Unique achievements in metagenomic analysis: Tokai University Institute of Medical Sciences takes on the challenge

Adapted from a roundtable discussion held by Tokai University, Institute of Medical Sciences (Japanese)

Tokai University Institute of Medical Sciences ("Soiken," Isehara City, Kanagawa Prefecture) was established in 1980 with the three pillars of genomics, regenerative medicine, and drug discovery as a core base for advanced medical science research and translational research that integrates basic research and applications, where research and development are conducted with the objective of contributing to society. The theme of this roundtable discussion was "metagenomic analysis."

Metagenomic analysis is steadily garnering attention as a novel method that can clarify the composition of bacterial species and the function of bacterial flora by comprehensively analyzing genomic DNAs extracted from clinical, fecal, and environmental samples; in addition, this novel method has recently made remarkable progress. At the roundtable discussion, under the direction of Director Kiyoshi Ando of Soiken, three lecturers who are actively conducting research and development on the front lines gathered, and based on the current state of their studies, exchanged opinions on the future development potential and discussed interesting topics related to COVID-19. In addition, the expectations of the University's President, Kiyoshi Yamada, for Soiken are presented, and the studies conducted by Soiken's five research divisions are also introduced.

■ Participants

Supported by the development of next-generation sequencing

Ando: The Tokai University Institute of Medical Sciences was established in 1980 as an affiliated laboratory to "promote basic research in medicine, develop novel technologies, and apply them to clinical practice." Particularly, in recent years, the researchers at the university have focused on developing research with the three pillars of "genomics," "drug discovery," and "regenerative medicine." Here, I would like to talk about the kind of research that is being conducted at the Institute of Medical Sciences, focusing on the field of "metagenomics". In the first place, how is metagenomics positioned in genomic research, and how should it be understood? First, I would like to ask Professor Imanishi, an expert in genomic research, for an overview.

Imanishi: In the case of normal genome analysis, an analysis is performed targeting a specific gene of a specific organism. When "meta" is added to it, it implies research conducted to acquire and completely analyze genomic information on multiple untargeted organisms and genes. The main purpose of research is to identify microorganisms related to some biological phenomenon using metagenomics, and elucidate the characteristics of biological composition. When the Human Genome Project was completed in the 2000s and large-scale genome research was established, one of the next major objectives was to apply genome analysis technology to the study of the bacterial flora. Recently, research on bacterial flora centered on intestinal bacteria has made remarkable progress. The relationship between various diseases and intestinal bacteria has been elucidated, and the obtained information has facilitated the development of novel treatment methods. In addition, large-scale metagenomic studies that do not target specific materials, but simultaneously obtain information on all living things such as bacteria, viruses, and fungi in the environment, are also widely conducted. The development of next-generation sequencing (NGS) has made this possible, and has become a major support for metagenomic research. Ando: NGS was pointed out as the background to the rise of metagenomic research. However, it seems that the power required for computers is also tremendous in informatics for processing the obtained information.

What was the breakthrough in informatics?

Imanishi: From an informatics perspective, the fundamentally important element is genome sequence data. As genome sequence data have been obtained for a large number of organisms, the genome database has been enhanced, and it has become possible to accurately determine the organism from which the sequence output from NGS is derived. Regarding gut microbiota, the sequence allows a fairly high proportion of organisms to be identified.

Ando: Recently, in the globalization trend, I believe that the awareness of sharing information, including human genome data, has emerged considerably in the world. Based on the explanations from Professor Imanishi so far, Dr. Nakagawa, do you have anything to add?

Nakagawa: The development of NGS technology and the enhancement of the database are simply crucial in my virology specialty, and regarding the currently prevalent novel coronavirus, the early discovery of virus variants with different properties such as the "delta" and "alpha" variants by genome analysis is a good example of the progress of sequencing technology in an obvious manner.

Ando: I really feel that advances in informatics support biological research.

Nakagawa: The capabilities of computers are advancing rapidly, thereby enabling large-scale global attempts such as human genome analysis.

Ando: That is right. Next, I would like to discuss each theory. Professor Imanishi is currently conducting research that adopts genomic information to quickly obtain the diagnosis of bloodstream diseases.

Imanishi: Originally, I was conducting research on the risk prediction of diseases in the field of bioinformatics. Among them, we have been conducting research on technological development to predict the kind of disease that is likely to occur using human genome information. There are genetic and environmental factors that cause illnesses, and some of the environmental factors include exercise and nutrition; however, I expected that the bacterial flora would account for a significant proportion of the environmental factors. The motivation for this approach originated from our research on human bacterial flora using metagenomic analysis as a means to predict the risk of individual diseases.

Then, to establish analysis technology, we started research on the theme of system development for the rapid diagnosis of infectious diseases. At that time, we considered a nanopore sequencing technology that can obtain DNA sequence data in a very short time. With the nanopore sequencing, DNA sequence data is written into a computer in actual time, and the data can be analyzed to quickly determine the bacteria or viruses that are in the obtained sample. Therefore, I believed that it would be possible to make a rapid diagnosis of infectious diseases via this approach. We published a paper, clarifying that it would be possible to identify the microorganisms that cause infectious diseases with a high degree of accuracy within 2 h, starting from the state in which the DNA was extracted.

Ando: For example, for severe pneumonia, if a nanopore sequencer is adopted one can identify the causative organism very quickly within approximately 2 h after the DNA extraction is completed.

Imanishi: That is right. With that amount of time, one can now identify the organism at the species level.

Ando: I think it is a remarkable achievement. Next, Associate Professor Imai is currently studying the relationship between intestinal flora and irritable bowel syndrome as a gastroenterologist. First, please give us an overview.

Inflammation of the gut worsens because of bacteria present in the oral cavity

Imai: Since my time as a trainee, while diagnosing patients with irritable bowel syndrome (IBD), I have been conducting research to cure Crohn's disease, which is often difficult to treat. Furthermore, bio-informatics and sequencing data have been released steadily, while the fecal data of patients with Crohn's disease and ulcerative colitis have begun to appear predominantly in Europe and the United States. Interestingly, it has been reported that the feces of untreated patients with Crohn's disease have significantly high proportions of oral bacteria. When I studied abroad at the University of Michigan (USA), which is famous for its research on intestinal bacteria, I made a model mouse for periodontal disease in the laboratory, and I am currently conducting research on how it actually affects gut inflammation (enteritis). In addition, while working on this research, we obtained data that showed evidence that periodontal disease bacteria in the oral cavity also appeared in the intestine, where they stimulated intestinal immunity and exacerbated enteritis. Currently, we are conducting joint research with the University of Michigan and Tokai University, and it has become clear that periodontal disease bacteria are also certainly found in feces in clinical specimens. Therefore, I believe that it could be a therapeutic objective in the future.

Ando: I believe that the relationship between irritable bowel syndrome and intestinal flora is an area of active research around the world; however, it is unique that the indigenous bacteria in the oral cavity can be found in the intestine, as they cause irritable bowel syndrome.

Imai: I have observed this being demonstrated in mouse models at my study destination abroad; hence, I believe this might be happening in actual clinical practice.

Ando: Considering the reason behind the phenomenon of bacteria in the oral cavity transporting into the intestine, the conventional understanding is that the acid in the stomach and bile of the duodenum are barriers; hence, it is unlikely that the bacteria will go beyond that. Is the fact that it passed through that barrier to the large intestine already abnormal in itself?

Imai: I believe that the reason why pathogenic symbiotic bacteria can pass through the digestive tract is within the scope of microbiology. Fungi itself have a mechanism via which they create a cover called a biofilm. For example, there is a phenomenon that the barrier function of the intestinal tract is lowered by taking medicine for stomach ailments, adopting a fat heavy diet, and using antibiotics; in addition, it becomes easier for bacteria that enter later to settle.

Ando: Is it okay to regard that as a trigger for irritable bowel syndrome?

Imai: There are patients with irritable bowel syndrome who do not have the bacterium. Therefore, I think it is both environmental and genetic factors.

Ando: One of the various events caused by the bacteria being able to settle in the intestine is irritable bowel syndrome.

Imai: That's right. In fact, it is quite difficult to examine with human specimens. However, in the mouth, when periodontal disease bacteria appear, the lymphocytes in the lymph nodes of the submandibular gland recognize that "this is a pathogenic bacterium" and release inflammatory cytokines. However, when oral bacteria move to the intestinal tract, these lymphocytes are said to conduct homing (automatically capture and track the target), and even go into the intestinal tract, where inflammation occurs. The intestinal tract contains regulatory T cells, and many cells suppress the immune response. Therefore, I believe that even if Enterobacteriaceae bacteria are in the intestinal tract, such lymphocytes cannot be produced. I hypothesize that the technology of trapping these lymphocytes somewhere may be a treatment objective.

Ando: In our field, ITP (immune thrombocytopenia) and MALT lymphoma, which are lymphomas of the gastrointestinal tract, are associated with Helicobacter pylori; however, treatment with Helicobacter pylori often improves the disease. I think there are several other diseases similar to this; hence, if we can take the same approach, I believe we will progress into an era where patients can be easily cured without much burden, which is an ideal objective in metagenomic research.

Now, I would like to talk to Dr. Nakagawa. I believe that virology is also widely regarded as one of the fields in metagenomics and is being researched. As you all know, this has played an active role in recent research on the novel coronavirus. I would like to hear about why you started researching viruses in the first place, and what you know about the novel coronavirus as a new topic.

Nakagawa: In the case of mammals, including humans, it is known that approximately 10% of genomic sequences are virus-derived sequences; however, at first, it was thought that most of them would not be related to its function. It has recently become clear that this is unlikely. Therefore, I became more interested in the kind of function that the genome sequence derived from the virus has, rather than the virus itself. As I proceeded with my research, I realized that viruses are very elusive. Each virus has its own evolution and own host. Although we use "virus" in a collective sense, they are really diverse, and the strains are all different; hence, they cannot be collectively referred to.

We started to work on virus research in earnest in 2017 when we joined the open recruitment group for a new academic research field called neo-virology. My main objective was to analyze the genome sequence inside the virus; however, after joining this research area, I started research to identify several viruses in the environment using metagenomic techniques. We have started analyzing metagenomic data to identify unknown viruses in small creatures, such as mosquitoes and mites that carry various viruses, as well as bats and birds. Mankind's battle with viruses will continue for decades to come.

As a motive for that research, I thought that the next outbreak would be a zoonotic disease, and that it would be extremely important to discover an unknown virus in animals. Although this was still before the outbreak of the novel coronavirus infection, I thought it would be logical to introduce these technologies in Japan. Samples from various animals were analyzed to obtain metagenome; in the case of RNA meta-transcriptome was obtained. However, when we analyzed such a large amount of base sequence data, we found that the sequence has viral characteristics, although it is not present in the genome sequence database of organisms and viruses. For example, a virus similar to morbillivirus, which causes measles, was found in the urine of cats, and a virus similar to adenovirus, which causes flus, was found in the feces of cranes.

Currently, the spread of the novel coronavirus is a crucial global problem. This virus is a zoonotic disease that is probably derived from bats; however, when examined, it turned out that in addition to bats, mammals covered with hard scales, such as pangolins, have a similar coronavirus. When the coronavirus was first found in the pangolin, I believe that researchers even did not pay much attention to it. Regarding a situation like this today, comparative analysis of the genome sequences of the pangolin and novel coronaviruses have provided various insights into, for example, how the novel coronavirus interacts with receptors in host cells. In addition, the genome sequence of this novel coronavirus was also identified for the first time using the metagenomic method.

COVID-19 has been spreading for about 2 years now; however, approximately 2 million or more genome sequences have been deciphered. This is the first time that the genome has been deciphered so rapidly for a single infectious disease. I believe that it was possible to respond more quickly when mutations such as the alpha variant were found because of this. If the genome sequence was not deciphered and if the viruses mutated suddenly, I believe that we could not have taken measures against infectious diseases so quickly.

Ando: From the perspective of virus research, how should we comprehend the problem of the novel coronavirus?

Nakagawa: There was an outbreak of the Ebola virus in West Africa in 2014, and the responses at that time and in this current novel coronavirus era are quite similar. The genome sequence of Ebola virus was published successively, and a project was started to adopt it for the development of therapeutic drugs. As soon as the mutations were identified, we conducted joint research to identify the mutations that are associated with the function based on that information. However, the members who worked on this research are also conducting research on the current novel coronavirus, and that experience is actually used as a base for this.

Ando: Fortunately, or unfortunately, you have had to devote your energy to COVID-19 countermeasures; however, after the outbreak of the novel coronavirus subsides, what are your plans for the research you intended to pursue, specifically on the involvement of the endogenous virus in various diseases?

Nakagawa: It has been reported that sequences derived from viruses that have entered the genome, the well-known endogenous viruses, express a large number of virus-derived genomic sequences even in cancer, and I started my research thinking that such insertions might be the cause of health issues. However, while there are some things that are considered to be actually bad, there are cases where the prognosis is rather improved by the expression. I wondered why such an outcome happened, and after various investigations, I realized that the relationship between cancer and immunity is crucial as I proceeded with my research. Most of the endogenous viruses do not usually appear in our bodies.

Approximately 10% of the human genome is derived from viruses, but most of them are not expressed as messenger RNA or protein. Therefore, when a virus-derived sequence is expressed as a protein, it is not recognized as self, but as non-self, and attacked even though it is derived from a sequence in its own genome. Therefore, when a virus-derived sequence is expressed in cancer, cancer cells may be recognized as non-self, and therefore, if the body attacks it as part of an immune response, it may be beneficial in the treatment of cancer, and we are currently conducting research based on this.

Imanishi: I think there is an impression that the novel coronavirus will be something that humanity will probably fight for decades to come. We would like to ask Dr. Nakagawa to comment on future predictions and how metagenomic or bioinformatics analysis can contribute to the development of treatment methods.

Nakagawa: One of the problematic properties of the novel coronavirus is that it can infect various animals with almost no mutation. In addition, although it cannot infect mice as it is, it has become clear that mice can be infected with a variant of the virus with a mutation in a single amino acid, and the host range is quite wide. Therefore, even if a vaccine or the like can resolve the outbreak temporarily, it is quite difficult to completely eliminate the virus from the natural world. It is also suggested that immunity due to vaccines and exposure infections may not be sustained, and many people expect the outbreak to be difficult to control, as the risk of infection will unfortunately be present even if the severity can be prevented. In addition, because mutations continue in the virus, it is important to continue surveillance because the mutant variants may change their properties in the future.

Various coronaviruses still exist in the natural world, and there is a possibility that a coronavirus will emerge by the exchange of genes between viruses gradually, which is called recombination. In that sense, unfortunately, it is difficult to completely eliminate the coronavirus. However, in the current situation where vaccines have become quite widespread, if it is socially acceptable to have flu-like symptoms even if infected, it is more realistic to think about creating a social system where this is the case and responding to it. There are certain limitations. People with underlying conditions that cannot be vaccinated, or there are regions where vaccines are expensive; hence, the question arises on the response of countries other than developed countries. There is also the aspect that multiple vaccinations cannot be administered because of mutations; hence, we must focus on these points. Accordingly, I think with these issues we are moving into the area of social sciences, beyond just the sciences.

The mRNA vaccine is also a result of metagenomics

Imai: Is this new messenger RNA vaccine one of the achievements of metagenomics? The vaccine allows the cell to know the genome sequence of the virus and so it is a system that recognizes what enters your cell as a spike protein and as "non-self"; however, we can hypothesize that the messenger RNA vaccine is also a novel technology in a series of research right?

Nakagawa: That is right. The messenger RNA vaccine was originally a technology that garnered attention from areas such as cancer immunity, but it was put into practical use for the first time in this manner. Moreover, I think that few people assumed that it would have been this effective. There are also coronaviruses that cause severe pneumonia, such as SARS and MERS. However, coronavirus is known as a virus that causes symptoms similar to the so-called common cold, and many people have wondered how difficult it is to prevent infectious diseases such as the common cold with vaccines. I think that it is a fairly advanced technology that can suppress the severity of the disease and the infection itself.

Ando: The fact that the messenger RNA vaccine originally had to be carefully advanced but was abruptly brought to clinical practice given the current situation, and even turned out to be effective, certainly feels like "turning misfortune into fortune."

Imanishi: There is a possibility that it will be applied more to other types of viruses.

Ando: Considering this, will the messenger RNA vaccine be the standard vaccine in the future?

Nakagawa: I believe that there is a good chance.

Ando: After making observations for a long time, I cannot predict what will happen yet, but this is revolutionary because one can now obtain a vaccine without having to consider proteins.

Nakagawa: However, there is a problem of mutation. As the coronavirus spreads from one source, there are few mutations, but for example, influenza and HIV are more diverse than coronaviruses, and we must consider what type of virus to combine in the future. Considering that Associate Professor Imai discussed the relationships with periodontal disease, and he mentioned that when periodontal disease occurs, bacteria does not only pass through the digestive tract from saliva, but also circulate in the blood and impair the function of important organs. From that perspective, are there any diseases that are attracting attention other than diseases of the large intestine?

Imai: It remains unclear whether its route goes directly through the digestive tract or a bloodstream infection. In fact, it seems that sometimes a patient without wisdom teeth is positive in a blood culture test and develops bacteremia; hence, there is a certain possibility of a blood route. However, my personal impression is that the gastrointestinal route is the main route. Steatohepatitis is a disease that has been garnering attention recently. Periodontal disease is also associated with steatohepatitis caused by lifestyle-related diseases, that is, non-alcoholic steatohepatitis, and the same is true for colorectal cancer. Probably not all of them are gastrointestinal routes, but I believe that the management of periodontal disease will receive more attention in the future.

Imanishi: To date, we have taken on the challenge of detecting bacteria in human blood, and we have analyzed the blood of patients with sepsis in several cases. In some cases, bacteria was detected, and in other cases, the bacteria could not be detected. The difference is the number of bacteria. Regarding bacteremia, the number of bacteria is usually very low, and current sequencing techniques are not always sufficient to detect it. Therefore, the detection of bacteria in blood has not yet been put into practical use. We are working on the development of technology that can detect a very small amount of bacteria; therefore, if this issue is cleared, I think we will be able to answer questions such as infection routes in the future.

Ando: Today, you all talked about your original research results on metagenomics and at the Tokai University Institute of Medical Sciences, we would like to continue research that contributes to the progress of medical treatment. Today, we discussed various interesting topics, including hot topics such as the novel coronavirus. Thank you for this lively discussion.