>> GOOD AFTERNOON, EVERYBODY.
THANK YOU SO MUCH FOR COMING.
I CANNOT TELL YOU, EVEN NOW, SO
MANY YEARS OUT, HOW WONDERFUL IT
IS TO SEE REAL HUMAN BEINGS IN A
ROOM.
BUT WELCOME AS WELL TO THOSE OF
YOU WHO HAVE JOINED US ON
VIDEOCAST.
OUR SPEAKER TODAY IS DR. DANIEL
CHERTOW.
DR. CHERTOW RECEIVED HIS
UNDERGRADUATE DEGREE IN
SOCIOLOGY FROM BOSTON COLLEGE
AND HIS MEDICAL DEGREE FROM
NORTHWESTERN UNIVERSITY.
HE THEN WENT ON TO GET A
MASTER'S DEGREE IN PUBLIC HEALTH
FROM THE UNIVERSITY OF
CALIFORNIA AT BERKELEY.
HE PURSUED RESIDENCY TRAINING IN
INTERNAL MEDICINE AT UCSD, AND
THEN HE WENT IN A SOMEWHAT
UNUSUAL MODE TO BECOME AN
EPIDEMIC INTELLIGENCE SERVICE
OFFICER THROUGH THE CDC.
HE THEN WENT ON TO A FELLOWSHIP
IN CRITICAL CARE MEDICINE HERE
AT THE NIH.
AND IN INFECTIOUS DISEASES AT
JOHNS HOPKINS UNIVERSITY.
DR. CHERTOW HAS WON MANY, MANY,
MANY AWARDS.
I'M JUST GOING TO READ OFF A FEW
OF THEM.
THE UNITED STATES PUBLIC HEALTH
SERVICE MERITORIOUS SERVICE
MEDAL, THE UNITED STATES PUBLIC
HEALTH SERVICE PRESIDENTIAL UNIT
CITATION, THE LASKER BLOOMBERG
PUBLIC SERVICE AWARD, THE LIST
REALLY GOES ON AND ON.
AND REALLY IS AN EMBLEM TO HIS
PASSION FOR TAKING PATHOGENESIS
INFORMATION, INFECTIOUS DISEASE
BASIC INFORMATION, AND BRINGING
THEM TO BEAR ON THE HEALTH AND
WELL-BEING OF THE FELLOW PEOPLE
WHO INHABIT THE PLANET WITH HIM.
WHEN I SAY PLANET, I MEAN
GLOBAL, INTERNATIONAL PLANET.
SO, DR. CHERTOW'S TRANSLATIONAL
RESEARCH PROGRAM EMPLOYS
ADVANCED ANIMAL MODELS AND
DETAILED NATURAL HISTORY STUDIES
IN PEOPLE, TO IMPROVE THE
UNDERSTANDING OF THE
PATHOPHYSIOLOGY AND THE
MOLECULAR PATHOGENESIS OF
EMERGING VIRAL INFECTIONS, AND
UP UNTIL RECENTLY THAT INCLUDED
INFLUENZA A, EBOLA, AND ZIKA
VIRUSES BUT AS YOU'LL HEAR WE
CAN ADD SARS-COV-2 TO THAT LIST.
AND THE INTENT IS REALLY TO USE,
AGAIN, THIS VERY BASIC
INFORMATION TO GUIDE CLINICAL
MANAGEMENT, DIAGNOSIS,
UNDERSTANDING OF HUMAN DISEASE.
SO HIS TALK TODAY IS ENTITLED
"REVIVING POSTMORTEM STUDIES TO
CLARIFY INFECTIOUS DISEASES
PATHOGENESIS."
WELCOME, DR. CHERTOW.
[APPLAUSE]
>> THANK YOU, DR. SCHOR, FOR THE
VERY KIND INTRODUCTION.
IT IS MY ABSOLUTE PLEASURE TO BE
HERE.
SO, GOOD AFTERNOON.
AND THANK YOU ALL FOR JOINING IN
PERSON AND ONLINE TODAY.
AS DR. SCHOR MENTION, THE TITLE
IS REVIVING POSTMORTEM STUDIES
TO CLARIFY INFECTIOUS DISEASES
PATHOGENESIS.
I SAY REVIVING BECAUSE AUTOPSIES
HAVE LONG BEEN A TOOL FOR
ESTABLISHING CLINICAL AND
PATHOLOGICAL CORRELATES OF
DISEASE.
PICTURED HER IS SIR WILLIAM
OSLER PERFORMING A BRAIN AUTOPSY
IN THE LATE 1800s AT A
PHILADELPHIA HOSPITAL, ALSO
RECOGNIZED AT THAT TIME
INVESTIGATING CAUTIONS OF DEATH
DIRECTLY INFORMS DISEASE
PREVENTION AND TREATMENT.
IN RECENT DECADES, HOWEVER, THE
FREQUENCY OF AUTOPSIES HAS
SIGNIFICANTLY DECLINED DESPITE
INCREASED AVAILABILITY OF MANY
NO POWERFUL TOOLS TO INVESTIGATE
DISEASE PATHOGENESIS WITHIN
TISSUES.
IN APRIL OF 2020, THROUGH APRIL
OF 2021, DURING THE FIRST YEAR
OF THE COVID PANDEMIC, WE, AND I
MEAN A LARGE CONSORTIUM WHICH
YOU'LL HEAR ABOUT TODAY,
PERFORMED AUTOPSY STUDY OF 44
FATAL COVID-19 CASES RECRUITED
FROM REGIONAL HOSPITALS, AND
THESE AUTOPSIES WERE PERFORMED
AT THE NIH CLINICAL CENTER, FOR
PURPOSE OF LEVERAGING SOME OF
THESE NEW TOOLS TO INFORM
COVID-19 PATHOGENESIS, A NEW
DISEASE THAT HAD SHOWN UP ON THE
SCENE.
THIS WORK WAS ACCOMPLISHED WITH
CLINICAL COLLEAGUES AT THE
UNIVERSITY OF MARYLAND, OUR
CLOSE COLLEAGUES AT THE NATIONAL
CANCER INSTITUTE, ONE OF THOSE
IS HERE TODAY, DR. HEWETT,
CO-COLLABORATOR AND LEAD ON
NUMBER OF THESE STUDIES, MANY
COLLABORATORS I HOPE TO
HIGHLIGHT.
THE OBJECTIVES WERE TO DEFINE
SARS-COV-2 DISTRIBUTION,
CELLULAR TROPISM, PERSISTENCE
AND EVOLUTION IN THE HUMAN BODY,
INCLUDING THE BRAIN, AND TO USE
STANDARD AND EMERGING TOOLS TO
CHARACTERIZE THE CELLULAR AND
MOLECULAR RESPONSES TO INFECTION
WITHIN TISSUES.
TO ACCOMPLISH THESE AIMS, WE
PROCURED TISSUES FROM ALL MAJOR
ORGANS IN 44 FATAL COVID-19
CASES WITH EXTENSIVE BRAIN
SAMPLING IN 11 OF THESE CASES.
WE PRESERVED GREATER THAN 10,000
SPECIMENS ACROSS 85 DISTINCT
ANATOMIC SITES AND BODY FLUIDS.
ADJACENT SPECIMENS WERE
PRESERVED IN RNA LATER, FOR
MOLECULAR STUDIES SUCH AS PCR
AND SEQUENCING, FLASH FROZEN FOR
CELL CULTURE SHOW PRESENCE OR
ABSENCE OF VIABLE VIRUS, AND
FIXED FOR DOWN EXTREME
MICROSCOPY-BASED ANALYSES.
OUR COHORT WAS DIVERSE ACROSS
AGE, SEX, RACE, ETHNICITY.
AT THIS STAGE OF THE PANDEMIC NO
ONE WAS VACCINATED.
THE WA-1, WILD TYPE STRAIN OF
SARS-COV-2 WAS CIRCULATING, KEEP
THOSE THINGS IN MIND US A THINK
ABOUT POTENTIAL GENERALIZABILITY
OF OUR FINDINGS.
77% OF OUR PATIENTS HAD TWO OR
MORE COMORBID CONDITIONS WITH
CARDIOVASCULAR DISEASE, CHRONIC
RESPIRATORY DISEASE, AND
DIABETES, HYPERTENSION, AND
OBESITY BEING THE MOST PREVALENT
CO-MORBIDITIES.
AS SHOWN IN THIS HEAT MAP OF ALL
RNA LEVELS ACROSS TISSUES IN
PATIENTS, WE DETECTED A HIGH
BURDEN OF SARS-COV-2 IN THE
DIGESTIVE TRACT, UPPER AND LOWER
RESPIRATORY TRACT, PREDOMINANTLY
EARLY AFTER ILLNESS, ALSO
IMPORTANTLY OBSERVED PERSISTENCE
OF VIRAL RNA ACROSS THESE
TISSUES FOR MONTHS AFTER ILLNESS
ONSET.
IN WORK LED BY BLAKE WARNER AND
COLLEAGUES IN THE DENTAL
INSTITUTE, THE CELLULAR
DISTRIBUTION OF SARS-COV-2 IN
SALIVARY GLANDS IS SHOWN ON THE
LEFT IMAGE, AND FREQUENT FRIENDS
OF ACTIVATED T- AND B-CELLS
SHOWN IN THE RIGHT IMAGE
PROVIDED INSIGHT INTO THE
MECHANISMS CONTRIBUTING TO THE
LOSS OF TASTE IN COVID-19
PATIENTS.
MOVING DOWN THE RESPIRATORY
TRACT, IN WORK LED BY JENNY
TINGE AND HER COLLEAGUES AT
UNIVERSITY OF NORTH CAROLINA,
THE PRIMARY CELL DEATH EVENT IN
SARS-COV-2 INFECTED AIRWAY
EPITHELIAL WAS WAS DETERMINED TO
BE NECROPTOSIS, A REPRESENTATIVE
IMAGE AMONG EIGHT AUTOPSY CASES
THAT THEY EVALUATED, SHOWING
CO-LOCALIZATION OF SARS-COV-2
NUCLEAR PROTEIN WITH
PHOSPHORYLATED MLK, A LATE
PROTEIN IN THE QUITE
PRO-INFLAMMATORY NECROPTOTIC
CELL-DEATH PATHWAY.
REMAINING IN THE AIRWAYS IN WORK
LED BY RICK BUCHEY AT UNIVERSITY
OF NORTH CAROLINA, MUCUS
PLUGGING DETECTED IN 60% OF 970
SMALL AIRWAYS ACROSS 15 AUTOPSY
CASES.
MUC 5B RATHER THAN 5 AS
PREDOMINATED, SHOWN ON THE RIGHT
IMAGE, REPRESENTATIVE IMAGE ON
THE RIGHT.
PROVIDING NOVEL INSIGHTS INTO
THE CONTRIBUTION OF MUCOUS
ACCUMULATION OF PLUGGING OF
SMALL AIRWAYS TO RESPIRATORY
INSUFFICIENCY AND FAILURE IN
SEVERE COVID-19.
WITHIN THE LUNG PARENCHYMA
ITSELF, WE AND OTHERS HAVE SHOWN
THAT DIFFUSE ALVEOLAR DAMAGE IN
THE EXUDATIVE PHASE IN PANEL A
ON THE LEFT AND PROLIFERATIVE
PHASE, LATER PHASE IN THE PANEL
IN B, OR FIBROTIC PHASE, NOT
SHOWN HERE, THIS DAD IS THE
PREDOMINANT PATHOLOGY IN FATAL
COVID-19 CASES.
BY TAKING OUR CASES, DAVID
KLEINER IN NCI PATHOLOGY LAB WAS
ABLE TO ANNOTATE PRESENCE OF DAD
IN THESE DIFFERENT STAGES ACROSS
EACH OF OUR PATIENTS.
AND THROUGH THAT ANNOTATION WE
DETERMINED THAT BY DAY 14 POST
ILLNESS ONSET, DAD WAS DETECTED
IN THE LUNGS OF ALL OF OUR FATAL
CASES, AND BY ONE MONTH LUNG
FIBROSIS WAS DETECTED IN OVER
50% OF OUR CASES.
WE USED SARS-COV-2 SUBGENOMIC
RNA, A SURROGATE FOR VIRAL
REPLICATION, TO DETERMINE THAT
SARS CoV STOP REPLICATED IN
OUR PATIENTS' LUNG SAMPLES OF
THESE FATAL CASES MEDIAN OF NINE
DAYS AFTER PATIENT RECORDED
ILLNESS ONSET.
DESPITE THE END OF VIRAL
REPLICATION AS NOTED EVERY
PATIENT AFTER DAY 14 DEVELOPED
OR HAVE PROGRESSIVE DAD IN THEIR
LUNGS.
SUGGESTING THAT IT'S NOT JUST
THE VIRUS THAT WE NEED TO TARGET
BUT THAT ACTUALLY WE NEED TO
TARGET THE HOST AND REPAIR
RESPONSES AFTER THE END OF VIRAL
REPLICATION WHEN WE'RE
THERAPEUTICALLY TREATING THEIRS
PATIENTS.
TO BETTER UNDERSTAND WE DESIGNED
AND VALIDATED A GREATER THAN 30
METAL TAGGED ANTIBODY PANEL AND
APPLIED IMAGING MASS CYTOMETRY
TO DEEPLY CHARACTERIZE INNATE
ADAPTIVE IMMUNE CELLS WITHIN
THEIR LUNG SPATIAL CONTEXT.
MYELOID AND PARENCHYMAL CELLS
CAPTURED BY OUR ASSAY INCLUDE
SUBPOPULATIONS OF NEUTROPHILS,
AND MACROPHAGES, AS WELL AS
EPITHELIAL, ENDOTHELIAL,
DENDRITIC CELLS.
WITH THIS PANEL, WE CAN ANALYZE
CHANGES IN CELL POPULATIONS AND
THEIR SPATIAL ORIENTATION
ACCORDING TO OUR WELL
CHARACTERIZED DEMOGRAPHIC
CLINICAL AND VIROLOGIC METADATA
VARIABLES.
IN THE EXAMPLES SHOWN HERE, WE
OBSERVED THAT THE NUMBER OR THE
COUNT OF LUNG MACROPHAGES IN
REGIONS WE EVALUATED, AND THAT
GRAPH IS SHOWN ON THE LEFT, AND
THE NEUTROPHILS, IN THE GRAPH
SHOWN ON THE RIGHT, THAT THOSE
COUNTS DO NOT SIGNIFICANTLY
CHANGE IN THE LUNGS WE EVALUATED
AFTER THE VIRUS STOPS
REPLICATING, AS DETERMINED BY
DETECTION OR NOT OF SUBGENOMIC
RNA IN ASSOCIATED LUNG SAMPLES.
SO THE CONCEPT THERE IS THE
VIRUS STOPS REPLICATING BUT
THESE INNATE IMMUNE CELLS OFTEN
THOUGHT TO CONTRIBUTE TO LUNG
INJURY AND PATHOGENESIS, THEY
PERSIST IN THE LUNGS.
AND I SHOULD NOTE THESE SAMPLES
WERE PARTICULARLY SELECTED TO
HAVE THE ABSENCE OF EVIDENCE OF
BACTERIAL CO-INFECTION IN THEM
WHICH WOULD BE A LOGICAL
RATIONALE TO SAY WHY DO THEY
HAVE ONGOING INNOVATE IMMUNE
CELL RESPONSES IN THESE SAMPLES,
AREAS FREE OF HISTOPATHOLOGIC OR
MOLECULAR EVIDENCE OF
CO-INFECTION.
TO VISUALIZE THE IMPLICATIONS OF
THIS, WE CAN EXAMINE THE LUNG
PARENCHYMA OF A 71-YEAR-OLD
FEMALE WHO DIED 33 DAYS POST
ILLNESS ONSET.
HER LUNG PATHOLOGY AS EXPECTED
SHOWED PROLIFERATIVE AND
FIBROSING DIFFUSE ALVEOLAR
DAMAGE WITH DISORDERED AND
SPARSE ALVEOLAR STRUCTURES,
WHICH ARE INDICATED IN THIS
IMAGE BY EPITHELIAL CELL MARKER
PAN CK, PSEUDOCOLORED MA GENERAL
DA. MAGENTA.
YOU CAN SEE SOME REMNANTS, BUT
VERY FEW, INSTEAD YOU HAVE
ALMOST REMNANTS OF WHAT USED TO
BE AN INTACT ALVEOLI, SCATTERED
THROUGHOUT THIS IMAGE.
WHILE SUBGENOMIC RNA FROM THIS
LUNG SAMPLE IS NEGATIVE, SHE HAD
A PRONOUNCED INTERSTITIAL
MACROPHAGE INFILTRATE INDICATED
IN THIS IMAGE BY CD14 STAINING
WHICH IS PSEUDOCOLORED RED, SO
YOU CAN SEE THIS INTERSTITIAL
MACROPHAGE INFILTRATE, OF COURSE
WE ARE ABLE TO GO ON AND FURTHER
CHARACTERIZE SUBPOPULATIONS OF
MACROPHAGES, I'M NOT SHOWING
THAT HERE.
IN THE SAME IMAGE IN DIFFERENT
SPATIAL ORIENTATION YOU'LL NOTE
THAT THERE ARE CLUSTERS OF
NEUTROPHILS WHICH ARE INDICATED
BY OUR CD15 MARKER, WHICH IS
PSEUDOCOLORED GREEN HERE, BUT
THAT THOSE NEUTROPHILS ARE
PRESENT IN WHAT APPEAR TO BE
ALVEOLAR REMNANTS.
TAKEN TOGETHER, MACROPHAGES AND
NEUTROPHILS ARE DETECTED, 33
DAYS AFTER SYMPTOM ONSET, AND
OCCUPY DISTINCTION SPATIAL
NICHES IN THIS PATIENT'S
SUBGENOMIC RNA AND AS I
MENTIONED BEFORE BACTERIAL
CO-INFECTION FREE, IF YOU WILL,
LUNG SAMPLE.
SUPPORTING THIS IDEA THAT
THERE'S THIS PERSISTENT INNATE
IMMUNE CELL IN THESE FATAL CASES
IN THE LUNGS.
NOW, AS DESIGNED OR IMC PANEL
ALSO ALLOWS US TO CHARACTERIZE
T- AND B-CELL POPULATIONS
INCLUDING NAIVE AND ANTIGENIC
EXPERIENCE, MEMORY OR EFFECTOR
CD4 AND CD8 T CELLS, THAT'S
REPRESENTED BY THIS BIORENDER
IMAGE HERE.
IN CONTRAST, CD4 T CELLS SHOWN
ON THE GRAPH ON THE LEFT, CD8 T
CELLS SHOWN ON THE GRAPH IN THE
MIDDLE, AND B CELLS SHOWN ON THE
GRAPH ON THE RIGHT, THOSE
POPULATIONS AND ALL THREE
INCREASE IN OUR LUNGS AFTER
SARS-COV-2 STOPS REPLICATING.
AND THAT'S, AGAIN, DETERMINED BY
PRESENCE OR ABSENCE OF OUR
SUBGENOMIC RNA AND OUR
ASSOCIATED LUNG SAMPLES.
THERE'S A BOOST IN IMMUNE
ADAPTIVE RESPONSES EVEN AFTER
THE VIRUS STOPS REPLICATING.
NOW, IF WE GO BACK TO OUR LUNG
OF OUR 71-YEAR-OLD FEMALE
PATIENT WHO DIED 33 DAYS POST
ILLNESS ONSET, WE OBSERVE AND
INTERSTITIAL T CELL INFILTRATE.
NOW, THAT IS SHOWN BY OUR CD3
STAINING WHICH IS BLUE, AND IN A
WAY THE DISTRIBUTION OF THOSE T
CELLS SOMEWHAT MATCHES THE
DISTRIBUTION OF OUR MACROPHAGES,
AND OF COURSE ACROSS ALL OF OUR
PATIENTS AND ACROSS ALL OF OUR
TIME WE'LL BE ABLE TO
CHARACTERIZE THE SPATIAL
ORIENTATION OF DIFFERENT CELL
SUBSETS.
WHILE WE HAVE MANY INFILTRATING
T CELLS, WE HAVE MANY FEWER
INTERSTITIAL B CELLS INDICATED
BY OUR CD20 STAIN AND THAT'S
CYAN.
YOU HAVE
YOU HAVE TO HUNT BUT THEY ARE
THERE.
THE WORK IN PROGRESS, THIS
SUMMARY, OVERALL AIMS TO PROVIDE
BETTER UNDERSTAND OF HOW INNATE
AND ADAPTIVE IMMUNE CELLS
INTERACT WITH EACH OTHER AND
OTHER PARENCHYMAL CELLS WE CAN
CAPTURE EPITHELIAL AND WE CAN
CAPTURE ENDOTHELIAL CELLS IN THE
LUNG PARENCHYMA DURING FATAL
COVID-19, THE HOPE IS THIS WILL
PROVIDE US INSIGHTS PERHAPS INTO
WHICH HOST RESPONSES MIGHT BE
THERAPEUTICALLY TARGETED IN
THESE SEVERE AND FATAL CASES,
AND AT WHICH POINT DURING
ILLNESS FROM EARLY TO LATE
DISEASE PROGRESSION.
SO, TO GAIN A BETTER INSIGHT
INTO SARS-COV-2 SPECIFIC T-CELL
RESPONSES IN THE LUNGS OF OUR
FATAL COVID-19 CASES, WE
PERFORMED T-CELL RECEPTOR BETA
SEQUENCE OF SEQUENCING OF LUNG
TISSUES ACROSS OUR COHORT.
V(D)J REARRANGEMENT OF THE CDR 3
VARIABLE REGION OF THE CHAIN
DETERMINES T CELL ANTIGEN
SPECIFICITY.
ADAPTIVE BIOTECHNOLOGIES WHICH
IS THE TOOL WE USED TO DO THE
SEQUENCING USES A PROPRIETARY
FUNCTIONAL MULTIPLEX ASSAY FOR
THE IDENTIFICATION OF T-CELL
RECEPTOR ANTIGEN SPECIFICITY.
THEY'VE DONE A NUMBER OF
FUNCTIONAL STUDIES.
THEY HAVE DETERMINED THE
DISTRIBUTION OF
SARS-COV-2-SPECIFIC CLONOTYPES,
AND THEIR COGNATE ANTIGENS AND
WITH THAT FUNCTIONAL DATASET
THAT IS A PUBLICLY AVAILABLE
DATASET, COULD BE USED AS A
RESOURCE FOR US TO COMPARE OUR
T-CELL RECEPTOR CLONOTYPES, AND
TO UNDERSTAND THE BREADTH, THE
DEPTH, ANTIGEN SPECIFICITY AND
SPECIFIC COV 2 ANTIGENS.
WITH THIS RESOURCE, AS A
COMPONENT OF A LARGER, AGAIN,
ONGOING PROJECT, WE OBSERVED
THAT THE RATIO OF SARS-COV-2
SPECIFIC CLONE TO NON-SPECIFIC
CLONES DOES NOT SIGNIFICANTLY
CHANGE AS A FUNCTION OF
SUBGENOMIC RNA POSITIVE OR
NEGATIVE STATUS, AND THAT'S THE
GRAPH SHOWN ON THE LEFT.
NOW, CLONES, YOU CAN THINK OF
THESE AS INDIVIDUAL T CELLS,
SAYING ALL YOUR T CELLS THAT ARE
SPECIFIC TO COV 2 OVER ALL OF
THE T CELLS PRESENT IN THE LUNG,
THAT'S THE RATIO, DID NOT
MEANINGFULLY CHANGE BEFORE AND
AFTER VIRUS STOPPED REPLICATING.
SIMILARLY THE SAME WAS OBSERVED
FOR SARS-COV-2 SPECIFIC
CLONOTYPES.
NOW A CLONOTYPE HAS AN
ANTIGEN-SPECIFIC TARGET, ONE
CLONOTYPE CAN HAVE MULTIPLE
CLONES EXPANDED.
BUT THE NUMBER OF CLONOTYPES
TELLS YOU BREADTH OF YOUR T-CELL
RESPONSE AGAINST ANTIGENS OF
INTEREST.
AND THE CLONOTYPE DATA ON THE
RIGHT TELLS US THAT THE BREADTH
OF THAT CLONOTYPE, THE RATIO OF
SARS-COV-2 OVER TOTAL DOESN'T
MEANINGFULLY CHANGE BEFORE AND
AFTER.
NOW, THERE'S A NUMBER OF WAYS TO
INTERPRET THIS DATA.
WE'RE STILL WORKING THROUGH IT.
BUT ONE WAY TO INTERPRET IT IS
THAT EVEN AFTER VIRUS STOPS
REPLICATING, WE'VE ALREADY SHOWN
VIRAL RNA PERSISTS IN THE LUNGS,
THAT THERE'S A PERSISTENT AND
DIVERSE T-CELL RESPONSE THAT'S
HAPPENING WITHIN THESE LUNGS AND
IN OUR COHORT THAT'S UP TO SEVEN
MONTHS AFTER INITIAL ILLNESS
ONSET.
NOW, MOVING OUT OF THE LUNGS,
AND INTO THE REST OF THE BODY,
WE DETECT VIRAL RNA IN ALL MAJOR
ORGAN SYSTEMS WITH THE HIGHEST
BURDEN DETECTED EARLY AFTER
ILLNESS ONSET WITH VARIABLE
PERSISTENCE OF VIRAL RNA,
ADDITIONALLY GREW VIRUS,
REPLICATION COMPETENT VIRUS FROM
SITES INCLUDING JEJENUM, HEART,
RENAL GLAND, THE EYE.
THERE'S GENERAL CONSENSUS IN THE
FIELD THAT, AGAIN, THERE'S STILL
SOME DEBATE BUT I THINK NOW
THERE'S GENERAL CONSENSUS IN THE
FIELD THAT SARS CoV HAS
ABILITY TO INFECT TISSUES
THROUGHOUT THE BODY, THERE'S
LESS CLARITY ON HOW THE VIRUS
DISSEMINATES TO ALL OF THESE
SITES.
OUR DATA IN OUR COHORT WOULD
SUGGEST THAT THE VIRUS TRAFFICS
IN THE NON-CELLULAR FRACTION OF
THE BLOOD EARLY IN INFECTION AND
PRIOR TO THE ONSET OF HUMORAL
IMMUNITIES.
WE DETECTED VIRAL RNA IN THE
PLASMA OF 11 OF 16, OR 69% OF
CASES WHO DIED WITHIN 14 DAYS OF
ILLNESS ONSET, THAT'S THE DATA
SHOWN ON TOP.
THE HIGHEST BURDEN OF THAT VIRAL
RNA IN THE PLASMA WAS DETECTED
IN EARLY CASES.
WE QUERIED THE PBMC FRACTION FOR
VIRAL RNA IN CASES WITH AND
WITHOUT VIRAL RNA DETECTED IN
THE PLASMA AND DETECTED ALMOST
NO VIRAL RNA IN THE CELLULAR
FRACTION.
AGAIN, SUGGESTING THAT THIS IS
VIRUS CIRCULATING IN THE PLASMA
AND NOT THE CELLULAR FRACTION.
WITHIN THIS GROUP OF PATIENTS,
PATIENT 27 STUCK OUT.
THEY STUCK OUT AS BEING THE
EARLIEST PATIENT THAT WAS
PRESENT IN OUR COHORT WHO SHOWED
UP AFTER ILLNESS ONSET AS HAVING
THE HIGHEST BURDEN OF VIRAL RNA
IN THEIR PLASMA.
AND IS BEING ONLY ONE OF THREE
PATIENTS IN OUR COMPLETE COHORT,
ALL 44 PATIENTS, WHO WAS FULLY
SERONEGATIVE AGAINST SARS-COV-2
NP AND SPIKE PROTEINS.
IN THIS PATIENT, WE SUCCESSFULLY
GREW VIRUS FROM THEIR PLASMA.
AND WE DETERMINED THAT THAT
VIRUS, THE NEAR FULL LENGTH
SEQUENCE OF THAT VIRUS, HAD
ESSENTIALLY THE EXACT SAME
SEQUENCE AS VIRUS THAT WE
EXTRACTED RNA FROM LUNG AND
HEART.
THIS CASE PROVIDES PROOF OF
PRINCIPLE THAT REPLICATION
SARS-COV-2 CAN TRAFFIC IN THE
BLOOD IN SOME CASES AND CAN SEED
DISTANT TISSUES.
ONE QUESTION THAT NATURALLY
ARISES RELATED TO DETECTION OF
VIRUS IN NON-RESPIRATORY TISSUES
IS POTENTIAL FOR BLOOD TO
CONTAMINATE THOSE TISSUES.
RATHER THAN THE TISSUES
THEMSELVES BEING INFECTED.
TO ADDRESS THIS QUESTION AND
VALIDATE OUR PCR FINDINGS, WE
ALSO UTILIZED IN SITU
HYBRIDIZATION ASSAY PROBING
SPIKE GENE TO CONFIRM INFECTION
OF TISSUES AND DETERMINE CELL
TYPE SPECIFICITY OF INFECTION
WITHIN THOSE TISSUES.
IN TOTAL, WE DETECTED SARS-COV-2
RNA WITHIN GREATER THAN 30 CELL
TYPES ACROSS 35 TISSUES.
NOTABLY, IN ALL TISSUES OUTSIDE
OF THE RESPIRATORY TRACT, WE
DETECTED A PAUCITY OF
INFLAMMATORY OR CYTO PATHOLOGY,
INFLAMMATION OR CYTO PATHOLOGY
DIRECTLY ATTRIBUTABLE TO VIRAL
INEFFECT.
ASIDE FROM THE RESPIRATORY TRACT
IN MAJORITY IF NOT ALL OF OUR
TISSUES WE NOTED AN ABSENCE OF
AN INFLAMMATORY CELL INFILTRATE
AND NOTED ABSENCE OF VIRALLY
ATTRIBUTED CYTOPATHOLOGY.
ALSO IN THE CASE IN EYE, A STUDY
WITH THE NEI, DETECTED
SARS-COV-2 SPIKE RNA IN
SCLEROFIBROBLASTS IN PANEL A,
RETINAL OUTER AND INNER NUCLEAR
LAYERS SHOWN IN PANEL C, AND
CORNEAL EPITHELIUM IN PANEL D
WITHOUT CYTO PATHOLOGY.
AND FINALLY, MOVING ON TO THE
BRAIN, WE DETECTED SARS-COV-2
RNA IN MULTIPLE NON-CONTIGUOUS
REGIONS IN 10 OF 11 BRAINS THAT
WE EVALUATED.
THIS NON-CONTIGUOUS DISTRIBUTION
SUGGESTS HEMO TOGENOUS SEEDING
RATHER THAN RETROGRADE SPREAD
VIA OLFACTORY TRACT ALONE, NOT
SAYING ONE HAPPENS AND THE OTHER
DOESN'T, THEY ARE NOT MUTUALLY
EXCLUSIONIVE, THEY CAN BOTH
HAPPEN.
BUT OUR DATA, BASED UPON THE
DISTRIBUTION, SUGGESTS THAT
INFECTION IN THESE INDIVIDUALS
WAS ATTRIBUTABLE TO HEMOTOGENOUS
SEEDING.
FROM PATIENT 38, THIRD FROM THE
LEFT, THE HASH MARKS ARE
SUBGENOMIC RNA, THAT WAS OUR
MOLECULAR MARKER OF REPLICATION,
SUGGESTING THERE WAS A LOT OF
RNA AND THERE WAS A SUGGESTION
THAT VIRUS WAS REPLICATING IN
THOSE TISSUES.
IT WAS WITHIN THAT TISSUE THAT
WE SUCCESSFULLY ISOLATED
SARS-COV-2 FROM THE BRAIN IN
PATIENT 38, PROVING THE
POTENTIAL FOR VIABLE SARS-COV-2
INFECTION OF THE BRAIN.
FURTHER, WE CONFIRMED SARS-COV-2
INFECTION OF NEURONS IN BRAIN
AND SPINAL CORD VIA FLUORESCENT
HEMO CHEMISTRY, VALIDATED,
CONFIRMING THESE, THOSE IMAGES
I'M NOT SHOWING YOU.
AND CONSISTENT WITH VIRAL
REPLICATION IN THE BRAIN, AND
INDICATIVE OF VIRAL EVOLUTION IN
THE BRAIN, WE DETECTED UNIQUE
SARS-COV-2 SPIKE GENE VARIANTS,
THIS IS LONG READ SEQUENCING
THAT WAS DONE BY ELY AND HIS
GROUP IN NIAID, THAT WE DETECTED
THESE UNIQUE SARS-COV-2 SPIKE
VARIANTS IN BRAIN TISSUE
SPECIFICALLY THALAMUS AND HYPO
THAT
HYPOTHALAMUS, PATIENT 38,
PATIENT WITH THE HOT BRAIN.
THOSE SEQUENCES, THOSE VARIANTS
IN THE BRAIN, WERE NOT DETECTED
IN THIS PATIENT'S LUNG SAMPLES,
SUGGESTING REPLICATION OR
PERHAPS VIRAL EVOLUTION IN
DIFFERENT ANATOMIC SITES.
DESPITE DETECTION OF VIRAL RNA
IN MULTIPLE BRAIN TISSUES WE
AGAIN OBSERVED A PAUCITY OF
INFLAMMATION THAT COULD BE
ATTRIBUTED TO THE - OR CYTO
PATHOLOGY ATTRIBUTED TO THE
VIRUS.
WE HYPOTHESIZE NEURONAL CHANGE
MIGHT BE OCCURRING AT
TRANSCRIPTIONAL LEVEL WITH
POTENTIAL PHENOTYPIC
CONSEQUENCES OR FUNCTIONAL
CONSEQUENCES, TO EVALUATE WE
SELECTED CEREBELLUM FOR A PILOT
STUDY USING SPATIAL
TRANSCRIPTOMICS TO QUERY
DIFFERENTIAL GENE EXPRESSION IN
CEREBELLAR TISSUE.
WE SELECTED CEREBELLUM FOR PILOT
STUDY BECAUSE OF DISTINCT
ANATOMY OF THE CEREBELLUM, THE
FOLIA SHOWN HERE, THAT HAS THIS
NEURON DENSE GRANULAR LAYER,
ADJACENT OUTER MOLECULAR LAYER,
CENTRAL WHITE MATTER LAYER.
WE USED 10X VISIUM PLATFORM UP
TO 14,000 SPOTS ON THIS 11 X
11-MILLIMETER SLIDE.
GIVEN AN APPROXIMATE NEURON CELL
BODY DIAMETER OF ABOUT FIVE
MICRONS, EACH OF THE SPOTS
REPRESENTS THE GENE EXPRESSION
PROFILE OF AROUND 10 NEURONS
WITHIN THE GRANULAR LAYER.
IN THIS EXAMPLE UNBIASED
CLUSTERING OF PATIENT 41
CEREBELLUM SHOWS DOMINANT
CLUSTERS ACCORDING TO GRANULAR
MOLECULAR AND WHITE MATTER
LAYERS, WITH MULTIPLE ADDITIONAL
LESS PROMINENT CLUSTERS THAT
WERE IDENTIFIED.
DECON CONVOLUTION OF CELL TYPES
WITHIN SPOTS FROM EACH LAYER
REVEALS THAT AS EXPECTED
EXCITATORY NEURONS PREDOMINATE
IN THE GRANULAR LAYER OF THE
CEREBELLUM.
FOCUSING ONLY ON THE EXCITATORY
NEURON DENSE CEREBELLAR GRANULAR
LAYER, WE WERE ABLE TO MANUALLY
ANNOTATE THE BURDEN OF
SARS-COV-2 INFECTION ACROSS OUR
PATIENTS ON ADJACENT 5 MICRO
METER SLICE OF TISSUE AS
DETERMINED BY ISH.
SOME PATIENTS INCLUDING 41 AND
42 HAD HIGH BURDEN OF INFECTION
IN CEREBELLAR GRANULAR LAYER,
OTHERS BURDEN OF INFECTION WAS
QUITE LOW.
WHILE WE WERE UNDERPOWERED TO
DETECT SIGNIFICANT GENE
EXPRESSION DIFFERENCES IN
INFECTED VERSUS UNINFECTED
REGIONS OF THE CEREBELLAR
GRANULAR LAYER AT INDIVIDUAL
PATIENT LEVEL, WHEN WE COMBINED
ALL HIGH INFECTION SPOTS ACROSS
ALL OF THE PATIENTS THAT WE
EVALUATED, AND COMPARED THAT
WITH MID-AND LOW INFECTION
SPOTS, WE REVEAL A HANDFUL OF
POTENTIAL -- PREDOMINANTLY
NEURONAL SIGNALING GENES THAT
WERE DIFFERENTIALLY INFECTED,
BETWEEN HIGH AND MID-VERSUS LOW.
NOW, THESE ARE PRELIMINARY DATA
WHICH WE INTEND TO PURSUE
FURTHER.
WITH ADDITIONAL SEQUENCING
APPROACHES AND ATTEMPT TO
CROSS-VALIDATE PHENOTYPIC AND
FUNCTIONAL IMPLICATIONS OF THESE
DIFFERENT -- OF THIS
DIFFERENTIAL GENE EXPRESSION?
IN A HUMAN BRAINING OR ORGANOID
MODEL, LET BY A POSTDOC, SOPHIA,
IN THE AUDIENCE TODAY.
IN CLOSING, I HOPE I CONVINCED
YOU THAT AUTOPSY TOOLS ARE --
AUTOPSIES ARE A POWERFUL TOOL
FOR GLEANING NEW INSIGHT INTO
INFECTIOUS DISEASE PATHOGENESIS,
AND THAT PROPER DESIGN AND
SAMPLE HANDLING BEYOND WHAT IS
ACCOMPLISHED IN STANDARD
AUTOPSIES IS FUNDAMENTAL FOR
SUCCESS, AND I HOPE I'VE
CONVINCED YOU THAT THE NIH
CLINICAL CENTER AND THE
INTRAMURAL RESEARCH PROGRAM AT
THE NIH ARE EXTREMELY WELL
POSITIONED TO MEANINGFULLY
ADVANCE THESE TYPES OF STUDIES
FOR BOTH INFECTIOUS AND
NON-INFECTIOUS DISEASES.
WITH THAT I'M GOING TO TAKE TWO
SECONDS TO JUST GO A LITTLE BIT
OVER MY ACKNOWLEDGMENTS BECAUSE
AS I SAID AT THE BEGINNING THIS
REALLY WAS TEAM SCIENCE.
AND WHILE I TRIED TO HIGHLIGHT A
NUMBER OF INDIVIDUALS WHO
CONTRIBUTED TO INDIVIDUAL
COMPONENTS OF THIS, I'D LIKE TO
JUST SPEND A MOMENT TO
ACKNOWLEDGE A FEW PEOPLE.
SO THESE STUDIES WOULD NOT HAVE
BEEN POSSIBLE WITHOUT OUR
RESEARCH VILLAGE.
I'D LIKE TO THANK AND
ACKNOWLEDGE LARGER NIH COVID
AUTOPSY CONSORTIUM, CALL OUT THE
NIH CLINICAL CENTER ADMISSIONS
DEPARTMENT THAT TOOK LATE NIGHT
PHONE CALLS AND FACILITATED
TRANSFER OF OUR NEXT INDIVIDUALS
FOR POSTMORTEM STUDIES.
I'D LIKE TO ACKNOWLEDGE OUR
CLOSE COLLABORATOR AND CO-LEADS
IN THE NCI PATHOLOGY GROUP, DR.
KLEINER AND HEWITT, AND ALL
THEIR STAFF AND TRAINEES,
ACKNOWLEDGE DR. SYDNEY STEIN
FROM MY GROUP, AND FIRST AUTHOR
ON "NATURE" STUDY AND OTHERS IN
THE CRITICAL CARE MEDICINE
DEPARTMENT PARTICULARLY SABRINA
RAMELLI, FOR HER ENDLESS HOURS
AND KEVIN VANILLA, WHO IS ALSO
IN ATTENDANCE TODAY, FOR HELPING
SO MUCH MOVE THIS WORK FORWARD,
AND DR. ALLISON GRAZIOLI, LEAD
CLINICAL PARTNER AT THE
UNIVERSITY OF MARYLAND, AND SO
MANY OF HER COLLEAGUES ACROSS
UNIVERSITY OF MARYLAND SYSTEM
WHO TIRELESSLY CARED FOR MANY OF
THESE PATIENTS, ULTIMATELY
FACILITATED RECRUITMENT OF
PATIENTS FOR THIS STUDY.
I'D LIKE TO ACKNOWLEDGE MY
COLLEAGUES IN NIAID AND THEIR
LABS FOR THE WORK THEY HELPED
WITH THE VIROLOGY, KAREN
PETERSON AND CLAYTON WINKLER
EVALUATING WHAT'S HAPPENING IN
THE BRAIN, ELY I MENTIONED
HELPED WITH THE LONG READ
SEQUENCING, I'D LIKE TO
SPECIFICALLY CALL OUT JEON ORGU
DIS
THE FREDERICK TEAM, NIDCR, PETER
AND BLAKE, AND CHI CHAN FROM
NEI.
WITH THAT I'LL CLOSE.
THANK YOU FOR YOUR ATTENTION,
AND I'LL TAKE ANY QUESTIONS YOU
MAY HAVE.
[APPLAUSE]
>> THANK YOU.
THAT WAS FABULOUS LECTURE.
A REMINDER OF HOW AUTOPSIES AND
PATHOLOGIES CAN REALLY ENHANCE
AND EXTEND OUR KNOWLEDGE OF SOME
VERY IMPORTANT DISEASES.
I'D LIKE THE AUDIENCE TO GO
AHEAD IF YOU HAVE QUESTIONS, GO
AHEAD AND COME TO THE
MICROPHONE, AND THOSE ONLINE I
THINK IF YOU WOULD LIKE TO TYPE
IN A QUESTION, IT WILL COME TO
MY E-MAIL HERE AND I WILL ASK
DAN THE THINGS YOU'RE INTERESTED
IN.
I'D LIKE TO TAKE THE OPPORTUNITY
TO ASK THE FIRST QUESTION, DAN,
WHICH IS I KNOW YOUR NUMBERS
WERE SMALL, BUT WHAT CAN YOU
TELL US ABOUT THE DEMOGRAPHICS
OF YOUR COHORT, AND COULD YOU
TELL US IF THERE'S ANY SORT OF
IMPACT OF THOSE DEMOGRAPHICS ON
YOUR FINDINGS, GENDER, AGE,
CO-MORBIDITIES, YOU HAVE
INTERESTING FINDINGS BUT WHAT
ELSE CAN YOU TELL US ABOUT HOW
THE INDIVIDUALS WHO ARE
AUTOPSIES MIGHT HAVE AFFECTED
YOUR RESULTS.
>> GREAT QUESTION, DR. CHAN.
ONE OF THE MOST IMPORTANT
ASPECTS OF THE DEMOGRAPHICS AND
COMORBIDITIES OF OUR COHORT
RELATES, AND WE WERE CABLE ABOUT
THIS, GENERALIZABILITY OF OUR
FINDINGS.
WE HAD AN OLDER AND SICKER
COHORT, WILD TYPE VIRUS
UNVACCINATED EARLY.
WE SAW REALLY A SEVERE PHENOTYPE
OF DISEASE, PARTICULARLY IN THAT
PATIENT POPULATION.
THERE'S NO DOUBT IN MY MIND, WE
ALL KNOW THAT OVER TIME THIS
DISEASE HAS CHANGED.
SO WHAT IT EMPHASIZES WE NEED TO
BE CAUTIOUS ABOUT HOW WE
INTERPRET OUR FINDINGS FOR THIS
COHORT.
I WILL MAKE ONE COMMENT ABOUT,
YOU KNOW, HOW MIGHT SOME OF OUR
CLINICAL OR DEMOGRAPHIC
VARIABLES PROVIDE SOME INSIGHT.
ONE OF THE THINGS I DIDN'T TALK
ABOUT IS THAT WE'RE ACTUALLY
LOOKING AT DIFFERENCES IN THE
T-CELL RECEPTOR BREADTH, DEPTH,
SARS-COV-2 SPECIFICITY, NOT JUST
IN THE LUNGS BUT ACTUALLY IN THE
LYMPH NODE COMPARTMENT, SPLEEN
COMPARTMENT AND PBMCs, I
DIDN'T PRESENT THAT PART.
ONE OF THE DEMOGRAPHIC VARIABLES
THAT STUCK OUT IN THAT DATA WAS
AGE.
SO, THE T-CELL RESPONSES IN
TISSUES ACROSS MULTIPLE OF OUR
PARAMETERS, BOTH COV 2 SPECIFIC
AND NON-VARIED AS A FUNCTION OF
AGE, A TEASER FOR WHAT
ULTIMATELY WILL COME OUT LATER.
>> GREAT TALK.
YOUR DATA SUGGESTS A WELL-TIMED
IMMUNOSUPPRESSION MIGHT HELP.
>> DR. OWENS, THANK YOU FOR THAT
QUESTION.
AND AS YOU KNOW, DURING THE
EARLY STAGES OF THE PANDEMIC,
THE NIH AND INTRAMURAL AND
EXTRAMURAL, YOU KNOW, REALLY
MADE SIGNIFICANT EFFORTS TO
BRING MULTIPLE POTENTIAL
THERAPEUTICS INTO CLINICAL
TRIALS AND EVALUATE EFFICACY,
AND A NUMBER OF THOSE HAVE SHOWN
TO BE EFFICACIOUS IN THE SICKEST
OF THE SICK INCLUDING STEROIDS
AND A NUMBER OF OTHER HOST
TARGET THERAPIES.
NOW, WHAT WE KNOW ABOUT THOSE
THERAPIES, AND LOGICALLY AND
BIOLOGICALLY IT MAKES SENSE, IN
THE CONTEXT OF OUR COHORT.
SO, I'M GLAD YOU POINT IT OUT
BECAUSE IF YOU'RE TARGETING ONLY
THE VIRUS, BUT THE VIRUS STOPS
REPLICATING, SOMEWHERE AROUND
DAY 9 OR 14 BUT IN SEVEREST OF
CASES, YOU KNOW, DISEASE
PROGRESSES, MAYBE REPAIR IS
DISORDERED, WE NEED TO BE
THINKING ABOUT HOW WE CAN
TARGET.
SO, I THINK MANY OF OUR
THERAPIES WERE EMPIRIC BUT THE
CONCEPT BEHIND THIS WORK IS THAT
MAYBE WE CAN GET INSIGHT INTO,
YOU KNOW, THE SPECIFIC CELLULAR
TARGET OR TARGETS, THE RIGHT
TIMING, BIOMARKERS THAT ARE
EASILY ACCESSIBLE IN THOSE
INDIVIDUALS, THAT KIND OF GIVE
YOU SENSE OF WHERE THEY ARE
ALONG THE DISEASE COURSE, THAT'S
ALL THE DIRECTION WE WANT TO GO.
WE'RE JUST GETTING STARTED.
>> THANK YOU.
>> SO, DAN, WE HAVE A NUMBER OF
ONLINE QUESTIONS.
THE FIRST ONE IS, WHERE WERE THE
AUTOPSIES PERFORMED AND DID THE
CLINICIANS TAKE SPECIAL
PRECAUTIONS?
>> ALL OF THESE STUDIES WERE
PERFORMED HERE WITHIN THE NCI
PATHOLOGY SUITE.
OUR PRIMARY LEADS ON THIS WERE
STEPHEN HEWITT, HERE WITH US
TODAY, CO-LEAD ON OUR STUDY, AND
DAVID KLEINER.
SO THEY WERE ALL DONE HERE.
AT THAT STAGE OF THE PANDEMIC,
AGAIN, NO VACCINES, SEVERE
PHENOTYPE, WE ALL WORE
ESSENTIALLY WHAT WOULD BE
CLASSIFIED AS BIOSAFETY LEVEL 2
WITH BIOSAFETY LEVEL 3
PRECAUTIONS, SO WE HAD TYVEK
SUITS, GOWNS, GLOVES, TO PREVENT
EXPOSURES.
I THINK VERONICA MAY HAVE HAD --
>> YEAH, I APPRECIATE, I'M
NOT -- THIS IS OUTSIDE MY FIELD
BUT SO FASCINATING, THOUGHT YOU
DID A REALLY GOOD JOB EXPLAINING
THIS STUDY.
BEAR WITH ME BECAUSE I CLEAR
LIAM NOT VERY KNOWLEDGEABLE.
MY QUESTION IS A LITTLE BIT OF A
FOLLOW-UP OF DR. OWENS TRYING TO
UNDERSTAND PROLONGED IMMUNE
RESPONSE.
BECAUSE YOU'RE DEALING WITH
AUTOPSIES, IT'S EASIER TO GET
ACCESS TO DNA SU INTO THERE ANY
WAY OF GETTING ACCESS TO
PROTEINS?
I WONDER DOES PROTEINS FROM THE
VIRUS, THEY ARE STILL PRESENT IN
SOME PATIENTS PROLONGED IN THE
IMMUNE RESPONSE, WHAT DO YOU
THINK IS THE MECHANISM YOU
DEMONSTRATED IN THE PATIENTS
THAT END UP WITH DEATH THERE IS
THIS PROLONGATION OF IMMUNE
RESPONSE BUT ONTO THE -- HOW
THIS IS CARRIED BY THE VIRUS
SEEMS TO BE TRANSFERRED THROUGH
THE BLOOD AND ALL THAT, BUT WITH
RELATES TO LATE ONGOING IMMUNE
RESPONSE YOU DO YOU HAVE ANY
MECHANISMS THAT YOU CAN PROPOSE?
>> I HAVE THOUGHTS ABOUT WHAT
MIGHT BE PROPOSED.
AND THAT'S THE MOST IMPORTANT
THING BECAUSE WE HAVE NOT --
WE'VE MADE OBSERVATION, WE'VE
NOT SHOWN OR PROVEN MECHANISMS,
SO WE ARE VERY CAUTIOUS.
YOUR QUESTION GETS TO THE HEART,
WE'RE ALSO CAREFUL TO SAY THAT
IN THIS STUDY WE DIDN'T STUDY
LONG COVID, RIGHT?
WE STUDIED INDIVIDUALS THAT HAD
SEVERE, YOU KNOW, MAJORITY OF
THEM HAD SEVERE ACUTE COVID,
MAJORITY OF OUR COHORT DIED FROM
SEVERE ACUTE COVID.
THERE ARE A FEW THAT RECOVERED
AND THEN WENT ON TO DIE, YOU
KNOW, AFTER RECOVERING FROM
COVID, BUT IMPORTANTLY THOSE
INDIVIDUALS DIDN'T MEET ANY
CLINICAL SYNDROMIC CRITERIA FOR
QUOTE/UNQUOTE LONG COVID.
NOW, WITH THAT IN MIND, YOU
KNOW, SO WE'RE CAREFUL NOT TO
SAY THAT OUR STUDY DIRECTLY
INFORMS LONG COVID.
WHAT WE DO SAY IS THAT OUR STUDY
PROVIDES PROOF OF PRINCIPLE THAT
PERHAPS IN ANOTHER, YOU KNOW, IN
THIS OTHER DISEASE ENTITY THAT
PERSISTENCE OF VIRAL COMPONENTS
MIGHT BE TRIGGERING OR MIGHT BE
CONTRIBUTING TO PROLONGED
INFLAMMATORY RESPONSE.
THEY MIGHT, YOU KNOW, OTHER
ASPECTS OF THE LONG COVID STORY,
ARE THEY RESULTING IN
RECREDESENCE OF OTHER VIRUSES,
HERPES VIRUSES, THAT MIGHT BE
CONTRIBUTING TO SOME OF THE
CLINICAL MANIFESTATIONS THAT ARE
OBSERVED IN LONG COVID.
I DON'T KNOW THAT YOU ABOUT WE
PROVIDE BIOLOGICAL
POSSIBILITIES, AND OTHERS, WE'RE
NOT THE ONLY ONES, IN HUMAN
STUDIES WHERE WE COLLECTED
TISSUES, IN THE G.I. TRACT, WERE
RNA OR PROTEINS IN INDIVIDUALS
THAT MEET SYNDROMIC CRITERIA WE
PROVIDED ADEQUATE BIOLOGIC
RATIONALE WE NEED TO CONSIDER
THAT, PERSISTENCE OF VIRAL
REMNANTS AND OTHER PROLONGED
AND/OR DISORDERED IMMUNE
RESPONSE CONTRIBUTING TO
CLINICAL MANIFESTATIONS.
WE DON'T HAVE ANSWERS BUT I
THINK WE HAVE RAISED SOME
ADDITIONAL QUESTIONS.
>> WE HAVE A NUMBER OF OTHER
ONLINE QUESTIONS.
WERE YOU ABLE TO VALIDATE SG RNA
FOUND IN THE GREATER THAN 31-DAY
COHORT IN THE SMALL INTESTINE
TISSUES?
>> SO, IF I WENT BACK TO MY HEAT
MAP, WHICH IS IN THERE, WHAT
I'LL SAY IS THAT THE MAJORITY OF
OUR SUBGENOMIC RNA POSITIVE
SAMPLES ACROSS RESPIRATORY AND
NON-RESPIRATORY TISSUES
PREDOMINATED IN OUR
QUOTE/UNQUOTE EARLY COHORT.
WE DEFINED OUR EARLY COHORT AS
LESS THAN OR EQUAL TO 14 DAYS
AFTER, YOU KNOW,
PATIENT-REPORTED ILLNESS ONSET.
SO THE MAJORITY OF RESPIRATORY
AND NON-RESPIRATORY SAMPLES
POSITIVE FOR SUBGENOMIC RNA FELL
IN EARLY COHORT.
NOW, THERE ARE A HANDFUL OF
INDIVIDUALS AND A HANDFUL OF
TISSUES MORE SPORADICALLY WHERE
WE FOUND EVIDENCE OF SUBGENOMIC
RNA LATER IN THE TIME COURSE.
ONE THAT WAS PARTICULARLY
INTERESTING, NOW WELL KNOWN AND
MORE OR LESS ACCEPTED IN THE
FIELD, IS WHAT IS PATIENT 33 IN
OUR COHORT.
PATIENT 33 DIED 76 DAYS AFTER
ILLNESS ONSET, THEIR CLINICAL
HISTORY WAS INTERESTING.
THEY HAD PREEXISTING LUNG
DISEASE, THEY HAD CO-MORBIDITIES
THEY HAD ACUTE COVID, AT THAT
TIME PATIENTS WERE BEING TREATED
WITH STEROIDS FOR SEVERE ACUTE
COVID.
IT WAS A CLINICAL OBSERVATION
THAT OFTEN WHEN YOU WITHDREW
THOSE STEROIDS WHEN YOU TAPERED
THEM, THE DISEASE WOULD FLARE.
SO THIS PARTICULAR INDIVIDUAL
WAS NOT ABLE TO BE TAPERED OFF
THOSE STEROIDS.
AND WAS ON A PROLONGED COURSE OF
STEROIDS AND DIED 76 DAYS AFTER,
EVIDENCE OF SUBGENOMIC RNA AND
NON-RESPIRATORY COMPONENTS,
THERE'S POTENTIAL FOR ONGOING
VIRAL REPRESENTLY TAIGS IN
REMINDERS AND NON-RESPIRATORY
COMPARTMENTS.
>> WERE THERE CLINICAL FEATURES,
SEVERITY OF ILLNESS, ET CETERA.
>> YES, IF I WERE -- YOU'RE
STRETCHING MY MEMORY A LITTLE
BIT.
THE ONE STANDS OUT, OBVIOUSLY,
THAT THE PATIENT 27 I POINTED
OUT IN MY TALK, THAT INDIVIDUAL
PRESENTED QUOTE/UNQUOTE ONE DAY
AFTER ILLNESS ONSET.
NOW, REMEMBER, ILLNESS ONSET FOR
US IS SOMEWHAT OF A CLINICAL
ANCHOR BUT IT IS NOT LIKE AN
EXPERIMENTAL CONDITION.
YOU KNOW, IT'S SUBJECT TO BIAS,
SUBJECT TO PATIENT REPORT.
AND IT'S ALSO -- IT DOESN'T
REFLECT TIMING OF INFECTION.
OF COURSE THERE'S INCUBATION
PERIOD WITH SARS-COV-2, SO THAT
INDIVIDUAL IS PROBABLY INFECTED,
YOU KNOW, AGAIN GUESSING FIVE OR
SEVEN OR EIGHT DAYS BEFORE THEY
HAD SYMPTOMS.
BUT DIRECTLY TO ANSWER THE
QUESTION, TIMING OF PRESENTATION
AFTER ILLNESS ONSET WAS A
DEFINING FEATURE OF INDIVIDUALS
THAT WERE COMPLETELY
SERONEGATIVE.
>> IF I RECALL CORRECTLY THERE
WAS ONE PATIENT THAT HAD A VERY
HIGH VIRAL LOAD IN PLASMA BUT
SEEMED TO HAVE NO ANTIBODY
RESPONSE.
WAS THIS A KINETIC ISSUE OR
DEMONSTRATION OF THE ROLE THAT
ANTIBODIES PLAY IN CONTROL?
HOW WAS THIS FOLLOWED UP?
>> YEAH, VERY GOOD QUESTION.
THAT'S PATIENT 27.
WE HYPOTHESIZE THAT INDIVIDUAL
PRESENTED TOO EARLY AFTER
ILLNESS ONSET, TOO EARLY AFTER
INFECTION FOR THEM TO HAVE AN
ADEQUATE HUMOROAL RESPONSE.
THE ANTIBODY MEDIATED ARM,
ANTIBODIES ARE QUITE GOOD,
OBSINISING VIRUS.
WE HYPOTHESIZE IN THOSE
INDIVIDUALS THAT PRESENTED EARLY
BEFORE THE ONSET OF HUMORAL
RESPONSE, VIRUS CAN CIRCULATE.
ONCE YOU HAVE AD QUIT HUMORAL
RESPONSE, ADEQUATE TITER AND
AFFINITY, CIRCULATING VIRUS IS
PRESUMABLY OBSINISING, CANNOT GO
ON TO AFFECT CELLS AND TISSUES
OUTSIDE RESPIRATORY TRACT.
>> THANKS FOR BRILLIANT WORK,
REALLY OUTSTANDING LECTURE.
I'M CURIOUS ON SOMETHING SIMPLE,
IN THIS COHORT WHAT WAS THE
DISTRIBUTION OF DISEASE IN THE
LUNGS, WAS IT FOCAL OR WAS IT
DIFFUSE, BOTH?
>> YEAH, THE ANSWER IS THAT IT
WAS DIFFUSE.
YEAH, THIS IS -- IT'S AN
OPPORTUNITY TO HIGHLIGHT ANOTHER
POINT WHICH IS AN INTERESTING
POINT IS THAT IN THE LUNG WHERE
WE UNDERSTAND AND BELIEVE THAT
VIRUS SPREADS CONTIGUOUSLY, YOU
KNOW, YOU'LL SEE WHOLE REGIONS
OF AIRWAY AND WHOLE REGIONS OF
LUNG PARENCHYMA THAT ARE
INFECTED.
IN OTHER ORGANS WHERE THE
HYPOTHESIS IS THAT THERE'S, YOU
KNOW, HENOTOGENOU, SEEDING
THAT'S NOT OBSERVED OUTSIDE THE
LUNG.
THE BRAIN, FOR EXAMPLE, WE HAD
INFECTION OF MULTIPLE REGIONS OF
THE BRAIN NOT ANATOMICALLY
CONTIGUOUS, EVEN IN THOSE
INDIVIDUAL REGIONS IT WASN'T
LIKE THE WHOLE SECTION WAS
POSITIVE FOR ISH.
NEIGHBORING CLUSTERS WOULD BE
INFECTED.
I USE THE ANALOGY OF OUR PLAQUE
ASSAY.
WHEN WE EXPOSE A CELL LAYER TO
VIRUS, A NUMBER OF CELLS BECOME
INFECTED, VIRUS SPREADS
CONTIGUOUSLY, YOU GET PLAQUES OF
VIRUS BUT NOT THE WHOLE
MONOLAYER IS INFECTED.
ONLY LATER IF THE VIRUS, YOU
KNOW, REPLICATES IN THOSE CELLS,
THERE'S ABSENCE OF INTERFERON
RESPONSE YOU GET MORE CONFLUENT
CONTIGUOUS SPREAD.
IN MANY TISSUES INCLUDING THE
BRAIN THAT'S NOT WHAT WE
OBSERVED.
WE OBSERVED MORE OF THESE WHAT I
WOULD CALL PLAQUES RATHER THAN,
YOU KNOW, CONTIGUOUS SPREAD BUT
IN THE LUNG, YEAH, THE LUNG DOES
GREAT.
>> SO, THERE'S A NUMBER OF
QUESTIONS HERE ABOUT NEURONAL
INVOLVEMENT.
AND QUESTIONS RELATED TO WHETHER
OR NOT NEURONS EXPRESS AC 2, AND
IF NOT WAS THE VIRUS MEDIATED
THROUGH AC 2, IF NOT HOW WOULD
IT BE SPREAD THROUGHOUT THE
NEURONAL AREAS?
>> SO, ACE-2 IS EXPRESSED ON
NEURONS, THAT WAS KNOWN BEFORE,
IT WAS KNOWN AFTER COVID.
THE EXACT MECHANISM BY WHICH
VIRUS INFECTS NEURONS, I'M --
THAT WORK MAY HAVE BEEN DONE, WE
DIDN'T DO THAT WORK BUT, AGAIN,
THAT WORK WOULD HAVE HAD TO HAVE
BEEN MODELED IN EITHER 2D OR 3D
NEURONAL SYSTEMS LIKE ORGANOIDS
OR OTHER SYSTEMS WHERE THEY CAN
DO, YOU KNOW, TARGETED KNOCKOUTS
OF DIFFERENT RECEPTORS, THOSE
TYPES OF THINGS.
SO, YES, NEURONS HAVE ACE-2.
WHAT THE MECHANISM OF INFECTION
OF NEURONS IS, I'M NOT -- I
DON'T THINK I KNOW THAT.
I KNOW I DON'T KNOW THAT ANSWER.
BUT THE OTHER PART OF THAT
QUESTION THAT'S IMPORTANT IS
EVEN IF A VIRUS INFECTS A CELL,
NOT ALL CELLS ARE CREATED EQUAL
SO SOME CELLS WILL UNDERGO
CYTOPATHIC EFFECT, HAVE A
RESPONSE.
SOME CELLS WILL GENERATE
INTERFERON RESPONSE THAT WILL
PRECLUDE VIRAL BUDDING AND
SPREAD TO NEIGHBORING CELLS, AND
SO JUST BECAUSE A CELL GETS
INFECTED THEY ARE NOT ALL GOING
TO ACT THE SAME WAY.
NEURONS SEEMED TO BE INFECTED AT
A RELATIVELY LOW LEVEL AND DON'T
AT LEAST BASED UPON OUR DATA
APPEAR TO UNDERGO CELL DEATH.
>> YOU MENTIONED INTERFERON.
I WAS WONDERING IF YOU KNOW THE
STATUS OF YOUR PATIENTS AND DID
THEY HAVE ANTI-TYPE I INTERFERON
ABC BECAUSE THAT WOULD MAKE THEM
EXTREME ARE -- ANTIBODIES
BECAUSE THAT WOULD MAKE THEM
EXTREMELY SUSCEPTIBLE.
>> WE DIDN'T PUBLISH BUT WE
WORKED WITH STEVE HOLLINS'
GROUP, THAT WAS QUERYING THE
ROLE OF INTERFERON ANTIBODIES
AND OTHER HOST-DIRECTED
AUTOANTIBODIES AND PERHAPS THEIR
CONTRIBUTION TO SEVERE COVID.
WE PROVIDED PLASMA SAMPLES TO
STEVE'S GROUP AND THEY QUERIED
THIS, WHAT I WOULD CALL A PILOT
STUDY, WAS NOT -- IT WAS NOT
SYSTEMATIC, DIDN'T INCLUDE ALL
OF OUR PATIENTS.
BUT THE RATES OF -- THE PRESENCE
OR QUANTIFICATION OF
HOST-DIRECTED AUTOANTIBODIES
DIDN'T MEANINGFULLY DIFFER IN
OUR COHORT --
>> OTHER PATIENTS.
>> YEAH, TO THEIR OTHER
PATIENTS.
>> ANOTHER QUESTION ABOUT T
CELLS.
I THINK THE THINKING IS THAT
THERE'S A LOT OF
CROSS-REACTIVITY BEFORE WE WERE
EXPOSED TO COVID DUE TO
DIFFERENT COLD VIRUSES.
SO, HOW YOU WERE ABLE TO
DISTINGUISH AND SAY THESE ARE
REALLY COVID-SPECIFIC T CELLS,
DO YOU THINK THAT THEY WERE SOME
RAISED FROM PREVIOUS EXPOSURE TO
DIFFERENT VIRUSES AND
PATHOLOGICALLY IN A WAY --
>> THAT'S TWO PARTS TO THE
QUESTION I'LL TRY TO ADDRESS.
ONE IS I THINK YOU'RE ABSOLUTELY
RIGHT.
THERE'S POTENTIAL FOR
CROSS-REACTIVE T-CELL RESPONSES
FROM EXPOSURE TO THE FOUR PRIOR
KNOWN ENDEMIC HUMAN
CORONAVIRUSES THAT DON'T INDUCE
SEVERE DISEASE.
AND MAYBE WE'RE SEEING SOME OF
THAT IN THE DIFFERENCES THAT
WE'RE SEEING AS A FUNCTION OF
AGE.
I MENTIONED TO YOU ABOUT OUR
T-CELL RESPONSES.
AS FAR AS THE ABILITY TO SAY ARE
THE INDIVIDUAL CLONOTYPES THAT
WE IDENTIFIED BY OUR TCR BETA
SEQUENCING COV SPECIFIC, TO THE
BEST OF MY KNOWLEDGE, YES BUT
THERE'S A CAVEAT, THE CAVEAT IS
THIS.
THAT ANSWER IS DEPENDENT UPON A
FUNCTIONAL ASSAY THAT WAS
PERFORMED BY ADAPTIVE
BIOTECHNOLOGIES USING
PROPRIETARY HIGH-THROUGHPUT
TECHNIQUE THAT DETERMINES
WHETHER OR NOT T CELLS, THESE
ARE T CELLS FOR THEIR ASSAY THAT
WORK FROM THE PBMC FRACTION,
RIGHT?
THOSE ARE THE ONES IMMEDIATELY
AVAILABLE.
WHETHER THEY ARE SPECIFIC
AGAINST, YOU KNOW, KNOWN COV 2
ANTIGENS.
NOW, I THINK THAT THE
SPECIFICITY OF THAT ASSAY IS
LIKELY GOOD, LIKE IN OTHER WORDS
IF THEY SAID THAT THE ASSAYS,
THIS IS COV 2 SPECIFIC AND WE
FOUND THE SAME TCR RECEPTOR THAT
RECOGNIZES WITH THE SAME MHC
CLASS YOU CAN GET CLOSE TO
SAYING OKAY MAYBE.
WHAT IS MISSING IN THE ASSAY IS
SENSITIVITY.
NOW, THEY CAN ONLY TEST SO MANY
T CELLS FUNCTIONALLY, RIGHT?
AND TIME HAS MOVED ON.
SO WHAT I'M CURIOUS ABOUT IN OUR
COHORT IS THE DENOMINATOR THAT I
SHOWED YOU IN THE COUPLE SLIDES,
THE NON-COV 2 SPECIFIC ANTIGEN,
T CELLS, SOME MAY MAY BE SPECIO
COS V 2, MAYBE GOING AGAINST HOT
OR OTHER PATHOGENS, BETTER
INSIGHT WOULD BE HELPFUL.
>> MAY BE AS IMPORTANT AS THE
COVID ONES.
>> A HUNDRED PERCENT.
>> THANK YOU.
>> SO WE'RE ABOUT OUT OF TIME
BUT I THINK NINA, DR. NINA
SCHOR, WOULD LIKE TO BRING US TO
A CLOSE.
>> HONESTLY, I JUST WANT TO
THANK YOU, DAN, FOR A FANTASTIC
DEMONSTRATION OF THE TRUE POWER
OF CLINICAL OBSERVATION AND
METICULOUS STUDY IN GENERATING
TESTABLE HYPOTHESES WITH
MECHANISTIC IMPORTANCE.
CONGRATULATIONS, AND THANK YOU.
>> THANK YOU SO MUCH, DR. SCHOR.
[APPLAUSE]
[END OF PROGRAM]