>> WELCOME, EVERYONE, TO THE
COVID-19 SCIENTIFIC INTEREST
GROUP SEMINAR SERIES.
OUR SPEAKER TODAY IS MICHAEL
LETKO, ASSISTANT PROFESSOR PAUL
G ALLEN SCHOOL OF GLOBAL HEALTH,
WASHINGTON STATE UNIVERSITY.
HIS RESEARCH FOCUSES ON
UNDERSTANDING ECOLOGICAL VIROME
AND ASSESSING POTENTIAL FOR
VIRUS ZOONOSIS TO HUMANS.
PRIOR TO STARTING HIS OWN LAB,
MICHAEL WAS A POSTDOC AT THE
ROCKY MOUNTAIN LABS WHERE HE
STUDIED BETA CORONAVIRUSES WITH
POTENTIAL TO EMERGE IN HUMANS.
IN 2020 PUBLISHED A PAPER
DEMONSTRATING NEWLY DESCRIBED
SARS-COV-2 ACE-2 RECEPTOR, AND
MADE THAT DISCOVERY USING
FUNCTIONAL VIROMIC STRATEGY HE
WILL INTRODUCE TO US TODAY.
TODAY HE WILL TELL US ABOUT HIS
CONTAINMENT EFFORTS TO SURVEY
USE BY BETA CORONAVIRUSES TO
PREDICT THE NEXT WITH PANDEMIC
POTENTIAL.
FOR THE AUDIENCE MEMBERS WHO
WOULD LIKE TO ASK A QUESTION USE
THE "SEND LIVE FEEDBACK" BUTTON
THROUGH THE NIH VIDEOCAST
APPROVE THE DESCRIPTIVE
PARAGRAPH.
LEAVE YOUR COMMENTS AT ANY POINT
DURING THE TALK.
WE'LL HAVE A FEW MINUTES TO DO A
Q&A WITH MICHAEL AFTERWARDS.
SO WITH THAT, I WANT TO THANK
MICHAEL FOR JOINING US, WE LOOK
FORWARD TO YOUR TALK.
MICHAEL, YOU ARE --
>> THERE WE GO.
THANK YOU FOR HAVING ME.
JUST TO GET RIGHT INTO IT, THIS
WORK STARTED MANY YEARS AGO NOW,
FOR ME STARTED AROUND 2018, BUT
REALLY THE PROBLEM THAT WE'RE
DISCUSSING IS A LONG-RUNNING
PROGRAM IN VIRAL, SARS EMERGED
IN 2003, TRACED BACK INTO ANIMAL
MARKETS, SHORTLY THEREAFTER WAS
FOUND IN BATS OR VIRUSES CLOSELY
RELATED TO SARS WERE FOUND IN
BATS.
AND REALLY SINCE THEN, THERE'S
WILL BE THIS KIND OF EXPLOSION
OF VIRUS DISCOVERY RESEARCH THAT
HAS KIND OF COME ALONGSIDE THE
NEXT GENERATION SEQUENCING
TECHNOLOGY GROWING SINCE THE
FIRST TIME SARS SPILLED INTO THE
HUMAN POPULATION.
THIS HAS LED TO A MOUNTAIN OF
DATA BASICALLY ABOUT DIFFERENT
VIRUSES, CORONAVIRUSES FOUND IN
DIFFERENT ANIMAL SPECIES,
BASICALLY ALL CORNERS OF THE
WORLD.
SO, IT'S JUST THIS LARGE PILE OF
DATA.
AND I MADE THIS SLIDE BEFORE THE
COVID-19 OUTBREAK, SO YOU CAN
IMAGINE THIS HAS GOTTEN EVEN
MORE EXPANSIVE SINCE THIS POINT.
BUT REALLY FOR MOST OF THESE
STUDIES THERE'S NOT A LOT OF
FOLLOW-UP AFTER THE SEQUENCING
IS DONE.
SO WE CAN DO FILE PHYLOGENETIC
ANALYSIS, CAN THEY TRANSMIT TO
HUMANS, DO THEY HAVE POTENTIAL
TO SPILL INTO OTHER ANIMALS,
WHAT DISEASE MIGHT THEY CAUSE?
AND SO LOOK AT THIS QUESTION A
LITTLE MORE VISUALLY, LIVE
VIRUSES CIRCULATE IN ANIMALS AT
THE MOMENT.
BUT THESE ARE NOT USUALLY
ISOLATED BECAUSE WHEN YOU'RE
SAMPLING HUNDREDS OF ANIMALS IN
THESE STUDIES IT'S NOT ALWAYS
FEASIBLE TO, YOU KNOW, TO KEEP
YOUR SAMPLES IN A WAY THAT WOULD
ALLOW YOU TO RECOVER LIVE VIRUS
FOR SAFETY REASONS, FOR LOGISTIC
REASONS.
OFTENTIMES WE'RE TRYING TO GET
SAMPLES OUT OF DIFFERENT
COUNTRIES, SO IT'S EASIER TO PUT
THEM IN BUFFERS AND DO GENETIC
SEQUENCING ON SAMPLES.
NEXT GENERATION SEQUENCING
ALLOWS A SCALABLE APPROACH,
UNBIASED TO GET WHOLE GENOME
SEQUENCES NOW OF VIRUSES,
BASICALLY ALL THE VIRUSES IN THE
SAMPLE.
SO WHEN I STARTED THIS WORK
THERE WERE 17 YEARS OF VIRUS
DISCOVERY, AND WE HAD ABOUT 200
VIRAL RELATIVES IDENTIFIED FOR
SARS-COV-1 ACROSS BATS IN ASIA.
WE HAD 12 VIRUSES ACTUALLY
ISOLATED OR CONSTRUCTED THROUGH
REVERSE GENETICS IN THE LAB,
VERY LITTLE DATA FOR MOST
VIRUSES RELATED CLOSELY TO ONE
WE KNOW HAD OBVIOUS PANDEMIC
POTENTIAL.
SO, ONLY ABOUT FIVE WERE
ISOLATED THOUGH.
SO REALLY HOW DO WE STUDY THINGS
WE DON'T HAVE VIRAL SAMPLES FOR
IN THE LABORATORY AND CAN WE DO
MORE OF THEM?
SO THIS GETS TO THE QUESTION OF
REVERSE GENETICS, BUILDING A
VIRUS FROM, YOU KNOW, BASICALLY
FROM NOTHING.
THE CORONAVIRUS GENOME IS
ARRANGED IN THIS SERIES OF NOW
WELL-CHARACTERIZED GENES THAT
MAKE UP DIFFERENT ASPECTS OF THE
VIRUS INVOLVED IN DIFFERENT
BIOLOGICAL PROCESSES.
YOU KNOW, WHILE WE HAVE ALL
THESE GENES THE ONE WE'RE MOST
INTERESTED IN IS THE SPIKE GENE
BECAUSE YOU CAN SEE FROM HERE
IT'S FOUND ON THE OUTSIDE OF THE
VIRUS.
IT'S WHAT INTERACTS WITH THE
IMMUNE SYSTEM AND THE HOST,
FIRST.
SO IT'S THE TARGET OF LOTS OF
ANTI-VIRAL STRATEGIES.
THE IDEA THEN IF YOU DON'T HAVE
A VIRUS YOU CAN RECONSTRUCT THIS
WHOLE GENOME OR AT LEAST PART OF
IT THROUGH REVERSE GENETICS IF
YOU WANT TO STUDY
UNCHARACTERIZED VIRUS.
NUCLEOTIDE SYNTHESIS IS ROUTINE,
USED FOR PRIMERS, 20 TO 60 BASE
PAIRS IS A FEW DOLLARS.
AS YOU GO UP IN SIZE YOU GET AN
INCREASE IN COST.
AND THE CORONAVIRUS GENOME IS
NOT EASY.
IT'S 30,000 BASE PAIRS.
SO THIS CAN COST $15,000 OR
MORE, MONTHS TO HAVE
SYNTHESIZED, LONGER TO ASSEMBLE.
SOMETIMES WE CAN SHORTEN THIS
DOWN TO THE SPIKE GENE, THE
OUTER MOST PROTEIN TO SEE WHAT
IT CAN DO.
AT A FEW THOUSAND BASE PAIRS CAN
COST MORE THAN A THOUSAND
DOLLARS TO HAVE SYNTHESIZED.
IF YOU WANT TO STUDY HUNDREDS OR
THOUSANDS OF VIRUSES IN
PARALLEL, YOU KNOW, YOU NEED
SOME OTHER WAY TO GET AT THIS
QUESTION BECAUSE THIS KIND OF
APPROACH WOULD BE -- IT'S NOT
FEASIBLE.
SO MY WORK COMES DOWN TO HOW CAN
WE STUDY MORE OF THESE VIRUSES
FOR LESS?
WHAT IS THE SMALLEST REGION OF
THE GENOME WE COULD SYNTHESIZE
AND ACTUALLY TEST IN SOME SORT
OF LABORATORY ASSAY TO LEARN
ABOUT A VIRUS'S ABILITY TO
INFECT MUSEUM -- HUMAN CELLS.
THE 2020 STUDY WAS ON THE
SARBICO VIRUSES.
WE HAD SOME STRUCTURAL
MECHANISTIC DATA FROM 2018, AND
THERE WERE SEVERAL LIVE VIRUSES
STUDIED.
ANYTHING WE WERE ABLE TO SHOW IN
THIS ASSAY WE'VE BEEN DEVELOPING
WE COULD THEN VERIFY WITH THE
PREVIOUSLY PUBLISHED LITERATURE.
SO, THE CORONAVIRUSES ARE BROKEN
DOWN INTO FOUR GENERA.
IT'S THE BETA CORONAVIRUSES THAT
HAVE PATTERNS OF ZOONOSIS AND
SEVERE HUMAN DISEASE, SO I'M
SHOWING DIFFERENT SUBJENERA
SARBE INCLUDES SARS, AND WE HAVE
EMBICOVIRUSES, BUT NOBICOVIRUS
IS FAIRLY BAT SPECIFIC.
HALF MIGHT HAVE POTENTIAL TO
INFECT ACROSS SPECIES BARRIERS
OR CAUSE DISEASE.
IF WE LOOK AT ARRANGEMENT OF
SPIKE GENE, THIS IS OUR SPIKE ON
THE OUTSIDE, INTERACTS WITH THE
RECEPTOR, THROUGH JUST A SMALL
REGION OF RECEPTOR BINDING
DOMAIN.
YOU CAN APPRECIATE IT'S THIS ONE
SINGLE CONTIGUOUS DOMAIN IN THE
BETA CORONAVIRUS SPIKE.
IF WE LOOK WHERE IT'S BEEN
MAPPED FOR MANY OF THESE VIRUSES
ACROSS THE DIFFERENT SUBGENERA
WE CAN SEE IT'S BASICALLY IN THE
SAME SPOT, ROUGHLY THE SAME
ADVISE FOR A LOT OF THESE
VIRUSES.
IF WE LOOK WITHIN, JUST SARBICO
IT'S MORE HOMOGENOUS, CAN WE
JUST TEST DOMAINS FROM THESE?
TO ILLUSTRATE THE APPROACH HERE,
THIS IS A SPIKE TRIMER FOR
SARS-COV-1 BOUND TO ITS RECEPTOR
HUMANASE 2.
WE
HUMAN ACE-2.
IT'S CAPABLE OF INDEPENDENTLY
FOLDING.
GROUPS CAN PURIFY THIS FRAGMENT
AND DO CRYSTALLOGRAPHY STUDIES.
WONDERING IF WE CAN EXCISE THE
SPIKE OUT OF SARS 1 AND REPLACE
WITH RECEPTOR BINDING DOMAIN
FROM OTHER VIRUSES.
AND SO THIS CHIMERIC SPIKE
APPROACH WE TOOK, CODON
OPTIMIZED SARS SPIKE FOR HUMAN
CARS, APPENDED C-TERMINAL SLIDE
TAG, INTRODUCED SILENT MUTATIONS
THAT ARE JUST OUTSIDE OF THE
RECEPTOR BINDING DOMAIN.
THEY DON'T CHANGE THE CODING
SEQUENCE BUT THEY ALLOW US TO AT
THE DNA LEVEL REPLACE RECEPTOR
BINDING DOMAIN FROM SARS 1 WITH
THAT OF ANY OF THE OTHER
SARBECOVIRUS OR LINEAGE B SPIKE
RBDs.
COMPARED TO PREVIOUS APPROACHES
IN THE COST OF SEVERAL THOUSAND
DOLLARS, FOR GENE TO TEST, WE
COULD TEST RECEPTOR BINDING
DOMAIN FOR UNDER $130, AND IT
ONLY TOOK ABOUT SIX DICE HAVE
THE SEQUENCE SYNTHESIZED,
SOMETIMES LESS.
SO FASTER MEANING WE COULD SCALE
IT UP.
ADJUST RECEPTOR BINDING DOMAIN
FROM SARS 1, REPLACE WITH
DIFFERENT SYNTHETIC RECEPTOR
BINDING DOMAIN, MAKE SINGLE
CELL -- SINGLE VEHICLEEL VSV
PARTICLES CAN CHIMERIC SPIKES,
ON THE OUTSIDE OF THE PARTICLE,
INFECT TARGET SELLS, MEASURE
LUCIFERASE PRODUCED FROM
INFECTION AS READOUT FOR CELL
ENTRY FROM RECEPTOR BINDING
DOMAIN, IT TOOK ABOUT A YEAR OR
SO OF DEVELOPMENT BECAUSE MAKING
A CHIMERIC SPIKE THAT COULD WORK
FOR ALL OF THE DIFFERENT VIRUSES
TOOK A LITTLE BIT OF TRIAL AND
ERROR.
TO SUM SUMMARIZE THIS WAS PUBLD
IN 2020, WE SCREENED
BASICALLY -- DEVELOPED THIS
CHIMERIC SPIKE APPROACH,
SCREENED A LARGE PANEL OF 20
VIRUSES, REPRESENTATIVE OF EVERY
SINGLE NATURAL PUBLISHED VARIANT
OF THE RECEPTOR BINDING DOMAIN
FOR SARBECOVIRUSS AT THE TIME
AND THESE DOMAINS COULD
PHYLOGENETICICALLY CLUSTER AND
CARRIED OVER TO CELL ENTRY
POTENTIAL, RECEPTOR TROPISM,
CLADE 1 SARBECOVIRUS USED HUMAN
ACE-2, CLADE 2 DON'T USE ANY OF
THE KNOWN CORONAVIRUS RECEPTORS
ALTHOUGH A FEW DO HAVE ABILITY
TO INFECT HUMAN CELLS.
AND BECAUSE THIS APPROACH WAS SO
QUICK AND CHEAP, WE WERE
ACTUALLY ABLE TO PRODUCE THE
FIRST LABORATORY DATA WITH
SARS-COV-2 12 DAYS AFTER THE
GENOME WAS RELEASED ONLINE, SO
WE WERE ABLE TO SHOW IN JANUARY
I THINK, END OF JANUARY, ACE-2
WAS RECEPTOR FOR THIS VIRUS AND
USED ITS HIGHER EFFICIENCY THAN
SARS 1.
SO WHEN STATERRED MY LAB THEN AT
WSU WE HAD A LARGE PILE OF
SEQUENCE DATA.
I PARTNERED WITH SOME FOLKS IN
OUR MATHEMATICS DEPARTMENT, WHO
USED NETWORK ANALYSIS TO
BASICALLY CHARACTERIZE AND
ANALYZE GENETIC SEQUENCES AND
WERE ABLE TO USE NETWORK
ANALYSIS TO CLUSTER THE
DIFFERENT VIRUSES BASED OFF OF
ENTRY PHENOTYPE.
AND SO THIS IS THE BEGINNING OF
A MODEL WE CAN USE TO PREDICT
WHERE NEW VIRUSES -- THEIR CELL
ENTRY POTENTIAL.
WHAT WAS IMPORTANT WE COULD
ACTUALLY DIG INTO THE GROUP WE
DIDN'T KNOW RECEPTOR FOR AND SAY
BASED OFF THE ENTRY DATA COULD
SAY WHICH OF THE NEWLY
DISCOVERED SARBECOVIRUSS IN THIS
CLADE WOULD BE ABLE TO INFECT
HUMAN CELLS OR NOT.
THAT'S THE POWER OF THIS
APPROACH WE DON'T HAVE TO KNOW
RECEPTOR FOR OUR MODELING
APPROACHES TO BE ACCURATE
BECAUSE WE'RE SCREENING SUCH A
LARGE GROUP OF VIRUSES.
I PARTNERED WITH THE INSTITUTE
OF VIROLOGY, REFINED TECHNIQUES
WE USED TO -- I GROW ACE-2
INDEPENDENT SARBECOVIRUSS AND
WERE ABLE TO SHOW REPLICATION OF
MOLECULAR CLONE AND LAST YEAR
WERE ACTUALLY ABLE TO USE WHAT
WE LEARNED ABOUT ACE 1 OF 2
INDEPENDENT VIRUSES, GOING TO
ISOLATING ONE OF THOSE VIRUSES
IN A HUMAN CELL LINE AS WE WERE
KIND OF PREDICTING.
WE ALSO CAN USE OUR TOOLS TO
CONTINUALLY SURVEY SEQUENCE
BASE, AS NEW VIRUSES ARE
DISCOVERED WE CAN PLUG THEM INTO
OUR SYSTEM AND GET A SNAPSHOT
FOR THEIR ABILITY TO INFECT
HUMAN CELLS AND HOW EFFECTIVE
ARE VACCINES FOR SARS 2 FOR
EXAMPLE AGAINST THESE OTHER
VIRUSES, SO A FEW YEARS AGO WE
FOUND A VIRUS THAT WAS
DISCOVERED IN RUSSIAN, IN 2021,
COULD USE HUMAN ACE-2, AND THEN
WE SHOWED THAT ACTUALLY A
CHIMERIC SPIKE BETWEEN SARS 2
WITH JUST RECEPTOR BINDING
DOMAIN FROM THIS RUSSIAN SPIKE
WAS ABLE TO RESIST BASICALLY
SERUM FROM VACCINATED
INDIVIDUALS AND PEOPLE WHO HAD
BEEN INFECTED WITH OMICRON.
SO SUGGESTING REALLY THAT
THERE'S NOT A LOT OF
CROSS-PROTECTION BETWEEN EVEN
RESPONSES TO NATURAL INFECTION
FOR SOME OF THESE OTHER
SARBECOVIRUSS LOOMING ON THE
HORIZON WITH HUMAN ACE-2 TROPISM
WITH SIMILAR EFFICIENCY TO SARS
2.
FINALLY WE JUST -- SO JUST
COLLECTING INFORMATION ISN'T
SUFFICIENT.
WE'RE EAGER TO TRY TO EXPAND ON
THESE RESULTS AND DEVELOP THEM
FURTHER INTO, YOU KNOW, AN
ACTUAL INTERVENTION TO BLOCK
TRANSMISSION OF SOME OF THESE
VIRUSES.
SO, WHAT WE WERE ABLE TO SHOW
LAST YEAR IN COLLABORATION WITH
A GROUP WITH WENDY'S GROUP AT
HARWARD IN THE PHARMACOLOGY
DEPARTMENT, THEY HAVE BEEN
DEVELOPING AN ACE-2 BASICALLY
VIRUS INHIBITORY DRUG, AND WE
WERE ABLE TO HELP THEM TEST AND
VALIDATE THEIR REAGENTS ACROSS A
WIDER SPECTRUM OF THE
SARBECOVIRUSES SHOWING THEIR
MOLECULE WAS ABLE TO INHIBIT
INFECTION BY A NUMBER OF THE
SARBECOVIRUSES, INCLUDING THE
RUSSIAN VIRUS WE HAD JUST FOUND
WITH ACE-2 USED THE YEAR BEFORE
THIS STUDY.
SO REALLY WE WENT FROM 2021 TO A
SEQUENCE ON A DATABASE TO HAVING
ANTI-VIRAL THERAPEUTIC DRUG WITH
EFFICACY IN SMALL ANIMAL MODEL
BY 2023 AND HAVING THE DATA
SHOWING EFFICACY AGAINST THAT
VIRUS IN PARTICULAR.
I THINK THAT'S THE BIGGER GOAL
FOR OUR WORK TO SCREEN THE
VIRUSES, IDENTIFY THREATS, AND
WORK AND PARTNER WITH LABS
DEVELOPING THERAPEUTICS.
CURRENTLY, WE'RE WORKING ON SOME
STUDIES TO IDENTIFY THE ENTRY
ROUTE AND RECEPTORS USED BY
THOSE ACE-2 INDEPENDENT VIRUSES.
MY LAB IS FUNCTIONAL VIROMICS.
WHAT DO I MEAN BY THAT?
IT'S REALLY THIS KIND OF
AGGREGATION OF DIFFERENT
DISCIPLINES WHERE WE CAN FIRST
SCREEN THE VIROME FOR DIFFERENT
PHENOTYPES, CURRENTLY LOOKING AT
VIRAL ENTRY AND VACCINE
RESISTANCE, TOOLS ARE DESIGNED
TO LET US LOOK AT PROTEASE USAGE
AND OTHER POST-ENTRY PHENOTYPES
AS WELL.
WE CAN THEN PERFORM BULK SCREENS
ON THESE CHIMERIC GENES, AND TRY
TO LEARN AS MUCH AS WE CAN ABOUT
THE GROUP THAT WE'RE AFTER.
AND WE CAN USE THOSE TOOLS THEN
TO CONTINUE SURVEILLANCE ON THAT
GROUP, AND REFINE OUR
PROTOTYPES, MAKE BETTER CHIMERIC
PROTEINS THAT LET US GET AT
DIFFERENT PHENOTYPES AND
CONTINUE THAT SCREENING AND
SURVEILLANCE.
BUT ONCE WE ALSO HAVE THIS --
ONCE WE HAVE THIS DATA THOUGH
FROM OUR INITIAL SCREENING, WE
CAN TRY TO EXTRAPOLATE TRENDS
ACROSS DIFFERENT TYPES OF
VIRUSES, IDENTIFY DIFFERENT
PHENOTYPES, FOR EXAMPLE, ACE-2
INDEPENDENT ENTRY OR RESISTANCE
TO SARS-COV-2 VACCINE SERUM.
WE CAN LOOK FOR MOLECULAR
DETERMINANTS, REITERATE ON THAT
PROCESS OF SEEING HOW FAR
PHENOTYPES ARE CONSERVED ACROSS
A LARGE GROUP OF VIRUSES.
ONCE WE ACTUALLY HAVE OUR
SCREENING DATA AND HAVE OUR
CHARACTERIZATION DATA, WE CAN
FEED THIS INTO COMPUTATIONAL
MODELS, IDENTIFYING TRENDS,
VALIDATING TRENDS WITH
COMPUTATIONAL MODELS AND REFINE
MODELS, USING WET LAB AND
COMPUTATIONAL LABS TO
CHARACTERIZE UNDERSTUDIED
VIRUSES AT LARGER SCALE THAN WE
KNOW WE CAN.
WITH THAT INTRODUCTION
TODAY'S -- THE BULK IS WITH THE
MERBECOVIRUSES, WHAT WE'VE BEEN
WORKING ON FOR THE PAST SIX OR
SEVEN YEARS, CAN WE ASK THIS
QUESTION FOR MERBECOVIRUSES?
THE CHARACTERISTIC VIRUS IS
MIDDLE EAST RESPIRATORY SYNDROME
CORONAVIRUS DISCOVERED IN HUMANS
IN 2012.
AND IT'S CURRENTLY ENDEMIC
BASICALLY IN THE MIDDLE EAST,
TRANSMITS FROM DROMEDARY CAMELS
REPEATEDLY.
THANKFULLY THERE'S LOW
HUMAN-TO-HUMAN TRANSMISSION BUT
IT HAS A HIGH MORTALITY OF 36%
IN REPORTED CASES.
SO, IT IS CONCERNING BUT
THANKFULLY FOR NOW IT'S NOT
SPREADING.
AND KIND OF SIMILAR TO WHAT WE
SAW WITH SARS 1 AND SARS 2 IS
REALLY AFTER THIS VIRUS WAS
DISCOVERED THE MORE WE LOOK IN
NATURE AND PERFORM VIRAL
SURVEILLANCE STUDIES WE FIND
MORE CORONAVIRUSES.
SO HKU 4 AND HKU 5 WERE THE
FIRST BAT MERBECOVIRUSES
DISCOVERED, ACTUALLY DISCOVERED
IN 2006 YEARS BEFORE MERS CAME
OUT, BECAUSE WE DIDN'T HAVE
TOOLS TO STUDY THESE VIRUSES,
YOU KNOW, THERE WAS JUST
SEQUENCE DATA.
AND REALLY SINCE MERS EMERGED IN
2012 WE FOUND THOUSANDS OF
RELATED VIRUSES ACROSS BATS,
HEDGEHOGS, PANGOLINS, IN ASIA,
AFRICA, EUROPE AND MIDDLE EAST,
A MORE DISTRIBUTION AND MORE
DIVERSE.
SINCE 2012 ONLY SIX STUDIED IN
THE LAB, WITH HIGH DIVERSE IT'S
MAKES MOST RELATIVES UNKNOWN.
ZOO NOTING RISK UNDERSTANDING IS
LOW.
TO HOW WE TACKLED THIS APPROACH,
THIS QUESTION, WE BASICALLY
STARTED WITH VERY SIMILAR DESIGN
TO THE SARS APPROACH, THE
SARBECOVIRUS APPROACH, NOW
WORKING WITH THE MERS SPIKE
PROTEIN, SILENT RESTRICTION
SITES, FLAG-tag ON IT TO
REPLACE RECEPTOR BINDING DO HE
MAIN, USE CHIMERIC SPIKES TO
RECOVER PSEUDOTYPES, LUCIFERASE
PSEUDOTYPES.
I STARTED THIS WORK IN 2018, BUT
SARBECOVIRUSES WERE EASIER AND
IT MOVED FASTER.
THEY ARE FOUND IN VERY FEW
SPECIES, AND SO THEY ARE QUITE
SIMILAR TO EACH OTHER.
SOMETIMES 99% AT THE NUCLEOTIDE
LEVEL, FOUND IN A LIMITED
GEOGRAPHIC REGION ALTHOUGH
THAT'S EXPANDING A LITTLE BIT.
BUT THE MERBECOVIRUSES ARE FOUND
IN A WIDE RANGE AND GEOGRAPHIC
DISTRIBUTION, QUITE DIFFERENT
FROM EACH OTHER, SOMETIMES ONLY
65% SIMILAR AT NUCLEOTIDE LEVEL
AT LEAST IN THE SPIKE GENE,
CONFOUNDS THE EFFORT WHEN YOU'RE
TRYING TO CALL WHERE EXACTLY TO
MAKE A CHIMERIC PROTEIN YOU NEED
TO KNOW EXACTLY AT THE AMINO
ACID LEVEL WHERE YOU CAN MAKE
THESE INTRODUCTIONS AND WHERE
THEY DON'T WORK.
AND SO WHAT I'M SHOWING IS
ALIGNMENT OF THREE
MERBECOVIRUSES, THERE'S KNOW ANY
STRETCH OF AMINO ACID WIDELY
CONSERVED ACROSS ALL OF THE
DIFFERENT VIRUSES.
AND SO FOR MANY YEARS EVERY TIME
WE MAKE A CHIMERIC SPIKE IT WAS
NON-FUNCTIONAL BASICALLY.
AND SO, YOU KNOW, THIS IS
BASICALLY A TRIAL AND ERROR
PROCESS TO FIGURE OUT WHAT AMINO
ACIDS WE CAN USE TO SAY THIS IS
WHERE WE CAN INTRODUCE THE AMINO
ACID SEQUENCE FROM ANOTHER
VIRUS.
SO WHILE THEY DON'T HAVE A LOT
OF CONSERVED MOTIFS THEY DO HAVE
CONSERVED GLYCINES IN YELLOW,
SHOWN IN YELLOW, FOUND IN EVERY
MERBECO VIRUS.
IT TOOK MANY YEARS BUT THAT'S
ALL WE COULD USE TO DEFINE THE
EDGE OF THE RECEPTOR BINDING
DOMAIN.
GLYCINES ARE AT THE BASE OF THE
SPIKE.
I MEAN BASE OF THE RECEPTOR
BINDING DOMAIN.
AND SO WE THOUGHT MAYBE THIS IS
A GOOD PLACE TO INTRODUCE BREAKS
FOR CHANGING THE RBD.
THEN THE QUESTION IS WHAT
VIRUSES DO WE USE?
WE'RE NOT GOING TO START MAKING
A LARGE CHIMERIC PANEL.
WE WANT GOOD CONTROLS THAT WOULD
GIVE CONFIDENCE THAT WE CAN
MEASURE DIFFERENT PHENOTYPES.
AND SO RIGHT AS WE WERE
FINISHING UP THE DESIGN OF OUR
CHIMERIC SPIKE, THIS STUDY CAME
OUT FROM CHINA SHOWING THAT
MANY -- AT LEAST SOME
MERBECOVIRUSES FOUND IN AFRICAN
BATS WERE CAPABLE OF USING HUMAN
ACE-2, WHICH AT THE TIME WAS
QUITE A PARADIGM SHIFT BECAUSE
WE THOUGHT THESE VIRUSES -- WE
KNEW OF ONLY MERS AND SOME OTHER
BAT CORONAVIRUSES THAT USED
DPP4, THE THOUGHT WAS ALL THE
MERBECOVIRUSES PROBABLY USED
DPP4 OR SOMETHING LIKE IT.
BUT THAT HASN'T HELD UP TO BE
TRUE.
AND THIS GROUP SHOWED ACTUALLY
SOME OTHER VIRUSES CAN USE ACE-2
SO COMPLETELY DIFFERENT RECEPTOR
USE ACROSS VIRUSES BOTH
TECHNICALLY MERBECOVIRUSES.
THIS BECAME A GREAT TARGET FOR
US, WITH THE IDEA OF CAN WE
REPLACE THE RECEPTOR BINDING
DOMAIN OF MERS WITH ONE OF THESE
ACE-2-USING VIRUSES IN ORDER TO,
YOU KNOW, EXCHANGE THE RECEPTOR
TROPISM.
AND SO I'M SHOWING A SMALL PANEL
OF CHIMERIC SPIKES I MADE.
AND WE HAVE THE MERS ORIGINAL
SPIKE.
WE HAVE THE HKU4 SPIKE, BAT
CORONAVIRUS, CLOSELY RELATED TO
MERS, THEY BOTH USE DPP4.
AND SO I MADE A CHIMERIC HKU4
SPIKE WITH RECEPTOR BINDING
DOMAIN HKU4 AND MERS, NOT EXPECT
BE THAT TO CHANGE THE TROPISM, A
SIMILAR WITH PDF 2180 SHOWN TO
USE HUMAN ACE-2.
AND BECAUSE I'M ALWAYS IN THE
QUEST TO SEE IF I CAN LOWER COST
OF STUDY, MOVE FASTER, I TRIED
SLIGHTLY SMALLER VARIANTS JUST
TO SEE IF WE COULD MINIMIZE THE
AMOUNT OF SEQUENCE TO HAVE
SYNTHESIZED.
AND SO HERE'S A PANEL OF
PSEUDOTYPES NOW, INFECTING THE
HUMAN 293T CELL LINE WHICH
DOESN'T SUPPRESS HIGH LEVELS OF
CORONAVIRUS VIRUS RECEPTORS, NOT
EXPECTING ENTRY CLEARLY.
THAT'S WHAT WE SEE.
THERE'S NO RELATIVE ENTRY.
IF WE TRANSDUCE THESE CELLS WITH
HUMAN DPP4 WE CAN SKI MERS AND
HKU4, SPIKES CAN INFECT A LITTLE
BIT.
WHEN WE TRANSDUCE WITH HUMAN
ACE-2, WE SEE THIS PDF 2180
AFRICAN MERBECVIRUS CAN GET IN.
THIS IS UNDER TEN-FOLD, NOT AS
RELIABLE AS WE WOULD LIKE.
SO ONE OF THE TRICKS IN OUR LAB
TO LOOK AT ENTRY A LITTLE BIT
MORE SPECIFICALLY IS INCLUDE
TRYPSIN IN THE EXPERIMENT.
TRYPSIN HELPS PROCESS THE SPIKE,
POST-RECEPTOR ENGAGEMENT.
NOT ALL CELL LINES EXPRESS
EFFICIENTLY, EFFICIENTLY EXPRESS
NECESSARY PROTEASE FOR
CORONAVIRUS ENTRY.
IF WE ADD TRYPSIN IT GREASES THE
WHEELS OF INFECTION AND HELPS US
DETECT LOW LEVEL INTERACTION
BETWEEN IS HE RECEPTOR AND SPIKE
GENE, DOESN'T CHANGE OUR STORY
WITH OUR CHIMERIC SPIKE BUT
SHOWS CLEAR STRONG ENTRY SIGNAL
ACE-2 DEPENDENT BY CHANGING
RECEPTOR DOMAIN, HIGHLY CONSERVE
GLYCINE RESIDUES FLANK THE
FUNCTIONAL VIRUS RBD.
GENE BANK, 2,000 ENTRIES FOR
MERS OR MERBECOVIRUSESS, MOST
COME FROM DIFFERENT VIRUS
DISCOVERY STUDIES WHERE THEY
DIDN'T ALWAYS SEQUENCE THE WHOLE
GENOME, SOMETIMES THEY ONLY
SEQUENCED A FRAGMENT, 842 SPIKE
GENES.
IF WE REMOVE THE DIFFERENT --
BASICALLY MERS ISOLATES TAKEN
OUT OF HUMAN PATIENTS OR MERS
DIRECT
DIRECTLY ISOLATEDs FROM CAMELS
THAT GAVE HUMAN MERS WE'RE LEFT
WITH 65 DIFFERENT SPIKES.
ALL ARE UNIQUE, ONLY 34 UNIQUE
VARIANTS OF THE RECEPTOR BINDING
DOMAIN SO THIS DAUNTING QUESTION
OF THOUSANDS OF ENTRIES REALLY
CAN BE WHITTLED INTO A SMALL
NUMBER THAT'S LARGELY
REPRESENTATIVE OF ALL THE
MERBECOVIRUSES WE HAVE THIS
INFORMATION FOR.
HERE IS A PANEL OF THE SPIKES,
DIFFERENT BINDING DOMAINS.
WE INCLUDED THE ORIGINAL.
SHOWN HERE IS AN RBD AMINO ACID
TREE, SO JUST LOOKING AT
PHYLOGENETIC RELATIONSHIP WE CAN
SEE THEY FORM -- SEEM TO FORM
CLADES OR CLUSTERS, THIS IS MORE
APPARENT WHEN WE LOOK AT AMINO
ACID ARRANGEMENT OF THE RECEPTOR
BINDING DOMAIN FROM
THOUSAND-FOOT VIEW, THIS IS JUST
N-TERMINUS TO C-TERMINUS,
RECEPTOR BINDING DOMAIN, SHOWING
PRESENCE OR ABSENCE OF INDELS IN
RELATIONSHIP TO EACH OTHER.
SO YOU CAN SEE CLADE 1s,
GENERALLY HAVE THESE COMMON
DELETIONS IN COMPARISON TO THE
OTHER MERBECOVIRUSES AND THAT'S
TRUE FOR ALL THESE CLADES.
SO WE MADE A LARGE PANEL THEN OF
SUED OF PSEUDOTYPES BEARING
DIFFERENT CHIMERIC SPIKES, USED
THEM TO INFECT CELLS.
LOOKING AT 293T CELLS WITHOUT
ANY RECEPTOR, WE'RE NOT SEEING A
LOT OF CLEAR ENTRY, VERY LOW
LEVEL ENTRY OF MERS CORONAVIRUS,
WHICH IS LIKELY BECAUSE 293 Ts
EXPRESS LOW LEVELS OF ACE-2 AND
DPP4.
WHEN WE EXPAND OUR INFECTION TO
INCLUDE CELLS TRANSDUCED WITH
DPP4, ACE-2 OR ANOTHER
CORONAVIRUS RECEPTOR THAT'S
FOUND IN HUMANS, APN, WE CAN SEE
THAT REALLY IT'S JUST THIS BLUE
GROUP OF VIRUSES DOWN HERE,
CLADE 1 VIRUSES, WE'LL CALL
THEM, CAN USE HUMAN DPP4.
WE CAN SHOW THE TWO AFRICAN
MERBECOVIRUSES FOUND IN THAT
NATURE PAPER CAN USE HUMAN
ACE-2, AND REALLY A LOT OF THESE
OTHER CLADES DON'T SEEM TO USE
ANY OF THESE HUMAN ORTHOLOGUES
OF RECEPTOR, TO ORIENT FOR FOR
POSITIVE CONTROLS, 229E WITH
APN.
IF WE ADD TRYPSIN IT'S NOT
GIVING A CLEAR ACKNOWLEDGES.
WE INCLUDED SPECIES ORTHOLOGS OF
NON-RECEPTORS, THIS BAT IS THE
NATURAL HOST FOR HKU5 VIRUSES,
THESE VIRUSES WERE ALL FOUND IN
THESE ABRAMUS BATS.
PINK IS FOUND IN EUROPEAN
HEDGEHOGS, WE WEREN'T ABLE TO
SEE CLEAR ENTRY.
WE CAN SEE A STRONG USE WHEN WE
ADD TRYPSIN OF BAT ACE-2 AMONGST
BAT CORONAVIRUSES, BUT AGAIN NO
REAL CLEAR RECEPTOR PATTERN.
CLADE 4 WAS STILL A QUESTION
MARK.
BUT THIS HKU5 QUESTION WAS NEW.
SO TO VALIDATE THIS STUDY, THIS
EXPERIMENT, WE THEN CHANGED OUR
CELL LINE BACKGROUND TO BABY
HAMSTER KIDNEY CELLS, COMPLETELY
DIFFERENT SPECIES, COMPLETELY
DIFFERENT CELL LINE, DOESN'T
EXPRESS COMPATIBLE FORMS OF
ACE-2 OR DPP4 FOR MERS OR SARS,
WE'RE COULD HAVE DENT WHEN WE
TRANSDUCE WE SEE CLEAR ENTRY OF
HKU 5 COMPLEX WITH THE ACE-2.
SO THAT'S NICE.
CHIMERIC SPIKES.
HOW DO THEY COMPARE TO FULL
LENGTH SPIKES?
AND SO HERE'S A SMALL PANEL
WE'VE MADE, SELECTED A COUPLE OF
DIFFERENT FULL LENGTH SPIKES,
AND WE'RE SHOWING FOR EXAMPLE
FULL LENGTH SPIKE FROM HKU ^5 20
IS ABLE TO INFECT CELLS THAT
SPECIAL PIPISTRELLIS ABRAMUS.
THAT'S CHIMERIC SPIKE, WHO ABOUT
WHOLE VIRUS?
WE PAIRED UP WITH RALPH'S GROUP
AT UNC, SPECIALIZES IN PRODUCING
REPLICATION COMPETENT MOLECULAR
CLONES OF CORONAVIRUSES, THEY
HAVE AN HKU 5 IN THEIR
COLLECTION THEY HAVEN'T BEEN
ABLE TO GROW.
THE IDEA WAS SIMILARITY TO MERS
WAS WORTH PRODUCING AND THOUGHT
THEY WORK ABLE -- WOULD BE ABLO
GET IT TO HE REPLICATE BUT
WITHOUT RECEPTOR IDENTITY IT
WASN'T ABLE TO GROW.
I SENT CELL LINES, TRANSDUCED
WITH PIPISTRELLUS ABRAMUS ACE-2,
NICELY SUPPORT REPLICATION OF
THEIR GFP EXPRESSING MOLECULAR
CLONE, CAN -- WE CAN DETECT
ACCUMULATION OF VIRAL PROTEINS
DURING REPLICATION SUGGESTING
ACE-2 IS SUPPORTING REPLICATION.
TAKING A CLOSE YOU ARE LOOK AT
HKU5, IT HAS A LOT OF DIVERSITY,
NOT JUST FROM OTHER CLADES IN
THE MERBECOVIRUSES BUT WITHIN
ITSELF.
I'M SHOWING CONTACT POINTS
IDENTIFIED BETWEEN THE AFRICAN
ACE-2 USING MERBECOVIRUSESS WITH
HUMAN AND BAT ACE-2, BUT IF WE
LOOK AT RESIDUES IN HKU5 ALMOST
EVERYONE IS DIFFERENT.
WE'RE NOT ALWAYS ABLE TO USE THE
DATA FROM EVEN ONE CLADE TO
PREDICT PHENOTYPE OF ANOTHER.
BECAUSE THESE VIRUSES CAN USE
REALLY WIDELY DIFFERENT
INTERACTING SURFACES WITH THESE
PROTEINS.
AND TO MAKE IT EVEN MORE
CHALLENGING HALF CONTACT POINTS
ARE IN A DELETION, A LOOP
DELETION BASICALLY ON THE
HKU5s.
SO THERE'S SOMETHING DIFFERENT
GOING ON THERE.
SO TAKING A CLOSER LOOK AT THE
STRUCTURAL DATA, THIS IS THE
CRYSTAL STRUCTURE FOR ONE OF THE
AFRICAN MERBECOVIRUSES WITH BAT
ACE-2, THIS IS A PREDICTED
STRUCK FEWER FOR PIPISTRELLIS
ACE-2 AND HKU5, AND WE CAN SEE
FOR THE VIRUSES THAT HAVE AN
INTACT SERIES OF BOTH OF THOSE
LOOPS WITHOUT ANY DELETIONS, CAN
LOOK SIMILAR, BUT FOR THE VIRUS
THAT HAS THAT DELETION IN ITS
LOOP IT'S ACTUALLY CONTRACTED
AWAY FROM THE SURFACE.
SO POTENTIALLY HAS LESS CONTACT
WITH ACE-2.
SO, TO STUDY THESE LOOPS AND
THEIR EFFECT ON ENTRY A LITTLE
BIT MORE WE SELECTED TWO
CRITICAL RESIDUES, BASICALLY IN
THE MIDDLE OF THOSE LOOPS,
INTRODUCED A SERIES OF LARGE
BULKY AND CHARGED AMINO ACIDS
AND MUTAGENESIS STUDY, TESTED
ENTRY OF MUTANT RECEPTOR BINDING
DOMAINS ON CELLS EXPRESSING
PIPISTRELLIS ABRAMU, OR HUMAN
ACE-2, HUMAN AND MA'AM CENTER
CELLS TO ELIMINATE CELL LINE
SPECIFIC DIFFERENCES HOPING TO
SEE DIFFERENCES ACROSS BOTH CELL
LINES REGARDLESS OF SPECIES
BACKGROUNDS SO WE CAN MORE
FIRMLY SAY WHERE THE PHENOTYPE
IS COMING FROM.
THIS IS A HEAT MAP SHOWING
ENTHIS TRY DATA IN COMPARISON
D. -- ENTRY DATA IN COMPARISON.
WHEN WE INTRODUCE MUTATIONS IN
LOOP 1 OR LOOP 2 FOR EITHER THE
TWO-LOOP VIRUS OR ONE-LOOP VIRUS
THEY ALL TAKE A HIT IN ABILITY
TO INFECT CELLS THAT ARE
EXPRESSING PIPISTRELLIS ABRAMUS
BAT ACE-2 REGARDLESS OF CELL
LINE BACKGROUND, STRONGER IF WE
MUTATE THE SECOND LOOK, THAT
MIGHT BE A CRITICAL POINT FOR
INTERACTION.
THE STORY IS QUITE DIFFERENT
WHEN WE TEST THIS MUTANT SET ON
HUMAN ACE-2.
NOW WE CAN SEE THE VIRUS THAT
HAS TWO LOOPS IS ACTUALLY ABLE
TO GAIN AN INCREASE IN ITS
ABILITY TO INFECT CELLS
EXPRESSING HUMAN ACE-2.
THE OTHER VIRUS THAT ONLY HAS
ONE LOOP DOESN'T SEEM TO HAVE
THAT SAME EFFECT.
IT MIGHT BE THAT THE LARGER LOOP
IN THIS INTERFACE MAY ALLOW THIS
VIRUS TO ADAPT BETTER TO OTHER
ACE-2s.
SO WHAT WE'RE LEARNING FROM THIS
IS WE NEED BETTER STRUCTURE AND
DEEP SCANNING MUTAGENESIS
STUDIES TO UNDERSTAND THE
FLEXIBILITY OF THIS INTERFACE.
AND SO WE'RE WORKING ON THAT.
WE HAVE A COLLABORATION WITH
PAMELA YORKMAN'S GROUP AT CAL
TECH, AND WE'VE BEEN ABLE TO
PRODUCE THE FIRST CryoEM
STRUCTURE OF THE WHOLE HKU520
SPIKE, WITH BOTH OF THE LARGE
LOOPS, ONE OF THE PROTOTYPICAL
HKU 5s IN LITERATURE IS HKU
5-1, BASICALLY THE FIRST
SEQUENCE, THAT WAS DESCRIBED.
HKU 5-1 WAS HUNDRED LOOP,
IMPORTANT TO UNDERSTAND POSSIBLE
DISTINCTION BETWEEN VIRUSES THAT
MAY IMPROVE THEIR CROSS-SPECIES
TRANSMISSION POTENTIAL.
WE'VE BEEN ABLE TO SHOW REALLY
SIMILAR TO OTHER BAT
CORONAVIRUSES THIS BAT HAS AN
ALL HVDs DOWN CONFIRMATION IN
OUR ASSAYS.
TO UNDERSTAND A LITTLE BIT MORE
WHAT MAKES THESE CLADES THE WAY
THEY ARE, WE'VE MADE DIFFERENT
CONSENSUS SEQUENCES
REPRESENTATIVE OF EACH OF THE
FOUR DIFFERENT CLADES, AT LEAST
THE CLADES WE HAVE MORE THAN TWO
SEQUENCES FOR.
AND THEN WE CAN TEST THEM IN OUR
CHIMERIC SPIKE BACKBONE.
AND SO WE CAN SHOW THAT CHIMERIC
SPIKE WITH CONSENSUS CLADE 1 RBD
CAN INFECT CELLS WITH HUMAN
DPP4, CLADE 2 RECEPTOR BINDING
DOMAIN CAN INFECT WITH THE BAT
ACE-2.
BUT NOT WITH THE HUMAN ACE-2.
WHEREAS THE CLADE 3 GROUP CAN
ACTUALLY INTERACT WITH BOTH,
WHICH IS IN AGREEMENT, CLOSE
AGREEMENT WITH PREVIOUS STUDY IN
"NATURE."
AND OUR CLADE 4 DOESN'T USE
THESE RECEPTOR MOLECULES.
WE'VE BEEN ABLE TO BREAK DOWN
THE MERBECOVIRUSES, IT'S THE
PRESENCE OF INDELS AND
SEQUENCE-SPECIFIC MOTIFS AND
ENTRY PHENOTYPE THAT DEFINE
THESE FUNCTIONAL CLADES, WE
CAPTAIN MAKE THESE
DETERMINATIONS FROM GEOGRAPHIC
ORIGIN FOR EXAMPLE, SOME RBDs
HAVE FOUND ACROSS A WIDE
GEOGRAPHIC RANGE TELLING US
THERE'S A SAMPLING BIAS IN THE
LITERATURE.
THE MORE WE LOOK FOR THESE
VIRUSES, THE MORE WE'RE GOING TO
FIND ACROSS A WIDER RANGE OF
SPECIES AND GEOGRAPHIC
LOCATIONS.
SO HKU 4 AND 4 AND 5 WERE DISCD
SIX YEARS BEFORE CORONAVIRUS, WE
DIDN'T KNOW WHAT TYPE OF
RECEPTOR THEY USED, DIDN'T KNOW
SPILLOVER PRO PRO TENSION.
POTENTIAL.
GROUPS STARTED GOING BACK AND
FINDING CLOSELY RELATED BAT
SEQUENCES, HKU 4 AND 5 LOOKED
SIMILAR TO MERS, ONLY ONE COULD
EAST THE SAME RECEPTORS, THE
OTHER COULDN'T.
IN THIS STUDY THEY ARE ONLY
LOOKING AT HUMAN AND ONE BAT
VARIANT OF DPP4, SO PROBABLY
MISSED THAT.
AFTER RALPH'S GROUP DEVELOPED
MOLECULAR CLONE IF YOU ADDED
TRYPSIN YOU COULD GET THE VIRUS
TO REPLICATE ON DIFFERENT CELL
LINES BUT WEREN'T ABLE TO PIN
RECEPTOR DOWN BECAUSE THEY ONLY
LOOKED AT DIFFERENT SPECIES
VARIANTS OF DPP4 AND ONLY
INCLUDED TRYPSIN IN THEIR ASSAY,
AGAIN MISSING IT.
IN 2022 WE LEARNED
MERBECOVIRUSES TO USE ACE-2,
MORE SPECIFICALLY HUMAN ACE-2,
THIS STUDY TESTED ACE-2 FROM 44
BAT SPECIES WITH HKU5 AND SOME
OTHERS, BUT THEY DIDN'T ACTUALLY
TEST PIPISTRELLIS ABRAMUS BAT,
SO RECEPTOR WAS ELUSIVE,
ENORMOUS SPECIES SPECIFICITY.
WE'VE SHOWN RBD IS FLANKED BY
TWO HIGHLY CONSERVED GLYCINES, A
GUIDE TO MAKE CHIMERIC SPIKES
AND SCREEN THE ENTIRE SUBGENUS,
PUBLISH DIVERSITY OF SUBGENUS
FOR ABILITY TO INFECT DIFFERENT
CELL LINES, USE DIFFERENT
RECEPTORS, SHOW THEY SORT INTO
DIFFERENT CLADES BASED OFF THEIR
SEQUENCE.
WE'RE ABLE TO CONFIRM CLADE 1
VIRUSES ALL USE DPP4, CLADE 3
CAN USE ACE-2.
HERE WE SHOWED CLADE 2 CAN ALSO
USE ACE-2, MORE SPECIES FASHION
AND CLADE 4 DOESN'T SEEM TO USE
ANY OF THESE KNOWN RECEPTORS.
SO, WE SHOWED DIFFERENT
MUTATIONS IN THE INTERFACE CAN
INFLUENCE THE RBD ABILITY TO USE
DIFFERENT ACE-2.
AND THEN JUST RECENTLY IN AUGUST
THIS YEAR STUDY CAME OUT WHERE
RESEARCHERS LOOKED ACROSS
BASICALLY FARMS, ANIMAL FARMS
ACROSS CHINA LOOKING AT DECEASED
ANIMALS THAT SEEM TO FIND FROM
DISEASE AND HKU5 WAS FOUND IN
SEVERAL DEAD MINK, WE'RE SEEING
ACROSS-SPECIES TRANSMISSION
EVENTS, ALL HAD SIMILAR VIRAL
SEQUENCE, AND VIRAL SEQUENCE ARE
THE TWO LOOP RBDs, PERHAPS THE
BIGGER RBD LOOPS ALLOW VIRUSES
TO TRANSMIT A LITTLE BIT BETTER.
THIS IS EARLY PRELIMINARY DATA
WHERE WE MADE CHIMERIC SPIKE,
CONTINUING OUR SURVEILLANCE
EFFORTS WE CAN CLONE THIS MINK
RECEPTOR BINDING DOMAIN INTO THE
CHIMERIC SPIKE AND TEST ENTRY ON
CELLS THAT EXPRESS MINK ACE-2 OR
HUMAN AILS 2, WE CAN NICELY
VERIFY THIS HKU5 USES MINK
ACE-2, WITH SIMILAR EFFICIENCY
AS SARS IF NOT BETTER.
BUT THAT IT HAS VERY LIMITED
ABILITY TO INFECT CELLS
EXPRESSING HUMAN ACE-2, SO EVEN
WHEN WE ADD TRYPSIN WE'RE NOT
GETTING A CLEAR STRONG SIGNAL.
BUT MAYBE A LITTLE BIT.
NOW AS OUR STUDY COMES OUT
SEVERAL GROUPS ARE WORKING ON
HKU 5, A GROUP HAS SHOWN THERE'S
AN HKU5 -- BASICALLY AN EXTRA
LINEAGE OF HKU 5 SEQUENCES, THEY
HAVE TAKEN A DEEPER DIVE INTO
THEIR SAMPLE DATABASE AND FOUND
HKU 5 VARIANTS THAT FORM A
DIFFERENT BRANCH ON THE TREE
THAT MAY HAVE IMPROVED OR HIGHER
ABILITY TO USE HUMAN ACE-2.
SO IT'S PRETTY LOW IN THIS ASSAY
STILL I THINK, BUT WHAT WE'RE
SEEING IS THAT THERE IS A WIDE
DISTRIBUTION OF ACE-2 USING
VIRUSES, THEY CAN USE DIFFERENT
REGIONS OF ACE-2 FOR THEIR
INTERACTIONS SO IT'S NOT LIKE
THEY ARE BINDING TO ONE
CONSERVED REGION WHICH IS WHY
INTERFACES CAN LOOK SO DIFFERENT
BUT REALLY FINDING VIRUSES WITH
EVEN TRACE POTENTIAL TO INFECT
CELLS IS A CONCERN.
SO WE SHOWED SEVERAL YEARS AGO
THAT MERS WAS CAPABLE OF REALLY
QUICKLY ADAPTING IN CELL CULTURE
TO A VIRUS EXPRESSING A
SUBOPTIMAL VARIANT OF DPP4, SO
WITHIN THREE PASSAGES THE VIRUS
PICKED UP ONE NUCLEOOH TIED
CHANGE CHANGING ITS ABILITY TO
REPLICATE ON A CELLS EXPRESSING
DPP4 FROM CON COGNATE SPECIES,
CAUSE FOR CONCERN.
WE'RE WORKING ON STRUCTURAL
EXPLORATION WITH PAMELA
BJORKMAN'S GROUP, DEEP
MUTAGENESIS WITH TYLER STARR'S
LAB AND PURSUING WORK WITH
MERBECOVIRUSES.
BARBARA HAHN IS USING MACHINE
LEARNING APPROACHES, CAN WE USE
THIS TO PREDICT DIFFERENT
PHENOTYPES.
AND SO WITH THAT I'D LIKE TO
THANK MY COLLABORATORS, THAT
HELPED US WITH THIS MERBECOVIRUS
WORK.
AND I WILL TAKE YOUR QUESTIONS.
>> THANKS, MICHAEL, THAT WAS A
WONDERFUL TALK.
THERE ARE LOTS OF QUESTIONS I
THINK.
AND I THINK THAT I COULDN'T STOP
THINKING OF QUESTIONS ACTUALLY
THROUGHOUT THAT TALK.
YOU INTRODUCED SO MANY ASPECTS
THAT COULD GENERATE INTERESTING
RESULTS IN THE FUTURE.
SO I'LL START BY ASKING A COUPLE
QUESTIONS.
BEFORE I DO THAT I WANT TO
REMIND THE AUDIENCE PLEASE ASK
YOUR QUESTIONS NOW BY CLICKING
ON THE SEND LIVE FEEDBACK
BUTTON, WHICH IS RIGHT IN THE
CENTER OF THE SCREEN UNDER THE
VIDEO, NIH VIDEOCAST SITE.
WE'LL RECEIVE THOSE QUESTIONS
AND RELAY THEM TO THE SPEAKER.
I WANTED TO ASK BECAUSE YOU
MENTIONED SOME OF YOUR FUTURE
DIRECTIONS ABOUT USING
MUTAGENESIS, SCANNING
MUTAGENESIS, TO UNDERSTAND MORE
ABOUT THE RBD AND RECEPTOR
USAGE.
SO DO YOU THINK THAT YOU WILL
MOVE INTO AN AREA WHERE YOU CAN
PLAN TO PREDICT WHICH RESIDUES
WOULD GIVE AN EXISTING RBD THE
POTENTIAL TO USE HUMAN ACE-2?
>> YEAH, I THINK THAT'S ONE OF
THE LARGER GOALS.
SO, YOU KNOW, TYLER STAR'S GROUP
HAS PIONEERED MUTAGENESIS
APPROACH WITH THE
SARBECOVIRUSES.
INITIAL CHARACTERIZATION OF
PANELS GAVE US INFORMATION IN
THE EARLY COVID DAYS ABOUT
RESIDUES THAT MIGHT EMERGE FOR
VACCINE RESISTANCE FOR EXAMPLE
AND ABILITY TO EVADE ANTIBODY
RESPONSE.
SO, YEAH, I THINK THAT KIND OF
APPROACH HAS A LOT OF POWER TO
IT.
AND SO I THINK ONE OF OUR BIGGER
GOALS IS TO AGGREGATE AND BUILD
DATA THAT'S ALL KIND OF IN A
SIMILAR PLATFORM BACKGROUND SO
WE HAVE A LOT OF COMPARABILITY
BETWEEN EXPERIMENTS, SO OUR
MODELS CAN BE A LOT MORE
ACCURATE.
THE IDEA I THINK IS TO DO THESE
MUTAGENESIS SCREENS AND THEN
FEED THAT TO OUR COMPUTATIONAL
TEAM TO HELP -- YOU KNOW, I
THINK PREDICTING IS ALWAYS HARD
TO SAY LIKE THIS VIRUS IS GOING
TO GO THIS WAY OR THAT WAY.
BUT I THINK WITH ENOUGH DATA WE
CAN AT LEAST LEARN CERTAIN
PATHWAYS THAT MIGHT BE LIKELY,
YOU KNOW, OR MORE LIKE BRACING
FOR IMPACT THAN HAVING TOOLS
THAT CAN -- OR THERAPEUTICS THAT
CAN HANDLE THOSE POSSIBLE
VARIANTS.
>> AND REGARDING THE VARIATION
IN AND OF ITSELF, WHAT DO YOU
THINK IS DRIVING A LOT OF THE
SEQUENCE VARIATION IN THE RBDs
THAT YOU'RE IDENTIFYING FROM
NATURE?
SO ARE RBDs UNDERGOING
SEQUENCE DIVERSIFICATION IN
RESPONSE TO -- ADAPTIVE IMMUNE
RESPONSE IN THE HOST AND/OR IS
IT RELATED TO SIMPLY RECEPTOR
USAGE?
>> YEAH, I THINK THAT'S -- A LOT
OF THE DIVERSITY WE SEE,
ESPECIALLY WITHIN SUBGENUS,
WITHIN THE SPECIFIC ANIMAL
POPULATION, A LOT OF HKU 5s
ARE FOUND IN BAT ROOSTS, SAME
WITH SARBECOVIRUS, OFTEN IN THE
SAME CAVE, THERE'S A LOT OF
PRESSURE ON THE POPULATION FROM
THE ADAPTIVE IMMUNE RESPONSE IN
THOSE ANIMALS, YOU KNOW, IN
ORDER FOR CIRCULATION TO STAY
PERSISTENT.
SO, YEAH, I THINK A LOT IS
DRIVEN BY ADAPTIVE RESPONSE BUT
THEN BECAUSE THERE'S SUCH A HIGH
ABUNDANCE OF VIRUSES IN SOME OF
THESE ANIMAL POPULATIONS I THINK
THERE IS PRESSURE THEN TO EXPAND
YOUR CELLULAR NICHE.
OF COURSE THIS IS ALL KIND OF,
YOU KNOW, JUST WHAT WE'VE BEEN
THINKING ABOUT FOR THE PAST
SEVERAL YEARS WITH ALL THIS
DATA, BUT THE FACT YOU DO SEE A
LOT OF THESE VIRUSES NOT
NECESSARILY CONVERGING ON THE
SAME RECEPTOR BUT ON THE SAME
TYPE OF RECEPTOR, THERE'S A LOT
OF REPEATED USE OF ACE 1 IN
THESE CORONAVIRUSES, AND DPP4,
AND APN, AND IT'S A HANDFUL OF
JUST A FEW MOLECULES THAT
VIRUSES FROM COMPLETELY
DIFFERENT BRANCHES OF THE TREES
CONVERGE ON AND LAND AT
DIFFERENT SPOTS, THAT GIVES US
CLUES ABOUT SOME UNDERLYING
BIOLOGY.
THESE VIRUSES HAVE TO USE NOT
JUST RECEPTOR BUT ALSO
PROTEASES, DIFFERENT ATTACHMENT
FACTORS, AND THOSE I THINK ARE
ALL CLUSTERED IN SPACE ON THE
CELL SURFACE SO THERE'S A LOT OF
PRESSURE TO, YOU KNOW, YOU HAVE
TO BE ABLE TO USE -- RETAINED
ABILITY TO USE ATTACHMENT
FACTORS, PROTEASES BUT IF YOU
WANT TO INFECT DIFFERENT CELL
TYPE YOU MIGHT NEED PRESSURE ON
THE RECEPTOR, THERE'S A COUPLE
DIFFERENT FORCES DRIVING HIGH
DIVERSITY IN THESE VIRUSES.
>> ENCOUNTERED A PAIRING WHERE
THE RBD SEQUENCE COULD NOT
COMPLETELY EXPLAIN ABILITY TO
INFECT CELL TYPE YOU'RE USING,
AND I THINK YOU DO SEE
INTERESTING DIFFERENCES BETWEEN
YOUR RBD CHIMERIC SPIKE AND YOUR
FULL LENGTH SPIKE.
SO WHAT ABOUT LIKE THE
N-TERMINAL DOMAIN OF SPIKE AND
--
>> OH, YEAH.
THAT'S SOMETHING THAT, YEAH, OUR
CHIMERIC SPIKES GENERALLY WORK
BETTER IN OUR ASSAYS THAN THE
FULL LENGTH WILD TYPE.
AND I THINK A LOT OF THAT COMES
DOWN TO THE CHIMERIC BACKBONE WE
USE ON THESE REALLY OPTIMIZED
MERS SPIKES, WHERE WE KNOW MERS
WORKS REALLY WELL IN CULTURE.
A LOT OF THESE OTHER
GASTROINTESTINAL-BASED BAT
CORONAVIRUS SPIKES SEEM TO TAKE
A DIFFERENT CONFIRMATION WHERE
ALL THE RBDs ARE DOWN.
THAT'S NOT WHAT MERS DOES IN
CULTURE, AT LEAST IN OUR
SYSTEMS.
SO, I THINK THAT OUR CHIMERIC
SPIKES JUST ARE POISED BETTER
FOR ENTRY ASSAYS BECAUSE THEY
PRESENT RBDs BETTER, I THINK
THEY ALSO HAVE, YOU KNOW, WE
KNOW THAT MERS GROWS WELL ON THE
CELL LINES, SO IT CLEARLY HAS AN
OPTIMAL KIND OF INTERACTION AT
ALL THE OTHER DOMAINS INCLUDING
N-TERMINAL DOMAIN, SOME BAT
VIRUSES WE'RE NOT SURE IF THEY
ARE GOING TO BIND TO, YOU KNOW,
HUMAN AND HAMSTER GLYCANS WITH
THE SAME EFFICIENCY AS THE SARS
VIRUS.
OTHER FACTORS PLAY INTO ENTRY,
IF IT'S NOT CONFIRMATION OF THE
PROTEIN THEN IT'S SOME OF THE
OTHER DOMAINS.
>> IT'S ALSO POSSIBLE THAT BY
MAKING THESE KIND OF UNNATURAL
CHIMERAS THAT YOU COULD ACTUALLY
STUMBLE UPON A FULL LENGTH SPIKE
THAT HAS AN ARRANGEMENT THAT
JUST SO HAPPENS TO INFECT BETTER
THAN ANY OF THE OTHER NATURALLY
EXISTING SPIKES, SO LIKE BY
COMBINING THE RDB WITH A GIVEN
REGION OUTSIDE OF THE RBD, YOU
KNOW.
SO THIS PERSON HAS ASKED A
REALLY INTERESTING QUESTION.
I WAS THINKING ABOUT IT MYSELF.
SO, FOR THESE MERBECOVIRUSES
THAT SEEM TO HAVE DUAL RECEPTOR
ACTIVITY WHAT CAME FIRST?
AND FOR THE MERBECOVIRUSES IN
GENERAL DO YOU THINK ACE-2 WOULD
BE MORE OF THE ANCESTRAL
RECEPTOR AND DPP4 WOULD HAVE
AROSE AFTER THAT?
>> IT'S HARD TO PINPOINT BECAUSE
IT'S SO VARIABLE IN THESE
VIRUSES.
BUT, YEAH, IF YOU TALK TO TYLER
STARR, THEIR DATA SUGGESTS THAT
ACE-2 IS AN ANCESTRAL BINDING
PROPERTY FOR THE SARBECOVIRUSES.
NEED TO DO SIMILAR TYPE OF
APPROACH TO DETERMINE THAT FOR
THE MERBECOVIRUSES.
BUT, YEAH, GIVEN SO MANY OF
THESE VIRUSES USE ACE-2 OR CAN
BE SWAYED TO USE IT SEEMS LIKE
THAT MIGHT BE A DEFAULT AND THEN
THERE'S DEVIATIONS IN PRESSURE
FROM BEYOND THAT TO DIVERSIFY.
BUT AGAIN WE'RE NOT 100% SURE,
MAYBE DBB 4 WAS THE ANCESTRAL
ONE BEING SWAPPED OUT BY THE
NEWER AIRS 2 VIRUSES BUT I DON'T
THINK PHYLOGENETIC DATA TELLS US
THAT.
YOU'RE LIMITED TO THAT REGION IF
YOU WANT TO STUDY HOW THAT PART
OF THE GENE EVOLVED.
>> ANOTHER QUESTION ASKS, WHY
DPP4 AND ACE-2, ARE THERE
STRUCTURAL SIMILARITIES BETWEEN
THE TWO, FOR EXAMPLE, WHAT WOULD
BE THE RATIONALE FOR A VIRUS
EVOLVING, ABILITY TO BIND ONE
AND THEN BOTH?
>> WELL THEY ARE NOT BINDING
BOTH.
THEY ARE BINDING ONE OR THE
OTHER.
SO, YEAH, YOU KNOW, KIND OF GETS
AT SOME WORK, REALLY COOL WORK
FROM TOM GALLAGHER'S LAB LOYAL.
THE SPIKE BINDS TO RBD, PROTEASE
PROCESSES THE SPIKE, AND THEN
THAT RELEASES THE FUSION
PEPTIDE, ALLOWS FUSION TO OCCUR
AT CELL SURFACE.
TOM SHOWS DPP4 IS STRUCTURALLY
COUPLED TO THE PROTEASE NEEDED
FOR THAT EVENT THROUGH A BRIDGE
MOLECULE, TETRA SPANNING
MOLECULAR RAFT IN THE MEMBRANE.
THEY NEED TO BE IN CLOSE
PROTECTION I FOR --
PROXIMITIESTY FOR ENTRY.
YOU CAN HAVE HIGH EXPRESSION IN
THE CELL BECAUSE BECAUSE THEY
ARE NOT LINKED THERE'S NO ENTRY.
THAT'S SOMETHING WE'RE ONLY
REALLY JUST STARTING TO SCRATCH
THE SURFACE ON WITH A LOT OF
THESE VIRUSES, BUT WHAT I
SUSPECT BECAUSE ALL THESE
CORONAVIRUSES USE THE SAME
PROTEASE OR SAME FAMILY OF
PROTEASES, I THINK THOSE
PROTEASES ARE NATURALLY COUPLED
TO DPP4 AND ACE-2, POTENTIALLY
SOME OTHER CORONAVIRUS
RECEPTORS.
>> I THINK THAT'S A COMPELLING
EXPLANATION.
>> SO, YEAH, THAT PUTS PRESSURE
THEN ON THAT VIRUS SO IF IT
EVOLVES TO USE A DIFFERENT
RECEPTOR CAN IT USE A TOTALLY
DIFFERENT RECEPTOR THAT TAKES
AWAY NEIGHBORING PROTEASE
BECAUSE THAT WOULD REQUIRE
EVOLUTION TO OCCUR AT BOTH
PROTEASE SITE AND RBD KIND OF
THEY SAME TIME SO IT'S EASIER
FOR A LITTLE POINT MUTATION AND
DELETION TO RESURFACE PROTEIN TO
STICK TO A NEIGHBORING MOLECULE
THAT STILL MAINTAINS THE REST OF
THE ESSENTIAL INTERACTIONS.
>> THIS MAKES ME THINK OF THE
RECENT PAPERS SHOWING HUMAN
SEASONAL CORONAVIRUS CAN USE
TMPRSS2 AS A RECEPTOR.
>> EXACTLY.
ANOTHER EXAMPLE OF A CORONAVIRUS
USING ONE OF FOUR OR FIVE
PROTEINS THAT SUSPECT IN SOME
WAY ARE PROBABLY COUPLED IN THE
CELL SURFACE.
AND VARYING DEGREES, MAYBE CELL
TYPES, YEAH, SOMETHING LIKE
THAT.
>> RELATING TO THESE QUESTIONS
THERE'S ONE ABOUT, YOU KNOW, IS
DPP4 UNDER POSITIVE SELECTION IN
MAMMALS AS THERE HAVE BEEN SOME
STUDIES SHOWING POSITIVE
SELECTION IN ACE-2?
>> YEAH, THERE IS A LITTLE BIT
OF -- THERE ARE SOME SIGNS OF
POSITIVE SELECTION BUT A LOT OF
THEM ARE AT SURFACES THAT
AREN'T -- WELL, THAT'S THE
THING.
WE DON'T KNOW ALL THE SURFACES
THAT WOULD INTERACT WITH THE
VIRUS BUT THEY ARE NOT AT A LOT
OF SURFACES WE KNOW WOULD
INTERACT WITH THOSE MOLECULES.
THEY ALSO DPP4 HAS ITS OWN
BIOLOGICAL FUNCTION IN
CHOLESTEROL PROCESSING, AND
THERE'S VARIATION IN SOME OF
THOSE POINTS.
WHICH IS KIND OF AN INTERESTING
THING.
SO, IT'S NOT ENTIRELY CLEAR IF
THERE'S A LOT OF -- I THINK
MAYBE THAT PART OF THAT PROBLEM
MIGHT BE WE DON'T KNOW THE EXACT
SPECIES THAT MERBECOVIRUSES
CIRCULATE IN.
WE KNOW SOME BAT SPECIES BUT WE
DON'T THINK IT CAUSES A LOT OF
PATHOLOGY IN THOSE ANIMALS WHICH
REDUCES OUR ABILITY TO SEE
PROBABLY A LOT OF STRONG
SELECTION.
>> THAT BRINGS UP ANOTHER
QUESTION.
WHAT ABOUT THE AGE OF THESE
VIRUSES?
FOR EXAMPLE, IN BATS, IF WE'RE
CONSIDERING THEM THE CLOSEST
THING TO A RESERVOIR HOW LONG DO
YOU THINK THEY HAVE BEEN
HARBORING THESE VIRUSES?
>> I'D HAVE TO GO BACK AND LOOK
AT SOME OLDER MOLECULAR CLOCKS,
WORKING WITH A GROUP NOW USING
THE MINK VIRUSES, SPILLOVER FROM
BATS OR MINK LIKELY OCCURRED IN
THE '90s.
SO, WE KNOW THAT FOR MERS
CORONAVIRUS THAT'S BEEN
RETROACTIVELY FOUND IN POSSIBLY
SERUM SAMPLES, SERUM RESPONSE IN
SAMPLES FROM THE '80s.
SO HUMAN SAMPLES FROM THE
'80s.
SO THESE VIRUSES HAVE BEEN
CIRCULATING FOR A WHILE,
DECADES, BUT I DON'T KNOW ABOUT
THE EXACT, YOU KNOW, HOW FAR IT
GOES BACK.
BUT LIKELY QUITE A WHILE.
>> I MEAN THE WHOLE QUESTION IF
THEY CAUSE DISEASE IN BATS
BRINGS UP THIS IDEA OF HAVE THEY
COEXISTED WITH BATS.
>> FOR A LONG TIME, EXACTLY.
>> FOR A LONG TIME, YEAH.
>> I'D HAVE TO LOOK BACK AT SOME
OF THOSE CLOCKS TO SEE IF PEOPLE
PINPOINTED EXACT SPILLOVER OR
MOST RECENT COMMON ANCESTOR
STUDIES.
>> THAT WAS GREAT.
THANKS FOR FIELDING THOSE
QUESTIONS.
AND AGAIN FOR A REALLY EXCELLENT
TALK.
EVERYONE REALLY ENJOYED THAT.
WE'VE COME UP ON THE HOUR.
SO WE AS A COMMITTEE WOULD LIKE
TO THANK YOU FOR PARTICIPATING
IN OUR SEMINAR SERIES.
>> THANKS FOR HAVING ME.
>> THANKS A LOT.
[END OF PROGRAM]