 Thank you so much, Eric. Good morning to everyone. I am delighted to be here, and I think that it's particularly important in a field that has moved as quickly as genomics to take these kinds of opportunities, the 10-year anniversary of the completion of the human genome sequence, the 60th anniversary, the discovery of the double helix, to pause and reflect back upon just how much has been accomplished. And so it's delightful to be part of the group doing that today. What I'm going to talk about today is the gut microbiota. It is an essential component of what it means to be human, and the information that we have about our microbial partners that make up the microbiota and all of the genes that they encode the microbiome represent another spectacular success that has come out of all of the work that went into getting the human genome sequence completed initially. Just a few little facts about what we know about the microbiota today, and those of you who are familiar with the field have probably heard these statistics ad nauseam, but they really do, I think, help put all of this in perspective. It's estimated that we carry with us as human beings hundreds of trillions of microorganisms that live in very close association with us. We are essentially born sterile, but immediately after birth, a very complex process of colonization begins. And within a couple of years, these bacterial, these multi-microbial communities mature and take on the characteristics that they have for most of the rest of our lives. It is estimated that we as individuals probably carry 10 times the number of bacterial microbial cells as human cells. Obviously that doesn't take mass differences into account, but if you think about this just in terms of cell number, we are really only 10 percent human. And if you do some quick calculations based on estimates of bacterial genome size and gene density compared to humans, the numbers suggest that we carry along with us 100 times more microbial genes than human genes. So just by sheer numbers alone, I think we need to pay attention to our microbial partners. And there is a concept that has emerged that I think has really taken hold and is critically important, and that is the concept of us as a superorganism, representing a combination of our human DNA and the DNA encoded by our microbial partners. What Eric didn't mention in his introduction today is that NIH has taken the lead in another international project to begin to characterize the microbes that live in association with humans. This is the Human Microbiome Project. It's finishing up five years. There have been some spectacular successes. There were a number of papers published last year summarizing the efforts of large numbers of laboratories, many of them that were also involved in the Human Genome Project. If you look at this PCA plot here, this just looks at the differences in the content of the microbial communities that you find in different human environments, and these are the environments are color coded. And you can see that we have co-evolved with our microbial partners to the point where we find now very distinct communities associated with different areas of the human body. There are a small number of bacterial phyla that you see associated with humans. This is a very, very high-level view, and when you get down to lower taxonomic levels to look at genus or sometimes species levels when you can, we see that there's tremendous variation among individuals. And I think at this point we believe, at least in terms of the types of organisms that are there that have come out of our ability to do a molecular census, I think there is good agreement that there is far more variability between any two individuals in terms of the microbes that we carry than there is difference in our human DNA. And the significance of that perhaps will become obvious as I go on in my talk. I'm going to now focus for the rest of the talk on what happens in the gastrointestinal tract. The GI tract is very heavily colonized. It represents the most diverse bacterial community, the estimates depending upon. And the methods used suggest that we have many hundreds if not thousands of different bacterial taxa that live with us in our GI tract. There are two major phyla, the bachroedetes and the firmacutes. And again, as I said before, we see a lot of variation among individuals, and in individuals we can see variation over time. And that is thought to be due to a lot of factors including aging, diet, exposure to antibiotics, and I'm going to talk a bit about this more in the talk. One of the important concepts that has come out of all of this work is the idea of a core microbiome or a core microbiota. The question is, do all of us as humans share key taxa that must be present in order for these communities to carry out the functions that are associated with health? So when you look at the great variability, there is another way to think about the core and that may be a core set of functions that can be carried out by different suites of organisms. And I think the jury is still out on that, but this has emerged as an important concept. Another concept that has come out of work on the human gut microbiota, this was originally reported by a group in Europe, the Metahit Consortium studying the GI microbiota and has been now picked up on and confirmed and refined by a number of other studies, and that's the idea of enterotypes, that there are distinct community types present in the human population, present in the human GI tract. Each of these different enterotypes can be defined by a dominant genus, and these are the three dominant genera here that were initially described by the Metahit study. If you don't know microbiology, it doesn't really matter. Another concept here is that perhaps we could take everybody in this room, do a characterization of your gut microbiota and place you into a limited number of community types. What's been suggested is that in part these different community types may be driven by diet, although that isn't seen in all studies and it may likely be driven by human genotype and the factors driving the concept of enterotypes is not known. That's also, I should say, somewhat controversial. There are some who believe that there are not distinct groups, but it's more of a gradient, but I think everyone would agree that if you go in and look at anybody's gut microbiota at any point in time, you will see that it is characterized by usually one dominant genus of bacteria. We have co-evolved with our gut microbiota very much, we think in large part, because of the important role that it plays in nutrition and metabolism. The bacteria that live in our guts have the capability to break down indigestible carbohydrates, the plant polysaccharides that we take in. This has important consequences, although they may be obvious. One is that it certainly increases the efficiency through which we can acquire nutrients. The ability to break down these compounds probably also ensures that these microbial communities have a constant source of energy themselves. In turn, there have been a number of very interesting studies that have begun to suggest that diet and nutrition can shape the microbiota. These communities are very dynamic. You see differences in omnivores versus carnivores versus vegetarians. If you look across the animal kingdom, you can see differences just by changing diet. These are all observations at this point, and I think a big question is what does all of this mean, and hopefully we'll continue to make some good progress in figuring that out. What I want to focus on for the rest of the talk is also the fact that the interactions between us and our gut microbiota go beyond metabolic functions, and very likely a critical role that these bacterial partners play is in modulating and helping to shape the immune system and vice versa. The gut is the largest immune organ in the body. In humans, if you think about this, there are probably a 10 micron layer of epithelial cells that separates the lumen of the gut where all of these organisms exist from the underlying immune cells, the lymphoid-associated tissue in the gut, and the surface area in the gut is estimated to be about 200 square meters, so that's a lot of area for interaction between our immune system and these commensal organisms that have to live in peace with us for the rest of our lives. There are a number of ways. If you look from the inside out that this happens, there is a mucous layer, a two-phase mucous layer that provides a physical barrier. We also know that antibacterial proteins can be secreted by some of the cells lining the GI tract as well as the release of secretory IGA by the immune system, so that helps to keep our microbiota in check. And conversely, the microbiota also impacts immune system development, and a lot of these insights have come from studies on germ-free mice. In the absence of any microbiota, you don't see normal formation of the organized lymphoid tissues that are associated with the GI tract. There is altered regulation of innate immune cells, and there are also changes in systemic immune cells and systemic immune responses. I don't have time in the presentation today to go into this in a whole lot of detail, but there is an incredibly well-balanced homeostasis between our commensal microbes and the GI immune system under normal, healthy conditions. So what I'm going to talk about today, then, is how we respond, how the gut microbiota responds to the presence of an enteric pathogen. In this case, we're going to be looking at Shigella, which causes the disease Shigella scelosis. This is a mucosally invasive bacterium. It's associated with food poisoning, terrible symptoms, bloody diarrhea, fever, stomach cramps. It's transmitted through a fecal oral route, and you see there are a lot of deaths, particularly in children, in developing countries from Shigella and other enteric pathogens. Shigella as a pathogen in terms of its ability to cause disease is restricted only to humans and non-human primates, and one of the most important models for studying Shigella in terms of non-human primates is in the cinemologous monkey. This is an important animal model that has served as a good system in a lot of vaccine development work, including attempts at development of vaccines against Shigella. Most of what I'm going to talk about now has to do with some studies in cinemologous monkeys. It's much easier to do intervention studies, to do challenge with wild-type Shigella in a non-human primate model as opposed to trying to do these studies in humans. When we look at what's been done in the efforts to develop an effective live attenuated vaccine against Shigella, and a lot of this work has been done by Mike Levine and colleagues at the Center for Vaccine Development at the University of Maryland. There has been some slow progress made, but successful vaccine development has been stymied by a continued finding that regardless of the vaccines that are tried, regardless of the vaccine regimen, there is tremendous variability in vaccine response, particularly if you look across global populations. This is not unique to Shigella vaccines. This is a somewhat more universal theme in vaccine development as a whole. The causes for this variability are not known, but we can begin to make some assumptions underlying genetic differences in the host, diet, environmental differences. It's been mentioned already today that many people in developing countries carry a great parasite burden in the intestine. This may, in part, contribute. One of the things that has not really yet been examined is the potential role of the gastrointestinal microbiota in vaccine response. This was the goal of the study that I'm going to tell you about. This was, we piggybacked onto Shigella vaccine trials that were being carried out in cinemologous monkeys. These were looking at efficacy of oral live attenuated vaccines. I will get to the study design, but we characterize the gastrointestinal microbiota in stool samples. Stool samples have been used very often in these kinds of studies as a surrogate for what's going on in the intestine. We can argue whether that's fully appropriate or not, but it's clearly much easier to collect stool samples and to go in and do biopsies, particularly over repeated sampling. We were interested in characterizing the gastrointestinal microbiota in these monkeys, post-immunization, and post-challenge with wild-type Shigella, with the big question being how does or does exposure to an enteric pathogen affect the intestinal microbiota, and conversely, does the composition of the intestinal microbiota possibly contribute to the outcome following immunization and challenge? What we used to characterize the gut microbiota was the 16S ribosomal RNA molecule. This allows us very quickly and easily to take a molecular census of all of the organisms that are present in the gut microbiota. Most of these organisms cannot successfully be grown in culture, so we need to be able to bypass that cultivation step, and we can go directly to characterization of these organisms through looking at DNA. The 16S ribosomal RNA gene is quite useful. This is a two-dimensional representation of the structure, and these loops here that you see that are highly conserved and are essential to the function of the ribosomal RNA molecule come about through the presence of highly conserved regions of the gene that are involved in base pairing here, interspersed with highly variable regions. So we can design PCR primers against the conserved regions of the 16S molecule, amplify across the variable regions, and this gives us incredibly useful information for making taxonomic assignments and constructing phylogenetic relationships. So we looked at a number, we looked at monkeys and a number of studies. I'll go through all of these, but we're only going to focus on a couple of studies then for the rest of the talk. In study one, this was a nearly a three-month study where two different live attenuated vaccines were being evaluated for efficacy compared to animals that received PBS as a control. In order to vaccinate these monkeys, they are anesthetized, and the bolus of Shigella vaccine is administered through oral gavage. So in the PBS control monkeys, they were getting PBS only as a vehicle. They were not getting any attenuated Shigella. There were two immunizations here shown in yellow, followed by a wild-type challenge at day 56 in red. Study two was a separate study with a different set of monkeys. This was looking at one of these two vaccines, but a different vaccination regimen, four doses given over the period of a week, again followed by wild-type challenge at a month. Study three was looking at naive monkeys that didn't receive any immunization. They were just challenged with wild-type Shigella, and study four was a group of control monkeys in quarantine that received no handling, no intervention whatsoever. But we are going to be focusing mostly on study one and study two. So we wanted to first characterize what was, what organisms, what types of organisms were present in the GI tract of the synomologous monkeys. This is the relative abundance here at the phylum level. We see the firmacutes and the bachrodides is the two major phyla, but they are present in very different proportions from what you see in humans. If anything in humans, the relative percentage is switched in more even. If we look then at the genus level, these are again looking at data that we collected from all of the monkeys over time. The most predominant genus that we see is Lactobacillus streptococcus. These are all in the firmacutes. Prevotella was the major member of the bachrodides, but the point here is that these, this community composition was different than humans in many respects. If we then looked at trying to ask what perhaps represents a core in the monkeys and we defined a core, I believe at 85 percent, any organism that was present in 85 percent of the samples at greater than 1 percent mean relative abundance. These are again, this is what we saw at different levels of prevalence, and these are monkeys from our different study groups here, color coded by the different study groups. We see that there were some differences, and I'll get into those differences in a bit more detail in a moment. Just to go back again to the concept of enterotypes in terms of the GI microbiota. This is what we see in humans, three different enterotypes. These are data from the meta-hit study that I mentioned previously. We've done a lot of work with the old order Amish in Lancaster County, Pennsylvania in an obesity study looking at the potential contribution of the gut microbiota. We see essentially the same enterotypes in the Amish. We and others have done other studies, and depending upon the populations, we don't necessarily see all three of these, but what we do see tends to fall into one of these three different groups. So, one of the first questions we wanted to ask was whether or not there were enterotypes in the CineMulgus monkey gut microbiota that we could identify as a way to begin to try and think about how to map the results from this study onto results from human vaccine trials. This is again looking at all of our samples here. This is a three-dimensional PCA plot. We saw four different community types here that we numbered one through four. These are the characteristics of the enterotypes, and again, they are each characterized by the presence of one or two more predominant genera. Very different from what we see in humans, but this concept still holds that this is not necessarily a continuous gradient of variability. When we looked at the four community types here, with a measure of overall diversity, this is the Shannon Diversity Index from, really derived from the field of ecology, we found that there was a very high diversity community type, community type two here, a low diversity community type three, and community types one and four were similar in diversity. Community type two, the very high diversity community is characterized by a high level of two bacterial taxa here, Prevotella and an unknown taxa that's given a number 2159. This contrasts with community type three, where this is a community type that is dominated to a very great extent by a single genus lactobacillus, therefore, because it's a single genus present at high level, the overall diversity is low. So our analysis revealed that we had these four community types each dominated, each characterized by a dominant genera, so the concept still seems to hold in the Cinemologus macaque model. We wanted to ask what might be driving this diversity in the monkeys, and certainly one of the suggestions in humans is that diet may play a role, but we didn't think that that was necessarily going to be quite so important in these monkeys because they had come in from different vendors but all were in quarantine for 90 days before these studies began, and then over the course of the studies were being housed under identical conditions, fed the exact same diet and same light dark cycle. So some of the potential variables that are thought to contribute in humans didn't seem like they would be playing such a big role in this particular model, so we immediately went to ask whether there was genetic diversity in the Cinemologus macaques that we might be able to identify, and this was based on a lot of previous work which has indicated that there are indeed allelic differences within Cinemologus macaques from different geographic origin, and in fact macaques from Indochina and Indonesia show a very high level of diversity, particularly within the MHC regions, one and two, and these are contrasted with macaques from Mauritius, which are geographically isolated and have been shown to have a restricted genetic diversity in the MHC region, and one can perhaps think about these different subpopulations of Cinemologus macaques almost in the same way that we think about outbred and inbred animals. The MHC haplotype in macaques has been shown to be very important in disease susceptibility, particularly in studies with Simeon immunodeficiency virus, and so we carried out both whole genome analysis using short tandem repeats and genotyping analysis looking specifically at the MHC region to try and get a sense of whether there was genetic diversity in our monkey populations, and this is what we found. This is a PCA plot here. This is looking at genotype analysis using 24 non-MHC short tandem repeats, and we found that indeed, as had previously been described, the Mauritius, the animals that were from Mauritius, and this was information that we both got from the vendor and also confirmed with these studies, clearly clustered separately from the rest of the monkeys from Indochina and the Philippines. So we knew that we were working with monkeys of different geographic and different genetic backgrounds. So when we looked at the MHC alleles, we looked at seven loci across the Class I and Class II region, and we then used that data to construct a phylogeny based on the MHC repertoire, and again, we saw that the monkeys from Mauritius shown here in green clearly cluster as a group distinct from the rest of the monkeys that we were using. So we knew that we were working with a mixed population of monkeys. So now we went on to ask, given that information, given that we see differences in the microbiota that actually map, and what I didn't say is that community type II, the very high diversity community type maps to the monkeys from Mauritius. So we now went to look at the effect of vaccination on the microbiota in these different animals. To ask the question, does vaccination or challenge with wild type Shigella alter the gut microbiota in any way, are the observed changes the same for all populations? One of the first things that we noticed was that in study one, and these were all monkeys from Indonesia and into China with greater MHC allele diversity. This is the Shannon Diversity Index, again, a measure of the diversity of the community. It takes the number of different organisms, the relative abundance, into account. This is the Shannon Diversity Pre-Treatment. We found that after the first immunization, the Shannon Diversity decreased, decreased yet again following the second treatment, and then came back following the challenge. And this is comparing the data to what we saw in study four monkeys, which were our control that didn't receive any handling. What we found in study two, however, was very different. This was, these were the animals that received four immunizations. There was no change in Shannon diversity whatsoever. So we saw that in terms of the response of the gut microbiota to immunization, to handling, there was indeed a distinct difference between the study one and the study two animals. Now this is going to be a very busy slide, and I will try and go through this step by step so it makes some sense. This is probably one of the most important slides in terms of all of the data here, and it's really a compilation of all of the data that we had putting our microbiota together with immune data. In our study one monkey, this first set of bars shows the community type in the monkeys, color coded here, one, two, three, four, you can see, or where we did white being where we had no sample. And what you see here, as I had just described, that after the first immunization in some of the monkeys, we begin to see a shift from community type one to community type three. Diversity community dominated by lactobacillus. Following the second immunization, essentially the communities in all of the monkeys shift to this low diversity community type shown by the light purple. When the animals are challenged with wild type chigella, we see a shift yet again to community type four shown in the dark purple. And this will describe the characteristics of that to you in another couple of slides. So we are seeing a continued shift. And what I should point out is that we see this regardless of whether the animals are given the vaccine or given PBS. And so we don't necessarily think that this is an effect of the vaccine per se. We think that this is what we're probably seeing here is an effect of the stress of the animals being anesthetized and having to go through the intervention. We actually had stored blood. We went back and were able to measure cortisol levels and found the cortisol levels peaked in the monkeys after each of these interventions. And stress has been described previously to potentially alter the composition of the gut microbiota. Next we look at clinical score. What happens following immunization or challenge with wild type chigella? Green is normal in terms of what's looking at being looked at here in the clinical score. This is diarrhea, diarrhea with blood. For the most part, nothing happens until the animals are challenged with wild type chigella. And then in some of the monkeys, we begin to see some clinical symptoms as shown by Gello or in a couple of cases here, orange or red, which was really severe clinical symptoms. And again, we see this across all of the monkeys and we see this in association with the emergence of this community type four. In study three, we see something similar. These are the animals that were just challenged with wild type chigella, no immunization. You immediately see the appearance of these altered communities and clinical symptoms in at least two of the animals. And now if you compare this with study two, these are the animals from Mauritius that are clearly different in terms of their genetic background. The community type here is represented at baseline by either one or a large number of the high diversity community type two. Even giving four immunizations over the course of a week, there is essentially little, if any, change in the gut community composition. That also nothing changes following challenge with wild type chigella with the exception of one sample here from one monkey. Now we fold in the immunology data. And we see that when the animals that are given the immunizations, when we look at IgG or IgA levels against LPS, we see an increase in antibody levels. We don't see this in the PBS monkeys and we see a more robust response following wild type challenge. This is the case as well in the study two monkeys. But what I think is most important here is that in these study two monkeys, it's not so much the immune response, but in these study two monkeys, particularly the monkeys that only receive PBS, they did not get sick following exposure to wild type chigella. And these are the monkeys that harbored a very different high diversity gut microbiota. So I think we've summarized all of that and in the interest of time, I just want to keep going. Community type four, we see an increase in very rare members of the community here that seem to be associated with the clinical symptoms. And one of the, so one of the I think very important conclusions that we have here is that only these study one monkeys exhibited clinical chigellosis. The immune response as we measure here, and we looked at antibodies to other components, does not seem to be a determinant of protection. So what is? And we are proposing that there perhaps is a potential role for the microbiota in mediating this protection. So we took, we did one more set of analyses. This is to look to see whether there are any correlations between the strength of the immune response, the type of the response, and the microbiota using the local similarity alignment, which allows one to look for time dependent correlations. In study one monkeys, this is the network that emerged. It's very dense. It's very complicated. It's probably in part related to the changes that we saw in community type. But we did see some correlations here, positive correlations between antibody level school stool scores and particular members of the gut microbiota. This is what we see in study two. And in the interest of time, I can't go through this in a lot of detail. But what we did see were some shared correlations among the studies and that a protective response was associated with three members of the gut microbiota in these monkeys. However, we realized that the differences in vaccine regimen may make it hard to figure out how all of these are related with these data sets. So in conclusion, what we have seen are the presence of different enterotypes in the monkeys. Two of these seem to be associated with health. Two of them seem to be transient that are associated with intervention or with challenge with an enteric pathogen. But I think what's most important here is that these different enterotypes are present in macaques from different geographic origins that are clearly different genetic backgrounds. Vaccination and challenge induced an immune response in both studies. But the immune response does not in any way explain the outcome. And so we are now very much interested in further exploring the potential role of genotype in shaping the composition of the microbiota. We saw that the high-diversity enterotype seemed to be protective against chigella or was at least associated with a lack of clinical symptoms. And one other comment and another unrelated set of studies in humans looking at response to live oral attenuated salmonella vaccination, we see that a very high diversity gut enterotype in humans in these studies seems to be associated with a more robust immune response. And I think I would then just argue that based on these data, particularly for studies like this, it is probably critically important to begin to think about folding in the microbiome and information on the microbiome on future studies. And I think this leaves open the possibility that some of the differences that have been well-described in efficacy from vaccine trials in populations around the world may be related to what's going on in our gut and the role of our microbial partners. And with that, I want to thank my collaborators on this study. And if there's any time, I'm happy to take questions. Anybody coming to the microphone? So Claire, technically, is the, at one point in time there was a lot of interest in getting the technologies for sequencing to be able to get at single microbes as opposed to populations. Is that still sort of a cutting-edge need or is the computational side of this equation probably the more challenging immediate need? I think that's a great question. And I would say that both of those represent very pressing needs. The ability to do the informatics is absolutely essential. But at some point, as we want to begin to further dissect what's going on, we need to be able to move away from studying these communities as assemblages of unknown organisms to begin to map function to specific cells or specific types of cells. Without the ability to culture them, you're going to want to be able to figure it out. Yes. One more question right there. Thank you for that talk. Wonderful, as always. Just wondering if anyone is looking at these enterotypes across phylogeny in the primates to see if there are any central evolutionary messages there. Those studies are just beginning, but I have not seen anything published yet in any sort of systematic way. Enterotypes have been looked at in rhesus macaques, and there is evidence for enterotypes. But in the broad kind of study that you're describing, I think that is work still to be done. Okay. Well, thank you, Claire. We will now move to the next talk, which is not the one showing up on this monitor, but rather Jeff Botkin is here from University of Utah to talk about whole genome sequencing and newborn screening. What are we screening for? Jeff.