WO2016164402A1

WO2016164402A1 - Methods of diagnosing and treating autoimmune disease

Info

Publication number: WO2016164402A1
Application number: PCT/US2016/026129
Authority: WO
Inventors: Patrick Gaffney; Judith JAMES
Original assignee: Oklahoma Medical Research Foundation
Priority date: 2015-04-06
Filing date: 2016-04-06
Publication date: 2016-10-13

Abstract

The present disclosure relates to methods of diagnosing and treating Systemic Lupus Erythematosus (SLE) based on microbe population alterations in the oral cavity and intestines. It has been discovered that SLE patients present with the following abnormalities as compared to healthy individuals: (1) decreases in the general microbial diversity in fecal samples; (2) decreases in the microbial phyla Bacteroidetes in fecal samples; (3) decreases in the Faecalibacterium genus in fecal samples; (4) decreased phylogenetic and species diversity in saliva samples; (5) increases in the microbial phyla Bacteroidetes in saliva samples; and (6) increased levels of the Prevotella genus in saliva. Thus, the present disclosure provides methods of diagnosing SLE by exploiting these abnormalities. Furthermore, the present disclosure provides methods of treating SLE by resolving the above microbe abnormalities.

Description

METHODS OF DIAGNOSING AND TREATING AUTOIMMUNE DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[01] The present application claims priority to U.S. Provisional Application No. 62/143,571, filed April 6, 2015, the entirety of which is incorporated herein by reference.

BACKGROUND

[02] Genome wide association studies have identified hundreds of genes associated with complex, autoimmune diseases such as Systemic Lupus Erythematosus (SLE). However, the genetic architecture and aggregate risk factor explained by these studies do not completely elucidate the cause of SLE. Complex disease can be caused not only by genetic factors, but also environmental stimuli as well. While it is not feasible to research the totality of the environment with which an SLE patient interacts, one may realistically begin assaying the intimate border at which the human immune system meets the environment, and the population of organisms that can both assist and hinder humanity in its environmental interactions. This is, of course, the human microbiome.

[03] Human beings play host to a large number of microorganisms on the various surfaces of their bodies. In aggregate the total number of these organisms outstrips the human somatic and germ-line cells by at least an order of magnitude. Many of these cells, such as those living in the intestinal microbiota, exist in a symbiotic nature with their hosts, assisting with the digestion of complex plant polysaccharides as well as providing a competitively hostile environment against microorganisms that would otherwise lead to illness. Given also that humanity has evolved an exquisitely complex system of cells to prevent infection and systemic movement of microorganisms it is then of little doubt that the human immune system and the human microbiome are in constant interaction.

[04] To date, there have not been any comprehensive studies to determine if SLE patients display any irregularities in their microbiome patterns or levels. As a result, the diagnostic and therapeutic potential of the microbiome have yet to be appreciated.

SUMMARY

[05] The present disclosure is based on the discovery that certain autoimmune disease patients, such as SLE patients, display the following compared to control individuals: (1) decreases in the general microbial content in fecal samples; (2) decreases in the microbial phyla Bacteroidetes in fecal samples; (3) decreases in the Faecalibacterium genus in fecal samples; (4) decreased phylogenetic and species diversity in saliva samples; (5) increases in the microbial phyla Bacteroidetes in saliva samples; and (6) increased levels of the Prevotella genus in saliva.

[06] The present disclosure is therefore directed to methods of diagnosing and treating autoimmune disease patients based on abnormalities in the microbiome.

[07] In an embodiment of the present invention, a method for diagnosing autoimmune diseases is provided. The method comprises the steps of: a) obtaining a biological sample from a subject; b) isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; c) quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level; d) determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with an autoimmune disease; and e) diagnosing the subject with the autoimmune disease if the target microbe population level is outside the established target microbe population level range, or diagnosing the subject as not having the autoimmune disease if the target microbe population level is within the established target microbe population level range. Additionally, the method above can be used to provide a prognosis for a subject having an autoimmune disease based on the target microbe population level being within or outsi de of the established range. Even further, the method may further comprise, based on this diagnosis or prognosis, a step of administering a therapeutic composition sufficient to bring the target microbe population level within the established range that is not associated with disease.

[08] In another embodiment of the present invention, a method of diagnosing an autoimmune disease is provided. The method comprises the steps of: a) obtaining a first biological sample and a second biological sample from a subject, the first biological sample is from the oral cavity, and the second biological sample is a fecal sample; b) isolating and purifying a target molecular population from the first biological sample and the second biological sample to yield a first purified target molecular sample and a second purified target molecular sample, respectively; c) quantifying a first target microbe population in the first purified target molecular sample to generate a first target microbe population level and quantifying a second target microbe population in the second purified target molecular sample to generate a second target microbe population level; d) determining whether the first and second target microbe population levels are within a first established target microbe population level range and a second established target microbe population level range, respectively, the first and second established target microbe population level ranges are not associated with the autoimmune disease; e) diagnosing the subject with the autoimmune disease, if the first and second target microbe population levels are outside the first and second established target microbe population level ranges, respectively, or diagnosing the subject as not having the autoimmune disease if the first and second target microbe population levels are within the first and second established target microbe population level ranges, respectively.

[09] In yet another embodiment of the present invention, a method for treating autoimmune disease comprises administering to a subject with an autoimmune disease a composition comprising microbes or spores thereof from one or both of the phyla Bacteroidetes and Firmicutes in an amount sufficient to increase the Bacteroidetes or Firmicutes microbe population to levels associated with individuals not having the autoimmune disease.

[10] In yet another embodiment of the present invention, a method for treating an autoimmune disease comprises administering to a subject with the autoimmune disease a composition comprising an anti-microbial agent in an amount sufficient to decrease the level of the phyla Bacteroidetes generally or more specifically, to the genus Prevotella in the oral cavity of the subject.

[11] In another embodiment, a method for treating a subject with an autoimmune disease based on the subject's microbiome is provided. The method comprises the steps of: a) obtaining a biological sample from a subject; b) isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; c) quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level; d) determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with autoimmune disease; and e) administering to the subject a therapeutic composition if the target microbe population level is outside the established target microbe population level range, wherein the therapeutic composition comprises the target microbe population if the target microbe population level is lower than the established target microbe population level range, and wherein the therapeutic composition is an anti-microbial agent specific to the target microbe population if the target microbe population level is higher than the established target microbe population level range.

[12] This summary is provided to introduce disclosure, certain aspects, advantages and novel features of the invention in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

DRAWINGS

[13] Fig. 1A illustrates a decrease in microbial richness in the lupus fecal samples compared to the control individuals using phylogenetic and non-phylogenetic metrics as explained in Example 2.

[14] Fig. IB illustrates a decrease in the bacterial group Bacteroidetes in the lupus fecal samples compared to the control individuals as explained in Example 2.

[15] Fig. 2A illustrates decreased diversity of the lupus saliva both phylogenetically and in observed species as explained in Example 3.

[16] Fig. 2B illustrates higher proportions of Bacteroidetes in lupus saliva as compared to the control individuals as explained in Example 3.

[17] Fig. 3 illustrates highest abundance of Prevotella reported in the current data as explained in Example 3.

DESCRIPTION

[18] The present disclosure is directed to methods of diagnosing and treating autoimmune disease patients based on abnormalities in the microbiome.

[19] In an embodiment of the present invention, a method for diagnosing an autoimmune disease, comprises the steps of: a) obtaining a biological sample from the oral cavity of a subject; b) isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; c) quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level, wherein the target microbe population is from the phyla Bacteroidetes; d) determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with an autoimmune disease; and e) diagnosing the subject with the autoimmune disease if the target microbe population level is outside the established target microbe population level range, or diagnosing the subject as not having the autoimmune disease if the target microbe population level is within the established target microbe population level range.

[20] In one aspect of the above embodiment, the target microbe population is of the genus Prevotella. In yet another aspect of the embodiments of this disclosure, the autoimmune disease includes, but is not limited to SLE, Sjogren's Syndrome, and multiple sclerosis. In certain aspects of this disclosure, methods for diagnosing may also be used for providing a prognosis.

[21] In yet another aspect, the method of the above embodiment further comprises administering a therapeutic composition to normalize the target microbe population level with the established range for not being associated with disease.

[22] In another embodiment of the present invention, a method of diagnosing SLE is provided. The method comprises the steps of: a) obtaining a first biological sample and a second biological sample from a subject, the first biological sample is from the oral cavity, and the second biological sample is a fecal sample; b) isolating and purifying a target molecular population from the first biological sample and the second biological sample to yield a first purified target molecular sample and a second purified target molecular sample, respectively; c) quantifying a first target microbe population in the first purified target molecular sample to generate a first target microbe population level and quantifying a second target microbe population in the second purified target molecular sample to generate a second target microbe population level; d) determining whether the first and second target microbe population levels are within a first established target microbe population level range and a second established target microbe population level range, respectively, the first and second established target microbe population level ranges are not associated with SLE; e) diagnosing the subject with SLE, if the first and second target microbe population levels are outside the first and second established target microbe population level ranges, respectively, or diagnosing the subject as not having SLE if the first and second target microbe population levels are within the first and second established target microbe population level ranges, respectively.

[23] In another embodiment, a method for treating a subject with SLE based on the subject's microbiome is provided. The method comprises the steps of: a) obtaining a biological sample from a subject; b) isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; c) quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level; d) determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with SLE; and e) administering to the subject a therapeutic composition if the target microbe population level is outside the established target microbe population level range, wherein the therapeutic composition comprises the target microbe population if the target microbe population level is lower than the established target microbe population level range, and wherein the therapeutic composition is an anti-microbial agent specific to the target microbe population if the target microbe population level is higher than the established target microbe population level range.

[24] In an embodiment of the present invention, a method for diagnosing or determining the prognosis of an autoimmune disease, comprises the steps of: a) obtaining a biological sample from a subject having the autoimmune disease or suspected of having the autoimmune disease, wherein the biological sample is of gastrointestinal origin; b) isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; c) quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level, wherein the target microbe population is from the phyla Bacteroidetes or Firmicutes or a combination of both; d) determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with an autoimmune disease; and e) diagnosing the subject with the autoimmune disease or otherwise determining a prognosis if the target microbe population level is outside the established target microbe population level range, or diagnosing the subject as not having the autoimmune disease or otherwise determining a prognosis if the target microbe population level is within the established target microbe population level range. In another embodiment, the method further comprises administering to the subject a therapeutic composition sufficient to bring the target microbe population level to be within the standard range. In some embodiments, the therapeutic composition comprises microbes or spores thereof from the phyla Bacteroidetes or Firmicutes or a combination of both, or more specifically, microbes or spores thereof from the genus

Faecalibacterium.

[25] In any of the above embodiments, the biological sample may be a fecal sample, and the target microbe population may selected from the group consisting of general microbe content, the phyla Firmicutes generally, the phyla Bacteroidetes generally, various genus of the phyla Firmicutes, including Faecalibacterium, and any combination thereof. In another instance, the biological sample is an oral sample, and the target microbe population is selected from any of the genus in the phyla Bacteroidetes, including but not limited to Prevotella, and more generally to the overall Bacteroidetes content.

[26] In yet another instance, the target molecular population is metagenomic DNA, and the purified target molecular sample is a purified metagenomic DNA sample. The step of quantifying the target microbe population is performed by subjecting the purified metagenomic DNA sample to metagenomic analysis, or otherwise described in the Examples herein below. In yet another instance, the target molecular population is DNA, and the purified target molecular sample is a purified DNA sample. The step of quantifying the target microbe population is performed by subjecting the purified DNA sample to polymerase chain reaction. In certain aspects, the polymerase chain reaction uses primers sufficient to amplify nucleic acid sequences specific to one or both of Bacteroidetes and Firmicutes. In yet another instance, the target molecular population is metabolites and the purified target molecular sample is a purified metabolite sample. The step of quantifying the target microbe population is performed by subjecting the purified metabolite sample to metabolomic analysis. In another instance, the step of quantifying the target microbe population is performed by subjecting the purified metabolite sample to mass spectrometry.

[27] In yet another embodiment of the present invention, a method for treating SLE comprises administering to a subject with SLE, a composition comprising microbes from one or both of the phyla Bacteroidetes and Firmicutes in an amount sufficient to alter the Bacteroidetes or Firmicutes microbe population to levels associated with individuals not having SLE.

[28] In yet another embodiment of the present invention, a method for treating SLE comprises administering to a subject with SLE, a composition comprising an anti-mi crobial agent in an amount sufficient to decrease the level of the phyla Bacteroidetes generally or more specifically, to the genus Prevotella in the oral cavity of the subject.

[29] As used herein, the term "microbe" refers to one or more of the microorganisms found in the human body that are considered to be, at appropriate levels, commensal and symbiotic, and in some instances can also be pathogenic.

[30] As used herein, the term "target molecular population" refers to the molecular entity being targeted for isolation and purification from one or more microbes in a biological sample including, but not limited to proteins, peptides, nucleic acids, chemical substances, lipids, metabolites, and other biological entities that the one or more microbes may express.

[31] As used herein, the term "target microbe population" refers to the amount or population of the target molecular population from a particular microbe phyla, genus, or species found in a biological sample. Specific examples include microbes form the phyla Bacteroidetes or Firmicutes, or more specifically, microbes from the genus Faecalibacterium or Prevotella. This term may also refer to the general microbiome of a biological sample.

[32] As used herein, the term "target microbe population level" refers to the amount or quantity of the target microbe population present in a given biological sample. For example, in instances where the target molecular population is DNA and the target microbe population is Prevotella, then the target microbe population level is a quantification of Prevotella in a given biological sample based on a DNA indication specific to Prevotella.

[33] As used herein, the term "an established target microbe population level range" refers to a predetermined range of the target microbe population level that is found to be associated with an indication of a normal target microbe population level found in a healthy patient or otherwise not indicative as being associated with Systemic Lupus Erythematosus. This established range may not be the same across all demographics, such that a male and a female may have different established target microbe population level ranges, or such ranges may vary by age, ethnicity, and the presence of other disease states. The established target microbe population level range may be established by compiling a statistically compelling demographic of biological samples from individuals that do not have disease and from individuals that do have disease, and based on such, calculating a range of target microbe levels that are indicative of a disease-free state.

[34] With respect to the methods of diagnosis described herein, the step of obtaining a biological sample can be performed directly with the subject by collecting a saliva sample, biopsy, oral swab, gingival plaque sampling, stool sampling, skin swab, and vaginal swab, or any other means known to one of skill in the art for obtaining a biological sample having a target microbe population. However, the step of obtaining the biological sample can be indirect, for example, receiving a biological sample from a separate party that is not performing the remaining steps. However, this step should not be construed to encompass any action taken by the separate party.

[35] The step of isolating and purifying the target molecular population from the biological sample may be performed by any method known in the art by which microbe molecules may be separated from the non-microbe components of the biological sample. One example of a purification and isolation method is discussed in detail below in the Examples.

[36] Given that the need to raise or lower the level of a particular microbe may be different with respect to the oral cavity and the intestinal region, some of the therapeutic embodiments here relate to administration of anti-microbial agents specific for the microbe population targeted for decrease while other compositions may comprise the microbe desired for increase. Examples of anti-microbial agents sufficient to reduce the population of Bacteroidetes generally, or more specifically, the Prevotella genus in the oral cavity include erythromycin and tetracycline. With respect to the methods of treatment, the desired microbe or anti-microbial agent to be administered to the patient may be formulated in any manner presently established in the art sufficient for delivering microbes to humans via gavage, capsule, enema, lozenge, wafer, or fecal transplant. The dosing regimen may require daily or weekly administration depending on the starting level of microbe. Thus, the dosing may be required to continue throughout the life of the subject in order to maintain the appropriate level of microbe.

[37] In other embodiments, the therapeutic composition may comprise microbes or spores thereof from the phyla Bacteroidetes or Firmicutes, or more specifically, microbes or spores thereof from the genus Prevotella or Faecalibacterium. The composition may be formulated in a number of different manners for formulating microbe-containing compositions generally known to those skilled in the art. In some instances, the composition may be a capsule that releases its contents upon entry into an enteric environment. In other instances, the composition may be formulated in a manner suitable for administration as an enema. Other formulations and routes of administration are contemplated and are readily appreciated by those skilled in the art.

[38] It is to be understood that the foregoing embodiments and aspects may be combined, as applicable, without departing from the spirit of this disclosure.

EXAMPLES

[39] The following examples are provided in order to demonstrate and further illustrate certain embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

Materials and Methods

[40] A cohort of patients from the Oklahoma Lupus Cohort, who are actively engaged in the provision of samples, were identified. Current antibiotic use, current disease status, physical fitness, ethnicity, sex, BMI and pregnancy were assessed in this cohort to evaluate fitness for inclusion into this study. These criteria were used to ensure that the intestinal microbiota was as similar as possible, given their possibility of skewing microflora abundances.

[41] Fecal and saliva samples were collected from 100 patients with lupus and 100 healthy control subjects matched by gender, age, ethnicity and BMI. Fecal samples were collected using FISHERBRA D commode specimen collection systems (Therm oFisher, Waltham, MA) followed by immediate freezing of the specimens. Following return of the specimens to the researcher two 500 mg subsamples of stool were made with the remaining specimen being stored at -20°C in 30mL containers. The two 500 mg stool samples were initially broken up using bead beating with 1 mm zirconia/silica beads on a horizontal vortexer in the presence of MoBiolysis buffer.

[42] Saliva and possible gingival plaque sample were harvested using defined protocols already in place in our Sjogren's syndrome research clinic at OMRF. Samples were processed and stored in parallel with fecal specimens. [43] Metagenomic DNA was isolated through the use of the MO BIO POWERSOIL DNA isolation kit (MO BIO Laboratories, Carlsbad, CA). Briefly, the lysed stool samples were incubated with a series of solutions designed to break down organic materials and remove humic acids, cellular debris, and proteins. Nucleic acids were then chromatographically purified away from any other contaminants through a series of spin columns and washes. Purified DNA was then ready for metagenomic sequencing.

[44] To identify the microorganisms present in each sample sequencing libraries compatible with the ILLUMINA SBS chemistry was generated using KAPA Library Preparation Kit (KAPA BIOSYSTEMS, Woburn, MA). Metagenomic DNA was first sheared using a COVARIS S2 sonicator, followed by an enzymatic end-repair reaction. Blunt-ended DNA molecules were then be given a single A-base tail via another enzymatic reaction and then ligated to the ILLUMINA chemistry compatible adapters. Ligated products were then enriched by a short, 8-cycle PCR amplification reaction. During the ligation reaction step, individual samples were ligated with adapters containing DNA sequences unique to their respective sample. This sample-specific barcode sequence allowed each sample, after precise quantification via fluorometry and/or qPCR, to be pooled together in sets of 10 in equimolar amounts prior to sequencing.

[45] Each pool of metagenomic samples were then loaded onto an ILLUMINA HISEQ 2500 and sequenced using paired-end lOObp reads in the rapid read mode, with one flowcell being loaded per metagenomic pool. Base quality scores and raw data filters were monitored during sequencing. After sequencing the pool of reads was broken down and binned according to their respective samples via the sequence of the ligated adapter tag incorporated during the library preparation process. Tag delineation was allowed to have one mismatched base during the binning process to account for possible sequencing errors during the index read. Data quality for each sample was monitored using the program FastQC, identifying any sequencing artifacts, overly represented populations of low complexity reads, and overall quality across the length of each read. Any reads that did not meet the quality thresholds were either removed or trimmed back through the use of the program Trimmomatic.

[46] Sequence reads for each sample were analyzed using the Ray bioinformatics suite for metagenomic studies. Ray Meta was used to generate a de novo sequence assembly of contigs and scaffolds. This assembly was used by Ray Communities to recapitulate, via sequence homology, the overall taxonomic structure of the microbial community. Ray Ontologies also used the de novo generated contigs to deduce the overall metabolome of the microbiotic community. Through the use of the metabolomic reconstruction, it was possible to identify enriched pathways in the community. The taxonomic and metabolomic reconstructions of the intestinal and oral microflora were compared between cases and controls. Furthermore, the differences between case and control samples were checked for association with genetic data for each sample from previously completed studies containing these samples.

EXAMPLE 1

[47] In order begin characterizing the microbiome of SLE, 18 patients with SLE from the Oklahoma Lupus Cohort were enrolled; a longitudinal cohort of over 400 SLE patients were followed every 3 months at the OMRF. In parallel, a group of healthy controls were enrolled after determining the absence of autoimmune disease using a screening questionnaire. Cases and controls were matched by age, gender and ethnicity. All subjects were enrolled using consent forms approved by the OMRF institutional review board. Stool and saliva specimens were collected from each subject and processed according to established protocols for tissue preservation and banking.

[48] Following isolation of DNA from both aliquots of feces and saliva, samples were PCR amplified for 16S ribosome sequences using the Earth microbiome project protocol for 30 cycles, pooled and sequenced. Resulting reads were split by barcode, and quality filtered to remove low quality bases (q<30), and reads with ambiguous bases (TNT). Read pairs were merged to reconstruct the full ~250bp V4 ribosome fragment, and Operational taxonomic unit (OTU) clustering was performed using the Greengenes 13.8 database as reference. A clustering threshold of >97% was used to assign sequences to OTUs. Resulting OTU tables were rarefied to a depth of 10,000 reads per sample, and alpha diversity, and taxonomic summaries were generated. Non-parametric Kruskal-Wallis tests were used to test for significant differences, followed by false discovery rate adjustment of p values in R. Only taxa with a median abundance of 10 counts (0.1% rel. abundance) within at least one population were filtered out. A p-value of <0.05, and FDR of <0.1 was used to identify taxa showing significant differences. [49] Lupus Fecal Samples. Microbial richness was estimated using phylogenetic (Faith's PD) and non-phylogenetic (observed species) metrics. Fig. 1A illustrates a statistically significant decrease in microbial richness in the lupus samples compared to control individuals. These results were consistent with the existence of a dysbiosis state in SLE.

[50] Relative abundance of microbial phyla was obtained from rarefied taxon tables. Fig. IB illustrates that at the phylum level, a decrease in the bacterial group Bacteroidetes was evident in the SLE samples compared to controls. Table 1 illustrates while both the SLE and control groups have high level of Firmicutes, they show differences when examined at the genus level. In particular, the genus Faecalibacterium demonstrated a significant reduction in SLE compared to control samples (median counts - SLE=19, control=339, FDR p-value=0.0421).

TABLE 1

Fecal Samples Median Counts

Phylum Family Genus pval FDR- Lupus Lupus Control

Adjusted

Bacteroidetes Rikenellaceae Unknown genus 0.0002 0.0171 1 131.5

Firmicutes Unknown Unknown genus 0.0003 0.0171 92.25 327.5

Clostridiales

Firmicutes Ruminococcaceae Faecalibacterium 0.0026 0.0421 19 339

Bacteroidetes Bacteroidaceae Bacteroides 0.0030 0.0421 65 746.5

Actinobacteria Coriobacteriaceae Unknown genus 0.0077 0.0886 3.5 20

Protereobacteria Alcaligenaceae Sutterella 0.0087 0.0912 1 15

Protereobacteria Desulfovibrionaceae Desulfovibrio 0.0094 0.0912 0 13

Firmicutes Christensenellaceae Unknown genus 0.0105 0.0942 2.5 18

Firmicutes Streptococcaceae Streptococcus 0.0118 0.0993 107.5 35

Actinobacteria Bifidobacteriaceae Bifidobacterium 0.0152 0.1070 356.25 67

Firmicutes Mogibacteriaceae Unknown genus 0.0153 0.1070 3.5 10

Firmicutes Lachnospiraceae Roseburia 0.0168 0.1116 4 10

Bacteroidetes Porphyromonadaceae Parabacteroides 0.0521 0.2296 2.5 31

Firmicutes Ruminococcaceae Unknown genus 0.0654 0.2296 536.5 1037

Firmicutes Lachnospiraceae Lachnospira 0.0912 0.2723 5 13

Firmicutes Ruminococcaceae Ruminococcus 0.0963 0.2723 313.75 477.5 [51] Faecalibacterium species was known to be associated with anti -inflammatory properties; therefore a reduction of this genus could influence SLE inflammation systemically. Low levels of Faecalibacterium prausnitzii have been associated with the autoimmune inflammatory bowel disorder, Crohn's Disease.

EXAMPLE 2

[52] Lupus Saliva Samples. Microbial diversity plots from saliva for SLE and controls are shown in Fig. 2A. A trend was observed for lupus saliva to show decreased diversity both in phylogenetically, and in observed species, however, this did not attain statistical significance. Fig. 2B demonstrates higher proportions of Bacteroidetes in lupus saliva, by taxanomic comparisons, compared to controls. Table 2 illustrates that, at the genus level, a predominance of the genus Prevotella in SLE saliva (median counts - SLE=2960, control=1609) was observed that is trending toward significance, however, with the current sample size the FDR corrected p- value is 0.225.

TABLE 2

Saliva Samples Median Counts

Phylum Family Genus pval FDR- Lupus Lupus Sjogren

Adjusted Control

Proteobacteria Pseudomonadaceae Pseudomonas 0.0001 0.0120 0 2 53

Firmicutes Lachnospiraceae Oribacterium 0.0020 0.0396 59 87 33.25

Fusobacteria Fusobacteriaceae Fusobacterium 0.0042 0.0539 221 505.5 661.75

Bacteroidetes Flavobacteriaceae Capnocytophaga 0.0043 0.0539 6 64 55

Proteobacteria Campylobacteriaceae Campylobacter 0.0048 0.0539 53 122 109.25

Firmicutes Gemellaceae Unknown genus 0.0050 0.0539 15 51 125.5

Firmicutes Lachnospiraceae Catonella 0.0059 0.0587 7 13 1.5

Actinobacteria Micrococcaeae Rothia 0.0067 0.0619 560 412.5 171

Spirochaetes Spirochaetaceae Treponema 0.0326 0.1798 3 18 12.25

Firmicutes Lachnospiraceae Unknown genus 0.0469 0.2243 57 44.5 14.5

Bacteroidetes Prevotellaceae Prevotella 0.0644 0.2554 2959.5 1609 1422.5

Bacteroidetes Porphyromonadaceae Porphyromonas 0.0653 0.2554 19 70 85.25

Proteobacteria Neisseriaceae Unknown genus 0.0716 0.2565 1 14 5.25

Bacteroidetes Weeksellaceae Unknown genus 0.0756 0.2567 7 12 23

Proteobacteria Pasteurellaceae Aggregatibacter 0.0846 0.2641 2 12 2 Actinobacteria Corynebacteriaceae Corynebacterium 0.0909 0.2641 6 26 25.5

Firmicutes Lachnospiraceae Moryella 0.0942 0.2641 28 20 10.75

[53] Referring to Fig. 3 and Table 3, it is observed that despite the low power of the study to detect statistically significant differences in Prevotella abundance in SLE saliva, a review of the current published literature demonstrated that of all published studies to date, that the current Lupus data demonstrates the highest abundance of Prevotella reported thus far. These results suggest that a larger study would likely define a significant difference in Prevotella in the SLE oral microbiome.

TABLE 3

Authors, Subjects Type Location Avg Seq/ 16S Seq Platform Notes Year Individual Region

I Lazarevic 753 HOME) Various 1149 V5-6 ILLUMINA

et al., 2011 USA

II Cephas et 5 Mothers USA (IL) 80,000 V4-6 ROCHE/454

al., 2011 Caregivers

II Cephas et 5 Infants USA (IL) 80,000 V4-6 ROCHE/454

al., 2011

III LMAMR 23 Healthy USA (OK) V4 ILLUMINA *35cy

III LMAMR 40 Healthy Adults USA (C&A, V4 ILLUMINA *35cy

OK)

III LMAMR 20 Adults Huanaco, V4 ILLUMINA *35cy

Peru

IV Stahringer 107 Longitudinal USA (CO) 1618 VI -2 ROCHE/454

et al., 2012 Twin Study

V Segata et 199 Adults HMP 9072 Full ROCHE/454

al., 2012

VI Pushalkar 2 Healthy Adults USA (NY) 19472 V4-5 ROCHE/454

et al., 2011

VII Nasidze et 13 Sierra Leone Sierra Leone 1370 VI -2 ROCHE/454

al., 2011

VIII Zaura et 3 Healthy Male Netherlands 10000 V5-6 ROCHE/454

al., 2009 Adults IX Lazarevic 5 Healthy Adults Switzerland 6372 V3 ILLUMINA et al, 2010

X Crielaard et 74 Healthy Netherlands 10515 V5-6 ILLUMINA

al., 2011 Children

XI Lazarevic 1 Healthy Adult Switzerland 555152 Vl-3 ROCHE/454

et al., 2012

XI Lazarevic 1 Healthy Adult Switzerland 541687 Vl-3 ROCHE/454

et al., 2012

XII Keijser et 71 Healthy Adults Netherlands 1035 V6 ROCHE/454

al., 2008

XIII Yamanaka 19 Pre-Periodontal Japan 3356 VI -2 ROCHE/454

et al., 2012 Therapy

XIII Yamanaka 19 Post- Japan 3356 VI -2 ROCHE/454

et al., 2012 Periodontal

Therapy

XIV Said et al., 24 Healthy Adults Japan 3000 VI -2 ROCHE/454

2013

XIV Said et al., 21 Crohn's Japan 3000 VI -2 ROCHE/454

2013 Disease

XIV Said et al., 14 Ulcerative Japan 3000 VI -2 ROCHE/454

2013 Colitis

XV Yang et al., 26 Healthy China 9458 V4-5 ROCHE/454

2012

XV Yang et al., 19 Active Caries China 9458 V4-5 ROCHE/454

2012

XVI LMAMR/ 12 Sjogrens USA (OK) V4 ILLUMINA *30cy OMRF

XVI LMAMR/ 13 Lupus Control USA (OK) V4 ILLUMINA *30cy OMRF

XVI LMAMR/ 19 Lupus USA (OK) V4 ILLUMINA *30cy OMRF

[54] The foregoing description of specific embodiments of the present disclosure has been presented for purpose of illustration and description. The exemplary embodiments were chosen and described in order to best explain the principles of the invention and its practical application, to thereby enable others skilled in the art to best utilize the invention and various embodiments with various modifications are suited to the particular use contemplated.

Claims

What is claimed is:

1. A method of diagnosing Systemic Lupus Erythematosus in a subject comprising the following steps: obtaining a biological sample from the subject; isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level; determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with Systemic Lupus Erythematosus; and diagnosing the subject with Systemic Lupus Erythematosus if the target microbe population level is outside the established target microbe population level range, or diagnosing the subject as not having Systemic Lupus Erythematosus if the target microbe population level is within the established target microbe population level range.

2. The method of claim 1, wherein the biological sample is a fecal sample and the target microbe population is Faecalibacterium.

3. The method of claim 1, wherein the target microbe population is selected from the group consisting of general microbe content, Bacteroidetes, Firmicutes, and any combination thereof.

4. The method of claim 1, wherein the biological sample is an oral sample, and the target microbe population is Prevotella.

5. The method of claim 1, wherein the target microbe population is selected from the phyla Bacteroidetes.

6. The method of any of claims 1-5, wherein the target molecular population is metagenomic DNA and the purified target molecular sample is a purified metagenomic DNA sample.

7. The method of any of claims claim 1-5, wherein the target molecular population is metagenomic DNA and the purified target molecular sample is a purified metagenomic DNA sample, and wherein the step of quantifying the target microbe population is performed by subjecting the purified metagenomic DNA sample to metagenomic analysis.

8. The method of any of claims 1-5, wherein the target molecular population is DNA and the purified target molecular sample is a purified DNA sample.

9. The method of any of claims 1-5, wherein the target molecular population is DNA and the purified target molecular sample is a purified DNA sample, and wherein the step of quantifying the target microbe population is performed by subjecting the purified DNA sample to polymerase chain reaction.

10. The method of any of claims 1-5, wherein the target molecular population is metabolites and the purified target molecular sample is a purified metabolite sample.

11. The method of any of claims 1 -5, wherein the target molecular population is metabolites and the purified target molecular sample is a purified metabolite sample, and wherein the step of quantifying the target microbe population is performed by subjecting the purified metabolite sample to metabolomic analysis.

12. The method of any of claims 1-5, wherein the target molecular population is metabolites and the purified target molecular sample is a purified metabolite sample, and wherein the step of quantifying the target microbe population is performed by subjecting the purified metabolite sample to mass spectrometry.

13. A method of diagnosing Systemic Lupus Erythematosus in a subject comprising the following steps: obtaining a first biological sample and a second biological sample from the subject, wherein the first biological sample is from an oral cavity, and wherein the second biological sample is a fecal sample; isolating and purifying a target molecular population from the first biological sample and the second biological sample to yield a first purified target molecular sample and a second purified target molecular sample, respectively; quantifying a first target microbe population in the first purified target molecular sample to generate a first target microbe population level and quantifying a second target microbe population in the second purified target molecular sample to generate a second target microbe population level; determining whether the first and second target microbe population levels are within a first established target microbe population level range and a second established target microbe population level range, respectively, wherein the first and second established target microbe population level ranges are not associated with Systemic Lupus Erythematosus; and diagnosing the subject with Systemic Lupus Erythematosus if the first and second target microbe population levels are outside the first and second established target microbe population level ranges, respectively, or diagnosing the subject as not having Systemic Lupus

Erythematosus if the first and second target microbe population levels are within the first and second established target microbe population level ranges, respectively.

14. The method of claim 13, wherein the first target microbe population is selected from the group consisting of general microbial content, Firmicutes, and any combination thereof.

15. The method of claim 13, wherein the first target microbe population is selected from the group consisting of Bacteroidetes.

16. The method of claim 13, wherein the target molecular population is DNA and the first and second purified target molecular samples are a first purified DNA sample and a second purified DNA sample, respectively.

17. The method of claim 16, wherein the step of quantifying the first and second target microbe population is performed by subjecting the first and second purified DNA samples to polymerase chain reaction.

18. The method of claim 17, wherein the polymerase chain reaction for the first purified DNA sample uses primers sufficient to amplify nucleic acid sequences specific to one or both of Bacteroidetes and Firmicutes.

19. The method of claim 17, wherein the polymerase chain reaction for the second purified DNA sample uses primers sufficient to amplify nucleic acid sequences specific to one or both of Bacteroidetes and Firmicutes.

20. A method for treating Systemic Lupus Erythematosus comprising administering to a subject with Systemic Lupus Erythematosus a composition comprising one or both of

Bacteroidetes and Firmicutes in an amount sufficient to alter Bacteroidetes or Faecalibacterium to levels associated with individuals not having Systemic Lupus Erythematosus.

21. The method of claim 20, wherein the composition is administered as an enema.

22. A method for treating Systemic Lupus Erythematosus comprising administering to a subject with Systemic Lupus Erythematosus a composition comprising an anti-microbial agent in an amount sufficient to decrease the level of Bacteroidetes or Prevotella or both in an oral cavity of the subject.

23. The method of claim 22, wherein the composition is in the form of a lozenge.

24. The method of claim 22 or 23, wherein the composition comprises erythromycin or tetracycline.

25. A method of treating a subject with Systemic Lupus Erythematosus comprising the following steps: obtaining a biological sample from the subject; isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level; determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with Systemic Lupus Erythematosus; and administering to the subject a therapeutic composition if the target microbe population level is outside the established target microbe population level range, wherein the therapeutic composition comprises the target microbe population if the target microbe population level is lower than the established target microbe population level range, and wherein the therapeutic composition is an anti-microbial agent specific to the target microbe population if the target microbe population level is higher than the established target microbe population level range.

26. The method of claim 25, wherein the biological sample is a fecal sample and the target microbe population is Faecalibacterium.

27. The method of claim 25, wherein the target microbe population is selected from the group consisting of general microbe content, Bacteroidetes, Firmicutes, and any combination thereof.

28. The method of claim 25, wherein the biological sample is an oral sample, and the target microbe population is Prevotella.

29. The method of claim 25 wherein the target microbe population is selected from the phyla Bacteroidetes.

30. The method of any of claims 25-29, wherein the target molecular population is metagenomic DNA and the purified target molecular sample is a purified metagenomic DNA sample.

31. The method of of any of claims 25-29, wherein the target molecular population is metagenomic DNA and the purified target molecular sample is a purified metagenomic DNA sample, and wherein the step of quantifying the target microbe population is performed by subjecting the purified metagenomic DNA sample to metagenomic analysis.

32. The method of any of claims 25-29, wherein the target molecular population is DNA and the purified target molecular sample is a purified DNA sample.

33. The method of any of claims 25-29, wherein the target molecular population is DNA and the purified target molecular sample is a purified DNA sample, and wherein the step of quantifying the target microbe population is performed by subjecting the purified DNA sample to polymerase chain reaction.

34. A method of diagnosing or providing a prognosis in a subject with an autoimmune disease comprising the following steps: obtaining a biological sample from an oral cavity of a subject; isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level, wherein the target microbe population is of the genus Prevotella; determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with the autoimmune disease; and diagnosing the subject with the autoimmune disease or otherwise providing a prognosis for the subject if the target microbe population level is outside the established target microbe population level range, or diagnosing the subject as not having the autoimmune disease or otherwise providing a prognosis if the target microbe population level is within the established target microbe population level range.

35. The method of claim 34, further comprising the step of administering to the subject a therapeutic composition sufficient to bring the target microbe population level within the established target microbe population level range.

36. The method of any of claims 34-35, wherein the autoimmune disease is Systemic Lupus Erythematosus.

37. A method of diagnosing or providing a prognosis in a subject with an autoimmune disease comprising the following steps: obtaining a biological sample from a subject, wherein the biological sample is of gastrointestinal origin; isolating and purifying a target molecular population from the biological sample to yield a purified target molecular sample; quantifying a target microbe population in the purified target molecular sample to generate a target microbe population level, wherein the target microbe population is of the phyla

Firmicutes; determining whether the target microbe population level is within an established target microbe population level range, wherein the established target microbe population level range is not associated with the autoimmune disease; and diagnosing the subject with the autoimmune disease or otherwise providing a prognosis for the subject if the target microbe population level is outside the established target microbe population level range, or diagnosing the subject as not having the autoimmune disease or otherwise providing a prognosis if the target microbe population level is within the established target microbe population level range.

38. The method of claim 37, further comprising the step of administering to the subject a therapeutic composition sufficient to bring the target microbe population level within the established target microbe population level range.

39. The method of claim 38, wherein the therapeutic composition comprises microbes from the phyla Firmicutes or spores thereof.

40. The method of claim 38, wherein the therapeutic composition comprises microbes from the genus Faecalibacterium or spores thereof.

41. The method of claim 37, wherein the target microbe population is of the genus

Faecalibacterium.

42. The method of any of claims 37-41, wherein the autoimmune disease is Systemic Lupus Erythematosus.