Methanobrevibacter smithii (M. smithii) for use as a biomarker and in the diagnosis and treatment of disorders associated with abnormal microbiota and / or archaeal deficiency

Abstract

The present invention generally relates to the field of gut health, disorders associated with abnormal microbiota and / or archaeal deficiency, and immune system development in early life. More specifically, the present invention relates to M. smithii and its components for use as biomarkers, methods for detecting M. smithii and / or monitoring the colonization status of M. smithii, biosensors and kits for detecting M. smithii or its markers, and M. smithii for promoting immune system homeostasis and gut health and for treating disorders associated with abnormal microbiota and / or archaeal deficiency.

Description

[Technical field]

[0001] The present invention generally relates to the field of gut health, disorders associated with abnormal microbiota and / or archaeal deficiency, and immune system development in early life. More specifically, the present invention relates to M. smithii and its components for use as biomarkers, diagnosis of disorders associated with M. smithii and archaeal deficiency, methods for detecting M. smithii and / or monitoring the colonization status of M. smithii, biosensors and kits for detecting M. smithii or its markers, and M. smithii for promoting immune system homeostasis and gut health and for treating disorders associated with abnormal microbiota (dysbiosis) and / or archaeal deficiency.

[0002] [Background Art and Problems Solved by the Invention] Humans have evolved to develop a high degree of mutualism with gut bacteria. Mutualistic microbes colonize the human intestinal tract during early life in coordination with immune system development. More recently, it has been found that microbiota composition influences immune cell regulation and mucosal antibody secretion, which in turn promotes microbiota diversification, which creates a mutualistic feedback loop with the host. On the other hand, the formation of abnormal microbiota during early life is associated with insufficient immune system development and may favor the emergence of various disorders, including autoimmune disorders.

[0003] The human intestinal tract harbors a diverse community of fiber-fermenting bacteria. These commensal organisms release fermentation by-products such as acetate and butyrate, which in turn promote immunological tolerance.

[0004] Archaea, which are evolutionarily distinct from bacteria, help maintain a gut microenvironment that supports beneficial bacteria.

[0005] M. smithii is the dominant (>90%) archaeal species in humans, accounting for an estimated 10.24±4.58% (mean±standard deviation, SD) of the healthy adult gut microbiome (Joon Yong Kim et al., The human gut archaeome: identification of diverse haloarchaea in Korean subjects, BMC Microbiome, August 2020).

[0006] Archaea have been neglected by the scientific community, likely because they are more difficult to identify and culture than bacteria. This is due to their stronger cell walls, which make it more difficult to extract DNA for analysis. Furthermore, anaerobic bioprocesses for culturing archaea remain a challenge.

[0007] Barnett et al., J Allergy & Clinical Immunology, 2019, found that intestinal M. stadtmanae was inversely associated with asthma in children aged 6 to 10 years.

[0008] Ghavami et al., Microb Pathology, 2018, found that M. smithii is inversely associated with diarrhea in IBD in adults.

[0009] Coker et al., Gastroenterology, 2019, found that M. smithii was inversely associated with colorectal cancer in individuals over the age of 50.

[0010] Camara et al., Scientific Reports, 2021, reported that M. smithii was absent from malnourished infants, while Grine et al., European J of Clinical Microbiology & Inf. Diseases, 2017, reported the presence of M. smithii in gastric fluid of newborns.

[0011] M. smithii is the predominant archaeal species resident in the adult colon and is the main hydrogen sink that allows bacteria to ferment and grow. Despite this important function in the adult microbiota, the role of M. smithii in the development of the infant microbiota and its association with immune system development in early life has not been previously explored.

[0012] It is not known whether M. smithii colonizes the intestinal tract in an age-dependent manner in coordination with the immune system and whether M. smithii colonization is timely coordinated with normal immune system development.

[0013] Colonization of the infant gut plays an important role in immune system development during early life (Tamburini S, Shen N, Wu H, Clemente J, Microbiome in early life: implications for health outcomes, Nature Medicine, 2016). Thus, there is a need for pediatricians to monitor the colonization status associated with normal immune system development.

[0014] Despite extensive research efforts, monitoring the status of the microbiome remains complex, costly, and often unclear, as thousands of bacterial strains exist and there is a high degree of epidemiological variation between different populations.

[0015] The present inventors find that M. smithii archaea is a keystone species of the human microbiota that serves as a hydrogen sink for the fiber-associated microbiota, which plays a known role in regulating the immune system and preventing asthma and allergies in infants (Maslowski K. et al., Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43, 2009, Nature).

[0016] In view of the prior art, there is a need to better understand the role of M. smithii and disorders associated with intestinal health, immune system homeostasis, and abnormal microbiota, archaeal deficiency, and M. smithii deficiency. There is also a need to treat and / or prevent such disorders.

[0017] There is a need for a test that allows monitoring of the intestinal colonization state associated with normal immune system development. Tests that can more easily measure archaeal levels in stool samples are needed to more easily monitor the role of M. smithii archaea in various health settings, particularly in children.

[0018] Further objects and problems addressed by the present invention will become apparent from the description of aspects and embodiments of the invention herein below.

[0019] [Summary of the Invention] The present invention provides uses of M. smithii and markers for detecting M. smithii as biomarkers. The present invention also provides methods for treating and / or preventing one or more of the conditions disclosed herein.

[0020] In one aspect, the present invention provides a marker for detecting and / or determining M. smithii, comprising any one of the amino acid sequences set forth in any one of SEQ ID NOs: 1 to 8, or comprising an amino acid sequence having at least 80% sequence identity to any one selected from SEQ ID NOs: 1 to 8. In one aspect, the present invention provides HmtA as a preferred marker for M. smithii.

[0021] In one aspect, the present invention provides the use of a protein comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 8, or an amino acid sequence having at least 80% sequence identity to any one selected from SEQ ID NOs: 1 to 8, as a marker for M. smithii.

[0022] In one aspect, the invention provides binding molecules, e.g., probes or antibodies or fragments thereof, for detecting, assessing, monitoring and / or determining (e.g., quantitatively and / or semi-quantitatively) M. smithii. Preferably, the binding molecules are specific for M. smithii.

[0023] In one aspect, the present invention provides a binding protein that specifically binds to a marker protein of M. smithii, wherein the marker protein is selected from proteins comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 8, or an amino acid sequence having at least 80% sequence identity to any one selected from SEQ ID NOs: 1 to 8.

[0024] In a further aspect, the present invention provides kits or assays, such as diagnostic kits, for detecting, determining and / or assessing M. smithii.

[0025] In one embodiment, the diagnostic kit is for monitoring and / or assessing one or more selected from the group consisting of immune system homeostasis, gut immune homeostasis, and gut health, preferably in human newborns, infants, and / or toddlers up to 6, 5, or 4 years of age.

[0026] In one embodiment, the diagnostic kit is for monitoring and / or assessing one or more selected from the group consisting of gut colonization status, mucosal immune barrier function, gut immune system status, and gut adaptive immune system status, preferably in human newborns, infants, and / or toddlers up to 6, 5 or 4 years of age.

[0027] In one aspect, the diagnostic kit is for diagnosing one or more selected from the group consisting of abnormal microbiota, dysbiosis, archaeal deficiency, and M. smithii deficiency in human newborns, infants, or toddlers up to 6, 5, or preferably up to 4 years of age.

[0028] The diagnostic kits of the present invention preferably comprise binding molecules for detecting and / or determining or estimating the amount of M. smithii in a sample obtained from an individual.

[0029] In one aspect, the present invention provides the use of M. smithii or a marker thereof to assess whether an individual suffers from or is at risk of developing one or more selected from the group consisting of colic, allergies, asthma, IBD (inflammatory bowel disease) and diarrhea, wherein the human individual is selected from the group of newborns, infants, and toddlers up to 6, 5 or 4 years of age.

[0030] In one aspect, the present invention provides the use of M. smithii to monitor and / or assess immune system homeostasis, intestinal immune homeostasis, and intestinal health in human newborns, infants, or toddlers up to 6, 5, or 4 years of age.

[0031] In one aspect, the present invention provides the use of M. smithii to promote one or more selected from the group consisting of immune system homeostasis, immune homeostasis in the intestine, intestinal health, intestinal colonization status, mucosal immune barrier function, immune system status in the intestine, adaptive immune system status in the intestine, modulating IgA homeostasis, and modulating T cell homeostasis.

[0032] In one aspect, the present invention provides a use of M. smithii for treating and / or preventing one or more selected from the group consisting of dysbiosis, abnormal microbiota, archaeal deficiency, and M. smithii deficiency.

[0033] In one aspect, the present invention provides the use of M. smithii for treating and / or preventing one or more selected from the group consisting of colic, allergies, asthma, colon cancer, IBD and diarrhea.

[0034] In one aspect, the present invention provides the use of M. smithii for modulating the development of the microbiota and / or the immune system.

[0035] In a preferred embodiment, the present invention concerns individuals, preferably human individuals, who are of a young age, such as newborns, infants, and young children, particularly young children up to 6, 5, or 4 years of age.

[0036] The present invention also provides the use of the present invention to fate an individual, preferably a human individual, who is 50 years of age or older. For example, the M. smithii, markers and / or binding molecules of the present invention are used to assess the risk of an individual developing colon cancer. Furthermore, the preparations containing M. smithii can be used to prevent and / or treat colon cancer. In some embodiments, M. smithii is used to promote one or more selected from immune system homeostasis, immune homeostasis in the intestinal tract, intestinal health, etc. in patients suffering from colon cancer.

[0037] In some aspects, the present invention provides methods for detecting, determining, assessing and / or monitoring M. smithii, for example, by detecting and / or assessing a marker of M. smithii. An exemplary marker is HmtA.

[0038] In one aspect, the present invention provides a method for monitoring and / or assessing one or more selected from the group consisting of immune system homeostasis, intestinal immune homeostasis, and / or intestinal health in a human individual.

[0039] In one aspect, the present invention provides a method for diagnosing one or more selected from the group consisting of an abnormal microbiota, a dysbiosis, an archaeal deficiency, and an M. smithii deficiency in a human individual.

[0040] The method of the present invention preferably comprises the steps of determining whether M. smithii is present in a sample collected from a human; and if present, determining or estimating the amount of M. smithii in the sample, as well as detecting insufficient immune system homeostasis, intestinal immune homeostasis, and / or intestinal health if M. smithii is absent or present in an amount below a threshold level, and diagnosing an abnormal microbiota, dysbiosis, archaeal deficiency, and / or M. smithii deficiency, respectively, if M. smithii is absent or present in an amount below a threshold level.

[0041] In some aspects, the present invention relates to treating and / or preventing one or more selected from the group consisting of colic, allergies, asthma, colon cancer, IBD, diarrhea, abnormal microbiota, archaeal deficiency, and M. smithii deficiency, and / or promoting one or more selected from the group consisting of immune system homeostasis, immune homeostasis in the gut, gut health, gut colonization, mucosal immune barrier function, gut immune system, and gut adaptive immune system, the method comprising administering to an individual in need thereof a composition or formulation comprising M. smithii.

[0042] Further aspects and preferred embodiments of the present invention are defined herein below and in the appended claims. Further features and advantages of the present invention will become apparent to those skilled in the art from the description of the preferred embodiments set forth below. [Brief description of the drawings]

[0043] [Figure 1A]Figure 1A shows the age-dependent increase of M. smithii biomarkers in a cohort of healthy infants. Microbial cells were gated using forward and side scatter, and then the percentages of IgA+ and F420- populations, as well as IgA+F420+ populations, were determined using a four-quadrant gate. The individual ratios of each population were plotted. Samples with a percentage of M. smithii biomarkers above 3.8% were marked as "M. smithii high" with a circle, and those below were marked as "M. smithii low" with a diamond shape. [Figure 1B] Figure 1B shows the age-dependent increase in M. smithii biomarkers in a cohort of healthy infants. The percentage of cells positive for the M. smithii biomarker F420 from each sample was measured, and the mean of the samples for each age category was then calculated and plotted (SD and statistics not shown). [Figure 2A] Figure 2A shows the positive correlation of relative abundance of M. smithii with microbiota sIgA coverage in infants. The percentage of cells positive for M. smithii biomarkers was plotted (x-axis) against the percentage of IgA-covered microbiota cells (y-axis) and a logarithmic trendline was calculated by excel formula to determine the correlation between M. smithii levels and the percentage of IgA-covered microbiota cells. [Figure 2B] Figure 2B shows the positive correlation of the relative abundance of M. smithii with microbiota sIgA coverage in infants. The graph compares the percentage of IgA-coated microbiota cells between "M. smithii high" vs. "M. smithii low" from the 18-24 month age group. [Figure 3A] 3A-3E show the binding of five scFv fragments based on the five monoclonal antibodies mAb1-mAb5 to HmtA according to an embodiment of the present invention. The binding of the scFvs was demonstrated in an ELISA setting as described in the Examples. [Figure 3B] 3A-3E show the binding of five scFv fragments based on the five monoclonal antibodies mAb1-mAb5 to HmtA according to an embodiment of the present invention. The binding of the scFvs was demonstrated in an ELISA setting as described in the Examples. [Figure 3C] 3A-3E show the binding of five scFv fragments based on the five monoclonal antibodies mAb1-mAb5 to HmtA according to an embodiment of the present invention. The binding of the scFvs was demonstrated in an ELISA setting as described in the Examples. [Figure 3D] 3A-3E show the binding of five scFv fragments based on the five monoclonal antibodies mAb1-mAb5 to HmtA according to an embodiment of the present invention. The binding of the scFvs was demonstrated in an ELISA setting as described in the Examples. [Figure 3E] 3A-3E show the binding of five scFv fragments based on the five monoclonal antibodies mAb1-mAb5 to HmtA according to an embodiment of the present invention. The binding of the scFvs was demonstrated in an ELISA setting as described in the Examples. [Figure 4] FIG. 4 shows colonization of M. smithii in gnotobiotic mice following oral administration of viable M. smithii as determined by flow cytometry. [Diagram 5] FIG. 5 shows colonization of M. smithii in gnotobiotic mice following oral administration of viable M. smithii as determined by PCR. [Figure 6] FIG. 6 shows the mean relative abundance of fecal microbiome composition from OligoMM12 mice treated versus untreated with M. smithii, as determined based on 16S rRNA sequencing.

[0044] Hereinafter, preferred embodiments of the device of the present invention are described to illustrate the present invention, without any intention of limiting the scope of the present invention.

[0045] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to M. smithii and markers that allow the detection of M. smithii in samples, particularly samples obtained from human or animal individuals. M. smithii and its markers can be used for diagnostic and prognostic purposes and as biomarkers, based on the role of M. smithii in, among other things, immune system homeostasis and intestinal health. The present invention also encompasses prophylactic, therapeutic and other methods, including, for example, the administration of M. smithii in the form of viable M. smithii or inactivated M. smithii.

[0046] The invention also relates to binding molecules for detecting M. smithii, for example by detecting binding of the binding molecule to a marker for M. smithii. The invention also includes the use of binding molecules that are specific for M. smithii in the uses, methods and kits of the invention.

[0047] The term "marker" for the purposes of this specification refers to a molecule, generally a protein or nucleic acid molecule, produced by M. smithii and which allows to detect M. smithii in a sample, preferably in a specific manner. The specificity is preferably such that the "marker" allows to distinguish between M. smithii and other microorganisms of the intestinal microflora, preferably non-archaeal microorganisms, such as gram-positive and / or gram-negative bacteria. More preferably, the "marker" also allows to distinguish, for example, between M. smithii and other archaea, such as M. boviskoreani and M. stadtmonae. In other words, the "marker" is preferably a molecule of detection that allows to conclude, preferably with high certainty, that M. smithii is present in a sample.

[0048] For purposes of this specification, the term "biomarker" generally refers to the use of M. smithii and its markers to make predictions about health conditions and / or risks associated with a deficiency of M. smithii in an individual. According to certain embodiments, M. smithii and its markers are used as biomarkers.

[0049] The present invention is based, in part, on the surprising finding that the spread of M. smithii in the infant microbiome appears to be age-dependent and correlates with immune system development. In various embodiments, the present invention relates to assessing, determining and / or monitoring M. smithii in one or more selected from newborns, infants, and toddlers up to 6 years of age, preferably up to 5 years of age, for example up to 4 years of age.

[0050] Furthermore, it is known that archaeal deficiency, particularly M. smithii deficiency, is associated with certain health risks in adults, e.g., human individuals, over the age of 50. In some embodiments, the invention encompasses assessing, detecting and / or monitoring M. smithii in adults, particularly human individuals, over the age of 50.

[0051] To assess, determine and / or detect M. smithii, a sample of the individual is preferably obtained. The sample may be a previously obtained sample. The sample may be selected from a stool, oral saliva, vaginal mucosa (prenatal), breast milk or colostrum sample.

[0052] Preferably, the sample is a stool sample. The stool sample is preferably processed as necessary to allow the marker to interact with the binding molecule and to detect the interaction. Preferably, the sample is processed to allow a conclusion to be made about the concentration of M. smithii in the sample, for example based on the characteristics of the sample, such as weight, volume, etc.

[0053] M. smithii or its marker can be determined qualitatively, for example, to know whether M. smithii is present in a sample or not. In a preferred embodiment, M. smithii is determined semi-quantitatively or quantitatively, preferably to allow the evaluation of the concentration described above. Preferably, in some embodiments, binding molecules are used to determine M. smithii qualitatively, semi-quantitatively and / or quantitatively. For example, the level of a marker, for example HmtA or any other marker, is evaluated semi-quantitatively or quantitatively.

[0054] To monitor M. smithii, M. smithii is preferably determined as described above repeatedly. For example, M. smithii may be determined twice, three times or more at different times. For example, M. smithii is determined once or more frequently per year, once or more frequently per two years. For example, M. smithii is determined once, twice, three times or more frequently per year. In some embodiments, M. smithii is determined and / or evaluated, for example, every two months or every month.

[0055] The present invention provides a marker suitable for detecting M. smithii. The marker is preferably selected from proteins and nucleic acid molecules, such as polynucleotides. The nucleic acid molecule may be selected from DNA and RNA molecules. Preferably, the marker is present only in a specific form, such as having a specific amino acid or nucleotide sequence, only in M. smithii or in other microorganisms that are possibly not present in the microbiome of humans and / or animals.

[0056] The present inventors have analyzed the proteome of M. smithii and identified several proteins that can be used as markers. Table 1 below lists eight different proteins that can be used as markers for the purposes of the present invention. F420+ (SEQ ID NO: 6), a protein previously reported as a marker for M. smithii, can also be used. In some embodiments, F420+ (SEQ ID NO: 6) is excluded from the markers, kits, uses and methods of the present invention.

[0057] In a preferred embodiment, the marker is selected from a protein comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 1 to 8 or comprising an amino acid sequence having at least 80% sequence identity, preferably at least 90% identity, most preferably at least 95%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 8. Details regarding sequence identity are further provided elsewhere herein, particularly below before the Examples section.

[0058] Preferably, the marker remains specific to M. smithii by virtue of proteins having amino acid sequences that do not have 100% identity to SEQ ID NOs: 1-8, including modified proteins linked to other molecules, peptides and / or proteins.

[0059] In certain embodiments, the markers are selected from the proteins listed below in Table 1. The sequences of these markers are further shown below in Table 2.

[0060] [Table 1]

[0061] It should be noted that marker proteins may naturally occur in association with other molecules, for example, fused to or otherwise covalently bound to other peptides, proteins, sugars, lipids, and other molecules. Furthermore, markers may be artificially generated. To this end, markers may be purposefully fused to other peptides, proteins, sugars, lipids, and other molecules, which may be useful for various purposes, for example, to generate, detect, and isolate the marker.

[0062] In a preferred embodiment, the marker comprises an isolated protein.

[0063] In certain embodiments, M. smithii or markers thereof may be used to monitor and / or assess one or more selected from immune system homeostasis, immune homeostasis in the intestinal tract, intestinal health, intestinal colonization status, mucosal immune barrier function, the status of the immune system in the intestinal tract, and the status of the adaptive immune system in the intestinal tract.

[0064] In certain embodiments, M. smithii or markers thereof may be used to assess whether an individual suffers from or is at risk of developing one or more selected from the group consisting of colic, allergies, asthma, colon cancer, IBD and diarrhea, preferably said individual being a human newborn, infant and / or toddler up to 6, 5 or 4 years of age.

[0065] In certain embodiments, M. smithii or markers thereof may be used to diagnose one or more selected from the group consisting of dysbiosis, abnormal microbiota, archaeal deficiency, and M. smithii deficiency.

[0066] In particular, the markers may be used in methods including diagnostic or prognostic methods, risk assessment methods, and the like, as well as in biosensors and / or diagnostic kits.

[0067] In one embodiment, the marker for M. smithii is selected from HmtA and F420+. In one embodiment, the present invention provides the use of HmtA for detecting M. smithii in a sample.

[0068] In a preferred embodiment, the marker is or comprises HmtA, a protein comprising or consisting essentially of the amino acid sequence of SEQ ID NO: 1, or a protein having at least 80% sequence identity to SEQ ID NO: 1. SEQ ID NO: 1 is a histone having 65 amino acids found in M. smithii, encoded by the gene Msm_0213.

[0069] Preferably, the HmtA is present in the intestinal tract of an individual or in the faeces of an individual.

[0070] Preferably, HmtA is detected in a sample obtained from within the intestinal tract of an individual, or in a sample of the individual's feces.

[0071] In one embodiment, HmtA is used as a biomarker.

[0072] In certain embodiments, HmtA is used as a biomarker for one or more selected from the group consisting of immune system homeostasis, intestinal immune homeostasis, and intestinal health.

[0073] In one embodiment, HmtA is used to assess the risk of developing one or more selected from the group consisting of colic, allergies, asthma, colon cancer, IBD and diarrhea.

[0074] In one embodiment, HmtA is used to assess the risk for a human individual of developing one or more selected from the group consisting of colic, allergies, asthma, IBD and diarrhea, wherein the human individual is selected from newborns, infants, and young children up to 6, 5, or 4 years of age.

[0075] In one embodiment, the invention provides the use of HmtA to assess the risk of having or developing colon cancer in a human individual aged 50 years or older.

[0076] In one embodiment, the present invention provides the use of HmtA for detecting M. smithii in a sample.

[0077] In one embodiment, the present invention provides a method for the preparation of a determining the presence or absence of M. smithii in a sample collected from the individual; and / or Determining or estimating the amount of M. smithii in a sample obtained from an individual The present invention includes the use of a binding molecule for

[0078] The phrase "estimating the amount of M. smithii" is intended to encompass a semi-quantitative assessment of M. smithii and / or its markers.

[0079] In one embodiment, the binding molecule comprises: Oligonucleotides or polynucleotides; A binding protein, preferably an immunoglobulin superfamily (IgSF) protein or a fragment thereof, preferably an antibody or a fragment thereof. The present invention includes one or more selected from the following:

[0080] When the binding molecule is an oligonucleotide or polynucleotide, it is preferably single stranded, hi certain embodiments, the binding molecule is a probe, preferably an oligonucleotide or polynucleotide probe.

[0081] When the marker comprises a protein, for example HmtA, it is preferably detected by a binding protein. The binding protein is preferably selected from the binding proteins of the immunoglobulin superfamily (IgSF), or fragments of such proteins. In some embodiments, the binding protein comprises one or several fragments of one or more IgSF proteins. The IgSF protein may be selected from an antibody, a TCR (T cell receptor), a BCR (B cell receptor), a CAR (chimeric antigen receptor), or a fragment of any one of the above. In a preferred embodiment, the binding protein is or comprises an antibody or a fragment thereof.

[0082] A fragment of a binding protein may be selected from, for example, a polypeptide comprising one or more selected from the group consisting of an Fc fragment (fragment crystallizable), a Fab fragment (fragment antigen-binding), an Fv fragment (variable fragment), an scFv (single-chain variable fragment), an Ab heavy chain, an Ab light chain, a VH domain (heavy chain variable region), a VL domain (light chain variable region), a CH (heavy chain constant region), and a CL (light chain constant region). Exemplary Fab fragments include Fab, Fab', and F(ab')2 fragments.

[0083] In certain embodiments, the binding protein is or comprises a human or humanized IgSF protein or fragment thereof, such as a human or humanized antibody or fragment thereof.

[0084] In a preferred embodiment, the binding protein binds to, and is preferably specific for, a marker, In a preferred embodiment, the binding protein binds to, and is preferably specific for, HmtA.

[0085] As used herein, the term "specifically binds" should be interpreted to mean that the binding interaction between a binding molecule (e.g., a probe, Ab, TCR, BCR, CAR, or fragment thereof) and a marker, e.g., HmtA, or a nucleic acid molecule, is dependent on the presence of an antigenic determinant or epitope of the marker protein bound to the binding protein, or a specific nucleotide sequence in the case of a nucleic acid molecule. Thus, the binding molecule preferentially binds to or recognizes an antigenic determinant or nucleotide sequence of the marker molecule, even when present in a mixture of other molecules. In one example, the binding molecule reacts or associates with one or several specific markers, or cells expressing said specific markers, more frequently, more rapidly, for a longer period of time, and / or with a higher affinity than it does with alternative antigens, cells, or nucleic acid molecules. By reading this definition, it is also understood that, for example, a binding molecule that specifically binds to one or more specific markers may or may not specifically bind to a second antigen or nucleic acid molecule. Thus, "specific binding" does not necessarily require exclusive or undetectable binding to another molecule. Generally, references herein to binding refer to specific binding, and each term should be understood to provide explicit support for the other term. Methods for determining specific binding will be apparent to those skilled in the art. For example, the binding molecules of the present disclosure are incubated with a specific marker, such as HmtA, or cells expressing said specific marker, or mutant forms thereof, or unrelated antigens or nucleic acid molecules. The binding of the binding molecules to said specific marker, its mutant forms, or unrelated molecules is then determined, and binding molecules that bind to said specific marker, rather than mutants, unrelated molecules, as shown above, are considered to specifically bind to said specific marker.

[0086] The "binding" of the binding molecule of the invention to a marker, e.g., HmtA, is preferably a non-covalent bond. The binding is preferably a specific bond. The molecular forces involved in the binding molecule-marker binding are preferably selected from one or more of the group consisting of electrostatic forces, hydrogen bonds, hydrophobic interactions, and van der Waals forces.

[0087] In a preferred embodiment, the binding molecule is a binding protein, preferably an IgSF protein or a fragment thereof. The binding protein preferably specifically binds to a marker for M. smithii.

[0088] In one embodiment, the binding protein specifically binds to a protein comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs:1-8.

[0089] The present invention provides monoclonal antibodies that specifically bind to HmtA. In certain embodiments, the binding protein comprises these antibodies or a fragment thereof, such as the VH, VL, and / or one of one or more CDRs of any one of these antibodies. Such fragments are listed in Table 2 below. In certain embodiments, the binding protein comprises at least one amino acid sequence selected from SEQ ID NOs: 9-49.

[0090] In one embodiment, the binding protein comprises a VH region comprising an amino acid sequence selected from SEQ ID NO: 9, 11, 13, 15, 17, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, 97%, 98% or 99% sequence identity, preferably at least 95% sequence identity, to any one of SEQ ID NOs: 9, 11, 13, 15, 17.

[0091] In certain embodiments, the binding protein comprises a VL region comprising an amino acid sequence selected from SEQ ID NOs: 10, 12, 14, 16, and 18, or an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95%, 97%, 98% or 99% sequence identity, preferably at least 95% sequence identity, to any one of SEQ ID NOs: 10, 12, 14, 16, and 18.

[0092] In one embodiment, the binding protein comprises one, two, three, preferably up to six CDR regions having a sequence selected from any one of SEQ ID NOs: 20-49 or a sequence having at least 80%, preferably at least 90%, more preferably at least 95%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 20-49.

[0093] In certain embodiments, the binding protein is a. SEQ ID NO: 20 to 22 and / or SEQ ID NO: 23 to 25, b. SEQ ID NO: 26 to 28 and / or SEQ ID NO: 29 to 31, c. SEQ ID NO: 32 to 34 and / or SEQ ID NO: 35 to 37, d. SEQ ID NOs: 38 to 40 and / or 41 to 43, and e. SEQ ID NO: 44 to 46 and / or SEQ ID NO: 47 to 49 or a sequence that independently has at least 80% sequence identity with any one of these sequences. and / or L-CDR1, L-CDR2 and L-CDR3 amino acid sequences selected from the group of: The further preferred sequence identity percentages given above also apply to this and other embodiments.

[0094] Preferably, the binding protein is an artificial protein or a naturally occurring protein that has been modified to be different from the naturally occurring molecule. Preferably, the binding protein is conjugated to one or more tags (gold, latex, fluorophore, peptide tag) to allow one or more of the production, isolation, manipulation of the binding protein and / or to allow reading in a test kit containing the binding protein.

[0095] In a preferred embodiment, the binding protein is or comprises an scFv protein, where the VH region is fused to the VL region via a suitable linker, preferably an artificial linker. The linker preferably comprises 5 to 40, preferably 10 to 25 amino acids. Preferably, the linker is flexible enough to provide a functional scFv with suitable binding properties. Preferably, the linker comprises a glycine for flexibility, and preferably a serine and / or threonine moiety, e.g. for solubility reasons. An exemplary linker is shown in SEQ ID NO: 19.

[0096] For example, an scFv may comprise the VH-VL pairs of SEQ ID NOs: 9 and 10; 11 and 12; 13 and 14; 15 and 16; 17 and 18. The variable domains may optionally be linked in, for example, an N-VH-VL-C or N-VL-VH-C format, where N and C indicate corresponding ends of the fusion protein.

[0097] Binding molecules, e.g., binding proteins, can be used to detect, determine, and / or assess M. smithii, e.g., to detect markers of M. smithii. Binding proteins can be used to determine and / or assess the presence of M. smithii qualitatively, semi-quantitatively, or quantitatively.

[0098] In certain embodiments, the binding molecule, e.g., binding protein, is used to assess one or more selected from immune system homeostasis, immune homeostasis in the gut, gut health, gut colonization status, mucosal immune barrier function, status of the immune system in the gut, status of the adaptive immune system in the gut. Preferably, the binding protein is specific for HmtA, and more preferably, is a binding protein according to an embodiment described herein.

[0099] In certain embodiments, the binding molecule, e.g., binding protein, is used to assess whether an individual suffers from or is at risk of developing one or more selected from the group consisting of colic, allergies, asthma, colon cancer, IBD, and diarrhea. Preferably, the binding protein is specific for HmtA, and more preferably is a binding protein according to an embodiment described herein.

[0100] Assessing the risk of an individual to develop a particular disorder is an embodiment of a prognostic use of M. smithii, its markers, and / or binding molecules to the markers. Preferably, the binding molecule is a binding protein specific for HmtA, more preferably a binding protein according to the embodiments described herein.

[0101] In certain embodiments, the binding molecule, e.g., binding protein, is used to diagnose one or more selected from the group consisting of dysbiosis, abnormal microbiota, archaeal deficiency, and M. smithii deficiency. Preferably, the binding protein is specific for HmtA, and more preferably, is a binding protein according to an embodiment described herein.

[0102] In certain embodiments, the binding molecule, e.g., binding protein, is used to assess whether a human individual suffers from or is at risk of developing one or more selected from the group consisting of colic, allergies, asthma, IBD, and diarrhea, wherein the human individual is selected from the group consisting of newborns, infants, and toddlers up to 6, 5, or preferably 4 years of age. Preferably, the binding molecule, e.g., binding protein, is specific for a marker, preferably a marker disclosed herein, e.g., in Table 1, most preferably HmtA. Preferably, the binding molecule is a binding protein according to an embodiment described herein, more preferably a binding protein specific for HmtA.

[0103] In one embodiment, the binding molecule, e.g., the binding protein, is used to assess an individual's risk of having or developing colon cancer, where the individual is 50 years of age or older, and the binding protein is specific to a marker for M. smithii, preferably a marker selected from the markers listed in Table 1. Preferably, the marker is HmtA. Preferably, the binding molecule is a binding protein according to an embodiment described herein, e.g., a binding protein that specifically binds to HmtA.

[0104] The present invention provides test kits, including diagnostic tests, and biomarkers for assessing and / or determining M. smithii, preferably qualitatively, semi-quantitatively or quantitatively.

[0105] The test kit may be in the form of a binding molecule, e.g., a binding molecule-based test, e.g., an antibody-based test. Such tests include rapid tests, rapid diagnostic tests that directly detect the presence or absence of an antigen, particularly a marker. Preferably, rapid tests provide results within 5 minutes to 1 hour.

[0106] In some embodiments, the test is a lateral flow test.

[0107] Preferably, the test comprises at least a housing, and detection agents, reagents, e.g., binding molecules, e.g., the binding proteins disclosed herein, are placed in suitable locations within the housing and / or in separate receptacles, e.g., tubes, etc.

[0108] In some kit applications, it may be advantageous to apply cell lysis solutions and buffers to prepare the sample in order to increase the sensitivity of detection of the biomarkers. One or more lysis solutions and / or buffers may be present in the test kit.

[0109] The test preferably comprises a sample receptacle or well into which a sample, e.g., a stool sample or a sample prepared from a stool sample, is added. Preferably, a predetermined amount, volume or weight of sample is added. The receptacle or well is preferably located in a housing.

[0110] Preferably, the test comprises a binding molecule according to the invention, preferably a binding protein, such as an antibody or fragment thereof, that specifically binds to HmtA. The binding protein may be immobilized or may be provided such that it is capable of diffusion or flow, typically upon exposure to the sample.

[0111] Preferably, the test further comprises one or more further binding molecules, e.g. further binding proteins, for the purpose of providing a negative control and / or for the purpose of immobilizing, in the event of a positive test result, the complex of analyte (marker) and binding molecule, e.g. binding protein, if the marker is present.

[0112] Typically, the assay involves a substrate, preferably an artificial substrate, e.g., a membrane, e.g., a nitrocellulose membrane, onto which a binding molecule, e.g., a binding protein, is deposited, typically to allow a readout at a predetermined location on the substrate. The substrate is preferably mounted, e.g., supported or immobilized, within a housing.

[0113] The test preferably includes one or more receptacles, such as Eppendorf tubes, in which additional reagents required for using the test are placed.

[0114] The test kit may be used to assess, determine and / or monitor M. smithii. The test may also be used for one or more of the diagnostic and / or prognostic purposes disclosed herein.

[0115] The kits and methods of the present invention may provide a negative result if M. smithii is absent or present in an amount considered to be below a given level, which may be considered a threshold level for distinguishing healthy from unhealthy and / or poor M. smithii colonization conditions.

[0116] In one embodiment, the threshold level is defined by a 90% or greater reduction in the relative abundance of M. smithii in stool samples compared to the average abundance of M. smithii in the same age group as determined for healthy individuals. In other words, the threshold level can be defined as a 10-fold (-1 log10) reduction in M. smithii count compared to the average in the age group of the individual being evaluated.

[0117] When M. smithii is assessed by DNA analysis, infants with M. smithii deficiency have a 1 log reduction in M. smithii specific DNA relative to total bacterial DNA in stool samples compared to the average of healthy infants from the same age bracket. When viable bacteria is assessed, infants with M. smithii deficiency have a 1 log10 reduction in M. smithii viable cells relative to total viable bacterial cells (per gram of stool) in stool samples compared to the average of healthy infants from the same age bracket.

[0118] In a preferred embodiment, the binding molecules of the invention are used to qualitatively and / or semi-quantitatively assess and / or determine the colonization status of M. smithii.

[0119] The age categories are preferably defined by the following time periods, each of which defines the age category: 6-12 months, 12-18 months, 18-24 months, 24-30 months, 30-36 months, 3-4 years, 4-5 years, 5-6 years.

[0120] Microbiota density (i.e., the number of viable microbial cells per gram of stool) can be estimated using anaerobic culture methods based on the number of colony forming units (CFU) per gram of stool. Microbiota density is approximately 10 11 ~10 12 This is the range of individual cells.

[0121] If the microbiota density is known, the relative abundance of M. smithii (e.g., determined by flow cytometry or qPCR) can be used to calculate the absolute amount of M. smithii cells per gram of stool.

[0122] In order to compare values ​​for an individual being evaluated with the average values ​​for the corresponding age category, M. smithii is preferably determined in the same way to ensure that a meaningful comparison is possible.

[0123] In other embodiments, unhealthy and poor M. smithii colonization status resulting in a negative diagnostic evaluation and / or test result is found when M. smithii represents 5% or less of the gut microbiome, preferably 3%, 1.5% or 1% or less. These percentages may refer to either or both of the following: viable cells (in comparison with the CFU of other bacteria) and biomass (in comparison with the biomass of other bacteria in the gut microbiome). According to this embodiment, no comparison with the average value of M. smithii in healthy individuals of the corresponding age group is necessary, and the threshold level is determined with reference to the individual's own microbiome.

[0124] A negative test result is interpreted according to the individual embodiment to mean: unfavorable and / or insufficient immune system homeostasis and / or immune homeostasis in the gut, and / or poor gut health. Additionally, a negative test result may mean: insufficient gut colonization status, mucosal immune barrier function, gut immune system status, and gut adaptive immune system status, presence of abnormal microbiota, dysbiosis, archaea deficiency, and M. smithii deficiency, and / or increased risk of suffering from and / or developing one or more selected from the group consisting of colic, allergies, asthma, IBD, colon cancer, and diarrhea.

[0125] In a preferred embodiment, the present invention also provides a method of administering M. smithii.

[0126] M. smithii is preferably administered to treat and / or prevent conditions and / or disorders as defined herein. In some embodiments, M. smithii is administered to address archaeal deficiency, including, but not necessarily limited to, M. smithii deficiency in an individual.

[0127] In some embodiments, M. smithii is administered not necessarily to treat and / or prevent a disorder, but generally to promote and / or improve intestinal health.

[0128] In some embodiments, M. smithii is administered to promote and / or improve immune system homeostasis, immune homeostasis in the intestinal tract, intestinal health, intestinal colonization status, mucosal immune barrier function, immune system status in the intestinal tract, or adaptive immune system status in the intestinal tract.

[0129] In some embodiments, M. smithii is administered to regulate immune status and / or homeostasis, for example in infants. In some embodiments, inactivated M. smithii is administered to modulate immune status, immune homeostasis, for example, one or more selected from IgA homeostasis and T cell homeostasis, in neonates, infants and young children.

[0130] In certain embodiments, M. smithii is administered to treat and / or prevent abnormal microbiota, dysbiosis, archaeal deficiency, and M. smithii deficiency.

[0131] In certain embodiments, M. smithii is administered to treat and / or prevent one or more selected from the group consisting of colic, allergies, asthma, colon cancer, IBD, and diarrhea.

[0132] In certain embodiments, M. smithii is administered to an individual, such as a human, in need thereof in the form of a suitable formulation.

[0133] In some embodiments, M. smithii is administered to individuals at a young age, hi some embodiments, M. smithii is administered to one or more selected from newborns, infants, and children up to 6, 5, or 4 years of age.

[0134] A newborn is a human individual up to 28 days of age. For purposes herein, an infant is a human individual over 28 days and up to 1 year of age. A toddler is a human individual over 1 year and up to 3 years of age. For purposes herein, infants and children up to 6 years of age, preferably up to 5 years of age, most preferably up to 4 years of age, are the primary subject of the present invention. Thus, the term "toddler" is used herein generally to include children up to the ages indicated above.

[0135] However, it should be noted that human individuals over the age of 50 are also subject to the present invention. For example, the present invention provides for the assessment and / or determination of M. smithii in these individuals to determine their risk of developing colon cancer, as well as the use of live and / or inactivated M. smithii for the prevention and / or treatment of colon cancer in these individuals.

[0136] In one embodiment, M. smithii is used to modulate the development of the microbiota and / or immune system in human individuals during early life, particularly those selected from newborns, infants, and young children up to 6, 5, or 4 years of age.

[0137] M. smithii is preferably administered in the form of a formulation and / or composition comprising M. smithii. In the formulation, M. smithii may be live or viable. Alternatively, inactivated M. smithii may be administered. In some embodiments, the formulation comprises live and inactivated M. smithii.

[0138] The formulation preferably comprises M. smithii and at least one pharma- ceutically acceptable carrier. In some embodiments, the formulation is provided in the form of a tablet, pill, capsule, formula, nutritional composition, powder form in a sachet, or liquid drop. The nutritional composition may be powdered or liquid, e.g., ready to drink.

[0139] M. smithii or preferably a formulation containing it is preferably administered by enteral, sublingual, buccal, oral, rectal, intrarectal, or intranasal administration.Preferably, the formulation, in particular the carrier or carriers, are adapted to a suitable administration route.It should be noted that the administration route also determines the dosage and / or frequency of administration of M. smithii.

[0140] In the case of administration of live M. smithii, dosage is preferably determined in terms of viable cells, which may be expressed as a number per dosage unit and / or per gram of carrier material. As used herein, the terms "live M. smithii" and "viable M. smithii" are used interchangeably and refer to M. smithii cells that are capable of growing and proliferating when exposed to the appropriate conditions.

[0141] In one embodiment, the formulation provided for administration contains 10 mg of 10 ... 7 ~10 12 , preferably 10 8 ~10 11 In the case of infants receiving live M. smithii, the formulation may contain, for example, 5×10 viable cells per unit of administration. 7 ~30×10 12 Viable cells, preferably 5 x 10 8 ~30×10 11 The culture may directly contain viable cells.

[0142] The number of viable M. smithii per gram of preparation is preferably determined by staining protocols reported in the literature, in particular the live / dead staining protocol disclosed in WO 2020 / 002543.

[0143] The dosage will preferably depend on factors such as frequency of administration, delivery vehicle (administration form and / or route), and the health condition of the individual, which need to be assessed when administration is prescribed or recommended.

[0144] M. smithii can be provided by known processes. To obtain a high amount or ratio of live M. smithii, freeze-drying is the preferred method. Freeze-drying can result in up to 40% viable cells when the freeze-drying process is optimized. Alternatively, spray-drying protocols can also be performed to obtain M. smithii containing live M. smithii. To generate inactivated M. smithii, inactivation protocols can be used, for example, as disclosed in WO2020 / 002543.

[0145] Table 2 below relates to certain embodiments of the present invention. Experimental examples to illustrate the present invention are further disclosed below.

[0146] [Table 2] TIFF2025508105000004.tif203149 TIFF2025508105000005.tif217149

[0147] The percentage of "sequence identity" can be determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) due to optimal alignment of the two sequences. The percentage is calculated by determining the number of positions where identical amino acid residues are present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for comparison is performed by global pairwise alignment, for example, using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443. The percentage of sequence identity can be easily determined, for example, using the program Needle with the BLOSUM62 matrix and the following parameters: gap-open=10, gap-extension=0.5.

[0148] In certain embodiments, the replacement and / or substitution of any amino acid residue in a sequence having a certain sequence identity with any one of the sequences disclosed herein is preferably under the condition that any amino acid that is not identical to the amino acid at the corresponding position in the given sequence is conservatively substituted according to the substitutions set out in Table 3 below, which shows the amino acid residues that can be substituted for one another.

[0149] [Table 3]

[0150] Although certain preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the present invention be limited to such embodiments. Various modifications can be made thereto without departing from the scope and spirit of the present invention as set forth in the following claims. Hereinafter, examples of the present invention are disclosed. These examples are merely illustrative and are not intended to limit the scope of the present invention.

[0151] [Example] Example 1: M. smithii in infancy and early childhood Introduction It is not known whether M. smithii colonizes the intestinal tract in an age-dependent manner in coordination with the immune system and whether timely colonization of M. smithii correlates with normal immune system development.

[0152] Mucosal antibodies, called immunoglobulin A (sIgA), are continually secreted in the intestinal tract and coat the surfaces of mutualistic gut bacteria, a process that aids in the development of immune tolerance and microbiota diversity in the host.

[0153] The secretion and quality of sIgA is regulated by the microbiota composition, and in turn the sIgA coverage of the microbiota aids in the development of tolerance and symbiotic interactions (Sutherland DB, Suzuki K, Fagarasan S. Fostering of advanced mutualism with gut microbiota by Immunoglobulin A, 2016, Immunological Reviews; Sutherland DB, Fagarasan S. IgA synthesis: form of functional immune adaptation extending beyond gut, 2012, Current Opinion in Immunology).

[0154] Microbial species that preferentially drive adaptive sIgA secretion and microbiota coverage in early life are of great interest to achieve host-microbiota symbiosis and immune system homeostasis.

[0155] Insufficient sIgA coverage is associated with abnormal microbiota, dysbiosis and inflammation. Therefore, there is a need to identify effective biomarkers to monitor the development of abnormal microbiota and the immune system.

[0156] The correlation between M. smithii colonization and sIgA coverage of the microbiota in early life is evaluated here. The first goal is to understand whether M. smithii colonization status can potentially be used as a biomarker to evaluate normal vs. abnormal microbiota and poor immune system development in infants.

[0157] method Ethical approval was obtained according to the standard CER-VD in Vaud, Switzerland. A healthy cohort of 30 individuals from a kindergarten in Lausanne consented to participate in the study. Participants were divided into three age groups (n=10 per group): 6-12 months, 12-18 months, and 18-24 months. Faeces were collected from participants.

[0158] One gram of feces was collected and suspended in 1 mL of 4% PFA in PBS for 10 min. The suspension was mixed vigorously and diluted with 10 mL of PBS. The suspension was centrifuged at 1000×g for 10 min and the supernatant was collected. The number of cells per mL was counted and the cell density was determined to be 10 cells / mL. 7 The staining protocol was performed with Invitrogen goat anti-human IgA Fc secondary antibody FITC (catalog H14101). Flow cytometer analysis was performed using a BD LSRFortessa cell analyzer. M. smithii biomarker F420 was excited by a 405 nm laser (purity of the gated population of M. smithii was confirmed by cell sorting F420+ gating events using a BD FACSAria II and PCR testing). The percentage of F420+ events and the percentage of IgA+ coated bacterial cells were analyzed by flow-jo software and plotted using excel software.

[0159] result Infant stool samples were collected from healthy infants in Lausanne, Switzerland in separate groups aged 6-12 months, 12-18 months, and 18-24 months.

[0160] The microbial cell population extracted from the stool samples was analyzed by flow cytometer using two key parameters: i) % IgA coated microbial cells, ii) % F420+ 405 nm excited cells.

[0161] F420 is a coenzyme that is exclusively produced by M. smithii in the human microbiota, with fluorescence excitation at 405 nm, as further confirmed by cell sorting and PCR identification, as well as by fluorescence microscopy analysis.

[0162] The data show the absence or low levels of M. smithii biomarkers in infants aged 6-12 months, as shown in Figures 1A and 1B. An explosion in M. smithii biomarkers was observed in the 12-18 month age group, and a significant increase in M. smithii biomarkers was observed in the 18-24 month age group.

[0163] Seventy percent of subjects aged 12–24 months showed a clear elevation of M. smithii biomarkers, labeled “M. smithii high” versus the 6–12 month age group, labeled “M. smithii low.”

[0164] Increases in flow cytometer M. smithii biomarkers positively correlated with levels of IgA-coated microbiota cells, as shown in Figure 2 A. Figure 2 B shows that infants in the 18–24 month group who were “M. smithii high” had twice the percentage of IgA-coated microbiota cells, on average 40.7% IgA-coated microbiota cells, versus “M. smithii low” infants from the same age group who had only 19.4% IgA-coated microbiota cells.

[0165] Observations and Conclusions The data show a correlation between the age of the infants and the percentage of M. smithii biomarkers monitored in the stool samples. The absence of M. smithii in the tested infants is associated with lower levels of sIgA microbiota coverage. The data reveal that M. smithii levels in stool samples correspond directly with sIgA coverage levels, thus providing information about the state of the immune system in the intestinal tract during early life. Clinical studies suggest that intestinal archaea are inversely correlated with asthma (Barnett et al., Intestinal archaea inversely associated with asthma, 2019, Journal of Allergy and Clinical Immunology), raising the possibility of either an indirect or direct interaction between M. smithii and immunological function. Our study shows the first relationship between M. smithii and mucosal immune phenotype in infants.

[0166] IgA coating is a T cell-dependent process (Sutherland DB, Suzuki K, Fagarasan S, Fostering of advanced mutualism with gut microbiota by Immunoglobulin A, 2016, Immunological Reviews), suggesting a link between M. smithii colonization and T cell differentiation and immune system phenotype. T cells are master regulators of the adaptive immune system that underpin immunological homeostasis and human health. Further studies in gnotobiotic mice inoculated with M. smithii and minimal bacterial consortia will allow elucidation of the cellular pathways involved.

[0167] Surprisingly, the expansion of M. smithii in the infant microbiota is age-dependent and correlates with immune system development. Studies demonstrate that M. smithii can be used as a biomarker to infer the state of the immune system in the intestinal tract in infants.

[0168] Example 2: Selection of markers to identify M. smithii To develop a rapid test that would allow monitoring of M. smithii , the proteome of M. smithii (strains ATCC 35061; DSM 861; OCM 144; PS) was harvested and evaluated.

[0169] Eight candidate marker proteins were identified and are shown in Table 2. Several markers were selected based on the presence of strong and distinctive 3.5 kD, 4.3 kD and 5.6 kD peaks seen in mass spectrometry, for which the RNA polymerase subunit is hypothesized to be a possible match since it is highly expressed.

[0170] Histone HmtA (SEQ ID NO:1), a histone with 65 amino acids and a MW of approximately 6.9 kDA, was selected for further evaluation as a marker specific for M. smithii. HmtA has a homodimeric structure with 71.88% sequence similarity to histone HMFA of Methanothermus fervidus.

[0171] Example 3: Preparation of antibodies specific to HmtA Five monoclonal antibodies, mAb1 to mAb5, were selected from the Tomlinson I,J library, a semi-synthetic naive library of human origin kindly provided by the MRC, Cambridge, UK. This library contains approximately 109 independent scFv recombinant antibodies inserted into the pIT2 vector (de Wildt et al., 2000). The GST-fusion proteins used to select the recombinant antibodies were prepared according to the procedure disclosed in C. Blanc et al. "Use of In Vivo Biotinylated GST Fusion Proteins to Select Recombinent Antibodies", ALTEX. 2014, Vol. 31, No. 1, pp. 37-42.

[0172] The scFv (single chain variable fragment) recombinant antibodies were prepared according to the general formula N-VH-VL-C (scFV) and using the linker shown in Table 2 (SEQ ID NO: 19) between the VH and VL regions.

[0173] The VH and VL sequences and the amino acid sequence of the linker are shown in Table 2.

[0174] Example 4: ELISA test The binding of all antibodies was tested by ELISA. A glutathione S-transferase (GST)-HmtA chimeric construct was prepared and further bacterially biotinylated in vivo at the N-terminus of the HmtA construct (GST) (GCJ construct). The construct was immobilized on coated polystyrene plates. A control chimeric GST construct lacking HmtA (GCH) was similarly prepared, biotinylated, and immobilized.

[0175] HmtA and control constructs were exposed to each of the five scFVs of Example 3 under binding conditions.

[0176] Binding was revealed by a secondary HRP polyclonal antibody specific for the (GST)-HmtA chimeric construct, measuring the colorimetric reaction.

[0177] The results are shown in Figures 3A-3E for each of the scFVs. All scFVs bind to the HmtA-containing construct but not to the control construct, and the colorimetry is dependent on the antibody concentration as shown in the figures.

[0178] Example 5: Detection of M. smithii in gnotobiotic mice We use gnotobiotic mice colonized with M. smithii and 12 defined commensal bacterial species to compare microbiota samples containing M. smithii (HmtA positive) versus microbiota samples not containing M. smithii (HmtA negative).

[0179] As a control, gnotobiotic mice are lacking any M. smithii but containing only the 12 defined commensal bacterial species. The antibodies of the invention give a positive reading on stool samples obtained from M. smithii positive mice.

[0180] Example 6: Differentiation between M. smithii and Methanobrevibacter boviskoreani M. bovis coreanii is the predominant archaeal species in adult pigs and is related to M. smithii. A sample containing M. bovis coreanii that does not contain M. smithii is obtained. The HmtA antibody works only in samples containing M. smithii archaeal species, but no binding is detected in samples containing only M. bovis coreanii.

[0181] Example 7: Discrimination between M. smithii and M. statmonae M. statomonae is grown as a monoculture, and the anti-HmtA antibodies of the invention do not detect this alternative archaeal species.

[0182] Example 8: Treatment of Archaeal Deficiency by Oral M. smithii Administration Objectives and Background The goal of this example is to discover whether orally administered archaea can treat archaeal deficiency. Specifically, gnotobiotic mice with a defined microbiome composition that are archaeal deficient are treated with oral administration of M. smithii archaea, and the archaeal colonization status in the intestinal tract of the treated mice is determined.

[0183] Infants who lack archaeal colonization at a young age due to factors that may include type of birth, lack of human milk, and nutritional disorders are deficient in the keystone species M. smithii, which serves as the primary hydrogen sink for fermenting bacteria in the human gut. It is not known whether oral administration of M. smithii archaea can stimulate archaeal colonization and treat archaeal deficiency. It is not known whether orally administered archaea can efficiently anchor and colonize themselves in the intestinal mucosa, a requirement for stable colonization and incorporation into the gut microbiome. The early life stage (0-4 years) provides a window of opportunity to colonize a diverse microbiome associated with immune system development. Experimental mouse models are the gold standard in preclinical screening capable of evaluating therapeutic efficacy of M. smithii for the treatment of archaeal deficiency in humans. A gnotobiotic mouse maintained in a germ-free facility, called Oligo MM12, with a defined and controlled microbiome is described, providing an experimental model to evaluate treatments for archaeal deficiency.

[0184] Oligo MM12 mice were originally defined by Brugiroux, S. et al. (2016) Genome-guided design of a defined mouse microbiota that confers colonization resistance against Salmonella enterica serovar Typhimurium. Nature Microbiology 2:16215. doi:10.1038 / nmicrobiol.2016.215

[0185] Methods and Results The bacteria lacked archaea and were isolated from Akkermansia muciniphila YL44, Bacteroides caecimuris I48, Muribaculum intestinale YL27, Turicimonas muris YL45, Bifidobacterium longum subsp. animalis YL2, Enterococcus faecalis KB1, Acutalibacter muris KB18, Clostridium clostridioforme YL32, Blautia coccoides YL46, and Bacteroides cereus YL47. We established C57BL / 6 Oligo MM12 gnotobiotic mice colonized with a defined bacterial consortium consisting of Lactobacillus coccoides YL58, Flavonifractor plautii YL31, Lactobacillus reuteri I49, and Clostridium innocuum I46. Experimental mice were maintained in a pathogen-free facility.

[0186] Six-week-old archaea-deficient C57BL / 6 OligoMM12 mice (n = 5 males, n = 3 females) were treated with 10 mL of orally administered freeze-dried viable M. smithii archaeal organisms. 9 Animals were treated with a single dose of 0.01 mg / kg / day of M. smithii. Successful colonization of animals with M. smithii was confirmed by screening fecal samples at weekly intervals for 8 weeks by PCR, FACS and / or 16s rRNA sequencing (Oxford nanopore). All three methods confirmed colonization with M. smithii. Two breeding pairs were set up. The resulting offspring were again tested at 4, 6 and 8 weeks of age to confirm successful vertical transfer of M. smithii.

[0187] Figure 4 shows the amount of M. smithii in the feces. Feces of naive Oligo MM12 mice, as well as adult mice inoculated with a single dose of M. smithii and their offspring, were washed and analyzed by flow cytometry. + Events (biomarkers of M. smithii) are expressed as a percentage of the parent gate (i.e., total single bacteria). Data shown are from two independent experiments. Parent and offspring mice are stably colonized with an average relative abundance of M. smithii in their microbiome of 12%, whereas naive control mice have a relative abundance of M. smithii of 0% in their microbiome.

[0188] Figure 5 shows colonization of M. smithii confirmed by fecal PCR testing. A 222 bp PCR product from M. smithii nifH gene is amplified for 30 cycles using the primers shown below according to a method adapted from Ufnar, JA et al. (2000). Detection of the nifH gene of Methanobrevibacter smithii: a potential tool to identify sewage pollution in recreational waters. Journal of applied microbiology, Vol. 101(No. 1), pp. 44-52. Forward AACAGAAAACCCAGTGAAGAG-3' (SEQ ID NO:50) Reverse AGTAAAGGCACTGAAAAACC-3' (SEQ ID NO:51)

[0189] In Figure 5, columns 1-3 show the positive control for M. smithii, columns 4-7 are representative M. smithii colonized Oligo MM12 mouse fecal samples, column 8 is an archaea-depleted Oligo MM12 mouse fecal sample, and column 9 is the negative control (H20).

[0190] For Figure 6, fecal samples were collected from untreated (n=8) or M. smithii treated (n=12) OligoMM12 mice and 16S rRNA sequencing Oxford Nanopore qPCR was performed to determine the microbiota composition as the mean percentage of relative abundance. Treatment with M. smithii resulted in a highly significant increase in Bacteroides cessimulis (I48) and a significant increase in Clostridium innocuum (I46) compared to untreated mice. Meanwhile, there was a significant reduction in Enterocloster clostridioformis (YL32) in M. smithii (DSM861) treated mice versus untreated mice. Statistical T-tests were calculated by excel software ( *** P value < 0.001, and ** P value < 0.01, and * P value < 0.05).

[0191] [Table 4]

[0192] conclusion Orally administered lyophilized M. smithii demonstrated that it treats archaeal deficiency by allowing stable archaeal colonization. The data demonstrate that infants with poor growth and archaeal colonization in early life can be effectively treated by orally administering M. smithii to allow archaeal colonization within the normal time window (0-4 years) during which the immune system is developing.

[0193] The qPCR data show that Archaea treatment significantly shifts the microbiome composition (Figure 6, Table 4). Archaea treatment allows modulating the developing microbiome in favor of an Archaea-associated microbiome that supports fiber fermentation, production of short-chain fatty acids, as well as normal development of the immune system, including T cell homeostasis and IgA regulation. Notably, the species Bacteroides cessimuris (I48) and Clostridium innocuum (I46), associated with carbohydrate and polysaccharide metabolism, were significantly enhanced in OligoMM12 mice treated with M. smithii. Interactions between the microbiome and the immune system in early life have been shown elsewhere to develop immune system homeostasis and microbiome resilience (higher resistance to dysbiosis), with lasting effects into adulthood.

Claims

1. A method for collecting data to monitor and / or evaluate one or more of the following selected from the group consisting of immune system homeostasis, intestinal immune homeostasis, and intestinal health in individuals that are human neonates, infants and / or children up to 4 years of age, comprising using M. smithii as a biomarker.

2. The method according to claim 1, wherein the intraintestinal immune homeostasis and / or intraintestinal health comprises one or more selected from the group consisting of the colonization state of the intestines, the immune barrier function of the mucosa, the state of the intraintestinal immune system, and the state of the intraintestinal adaptive immune system.

3. A method for collecting data to diagnose one or more conditions selected from the group consisting of abnormal microbiome, dysbiosis, archaeal deficiency, and M. smithii deficiency in a human neonatal, infant, or child up to 4 years of age, comprising using M. smithii as a biomarker.

4. A method for collecting data to assess whether a human neonatal, infant, and / or child up to four years of age has one or more conditions selected from the group consisting of colic, allergies, asthma, IBD, and diarrhea, or is at risk of developing them, the method comprising using M. smithii as a biomarker.

5. This includes using a binding molecule that is specific to M. sumisi, wherein the binding molecule is To determine the presence or absence of M. sumisi in the sample collected from the aforementioned individual, and / or The method according to any one of claims 1 to 4, used to determine or estimate the amount of M. sumisi in a sample obtained from the aforementioned individual.

6. The aforementioned binding molecule, oligonucleotides or polynucleotides; Binding proteins or fragments thereof that are immunoglobulin superfamily (IgSF) proteins, The method according to claim 5, comprising one or more selected from the following.

7. The method according to claim 6, wherein the binding protein specifically binds to a marker for M. sumisi.

8. The method according to claim 6, wherein the binding protein specifically binds to a protein containing an amino acid sequence having at least 90% sequence identity with any one of sequence numbers 1 to 8.

9. The method according to claim 6, wherein the binding protein specifically binds to HmtA containing the amino acid sequence of SEQ ID NO:

1.

10. A binding protein that specifically binds to a marker protein of M. sumisi, wherein the marker protein is selected from proteins containing any one of the amino acid sequences of SEQ ID NOs: 1 to 8.

11. The binding protein according to claim 10, wherein the marker protein is HmtA containing the amino acid sequence of SEQ ID NO:

1.

12. Use of a protein selected from proteins containing any one of the amino acid sequences of SEQ ID NOs: 1-8 as a marker for M. sumisi.

13. A diagnostic kit for monitoring and / or evaluating one or more of the following in human neonates, infants and / or children up to four years of age: immune system homeostasis, intestinal immune homeostasis, and intestinal health, wherein the diagnostic kit contains a binding molecule for detecting and / or determining or estimating the amount of M. smithii in a sample obtained from an individual.

14. A formulation or composition containing M. smithii for administration to one or more human individuals selected from neonates, infants, and children up to four years of age, for use in treating and / or preventing abnormal microbiomes, archaeal deficiencies, and M. smithii deficiencies.

15. Preparations or compositions containing M. smithii for administration to one or more human individuals selected from neonates, infants, and children up to four years of age, for use in promoting one or more of the following: immune system homeostasis, intestinal immune homeostasis, and intestinal health.

16. A preparation or composition containing M. smithii for administration to one or more human individuals selected from neonates, infants, and children up to four years of age, for use in treating and / or preventing one or more conditions selected from the group consisting of colic, allergies, asthma, colon cancer, IBD, and diarrhea.

17. A formulation or composition containing M. smithii for administration to one or more human individuals selected from neonates, infants, and children up to four years of age, for use in modulating the development of the microbiome and / or immune system.

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