2-15-24 MSC Perspectives on Medicine - Akiko Iwasaki

February 21, 2024

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00:10OK, I think we've got a quorum here,
00:12so we'll go ahead and get started.
00:13Welcome everyone for joining
00:15their Perspectives on Medicine
00:17series February EDITION.
00:18Hope everyone's staying warm.
00:21I'm very excited today to introduce
00:23our speaker for today's session,
00:25Doctor Akiko Wasaki. Dr.
00:27Wasaki is the Sterling Professor of
00:29Immunobiology and Professor of Molecular,
00:32Cellular and Developmental Biology,
00:35dermatology, and of epidemiology
00:37at the Yale School of Medicine,
00:39and she is also a Principal Investigator
00:42at the Howard Hughes Medical Institute.
00:45Long recognizes.
00:46One of the most distinguished
00:47and respected immunologists,
00:48she has helped develop new methods
00:50of vaccine delivery and contributed
00:52greatly to our understanding of
00:54mucosal immunity and the immune
00:56response to the herpes simplex viruses,
00:58human rhinovirus,
00:59Zika virus and most recently
01:02the SARS COV Two virus.
01:04Her recent work led to the development
01:06of a mouse model for COVID-19 and then
01:08she is also credited with proposing
01:10hypothesis for the causes of long COVID,
01:12the post acute squali of
01:14SARS COVID 2 infection.
01:16She also currently Co leads the
01:18Yale Paxilvid for long COVID trial,
01:20a double-blind randomized control trial
01:22testing the efficacy of Paxilvid and
01:24treating people with long COVID alongside Dr.
01:27Harlan Krumholz.
01:28But above all,
01:29her contributions to science,
01:30which are impossible to cover all
01:32of them here Doctor Wassaki is a
01:34beacon of inspiration for women
01:36in science and a vocal advocate
01:38for combating sexism in academia.
01:40Dr.
01:40Wassaki completed her Bachelor's and
01:42PhD in immunology at the University
01:44of Toronto and her post doctorates
01:46at the National Institutes of Health.
01:48We're very excited to have her join
01:50us today on her talk titled The
01:53Immunology of Post Acute Infection Syndrome.
01:56Doctor Asaki,
01:56thank you so much for joining us.
01:58And I'll turn over to you and
01:59whenever you are ready.
02:01Thank you so much, Wilton.
02:03It's really my pleasure to be here today.
02:05And I'd like to just give you
02:08the most recent update of
02:09what we're doing in the lab.
02:11So let me share my screen.
02:18Can you see this? Yes. OK, great.
02:23So yeah, we've been studying many
02:26different types of viral infections
02:29and thinking about disease
02:31pathogenesis of infectious diseases.
02:33And so today I'd like to give our current
02:36thinking about what may be happening
02:38in post acute infection syndromes,
02:40which includes long COVID.
02:44So the this year's theme for the
02:48seminar series is Shaping the Future,
02:51Exploring Medicine,
02:52Research Society and Beyond.
02:53And I think I'll try to highlight
02:56how I think our research might
02:59contribute to this goal of shaping
03:02the sort of the right kinds of future
03:07where medicine incorporates patients
03:10perspective and true collaboration
03:12with patients to learn about complex
03:15diseases that are not yet clearly
03:18understood and how that can contribute
03:21to both rigor in research as well
03:25as a diagnosis and therapeutics for
03:28post acute infection syndromes.
03:32So before I get there,
03:34I just wanted to kind of pose this question.
03:37How do viruses cause disease?
03:39And I think most of you are probably
03:42think this is such an obvious
03:44question what the answer might be,
03:46but it's a little bit more complicated
03:49than a simple straight answer.
03:51We are constantly exposed
03:53to numerous viral agents,
03:56not all of them are pathogens.
03:59Some of them are in fact encoded
04:02in our genome.
04:038% of our genome is occupied
04:06by endogenous retroviruses,
04:07seemingly not really causing
04:09diseases of steady state.
04:12However,
04:12we've shown that in the case
04:15of lupus patients,
04:16these endogenous retroviral onogens become
04:19target of pathologic antibody responses.
04:22And so you know,
04:23right there are,
04:25we have this endogenous viruses that
04:27could be a trigger of autoimmune
04:29diseases and there are other viruses
04:32that we occasionally encounter
04:34through seasonal exposures,
04:36respiratory infections as well
04:38as those that we have.
04:41You know,
04:42vast majority of us carry as latent
04:44forms a herpes virus family members.
04:47So the relationship that we have
04:50as humans to viruses are you
04:52know really very dramatically
04:54between these kinds of agents.
04:58And so it's possible that the
05:01viruses are causing disease
05:03as a result of damage that is
05:06caused by the viral life cycle.
05:08For instance,
05:09if it's a lytic virus that causes a
05:12license and death of the host cells,
05:15that would be sufficient to cause disease.
05:20Whereas if immune response to an
05:23otherwise innocuous virus infection
05:25could be triggering the disease.
05:27An extreme example I gave of the lupus case,
05:30but there are many other pathogens
05:33that we may be reacting to overtly
05:36in a inadvertently or overtly
05:40to to cause diseases.
05:42And the real answer to this question is
05:45likely a combination of these features
05:47of virus itself causing some damage,
05:49but then the immune responses that
05:52are amplifying such pathologies.
05:56So we've been applying this question
05:59to multiple pathogens including the
06:02herpes simplex virus as well as Zika
06:06virus and rhinovirus and many others.
06:09And of course when the pandemic hit,
06:11it was,
06:12you know,
06:12we were in a in a perfect situation
06:15to be addressing how does SARS
06:18COV two infection cause disease.
06:21And this question has been sort of
06:25tackled by numerous laboratories
06:27around the world.
06:29And the current insights that we have
06:32is essentially a sequence of event
06:36that leads to severe acute disease.
06:38And this happens most dominantly in
06:42older adults and males over females.
06:46And what's happening here is that
06:49there is starting off with an
06:51inability to mount a timely response.
06:54This happens due to aging due to
06:58delayed interferon response as a
07:01result of having genetic lesions in
07:04these pathways or having impaired
07:07on neutralizing antibody responses.
07:10And this leads to replication
07:12and persistence of virus that
07:15sees multiple different organs,
07:17including the obviously from the lung
07:19but including many other organs.
07:21And it's really the excess myelopoiesis and
07:26release of these myeloid cells that are,
07:29you know, 'cause vascular damage,
07:31leakiness as well as blood clot
07:35and tissue damage,
07:36leading to severe cases of COVID.
07:40And so the the world of scientists
07:43really got together and figured
07:46out small pieces of this puzzle.
07:48And now we kind of have a pretty
07:50good idea of what's going on.
07:52And having this kind of insight at the
07:55cellular and molecular level really
07:57help the field to come up with an
08:00appropriate therapeutic modalities to
08:02target each of these defective pathways.
08:07And why is this important?
08:09To really think about? How does
08:11an infectious agent cause disease?
08:15To treat an infectious disease,
08:18we really need to understand
08:19which of the scenarios is
08:21responsible for the pathogenesis,
08:23because depending on that answer,
08:26the treatment strategy will greatly
08:28differ based on that knowledge.
08:30For instance, do we want to
08:31target the virus or do we want
08:33to target the immune cells?
08:34And if So, what part of the immune cells,
08:37the immune system should we be going
08:40after and also finding unexpected
08:43causes of disease will dramatically
08:45change the way we treat patients.
08:47For instance,
08:48the most recent and strong evidence
08:51linking Epstein Barr virus to multiple
08:54sclerosis and and systemic lupus
08:57erythematosis really, you know,
09:00makes us think differently about
09:03prevention of these diseases.
09:05So there's a lot of good reasons why
09:07we need to be thinking about this.
09:12But today I want to focus on this post
09:14acute phase of infectious disease,
09:16which is really these medically
09:18unexplained chronic diseases that happen
09:21after a number of different infections.
09:24And right now the, you know,
09:27underlying mechanisms is unclear.
09:31But it's important to note that
09:33there are number of infectious agents
09:36that can cause these unexplained
09:39post acute infection syndrome,
09:41including many viruses, Ebola,
09:44dengue, Poliosaurus, chikunya ebb,
09:46many others, as well as some
09:50bacterial and parasitic pathogens.
09:52And this is a review that I had the
09:55fortune of riding together with Yan Chokka,
09:57who himself is an MECFS patient.
10:02And this is one of the examples in which,
10:05you know, as I was saying in the beginning,
10:07how do we kind of plot out the
10:10future of medicine?
10:11It really requires collaboration,
10:13a true collaboration and learning
10:15from the patients themselves,
10:17who many of them are experts in the
10:21diseases that they're suffering from.
10:25So what are these post
10:27acute infection syndromes?
10:28There are over 200 symptoms
10:31reported for long COVID alone,
10:33but there are some core symptoms
10:35that most of these patients
10:38share which are listed here.
10:40Exertional intolerance,
10:41severe fatigue and many of these symptoms
10:46also involve neurocognitive impairment,
10:48sensory impairment,
10:50flu like symptoms,
10:52sleep disturbances.
10:53This is one of the major things that happen
10:57in these place conditions Dysautonomia,
10:59myalgia, arthralgia.
11:00These are some of the main
11:03sort of symptoms that are being
11:06reported by people regardless of
11:08how the disease was triggered.
11:13And the reality of these Long post Acute
11:17infection syndrome is quite serious.
11:19It's estimated that a 65 million people
11:22in the world are currently living with
11:26long COVID and about 20 million people
11:30with my Myalgic encephalomyelitis
11:32or Chronic Fatigue syndrome,
11:3525% of whom are bed bound or house bound.
11:40And any CFS can strike people of all ages
11:43and backgrounds and so can long COVID.
11:46And what what we do know is that it does
11:50strike people of female sex as well as
11:55ages between 30 to 50 more dominantly
12:01than in other demographic groups.
12:03But it doesn't mean that people outside
12:06of that cannot be susceptible to this.
12:09So it's really a problem that's
12:12plaguing the world right now and
12:15not only is the labor workforce and
12:21my schools are being impacted,
12:22but also becoming a social,
12:24economic and national security
12:28problem because it impacts everyone.
12:31It you know obviously shortage in the
12:34national security issues labor force will
12:38be a problem for World Peace I would say.
12:42So this is something that I think
12:45the world needs to pay more attention
12:47to and start to really put some
12:50resources to try to figure this out and
12:53provide therapeutics and diagnostics.
12:58So as a basic scientist,
13:00you know we are thinking about four
13:03possible root causes of disease.
13:05There are many sort of downstream
13:08pathologies that you find such as clotting
13:11issues or muscle damage and so on.
13:15But what is actually driving these diseases?
13:17The root causes.
13:19So we I listed four of them here.
13:22Viral reservoir of SARS COV two
13:24could be one of them for long.
13:27COVID autoimmunity that involves T
13:29cells and antibodies may be another
13:32one that can be triggered triggered as
13:35a result of SARS, COV two infection,
13:37tissue damage and dysfunction,
13:39and latent viral reactivation.
13:43As I mentioned earlier,
13:45we are all colonized with the
13:47number of viruses which are mostly
13:49latent and healthy individuals,
13:51but these these viruses can reactivate.
13:54So based on these hypotheses,
13:57we are now using every tools under
14:00the sun to try to figure out what,
14:03if any,
14:04of these root causes might
14:07be resulting in long COVID.
14:09And I'll give you snippets of several
14:12different studies to illustrate
14:14evidence for each of these hypothesis.
14:17So this first study is a Mount Sinai
14:20Yale long COVID study where I have
14:24the fortune of collaborating with
14:26Doctor David Petrino who sees patients
14:28thousands of patients with long COVID and
14:32he's at the Mount Sinai School of Medicine.
14:34These team members, Jamie,
14:36Laura and Dana contributed
14:38significantly to the study.
14:40And on the Yale side, John Klein,
14:43who some of you may know is an MDPHD
14:47student about to to graduate Rahul,
14:50he was an MD student here at Yale.
14:54He's already gone to presidency in California
14:57pay when a post doctor fell on the lab.
15:01Jill J Cox,
15:02a graduate MSDP student here at
15:05Yale in Aaron Ring's lab.
15:07Jeff Gelhausen,
15:08he is a dermatology fellow in my lab.
15:11And Sasha Tabachnikova,
15:13she is a graduate student in the lab,
15:15And David Van **** also contributed
15:18his computational approaches.
15:20And Aaron Ring developed this rapid
15:23extracellular antigen profiling,
15:25which we used to identify
15:27antibody reactivity.
15:30So in this study,
15:32we've recruited five sets of participants,
15:35long COVID participants,
15:3799 people convalescent control.
15:39These are the people who got COVID
15:42around the same time as those who
15:44got long COVID but then recovered.
15:47We have 39 healthy controls who weren't
15:49infected at the time of the study.
15:52There are also healthcare workers from
15:55Yale that contributed blood for a vaccine
15:58study and external long COVID participants.
16:00These are the patients that are seen by
16:03the Yale Pulmonary long COVID Clinic.
16:07And so we collected their blood and analyzed
16:11variety of factors including cell subsets,
16:16activation status using full cytometry,
16:19looking at linear epitope mapping using
16:22serumen psoroscovie 2 antigen profiling,
16:26peptide display, oh sorry,
16:27this is the the saramine peptide
16:29display library and the human
16:31extraprotium library is the one that
16:33I mentioned that Aaron Ring developed.
16:35And then proteomics of plasma as
16:39well as EMR and symptom survey.
16:42And combining these things we did
16:45find significant changes in immune
16:47profiles of those with long COVID.
16:50First, the types of participants
16:53that were recruited.
16:54We're the age group of about
16:5730 to 50 years of age.
17:00That's the the,
17:01the most sort of high risk factor
17:04group that that are seen by the
17:06Mount Sinai long COVID clinic.
17:08We have dominantly female participants
17:12and again this Pais and long COVID
17:16are female dominant disease.
17:18We also focused on acute severity that
17:21was not hospitalized because vast
17:24majority of people with long COVID
17:26were not hospitalized to begin with.
17:29And then based from acute COVID were
17:32were around 400 days plus or minus.
17:35So this is a much later time point than
17:37other studies that have been published.
17:40Looking at the full cytometry data,
17:42we identified that there are increases
17:47in non conventional monocyte populations.
17:50In people with long COVID we have
17:53reduction in circulating dendritic
17:54cell type one.
17:56These are the cells that are very
17:59important for priming T cell immune
18:01responses and we see increased activation
18:03of B cells and double negative B cells.
18:08On the T cell side,
18:10we see reduced circulating tissue,
18:14central memory T cells of the CD4 type,
18:17as well as increases in exhausted T cells.
18:20And another study from UCSF identified
18:24that this increase in exhausted
18:26T cells are seen in SARS COV,
18:29two specific T cells,
18:31so suggestive of persistent virus
18:33driving the Exhaustion phenotype.
18:38We also saw an increases in
18:42cytokine secretion from T
18:44cells including Illinois 2,
18:45Illinois four and Illinois 6.
18:47And long COVID participants were
18:49pretty much the only people who
18:52had diesels that secreted both
18:53Illinois four and Illinois 6.
18:58Looking at the long COVID patients is
19:02antibody responses to SARS COV 2 antigens.
19:06We noticed that the anti spike antibodies
19:09were elevated in those with long
19:12COVID whether we looked at the whole
19:15spike at the S1 region or the RBD.
19:19So this suggested that there again
19:21there may be a viral reservoir that's
19:24driving the increases in these anti
19:28antiviral antibodies over time.
19:30Notice that there is a mark X2.
19:33This denotes the fact that we only
19:36included participants who had two doses
19:39of mRNA vaccine just to remove any impact
19:42of the vaccines in what the antibodies
19:45that we're you know looking at here.
19:48So T cell site and B cell site are
19:51telling us that there may be a SARS
19:53Scooby 2 viral reservoir that may be
19:56driving a chronic immune responses
20:00when we looked at the
20:02levels of plasma factors.
20:04So the way in which you read this
20:07graph is to look at the the right
20:09side which is elevated in people
20:11with long COVID and you see things
20:14like complement C4B elevated and
20:16several different chemokines and
20:19cytokines that are upregulated.
20:21Whereas one factor that really stood
20:24out for us was the cortisol level.
20:27So cortisol level was almost uniformly
20:30reduced in people with long COVID,
20:34and there's also slight reduction of
20:36Illinois 5 S The cortisol was by far
20:40the most impressively different factor
20:42that we find in the people with long COVID,
20:44and this is sort of AZ score
20:46representation of that data.
20:48So the long COVID participants
20:50had much lower levels,
20:51almost half of the level of cortisol
20:55in compared to healthy controls.
20:58And of course cortisol is the urinal
21:01hormone and we wanted to ensure that
21:03the time of collection of blood from
21:05these participants were similar and
21:08indeed they were pretty similar people.
21:11So.
21:12So because this was quite striking,
21:15we wanted to validate this
21:17finding in another cohort.
21:19And so we looked at the blood of
21:22this Yale Pulmonary Clinic patients
21:25and they also exhibited significant
21:28reduction in the levels of cortisol.
21:33So cortisol is a very important hormone.
21:37It's known as a stress hormone but
21:40it's actually very important for the
21:42physiological functioning of the of
21:44the of the Organism on a daily basis
21:47and it's regulated by the central
21:51hypothalamic and pituitary axis.
21:53Hypothalamus secrets the CRH which
21:56then trigger ACTH secretion from
21:58the pituitary which then acts on the
22:01adrenal glands to produce cortisol
22:03which then has a negative feedback
22:06regulation of these upstream hormones.
22:11So we wanted to understand whether the
22:15there was any kind of impact of having
22:17long COVID on the adrenal hormone ACTH level.
22:21And we thought that because the
22:24level of the cortisol is so much
22:27lower than the healthy control,
22:29it would expect an elevated level of
22:31ACTH to compensate for that low level,
22:34but that's not what we saw here.
22:37So this suggests that there may
22:40be a central dysregulation of the
22:43hypothalamus pituitary axis and that's
22:45something that we are now investigating
22:48using MRI and other models that the
22:51animal models that we're creating.
22:55So what about the latent
22:58viral reactivation hypothesis?
23:00To this end, we looked at the antibody
23:04reactivity against lytic proteins
23:06of herpes virus family members.
23:08And what we noted is that there is
23:11an elevated level of IgG against
23:14Epstein Barr virus onnogens P23 and
23:17GP42 as well as varicella zoster
23:20virus GE in patients with long COVID.
23:24And and and this difference cannot
23:27be accounted for by the latent virus
23:32status in these people because when we
23:35looked at the chronic or latent antigens,
23:38antibody reactivity were equal between
23:41controls and long COVID participants.
23:45So this really suggested that there may
23:47have been a recent reactivation of EVV
23:50and VCV in a subset of participants.
23:53And since we reported this study,
23:56there have been two other studies
23:58that have confirmed EBB reactivation
24:01in patients with long COVID.
24:05So the long COVID patients here
24:07again are in purple.
24:09This is the REAP score that illustrates
24:12how much higher the antibody levels
24:15are in people with long COVID
24:18against the ebb P23 and and.
24:22But we use two other types of
24:24approaches to confirm this.
24:26One is to look at the the ceremian,
24:29the linear epitope mapping strategy
24:32to map a particular epitope the
24:35peptide within the GP 42 of EBV.
24:39And here again you see that increase
24:42in the Z score for this antibody
24:46in patients with on COVID BZ Beach.
24:50GE is similarly elevated for reactivity.
24:54And curiously the the EBB reactive antibody
24:59score correlated positively with Illinois 4,
25:02Illinois 6 double positive CD4T cells
25:04which I showed you is pretty much
25:07only present in those with long COVID.
25:09So whether there's a functional link
25:12between the the cytokine secreting
25:14T cells and the antibody score as
25:16something that we're investigating.
25:22And so with David Van **** and Rahul,
25:26they they looked at the our immunological
25:30phenotyping and determined that a handful
25:33of immune factors alone can distinguish
25:36people with long COVID with 94% accuracy.
25:39And those factors turned out to be
25:42things like cortisol reduced levels of
25:45CD4TCM and dendritic cell type one,
25:49as well as increased levels of EBB GP42,
25:52reactive antibody and Galactin
25:54one and a handful of other sort
25:58of cellular and cytokine markers.
26:00So this really kind of painted the
26:03picture that long COVID can be explained
26:06by immunological perturbations and
26:08endocrinological perturbations alone.
26:10And this is something that we really need
26:13to pay attention to with respect to both
26:16biomarker for diagnosis and for treatment.
26:21So we took the same set of data
26:24and now asked the question,
26:26what are the sex differences in
26:28long COVID immune phenotype, if any?
26:30And this is a work done by Julio Silva
26:33who is a current MDPHD student here,
26:36and Takahira Takahashi who's a former postdoc
26:39who who now runs his own lab in Japan.
26:43So taking the same set of data,
26:45we are now dividing people into male
26:48versus female and control versus
26:50long COVID and applying some machine
26:53learning analysis to understand you know
26:56what are the the different features
26:59that are found in these two sexes.
27:02So females with long COVID A females
27:04here are always indicated in blue,
27:06males are in red.
27:08You can see that the symptom burden
27:11is higher in females with long COVID
27:13compared to the males and organ
27:15system involvement is also higher
27:17in the female compared to the males.
27:22This is a very interesting
27:24chart showing that the distinct
27:27symptoms affect males and females.
27:29So at the top, the relative frequency
27:32of symptoms such as sleep and
27:35disorientation and urinary issues are
27:38equally reported in males and females.
27:41However, if you go down this list
27:43further and further on the bottom,
27:45here are female dominance symptoms.
27:48That includes things like dizziness
27:51and body temperature issues, throat,
27:53chest pain, pins and needles,
27:55and so on, which is more frequently
27:58reported in females over males.
28:00And the most sexually dimorphic
28:03symptom was the sexual dysfunction
28:06in male and hair loss in female.
28:10So this this alone tells us that people
28:13are experiencing long COVID differently,
28:15that based on their biological sex and that
28:19there may be different drivers of disease.
28:25Looking at the immune features,
28:27we saw things like the exhausted
28:30T cells being elevated,
28:32particularly in the female cohort.
28:36Females with bone COVID and cytokine
28:38secreting T cells were also
28:41dominantly in females with bone COVID.
28:44Ebb reactive antibodies were
28:45also found mostly in females with
28:48bone COVID indicating that those
28:50features that I showed you just a
28:53few slides ago that distinguished
28:55male distinguished long COVID versus
28:57non long COVID that many of them
28:59are driven by the female patients.
29:04Males also do have differences in
29:07their immune features including
29:09elevated natural killer cells,
29:11TGF beta and April.
29:13These are cytokine levels that are
29:15higher in males with long COVID.
29:21And then when we looked at different
29:25hormones that are circulating
29:27in patients versus control,
29:30obviously testosterone is much higher
29:32in males compared to the females here.
29:35But what you know is that the
29:38females with long COVID have lower
29:41levels of testosterone compared
29:42to their healthy counterpart.
29:44And this is interesting because testosterone,
29:47even though it's mostly thought
29:49of as a male hormone,
29:51has important physiological function in
29:54females and reduced levels of testosterone.
29:56Testosterone could be leading to
29:58some of the symptoms that people
30:01are experiencing and doing this
30:04logistic regression analysis.
30:06Accounting for HDMI and other Co founders,
30:09we noted that the test the per
30:12unit changes in the testosterone
30:14is the top factor that can predict
30:17long COVID status within females.
30:23And conversely, we see that males have
30:25lower levels of estradiol compared
30:28to males with long COVID compared
30:30to their control male counterpart.
30:33And estradiol per unit change
30:37is again the top predictor of
30:40long COVID status in males,
30:42which is better than say,
30:44something like cortisol here.
30:48So this already told us that there's a
30:50very big difference between the sexes
30:53with respect to EBV reactivation,
30:56the cytokine secreting T cells and NK cells,
30:59as well as hormonal changes
31:01that are seen in these people.
31:04And I haven't said much about autoimmunity,
31:07but so when we use the read to identify
31:12broadly what patients might be
31:14reacting to with respect to their IgG,
31:16we did not see a public auto antigen that
31:20like everyone in long COVID group had.
31:24However, we didn't give up.
31:28We, meaning Kayla, saw a postdoc
31:30who's very talented in the lump,
31:32decided to do a functional experiment
31:35where she purified IgG from healthy people,
31:39homeless and control,
31:41severe acute COVID patients
31:43and long COVID patients,
31:45and possibly transfer these into B6 mice.
31:48And she did a battery of behavioral
31:51tests to look at anxiety and
31:55other sort of behavioral issues
31:57that we could measure in mice.
32:02She did many, many tests,
32:04but I'm only going to show you what's
32:06different in the recipients of these animals.
32:10One thing Kayla found was that the
32:13balance and coordination of mice that
32:17are injected with IgG from patients
32:20from with long COVID were quite reduced.
32:23So this is a rotorod analysis where you
32:27let the mice hang on to this rotating
32:30rod and the mice have to kind of keep
32:33balanced in order to hang on to it.
32:36When they lose balance,
32:37they fall off and the latency
32:39to fall is measured in.
32:40Here you can see that the PBS or the
32:44healthy control IgG injected animals were
32:47able to hang on for 200 seconds or so.
32:51However, those who were injected
32:53with IgG from long COVID patients
32:56had a much reduced latency to fall,
32:59indicating that auto antibodies
33:01are sufficient to induce loss
33:03of balance and coordination.
33:07Another change that she noticed
33:09is a grip strength of mice.
33:11Again, animals are allowed to
33:14hang on to this sort of a mesh
33:18and we're measuring the amount of
33:20grip strength that these animals
33:22have until they kind of fall off.
33:24And this indicated that auto antibodies
33:26can induce reduction in muscle
33:28strength because you can look at the
33:30peak force being quite diminished
33:32in those animals that received IgG.
33:37And thermal sensation using hot plate
33:40test also demonstrated that latency
33:43to pain behavior is much shortened,
33:46meaning that auto antibodies are sufficient
33:49to increase sensitivity to thermal pain.
33:53So these tests are now indicating
33:55to us that there's a physiological
33:57change that happened only by injecting
34:00IgG from patients into the mice.
34:02And it also implies that whatever auto
34:04antigens that are being detected are
34:07shared between these two two species.
34:11And finally, I'd like to give you
34:14a little example of tissue damage
34:16and dysfunction that can happen
34:18after even a very mild acute COVID.
34:21This is a study that we had the fortune
34:24of doing with Professor Michelle Monje's
34:26group at Stanford where she's a a
34:29neuroscientist and A and a psychiatrist
34:32who have been studying the impact of
34:35chemotherapy on brain dysfunction.
34:38And based on her knowledge of this area,
34:43we collaborated to look at the impact of
34:47mild respiratory only infection on the CNS.
34:51So a respiratory infection can
34:54cause CNS impact by either directly
34:57infecting brain cells or generating
35:00autoimmunity that targets the brain.
35:03Or it's possible that distal
35:05inflammation alone is capable of
35:08making chronic changes in the brain.
35:11So this study really probes
35:14the third hypothesis.
35:15And the way in which we did this
35:18is to we had an established animal
35:20model in which we can introduce
35:22the viral entry receptor,
35:24human ACE 2 into any organ of choice.
35:28Here we're doing intratracheal
35:30administration so that the lung
35:32is the only organ in which the
35:35Tsarsco V2 can infect and replicate.
35:37Then we give a Tsarsco V2 intranasally
35:40after about a couple weeks.
35:42And then we looked at the brain
35:45sections here and of course there
35:46is no entry receptor in the brain.
35:48So we don't see any Tsarsco
35:50V2 antigen in the brain.
35:52Animals don't lose much weight either.
35:54This is a very mild infection,
35:56but we know that the viral antigen
35:59is abundantly expressed in the lung
36:02around 7:00 and which is cleared
36:04within about seven days post infection.
36:07So using this respiratory only
36:09mild COVID model,
36:11we then went on to look in the
36:13cytokine in the serum and within
36:16the cerebral spinal fluid of these
36:19animals and at seven days post
36:21infection on the left and seven
36:23weeks post infection on the right.
36:25And what we noted is that even at
36:287 weeks post infection you see a
36:31still elevation of cytokines and
36:34chemokines in the both the serum
36:37and serial spinal fluid.
36:38And one thing to know is the
36:41OR elevated CCL 11 expression.
36:45Now CCL 11 has been shown by others
36:48in the past to correlate with
36:51neurocognitive decline during aging.
36:55And so we're seeing the elevated
36:56levels of that here.
37:00And interestingly, when we look in
37:03the brain of these animals either
37:06seven days on on the top or seven
37:08weeks post infection on the bottom,
37:09we see a a consistently elevated
37:13reactive microglia in the subcortical
37:15white matter of two strains of animals.
37:18We tested CD1 and Bob C and you can
37:22see the micrograph showing here.
37:27We also validated or looked at humans.
37:30We validated in mice, we look at humans.
37:33We looked at humans who
37:35have had died with COVID.
37:38So these people had source COVID 2 PCR
37:41positive at the time of death but they
37:44weren't suffering from acute respiratory
37:47distress syndrome at that point.
37:49They they they died of some other causes.
37:52And even though that's the case,
37:55the mild COVID patients autopsy
37:58results show that there is increases
38:00in the reactive Mycoglia in the white
38:03matter of these these patients who
38:05or people who passed away with COVID.
38:10We also see an accompanying impairment
38:14of the hippocampal neurogenesis in seven
38:17days and seven weeks post infection.
38:19You can see that these neuroblasts
38:22are significantly reduced in those
38:24animals that had these mild COVID as
38:28well as elevated levels of CCL 11
38:31that I noted if a few slides ago.
38:34We see this not only in the mouse cerebral
38:37spinal fluid at 7 weeks post infection,
38:40but also in human serum.
38:42This was elevated in long COVID
38:45participants who who had brain fog,
38:51and we also examined some of the
38:53sort of downstream consequences of
38:56having these reactive microglia,
38:58which is expected to impact
39:01other glial cells and neurons.
39:04For instance, the oligodendrocytes
39:06in the corpus callosum of these
39:09animals were also reduced,
39:12as you can see at 7 weeks post infection.
39:16And as a result of that we also
39:20see loss of myelination of axons
39:23within the cingulum of the corpus
39:27callosum as indicated here at 7
39:29weeks and seven days post infection.
39:32So All in all this study that
39:37combines mouse model and human
39:39brain illustrated that even a mild
39:42respiratory psoroscovito infection can
39:44have lasting impact on the CNS both
39:48within the subcortical white matter
39:50as well as within the hippocampus.
39:53And then specifically received
39:55reactive mycoglia elevated in these
39:58tissues accompanied by a reduction
40:01in oligodendrocyte numbers as well as
40:04myelination of axons and a reduction in
40:08neurogenesis within the hippocampus.
40:10So this illustrates that you know,
40:13you don't have to have direct infection
40:16or direct immune pathology in the
40:18brain to have a chronic changes that
40:20could result in cognitive impairment.
40:25So collectively our findings
40:26show that reactivation of latent
40:28viruses like ebb is happening in a
40:30subset of people with long COVID.
40:32We see elevation of auto antibodies
40:35that are functionally pathological in
40:37in the mouse model that we created.
40:40And we also see that hormonal
40:43dysregulation is happening both
40:46likely stemming from the hypothalamus
40:49pituitary axis as well as sex
40:51hormone differences that we see.
40:53And there's a significant difference
40:55in male and female with respect to
40:58the symptoms and organ involvement
41:00and immune features that are
41:02happening in people with long COVID.
41:06So what do we do about these?
41:08Well, we'd love to kind of bring
41:11this forward to something that's
41:13useful in patients.
41:15So now that we kind of see evidence
41:17for these four types of root causes,
41:20one thing that one can do is to
41:22do a randomized clinical trial
41:24coupled with biological analysis.
41:27So for example, autoimmunity,
41:29there are many very useful biologics
41:32and drugs that are currently
41:34being used to treat autoimmunity.
41:36And whether we can use those types of
41:39approved drugs for treatment of long COVID,
41:43it's something to be seen.
41:47As Wilton already mentioned,
41:49we are collaborating with Harlan Krumholz
41:52group to carry out this Yelpak Slovak trial.
41:55This is a decentralized trial in that
41:58anyone in the contiguous US can participate.
42:02It's an online recruitment.
42:05We send the drugs to the the
42:07homes of the participants.
42:08We send phlebotomist to collect blood and
42:12saliva before and after the treatment.
42:14And we are doing a deep symptom
42:18survey all kind of electronically.
42:20And this is a revolutionary thing that
42:23Harlan is doing with this trial and I'm
42:26really kind of excited to be part of it.
42:29We are contributing more of the
42:33biological analysis that accompanies
42:35this and I think that's very important
42:37because let's say Pakslovid improves
42:40the symptoms in 10% of the participants.
42:43We should be able to find biomarkers
42:47of success in those temptations that
42:51then would help others who have those
42:55biomarkers as potential sort of you know
42:58candidates for treatment with Pakslovid.
43:00And it also gives us an empirical
43:03biomarker for success with Paxlovid
43:05which really implies persistent
43:07virus replication as being the the
43:10root cause for those patients.
43:12So this is a 15 day oral Paxlovid trial
43:14which is much longer than the five day
43:17course that we're using for the acute phase.
43:19We think it's 15 days are necessary
43:22because of the slowly replicating
43:24nature of these persistent reservoir.
43:28We're also very excited to have another
43:31trial ongoing which we're just starting.
43:33Actually Doctor was Eli from Vanderbilt
43:36is leading this anti-inflammatory agent
43:39study well Jack inhibitor this is called
43:43reverse LC trial which we are beginning
43:45soon and again we will be contributing some
43:49immunological analysis and insights there.
43:52And finally with David Petrino we're
43:56doing antiviral randomized clinical
43:58trial targeting latent EBB reactivation.
44:01So I think you know knowing some of these
44:04key features are really helping us to
44:07target the right kinds of pathways with
44:10an already approved drug that can we we
44:14are hoping to help patients with on COVID.
44:17So the mystery is there,
44:20but it's being solved and we are
44:22scratching the surface of this mystery,
44:24but at least we're seeing
44:26something underneath this the the,
44:28the thin layer that we are scratching and
44:31very excited to be able to progress in this
44:35both for the diagnosis and therapeutics
44:38and looking further into the future,
44:40of course we want to prevent these
44:42diseases from ever happening and we are
44:45pursuing mucosal vaccine strategies
44:47that would enable people not only to
44:50be preventing severe disease but also
44:52infection and transmission altogether,
44:55which would by definition prevent
44:58post acute infection syndromes.
45:00And we're also looking at therapies
45:02for post acute infection syndromes that
45:05hopefully will inform things like MECFS.
45:07Once we identify the root causes in one pies,
45:11we know we should be able to apply
45:14the same strategy for others.
45:16So I want to acknowledge the the,
45:19the amazing team that I have
45:20the fortune of working with.
45:22I have only was able to tell
45:23you a couple of stories,
45:25but they're just like amazing things
45:27going on right now and the very generous
45:29funders that support our research.
45:32And finally,
45:32just a special thank you to all
45:34the participants and the patients
45:37who provided efforts,
45:38time and their specimen in order
45:40for us to learn together.
45:42So thank you so much for your attention.
45:49Some thank you so much Doctor Rasaki,
45:50that was a great presentation for those
45:53of us who are still in the on the call.
45:55If you have a question,
45:56you can use the Q&amp;A function,
45:58you can just type in your question
46:00and then we'll relay them over.
46:02But again, thank you so much.
46:03I just had a quick question, you know,
46:06especially regarding you know some of
46:08the data you showed about you know,
46:10lower levels of cortisol and like
46:12testosterone or estradiol and some of
46:14those patients who had long COVID.
46:15I think given since long COVID has
46:18all these different symptoms and it's
46:20really hard to sort of diagnose and
46:22and sort of sort of spot like how
46:24feasible would it be to use those sorts
46:26of levels to as like a screening tour
46:28or like a bio marker that people could
46:31clinically use to sort of indicate, hey,
46:33maybe this person does have long COVID.
46:34So just wanted to kind of hear
46:36your thoughts on that.
46:38Yeah. So in in our own cohort,
46:41those handful of markers were sufficient
46:44to very accurately diagnose long
46:47COVID using the machine learning.
46:49So I I think we can get there.
46:52It's just that because every
46:55physician diagnosed this long
46:56COVID slightly differently.
46:58So that that's really kind of the
47:00problem is that because the diagnostic
47:03criteria used are not uniform,
47:07you know we may not be able to use one type
47:11of biomarkers to as a diagnostic tool.
47:14So the Mount Sinai clinic where
47:17these patients were recruited from,
47:19they have pretty strict guidelines
47:21as to how they define long COVID.
47:23And I think that's helping us
47:25come up with these markers.
47:26But so yeah,
47:27it's kind of a chicken and egg question,
47:29right, 'cause if you're not
47:32diagnosing them properly,
47:34those patients cannot be used as a sort
47:37of platform to generate a biomarker.
47:40And so it's kind of a problem both ways,
47:44but we are hoping to be able to,
47:46for instance,
47:47let's say if these four drivers are,
47:50you know, creating the disease,
47:52we should be able to get biomarkers
47:54for each of these.
47:55I mean there's some of them are pretty
47:57simple, right, Like if it's SARS,
47:58COVID 2 that's persistent,
48:00we should be able to just
48:02measure their you know,
48:04viral antigens and circulations or
48:06something like this which some people
48:08are doing already autoimmunity.
48:10You know if we had a panel of auto
48:12antibody we can order right from Quest
48:14that that that should also cover it.
48:16So I think if we can identify more the
48:20molecular sort of agents that we can target,
48:24we're hoping to to be able to
48:25do that as a diagnostic tool.
48:29Awesome. Thank you so much.
48:30It looks like we do have one
48:33question as you know from someone
48:35related to my question was would
48:37you consider the clinical syndrome
48:39of adrenal fatigue to be some sort
48:41of a post acute infection syndrome?
48:44Yeah, great question.
48:45So adrenal fatigue is something
48:47that we are really interested in.
48:50I mean that was a basically the the
48:52biggest thing that hit us in the in
48:55the face right with this analysis.
48:57So right now what we are doing is we
49:01collected A saliva from long COVID
49:04patients over the 48 hours time window.
49:07There are 12 collections and and we're
49:10doing a very kind of detailed high
49:13resolution mapping of the cortisol level,
49:16you know over that time period.
49:18And so we should be able to tell,
49:20you know, is it like a general, you know,
49:23adrenal fatigue and insufficiency or is
49:25there some sort of circadian, you know,
49:28this regulation that's leading to this?
49:32I think there's circadian rhythm is
49:34quite disrupted in these people.
49:36We know that from not only survey but
49:39also from other people's analysis.
49:41Jim Heath for example published
49:43this early on.
49:44So it could be many things happening.
49:47But yeah,
49:47that that that's a really key thing
49:49to nail down and and we're excited.
49:51We just shipped the saliva samples yesterday.
49:54So very,
49:56very exciting. It really is very,
49:58you know, hot off, depressed, awesome.
50:03Next question, are there any lessons
50:05learned from long COVID that could
50:07inform will be relevant to other
50:08conditions like chronic fatigue syndrome?
50:11I really hope so.
50:13That's why we are now including
50:16MECFS patients as a as a separate
50:19cohort as well as you know chronic
50:23Lyme patients because you know as
50:27I mentioned these Pais syndromes,
50:29they are not unique to COVID and the
50:33more we can understand the overlap and
50:36distinction between these diseases,
50:37the more we can sort of
50:39learn from each other.
50:41And so for instance it let's say
50:43you know a third of the people
50:45reactivated EBV and that's sort
50:47of causing some of the symptoms.
50:50I would expect similar kinds of
50:53impact in other conditions like
50:56MECFS and if if a drug works in EBV
51:01long COVID patients that should
51:04also work in EBVME patients.
51:06So, you know,
51:07we're hoping that these are going to
51:09be cross fertilizing and you know,
51:11able to come up with proper treatment
51:14that that's another thing that really
51:16drives me and others in the lab
51:19is the suffering that's happening
51:20and in people with ME it's just,
51:22it's just it's devastating.
51:24People are taking their lives.
51:26So this is an urgent and really
51:29severely unmet medical need.
51:34Yeah. Next question asks,
51:35do you know how diverse the
51:37four factors that you mentioned
51:39are with persistent virus,
51:40auto antibodies, reactivation and
51:42the dysregulation of the hormones?
51:44And what do you think is sort of
51:46the relationship with a chain of
51:48causality between being being between
51:49those four different factors? Yeah,
51:52great question. I wish I had a
51:55longitudinal sampling of all these
51:57people so I can see like what actually
52:00happened over the time course.
52:02But my thinking is that it
52:05all starts with SARS Co V2.
52:07Of course that infection,
52:10if it's not being properly
52:12managed in the body,
52:14the the virus can seed other organs,
52:18things like small intestine or large
52:20intestine where people are finding
52:21viral antigens or in the meninges
52:23there's spike proteins everywhere.
52:25So you know,
52:26these types of events can happen if
52:28you don't control it at the respiratory
52:31tract and that can certainly trigger,
52:34you know,
52:35tissue dysfunction easily imaginable
52:37that can also trigger activation of
52:40bystander immune cells that can start
52:43to create these auto antibodies.
52:46And of course these old antibodies then
52:48can target things like hypothalamus,
52:51brain stem,
52:52whatever that then trigger these
52:55adrenal insufficiency, right.
52:56So I mean there are lots of
52:58arrows that one can draw,
53:00but that's sort of the way
53:01we're kind of thinking about it.
53:02It's just starting with the virus
53:04and all these other triggers
53:05that come come forward.
53:09Awesome. And then a follow up question
53:12for the very first question was there,
53:13are there any connections to long
53:15bacterial infections as well like
53:17the controversial like chronic Lyme
53:20syndrome. Yeah, exactly.
53:21So that that is why we're using this,
53:24we're we're kind of recruiting
53:27people with chronic Lyme.
53:29It is controversial unfortunately,
53:32but I I think we can sort of weed
53:35out some of the controversy by
53:37really focusing on the science,
53:41you know, science alone.
53:42Like it's just ensuring that what they
53:45have is explainable by biological factors.
53:49And if those factors are overlapping,
53:51say with long COVID,
53:52that will bring some sort of insights and
53:55legitimacy to these kinds of conditions.
53:58So, you know,
54:00we're completely open minded.
54:02We're just want to know what's
54:03going on and science will tell us,
54:06you know what way we need
54:07to interpret things but
54:09right. Awesome.
54:10The next question wanted you to
54:12ask if you could sort of elaborate
54:15regarding the the sex hormones,
54:17testosterone and and estrogen
54:18and do you think these are
54:20correlated or are they causative
54:21of the long COVID syndrome?
54:24Yeah. So there's a huge amount of
54:27correlation that I said already.
54:29The lower levels of testosterone really
54:31seems to be a a, a bad, you know,
54:36prognostic factor for developing
54:37all kinds of symptoms and and and
54:40other other immune pathologies.
54:42So testosterone has long been known
54:46to be have a immunosuppressive
54:48impact and it's also thought to be
54:50one of the reasons why women develop
54:53autoimmunity more often than men.
54:56And actually this whole thing started
54:59with a a parent of a a a trans child
55:02contacting me and telling me that the
55:05this child who has long COVID when they
55:08went on to a testosterone treatment,
55:10completely resolved in their symptoms
55:13and then when the testosterone levels
55:16were reduced for the treatment,
55:18they went back back onto the
55:20sort of original symptoms.
55:22So we know that there's anecdotal
55:25evidence of cause and effect,
55:27but obviously you know it's just end of one,
55:30but that that's sort of the the kind
55:32of driving force is by learning
55:34about these types of anecdotes from
55:36patients and really kind of trying to
55:40understand that at the population level.
55:42So yes,
55:43I think there may even be a
55:45cause and effect there,
55:46but something we really need
55:48to do a large scale study,
55:51yeah, awesome.
55:51And I think we'll probably only have
55:54time for one more question just
55:56to make sure we have, you know,
55:57we respect Doctor Osaki's time,
55:59but the next question that was
56:00on the list is, you know,
56:02thank you for your work on long COVID.
56:03Do you find that individual
56:05participants had a combination of
56:07the findings from each of the four
56:08root causes and or do you think,
56:10you know, maybe there's certain
56:11individuals who have a greater
56:13dominance of 1 cause over another?
56:15Yeah, that's definitely our next step.
56:18I mean we we have you know like 100
56:20people in the Mylan COVID study which
56:22is a little bit too small to start
56:25doing this kind of cluster analysis.
56:27But cortisol reduction was a pretty uniform,
56:32so that that that's probably
56:34happening in most people.
56:35But the persistent virus,
56:37persistent source could be two
56:39again it may be happening in
56:41like a third 40% of the people.
56:43Ebb reactivation happening mostly
56:45in females and not in every female.
56:48So you start to see these clusters,
56:50but you know there's definitely
56:52overlapping as well.
56:53So it's not that one person has one
56:55and the other person has another.
56:57It's it's an overlapping cluster.
56:58But we're starting to see these separate.
57:02We need thousands of people to be
57:03able to do that cluster analysis.
57:05But we're hoping to get there.
57:07Yeah,
57:08awesome. And we're just out of
57:09time and we're very sorry that we
57:11couldn't answer every single question,
57:13but want to thank everyone for
57:15joining during their lunch time today.
57:16And thank you for those of you
57:18who asked such great questions.
57:19And then thank you again,
57:20Doctor Saki, for joining us.
57:22That was a very,
57:23very great presentation and
57:24we're glad that please join us.
57:26If anyone has any additional questions,
57:28they can always contact her.
57:29But otherwise,
57:30have a great rest of your Thursday
57:32and and a great weekend.
57:34Thank you so much.
57:35Thank you. Bye, bye.