OUT
FRAGMENTS OF RNA TO COME FROM
ANY OF THESE, JUST FROM A FRAG
FRAGMENT OF RNA, YOU CAN TELL
EXACTLY WHAT THE CODING CAPACITY
OF THAT PROVIRUS IS.
IT'S A VERY POWER TESTIMONY TOOL
POWERFUL TOOL TO USETO STUDY EXN
TUMORS AND SO ON.
SO THE PROVIRUS IN PARTICULAR
MARKED BY THE BLUE CIRCLE, I HAD
TO GET A BIT OF NOMENCLATURE,
I'LL SHOW YOU MORE ABOUT THIS IN
A MINUTE, YOU CAN SUBDIVIDE
THESE INTO THREE GROUPS,
TREE IS THAT THESE ARE
INTEGRATED INTO -- THESE
APPEARED IN THE TREE AT A TIME
THAT'S CONSISTENT WITH BEING
INTEGRATED INTO COMMON ANCESTOR
HUMANS AND GORILLAS.
SO THAT'S ON THE ORDER OF
MILLIONS OF YEARS AGO.
THE OTHER ONE, THE 22Q11.3 IS IN
PART ANCESTRAL TO THE -- THIS ON
THE LEFT SHOWS YOU ANALYSIS OF
ALL OF THESE WITH THE SAME COLOR
CODING AS BEFORE, AND YOU CAN
SEE THAT THE PICK ONES, THE
LTR5V, ARE ANCESTRAL TO THE
OTHER TWO GROUPS, THE 5HS IN
BLUE, AND SOMETHING ELSE I HAVE
TO TELL YOU IS THAT PAUL
BIENIASZ'S LAB SOME YEARS AGO
DID A VERY CLEVER EXPERIMENT,
THE VERY, VERY SHORT BRANCHES,
THOSE ARE THE MOST RECENT
INTEGRATED IN OUR DNA.
ON THE PRINCIPLE THAT THESE WERE
ALL INFECTIOUS AT THE TIME THEY
WERE INTEGRATED, SO THEY
PROBABLY ARE ALL VERY CLOSELY
RELATED TO ONE ANOTHER, VERY
SIMILAR TO ONE ANOTHER, AND WITH
ALL THE MUTATIONS THAT MAKE THEM
DIFFERENT HAVE OCCURRED SINCE,
SO THE CONSENSUS SEQUENCE FROM
ALL OF THOSE PROVIRUSES, AND
THEY'RE ABLE TO RECONSTRUCT AN
INFECTIOUS VIRUS FROM THAT,
WHICH IS CALLED HERV KCON, FOR
CONSENSUS, AND THIS IS INDEED
INFECTIOUS PROVIRUS, SO WE'VE
USED THIS FOR QUITE A NUMBER OF
OUR EXPERIMENTS.
WE USE IT IN A WAY THAT OFTEN
HAS IN THE PLACE OF THE ENVELOPE
GENE, THERE'S ACTUALLY BEEN PUT
A GFP OR SOME OTHER MARKER, AND
IT'S MADE BY TRANSFECTING CELLS
WITH THIS CLONE AND BY
TRANSFECTING VSVG ENVELOPE SO IT
CAN BE EASILY USED TO INFECT
CELLS.
SO THIS PARTICULAR PROVIRUS NOW,
WE'RE BACK TO 3Q12.3, HAS A MORE
INTERESTING CHARACTERISTIC.
8 TO 12 MILLION YEARS OLD,
AND -- A COMPLETE GAG PROTEIN,
BUT -- ARE ALL DISRUPTED.
BUT IT HAS GAG.
AND GAG FOR THE NON-RETRO
VIEROLOGISTS, EC EXPRESSION ALOE
IS ENOUGH TO MAKE GOOD LOOKING
VIRUS PARTICLES.
REMARKABLY, THERE IS A FOUR-BASE
PAIR DUPLICATION IN THE 5 PRIME
LTR BUT NOT IN THE 3 PRIME LTR.
THE GOOD RETROVIROLOGISTS WILL
IMMEDIATELY RECOGNIZE THAT
DUPLICATION MUST HAVE OCCURRED
AFTER THE LTRs WERE MADE, MOST
LIKELY AFTER INTEGRATION.
WE DON'T YET HAVE GOOD EVIDENCE
FOR REASONS I DON'T WANT TO GET
INTO, THAT THIS ACTUALLY
HAPPENED RELATIVELY RECENTLY,
PROBABLY WITHIN THE LAST 5 1/2
MILLION YEARS OR SO BUT WE DON'T
KNOW THAT FOR SURE.
IT'S SOMETHING I REALLY WANT TO
KNOW AND SHOULD BE ABLE TO KNOW
BUT WE DON'T KNOW IT YET.
BUT THE OTHER THING, REMARKABLE
THING ABOUT THIS DUPLICATION,
IT'S A DUPLICATION OF A --
SEQUENCE.
THE GATTGATT IS A BINDING SITE
FOR A FAMILY OF TRANS KRIPTION N
FACTORS. TRANSCRIPTION
FACTOR.
IF YOU TAKE AN LTR, SORT OF --
ASSAY, TACK ON LUCIFERASE AND
LOOK FOR LUCIFERASE ACTIVITY AND
YOU THEN REVERT THAT -- YOU TAKE
OUT THE DUPLICATION BASICALLY,
SO THAT GOES BACK TO WHAT --
ALMOST THE ANCESTRAL TYPE IN THE
3 -- YOU SEE THAT GREATLY
REDUCES ACTIVITY.
SO THIS CREATION OF THIS
REDUPLICATION WAS CLEARLY
IMPORTANT IN -- THE EXPRESSION
OF THIS PROVIRUS.
YOU CAN SEE THAT REALLY HERE.
THIS IS A STUDY THAT WAS DONE
PUBLISHED ABOUT A YEAR AGO WHERE
TOP TO BOTTOM ARE ALL THE
DIFFERENT PROVIRUSES,
COLOR-CODED, AND ACROSS ARE ALL
HUMAN TISSUES.
THIS IS A VERY LARGE DATASET OF
EXPRESSION -- OF RNA SEQ
ANALYSIS FROM DIFFERENT HUMAN
TISSUES.
AND WHAT CAN IMMEDIATELY CATCH
YOUR EYE IS 3Q12.3 IS EXPRESSED
IN EVERY SINGLE SOLID TISSUE IN
THE BODY.
IT'S NOT EXPRESSED,
INTERESTINGLY, IN WHOLE BLOOD --
ACTUALLY IS A LITTLE BIT, WE'VE
LOOKED MORE SPECIFICALLY AT
PBMCs, THERE'S A VERY LOW
EXPRESSION BUT YOU DON'T SEE IT
IN WHOLE BLOOD, BUT EVERY OTHER
TISSUE IS EXPRESSED -- THIS IS
THE SAME PROVIRUS WITH THE
INTACT GAG GENE.
IT'S A TINY FRAGMENT, ACTUALLY
NOT EXPRESSED FROM THE LTR, IT'S
EXPRESSED AS PART OF ANOTHER
GENE.
SO THAT'S ONE INTERESTING
FEATURE OF THIS PROVIRUS.
THE SUPPRESSION OF THE GAG GENE
OF EVOLUTION, IF YOU LIKE,
ANCESTRAL -- THERE'S STILL MORE
YET TO COME.
SO IF WE HAVE A LOOK AT THE
TREES THAT RELATE, I SHOWED YOU
ONE OF THESE BEFORE BUT THIS NOW
IS SEPARATED BY GENES.
IF WE NOW RELATE GAG, RT AND
ENVELOPE GENES, WE ALSO SEE
ABOUT THE SAME GENERAL PATTERN,
AND EVERYTHING IS ABOUT THE SAME
PLACE AS YOU MIGHT EXPECT, AND
YOU CAN SEE HOW THE 5B
PROVIRUSES ARE ANCESTRAL IN BOTH
THE 5HS AND 5A.
THE BLUE EARMARKS THE 3Q12.3.
THERE'S A VERY LONG BRANCH LIKE
WITHIN THE GAG GENE, BUT
EVERYTHING ELSE -- BUT -- AND
THAT THE PAL GENE ACTUALLY GOES
WITH 5B PROVIRUSES.
IT'S ACTUALLY RIGHT NEXT TO THE
ONE I SORT OF MENTIONED BEFORE,
22Q11.23.
BUT WHEN YOU GO BACK TO END, YOU
COME BACK TO -- YOU COME BACK UP
TO BEING NESTLED IN WITH THE
OTHER 5HS PROVIRUSES.
YOU'LL NOTICE ALSO THE RED ARROW
POINTS TO 22Q11.3 AND THOSE ARE
RIGHT NEXT TO EACH OTHER, SO IT
SUGGESTS THERE'S BEEN A
RECOMBINATION OF THAT.
THERE WAS AN HISTORICAL
RECOMBINATION EVENT THAT JOINED
THESE TWO PRO VICE PROVIRUSES.
YOU CAN SEE THAT IN THE SEQUENCE
OF THESE TWO, YOU CAN SEE THE
CROSSOVER PATTERN DOWN BELOW AND
YOU CAN SEE THE POINTS OF THE
CROSSOVER.
ABOUT IN THE MIDDLE OF THE GAG
GENE, GO THROUGH 5HS TO MIDDLE
OF THE GAG GENE TO 22Q11.23 AND
BACK TO A 5HS PROVIRUS IN THE
MIDDLE OF THE POL GENE.
THE POSITION IS TURNS OUT THAT
CROSSOVER IN GAG IS ACTUALLY
QUITE IMPORTANT IN THE PHENOTYPE
OF THIS PROVIRUS.
SO IT'S AN OLD PROVIRUS, AND
3 IM. 12-POINT #AND WE'VE GOT AN
HERE FOR DOING SOMETHING
INTERESTING.
SO AGAIN, WE HI HYPOTHESIZE, THS
HAS TURNED INTO A TRANSCRIPTION
FACTOR.
YOU SEE EXACTLY THIS KIND OF
THING HAPPEN, THERE ARE SEVERAL
MECHANISMS I ALREADY DISCUSSED.
AND SO IS THIS, IN FACT -- IS
EXPRESSION OF THIS GAG GENE IN
SOME WAY SUPPRESSIVE OR
REPLICATION OF THE ANCESTRAL
HML-2 VIRUS, WHICH IS NOW
REPRESENTED -- AND WHAT -- IF IT
IS, WHAT STAGE DOES IT WORK, AND
WHAT DOMAINS IN GAG ARE
IMPORTANT AND SO ON.
AND IS IT EVIDENCE, FOR EXAMPLE,
FOR PURIFYING SELECTION OF THIS,
WHICH WOULD BE A CHARACTERISTIC
YOU WOULD EXPECT TO SEE FOR
SOMETHING THAT WAS BEING
STRONGLY SELECTED IN THIS WAY.
SO WE'LL START WITH -- DOESN'T
DO ANYTHING AS FAR AS WE KNOW
BUT IT'S ABOUT THE SAME AGE.
AND ON THE LEFT, ON THE TOP IS
THE WHOLE PROVIRUS, 3Q12.3, AND
YOU CAN SEE ABOVE GAG, THERE'S
ACTUALLY A SPOT.
SO THE PA BARS HERE, WE SCANNEDE
GTEX -- WE SCANNED THE THOUSAND
GENOMES PROJECT WHICH IS
ACTUALLY ABOUT 2500 HUMAN GENOME
SEQUENCES, ABOUT 5,000 SEQUENCES
ACTUALLY, IF YOU THINK EVERYBODY
IS DUPLICATING, AND ON THE -- IS
THE FREQUENCY OF THE -- ALLELE
OF ALL THESE PEOPLE.
SO THESE 5,000 SEQUENCES, LOOK
FOR SEQUENCES THAT ARE DIFFERENT
FROM THE MAJORITY.
THE FREQUENCY OF THOSE, IT GOES
UP -- IT NEVER GOES ABOVE 50%
OTHERWISE THEN IT BECOMES A
MAJOR ALLELE, BUT IT GOES UP
PRETTY CLOSE IN SOME OF THESE.
BUT YOU'LL NOTICE PARTICULARLY
IN THE CAPSID REGION, THE GAG
GENE AT THE BOTTOM, THERE IS NOT
A SY SINGLE DIFFERENT NUCLEOTIDE
AMONG ANYBODY IN THE WORLD AS
FAR AS WE CAN TELL FROM THIS.
LESS THAN 5,000 MUTATIONS HAVE
OCCURRED SINCE THIS WAS LAST
COALESCED, IF YOU LIKE.
SINCE EVERY GENE COMES FROM ONE
PERSON AND AT SOME POINT IN THE
DISTANT PAST, WHICH IS CALLED
COALESCENCE POINT.
SINCE THAT POINT, THERE HASN'T
BEEN A SINGLE CHANGE.
WHICH -- QUITE RECENT.
AND SIMILARLY THE SAME THING IS
TRUE OF THE MATRIX PROTEIN, ALL
THOSE THAT'S FAIRLY SMALL SO
IT'S NOT QUITE AS SIGNIFICANT,
THEN BY COMPARISON, YOU CAN SEE
THIS NON-CODING PROVIRUS OVER --
ON THE RIGHT THAT SHOWS LOTS OF
ACCUMULATION CHANGE.
ALL RIGHT.
SO TO SEE HOW RESEARCH -- FACTOR
WORK, WE'LL TAKE THE FOLLOWING
EXPERIMENTAL APPROACH.
IF YOU -- TRANS FEX WITH THE --
AND AL also you make pseudovirus
with various gag genes, cores of
interest or just gag genes as
the case may be with gag --
and -- and then often with a --
you get infectious voi R virus d
assay, so you do the cotrans --d
do western blots and electron
microscopy and so on in the
cells to see what the proteins
and what the cells look like.
SO THE REST OF THE DATA WILL
SHOW YOU THAT.
SO THIS SHOWS INCREASING AMOUNTS
OF THE WILD TYPE CORE TO THE
MUTANT GAG 3Q12.3 GAG, AND SO
WHERE YOU HAVE -- AND SO WHAT IT
SHOWS IS THAT LOW LEVELS, LIKE G
SUPPRESSION OF THE NOW
INFECTIOUS VIRUS RELEASE, WHEN
YOU COTRANSFECT THOSE UP TO
ABOUT -- A RATIO OF 1 TO 16, I
THINK IT WAS HERE, WHERE WE GET
ABOUT 50% REDUCTION.
SO IT'S VERY STRONG SUPPRESSION
OF RELEASE OF PARTICLES, REVERSE
TRANS SCRIP TRANSCRIPTASE PARTI.
I STOLE ONE OF HIS MOST FAMOUS
DRAWINGS, I WOULD SAY.
FROM THE NON-RETRO VIROLOGISTS
IN THE AUDIENCE, I WANT TO POINT
OUT THE GAG PROTEIN IS ALL
THAT'S IMPORTANT RIGHT HERE AS
FAR AS I'M CONCERNED AT THE
MOMENT.
AND THE GAG PROTEIN CONSISTS
PRINCIPALLY, THERE'S SOME OTHER
BITS AND PIECES I'M NOT GOING TO
TALK ABOUT, OF THREE DOMAINS:
MATRIX MA, CAPSID CA, AND KNEW
CLEE CAPSID NC.
MATRIX INTERACTS WITH THE CELL
MEMBRANE USUALLY.
CAPSID INTERACTS ONE TO ANOTHER
TO FORM THE STRUCTURE -- THE
INTERNAL STRUCTURE -- TO BRING
THE VIRUS RNA -- WHEN IT'S THERE
AND FORMS A CODING ON THE RNA
BASICALLY.
ONE MORE PIECE OF NOMENCLATURE,
THE CAPSID DOMAIN IS DIVIDED --
YOU CAN DIVIDE IT INTO THREE
REGIONS.
NTD --, CTD.
SO THE REGION RESPONSIBLE FOR
THIS PHENOTYPE, HE MADE MADE HYD
STRUCTURES BETWEEN THE WILD TYPE
AND THE 3Q12.3 GAG, AND THEN
LOOKED AT THE ABILITY OF THESE
TO FORM PARTICLES ON THE
COTRA -- THECAPSID DOMAIN IN THS
VERY -- WAS THE SUPPRESSIVE --
DOMINANT SUPPRESSIVE DOMAIN AND
IN PARTICULAR, THE END TERMINAL
HALF OF THE CAPSID DOMAIN, IN
THIS STUDY INCLUDED THE LINGER.
SO THIS REGION FOR THE
SUPPRESSIVE EVENT -- DEFINE THAT
DOWN A LITTLE BIT, THIS NOW
SHOWS THE STRUCTURE OF THE -- OF
CAPSID, AND AGAIN THE INTERNAL
DOMAIN IS IN DARK GREY, THE Y
TERMINAL DOMAIN IS IN LIGHT GREY
AND YOU CAN SEE THE LINGER KIND
OF WINDING IN BETWEEN THEM.
WHEN HE LOOKED AT THAT, JUST
LOOKING AT THE SEQUENCE, THE
BIGGEST DIFFERENCE IS IN THE
MIDDLE OF THE LINGER IN THE
3Q12.3, THERE'S -- OF THE SER
SERINE.
IT'S WHAT ALLOWS THE FLEXIBILITY
OF THE CAPSID PROTEIN TO
ASSEMBLE AND THAT'S WHAT ALLOWS
THE VIRUS TO FORM THIS RATHER
INTERESTING SORT OF SPACE
CAPSULE-LIKE STRUCTURE WHERE
EVERY SINGLE HEXAMER, WHICH IS
THE -- SIX OF THESE IS A
FUNDAMENTAL UNIT, HAS TO BE AT A
SLIGHTLY DIFFERENT ANGLE WITH
RESPECT TO ONE ANOTHER.
SO THERE HAS TO BE SOME
FLEXIBILITY IN THE WAY THEY
INTERACT.
I THINK -- I BELIEVE AND I THINK
MOST PEOPLE BELIEVE THAT'S
ALLOWED BY THE PRESENCE OF THIS
LINGER SCWEANS.
SEQUENCE.
SO THE DELETION -- MIGHT WELL
CHANGE THE WAY THE CAPSID IS
ALLOWED TO FORM.
SO TH THIS EXPERIMENT SHOWS
DIRECTLY THAT IF YOU SUBSTITUTE
EITHER THE WHOLE LINGER THAT'S
THE THIRD BAR FROM THE LEFT, YOU
GET A VERY -- YOU GET A STRONG F
YOU JUST TAKE THE WILD TYPE GAG
GENE AND JUST DELETE THAT ONE
SERINE, YOU ALSO GET IN THIS
EXPERIMENT ABOUT A 54 OR SO
SUPPRESSION OF EXPRESSION.
SO THAT -- IT TURNS OUT IF YOU
TAKE THAT -- IF YOU REPAIR THE
SERINE DOMAIN IN 3Q12.3, THAT
ACTUALLY DOES NOT COMPLETELY
RECALL THE DEFECT SO THERE'S
SOMETHING ELSE GOING ON BUT THIS
IS CERTAINLY THE PRINCIPAL
REASON FOR THIS.
GIVEN THAT, WE TOOK ADVANTAGE OF
OUR ASSOCIATION WITH DRP AND
WITH OUR VERY GOOD FRIEND ALLAN
WHO'S ASSOCIATED WITH A VERY
GOOD ELECTRON MY CR MICROSCOPISE
PASSED THEM ON TO FERRI AND TOOK
SOME PICTURES.
SO THE TOP LINE HERE IS JUST THE
WILD TYPE, IT ACTUALLY MAKES
VERY NICE PARTICLES.
THIS IS JUST THE GAG PROTEIN, SO
THERE'S NO PROTEASE, SO YOU'RE
JUST GETTING MATURE PARTICLES IN
THE ABSENCE OF PROTEASE.
THEY'RE VERY NICE LOOKING
IMMATURE PARTICLE, TRUST ME.
S, TRUST ME.
THE SECOND LINE HAPPENS IF YOU
TRY AND COTRANSFECT IT WITH
ITSELF.
SO YOU SEE AGAIN, IT WORKS VERY
WELL.
SO NOW WHAT HAPPENS IF -- WHAT
HAPPENS NOW WITH THE 3Q12.3 GAG
ALONE, AND WHEREAS YOU DON'T SEE
ANY OF THOSE NICE BUDDING
PARTICLE, ALL YOU SEE ARE ONE OF
THE ARROWS THERE, YOU JUST SEE
THICKENINGS IN THE CELL
MEMBRANE, SO SORT OF TRYING TO
ASSEMBLE AND COULD ASSEMBLE BUT
IT COULDN'T CURVE ITSELF AROUND.
THEN ON THE SECOND LINE, YOU SEE
WHAT HAPPENS IF YOU NOW
COTRANSFECT WITH THE WILD TYPE,
AND NOT ONLY YOU GET ITSELF
AROUND, BUT NOW YOU FORM SOME
VERY ABERRANT LOOKING
STRUCTURES.
THESE ARE WHAT WE LIKE TO CALL
VIRAL UNLIKE PARTICLE, VUPs,
AS OPPOSED TO VIRAL LIKE
PARTICLES OR VLPs.
AND SO -- AND NOW IF YOU NOW
TAKE THE HS GAG AND PUT IN THE
CAPSID FROM 3Q12.3, AGAIN YOU
GET THE SAME COLLECTION OF
PHENOTYPES, AND THEN HOW IT
LEADS AGAIN TO THESE VUPs IN
THE C COTRANSFECTION EXPERIMENT.
>> [INAUDIBLE]
>> THE ANSWER IS NO.
BECAUSE OTHER MUTATIONS THAT ARE
ALSO INVOLVED IN THIS THAT WE
HAVEN'T TRACKED DOWN YET.
BUT IF YOU MUTATE THE WILD TYPE
SERINE, YES, IT DOES BUT WE
DON'T HAVE PICTURE OF THAT.
SO WHATEVER THE OTHER DEFECT IS,
IT'S STILL IN CAPSID BECAUSE YOU
PUT THE WILD TYPE CAPSID BACK
INTO 3Q12.3, NOW YOU GET WILD
TYPE BUDDING AGAIN.
SO NOW WHAT I SAID BEFORE IS THE
CAPSID DOMAIN OF 3Q12.3 IS
IDENTICAL EXCEPT FOR ONE AMINO
ACID THAT'S ALMOST CERTAINLY
IRRELEVANT, ANOTHER PART THAT'S
NOT PARTICULARLY INTERESTING TO
US RIGHT NOW, SO THE QUESTION
IS, DOES 22Q11.23 HAVE THE SAM
SAME -- CAN THAT ALSO SUPPRESS
KKAN.
I SHOWED YOU THIS BEFORE.
THE ANSWER, IF YOU LACK, IS NO.
THIS DOES NOT -- THE ANSWER IS
IF YOU PUT GAG CAPSID PROTEIN --
I'M SORRY, IF YOU PUT THE CAPSID
PROTEIN INTO HS GAG THEY'RE
MOVING CAPSID PROTEIN HAS THE
SAME PHENOTYPE.
IT'S BASICALLY THE SAME PROTEIN.
HOWEVER, IF YOU TAKE 22Q11.23
ITSELF AND EXPRESS GAG AND
EXPRESS IT, IT DOES NOT SUPPR
SUPPRESS.
SO WHAT'S GOING ON HERE?
WHAT'S GOING ON HERE IS REVEALED
IN ANOTHER ELECTRON MICROGRAPH,
AND THAT IS THAT IF YOU LOOK ON
THAT -- I KNOW I DON'T HAVE A
GOOD PICTURE OF IT, BUT IF YOU
LOOK AT THE CELL MEMBRANE, YOU
DON'T SEE ANYTHING.
IN HOW WE'RE EXPRESSING THIS
ANCESTRAL PROVIRUS FOR THE GAG
GENE IN 3Q12.3.
WHAT YOU DO SEE INSIDE THE CELL
ARE LOTS AND LOTS OF CAPSIDS,
IMMATURE CAPSIDS.
THESE -- AS SOME OF YOU OLD TIME
VIROLOGISTS REMEMBER, THESE ARE
CALLED A PARTICLES.
AND IT IS A KNOWN ROOT OF -- IN
FACT MOUSE MEMORY TUMOR VIRUS,
FOR EXAMPLE, OR MASON PFIZER
MONKEY VIRUS, ASSEMBLY AT THE
CELL MEMBRANE, INTRACELLULARLY
INTO THESE A PARTICLES, MAKE
THEIR WAY TO THE CELL MEMBRANE
AND BUD OUT AND GET PROCESSED.
SO THIS IS ACTUALLY A BIG
SURPRISE, WE'VE KNOWN ABOUT THE
DIVISION OF SOME OF THESE
VIRUSES FOR YEARS AND YEARS AND
YEARS, NOBODY LOOKED TO SEE WHAT
THE GAG PROTEIN ACTUALLY MADE
AND WHERE THEY WERE.
AND WHY IS THAT?
WELL, NOW WE LOOK AT THE MATRIX
PROTEIN.
THE MATRIX PROTEIN COMES FROM
THE 22Q11.23 TYPE B PARENT.
WE CAN SEE A VERY BIG DIFFERENCE
IN THE NUMBER OF BASIC AMINO
ACID RES KNEWS.
THE BASE NUMBER -- NUMBER OF
BASIC AMINO ACID RESIDUES.
THERE ARE MANY FEWER, A FEW NOR
ACIDIC RESIDUES, AS COMPARED TO
THE 5HS.
DOWN A BOTTOM YOU CAN SEE THE
CALCULATED PI OF THESE PROTEINS.
FOR THE MATRIX IS LTR5A -- L
LTR -- I GOT THEM REVERSED.
DO A LITTLE RECOMBINATION THERE.
IT'S 9.122 ROUGHLY AND FOR 5A OR
B -- I'M SORRY, THEY'RE RIGHT.
THEY'RE RIGHT.
IT'S -- NO, THEY'RE WRONG.
IT'S 4.8.
IT GOES FROM BEING BASIC TO
BEING ACIDIC BASICALLY.
SO THESE BASIC RESIDUES INTERACT
WITH THE ACIDIC PHOSPHATE HEAD
GROUPS OF PET 2 AT THE CELL
MEMBRANE AND ARE WHAT DRIVE THE
CELL MEMBRANE ASSOCIATION.
ERICA COULD LECTUI COULD LECTURR
HOURS.
SO THIS CERTAINLY IS TH THE
REASON -- THE FACT THEY ASEMI AE
IN THE CYTOPLASM MEANS THEY
NEVER GET TO INTERACT WITH THE
GAG PROTEIN, BUT THEY PROBABLY
DID RESTRICT -- I'LL COME BACK
TO THAT IN A MINUTE -- IT
PROBABLY WILL RESTRICT A VIRUS
THAT REPLICATED THROUGH THE SAME
MECHANISM AS MANY --
RETROVIRUSES DO, FOR EXAMPLE,
MAMMARY TAU M TUMOR VIRUS.
SO, FINALLY, LET'S LOOK AND
SATISFY SEEWHAT VIRUSES THESE D.
YOU CAN SEE HERE WHAT HAPPENS,
THE EMPTY VECTOR EXPRESSION OF
3Q12.3 GAG HS, THE 5HS GAG,
22Q11.23 GAG, AND YOU CAN SEE
THAT -- MLV AND ALV ARE NOT
TOUCHED BY ANY OF THAT'S,
THEY'RE OBVIOUSLY BUDDING
INDEPENDENTLY SO YOU DON'T
INTERACT WITH THIS PROTEIN.
HOWEVER, MASON -- MONKEY VIRUS
WHICH ALSO -- IS SUPPRESSED BY
THE 3Q12.3.
IN FACT, WE POSTULATE THAT THIS
SUPPRESSIVE GENE EVOLVED OR AT
LEAST WAS STRONGLY SELECTED BY
ITS RESISTANCE TO THE VIRUS AS
REREPLICATED IN THIS WAY.
SO -- BUT THEN NOW OF COURSE --
FOW LOOK AT THE GUERILLA IN THEE
ROOM HERE, HIV WHICH I AVOIDED
TALKING ABOUT UNTIL NOW, HIV IS
ALSO STRONGLY SUPPRESSED BY
THIS -- BY THE
3Q12.3 COEXPRESSION.
OKAY?
AND HERE'S JUST ANOTHER PLOT
THAT SHOWS THE DOSE-RESPONSE TO
THAT.
ON THE LEFT-HAND PLOT.
ALV, I THINK.
>> YOU SAID THIS WAS
UBIQUITOUSLY -- NOT T CELLS?
>> QUITE A LOW LEVEL.
IT WASN'T SEEN AT ALL IN WHOLE
BLOOD IN GTEX.
SO YES, GOOD POINT.
SO WHY ARE WE INFECTED BY HIV?
I COULD ARGUE THAT THE
EXPRESSION IN T CELLS IS JUST
TOO LOW FOR THAT TO HAPPEN.
>> [INAUDIBLE]
>> WE HAVEN'T LOOKED AT THE
EXPRESSION.
THAT'S A VERY GOOD QUESTION BUT
WE HAVEN'T LOOKED AT THE
EXPRESSION LEVEL IN -- CELLS.
THOSE ARE, OF COURSE -- I DON'T
KNOW.
IS THE ANSWER TO THAT.
WE DON'T KNOW, WE'RE IN THE
PROCESS ACTUALLY OF PUTTING THIS
IN FRONT OF A TUNEABLE PROMOTER
SO WE CAN SEE WHAT LEVEL OF
EXPRESSION IS NECESSARY.
THESE ARE WAY -- CMV LINK
PROMOTER.
SO, AGAIN YOU CAN SEE THE HRV ON
THE RIGHT-HAND PANEL, THERE YOU
GET THE MESSY PUV PHENOMENON
THEN, SUGGESTING THE MECHANISM
OF SAME.
AND QUITE STRIKINGLY, THIS IS A
PAPER THAT APPEARED FROM JENG'S
LAB RECENTLY, AND THIS IS THE
C-TERMINUS OF GAG AND THIS IS
THE ASSEMBLY OF THE IMMATURE GAG
PROTEINS FORMS, ERIC CAN TALK TO
YOU FOR HOURS ABOUT THIS ONE
TOO.
AND IN ORANGE IS THE HML-2 AND
GREY IS THE HIV1, AND LOOK HOW
THOSE OVERLAY.
SO LOOKING AT THIS, THIS
REALLY -- I MEAN, IT'S NOT --
IT'S EASY TO HYPOTHESIZE, WE'RE
NOT PROVING IT BUT IT'S EASY TO
HYPOTHESIZE THESE MIGHT
COASSEMBLE VERY WELL.
EVEN THOUGH -- THERE ARE SOME
CONSERVED AMINO ACID SEQUENCES
BETWEEN THEM AND MAYBE IN THE
IMPORTANT PLACES WE HAVEN'T DONE
A REALLY DETAILED ANALYSIS, BUT
CERTAINLY WORKING HYPOTHESIS IS
WHAT HAPPENS HERE, THESE
COASSEMBLE BECAUSE OF THIS VERY
STRONG SIMILARITY IN WHAT HAVE
THE IMPORTANT -- I THINK WE'LL
ALL AGREE IS THE IMPORTANT
COASSEMBLY POINT, ASSEMBLY POINT
FOR IMMATURE VIRUS PARTICLES.
AND I THINK THIS IS MY LAST
SLIDE, JUST TO SHOW YOU, SO FROM
THIS, WE CAN INFER QUITE A BIT
AND HYPOTHESIZE QUITE A BIT
ABOUT HOW THE EVOLUTION OF THIS,
WHAT CLEARLY HAS TO BE A
RESTRICTION FACTOR.
THERE'S NOTHING ELSE THAT CAN
REALLY EXPLAIN ALL THAT WE HAVE,
I THINK.
WHAT HAPPENED IS YOU STARTED OUT
WITH BOTH POINTS 22Q23.3 AND
3Q12.3 WERE INTEGRATED AT SOME
TIME WITHIN THIS 4 MILLION
YEAR -- OF COURSE THEY COULD
HAVE BEEN 2 MILLION YEARS APART,
3 MILLION YEARS APART, WE CAN'T
TELL THAT, BUT OUR WORKING
HYPOTHESIS IS THAT THE MUTATION,
THE KEY GAG MUTATION AROSE IN
THE 22Q11.23 AND WAS STRONGLY
SELECTED SO BY GIVING RESISTANCE
TO A VIRUS LIKE THE 5A OR 5B
TYPES THEY'RE REPLICATING
THROUGH THIS INTRACELLULAR
ASSEMBLY.
AND THAT PERHAPS IN RESPONSE TO
THAT, I CAN REALLY SPECULATE
LIKE CRAZY NOW BECAUSE THIS IS
THE SPECULATION PART OF THE
TALK, AS A RESPONSE TO THAT TO
FIND HS TYPE, NOW FOUND A WAY
AROUND IT BY PUTTING IN ALL
THOSE BASIC AMINO ACIDS WHICH
GAVE IT THE PLASMA MEMBRANE
INTERACTION, SO IT SNEAKED ITS
WAY AROUND WHAT WAS NOW THIS
RESTRICTION FACTOR.
SO THIS IS A HOST OF -- THIS IS
SORT OF THE HOST-VIRUS -- IT
TOOK PLACE LIKE 10 MILLION YEARS
AGO.
AND THEN 3Q12.3 IS THE ONLY
REIMI NENT -- IN OUR BODY, IN
OUR GENOME, PICKED UP THAT GAG
GENE AND THAT THEN WAS SELECTED
BECAUSE NOW THAT WAS ABLE TO
RESIST INFECTION BY -- THAT WAS
ABLE TO RESIST INFECTION BY THE
HML-2 TYPE AND 5HS TYPE AND --
BUT ONLY ACTUALLY WHO'S
EXPRESSED WELL ENOUGH, AND SO AT
SOME POINT, WHICH I THINK
ACTUALLY -- OUR INITIAL DATA
SUGGESTED THAT THE FOUR BASE
DUPLICATION THAT CREATES THIS
VERY STRONG PROMOTOR FOR THE OX
PBX FACTOR -- SITE HAPPENED IN
THE HUMAN LINEAGE.
AND I THINK -- WHAT I WOULD LOVE
TO THINK, THIS IS NOW REAL
SPECULATION, IS THAT HAPPENED
ONLY QUITE RECENTLY, AND IT WAS
THE OCCURRENCE OF THAT MUTATION
THAT GAVE THIS VIRUS ENOUGH
RESISTANCE POWERS, WE BOOSTED
THE LEVEL OF EXPRESSION HIGH
ENOUGH SO THAT IT WAS NOW -- IT
NOW HAD THE PATTERN THAT YOU SEE
IN VIRTUALLY EVERY CELL IN THE
BODY.
VERY INTERESTINGLY, NOT IN
LYMPHOCYTES APPARENTLY.
AND THAT'S WHAT -- ACTUALLY ONE
REASON BECAUSE HIV IS SENSITIVE
TO IT, THAT SORT OF DIRECTED THE
INFECTION LEVEL OF HIV BUT
THAT'S GETTING A LITTLE TOO FAR
AFOOT, BECAUSE HIV HAD THAT
BEFORE IT SAW HUMANS, OF COURSE.
SO THAT I THINK IS THE END OF MY
STORY, AND I WILL THANK ALL OF
THESE PEOPLE VERY MUCH.
PARTICULARLY MIKE FREEMAN,
REALLY DID ESSENTIALLY --
VIRTUALLY ALL OF THE WORK EXCEPT
FOR THE ELECTRON MICROGRAPHS,
AND I WILL THANK YOU FOR YOUR
ATTENTION, I'LL THANK EVERYBODY
FOR A REALLY WONDERFUL DAY.
[APPLAUSE]
>> SO -- MATRIX HAS A --
RETENTION SIGNAL.
I'M WONDERING IF THERE'S ANY
ELEMENT IN THIS --
>> THEY'RE PROBABLY IS.
WE HAVEN'T DONE -- IT COULD --
INCLUDES THAT, WE HAVEN'T DONE
ANYTHING -- IT'S A GOOD POINT.
AS I RECALL IT WAS VERY EASY TO
CONVERT IT BACK TO -- RIGHT.
ALTHOUGH THIS ONE, WITH ALL OF
THOSE -- ON THAT VERY HIGH LOW
P.I., IT VERY HARD TO SEE HOW IT
COULD GET BACK TO THE PLASMA
MEMBRANE.
MARY.
>> SO PRESUMABLY, WE HAVE HIV
BECAUSE IT'S NOT EXPRESSED IN
LYMPHOCYTES AND --
>> IT COULD BE DRK CERTAINLY
COULD BE ONE THING THAT ALLOWED
HIV TO --
>> THERE'S OTHER RETROVIRUSES
WHICH MAY HAVE JUMP AS MANY
TIMES BUT NOT BEEN ABLE TO CAUSE
A PANDEMIC.
>> MIGHT WANT TO THINK ABOUT
WHAT WOULD HAPPEN IF WE COULD
EXPRESS IT.
>> KIND OF GOING ON MARY'S
QUESTION, SO A LOT OF
RETROVIRUSES INFECT AND
REPLICATE IN --
>> NOT THAT MANY.
THEY'RE MOSTLY --
>> --
>> MLV -- I DON'T -- REPLICATE
IN T CELLS?
OH, YEAH.
IN LYMPHOCYTES.
NLCs, IT IMMUNE TO THIS.
I SHOWED YOU THAT.
>> OH, OKAY.
>> ANY VIRUS THAT -- WHERE THE
GAG GENE IS CAPABLE OF ASSEMBLY.
>> THERE SHOULD HAVE BEEN AT
SOME POINT -- MAYBE THERE'S NOT
ENOUGH TIME TO MAYBE TURN ON
EXPRESSION OF LYMPHOCYTES.
>> WELL, HIV HAS NOT PUT -- FOR
A HUNDRED AND SOME YEARS.
>> -- AND VIRUSES LIKE THAT.
>> WE HAVE OTHER MECHANISMS TO
PROTECT US AGAINST -- WE DIDN'T
HAVE AGOVEK, WE'D PROBABLY ALL
BE DEAD FROM IT BY NOW.
>> YOU'RE FOCUSED ON RESTRICTION
FACTORS BUT -- SPHERICAL BALLS
IN THE CELL -- DO A HECK OF A
LOT OF OTHER THINGS.
SO NOT OTHER -- MAYBE 22?
>> WELL, PERHAPS.
THAT COULD BE.
>> FROM YOUR PERSPECTIVE --
>> WHEN WE LACK CLOSELY AT
THOSE, YOU PROBABLY COULDN'T --
VEVERY EASILY -- THERE'S AN
OPENING AT THE END.
BECAUSE THEY STILL HAVE THAT
SAME MUTATION, THAT SERINE
DELETION IN THEM, SO MY GUESS IS
THEY CAN FORM A SPHERE IF
THEY'RE SORT OF UNIMPEDED BUT
WHEN THEY HAVE TO PULL THE CELL
MEMBRANE AROUND WITH THEM, IT'S
JUST NOT A STRONG ENOUGH
ASSOCIATION TO DO THAT.
THERE'S SOME INTERACTION
PROBABLY THAT SERINE -- THAT IT
NEEDS TO FORM THE -- SOME EFFORT
NEEDED IF YOU LIKE ON THE PART
OF -- TO ACTUALLY FORM THAT
SCWEER.
FRANK, I THINK?
>> JUST REAL QUICK, YOU SAID IT
WASN'T EXPRESSED IN HUMAN --
WHAT ABOUT IN THE GORILLA OR --
>> WE HAVEN'T LOOKED AT THAT.
>> IS IT POSSIBLE IT'S
PREVENTING TRANSMISSION FROM
OTHER MAYBE LOWER PRIMATES?
>> THIS DOESN'T GO VERY LOW.
THESE PARTICULAR -- THESE PLR
PROVIRUSES ARE IN GORILLAS,
HUMANS, CHIMPANZEES, AND -- BUT
THAT'S IT.
>> BUT WOULD IT PREVENT IT
FROM -- BUT WHO IT PREVENT
THINGS COMING OVER FROM RHESUS?
>> POSSIBLY.
OH, IT WOULD PREVENT THINGS
THAT -- THINGS THAT WERE CAPABLE
OF COASSEMBLY WOULD BE BLOCKED.
>> SO THEY WOULD BE UNDER A GAG?
>> SO IF THEY WERE -- OTHER
RELATIVES OF HERV-K, AND THERE
ARE -- LOWER PRIMATES IF YOU
LIKE, MONKEYS AND SO ON, THAT
PROBABLY WOULD COASSEMBLE AND IT
PROBABLY WOULD BE --.
>> SO -- HAS A QUESTION ONLINE.
CAN YOU JUST UNMUTE AND ASK?
>> SURE.
I WAS JUST WONDERING, DOES THIS
MEAN THAT IF YOU HAVE
COINFECTION FROM -- RETROVIRUSES
A CELL THAT THEY WOULD RESTRICT
EACH OTHER IF THEIR GAG PROTEINS
ARE CLOSE ENOUGH?
>> IT'S UNLIKELY THE TWO
RETROVIRUSES JUST INDIVIDUAL
PARTICLES INFECTING THE SAME
CELL WOULD EVER MEET.
THEY'RE VERY -- THE CELL IS VERY
BIG, THE -- ARE QUITE SMALL.
WHAT WE DON'T KNOW, WE JUST
HAVEN'T DONE IT YET, IS WHETHER
IT IS ACTUALLY -- THE EXPRESSION
OF THIS GAG PROTEIN HAS AN
EFFECT ON INCOMING VIRUS.
BECAUSE THERE'S POSSIBLE.
PROTEIN FRAGMENT THAT DOES HAVE
AN IMPACT ON ONCOMING VIRUS.
SO WE JUST DON'T KNOW THAT.
WE'LL TRY TO GET THE EXPERIMENT
DONE SOON.
>> YOU SAID THE NTD IS HAVING AN
EFFECT?
>> THAT WAS NTD PLUS LEAKER IN
THAT EXPERIMENT.
THE DATA SUGGESTS THAT THERE IS
SOME OTHER -- THERE ARE SOME
OTHER MUTATIONS INVOLVED AS
WELL.
WE DON'T HAVE THOSE SORTED OUT
YET.
>> TO DECEMBER TE THAO T THESE A
LOT OF HOST FACTORS.
>> [INAUDIBLE]
>> RIGHT.
>> SO WILL THEY -- DONE
ENDOGENOUS RETROVIRUSES ARE
EASIER TO INFECT?
>> THAT'S ACTUALLY -- THERE'S A
HISTORY -- BLAME IT ON CHICKENS.
WHERE THE EXPRESSION OF
ENDOGENOUS RETROVIRUSES DOESN'T
LOSE TOLERANCE, BUT IT ACTUALLY
MAKES INCOMING VIRUSES -- IT
OBVIOUSLY HELPS THE REPLICATION
OF INCOMING VIRUSES, BUT IN THAT
PARTICULAR CASE, IT MAKES THE
MOST PATHOGENIC, BECAUSE THE
MAJOR PATHOGENIC EFFECT OF THIS
PARTICULAR VIRUS IS -- MEDIATED,
SO YOU GET IMMUNE RESPONSE
AGAINST VIRUS, THAT KILLS OFF
IMPORTANT CELLS WHEN YOU GET
SICK.
SO IT CAN WORK BOTH WAYS.
IN THIS CASE, I JUST DON'T KNOW.
>> THE FINAL LAST QUESTION?
>> HAVE YOU KNOCKED DOWN THE
EXPRESSION IN DIFFERENT CELLS
AND TRIED TO INFECT HIV?
DOES IT CHANGE THE TROPISM?
>> WE'RE AT THE VERY BEGINNING
ON THAT KIND OF EXPERIMENT.
YOU SEE MOST ALL THE DATA WE
HAVE ON THAT.
THOSE ARE ALL ON THE DRAWING
BOARD.
TURNING IT DOWN, TURNING IT UP,
SO ON, SO FORTH.
>> CAN I ASK ONE LAST?
>> THANK YOU.
>> THERE'S SOMEONE ONLINE.
>> GOT ANOTHER ONE.
>> ONLINE, GO FOR IT.
>> I JUST -- YOU MENTIONED
LYMPHOCYTES AND YOU SAID THEY'RE
NOT EXPRESSED IN T-CELLS.
WHAT ABOUT MACROPHAGES?
>> WE DON'T KNOW.
WE'VE LOOKED AT -- THERE'S WHOLE
BLOOD IN GTEX AND THEN WE'VE
LOOKED AT PDMCs, TOTAL
PDMCs, WE LOOKED AT THAT
PURIFIED CD4 CELLS, JUST -- I
THINK JUST NAIVE CELLS IS WHAT
WE HAPPENED TO HAVE AROUND IN
THE LAB AT THE TIME WE DID THAT.
SO WE HAVEN'T DONE AN EXHAUSTIVE
SEARCH ON THAT.
WE HAVEN'T LOOKED AT MACROPHAGES
OR OTHER CELL TYPES.
THAT, WE WILL CERTAINLY DO, BUT
WE HAVEN'T DONE THAT YET.
>> OKAY.
THANK YOU.
BECAUSE A LOT OF THE --
>> WHERE DO WE PICK UP OUR GIFT
BAGS OF CRANBERRIES?
>> IT'S ABOUT 400 MILES NORTH OF
HERE.
>> ALL RIGHT.
I THINK WE'RE AT THE END OF THE
DAY.
THANK YOU VERY MUCH.
[APPLAUSE]
>> SO I'LL BE VERY BRIEF.
IT BEEN A REALLY FANTASTIC DAY.
I HOPE EVERYBODY ENJOYED IT AS
MUCH AS I DID.
I THINK A FEW THANKS ARE IN
ORDER.
I'D LIKE TO THANK ALL THE
SPEAKERS FOR FANTASTIC TALKS.
[APPLAUSE]
AND ALL OF YOU FOR A GREAT
DISCUSSION AND STAYING UNTIL THE
END.
THANK YOU FOR OUR ONLINE
AUDIENCE.
I'D LIKE TO THANK OUR
ADMINISTRATIVE STAFF FOR
LOGISTICS AS WELL AS THE
CONFERENCE PLANNERS.
I THINK A LOT OF YOU PROBABLY
GOT EMAILS FROM TERRY AT SOME
POINT ALONG THE WAY.
WE APPRECIATE THAT HELP.
[APPLAUSE]
AND I HOPE YOU'LL JOIN US FOR
UPCOMING AND FUTURE DRP EVENTS.
NEXT UP IS THE DERSEY MEMORIAL
SYMPOSIUM WHICH WILL BE NEXT
THURSDAY.
YOU CAN ATTEND IF YOU'RE LOCAL
IN PERSON OR ONLINE IF YOU'RE
NOT LOCAL.
SO AGAIN, THANKS VERY MUCH, AND
ENJOY THE REST OF YOUR DAY. 8666 05:58:08,567 [APPLAUSE]