>>HELLO, EVERYONE. I WANT TO
THANK EVERYONE FOR COMING. IT
IS GREAT TO SEE EVERYONE IN
PERSON FOR A CHANGE. I WANT TO
GIVE A FEW COMMENTS ABOUT THE
CENTER THAT THE CENTER WAS
ORIGINALLY ORGANIZED TO GET
COLLABORATIONS GOING AND GET
PEOPLE TALKING ABOUT HIV AND
CANCER VIROLOGY. THIS YEAR
VINAY AND I WERE ASKED TO BE
PART OF IT WE ARE WELCOMING
SUGGESTIONS AND COMMENTS AND
IDEAS AND WOULD LIKE TO GROW THE
CENTER GETTING PEOPLE MORE
INVOLVED AND COMMUNITY ORIENTED.
GIVING A FEW LOGISTICS. MAIN
SEMINARS ARE IN THIS ROOM.
THERE IS AN OVERFLOW ROOM G1 AND
G2 AND THERE ARE REFRESHMENTS
THERE. FEEL FREE TO COME AND GO
IN THE ROOMS AND POSTER SESSIONS
THIS AFTERNOON WILL START AT 1
O'CLOCK IN THE LEFT ROOMS C1/C2
AND AB AND PRESENTERS AND JUDGES
WILL COME AROUND AND LOOK AT
YOUR POSTERS. PLEASE BE THERE
AT 1 O'CLOCK. SCHEDULE IS
TIGHT. LET KEEP THINGS MOVING.
WITH THAT I WILL HAND IT TO VEFA
WHO WILL INTRODUCE DREW TO HIS
TALK UP.
>>RESILIENCE AND CONTINUOUS
RIGOROUS EFFORT. THESE PAPERS
ARE RIGOROUS AND SCIENCE IS
BASIC SCIENCE BUT SHOULD BE AN
INCREDIBLE EXAMPLE FOR YOU YOUNG
PEOPLE IF YOU HAVE A PASSION AND
IDEA YOU WANT TO PURSUE, YOU
SHOULD REALLY DO IT. YOU NEVER
KNOW WHETHER IT WILL WORK WELL
OR NOT AND DREW SAID THIS
MORNING, IT COULD ALSO NOT HAVE
WORKED AND I WOULD HAVE SPENT
ALL THESE YEARS TO WORK.
ONE MORE THING TO TELL YOU.
DREW TRAINED AT NIH WITH TONY
FAUCI AND CAREER TOOK OFF
TREMENDOUSLY IN THE LAST SEVERAL
YEARS AND OBTAINED AN IMPORTANT
PRIZE LIKE JAPAN PRIZE AND
ALASKA PRIZE AND THOSE THAT GET
ALASKA PRIZE MIGHT GET A NOBLE
PRIZE AND HAPPY HE AGREED TO
TALK WITH YOU YOUNG PEOPLE. HE
TOLD ME THIS MORNING HE LIKES TO
TALK TO YOUNG PEOPLE. REALLY,
YOU ARE THE FUTURE FOR SCIENCE
AND THANK YOU DREW FOR COMING.
THANK YOU VERY MUCH FOR INVITING
ME. I TOLD BETHEL THIS MORNING
THAT ONE THING I PUT HIGH ON MY
LIST IS TALKING TO YOUNG
SCIENTISTS.
YOU -- YOU KNOW, IT IS GREAT, US
OLD FOLKS, TO BE DOING RESEARCH.
WE ARE HAPPY TO BE RECOGNIZE THE
FOR THAT. OUR MAIN JOB REALLY
IS THAT OUR CRITICAL JOB IS
TRAINING YOUNG SCIENTISTS NOT
ONLY TO TAKE OVER FOR US BUT TO
ADVANCE SCIENCE AND BE THE
FUTURE OF SCIENCE. SO, THANK
YOU FOR INVITING ME HERE.
I WANTED TO TALK TO YOU FIRST
ABOUT THE VACCINES. I WANTED TO
CONCENTRATE ON HOW THE VACCINES
WORK.
PEOPLE DON'T REALIZE THE
MECHANISMS THAT ARE BEHIND THE
POTENCY OF THE RNALNP VACC-LNP .
I WILL TALK ABOUT THE
BACKGROUND. I TALK TO LAY
AUDIENCES AND ONE THING THEY ARE
GUARANTEED TO TELL ME IS I DON'T
WANT TO TAKE THE RNA VACCINE
BECAUSE IT WAS INVENTED IN 10
MONTHS AND I DON'T TRUST IT.
THAT IS, OF COURSE, RIDICULOUS.
MRNA WAS DISCOVERED IN 1961.
FIRST TIME IT WAS INJECTED INTO
ANIMALS WITH IDEA OF USING IT TO
DELIVER THERAPEUTIC PROTEINS WAS
IN 1990. AFTER THAT, IT WAS
MAINLY STUDIED IN AN UNUSUAL
VACCINE PLATFORM TAKING
DENDRITIC CELLS OUT OF ANIMALS
AND OUT OF HUMANS AND PULSE THEM
WITH MRNA AND GIVE THEM BACK.
THAT MRNA COULD BE IN VITRO
TRANSCRIBED OR PURIFIED FROM
TUMOR CELLS OR A VARIETY OF
DIFFERENT SOURCES.
THE REASON THAT WAS DONE WAS
BECAUSE OF THIS. THERE ARE
CURRENTLY 7 TAENDIFFERENT INNATE
IMMUNE CENSORS THAT RECOGNIZE
RNA AND THEY PRODUCE A VARIETY
OF MEDIATORS INCLUDING
INFLAMMATORY CYTOKINES AND TYPE
1 INTERFERONS AND DIRECTLY
INHIBIT TRANSLATION BY ENACTING
ON RIBOSOMES AND INDUCE
APOPTOSIS AND CELL DEATH. LATER
WHEN KATIE AND I STUDIED MRNA,
WE WOULD INJECT IT INTO AN
ANIMAL WHICH WOULD GET SICK.
THIS GREATLY, OF COURSE, UPSET
KATIE.
YOU CAN'T MAKE SOMEBODY SICK
WHEN YOU GIVE THEM A DRUG. I
WAS LESS CONCERNED BECAUSE
STUDIES OF IT SHOWED REASONS
THAT THEY GOT SICK WAS BECAUSE
OF INNATE IMMUNE CENSORS AND
THEY RECOGNIZE THE IN VITRO
TRANSCRIBED RNA INDUCING ADJUNCT
ACTIVI
ACTIVITIES.
WE WANTED TO DEVELOP AN RNA THAT
DIDN'T MAKE ANIMALS SICK. WE
HAD TO STUDY WHY RNA WAS SO
INFLAMMATORY AND TELLING
EXPERIMENT AFTER 7 YEARS OF
WORK, WHAT WE DID WAS MAKE A
VARIETY OF DIFFERENT TYPES OF
RNA FROM BACTERIA MAMMALIAN
CELLS THAT AT THE TIME WERE THE
WAYS THAT YOU COULD SUBDIVIDE
RNA FROM A CELL. WHEN WE TESTED
THEM ON DENDRITIC CELLS THAT
HAVE MOST INNATE IMMUNE CENSORS,
THE TWO POSSIBILITIES WERE THAT
ALL OF THE RNAS WOULD BE
INFLAMMATORY AND THAT IS BECAUSE
RNA IS INFLAMMATORY. THIS IS
THE ALTERNATIVE. IT SURPRISED
US. CERTAIN TYPES OF RNA,
PARTICULARLY TRANSFER RNAS WERE
NOT INFLAM.
THAT LED US TO A HYPOTHESIS.
TRANSFER RNAS HAVE 25% OF THIR
NUCLEOSIDES ARE MODIFIED. THERE
IS OVER 100 DIFFERENT TYPES AND
THEY ARE PUT IN AFTER RNA IS
MADE BY THE CELL BY VARIETY OF
RNA-DRIVEN ENZYME SYSTEMS THAT
ARE DONE IN A SEQUENCE-SPECIFIC
MANNER OF THESE ARE ICE
OMERIZATIONS LIKE URIDINE AND
METHYLATIONS OF THE BASE OR OF
THE SUGARS AND DIFFERENT
ADDITIONS OF AMINO ACIDS AND
COMPLEX MODIFICATIONS THAT OCCUR
IN CONSERVED POSITIONS.
THAT MEANS IF YOU LOOK AT A
BACTERIAL RIBOSOMAL RNA, ITS
MODIFICATION IS AT THE SAME TIME
OF A MAMMALIAN RIBOSOMAL RNA AND
THEY HAVE OBVIOUS BIOLOGICAL
FUNCTIONS, MANY OF WHICH WE
DON'T KNOW. M6A WAS RECENTLY
SHOWN TO BE REVERSIBLE. OUR
PROBLEM WAS WE HAD TO MAKE
MODIFIED M RCHLT NRNA.
MOST OF THE ENZYMES FOR
MODIFICATION WEREN'T UNDERSTOOD.
EACH IF THEY WERE LIKE PSEUDI
YOU'RE IDDINE MODIFICATION
SYSTEMS THEY WERE RNA DEPENDENT.
OUR IDEA WAS LET'S TRICK
THEPHAGE HOPPIM RACE. YOU TAKE
A DNA TEMPLATE THAT HAS A
PROMOTER RECOGNIZED BY A PHAGE
RNA POLYMERASE. AFTER IT BINDS
MAKING AN RNA COPY OF THE DNA
TEMPLATE. IT FALLS OFF AND
KEEPS DOING IT OVER AND OVER AND
OUR THOUGHT WAS COULD WE
EXCHANGE COGNATE NUCLEOTIDE
TRIPHOSPHATES FOR MONOSIDE
NUCLEOTIDE TRIPHOSPHATES?
WE TRIED ABOUT 10 DIFFERENT
MODIFIED NUCLEOSIDE
TRIPHOSPHATES THAT WERE
AVAILABLE AT THE TIME. FIVE OF
THEM WORKS THAT WE TESTED ON
DENDRITIC CELLS. SOME HAD
REDUCED INFLAMMATORY ACTIVITY
THAT WAS DONE AT THE TIME ON
PRIMARY DC. SO, PURIVEFIED FROM
LEUKO FORESIS SAMPLES FROM
HUMANS.
WHAT IS INTERESTING IS ONLY
URIDINE MODIFICATIONS WORKED AND
WE AND OTHERS LATER FOUND OUT
THAT URIDINE IS CRITICAL FOR
INFLAMMATORY INFLAMMATION
RECOGNITION BY THE CENSORS.
WE HAD A SURPRISE. DO MODIFIED
RNAS, HERE, 100% OF THE YEWS OF
YOU'RI SEER ADEAN AND
[INDISCERNIBLE] COULD THEY WE
TRANSLATED? NO. WE WERE WRONG.
WASN'T ONLY TIME THAT OUR
HYPOTHESIS WAS WRONG, BUT NOT
ONLY WAS IT TRANSLATED BUT IT
WAS TRANSLATED MUCH BETTER BY
CELLS, ESPECIALLY PRIMARY CELLS
THAT GAVE AN RNA THAT WASN'T
INFLAMMATORY THAT DIDN'T MAKE
ANIMALS SICK AND MADE MUCH MORE
PROT
PROTEIN.
I WOULD GO TO MEETINGS AND TALK
TO TONY AND SENIOR PEOPLE IN THE
FIELD. THEY WOULD LISTEN TO MY
RESEARCH SAYING THAT IS VERY
NICE. NOBODY WILL EVER USE RNA.
IT FAILED CLINICAL TRIALS
EARLIER ON. WHY DON'T YOU DO
SOMETHING USEFUL WITH YOUR LIFE?
WHY DON'T YOU TRY DOING RESEARCH
THAT MIGHT ACTUALLY HELP THE
FIE
FIELD.
I'M A VERY CAN QUIET GUY AND
WOULD SAY THANK YOU AND TRY TO
ARGUE WITH HIM POINTING OUT IN
PHARMACEUTICAL DEVELOPMENT
PROTEIN THERAPEUTICS ARE MOST
RAPIDLY GROWN FIELD MONOCLONAL
AUTO BODY THERAPIES AND THEY
KEEP DEVELOPING MORES.
TO MAKE THESE DRUGS, YOU START
WITH A 50,000 LITER DRUM FULL OF
KHO CELLS OR OTHER CELLS AND
HAVE TO FIGURE OUT HOW TO PURIFY
THAT PROTEIN AWAY FROM THE CELL
CULTURE CONTAMINANTS.
EVERY NEW DRUG OR PROTEIN YOU
START OVER AND MAKE A NEW CELL
AND FIGURE OUT HOW TO GROW IT
AND HOW TO PURIFY IT. THE COST
OF A PROTEIN THERAPEUTIC FOR
THESE MONOCLONALS ARE IN
TYPICALLY 50 TO $100,000 A YEAR
RAN
RANGE.
THEY ARE COSTS MANY IN THE
UNITED STATES AND ACROSS THE
WORLD CAN'T AFFORD. MY ARGUMENT
IS RNA IS A SIMPLE REACTION THAT
DOESN'T USE MAMMALIAN CELLS BUT
ENZYME AND MODERNA AND BIOTECH
WHEN USING THEIR VACCINE USE 100
LITER REACTORS THAT THEY MAKE AS
MUCH VACCINE OF 50,000 LEADERS
OF CHO CELLS MAKING ADENO
VIRUSES OR PROTEINS. IT IS
SIMPLE. ONLY THING YOU HAVE TO
CHANGE IS THE CODING SEQUENCE
WHEN YOU WANT TO MAKE A NEW
PROTEIN OR VACCINE. YOU DON'T
HAVE TO REINVENT ANYTHING. YOU
PLUG IN THE NEW DNA TEMPLATE AND
EVERYTHING IS THE SAME IDT
REACTION, PURIFICATION, LIPID
COMPLEXING IS IDENTICAL. IT IS
SIMP
SIMPLER. IT IS QUICK. YOU CAN
RAPIDLY EXPAND THESE, AS WE HAVE
SEEN.
IT IS SAFER. INSTEAD OF HAVING
A CHO CELL MAKE YOUR PROTEIN,
SOMETIMES IT DOESN'T QUITE
MODIFY THE PROTEIN OR DOESN'T
GLYCOSCILLATE CORRECTLY AND BAD
EVENTS HAVE HAPPENED FROM THAT.
HOST CELLS MAKE THE PROTEIN.
HOST CELLS KNOW HOW TO MAKE
PROTEINS OFF OF RNA. IT SHOULD
BE GREATLY REDUCED COST AND EASE
OF PRODUCTION. THE PROBLEM WAS:
HOW DO YOU DELIVER THE RNA?
JUST ABOUT EVERY FLUID AND CELL
IN OUR BODY HAS RNASES IN IT.
IF YOU INJECT UNCOMPLEX NAKED
RNA INTO AN ANIMAL OR HUMAN, 30
SECONDS LATER, IT IS GONE. IT
IS DEGRADED.
WE LOOKED AT PROBABLY 50
DIFFERENT FORMULATIONS DESCRIBED
FOR DELIVERY NUCLEIC ACIDS
IDENTIFYING LIPID NANOPARTICLES
AS BEING THE BEST. LNPS WERE
DEVELOPED FOR SRNA DELIVERY THAT
WAS DRIVEN BY A COMPANY AD-NILUM
WITH 3FDA APPROVED DRUGS AND
FOUR LIPIDS SELF-ASSEMBLING WITH
RNA FORMING 60 TO 100
NANOPARTICLES AND THEY HAVE NO
CHARGE. THEY ARE NOT TOXIC AND
BIND APOE AND GO TO THE LIVER
AND FOUND THAT FOR MRNA THEY
WORK BEST AND WE P MAKE THIS
LUCIFERASE CODEINE AND
LUCIFERASE ACTS ON SUBSTRATE AND
YOU CAN GIVE MICE THAT AND THEY
WILL GLOW. YOU STICK THEM UNDER
A CAMERA AND MEASURE LIGHT
PRODUCTION. WE GAVE LUCIFERASE
RNALMPS BY VARIETY OF INJECTION
ROOTS. EVERYTHING WAS SYSTEMIC
CIRCULATION THAT WENT TO THE
LIVER AS EXPECTED AND WAS
TRANSLATED FOR A DAY AND
DISAPPEARED. WHAT WAS
INTERESTING FROM AN
VACCINOLOGIST POINT OF VIEW WHEN
WE INJECTED PERIPHERALLY IDIM
SUB Q WE HAD PROTEIN INJECTION
AT SITE OF THE INJECTION AND
DRAINING LYMPH NODES FOR ABOUT
10 DAYS.
KNOWING VACCINES, BEST VACCINES
ARE TYPICALLY LIVE VIRUS THAT
INFECT CELLS AND PRODUCE PROTEIN
FOR A FEW DAYS LOADING GERMINAL
CELLS.
PROTEIN THERAPEUTICS LIKE TET
ANNIS TOX YOID, YOU PUT THEM IN
A FEW HOURS LATER AND THEY ARE
GONE.
WE LOOKED AT RNA-LNP MODIFIED
VACCINES. SHOWING YOU HOW EASY
IT IS, FOR COLLABORATORS, EASY
IS A SIMPLE THING TO PUT ON THE
SLIDE. THERE IS LOTS OF SCIENCE
THAT GOES BEHIND IT. WHEN YOU
HAVE SEQUENCE OF IMMUNOGENERAL,
IN EARLY JANUARY OF 2020 WHEN
CHINESE RELEASED COVID-19
SEQUENCE, WE TOOK THE SPIKE AND
STARTED TO MAKE RNA VACCINES
WITH IT. YOU DON'T NEED THE
VIRUS OR PROTEINS JUST THE
SEQUENCE TO MAKE AN RNA VACCINE.
YOU CODON OPTIMIZE SEQUENCE TO
INCREASE PROTEIN PRODUCTION
MAKING A D-DNA TEMPLATE AND
STICK IN REACTION WITH PHAGE RNA
POLYMERASES AND YOU HAVE RNA TWO
HOURS LATER. -- MIX IT WITH
LIPIDS AND ETHANOL
SELF-ASSEMBLING INTO
NANOPARTICLES AND FILTER AWAY
ETHANOL IN SALTS AND STICK IN A
VILE AND YOU MADE A VACCINE. IT
IS VERY QUICK AND EASY. WE HAVE
A ROBOT THAT CAN MAKE 96RNAS AT
A TIME. WE CAN MAKE 1,000 RNAS
IN A COUPLE DAYS VERY EASILY.
THAT IS HOW MODERNA AND BIOTECH
MAKE VACCINES ON A BIGGER SCALE
OF 100 LITER BIOREACTORS
STARTING FIRST WITH HIV AND
INFLUENZA. I WILL SHOW YOU
INFLUENZA BECAUSE THEY WERE
SUCCESSFUL. INFLUENZA IS A
SEASONAL RESPIRATORY DISEASE
MAINLY IN THE WINTER TIME THAT
KILLS ABOUT 30 TO 60,000 PEOPLE
A YEAR IN THE UNITED STATES.
THERE IS PANDEMIC YEARS THAT ARE
MUCH MORE SEVERE THAT MAINLY
KILLS PEOPLE OVER AGE OF 65 AND
PEOPLE WITH IMMUNE DEFICIENCIES.
THIS IS A REVIEW OF VACCINES IN
DEVELOPMENT WHEN WE WORKED ON
RNA VACCINES I WANTED TO SHOW
YOU IN INBRED MOUSE DRAINS WITH
H1N1 VIRUS TWO IMMUNIZATIONS OF
A MOUSE GIVE TIGHTERS PSEUDONEWT
TRILLIZATION TIGHTER OF AROUND
100. IT THAT IS PRETTY GOOD. 1
TO 40 IS PROTECTED. WHEN WE DID
OUR ARE. NA VACCINE THAT IS AN
MRNA THAT ENCODES HRMN MOLECULE
IN LIPID PROTOCOLS IN A MOUSE
GAVE A TIGHTER OF -- 5 TIMES
BETTER THAN A LIVE VIRUS
INFECTION. I GAVE SERUM TO
SCOTT HENSLY WHO IS AT PENN AND
TOOK HIM A MONTH TO GIVE ME THE
RESULTS WHEN HE FINISHED HE WAS
UPSET AND STORMED IN MY OFFICE
AND SAID WHAT DID YOU DO? DID
YOU PUT MONOCLONAL ANTIBODIES
INTO THE SAMPLES I HAVEN'T SEEN
TIGHTERS THIS HIGH. TOOK A
MONTH HE HAD TO DO DILUTION
AFTER DILUTION TO MEASURE
TIGHTERS.
THIS DATA IS REAL AND EXCITED
COMPANIES LIKE MODERNA AND
BIOTECH AND PFIZER AND OTHERS
BECAUSE IT WAS A SIMPLE AND
INCREDIBLY POTENT VACCINE. AS A
BASIC SCIENTIST, I WANTED TO
KNOW HOW IT WORKED. THIS IS HOW
B CELLS RECOGNIZE ANTI-GENERALS,
VIRUSES, PARTICLES, ET CETERA.
B CELLS PROLIFERATE RAPIDLY
SHIFTING BETWEEN LIGHT AND DARK
CELLS OF GERM CENTER AND
AFFINITY MATURE AND CLASS SWITCH
AND FORM PLASMA CELLS AND MEMORY
B CELLS.
SO, WE MEASURED THESE
POPULATIONS.
THIS IS AN ANTI-GENERAL SPECIFIC
MANAGER AND ADDED THIS TO FLOW
COCKTAIL AND STAINED FOR THE B
CELL POPULATIONSGEN SPECIFIC
MANAGER AND ADDED THIS TO FLOW
COCKTAIL AND STAINED FOR THE B
CELL POPULATIONS.
WE SAW A 50-FOLD INCREASE IN
ANTIBODY TIGHTERS.
WE FORGOT ABOUT MICE FOR A YEAR
AND WENT BACK AND LOOKED AT BONE
MARROW WHERE LONG-LIVED PLASMA
CELLS RESIDE AND MEASURED
HA-SPECIFIC PLASMA CELLS AND
TIGHTERS AND NUMBERS WERE
ENORMOUS, .05% OF NUCLEATED
CELLS IN BONE MARROW WERE MAKING
HAA ANTIBODY THAT IS MORE THAN
ANY PROGENITOR CELL IN THE BONE
MARROW.
IT DIDN'T EXPLAIN HOW THE
VACCINE WAS WORKING AND WE HAD A
PROBLEM. RNA HAD NO ADJUNCT
ACTIVITY MAKING NO INFLAMMATORY
CYTOKINES OR STIMULATED
RECEPTORS OR INNATE IMMUNE
CENSORS AND LNPS AT THE TIME
DIDN'T BIND INNATE IMMUNE
CENSORS AND WEREN'T THOUGHT TO
HAVE AJEFFANT ACTIVITY. WHERE
WAS IT COMING FROM VACCINES
REQUIRE AJIFIENTS YOU HAVE TO
STIMULATE IMMUNE SYSTEM TO MAKE
AN IMMUNE RESPONSE FROM
ANTIBODIES WE WERE SEEING WE
THOUGHT T FOLLICULAR CELLS WERE
INVOLVED IT IS A CD4 HELPER THAT
MAKES GERMINAL CENTERS IF YOU
DON'T HAVE THEM A FEW DISEASES
WHERE THEY ARE DEAFISHIENT YOU
DON'T MAKE GERMINAL CENTERS
MAKING LOUSY ANTIBODY RESPONSES
THEY SUPPLY HELP TO GERMINAL
CENTER B CELLS ALLOWING HIGH
AFFINITY B CELLS TO PROLIFERATE
AND EXPAND AND LOW AFFINITIES
DIE OUT. THAT IS THEIR JOB.
THEY DRIVE AFFINITY INCREASES
AND CLASS SWITCH AND FORMATION
OF N STAGE MEMORY CELLS.
WE DID A COMPARISON. WE DID AN
MRNA-LNP ENCODING HIV ENVELOPE
IMMUNOGEN AND COMPARED TO
IDENTICAL ENVELOPE PROTEIN
ADGENTLEMAN VENTEDED WITH DOUBLE
STRANDED RNA A POTENT T INDUCING
ADGENTLEMAN VENTS YOU CAN SEE NO
COMPARISON. THEY PRODUCED
ENORMOUS LEVELS OF THHS IN
SINGLE VACCINATION IN MONKEYS
AND RABBITS AND FERRETS AND
CHICKENS AND PIGS AND FISH.
EVERY IMAGINABLE ANIMAL SPECIES
WE HAVE VACCINATED SO FAR AND
HUMANS.
WE DID A CALCULATION IN A
TYPICAL ADGENTLEMAN VEPTED
VACCINE WITH ANY A SERIES
ADGENTLEMAN VENTS 5% OF CD4
HELPERS ARE TFHS AND RNA-LNPS IS
OVER 50%. THIS IS AN AGENTLEMAN
VENT THAT DRIVES THF RESPONSES
THAT DRIVE ANTIBODY RESPONSES
AND WHY VACCINE TIGHTERS ARE 5
TIMES HIGHER THAN CONVALESCIENT
PATIENT ANTIBODY LEVELS.
WE MADE THIS INFLUENZA VACCINE
WITH TIGHTERS THAT WERE THROUGH
THE ROOF. WE WANTED TO
UNDERSTAND HOW GOOD WAS IT?
INFLUENZA, WE MAKE A NEW VACCINE
EVERY YEAR. HEAD OF THE HA
MUTATES. IT AVOIDS LAST YEAR'S
VACCINES AND IMMUNE RESPONSES
AND THERE ARE PANDEMIC STRAINS
THAT ARE H5 AND H7 AND A FEW
OTHERS AND OCCASIONALLY SHOW UP
IN THE WORLD WITH NO IMMUNITY IN
THOSE AND CAUSES A WORLDWIDE
INFECTION AND WE IMMUNE IIZE WI
H1 AND HAD 100% PROTECTION.
HEADS AND STRAINS.
ANIMALS LOST NO WEIGHT AND
SURVIVED THE CHALLENGE.
REASONING WAS RNA-LNP VACCINES
MAKE RESPONSES AGAINST
SUBDOMINANT EPITOEPS WITH IMMUNE
SYSTEM THAT DOESN'T RESPOND TO
IT C
IT. GERM NAL CENTERS ARE BIG
THAT SUBDOMINANT RESPONSES CAN
SURV
SURVIVE.
MECHANISMS OF IT WERE TWO MAIN
ONES.
FIRST, THEY INDUCED POTENT TFH
RESPONSES. SECOND IS THEY
PRODUCED IMMUNOGEN FOR UP TO 10
DAYS LOADING GERM NAL CENTERS TO
MAKE A GOOD B CELL RESPONSE. INO
MAKE A GOOD B CELL RESPONSE.
WE HAVE WORKED ON 30 + PATHOGENS
WITH COLLABORATORS AROUND THE
WORLD WE WANT TO IMPROVE
IMMUNOGENS WE ARE DELIVERING AND
KNOW THAT OTHERS HAVE SHOWN THAT
VRPS VIRAL PARTICLES ARE BETTER
IMMUNOGENS THAT ARE SECRETED OR
CELL SURFACED PROTEINS AND
PROBLEM IS HOW DO YOU MAKE A VLP
WITH MRNAS? WE HAVE DONE IT A
FEW DIFFERENT WAYS AND NEWEST IS
FOR HCV AND NUMBER OF TRIALS
WITH IT THAT HAVE FAIL AND
DIDN'T MAKE STRONG ENOUGH AND
CROSS-REACTIVE RESPONSES.
HCV IS AN RNA VIRUS WITH A
SINGLE RNA PRODUCT CLEAVED BY
CELLULAR AND ENVIRONAL PROETASS
AND ARE NORN STRUCTURAL AND CORE
STRUCTURAL PROTEINS AND THERE IS
ONE PROTEIN OF INTEREST CALLED
P7. IT LOOKS LIKE AN ION
CHANNEL.
IT WAS ASSOCIATED WITH VIRAL
VIRION FORMATION AND IF YOU TOOK
IT OUT, VIRUS INFECTED JUST AS
WELL. OUR IDEAS AND IDEA OF A
GRAD STUDENT IN MY LAB IS
INCLUDING P7 IN MRNA WOULD MAKE
BETTER VIRAL PARTICLES IN A
BETTER VACCINE. SHE MADE TWO
RNAS ONE WITH CORE E12 ONE
WITHOUT P7 AND PUT THEM INTO A
VARIETY OF CELL TYPES THAT ARE
HELPA TOMORROWA CELLS WE HAVE
DONE SAME IN DENDRITIC CELLS AND
WHEN P7 WAS PRESENT, AMOUNT OF
SECRETED C PROTEIN OR CORE
PROTEIN WAS MUCH HIGHER. THIS
SUGGESTED THAT WE WERE GETTING
BETTER VLP RELEASE FROM VIRUSES.
WE LOOKED AT IT WITH CRYO-EM.
YOU CAN'T QUANTITY TATE WITH
CRYO-EM. THEY ARE SAMPLE IMAGES
AND RED ARROW ARE VHPS FOR HCLV
AND LOOKING AT NUMBERS THERE IS
ABOUT 5 TIMES MORE VLPS WHEN P7
WAS PRESENT AND PUT INTO MOUSE
SYSTEMS LOOKING AT IMMUNOGENISTY
AND SAW HIGHER TIGHTERS AND
CROSS-REACTIVITY WITH P7
CONTAINING IMMUNOGEN. THEY ARE
GOING FORWARD TOWARDS HUMAN
CLINICAL TRIALS THAT WE HAVE
ALSO DONE THIS FOR HIV.
EVERYBODY KNOWS WE HAVE BEEN
WORKING ON HIV VACCINES FOR 30
YEARS TI TRIAL HAD A SIGNAL THAT
MAYBE COULDN'T BE REPLICATED IN
THE AFRICAN TRIAL IN MY VIEW WE
HAVEN'T MADE A VACCINE THAT MADE
A STRONG PROTECTIVE RESPONSE.
THAT IS A GOAL RETIRING HEAD OF
NAID HAS MADE IT A GOAL HIS
ENTIRE CAREER. OUR IDEA WAS
LET'S MAKE VLPS WITH HIV VACCINE
AND THAT WAY YOU HAVE ENVELOPE
ON VIRAL PARTICLE THAT WILL BE
MORE IMMUNOGENIC AND YOU HAVE
GAG CD4 AND CD8 RESPONSES IT
THAT WILL AID IN PROTECTION AND
WE COMBINE TWO RNAS, ONE FOR HIV
OR SIV GAG AND ONE FOR HIV
ENVELOPE AND PUT THEM INTO CELLS
COLLECTING SUPER-NATANT AND
STAINED FOR GAG AND ENVELOPE.
HERE, THIS IS HUMAN PLASMAS
MEASURING HIV GAG AND SEE GAG IN
ENVELOPE PRESENT. THIS IS SIV
GAG SIRA AND SEE SIV GAG AND
ENVELOPE PRESENT AT HIGH LEVELS.
WE WERE ABLE TO MAKE A SECRETED
VIRAL PARTICLE THAT EXPRESSED
GAG AND ENVELOPE AND SHOWING
THAT WE HAVE VIRAL PARTICLES BUT
THIS IS PRETTY CONVINCING.
BUT, HIV IS NOT SO
STRAIGHTFORWARD. THE PROBLEM
IS, IS THAT THE ENVELOPE IS
ESSENTIALLY LOOKING AT IT AS A
BAG OF JELL-O THAT IS A
CONSTANTLY MOVING STRUCTURE THAT
IS OFTEN IN CONFIRMATIONS THAT
HAVE NON-NEUTRALIZING EPITOEPS
AND WE AND OTHERS HAVE LOOKED AT
WAYS OF STABILIZING THE ENVELOPE
SO IT RETAINS BROADLY
NEUTRALIZING EPITOEPS AND
DOESN'T EXPRESS DISTRACTING
EPITOEPS.
THIS IS A SERIES OF 5
MODIFICATIONS.
WE STARTED WITH AN ENVELOPE.
CH848 AND IT IS A CHILD D
PATIENT FROM AFRICA WHO MADE
STRONG GLYCAN BNAB RESPONSE.
THEY FOLLOWED THE PASH YENTD FOR
YEARS IDENTIFYING VIRUSES AND
ANTI-VIRUSES OVER TIME MAKING A
BNAB RESPONSE AND DT ARE V1
GLYCANS THAT HAVE BEEN REMOVED
FROM THE IMMUNOGEN ALLOWING IT
TO BIND TO UCA, UNMUTATED COMMON
ANCESTOR OF THE ANTIBODY WHICH
IS HOW YOU START AN ANTIBODY
RESPONSE AND WE ADDED A SERIES
OF MUTATIONS FILLING GLYCAN
HOLES AND PUT MUTATIONS LOCKING
DOWN CD4 AND LOCKING DOWN V3 AND
V1 AND V2. THIS IS THE SERIES.
WE TOOK OUT THE CLEAVAGE SITE.
WE STUDIED THEM FOR
ANTIGENICITIY. TOP 3 ARE COMMON
BROADLY NEUTRALIZING ANTIBODIES
RECOGNIZING EPITOEPS AT BINDING
SITE AND 7 TAEN-B IS A CDI SITE.
374 IS A LINEAR V3 EPITOEP.
BOTH OF THESE ARE BAD. THESE
ARE DISTRACTING EPITOEPS.
WE HAVE DONE A VARIETY OF CODON
OPTIMIZATIONS TO THE RNA.
WE DID THIS TO TEST FOR MAKING
THE MOST ENVELOPE. YOU CAN
CLEARLY SEE THAT CODON
OPTIMIZATION IS IMPORTANT. RED
IS ENVELOPE-BINDING WITH THESE
DIFFERENT ANTIBODIES.
GOING THROUGH THE SERIES OF
MODIFICATIONS, WE WERE ABLE TO
GREATLY INCREASE AMOUNT OF BNAB
BINDING TO THEM. WE WERE ABLE
TO GET RID OF THE CD4I SITE. WE
HAVEN'T BEEN ABLE TO GET RID OF
THE V3 SITE. V3 IS STILL
STICKING OUT IN THIS IMMUNOGEN
AND WE ARE CONTINUING WITH MORE
MODIFICATIONS TO GET RID OF V3.
WE HAVE REDUCED IT BUT HAVEN'T
GOTTEN RID OF IT YET. THIS
IMMUNOGEN [INDISCERNIBLE] TO
INDUCE BNAB RESPONSES HOPEFULLY
AND WE DO IT WITH MANY HIV
ENVELOPES WE MAKE. PLAN IS TO
MAKE ENVELOPES THAT STIMULATE AT
LEAST THREE DIFFERENT BNAB
EPITOEP. CD4 INTERFACE V3
GLYCAN V1 AND V2NPER TO PUT THEM
TOGETHER INTO A FINAL VACCINE
THAT WILL HOPEFULLY PROTECT
PEOPLE. I WILL STOP THERE AND
TAKE A FEW QUESTIONS BEFORE I
MOVE ON TO THE SECOND PART OF MY
TALK.
>>HI. THANK YOU SO MUCH. THIS
IS SO INTERESTING. I REMEMBER
IT THAT I VISITED PENN A FEW
YEARS AGO TO GIVE A TALK AND YOU
MET WITH ME AND SHOWED ME YOUR
DATA AND I WAS BLOWN AWAY. I
HAD A QUESTION. YOU KNOW, YOU
SHOWED US HOW IN THE ANIMAL
MODELS THAT THE DURATION WAS
LONGER THAN ANTIBODIES COMPARED
TO OTHER VACCINE PLATFORMS AND
MY QUESTION IS BASICALLY WHY DO
YOU THINK IT IS SO SHORT IN
HUMANS WITH COVID-19 VACCINES?
>>I DON'T THINK IT IS SO SHORT
BUT WHAT HAS HAPPENED IS THAT
VARIANTS HAVE APPEARED AND
PROBLEMS IS THAT EVERY VARRANT
IS LESS SENSITIVE TO THE
VACCINE.
YOU KNOW, DELTA VARIANTS WERE
ABOUT 30 OR 35% LESS SENSITIVE
AND OMICRONS ARE 80% LESS
SENSITIVE AND ONLY WAY WE HAD TO
ADDRESS THAT WAS TO BOOST THE
OVERALL TIGHTER.
IDEA WAS IF AT BASELINE THEY ARE
20% SENSITIVE, IF YOU GIVE A
BOOSTER YOU CAN INCREASE IT TO
50 OR 60%.
WE WERE BOOSTING THE OLD
ANTIBODIES THAT BARELY
NEUTRALIZED.
BOOSTERS ARE NOT BECAUSE
DURABILITY IS POOR BUT VARIANTS
KEPT APPEARING. WE NEEDED A LOT
OF ANTIBODY TO PROTECT AGAINST
THEM.
>>I PRESUME WE ARE NOT LOOKING
AT RIGHT REDIAL. IT IS NOT
NECESSARILY SERUM TIGHTERS BUT
PLASMA BLASTS OR B CELLS THAT
PROBABLY WOULD STAY LONGER BUT
ARE NOT NECESSARILY ACTIVELY
PRODUCED?
>>HUMAN VACCINES, WAY THAT YOU
DETERMINE WHEN YOU NEED A
BOOSTER IS FOLLOW PEOPLE WHEN
THEY GIVE A DISEASE AGAIN YOU
GIVE A BOOSTER AND FOR TET AN US
IT IS 10 YEARS AND SMALL POX IT
IS A LIFETIME. WE WON'T KNOW
WITH COVID BECAUSE OF ALL
BOOSTERS WE HAVE GIVEN.
>>COULD YOU COMMENT ON YOUR
EXPERIENCE ON THE ABILITY OF
DIFFERENT IMMUNOGEN TO ACTUALLY
GIVE YOU THIS LONG-LIVED PLASMA
CELLS OR -- SO, DO YOU KNOW WHAT
-- YOU MUST HAVE DONE
COMPARATIVE STUDIES.
>>YEAH. IT IS REALLY TWO
PROBLEMS.
IF YOU LOOK AT GERMINAL CENTERS
AFTER RNA-LNPS WE HAVE DONE IN
MICE AND MCKAKS. GERMINAL
CENTERS LAST 3 OR 4 + MONTHS
GERMINAL CELL ACTIVITY CONTINUES
FOR A LONG TIME. WHAT WE HAVE
SEEN WITH HIV IS WE GET A BIG
INCREASE IN ENVELOPE-BINDING
ANTIBODIES AND LOOKING AT BNABS
WE SEE AN INCREASE AND THEY
START TO DROP FAIR WILL I
QUICKLY AND LIKELY IS GERMINAL
CENTERS CONTINUING TO MUTATE
THOSE CELLS AND ARE LOSING BNAB
SPECIFICITY OVER TIME. LOOKING
AT ZIKA IN MCCABBINGS WE
IMMUNIZED ONCE AND FOR A YEAR
TIGHTERS NEVER DROPPED. THERE
IS LOTS OF IMMUNOGEN INFLUENCE
ON DURABILITY OF THE RESPONSE
FOR MANY REASONS.
PART OF IT IS SOME IMMUNOGENS
INDUCE NOT VERY DURABLE
RESPONSES AND FLU IS
CHARACTERISTIC FOR THAT SOME
IMMUNOGENS GERMINAL CENTER
REACTION YOU LOSE BNAB BROADLY
NEUTRALIZING RESPONSES YOU WANT
BECAUSE OF BNAB AND GC ACTIVITY.
>>WE DON'T KNOW WHY?
>>WE DON'T KNOW WHY YET.
ALL RIGHT. I WILL MOVE ON. SO,
WHAT I WANTED TO TALK TO YOU
ABOUT NEXT WAS THE FUTURE OF RNA
THERAPEUTICS.
WE HAVE GOT A LOT OF VACCINES.
WE HAVE 7 PHASE 1 CLINICAL
TRIALS GOING ON RIGHT NOW FOR
HIV, INFLUENZA, GENITAL HERPES,
MALARIA, CDIF, NEUROVIRUS AND
MORE ON THE WAY.
WE HAVE BEEN LOOKING AT NEW WAYS
OF USING RNA.
RNA CAN BE USED TO DELIVER
PROTEIN THERAPEUTICS.
IT CAN TREAT ACUTE AND CHRONIC
DISEASES.
TO ME, MOST IMPORTANTLY IT CAN
MEDIATE GENE THERAPY SAFELY.
BIGGEST PROBLEM WITH RNA AND ANY
NUCLEIC ACID THERAPEUTIC IS
DELIVERY GETTING THEM TO CELLS
OF INTEREST AND LNPS GO TO THE
LIVER AND THERE HAS ALREADY BEEN
A PHASE 1 TRIAL DELIVERING CAS9
IN A MODIFIED RNA-LNP FOR
TRANSPI REITANT AM ILLOID DOSIS
THAT WAS SUCCESSFUL. LNPS GO TO
THE LIVER AND TRANSFAT 98% OF
THE CELLS AND GAVE GOOD
KNOCKOUT. WHAT IF YOU WANT TO
DELIVER TO BONE MARROW STEM
CELLS OR T CELLS OR LUNGS OR
HEART OR BRAIN WE DON'T HAVE A
WAY OF DOING THAT. THERE IS NOT
A LIPID SYSTEM THAT DOES THAT
WELL.
SO, MY LAB YEARS AGO STARTED TO
STUDY WAYS OF TARGETING LNPS TO
OTHER TISSUES, ORGANS AND CELLS
AND WE CONCENTRATED ON THREE
TYPES OF TARGETING FOR
ENDOTHELIAL AND T CELLS AND BONE
MARROW STEM CELLS.
APPROACH THAT WE TOOK WAS TO
MODIFY THE SURFACE OF THE LIPID
NANOPARTICLE. LNPS HAVE A DSPC
OR OTHER CARRIER LIPID PLASMA
MEMBRANE-LIKE STRUCTURE.
IMBEDDED IN THAT ARE PEGGOLATED
LIPIDS AND INSIDE OF IT ARE CATI
ONIC LIPIDS BINDING RNA AND
CHOLESTEROL THAT STABILIZES WE
ADDED ANTIBODIES AND LIGANDS TO
PEG MOLECULES ON SURFACE LNP.
WHEN WE DID THAT WE MAINTAINED
MORPHOLOGY OF LNP AND THEY GOT A
LITTLE BIGGER THAT IS ABOUT SIZE
OF THE N BODY CODEINE.
IMPORTANTLY, THEY MAINTAIN
SURFACE CHARGE. YOU CAN'T GIVE
A CHARGED PARTICLE TO A PERSON.
IT ACTIVATES COAGULATION AND
ACTIVATES A VARIETY OF TOXICITY
PATHWAYS.
WHEN WE ADDED ANTIBODIES, THEY
REMAIN NON-CHARGED.
WE INJECTED THEM INTO ANIMALS.
FIRST, WE TRIED THEM ON PRIMARY
HUMAN CELLS THAT ARE UVACS,
ENDOTHELIAL CELLS AND ABSENCE OF
TARGETING, YOU GET NO RNA
UPTAKE. LNPS ARE NOT TAKEN UP
BY UVACS. PCAN IS HIGHLY
EXPRESSED ON ENDOTHELIAL CELLS
TARGETING LNPS WITH ANTIBODY AND
DID GFP ENCODING RNA AND YOU SEE
EVERY CELL IN THE CULTURE IS
MAKING GNP. TOOK UP LNPS AND
TRANSLATED RNA. WHEN WE
INJECTED PARTICLES INTO THE
ANIMALS, WE SAW TWO THINGS.
FIRST, WHEN WE ADDED ANTIBODY TO
THE SURFACE THAT IS CONTROL IGG
OR ANTI-PCAM, WE REDUCED LIVER
UPTAKE THAT IS LIKELY BECAUSE WE
ARE BLOCKING AND BINDING AND
REDUCING LIVER TARGETING AND
ADDING PCAM, LUNGS NOW LIT UP
AND ABOUT HALF OF THE ACTIVITY
IS NOW IN THE LUNGS.
PCAM IS HIGHLY EXPRESSED ON
ENDOTHELIAL CELLS WITH LUNGS
WITH HIGHEST DENSITY OF
ENDOTHELIAL CELLS AND TARGET
LUNGS WITH ANTI-PCAM ANTIBODY
AND I'M ALWAYS ASKED ABOUT
NEUROLOGIC TARGETING. I DON'T
-- THERE IS A VARIETY OF REASONS
PROBABLY BUT WE DON'T HAVE TO
GET INTO THAT.
AS A PHYSICIAN, WHEN I WAS A
RESIDENT, MY BIGGEST NIGHTMARE
CAME WHEN A STROKE PATIENT CAME
IN. THIS IS WHAT A CT SCAN OR
MRI OF A STROKE PATIENT LOOKS
LIKE. THIS IS MIDDLE CEREBRAL
ARTERY DISTRIBUTION HERE. WHEN
THEY FIRST COME IN THE DOOR WITH
TYPICALLY HEMIPORESIS IT IS
BECAUSE MIDDLE CEREBRAL ARTERY
GOT PLUGGED UP. YOU FOLLOW THEM
FOR A FEW DAYS.
ALSO, WHAT OFTEN HAPPENS IS
THIS. THE STROKE EXPANDS.
YOU WIPE OUT ONE ENTIRE SIDE OF
THE BRAIN. THIS IS BECAUSE ALL
OF THE DEAD CELLS ARE RELEASING
INFLAMMATORY MEDIATORS AND A
VARIETY OF DAMAGE-INDUCING
EFFECTS CAUSING VASCULAR LEAKAGE
AND EXPANSION OF THE STROKE.
SO, THERE ARE WAYS OF
APPROACHING THIS. THERE IS
ENZYMES THAT CAN DESTROY CLOTS
AND THERE ARE SURGERIES THAT CAN
REPAIR.
BUT, THERE IS NO WAY TO TREAT
THIS. STEROIDS DON'T WORK AND
OTHER THINGS DON'T WORK.
WE HAD THE IDEA. WE HAD A MODEL
SYSTEM WHERE WE COULD INJECT TNF
INTO ONE HEMISPHERE OF A MOUSE
BRAIN. IT THAT CAUSED LOCAL
INFLAMMATION AND IT WAS IN HALF
OF THE BRAIN OF THE MOUSE.
WE DEVELOPED A THERAPEUTIC. WE
ENCODED TLOEM BOW MODULIN THAT
INHIBITS COAGULATION AS
ANTIINFLAMMATORY MOLECULE AND
PUT ON LNP WE TARGET TO THE
BLOOD BRAIN BARRIER USING ANTI-V
CAM ANTIBODIES THAT WE ANALYZE
BOTH SIDES THIS IS A SIDE THAT
TNF WAS INJECTED AND THIS IS THE
OTHER SIDE. ON OTHER SIDE, YOU
SEE THAT TREATMENT HAD SOME BUT
NOT MUCH EFFECT. THERE WASN'T
MUCH VASCULAR LEAKAGE. WHERE
TNF WAS INJECTED WE HAD HUGE
AMOUNTS OF VASCULAR LEAKAGE.
WHEN WE DELIVERED TLOEMO MODULIN
WITH TARGETED LNP, WE COULD GET
RID OF IT AND ALL VASCULAR
LEAKAGE FROM THAT INFLAMMATORY
INSULT. WE ARE TAKING THIS
FORWARD INTO LARGER ANIMAL
MODELS AS A WAY OF TREATING
BRAIN INFLAMMATION AND NEUR
LOGIC INFLAMMATION DUE TO
INFLAMMATORY INSULTS. MY LAB
HAD LONG INTEREST IN HIV SINCE I
WAS AT NIH IN TONY'S LAB WANTING
TO KNOW IF WE COULD TARGET CD4
CELL AS WAY OF ADDRESSING HIV
LATENCY. WE DID THE SAME THING
AND ADDED ANTI-CD4 ANTIBODIES TO
THE LNPS.
WHAT YOU HAVE TO KNOW ABOUT T
CELLS IS THEY HAVE NO ENDO
CRITIC ACTIVITY AND IF YOU GIVE
THEM A PARTICLE OR ANY LIPID
CARRIER, THEY DO NOT -- THEY
HAVE NO ENDOCYTIC ACTIVITY AND
CAN'T PRELY SITE OES NUCLEIC
ACIDS. YOU CAN'T ELECTROPORATE
ALL T CELLS IN A BODY.
OUR HYPOTHESIS WAS THAT IF WE
BOUND AN ANTIGEN THAT
ENDOCYTOSIS AFTER BINDING WOULD
BRING LNP INTO THE CELL RNA
COULD BE RELEASED AND TRANSLATED
AND WE LOOKED AT CD4 FIRST.
THESE ARE PURIFIED HUMAN CD4T
CELLS AND ONLY TIME YOU GET
ACTIVITY FROM RNA UPTAKE OF LNPS
IS WHEN THEY ARE TARGETED WITH
-- AGAINST THEIR CD4 MOLECULE.
WE HAD EFFECTIVE DELIVERY WITH
ANTI-CD4 ANTIBODIES.
WE INJECTED THESE INTO A MOUSE.
WE SEE NOW UPTAKE IN THE SPLEEN
THAT HAS DECENT NUMBERS OF CD4
HELPERS IN IT.
WHAT WE DID WAS SOMETHING
INTERESTING.
WE IMAGED MICE AGAIN AFTER WE
REMOVED ALL OF THE ORGANS ON THE
PREVIOUS SLIDE.
WHAT WE SAW IS THAT LYMPH NODES
WERE NOW LIGHTING UP PARAAORTIC
[INDISCERNIBLE] LYMPH NODES WHEN
THIS LNP ESCAPED FROM
CIRCULATION AND WENT INTO
TISSUES AND INTO DRAINING
LYMPHATICS BACK TO DRAINING
LYMPH NODES WHERE THEY WERE
TAKEN UP BY CD4 POSITIVE T CELLS
WHERE RNA COULD BE EXPRESSED.
HERE, WE PURIFY T CELLS SHOWING
THEY HAD ACTIVITY AND THAT MEANS
LNPS COULD BE GIVEN IV FOR
SYSTEMIC TISSUE THERAPEUTICS.
WE SWITCHED TO A PRELOCKED
SYSTEM THAT ALLOWS US HIGHER
SENSITIVITY.
PRELOCKS, YOU HAVE USUALLY A
STOP CODON IN FRONT OF A
FLUORESCENT PROTEIN SURROUNDED
BY LOCKS P SITES AND IT CUTS OUT
STOP CODON TURNING ON PROTEIN.
WE INJECTED MICE AND LOOKED AT
SPLENOCYTES AND LYMPH NODES AND
Y AXIS IS THE COLOR OF THE GREEN
FLUORESCENT PROTEIN AND X AXIS
IS AN ACTIVATION MARKER.
HIV, WE KNOW PROVIRAL HIV
LATENCY IS IN UNACTIVATED T
CELLS.
WE SAW WE HAD EQUAL LEVELS OF G
RECOMBINATION IN RESTING T CELLS
THAT IS CRITICAL FOR ANY HIV
LATENCY THERAPEUTICS THAT SHOWS
THAT LNPS CAN TARGET RESTING T
CELLS AND MEDIATE ACTIVITY.
OUR CONCLUSIONS WERE WE FIGURED
OUT HOW TO TARGET OTHER CELLS
PART OF TARGETING REDUCED LIVER
UPTAKE AND INCREASED SPECIFIC
UPTAKE IN CELLS AND TISSUES OF
INTEREST. YOU CAN USE TO
DELIVER THERAPEUTIC PROTEINS TO
GENE EDITING TECHNOLOGIES AND
WHATEVER YOU WANT.
COMING FROM PEN, PEN HAS A LOT
OF NOTABLE THINGS AND TWO OF
THEM ARE RECLINICALLY DEVELOPED
FIRST CAR T CELLS AND FIRST 2FDA
APPROVED T CELL CAR T THERAPIES
THAT CAME OUT OF PEN WORK. WE
ALSO DEVELOPED THE FIRST FDA
APPROVED GENE THERAPY FOR
BLINDNESS.
SO, OUR IDEA WAS COULD WE
DELIVER RNA'S THAT MODULATE T
CELL FUNCTION?
IN THIS CASE, IT WAS CAR T
CELLS.
SO, I'M SURE THAT YOU GUYS KNOW
WHAT CAR T CELLS ARE. I WILL
QUICKLY GO OVER IT. WAYS THAT A
T CELL RECOGNIZES A TUMOR OR
INFECTED CELL IS IT SEES A
PEPTIDE IN ASSOCIATION WITH MHC
ON A T CELL RECEPTOR WITH
SIGNALING [INDISCERNIBLE] THAT
ACTIVATE THE T CELL. WHAT A CAR
DOES IS IT REPLACES THE EXTERNAL
REGION WITH A SINGLE-CHAIN ANTI
BODY OR OTHER ANTI BODIES THAT
BIND A TUMOR-ASSOCIATED OR
TUMOR-SPECIFIC ANTIGEN AND FIRST
DEVELOPED BOUND CD19 THAT WERE
USE THE FOR B CELL LYMPHOMAS AND
LEUKEMIAS THAT ARE INCREDIBLY
SUCCESSFUL WITH CURE RATES IN 70
TO 80% RANGE WITH CAR T
THERAPIES.
PROBLEM IS MAKING CAR TS.
TO MAKE A CAR T, YOU HAVE TO
FIRST LUCHO FORRESE THE PATIENT
MEANING PUTTING THEM ON A
MACHINE FOR A COUPLE HOURS
TAKING OUT A FEW BILLION T CELLS
AND YOU INFECT T CELLS WITH A
LENGTHY VIRUS THAT DELIVERS THE
CAR GENE AND YOU EXPAND THOSE T
CELLS FOR 10 + DAYS TO MAKE
TRILLIONS OF CELLS THAT YOU GIVE
BACK TO THE PATIENT.
THE TWO FDA APPROVED CD19 DRUGS
COST I THINK AROUND $450,000 A
DOSE.
THIS IS TWO WEEKS OF GMP CELL
CULTURE FACILITY WORK.
THERE THEY ARE LIMITED WHERE IT
IS GIVEN MAINLY US AND WESTERN
EUROPE AND THERE IS ONE SITE IN
CHINA BUT REST OF THE WORLD HAS
NO ACCESS TO CAR T CELLS.
OUR IDEA WAS COULD WE TARGET CAR
T CELLS WITH LNPS DELIVERING AN
MRNA THAT CODED CAR MOLECULE AND
PRODUCE CAR PROTEINS T CELL
WOULD EXPRESS AND TARGET
WHATEVER CAR BOUND. WE STARTED
WITH CAR CALLED AGAINST -- A
PROTEIN CALLED FAP THAT IS
EXPRESSED BY ACTIVATED
FIBERGLASS USED IN VARIETY OF
CANCER THERAPIES AND FOR
FIBROSIS.
WE ALSO CHOSE CD5 FOR TARGETING
WHICH IS A SCAVENGER RECEPTOR ON
T CELLS MEANING IT IS ENDOCITE
OESED AFTER BINDING.
SO, WE TESTED THIS AND MADE CD5
TARGETED LNPS DELIVERING P
PREECOMBINATES TO LOXENE MICE
AND SAW 80% OF CD4S AND CD8S
WERE GENE MODIFIED AFTER A
SINGLE TREATMENT AND WE COULD
EFFECTIVELY DELIVER TO T CELLS
IN TISSUES AND SPLEEN AND LYMPH
NODES AND WE COLLABORATED WITH A
CARDIOLOGIST WHO HAD A FIBROSIS
MODEL AND WHAT THEY DID WAS TOOK
PUMPS AND PUT HYPERTENSIVE
AGENTS IN THE PUMPS. THEY MADE
THE MICE HYPERTENSIVE. WHEN YOU
DO THAT, HEART IS FIBR ROWS.
THE WAY THAT YOU MEASURE
FIBROSIS IS AN ECHO CARDIOGRAM
THAT TAKES PICTURES OF THE HEART
AND HEART WALL FIBROSIS DOESN'T
MOVE AS WELL. IT IS STIFF.
VOLUMES OF THE HEART INCREASE
AND WHAT WE ARE MEASURING HERE
AND THIS IS AFTER BEATING AND
BEFORE BEATING THE SIZE OR
VOLUME OF THE HEART. THEY ARE
MUCH HIGHER AND HEART IS STIFF
AND IT CAN'T MOVE. THIS IS THE
INJECTION FRACTION THAT IS HOW
MUCH BLOOD DOES THE HEART FORCE
OUT WITH EACH BEEP. WHEN THE
HEART IS STIFF, IT CAN'T PUMP
BLOOD OUT AND INJECTION FRACTION
DROPS.
GRAY, IT IS UNTREATED ANIMALS.
THIS IS NORMAL. YELLOW IS
HYPERTENSIVE MICE AND RED
TREATED HYPERTENSIVE MICE WITH
SINGLE TREATMENT OF T CELL
TARGETED RNA-LNPS ENCODING FAT
CAR. ECHOCARDIO GRAPHIC
MEASURES HEART FUNCTION RETURNS
TO NORMAL WE TOOK HEARTS OUT
STAINING THEM FIBROSIS IN RED
AND CONTROL ANIMALS HAVE MINIMAL
AND HYPERTENSIVE HUGE AMOUNTS OF
FIBROSIS IN MIOCARD YUM. SINGLE
TREATMENT FIBROSIS IS RETURNING
TO NORMAL. OFF THE SHELF LNP WE
CAN DELIVER A CAR T TO AN ANIMAL
AND HAVE A THERAPEUTIC OUTCOME
IN THEORY THIS MAKES CAR TS
AVAILABLE TO THE ENTIRE WORLD
AND TREATING CD19 LEUKEMIA
HEALTH SYSTEM SAYS A HALF A
MILLION IS NOT A HIGH PRICE
COMPARED TO COST OF TREATING
THAT PATIENT. IF TREATING H
ICHHIV
YOU CAN'T DO EX-VIVO THERAPY IN
AFRICA TO CURE HIV OR IN ANY
OTHER PARTS OF THE WORLD AND
WITH OFF THE SHELF FROZEN LNP
YOU INJECT INTO THE PERSON, YOU
CAN DO THAT MOVING THIS FORWARD
PART OF HIV CURE STRATEGY AS AN
IMMUNOLOGIST I WAS INTERESTED IN
BONE BARROW STEM CELLS AND
THOUSANDS OF GENETIC DISORDERS
OF BONE MARROW STEM CELLS. NO.
1 ON THE LIST IS SICKLE CELL
ANEMIA. IN SICKLE CELL 300,000
PEOPLE A YEAR ARE BORN WITH THE
DISEASE THAT ARE MAINLY IN SUBIS
A HARRANT AFRICA BUT ARE
SCATTERED THROUGHOUT THE WORLD
AND THERE ARE GENE THERAPIES
BEING DEVELOPED FOR SICKLE CELL
THAT INVOLVE TAKING LOTS OF BONE
MARROW OUT OF A PATIENT
INFECTING THEM WITH LENGTHY
VIRUS TO FIX THE GENE AND GIVING
BACKBONE MARROW IS PROBABLY A
MILLION A SHOT. YOU CAN'T TREAT
300,000 PEOPLE IN AFRICA WITH
THAT KIND OF TECHNOLOGY THAT IS
NOT AVAILABLE AT A MILLION A
SHOT OUR IDEA WAS TARGETING BONE
STEM CELLS IN VIVO -- WE COULD
CURE SICKLE CELL WITH SIMPLE
INJECTION OF RNA-LNPS AT A COST
OF I DON'T KNOW A COUPLE HUNDRED
DOLLARS PER AND EASY TO DELIVER
IT. FIRST THING, WE HAD TO
TARGET BONE MARROW STEM CELLS
AND THERE ARE VERY FEW OF ANY
MARKERS THAT IDENTIFY
EPOPULATING BONE MARROW STEM
CELLS WE STARTED WITH CD117 OR
EXPRESS KIT AND THEY HAVE STEM
CELLS AND MANY PROGENITORS AND
VARIETY OF TISSUES AND GAVE MICE
A SINGLE TREATMENT 85% OF
LINEAGE NEGATIVE BONE MARROW
CELLS WERE GENE MODIFIED. WE
FOLLOWED MICE FOR SIX MONTHS AND
THEIR NUMBERS DIDN'T DROP. WE
THEN DID A SECONDARY TRANSPLANT.
THAT INVOLVES TAKING BONE MARROW
FROM TREATED MICE AND PUTTING
THEM INTO A RADIATED RECIPIENT
MICE AND YOU ASK WHAT DO THE
BONE MARROW STEM CELLS OR
PRODUCT CELLS LOOK LIKE?
100% OF RED CELLS AND WHITE
CELLS AND GRANULOCYTES WERE GENE
MODIFIED IN SECONDARY
TRANSPLANTS AND WHAT IT MEANS IS
WE GENE MODIFIED 100% OF
REPOPULATING BONE MARROW STEM
CELLS WITH .1 MICROGRAMS THAT IS
.005MGS PER GIG. VERY LOW DOSE
TAKING TECHNOLOGY FORWARD
DEVELOPING VARIETY OF GENE
EDITING APPROACHES TO ADDRESS
SICKLE CELL AND IS A BC11A
LOCUST THAT TURNS ON FETAL
HEMOGLOBIN THAT YOU CAN ALSO USE
PRIME EDITORS TO FIX BETA
HEMOGLOBIN GENETIC MUTATIONS AND
ALL THAT IS TAKEN FORWARD WITH
THE PLAN OF DOING CLINICAL
TRIALS TO TREAT SICKLE CELL.
THAT HAS EASY EXPANSION TO
THOUSANDS OF OTHER BONE MARROW
GENETIC DISEASES THAT I
MENTIONED EARLIER THAT THEY HAVE
ALREADY USE RNA-LNPS. WITH
INCREASED TARGETING WE CAN TREAT
MORE DISEASES WITH GENE
TECHNOLOGY. POTENTIAL FOR LNP
TECHNOLOGY IS ENORMOUS AND SPANS
JUST ABOUT EVERY DISEASE AND
EVERY THERAPEUTIC POTENTIAL.
SO, WHAT WE HAVE DEVELOPED IS A
WAY TO HIGHLY EFFICIENTLY GENE
EDIT CELLS IN VIVO WITH THE HOPE
OF MOVING THAT FORWARD BOTH FOR
THERAPEUTIC APPLICATIONS
DELIVERING THERAPEUTIC PROTEINS
AND CELLS OF INTEREST OR GENE
EDITING TECHNOLOGY.
WE STARTED THE PROCESS OF
DELIVERING THE BRAIN. WE NEED A
LOT MORE WORK TO DO THAT
EFFECTIVELY. WHEN YOU CROSS THE
BLOOD BRAIN BARRIER, NOW YOU
HAVE TO TARGET PLAQUES OR TARGET
DOPAMINERGIC CELLS OR OTHER
REGIONS OF THE BRAIN AND WE ARE
WORKING ON THAT AND THINK IN THE
NEAR FUTURE THAT WE CAN TREAT A
VARIETY OF NEURODEGENERATIVE
DISORDERS IN THE BRAIN.
AND LET'S SEE.
I THINK I WILL STOP THERE. I
USUALLY LIKE TO GIVE A QUICK
TALK ON EQUITY. WE DON'T HAVE
TIME. I NEED TO THANK ALL OF
THE PEOPLE IN MY LAB AND ALL
LABS THAT WE COLLABORATE THAT
HAVE DONE THIS WORK. THANK YOU.
>>[APPLAUSE].
>>IF THERE ARE ANY QUESTIONS,
PLEASE COME UP TO THE MIC.
>>YOU THOUGHT ABOUT OBVIOUSLY
SHOWED YOU CAN TARGET CELLS.
CAN YOU THINK ABOUT POSSIBLY
TARGETING VIRUSES OR VIRUS
SEQUENCE INSIDE OF CELLS. CAN
YOU ERADICATE VIRUSES FROM THE
GENOME?
>>WE ARE TRYING WITH HIV. WHAT
WE ARE ABLE TO TARGET RESTING
CD4 CELLS AND WE ARE DELIVERING
-- THERE IS AN ENZYME KNOWN AS
BREK1 THAT IS A PRERECOMBINACE
PROGRAM FOR RLT SEQUENCES AND
DELIVERED CAS9 THAT PEOPLE FROM
PHILADELPHIA HAD DEVELOPED AS A
WAY OF INACTIVATING HIV AND WE
ARE LOOKING AT IT AS PART OF A
CURE STRATEGY AND THE PROBLEM IS
THAT AROUND 1 IN A MILLION
RESTING T CELLS HAVE LIVE
REPLICATION COMPETENT VIRUS IN
IT MEANING YOU HAVE TO HAVE AN
EFFICIENCY IN THE 99.99999
RANGE.
I DON'T THINK WE WILL EVER GET
THAT HIGH WITH LNP DELIVERY.
IF IF WE COMBINE IT WITH HIV
CARS AND DELIVERY OF BNABS AND
OTHER HIV THERAPIES, WE MIGHT BE
ABLE TO CURE OR TO MAKE PATIENTS
NOT REQUIRE ART THERAPY.
>>FOR STEM CELL THERAPY RNASE
COULD MAINTAIN FOR A LONG TIME.
AT EVERY SINGLE PROGENITOR CELLS
AND HOW THAT CAN BE ACHIEVED.
IF THAT MEANS IT HAS BEEN
INTEGRATED INTO DNA?
>>NO. SO, WE ARE -- RNA
DOESN'T LAST VERY LONG IN A
CELL.
MAYBE 24 TO 36 HOURS BUT WE
ENCODE GENE EDITING AND CAS9 AND
OTHER GENE EDITING TECHNOLOGIES
IN THE RNA THAT CHANGE DNA AND
AFFIX GENETIC DEFECTS.
>>OKAY. THAT PHENOTYPE?
>>YEAH. THE RNA ENCODES
ENZYMES THAT FIX DNA SCOMBL
OKAY.
>>RNA DOES NOT EFFECT. I SAY
THIS BECAUSE I -- YOU KNOW,
CRAZY PEOPLE ON THE INTERNET SAY
THAT RNA CHANGES THEIR GENOME
AND MAKES YOU INFERTILE AND
GIVES YOU CANCER AND MAKES YOU
MAGNETIC. I HAVE HEARD THEM
ALL. RNA DOES NOT CHANGE DNA
AND DOESN'T GO INTO THE NUCLEUS.
IT CANNOT CHANGE OR CANNOT
INTEGRATE.
>>JUST A FOLLOW-UP QUESTION
FROM SOME OF THE DISCUSSION.
YOU MENTIONED TARGETING OF CD4
POSITIVE T CELLS.
AND THAT YOU WOULD GET TO
EFFICIENCY OF 99.999%. WHAT IS
CURRENT EFFICIENCY OF DELIVERY
TO C4 CELLS IN VIVO IN YOUR
EXPERIMENTS? WE HAVE SEEN SO
FAR IN MICE THAT THERE IS ABOUT
80% GENE EDITING WITH A SINGLE
TREATMENTWE HAVE SEEN SO FAR INT
THERE IS ABOUT 80% GENE EDITING
WITH A SINGLE TREATMENT>WE HAVE
THAT THERE IS ABOUT 80% GENE
EDITING WITH A SINGLE TREATMENT
THAT THERE IS ABOUT 80% GENE
EDITING WITH A SINGLE TREATMENT
THAT THERE IS ABOUT 80% GENE
EDITING WITH A SINGLE TREATMENT.
THE ADVANTAGE WITH 4 OR 5
VACCINE TREATMENTS IS YOU CAN
REPEAT TREATMENTS. IN THEORY,
WE CAN TREAT PEOPLE WEEKLY FOR
HOWEVER LONG WE NEED TO, TO GET
THAT EFFICIENCY UP AND DON'T
THINK WE WILL EVER GET TO THAT
NEEDED TO GET TO 1 OVER A
MILLION EFFECTORATE CELLS BUT WE
CAN GET VERY HIGH.
>>RIGHT. IN TERMS OF GENE
EDITING, IT MIGHT BE POSSIBLE TO
TARGET THE VAST MAJORITY OF CD4
POSITIVE T CELLS AND EDIT THEM
FOR SOME OTHER GENE MAYBE NOT
PROFILE SEQUENCES BUT FOR OTHER
GENES.
>>WE ARE ACTUALLY LOOKING AT
BONE MARROW STEM CELLS TO DO
THAT FOR KNOCKING OUT CCR5.
>>YEAH.
>>OR INSERT IING AN HIV GENE
INTO. PROBLEM WITH THAT AND T
CELLS IS THEY ARE MOSTLY TERMIN
ALLEY OR CLOSE TO TERMIN ALLEY
DIFFERENTIATED.
THEY ARE CONSTANTLY REPLACED.
STEM CELLS WILL BE BETTER FOR
THAT.
>>I AGREE. THANK YOU. THANK
YOU FOR AN INSPIRING TALK.
>>THANK YOU.
>>THANK YOU VERY MUCH.
>>[APPLAUSE].
>>I WILL INTRODUCE CO-CHAIR FOR
>>GOOD MORNING AND WELCOME TO
THE SESSION AND I'M A SCIENTIST
IN THE HIV [INDISCERNIBLE] AND
FIRST SPEAKER IS [INDISCERNIBLE]
A PHD CANDIDATE IN
[INDISCERNIBLE].
>>THANK YOU. GOOD MORNING,
EVEN. THANK YOU FOR BEING HERE
TO ATTEND I'M IN THE HIV AND
AIDS MALIGNANCY BRANCH AND
PLEASED TO SHARE MY WORK.
IN THE LAB WE STUDY
GAMMAHERPESVIRUSES A SUBFAMILY
OF CANCER CAUSING VIRUSES.
-- EBV IS UBIQUITOUS WORLDWIDE
INFECTING OVER 95% OF THE
POPULATION THAT CAN LEAD TO
VARIOUS LYMPHOMAS AS WELL AS
GASTRIC AND NASAL CARCINOMAS AND
TRANSZ PLANT PROLIFERATION -- IT
CAN LEAD TO PRIMARY EH FUSION
LYMPHOMA AND [INDISCERNIBLE]
DISEASE.
HERPES VIRUSES ARE LARGE DOUBLE
STRAND ND DNA VIRUSES ENCODING
OVER 80 FUNCTIONAL PROTEINS AND
BECAUSE OF THIS BRING WITH THEM
THEIR OWN CORE REPLICATION
MACHINERY CONSISTING OF VIRAL
DNA POLYMERASE AND SINGLE STRAND
DNA BINDING PROTEINS AND
HELICASE PRIMARY COMPLEX. CORE
REPLICATION MACHINERY IS
ESSENTIAL FOR REP MRIKS AND
KNOCKING OUT IN CELLS AND
PATHOGEN ESIS IN VIVO LEADING TO
NONREPRODUCTIVE VERSION OF THE
VIRUS AND HERPES CODE WORD
TERMED AS ACCESSORY PROTEINS
INVOLVED IN NUCLEOTIDE
METABOLISM IN DNA REPAIR AND
HOMOLOGS OF HIGHLY CONSERVED
ENZYMES AND ACCESSORY PROTEINS
ARE THOUGHT TO BE DISPENSABLE
FOR CELL CULTURE NOT UNTIL WE
INFECT IN VIVO MOUSE MODELS FOR
EXAMPLE IN OUR CASE THAT
REPLICATION PHENOTYPES ARE
OBSERVED AND AT THIS POINT I
WANT TO PAUSE BRIEFLY TO EXPLAIN
WE USE THE MOUSE GAMMA HERPES
MHV68 THAT IS A MODEL SYSTEM
STUDYING HUMAN GAMMA HERPES
VIRUSES AND NICE SYSTEM THAT
SHARES GENETIC AND BIOLOGICAL
PARALLELS WITH GAMMA HERPES
VIRUSES ALLOWING US TO UNVAIL
PHENOTYPES OF ACCESSORY PROTEINS
THAT INCLUDE THOSE ENZYMES
INVOLVED IN NUCLEOTIDE
METABOLISM AND RIBONUCLEOTIDE
REDUCTASE SMALL AND LARGE
SUBUNITS AND OUR LAB IS FOCUSED
ON DNA REPAIR ENZYME URACIL DNA
GLYCOL ASIS AND IT IS ACTIVE AND
CAN COMPLEMENT FOR INTERESTING
FUNCTIONS OF HOST UNG AND MOST
IMPORTANT FOR THIS TALK PREVIOUS
WORK IN LAB HAS SHOWN THAT A
KNOCKOUT VIRUS WITH SEVERELY
ATTENUATED FORPATHOGEN ESIS IN
VIVO.
WHAT IS URACIL DNA GLYCOLYSIS
AND WHAT DOES IT DO?
URACIL CAN ARISE IN DNA THROUGH
MISIN-CORPORATION AND -- IN CASE
OF MISIN-CORPORATION DNA
POLYMERASE MAY NOT DISCRIMINATE
AND IN CELLS WITH SCREENED --
THERE IS A HIGHER CHANCE OF
MISINCORPORATION OF URACIL
RESIDUES AND CYTOSINE -- SO, UNG
THROUGH ITS HIGHLY CONSERVED
ENZYMATIC DOMAINS SCANS DNA
EXCISES URACIL FROM DNA LEADING
TO A BASIC SITE IT THAT IS
FILLED IN BY HOST BAITS EXCISION
REPAIR PATHWAY. SO, IT IS HYPE
OJ CRIED IN HERPES FIELD THAT
HERPES VIRUSES CO-OPTED URACIL
DNA GLYCOS ILLACE TO KEEP THEM
FREE OF GLYCOLIZATION. WE ARE
INTERESTED IN ENZYMATIC
FUNCTIONS FOR VIRAL UNG.
SO, JUST SOME BACKGROUND AND
PREVIOUS DATA IN THE LAB, WE
HYPOTHESIZED VIRAL UNG ENZYMATIC
ACTIVITY WAS IMPORTANT IN
REPLICATION IN OUR MOUSE SYSTEM
AND WE CONSTRUCTED A VIRAL UNG
CATALYTIC MUTANT OR VIRAL UNG.CM
MAKING KEY CHANGES IN UNG
ENZYMATIC DOMAINS AND SHOWED
THIS VIRUS WAS ABERGATED FOR ITS
ABILITY TO EXCISE URACIL
RESIDUES IN VITRO. WHAT IS
PHENOTYPE OF CATALYTIC MUTANT
VIRUS? INFECTING MICE WITH THIS
MHV68 VIRUS AND CASE OF STOP OR
KNOCKOUT VIRUS IT WAS SEVERELY
ATTENUATED IN ACCUSE PHASE OF
REPLICATION IN LUNGS AND LATENCY
ESTABLISHMENT IN THE SPLEEN
COMPARED TO WILD TYPE VIRUS
INFECTING MICE WITH UNG VIRAL
CATALYTIC -- OR IN LATENCY
ESTABLISHMENT IN THE SPLEEN.
THESE RESULTS WERE SURPRISING TO
US AND INDICATED THAT VIRAL UNG
MUST HAVE IMPORTANT NONENZYMATIC
FUNCTIONS TOWARDS PATHOGEN ESIS
IN VIVO. BEGINNING TO
UNDERSTAND WHAT VIRAL UNG MIGHT
DO OUTSIDE OF ENZYMATIC ACTIVITY
WE WERE INTERESTED IN STUDYING
POTENTIAL PROTEIN PROTEIN
INTERACTIONS WITH OTHER VIRAL
FACTORS COLLABORATING WITH
KEVIN'S GROUP AT THE CANCER
CENTER TO PERFORM
CO-IMMUNOPRECIPITATION MAS SPECK
TROMTRY AGAINST ANTIVIRAL UNG I
INFECTED PERMISSIVE YOU'RE EVEN
FIBERGLASS CELLS WITH VIRUS THAT
EXPRESSES YFP SENDING CELL
PALETTES TO KEVIN'S GROUP FOR
ANALYSIS USING FLOW CITOMETRY 36
HOURS POST INFECTION WAS HIGHEST
LEVEL OF INFECTED AND UNG
POSITIVE CELLS USING THIS TIME
FOR CO-IP/MS. THESE ARE RESULTS
OF CO-IP/MS ANALYSIS AND KEVI'S
GROUP HAD A RANGE OF HOST
FACTORS AND IMPORTANTLY TWO
VIRAL PROTEINS WERE MUCH HIGHER
IN HITS THAN A FEW OTHER VIRAL
PROTEINS THAT I WILL FOCUS ON IN
THIS TALK THOSE WERE VIRAL DNA
POLYMERASE AND PROCESSIVETY
FACTOR SEEMING LIKE VIRAL UNG
MIGHT INTERACT PHYSICALLY WITH
VIRAL REPLICATION COMPONENTS AND
BASED ON THIS AND DATA THAT I
DON'T HAVE TIME TO GO OVER, WE
HYPOTHESIZED THAT MHV68 -- VIRAL
DNA POLYMERASE PROCESS OF YOUTH
FACTOR THINKING THIS INTERACTION
IS INDEPENDENT OF ENZYMATIC
ACTIVITY I WILL REFER TO VIRAL
DNA POLYMERASE AS B POLL AND
PROCESS FACTOR IS BPS AND HOPE
THAT MAKES SENSE MOVING FORWARD
AND HAPPY TO PRESENT TWO
APPROACHES -- SO, THIS IS THE
CO-IPMS DATA FOR
IMMUNOPRECIPITATION FOLLOWING
VPF LOOKING AT THIS YOU GET TO
FIGURE A IN HIGHLIGHTED REGION
ABLE TO SHOW THAT IT PULLED DOWN
BOTH VIRAL POLYMERASE AND VIRAL
UNG AND IN FIGURE B, THIS IS A
REPEAT OF THE EXPERIMENT THAT I
TRANSFEKTED IN VIRAL UNG
CATALYTIC PLAS MANT AND IT LEADS
TO PULLDOWN OF PULL AND UNG AND
AGAIN ECHOING WHAT WE SAW WITH
CO-IP/MS DATA AND I PERFORMED
THIS EXPERIMENT IN THE CONTEXT
OF IMMUNOPRECIPITATION AGAINST
VIRAL UNG AND LOOKING AT
HIGHLIGHTING REGION THIS IS
TRIPLE TRANSVEKS REGION AND
POLYMERASE AND [INDISCERNIBLE]
IN PULLDOWN AND THIS HOLDS TRUE
IN [INDISCERNIBLE] AND THESE
DATA SEEM TO CONFIRM CO IPMS
DATA AND IT MIGHT NOT BE
REQUIRED FOR INTERACTIONS.
SO, THE NEXT APPROACH THAT WE
PERFORMED WAS IMMUNOFLUORESCENCE
TO EXAMINE POTENTIAL
COLOCALIZATION BETWEEN VIRAL UNG
WITH V POLL AND UPPF AND
INFECTED CELLS WITH VIRUSES THAT
EXPRESS GFP TAG POLL TO LOOK AT
COLOCALIZATION WITH UNG AND
INFECTED CELLS WITH VIRAL UNG
CATALYTIC MUTANT VIRUS.
HERE IS RESULTS OF THE
EXPERIMENT THAT I INFECTED
NIH3G12 FIBERGLASS WITH HIGH MOI
AND IF WE LOOK AT RED THIS IS
VIRAL UNG STAIN AND IN THE GREEN
IS THE POLL OR PPF AND AS TIME
INCREASES SIZE OF THE VIRAL
REPLICATION COMPARTMENTS
INCREASES AND TOP ROW SHOWS OR
EMERGE OF VIRAL UNGPOL AND PPF
AND THERE IS LARGE
COLOCALIZATION BETWEEN VIRAL
FACTORS AT EACH TIME POINT IN
THE STUDY.
NEXT, I WANT TO DETERMINE
WHETHER CATALYTIC ACTIVITY OF
VIRAL UNG IS REQUIRED FOR
COLOCALIZATION AND INFECTED
CELLS WITH CATALYTIC MUTANT
VIRUS AND WILD TYPE VIRUS AND IF
WE LOOK AT TOP ROWS THIS IS THE
MERGE SHOWING VIRAL UNG AND
PROCESSIVETY FACTOR THAT
OVERLAPPED AND AT EACH TIME
POINT WE ANALYZE THERE IS LARGE
OVERLAP BETWEEN VIRAL UNG AND
PROCESSIVETY FACTOR THAT IS
INDEPENDENT WHETHER I INFECTED
WITH CATALYTIC MUTANT WITH WILD
TYPE VIRUS.
HERPES VIRUS LITERATURE
INDICATES VIRAL UNG CAN INTERACT
WITH VIRAL POLYMERASE AND
PROCESSIVETY FACTOR FOR HSV AND
EBV AND SUGGESTS THAT ENZYMATIC
ACTIVITY OF IT IS NOT REQUIRED
FOR INTERACTIONS MOVING FORWARD
WE ARE REALLY EXCITED TO TRY
SOME OF THIS IN KSHV WHERE LESS
IS KNOWN BETWEEN INTERACTION OF
VIRAL UNG AND REPLICATION
MACHINERY AND BASED ON THIS AND
OTHER DATA I HAVEN'T HAD TIME TO
SHOW WE PROPOSE A MODEL WHERE
VIRAL UNG IS PRESENT IF ACTIVE
SITES OF VIRAL REPLICATION
POTENTIALLY INVOLVED IN A
SCAFFOLDING FUNCTION WITH VIRAL
DNA POLYMERASE AND VIRAL DNA
POLYMERASE PROCESS FACTOR
EXCISING VIRAL GENOMIC URACILS
TO MAINTAIN FIDELITY OF VIRAL
REPLICATION.
SO, IN SUMMARY, I HAVE SHOWN CO
IPMS DATA DEMONSTRATING VIRAL
POLYMERASE AND PROCESSIVETY
FACTOR INTERACT WITH THIS AND IN
COLLABORATION WITH KEVIN MCBRIDE
AT MD ANDERSON AND PRECIPITATION
THAT SUPPORTS THIS SUGGESTING
ENZYMATIC ACTIVITY MIGHT BE
DISPENSABLE FOR THE INTERACTION
SHOWING VIRAL UNG COLOCALIZES
WITH CORE REPLICATION FACTORS IN
CASE OF PPF COLOCALIZATION
APPEARED INDEPENDENTLY WITH THE
ENZOMATIC DOMAIN AND IN FUTURE
DETERMINING THIS IS IMPORTANT
FOR INTERACTION AND
COLOCALIZATION AND MUTATE THOSE
DOMAINS TO DETERMINE THE EFFECT
ON THESE PHENOTYPES AND
ULTIMATELY PATHOGEN ESIS.
WE THINK ELUCIDATING THESE
INTERACTIONS WITH VIRAL
REPLICATION MACHINERY MIGHT BE
IMPORTANT TO PRODUCE
THERAPEUTICS TO TAFRG ET KSHV IN
COMBINATION WITH RASE SUCCESSFUL
NUCLEOANALOGS.
SO, WITH THAT, I WOULD LIKE TO
THANK MY LAB AND MENTOR LORI
CREW. I'M A GPP STUDENT FROM
STONEY BROOK UNIVERSITY AND
WOULD LIKE TO THANK MY COMMITTEE
AND FUNDING SOURCES AND KEVIN
MCBRIDE'S GROUP AT MD ANDERSON
WHO IS HECHFUL IN THE ENTIRE
PROJECT AND WITH THAT I'M HAPPY
TO TAKE ANY QUESTIONS.
>>ANY QUESTIONS?
>>I HAVE ONE. SO, THE VIRAL --
IT IS ONE OF THE HOST. THE HOST
PROBABLY CAN'T DO THIS AND WHAT
IS DIFFERENCE BETWEEN TWO?
>>HOST AND VIRUS AND UNGS
ACROSS THESE ARE HIGHLY
CONSERVED AT ENZYMATIC DOMAINS
AND ARE DIFFERENCES FOR EXAMPLE
HOST HAS A LONGER END TERMINUS
AND WHAT COLLABORATOR KEVIN
MCBRIDE IS INTERESTED IN AND
HOST REPLICATION FACTORS AND
HOST UNG ACTUALLY LEADS TO ERROR
PRONE REPAIR OF MUTATIONS IN
VEET TROE AND WE THINK IT IS
POSSIBLE THAT VIRAL UNG MIGHT BE
SHIELDING BASIC SITES FROM
POTENTIALLY DELETERIOUS HOST
FACTORS THAT ARE THINGS THAT WE
ARE INTERESTED IN LOOKING AT
MOVING FORWARD.
>>ALL RIGHT. THANK YOU.
>>I CAN INTRODUCE YOU. SHE IS
A RESEARCH FELLOW IN THE
LABORATORY TUMOR VIRUS VIROLOGY
SECTION IN CCR. SHE WILL
PRESENT DATA ABOUT SELECTIVE
EXPRESSION ABOUT ONCOGENES
[INDISCERNIBLE] IN LINE WITH
MULTIPLE INTEGRATION SITES. SHE
IS CONNECTING THROUGH ZOOM. YOU
CAN GO. EVERYTHING IS FINE.
>>OKAY. THANK YOU FOR THE
INTRODUCTION. GOOD MORNING,
EVERYONE. THANK YOU TO MEETING
ORGANIZERS TO GIVE ME THE
OPPORTUNITY TO INTRODUCE WORK
HERE. HUMAN PAPILLOMA VIRUS
[INDISCERNIBLE] DISEASE IS NOT
PART OF THE VIRUS LIFE CYCLE.
IN FACT, INTEGRATION IS THAT END
FOR THE VIRUS. IT IS NO LONGER
ABLE TO REPLICATE AND MAKE A NEW
VIRUS. HPV INTEGRATION BY
DISRUPTION OF E1 AND E2 GENES
RESULTS IN INCREASED EXPRESSION
OF E6 AND E7 ONCOGENES AND HAS
DEGRADATION OF [INDISCERNIBLE].
CONSEQUENTLY PROMOTING CELL
[INDISCERNIBLE] THIS GAVE CELLS
A SELECTIVE ADVANTAGE AND
PROMOTES ONCHOGENIC PROGRESSION
AND HPV ASSOCIATED CANCERS ARE
DEPENDENT ON EXPRESSION OF
VIRUSES ONCOGENES FOR CANCER
CELL PROLIFERATION AND SURVIVAL.
IT IS THAT THERE IS INTEGRATED
VIRAL GENOME IS OFTEN DISRUPTED
NEXT 1 AND E2 GENES IN CANCER
TISSUES THAT MIGHT BE A
SELECTIVE FEATURE OF CANCER
PROGRESSION AND DISRUPTION OF E1
AND E2 DESTROY SIGNAL FOR E6 AND
E7 EXPRESSION.
E6 AND E7 USES THEM AND THEY ARE
EARLY PROMOTER P97 AND
TRANSCRIBES WITH [INDISCERNIBLE]
CALLED E6 PROTEIN AND OTHER TWO
TRANSCRIPTS WITH SPLICED OPEN
READING FRAME. ALL THESE
TRANSCRIBE TO USE THIS EARLY AT
DIVISION SITE VIRUS INFECTION.
FROM THIS FIGURE, WE CAN SEE IF
E1 AND E2 IS DISRUPTED THAT ALL
THESE WILL LOSE EARLY POLY A
SITE THAT WON'T MAKE E6 AND E7
ONCHOPROTEIN.
QUESTION IS HOW DOES HPV
INTEGRATE TO EXPRESS THESE GENES
IN CANCER CELLS?
FIRST, WE ANALYZE THIS DATA FOR
HPV INTEGRATION FROM TCG DATA
BASE AND THESE DATA BASES.
HERE IS PRINCIPLE FOR DATA
ANALYSIS IN JUNE SEQUENCING 1D
FRAGMENTED IS SEQUENCED TO FIRM
BOTH DIRECTIONS AND INTEGRATION
ANALYSIS FOCUS DOWN THIS VIRUS
HOST JUNCTION RATES CALLED CGRS
THAT REFLECT THIS INTEGRATION
SITE IT THAT IS VIRAL.
7 TAENCASES OF 55HPV16 AND 18
POSITIVE CANCER TISSUES SHOWS
MULTIPLE DENATURE INTEGRATION
SITE AND ONE SITE EXPRESSES
[INDISCERNIBLE] AND EXEMPLIFIED
IN THIS TISSUE. HERE IS HP16
RISK COVERAGE SHOWING GENOME IS
COVERED BY THIS FROM ALL
INTEGRATED SIZE SHOWING RATES
WITH NO RATES FROM VERY EARLY
POLYINSIGHT AND DAZE AH
INTEGGATED INTEGRATION AT TISSUE
LED TO THIS.
DNA SEQUENCE CGR MAPPING
IDENTIFIED INTEGRATION SITES IN
THE TISSUE.
IT SHOWS THIS WAS EXPRESSED.
BASED ON CGR DETECTED WE CAN MAP
HOW IT WAS INTEGRATED AND
EXPRESSED.
THIS IS HOST GENOME MAP FIELD
AND SHOWN IN THE FIGURE
[INDISCERNIBLE] INSERTED INTO
[INDISCERNIBLE] REGION ON
CHROMOSOME 4. POLY A SIGNAL
DOWNSTREAM WAS LOST TOO. E6 AND
E7 TRANSCRIPTS WOULDN'T BE
[INDISCERNIBLE] SUBSEQUENTLY WE
CONSTRUCT HOST GENOME THAT IS
BASED ON 2 CHEMMER JUNCTION
SITES. SPLICING OF THIS E6 AND
E7 AND A TO DOWNSTREAM HOST POLY
A SITE LOCATED IN CHROMOSOME 4.
THIS SPLICING PRODUCED TO THESE
TRANSCRIPTS AND TRANSCRIPT 2
PRODUCING E7 PROTEIN.
EXAMPLE, ALL OF THESE TISSUE
SAMPLES DISPLAYED IN THIS TABLE
AT MULTIPLE INTEGRATION SITES
AND BY ANALYSIS AND SURPRISINGLY
IN EACH CASE THIS SHOWS THAT
ONLY ONE OF THE INTEGRATED
CHROMOSOMES THAT WERE EXPRESSING
CHEM ERIC -- AFTER THIS ANALYSIS
WE FOUND ONLY ONE INTEGRATED
SITES ON THE CHROMOSOME THAT IS
EXPRESSING CHEM ERIC HOST
PROGRAM.
IN ORDER TO VERIFY OBSERVED HPV
INTEGRATION EXPRESSION, WE
PERFORM THIS ANALYSIS AND ANSWER
IS CANCER CELL LINES WITH THIS
DATA YOU THAT IS AVAILABLE FROM
PUBLIC DATA BASE AND VERIFIED IS
RESULTS BY TRADITIONAL TECHNICS
INCLUDING THIS 3 PRIME RATES AND
NONBLOT.
WE FIRST MAPPED CASCADE
[INDISCERNIBLE] TO HPV16 GENOME
AND LOWER PART HAS FRAMES IT
THAT WE CAN SEE E6 AND E7 AND E1
READING FRAME IN TACT THAT
TRANSCRIPTION IS TERMINATED AT
STUDY 7 AND 29 AT E2 RATING THAT
IS INDICATING THAT INTEGRATION
REMOVES THIS POLY A SIGNAL AND
HOST POLY A SIGNAL DOWNSTREAM
MUST BE AVAILABLE FOR E6 AND E7
EXPRESSION AND THESE CELLS HAVE
-- MAPPING THE [INDISCERNIBLE]
TO HUMAN AND HPV16 GENOME WITH
MOST OF THE CGR JUVENGSS CAME
FROM HOST CHROMOSOME 6 AT
SPECIFICATION AND THIS
TRANSCRIBES AND THIS IS E2 GENE
INDICATING IT THIS IS ONLY
CHROMOSOME REGION EXPRESSING
THIS.
NEXT, WE MAP TWO CHEM ERIC
JUNCTION SITES FROM THIS TO
CHROMOSOME 6 IN THESE CELLS IT
THAT IS SHOWING IN THE FIGURE
THE LINEAR [INDISCERNIBLE] HPV16
WAS INSERTED INTO INTERGENIC
REGION IN CHROMOSOME 6 AND
INTEGRATION JUNCTION SITES
VERIFIED IN PCR SEQUENCING
ACCORDING TO THESE SUBSEQUENTLY
CUBED VARIOUS HOST OF CHEM ERIC
GENOME AND RATES TO
[INDISCERNIBLE] AS SHOWN HERE,
WAS TRANSCRIBED FROM EARLY
PROMOTER P97 AND SPLICED TWICE
BEFORE REACHING TO HOST POLY A
SITE. THIS IS VERIFIED BY 3
PRIMASE. THESE ALTERNATIVE
SPLICING PRODUCED THIS
TRANSCRIBES AND WE CONFIRM
DISEASE PRODUCTS BY THIS USING A
VERY SPECIFIC PROBE, C2 FROM E7
REGION.
TO CONFIRM THIS EXPRESSION FROM
IDENTIFIED INTEGRATION SITE ON
CHROMOSOME 6, WE DESIGNED A HOST
THAT IS AS BIG AS
[INDISCERNIBLE]. AS I SEE, IT
THIS IS HOST GENE NEXT TO CGR
JUNCTION ON CHROMOSOME 6 AND
THIS BLOT ANALYSIS CONFIRMED
THIS PROTEIN EXPRESSION WAS
ABOLISHED UPON THIS SPECIFIC AS
KNOCKED DOWN WHEN COMPARED TO
TARGETING CONTROL AND SHOWN IN
CELL PROLIFERATIONS HERE THIS
GROWS AND NOT TO HPV NEGATIVE
THIS CELL WAS INHIBITED BY
SPECIFIC KNOCKING DOWN WHEN
COMPARED TO THIS.
LIKE WE DID IN THESE CELLS WE
DESIGNED HOST SPECIFIC
[INDISCERNIBLE] TARGETING HOST
[INDISCERNIBLE] NEXT TO CGR
JUVENGSS CHROMOSOME 18 CELLS AND
WESTERN BLOT ANALYSIS CONFIRM
THESE PROTEIN EXPRESSION WAS
ABOLISHED SPECIFIC AS IT WAS
KNOCKED DOWN WHEN COMPARED TO
THESE.
AND HEELO CELL GROWS BUT NOT
THIS A CELLS INHIBITED BY
SPECIFIC WHEN KNOCKED DOWN WHEN
COMPARED TO THIS.
SO, FROM THE CLINICAL STANDPOINT
AND CELL INTEGRATION ANALYSIS,
WE CAN SEE HPV INTEGRATION
HAPPENS RANDOMLY AND HOST GENOME
REGION WITH INTEGRATION HAS
DELETION ARRANGEMENTS AND
INTEGRATES INTO THE GENOME AND
IF THIS POLY A SITE WAS LOST
THIS IMPORTANT THING FOR THE
VIRUS IS FINDING HOST POLICY A
SITE NEARBY FOR THIS GENE
EXPRESSION AND HIGH STABLE E6
AND E7 EXPRESSION FROM
INTEGRATION SITE IS ESSENTIAL
FOR FURTHER [INDISCERNIBLE]
EXPANSION AND [INDISCERNIBLE].
IN THIS REPORT, WE SHOWED THAT
ONCOGENES ARE EXPRESSED IN
SINGLE INTEGRATION SITES IN
CANCER TISSUES AND CELL LINES
WITH MULTIPLE [INDISCERNIBLE]
INTEGRATION SITES AND INTEGRATED
[INDISCERNIBLE] ARE FRAGMENTED
AND EXPRESSED GENOMIC DNASE
LACKING PROMOTERS SPLICING SITES
OR [INDISCERNIBLE]. E1 AND E2
REGION FOR INTEGRATION AND
EXPRESSION OF ONCHOGENIC E6 AND
7 MUST UTILIZE HOST POLY A
SIGNAL DOWNSTREAM OF INTEGRATION
SITE AND CELL PROLIFERATION AND
CLONAL CELL EXPANSION. I WANT
TO THANK ALL MEMBERS IN OUR LAB.
OUR [INDISCERNIBLE] TEAM, ALEX
AND MAGGIE AND [INDISCERNIBLE]
FOR HIS ADVICE ON OUR ANALYSIS
AND [INDISCERNIBLE] FOR
PROVIDING E6 ANTIBODIES AND
THANKS ALL FOR LISTENING. I'M
HAPPY TO TAKE ANY QUESTIONS.
>>[APPLAUSE].
>>>>THAT WAS A NICE TALK.
WHAT DO YOU THINK EFFECTS COULD
BE ON CELL BEHAVIOR, FOR
EXAMPLE?
>>WE DID LOTS OF E6 AND E7
KNOCKDOWN EXPERIMENTS BEFORE.
IF WE DO THAT, CELL GROWS
INHIBITED AND CELL WILL DIE.
>>THANK YOU.
>>I ACTUALLY HAVE AN ADDITIONAL
QUESTION. DO YOU KNOW IF THERE
IS A DIFFERENCE BETWEEN
INTEGRATION SITES IN PEOPLE THAT
PROCEED OR NOT TOWARDS
DEVELOPMENT OF THE CONCERT OF
THE KREFB CAL LEVEL?
>>VERY GOOD QUESTION. WE DON'T
HAVE THE DATA, UNFORTUNATELY.
WE ONLY HAVE DATA FROM CANCER
STAGE.
>>THANK YOU.
>>NEXT SPEAKER IS SARAH DREMEL.
SHE WILL TALK ABOUT ALTERNATIVE
SPLICING AND CIRCRNA BIOGENESIS
DRIVEN BY ALPHA AND GAMMA HERPES
VIRUSES.
>>>>GOOD MORNING. SORRY FOR
THAT. I'M SARAH IN THE LAB IN
HIV AND AIDS MALIGNANCY BRANCH
AND KYLE GAVE AN INTRODUCTION
EARLIER ON HERPES VIRUSES THAT I
STUDY THEM ALSO AND WON'T TALK
MUCH ABOUT THEM THEMSELVES MY
PROJECT FOCUSES ON CIRCULAR RNAS
THAT ARE A NOVEL CLASS OF
TRANSCRIPTS FORMING CONTINUOUS
LOOP DUE TO 5 PRIME TO 3 PRIEF
CO-VAILANT LINKAGE TO BACKMRASH
JUNCTION.
THROUGHOUT THE TALK YOU WILL SEE
BACKSPLASH JUNCTION THEY HAVE
EXTENDED HALF LIVES AND ARE NOT
GENERALLY MUTAGENIC. THEY
FUNCTION TO MODULATE GENE
EXPRESSION AND -- IN SOME
INSTANCES THEY ACTUALLY
THEMSELVES CONSERVE AS
TRANSLATIONAL TEMPLATES.
IN LAST 5 YEARS THESE WERE
DISCOVERED STARTING WITH HERPES
VIRUSES THEMSELVES AND LIST
EXTENDEDS TO DOZENS OF VIRUSES
BOTH RNA AND DNA GENOME. MY
PROJECT IN PARTICULAR FOCUSES ON
CIRCULAR RNAS EXPRESSED BY
DIFFERENT HERPES VIRUSES AND
DEFINING CHARACTERISTIC OF
HERPES VIRUSES IS ABILITY TO
UNDERGO LYTIC AND LATIENT MODES
OF INFECTION REACTIVATES TO
VARIOUS RESPONSES OF STIMULI.
LOOKING AT TRANSCRIPTS GENERATED
IN SPECIFIC PHASES OF LIFECYCLE.
HERPES VIRUS AND THIS VIRUS 68
AND ALPHA VIRUS SIMPLEX VIRUS
TYPE 1 AND LOOKED AT CELL
CULTURE AND ANIMAL MODELS OF THE
VIRUSES TAKING TOTAL RNA -- SO,
THESE ARE ANALYZED WITH SPECIFIC
BIOINFORMATIC PIPELINE ALLOWING
US TO DIFFERENTIATE LINEAR
VERSUS CIRCULAR REEDS BASED ON
PRESENCE OF SOMETHING CALLED A
BACKSPLICE JUNCTION THAT IS ONLY
UNIQUE SEQUENCE COMPONENT TO A
CIRCULAR RNA FROM LINEAR
COUNTERPART MADE FROM THE SAME
GENE. USING THAT WE CAN
QUANTIFY LINEAR AND CIRCULAR
TRANSCRIPTS.
OKAY. HERE, I'M SHOWING YOU
RAW COUNTS FROM PART OF MY DATA
SITE LOOKING AT REEDS CONTAINING
BACKSPLICING JUNCTION. CIRCULAR
NRA READS -- HERE ARE THOSE
DERIVED FROM THE HOST GENOME. I
WAS SURPRISED TO SEE THOUSANDS
OF VIRAL BACKSPLICE JUCHGSS
PRESENT IN DATA SIS CIRCRNAS ARE
THOUGHT OF AS LOW INCIDENCE
EVENTS PRESENT DURING LYTIC
PHASE OF THE LIFECYCLE.
UNSURPRISING THAT IS WHEN
MAJORITY OF TRANSCRIPTIONAL
ACTIVITY OCCURS THINK OF THIS AS
FUNCTION OF GENOME SIZE AND
INCIDENCE OF BACKSPLICING GENOME
INCIDENCE FOR HERPES VIRUSES IS
ANYWHERE FROM 800 TO 30,000 FOLD
GREATER FROM INCIDENCE OF
BACKSPLICING FROM THE HOST
GENOME. IN DATA I DON'T HAVE
TIME TO TALK ABOUT TODAY.
SEQUENCING RESULTS CONFIRMED FOR
MODELS USING RIBONUKE LEEACE
RESISTENT DNAS TO CONFIRM
CIRCULAR NATURE OF THESE
TRANSCRIPTS AND APPLYING
ORTHOGONAL APPROACH TO VALIDATE
FINDINGS.
SO, WHAT DO THESE CIRCULAR NRA
READS LOOK LIKE?
I WILL FOCUS ON TWO MODELS OF
LATENT INFECTION AND VIRUS AND
HSV1. HERPES VIRUSES ENCODE A
SMALL HANDFUL OF TRANSCRIPTS
DURING LATENT PHASE OF THE LIFE
CYCLE AND TRANSCRIPTS
SURROUNDING THIS AND HSV1 THIS
INCLUDES LATENCY ASSOCIATED
TRANSCRIPT LONG NONCODING RNA IN
ITSELF CONTAINS STABLE INNERIM
THAT WE WERE PARTICULARLY
INTERESTED IN CIRCULAR RNAS
BEING DERIVED FROM THE LOCUST.
SO, HERE I'M SHOWING YOU
SOMETHING CALLED A PLOT THAT HAS
A DEPTH OF THE ARC THAT
INDICATES NUMBER OF READS FOR
THE BACKSPLICE JUVENGS AND
ACTUAL POSITIONS IS SLICE DONOR
AND ACCEPTOR FOR CIRCULAR RNA
I'M SHOWING MODELS FOR LATENT
INFECTION AND HERE WE IDENTIFIED
A NUMBER OF DIFFERENT CIRCULAR
ARE. NAS DERIVED FROM K12 AND
THIS 72 LOCUST AND WE WERE
EXCITED TO SEE CIRCULAR RNAS
DERIVED FROM THIS STABLE INTRON
DURING HSV1 MODEL LATENCIES.
WE CAN ACTUALLY FIND THAT SAME
CIRCULAR RNA IDENTIFIED IN LATE
MODELS OF LYTIC INFECTION.
SO, TO NOTE, THIS ADDS
ADDITIONAL TRANSCRIPTS THAT ARE
PRESENT HAVING A FUNCTIONAL ROLE
DURING HERPES VIRUS LATENCY TO
DATE IT IS FIRST REPORT OF THIS.
SO, NOW, MOVING ON I WILL SHOW
YOU DATA SPECIFICALLY FOR LYTIC
MODELS OF INFECTION AND I'M
SHOWING YOU REED TRACES FOR
LINEAR AND BACKSPLICE READS
MAPPED TO KSHV GENOME WITH
SELECT GENES ANNOTATED BELOW AND
PATTERNS OF REEDS IN BLACK AND
RED YOU WILL SEE THEY LOOK
IDENTICAL. PATTERNS LOOK
SIMILAR AND IS SURPRISING AND
INSTANCES OF FORWARD SPLICING
SEE IF THEY CORRELATE WITH
BACKSPLICING AND IN GRAY I
PLOTTED REEDS THAT ARE FORWARD
SPLICE AND DON'T CORRELATE WITH
REGIONS OF BACKSPLICING AND
PLOTTING HOST REEDS MAPS THE
SAME WAY YOU DON'T SEE THIS
PHENOTYPE. I WENT AHEAD AND
QUANTIFIED THIS OBSERVATION. I
PERFORMED LINEAR REGRESSION
ANALYSIS LOOKING AT LINEAR AND
BACKSPLICE REEDS MAPPING TO
VIRAL GENOME IN PURPLE OR HOST
GENOME IN YELLOW IT IS A BUBBLE
BLO THE AND R2 VALUE BACKSPLICE
TRANSCRIPT ABUNDANCE AND CIRCLE
SIZE ITSELF INDICATES GIVEN LOCI
WILL GIVE RISE TO CIRCULAR RNA.
SMALLER BUBBLE MORE LIKELY THAT
GENE IS -- IN PURPLE LINEAR
REGRESSION ANALYSIS HIGH
STRENGTH OF CORRELATION FOR
VIRAL REEDS INDICATING CIRCULAR
ABUNDANCE IS CORRELATED WITH
LINEAR TRANSCRIPT ABUNDANCE SAME
OBSERVATION IS NOT TRUE FOR HOST
GENOME. THIS ANALYSIS CAN TELL
US LIKELIHOOD GIVEN REGION GIVES
RISE TO CIRCULAR RNA IN THIS
MODEL KHV LYTIC INFECTION 4 --
THIS MEANS IN THE DATA SET .2%
OF VOOIRL READS WERE CIRCULAR
RNASE. THIS INDICATES IT MIGHT
BE A SEQUENCE INDEPENDENT
MECHANISM MAKING THESE. I WENT
AHEAD AND DID THIS. I'M SHOWING
VENN DIAGRAMS AND CENSUS MOTIFS
FOR THESE. WE SEE CANON CAL
SPLICE -- PRESENCE IN MOST HOST
CIRCULAR RNAS MAKING SENSE THAT
THEY HAVE BEEN SHOWN TO BE MADE
BY HOST LYSOSOME WE DON'T SEE
FOR VIRAL -- THIS IS SEQUENCE
INDEPENDENT SYNTHESIS AND QUICK
SUMMARY, THEY DON'T CORRESPOND
BEFORE SPLICING INJUNCTIONS AND
DON'T USE CANON CAL SPLICE DONOR
AND ACCEPTOR AND DON'T HAVE TIME
TO SHOW TODAY. EXPRESSION IS
RESISTANT TO MAJOR SPLICEOSOME
INHIBITION. IS SOMETHING
SPECIFIC TO LYTIC PHASE OF THE
LIFE CYCLE PROMOTING THIS UNIQUE
RNA SYNTHESIS MECHANISM?
TESTING IT THAT TOOK ADVANTAGE
OF THIS ACTIVE REPORTER PRESENT
IN THE KSHV GENOME. IT IS UNDER
THE EF1 ALPHA PROMOTER AND IN
THIS WAY I CAN LOOK AT CIRCULAR
RNASE DURING LYTIC AND NATIVE
PHASE OF THIS. HERE, I'M
PLOTTING CIRCULAR A TRANSCRIPT
RATIO OF CIRCULAR TO LINEAR
REEDS FROM THE LOCUST AND GRAY
IS LYTIC REACTIVATION AND BLUE
IS LATENT REINFECTION.
IN NASCENT RNA YOU SEE TEN FOLD
INCREASE AND SEEMS LYTIC PHASE
OF THIS LIFE CYCLE IS DRIVING
THIS. SO, THAT BROUGHT ME TO
QUESTION WHAT UNIQUE EFFECTERS
ARE PRESENT DURING THE LYTIC
PHASE PROMOTING CIRCULAR PHASE?
VIRAL RNA BINDING PRO E- TEEN OR
RNA57 AND HOMOLOG IN ALL HERPES
VIRUSES FANTASTIC WORK IN THE
FIELD WE KNOW INTERACTS WITH
HOST SPLICING MACHINERY AND HAS
ROLE TO PREVENT RNA DECAY IN
CONTEXT OF PROJECT I WANT TO SEE
IF IT BINDS TO THIS. SECOND OF
ALL, HOW MIGHT IT EFFECT
ACCUMULATION IS IT THIS
IMPACTING SPLICING OR IS IT POST
TRANSCRIPTIONAL IN THAT IT MIGHT
PREVENT DEGRADATION IN
COLLABORATION WITH NICK CONRAD
AT SOUNL WESTERN WE ANALYZED
457E CLIP DATA IN THE PIPELINE
WE CAN SEE IF THEY BIND THESE I
AM SHOWING FORWARD AND
BACKSPLICE JUNCTION REEDS IN
SIZE MATCH INPUT OR 457IP AND
YOU SEE 11-FOLD ENRICHMENT IN IP
INDICATING 57 BINDS FOR CIRCULAR
RNASE AND WHEN WE LOOK AT HUMAN
REEDS WE SEE THAT 57 SEEMS TO
PREFERENTIALLY BIND ADJACENT TO
FORWARD AND BACKSPLICE JUVENGSS
AND INVESTIGATING WHAT IMPACT OF
THESE MIGHT BE AND I EMPLOYED
TOTAL AND NASCENT RNA SEQUENCING
AND IN THIS TIME DID IN CONTEXT
COMPARING WILD TYPE OR R57 NULL
MUN. I CAN LOOK AT CHANGESES IN
NASCENT AND TOTAL POPULATION TO
SEE WHICH STAGE N57 MIGHT HAVE A
ROLE. I CLUSTERED TRANSCRIPTS
AND AGAIN YOU ARE SEEING HERE A
PLOT LOG TWO FOLD CHANGE OF NULL
OVER WILD TYPE X AXIS LOOKS AT
TOTE AT RNA ABUNDANCE AND Y AXIS
LOOKING AT CO-TRANSCRIPTIONAL
ABUNKED ANSZ. IF SOMETHING
FALLS IN TOP RIGHT QUADRANT IT
IS R57 REPRESSED BEYOND THAT
LOOKING AT LEVELS ARE MORE
IMPACTED IN NASCENT ARE TOTAL
RNA IF SOMETHING FALLS IN
QUADRANT ABOVE DIAGONAL OR 57
ENHANCES ACCUMULATION POST
TRANSCRIPTION ALLEY I APPLIED
DATA AND CLUSTERING TECHNIQUE TO
LINEAR TRANSCRIPTS YOU CAN SEE
EVERY TRANSCRIPT FALLS IN BOTTOM
LEFT QUADRANT AND BEYOND THAT
THEY FALL ABOVE THE DIAGONAL
INDICATING ENHANCED POST
TRANSCRIPTION ALLEY THAT IS IN
LINE WITH WHAT IS PUBLISHED
ABOUT R57'S ROLE IN MODULATING
RNA DECAY THERE WERE NOTABLE
EXCEPTIONS THAT WOULD BE
CLASSIFIED AT OR 57 REPRESSED
THAT IS IN LINE WITH RECENT PUB
KAITION IN CONRAD LAB THAT
DEMONSTRATES THAT IT WILL
PROMOTE DEGRADATION FOR LATE
TRANSCRIPT MADE PRIOR TO DNA
REPLICATION PROVIDING CONFIDENCE
IN CLUSTERING METHOD AND
TECHNIQUES USED APPLYING TO
CIRCULAR REEDS THIS IS HOW DATA
LOOKS MAJORITY OF THESE FALLS IN
BOTTOM LEFT QUADRANT OR 57
ENHANCED ABOVE THE DIAGONAL.
THESE SEEM TO BE REPRESSIONED IN
LOW SIZE SPECIFIC FASHION. WITH
THAT I WOULD LIKE TO THANK
EVERYONE IN THE LAB WHO MADE IT
POSSIBLE AND COLLABORATORS NICK
AND CISTY AND BIOINFORMACIST WHO
HAS BEEN PHENOMENAL WITH THIS
PROJECT AND WITH THAT I WOULD
LOVE TO TAKE ANY QUESTIONS.
>>ANY QUESTIONS?
>>I MAY ASK.
>>UH-HUH.
>>DATA NEEDED SPLICING YOU HAVE
THOUGHT ABOUT WHAT MECHANISMS TO
USE?
>>MY DIAGRAM IS SHOWING IT
SLICES INDEPENDENT THAT IS
OPPOSITE WHAT IS SHOWN FOR HUMAN
CIRCULATED RNAS AND
INVESTIGATING A NUMBER OF
DIFFERENT METHODS FOR THIS THERE
ARE RNA LYINGASES THOUGHT OF IN
TERMS OF TRNA BIOLOGY AND NOVEL
ONE FOUND FOR HOST BONA FIDE
LYINGACE THAT IS POSSIBLE IT IS
RESPONSIBLE FOR CO-VAILIENT
LINKAGES.
>>ANY OTHER QUESTIONS?
OKAY.
>>I WILL INTRODUCE YOU TO JAMES
STAMOS WHO WORKS IN THE LAB.
HE RECENTLY GRADUATED. WHEN I
SAY RECENT, IT WAS MONDAY. VERY
RECENT AND WILL PRESENT TO YOU
DATA ABOUT OPPOSING EFFECT ON
SIVMAC 251 ACQUISITION OF
MONOCLONAL ANTIBODIES
RECOGNIZING SIV V2 IN AN A HELIX
AND B SHEET CONFORMATION.
>>AS DR. WOISMAN NOTED THIS
MORNING THIS PHASE 3 CLINICAL
TRIAL WAS ONLY EFFICACIOUS
VACCINE CLINICAL TRIAL TO DATE
THEY ANALYZED IMMUNE CORRELATES
IN THE STUDY AND FOUND
NONNEUTRALIZING IGG BINDING
BOOPS OF THIS REDUCED RISK OF
ACQUISITION AND RIGHT SIDE AND
YOUR LEFT SIDE YOU SEE HIV
VARRANT AND SURFACE OF GP120
TRIMERS AND BOTTOM 3-DIMENSIONAL
STRUCTURE OF IT AS IF LOOKING
FROM TOP DOWN AND RED IN GREEN
ARE V2 LOOPS POSITIONED AT APEX
OF TRIEMER AND V1 DIRECTLY
ADJACENT TO IT SORT OF COVERING
IT.
IN THE TRIAL ALSO AS A SECONDARY
CORRELATE IN A VAXINESE WITH LOW
THESE ANTIBODIES ARE
NONNEUTRALIZING AND BINDING TO
INITIAL INFECTED CELLS IN MUCOSA
CLEARING HELLS FROM HOST BEFORE
IT GOES ON TO SEE SYSTEMIC
INFECTION.
SUBSEQUENT ANALYSIS V2 POSITION
166 TO 178 FOUND TO CONFORM TO
ALPHA HELICAL STRUCTURE ANTIBODY
HOTSPOT FOR ADCC. V2 LOOP IS
PARTICULARLY ATTRACTIVE HIV
VACCINE TARGET. THE LOOP BINDS
TO MUCOSA -- ACTIVATES CD4
POSITIVE T CELLS AND
PREFERENTIALLY EFFECTS THIS
EARLY ON AND POSITIVE MUCOSAL T
CELLS V2 LOOP MIGHT BE ABLE TO
TARGET VIRUS CELLS ACTIVATING
THEM CREATING MORE TARGET TO
CELLS CREATING FUEL INFECTION
AND THROUGH MANY VACCINE STUDIES
WHERE WE TOOK THIS VACCINE AND
TRANSLATED BACK TO PRECLINICAL
MODEL AND THIS IS A REPRODUCIBLE
CORRELATE RISK OF INFECTION THAT
SHOWS MCCOCK MODEL IS PREDICTIVE
MODEL FIGURING OUT HIV VACCINES
WE GET SAME CORRELATES AS WE DO
IN HUMANS AND HYPOTHESIS THAT
V1V2IDG CORRELATE NEEDS TO BE
REFINED AND WE BELIEVE ADJACENCY
OF V1 TO V2 INTERFERE WITH V2
ANTIBODIES AND SO DOWN HERE YOU
ARE SEEING THAT V1 NEXT TO V2
STRUCTURE AND 3-DIMENSIONAL
STRUCTURE YOU SEE V1 IN GRAY IS
PARTLY MASKING VERY 2 THAT IS
DOWN HERE IN RED.
AND IN FACT WHEN WE LOOKED AT A
LARGE NUMBERS OF VACCINATED
ANIMALS, WE FOUND THAT ANIMALS
WITH LOW AND WITH HIGH LEVELS OF
ANTI-V1 ANTIBODIES SHOWN IN
DASHED CAPITAL MEYER CURVE
COMPARED TO ANIMALS OF LOW V1
ANTIBODIES ACQUIRE INFECTION
MUCH EARLIER AND V1 IS
INTERFERING WITH VACCINE
PROTECTION INCREASING RISK OF
ACQUISITION IN MCKOCHL. WHAT WE
DECIDED TO DO WAS DELETE OUT THE
V1 FROM THE RV144 LIKE VACCINE
WE DEVELOPED AND IN
COLLABORATION WITH STRUCTURAL
[INDISCERNIBLE] KOR DOZO WE
GENERATED DELTA V1GP120
IMMUNOGEN EXCISING OUT V1 LOOP
AND PREDICTED V2 LOOP WOULD BE
IN ALPHA HELICAL STRUCTURE
TARGETED BY VACCINE AND LOOKING
AT THIS YOU SEE THAT THEY
ACQUIRE THE INFECTION LATER OR
ARE PROTECTED FROM INFECTION
COMPLETELY ALL TOGETHER AND
MIRACULOUSLY IN SAME STUDY HE
INSERTED GPG GLYCINE MUTATION AT
EXCISION SITE OF V1 PREDIKTSING
IT WOULD FORM A BETA STRAND
INSTEAD OF A-HELIX TARGETING
ACC. JUST THREE AMINO ACID SITE
COMPLETELY DESTROYING THIS.
IN THE PLATFORM WE FOUND THROUGH
MANY DIFFERENT STUDIES WITH SAME
VACCINE V2 SPECIFIC ADCC IS A
STRONG CORRELATE OF REDUCED RISK
THIS APPEARS TO BE AUGMENTING
THESE CAPABILITIES OF A VACCINE
AND THIS COLLECTIVELY SHOWS
CONFIRMATION OF V2 IS CRITICAL
TO VACCINE EFFICACY SHIFTING
FROM ALPHA HELICAL TO ß-SHEET
CONFIRMATION TO COMPLETELY
DESTROY THE VACCINE.
OVERALL, THIS MODEL REVEALED
SYSTEMIC IMMUNE RESPONSES
REPRODUCIBLE ACROSS MANY STUDIES
NO. 1 ANTI-V2 ANTIBODIES THAT
ARE FOUND IN THE PLASMA AND AS
WELL AS CHARACTERIZING BY EPI
GENETIC TECHNIQUES INCREASING
CHROMATIN ACCESSIBILITY IN KREB
1 GENE ACCESSIBILITY THERE
ASSOCIATED WITH PROCESS CALLED
EF ROWCYTOSIS AND THESE ARE
PRODUCED IN PLASMA AS WELL
ANTIINFLAMMATORY MOLECULES AND
WE HYPOTHESIZED THAT THESE
EFFECTORS GET THERE AND THERE IS
EARLY INFECTION OF TARGET CELLS
FOCUS OF INFECTION AND V2
ANTIBODIES RECOGNIZE A-HELIX OF
GP120 AND MEDIATE THESE THROUGH
N CASE CELLS AND EVERIO CITES
COMING FROM KREB ONE INCREASED
ACCESSIBILITY GET TO MUCOSA
CLEARING UP DEAD APOP TO THEIC
CELLS FROM MUCOSA MAINTAINING
ANTIINFLAMMATORY ENVIRONMENT IN
MUCOSA WITH THE INFECTION
WITHOUT CREATING MORE TARGET
CELLS.
-- PRODUCING IL-7 TAENSTRONG
CORRELATE REDUCED INFECTION
ACROSS SEVERAL STUDIES AND FIND
IN MANY STUDIES THESE TH1 AND
TH2 CELLS CORRELATE WITH REDUCED
RISK IN THE BLOOD AND FIND CELLS
ARE GETTING TO MUCOSA CREATING
LESS TARGET CELLS FOR THE VIRUS
AND MORE PROTECTION THAT LED ME
TO ASK THE QUESTION THAT
RESEARCH HYPOTHESIS WAS
MONOCLONAL ANTIBODIES TO PROTECT
THESE AGAINST SIV ACQUISITION
AND DOING THIS USE PAST
IMMUNIZATION AND THESE CHALLENGE
WITH SIV251 AND DOING THIS I
USED TWO ANTIBODIES THAT WERE
CLONED IN COLLABORATION BETWEEN
OUR LAB AND VRC.
THEY CLONED OUT B CELLS AND
VACCINATED AND PROTECTED MCCOCK
THAT IS CHALLENGED 22 TIMES AND
NEVER BECAME INFECTED CHOSE
ANIMAL TO PROFILE V2 SPECIFIC
RESPONSES FOUND THAT THESE B
CELLS BY USING PROBES WITH B1
WITH B2 PROBES WITH DIFFERENT
STRAINS OF SIV AND THEY FOUND
TWO ANTIBODIES NCI05 AND NCI09.
-- OVERLAPPING EPITOEP IN V2C
REGION DOWN HERE OVERLAPS WITH
THIS BINDING SITE DOWN HERE.
VERY IMPORTANTLY FOR THIS STUDY
IS THAT THESE TWO ANTIBODIES
HAVE COMPLETELY DIFFERENT FABS
BUT IGG1 EFFECTIVE FUNCTION
SHOULD BE THE SAME THAT IS
IMPORTANT AS I PROGRESS IN THE
TALK.
SO, THEY CHARACTERIZED -- WE
CHARACTERIZED THE THREE
DIMENSIONAL EPITOEP STRUCTURE
RECOGNIZED BY TWO DIFFERENT
ANTIBODIES THIS RECOGNIZES V2 IN
ROUGHLY LINEAR EPITOEP
STRUCTURE.
THIS IS IN CONTRAST TO THE NCI05
RECOGNIZING A HELICAL COIL
STRUCTURE IN V2. YOU CAN SEE
THESE TWO ANTIBODIES RECOGNIZE
VERY DIFFERENT EPITOEP
CONFIRMATIONS FROM ONE ANOTHER.
NEXT, WE COMPARE DIFFERENT
EFFECTOR FUNCTIONS OF ANTIBODIES
BETWEEN NCI05 IN RED AND NCI09
IN BLUE.
WE TAKE STEM CELLS CODING WITH
GP120 AND THEY KILL CODED CELLS
AND WERE ABLE TO MEASURE THAT
AND NCI05 AND NCI09 MEDIATE
EQUIVALENT LEVELS OF ADCC.
DIFFERENT ASSAY PERFORMED BY
DAVID AT UNIVERSITY WISCONSIN
MADISON WITH INFECTED CELLS IN
PRESENCE OF SOLUBLE CD4 THAT
BINDS TO TRIMERIC GDP120 ON
VIRUS INFECTED CELLS OPENING THE
STRUCTURE AND WHAT WE FIND IS
THAT NCI05 IS ABLE TO MEDIATE
THESE INFECTED CELLED WITH --
NCI09 DOES NOT AND NUANCED
DIFFERENCE IN WAY THAT THIS
PERFORMS BASED ON WHETHER
CONFIRMATION EPITOEP
CONFIRMATION IS EXPOSE THE IN
PRESENCE OF CD4 OR NOT AND NCI09
DOES NOT DO THAT AND HOWEVER
NCI09 ENGAGES PROCESS CALLED
[INDISCERNIBLE] AND
IMMUNOEVASION MECHANISM USED BY
PATHOGENS AND THIS MEASURES STEM
CELLS CODED WITH GDP120.
THE THP1 CELLS ARE ABLE TO
EXTRACT OFF BOTH ANTIGEN AND
ANTIBODY FOR SURFACE OF THE CELL
REDUCING IMMUNE RECOGNITION.
FURTHER, THERE WAS ANOTHER
DIFFERENCE THAT THE NCI09
PROMOTES ANTIBODY CELLULAR
PHAGOCYTOSIS STRONGER THAN NCI05
-- INFECTION OF MORE TARGET
CELLS YOU CAN SEE HERE I
MENTIONED BOTH ANTIBODIES HAVE
SAME IGG1 FC REGION HOWEVER
DIFFERENT FAB AND WAY THAT
ANTIBODY BINDS TO SURFACE OF
GP120 CODED CELLS ARE INFECTED
CELLS COMPLETELY CHANGES PROFILE
OF THE ANTIBODIES WE BELIEVE
THEY ARE BALANCED IN DIFFERENT
DIRECTIONS AND NCI09 APPEARS TO
HAVE COMPETING EFFECTOR
FUNCTIONS TAKING AWAY FROM ADCC.
FURTHER, WE LOOKED AT ABILITY OF
ANTIBODIES TO BLOCK V2 BIENTDING
TO ALPHA 4 BETA 7 AND THEY
EQUIVALENTLY BLOCK -- NCI09
APPEARS TO BLOCK T CELL
ACTIVATION MARKED BY CC5 UP
REGULATION MORE TARGET CELLS FOR
VIRUS AND NCI09 DOESN'T BLOCK T
CELL ACTIVATION BY B2. SO, I
DESIGNED A MASSIVE IMMUNOIZATION
STUDY AT MCKOCHL AND TOP IN RED
ARE NCI05 ANIMALS AND DOWN HERE
NCI09.
WHAT WE DID IN THE GROUPS IS
PASSIVE IMMUNIZED ANIMALS 4 DAYS
PRIOR TO CHALLENGE TO HAVE
ANTIBODIES REACH HIGH LEVELS OF
MUCOSA AND 3 TIMES AND
REINOCULATED ANIMALS AGAIN WITH
ANTIBODY TO MAINTAIN HIGH LEVELS
OF ANTIBODIES CHALLENGING
ANIMALS UNTIL THEY WERE INFECTED
AND NEITHER OF THEM PROTECTED
MCKOCHL AGAINST ACQUISITION OF
THESE COMPARED TO CONTROLS AND
V2 MONOCLONAL ANTIBODIES
THEMSELVES AND THE V2 REQUIRES
PRESENCE OF OTHER VACCINE
INDUCED INNATE IMMUNE RESPONSES
TO HARNESS PROTECTIVE
CAPABILITIES AND HOWEVER THAT IS
NOT THE FULL STORY WHEN I LOOKED
CAREFULLY IN RECTAL MUCOSA
MEASURING LEVEL OF MONOCLONAL
ANTIBODY IN THIS WHEN LOOKING AT
THIS GROUP THERE WAS A STRONG
CORRELATION WITH DELAYED
ACQUISITION WITH THIS SPECIFIC
ANTIBODY AND MIGHT BE PART OF
VULNERABILITY SITE EXPOSED UPON
CD4 BINDING INDUCING ADCC AND WE
SEE THIS AND NO CORRELATION WITH
NCI09 GROUP AND HOWEVER WHEN WE
LOOK AT PLASMA THIS IS
CORRELATED WITH INCREASED RISK
OF ACQUISITION. FURTHER IN THE
ADCC MEASURED IN PLASMA IS ALSO
TRENDING TOWARDS REDUCED RISK IN
THE NCI05 GROUP AND NOT NCI09
GROUP AND THISCYTOSIS MEASURED
IN BASELINE POSITIVE SITES
STRONGLY CORRELATED REDUCED
RISK.
SO, WHAT IS MISSING FROM THE
PASSIVE IMMUNIZATION STUDIES WE
HAVE CRYTOCITAL RESPONSES THAT
ARE REDUCED BY LBAC VACCINATION
AND CELLS ARE ABLE TO GET INTO
MUCOSA AND WHEN THERE IN
PRESENCE OF V2 ANTIBODIES CAN
THEY MEDIATE AUGMENTED ADCC IS
WHAT WE BELIEVE AND THESE
CLEANING UP APOP TO THEIC CELLS
ARE NOT THERE PASSIVE
IMMUNOIZATION EXPERIENCE YOU
NEED THESE LOCALIZED THERE TO
HARNESS CAPABILITIES OF V2
ANTIBODIES AND HELICAL
COILS/ALPHA HELICAL G2 APPEAR TO
BE VERY IMPORTANT IN OUR
VACCINE.
THERE IS ALSO WE ARE MISSING
NKP44 POSITIVE ILC17 POSITIVE
CELLS AAND REDEUCED NUMBER OF
TARGETED CELLS YOU NEED SYMPHONY
OF IMMUNE RESPONSES IN MUCOSA TO
PRODUCE RISK OF SIB ACQUISITION
AND WITH THAT THAFKING LAB AND
EVERYONE ELSE IN MY LAB I DON'T
HAVE TIME TO NECESSARILY GO
OVER.
VACCINE RESEARCH CENTER
PARTICULARLY [INDISCERNIBLE]
HELPED RESOLVE THE STRUCTURE OF
NCI05 AND MHRP AND UNIVERSITY OF
WISCONSIN MADISON NIAD AND NYU
AND EMORY HELPED TO DO THE
STUDIES AND WITH THAT I WILL
TAKE ANY QUESTIONS YOU HAVE.
>>[APPLAUSE].
>>OKAY.
>>THANK YOU.
>>I WOULD LIKE TO THANK
SPEAKERS AGAIN.
>>[APPLAUSE].
>>WE HAVE A 15 MINUTE BREAK SO
SEE YOU AT 11:15. THANK YOU.
>>WELCOME EVERYONE TO THE
SECOND SESSION. KEEPING THINGS
ON TIME, WE HAVE THE FIRST
SPEAKER, RAMONA MOLS WHO WILL
TALK ABOUT ROLE OF MONOCYTES,
CTL, AND NK CELLS IN PRIMARY
HTLV-1 INFECTION.
>>THANK YOU. I WANT TO THAFK
THE COMMITTEE GIVING ME
OPPORTUNITY TO PRESENT MY WORK
ON ROLE OF MONOCYTES IN HTLV-1
INFECTION. BRIEF INTRODUCTION
OF THE VIRUSES HTLV-1 IS RETRO
VIRUS AND ONCOVIRUS THAT EFFECTS
10 TO 20 MILLION PEOPLE
WORLDWIDE EVEN IF REAL NUMBER IS
HIGHER DUE TO LACK OF TESTING IN
MANY COUNTRIES.
HTLV-1 IS AGENT OF HUMAN T CELL
LEUKEMIA VIRUS TYPE 1.
IT IS COMMONLY OBSERVED IN
NATURED D1 INFECTED INDIVIDUALS
AND THIS IS REPRESENTATION OR
PICTURE OF HTLV-1 PATIENT
DISPLAYING INVEKTED DERMATITIS
THAT IS INDUCED BY THE VIRUS.
HTLV-1 TARGET PRIMARILY CD4T
CELL TO LESS EXTENT
[INDISCERNIBLE] CELL AND VIRAL
DNA FOUND TO BE DYE SECTABLE IN
PATIENT AND MONOCYTE AND DEN
DRIDIC CELLS AND OPTION OF THESE
CD4 CELLS AND CELLULAR
TRANSFORMATION THAT WILL
ULTIMATELY LEAD TO THE
DEVELOPMENT OF THIS LYMPHOMA AND
H1 INFECTED INDIVIDUAL IS
DEREGULATION OF IMMUNE RESPONSE
WITH ALTERATION AND PERTUBATION
OF [INDISCERNIBLE] AND HTLV-1
INFECTION IS LIFELONG DISEASE
AND VIRUS PERSISTS IN HOST OF
FACE OF STRONG ADAPTIVE
RESPONSES AND IN VIRUS OF
[INDISCERNIBLE] AND VIRAL
PROTEIN P12 AND MP8 PLAY AN
IMPORTANT ROLE THAT IS A
SCHEMATIC REPRESENTATION OF THIS
GENOME THAT ENCODES FOR VIRAL
PROTEIN.
P12 LOCALIZE ON ENDOPLASMATIC
RETICULUM AND IN GOLGI
UNDERGOING CLEAVAGE AND THEY
LOCALIZE ON CELLULAR MEMBRANE.
THE VIRAL PROTEIN P12 IS PLAYING
AN IMPORTANT ROLE IN PROTECTING
INFECTED CELL FROM IMMUNE
RECOGNITION BY DOWNREGULATING
THEM ON C + 1 ON SURFACE OF
INFECTED CELL THAT PROVIDES
PROTECTION AGAINST CD8 + --
INFECTED CELL MORE SUSCEPTIBLE
TO THE KILLING AND P12 IS
DOWNREGULATING INCOME 1 AND 2
THAT ARE MOLECULES THAT PROTECT
INFECTED CELL FROM THIS KILLING.
VIRAL PROTEIN P12 IS PROMOTING
[INDISCERNIBLE] OF VIRUS
INDUCING TRANSCRIPTION FACTOR
INVOLVING T CELL GROWTH THAT
INCLUDE STAT 5 AND NFAT.
VIRAL PROTEIN P8 IS RELEASE BY
-- FROM CLEAVAGE FROM THE VIRAL
PROTEIN P12 THAT LOCALIZED ON
CELLULAR MEMBRANE AND PROMOTING
TRANSMISSION OF THE VIRUS. YOU
CAN SEE HERE IN MICROSCOPY
PICTURE.
VIRAL PROTEIN PH IS POSITION OF
THE VIRUS FROM INFECTED CELL TO
UNINFECTED CELLS AND ALSO VIRAL
PROTEIN P12 IS UPREGULATING LFA1
ON SURFACE OF INFECTED CELL THIS
IS CLASSROOM MARKET PROMOTING
RECRUITMENT OF UNINFECTED CD4 TO
SITE OF INFECTION WHERE THEY
WILL GET INFECTED THAT IS
MECHANISM THAT INVOLVES USE TO
PROMOTE SPREADING ON VIRUS AND
VIRAL PROTEIN P8 PROTECT
INFECTED CELL FROM CDA MEDIATED
KILLING DOWNBINDING ON CD4 ON
CELL SURFACE AND [INDISCERNIBLE]
RESPOND TO BE PARTICULARLY
IMPORTANT FOR CONSISTENCE OF THE
VIRUS. IN ANIMAL STUDY
PERFORMED IN OUR LAB, WE SHOWED
THAT HLB1P KNOCKOUT VIRUS THAT
IS LACKING EXPRESSION OF VIRAL
PROTEIN P12 AND P8 GIVE
UNININSTRUCTION IN THE INFECTION
IN [INDISCERNIBLE] IN THIS STUDY
WE INOCULATED [INDISCERNIBLE]
WITH WILD TYPE VIRUS AND P12KO
VIRUS AND FOLLOWED 20 WEEK POST
INOCULATION OF THE VIRUS AND
USED WESTERN BLOT TO DETECT
PRESENCE OF THE ANTIBODY AND
POSITIVE AND NEGATIVE WESTERN
BLOCK TO LOOK LIKE AND PRESENCE
OF ANTIBODY WE HAVE ACTIVITY
AGAINST HTLV-1 ANTIGEN AND
ABSENCE OF ANTIBODY THERE IS NO
ACTIVITY AGAINST THESE ANTIGEN
AND THESE REMAINED COMPLETELY
BLANK. BASED ON THIS STUDY AND
OUR STUDY PERFORMED IN THE LAB
WE ASSESS INFECTIVITY OF WILD
TYPE VIRUS TO BE 50 TO 75%. ON
OTHER END KNOCKLATING INDIVIDUAL
WITH THIS VIRUS NONE OF THE
ANIMALS CONVERTED AND WE
COULDN'T DETECT PRESENCE OF
VIRAL DNA IN THIS OF ALL ANIMAL
AT ANY TIME POINT DURING THE
STUDY THAT ASSESS INFECTIVITY OF
P12 KNOCKOUT VIRUS TO BE 0%.
OUR LAB SHOWS IMPORTANCE IN THE
PERSISTENCE AND ORF1 IS
PROTECTING DEVELOP FROM MEDIATED
KILLING IN VITRO AND THE
EXPRESSION IS REQUIRED FOR
PERSISTENCE OF THIS AND VIRAL
DNA WAS FOUND TO BE DETECTIBLE
IN A MONOCITE ISOLATED FROM HV1
INFECTED PATIENT AND LAB SHOWS
P12 KNOCKOUT VIRUS LACKING
EXPRESSION OF ORF-1 UNABLE TO
GIVE -- ALL THIS EVIDENCE LEADS
US TO OUR HYPOTHESIS THAT IS
THAT NKCTL OR MONOCITE INFECTION
IN VIVO AND THIS KNOWLEDGE IS
PARTICULARLY IMPORTANT FOR OUR
LAB. FINAL GOAL OF THE LAB IS
TO DEVELOP A PROTECTIVE HLV1
[INDISCERNIBLE] SO, TO STUDY
IMPORTANCE OF THE CD8 AND NK
MONOCYTE IN EARLY HTLV-1
INFECTION WE INOCULATED
[INDISCERNIBLE] WITH THE VIRUS
FOLLOWING THE DEPLETION OF THOSE
SPECIFIC SUBSET. THIS
SLIDESHOW, OUR STUDY DESIGN AND
DIVIDED ANIMAL IN THREE GROUP
AND FIRST GROUP WE DEPLETED
ANIMAL CD8 AND NK AND SECOND
GROUP DEPLETE IT ALONE AND THIRD
GROUP DEPLETE MONOCYTE AND
MACROPHAGES AND INOCULATE ANIMAL
WITH WILD TIEF OR P12 KNOCKOUT
VIRUS AND FOLLOW INFECTION FOR
20 WEEKS GROUP OF DATA SHOWS --
INOCULATED WITH WILD TYPE VIRUS
SHOWS ALL ANIMALS FULLY
CONVERTED WITH CONVERSION START
AS EARLY AS WEEK 4 POST
INOBJECTION OF THE VIRUS AND
DEPLETED WITH CD8 ALONE WE SHOW
ANIMALS CONVERTED WITH
[INDISCERNIBLE] AS EARLY AS WEEK
8 POST INOCULATION VIRUS AND
GROUP OF ANIMAL DEPLETED WITH
MONOCYTE AND MACROPHAGES ALL
ANIMALS CONVERTED AND END OF
STUDY AND WEEK 21 WE START TO
SEE DECREASED LEVEL OF ANTIBODY
IN PLASMA OF ANIMAL SUGGESTING
IN THIS CONDITION THAT INFECTION
IS NOT SUSTAINED AND WE THEN
CONFIRM OUR DATA BY TESTING
ANTIBODY TIGHTER AGAINST HW1
ANTIGEN. THIS DILUTES PLASMA IN
INDIVIDUAL AND WE PLOTTED
HIGHEST DILUTION AND ACT AGAINST
HTLV-1 ANTIGEN IN THIS CONTEXT
HIGHER DILUTION IS ASSOCIATE THE
WITH HIGHER ANTIBODY IN THE
PLASMA OF THE INDIVIDUAL AND
STUDIES SHOW GROUP OF ANIMAL
THAT IS DOUBLE DEPLETED FOR CD8
AND NK IS A GROUP OF ANIMAL WITH
HIGHER LEVEL OF ANTIBODY
SUGGESTING THAT THIS DEPLETION
IS DEPLETION THAT EXACERBATES
THE INFECTION THE MOST AND SHOW
THAT IN GROUP OF ANIMAL DEPLETED
WITH MONOCYTE AND MACROPHAGES
END OF THE STUDY IN WEEK 21 WE
START TO SEE DECREASE IN
ANTIBODY TIGHTER SHOWING
INFECTION IS NOT SUSTAINED AND
PRESENCE OF INFECTION AND DETECT
VIRAL DNA IN THIS ANIMAL AND
DATA SHOW ALL ANIMALS FULLY
CONVERTED ARE FOUND TO BE
POSITIVE FOR VIRAL DNA IN PBNC
AND IN GROUP OF ANIMAL THAT IS
DOUBLE DEPLETED FOR THIS
INOCULATED WITH P12 KNOCKOUT
VIRUS ALL ANIMALS ARE FULLY
CONVERTED SHOWING FIRST TIME
DOUBLE DEPLETION NK FULLY RESCUE
LACK OF [INDISCERNIBLE] OF P12
KNOCKOUT VIRUS AND CONVERSION
STARTING AS EARLY AS WEEK 8 POST
INOCULATION OF THE VIRUS AND WE
SHOW ONE ANIMAL FULLY CONVERTED
OUT OF 4 AND GROUP OF ANIMAL
DEPLETED WITH MONOCYTE
MACROPHAGES ONE ANIMAL CONVERTED
OUT OF 5 AND ONCE AGAIN BY END
OF THE STUDY WEEK 21 WE HAVE
DECREASED LEVEL OF ANTIBODY THAT
IS AGAINST IN THE PLASMA OF THE
ANIMAL THAT IS SUGGESTING IN
THIS CONTEXT THAT INFECTION IS
NOT SUSTAINED.
WE CONFIRMLE OUR DATA BY USING
THIS P24 ANTIBODY TIGHTER AND
SHOW THAT GROUP OF ANIMAL DOUBLE
DEPLETED FOR CDNK HAS HIGHER
LEVEL OF ANTIBODY AND CONFIRMING
PRESENCE OF VIRAL DNA IN THIS
PVC OF THE ANIMAL AND SHOW ALL
ANIMAL CONVERTED TO BE FOUND
POSITIVE FOR VIRAL DNA IN PBNC
AND INSIST P12 KNOCKOUT VIRUS AS
POINT MUTATION DESTROYING ATG OF
ORF1 AND DECIDE TO SEQUENCE
VIRUS TO MAKE SURE NO REVERSION
OF P12 KNOCKOUT MUTATION INTO
THE WILD TYPE AND DATA SHOWS NO
REVERSION DEMONSTRATING THIS IS
A TRUE INFECTION OF THE P12
KNOCKOUT VIRUS.
SO, IN SUMMARY, OUR DATA SHOWS
THAT IN CONTEXT OF THE WILD TYPE
VIRUS ALL DEPLETION RESULTS IS
EXACERBATING INFECTION SHOWING
100% INFECTIVITY AND DOUBLE
DEPLETION OF CDNK IS DEPLETION
THAT EXACERBATES INFECTION MOST
WITH AS EARLY AS WEEK 4 POST
INOCULATION OF THE VIRUS AND
[INDISCERNIBLE] BY END OF THE
STUDY DECREASED LEVEL OF
ANTIBODY SUGGESTING THAT IN THIS
CONDITION THAT INFECTION IS NOT
SUSTAINED.
IN THE CONTEXT OF THE P12
KNOCKOUT VIRUS SHOWING FIRST
TIME DEPLETION OF CD8/NK -- WE
SHOW THAT DEPLETION OF THIS
ALONE RESULTS IN 25% OF
INFECTION AND DEPLETION OF
MONOCYTE 20% INFECTION AND AGAIN
IN CONTEXT OF P12 KNOCKOUT WE
SHOW MONOCYTE DEPLETION RESULT
IN SUSTAINING INFECTION AND IN
SUMMARY, DATA SHOWS THAT
DEPLETION OF CD8NK MONOCYTE
MACROPHAGES -- INFECTION AND
DATA POINTS OUT CRUCIAL ROLE OF
K CELL RESTRICTING EARLY HIV-1
INFECTION SUGGESTING THAT
VACCINE SHOULD PRODUCE AN
ADAPTIVE RESPONSE AND STRONG
INNATE RESPONSE TO PROMPTLY
ELIMINATE THE CELLS AND DATA
HIGHLIGHTS DOUBLE ROLE OF
MONOCYTE ON ONE SIDE. MONOCYTE
AND MACROPHAGE RESTRICT EARLY IN
ONE INFECTION AND OTHER SIDE
CELL TYPES ARE NEEDED FOR VIRUS
TO PERSIST IN THE HOST AND DATA
THAT I'M PRESENTING TODAY ARE
RECENTLY ACCEPTED FOR
PUBLICATION ON [INDISCERNIBLE]
AND I'M A CO-AUTHOR IN THIS
PUBLICATION TOGETHER WITH
[INDISCERNIBLE] AND WOULD LIKE
TO THANK MENTORS FOR SUPPORTING
THIS PROJECT. AND MY LAB AND
COLLABORATORS TO MAKE THE
PROJECT POSSIBLE AND I'M HAPPY
TO TAKE ANY QUESTIONS.
>>SO, GIVEN IMPORTANCE OF THE
P12 AND P8 PROTEINS AND THAT
CLEAVAGE SITE I'M ASSUMING IT IS
PRETTY CONSERVED PEOPLE TRYING
TO DEVELOP SMALL MOLECULE
INHIBITORS TO THAT SITE ARE THEY
SAME AS KNOCKOUT?
>>THERE ARE MUTATIONS AND SOME
HAVE PRIMARY EXPRESSION DEPENDS
ON LIMITATION AND EXPRESS P12
AND P8 OR BOTH. BOTH OF THEM.
>>RIGHT.
>>IN PATIENT WE KNOW THIS LOAD
IS HIGHER WHEN THEY EXPRESS BOTH
AND WE THINK THIS IS BIOLOGICAL
ADVANTAGE AT THE SAME TIME.
>>OKAY.
>>I HAVE A COUPLE QUESTIONS.
>>WHEN YOU GET OR DO THE
INFECTION, YOU GET ZERO
CONVERSION. IS THAT LONG AFTER
THAT, EVEN IN HUMANS THAT THEY
WOULD SHOW THESE ANTIBODIES?
>>YEAH. NORMALLY WHEN THEY
START OR HAVE SYMPTOMS THEY
START TO HAVE HIGH LEVEL OF
ANTIBODIES AND THEY STAY HIGH
AND IN MCCOCK NORMALLY WHAT WE
SEE WHEN WE HAVE CONDITION OF
DEPLETION IN CONDITION OF WILD
TYPE VIRUS WITH HIGH LEVEL OF
ANTIBODY THEY STAY HIGH SO THEY
DON'T LOSE IT USUALLY AROUND 20
WEEKS.
>>CONVERSION OF WESTERN BLOTS
YOU SEE ONE COME OUT AT THE
BOTTOM YOU ARE NOT CALLING IT
[INDISCERNIBLE] CONVERSION IS
THAT BECAUSE IT IS INDUCED
ITSELF VIRUS COMING IN NOT NEW
VIRUS?
>>THEY ARE WESTERN BLOT
[INDISCERNIBLE] FOR PATIENTS AND
BASED ON KIT YOU CAN TALK ABOUT
PATIENT BEING POSITIVE WHEN YOU
HAVE THESE THREE BANDS.
OTHERWISE IT MIGHT BE YOU COME
ACROSS THIS. YOU CAN DO
SOMETHING. WE ARE NOT
CONFIDENT.
>>OKAY.
>>OKAY. THANK YOU.
>>OKAY. NEXT SPEAKER IS HANA
WHO WILL TALK ABOUT GUANO LATE
BINDING PROTEIN 5 IMPAIRING
VIERI ON INFECTIVITY BY
TARGETING ENVELOPE GLYENVELOPE
>>SOME OF THEM IN PARTICULAR
CAN TARGET ENVELOPE
GLYCOPROTEINS. AMONG THOSE FOR
EXAMPLE ARE IF DOMS DOWNREGULATE
AND BLOCK ENVELOPE FOLDING AND
WE HAVE LARGE PROTEINS AND WE
HAVE GUANYLATE BINDING PROTEINS
THAT I WOULD LIKE TO DEDICATE
THESE TO THE TALK. BROADLY
SPEAKING GUANYLATE -- THERE IS 7
MEMBERS OF THE FAMILY GBP1 TO
GBP7. THEY HAVE A COMMON
FEATURE THAT IS POST IN HOST
DEFENSE AGAINST IMPORTANCE
PATHOGENS FOR EXAMPLE
MICROBACTERIA CLIM IDDIA AND
HYSTERIA AND GBP5 GETS ATTENTION
IN FIELD OF VIROLOGY IT IS SHOWN
TO SHOW BROAD ANTIVIRAL
ACTIVITY. NAMELY BLOCKING
REPLICATION OF DIFFERENT VIRUSES
INCLUDING HIV AND HIGHLY
PATHOGENIC VIRUSES LIKE MEASLES
AND HIV AND IN PARTICULAR GBP5
REPORTABLY SHOWN TO -- SO WHAT
RESEARCHERS HAVE PROPOSED IS IN
PRESENCE OF GDP5 THIS ACTIVITY
IS SIGNIFICANTLY REDUCED AND
RIGHT-HAND SIDE YOU SEE A
SCHEMATIC WHAT MIGHT BE GOING ON
IN CELL IN PRESENCE OF GBP5 AND
THIS PROTEIN REQUIRES THIS
CLEAVANCE TO GET PROCESS INTO
GP100 -- NOW IN PRESENCE OF GBP5
SINCE ACTIVITIES IS INHIBITED OR
SIGNIFICANTLY REDUCED WE GET
THIS AND CONSEQUENTLY LEVEL OF
THIS IS REDUCED. NOW, THAT
MEANS NEWLY ASSEMBLED VIRIONS
AND AS A CONSEQUENCE THEIR
INFECTIVITY IS REDUCED.
HOWEVER, AS SEEMS -- YEAH.
HOWEVER. IT MIGHT NOT BE AS
STRAIGHTFORWARD AS IT SEEMS AND
PREVIOUS POST DOCK RESEARCH
FELLOW IN OUR LAB OBSERVED
RUNNING SAMPLES ON THIS TRIAL
AND INCREASE CONCENTRATION OF
GDP5 MOEKT SHIFTS AND YOU CAN
HOPEFULLY APPRECIATE ON LAB HAND
SIDE OF THE CELL SAMPLES ON THE
PLOT AND IN FACT GBP5 MIGHT HAVE
EFFECT ON GLYCOLIZIZATION OF
PROTEINS AND ASK OURSELF HOW GBF
INHIBIT GLYCOSCILLATYLATION.
WE COULD PSEUDOTYPE WITH
ENVELOPES FROM HIV OR SPIKE
PROTEIN FROM SARZ COV OR SARZ
COV2 VIRUS.
WE NORMALIZE INFECTIOUS VARIOUS
SEALED TO REVERSE TRANSCRIPTASE
VALUE AND USE THIS TO -- TWO
DAYS AFTER INFECTION, WE WERE
THEN ABLE TO MEASURE LUCIFEROUS
ACTIVITY USE TOTD DETERMINATE
INFECTIOUS YIELD OF PSEUDOTYPE
VIRUSES USED TO DETERMINE
INFECTIVITY IN PRESENCE OR
ABSENCE OF GBP5 AND BEFORE
PERFORMING THESE EXPERIMENTS I
WANT TO UNDERSTAND WHAT IS
PHYSIOLOGICAL RELEVANT AMOUNT OF
GBP5 AND ANSWERING THIS QUESTION
I TREATED THESE CELLS WITH --
INDUCIBLE GENE AND I WAS
SUCCESSFULLY ABLE TO DETECT GDP5
-- MOST IMPORTANTLY I WAS ABLE
TO SHOW THIS TRANCE INSPECTION
OF THESE CELLS CAN MIMIC
ENDOGENOUS EXPRESSION OF GBP5
UPON TREATMENT WITH INTERFERON
GAMMA. GOING BACK TO DATA WHERE
I HAVE MENTIONED THAT SHE
OBSERVED SHIFT IN GB20 AND 41
AND OBSERVING CONCENTRATION
INCREASES CONCENTRATION
INDEPENDENT INCREASE IN
INFECTIVITY OF VIRUS THAT IS
CONSISTENT WITH ALREADY
PUBLISHED DATA REPRODUCING DATA
THIS TIME USING PHYSIOLOGICAL
RELEVANT AMOUNT OF GDP5 AND
SIMILAR TO DATA SAW SHIFT IN
GB120 -- CONCENTRATION OF GBP5
INCREASED NOW UNDERSTANDING
WHETHER THIS IS REALLY AS A
CONSEQUENCE BECAUSE OF GDP5
MIGHT HAVE ROLE IN GLYCOLIZATION
OF PROTEINS TREATED SAMPLES
WHICH IS A [INDISCERNIBLE] THAT
CLEAVES OFF ALL N LINK GLYCO --
N LINK SACCHARIDES FROM PROTEINS
AND IN SAMPLE YOU TREAT THE
SAMPLE YOU GET A VERY HIGH
MOBILITY SHIFT FOR ALL BANDS
REPRESENTING GP60 AND THEY HAVE
BEEN SHOPPED OFF FROM ENVELOPE
GLYCOPROTEIN AND WITHOUT
TREATMENT AND IN PRESENCE OF
GBP5 WE SEE MOBILITY SHIFT IN 20
AND 41 BANDS ASSUMING THAT
[INDISCERNIBLE] THAT IS INDEED
THE CASE WE WOULD EXPECT TO SEE
UPON TREATMENT WITH PNJF SAME
MOBILITY SHIFT IN TREATED
CONTROL SAMPLE. YOU SEE BOTH
BANDS REPRESENTING GBP120 AND 41
AND 160 ARE SAME MOLECULAR WAIT
AND GBF PIEF REDUCES
[INDISCERNIBLE] ENVELOPE
GLYCOSYLATION.
SO, I PSEUDOTYPE WITH SPIKE
PROTEIN WITH COV AND COV2
VIRUSES SPIKE FROM COV2 GETS
CLEAVED AND SARZ COV SPIKE
PROTEIN REQUIRES A DIFFERENT
HOUSE PROTEASE. I ALSO OBSERVED
A MOBILITY SHIFT AS
CONCENTRATION OF GBP5 INCREASED
AND INTERESTINGLY ENOUGH,
ACTIVITY OF VIRUS WAS INCREASED
IN CONCENTRATION DEPENDENT
MANAGER OF GBP5 INCREASED AND
SIMILAR RESULT OBSERVED IN THIS
SPIKE AND YOU SEE HERE NICE
MOBILITY SHIFT AS THIS BAND
CLEAVED IN HIGH PRESENCE OF GBF5
AND LIKEWISE INFECTIVITY OF
VIRUS REDUCED IN CONCENTRATION
DEPENDENT MANNER AND THIS IS THE
CASE BECAUSE I TREATED SAMPLES
WITH THIS ENZYME AGAIN AND THIS
IS IN CASE OF SARZ COV SPIKE AND
CONTROL SAMPLE WE SEE MOBILITY
SHAPE SPIKE WITH TREATMENT OF
THIS BECAUSE SACKARICHARIDES HA
BEEN CHOPPED OFF AND WITHOUT
TREATMENT WE SEE MOBILITY SHIFT
BECAUSE OF REDUCTION OF
GLYCOSYLATION AND WE SEE WEIGHT
BANDS AS IN CONTROL TREATED
SAMPLE AND SAME DATA WAS
OBSERVED WHEN I DID THAT WITH
SARZ COV2 SPIKE YOU CAN
APPRECIATE MOBILITY SHIFT IN
PREFERENCE OF GBP5 WITHOUT
TREATMENT AND WE GET SAME
RESPONSES IN CONTROL SAMPLE AND
REDUCES GLYCOSYLATION OF
GLYCOPROTEINS AND USING COV AND
COV2 SPIKE PROTEINS WHETHER FOR
INTOOE PENDIENT PROCESSING AND
SUMMARIZING OUR WORK, WE SHOW
THAT GBP5 -- WE SHOW THAT IT
REDUCES HIV-1 ACTIVITY BY
INTERFERING WITH ENVELOPE
PROCESSING AND KRORNGS AND SHOW
IT REDUCES CORPORATION AND
VARIOUS INFECTIVITY FOR SARZ COV
AND COV2 VIRUSES AND IMPORTANTLY
AND NOT LASTLY SHOWING THAT IT
INHIBITS GLYCOPROTEINS
INCREASING MOBILITY OF SHIFT
THAT ANALYZES IT EFFECTS
GLYCOLIZATION AND LASTLY
PSEUDOTYPING COV AND SPIKE
PROTEINS WE SHOW THIS EFFECT
HAPPENS REGARDLESS WHETHER
GLYCOPROTEINS REQUIRE FOR
DEPENDENT OR INDEPENDENT
CLEAVAGE AND WITH THAT I WOULD
LIKE TO THANK LAB IN PARTICULAR
DR. ERIC AND BONNIE AND GERALD
EVEN THAT WERE HEAVILY INVOLVED
IN THE PROJECT AND ALL IN THE
AUDIENCE FOR YOUR ATTENTION. IF
YOU HAVE QUESTIONS, I WILL BE
HAPPY TO TAKE THAT.
>>HAVE YOU LOOKED AT OTHER
RETRO VIRUSES LIKE MLBS? ARE
THEY INFECTED?
>>WE HAVE LOOKED AT
[INDISCERNIBLE] GLYCOPROTEIN AND
WORKING ON FINALIZING DATA
DOESN'T SEEM TO BE EFFECT ON
ACTIVITY BUT NOT SURE BECAUSE OF
AMOUNT OF GLYCOPROTEIN THAT HAS
TO BE [INDISCERNIBLE] AND
INCORPORATION SEEMS TO BE
EFFECTED.
WE LOOK IN TUMORS IN TERMS OF
[INDISCERNIBLE] WITH SARZ CV AND
SARZ CO-V2 VIRUSES.
>>YOU HAVE LOOKED AT OTHER
PROTEINS?
>>TREATED CD4 -- THERE IS A
FACT. WE DON'T THINK IT
PARTICULARLY ONLY TARGETS VIRI
ON GLYCOPROTEINS.
>>HAVE YOU LOOKED AT VIRUSES
THAT USE CELL TO CELL
TRANSMISSION AND LOOKING AT
RECEPTORS THAT BIND AND HOW
ABOUT CELL TO CELL?
>>WE HAVE NOT LOOKED AT OTHER
VIRUSES ONLY OTHER ONES WE
LOOKED AT WERE GLYCOPROTEINS COV
AND [INDISCERNIBLE] MORE WE
COULD EXPAND INTO TESTING MORE
WITH THE SYSTEM IT THAT WE HAVE.
THANK YOU.
>>HELLO. EVERYONE IS DOG A
GREAT JOB STAYING ON TIME. I'M
AMAZED BY THAT. NEXT TALK IS BY
CONSTANCE RINK. HIS TITLE IS
EVALUATING HIV-1 GAG KINETICS
WITH RNAS USING SWITCHSENSE
TECHNOLOGY.
>>THANK YOU.
>>>>THANK YOU FOR THAT
INTRODUCTION. I'M CONNIE AND A
POST DOC IN ALLAN RYAN'S LAB AND
WILL TALK ABOUT KINETICS OF GAG
BINDING TO DIFFERENT RNAS AND
PROJECT FOCUSES ON VIRAL
ASSEMBLY THAT IS QUITE SIMPLY
THE FORMATION OF NEW VIRAL
PARTICLES.
AND THERE ARE TWO COMPONENTS IN
VIRUS ASSEMBLY, STRUCTURAL
PROTEIN GAG AND VIRAL RNA.
GAG SELECTS DIMERS OF VIRAL
GENOME TO PACKAGING TO VIRAL
PARTICLES AND PROCESS OCCURS AT
PLASMA MEMBRANE AND LONGSTANDING
QUESTION IN THE FIELD IS HOW
GENOMIC RNA IS SELECTED FOR
INCLUSION INTO ASSEMBLING
PARTICLES AND PROBLEM IS VIRAL
GENOME IS IN VAST PRESENCE OF A
BUNCH OF CELLULAR MRNAS AND TO
QUITE EASILY DEPICT THIS,
IMAGINE IT IS LIKE WHERE'S
WALDO. THE GENOME IS LIMITING
IN A VAST POOL OF CELLULAR MRNA.
IT HAS BEEN ESTABLISHED IN THE
FOO ELD THAT VIRAL GENOME IS
EXCLUSIVELY PACKAGED IN PRESENCE
OF CELLULAR MRNA AND VIRAL
GENOME WHEN IT IS NOT IN THE
CELL THAT GAG LEAVISHIENTLY AND
INDISCRIMINANTLY PACKAGE
CELLULAR MRNA.
SO, HOW DOES GAG ABLE TO
RECOGNIZE AND PACKAGE VIRAL
GENOME?
SIMPLY IT IS ABLE TO SELECTIVELY
PACKAGE 9KGB GENOME IT CONTAINS
PACKAGING SIGNAL OR
[INDISCERNIBLE] IT IS LOCATED IN
THAT PACKAGING SIGNAL IS CONTAIN
THE IN 5 PRIME UTR SHOWN IN THE
SECONDARY STRUCTURE THAT IS A
SECONDARY REGION WITH STEM LOOPS
AND IS ONLY A VERY SMALL REGION
WITHIN THE LARGE 9KB GENOME ONLY
356 NUCLEOTIDES AND SPECIFICALLY
THE PACK ABLGING SIGNAL IS A
SMALLER SUBSET IN THE 5 PRIME
UTR THAT IS COMPRISED OF LOOPS
1, 2, AND 3 AND POINTING OUT
ANOTHER IMPORTANT FEATURE WITHIN
PSI IS IN STEM LOOP 1 AND THIS
REGION SUPPORTS GENOME
DIMERIZATION THROUGH DIS THAT IS
DIMERIZATION INITIATION SITE AND
THIS IS A SMALL REGIONALLY 6
NUCLEOTIDES THAT IS A PALINDROME
SEQUENCE AND THIS IS WHERE
GENOME DIMERIZATION OCCURS AND
WE KNOW PSI CONFERS SELECTIVE
PACKAGING AND NATURE WHICH
OUTCOMPETE CELLULAR PACKAGING IS
UNKNOWN AND MANY THEORIES COULD
THEY HAVE A HIGH BINDING
AFFINITY FOR PSI AND IT LEAD TO
ASSEMBLY EFFICIENTLY BINDING TO
NON-PSIRNA OR DOES IT HAVE A
KINETIC ADVANTAGE OF IT COULD
ONE OF THESE OR COMBINATION OF
ANY ONE OF THESE THEORIES?
FIRST STARTING OFF PREVIOUSLY
OUR LAB AND OTHERS HAVE LOOKED
AT WHETHER OR NOT GAG COULD HAVE
HIGH AFFINITY FOR PSIRNA DONE
WITH IN VITRO BINDING STUDIES
DONE WITH RECOMBINANT GAG AND
DIFFERENT FEATURES OF RNA
TRANSCR
TRANSCRIPTS.
ITERATING THAT POINT THIS IS
DATA FROM OUR LAB AND QUENCHING
ASSAYS AND COMPARING MONOMERIC
AND DIPSI AND CONTROL GRAP THAT
IS REGION FURTHER UPSTREAM IN
THE GENOME AND SEQUENCE FROM
ANOTHER RETRO VIRUS AND YOU SEE
TAKE HOME POINT IS BINDING
CURVES OVERLAP OR ARE QUITE
CLOSE TO ONE ANOTHER AND WHEN
KDS ARE DERIVED FROM THERE,
THERE IS NO DIFFERENCE FROM
AFFINITY FROM THESE DIFFERENT
RNAS.
SO, SELECTIVE PACKAGING CANNOT
BE EXPLAINED SIMPLY BY JUST
HIGHER BINDING AFFINITIES.
SO, OUR LABORATORY HAS COME UP
WITH A HYPOTHESIS TO EXPLAIN
SELECTIVE PACKAGING THAT PSIRNA
IS SELECTIVELY PACKAGED DUE TO
EFFICIENCY OF THIS PROCESS.
THAT MEANS WHEN GAG BINDS TO
PSIRNA NUKE LEEATION KINETICS OR
FEW GAGS THAT BIND OR CREATE A
SEED KINETICS OCCUR FASTER THAN
PSIRNA AND OTHER RNAS IN THE
CELL THAT LEADS TO MORE
EFFICIENT LATTICE FORMATION ON
PSIRNA THAN ON OTHER CELLULAR
RNAS AND UNANSWERED QUESTION IS
WHETHER OR NOT GAG BINDS WITH
DIFFERENT KINETICS TO PSI AND
NONPSIRNA AND ADDRESSING
QUESTION WE UTILIZE SWITCHSENSE
TECHNOLOGY. SWITCHSENSE USES A
BIOCHIP THAT ON THE BIOCHIP ARE
FOUR FLUIDIC WELLS AND WITHIN
EACH OF THE WELLS IS 6 DETECTION
SPOTS. ON THOSE DETECTION SPOTS
IS MONOLAYER OF DNA NANOLEVERS
THAT ARE UNIQUE SEQUENCE THAT
EACH OF THEM IS LABELED RED OR
GREEN AND FROM THERE YOU ARE
ABLE TO HYBRIDIZE YOUR RNA OF
INTEREST.
AND AN IMPORTANT FEATURE OF THIS
TECHNOLOGY IS WE ARE ABLE TO
DIRECTLY COMPARE AT THE SAME
TIME TWO DIFFERENT RNAS TO IN
THIS CASE BINDING OF GAG
PROTEINS.
SO, WHAT THE -- AN EXAMPLE OF
KINETIC REED OUT OF THE KINETIC
MEASUREMENTS IS FOR SWITCHSENSE
WE ARE OBSERVING CHANGE IN
FLUORESCENCE OVER TIME AND
INITIALLY YOU WILL HAVE A
BASELINE FLUORESCENCE VALUE FOR
RNAS HYBRIDIZED TO THE SURFACE
AND INTRODUCE PROTEIN OF
INTEREST THAT BINDS TO RNA AND
YOU WILL OBSERVE QUENCHING OF
SIGNAL OVER TIME AND THIS CURVE
YOU WILL BE ABLE TO DRIVE A KON
OR ASSOCIATION RATE AND LOOKING
AT DISASSOCIATION WE WILL
INTRODUCE BUFFER ALONE. WE WILL
LOOK AT UNQUENCHING OR
RESTORATION OF FLUORESCENCE AS
YOUR PROTEIN UNBINDS FROM THE
RNA. FROM THERE, THESE CURVES
DERIVE A DISSOCIATION KOFF RATE.
SO, TO SHOW SOME RAW DATA, THIS
IS SHOWING GAG BINDING KINETICS
TO PSIRNA. YOU CAN SEE WE ARE
ABLE TO SUCCESSFULLY MONITOR GAG
RNA BINDING GAGS USING
SWITCHSENSE AND HERE WE MEASURE
WITH INCREASING GAG
CONCENTRATION TO 5 TO 0
NANOMOLAR OF GAG AND SEE
QUANTIFIABLE CHANGE IN
ASSOCIATION OF CURVES AND FASTER
RATES AS GAG IS TIGHT TREATED IN
AND POINTING OUT IN
PHYSIOLOGICAL CELLS BUFFER,
INTERACTION BETWEEN GAG AND RNA
IS A STABLE INTERACTION. WE CAN
CONCLUDE THAT BECAUSE FOR
DISSOCIATION CURVES THERE IS NO
QUANTIFIABLE DISSOCIATION BEING
ABLE TO BE MEASURED BECAUSE
THERE IS NO RESTORATION IN
FLUORESCENCE AND REST OF THE
DATA WE ARE SHOWING IT IS ONLY
ASSOCIATION RATES THAT WE WILL
TALK ABOUT.
SO, FIRST OFF, WE WANTED TO
COMPARE DIFFERENT FULL LENGTH
PSI. IT IS 145 NUCLEOTIDES IN
LENGTH CONTAINING STEM LOOPS 1,
2, AND 3 AND MAKING IT EASIER
MUTATED DIS SIGNAL CONTAINING
6CS INHIBITING DIMERIZATION WITH
ANOTHER PSI MOLECULE THAT WE
COMPARED TO A MULTIPLE BINDING
SITE MUTANT. WHAT EXACTLY THAT
MEANS IS IT HAS BEEN SHOWN
PREVIOUSLY THAT THERE ARE
NUMEROUS UNPAIRED GS THAT ARE
PROBABLY 16 OF THEM THAT ARE
LOCATED WITHIN PSI.
THEY HAVE BEEN LINKED TO BE
IMPORTANT IN PACKAGING AND WE
MUTATED GS TO AS AND ANOTHER
CONTROL IS WE HAVE A SCRAMBLED
UP PSI THAT WE LOOKED AT BINDING
KINETICS AND SHOWING COMPARISON
OF GAG ASSOCIATION RATES BETWEEN
PSI MULTIBINDING SITE MUTANT
UNSCRAMBLED AND WE SEE K ON RATE
FOR PSI IS SIGNIFICANTLY FASTER
THAN SCRAMBLED AND MULTIPLE
BINDING SITE MUTANT.
SO, LOOKING AT OVERALL TREND OF
THE DATA WE INTERESTINGLY FOUND
THAT A CONCENTRATION DEPENDENT
INCREASE IN GAG ASSOCIATION
RATES MEANING WE FOUND A FASTER
RATE PER GAG MOLECULE AND TREND
IN DATA MAY SUGGEST
COOPERATIVITY OF GAG FINDING AND
IMPORTANT TAKE-HOME MESSAGE IS
THAT YOU CAN SEE PSIRNA IS FAR
MORE SENSITIVE TO INCREASING GAG
CONCENTRATIONS THAN SCRAMBLED IN
MULTIPLE BINDING SITE MUTANT.
SO, NOW THAT WE HAVE FOUND THAT
THERE IS A DIFFERENCE IN
KINETICS BETWEEN PSI AND OTHER
RNAS WE WANTED TO SEE WHETHER OR
NOT FAST KINETICS COULD BE
MAPPED TO A SPECIFIC REGION OF
PSI. DOING THAT WE TOOK IT AND
BROKE IT DOWN INTO STEM LOOP
PAIRS CONTAINING SL1 AND 2 OR
COMPARING IT TO SL2 AND 3 AND WE
LOOKED AT ALSO IF YOU TOOK PSI
AND BROKE IT DOWN FURTHER INTO
INDIVIDUAL STEM LOOP COMPONENTS
LOOKING AT GAG BINDING KINETICS
AND SHOWING FIRST OFF LOOKING AT
THE DISCRETE REGIONS OF PSI
COMPARING STEM LOOPS 1 AND 2 AND
PSI2 AND 3 SCRAMBLED WITH
INCREASING GAG CONCENTRATION
THAT IS SHOWN IN THE GREEN THAT
IN FACT PSI2 AND 3 HAS
SIGNIFICANTLY FASTER GAG
ASSOCIATION RATES THAN SCRAMBLED
RNA IN PURPLE AND INTERESTINGLY
LOW RATE OF CONCENTRATION OF GAG
WE FOUND LOWEST DIFFERENCE IN
ASSOCIATION RATES SCRAMBLED IN
125 NANOMOLAR OF GAG. WE THEN
LOOKED AT THE INDIVIDUAL STEM
LOOPS.
YOU CAN SEE HERE THAT ACROSS THE
INDIVIDUAL STEM LOOPS WITH
INCREASING GAG CONCENTRATION
THAT THESE RNAS HAVE SIMILAR GAG
ASSOCIATION RATES TO ONE
ANOTHER. AND TO SCRAMBLE.
SO, THE INDIVIDUAL STEM LOOPS,
THEY ARE NOT REALLY CONTRIBUTING
TO THE FAST FINDING KINETICS WE
ARE OBSERVING WITH COMBINATION
OR WITH THE LENGTH PSI.
SO, IN CONCLUSION I HAVE SHOWN
YOU WE SEE A MODEST AND
REPRODUCIBLE GAG ASSOCIATION IN
KINETICS BETWEEN PSI AND
SCRAMBLED ARE. NA AND RESULTS
CONSISTENT WITH HYPOTHESIS
KINETIC EXPLANATION WITH
SPECIFIC PACKAGING OF VIRAL
GENOME AND INCREASE IN GAG
ASSOCIATION RATES FASTER RATE
PER GAG MOLECULE AND
INTERESTINGLY, WE SEE THAT THE
SPECIFIC UNPAIRED GS WITHIN PSI
ARE CONTRIBUTING TO RAPID
BINDING KINETICS WHEN MUTATED
THEY HAVE SIMILAR KINETICS TO
UNSCRAMBLED RNA AND GAG
ASSOCIATION RATES ARE ACTUALLY
LOST WHEN BROKEN DOWN INTO
INDIVIDUAL STEM LOOPS THAT THESE
SEQUENCES ARE NOT IMPACTING
BINDING KINETICS INTERESTINGLY
TWO STEM LOOPS TOGETHER
PARTICULARLY 2 AND 3 HAS FASTER
ASSOCIATION RATES THAN ANOTHER
REGION WITHIN PSI IMPLYING THAT
GAG BINDING KINETICS IS
DETERMINED BY 3D STRUCTURAL
ORGANIZATION OF PSI EXPLAINING
WHY PSI HAS FASTER K ON RATES
THAN A SCRAMBLED RNA AND LASTLY
ONGOING EXPERIMENTS WE WANT TO
DETERMINE BINDING AFFINITIES OF
KDS THAT COULDN'T BE DERIVED
FROM SWITCHSENSE STATUS WE
WEREN'T ABLE TO OBSERVE
DISSOCIATION AND LOOKING AT
MICROSKWAL THERMOFORESIS FOR
THAT AND WANT TO DETERMINE GAG
MOLECULES FOR RNAS WE HAVE BEEN
TESTING USING A NEW TECHNOLOGY
COMING TO NCI WHICH IS MASS
SPECK TROMTRY AND WE WILL LOOK
AT IN VIVO EFFECTS LOOKING AT
STEM LOOP FOR IN VIVO PACKAGING
EFFICIENCY AND WITH THAT
THANKING MENTOR FOR ALL THEIR
HELP ON THE PROJECT AND
BIOPHYSICS RESEARCH SURVEY IN
MARZENA FOR HELP WITH
TECHNOLOGIES USED IN THE STUDY
AND WITH THAT I WILL TAKE ANY
QUESTI
QUESTIONS.
>>DO YOU KNOW IF THERE ARE
CHANGES IN DIFFERENT
[INDISCERNIBLE] OF HIV IN STEM 2
AND 3?
>>SO, THAT REGION IS HIGHLY
CONSERVED. SO --
>>[INDISCERNIBLE].
>>WELL, NOT EXACTLY COMPLETELY.
THERE IS A FEW MUTATIONS THAT
ARE HERE AND THERE AND GENERALLY
IN THAT SPECIFIC REGION IT IS
QUITE CONSERVED.
>>GOING BACK TO THE FIRST TALK
THIS MORNING, WHAT ARE YOUR
THOUGHTS ABOUT USING CD4
TARGETING LIPID NANOPARTICLES
DELIVERING THESE THAT WOULD
EITHER DISRUPT STEM LOOP
STRUCTURE OF PSI OR PREVENT GAG
INTERACTION WITH PSI?
>>IT IS A POSSIBILITY. I KNOW
THAT SMALL MOLECULE INHIBITORS
HAVE BEEN DEVELOPED TO TARGET
INTERACTIONS OF NC AND STEM LOOP
3.
SO, GS SORT OF THE ONLY ISSUE
WITH THAT IS THAT THERE IS HIGH
TOXICITY AND OTHER THINGS.
THOSE HAVE NOT COME TO THE
FOREFRONT. IT IS A POSSIBILITY
IF --
>>THANK YOU.
>>YEAH.
>>I HAVE A QUICK QUESTION. IS
PSIRNA PART OF GAG RNA AS ONE
PART OF IT OR SEPARATE RNA THAT
IS -- YOU KNOW WHAT I MEAN?
>>SORRY.
>>THE PSIRNA.
>>YES. THAT REGION.
>>IS IT PART OF THE ENTIRE
VIRAL RNA?
>>YES.
>>OKAY. SO, THEN DURING
INFECTION, THIS INFECTION, IS
THERE -- YOU HAVE A MECHANISM
YOU POSTULATE HOW IT LET'S GO OF
HOW GAG LET'S GO OF PSIRNA
DURING INFECTION AND YOU HAVE A
MECHANISM HOW IT GETS IT IN AND
MECHANISM HOW IT GETS IT OFF?
>>WELL, SO -- IT COMES IN. SO,
WHEN IT IS IMMATURE VIRAL
PARTICLE IN THERE YOU WILL HAVE
CLEAVAGE AND THAT PROTEIN AND
GAG IS CLEAVED INTO MULTIPLE
PARTS AND YOU WILL HAVE
REORGANIZATION.
SO, THEY ARE LOOKING AT HOW THAT
RE-ORG.
>>I SEE. IT IS IMAGE OF GAG
THAT IS BINDING.
>>YES.
>>PROCESS TO SEPARATE BINDING?
>>YES.
>>I GET IT.
>>GETS REVERSE TRANSCRIBED AND
BECOMES DNA.
>>THANK YOU.
>>LAST TALK IN THE SESSION AND
THEN LUNCH BREAK IS BY CHUEN-YEN
LAU.
>>PRETTY GOOD.
>>TALK IS INNATE IMMUNE
CORRELATES OF CELL-ASSOCIATED
HIV RNA AND DNA DURING LONG-TERM
SUPPRESSIVE ART.
>>THANKS SO MUCH. EVERYONE,
CAN YOU HEAR ME OKAY?
>>YEAH.
>>IS THIS A POINTER? DO YOU
THINK IT IS?
THAT IS GOOD.
WELL, WHILE SHE IS PULLING UP
THE SLIDE, I WILL TELL YOU I
WILL SHARE WITH YOU A STORY THAT
WE HAVE TAKEN A LONG TIME TO
COME TO TALKING ABOUT INNATE
IMMUNE CORRELATES OF CELL
ASSOCIATED HIVRNA IN PATIENTS
THAT ARE LONG TERM ART.
THIS IS EXCITING AND COMPLEMENTS
TALKS WE HAVE HEARD ALREADY
ABOUT ROLE OF INNATE IMMUNITY IN
VIRAL INFECTIONS NO THE JUST
HIV.
JUST AS A REMINDER, MOST OF THIS
AUDIENCE IS FAMILIAR WITH THIS.
WE ARE CONCERNED ABOUT HIV
PERSISTENCE AS IMPEDIMENT TO
CURING HIV. WE DON'T HAVE
REPLICATION DURING A LONG TERM
SUPPRESSED INFECTION AND WE
STILL HAVE PERSISTENCE OF CELLS
AND THEY ARE DIVERSE AND HEAVY
PORTION OF DEFECTIVE PROVIRUSES
AND DON'T FULLY UNDERSTAND
MECHANISMS DRIVING THIS
FORMATION AND MAINTENANCE OF
POPULATIONS AND PERSISTENCE
ASSOCIATED WITH IMMUNE
ACTIVATION IN INFLAMMATORY STATE
LEADING TO LONG-TERM MOBILITY
AND MORTALITY AND PROVIRAL
POPULATIONS THAT CAN EFFECT OR
CLONE ALLEY EXPAND AND CAN
EXPRESS RNA THAT CONTRIBUTES TO
INFLAMMATORY STATE AND DON'T
UNDERSTAND SOURCES OF IMMUNE
ACTIVATION AND CHARACTERIZE
RELATIONSHIP BETWEEN PERSISTENCE
AND INFLAMMATORY STATE TO HELP
WITH CURE AND SOUGHT TO DEVELOP
A COHORT OF LONG TERM SUPPRESSED
HIV PATIENTS AND WE HAVE 73 IN
THE STUDY I WILL SHOW YOU DATA
IN A SECOND MEASURING DNA AND
RNA USING DDC PEL CR AND FOCUSED
ON LTR AND GAG GENES. YOU WILL
SEE 2LTRS PER GAG THAT IS
CRITICAL WITH OUR ANALYSIS AND
LOOKED AT RELATIVE LEVELS OF
SITES BY FLOW FOTOMETRY.
IDENTIFYING FACTORS ASSOCIATED
WITH HIV, DNA AND RNA LEVELS AND
USED 13 CORRELATION REGRESSION
AND CLASSIFICATION TESTS WITH 31
BIOMARKERS TO IDENTIFY
PARAMETERS RELEVANT IN THE MOST
TEST. THEN WE TOOK PARAMETERS
AND WE DID MULTILINEAR
REGRESSION TO LOOK AT
ASSOCIATIONS AND DID EXHAUSTIVE
SEARCHES OF LINEAR MODELS WITH
THOSE PARAMETERS OR JUST WITH
TWO OR THREE SEPARATE PARAMETERS
TO LOOK AT A MODEL AND IDEA
BEING AUTHENTIC BIOMARKERS WOULD
BE PRESENT IN MULTIPLE
ALGORITHMS THAT THOSE WOULD BE A
DISCRIMINATORY FEATURE.
HERE, YOU CAN SEE THE
CHARACTERISTICS OF PATIENTS AND
I WILL TELL YOU 73 OF THEM HAD
DNA RESULTS.
58 OF THOSE HAD RNA RESULTS
ALSO. THOSE GROUPS ARE FAIRLY
SIMILAR. THEY WERE ABOUT AROUND
50 YEARS OLD AND THEY WERE
MOSTLY WHITE MALES.
WE WERE UNDER-REPRESENTED IN
TERMS OF FEMALES IN A LITTLE BIT
AND WE WERE OVERREPRESENTED IN
WHITES AND BLACKS AND AFRICAN
AMERICANS WERE ABOUT WHERE WE
ARE IN THE EPIDEMIC IN TERMS OF
PERCENTAGE AND THEY WERE LESS
LATINX PEOPLE. YOU CAN SEE FROM
THE -- LET'S SEE IF THIS --
WELL, OKAY. I WILL WALK OVER
HERE AND POINT TO IT. YOU CAN
SEE HERE THEY WERE PRETTY WELL
RECONSTITUTED IN TERMS OF CD4
COUNTS ABOUT YOU DOESN'T MEAN
THEY HAVE FULLY NORMAL IMMUNE
SYSTEMS AS YOU KNOW.
LOOKING AT DNA RESULTS HERE I'M
SHOWING YOU HISTOGRAMS OF THE
LTRDNA AND GAG DNA.
Y AXIS IS NUMBER OF PARTICIPANTS
AND YOU SEE THERE A BIT OF
VARIATION IN LEVELS AMONGST
PARTICIPANTS AND ALSO THAT THERE
ARE FEWER COPIES OF GAG THAN
LTR.
YOU CAN SEE IF YOU LOOK AT
MEDIAN COPIES THERE IS 2.8 IN
TERMS OF LOGS AND IN 3.4 IN
TERMS OF LTR AND WE WILL LOOK
AND TAKE CLOSER DIVE INTO THE
DNA RESULTS.
THINKING ABOUT EXPECTED RATIO
SHOWING TWO LTRS PER GAG YOU SEE
YOU EXPECT A RATIO OF 2LTRS TO
GAG THAT IS SHOWN ON Y AXIS AND
ON HISTOGRAM YOU SEE THAT WE ARE
HEAVILY SHIFTED.
THERE IS A LOT OF PEOPLE WHO
HAVE MORE THAN EXPECTED -- WELL,
I GUESS YOU CAN SAY THEY HAVE
MORE GAG DELETED PRO-VIRUSES AND
YOU MIGHT SAY YOU MIGHT UNTAP
THE GAG AND YOU MISSED THAT AND
SINGLE GENOME SEQUENCING WAS
DONE TO SHOW PRIMERS ARE
ACTUALLY RECOGNIZING THE TARGET
AND WE DON'T THINK THAT IS WHAT
IS RESPONSIBLE FOR THE RESULT
AND IN THIS OTHER PLOT, YOU ARE
LOOKING AT LTRDNA TO GAG DNA ON
THE Y AXE YIGS AND LOOKING AT
THE SLOPE IT IS DIFFERENT FROM
EXPECTED SLOPE WITH USUALLE TWO
LTRS TO GAGS AND IS PRETTY
CONSISTENT THAT YOU SEE IT IS A
GOOD FIT IN TERMS OF THE
EQUATION. HERE, I'M SHOWING
AGAIN THAT GAG DELETED
PROVIRUSES ARE PRESENT AT AN
ALMOST CONSTANT RATE THAT IS
ACTUALLY VERY INTERESTING TO US.
WHAT FACTORS ARE ACTUALLY
ASSOCIATED WITH DNA LEVELS?
IF YOU DO THE ANALYSIS THAT I
HAD MENTIONED EARLIER AND THIS
REGRESSION ANALYSIS AND YOU CAN
SEE HERE THAT LOOKING AT GAG DNA
AND THE BEST FIT ONE PARAMETER
MODEL HAD MEMORY CD8 CELLS AS
THE BEST PREDICTOR.
AND THAT HAD AN R VALUE OF .05.
IF YOU LOOK FURTHER AT MORE
PARAMETERS, WHAT WE FOUND WAS
THAT A 2 PARAMETER SHOWED PRETTY
GOOD IMPROVEMENT OVER THE 1
PARAMETER MODEL AND ADDING
FACTORS DIDN'T HELP MUCH. WE
SETTLED ON TWO PARAMETER
COMPOSITE MODEL WITH NADARCD4
AND MEMORY CELLS AS BEST
PREDICTORS AND R IMPROVED TO .6
THAT IS SIGNIFICANT WITH BOND
[INDISCERNIBLE] AND THAT IS DNA
RESULTS. YOU CAN SAY THAT IS
JUST DNA. WHAT ABOUT
EXPRESSION? RNA WE USE AS
REPRESENTATION OF EXPRESSION.
YOU CAN SEE HERE I'M SHOWING ON
THIS SIDE BASICALLY MORE LTRNA
THAN GAG RNA SAME GOING ON AS
YOU SAW WITH DNA AND IN THIS TOP
PLOT OR TOP HISTOGRAM YOU HAVE
THIS AND ON BOTTOM YOU HAVE GAG
RNA PER MILLION CD4 CELLS YOU
SEE AGAIN THE SHIFT IN THE PLOTS
AND MEDIAN FOR LTR IS MUCH
HIGHER ON THE LOG SCALE THAN FOR
GAG. OKAY. THAT IS TOTAL.
WHAT IF YOU LOOK AT EXPRESSION
IN TERMS OF HOW MANY PROVIRUSES
ARE PRESENT? YOU HAVE SOME
PROVIRUSES WITH TWO LTRS AND
SOME PROVIRUSES -- SOME
PROVIRUSES HAVE LTR AND GAG AND
SOME WILL NOT HAVE GAG. IF YOU
LOOK AT TOTAL AMOUNT OF
PROVIRUSES THAT IS LTRRNA OVER
TOTAL PROVIRUSES ON TOP IS THE
LEFT-HAND SIDE AND YOU COMPARE
TO WHAT YOU HAD FOR GAG ARE. NA
EXPRESSION PER GAG CONTAINING
VIRUS IT IS PRETTY SIMILAR THAT
SUGGESTS THERE IS COMPARABLE
EXPRESSION AND ANOTHER WAY TO
LOOK AT IT I PLOTTEDED
PROPORTION OF THIS WITH HIV GAG
RNA GAG CONTAINING VIRUSES AND
LOOKING AT SLOPE HERE THIS SLOPE
IS ALMOST 1 THAT IS PRETTY
INTERESTING SUGGESTING THAT
RELATIVE LEVELS ARE COMPARABLE
IN TERMS OF EXPRESSION.
TAKING THAT A STEP FURTHER MEANS
TRANSCRIPTIONAL ELONGATION
GENOME WE LOOKED AT
TRANSCRIPTIONAL ELONGATION IS
EXTENDING INTO GAG.
WHAT IS REALLY ALSO KIND OF
CURIOUS HERE IS IF YOU LOOK AT
BLUE ARROWS YOU SEE HERE SOME
PEOPLE ARE NOT REALLY ON THAT
LINE.
SOME PEOPLE HAVE MUCH, MUCH
LOWER GAG LEVELS THAN LTRA. I
DON'T HAVE AN ANSWER YET BUT AT
THE END OF THIS YOU MIGHT HAVE
SUGGESTIONS FOR US TO LOOK INTO
THAT AND OTHER THINGS AND WE
WILL TAKE THE R NCHLT ANA A STER
AND SEE WHAT IMMUNE CORRELATES
WOULD HELP US PREDICT WITH WHAT
IS GOING ON WITH RNA SO YOU CAN
KNOW AND TARGET THINGS
DEVELOPING CURE STRATEGIES AND
WHEN WE DID ANALYSIS THAT WAS
BRIAN BUT ANYWAY WE HELPED AND
YOU LOOK AT COMPOSITE FUNCTIONS
PREDICTING WHAT YOU WILL SEE IN
TERMS OF LTR RNA FOR PER TOTAL
VIRUSES OR GAG RNA FOR GAG
CONTAINING VIRUSES AND BOTH OF
THOSE WERE RELATED TO PROPORTION
OF NK CELLS AND AGE. THAT IS
REMARKABLE. RNA VALUES RELATE
TO INNATE IMMUNITY AND WE DIDN'T
SEE THE SAME RELATIONSHIP WITH
OTHER PARAMETERS WE WERE LOOKING
AT AND ALSO JUST AS AN ASIDE YOU
MIGHT LOOK AT THIS AND OKAY WHAT
CURVE IS ACTUALLY FITTING THIS?
WELL, PROBABLY WHAT MAYBE THERE
ARE DIFFERENT POPULATIONS.
HERE, YOU KNOW, ON THE
RIGHT-HAND SIDE OF THE CURVE,
YOU HAVE A LITTLE ALMOST A
FLATTER LINE THAT SEEMS TO
REALLY SLOPE UP WHEN YOU GET TO
THE LEFT-HAND SIDE AND MAYBE
THERE IS DIFFERENT POPULATIONS
AND I HAVE A FEW WRAP-UP SLIDES
HERE.
IN SUMMARY, THE LTR RNA PER
TOTAL PROVIRUSES AND GAG RNA PER
GAG CONTAINING PROVIRUSES. THAT
EXPRESSION IS COMPARABLE.
THESE THINGS CORRELATE WITH
PROPORTION OF NK CELLS OR INNATE
IMMUNITY AND AGE. WE THINK THAT
HIV RNA IS A DRIVER OF INNATE
IMMUNE ACTIVATION THAT IS
SPECIFICALLY RNA NOT DNA.
WE ARE VERY EXCITED ABOUT THE
STATISTICAL APPROACHES THAT HAVE
BEEN USED TO IDENTIFY THESE
COMPOSITES.
IN TERMS OF THINKING OF THIS
CONCEPTUALLY, HERE IS A MODEL
FOR YOU TO KEEP IN YOUR MIND IF
YOU CAN'T REMEMBER ALL THAT
DATA. SORRY. I CAN'T REMEMBER
DATA YOU PRESENTED BUT REMEMBER
SOME OF IT. IF YOU NEED A
CONCEPTUAL MODEL YOU CAN SEE
HERE IN A PERSON WHO IS INFECTED
INITIALLY STARTS WITH HIGH VIRAL
MODE LOTS OF PROVIRUSES GAG
CONTAINING PROVIRUSES UP THERE
YOU START THEM ON ART AND VIRAL
LOAD COMES DOWN ASSUMING THEY
ARE TAKING IT IN RESPONSE.
VIRAL LOAD COMES DOWN AND
PROVIRUSES START TO TRACK DOWN
TOO. BUT, YOU SEE GREATER
DECREASES IN GAG CONTAINING
PROVIRUSES THAN YOU SEE IN TOTAL
PROVIRUS AND AS YOU GET FURTHER
ON AND LIKE IN OUR PARTICIPANTS
WHO HAVE BEEN SUPPRESSED FOR A
LONG TIME WHAT WE THINK IS GOING
ON IS YOU HAVE A BUNCH OF CELLS
WITH PROVIRUSES AND SOME ARE
EXPRESSING RNA AND THOSE
EXPRESSING RNA ARE STIMULATING
INNATE IMMUNITY IN PARTICULAR NK
CELLS.
THIS MEANS THAT AGE-RELATED
FACTORS I SHOWED IN THE PREVIOUS
PLOT AND AGE-RELATED FACTORS MAY
INFLUENCE EXPRESSION OF BOTH
DELETED AND NON-GAG DELETED
PROVIRUSES AND NK CELLS SENSING
POPULATION OF RNA-CONTAINING
CELLS AND EXPRESSION OF BOTH
DELETED AND NON-DELETED
PROVIRUSES STIMULATE INNATE
IMMUNITY THAT HELPS TO DRIVE
DYNAMICS OF PROVIRUS EXPRESSION
AND VICE VERSA. NOT A CYCLE.
IT IS NOT ONE THING BOTH
INFLUENCE EACH OTHER AND OTHER
IMMUNE FACTORS INVOLVED AND IN
TERMS WHERE WE GO WITH THIS AND
WOULD LOVE TO HEAR INPUT AND
SUGGESTIONS ALSO WE WANT TO
PROCEED WITH FUNCTIONAL
EVALUATION OF INNATE IMMUNE
FACTORS INTERACTING WITH THE
PROVIRUS AND FOR EXAMPLE WE
MIGHT DO A STUDY WHERE WE GIVE A
MONOCLONAL ANTIBODY TO PATIENTS
THAT MODULATES NK AND CD8
EXPRESSION. WE WILL ALSO DO
LONGITUDINAL ASSESSMENTS FROM
OUR POPULATIONS THAT HAVE BEEN
INFECTED AND SUPPRESSED
LONG-TERM AND LOOK AT PROVIRUS
DURING TREATMENT INTERRUPTIONS
AND I'M ENROLLING IN A STUDY
DOING ANALYTIC TREATMENT
INTERRUPTION FOR PATIENTS AND
WILL COLLECT SAMPLES FROM
ANO-TOMIC SITES AND PROIVERY AND
LOOK AT WHAT IS GOING ON WITH
VIRUS THERE AND WILL LOOK AT RNA
AND DNA FROM THOSE AS WELL AND
LOOK AT DIFFERENT POPULATIONS
AND WE ARE JUST NOW
COLLABORATING NOW WITH MAX WEISS
COHORT TO LOOK AT MORE
REPRESENTATIVE SPECIMENS
ESPECIALLY IN FEMALES.
WITH THAT, THIS IS A COMMUNITY
EFFORT AND IS AN EFFORT OF A
LARGE GROUP OF PEOPLE THAT I
STAND HERE REPRESENTING MY TEAM.
THEY HAVE BEEN FABULOUS.
ESPECIALLY, YOU KNOW, FRANKS
GROUP AND THE DRP AND NIAD OR
EXTERNAL COLLABORATORS AND TUFTS
AND WALTER REID AND FREDERICK
LAB WITH THE STATISTICIAN BRIAN
LUKE WHO IS IN THERE AND STUDY
PARTICIPANTS AND LOTS OF US
COULDN'T DO WORK WE DO WITHOUT
STUDY PARTICIPANTS AND WHAT THEY
GIVE US AND WITH THAT I INVITE
YOU TO PROVIDE SUGGESTIONS AND
COMMENTS AND THANK YOU VERY M
MUCH.
>>NOT A SUGGESTION BUT A
QUESTION.
>>OKAY.
>>YOU HAD CORRELATION FOR NK
AND AGE.
>>YES.
>>SO, IS THAT -- I GUESS THE
AGE AT THE TIME YOU SAMPLE OR
DETERMINE NK COUNTS?
>>CORRECT.
>>AGE AND ACQUISITION AND HIV
DURATION AND ART AND HOW IT TIES
INTO IMMUNOLOGICAL AGING I
GUESS.
>>I STRUGGLE WITH THAT ALL THE
TIME. YOU KNOW IT IS HARD TO
DETERMINE WHEN EXACTLY SOMEONE
WAS INFECTED LOOKING AT
DIVERSITY OF PROVIRUS AND IF
THEY TELL YOU OH, YEAH LIKE I
ONLY HAVE ONE EXPOSURE THAT IS
AT SUCH AND SUCH OF TIME AND
BEING THAT IS NOT ALWAYS EXACT.
WE TRIED TO PUT IT IN AND
DURATION OF THERAPY INTO THE
MODEL.
AND THE -- THOSE TYPES OF
THINGS, FIRST, WE DIDN'T THINK
THEY WERE FULLY RELIABLE. THEY
DIDN'T REALLY PAN OUT LIKE AGE.
BUT, BUT TIME -- YOU KNOW, THEY
ARE TIME-RELATED FACTORS THAT
ARE ALL TOGETHER. YOU KNOW, IF
WE HAD REALLY, REALLY ACCURATE
DATA THAT IT WERE -- YOU KNOW,
IT COULD BE SOMETHING THAT WE
WOULD LOOK AT. SO, WE DID SEE
THAT THOSE THINGS CAME OUT.
THEY WERE ALSO TIME-RELATED THAT
WE WRAPPED THEM TOGETHER IN AGE.
DOES THAT HELP?
WHAT WOULD YOU HAVE DONE?
>>THAT IS WHAT I'M SAYING.
>>OH, MY GOSH. WE TRIED BUT,
YOU KNOW, I'M SURE WE MISSED
SOMETHING.
>>JUST QUESTIONS AND
SUGGESTIONS KIND OF.
>>YEAH.
>>YOU SEE THIS NK CORRELATION
THAT SEEMS TO BE VERY
CONVINCING.
>>UH-HUH.
>>THINKING ABOUT NK YOU THINK
OF ENVELOPE EXPRESSED ON SURFACE
OF CELLS AND PLANNING TO LOOK AT
ENVELOPE EXPRESSION AND ASSUMING
IF YOU SEE GAG EXPRESSION YOU
ENVELOPE EXPRESSION AND DON'T
THINK YOU COULD REALLY. TWO
THINGS COULD BE QUITE SEPARATED.
>>I TRY TO MAKE THOSE KINDS OF
ASSUMPTIONS.
>>SUGGESTION IS LOOK FOR RNA
EXPRESSION AND THAT COULD BE
INTERESTING KIND OF.
>>GREAT PLAN. THANK YOU.
THANK YOU FOR THAT SUGGESTION.
>>OKAY. TWO QUESTION SGLZ YES.
>>WHEN IS HOW BIG IS YOUR
MEASUREMENT AND CAN THEY DETECT
OTHER MUTUAL VIRUS FOR FIRST
QUESTION AND SECOND QUESTION DID
YOU MEASURE ADAPTIVE IMMUNITY?
SO, IN THE MODEL, YOU ONLY
COMPUTED INTO THE INNATE
IMMUNITY.
>>RIGHT.
>>HOW MUCH IS ADAPT IMMUNITY
CONTRIBUTING?
>>ALL RIGHT. THAT IS A REALLY
GREAT QUESTION.
WE DID PUT IN FACTORS FOR
ADAPTIVE IMMUNITY.
WE PUT IN THERE WERE 31
BIOMARKERS AND I SHOWED YOU ONES
THAT ACTUALLY CAME OUT AS
SIGNIFICANT IN EQUATIONS AND
CERTAINLY THERE WERE, YOU KNOW,
MANY -- THERE WERE MANY OTHER
CELL TYPES WE LOOKED AT AND WE
DIDN'T HAVE ANTIBODY LEVELS IN
THERE. YES. WE DID CONSIDER
ADAPTIVE IMMUNITY AND I'M HAPPY
TO SHOW YOU AND GO THROUGH THE
OTHER MODELS.
THEY REALLY WEREN'T AS GOOD.
SURE. THANKS. GOOD POINT.
>>[INDISCERNIBLE].
>>OH, YES. YOU KNOW, I CAN'T
FULLY ANSWER THAT QUESTION.
MAYBE SOMEONE ELSE IN MY LAB CAN
HELP WITH THAT. BUT, IT SHOULD
BE SPECIFIC FOR HIV. I DO NOT
KNOW THE SPECIFIC SEQUENCES.
>>OKAY. GREAT.
>>GREAT JOB.
>>THANK YOU.
>>YEAH. I JUST WANT THE TO
MAKE A COMMENT AND SUGGESTION, I
GUESS. ASSOCIATION IS NOT
POSSESSION. YOU MENTIONED THAT
AT END AND FACT YOU SEE
ASSOCIATION DOESN'T MEAN IT MAY
BE THAT HIV RNA IS DRIVING THAT
OR VICE VERSA AND WOULD SAY
SUGGESTION PERHAPS MOVING
FORWARD WHEN WE SAY SOMETHING
CORRELATES WITH NK CELLS IS
SAYING CORRELATES WITH STARS.
CELLS HAVE DIFFERENT VARIETIES
AND VERSIONS AND SOME ARE
CYTOTOXIC AND OTHERS MAKING
CYTOKINES AND MIGHT BE
INTERESTING TO LOOK MORE IN
DEPTH INTO EXACTLY WHICH NK
CELLS WE ARE TALKING ABOUT HERE.
>>WE TOTALLY AGREE WITH YOU ON
THAT. LOTS OF PEOPLE WILL WANT
TO LOOK INTO THAT. SO, WE ARE
GOING TO TRY TO DO THAT.
>>HAVE YOU LOOKED AT MONOCYTES
AND [INDISCERNIBLE] OR ANY OTHER
CYTOKINES?
>>WE HAD MONOCYTES IN THERE.
WE DON'T HAVE IL15. BUT, WE CAN
-- WE CAN LOOK MORE INTO THE
CYTOKINES.
THIS IS MOSTLY ON CELL TYPE.
>>THANK YOU.
>>THANK YOU SO MUCH, EVERYONE.
>>ALL RIGHT. THANKS TO ALL
PARTICIPANTS THIS MORNING. WE
WILL GO TO LUNCH AND POSTER
SESSIONS START AT 1 AND START
BACK UP HERE WITH PRESENTING
TALKS AT 2 AND GIVING EVERYBODY
ANOTHER HAND. POSTERS ARE NEXT
DOOR OVER HERE.
THE SECOND SESSION. FIRST
SPEAKER IS AIMÉE KREIMER. SHE
WILL GIVE AN UPDATE ON HPV.
SHE IS NCI PRINCIPLE
INVESTIGATOR ON SEVERAL
PROPHYLACTIVE HPV VACCINES.
RESEARCH INITIATIVES, ONE THAT
HAS COME BY THE CANCER MOON SHOT
AND WILL LET AIMÉE TAKE OEVER.
>>GOOD AFTERNOON, EVERYONE.
CAN YOU HEAR ME?
I'M PRETTY LOUD. EVERYONE CAN
USUALLY HEAR ME. I'M ALSO
INFORMAL. JUMP IN AND ASK
QUESTIONS IF SOMETHING IS NOT
CLEAR. IT IS YOUR TALK. I'M
AIMÉE KREIMER, AN INFECTIOUS
DISEASE EPIDEMIOLOGIST. I'M SO
FOCUSED ON HPV THAT I DON'T
THINK I KNOW ANYTHING OTHER THAN
HPV. I HAVE IT COVERED AND I'M
NOT AN EXPERT IN HIV. I DO SEE
RELEVANCE TO HPV ASSOCIATED
CANCERS AND I'M PREPARED TO TALK
ABOUT THAT.
SO, JUST A LITTLE ABOUT MY TALK,
I WILL GO OVER EPIDEMIOLOGY OF
HPV AND ASSOCIATED CANCERS AND
WILL STRONGLY FOCUS ON HPV
VACCINATION. I THINK IT IS THE
FUTURE. FIRST PART OF TALK IS
THE FUTURE AND NOW AND YESTERDAY
BUT ALSO THE FUTURE. I WILL
TALK ABOUT IT IN THE SETTING OF
PEOPLE LIVING WITH HIV.
SO, JUST TO REMIND EVERYONE WHY
WE KEEP TALKING ABOUT HPV AND
KEFSH CAL CANCER, IT IS A
NECESSARY CAUSE OF IS CERVICAL
CANCERS. THEY HAVE HPV DRIVING
TOWARDS CERV LOGICAL CANCER.
THEY ARE NOW IN RESOURCE LIMITED
COUNTRIES AND LOW-INCOME
COUNTRIES AND IS SECOND MOST
COMMON CAUSE OF CANCER IN AFRICA
AND 4TH AMONG WOMEN WORLDWIDE
AND IS AN ENORMOUS PROBLEM AND
HALF OF WOMEN WHO GET CERVICAL
CANCER IN LOW INCOME COUNTRIES
DIE FROM IT. FURTHER THING THIS
SLIDE DOESN'T CAPTURE IS TAKES
WOMEN OUT AT THE PEAK OF WHEN IN
MOERJHOOD AND CENTER OF FAMILIES
COULD BE BREAD WINNERS AND IS
NOT DESTRUCTIVE TO THE
INDIVIDUAL BUT IS REALLY HARMFUL
TO FAMILY UNITS AND COMMUNITIES.
AND THIS IS A CANCER THAT IS OF
INEQUALITY. IT IS A CANCER
WHERE 90% OF CASES AND DEATHS
OCCUR IN LOW AND MIDDLE INCOME
COUNTRIES.
AND IT THAT IS BECAUSE WE REALLY
KNOW HOW TO PREVENT CERVICAL
CANCER AND KNOW HOW TO VACCINATE
AND THAT THEY WORK AND KNOW HOW
TO DO CERVICAL CANCER SCREENING.
IT IS NOT EFFICIENT AND REQUIRES
LOTS OF INFRASTRUCTURE, BUT IT
WORKS.
SO, I THINK THAT IS WHY AT THIS
POINT SO MANY OF US ARE SO
REALLY FOCUSED ON HPV. WE SEE
THE ENDGAME AND HOW TO, IN FACT,
REALLY CONTROL THESE CANCERS AND
IT IS NOT GETTING DONE YET. WE
KNOW FROM REAL-WORLD DATA THAT
HAD HPV VACCINES REDUCE CERVICAL
CANCER BY 90%. WE CAN TALK
ABOUT TYPES AND VACCINE AND
INCREASING VAILENCY. LOW
VAILENCY TIME YOU GET TO
CERVICAL CANCER, THESE VACCINES
JUST WORK AND SECONDARY
PREVENTION SCREENING WORKS AND
IF PEOPLE HAVE CERVICAL CANCER,
THEY DESERVE THERAPY FOR THOSE
CANCERS.
SO, THERE WAS A CALL TO ACTION
BY THE WHO THAT HAPPENED A FEW
YEARS BACK. MUCH LIKE NCI I
FEEL HAS BEEN SAYING FOR A LONG
TIME NOW WHICH IS TO SAY WE HAVE
TOOLS AND SHOULD MAKE AND
CONTINUE TO IMPROVE ON THEM AND
SHOULD ALSO MAKE USE OF THEM.
THEY PUT FORTH A STRATEGY WITH
THREE PILLARS AND WE SHOULD
VACCINATE 98% OF GIRLS BY AGE 15
AND SCREEN 70% OF WOMEN AND
MAKING A NOTE HERE ABOUT
SCREENING THAT GETS ATTENTION ON
BIOMARKER AND UP FRONT PART OF
SCREENING IS A RISK STRATIFIER
AND LUMPS PEOPLE INTO HIGHER
RISKS FOR CERVICAL PRECANCER AND
CANCER VERSUS LOWER RIFSHG AND
DOESN'T REDUCE INCIDENTS OF
CERVICAL CANCER BUT TREATING
PEOPLE WITH SCREEN POSITIVE AND
STANDING UP THAT TREATMENT AND
SCREENING IS A PROCESS AND
SCREEN FOR ANYTHING COLON OR --
IT IS NOT JUST BIOMARKER BUT
DIAGNOSTICS AND TREATMENT OF
PRECANCER THAT PULLS DOWN CANCER
AND WE SEE TIME AND AGAIN THAT
EVEN IN US25% OF WOMEN SCREEN
POSITIVE AND SHOULD GO ON FOR A
TREATMENT WHO DON'T DO THAT IN
THE US WHERE WE HAVE ALL
RESOURCES IN THE WORLD AND LIKE
TO OVERMANAGE PEOPLE FROM A
MEDICAL PERSPECTIVE. IT IS EVEN
MORE COMPLICATED IN COUNTRIES
WHERE THERE IS A SINGLE PERSON
TO TREAT THESE DISEASES, YOU
KNOW?
AND ALSO IN MY OPINION, NOT IN
MY OPINION, IF YOU CAN'T OFFER
FULL PROCESS FIND PEOPLE WITH
HIGH RISK AND DIAGNOSE THEM AND
SCREEN OR TREAT THEM
APPROPRIATELY AND UNETHICAL TO
HAVE BIOMARKER IF YOU CAN'T
COMMIT TO THE WHOLE PROCESS.
THIS STRATEGY AND GOAL IS TO BE
PUT IN PLACE BY 2030.
SO, WE WILL TALK ABOUT HPV
VACCINATION AND GOAL IS 90% OF
GIRLS UP TO AGE 15 VACCINATED BY
2030. WE WILL SEE WHERE WE ARE.
VACCINES WERE LICENSED AND
PROVEN TO BE SAFE AND
EFFICACIOUS 16 YEARS AGO AND
RECOMMENDED AND IS BOTH IN THE
UNITED STATES AND GLOBALLY AT
WHO LEVEL. AT THIS POINT FAST
FORWARD 16 YEARS AND GLOBAL
COVERAGE OF AGE-APPROPRIATE
GIRLS IS 13%. IF WE RESTRICT
THAT TO WOMEN OR GIRLS LIVING IN
LOW AND MIDDLE INCOME COUNTRIES,
IT IS 8%. YOU CAN SEE, AGAIN,
WE ARE TALKING ABOUT A CANCER
THAT MARKS DISPARITY AND HEALTH
INEQUALITY. I CAN'T THINK OF A
BETTER EXAMPLE THAN THIS.
HIGH-INCOME COUNTRIES ARE
MARCHING TO 80 OR 90% AND
LOW-INCOME COUNTRIES IN BLUE ARE
AT VERY LOW PROPORTIONS AND WE
NEED SOMETHING LIKE THAT TO MAKE
IT BETTER.
WE TAKE PROPORTIONS AND WILL SAY
IT IS FIXED AND WE HAVE BEEN
WORKING 16 YEARS TO GET THIS
UPTAKE UP. WE ARE AT 8% AT LMIC
WHICH IS IMPORTANT AND IS WHERE
THE BURDEN OF CERVICAL CANCER
IS. WHAT HAPPENS WHEN WE FAST
FORWARD THOSE NUMBERS?
WE PROJECT OUT TO SEE HOW MANY
LIVES WE WILL SAVE. SO, OVER
THE NEXT 65 YEARS, OF COURSE, IS
MODEL OR PROJECTS TO A HALF END.
WHEN WE LOOK AND ARE AGE GROUPS
OF GIRLS IN 2014 WITHIN HIGH
INCOME COUNTRIES AND UPPER
MIDDLE INCOME LOW INCOME AND WE
HAVE ESTIMATES OF CERVICAL
CANCERS THAT WILL HAPPEN BEFORE
AGE OF 75 AND LOWER MIDDLE
INCOME COUNTRIES WE TALKED ABOUT
THIS GROUPING HAVING 80 TO 90%
OF BURDEN OF CERVICAL CANCER AND
90% OF THE DEATHS.
IF WE APPLY THAT VACCINATION
PROGRAM THAT IS, YOU KNOW, AS IT
CURRENTLY IS, HOW MANY CERVICAL
CANCERS AND DEATHS WILL WE
AVERT? IN TOTAL CURRENT
VACCINATION STRATEGIES WILL
AVERT 365,000 CASES AND
$150,000.
SO, WE NEED TO DO BETTER.
WE HAVE BEEN WORKING USING HPV
VACCINE FOR MANY YEARS NOW AND
MY WORK IS IN COSTA RICA I WILL
FOCUS ON MY WORK AND HAPPY TO GO
BIG PICTURE AS WELL AND JUST
WITH A LITTLE BIT OF HISTORY HPV
WAS DISCOVER THE WITH CERVICAL
CANCER IN THE 80S AND NCI I
WASN'T PART OF IT OR AROUND BUT
NCI WAS INSTRUMENTAL IN THAT AND
SAYING NOW THAT THEY KNOW HPV
CAUSES CANCER WHAT DOES NATURAL
HISTORY LOOK LIKE? STUDIES
LAUVENLGED ONE IN COSTA RICA TO
DEFINE NATURAL HISTORY AND
ONGOING AND DOCTORS WERE DOING
ASSAYS FORMING FOUNDATION OF THE
VO OR VIRUS LIKE PARTICLE OF HPV
VACCINE AND ARE ONE OF THE
INVENTORS OF THE VACCINE AND
THROUGH COMMITMENT WE SAID
VIRUS-LIKE PARTICLE VACCINES
LICENSED TO PHARMACEUTICAL
COMPANIES RAN PHASE 3 CLINICAL
THEY DID PRECLINICAL AND
CLINICAL TRIAL WORK AND FROM
SUITABLE COMPANIES LOOKING AT
THESE, WE THINK THERE SHOULD BE
A PRIVATE OR NOT PRIVATE BUT
PUBLIC EFFORT AS WELL. AGAIN,
NOT ME. I'M STILL NOT HERE YET
TO WLOOK AT THESE AND LAUNCHED
THIS HPV VACCINE TRIAL THAT IS
STILL ONGOING AND LOTS OF
CONTRIBUTIONS TO SCIENCE AND WE
PRESENT DATA ALL OVER THE WORLD
AND ENCOURAGE COLLABORATIONS.
IF ANYONE IS INTERESTED, WE WORK
WITH MANY PEOPLE IN THE ROOM AND
COME FIND ME.
LET ME TELL YOU A LITTLE ABOUT
IT. WHY IS THIS COSTA RICA
VACCINE TRIAL IMPORTANT?
WE MADE A DISCOVERY THAT MAYBE
ONE DOSE OF THE HPV VACCINE
MIGHT BE ENOUGH. WHEN I'M
TALKING ABOUT 8% OF LMICS ARE
VACCINATING, WE ARE THINKING
ABOUT THEY ROLLED OUT AS
THREE-DOSE REGIMENS AND SCALING
THAT BACK TO 1 DOSE AND CUTS
COST OF VACCINE BY 1/3 OR HALF
IF IN ONE DOSE AND ALSO REALLY
RELEASES REQUIRED INFRASTRUCTURE
THAT IS ASSOCIATED WITH A
VACCINE PROGRAM. THIS IS NOT AN
ADOLESCENT PLATFORM OR NOT WHERE
MOST PEOPLE -- MOST GET PART OF
EPI PROGRAM AND GETTING VACCINES
UP TO AGE OF 2 WORLDWIDE. THIS
STICKS OUT AND GETTING IT DOWN
TO A SINGLE DOSE MAKES IT
AFFORDABLE AS WELL AS JUST
REALLY DOABLE.
SO, 2004, WE LAUNCHED THE COSTA
RICA VACCINE TRIAL AND DIDN'T
KNOW IT WAS SAFE. THOUGHT SO.
SAFETY AND EFFICACY WERE MAIN
NAMES OF TRIAL AND RANDOMIZE THE
TO RECEIVE HEPATITIS A VACCINE
OR CERVARIX ATHAVENTED WITH TLR
AGONIST AND 18 TO 12 AND GIRLS
OR WOMEN THAT WERE 18 TO 25
YEARS AND WERE GETTING PREGNANT
AND DON'T VACCINATE PREGNANT
WOMEN AND HAD COLPOS COPY AND
20% OF WOMEN RECEIVED FEWER THAN
THREE DOSES AND WE MADE THIS
DISCOVERY USING WOMEN IN POST
DOC WAY AND NOT RANDOMIZE THE
WINDOWS HOPING THREE WOULD WORK
AND THIS PROTECTS THE SAME AS
TWO OR THREE DOSES SAYING WE
HAVE TO CHECK NOW FOR DURABILITY
AND FOLLOWED THIS HPV ARM FOR
VIER OL OJIC ENDPOINTS AND NOW
FOLLOWING FOR IMMUNOLOGICAL
ENDPOINTS AND I WILL SHOW RECENT
DA
DATA.
WHEN CHECKING TO SEE IF ONE DOSE
STILL WORKS DOING A TRIAL
LOOKING AT CUMULATIVE ENDPOINTS
IN THE FOLLOWUP AND FOUR YEARS
AND BLINDED RANDOMIZED PHASE IT
WAS A CUMULATIVE ASSESSMENT AND
ASKING THE QUESTION IS IT
WORKING AT 10 YEARS, YOU DO AN
ANALYSIS AT THAT POINT AND KEY
QUESTION WE CAME OUT AND SAID,
HEY, EVERYONE. LOOK. ONE DOSE
WORKS AND WE WERE EXCITED THAT
TWO DOSES WORKED AND EVERYONE
SAID NO WAY THESE UNIT VACCINES
MUST HAVE MULTIPLE DOSES IF YOU
ARE -- WE SAID, OKAY. DATA
SHOWS IT REALLY WORKS AND ONCE
WE PROVIDED ENOUGH DATA TO GET
REALLY SOME TRACTION. YEAH.
TRACTION GOING THAT ONE DOSE
WORKED THEY SWITCHED TO SAYING
WE SEE IT WORKS IN SHORT-TERM
BUT WON'T BE DURABLE OR AMOUNT
TO ENOUGH ANTIBODIES.
DURABILITY CHECKS HAVE BEEN
IMPORTANT AND GOING QUICKLY
THROUGH IT WE BENCHMARKED
AGAINST THREE DOSES THAT IS
STANDARD OF CARE IN WOMEN THIS
AGE. WE ARE ASKING HOW DOES ONE
DOSE COMPARE TO THREE DOSES?
LOOKING AT VERY SMALL NUMBER OF
ONE DOSE THAT WERE NOT
RANDOMIZED AND THEY DIDN'T GET
ADDITIONAL DOSE THEY WERE
INTENDED TO GET AND HAVE DONE
LOTS OF WORK TO SHOW BIAS
DOESN'T DRIVE FINDINGS.
I WON'T GET INTO IT NOW. WE
SHOWED HPV16 AND 18 TYPES IN
BIVALENT HPV VACCINE.
PROTECTION FOR 1 AND 3 DOSES IS
THE SAME CONFIDENCE INTERVAL IS
BIG HERE BECAUSE OF SMALL
NUMBERS AND POINT ESTIMATES ARE
THE SAME AND WE SHOWED AT 10
YEARS IT THAT CROSS-PROTECTION.
REMEMBER, VACCINES PROTECT
AGAINST HOMOLOGOUS TYPES AGAINST
THE VACCINE AND TYPES FILO
GENETICALLY RELATED TO 18 AND 16
AND WE SEE EVIDENCE OF
CROSS-PROTECTION.
SO, THESE ARE -- THESE DATA ARE
BOLSTERED BY ANTIBODY PATTERNS
AND SHOWING HPV16 HERE AND SHOW
ON X AXIS TIME OUT TO 11 YEARS
THIS ANTIBODY GMT THAT SAMPLES
ARE TESTED AT FRED ERIC NATIONAL
LAB BY LISHA PINTO WHO RUNS THE
LABORATORY AND DOES ALL OF THE
ANTIBODY WORK. WHAT WE SEE AND
FIRST THING THAT GRABS YOU IS
ONE DOSE STABLE OUT TO 11 YEARS
WHICH WAS ABSOLUTELY UNEXPECTED
AND IS ABOUT FOUR-FOLD LOWER
THAN THAT ELICITED BY THREE
DOSES BUT DON'T HAVE CORRELATIVE
PROTECTION AND DON'T KNOW
MINIMUM ANTIBODY LEVEL IS THAT
IS MAGIC NUMBER YOU GET
PROTECTION OR NOT. PAIRING UP
WITH DATA ON PREVIOUS SLIDE WE
CAN SAY LOWER ANTIBODY LEVELS
DON'T ALWAYS EQUATE TO INFERIOR
PROTECTION AND BENCHMARK AGAINST
NATURAL IMMUNITY THAT IS AGAINST
HPV IS WEAK. IT IS NOT VERY
STRONG BUT PROVIDES A BENCHMARK
AND ONE-DOSE LEVELS ARE ABOUT AN
ORDER OF MAGNITUDE HIGHER AND WE
WILL FOLLOW TO 20 YEARS POST
DOSE 1 ON THESE WOMEN. WHY IS
20 YEARS MAGIC NUMBER? HPV
VACCINES WE DON'T HAVE TO INDUCE
STERILIZING IMMUNITY. DOESN'T
ARE TO BE LIFELONG. SEXUALLY
TRANSMITTED INFECTION WE HAVE TO
COVER GIRLS WE CAN TALK BOYS TOO
BUT I'M FOCUSED ON GIRLS TODAY.
GIRLS FOR THOSE PEAK PERIODS AND
AGES OF SEXUAL ACQUISITION THAT
IS SHORTLY AFTER SEXUAL DABU AND
HPV ACQUISITION EXPLODES AND
STARTS TO COME DOWN AND
PROTECTING 10 TO 30 YEAR OLDS WE
WILL REALLY INCREDIBLY REDUCE
INCIDENCE OF CERVICAL CANCER.
OF COURSE, IN THE US WE
VACCINATE UP TO 45, SURE. YOU
KNOW, YOU WILL GRAB A FEW AND
ALWAYS WE HAVE TO BE CAREFUL
WHEN THINKING PUBLIC HEALTH THAT
IS TO SAY LET'S MAKE A VERY,
VERY BIG IMPACT LET'S NOT BE AT
THE INDIVIDUAL LEVEL THINKING
ABOUT PERFECTION. WE WILL MAKE
THE BIG IMPACT FIRST AND CAN
CLEAN UP EVERY SINGLE PERSON
LATER WE HAVE FOUR CLINICAL
TRIALS ONGOING LOOKING AT SINGLE
DOSE HPV VACCINATION THAT AT
THIS POINT I HOPE I REALLY
CONVINCED YOU HOW THIS COULD BE
SOLUTION TO BARRIER OF POOR
UPTAKE AFTER 16 YEARS BEING ON
THE MARKET. I TOLD YOU ABOUT
THE COSTA RICA HPV VACCINE TRIAL
NOW IN 18TH YEAR FOLLOW UP AND
WE MADE A DISCOVERY OF ONE DOSE.
I WOULD LIKE TO POINT OUT 1 DOSE
IS NOT POPULAR WITH
PHARMACEUTICAL COMPANIES AND
THEY DON'T WANT TO SELL LESS OF
THEIR VACCINE. I THINK THIS
REALLY SPEAKS TO THE IMPORTANCE
OF PUBLICALLY FUNDED EFFORTS
WHERE ANALYSIS AND CONTROL IS IN
HANDS OF THE PUBLIC AND WE
REALLY INTERROGATED THIS TRIAL
LIKE WHAT OTHER PHARMACEUTICAL
COMPANIES ARE POISE TODAY DO AND
MADE IMPORTANT HEALTH
DISCOVERIES AS A RESULT AND WE
CONSIDER OUR COSTA RICA HPV
VACCINE TRIAL AS HYPOTHESIS
GENERATING AND SHOWED YOU WOMEN
WERE NOT RANDOMIZE TODAY GET ONE
DOSE AND THERE IS A SMALL
NUMBER, YOU KNOW?
WE LAUNCH THE ENORMOUS STUDY
CALLED ESCUDDO TRIAL
NONINFERIORITY TRIAL WITH 1
VERSUS 2 DOSES.
THAT IS THE AMERICA PRODUCT WITH
95% OF GLOBAL HPV VACCINE MARKET
SHARE AND NOT A GREAT THING AND
HEALTHY MARKETS REQUIRE MULTIPLE
COMPETITORS, AS YOU KNOW. THAT
IS A SEPARATE TALK. WE CAN TALK
ABOUT DEVELOPING COUNTRY
MANUFACTURERS AND SERUM
INSTITUTE AND THAT IS ALL ON THE
TABLE AND WE COMMENT ONE DOSE A
COUPLE DIFFERENT WAYS PRIMAVERA
IS IMMUNOBRIDGING TRIAL THAT
GETS 1 DOSE SUCCESSFUL AND FOCUS
IS REALLY ON GIRLS NOW GETTING
ONE-DOSE RECOMMENDATION TO GIRLS
BUT SEE HPV VACCINATION AS AN
ACCELERATION PLAY. WHEN WHO,
FOR EXAMPLE, TALKS ABOUT
CERVICAL CANCER ELIMINATION,
THEY TALK ABOUT A 100-YEAR TIME
SCALE TO THAT GOAL, WHICH IS A
LONG TIME. WE THINK IF YOU
VACCINATE UP AN AGE RANGE WITH
ONE DOSE IT REALLY PULLS THAT
TIMELINE FASTER. YOU TAKE HPV
ENDOM NISTY AND CRUSHING IT.
THIS WILL LOOK TO READ OUT LATER
ON IN THIS DECADE.
WHAT TIME IS IT?
OKAY. I WILL PASS ALL TRIALS
ARE IN CLINICAL TRIALS.GOV.
I WANT TO FAST FORWARD A LITTLE
BIT. IN ADDITION TO OUR FOUR
TRIALS SO THE WORLD HAS REALLY
GOTTEN -- THIS HAS GOTTEN A LOT
OF -- WHAT IS THE WORD?
PEOPLE AROUND THE WORLD SEE
POTENTIAL IN THIS AND HAVE
SIMILARLY LAUNCHED STUDIES
WORKING BACKWARDS UP THE SLIDE
AGE-RAGE DOWN IN GAMBIA THAT IS
A 4 AND EFFECTIVENESS THROUGH
IMMUNOBRIDGING EASIER TO
IMMUNOBRIDGE DOWN AGE RANGE AND
YOU CAN SHOW IMMUNE LOGIC NOT
INFERIORITY AND THERE IS AN
EFFECTIVENESS STUDY IN THAILAND.
THIS IS THE FIRST STUDY WITH A
SINGLE DOSE AMONG PEOPLE LIVING
WITH HIV THAT IS GOING ON IN
SOUTH AFRICA AND IS AN
EFFECTIVENESS STUDY NOT EFFICACY
KEYS YOU INTO AN INDIVIDUAL
LEVEL RANDOMIZATION AND
EFFECTIVE N NGS STUDY CAN DO
LARGER GROUPS OF PEOPLE NOT
ROBUST TYPE OF EVIDENCE BUT
OFTEN TIMES EASIER TO GET AT.
THIS IS IMPORTANT. IT IS A
SINGLE DOSE STUDY LOOKING AT 8
WOMEN LIVING WITH AND WITHOUT
HIV.
WE SAW PROGRESS THIS YEAR AND
WHO SAGE PUT OUT PERMISSIVE
RECOMMENDATION FOR ONE OR TWO
DOSES OF HPV VACCINE THAT IS A
HUGE ADVANCE.
IT WILL NOW BE UP TO COUNTRIES
TO SAY WHETHER THEY FEEL IT IS
ROBUST ENOUGH. TRIALS WILL
CONTINUE TO READ OUT IN GOAL
OVER NEXT 2 TO 3 YEARS IS PUSH
TO A FULL RECOMMENDATION AND UK
ALSO CAME OUT AND WENT WITH A
FULL RECOMMENDATION AND THEY
TEND TO BE BOLD AND PROGRESSIVE
WHEN RECOMMENDING VACCINE POLICY
THEY WERE BOLD HERE AND IN BOTH
INSTANCES PEOPLE WITH KNOWN
STATUS OF HIV SHOULD CONTINUE TO
GET A THREE-DOSE SCHEDULE. I
WANT TO SHIFT AND TALK ABOUT HIV
AND CERVICAL CANCER AND THINK
THE KEY THING WHEN I THINK ABOUT
HIV AND CERVICAL CANCER WHEN WE
LOOK AT GLOBAL LEVEL PEOPLE
LIVING WITH HIV CONTRIBUTE 5 OR
6% TO TOTAL CERVICAL CANCER
BURDEN THAT IS A RELATIVELY
SMALL PROPORTION AND FLIPPING
LOOKING AT SOME COUNTRIES SOUTH
AFRICA AND COUNTRIES IN SUBIS A
HARRANT AFRICA IT IS
OVERWHELMING MAJORITY AND WE
HAVE TO THINK ABOUT WOMEN LIVING
WITH HIV AND PROTECTING THEM AS
WELL AND THIS WILL COME TWO WAYS
COMING DIRECTLY WITH VACCINATION
OF HPV AND INDIRECTLY BUILDING
HERD IMMUNITY IN COMMUNITIES
THEY LIVE. I AM COGNIZANT OF
THE TIME AND I WANT TO TALK
ABOUT SOME OF THE DATA THAT HAVE
COME OUT. I AM PUTTING IT UP.
IN NO WAY DO I THINK YOU SHOULD
BE ABLE TO READ THIS SLIDE BUT
IS TO SAY THERE ARE MANY, MANY
TRIALS LOOKING AT PROTECTION IN
PEOPLE WITH KNOWN HIV STATUS, SO
WOMEN LIVING WITH HIV LOOKING AT
ZERO POSITIVITY AS AN ENDPOINT.
WHEN WE THINK ABOUT THIS WE DO
IN CONTEXT OF IN MY OPINION OF
ONE DOSE AND PEOPLE WITH HIV ARE
MAKING A LESS ROBUST RESPONSE TO
THREE DOSES OF HPV VACCINE.
THINKING ABOUT IMMUNE LEVELS
SOLICITED BY ONE DOSE THOSE
LEVELS OR THAT QUANTITY IS
ENOUGH. OF COURSE, THAT DOES
NOTHING TO SPEAK OF THE QUALITY
OF THE IMMUNE RESPONSE AND
AINDIVIDUALITY OF ANY OF IT. IT
THAT IS STILL AN OPEN QUESTION
THAT WILL REQUIRE EFFICACY AS AN
ENDPOINT AND NEED TO KNOW
WHATEVER ANTIBODIES THEY ARE
GENERATING FROM A QUANTITY AND
QUALITY PERSPECTIVE ARE
SUFFICIENT TO PROTECT.
THESE ARE TRIALS LISTED IN
CLINICAL TRIALS.GOV THAT ARE
GOING ON AND WE NOTE ONE
EFFICACY TRIAL THAT IS A
RELATIVELY SMALL SAMPLE THOUGH I
ASSUME THE ATTACK RATE IS QUITE
HIGH AND 3 DOSES VERSUS PLACEBO
IS LOOKING TO READ OUT NEXT
YEAR. THAT WILL BE REALLY,
REALLY IMPORTANT.
AND ONE OTHER STUDY IN RWANDA
LOOKING AT QUADRO VAILANT
VACCINE THEY HAVE 3 VERSE 2
DOSES AND TRYING TO SEE IF WE
CAN OBTAIN ADEQUATE PROTECTION
WITH FEWER DOSES THAT ARE 2 NOT
1 THAT IS WELL AND LOOKS AND IS
A LARGE STUDY.
SO, ONE KEY THING, AS I SAID, WE
HAVE MOVED THE NEEDLE NOW DOWN
TO ONE OR TWO DOSE PERMISSIVE
RECOMMENDATION GLOBALLY BY WHO.
WE WILL NOW WAIT TO SEE IN THE
NEXT YEAR OR 2 WHAT COUNTRIES
PICK UP THIS RECOMMENDATION.
LOTS OF QUESTIONS IN COUNTRIES
ARE HIGH HIV BURDEN OR
PREVALENCE ABOUT WHAT THEIR
POPULATIONS ARE AND TWO WAYS TO
THINK ABOUT THIS AND TWO
POPULATIONS TO CONSIDER.
THERE IS PEOPLE WHO -- THERE IS
GIRLS WHO WILL GET HPV
VACCINATION THAT IS AT 10 OR 11
OR 12 OR WHENEVER THE
RECOMMENDATION IS AND MAYBE UP
TO 15. IN COUNTRIES WHERE
MEDIAN AGE OF HIV ACQUISITION IS
7 TAENOR LATER THEY WILL HAVE
HPV VACCINATION AND ONBOARD
ANTIBODIES PRIOR TO HPV
ACQUISITION AND KEY QUESTION
HARD TO GET AT IS WILL
VACCINATION OF POPULATIONS PRIOR
TO HIV ACQUISITION, WHAT WILL
ANTIBODY RESPONSE LOOK LIKE
AFTER HIV ACQUISITION?
FRANKLY, THAT IS THE BIGGER
PICTURE QUESTION. THAT IS WHAT
WILL HAPPEN THE WORLD AROUND.
THERE IS A SMALLER QUESTION OF
HPV VACCINATION THAT IS AMONG
PEOPLE LIVING WITH HIV AND I
SHOWED YOU TRIALS THAT ARE IN
FLIGHT TO LOOK AT THREE DOSES
AND TRIAL IN FLIGHT TO LOOK AT
TWO DOSES AND DON'T THINK WE
WILL EVER GET TO ONE DOSE. IF
YOU STAND THERE LOOKING AT A
WOMAN IN HIV CARE, WHY WOULD YOU
-- YOU KNOW, WHY WOULD YOU GIVE
THE NEXT DOSE WOULD BE MY GUESS.
THAT IS THE MAIN POINTS I WANTED
TO TALK ABOUT. THIS IS MY
RESEARCH TEAM IN COSTA RICA AND
PEOPLE I LEFT HERE. HOPE I HAVE
LEFT ENOUGH TIME TO TALK A
LITTLE BIT. THANK YOU.
>>ARE THERE ANY QUESTIONS?
>>I AM NOT AN IMMUNOLOGIST.
>>ME EITHER.
>>NO. I'M REALLY NOT. BUT,
WHAT ABOUT IMMUNIZE -- WE
IMMUNIZE BABIES AGAINST MANY
VIRAL DISEASES, SO WHY IS THAT
OUT OF THE QUESTION FOR HPV?
>>THERE IS A COUPLE ANSWERS TO
YOUR QUESTION. THEY DON'T HAVE
A LOT TO DO WITH IMMUNOLOGY.
FIRST ONE IS DURABILITY AND IF
WE HAVE TO COVER PEOPLE FROM
AGES OF MEDIAN AGE OF SEXUAL
DEBUT WE WILL PICK IT AT 17 OR
16 IN OUR COUNTRY IN LATER
COUNTRIES AND NEED TO COVER THEM
YOU KNOW FOR AT LEAST 10 TO 15
YEARS AFTER THAT DURING PEAK
ACQUISITION AND IT ADDS ANOTHER
DECADE OF ANTIBODIES AT THIS
TIME WHERE NOTHING SHOULD BE
HAPPENING AND THEY ARE NOT
GETTING CHALLENGED BY HPV
BETWEEN AGES OF 2 AND 12. SO,
THOSE ANTIBODIES ARE SUPERFLUOUS
AT THAT POINT IS ANSWER ONE.
OTHER ANSWER IS EPI PROGRAM IS
FILLED AND I THINK THAT THESE
KIDS GET -- YOU KNOW, A ZILLION
DOSES OF SHOTS WITHIN -- YOU
KNOW, WITHIN 18 MONTHS. AS I
UNDERSTAND, PHARMACEUTICAL
COMPANIES ARE NOT KEEN TO ADD
MORE BURDEN TO THAT. I THINK
THAT THEY ARE ACTUALLY LOOKING
FOR ANOTHER PLATFORM THAT IS NOT
EPI AGE. I DON'T KNOW IF WILL
BE AN ADOLESCENT PLAY. OH,
SORRY. EARLY PROGRAM FOR
IMMUNIZATIONS AND EARLY
SOMETHING IMMUNIZATIONS AND
DON'T REMEMBER BECAUSE WE ALL
SAY IT. CHILDHOOD VACCINATIONS
THAT SUPPORTED THE WORLD AROUND
IS THE PROGRAM WITHIN WHICH
SOMEBODY NEEDS TO GOOGLE THAT.
WITHIN WHICH ALL CHILDREN IN THE
GLOBE ARE VACCINATED.
DURABILITY IS A QUESTION THAT IS
CONGESTED AND SO, YEAH. HAVING
SAID THAT, THERE IS A TRIAL.
PEOPLE ARE THINKING OF A TRIAL
RIGHT NOW TO LOOK AT ONE DOSE
IN, YOU KNOW, TODDLERS AND
ANOTHER PROBLEM WITH THAT --
EVEN WITH THE STUDY DESIGN YOU
AGAIN NEED TO FOLLOW THOSE
CHILDREN FOR 15 YEARS TO SEE IF
IT WORKS AND WE DON'T LIKE TO DO
TRIALS FOR 15 YEARS.
YOU LOSE THEM. IT IS HARD TO
TRACK.
>>IS THE ESSENTIAL PROGRAM
[INDISCERNIBLE].
>>THANK YOU. THERE IS ZERO
CHANCE I WOULD HAVE FOUND THE
WORD ESSENTIAL.
>>THERE MIGHT BE SOCIETAL
ARGUMENTS TO BE MADE FOR
IMMUNIZING YOUNG GIRLS VERY
YOUNG IN TERMS OF THIS IS MORE
OF A SOCIAL CLINICAL QUESTION
ABOUT PARENTS BEING RELUCTANT TO
ENCOURAGE THEIR 15 YEAR OLD
GIRLS BEING SEXUALLY ACTIVE AND
[INDISCERNIBLE]?
>>YEAH. UNLINKING IT TO SOCIAL
STIGMA OF HPV IS A
CONSIDERATION.
I THINK THAT OUR -- FROM SURVEYS
THAT HAVE BEEN DONE, OUR GIRLS
UNDERSTAND PROTECTION AGAINST
ONE STI WHEN THERE IS A WHOLE
HOST OF THEM STILL DOESN'T
EFFECT BEHAVIOR. YOU STILL HAVE
TO PROTECT YOURSELF AGAINST
GONORRHEA AND CHLAMYDIA AND ALL
OF THE OTHERS AND THEY DID A
STUDY USING HARD ENDPOINT OF
PREGNANCY AND SAID IF YOU HAVE
HPV VACCINATION DID YOU HAVE
HIGHER PREGNANCY BECAUSE YOU
SUDDENLY WENT WILD?
ANSWER IS NO. IT WAS BALANCED.
SO, THAT HAS BEEN BROUGHT UP
OFTEN TIMES AND HASN'T BEEN BORN
OUT IN THE DATA.
>>ANY DATA ON [INDISCERNIBLE].
>>YEAH. IT IS LICENSED IN
MALES FOR PREVENTION AND WAS
ORIGINALLY LICENSED FOR
PREVENTION OF ANAL CANCER WHERE
STUDIES OBSERVED DIRECT EFFICACY
AND THEY SAW PROTECTION AGAINST
COMPOSITE ENDPOINTS OF PEENILE
HPV INFECTION AND RECENTLY ALSO
HAD AN INDICATION AGAINST
CANCERS CAUSED BY HPV IT THAT IS
FOR THREE DOSES IN THE UNITED
STA
STATES.
IN TERMS WHEN THE WHO MADE ONE
DOSE RECOMMENDATION THEY THREW
IN IN BOYS TOO. 25% OF THE
WORLD DOES GENDER-NEUTRAL
VACCINATION THAT HELPS A LOT AND
PEOPLE ARE SKEPTICAL OF
GIRLS-ONLY VACCINATION GLOBALLY.
NOT IN THIS COUNTRY BUT PEOPLE
QUESTION WHY ARE YOU JUST DOING
MY GIRLS? GENDER-NEUTRAL SEEMS
TO HELP THERE. WE ARE COMING
OFF A VACCINE SHORTAGE THAT WAS
PRETTY PROFOUND WHERE THERE JUST
WASN'T ENOUGH DOSES TO VACCINATE
THE GIRLS OF THE WORLD.
SO, WHEN THAT HAPPENED AND THERE
WASN'T ENOUGH VACCINE WE ASKED
COUNTRIES AND PUBLIC HEALTH
PEOPLE TO CONSERVE THEIR DOSES
FOR WOMEN. FOR EXAMPLE, IN
SUBIS A HARR AN AFRICA
VACCINATING 100 LITTLE GIRLS YOU
WILL PREVENT ONE CASE OF
CERVICAL CANCER BECAUSE
INCIDENCE IN BIRTH IS SO HIGH
AND UNITED STATES IF YOU
VACCINATE 12 TO 16,000 LITTLE
BOYS YOU WILL PREVENT 1 CASE OF
ORO PHARYNX CANCER ROOM WITH
STOCK PILE OF FIXED NUMBER OF
DOSES USE METRICS NUMBERS NEEDED
TO PREVENT ONE CANCER TO HAVE
DECISION MAKING AND WE ARE
COMING OFF THE SHORTAGE RIGHT
NOW AND ARE ACTUALLY FLIPPING.
THAT WAS AN UNHEALTHY MARKET.
WE DIDN'T HAVE ENOUGH AND
DEVELOPING COUNTRY MANUFACTURERS
IN CHINA ARE COMING FORWARD WITH
TWO HPV VACCINES AND SERUM
INSTITUTE IS COMING OUT WITH
THEIRS AS WELL AND THEY CAN MAKE
LOTS OF DOSES AND WE WILL FLIP
AND TOWARDS END OF DECADES WE
WILL HAVE TREMENDOUS SURPLUS OF
DOSES AND WE WILL SEE VERY
QUICKLY ONE DOSE OR
GENDER-NEUTRAL KIND OF THE WORLD
AROUND AND ALSO UP AN AGE RANGE
AND WILL TELL YOU ONE VIGNETTE
ABOUT COUNTRY-LEVEL DECISION
MAKING. I'M VERY CLOSE TO COSTA
RICA AND IN MODELING AGAIN WHEN
YOU TRY TO SAY WE HAVE EXTRA
DOSES COSTA RICA HAD EXTRA DOSES
-- GOING UP THE AGE RANGE WE
WILL BRING MORE CANCER
PREVENTION THAN DOING BOYS COSTA
RICA HAS ALWAYS DONE THEY DID
VACCINATION FOR A COUPLE YEARS
THAT IS GIRLS ONLY AGE 10 IN
SCHOOLS AND COSTA RICA SAID
THANK YOU. WE SEE THE DATA AND
EASIER WHEN SEEING GIRLS AGE 10
TO DO JUST THE BOYS AND EVEN
THOUGH PUBLIC HEALTH YIELD IS
HIGHER DOING GIRLS AND BOYS OR
DOING GIRLS AT AGE RANGE BECAUSE
OF FEASIBILITY OF DOING JUST
GIRLS WE WILL DO OR GIRLS AND
BOYS WE WILL DO THAT AND IS
INTERESTING THERE IS
EVIDENCE-BASED PART AND
FEASIBILITY.
>>MOST GIRLS GET HPV FROM BOYS
SO [INDISCERNIBLE] TRANSMISSION
FROM VACCINATED OR UNVACCINATED
BOYS TOO TO GIRLS.
>>IF GIRLS HAVE ANTIBODIES AND
THEY WORK TO NEUTRALIZE
INFECTION ALMOST 100% IT IS
DIRECT PROTECTION.
>>I UNDERSTAND. I GUESS WHAT
I'M SAYING IS FEEN LOGICALLY IF
YOU HAVE UNVACCINATED GIRLS AND
UNVACCINATED BOYS THERE WILL BE
SOME PROTECTION FOR GIRLS TOO?
>>YEAH. SO, IT THIS VACCINE I
TOOK IT OUT. SORRY I CAN'T SHOW
YOU. THIS VACCINE INDUCES MORE
HERD IMMUNITY THAN EXPECTED IN
THE US. WHAT WE HAVE SEEN,
AGAIN, IN GIRLS BUT IT WORKS TO
BOYS TOO. ONCE YOU IMPLEMENT
HPV VACCINATION AND THEY
IMPLEMENTED IN AUSTRALIA WITH
VERY HIGH UPTAKE AND WITHIN A
HANDFULL OF YEARS BEFORE THERE
WAS A MALE VACCINATION THERE
JUST WASN'T 16 AND 18 OR A POOL
FOR IT TO BE IN. IN US WE HAD
60 OR 65% UPTAKE OR A COUPLE
YEARS AGO WE STILL SEE
TREMENDOUS AMOUNTS OF HERD
IMMUNITY. SO, BOYS GET
PROTECTED AND IN FACT MODELING
SHOWS IF UPTAKE IS HIGH IN GIRLS
BOYS WILL ALL GET PROTECTED
EXCEPT FOR MEN WHO HAVE SEX WITH
MEN AND POCKETS OF THOSE AND
THINKING OF, YOU KNOW, THERE --
TO ME I THINK ABOUT WE HAVE TO
GET TO SUBIS A HARR AN AFRICA
AND BURDEN IS ENORMOUS AND
POCKETS ALSO WITH VERY HIGH
RATES OF HPV-ASSOCIATED CANCERS
GETTING THEM VACCINATED AS WELL
IS ALSO IMPORTANT. YES?
>>HAVING ONE DOSE VACCINE GETS
MORE EFFICACIOUS AND DOES IT
MAKE SENSE TO DO THAT IN
COUNTRIES WHERE HIV IS MORE
PREVALENT AND SUGGESTED IN
RESPONSE EVEN WITH THREE DOSES
AND IS NOT THAT GOOD?
>>YUP. SO, RIGHT. SO, JUST TO
BE CLEAR, WHEN WE ARE DOING ONE
DOSE, IT IS JUST ONE DOSE OF THE
EXACT SAME VACCINE NOT DIFFERENT
FILE OR MORE ANTIGEN. IT IS THE
EXACT SAME. SO, I THINK THAT IF
YOU HAVE A WOMAN OR SOMEONE IS
IN A CLINIC AND WOMAN HAS HIV
DIAGNOSIS STANDING IN FRONT OF
YOU YOU WILL CONTINUE TO DO
MULTIPLE DOSES FOREVER AND
BIGGER RESEARCH QUESTION OR
PUBLIC HEALTH QUESTION -- 10,
11, OR 12 GIRLS WHO AT AGE OF 18
ACQUIRE HIV YOU KNOW THEY ARE
ALREADY PART OF A GLOBAL PROGRAM
AND DO YOU NEED TO GIVE THEM
ANOTHER DOSE OR DO YOU NEED TO
-- YOU KNOW, IS THAT SECOND DOSE
6 TO 8 YEARS LATER A BOOSTER
THAT WILL HELP? YOU KNOW, THAT
IS WHERE PEOPLE ARE LOOKING TO
IMPLEMENT RESEARCH AT THIS
POINT.
>>THANK YOU.
>>THANK YOU, EVERYONE. IT WAS
FUN TALKING.
>>HI. I'M SIDDHARTHA. OUR
NEXT SPEAKER IS KRISTA DELL
VIKZ-FRANKENBERRY TALKING ABOUT
POTENT DUAL BLOCK TO HIV-1
INFECTION USING LENTI VIRAL
VECTORS EXPRESSING VIF RESIST
APOBEC3G AND FUSION INHIBITOR
PEPTIDE MC46.
>>ALL RIGHT. TREATMENT FOR HIV
INFECTION WITH POE TENT -- CAN
CONTROL VIRAL REPLICATION
DELAYING ONSET OF AIDS AND WE
KNOW THIS TREATMENT IS LIFELONG
AND ASSOCIATED WITH HIGH MEDICAL
COSTS DRUG TOXICITIES AND
SIDE-EFFECTS LEADING TO LACK OF
ADHERENCE IN PATIENTS. THERAPY
INTERRUPTION CAN LEAD TO
EMERGENCE OF DRUG RESISTANCE
VIRUS AND REKINDLE INFECTION IN
CELLS AND ALTERNATIVE FUNCTIONAL
CARE STRATEGIES ARE NEEDED FOR
PATIENTS THAT ARE THERAPIES
DEFINED AS THOSE CONTROLLING
PYREMIA MAYBE USING CRISPR CAS9
TO HAVE A PROVIRUS AND FOR TALK
TODAY WE WILL LOOK AT WAYS TO
HARNESS ANTIVIRAL ACTIVITY OF
VIRAL RESTRICTION FACTORS AND
OVERALL PROJECT GOAL IS ASKING
WHETHER WE CAN ENGINEER T CELLS
OR HEMOPOE ATTIC -- TO BLOCK
HIV-1 REP MRIKS.
THIS IS AN OVERALL SCHEMATIC
SYNTHESIZING LENGTHY VECTOR AND
PRODUCE VIRUS IN EX-VIVO TRANCE
DEUCE PATIENT T CELLS OR STEM
CELLS AND CELLS CAN BE REINFUSED
BACK INTO THE PATIENT WHERE THEY
CAN BE SELECTED AND EXPANDED AND
DEPENDING ON HIPV1 FACTOR WE
HAVE CHOSEN THAT CAN PROTECT THE
CELL FROM HIV EVER INFECTING OR
POST ENTRY FACTOR TARGETING CELL
TO REPLICATION AND BEGAN STUDIES
USING HOST RESTRICTION FACTOR
KNOWN AS -- THAT WILL
HYPERMUTATE AND INACTIVATE HIV-1
PROVIRUS AND INCLUDED IN VECTOR
SELECTABLE MARKER IN ANIMAL
STUDIES AND TISSUE CULTURE
STUDIES AS WELL WE CAN SELECT
AND EXPAND CELLS CARRYING HIV
ANTI-V1 GENE AND APOE VEX BOX
HIV-1 INFECTION AND ABSENCE OF
VIF PROTEIN AND UPON SUBSEQUENT
INFECTION INTO TARGET CELL IN
REVERSE TRANSCRIPTION INSIDE
CAPSI
CAPSID APOEB /* APOBEC --
HOWEVER HIV HAS EVOLVED TO
COUNTERACT APOBEC RESTRICTION
FACTOR EXPRESSING VIRALLY
ENCODED VIF PROTEIN THAT WILL
BIND TO APOBEC AND THEREFORE
EXCLUDE IT FROM VIRION AND VIRUS
CAN CONTINUE ON LIFECYCLE.
HARNESSING THIS ACTIVITY SOMEHOW
MAKING APOBEC ALWAYS
INCORPORATED INTO VIRUS WOULD
INACTIVATE IT SO WE COULD USE AS
POTENTIAL THERAPY.
THAT IS EXPRESSING APOBEC GENE
AND INADVERTENTLY SOME AMOUNT OF
PROTEIN EXPRESSED FROM THIS
VECTOR MENTIONING BEFORE THAT
APOBEC WANTS TO BE NICELY
INCORPORATED INTO VIRION AND
VIRUS WILL CARRY THIS AND ALSO
THIS HYPERMUTATION OR WE ARE
ACTUALLY INACTIVATING A GENE WE
WANT TO DELIVER AND NEED A WAY
TO HAVE APOBEC3G IN HELPER CELL
LINE IN INACTIVE FORM AND WHEN
WE DELIVER TO TARGET CELL WE
WOULD LIKE TO HAVE IT FULLY
FUNCTIONING.
TO DO THAT OUR LAB FOR A NUMBER
OF YEARS STUDIED USING DIRECT
REPEATS TO ACCOMPLISH THIS THAT
IS A QUICK EXAMPLE USING GFP AND
WE CAN DUPLICATE INTERNAL
PORTION OF ANY GENE OF CHOICE.
WE HAVE GFFP JUST MAKING A
DUPLICATION AND HELPER CELL LINE
IF WE HAVE ANY EXPRESSION FROM
THE PROMOTER, THAT PROTEIN
TRANSCRIBED AND TRANSLATED WOULD
BE NONGARBLE THE AND
NON-FUNCTIONAL AND THIS VIRUS
DELIVERING TO TARGET CELL DURING
REVERSE TRANSCRIPTION THAT
DIRECT REPEAT IS DELETED ENDING
UP WITH INTEGRATED PRO VIRUS IN
TARGET CELL WITH FULLY
FUNCTIONING GFP GENE AND WHAT WE
DID FOR APOBEC VECTORS AND IN
THIS CASE WE CREATED A
DUPLICATION IN INTERNAL PORTION
OF THE GENE AND THIS CASE 900
NUCLEOTIDES IN LENGTH AND A33G
AND ANY TRANSCRIPTION OR
TRANSLATION AND THAT PROTEIN IS
NON-FUNCTIONAL AND VECTOR HAS
P2A CLEAVAGE SITE AND THIS
SELECTABLE MARKER TAGGED WITH
GFP IN HELPER CELL AND VIRUS
TARGET CELL -- TO DOUBLE
STRANDED DNA DIRECT REPEAT IS
DELETED WITH A FULLY FUNCTIONING
APOBEC IN TARGET CELLS THAT IS
CLEAVED AWAY FROM GFP-TAGGED IS
SELECTABLE MARKER AS NOTICED WE
WON'T DELIVER WILD TYPE APOBEC
TO TARGET CELLS -- SO, WE ARE
USING POINT MUTANT NOTED AS
THIS. THIS IS KNOWN TO BE
RESISTANT TO HIV-1 MEDIATED
DEGRADATION. THIS IS PROOF OF
PRINCIPLE SHOWING DIRECT REPEATS
ARE DELETED WHEN WE SELECT FOR
CELLS WITH MGMT SELECTION, WE
CAN USE BASIC PCR ANALYSIS TO
SHOW HERE THAT BOTTOM BAND WOULD
BE CORRECT SIZE IF DIRECT REPEAT
IS DELETED DON'T SEE UPPER BAND
THAT IS INDICATIVE UNDELETED
GENE WESTERN ANALYSIS NICE
EXPRESSION OF THIS APOBEC3G GENE
THAT IS EXCISED AND CLEAVED AWAY
FROM THE SELECTABLE PROTEIN AND
WENT AHEAD MAKING T CELL LINES
EXPRESSING VECTOR. WE HAVE
APOBEC WITH SELECTABLE MARKER
AND CELL LINES WITHOUT APOBEC
AND ORIGINAL PARENTAL CELL LINES
AND ASKED OVERTIME BY MEASURING
P24 CAPSID DO WE SEE ANY VIRAL
REPLICATION?
THIS IS SHOWING CEM CELLS AS
EXPECTED IN GRAY IS PARENTAL
CELL LINE AND ORANGE IS JUST
EXPRESSING SELECTABLE MARKER AND
THOSE CELLS WERE EASILY INFECTED
BY NL43 AND SEE CULTURES CRASH
OVER TIME AND VECTORS AND CELL
LINES EXPRESSING APOBEC3G D28K
BLUE LINE BELOW AFTER 41 DAYS IN
CULTURE WE SEE NO EVIDENCE FOR
VIRAL REPLICATION AND TRUE FOR
OTHER CELL LINES AND T LINES
BLUE LINE ON BOTTOM 45 DAYS POST
INFECTION WE SEE NO EVIDENCE OF
VIRAL REPLICATION.
IT IS A POTENT INHIBITOR OF
HIV-1 REPLICATION. WE DON'T
ONLY WANT TO DELIVER ONE HOST
RESTRICTION FACTOR AND WOULD
LIKE TO IMPROVE THERAPY. WE
WILL DO A DUAL BLOCK AND ADD A
SECOND VAKTOR TO INHIBIT THAT IS
ENTRY TO PEPTIDE INHIBITOR THAT
IS MC46 WHICH PEPTIDES CAN BLOCK
H1C FUSION AND SHOWN HERE WE
KNOW THAT HIV ENVELOPE WILL BIND
TO TARGET CELLS CD4 AND
CORECEPTOR WITH REARRANGEMENTS
AND GP41 WILL EXPOSE EDGE AND
TRIMERIC PEPTIDES AND THESE WILL
COME TOGETHER TO FORM OR ALLOW
FOR FUSION. WE CAN EXPRESS
EXOGENOUS LITTLE C PEPTIDES THAT
COMBINE TO N TRIEM ERIC PEPTIDE
AND BASICALLY BLOCK THIS
REACTION. THESE C PEPTIDES HAVE
BEEN STUDIED WITH DIFFERENT
LINKS AND POSITIONS AND CAN BE
DELIVERED EXOGENOUSLY AND ALSO
THEY CAN BE MEMBRANE-BOUND AND
EXPRESSED ON OUTSIDE OF YOUR
TARGET CELL AND WE WILL USE THIS
C46 FOR THIS PURPOSE. WE WENT
AHEAD AND EMODIFIED VECTORS TO
CARRY MC46 ALONE OR CARRY D128K
AND P2A CLEAVAGE WE HAVE
SELECTABLE MARKER ADDING A
SECOND CLEAVABLE SELF-CLEAVING
PEPTIDE TO EXPRESS MC46 AND
CELLS WILL HAVE A DUAL BLOCK FOR
HIV-1 WE LOOKED HOW EFFICIENT
NEW SYSTEM WAS AND MT4 CELLS
PARENTAL CELLS NICELY EFFECTED
WITH THESE AND CULTURES CRASH
AND CELLS RED LINE EXPRESSING
MC46 THAT BLOCKS ENTRY AND WE
SEE NICE PROTECTION A MONTH OR
MONTH AND A HALF LATER THERE IS
A BLIPPO VIRUS WE SEE COMING UP
THAT IS NOT SUPPORTED HAVING
BOTH MC46 AND THIS, THE BLUE
LINE, AGAIN, 45 DAYS POST
INFECTION WE SEE STILL NO
EVIDENCE OF VIRAL REPLICATION.
THIS IS SHOWING THE EXPRESSION
OF MC46 ON MEMBRANE-BOUND CELLS.
WE LOOKED AT OTHER T CELLS AND
PARENTAL CELLS NICELY INFECTED
AND WE SEE THESE CELLS IT THAT
WERE ONLY EXPRESSING MC46 AND
SEE ONE FLASK WHERE YOU GET A
LITTLE OF THE SCAPE VIRUS AND
AGAIN HAVING BOTH GENES PRESENT
MC46 AND D128K WE DON'T SEE ANY
REPLICATION AND FOR CEMS AGAIN
PARENTALS ARE INFECTED AND IN
THIS CASE WE SAW THREE OUT OF 6
FLASKS WE HAD ESCAPED VIRUS AND
AGAIN HAVING BOTH DUAL BLOCHL
AND NO EVIDENCE FOR VIRAL
REPLICATION. ALL RIGHT. WHY IS
IT IMPORTANT TO HAVE BOTH
FACTORS PRESENT?
SO, AGAIN, IN A NORMAL WILD TYPE
INFECTION HIV WON'T INFECT THE
TARGET CELL AND PRODUCTIVE VIRUS
BEING RELEASED THAT IN TERN WILL
LEAD TO CELL DEATH. IF WE ONLY
INCLUDE HAVING D128K PRESENT
THOSE CELLS CAN STILL BE
INFECTED BY HIV-1 AND VIRUS
COMING OUT WILL CARRY APOBEC --
WE WOULD HAVE TO REPLENISH
GENETICALLY MODIFIED CELLS IF IT
WAS TO LEAD TO THAT THERAPY. BY
ADDING MC46 PEPTIDE FUSION
INHIBITOR, NOW CELLS ARE
INITIALLY BLOCKED BY HIV-1
INFECTION.
IF WE HAVE ANY ESCAPE VIRUS
GETTING THROUGH OR PASSED MC46
BLOCK WE HAVE A BACKUP AND
SECOND LINE OF DEFENSE AND
APOBEC IS THERE READY TO BE
INCORPORATED INTO THE VIRUS AND
ACTIVATED ON SECOND ROUND OF
INFECTION AND THAT WAY TO
MAINTAIN LONGER CELLS FOR THESE
GENETICALLY MODIFIED CELLS AND
SHOWING THIS EXPERIMENTALLY AND
IN TISSUE CULTURE WE WILL TAKE
AND DO A MIXING EXPERIMENT
MIXING 95% PARENTAL CEM CELLS
WITH 5% EXPRESSING D128K ALONE
OR D128K PLUS MC46 PEPTIDE
INHIBITOR AND MONITOR OVER TIME
POST NL4-3 INFECTION. FOR TIME,
TLEEZ BLUE LINES HERE ARE
PERCENT GFP AND 95% ARE PARENTAL
IMAGINE WITH NL4-3 INFECTION
PARENTAL CELLS ARE KILLED AND 5%
ARE PROTECTED AND START TO GROW
OUT IN CULTURE AND ARE
MAINTAINED AND WE HAVE A
PREFERENTIAL SURVIVAL OF CELLS
WHEN THEY HAVE EXPRESSION OF
MC46 AS WELL.
IN CONCLUSION WE HAVE -- FOR
EXPRESSION OF APOBEC3G D128K.
WE HOPEFULLY CAN MOVE TO
EXPERIMENTS IN MICE AND T CELLS
TRANCE DEUCED WITH VECTORS
EXPRESSING FUSION PEPTIDE
INHIBITOR AND APOBEC3G D128K AND
PREVENTING ENTRY MORE
IMPORTANTLY CONFERRED A
SELECTIVE SURVIVAL ADVANTAGE AND
ACCUMULATION OF GENETICALLY
MODIFIED CELLS AND DUAL BLOCK
VECTORS SHOULD REDUCE
PROBABILITY OF ESCAPE MUTANTS
GAINING RESISTANCE AND HOPEFULLY
COULD PROVIDE NOVEL STRATEGY FOR
HIV-1 TREATMENT IN THE FUTURE
AND WOULD LIKE TO THANK MENTOR
AND MEMBERS OF THE LAB AND DR.
WHO IS A CO-AUTHOR ON THE STUDY
AND LAB MEMBERS IN HER LAB FOR
HELPFUL SUGGESTIONS AND BRUCE
AND KIP HERMAN FROM UNIVERSITY
OF WASHINGTON WHO WILL HOPEFULLY
CONTINUE WITH MOUSE STUDIES TO
VERIFY OUR FINDINGS.
THANK YOU.
>>THAT WAS A REALLY GOOD TALK.
VERY INTERESTING. TRYING TO
THINK THERAPIWISE, WOULD IT BE
POSSIBLE IF CELLS WERE
EXPRESSING MUTANT APOBEC ALL THE
TIME AND THAT WOULD ELIMINATE?
OBVIOUSLY, IT COULD PREVENT HIV
INFECTION AND PEOPLE THAT ARE
HIV INFECTED WOULD IT ELIMINATE
REPLICATION OR BE SOMETHING LIKE
CRISPR CAS SYSTEM WITH MUTANT
APOBEC BE EXPRESSED ALL THE TIME
OR SOME BAD REASON FOR WHY YOU
DON'T WANT TO DO THAT.
>>INITIAL STUDIES FLIPPING IN
PUTTING D128K AS A NATURAL
MUTATION IN THE CELLS AND DON'T
KNOW IF THEY WILL HAVE
IMMUNOLOGICAL EFFECTS OR NOT AND
SOMETHING TO THINK OF PUTTING
POINT MUTATION IN THERE HIV IF
IT INFECTS IT WOULD BE --
>>ONE OTHER THING, IF YOU MAKE
VIRUSES THAT PUT IN THIS APOBEC,
IS -- ARE THEY INTEGRATING INTO
THE GENOME IN THE CELLS AND
EXPRESSING THE --
>>YES. THEY ARE INTEGRATING.
>>JUST WANTED TO MAKE SURE.
THANK YOU.
>>DO YOU HAVE [INDISCERNIBLE].
>>INTERESTING. RIGHT. IN THE
LITERATURE THERE IS NO ESCAPE
MUTANTS. ONE PERSON DID A 200
DAY TISSUE CULTURE EXPERIMENT
AND GOT ONE MUTANT 10% RESISTANT
AND 5 MUTATIONS. WE ARE EXCITED
OR NOT EXCITED TO SEE ESCAPE TO
IDENTIFY NEW MUTATIONS BUT WENT
BACK AND SEQUENCED THE VIRUS.
THERE IS NOTHING THERE. NOT
LIKE EVERYBODY IN THE CULTURE
HAD A MUTATION AT POSITION
FIFTEEN THAT WE COULD ACCOUNT
FOR IT WAS ALL SINGLETS AND NOW
WE ARE UNSURE AND THINK IT MIGHT
BE DUE TO THE FACT THAT THE CELL
MIGHT HAVE LOST EXPRESSION OF
MC46 THAT GOT TURNED OFF
ALLOWING THE VIRUS.
>>[INDISCERNIBLE].
>>WE WOULDN'T THINK SO BUT WITH
CM CELLS WE GOT 3 OUT OF 6
CULTURES BUT WE DON'T --
>>REPASSAGE THAT VIRUS --
>>RIGHT. WE HAVEN'T DONE THAT
OR GONE BACK TO TAKE THAT VIRUS
REPASSAGING IT BUT IS SOMETHING
WE WOULD LIKE TO DO.
>>OKAY.
>>THANK YOU.
>>WE WILL HAVE OUR NEXT SPEAK
JIK JUAN YIQUAN WU.
THE STAGE IS YOURS.
>>GOOD AFTERNOON, EVERYONE. I
WOULD LIKE TO THANK THE
ORGANIZER GIVING ME AN
OPPORTUNITY TO PRESENT MY WORK
HERE. ALSO, OF COURSE, FOR YOUR
STAYING HERE.
[INDISCERNIBLE] ONCHOGENIC
HERPES VIRUS COULD CAUSE
[INDISCERNIBLE] P MULTI KRCENTR
DISEASE. THAT IL PATHWAY IS
ACTIVE IN THE CELLS AND VERY
IMPORTANT FOR RELATED MCD
CURRENTLY IT HAS TREATMENTS AND
[INDISCERNIBLE] CONVENTIONAL
ANTIVIRUS THERAPY AS WELL JACKS
JAK INHIBITORS HAVE BEEN PROVEN
TO BE SAFE. EVERYTHING HAPPENED
UNDER CLINICAL TRIALS OR FOR
TREATMENT OF ADVANCED
[INDISCERNIBLE] AND PURPOSE HERE
IS TO EXPLORE WHETHER JAK
INHIBITORS CAN SERVE AS MORE
EFFECTIVE AND LESS TOXIC
TREATMENT FOR CASE ASSOCIATED
WITH MCD OR KICS.
5NJAK INHIBITORS CLINICALLY
APPROVED OR IN CLINIC STAGE
PICKED AND WERE TESTED. HERE,
WE CHOOSE THREE POC9S AND
[INDISCERNIBLE] DIFFERENT DOSES
OF THOSE INHIBITORS AND TESTED
ON THESE [INDISCERNIBLE] YOU CAN
SEE HERE COMPARED WITH OTHER
FOUR INHIBITORS SHOWS VERY
IMPORTANT INHIBITION OF THE CELL
GROWTH.
WE FURTHER EVALUATE INHIBITION
EFFECT USING FLOW SYMMETRY AND
THOSE CELLS WERE TREATED WITH
DIFFERENT DOSES AND APOPTOSIS
AND THIS WAS EVALUATING USING
FLOW SYMMETRY. YOU CAN SEE HERE
IT CAN INDUCE APOPTOSIS.
THOSE DATA ARE INTERESTING ON
WHY THIS IS SO DIFFERENT IN
GROSS INHIBITION AND WITH
COMPARISON WITH OTHER INHIBITORS
AWAY FROM THAT AND IRAQ 1 AND
[INDISCERNIBLE] K4 MIGHT BE THE
PLAYER CHOOSING INHIBITORS AND
[INDISCERNIBLE] TO TEST THEM ON
A DIFFERENT [INDISCERNIBLE].
AS YOU CAN SEE HERE, ONLY THESE
FLT3 INHIBITORS SHOW SIMILAR
INHIBITORY EFFECT AND TO FURTHER
CONFIRM THAT, WHICH WE PICK
SEVERAL MORE DIFFERENT AND OTHER
INHIBITORS AND TO COMPARE EFFECT
WITH [INDISCERNIBLE].
AS YOU CAN SEE, THOSE FT3
INHIBITORS INHIBITED CELL GR
GROWTH.
WE FURTHER EXPLORE THEM.
EFFECT ON CELL GROWTH. SO, WE
USE THIS LOCKDOWN OF THIS AND ON
LEFT PANEL SHOWS THIS EXPRESSION
DATA AFTER IT KNOCKED DOWN.
DATA SHOWS COMPARISON OF LT AND
THIS EXPRESSING SIMILAR GABA DH
SHOWING POSITIVE CONTROL OF
LOCKDOWN AND I WILL JUST -- I
DON'T KNOW WHAT HAPPENED IN THE
POWERPOINT.
ON THE RIGHT IT SHOWS THIS
VARIABILITY OF THE CELLS AFTER
KNOCKDOWN OF FLT3. YOU SEE RATE
OF CELLS HERE AND ALSO THE
[INDISCERNIBLE] ARE REDUCED
AFTER SIRNA KNOCKDOWN. HAVING A
GLOBAL OVERVIEW OF THE GENE
EXPRESSION AFTER TREATMENT WE
PERFORM [INDISCERNIBLE] OF THE
CELLS TREATED WITH
[INDISCERNIBLE] AND ON RIGHT
PANEL SHOWS GENE EXPRESSION
PROFILE AND ALSO PASS RATE
ENRICHMENT OF THE REGULAR GENES
AND ON RIGHT SHOWS DATA FOR THE
BCBL-1 CELL. YOU CAN SEE A LOT
OF THEM ARE HERE FALLING TO A
CELL CYCLE OR CELL PROLIFERATION
OR CELL DIVISION. RELATED AND
SO IT IS OVERLAPPED IN TWO
GROUPS.
SO, THAT IS THE INDICATION.
ELATED TO CELL PROLIFERATION
DOWNREGULATED BY
[INDISCERNIBLE].
WE ARE INTERESTED IN WHETHER
SOME GENES ARE CRUCIAL FOR CELLS
SURVIVAL IN DISREGULATED GENES
BY THIS ON LEFT SHOWS THIS AND
ON UPSIDE IS DOWN-REGULATED
GENES OF THE TWO GROUPS --
GROUPS WITH THIS [INDISCERNIBLE]
AND WE COMBINE IT WITH CRISPR
[INDISCERNIBLE] THAT WAS
PUBLISHED BEFORE. SO, WE TRIED
TO FIND OVERLAP GENES THAT CAME
OUT WITH CERTAIN GENES THAT ARE
PEL-SPECIFIC DEPENDENT GENES.
IT SHOWS A RANK AND FULL CHANGE
FOR OUR DATA. SO, THOSE GENES
ARE FURTHER TARGETED AND WE
WOULD LIKE TO FURTHER EXPLORE
AND BESIDES HOST GENES WE WOULD
LIKE TO CHECK THESE GENES WITH
RESPONSE TO THE TREATMENT AND
THIS LEFT PANEL SHOWS DATA OF
ACCOUNTS FOR [INDISCERNIBLE]
GENES AND HERE SEX GENES ARE
RELATIVELY MORE ABANDONED.
AS YOU CAN SEE ON VIRAL RL6 AND
[INDISCERNIBLE] IS NOT MUCH
EFFECTED AND THIS IS K2 HERE AND
SLIGHTLY DECREASED AND OTHER
THREE GENES ARE DOMINANT AND
SIGNIFICANT AND KPCR ON RIGHT
FURTHER INFORMATION WITH DATA
USING SAME EXPERIMENT SUBMITTED
FOR RSEC THAT IS REFLECTED SAME
AS RESULTS IN RSEC DATA AND
VIRAL F3 AND [INDISCERNIBLE] CAN
DOWNREGULATE IN RT AND ARE NOT
MUCH EFFECTED AND ALSO
[INDISCERNIBLE] IS NOT THAT MUCH
EFFECTED AND THE VIRO-6 CARRY
INDUCE WHOM IO6 EXPRESSION AND
PATIENT WITH MECD HAS HIGH MO6
PRODUCTION AND PATIENTS CAN
BENEFIT FROM TREATMENT OF IO6.
IT WOULD LIKE TO SEE WHETHER
[INDISCERNIBLE] CAN DOWNREGULATE
WITH [INDISCERNIBLE] AND IO6
INDUCED PRODUCTION AND HERE WE
CHOOSE MACROPHAGES THAT ARE ONE
OF THE MAIN PRODUCER OF HUMAN
IO6 AS OUR TARGETS.
SO, HERE, WE CHOOSE TWO
DIFFERENT MACROPHAGES. THP1 AND
U937. ON THE LEFT SHOWS
VARIABILITY ASSAY WITH RESPONSE
TO THE [INDISCERNIBLE] TREATMENT
AND ON THE RIGHT SHOWS DATA OF
IO6. BASICALLY, WE TREAT CELLS
WITH IO6 AND [INDISCERNIBLE] AND
COLLECT THIS OF THE CELLS AND
THIS VIRUS AND GOES IN THE
ELIZA. YOU CAN SEE
[INDISCERNIBLE] INHIBIT IO6
PRODUCTION IN NON-TOXIC DOSAGE
THAT IS HIGHLIGHTED BY RESULTS.
[INDISCERNIBLE] WE WOULD LIKE TO
SEE CHANGE OF KINASES IN CELLS
AFTER TREATMENT AND CHOOSE HUMAN
FIRST [INDISCERNIBLE] AND TO
TEST CHANGE IN THESE CELLS. YOU
CAN SEE HIGHLIGHT IN THE SQUARE
AND SO THESE ARE BOTH
DOWNREGULATED IN THIS JSC-1 AND
BCBL-1 AND THIS ACTIVATOR OF IO6
AND WAIT TO CONFIRM WITH WESTERN
BLOT AND YOU CAN SEE IN THESE
CELLS BOTH IN JSC-1 AND BCBL-1
AND STAGE 3 IS DOWNREGULATED AND
TARGETS START TO [INDISCERNIBLE]
THAT IS WHY WE WENT TO LOOK UP
THE ACTIVITY. SO, USING THIS
PROTEIN ARRAY, WE FOUND THAT
GENERALLY BIG ACTIVITY ARE
INHIBITED IN THESE. WE CHOOSE
ONE MAIN COMPONENT OF GABA B
COMPLEX AND P6 DEFINE AND CHECK
PROTEIN EXPRESSION BY WESTERN
BLOT. IT IS LOUSY HERE BUT YOU
CAN HAVE A GLANCE IN JC1 IT IS
DOWNREGULATED BUT HAVE TO BE
FURTHER CONFIRMED AND BASED ON
DATA WE PROPOSE POSSIBLE
MECHANISM OF ACTION OF
[INDISCERNIBLE] RELATED MCD.
SO, IN CASE INFECT AND PROMOTE B
CELLS DIFFERENTIATION IN PLASMA
CELLS AND FURTHER INTO THESE MCD
PLASMA BLAST. AND IN MRAZ MA
BLAST VIRUS PRODUCER OF IO6 AND
THOSE VIRAL IO6 AND
[INDISCERNIBLE] IN MONOCYTES AND
YOU CAN SEE TARGETS OF THESE
[INDISCERNIBLE] THEY CAN BE
INHIBITED. EXCUSE ME. THEY CAN
BE INHIBITED BY THIS. AND
REDUCING THIS IO10 EXPRESSION.
CONCLUSION IS [INDISCERNIBLE]
TIME IN DEPENDENT INHIBITED CELL
GROWTH AND THIS MAY COUNT FOR
[INDISCERNIBLE] CELL GROSS
INHIBITION EFFECT.
[INDISCERNIBLE] DOWNREGULATE A
LOT OF GENES CELL CYCLE
PROLIFERATION AND REDUCE MI
EXPRESSION IT ALSO INHIBITS
MULTIPLE DINACES INCLUDING THE
STATS 3 AND GAMA B.
IT MIGHT BE USEFUL FOR TREATMENT
AND FUTURE PLAN IS TESTING
EFFECT OF CYTOKINE PRODUCTIONS
AND EFFECT ON THESE PATHWAYS.
NOW CURRENTLY PROTOCOL IS
TREATING THIS AND BEING
DEVELOPED BY OUR TEAM MEMBER.
WITH THAT I WOULD LIKE TO THANK
MENTOR DR. ROBERT AND OUR GROUP
MEMBERS AND ALSO OUR BRANCH AND
THANK YOU. I WOULD LIKE TO TAKE
QUESTIONS.
>>THANK YOU. WE DON'T HAVE
ENOUGH TIME. SO, NOW WE WILL
MOVE TO THE NEXT. UP NEXT, NEXT
SPEAKER IS TARA. YEAH. SO, THE
TITLE IS EXPRESSION OF HUMAN
ENDOGENOUS RETROVIRUSES DUE TO
DEFECTS IN CHROME ATTIN
REMODELING IN CLEAR CELL
MENINGIOMA. SO, NOW IS YOUR
STAGE.
>>THANK YOU.
>>GOOD AFTERNOON, EVERYBODY.
I HAVE NO DISCLOSURES TO REPORT.
SO, FIRST, I WILL TELL YOU A
LITTLE ABOUT CLEAR CELL
MENINGIOMA WHO IS EFFECTED WITH
IT AND WHAT IS PROGNOSIS FOR
THIS TUMOR. MOST PATIENTS
DIAGNOSED WITH CLEAR CELL
MENINGIOMA ARE YOUNG AND RARE
TUMOR AND ABOUT 100 PEOPLE IN
THE UNITED STATES ARE DIAGNOSED
WITH IT AND MAJORITY ARE YOUNG
CHILDREN AND ADULTS DIAGNOSED
ARE YOUNG IN AGE STILL. PROBLEM
WITH THE TUMOR IS THERE IS NO
GOOD TREATMENT FOR IT OTHER THAN
STANDARD OF CARE CIRCCAL
[INDISCERNIBLE] AND THERE IS
NOTHING THAT IS TARGETED. SO,
THE PROGNOSIS IS DISMAL FOR MOST
PATIENTS IT THAT GET THIS TUMOR.
SOMETHING THAT CHARACTERIZES THE
TUMOR IT HAS BEEN KNOWN SINCE
2013 IS LOSS OF SMARKY PART 1.
IN STUDY DONE IN 2016 THEY FOUNT
SMASHGE 1CHARACTERIZES THE TUMO
BEEN KNOWN SINCE 2013 IS LOSS OF
SMARKY PART SMARCE1. IN STUDY
DONE IN 2016 THEY FOUNT SMASHGE
SMAR
SMARCE1.
IT IS A CHROMATIN REMODELING
PROTEIN AND HAS TO BE IN NUCLEUS
TO DO ITS JOB. IF NO
EXPRESSION, THERE IS NO
FUNCTION. THIS TUMOR TYPE WAS
INTERESTING TO ME. WE WORKED ON
A PROJECT LOOKING AT ATYPICAL
TERTOID TUMORS WITH KEY PART OF
CHROMATIN REMODELING.
THERE IS INTERESTING PARALLELS
BETWEEN TWO TUMOR TYPES ONE THEY
BOENT HAVE INTEE STIT SHAL --
THE HUMAN ENDOGENOUS -- IT IS
SPECIFIC TO HUMANS WITH OPEN
READING FRAMES MAKING VIRAL
PROTEINS ONE THAT SEEMS TO BE
MOST PROBLEMATIC IS ENVELOPE.
ENVELOPE CAN BE EXPRESSED IN
MEMBRANE LIKE OTHER VIRAL
PROTEINS AND OTHER VIRAL
ENVELOPES I SHOULD SAY AND 95%
OF AT/RT CASES PREDOMINANTLY IN
YOUNG CHILDREN THERE WAS HIGH
EXPRESSION OF HML-2 ENVELOPE
PROTEIN AND SAW IN CELL LINES
MADE IN CHILDREN'S HOSPITAL OF
LOS ANGELES FROM TWO DIFFERENT
PATIENTS IT WAS EXPORTED IN
EXTRA CELLULAR VESICALS THAT WE
SAW THEM BY ELECTRON MICROSCOPY.
WE SEE HML-2 ENVELOPE THAT IS IN
THE PARTICLES.
HOW DOES IT RELATE TO CLEAR CELL
MENINGIOMA? 8 TUMORS BUNCH OF
DIFFERENT INSTITUTIONS AND FOUND
EXPRESSION OF HERV-K. THEY ARE
SHARING MRAZ MA MEMBRANE AND
WHERE ARROWS ARE YOU SEE ACTUAL
EXTRA CELLULAR VESICLES WITH
PROTEIN IN THEM AND CHECK TO
MAKE SURE TUMORS HAD LACK OF
SMASHGE SMARCE1 EXPRESSION.
THEY DID AND LOOKED FOR MRURY
POTENCY FACTOFACTOR OCT4.
HML6 IS IN OLDER HERBV-K -- NEX
STEP LOOKING AT COUPLE
ENDOGENOUS RETRO VIRAL PROTEINS.
[INDISCERNIBLE] IS NOT ONLY ONE
THAT CAN INTERSTITIAL CELLS.
HEFSH K IS ACTUALLY BUT LESS IS
KNOWN ABOUT THAT. SIN CRITTIN
SMARCE1 A -- SIN CRITTIN SMARCE
AND SIN CRITTYNCY
-- SO, IT COULD ACTUALLY BE A
VERY BIG KEY FOR PROTECTION OF
FETUS FROM MOTHER DURING
DEVELOPMENT AND COULD BE
CO-OPTED BY TUMOR TO HELP IT BE
PROTECTED FROM THE IMMUNE SYSTEM
AND WANTED TO LOOK FOR CD63 A
MARKER FOR EXOSOMES AND IF EXTRA
CELLULAR VESICLES WERE EXOSOEMS.
WE ARE CONFIDENCE THEY ARE
EXOSOMES TRACKING WHAT WE HAVE
SEEN IN ATYPICAL TUMORS AS WELL.
WE FOUND THAT WE COULD
SIGNIFICANTLY INCREASE
TRANSCRIPTION OF SYNCYTIN-11.
NEXT THING TO TALK ABOUT I
MENTIONED EARLIER LOOKING FOR
OCT4 IN THESE TUMORS AND IF
THERE ISL
THERE IS CORRELATION. -- WE ARE
LOOKING FORWARD TO TESTING THEM
TO SEE IF WE CAN PULL DOWN
PROMOTERS USING OCT-4 ANTIBODY
AND MIGHT WONDER WHAT ROLE OF
THE VIRUSES IS IF IN PATHOGEN
ESIS TUMOR AND QUESTION WE HAVE
TO EXPLORE IS IF ONE OF TWO
THINGS IS HAPPENING AND IF THESE
RETROVIRUSES ARE EXPRESSED IN
DEVELOPMENT AND ARE IMPORTANT IN
PROCESS OF DEVELOPMENT IF
TURNING THEM OFF PREMATURELY
DEVELOPMENT WON'T PROCEED. THEY
ARE REMAINING ON AFTER
DEVELOPMENT OR GETTING TURNED
BACK ON SOMEHOW UNDERSTANDING
WHICH OF THE TWO POSSIBILITIES
IS HAPPENING IS SOMETHING TO
MODEL IN IN VIVO SYSTEM AND
TELLING YOU MORE ABOUT THESE
VIRUSES.
THEY INTEGRATED INTO THE GENOME
AND INTEGRATED INTO GERM LINE
ANIMAL MOUSE OR HUMAN ANCESTORS
AND ARE PASSED ON THEY ARE THE
GERM LINE AND LIKE ANY OTHER
MENDELIAN GENE AND LOTS OF TIMES
THEY HAVE BEEN THERE MILLIONS OF
YEARS AND CAN PICK UP MUTATIONS
AND RECOMBINE AND YOU CAN END UP
WITH SOLO LTRS THAT ARE FLANKING
BITS OF THE VIRUS.
AND LOTS OF OTHER CONSEQUENCES
OF RECOMBINATION.
BUT MOST IMPORTANT THING IS OPEN
READING FRAMES. SO, ENVELOPE
GENE HERE IS THE ONE I FOCUSED
ON THE MOST TODAY. THERE IS
EVIDENCE FOR EXPRESSION OF OTHER
ENDOGENOUS RETRO VIRAL GENES AS
WELL IN TUMORS.
SYNCYTIN-1 IS IMPORTANT FOR
MAMMALS AND PLACENTA.
THIS IS SYNCYTIN-2 AND YOU SEE
IT IN THE GENOME AND IT IS
EXPRESSED EARLY IN DEVELOPMENT
DAYS 6 TO 7 AND CONTINUES TO BE
EXPRESSED THROUGHOUT DEVELOPMENT
AND MANY HAVE SLIGHTLY DIFFERENT
TIMINGS. THEY ARE ALL
EXPRESSED AT SOME POINT AND ARE
MANY OF THEM. KNOCKING ONE OF
THEM DOWN OR OUT OTHERS COULD BE
THERE TO COMPENSATE AND OTHER
THING INTERESTING TO NOTE IS
SYNCYTIN-1 AND SYNCYTIN-2
INTEGRATED SLIGHTLY DIFFERENT
TIMINGS. SYNCYTIN-1 IS KNOWN
FOR IMMUNOSUPPRESSIVE PROPERTIES
AND [INDISCERNIBLE] ALTHOUGH IT
IS NOT STRONGLY REFLECTED IN THE
LITERATURE AT THIS MOMENT.
IN FUTURE, WE WANT TO REALLY
UNDERSTAND HOW HERV EXPRESSION
IS REGULATED AND OCTOBE-4 -- CAE
CONTRIBUTING TO EITHER
DEVELOPMENTAL ISSUES OR CLEAR
CELL MENINGIOMA SPECIFICALLY IN
VIVO. WITH THAT I WOULD LIKE TO
THANK PI AND TWO MEMBERS OF THE
LAB THAT WORKED HARD ON THIS
PROJECT AND COLLABORATORS THAT
SENT SAMPLES FROM ALL ACROSS THE
COUNTRY AND OUR VERY ESTEEMED
COLLEAGUE SOPHIE THAT WILL HELP
WITH OUR CHICK EMBRYO MODEL AND
WOULD BE HAPPY TO TAKE ANY
QUESTIONS.
>>ANY QUESTIONS?
>>IS [INDISCERNIBLE] SUPPRESSED
IN THE TUMORS AND OTHER THINGS
YOU CALL EXOSOMES?
>>WE CHECKED TO SEE IF THEY
WERE VLPS IN ATRT PROJECT. THEY
DID NOT LOOK LIKE VLPS AT ALL.
SO, I DOUBT THEY ARE VLPS AND
COULD BE SOME GAG EXPRESSION I
HAVEN'T STAINED FOR IT BUT
ANTIBODIES FOR HEFSH K AND MAIN
PROTEIN THEY EXPRESS IS
ENVELOPE.
>>[INDISCERNIBLE].
>>WE HAVEN'T CHECKED YET. CAN
YOU IN SOME LABS AND ONE POST
DOC IS LOOKING AT THAT WITH ALS
ACTUALLY.
>>OTHER QUESTIONS?
OKAY. THANK YOU.
>>HE HAS A QUESTION.
>>IF YOU LOOK IN THE TUMOR AND
[INDISCERNIBLE].
>>NOT TO MY KNOWLEDGE. WE
STARTED THE PROJECT A COUPLE
MONTHS AGO AND LOOKING AT
AVENUES AND I WAS THINKING ABOUT
E-MAILING AND ASKING HIM. THAT
IS ONLY ONE I KNOW OF THAT
EXISTS AND WOULD BE USEFUL.
THANK YOU.
>>THANK YOU.
>>NOW LET'S MOVE TO THE NEXT
SPEAKER.
CHAD HOGAN. TALK IS LOSS OF
STAT3 IN B CELLS HEIGHTENS
EXPRESSION OF ANTIVIRAL GENES
AND REDUCES LATENCY OF MURINE
GAMA HERPES VIRUS 68 IN VIVO.
>>THANK YOU FOR STICKING AROUND
FOR MY TALK AND ORGANIZERS FOR
GIVING ME OPPORTUNITY TO SHARE
MY WORK. I'M CHAD HOGAN AND
TODAY I WILL GIVE YOU A
HIGHLIGHT OF MY DISSERTATION
RESEARCH I'M CHARACTERIZING ROLE
OF STAT3 IN B CELLS IF IN
GAMMAHERPESVIRUS LATENCY.
I KNOW WE HAD HERPES VIRUS TALKS
EARLIER IN THE DAY. REFRESHER,
HERPES VIRUSES ARE LARGE DOUBLE
STRANDED DNA VIRUSES CHARACTERED
BY ABILITY TO ESTABLISH
LIFE-LONG INFECTION IN THE HOST
TWO PHASES OF INFECTION. LYTIC
PHASE AND LINEAR GENE
REPLICATION IN THE NUCLEUS AS
WELL AS PRODUCTION OF INFECTIOUS
PARTICLES AND OTHER HAND LATENCY
A DORMANT OR QUIET STATE AND
GENOME IS CENTERED IN NUCLEUS AS
EPISOME AND NO INFECTIOUS
PARTICLES PRODUCED WE HAVE
VACCINES AGAINST ONE VISA VEE AS
WELL AND ONE CLASS OF ANTIVIRAL
DRUGS.
OF THREE SUBFAMILIES OF HUMAN
HERPYS VIRUSES OUR LAB IS
FOCUSED ON GAMMA VIRUSES EBV AND
EPSTEIN VIRUS BECAUSE OF -- THEY
ARE ASSOCIATED WITH CANCERS AND
INFECT A LARGE PORTION OF THE
GLOBAL POPULATION AND BECAUSE OF
IT THAT OUR LAB FOCUSES ON GAMMA
HERPESVIRUSES MY PROJECT IN
PARTICULAR FOCUSED ON HOST
PROTEIN STAT3 TRANSCRIPTION
FACTOR. WE CAN TRANSLOCATE TO
NUCLEUS TO ACT AS TRANSCRIPTION
FACTOR REGULATING PROCESSES
PROLIFERATION SURVIVAL AND NO
SURPRISE IT IS A MAJOR PLAYER IN
CANCER AND IN FACT IS STAT3
ACTIVATION IS FOUND IN MANY
HUMAN CANCERS INCLUDING THOSE
THAT ARISE DUE TO EPV AND KSHV
AND STAT3 IS ACTIVATED ON GAMA
HERPES VIRUS ACTIVATION AND
BENEFIT AND MULTIPLE FRONTS ON
CELL SURFACE AND DIRECTLY IN
NUCLEUS FOR TRANSCRIPTIONAL
ACTIVITY AND DRIVING QUESTION OF
MY RESEARCH IS WHAT IS ROLE OF
STAT3 IN GAMA HERPES VIRUS
LATENCY ESTABLISHMENT?
TO STUDY THAT ROLE WE UTILIZE
THIS HERPES VIRUS THIS MODEL
PATHOGEN AND HOMOLOGS OF MASS
GENES AND -- REPLICATION AND
LATENCY. REAL POWER OF OUR
MODEL COMES WITH THE ABILITY TO
INFECT IN NATURAL LIVE HOST AND
TO OBSERVE PRIMARY INFECTION TO
LOOK AT HOST DETERMINANTS OF
ESTABLISHMENT OF INFECTION THAT
IS HARDER TO DO WITH HUMAN GAMA
HERPES VIRUSES THEMSELVES AND
PREVIOUS RESEARCH IN LAB SHOWED
NO ROLE IN REPLICATION AND IN
LUNGS OF MICE IN THE MEMORY OR B
CELL IN GENERAL AND DETERMINING
THIS ROLE SPECIFICALLY IN B
CELLS WE NEED A MODEL TO KNOCK
OUT PROTEIN SPECIFICALLY IN THAT
COMPARTMENT AND WE HAVE THIS
DOWNSTREAM OF INDIGENOUS
PROMOTER KNOCKING OUT THIS AND
FOR ALL EXPERIMENTS SHOWING YOU
UTILIZING MICE THAT WOULD BE
CONSIDERED WILD TYPE MICE AND
HAVE STAT 3 IN B CELLS AND AS
WELL WE ARE UTILIZING FLOW
CITOMETRY TO TRACK PHENOTYPE AND
CELLS AND WE HAVE A REPORTER
VIRUS AND ABLE TO TRACK
PERCENTAGE OF INFECTED B CELLS
IN STAT 3 WILD TYPE I WILL REFER
TO THEM AS AND YOU CAN SEE WE
HAVE A SIGNIFICANT FREQUENCY OF
POSITIVE B CELLS IN IN TWO
DISTINCT STRAINS OF STAT 3
KNOCKOUT MICE THAT WAS FIRST
INDICATION TO US THAT STAT 3 IS
PLAYING ROLE IN VIRAL LATENCY
ESTABLISHMENT AND MENTIONING IN
DATA NOT SHOWING NOW BUT DEFECT
VALIDATED BY LIMITING DILUTION
PCR DIDN'T SEE FURTHER
REACTIVATION DEFECT WITH THESE
MICE.
SO, ONE IMPORTANT ASPECT --
GERMINAL CENTER IS MICRO
STRUCTURE IN -- ACTIVATED B
CELLS GO TO MUTATE B CELL
RECEPTOR AND GET TESTED.
SO, HERE -- IT IS KNOWN THAT
WITH THIS YOU NEED A PROPER
GERMINAL CENTER FORMATION FOR
VIRUS TO ACTUALLY PERSIST. HERE
I'M UTILIZING TWO MARKERS TO DEN
OAT GERMINAL CENTER B CELLS AND
SUBPOPULATION AND TO SURPRISE WE
SAW INCREASE IN KNOCKOUT MICE
THAT IS OPPOSED TO DECREASE IN
LATENCY THAT WE ACTUALLY SAW.
IN ORDER TO GET A BETTER -- FLOW
CITOMETRY IS REPRESENTATIVE OF
THE POPULATION WE WANT TO LOOK
AT OVERALL STRUCTURE AND
ARCHITECTURE OF SPLEENS AND WITH
THE LAB AND NIAD WE HAD NAÏVE
INFECTED SPLEENS OF KNOCKOUT
MOOIS TO DO [INDISCERNIBLE] AND
YOU CAN APPRECIATE IN PURPLE I
HAVE B CELL MARKER B220 DENOTING
A FOLLICULAR REGION OF GERMINAL
CENTERS APPRECIATING IN NAÏVE
CASE COMPARING WILD TYPE AND
KNOCKOUT FOLLICLES THAT ARE
FRAGMENTED AND SMALLER IN WILD
TYPE CASE AND UPON EXPRESSION WE
SEE EXPRESSION OF ACTIVATION OF
WILD MARKER. GERMINAL CENTER B
CELLS THAT ARE TYPICAL AND
EXPRESSION OF MARKER IS MORE
DISBURSED AND OUT OF NORM AND
NOT REALLY COMPACT AND AGAIN IS
AN INDICATION TO US THERE IS
SOMETHING WRONG WITH STRUCTURE
OF GERMINAL CENTERS IN THESE
MICE.
SO, BECAUSE WE ARE SEEING
DISREGULATION OF GERMINAL CENTER
ITSELF WE LOOK AT DOWNSTREEMENT
EFFECTS OF THAT AND WE SEE A
SIGNIFICANT DECREASE IN THIS
POPULATION IN KNOCKOUT MICE.
WE COLLECT SERUM AND IN LAB
UNIVERSITY OF ARKANSAS MEDICAL
SCHOOL THERE IS DECREASE IN
ANTIBODIES AND CAPACITY TO
NEUTRALIZE VIRUS IN COMING
KNOCKOUT MICE AND AS WELL
LOOKING AT OTHER ARM OF ADAPTIVE
RESPONSE AND T CELLS 42 DAYS
POST INFECTION WE SEE
SIGNIFICANT INCREASE IN VIRUS
SPECIFIC T CELLS FIRST
RESPONDERS TO INFECTION CLEAR
INFECTION AND DIE OFF AND
MINIMAL ACTIVE REPLICATION WE
SEE INCREASE IN THIS POPULATION
AND SO FROM TWO SORT OF THINGS I
HAVE SHOWN SO FAR WE HAVE
DISFUNCTION IN B AND T CELL
RESPONSES WITH INFECTION AND
WANTED TO SEE IF REDUCTION IN
LATENCY IS DUE TO IMMUNE
RESPONSE OR WHAT IS NECESSARY IN
B CELL ITSELF FOR VIRUS TO GO
LATENT WE TAKE B CELLS WITH STAT
3 AND COMBINE TO HOST AND THAT
WAY WE HAVE A COMPETITIVE MODEL
WE CAN DETERMINE WHETHER OR NOT
STAT 3 IS NECESSARY WHILE CELLS
ARE RESPONDING TO SAME IMMUNE
RESPONSE.
IN ORDER TO DO THIS, WE LAYERED
ON TOP OF THE ORIGINAL STAT3
MUTATION TOMATO RED FLUORESCENT
PROTEIN UNDER REGULATION OF
PRECOMBIN NANT -- KNOCKOUT STAT
3 AND TURN ON THIS NICE RED
PROTEIN AND FLOW CITOMETRY WE
CAN TAKE ADVANTAGE OF THE MARKER
AND KNOCKOUT IN POSITIVE CELLS
LOOKING AT FREQUENCY OF THIS.
WE SAW SIGNIFICANT DECREASE IN
LATENCY ESTABLISHMENT AND NOT
SHOWING DATA HERE BUT WERE
VALIDATED BY OTHER MEANS HERE AS
WELL. KNOCKING IMPORTANT
TRANSCRIPTION FACTOR AND NEXT
IDEA SEEING WHAT IS HAPPENING TO
TRANSCRIPTIONAL LANDSCAPE IN
ABSENCE OF STAT3 AND TOOK
POPULATIONS UTILIZING THESE
MARKERS SORTING WILD TYPE
KNOCKOUT UNINFECTED AND ENFEKTED
CELLS PERFORMING RNA SEQUENCING
PRINCIPLE COMPONENT -- AND
PERFORMING GENE CENTER
ENRICHMENT ANALYSIS WITH
KNOCKOUT WILD TYPE CELLS WE SEE
SOME PATHWAYS THAT MAKE SENSE
MTOR AND MIXED SIGNALING THAT
HAVE GROWTH PATHWAYS AND SEE
INCREASE IN INFLAMMATION AND
INFLAMMATORY PATHWAYS AND IT IS
KNOWN THAT IT HAS MECHANISMS FOR
DAMPENING THIS RESPONSE AND
LOOKING AT WHAT IS GOING ON IN
THIS CASE. HERE I'M SHOWING YOU
A COMPARISON OF INFECTED AND
UNINFECTED WILD TYPE GERMINAL
CELLS AND WE ARE SEEING NEGATIVE
ENRICHMENT SCORE THAT MEANS THAT
GENES INVOLVED IN TYPE 1
INTERFERON RESPONSE ARE
DOWNREGULATED IN INFECTED CELLS
COMPARED TO WILD TYPE
COUNTERPARTS AND THAT THIS
RESPONSE IS DAMPENED AND ON
OTHER HAND COMPARING WILD TYPE
INFECT TODAY KNOCKOUT INFECTED
CELLS POSITIVE ENRICHMENT SCORE
I SEE AND GENES ARE HEIGHTENED
ACTUALLY IN EXPRESSION.
AND SO IT IS KNOWN THAT STAT3
CAN PLAY A ROLE IN DAMPENING OF
THIS COMPLEX A MAIN DRIVER OF
ISGS IF IN TYPE 1 INTERFERON
RESPONSE REGULATION HAS
COMBINING WITH OTHER STATS AND
REPRESSION ON IRF9 AND MADE A
MODEL WITH LOSS OF STAT 3 LEADS
TO INCREASE IN SIGNALING
MOLECULES LEADING TO OVERALL
INCREASE IN FORMATION OF THIS
COMPLEX AND LEADING TO INCREASED
ISGS THAT ARE ACTUALLY EFFECTING
VIRAL OR VIRUS ITSELF IN MANY
DIFFERENT ASPECTS OF LIFECYCLE
HOST KRENSING AND DIRECT EFFECTS
ON VIRAL GENOME AND HERE TO THE
RIGHT I'M SHOWING THIS DATA IT
THAT IS VALIDATING ISGS IT THAT
WE FOUND MOST INTERESTING AND
AGAIN WE ARE -- GPPS ARE GENES
POINTED OUT TO US MOST HEAVILY.
SO, NEXT, WE WANTED TO ALSO
VALIDATE ANOTHER ISG THAT WE HAD
IN OUR HIT THAT ARE IF IT
PROTEINS AND IF 1, 2, AND 3 HAVE
DIFFERENT ANTIVIRAL ACTIVITIES
THAT ARE KNOWN AND FOUND THAT IF
IT 3 WAS ISG HEAVILY UPREGULATED
IN KNOCKOUT CELLS THEMSELVES AND
IN COLLABORATION WITH NANCY
REISH LAB AT STONEY BROOK
UNIVERSITY IF [INDISCERNIBLE]
YOU BLOCK REPLICATION OF MHV68
IN THIS DATA YOU SEE SHOWING A
24-HOUR TIME COURSE. SO, IN
CONCLUSION, LOSS OF STAT 3 AND B
CELLS LEADS TO GLOBAL IMMUNE
DEFECTS ON B AND T CELL
RESPONSES IN TWO DISTINCT ANIMAL
STRAINS AND THERE IS INTRINSIC
REQUIREMENT FOR THESE FOR -- AS
WELL THIS INFECTION OF GERMINAL
CELL B CELLS ALTERED
TRANSCRIPTIONAL LANDSCAPE AND
UPREGULATION OF GROWTH OF
PATHWAYS AND [INDISCERNIBLE]
RESPONSE AND SILENCING OF TYPE 1
INTERFERON RESPONSE AND THESE
STUDIES I COVERED SOME NOVEL
PROVIRAL RULES FOR STAT 3 WE ARE
LOOKING FURTHER INTO IN HOPES OF
PREVENTING LATENCY AND BY THAT
WAY PREVENTING VIRUS DRIVEN
MALIGNANCIES FROM OCCURRING AND
WITH THAT I WOULD LIKE TO THANK
MY LAB AND BRANCH AND MANY
COLLABORATORS THAT HELPED TO PUT
THIS WORK TOGETHER. WITH THAT,
I WILL TAKE ANY QUESTIONS.
>>QUESTIONS?
>>OKAY. I HAVE A QUESTION.
>>WE HAVE. THAT ACTUALLY WAS
NOTHING. THERE WAS NEVER
ANYTHING THAT CHANGED BETWEEN
THEM BETWEEN WILD TYPE AND
KNOCKOUT MICE. GOOD QUESTION.
>>OKAY. SO, I GUESS WE CAN
MOVE TO THE NEXT SPEAKER.
>>FOR THE LAST TALK IN THE
SESSION BEFORE OUR LITTLE BREAK,
GABRIEL STARRETT WILL TALK ABOUT
OR TELL US ABOUT VIROMES OF
BLADDER CANCERS OF SOLID ORGAN
TRANSPLANT RECIPIENTS AND PEOPLE
LIVING WITH HIV REFLECT RELATIVE
DISEASE RISK.
>>THANKS FOR STICKING AROUND
BEFORE THE BREAK. INTERESTING
STORY TO SHARE WITH YOU TODAY.
WHAT EMERGED AS A PILLAR OF
CANCER VIROLOGY IS
IMMUNOSUPPRESSED PEOPLE
TRANSPLANT RECIPIENTS OR PEOPLE
LIVING WITH HIV/AIDS ARE IN HIGH
RISK OF RESOLVING NUMEROUS TYPES
OF CANCERS THAT IS MOST WELL
SEEN WITH [INDISCERNIBLE] SORE
COMBA AND MERC CELL CARCINOMA
AND FREQUENCIES OF POPULATIONS
AND STUDIES LOOKING AT VARIOUS
CANCERS THAT INCREASED RISK FOR
FURTHER HAVE CONFIRMED THIS
SHOWING THAT THIS SARCOMA YOU
HAVE AT TOP AND INCLUDES HPV
CANCERS WE HEARD ABOUT
PREVENTION TODAY AND OTHER
CANCERS DOWN HERE THAT DON'T
HAVE CLEAR ASSOCIATIONS WITH
INFECTIOUS DISEASE AND INCREASED
INCIDENCE WITHIN POPULATION
SUGGEST THEY MIGHT BE WITH
INFECTIOUS AGENT AND GOING ALONG
WITH THIS FOCUSING ON KIDNEY
TRANCE CRIPPENTS AND PATIENTS
WITH PREVIOUS HISTORY OF
VIEREMIA ARE A FURTHER INCREASED
RISK FOR DEVELOPING BLADDER
CANCER SUGGESTING THAT THIS
PARTICULAR VIRUS IS ASSOCIATED
WITH DEVELOPMENT OF THIS TYPE OF
CANCER AND THERE IS A CONFUSING
RESULT AS WELL AND LOOKING AT
PEOPLE LIVING WITH HIV AIDS AND
RISK FOR DEVELOPING KIDNEY OR
SPECIFICALLY BLADDER CANCER SEEM
TO BE AT A LOWER RISK FOR
DEVELOPING BLADDER CANCER AND
OTHER STUDIES HAVE SHOWN THEY
HAVE NO INCREASED RISK FOR
DEVELOPING BLADDER CANCER. WHAT
DOES IT MEAN FOR ASSOCIATION OF
BEING CAUSED BY INFECTIOUS
AGENT? THAT IS GIST WHAT I WILL
TALK ABOUT TODAY. THIS IS
COVERING TWO CASE SERIES. ONE
OF WHICH IS 43 CASES FROM
TRANSPLANT RECIPIENTS FROM
VARIOUS TYPES OF ORGANS AND WE
WERE ABLE TO COLLECT 43 PRIMARY
TUMORS AND TISSUES MATCHED WITH
15 CASES HERE AND MET AFT ASEIZE
AND 15 PRIMARY TUMORS FROM
PEOPLE LIVING WITH HIV AND AIDS.
I WANT TO HIGHLIGHT THIS IS
RETROSPECTIVE AND ARCHIVED AND
WE HAVE DONE TOTAL RNA
SEQUENCING AND GENOME SEQUENCING
AND GIVING PATIENT
CHARACTERISTICS OF POPULATIONS
TO HIGHLIGHT CAVEATS AND HOW TO
INTERPRET THIS. MEDIAN AGE
SIMILAR 65 AND 59 AND GENERAL
BLADDER CANCER IS INCREASED
FREQUENCY IN MEN THAT IS 3 TO 4
OR 1 TO WOMEN AND TRANSPLANT
RECIPIENT POPULATION IS FAIRLY
REPRESENTATIVE OF BEING 7 TO 3
AND HIV POPULATION IS ABOUT 13
TO 1. IT IS SKEWED VERY MUCH TO
MALE AND MAJOR DIFFERENCE
BETWEEN TWO GROUPS WE HAVE
TOGETHER IS WE HAVE HIV POSITIVE
POPULATION THAT SKEWS TOWARDS
HAVING MORE ADVANCED DISEASE.
THESE ARE MORE -- SO, IN
GENERAL, THEY HAVE SIMILAR
PROPORTIONS TO INVASIVE BLADDER
CANCERS VERSUS NONINVASIVE AND
SKEWED TOWARDS METASTATIC AND
DEEPER INVADING TUMORS AND IN
GENERAL WE HAVE HIGHER-GRADE
TUMORS WITHIN THIS COHORT VERSUS
ORGAN TRANSPLANT RECIPIENTS IN
GENERAL REFLECTS WHAT LARGER
STUDIES HAVE DONE LOOKING AT
OVERALL EPIDEMIOLOGY OF THE TWO
DIFFERENT GROUPS AND ADDITIONAL
CHARACTERISTICS THAT MIGHT BE
RELEVANT HERE AND COUNTS ON
AVERAGE WERE 493 CELLS PER MIL.
AS WELL AS HIV VIRAL LOAD THAT
WAS AT 50. OTHER IMPORTANT
POINT IS ALL PATIENTS WERE ON
ANTIVIRAL THERAPY AT TIME THEY
WERE DIAGNOSED WITH BLADDER
CANCER AND IN GENERAL
TRANSPLANTED POPULATION MOSTLY
KIDNEY TRANSPLANT RECIPIENTS
HEART AND OR LUNG TRANSPLANT
RECIPIENTS IS GENERAL WORK FLOW
HOW TO PROCESS SAMPLE AND EVERY
SAMPLE HAD SECTIONS SENT OFF FOR
DNA ISOLATION AND RNA ISOLATION
AND DID WHOLE GENOME AND TOTAL
SEQUENCING [INDISCERNIBLE].
SO, I WILL SHOW THIS TO
BASICALLY BRIEFLY GLOSS OVER IT
THAT COULD BE SUMMARIZE THE IN
THIS PIE CHART THAT IS SHOWING
OVER 50% OF TUMORS FROM SOLVENT
TRANSPLANT RECIPIENTS CONTAIN
THIS AS A RESORT AND SEEMS BK
MAY PLAY A ROLE WITHIN THESE. I
WILL SHOW ADDITIONAL EVIDENCE
FOR THAT AS WELL.
NEXT MOST ABUNDANT WERE
INTERESTING SINGLE-STRANDED
VIRUSES CALLED YELLOW VIRUSES
MOST ABUNDANT VIRUSES IN US OR
OUR BLOOD STREAM INCREASING
FREQUENCY OF [INDISCERNIBLE] AND
[INDISCERNIBLE] PAPILLOMA
VIRUSES AS WELL IN THE TUMORS AS
WELL. HIV AND PEOPLE LIVING
WITH HIV POPULATION, THERE WERE
VERY FEW VIRUSES AND TWO
PATIENTS BOTH HAD
[INDISCERNIBLE] VIRUSES IN THEM
AND MORE THAN ONE VIRUS WITHIN
EACH TUMOR AND IT IS EXTENT OF
IT VIRIONS OF THESE POPULATIONS
LOOK STRIKINGLY DIFFERENT THIS I
WILL TRY TO ARGUE MAY REFLECT
INCREASED RISK WE SEE AT
EPIDEMIOLOGICAL LEVEL. I WANT
TO TAKE A MOMENT TO TALK ABOUT
[INDISCERNIBLE] VIRUSES WHICH
ARE DOUBLE STRANDED CIRCULAR
VIRUSES AND WE ARE COVERED IN
THEM AND IN GENERAL WITH -- THEY
DON'T CAUSE PROBLEMS IMMUNE
SUPPRESSION COMING INTO PICTURE
YOU CAN HAVE VARIOUS SKIN
DISEASES OR FATAL BRAIN
INFECTION AND MORE COMMONLY WHAT
WE KNOW ARE NEVER ROP OJY AND
CYSTITIS AND AMERICALE CELL
POLYOMA VIRUS WHICH IS EMERGING
ONCOGENIC VIRUS. WE ARE COVERED
IN THESE THINGS AND THEIR
INFECTIONS ARE LIFELONG AND
SUBCLINICAL AND USUALLY
INFECTION HAPPENS SOMETIMES IN
EARLY CHILDHOOD.
YOU HAVE INLT MITTIENT SHEDDING
OF VIRUSES DEPENDING WHAT TISSUE
THEY ARE IN.
SKIN VIRUSES COMING OUT OF
YOU'RIN AND 10% OF PEOPLE ARE
SHEDDING SOME SF THE VIRUSES AND
SHEDDING INCREASES AND VIRUS
REPLICATION INCREASES DURING
IMMUNE SUPPRESSION AND INCLUDING
PREGNANCY AND ALSO HIV AND
INCLUDING DRUGS AND AGING
INCREASING VIRAL SHEDDING AND
WHEN WE TEND TO SEE AMERICALE
CELL CARCINOMA IS CANCER WITH
PEOPLE WITH VERY ADVANCED AGE
AND POLYOMA VIRUSES ARE SMALL
SINGLE STRANDED DNA VIRUSS THAT
ARE SIMPLE YOU CAN DIVIDE GENOME
INTO THREE PIECES EARLY REGION
VIRAL T ANTIGENS AND YOU HAVE
SAME FUNCTIONS OF THESE THAT HPV
HAS AND THEY CONTAIN VIRAL
HELICASE THAT HAS THIS AND
CAPSID PROTEINS AND NONCODING
CONTROL REGION BIDIRECTIONAL
PROMOTER THAT CONTROLS BOTH OF
THESE AND NOW LOOKING AT THIS
TRANSPLANT RECIPIENT POPULATION
LOOKING SPECIFICALLY AT
CONFIRMATION OF THE GENOME WE
SEE PRETTY HETEROGENOUS BOTH RNA
EXPRESSION AND WHAT IS
REPRESENTATIVE OF THIS WITHIN
SOME OF THE TUMORS AND WHAT WE
SEE IN LIKE THIS TUMOR IS WE SEE
VERY, VERY HIGH AND CONSISTENT
COVERAGE OF THIS VIRUS GENOME
ACROSS THE BOARD AND SEE HIGH
EXPRESSION OF BOTH OF THE LATE
AS WELL AS EARLY REGIONS AND
WHAT WAS UNUSUAL WITHIN TUMOR IS
1% OF TUMORS EXPRESSED T ANTIGEN
WE COULD SEE AND ADDITIONAL
AMOUNTS SUGGEST THIS IS A
PRIMARY TUMOR INFECTED WITH BK
THAT DOESN'T CONTAIN INTEGRATED
FORM THAT WE WOULD EXPECT WITH
HPV ASSOCIATED CANCERS AND OTHER
HAND HAVE THIS TUMOR THERE IS
DRASTIC DIFFERENCES IN COVERAGE
PLOTS IN THIS GENOME AND LARGE
DELETION IN MIDDLE OF THIS.
THIS IS CONSISTENT WITH WHAT YOU
WOULD SEE WITH HPV INTEGRATED
INTO CERVICAL CANCER AND THERE
IS A BREAK POINT WITH
DUPLICATIONS AND THIS LEADS TO
VERY HIGH EXPRESSION OF T
ANTIGEN REGION INACTIVATES
HELICASE AS WELL WRU HAVE A
JUFRPGS HERE AND IN THIS TUMOR
AND OTHER ONE WE SEE 100% OF
TUMORS EXPRESSING VIRAL LARGE
ANTIGEN SUGGESTING THAT THIS
VIRUS IS VERY IMPORTANT FOR
DEVELOPMENT OF THIS TUMOR
FURTHER SUPPORTING IT THIS WE
HAVE A PRIMARY AND METASTATIC
TUMOR PAIR WE SEE A SIMILAR
THING AND MAINTENANCE OF THIS
VIRUS DNA AS WELL AS RNA
EXPRESSION THAT IS IDENTICAL
BETWEEN THESE WITH
REARRANGEMENTS AND DELETIONS
THAT IS HARD TO SEE ON THIS.
THESE HAVE VERY DIM BROWN STAIN
FOR LARGE T ANTIGEN AND THESE
INCREASE A LITTLE IN INTENSITY
FOR THIS FURTHER SUGGESTING
COURSE OF METASTASIS AND THESE
ANTIGENS MIGHT BE IMPORTANT FOR
SURVIVAL OF THE TUMOR AND WE
WERE ABLE TO GO THROUGH AND
CHARACTERIZE NUMEROUS
INTEGRATION SITES SOME TUMORS
HAD MANY INTEGRATION SITES AND
OTHERS HAD SINGLE INTEGRATION
SITE WE COULD SEE WITHIN THEM
AND THESE INTEGRATIONS ALSO TEND
TO OVERLAP WITH AMPLIFICATIONS
OF HOST GENOME INDICATING YOU
GET BREAKS AND REARRANGEMENTS IN
VIRAL GENOME AND BREAKS AND
REARRANGEMENTS IN AMPLIFICATIONS
OF HOST DNA CLEARLY SHOWN HERE
EXAMPLE OF CHROMOSOME 1 AND
WHERE THIS POLYOMA VIRUS IS
INTEGRATED AND 11 COPIES OF THIS
SECTION OF HUMAN GENOME AND 8
COPIES HERE AND BLUE LINE IS
DIPLOID COPY OF HUMAN ADJACENT
DNA AND SHOWN FOR THIS TUMOR
THIS IS SIMILARLY SIZE THE
REGION AND SINGLE COPY GAIN AND
LARGE REGION WITH TWO-COPY GAIN
WITH 200 KILOBASES AND DON'T
KNOW FUNCTION WITHIN THESE
TUMORS DON'T SEE DISTINCTION IN
THIS -- THESE CAN CREATE
SUPERENHANCERS CHANGING -- THAT
CAN EFFECT TUMOR BIOLOGY AND
FUTURE WORK BY MY GROUP IS
UNDERSTANDING HOW BK CAN CAUSE
CANCER THROUGH SOME OF THE OTHER
MECHANISMS.
AND MY LAST LITTLE POINT THAT IS
KIND OF SUPPORTING FACT THAT
THIS VIRUS IS PLAYING A VERY
LARGE ROLE IN TUMORS OF THESE
TRANSPLANT RECIPIENTS IS SEEING
FINGERPRINTS OF LARGE T ANTIGEN
ACTIVITY BRIEFLY TALKED ABOUT
THAT IS IMPORTANT FUNCTIONS IN 6
AND 7 AND IT BINDS AND
INACTIVATES P53 AND RB AND SEE
STRONG WITHIN BK POSITIVE TUMORS
HERE IN RED WE SEE DRASTIC
EFFECTS ON CELL CYCLE GENES THAT
ARE RELATED TO TRANSCRIPTION
FACTORS CONTROLLED BY RB-LIKE
FAMILY MEMBERS AND GOT TALK
ABOUT APO BECK 3 THAT IN WORK IS
SHOWN TO BE SUPPRESSED BY THE
DREAM COMPLEX THAT IS VERY
HIGHLY EXPRESSED AND WANT TO
HIGHLIGHT THAT IS THE DOMINANT
MUTATION SIGNATURE WITHIN
BLADDER CANCER AND SUGGESTING
THAT, THAT IS A POTENTIAL
MECHANISM FOR HOW THE VIRUS IS
CONTRIBUTING TO CARCINOGEN ESIS
AND CONCLUDING THIS TALK AND SO
REALLY THE MAIN TAKEAWAY IS DNA
VIRUSES ARE RELATIVELY COMMON
BLADDER CANCERS WITH TRANSPLANT
RECIPIENTS RARELY IN PEOPLE WITH
HIV AND PROVIDING EXPLANATION
WHY THERE IS SIFRNS IN OVERALL
RISK WITHIN POPULATIONS FOR
DEVELOPING BLADDER CANCER AND
SUPPORTING THIS IDEA THAT
VIRUSES CAN CAUSE BLADDER CANCER
WITHIN OUR COHORTS WE ARE SEEING
MAINTENANCE WHAT INTEGRATE VIRAL
GM IN METASTATIC LEGIONS AND
MECHANISMS SIMILAR TO WHAT WE
SEE FOR HPV AND VIRUS MEDIATED
CANCERS AND ALSO WANT TO SAY WE
SAW WITHIN ONE HPV -- PRIMARY
AND METASTATIC TUMOR AND NOW I
WANT TO PAUSE FOR QUESTIONS TO
EVERYONE IS SOMETHING THEN
INTRINSIC ALLEY DIFFERENCE IN
IMMUNE SUPPRESSION BETWEEN TWO
POPULATIONS EXPLAINING THIS
PHENOMENON? WE KNOW THERE ARE
DIFFERENCES AND IS IT ENOUGH TO
EXPLAIN WHY PEOPLE LIVING WITH
HIV WE DON'T SEE SAME TYPES OF
VIRUSES WE SEE IN THIS OTHER
POPULATION OR SOMETHING
DIFFERENT WITH THE WAY THAT WE
SURVEIL THESE PATIENTS I SHOW
DIFFERENCE IN ADVANCE BETWEEN
TUMORS IN THESE POPULATIONS AND
ACTIVELY SCREENED FOR IN
INDIVIDUALS THAT ARE SOLVENT
TRANCE SIPPENT -- IS THERE A
GREATER ROLE OF TUMOR VIRUS AS
CROSS THE BOARD WITH BLADDER
CANCER I ARGUE PROBABLY
HOPEFULLY EVERYTHING TURNS OUT
WITH RESEARCH.
ACKNOWLEDGING THOSE IN MY GROUP
AS WELL AS CLOSE COLLABORATORS
ESPECIALLY ERIC WHO IS
EPIDEMIOLOGISTS I WOULDN'T HAVE
GOTTEN ANY SAMPLES WITHOUT HIM
AND VERY USEFUL RESOURCES FROM
BIOWOLF OUR SEQUENCING
FISTILITIES AND COLLABORATORS AT
HOSPITALS THROUGHOUT THE US THAT
HELPS US GET THESE SAMPLES AND
WITH THAT I WILL TAKE QUESTIONS.
>>[APPLAUSE].
>>[INDISCERNIBLE].
>>THAT WOULD BE INTERESTING TO
LOOK AT IN GENERAL POPULATIONS
IN ABSENCE OF CANCER AND WITHIN
POPULATIONS WE DIDN'T SEE A
GREATER ENRICHMENT OF MALE
TUMORS HAVING MORE OF A
PARTICULAR VIRUS THAN WHAT WE
SAW IN FEMALE TUMORS THAT WAS
SURPRISINGLY EVEN.
YEAH.
>>[INDISCERNIBLE]. VIRENIA IS
NOT IN HEALTHY PEOPLE. VIRURIA
IS ABOUT 10-ISH PERCENT OF THE
POPULATION AT ANY GIVEN MOMENT.
>>FAIRLY INTERESTING TRANSPLANT
RECIPIENTS HAVE [INDISCERNIBLE].
>>I SKIMMED OVER THAT A LITTLE
BIT AND LONG TIME THIS IS
EMERGING HYPOTHESIS IN KIDNEY
TRANCE PLANTS AND THESE VIRUSES
REMAIN PERSISTENT AND SEEMS IT
IS QUITE POSSIBLE A NEW VIRAL
GENOTYPE INTRODUCED FROM THE
GRAFT IS WHAT CAN INCREASE RISK
FOR THAT PERSON DEVELOPING
BLADDER CANCER AND HOLDS UP WITH
THAT HYPOTHESIS SHOWS TRUE FORM
OF NEVER OPATHY MAKES SENSE. IT
CAN REPLICATE AND LOSE THE
KIDNEY AND DOESN'T HOLD UP FOR
BLADDER CANCER. ALSO WHAT WAS A
BIG ADVANTAGE OF THE STUDY IS IT
WASN'T JUST KIDNEY TRANSPLANT
RECIPIENTS PREVIOUS CASE STUDIES
FOCUSED ON WE HAVE HEART LUNG
AND HEART LIVER AND COUPLE ARE
FROM HEART LUNG ONE IS A LIVER
TRANSPLANT RECIPIENT AND THEY
DON'T FULLY EXPLAIN IDEA OF
INTRODUCTION OF NEW GENOTYPE
FROM THE GRAFT ORGAN AS FAR AS
WE UNDERSTAND THIS VIRUS IT
DOESN'T JUST RESIDE WITHIN THE
ORGANS.
>>THANK YOU.
>>[APPLAUSE].
>>SHORT BREAK. BE BACK AT
4:10.
IT IS MY PLEASURE TO INTRODUCE
DR. IRINI SERETI WHO IS THE NEXT
SPEAKER AND SENIOR INVESTIGATOR
AND CHIEF OF -- AT NIAD.
SO, SHE GOT HER MD IN ATHENS
GREECE AT UNIVERSITY OF NATIONAL
CAPODISTRIAN UNIVERSITY AND
COMPLETED MHS AT DUKE UNIVERSITY
AND CAME TO THE NIAD IN
INFECTIOUS DISEASES FELLOWSHIP
AND STAYED ON AS A CLINICAL
INVESTIGATOR RECEIVING HER
TENURE IN 2015 AND PUBLISHED
MORE THAN 200 PAPERS AND
RECENTLY WAS NAMED AN EXECUTIVE
LEADERSHIP IN ACADEMIC MEDICINE
FELLOW THIS YEAR.
SHE IS AN UPCOME AGO ASSOCIATE
EDITOR IN INFECTIOUS DISEASES
AND EXECUTIVE MEMBER OF NIH
OXFORD CAMBRIDGE SCHOLARS
PROGRAM AND ASSOCIATED
INFLAMMATION AND IN BOTH CLINIC
AND LABORATORY.
WELCOME. WE LOOK FORWARD TO
YOUR TALK.
>>I WILL GET MY FACE OUT OF IT.
>>THAT IS A GREAT PICTURE.
>>I HAVE THE NAME OF THE PERSON
WHO TOOK IT. IT WAS FROM HERE
FROM NIH.
GREAT. THANKS FOR THE KIND
INTRODUCTION AND SO MUCH FOR THE
INVITATION FOR SUCH A WONDERFUL
MEETING. I GOT IMPOSTER
SYNDROME AND FELT I SHOULD BE
ACROSS THE HALL IN THE
IMMUNOLOGY MEETING. ANYWAY,
FORGIVE ME FOR BEING
UNAPOLOGETICALLY CLINICAL IN
IMMUNOLOGY THAN VIROLOGY I WILL
DISCUSS INFLAMMATORY SYNDROMES
WE SEE IN PEOPLE WITH ADVANCED
HIV STARTING ANTIRETROVIRAL
THERAPY AND WAYS FOR
INTRODUCTION REMINDING WHERE WE
ARE IN HIV EPIDEMIC.
HALF OF THEM ARE WOMEN AND GIRLS
AND IN TREATMENT CASCADE, WE ARE
TRYING TO REACH -- AND ARE
CLOSER TO 85, 73, 66 AT THE
MOMENT AND IMPORTANTLY
POPULATION WITH HIV IS AGING AND
ESTIMATED THAT ABOUT 75% OR 73%
WILL BE OVER 50 YEARS OF AGE BY
2030.
UNFORTUNATELY, WE KNOW THAT THE
BEST OUTCOMES IN HIV ARE WHEN
PEOPLE GET STARTED ON THERAPY AT
HIGH -- CURRENT TREATMENT
GUIDELINES INCLUDING WHO ARE TO
START THERAPY AT CD4 COUNTS OF
500 AND UPON HIV DIAGNOSIS
BASICALLY. WE ARE DEALING WITH
A PERSISTENT PROBLEM OF LATE
PRESENTATION TO CARE AND PEOPLE
PRESENTING WITH ADVANCED DISEASE
THAT ARE DATA FROM EUROPE
ACTUALLY SHOWING 50% OF PATIENTS
ACCOUNT FOR LESS THAN 350 AND
START THERAPY AND IF YOU THINK
IT IS BETTER, YOU ARE WRONG AND
MEDIAN CD4 CURRENTLY ARE FAIRLY
RECENT DATA FROM FEW YEARS AGO
PEOPLE THAT ARE 50 YEARS OR
OLDER AND CLOSE TO 400 FOR
PEOPLE YOUNGER THAN 50 YEARS OF
AGE.
SO, THAT OBVIOUSLY MATTERS AND
THIS IS ALSO DATA FROM THE CDC
AND FROM THE US THAT IS SHOWING
A VERY -- AGAIN, PERSISTENT
PROPORTION THAT IS CLOSE TO 20%
OF PEOPLE PRESENTING WITH STAGE
3 OR AIDS CLASSIFICATION.
AND THIS EFFECTS MORE MINORITIES
THAT ARE COMPARED TO WHITE
PATIENTS NOT HISPANIC PATIENTS
AND ARE PEOPLE THAT WE STUDY IN
THE CLINIC AND THAT HAVE AN
INTEREST IN ADVANCED HIV DISEASE
AND I COLLABORATE CLOSELY WITH
BOB'S GROUP AND RAMASWANI'S
GROUP AND PATIENTS WITH RISK FOR
ACUTE INFLAMMATORY RESPONSES
STARTING INTRAVIRAL THERAPY AND
CALL THIS IRIS AND IMMUNE
RECONSTITUTION INFLAMMATORY
SYNDROME THAT ARE PARADOXICAL
REACTIONS HAPPENING IN PEOPLE
WITH HIV WHO START ANTIVIRAL
THERAPY AND HAVE AN UNDERLYING
INFECTION OR TUMOR AND CALL THEM
PARADOXICAL AND EXPECTATION IS
WHEN PEOPLE START UNDER THIS
THERAPY EVERYTHING GETS BETTER
AND IN THIS CASE, THEIR
INFECTION OR MANIFESTATIONS OF
INFECTION OR TUMORS MAY GET A
LOT WORSE AFTER STARTING
ANTIVIRAL THERAPY AND INCIDENTS
CAN BE APPROXIMATELY 20% WITH
LOW CD4 COUNTS STARTING THERAPY
AND THEY HAPPEN EARLY WITHIN
FIRST 6 MONTHS OF ART AND
SUCCESSFUL HIV VIER LOGIC
SUPPRESSION AND UNDERLYING
INFECTION SEEMS TO BE UNDER
CONTROL IF HAVE TUBERCULOSIS
CULTURE IS MORE NEGATIVE AND
THREE MAJOR CLINICAL PREDICTORS
IDENTIFIED ARE HAVING LOW CD4
COUNTS AND HAVING PREEXIST
OPPORTUNISTIC INFECTION IF IT IS
A CLINICAL SUCH AS TUBERCULOSIS
FOR EXAMPLE AND PRETREATING
INFECTION FOR SHORTER PERIOD OF
TIME PRIOR TO STARTING ANTIVIRAL
THERAPY OR IMMUNOLOGICAL TERMS
HIGHER ANTIGEN LOAD AND WHAT IT
LOOKS LIKE IN CLINIC AND
PATIENTS WE HAVE SEEN AT NIH AND
WOMAN WITH RECURRENT CERVICAL
[INDISCERNIBLE] AND WOULD
CONTINUE TO ACCUMULATE WITH
NEGATIVE CULTURES FOR MTB AND
THIS PERSON FROM MEXICO HAD
HISTOPLASMA IRIS AND AFRICAN
AMERICAN PATIENT HERE AND
ANOTHER PATIENT WHO HAD MAC --
AND THIS WAS A PATIENT WITH JC
VIRUS PROGRESSIVE
[INDISCERNIBLE] WHO DEVELOPED AN
IRIS AFTER STARTING ANTI VIRAL
THERAPY. I WILL SUMMARIZE
PREVIOUS OVENGSS AND WE DID 500
PATIENT STUDY IN US, KENYA AND
THAILAND AND IRIS WAS
INDEPENDENT PREDICTOR OF
MORTALITY. MOST WAS ABROAD. 3%
IN US AND CLOSER TO KENYA AND
THAILAND AND IN ALL THREE
COUNTRIES ACTUAL INCIDENTS OF
VIRUS WAS IDENTICAL. 23% FROM
ALL COUNTRIES AND LOW HEMOGLOBIN
WAS [INDISCERNIBLE] WHICH WAS
EASILY DONE CLINICAL LABORATORY
TESTING AND DEATHS LOW BMI SO
LOW BODY MASS INDEX AND HIGH
REACTIVE PROTEIN AND D DIMER
WERE CLOSELY ASSOCIATED WITH
DEATH AND FOUND FROM LABORATORY
ASPECT WE AND OTHERS HAVE SHOWN
EXPANSION OF ACTIVATED CD4
POSITIVE ANTIJ ENSPECIFIC CELLS
RECOGNIZING UNDERLYING INFECTION
AND UNDERLYING TB OR MAC THAT
SEEM TO BE KEY COMPONENT OF
PATHOGEN ESIS AND EFFECTIVE
PHENOTYPE AND CYTOTOXIC
PHENOTYPE MAKING LARGE AMOUNTS
OF GAMA AND TNF AND WE DO IT BY
SIMULATION THAT I WILL SHOW YOU
AN EXAMPLE OF WE HAVE ALSO FOUND
IN PREVIOUS WORK THERE IS A
HYPERMETABOLIC STATE IN PATIENTS
AND EXPRESSION ON CD4 CELLS AND
MONOCYTES AND UPTAKE THAT COULD
BE VISUALIZED IN CLINIC WITH FTG
PET SCANNING AND FINALLY I WILL
DELVE FURTHER INTO THIS AND
THERE IS SIGNIFICANT MYELOID
CELL ACTIVATION PRONOUNCED IN
IRIS AND BOTH LOOKING AT PLASMA
BIOMARKERS AND INTERLEUKIN 18
AND INTERFERON AND GAMA AND
FOUND HIGH PROPORTION OF
INFLAMMATORY MONOCYTES.
I WILL SHOW YOU SOME DATA THAT
IS ON WHAT WE ARE FINDING NOW
THAT IS ROLE OF NRP3
INFLAMMATORY ACTIVATION AS WELL
AS CLASSICAL COMPLEMENT
ACTIVATION IN PEOPLE WHO DEVELOP
MICROBACTERIALIZE SPECIFICALLY
AND IS AN EXAMPLE WHAT WE FIND
WHEN WE DO T CELL STIMULATIONS
IN LAB IN VITRO AND PERSON THAT
PRESENTED WITH THIS AND THIS IS
LYMPHOID OPATHY FOR THOSE NOT
USE TOTD SEEING CAT SCANS AND
TUBERCULOSIS WAS IN LYMPH NODES
HERE AND AFTER STARTING
ANTIVIRAL THERAPY PATIENT
DEVELOPED THIS. THIS IS BIGGER
AND DESPITE FACT THAT CULTURES
WERE NOW NEGATIVE AND
STIMULATING THIS FOR T CELLS IN
VITRO USING TB ANTIGENS YOU CAN
SEE IS ACTUALLY SIGNIFICANT TNF
IN PRODUCTION OF BASELINE THAT
IS THE NORM OF THESE PERCENTAGE.
DURING THE IRIS EVENT THIS IS
TRULY SKUBERRANT AND HIGHER WHAT
WE SEE WITH POSITIVE CONTROL AND
WE USE IT A LOT OBVIOUSLY AND
OTHER GROUPS HAVE DESCRIBED THIS
SKUBERRANT DISREGULATED T CELL
ACTIVATION DURING IRIS AND WE
FOUND NOT VERY HELPFUL IS THAT
EVEN IF OBVIOUSLY WE USE IMMUNE
SUPPRESSION WHEN THINGS
OBVIOUSLY GO AND WHEN
SYMPTOMATOLOGY IS TOO INTENSE
AND TREAT PATIENTS CLOSELY AND
IF YOU PUT THEM ON COURTI CO
STEROIDS FOR EXAMPLE EX-VIVO
THEY WOULD HAVE HIGH T CELL
STIMULATION AND STIMULATING THEM
WITH ANTIGENS AND WE WANTED TO
LOOK AS I MENTIONED AT MYELOID
CELLS AND HAD A VERY CLEAR
SIGNAL BY HIGH LEVELS OF IL1 AND
18 THAT WAS INFLAMMATORY
ACTIVATION IN PATIENTS AND QUICK
REMINDER FOR INFLAMMATORY
ACTIVATION THERE ARE SIGNALS
FROM PATHOGENS AND DAMAGE
ASSOCIATED MOLECULAR PATTERNS
AND ONE OF THE RECEPTORS IS THE
NLRP3 THAT IS CLASSIC
[INDISCERNIBLE] THAT ACTIVATES
FURTHER COMBINING APOPTOSIS
ASSOCIATED SPECK-LIKE PROTEIN
CONTAINING CARD THAT THEY CREATE
ALC COMPLEX FURTHER ACTIVATING
CAL SPACE 1 THAT CONVERTS PRO-IO
TEAM TO ACTIVE CYTOKINES AND
IMPORTANTLY IO1 BETA CAN LEAD TO
CLINICAL MANIFESTATIONS SUCH AS
FEVER AND PRODUCTION OF IL-6 AND
CLP AND IT IS CRITICAL FOR
ACTIVATION THAT WE KNOW HAPPENS
IN IRIS AND GAM INTERFERON
ACTIVATION AND SO THE SPECKS AND
AFC SPECKS ARE CONSIDERED REALLY
A HALLMARK OF INFLAMMATORY
ACTIVATION IF YOU VISUALIZE
THEM. YOU CAN USE OBVIOUSLY
PHOTON MICROSCOPY AND WE USE THE
IMAGE STREAM ANALYSIS A LOT THAT
COMBINES VISUALIZATION
ADVANTAGES AND PHOTON MICROSCOPY
BACK WITH ADVANTAGE OF FLOW
CITOMETRY AND CAN YOU MEASURE
HOW MANY CELLS HAVE AFC SPECKS
OPPOSED TO JUST FINDING THEM
QUALITATIVELY AND WHAT WE FOUND
WAS THAT BY LOOKING AT THE CELLS
THAT HAVE THESE SPECKS AND THESE
SPECKS I NEED TO MENTION THAT
FLICA IS A SUBSTRATE FOR CAS
SPACE 1 AND THIS MEANS THEY HAVE
ACTIVATED CAS SPACE 1 AND WE
HAVE EVIDENCE OF HIGH LEVELS OF
THE MONOCYTES THAT ARE BOTH ASC
SPECK POSITIVE AND YOU SEE THEY
COLOCALIZE IN CITES HERE AND
MONOCYTES AND THAT INTERESTINGLY
ENOUGH WAS A BIG PLUS FOR US AND
WHEN WE TREATED PATIENTS WITH
COURTI CO STEROIDS THAT
ALLEVIATED PRESENCE OF THESE
COMPLEXES.
WE WERE ABLE TO SEE THERE WAS
FOR EXAMPLE RESPONSE TO THERAPY
WHICH IS IMPORTANT FOR OUR
PURPOSES AND WE FOUND THAT ALSO
BY USING THIS RP3 INHIBITOR THAT
IS HERE THE MCC950, WE WERE ABLE
TO ABROW GATE RELEASE OF
INTERLEUKIN 1. ALSO, WE COULD
BLOCK THAT BY BLOCKING POTASSIUM
INFLUX THE IMPORTANT SECOND
SIGNAL FOR INFLAMMATORY
ACTIVATION AND WE SHOWED IT WAS
AN RP3 INFLAMMATORY THAT IS
INVOLVED IN PATIENTS WITH MICRO
BACTERIAL VIRUS AND CELLS WITH
FLICA EXPRESSIONS AND ACTIVATED
KAS 1 HAS POSITIONS AND EVIDENCE
OF COMPLEMENT ACTIVATION IN
PATIENTS ALONG WITH CAS SPACE 1
AND INFLAMMATORY ACTIVATION.
SO, WHAT I -- WHAT WE -- OUR
MODEL SO FAR WE HAVE ACTIVATED
MYELOID CELLS THAT PRODUCE
CERTAIN BIOMARKERS AND
INFLAMMATORY CYTOKINES AND
EXPANSION OF ANTIGEN SPECIFIC
CELLS THAT PRODUCE MASSIVE
AMOUNTS OF INTERFERON AND GAMMA
AND WERE STILL SEEING IN CLINIC
SUBSET OF PATIENTS THAT HAVE
MORE SEVERE DISEASE
MANIFESTATIONS AND WERE ONES
THAT HAD MORE PERSISTENT SIM
TOMORROWTOLOGY THAT REQUIRED
PROTECTIVE COURSES OF COURTI CO
STEROIDS REMINDING US OF
PATIENTS WITH SYNDROME OF HLH
THAT IS A HEMOPHAGOCYTIC HISTORY
OFCYTOSIS THAT IS PRIMARY IN
PEOPLE WHO HAVE MUTATIONS IN
CYTOTOXIC GENES AND SECONDARY TO
INFECTIONS AND TO AUTO IMMUNE
DISEASES AND YOU HEAR THE TERM
MICROPHAGE ACTIVATION SYNDROME
AND COHORT OF 80 PATIENTS WITH
MICRO BACTERIAL DISEASE AND WE
WENT BACK AND APPLIED CRITERIA
FOR HLH AND THOSE PRESENTING
EVENTUALLY FOLLOWING THEM
LONGITUDINALLY WITH IRIS
FEATURES AND WERE ABLE TO
CLASSIFY THOSE WHO PRESENTED
WITH SAY REGULAR IRIS OR SIMPLE
IRIS AND MEETING HLH CRITERIA
AND ONE IS HIGH FERA TEEN AND
OTHER CRITERIA WITH CRYTOPINA
AND [INDISCERNIBLE] AND WANTED
TO SEE IF THAT RETROSPECTIVELY
APPLIED CLASSIFICATION THAT
WOULD HELP US TO IDENTIFY MORE
UNIQUE PATHOPHYSIOLOGY OR WORSE
OUTCOMES OF PATIENTS THAT SO
HAPPENED THEY WERE EVENLY SPLIT
BETWEEN THREE GROUPS AND NO IRIS
SIMILARLY IN THESE ACCOUNTS AND
STRIKINGLY DIFFERENT IN PATIENTS
THOSE FOR HLH THAT REQUIRED MUCH
MORE FREQUENTLY WERE REQUIRING
COURTI CO STEROID TREATMENT AND
DON'T USE TREATMENT FOLLOW THEM
AND USE N SETS FOR EXAMPLE AND
THEY DEFINITELY NEEDED MORE
CORTICO STEROIDS AND NEEDS
ADDITIONAL IMMUNOSUPPRESSION AT
TIMES AND NEEDED MORE PROTECTED
AND MORE SEVERE COURSE THAT
LASTED LONGER AND IN QUEST TO
TRY TO FIND BIOMARKERS TO
PREDICT OR IDENTIFY PATIENTS AT
BASELINE BEFORE STARTING
ANTIVIRAL THERAPY WE WANTED TO
LOOK AT MARKERS EASILY DONE IN
CLINICAL LABORATORIES LOTS OF
PATIENTS ARE IN RESOURCE LIMITED
SETTINGS IN LOW AND MIDDLE
INCOME COUNTRIES AND FINDING
HEMOGLOBIN AND FER ACONTINUE WE
HAD A DECISION TREE ANALYSIS TO
IDENTIFY MOST PATIENTS THAT
WOULD GO ON TO DEVELOP HLH TYPE
OF IRIS.
NOW, IMPORTANTLY ALSO WHEN THEY
PRESENTED WITH IRIS WE FOUND HLH
IRIS HAD A VERY CLEAN
DISTINCTION BASED ON THIS WHICH
IS GAMMA INTERFERON BIOMARKER
AND THIS HAPPENS TO BE OF COURSE
ONE OF THE CRITERIA OF HLH THAT
ARE USED AND ALSO SIGNIFYING
REALLY MOSTLY T CELL
PROLIFERATION AND ACTIVATION.
ALTHOUGH THIS WAS STATISTICALLY
SIGNIFICANT AND CLEARLY IT WAS A
VERY CLEAN DISTINCTION OF HLH
GROUP AND IMPORTANTLY WE HAD
ALWAYS PUZZLED AND WERE ALWAYS
VEXED NOT FINDING ANYTHING WITH
REGULATORY T CELLS IN IRIS AND
NOW THAT WE DISTINGUISH MORE
SEVERE TYPE WE FIND
PARADOXICALLY THEY HAD LOWEST
PROPORTION OF T. REX AND AREA WE
ARE MORE INTERESTED TO LOOKING
FURTHER INTO WHY THAT IS THE
CA
CASE.
LOOKING AT ANALYSIS BIOMARKERS
AND PHENOTYPES GOOD SDICHGS
PEOPLE DEVELOPING THIS AND
COMPARING IRIS AND NO IRIS AT
ALL AND EVERYBODY HAD
MICROBACTERIAL DISEASE WHEN
INCLUDED IN COHORT I DON'T HAVE
DATA HERE BUT LOOK AT GENETICS
OF HLH AND PATIENTS DEVELOPING
IRIS HAVE A HIGHER PROTEIN
VARIANCE IN GENES RELATED TO HLH
AND WE FOUND SIGNIFICANT
DIFFERENCE 20% OF PATIENTS
DEVELOPING IRIS HAD LOW HLH
VARIANCE AND MENTIONED WE USE
THE COURTI CO STEROIDS TO TREAT
THE PATIENTS AND TREATING
MICROBACTERIA PATIENTS WITH
CORTICO STEROIDS WE SAW
EMERGENCE OF [INDISCERNIBLE]
SARCOMA NOT SUSPECTED AT
BASELINE AND WE SCREENED ALL
PATIENTS COMING IN WITH LOW CD4
COUNTS WITH PCR TO MAKE SURE WE
DON'T GET THESE SURPRISES AND
INTERESTING ABOUT KS HERPES
VIRUS THAT I WILL MENTION IS
REALLY PRELIMINARY WORK WE ARE
DOING WITH THIS GROUP AND
INTERESTING THING ABOUT KS IS IT
CAN CAUSE INFLAMMATORY RESPONSES
AND SYNDROMES IN PEOPLE WITH HIV
THAT COULD BE SOMETIMES
DIFFICULT TO DISTINGUISH FROM
WHAT WE SEE IN OTHER INFECTIONS
THAT ARE A VIER YUGS AND YOU HAD
A FEW TALKS TALKING ABOUT KSHV.
THANKFULLY I DON'T HAVE TO GO
THROUGH SPECIFICS.
IF YOU HAVE QUESTIONS ABOUT
VIRUS YOU CAN ASK BOB. WHAT IS
FASCINATING ABOUT KSHV MASSIVE
AMOUNT OF OPEN READING FRAMES.
I THINK OVER 80. WHAT IS
FASCINATING IS HAS AT LEAST 14
CELLULAR HOMOLOGS AND VIRUS
ITSELF PRODUCES IL-6 AND
CHEMOKINDS AND PRODUCES BCL2.
INTERESTINGLY IT CAN ACTIVATE
CAPPA B AND CELLS FURTHER
PRODUCE INFLAMMATORY CYTOKINES
AND SPECTRUM OF DISEASES ARE
BROAD AND RANGE EVERYWHERE FROM
SKIN DISEASE IN OUR PATIENTS TO
MULTICENTRIC CASTLEMAN'S DISEASE
THAT ALREADY HAS BEEN MENTIONED
AND KICKS THAT IS INFLAMMATORY
SYNDROME WITH KS AND PRIMARY
INFUSION LYMPHOMA IS ONLY AND
BOB CAN CORRECT ME TRUE CANCER.
MOST OF THESE CAN BE -- MOST ARE
POLYCLONAL AND ANGIOPER
LIVERATIVE OPPOSED TO MONOCLONAL
DISEASE AND WE WERE INTERESTED.
CLEARLY PATIENTS CAN SOMETIMES
NOT SOMETIMES BUT MANY TIMES
COME FROM AFRICA AND THEY DO
HAVE -- WE SEE LOTS OF KS AND WE
DON'T SEE LOTS OF BRIGHT PEOPLE
BUT LOTS OF KS BECAUSE OF
AFRICAN ORIGIN PATIENTS AND
DECIDED TO DO SOME STUDIES THAT
WR DONE MICRO BACTERIALIZE NTD
PATIENTS WITH KS AND MALIGNANCY
BRANCH WITH OPPORTUNITY TO STUDY
PATIENTS ASSOCIATED DISORDERS
AND HIV PATIENTS CONTROLLED IN
ANTIVIRAL THERAPY AND DIDN'T
HAVE THESE INFLAMMATORY
SYNDROMES YOU CAN SEE ARE FAIRLY
SIMILAR WITH RESPECT TO OVERALL
CD4 COUNTS AND VIRAL LOAD.
SO, WE DECIDED TO DO EXACTLY THE
SAME ANALYSIS AND LOOK AT
EVIDENCE IF THERE IS ANY
EVIDENCE OF INFLAMMATORY
ACTIVATION IN PATIENTS WITH
CAPPA ASSOCIATED DISORDERS, ONE.
2, IF THERE IS ANYTHING THAT CAN
DIFFERENTIATE THE DIFFERENT
GROUPS AND CLEARLY WHAT
ONCOLOGISTS ARE STRUGGLING WITH
IS WHO HAS PRIMARY INFUSION
LYMPHOMA REALLY WHERE THE
DIFFERENCE OF TREATMENT AND
PROGNOSIS COMES IN DIFFERENCE TO
A GREAT EXTENT AND FOUND HIGH
LEVELS OF THIS AND OF EXPRESSION
ON MONOCYTES BOTH SUGGESTING
THERE IS INFLAMOSOME ACTIVATION
IN PATIENTS WITH THIS KHV
ASSOCIATED DISORDERS AND WE
APPLIED SAME TECHNIQUES WITH
IMAGE STREAM ANALYSIS AND FOUND
REALLY MASSIVE AMOUNTS OF
MONOCYTES THAT ARE EXPRESSING
SPECKS AS WELL AS
[INDISCERNIBLE] BEING
[INDISCERNIBLE] POSITIVE
COMPARED TO HEALTHY CONTROLS AND
YOU KNOW WE THOUGHT WELL THESE
PATIENTS HAVE ALSO HIV.
ALTHOUGH THEY ARE TREATED THEY
MIGHT HAVE INFLAMOSOME
ACTIVATION AND WENT TO COMPARE
WITH HIV PATIENTS AND THEY WERE
HIGHER AND MICRO BACTERIA
PATIENTS WITH IRIS WERE LOWER
THAN THOSE WITH KS DISEASE AND
THERE IS CLEARLY MASSIVE
INFLAMOSOME ACTIVATION IN THESE
PATIENTS AND THIS IS AGAIN
SHOWING COMPARISON WITH
SUPPRESSED HIV NOT ONES WITH
MICRO BACTERIAL IRIS I SHOWED
YOU BEFORE AND THERE IS
SIGNIFICANT DIFFERENCE AND
TREATED HIV DIDN'T SEEM
DIFFERENT FROM HEALTHY CONTROLS
AND WE FOUND THERE WAS
SIGNIFICANT ASSOCIATION IN
INFLAMOSOME ACTIVATION
PREDOMINANTLY WITH KS HV VIRAL
LOAD AND NOT HV VIRAL LOAD.
NOW, UNFORTUNATE THING IS WE
COULD NOT REALLY DISTINGUISH
BETWEEN DIFFERENT KS SYNDROMES
AT ALL. IT WAS REALLY A BLUR AS
YOU CAN SEE HERE. SO, WE PUT
ALL INFLAMMATORY MARKERS AND
MONOCYTE INFLAMOSOME MARKERS AND
COULD NOT REALLY DISTINGUISH WHO
HAD PRIMARY LYMPHOMA VERSUS SKIN
KS AND KEEP IN MIND THERE IS
LOTS OF OVERLAP AND THAT SOME
PATIENTS HAVE BOTH SKIN KS'S AND
MULTICENTRIC CASTLEMAN'S DISEASE
AND WE SEE THOUGH LOTS OF
INFLAMMATORY SIGNAL IS COMING
AND IN RED IS PATIENTS WITH
MULTICENTRIC CASTLEMAN'S DISEASE
AND THEY TEND TO CLUSTER BASED
ON DIFFERENT MARKERS COMING TO
INFLAMMATORY MARKERS THAT ARE
SHOWN HERE TOWARDS THE END WITH
CRP AND IL18 AND PHEROCONTINUE
IN I LICHLT 1.
NOW, WE WERE THOUGH VERY
RELIEVED WHEN WE SAW THAT WE
COULD SEE A SIGNAL OF
DIFFERENTIATION BETWEEN PATIENTS
WITH MICROBACTERIAL IRIS AND
PATIENTS WITH INFLAMMATION OR
INFLAMMATORY SYNDROMES RELATING
TO KS AND SEEMING THAT KS WAS
MORE IL18 RELATED AND MICRO
BACTERIAL IRIS WAS MORE GAMMA
INTERFERON ASSOCIATED THAT IS
BECAUSE PATIENTS DEVELOP IRIS
WITH A T CELL RESPONSE AND
DRIVES PATHOLOGY HERE AND
PATIENTS HAVE MORE OF IT PURE
INFLAMOSOME WITH IL-6 ON TOP OF
CYTOKINES AND CHEMOKINDS OF THE
VIRUS.
I SHOWED WE HAVE FOUND SYSTEMIC
INFLAMMATORY ACTIVATION IN
INDIVIDUALS WITH HIV WITH
MICROBACTERIAL IRIS AND KHV
ASSOCIATED CONDITIONS AND SEVERE
MICRO BACTERIAL VIRUS WITH
UNIMPOSED T CELL REGULATIONS.
THAT CASE HB IS POTENTIALLY
DRIVING INFLAMMATION DUE TO
STRONG ASSOCIATION WITH THIS
ACTIVATION WITH IL18 AND CURIOUS
DIGGING INTO MECHANISM OF IT AND
OPEN READING FRAME OF KS SEEMS
TO INHIBIT NLRP1 AND NLRP3 AND
BIT OF A CONUNDRUM THERE AND
WORTH DIGGING INTO THAT AND WE
SEE MULTICENTRIC CASTLEMAN'S
DISEASE DRIVING THIS PROFILE AND
CAN HAVE DISTINCT SIGNATURE TO
DIFFERENTIATE BETWEEN DIFFERENT
CASE AND KS SYNDROMES AND IN
CONTRAST TO THAT WE FOUND MACRO
BACTERIAL CASE AND INFLAMMATORY
SYNDROMES AFRICAN POPULATIONS.
TREATMENTS ARE VERY DIFFERENT.
I WOULD LIKE TO FINISH THANKING
MY TEAM AND SYLVIA IS AN EXPERT
IN THE GROUP AND JOE IS
INFECTIOUS FELLOW WITH LOTS OF
WORK AND ANALYSIS I SHOWED YOU
AND CLINICAL PEOPLE AND BOB'S
GROUPS THAT WERE INCREDIBLE
COLLABORATORS AT THE BEDSIDE AND
ALSO IN THE BENCH. THANK YOU.
>>VERY NICE.
WHAT HAPPENS IF YOU BLOCK CAS
SPACE 1 WITH INHIBITORS OR IL-1
BETA WITH INHIBITORS?
>>VERY GOOD QUESTION. THAT
HASN'T BEEN DONE YET IN THE
CLINIC. WE DO THINK THAT, THAT
IS ONE OF THE DIRECTIONS THAT WE
WILL BE LOOKING AT.
YEAH. THERAPIUTICALLY SPEAKING.
>>OKAY.
>>AND JAK INHIBITORS MENTIONED
IN A TALK BEFORE MIGHT BE
ANOTHER WAY TO GO.
>>THANK YOU.
>>HI. IS THERE DATA WITH
[INDISCERNIBLE] WITH IRIS AND
GLOBAL DEPLETION IN BLOOD.
>>SORRY?
>>OCCURRENCE OF IRIS PATIENTS
WITH GLUTEA THIGH OWN DEPLETION
IN BLOOD?
>>IF HAPPENS DURING IRIS YOU
MEAN.
>>OCCURRENCE OF IRIS IS WITH
DEPLETION.
>>GOOD QUESTION. WE HAVEN'T
DONE IN MICRO BACTERIALIZE AND
WE FOUND IT IN COVID AND DID
SIMILAR STUDIES LOOKING AT
INFLAMMATORY ACTION IN PATIENTS
AND FOUND SIGNIFICANT DEPLETION.
>>IT OCCURS IN VITRO INFECTION.
>>THAT IS GOOD.
>>WE HAVEN'T DONE FOR THESE
PATIENTS YET BUT COVID COHORT
AND REMARKABLE FOUND OXIDATIVE
STRESS AND [INDISCERNIBLE] SO WE
HAVEN'T REPEATED THAT WITH OUR
--
>>MOST PREDOMINANT ANTIOXIDANT.
>>GREAT SUGGESTION. THANKS
VERY MUCH.
>>YEAH.
>>THANK YOU.
>>>>OKAY. GREAT. THANK YOU.
>>SORRY. I JUST HAVE TO DO
THIS.
OKAY. SO, I JUST WANT TO END BY
THANKING EVERYONE WHO
CONTRIBUTED TO MAKING THIS DAY
SO EXCITING AND FUN DAY WITH A
LOT OF GREAT TALKS AND FIRST OF
ALL WANT TO THANK MY
CO-ORGANIZER CINDY WHO HAS BEEN
A PLEASURE TO WORK WITH HER IN
ORGANIZING THIS IN THE LAST FEW
MONTHS AND THANKING INVITED
SPEAKERS.
AND ALL OF THE ORAL AND POST
REPRESENTERS WHO SUBMITTED
ABSTRACTS SHARING THEIR STUDIES
I THINK WITHOUT THEM THIS
WOULDN'T BE SUCCESSFUL.
ALSO, I WANT TO THANK ALL OF THE
PEOPLE WHO ATTENDED IN PERSON
AND VIRTUALLY.
FINALLY, I WANT TO THANK THE
ADMINISTRATIVE STAFF WHO
SUPPORTED US DURING THE
ORGANIZATION, PARTICULARLY
JACKIE AND JULIA LAM AND TERRY
AND VALERIE TURN KWIFT. AND THE
ONLY THING THAT REMAINS THEN IS
ANNOUNCING TRAVEL AWARD WINNERS.
SO, DRUM ROLL, PLEASE.
OKAY. SO, THE FIRST FOR THE
ORAL PRESENTATIONS, THE WINNERS
ARE IN NO PARTICULAR ORDER
RAMONA MOLES AND TRINYIN LAU,
AND KRISTA A /* FRANKEN BERRY
AND FOR THE POSTER
PRESENTATIONS, THE WINNERS ARE
UTAH AND ALEX FLINE PETER AND
SARCAS SARCAS.
OKAY. CONGRATULATIONS,
EVERYONE.
I SHOULD NOTE THAT PARTICULARLY
SINCE KRISTA AND I ENDED UP
WINNING THAT I WASN'T INVOLVED
IN THE DECISION-MAKING PROCESS
AND WE HAD A NUMBER OF JUDGES
THAT RANKED ALL OF THE TALKS
AND.
>>THANK YOU TO THE JUDGES.
>>THANK YOU TO THE JUDGES FOR
ALL OF THE HARD WORK AND OKAY.
BYE, EVERYBODY. SEE YOU NEXT YEAR