colitis

>>> good afternoon, everyone. i would like to welcome everyone who is here and who is watching from abroad or wherever you are watching the videocast, to the rola e. dyer lecture established in 1950 in honor of the former nih director, rola dyer, the lecture features internationally

renown researchers who have contributed to infectious diseases. my name is brian kelthal, and i have the honor of introducing this speaker her better w. verger, iv, known as skip. he's the professor and chairman and department of pathology and

immunology, professor of molecular and microbiology and professor of medicine as well as a director and principal investigator of the midwest regional center of excellence in biomedical and emerging infectious disease research at the washington university school

of medicine. skip grew up in florida where he actually sailed competitively and where his father and he were in multiple races together and where he became the real skip, we found out last night. skip was an undergraduate at harvard, graduated magna

cum laude in biology, went to harvard medical school, got his ph.d., doing residency and internal medicine at brigham women's hospital r worked with bernie field, one of the giants, and did a fellowship in infectious disease at washington university in 1990 and has been

there ever since. he's had multiple academic appointments and has recently become professor of pathology and immunology in 2002 and became the professor and chair of proceed pathology and immunology in 2006 and professor of medicine in 2008.

dr. virgi is a field of virology and infectious diseases his laboratory cr studies the are are pathogenesis of acute and chronic diseases, using several experimental models of infection with dna and rna viruses including herpes simplexvirus, a number of studies with gamma

herpes virus 68 and neuro virus together with genetic structural computational sequencing methods his laboratory really defined the mechanisms of many viral and host genes and viral pathogenesis latency, oncogenesis, recently inflammatory bowel disease.

in addition, they discovered the first mirianuro virus, discovered the first culture system for a neurovirus and showed neuro virus infection can trigger cr intestine inflammation in mice who have a hypomorphic mutation in 1601 and this really established the

concept the cooperative roles for viral infection and genetic susceptibility that can result in the development of inflammatory diseases. more recently, they have developed next generation sequencing technology to define the intestinal virome and have

been pioneers in identifying potential roles for virome and diseases such as inflammatory bowel disease and hiventeropathy. adaptive immunity, pathogen persistence and protein secretion. they were one of the first

laboratories to identify nonconical roles of proteins, in this case immunity, b cell development, panocell development and cellular pathogens. they have recently provided fascinating evidence for transkingdom interactions in

regulating immunity. he's recently published five or six really brilliant manuscripts that really have described multiple interactions between organisms in regulating immune systems, and this has included the demonstration of chronic herpes virus infection can

actually be beneficial to its host and stimulate innate immunity and to provide innate immunity to other inner infectious agents such -- other infectious agents such as bacteria. they've destroyed that helmuth infection can activate, for

sight kind action on viral replication. given the fact we all harbor a vast virome in the form of lay tent infections and bacteriophage populations, i think his studies have broad implications for understanding the are susceptibility to

chronic inflammatory and infectious diseases. he's published over 200 papers and chapters, he's won numerous awards and honors including a welcome young investigator award in virology, melancroft scholar, member of the american associates of physicians, member

of the american academy of microbiology and it's recently inducted into the henry kunkle society. he actually performs a number of roles both at washington and abroad and in other institutions in the united states. he has been on steering

committees for the postere institute, for the rogan institute, harvard and mit and he's also been on the steering committee of the nih genome centers for infectious diseases, he's been advisory board member at stanford in immunology program as well as others.

he's really a very well-recognized expert in virology and its interactions with the immune system. finally i should mention that in addition to science he's very committed to education and performs a number of roles at washington university,

administrative roles, as well as teaching and mentoring. this is evidenced by his awards for mentorship and teaching at wash u as well as his contributions to training programs. as you'll find out, he's a very engaging, enthusiastic person

and colleague and speaker really it's a joil to have him visit the nih and to present the dyer lecture today. skip? [applause] >> you can hear me? i'm not 100% sure who brian just introduced.

[laughter] but it is indeed a great honor to be here and to speak in the name of dr. dyer. as you will find, i love infectious diseases and this is one of those things that you are get to do, it's a particular honor to be at the nih to give

this lectureship. so it's much appreciated. plus in each of the meetings today, i have learned something new that i did not know before, and that's why we do this. right? so i'm going to, today, make what's really a genetic

argument, and i'm going to say some things that may be controversial to those who think that all the genes that matter to us are on our own chromosomes. and i'm going to make an argument that you've heard before in terms of the bacterial

microbuy ohm but i'm going to try to add the viruses that live on and in us as genetic elements that create the composite organism that we are and make the argument that some of the dark matter of inheritance and the fact that we don't always understand everything when we

analyze a chromosomal gene might be just because you're ignoring some of the genes in you that don't happen to be on your own chrome tion. so that's the arg -- your own chromosomes, that's the argument i'll make here. i'll start by how we got there

and summarize two studies that gave u.s. the idea that we should pursue an avenue of research towards understanding how viruses that are present in or on us might influence or fundamental biology and immunity and then tell you a series of more recent stories that are

published and then i will end with a set of unpublished data which i think is interesting in regards to how we think about species differences between immune responses. that's the outline of what i will try to accomplish today. so everything is about the

people who work in your lab, and we have groups working in viral persistence, the virome which i'll define and inflammatory bowel disease, a number of different genes and i'll refer to the work done by these people but i want to start by telling you that i have three people who

are on the job market right now, donna mcduff who published this e-life paper showing that herpes viruses can complement immunodeficiency, norman who showed disease in descr bacteria phases, tim nice who found there is such a thing as sterilizes innate immunity.

i used to think that b cells and t cells were required to get rid of viruses, it turns out at least in my set it's not true. this smart man covered that. megan will come out on the job market next year. she's found that bacteria control virus persistence in the

gut and also that inherited changes in the rin heterod microbiome control in the phenotype of the host. these are the kind of young people who if you can, you should hire. so the virome. this is from 2009.

at that time the number of people on efort, at that time about 7 and 3/4 billion, this is a proportion of the total people on efort to be infected with these viruses, and you meadely see there are a number of viruses, amongst them herpes viruses, that are present in

everyone and in fact in this room right now each of you has around ten of these viruses. they are inside your body, you will never get rid of them. they are things like ebv. if i were to swab your throats, most of you i would be able to detect ebv dna in the swab from

your ton singles. it's occurred to us from the two stories i'll start with that we haven't thought enough about this because we know that when you immunocompromise a person, for instance with a i ds, you can get cmv retinitis. the cmv is in equilibrium with

the immune system, in fact all these viral infections are in equilibrium with the immune system, but if that's true, then this is a dynamic interaction. these elements are permanent to us and they're in constant motion stimulating the immune system.

since being recognized by the immune system, and since cytokines made by c cells and t cells and other innate cells, don't just -- it's possible there are substantial effects on the host itself and that's the concept we have come to and that i will be talking to you about.

then i will talk to you about what might be regulating some of these things. is it only our genes? or is it also genes that would be present in other organisms that inhabit us? so this is the concept, that there is a virome and that the

virome is individual to different people and it's constantly in exchange with other mammals, birds, bats, et cetera, and that it evolves very quickly. that they're eukaryotic viruses, endogenous elements such as retrovirus derived elements,

prokaryotic and arkao organisms and that they interact with each other in transkingdom interactions as a virologist forgive me, i'm going to call viruses alive for the sake of this argument, and that this in turn, these interactions interact with genetic variations

to define our genotype/phenotype relationship in terms of systemic inflammation, immuneo phenotype of the host, the basal immune system, we think of how the activation of the immune system works, when a new agent comes in, that would be the immunophenotype of the

individual at the time of challenge, disease susceptibility i think some of these things are clearly good for you, they're mutualistic symbiosis and perhaps individual intervariation would come from the interaction of these viers and genes and of course

traditional viral pathogens which cause disease in the majority of people who are infected with them. that's the concept we've come to test. so how do we get there? so we thought about the virome, changing the immunophenotype and

changing the relationship of the phenotype of the individual and the genotype based on two studies that were published about seven or eight years ago. one of them, a virus we discovered, a chronic nor oh virus interacted with a gene to change the transcriptional

pattern of epithelial cells and generated phenotype tion in mice that actually look just like a subset of patients with crohn's disease. i will show you a few slides from the study to introduce it. and in another, we found that chronic herpes virus infection

could change gene expression in the host and actually protect against bacterial infection and tumors. so in each of these cases we had a chronic virus altering transcription leading to very substantial new phenotypes that did not exist, just with the

mutation or just with the organism. and that's what's made us think about this idea that there's this interaction with the host and the virome that changes biology. so some preliminary slides of those data.

so this is the study here, it's a very simple experiment. we took mice and latently infected them with a gamma herpes virus shown in red here and then challenged them with a very high dose of listerium, an intracellular bacteria, this bacteria killed mice except when

they were latently infected. this lasted for many months, up to six months it's been oabled and in fact -- it's been observed. and we engineered versions of this virus by deleting some genes so we could get acute infection and not latent

infection and this effect disappeared. this is chronic infection protecting the host. this is a class of viruses which most of you have four or maybe five of. we think it's reasonable to consider the possibility that

that chronic infection might alter your susceptibility to bacterial disease, for example. the mechanism was published but i'll just outline it, it turns out that this chronic viral infection sets the innate immune system by stimulating interferon gamma secretion, activating

macrophages, we show in k cells and other studies, those ak vaitd macrophage tion -- activated macrophages protect against infection. they protect the host. if you do a very simple experiment and take an animal and latently infect it and just

harvest the organs and gene shape them, liver, spleen and brain, the first three are from the virus that can establish infection, next three the mutant that's only acute, chronic, acute, chronic, acute, and look at the genes, you can immediately see that the

latently infected animals have substantial changes in just basal gene expression. this is different in the liver, spleen and brain. in fact, if you look at these genes, there's several genes which have been linked in studies to susceptibility to

human diseases, celiac disease, crohn's disease, multiple sclerosis, regulated by these chronic infections. so what this says is that we as transcriptional entities are not the same when we are chronically infected and yet we are all chronically infected by multiple

viruses. so this leads to the idea that maybe these changes in gene expression have very important phenotypic effects on us. so that's an example of the virome regulating the immunophenotype of the host. second story is this chronic

norovirus, which are the cause of 85% of the cr gastroenteritis in the world, not been cultured, we discovered a culture system, reverse genetics and such. so we have a nice animal model. we were studying the genes and found out that we got this interesting crohn's disease-like

phenotype when we combined this virus with this mutation. so this is a virus plus gene interaction. this is a combinatrial concept, the idea you don't get the phenotype with just the gene or virus, it's only the two together.

mice mu taitd for the gene have normal pennet cells, they get colitis when you give them dss and they have normal small intestine, this is the ileum. you take this mutant animal and infect with the virus, you get abnormal pennat cells, much changed response to dss

including some phenotypes that look more like crohn's disease, now we have a completely new phenotype which is ileal atrophy. if you were looking for a genetic effect on ileal atrophy, you would only see it in the presence of this chronic virus

infection. that pathology, if you now look in humans with crohn's disease who are wild type 3at16, these these nice pennat cells, the human with the mutation in the gene have the same panocell abnormality we found in the mouse.

think about what we did, we took mouse with a gene mutation, normal pana cells, added a chronic infection, got a phenotype which is duplicated in humans. here is shown the stain being for those panocell granules, you can see the an although inside

in inside -- the abnormality inside the cell. a similar message is if you look at transcription in the presence of the virus in wild type versus presence of the virus in these mutant animals you get substantial changes in

transcription and it isn't just quantitative, this is for sets of genes, for instance, protein localization, you get complete inversion, things go up instead of down. so what that means is that our transcriptional identity can be influenced in a commonatorial

fashion between a virus and a gene. that's an example of a virome enter acting with a host interaction to cause pathology. i want to respond to a question i was asked at a meeting in argentina which is, dr. virgin, isn't that terribly complicated?

and the answer is yes. but it's not my fault. okay? this is the way biology is. and if we try to look in silos at one picture, we're not going to get the whole picture. so i know it's complicated, but it's not my fault.

so with regard to the immunophenotype f a virus could make a wild type animal more resistant to a bacteria, it occurred to us that a chronic virus infection might actually be able to complement an immunodeficiency, and since people with immunodeficiencies

have broadly variant phenotypes, we thought maybe the presence of a virus could alter such a phenotype. so we collaborated with casanova's group and identified this gene called hoyle in two families who had three children who died from disease both

immunodeficient and hyperinflammatory, we obtained the mice, and i'll summarize, these mice are very immunodeficient, they died when infectdz with tox oplasma and with listeria, but they're the only animals we've studied which have a rather unique phenomena

which is that they're resistant to microbacterium, and chronic herpes infection limiting the activation of this virus. this gene seems to chrome the relationship between inflammation and immunodeficiency, but it allowed us an experimental tool to test

a hypothesis. so this animal doesn't die from so we could look at the effect of this on the genetic susceptibility. so the mechanism of this gene is shown here, it's responsible in this case after bacterial infection for generating

cytokines such as 6, 12, knockouts are in red, at the transcriptional level, each of these genes is essential to resistance are in a mouse. basically in the absence of hoyle, you don't make the cytokines that protect you from listeria infection.

the reason we did this experiment came here. the first three patients that were identified with hoyle deficiency had this disease i referred you to plus a myopathy, but then a number of other patients were published who had mutations, many of which looked

like null mutations but they didn't have the same phenotype. they all, cluck the mice, have the my op -- including the mice, have the myopathy but didn't have the immunodeficiency and inflammation. there are a lot of explanations, maybe the data are not correct,

maybe there's another gene thatted month fiez this, but the other possibility is that maybe these patients and these patients have a different environmental exposure and these patients are lacking something that these patients have that complement the immunodeficiency.

so we developed the hypothesis that chronic herpes virus infection to which these mice are resistant will complement that severe immunodeficiency, and generate autoinflammation the second phenotype. so we did a very simple experiment, we took the knockout

animals infected with the herpes virus and looked for sight kinds and sure -- cytokines and sure enough, these animals when chronically infectdz with the herpes virus are hyperinflammatory. but they are not hyperinflammatory in the absence

of the virus. then we did the key experiment, from our point of view, we took a number of different knockout animals and we asked can the virus complement and transthe genetic immunodwisht -- genetic immunodeficiency. here is data for the hoyle

knockout. here are the animals that died, the hoyle knockout die, wild type die. if you chronically infect the hoyle knockout, you can complement. if you do the same thing for aisle, it complements.

we can make an animal resistant to a virus by many orders of magnitude to a bacterial infection by chronic viral it makes us wonder whether some of the variation in clinical presentation of genetic immunodeficiency might be due to elements of the virome.

so that's an example of phenotypic complementation of a genetic immunodeficiency by influencing the genotype/phenotype relationship in that animal model. now, i introduce #-d the idea and brian mentioned, what croasms the virome -- what

controls the virome, is it just our genes and the immune system which we all know and love? or are there other elements? so we developed a hypothesis that transkingdom interactions, clug all of the sequences in and around the host will alter pathogenesis and immunity and we

did that because that virus plus gene phenomena that i showed you that was the atg16 plus the norovirus, we can actually cure that with antibiotics, so bacteria were required for a virus phenotype that only occurred in the presence of a host gene mutation that really

suggested to us that bacteria were interacting with viruses in a manner controlled by the host immune system. so in each of these stories, it turns out that exploring this concept has led us to some interesting discoveries. interaction between a virus and

an otophagy gene, it turns out that the bacterial microbiome actually controls viral persistence, that's a mechanism by which antibiotics can change chronic virus infection. i'll show you those data. we found that helman infection substantially altered chronic

herpes virus infection including i'll show you some human viral data, and it's threw a sight kind. so since -- through a cytokine. so it's potentially important that there would be an evolutionarily conserved genetic interaction between the inducer

of aisle4 and 13 and a viral promoter and that's what i'll tell you. the concept here is that the virus is controlled by the worm through something we call cytokine competition at a viral promoter, and i'll explain that. i want to prestaining the

argument -- prestage the argument that the preancestral herpes virus is shared between birds, animals and animals, the herpes virus has been studying you far longer than you've been studying the herpes virus. there's been co-evolution with each species from the time the

species separated. so when mammals speciate, their herpes viruses come along with them and co-evolve. so i will show you that that gives the chance for the virus to do some pretty remarkable stuff. experiment.

we took a virus which was engineered when it was latent in a host, if you reactivated it, it would express luciferase and we could measure the amount of reactivation by imaging the amount of light that came out of the mouse and infected with the helmith and looked to see what

happened. the helmith infection reactivated the virus. this is 7 days after the infection, there's significantly more light coming from the animals that have this virus so this is reactivation of the virus.

we showed it by other methods as well. so the worm is reactivating the virus from latency. so our hypothesis was that there's something about the immune response from the worm that actually triggered the virus and that therefore aisle4

which is induced by helmuth infection as a result of th2 response, might increase replication, i won't show you all the data, but testing the hypothesis led to the discovery that aisle4 and 13 make the virus grow faster, require the 6 which is downstream from the

receptor from these, and very interestingly, this is because the viral gene 50 which is essential for growth and reactivation, it's a switch gene basically, when the virus is latent, this gene is off. when the gene turns ork the virus emerges from latency and

makes infectious virus, it turns out these cytokines turn on that that's the mechanism we believe by which the cytokines reactivate the virus. that's shown here. this is just expression of gene 50 from the virus, and if you treat, this is the expression of

the gene without cytokines, if you put interferon gamma which blocks reactivation, you can inhibit the expression of the gene but aisle 4 added here counters. so this gene somehow is sensing both host cytokine that turns it off and a host cytokine that

turns it on. so how does that work? it turns out that there are five different promoters in the virus for this gene, gene 50, and one of them is directly responsive to aisle 46789 this is not the immune system controlling the virus, this is the virus acting

as a sensor by having its own promoters actually respond to host cytokines and we showed that this is through stat 6 binding using chip assays. if this is a true general phenomenon narks we should be able to reactivate a human herpes virus with aisle 4 and we

should be able to use in mice both cytokines and get a greater effect. what i mean by that is we should be able to, if we inhibit reactivation with interferon gamma, werbled see limited reactivation. if we treat the animals with

aisle 4, limited, but if you do both, you get huge amounts of reactivation, this is a logged scale. so it means that the virus is actually a two-signal model. it's using a promoter to sense one thing to turn it off, one thing to turn it on, so it's

actually learned how to sense the immunologic milieu of the host. with regard to the human virus, if we add aisle 4 to cultured cells with cap at that c herpes virus, we induce the expression of similar genes and reactivation of the virus, that

was done at the university of florida. so this is an evolutionarily conserved mechanism by which part of our virome, herpes virus, actually senses the immunologic milieu of the host. helmuth co-infection reactivates tweern a transkingdom

interaction between the worm and virus and the same virus croams the immunophenotype of the host and resistance to bacterial we also at the same time working with david artiss found that this helmuth infection, same infection actually inhibited the cd8 tc response to another

chronic virus, so these interactions are very complicated. the same worm reactivated a herpes virus and inhibited the cd8 response to our norovirus. all right. the second transkingdom interaction story.

the bacterial microbiome controls chronic norovirus there are two effects. one is that the bacteria are actually required for this virus to establish chronic infection and the other is that a specific cytokine and cytokine receptor are involved in that and we can

lef rang that to cure -- leverage that to cure chronic virus infection. this story starts with our discovery of the mary norovirus. it turns out human norovirus, a certain proportion of people chronically shed these viruses for long periods of time,

explaining howmp the virus can cause epidemics even though there's no animal reservoir. some proportion of you in the audience or wherever you're watching this lecture are probably shedding chronic norovirus infection, and occasionally someone shedding

the virus makes food for a picnic and you generate an epidemic for cam nathaning the food or a cruise ship or military base, all of them are susceptible to he dix of this it's -- epidemics of this virus. it's. when we discovered the myriad

virus, we discovered some viruses in orange here which can consistently be shed in the stool and other viruses which cannot. this virus, the blue virus here, it's not attenuated virus, it actually spreads systemically, goes through the spleen and

lymph nodes. this one doesn't. this virus is adapted to the intestine, grifg us a model for understanding the persistent cearng of the norovirus. that's where the story begins. we did a simple experiment, we treated with intoctsz for 14

days -- with antibiotics for 14 days and then infected with the during the reception afterwards, you can ask me why we would ever do this experiment. there was a good reason. it wasn't just luck, i don't think. what happened was when we

treated with antibiotics, the virus which would normally become persistent was actually cleared, and here is shown across many experiments many of the animals are cured, they don't get this persistent infection, even though they clearly have the virus early,

they don't get the infection. so something about the antibiotics is preventing the virus from establishing a chronic infection. that's a transkingdom interaction, bacteria, the is it the bacteria? well, we just did a simple

experiment again, we stopped the antibiotics and we fed fecal pellets, did a fecal transplantation and we restored, this is untreated, antibiotics, fecal feedback, restores the virus and colon, lymph node and shedding in the stools, it's really something in the fecal

material that the antibiotics are removing, and to understand the mechanism, we decided we would assess the requirement for host genes in this effect. we took a broad range of knockout mice and did the same experiment, and these are all genes which are not required for

the antibiotics to work. this is not projecting. that's the interferon alpha-beta receptor, gamma receptor, both the interferon alpha and gamma receptor, are poll like receptors, sensors for the, the antibiotics are fine. there are three genes,

interferon lambda receptor, the antibiotics that did not work. so what this means is that the immune system is somehow involved in this transkingdom that led us to do the following interferon lambda is what is also called type 3 interferon. it's less well studied, it's

been used to treat hepatitis c in humans, it's less well studied than type 1 and type 2 interferons, interferon alpha-beta and gamma and the other. it's stat 1, another one of the genes required for our experiment, we took type 3

interferon supplied by bristol-myers squibb and treated before ferkz and the animals -- before infection and the animals do not become persistently infect. you can pretreat with interferon lambda and the infection does not occur.

we did what i cawsm the key experiment, we waited for the persistence to be established, this is day 21, and the virus is shedding, this is a wild type animal and we treated with interferon lambda and we could cure the persistent infection. in an animal lack being the type

-- lacking the type 1 interferon receptor, it still works, but in an animal lack being the interferon receptor, there's no effect. interferon lambda can cure a persistent virus infection with this norovirus. now, i was always taught that

the innate immune system controls infection until t cells and b cells can come into the fray and clear an infection out and it's sterilizing immunity is due to the effect of t and b cells. but one of the mice that we had studied in the antibiotic

treatment was a rag knockout, which suggested the innate immune system actually can do whatever it is that we're studying here, so we did the following experiment. this is tim nice's key last experiment in his paper. we took persistently infected

rag knockouts and treated them with interferon lambda and cured them. this is by pcr but we also took the fecal material here and put it back into stat knockout and rag knockout mice so we think that is an actual cure of a virus infection in the absence

of t and b cells and that that suggests to us that there is sterilizing innate immunity, which suggests in turn that we might be able to induce this kind of immunity to eliminate virus infection even when t and b cells are compromised. it's an interesting concept to

us. so these are examples of transkingdom interactions where commensal my exproabz a host molecule interferon lambda are responsible for controlling norovirus infection and where we can discover something new about the immune system by leveraging

the initial studies of the transkingdom interactions to identify what parts of the immune system might then in turn be involved in controlling virus infection in the intestine. now, we should be able to demonstrate changes in the virome in disease states.

so we began to look at that in human studies and we performed a study together with miles parks in the united kingdom, ron xavier and ali at rush where we looked at the enteric virome in patients with inflammatory bowel diseetz. this cohort is notable.

because they have a remarkable clinical scenario there, setting there, they could get us household controls and the data is much more powerful when you do shotgun sequencing or metagenomic comparisons of patients, a number of studies have shown that our microbiomes

are related to the people we live with. this was a particularly powerful cohort. it has been long published that disease have abnormalities in their bacterial microbiome. in fact, these patients have decreased species diversity and

species richness compared to controls. so there's something not complicated, decreased complexity of their bacteria, so the first thing we did in our cambridge, chicago and boston cohorts is look at the number of species and the diversity and we

showed that both for crohn's disease and ulcerative colitis, purple and red here, we got exactly what everyone else had published. so this is bacterial 16s data and these patients have an abnormality of the bacterial microbiome, their bacteria are

less complex. so then we purified virus pardon mes from the fecal material -- pardon mes from the fecal material from these tr patients, we desequenced them. what we found was the following, that these are household controls, these are ulcerative

colitis patients and these are crohn's disease patients, and this is the number of different species of bacteria, so the bacteria phages are becoming more diverse and richer, there are more viruses even when there are less bacteria. we believe that it is a stretch

that this decrease in bacterial diversity could explain the presence of greater numbers of different bacteria ya phages. this signature is disease specific in that the viruses in ulcerative colitis are different than the viruses in crohn's. this repeated to a greater or

lesser degree in all of our cohorts. so acroches the ocean and across -- across the ocean and across three different cohorts. so the bacteria which are changing in a downward direction and viruses which are now greater in number.

is there a relationship? so we developed an approach to take the discriminate bacteria, bacteria in the uk cohort which correlate with disease and put them statistically against the top 30 or so viruses and a red dot means there's a negative correlation, that means when

there's more of this phage, there's less of that bacteria, so on, a blue is a positive relationship. these are the household then we did crohn's disease. what's really striking is that the pattern of relationships here and here are completely

different. so there is in fact a signature of the relationship between these bacteriorphages and the bacteria which is in crohn's disease compared to controls and in turn ulcerative colitis is different than either. so these data demonstrate that

there is a correlation between the enteric virome and a human the way we are thinking of this is shown in this picture. this is a picture of a virus. here is another virus. this is a bacteria. and another bacteria. we think there could be a

predator/prey relationship between the bacteria -- between the viruses and the bacteria such that maybe inflammatory bowel disease is actually a viral disease in some senses, that bacteriorphages either emerge or are introduced, that that changes the bacterial

microbiome into a new equilibrium state and that that may, in fact, be how have the viruses might trigger this is purely hypothetical and we're in the process of setting up animal models to try to understand this. now, that's an example of a

transkingdom interaction between a virus and a bacteria. so we've seen worms interacting with viruses, we've seen bacteria interacting with viruses in a positive sense where bacteria were required for persistent virus, and this is a negative relationship, we think,

between a virus and a particular group of bacteria. so that we think is the first example of a relationship between the virome, in this case bacteriorphages and the human what do we think are the implications? we wonder whether this is

relevant to other diseases where the bacterial microbiome has been 50eu6d to be a risk factor, type 1 diabetes, cardiovascular disease, nutritional deficiencies, there's been extensive type 1 and type 2 diabetes, there's extensive data relating the bacterial

microbiome to she's diseases but no one has looked at the viruses yet. so we wonder whether that's a good idea. we also think that probiotic therapies added into the intestine might fall prey to the same bacteriorphages which we

identified and speculative but i wonder whether one could make probiotics, that would be antibiotics which you could put into the host to manipulate the bacteria in a way that was useful for the health of the maybe that could correct some of the abnormality tion in bacteria

that we see in different so again, we're trying to develop animal models to look at these ideas. so coming back to the immunophenotype, i've made an argument that chronic infections can alter the fundamental nature of the immune system.

now, i'm going to show you some unpublished data. this is one of the experiments where i've always wanted to do this experiment and it was merely a matter of me finding someone i could convince to do it because i tried to convince a number of people to do this

experiment and no one was brave enough except for tiffany reese who published that worm paper and who is now a new faculty member at ut western on the tenure track and she deserves credit for this experiment. thrms a bias that mice are not humans and i completely

understand that mice look very different than humans. but there's also a bias that the mouse immune system is not reflective of the human immune system and therefore human immunologists at times look down on the mouse model because they say the mouse model is not

reflective of human immune responses. i cannot get a prediction from a mouse model that is relevant for my human vaccine study. so i understand that concern. but what if there was a way to look mice look more like humans instead of transplanting human

tissues into them, altering their environment? so now think about what we've done with experimental mice. we put them in ever-cleaner environments and then we have made the data from them very reproducible, the same thing happens every time more or less.

but at the same time we've removed them from all of their environmental triggers. but all of us are sequentially infected by herpes viruses and worms and other things, so is it possible that the reason that the immune system in a mouse is different than the immune system

in human is in part not just the chromosomal abnormalities but something about the environment of the mouse? so that's the hypothesis. the idea is that serial co-ferkzs that limb -- co-infections that mimic the early life exposure to humans

might alter the imization. we did a truly massive experiment with a lot of mice and four different replicates and we left some mice uninfected and then just sequentially infected mice with different pathogens, gamma herpes virus, beta herpes virus, then a worm,

we waited various periods of time, then we gave yellow fever vaccine, bled the mice at day 0 and bled them 3, 7, 21 and 34 days after vaccination, we picked this vaccination for a reason that will be clear in a moment. we asked first the question is

the immune system different when the mouse has had a history of being infected with some of these chronic viruses and other pathogens? firstly, if you take the animals, this is mock or co means co-infected, these are the animals sequentially infected,

they all make a nice immune response, early, but the co-infected animals lose their antibody responses over time. so we just looked at what genes are expressed in response to vaccination. this is the mock, so if you were to do this with mice from a

clean mouse facility, give them 17d yellow fever on day 3, 318 genes would be up, 142 down, et cetera. this is what a non-- a control mouse would look like. if you take the co-infected mice, genes are changed, but there is negligible overlap.

so what this figure shows is that the immune response of the mouse is completely different if it had those environmental infections is he quecialgly when it was -- sequentially when it was growing up. now, is this more like a human immune response?

it could be that those serial infections would alter the mouse immune response so that it looked more human. so that's the second hypothesis. co-infection induced changes will include the expression of genes related to human vaccine so if we looked at the genes in

the co-infected mice versus the nonco-infected mice, we would find the resemblance between humans in the co-infected mice that was not seen in the mock infected mice. so let me introduce you to this kind of a figure. this is a figure looking at the

genes which are -- let me come back to the cohort. i'm so excited about this, i jumped ahead. this is a collaboration between po, are yellow fever are virus, one is in switzerland and in each they did exactly the same experiment that i showed you in

that they bled the patients at day 0 and then day 3, day 7 and then sequentially afterwards and did array analysis to look at the gene expression patterns. so we had the mouse data and now we have human data from two different cohorts. we're going to ask is there a

relationship? so this is in tebby day 3 sub subtracting out baseline on day 0 we're looking at the genes co-infection regulated on day 3 in the mouse and the graphs will all be the same. these are genes that are up in both cohorts, down in both

cohorts, and are inverted, and you can see a very significant overlap between the co-infected regulated genes and the genes expressed in patients in uganda after vaccination. if you look at the day 0 data, this is corrected for day 0, the genes expressed in lasin on day

0 are highly related to the co-infected genes, lasin on day 3, and if you now look at this set of genes in a network analysis, you discover that the genes which are here are things like stout 1, trafts, chemokines, this is actually the mouse immune response being made

to overlap with the human immune response. similarly if you look at in tadi on day 7 and day 3 and you look at the network, again you find stat 1-rbgs stat -- stat 1-rbgs stat 2, hla molecules, so what we're seeing here is that the immune response of the mouse was

made to be more similar in response to a vaccine to the immune response in a human by serial co-infection. so that is an example we think that the genotype/phenotype relationship in this case measured by a vaccine response may be altered by co-infections.

so today what i've shown you or i've tried to argue to you is a genetic argument, that when we look at our own genotype/phenotype relationship, that we need to account for the virome, that in fact there are additional interactions in the metagenome between bacteria and

viruses, between worms and viruses, between viruses and bacteria which can influence or be related to inflammation or human diseases. that host genetic variation plays a role. when we're mutated in a gene, it could be that the expression of

that mutation is not solely defined by its presence on our chromosomes and homozygous or heterozygous state, that it may take a trigger or an element from the virome in order to have that host genetic variant actually generate a phenotype. i've given you multiple examples

of how far the can be changed. we can complement immunodeficiency, we can create new pathologies that mimic human pathologies, i've argued for each of these, systemic inflammation changes the immunophenotype a relation to

disease susceptibility, symbiosis of viruses, it's not all bad news. enter individual phenotypic variation and profound changes in the transcriptional milieu when you have a chronic virus or you don't in multiple organs, the brain, spleen, liver.

and i've shown you that multiple different elements of the virome can play a role and i think we're in the very infancy of this field. the number of sequences we can actually annotate with current databases is very limited, so i think there's a huge opportunity

to define the virome and its relationship to these important pieces of biology in the future. and i'm going to end by telling you that megan b -- baldridge was involved inned studies controlling chronic infection. simon park was involved with donna mcduff.

tim nice found sterilizing jason norman and scott handly with able assistance from a number of others were the authors and the discoverers of the relationship between the enteric virome and inflammatory bowel disease. this is why i love this job,

these are smart people, they challenge you every day, they don't believe what you say, and they try to disprove you and in so doing you discover sometimes exciting things. so i am very indebted to them. and with that, i'll stop and be happy to take any questions.

>> thank you very much. we have time for a couple of questions and then i want to make sure everyone knows there is a reception in the library afterwards if you would like to talk more with skip. julie? >> so skip, really nice talk.

you talk about it like there's two worlds, there's the viergses that -- the viruses that infect bacteria and there's the viruses that infect the mammalian cells and i wonder if you're thinking that there's going to be some community, some assembly and some way that they sense each

other, or is it really through who they interact with rather than their recognition of each other? >> skip: so i don't have any data with which to answer that, so that gives me permission to explain my bias. i think that these things are

all going to interact with one another. it's impossible for me to believe that viers that change -- viruses that change the inflammatory milieu of the host do not then in turn change bacteria and that that doesn't in turn interact with other.

so i think there will be a very complex and not grated matrix, and i believe that this is an evolved process. these viruses have evolved with us, as has the bacteria. so it would be very surprising if certain interactions hadn't developed genetically which

allow those community interactions that you're talking about. a simple answer is that we haven't published this but there are a few papers in the literature that show that host cells, eukaryotic cells, can actually sense bacteriorphages,

so you could also argue that the bacteriorphage could interact with the bacteria but it could also be interacting with the host and it was actually shown i believe here in the 1960s that bacteriorphages can be immunogeneral nick, studies of fy x.

i think there's a lot yet to be discovered but my bias is that there's going to be the network that you described. >> yes. with respect to asthma and the hygiene hypothesis, i've seen that there are contrasting points of view in terms of

exposure. some people say that leads to it, some say it's the other. they talk about th1 and th2. does your work in any way relate to this? i know you've talked about inner inflammatory bowel disease, have people talked about that and the

microbiome, et cetera, so i guess i'm trying to say, could you shed some light on how this relates to asthma and the hygiene hypothesis? >> skip: so i think this is the hygiene hypothesis except what i'm saying is that in addition to the things that one would

normally perhaps have thought about when the hygiene hypothesis was originated, that the viruses which live in us permanently and on us are a part of that interaction between organisms that sets up our immune system, chmtion the heart of the high -- which is the

heart of the hygiene hypothesis. if we change that is correct we could change our immune responses, that could leave to inflammatory diseetz. we have no direct data to support that hypothesis. itfor viruses. it is a very attractive

hypothesis. there are clear changes in the epidemiology of these chronic viruses that occur if you compare what we think of as westernized countries to nonwesternized countries. when you compare environments in which one is more exposed to the

natural world versus more sterile from the natural world. all of those things do in fact change virus infection. so i think it's the same general idea and i'm saying that you should think about viruses but i don't have any data which would specifically say that a certain

viral infection occurred early versus late would predispose or not predispose to asthma. >> thank you. >> it looks like you covered all the genomes. and also you showed the beneficial effect and also adverse effects of this

combination. so when do you think you will make some cocktails for people who want to take a -- >> the question is, if i may paraphrase, if it's that complicate, how is it possible that you'll get a specific change in the host with one

particular approach to altering the viruses? i think that's a real challenge. i think that probably the bacterial microbiome is the best early target for doing this. but i would point out that even in primate studies there are magnificent data showing that

certain viral vectors, cmv, can induce very valuable immune responses when other viral vectors do not. and that some of those viral vectors, cmv, the work of luf wis picker and others -- louis picker and others, are actually chronic viruses and so i'm i

don't think that we could today design a cocktail of viruses which would get rid of asthma or change your susceptibility to ibd, but neither do i think that there's no hope that specific results could occur if one could just understand the fundamental relationship between the virus,

its host cell and then the tissue for which it has trouble. >> when do we think we will have fecal tests for patients with inflammatory bowel disease and crohn's disease? >> this is not an area that i work in. i can tell you that i've just

come from a keystone microbiome meeting and there was a lot of interest interesting data in fecal transplantation. i think you're probably aware of the data of human fecal transplantation, in c colitis is nothing short of stunning. there have been some trials of

feek al translation in ibd which has been less than impressive, it's also possible that's evolving and some people believe they have positive data. it's interesting. i don't think there's anything published that says that we should drop everything, stop

immunomodulatory drug therapies and fecally transplant people, but it could have a role. >> good luck. >> i was wondering about the demographics of the bacteriorphages and have you looked at cohorts of family members and whether or not they

have similar profiles, or do they differ, or taking it further, if i go to africa, i imagine i would have one profile and if i come to the united states i would have another, if i go to india, another. i'm just curious to know. >> this is a really great

question, and the answer is that there are profound differences in the data if you compare chicago, boston and the united kingdom. now, the caveat to that statement that i just made is that at the current level of capacity to annotate viruses,

given the weakness of current databases, okay, it could be that some of that apparent variation is due to failure to fully annotate the sequences, and there is no question that the bacterial metagenomic world is leagues ahead of the viral metagenomic world and in my

view, given data like this and data from others, that difference needs to be fixed. we need to be able to annotate these things as well as we can annotate bacteria today. now, the most interesting difference is between boston and the other two sites and there

are more bacteriorphages in people in boston. now, i have a number of colleagues in boston, and as a virologist, we occasionally get the permission to name a virus and i'm strongly considering naming the extra viruses in boston choudaviruss for the fact

that these are people who eat clam chowder and maybe they have chowderviruses. it's striking. if you look at the controls there. i don't have an explanation for why that might be. but there will definitely be

geographic variations. >> how about family members? >> that's been done in some recently studied work and it is clear and we have some other data that's unpublished tion it is clear that we share not only our bacteria with our family members but also our viruses

with our family members. and you know that because children get cmv or chickenpox from, you know, grandparents with zoster so you know there is this kind of transfer but that clearly happens with bacteriophages as well and with the environment.

if you go into a household and you sequence the environment, doorjambs, you will find that the family microbiome and very likely virome are significantly controlled by the immediate family environment. >> i have a related question to ibd because of the diversity of

bacteriophages with ibd, have you looked at -- if you look at family members, is that a source of the diversity that precedes the disease or is this something that develops as you're developing it? >> brian asks a very good question.

in the cohort where we have more viruses and we have household controls, what is the overlap between the viruses in the two? our data is it's a really great question, our data is not robust enough to answer that question because what we annotate is not the full genomes of viruses.

what we annotate are sections of the virus. so in order to really know whether i share a virus with my daughter or my wife, we would actually have to sequence the virus and show that unique sequence variations within the overall sequence of that virus

are shared between the two of us, and our data is just not robust enough to do that at the current time. as we get bigger and bigger datasets, this is a key >> because if your hypothesis is really that the diversity or predator/prey relationship is

something that relates to that diversity which relates to the prey, so what is it that creates that initial diversity if it's not for some other diversity within the bacterium? >> first of all, there's clearly a genetic element to the risk for crohn's disease, for

example, so one could argue that all the people in the household were exposed to the same viral milieu but the genetic susceptibility selected out certain patterns within the patient who was predisposed to the disease. but it's also possible that one

person ate at a salad bar that the other person didn't eat at and got different viruses introduced into them. our data can't address those two variant hypotheses at the >> thank you for the excellent presentation. are a question about bacteria in

the gut can translocate into the blood and which would be associated with inflammation, with aging and other things. i wonder, like viruses in the gut, could this same thing be happening with viruses or not? >> first of all, the nih is the source of this hypothesis, danny

duex's group was the first to publish that, that's a paper we paid a lot of attention to. we've fundamentally confirm those same findings in primates with hiv infection advancing to aids. we've found with regard to the virome there are many hypotheses

by which you would get translocation into the systemic circulation and that would drive you could change the nature of the immune response to bacterias that's one good hypothesis. what we found when we deep sequenced the primates, and this was published a couple years

ago, is 21 novel viruses in the primates that were advancing to aids and they were never discovered before, you wouldn't find them by pcr necessarily, and they were from groups of viergses which are known -- of viruses which are known some of the viruses we

discovered could actually be seen in lesions in the gut where there was an abnormality and a break in the epithelium and we could stain and show that a novel had no virus was actually present in the lesion. so our hypothesis would be that some translocation is due to the

presence of as yet diagnosed viruses that are present, which actually may be acting in a more classical fashion. they have a tropism for cells in the intestine, cause inflammation, cause break down of the barrier and some of the translocation you ever get could

be due to the presence of viral diagnostics in humans are wonderful things but they're very targeted to sequences that are in described viruses. if you do metagenomics in those settings you frequently find the presence of viruses that would be missed by standard pcr-based

diagnostics. so you really can't, in my view, fully diagnose, if you will, the enteric virome with current diagnostic tools so i think metagee mow mix -- metagenomics, shotgun sequencing, is a good way to know whether there's actually a virus there.

standard diagnostic, you do the diagnostics, you don't find a virus, you say there's no virus, i don't think that's accurate. >> do you have nonresponsive data linking cancer susceptibility and the viromes, for instance, cancer due to oncogenic viruses or other type

of cancer? >> the question is whether there's a linkage between the virus tion and cancer. we've not done those studies and so it's an attractive we'll be happy to send people the methods for how to do this >> we'll take one more question,

then i think we'll move to the reception. >> you've mentioned this difference between the ability to annotate are bacteria versus it's got to be partly due to the lack of marker gene in viruses like 16s. so how do you currently do

something where you want to given a dataset identify all the virus tion? >> so there are two parts to this question and it's one we think about a lot. so is the reason that it's difficult to annotate viruses because viruses do not share a

common gene that can be targeted by conserved pcr as is done for 18 bacterial 16s? the answer to that is in part yes but it is much more than that. bacteria, which differ by 5% at the nucleotide level or 10% at the nucleotide level are very

different from each other. viruses which vary by 5% within a quasi-species are the same so the real problem is that the databases don't have enough viral sequences to fully annotate. you cannot do viral metagenomics by nucleic acid homology.

it cannot be done. you can identify known viruses that way. but viruses vary so much from each other, even within a single individual, that you can't use strict conservation of nucleic acid, and that means you have to do protein based searches and

that's a lot of what we do. so not enough viruses, no common gene as an element, you need to do protein, and the last thing is, with due respect to my bacterial colleagues, the bacterial genomes which go into the databases, annotated as bacteria, contain prophages.

so if you take a prophage, if i make purified virus ps from the gut, i get eukaryotic viruses, i get some prokaryotic viruses, if i search the prokaryotic viral sequences, the database says they're bacteria. they're not bacteria. they're phages.

they're just matching phages in the bacterial database so there is a problem with the annotation of the database itself because they are sometimes not completely annotated for we're working hard to solve these problems. the last thing, and i think it's

a really important point, and it's a good one to end on, not all life is encoded by dna. not all life is encoded by dna. there are rna organisms. when you see -- i studied, i define the microbiome with dna sequencing, you need to read i did not define the microbiome by

doing only dna sequencing. all of the major viral pathogens, most of the -- the major viral pathogenless in the gut of human -- pathogens in the gut of humans are noroviruss, rotoviruss, astro viruses and adenoviruses, so there's a dna virus down there.

so we really have to get away from this idea that sequencing dna is the way to get at the metagenome because there are rna phages, rna viruses, and the methods are there. i think that it's just easier to make dna than it is dna and rna and i think that's a major flaw

in many current studies of the virome and the metagenome itself. >> thanks. >> so thank you for your attention.

Monday, January 9, 2017