WO2014127193A1 - Pdcs and herv cell signatures - Google Patents

Abstract

Provided herein are diagnostic and therapeutic methods based at least in part on the discovery that levels of pDCs or pDC-localized HERV, TLR-7 or TLR-9 inhibition, or PCLN2 upregulation occurs in a subject with ME/CFS is different from levels of pDCs and HERV cell expression in a healthy individual. One aspect provides diagnosing a subject with an autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimmune-associated disease according increased levels of pDCs, pDC-localized HERV, TLR-7 or TLR-9 inhibition, or PCLN2 upregulation in samples of the subject. Another aspect provides for treatment of an autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimmune-associated disease in a subject diagnosed according to methods described herein.

Description

PDCS AND HERV CELL SIGNATURES

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from U.S. Provisional Application Serial No. 61/765,000 filed 14 February 2013, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

MATERIAL INCORPORATED-BY-REFERENCE

Not Applicable.

FIELD OF THE INVENTION

The present disclosure generally relates to the differences in

Plasmacytoid dendritic cells (pDCs) and human endogenous retroviral (HERV) expression seen between healthy individuals and individuals with an

autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as myalgic

encephalomyelitis / chronic fatigue syndrome (ME/CFS).

In addition, the invention relates to the differences in pDC-localized activity of TLR-7 or TLR-9 or pDC-localized expression of PLCL2 seen between healthy individuals and individuals diagnosed with an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease, such as ME/CFS.

BACKGROUND OF THE INVENTION

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating illness or disorder of unknown etiology characterized by

neurocognitive dysfunction, neurocognitive impairments, inflammation, immune abnormalities, multisystemic neuropathology, gastro-intestinal abnormalities, gastrointestinal dysfunction or gastrointestinal distress, inflammation, innate immune dysfunction, and innate immune dysregulation (Carruthers, 201 1 ). Immunological symptoms often include viral reactivation, cytokine and chemokine irregularities, and decreased natural killer (NK) cell function (Choppa, 1998; Klimas, 1990; Caligiuri, 1987; Vercoulen, 1994; De Meirleir, 2002;

Suhadolnik, 1994). Additionally, reports of ME/CFS subjects expressing autoantibodies (Konstantinov, 1996; von Mikecz, 1997), and the successful treatment of ME/CFS subjects with the B-cell depleting drug Rituximab (Fluge, 201 1 ; Fluge, 2009), suggest that a subset of these individuals may suffer from an uncharacterized antibody-mediated autoimmunity.

Little is known regarding the pathophysiology of ME/CFS; therefore, diseases with similar or overlapping symptoms often serve as useful guides when exploring new experimental concepts. For instance, autoimmune diseases such as multiple sclerosis (MS) and systemic lupus erythematosus (SLE) have many symptoms that overlap with those of ME/CFS. Neurological manifestations often associated with ME/CFS (Buchwald, 1992) are analogous to the

neuroinflammation and cognitive abnormalities associated with MS and SLE (Compston, 2008; Utset, 2006). Additionally, gastrointestinal aberrations, which are common to individuals with MS and SLE (Apperloo-Renkema, 1994; Ebert, 201 1 ; Ghezzi, 2001 ), are among the most frequently reported symptoms by subjects with ME/CFS (Devanur, 2006; Maes, 2007). The gut-associated lymphoid tissue (GALT) represents the largest immune compartment in the body. In fact, it has been estimated that more than 60% of all T cells may reside within the small intestine (Guy-Grand, 1993), emphasizing the potential contribution of the gut to systemic immunity and when investigating dysregulation of the immune system. Indeed, increases in serum bacterial byproducts, particularly LPS resulting from bacterial translocation in the gut, can be associated with systemic immune activation in many diseases such as HIV/AIDS, inflammatory bowel disease, and acute graft-versus-host disease (Brenchley, 2006; Caradonna, 2000; Cooke, 2002). Extraintestinal immune dysregulation originating within the gut is described in such diseases as

HIV/AIDS and idiopathic lymphocytopenia (Brenchley, 2006; Lee, 2009). In HIV infected individuals, the gastrointestinal (Gl) tract is preferentially and

dramatically impacted during the acute and chronic phase of the infection, leading to compromised epithelial integrity and bacterial translocation. Although, its contribution to neuroimmune disease in humans is largely unknown, recent studies using animal models support a connection between gastrointestinal immunity and neuroinflammation. Lee and colleagues reported that intestinal microbiota significantly influence the balance between proinflammatory and antiinflammatory immune responses during the induction of experimental autoimmune encephalomyelitis, an animal model for MS (Lee, 2010).

Human endogenous retroviruses (HERVs) are remnants of ancient retroviral infections that integrated into the germ line and are now transmitted vertically (Belshaw, 2005). Not including the long interspersed elements (LINE) and other retrotransposons, HERVs constitute approximately 8% of the human genome (Weiss, 2006). Although HERV proteins are "self-antigens" and should not induce an immune response, HERV proteins and anti-HERV serum antibodies have been associated with a number of autoimmune diseases including MS and SLE (Lefebvre, 1995; Blomberg, 1994; Bengtsson, 1996; Rasmussen, 1997). Although associations are often difficult to render into definitive causation, it is widely believed that the humoral immune response to HERV proteins leads to autoimmunity through a process of molecular mimicry (Query, 1987). HERV proteins are also known to act as superantigens, which have the ability to cause polyclonal T cell activation and massive cytokine production (Sutkowski, 2001 ). In 2001 , Perron and colleagues reported the potential immunopathogenic properties of HERV-W envelope protein as a major proinflammatory and superantigenic determinant associated with MS (Perron, 2001 ). Yu et al. (Immunity, 2012) describes expression of HERV proteins by pDCs using a Toll-like receptor (TLR) 3-7-9 knockout mouse model.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision of a diagnostic method for detection of an autoimmune disease, an autoimmune- associated disease, a neuroimmune disease, or a neuroimmune-associated disease in a subject. The present disclosure is based at least in part on the observation that human endogenous retroviral (HERV) protein expression in plasmacytoid dendritic cells (pDC) in an individual diagnosed with chronic fatigue

syndrome/myalgic encephalomyelitis (ME/CFS) is different from HERV protein expression in pDCs expression in a healthy individual. Further observations include inhibited pDC-localized activity of Toll Like Receptor (TLR)-7 or TLR-9 in an individual diagnosed with ME/CFS; or upregulated pDC-localized

phospholipase C-like 2 (PLCL2) expression in an individual diagnosed with ME/CFS. One aspect provides a method of diagnosis. In some embodiments, the method of diagnosis includes: (a) determining in a biological sample of a subject one or more of (i) a level of plasmacytoid dendritic cells (pDCs) cell-localized immunoreactivity to an antibody specific for human endogenous retroviral (HERV); (ii) a level of pDCs; (iii) a level of pDC-localized activity of Toll Like Receptor (TLR)-7 or TLR-9; or (iv) a level of pDC-localized phospholipase C-like 2 (PLCL2) expression; (b) comparing one or more of (i) the level of pDC- localized immunoreactivity to the anti-HERV antibody with a first control, (ii) the level of pDCs with a second control, (iii) the level of pDC-localized activity of TLR-7 or TLR-9 with a third control; or (iv) the level of pDC-localized expression of PLCL2 with a fourth control; and (c) diagnosing the subject with an

autoimmune-associated disease when at least one of the following is satisfied (i) the level of pDC-localized immunoreactivity to the anti-HERV antibody is detected or is higher than the first control; (ii) the level of pDCs is higher than the second control; (iii) the level of pDC-localized activity of TLR-7 or TLR-9 is inhibited compared to the third control; or (iv) the level of pDC-localized PLCL2 expression is upregulated compared to a fourth control.

Another aspect provides a method of treating an autoimmune,

autoimmune-associated disease in a subject comprising: diagnosing an autoimmune-associated disease according to the method of any one of claims 1 -1 1 ; and administering an antiviral agent to a subject in need thereof. 13. The method of any one of claims 1 -12, wherein the autoimmune- associated disease is selected from the group consisting of chronic fatigue syndrome, fibromyalgia, myalgic encephalitis, atypical multiple sclerosis, autism, non-epileptic seizures, and Gulf War Syndrome.

Other objects and features will be in part apparent and in part pointed out hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

FIG. 1A-H is a series of images depicting immunoreactivity to HERV monoclonal and polyclonal antibodies in duodenum biopsy of a representative ME/CFS and control subject.

FIG. 1 A shows immunoreactivity with anti-HERV-K Gag lgG1 mAb

(Dylight594 donkey anti-mouse IgG secondary) in gut biopsy of ME/CFS subject.

FIG. 1 B shows immunoreactivity with anti-HERV-K18.1 Env IgG pAb (Dylight594 donkey anti-mouse IgG secondary) in gut biopsy of ME/CFS subject. FIG. 1 C shows immunoreactivity with anti-HERV-FRD Env IgG pAb

(rhodamine goat anti-rabbit IgG secondary) in gut biopsy of ME/CFS subject.

FIG. 1 D shows immunoreactivity to anti-HERV-R Env (rhodamine goat anti-rabbit IgG secondary) in gut biopsy of ME/CFS subject.

FIG. 1 E shows immunoreactivity with anti-HERV-K Gag lgG1 mAb

(Dylight594 donkey anti-mouse IgG secondary) in gut biopsy of control subject.

FIG. 1 F shows immunoreactivity with anti-HERV-K18.1 Env IgG pAb (Dylight594 donkey anti-mouse IgG secondary) in gut biopsy of control subject.

FIG. 1 G shows immunoreactivity with anti-HERV-FRD Env IgG pAb (rhodamine goat anti-rabbit IgG secondary) in gut biopsy of control subject.

FIG. 1 H shows imnnunoreactivity to anti-HERV-R Env (rhodamine goat anti-rabbit IgG secondary) in gut biopsy of control subject. Immunoreactivity to anti-HERV-R Env protein (rhodamine goat anti-rabbit IgG secondary) in gut biopsy of control subject (Bar represents 20 μιτι). TOPO3 was used to indicate nucleus localization in all images.

FIG. 2A-H is a series of images depicting immunoreactivity with rat anti- SFFV-gp-52Env and goat anti-MLV-Gag (gammaretroviral) antibodies shows colocalization in the cytoplasm of cells with a small, eccentric nucleus and granular inclusions.

FIG. 2A shows immunoreactivity with goat anti-Gag IgG pAb (Dylight488 bovine anti-goat IgG pAb secondary) in gut biopsy of ME/CFS subject.

FIG. 2B shows immunoreactivity with rat anti-Env lgG1 k mAb (rhodamine donkey anti-rat IgG pAb secondary) in gut biopsy of ME/CFS subject. FIG. 2C shows an overlay of images FIG. 2A and FIG. 2B (bar represents

20 pm).

FIG. 2D shows the morphology of the immunoreactive cells of ME/CFS subject.

FIG. 2E shows immunoreactivity with goat anti-Gag IgG pAb (Dylight488 bovine anti-goat IgG pAb secondary) in gut biopsy of control subject.

FIG. 2F shows immunoreactivity with rat anti-Env lgG1 k mAb (rhodamine donkey anti-rat IgG pAb secondary) in gut biopsy of control subject.

FIG. 2G shows the overlay of images FIG. 2E and FIG. 2F (bar represents 20 pm). FIG. 2H shows the morphology of the immunoreactive cells of control subject. TOPO3 was used to indicate nucleus localization in all images.

FIG. 3A-D is a series of images depicting the specificity of the rat anti- SFFV-gp-52Env and goat anti-MLV-Gag gammaretroviral antibodies in an antibody depletion experiment. Antibodies were incubated (1 hour, 25 °C, constant shaking) with purified and heat inactivated retrovirus derived from 22Rv1 prior to probing duodenum biopsy sections.

FIG. 3A shows duodenum biopsies probed with polyclonal goat anti-MLV- Gag antibodies. FIG. 3B shows duodenum biopsies probed with polyclonal goat anti-MLV-

Gag antibodies incubated with heat inactivated 22Rv1 .

FIG. 3C shows duodenum biopsies were probed with monoclonal rat anti- SFFV-gp-52Env antibodies. FIG. 3D shows duodenum biopsies probed with monoclonal rat anti-SFFV-gp-52Env antibodies incubated with heat-inactivated 22rv1 (bar represents 20 μm).

FIG. 4A-G is a series of images evaluating the specificity and potential cross-reactivity of the antibodies used by probing the biopsy of a ME/CFS subject.

FIG. 4A shows duodenum biopsies probed with rhodomine conjugated anti-SFFV-gp-52Env mAb (1 :500).

FIG. 4B shows duodenum biopsies probed with rhodamine donkey anti- rat IgG pAb (1 :1000) and Dylight488 bovine anti-goat IgG pAb (1 :500).

FIG. 4C shows duodenum biopsies probed with rat anti-HHV8 ORF73 mAb (1 :1000) followed by incubation with rhodamine donkey anti-rat IgG pAb (1 :1000).

FIG. 4D shows duodenum biopsies probed with rat lgG1 ,κ mAb isotype control (1 :500) followed by incubation with rhodamine donkey anti-rat IgG pAb (1 :1000) (Bar represents 20 μηη).

FIG. 4E shows duodenum biopsies probed with normal goat serum (1 :2000) followed by incubation with Dylight488 bovine anti-goat IgG pAb (1 :500).

FIG. 4F shows duodenum biopsies probed with rat anti-SFFV-gp-52Env IgGl K mAb followed by incubation with Dylight488 bovine anti-goat IgG pAb (1 :500). FIG. 4G shows duodenum biopsies probed with goat anti-Gag pAb followed by incubation with rhodamine donkey anti-rat IgGpAb (1 :1000). TOPO3 was used to indicate nucleus localization in all images.

FIG. 5A-H is a series of images showing the expression of the surface antigens CD45 (top row) and CD303 (bottom row) on cells immunoreactive with rhodamine conjugated rat anti-SFFV-gp-52Env IgGl k mAb.

FIG. 5A shows expression of CD45 in duodenum biopsy of a ME/CFS subject.

FIG. 5B shows immunoreactivity of rhodamine rat anti-SFFV-gp-52Env lgG1 k mAb in duodenum biopsy of ME/CFS subjects.

FIG. 5C shows merged images FIG. 5A and FIG. 5B, (bar represents 20 μm). TOPO3 was used to indicate nucleus localization.

FIG. 5D shows morphology of the immunoreactive cells.

FIG. 5E shows expression of CD303in duodenum biopsy of a ME/CFS subject.

FIG. 5F shows immunoreactivity with rhodamine rat anti-SFFV-gp-52Env lgG1 k mAb in duodenum biopsy of an ME/CFS subject. FIG. 5G shows merged images FIG. 5E and FIG. 5F (bar represents 20 μm). TOPO3 was used to indicate nucleus localization. FIG. 5H shows morphology of the immunoreactive cells.

FIG. 6A-H is a series of images showing localization of PLCL2 and CD303 in gut biopsy samples from an ME/CFS subject (FIG. 6A, FIG. 6C, FIG. 6E, and FIG. 6G) and a control subject (FIG. 6B, FIG. 6D, FIG. 6F, and FIG. 6H) showing TLR-7 and TLR-9 are inhibited in pDCs of gut samples from ME/CFS subjects.

FIG. 6A and FIG. 6B are composite images of PLCL2 and CD303 localization.

PLCL2 localization is shown in green in original images and featured in FIG. 6C and FIG. 6D. CD303 localization is shown in red in original images and featured in column FIG. 6E and FIG. 6F.

FIG. 6G and FIG. 6H are micrograph images of the samples.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein is a method of diagnosing an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease in a subject. The present disclosure is based at least in part on the observation that human endogenous retroviral (HERV) protein expression in plasmacytoid dendritic cells (pDC) in an individual diagnosed with chronic fatigue syndrome/myalgic encephalomyelitis (ME/CFS) is different from HERV protein expression in pDCs expression in a healthy individual. The present disclosure is also based at least in part on the observation that pDC-localized activity of Toll Like Receptor (TLR)-7 or TLR-9 is inhibited in an individual diagnosed with ME/CFS; or pDC-localized phospholipase C-like 2 (PLCL2) expression is upregulated in an individual diagnosed with ME/CFS. Such findings support that the presence or expression of HERV protein in pDCs can be associated with a pathological manifestation in at least a subset of ME/CFS subjects.

As shown herein, endogenous retroviral proteins were detected in Gl tissue of subjects with ME/CFS. Specifically, pDCs were found in the duodenum biopsies of ME/CFS subjects that exhibited specific immunoreactivity to monoclonal and polyclonal antibodies reactive with HERV and gammaretroviral proteins, while the same immunoreactivity was not observed in control biopsies. As shown herein, approximately 50% of duodenum-associated pDCs expressed human endogenous retroviral (HERV) proteins, whereas no HERV protein expression was observed in any of the control biopsies. Also shown herein, Toll Like Receptor (TLR)-7 or TLR-9 is inhibited in gut-associated pDCs of subjects with ME/CFS. While being under no obligation to provide a mechanism, and in no way limited the scope of the present disclosure, it is thought that HERV expression by gut-associated pDCs in ME/CFS subjects may result from TLR inhibition. In other words, expression of HERV proteins by pDCs may be a biomarker for TLR inhibition, and specifically, TLR inhibition of pDCs in the gut of ME/CFS subjects.

Thus, presence or expression of HERV in pDCs of a subject (e.g., pDCs in a sample from a subject) can be indicative of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease in a subject.

Experiments reported herein support that expression of endogenous retroviral proteins in the pDCs, increased number of pDCs, pDC-localized TLR-7 or TLR-9 inhibition, or upregulation of pDC-localized PLCL2 in a subject can be indicative of autoimmune, autoimmune-associated or neuroimmune diseases. For example, expression of endogenous retroviral proteins in pDCs of a subject can be indicative of ME/CFS.

International patent application publication number WO 2012/106,674, filed 03 February 2012, is incorporated herein by reference in its entirety. DIAGNOSIS

Levels of pDCs, pDC-localized expression of HERV (e.g., HERV immunoreactivity), pDC-localized TLR-7 or TLR-9 inhibition, or upregulation of pDC-localized PLCL2 as described herein can be used to diagnose an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease in a subject.

A detectable presence or expression of HERV in a biological sample, or in pDCs of a biological sample, can be indicative of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease, such as ME/CFS. Detection of HERV immunoreactivity in pDCs of subjects with an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease (but not control subjects) supports direct association between pDCs and HERV expression in human disease. For example, levels of pDCs or HERV

immunoreactivity described herein can be used to diagnose ME/CFS in a subject. In some embodiments, pDC-localized HERV immunoreactivity does not occur in a healthy subject, and so, mere detection of such immunoreactivity can be indicative of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS.

In some embodiments, HERV immunoreactivity levels that can be used to diagnose an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease (e.g., ME/CFS) can include an expression pattern in which HERV immunoreactivity is detected or pDC-localized HERV immunoreactivity levels are increased in a subject relative to a control. Control.

In some embodiments, pDCs and HERV immunoreactivity levels that can be used to diagnose an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease (e.g., ME/CFS) can include an expression pattern in which pDCs and HERV

immunoreactivity levels are increased in a subject relative to control. A control can be a standard representing absence of the autoimmune, autoimmune- associated disease, or neuroimmune disease in question. A control can be a single control sample for various parameters or multiple controls each for one or more parameters described herein. For example, a control can be a subject, or a sample therefrom, not having an autoimmune disease, an autoimmune-associated disease, a

neuroimmune disease, or a neuroimmune-associated disease . As another example, a control can be a subject, or a sample therefrom, not diagnosed as having an autoimmune disease, an autoimmune-associated disease, a

neuroimmune disease, or a neuroimmune-associated disease . As another example, a control can be a subject, or a sample therefrom, not having the autoimmune, autoimmune-associated disease, or neuroimmune disease at issue. As another example, a control can be a subject, or a sample therefrom, not diagnosed as having the autoimmune, autoimmune-associated disease, or neuroimmune disease at issue. As another example, a control can be a subject, or a sample therefrom, not having ME/CFS. As another example, a control can be a subject, or a sample therefrom, not diagnosed as having ME/CFS. pDCs.

Levels of pDCs can be indicative of an autoimmune disease, an

autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease in a subject.

Plasmacytoid dendritic cells (pDCs) are innate immune cells that circulate in the blood and are found in peripheral lymphoid organs. They are understood to constitute < 0.4% of peripheral blood mononuclear cells (PBMC).

Characteristics of pDCs include, but are not limited to, a small, eccentric nucleus and prominent granular inclusions.

Characteristics of pDCs include, but are not limited to, expression of surface markers CD303 (BDCA-2). Characteristics of pDCs include, but are not limited to, surface cell expression of CD123, BDCA-2 (CD303) or BDCA-4 (CD304). Characteristics of pDCs include, but are not limited to, expression of CD4 or CD45. Characteristics of pDCs include, but are not limited to, lack of high-level expression of CD1 1 c, B220, BST-2 (mPDCA) or Siglec-H or negative expression of CD1 1 b. The lack of CD1 1 c or CD14 expression distinguishes pDCs from conventional dendritic cells or monocytes, respectively. Mouse pDCs express CD1 1 c, B220, BST-2 (mPDCA) or Siglec-H and are negative for CD1 1 b. As components of the innate immune system, these cells express intracellular Toll-like receptors 7 and 9 which detect ssRNA and CpG DNA motifs,

respectively. Upon stimulation and subsequent activation, these cells produce large amounts of type I interferon (mainly IFN-α (alpha) and IFN-β (beta)), which are critical pleiotropic anti-viral compounds mediating a wide range of effects. A level (e.g., an amount, frequency, or density) of pDCs in a sample can be determined. A sample of a subject having substantially more pDCs than a control sample can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a

neuroimmune-associated disease, such as ME/CFS. For example, a sample of a subject having about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, or more, pDCs than a control sample can be indicative of the subject having an

autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects. As another example, a sample of a subject having 4.7 times more pDCs than a control sample can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a

neuroimmune-associated disease, such as ME/CFS (see Examples).

HERV.

HERV presence or expression in pDCs can be indicative of an

autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease in a subject In some

embodiments, HERV immunoreactivity levels that can be used to diagnose an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease (e.g., ME/CFS) can include an expression pattern in which HERV immunoreactivity is detected or pDCs immunoreactivity levels are increased in a subject relative to a control.

An HERV as that term is described herein can be any human endogenous retrovirus recognized as such in the art.

Endogenous retroviruses (ERVs) are sequences in the genome that are derived from ancient viral infections of germ cells in humans, mammals and other vertebrates; as such their proviruses are passed on to the next generation and now remain in the genome. An endogenous retrovirus is understood as a variant of a retrovirus that became permanently integrated with its host and is inherited from generation to generation as part of the genome of the host. A HERV can include a HERV-K (HML2) or variant thereof. A HERV can include a HERV-K106 or HERV-K1 16. A HERV can be a sequence encoding some or all of sequences known or reported to be a human endogenous retrovirus.

Detection of HERV can be according to detecting presence of an HERV nucleotide sequence in a sample. Detection of HERV can be according to detecting presence an amino acid sequence associated with expression of an HERV. Detection of HERV can be according to detecting presence a protein expressed from an HERV. Detection of HERV can be according to detecting immunoreactivity to an antibody specific for HERV or an expression product of HERV. For example, detection of HERV can be according to detecting immunoreactivity to an antibody specific for a protein expressed from an HERV. An antibody, such as a polyclonal antibody or monoclonal antibody, specific for HERV or an expression product of HERV can be as known in the art (see Examples).

A sample of the subject, for example a duodenum biopsy, can be examined for the presence or expression of HERV. Detection of HERV can be as discussed herein or any protocol known in the art. One of ordinary skill in the art, provided with guidance herein, can adapt conventional HERV detection protocols for use with methods herein. For example, an antibody specific for an endogenous retroviral protein can be used to detect the presence or expression of HERV in a sample. As another example, monoclonal or polyclonal antibodies reactive with HERV or gammaretroviral proteins can be used to detect the presence or expression of HERV in a sample. As another example, HERV- expressed nucleotides can be detected. As another example, HERV-associated polypeptides can be detected. The occurrence of non-specific binding can be excluded according to methods described herein or known in the art (see

Examples). As another example, monoclonal or polyclonal antibodies reactive with HERV or gammaretroviral Env or Gag proteins can be used to detect the presence or expression of HERV in a sample. Antibodies can be labeled (e.g., Rhodamine). Potential cross-reactivity of a secondary antibody with an incorrect primary antibody can be can be excluded according to methods described herein or known in the art (see Examples). An observed immunoreactivity according to one protocol can be verified through use of one or more additional detection protocols.

Detection of HERV in sample can include localization to a cell or cell type of the sample. For example, immunoreactivity can be colocalized in a particular cell type. As another example, immunoreactivity colocalized in cells with characteristics of pDCs, such cells having features as described herein. Such colocalization as described above can be verified through use of one or more additional detection protocols.

Diagnostic use of HERV can include detection of cells immunoreactive to anti-HERV antibodies. Diagnostic use of HERV can include detection of a level of cells immunoreactive to anti-HERV antibodies higher than a control.

Diagnostic use of HERV can include detection of cells immunoreactive to anti- HERV antibodies and detection of a level of cells immunoreactive to anti-HERV antibodies higher than a control.

An elevated level of pDCs immunoreactive to anti-HERV antibodies in sample can be used as a diagnostic criteria. A level of cells immunoreactive to anti-HERV antibodies in a sample can be determined. A detected level of cells immunoreactive to anti-HERV antibodies in a sample can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects. A level of pDCs immunoreactive to anti-HERV antibodies in sample can be compared to a control.

In some embodiments, detection of pDC-localized immunoreactivity to an antibody specific for human endogenous retroviral (HERV) can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as

ME/CFS subjects.

In some embodiments, a level of pDC-localized immunoreactivity to an antibody specific for human endogenous retroviral (HERV) higher than the control can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease, such as ME/CFS subjects. For example, a sample of a subject having substantially more pDCs immunoreactive to anti-HERV antibodies than a control sample can be indicative of the subject having a an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS. As another example, a sample of a subject having about 1 %, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or more, pDCs immunoreactive to anti-HERV antibodies than a control sample can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects.

Inhibition of TLR-7 or TLR-9.

Inhibition of pDC-localized activity of TLR-7 or TLR-9 in a sample can be used as a diagnostic criteria. A level of pDC-localized activity of TLR-7 or TLR-9 in a sample can be determined. An inhibited level of pDC-localized activity of TLR-7 or TLR-9 in a sample can be indicative of the subject having an

autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects. A level of pDC-localized activity of TLR-7 or TLR-9 in a sample can be compared to a control. In some embodiments, a level of pDC-localized activity of TLR-7 or TLR-9 in a sample that is lower than the control (e.g., inhibited activity) can be indicative of the subject having an autoimmune disease, an autoimmune- associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects. For example, a sample of a subject having significantly less pDC-localized activity of TLR-7 or TLR-9 than a control sample can be indicative of the subject having an autoimmune disease, an autoimmune- associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS. As another example, a sample of a subject having about 1 %, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% lower pDC-localized activity of TLR-7 or TLR-9 than a control sample can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease, such as ME/CFS subjects. Upregulation of PLCL2.

Upregulation of pDC-localized phospholipase C-like 2 (PLCL2) expression in a sample can be used as a diagnostic criteria. A level of pDC-localized expression of PLCL2 in a sample can be determined. Upregulation of pDC- localized PLCL2 expression in a sample can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects.

A level of pDC-localized PLCL2 expression in a sample can be compared to a control. In some embodiments, a level of pDC-localized PLCL2 expression in a sample that is higher than the control (e.g., upregulated) can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects. For example, a sample of a subject having significantly higher pDC-localized PLCL2 expression in a sample than a control sample can be indicative of the subject having an autoimmune disease, an autoimmune- associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS. As another example, a sample of a subject having about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 150 fold, 200 fold, 250 fold, 300 fold, 350 fold, 400 fold, 450 fold, 500 fold, 550 fold, 600 fold, 650 fold, 700 fold, 750 fold, 800 fold, 850 fold, 900 fold, 950 fold, 1 ,000 fold or more increased pDC-localized PLCL2 expression in a sample compared to a control sample can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects. As another example, a sample of a subject having about 500 fold or more increased pDC-localized PLCL2 expression in a sample compared to a control sample can be indicative of the subject having an autoimmune disease, an autoimmune- associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS subjects (see e.g., Examplel O). Any of the parameters described herein (e.g., pDC numbers, HERV immunoreactivity, TLE-7 or TLR-9 inhibition, or PLCL2 upregulation) can be separately or jointly used for diagnosis of an autoimmune disease, an

autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease, such as ME/CFS subjects. For example, HERV presence or expression in pDCs and number of pDCs versus control can be jointly used. For example, detection of HERV in a sample and detection of substantially more pDCs in the same sample or another samples of the subject (compared to control) can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease, such as ME/CFS. Such joint diagnostic use can increase certainty of a diagnosis of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease . For example, HERV immunoreactivity colocalized in sample cells with characteristics of pDCs in conjunction with detection of substantially more pDCs in the same sample or another samples of the subject (compared to control) can be indicative of the subject having an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS. Such joint diagnostic use can increase certainty of a diagnosis of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease . In some embodiments, the method of diagnosis includes: (a) determining in a biological sample of a subject a level of cells immunoreactive to an anti- human endogenous retroviral (HERV) antibody or a level of plasmacytoid dendritic cells (pDCs); (b) comparing the level of cells immunoreactive to the anti-HERV antibody in the sample with a first control or the level of pDCs in the sample with a second control; and (c) diagnosing the subject with an

autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease when a level of cells

immunoreactive to anti-HERV antibodies is detected; the level of cells

immunoreactive to anti-HERV antibodies is higher than the first control; or the level of pDCs is higher in the second control. Similar steps can be taken, independently or jointly, with other parameters, such as pDC numbers, HERV immunoreactivity, TLE-7 or TLR-9 inhibition, or PLCL2 upregulation. SAMPLE AND SUBJECT

Methods described herein are directed to diagnosis of a subject, generally through analysis of a biological sample from the subject.

As described herein, a sample of a subject can be used as the basis for diagnosing a disease or disorder disclosed herein. For example, a sample of a subject can be screened for the presence or expression of HERV or elevated pDC levels and detection of either or both can support a diagnosis of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease in a subject. A sample can be a biological sample. A sample can be a biological sample from a subject. A sample can be a blood sample, a serum sample, a plasma sample, a cerebrospinal fluid sample, or a solid tissue sample. For example, the sample can be a blood sample, such as a peripheral blood sample. As another example, a sample can be a connective tissue sample (e.g., shoulder connective tissue). As another example, a sample can be a solid tissue sample, such as a gut biopsy tissue sample. A sample can include gut-associated lymphoid tissue. Gut-associated lymphoid tissue represents the largest immune compartment in the body with an estimated 60% or more of all T cells residing within the small intestine. As another example, a sample can be a solid tissue sample, such as a duodenum or stomach sample. As another example, a biological sample of a subject can be a punch biopsy of a tissue. As another example, a biological sample of a subject can be a punch biopsy of a duodenum or stomach sample.

A sample can include cells. A sample can include cells of a subject. For example, a sample can include cells such as fibroblasts, endothelial cells, peripheral blood mononuclear cells, haematopoietic cells, or a combination thereof. As another example, a sample can contain pDCs.

A sample can contain or be suspected of containing pDCs or HERV. For example, a biological sample of a subject can be from a tissue containing, known to contain, or thought to contain pDCs. As another example, a biological sample of a subject can be from a tissue containing, known to contain, or thought to contain HERV.

A sample can be prepared, processed, or preserved as described herein (see Examples) or according to conventional protocols known in the art.

The subject can have, be thought to have, be diagnosed with, be suspected of having, or be at risk for developing an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease . For example, subject can have, be thought to have, be diagnosed with, be suspected of having, or be at risk for developing myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS). A subject can be tested for the presence of elevated pDCs or presence or expression of HERV where the subject exhibits signs or symptoms of an autoimmune disease, an

autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease . For example, subject can be tested for the presence of elevated pDCs or presence or expression of HERV where the subject exhibits signs or symptoms of ME/CFS. A subject can be considered at risk of developing an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease where the subject has, for example and without limitation, a familial history of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease or has resided in a region comprising a cluster of individuals with an autoimmune disease, an autoimmune-associated disease, a

neuroimmune disease, or a neuroimmune-associated disease . For example, a subject can be considered at risk of developing ME/CFS where the subject has, for example and without limitation, a familial history of an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a

neuroimmune-associated disease or has resided in a region comprising a cluster of individuals with an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease . As another example, a subject can be considered at risk of developing ME/CFS where the subject has, for example and without limitation, a familial history of ME/CFS or has resided in a region comprising a cluster of individuals with ME/CFS. In some cases, a subject can have a significant number of symptoms that are similar to those described in autoimmune diseases such as MS and SLE. In some cases, a subject can exhibit no persistent symptoms; i.e., they are apparently healthy. In other cases, subjects are diagnosed with ME/CFS. In other cases, a subject can be diagnosed with an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease ; where methods described herein can confirm previous diagnosis. In other cases, a subject can exhibit an altered immune response. Some subjects can develop multiple clinical symptoms, for example both

ME/CFS and another an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease .

A subject can be one which fulfills the 1994 CDC Fukuda Criteria for CFS (Fukuda et al., Ann Intern Med 1994;121 : 953-9); the 2003 Canadian Consensus Criteria (CCC) for ME/CFS (Carruthers et al, J Chronic Fatigue Syndrome 2003; 1 1 :1 -12; Jason et al., J Chronic Fatigue S 2004; 12:37-52), or both the Fukuda and CCC criteria. The CCC requires post-exertional malaise, which many clinicians believe is the sine qua non of ME/CFS. In contrast, the Fukuda and 1991 Oxford Criteria do not require exercise intolerance for a diagnosis of ME/CFS. The CCC further requires that subjects exhibit post-exertional fatigue, unrefreshing sleep, neurological/cognitive manifestations and pain, rather than these being optional symptoms. Further discussion of ME/CFS diagnostic criteria is provided below.

The subject can be an animal subject, preferably a mammal, more preferably horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters, guinea pigs, and chickens, and most preferably a human.

As another example, the subject can be an animal, such as a laboratory animal that can serve as a model system for investigating a disease or disorder described herein (see e.g., Chen, R. et al., Neurochemical Research 33: 1759- 1767, 2008; Kumar, A., et al., Fundam. Clin. Pharmacol. 23(1 ): 89-95, Feb 2009; Gupta, A., et al., Immunobiology 214: 33-39, 2009; Singh, A., et al., Indian J. Exp. Biol. 40: 1240-1244, 2002; Ford, R.J., et al. Blood 109: 4899-4906, 2007; Smith, M.R., et al., Leukemia 20: 891 -893, 2006; Bryant, J., et al., Lab. Invest. 80: 557-573, 2000; M'kacher, R., et al., Cancer Genet Cytogenet. 143: 32-38, 2003).

DISEASE

Methods described herein can be used to diagnose or treat an

autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease in a subject. For example, methods described herein can be used to diagnose or treat a neuroimmune disease (or neuroimmune-associated disease) having an autoimmune disease (or autoimmune-associated disease) component. As another example, methods described herein can be used to diagnose or treat an autoimmune disease (or autoimmune-associated disease) having a neuroimmune disease (or

neuroimmune-associated disease) component.

Neuroimmune Disease. Methods described herein can be used to diagnose or treat a

neuroimmune or a neuroimmune-associated disease.

A neuroimmune disease or a neuroimmune-associated disease can be an illness resultant from acquired dysregulation of both the immune system and the nervous system, often can result in lifelong disease and disability. Symptoms of a neuroimmune disease can include mild to severe cognitive impairment;

disordered sleep; severe headache; swollen lymph nodes; sore throat; malaise; postural orthostatic tachycardia; painful nerves, joints and or muscles; abdominal pain; nausea; and unusual fatigue. A neuroimmune disease can follow an infectious or flu-like illness that may not fully resolve after standard treatment or over a typical course of time. Challenges to the immune system, such as new or reactivated infections, major life stresses or exposure to toxins, can trigger a severe relapse or worsening of existing symptoms. Recent published evidence associates human gamma retroviral infections with those who suffer from ME and CFS. Many classic autoimmune diseases can have a treatable

neuroimmune or neuroimmune-associated disease component. In some embodiments, ME/CFS can be considered a neuroimmune- associated disease because while it may demonstrate aspects of a

neuroimmune disease, it may not meet all requirements of a classical

neuroimmune disease. A neuroimmune disease can be a chronic neuroimmune disease. A neuroimmune disease can be, for example, chronic fatigue syndrome, fibromyalgia, myalgic encephalitis, atypical multiple sclerosis, non-epileptic seizures, Lyme disease, Gulf War Syndrome or autism.

Autoimmune Disease. Methods described herein can be used to diagnose or treat an

autoimmune or an autoimmune-associated disease.

Autoimmune diseases can arise from an inappropriate immune response of the body against substances and tissues normally present in the body. In other words, the immune system can mistake some part of the body as a pathogen and can attack its own cells. This may be restricted to certain organs (e.g., in autoimmune thyroiditis) or may involve a particular tissue in different places (e.g., Goodpasture's disease which may affect the basement membrane in both the lung and the kidney). Conventional treatment of autoimmune diseases can be with immunosuppression— medication which decreases the immune response.

An autoimmune disease can be a disease, disorder, or condition that conforms to Witebsky's postulates (first formulated by Ernst Witebsky and colleagues in 1957 and modified in 1994) including: direct evidence from transfer of pathogenic antibody or pathogenic T cells; indirect evidence based on reproduction of the autoimmune disease in experimental animals; and

circumstantial evidence from clinical clues. An autoimmune disease can be a disease, disorder, or condition that partially or substantially conforms to a definition of an autoimmune disease (e.g., Witebsky's postulates).

As used herein, an autoimmune or an autoimmune-associated disease can be a disease or disorder that corresponding to the following type of hypersensitivity: type II hypersensitivity, type III hypersensitivity, or type IV hypersensitivity. It is generally understood in the art that no type of autoimmune disease mimics type I hypersensitivity. To the extent an autoimmune disease does mimic type I hypersensitivity, it can be considered an autoimmune or an autoimmune-associated disease as that term is used herein. As used herein, an autoimmune or an autoimmune-associated disease can include, but is not limited to, Acute disseminated encephalomyelitis (ADEM); Addison's disease; Agammaglobulinemia; Alopecia areata; Amyotrophic Lateral Sclerosis; Ankylosing Spondylitis; Antiphospholipid syndrome; Antisynthetase syndrome; Atopic allergy; Atopic dermatitis; Autoimmune aplastic anemia;

Autoimmune cardiomyopathy; Autoimmune enteropathy; Autoimmune hemolytic anemia; Autoimmune hepatitis; Autoimmune inner ear disease; Autoimmune lymphoproliferative syndrome; Autoimmune peripheral neuropathy; Autoimmune pancreatitis; Autoimmune polyendocrine syndrome; Autoimmune progesterone dermatitis; Autoimmune thrombocytopenic purpura; Autoimmune urticaria;

Autoimmune uveitis; Balo disease/Balo concentric sclerosis; Behget's disease; Berger's disease; Bickerstaffs encephalitis; Blau syndrome; Bullous pemphigoid; Cancer; Castleman's disease; Celiac disease; Chagas disease; Chronic inflammatory demyelinating polyneuropathy; Chronic recurrent multifocal osteomyelitis; Chronic obstructive pulmonary disease; Churg-Strauss syndrome; Cicatricial pemphigoid; Cogan syndrome; Cold agglutinin disease; Complement component 2 deficiency; Contact dermatitis; Cranial arteritis; CREST syndrome; Crohn's disease (one of two types of idiopathic inflammatory bowel disease "IBD"); Cushing's Syndrome; Cutaneous leukocytoclastic angiitis; Dego's disease; Dercum's disease; Dermatitis herpetiformis; Dermatomyositis; Diabetes mellitus type 1 ; Diffuse cutaneous systemic sclerosis; Dressler's syndrome;

Drug-induced lupus; Discoid lupus erythematosus; Eczema; Endometriosis; Enthesitis-related arthritis; Eosinophilic fasciitis; Eosinophilic gastroenteritis; Epidermolysis bullosa acquisita; Erythema nodosum; Erythroblastosis fetalis; Essential mixed cryoglobulinemia; Evan's syndrome; Fibrodysplasia ossificans progressiva; Fibrosing alveolitis (or Idiopathic pulmonary fibrosis); Gastritis;

Gastrointestinal pemphigoid; Giant cell arteritis; Glomerulonephritis;

Goodpasture's syndrome; Graves' disease; Guillain-Barre syndrome (GBS); Hashimoto's encephalopathy; Hashimoto's thyroiditis; Henoch-Schonlein purpura; Herpes gestationis aka Gestational Pemphigoid; Hidradenitis

suppurativa; Hughes-Stovin syndrome; Hypogammaglobulinemia; Idiopathic inflammatory demyelinating diseases; Idiopathic pulmonary fibrosis; Idiopathic thrombocytopenic purpura (See Autoimmune thrombocytopenic purpura); IgA nephropathy; Inclusion body myositis; Chronic inflammatory demyelinating polyneuropathy; Interstitial cystitis; Juvenile idiopathic arthritis aka Juvenile rheumatoid arthritis; Kawasaki's disease; Lambert-Eaton myasthenic syndrome; Leukocytodastic vasculitis; Lichen planus; Lichen sclerosus; Linear IgA disease (LAD); Lou Gehrig's disease (Also Amyotrophic lateral sclerosis); Lupoid hepatitis aka Autoimmune hepatitis; Lupus erythematosus; Majeed syndrome; Meniere's disease; Microscopic polyangiitis; Miller-Fisher syndrome see Guillain- Barre Syndrome; Mixed connective tissue disease; Morphea; Mucha-Habermann disease aka Pityriasis lichenoides et varioliformis acuta; Multiple sclerosis;

Myasthenia gravis; Myositis; Narcolepsy; Neuromyelitis optica (also Devic's disease); Neuromyotonia; Occular cicatricial pemphigoid; Opsoclonus

myoclonus syndrome; Ord's thyroiditis; Palindromic rheumatism; PANDAS (pediatric autoimmune neuropsychiatric disorders associated with

streptococcus); Paraneoplastic cerebellar degeneration; Paroxysmal nocturnal hemoglobinuria (PNH); Parry Romberg syndrome; Parsonage-Turner syndrome; Pars planitis; Pemphigus vulgaris; Pernicious anaemia; Perivenous

encephalomyelitis; POEMS syndrome; Polyarteritis nodosa; Polymyalgia rheumatica; Polymyositis; Primary biliary cirrhosis; Primary sclerosing

cholangitis; Progressive inflammatory neuropathy; Psoriasis; Psoriatic arthritis; Pyoderma gangrenosum; Pure red cell aplasia; Rasmussen's encephalitis;

Raynaud phenomenon; Relapsing polychondritis; Reiter's syndrome; Restless leg syndrome; Retroperitoneal fibrosis; Rheumatoid arthritis; Rheumatic fever; Sarcoidosis; Schizophrenia; Schmidt syndrome another form of APS; Schnitzler syndrome; Scleritis; Scleroderma; Serum Sickness; Sjogren's syndrome;

Spondyloarthropathy; Still's disease see Juvenile Rheumatoid Arthritis; Stiff person syndrome; Subacute bacterial endocarditis (SBE); Susac's syndrome; Sweet's syndrome; Sydenham chorea see PANDAS; Sympathetic ophthalmia; Systemic lupus erythematosis see Lupus erythematosis; Takayasu's arteritis; Temporal arteritis (also known as "giant cell arteritis"); Thrombocytopenia;

Tolosa-Hunt syndrome; Transverse myelitis; Ulcerative colitis (one of two types of idiopathic inflammatory bowel disease "IBD"); Undifferentiated connective tissue disease different from Mixed connective tissue disease; Undifferentiated spondyloarthropathy; Urticarial vasculitis; Vasculitis; Vitiligo; Wegener's granulomatosis.

In some embodiments, ME/CFS can be considered an autoimmune- associated disease because while it may demonstrate aspects of an

autoimmune disease, it may not meet all requirements of a classical autoimmune disease.

ME/CFS.

Methods described herein can be used to diagnose or treat Chronic fatigue syndrome, i.e., Myalgic Encephalomyelitis / Chronic Fatigue Syndrome (ME/CFS). Chronic Fatigue Syndrome (CFS) is the most common name used to designate a significantly debilitating medical disorder or group of disorders generally defined by persistent fatigue accompanied by other specific symptoms for a minimum of six months in adults (and 3 months in children/adolescents), not due to ongoing exertion, not substantially relieved by rest, nor caused by other medical conditions. The disorder may also be referred to as myalgic encephalomyelitis (ME), post-viral fatigue syndrome (PVFS), chronic fatigue immune dysfunction syndrome (CFIDS), or several other terms. Aside from CFS, some other names used include Akureyri disease, benign myalgic

encephalomyelitis, chronic fatigue immune dysfunction syndrome, chronic infectious mononucleosis, epidemic myalgic encephalomyelitis, epidemic neuromyasthenia, Iceland disease, myalgic encephalomyelitis, myalgic encephalitis, myalgic encephalopathy, post-viral fatigue syndrome, raphe nucleus encephalopathy, Royal Free disease, Tapanui flu and yuppie flu.

Symptoms of CFS can include post-exertional malaise; unrefreshing sleep; widespread muscle and joint pain; sore throat; headaches of a type not previously experienced; cognitive difficulties; chronic, often severe, mental and physical exhaustion; and other characteristic symptoms in a previously healthy and active person. Persons with CFS may report additional symptoms including muscle weakness, increased sensitivity to light, sounds and smells, orthostatic intolerance, digestive disturbances, depression, and cardiac and respiratory problems.

Physical symptoms of ME/CFS can include post-exertional malaise or fatigue, sleep dysfunction, and pain; have two or more neurological/cognitive manifestations and one or more symptoms from two or the categories of autonomic, neuroendocrine and immune manifestations. Autonomic

manifestations of ME/CFS can include orthostatic intolerance-neurally mediated hypotension (NMH); postural orthostatic tachycardia syndrome (POTS); delayed postural hypotension; light-headedness, extreme pallor; nausea and irritable syndrome; urinary frequency and bladder dysfunction; palpitations with or without cardiac arrhythmias; or exertional dyspnea. Neuroendocrine

manifestations of ME/CFS can include loss of thermostatic stability-subnormal body temperature and marked diurnal fluctuation; sweating episodes; recurrent feelings of feverishness and cold extremities; intolerance of extremes of heat and cold; marked weight change-anorexia or abnormal appetite; loss of adaptability and worsening of symptoms with stress. Immune manifestations can include tender lymph nodes, recurrent sore throat, recurrent flu-like symptoms, general malaise, new sensitivities to food, or medications or chemicals. To meet a criteria for CFS, these symptoms may have persisted for at least six months or usually have a distinct onset, although onset may be gradual. CFS symptoms can vary from person to person in number, type, and severity. A subject having ME/CFS can be a subject meeting one or more of the diagnostic criteria described below. A diagnosis according to criteria below can be confirmed according to diagnostic methods described herein. A diagnoses accord to diagnostic methods described herein can be confirmed according to criteria below. CDC definition (1994) is a widely used clinical and research description of

CFS, also called the Fukuda definition and was based on the Holmes or CDC 1988 scoring system. The 1994 criteria require the presence of four or more symptoms beyond fatigue, where the 1988 criteria require six to eight.

According to the CDC criteria, a subject can be diagnosed with ME/CFS where the following three criteria be fulfilled: A new onset (not lifelong) of severe fatigue for six consecutive months or greater duration which is unrelated to exertion, is not substantially relieved by rest, and is not a result of other medical conditions. The fatigue causes a significant reduction of previous activity levels.

Four or more of the following symptoms that last six months or longer: Impaired memory or concentration; post-exertional malaise, where physical or mental exertions bring on "extreme, prolonged exhaustion and sickness"; unrefreshing sleep; muscle pain (myalgia); pain in multiple joints (arthralgia); headaches of a new kind or greater severity; sore throat, frequent or recurring; tender lymph nodes (cervical or axillary). Other common symptoms can include: irritable bowel, abdominal pain, nausea, diarrhea or bloating; chills and night sweats; brain fog; chest pain;

shortness of breath; chronic cough; visual disturbances (blurring, sensitivity to light, eye pain or dry eyes); allergies or sensitivities to foods, alcohol, odors, chemicals, medications or noise; difficulty maintaining upright position

(orthostatic instability, irregular heartbeat, dizziness, balance problems or fainting); psychological problems (depression, irritability, mood swings, anxiety, panic attacks).

The Oxford criteria (1991 ) includes CFS of unknown etiology and a subtype called post-infectious fatigue syndrome (PIFS). Important differences can be that the presence of mental fatigue is necessary to fulfill the criteria and symptoms are accepted that may suggest a psychiatric disorder.

The 2003 Canadian Clinical working definition states that "A patient with ME/CFS will meet the criteria for fatigue, post-exertional malaise and/or fatigue, sleep dysfunction, and pain; have two or more neurological/cognitive

manifestations and one or more symptoms from two of the categories of autonomic, neuroendocrine, and immune manifestations; and [the illness will persist for at least 6 months]".

Clinical practice guidelines, as related to improving diagnosis,

management, and treatment, can be based on case descriptions. An example is the CFS/ME guideline for the National Health Service in England and Wales, produced in 2007 by the National Institute for Health and Clinical Excellence (NICE).

In some embodiments, ME/CFS can be considered a neuroimmune- associated disease because while it may demonstrate aspects of a

neuroimmune disease, it may not meet all requirements of a classical

neuroimmune disease.

In some embodiments, ME/CFS can be considered an autoimmune- associated disease because while it may demonstrate aspects of an

autoimmune disease, it may not meet all requirements of a classical autoimmune disease.

In some embodiments, ME/CFS can be considered a neuroimmune- associated disease and an autoimmune-associated disease because while it may demonstrate aspects of both a neuroimmune and an autoimmune disease, it may not meet all requirements of a classical neuroimmune or autoimmune disease.

One of ordinary skill will understand that one or more of the above diagnostic criteria can be used in conjunction with diagnostic methods described herein.

THERAPEUTIC METHODS

Also provided is a process of treating an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune- associated disease, such as ME/CFS. In some embodiments, a subject diagnosed according to methods describe herein with an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS, can be treated as described herein or as known in the art (e.g., antiviral treatment).

Methods described herein are generally performed on a subject in need thereof. A subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing an autoimmune disease, an autoimmune-associated disease, a neuroimmune disease, or a neuroimmune-associated disease, such as ME/CFS. A

determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art. The subject can be an animal subject, including a mammal, such as horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, guinea pigs, and chickens, and humans. For example, the subject can be a human subject. An effective amount of anti-viral agent is generally that which can inhibit viral development. For example an anti-viral agent can be a retroviral integrase inhibitors, gene silencing therapy, or vaccine. For example, in various

embodiments, an effective amount of anti-viral agent described herein can substantially inhibit viral development, slow the progress of infection, or limit the development of viral infection, development, or variation.

According to the methods described herein, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration. When used in the treatments described herein, a therapeutically effective amount of a therapeutic agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to ameliorate disease symptoms, substantially inhibit viral development, slow the progress of infection, or limit the development of viral infection, development, or variation.

The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or

experimental animals for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.

The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4^th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Sharqel (2004) Applied

Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired

therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

Administration of a therapeutic agent can occur as a single event or over a time course of treatment. For example, a therapeutic agent can be

administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for an autoimmune-associated disease, or a neuroimmune disease, such as ME/CFS.

A therapeutic agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an antiinflammatory, or another agent. For example, a therapeutic agent can be administered simultaneously with another agent, such as an antibiotic or an antiinflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a therapeutic agent, an antibiotic, an antiinflammatory, or another agent. Simultaneous administration can occur through administration of one composition containing two or more of a therapeutic agent, an antibiotic, an antiinflammatory, or another agent. A therapeutic agent can be administered sequentially with an antibiotic, an antiinflammatory, or another agent. For example, a therapeutic agent can be administered before or after administration of an antibiotic, an antiinflammatory, or another agent.

ADMINISTRATION

Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art. The agents and composition can be used therapeutically either as exogenous materials or as endogenous materials. Exogenous agents are those produced or

manufactured outside of the body and administered to the body. Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.

As discussed above, administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration. Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection {e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres (e.g., 1 -100 μm), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions. Other methods of controlled-release delivery of agents or compositions will be known to the skilled artisan and are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, an agent or composition is administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ). Carrier-based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.

KITS

Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to assays for the detection of pDCs, HERV, TLR-7 or TLR-9 activity, and PCLN2 expression. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components. Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.

Removable membranes may be glass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD- ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001 )

Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in

Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10:

0954523253).

Definitions and methods described herein are provided to better define the present disclosure and to guide those of ordinary skill in the art in the practice of the present disclosure. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about." In some embodiments, the term "about" is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value. In some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable. The numerical values presented in some embodiments of the present disclosure may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein.

In some embodiments, the terms "a" and "an" and "the" and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise. In some embodiments, the term "or" as used herein, including the claims, is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.

The terms "comprise," "have" and "include" are open-ended linking verbs. Any forms or tenses of one or more of these verbs, such as "comprises," "comprising," "has," "having," "includes" and "including," are also open-ended. For example, any method that "comprises," "has" or "includes" one or more steps is not limited to possessing only those one or more steps and can also cover other unlisted steps. Similarly, any composition or device that "comprises," "has" or "includes" one or more features is not limited to possessing only those one or more features and can cover other unlisted features. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. "such as") provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admission that such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustrate the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches the inventors have found function well in the practice of the present disclosure, and thus can be considered to constitute examples of modes for its practice.

However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

EXAMPLE 1: SPECIMEN SELECTION AND STUDY DESIGN

The following example describes the specimen selection and study design. An objective of the study was to confirm the association between pDCs and HERV expression in human disease.

Biopsy specimens from the duodenum of myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) and control subjects were probed using monoclonal and polyclonal antibodies to HERV and gammaretroviral proteins. Additionally, cellular phenotyping was performed using monoclonal antibodies to cell surface markers.

Immunoreactivity to HERV proteins was observed in duodenum biopsies in 8 of 12 subjects with ME/CFS. In contrast, no immunoreactivity was detected in any of the 8 control subjects. Immunoreactivity to HERV Gag and Env proteins was uniquely colocalized in hematopoietic cells expressing the surface markers CD303, consistent with plasmacytoid dendritic cells (pDCs).

Immunoreactivity was detected in pDCs of ME/CFS subjects, consistent with the expression of HERV proteins. In contrast, the same immunoreactivity was not observed in the pDCs of control subjects. This study shows expression of HERV in the pDCs of ME/CFS subjects suggests HERV/pDC involvement in ME/CFS pathology. The following examples describe the first direct association between pDCs and HERV expression in human disease.

EXAMPLE 2: SUBJECT CLINICAL EVALUATION

The following example describes the subject selection criteria. An objective of the study was to select specimens from subjects with ME/CFS and control subjects without symptoms of ME/CFS for evaluation of HERV

expression in pDCs.

Biopsies were acquired as deidentified surplus clinical diagnostic specimens under an exemption to the Institutional Review Board (IRB).

Therefore, subject information was limited to a general pathological description by which the specimens were grouped prior to deidentification. ME/CFS specimens were from 12 subjects fulfilling both the Canadian consensus criteria of ME (Carruthers, 2003) and the Fukuda criteria for CFS (Fukuda, 1994). Fecal microbial analysis established the presence of substantial disruption of gut microbiota composition with occurrence of a higher load of microbes with higher pathogenic potential in all ME/CFS subjects, particularly of the Enterococcus, Streptococcus, and Prevotella genera. Gastritis (mainly antritis) was present in all subjects and routine histological examination showed a lympho-plasmatic infiltrate in the submucosa in all specimens. All subjects tested negative for Helicobacter pylori. Control subjects were 8 anonymous individuals without symptoms of ME/CFS who underwent routine gastroscopy for epigastric pain.

EXAMPLE 3: TISSUE AND PREPARATION

The following example describes the specimen tissue preparation. Punch biopsies were obtained from the duodenum and stomach of ME/CFS subjects and control subjects. Fresh tissue was fixed in 4% paraformaldehyde for 4 hours at 4 °C and cryoprotected with a 30% sucrose solution in PBS.

EXAMPLE 4: IMMUNOHISTOCHEMICAL ANALYSIS

The following example describes the protocol for the

immunohistochemical staining of the tissue samples.

Immunohistochemical staining was performed on 0.5-3-μm thick tissue sections. Tissue slides were deparaffinized with xylene and rehydrated through a graded alcohol series. Antigen retrieval was carried out by boiling slides in sodium citrate (0.01 M, pH 6.0) at 95 °C for 10 minutes. The slides were next rinsed in PBS and incubated in cold methanol for 20 minutes at -20 °C. Tissue sections were incubated with serum (matching the host of the secondary antibody) to block nonspecific staining (1 hour at 37 °C) and then incubated with the primary antibody overnight at 4 °C in a humidified chamber. After washing 3 times with PBS containing 0.1 % Tween 20, the sections were incubated with the second antibody for 1 hour at 37 °C. All subjects were analyzed for the presence of HERV and gammaretroviral Env and Gag proteins. Isotype-matched controls and secondary only controls were included with all experiments. A summary of each antibody and the concentration at which it was used is presented in Table 1 . Slides were examined using a Zeiss LSM 7000 scanning laser confocal microscope (Carl Zeiss Microscopy, Thornwood, NY) and images were captured with Zeiss Zen 2009 analysis software.

TABLE 1 : ANTIBODIES AND DILUTIONS

EXAMPLE 5: DETECTION OF IMMUNOREACTIVE PROTEINS IN GUT BIOPSIES OF ME/CFS SUBJECTS

The following example describes the immunohistochemical analysis of gut biopsy samples. An objective of the study was to detect immunoreactive proteins in gut biopsy samples.

Immunochemical analyses of 12 ME/CFS gut biopsies probed for viral antigens showed that 8 samples of the duodenum were immunoreactive with antibodies raised against HERV proteins (see e.g., FIG. 1A-D). In contrast, no immunoreactivity was observed in any of the control duodenum samples (see e.g., FIG. 1 E-H).

Additional analyses were conducted using two anti-murine

gammaretroviral antibodies: goat polyclonal IgG antibody raised to the Gag protein of murine leukemia virus (see e.g., FIG. 2A) and a rat monoclonal lgG1 k (clone 7C10) raised to the Env protein of spleen focus forming virus (see e.g., FIG. 2B). The observed immunoreactivity was reproducibly consistent with the previous anti-HERV results, suggesting that the gammaretroviral antibodies were cross-reactive with the HERV antigen(s). Additionally, the immunoreactivity was observed to colocalize (see e.g., FIG. 2C) in cells with a small, eccentric nucleus and prominent granular inclusions (see e.g., FIG. 2D). Consistent with previous observations using anti-HERV antibodies, no immunoreactivity was observed in the control subject's biopsies using either anti-gammaretroviral antibody (see e.g., FIG. 2E-G). Matched stomach biopsies, collected from ME/CFS and control subjects, were also analyzed but were consistently unreactive when probed with the same anti-HERV and anti-gammaretroviral antibodies (data not shown).

Specificity of the primary anti-gammaretroviral antibodies was established when their ability to detect Env and Gag proteins was ablated by preincubation with purified and heat deactivated virus derived from 22Rv1 (see e.g., FIG. 3). In order to rule out nonspecific binding of the primary antibody, the rat monoclonal anti-Env IgG antibody (7C10) was directly labeled with Rhodamine and used to probe the tissue sections (Figure 4A). Potential nonspecific binding of the secondary antibody was excluded by using the Rhodamine conjugated donkey anti-rat IgG and the Alexa647 conjugated donkey anti-goat IgG secondary antibodies in the absence of the primary antibody (see e.g., FIG. 4B).

Nonspecific binding of the primary rat monoclonal antibody was further ruled out by the lack of immunoreactivity using an irrelevant rat monoclonal (rat anti- human herpes virus-8 UL84 IgG) and a rat lgG2K isotype control (see e.g., FIG. 4C and FIG. 4D). Potential nonspecific binding of the secondary antibody was excluded by replacing the primary antibody with normal goat serum (see e.g., FIG. 4E). Finally, to address the potential cross-reactivity of the secondary antibody with the incorrect primary antibody (e.g., donkey anti-goat IgG secondary antibody cross-reacting with the rat anti-Env IgG primary antibody) biopsies were probed with a primary antibody followed by incubation with mismatched secondary antibody see e.g., FIG. 4F and FIG. 4G).

In summary, patients with ME/CFS were observed to exhibit

immunoreactivity in the gut tissue using the anti-Env monoclonal and anti-Gag polyclonal antibodies; in contrast, no immunoreactivity was detected in biopsies of healthy control subjects. The ability of the monoclonal and polyclonal antibodies to detect Env and Gag proteins was ablated by preincubation with purified retroviral proteins. EXAMPLE 6: PHENOTYPE OF THE IMMUNOREACTIVE CELLS

The following example describes the characterization of the

immunoreactive cells. An objective of the study was to show the immunoreactive cells were pDCs. In order to determine the phenotype of the immunoreactive cells, immunohistochemical analysis was performed using the pan-lymphocyte marker CD45 (see e.g., FIG. 5A) in combination with the rhodamine conjugated rat anti- Env IgGl k monoclonal antibody (see e.g., FIG. 5B). Consistent with a

hematopoietic lineage, only cells expressing CD45 proved immunoreactive with the anti-Env antibody (see e.g., FIG. 5C).

The immunoreactive cells were determined to be plasmacytoid dendritic cells (pDCs). pDCs express CD45 and uniquely express the surface marker CD303 (BDCA-2) (Dzionek, 2000; Dzionek, 2001 ; Dzionek, 2002). The analysis confirmed that CD303 expression (see e.g., FIG. 5E) and immunoreactivity to the rhodamine conjugated rat anti-Env monoclonal antibody (see e.g., FIG. 5F) colocalized in the same cell (see e.g., FIG. 5G), consistent with the pDC phenotype.

In summary, the immunoreactivity was co-localized in cells

morphologically similar to plasma cells but negative for CD138 and common pan-B cell markers; however, the observed cells were strongly positive for CD303, consistent with plasmacytoid dendritic cells (pDCs).

EXAMPLE 7: IMMUNOREACTIVITY TO HERV

The following example describes the comparison of ME/CFS and control subjects. An objective of the study was to show pDCs are more prevalent in ME/CFS subjects. Another objective of this study was to show pDCs in ME/CFS subjects showed positive immunoreactivity to anti-HERV antibodies.

Five random microscopic fields of view for 8 subjects from each group were analyzed (see e.g., Table 2) to evaluate if the relative number of

duodenum-associated pDCs differed between ME/CFS and control subjects. The frequency at which the pDCs of ME/CFS are immunoreactive with the HERV antibodies was also determined. ME/CFS subjects were found to have approximately 4.7 times as many pDCs per field when compared to the control subjects (35.6 ± 10.9 vs. 7.5 ± 3.5, respectively, p < 0.0001 ). Additionally, it was observed that approximately 44% (15.7/35.6) of the duodenum-associated pDCs in the 8 ME/CFS subjects were immunoreactive to anti-HERV antibodies.

EXAMPLE 8: RULING OUT INFECTIOUS RETROVIRUS

The following example describes the detection of HERV expression using HERV-specific antibodies, next generation sequencing (NGS) and coculture methods to rule out an exogenous retroviral infection producing the observed results. Methods are as described above unless otherwise noted.

This study demonstrated that gut biopsies from 8 of 12 subjects with ME/CFS display immunoreactivity consistent with the expression of human endogenous retroviral proteins. The same immunoreactivity, however, was not observed in the biopsies of control subjects. Additionally, it was shown that the immunoreactivity was observed in cells with a phenotype that is consistent with pDCs. These observations suggest that the presence of HERV protein in pDCs may be associated with a pathological manifestation in at least a subset of ME/CFS subjects. The detection of a protein that reacts with HERV monoclonal antibodies is consistent with HERV expression.

Next generation sequencing (NGS) on both RNA derived from the duodenum and purified pDCs from one HERV positive ME/CFS subject (for which cryopreserved lymphocytes and preserved RNA from a duodenum biopsy were available) was performed to rule out an exogenous retroviral infection producing the observed results. Because the present study was conducted using surplus clinical biopsies from deidentified subjects, collection of additional biological material was not possible and the proper matched specimens were not available to conduct a rigorous transcriptional analysis. Results showed that multiple contigs of known HERV genes were observed; however, no open reading frames were identified that could account for an infectious retrovirus.

The identification of an infections MLV-related virus, by co-culturing lymphocytes and purified pDCs, using the DERSE indicator cell line was attempted (Lee, 201 1 ). The DERSE indicator cell line is derived from the prostate cancer cell line LNCaP and stably transfected with an MLV vector containing the green fluorescent protein (GFP) gene in reverse orientation. Only after rescue and transfer to new cells through reverse transcriptase and integrase enzymatic activity can the GFP be detected. In previous experiments, the sensitivity of this assay was established by detecting infectious murine leukemia related virus derived from 2 individual 22Rv1 cells per culture

(unpublished data). In spite of using this sensitive detection method an infections MLV-related retrovirus was not identified.

The results using HERV-specific monoclonal and polyclonal antibodies as well as NGS and co-culture methods strongly argue against the possibility that observations were the result of an infectious retrovirus.

In summary, co-culture methods using a cell line that fluorescently reports integrase and reverse transcriptase activity failed to identify an infectious retrovirus, as did transcriptome analysis by next generating sequencing (NGS), however, multiple human endogenous retroviral (HERV) transcripts were identified. EXAMPLE 9: ASSOCIA TION OF ME/CFS WITH PDCS AND AUTOIMMUNE

DISEASE

The following example describes the association of ME/CFS pathology with pDCs and autoimmune disease.

A number of immunological observations have been described in relation to ME/CFS. The data presented here, however, represents the first report of pDC association with ME/CFS. While the expression of endogenous retroviral proteins in the pDCs of ME/CFS subjects does not intrinsically explain pathology, the observation that the immunoreactive proteins are only observed in pDCs is supportive of this concept. The association of pDCs with ME/CFS is further supported by previous reports of the over-production of inflammatory cytokines in a cohort of ME/CFS subjects (Lombardi, 201 1 ). One subset of this cohort was characterized by elevated IL-8, IL-6, TNF-α, ΜΙΡ-1 β, MIP-1 α, IP-10 and depressed IFN-a production. Plasmacytoid dendritic cells are known to produce many of the same pro-inflammatory cytokines (Piqueras, 2006) as those described in a subset of subjects identified in a previous report (Lombardi, 201 1 ). Additionally, they are responsible for the production of over 95% of circulating IFN-a (Seigal, 1999). A pDC involvement is consistent with previous

observations of cytokine dysregulation in ME/CFS subjects.

A potential dysregulation of pDCs suggests an explanation for a number of clinical observations associated with ME/CFS. As stated previously, pDCs are most remarkable for their ability to produce copious amounts of type I IFN. In

1978, Trinchieri and colleagues had indirectly identified pDCs by their ability to activate NK-cell-mediated cytotoxicity through the production of IFN-a (Trinchieri,

1978) and several subsequent studies have expanded upon the importance of type I IFN in modulating NK cell function (Lodoen, 2006). Decreased NK cell activity can be a commonly reported observation associated with ME/CFS (Whiteside, 1998), therefore, an aberrant pDC response leading to a decrease in IFN-α, would be consistent with dysregulation of NK cells. Inflammatory cytokine and chemokine abnormalities have also been reported in association with ME/CFS by other researchers. For instance, Natelson et al. reported elevated levels of interleukin-8 (IL-8) and interleukin-10 (IL-10) in the spinal fluid of subjects with influenza-like onset ME/CFS (Natelson, 2005). Also, Vernon et al. reported that IL-8 gene transcription was elevated in ME/CFS subjects (Vernon, 2002). Finally, Chao et al. reported interleukin-6 (IL-6) to be upregulated in subsets of ME/CFS subjects (Chao, 1990). In previous studies, the expression of inflammatory cytokines was more prevalent in a subset of subjects characterized by a gamma-delta T cell clonality (Lombardi, 201 1 ). A similar clonality has been observed in other autoimmune diseases characterized by the expression of HERVs, such as rheumatoid arthritis and MS (Olive, 1994; Bieganowski, 1996). Additionally, the expansion of gamma-delta T cells in response to the expression of endogenous

gammaretroviruses has been reported in animal models (Sim, 1993). Some HERV proteins act as superantigens (Sutowski, 2001 ), promoting the expansion of T cell populations and, therefore, the observation of gamma-delta T cell clonality associated with ME/CFS subjects, as well as other diseases, may be the result of HERV superantigen stimulation.

Although the Canadian consensus criteria of ME and the Fukuda criteria for CFS do not include symptoms of autoimmunity, the recent study by Fluge et al. supports that at least a subset of individuals with ME/CFS may have an autoimmune element associated with their disease (Fluge, 2009). Autoimmune diseases, such as SLE, MS and rheumatoid arthritis (RA) have several symptoms that overlap with those of ME/CFS and all have been associated with pDC dysfunction. Moreover, the same autoimmune diseases are also reported to associate with the expression of HERVs, although a physical connection between HERVs and pDCs has not been reported. As Stoye points out in his recent review (Stoye, 2012), the role of HERVs in autoimmunity remains an unproven hypothesis; however, an increasing number of studies suggest that HERVs may have the capacity contribute to disease pathology (Wang- Johanning, 2008; Ariza, 201 1 ). It is noteworthy that work conducted in the laboratory of Bridget Huber showed that HERV-K18 expression could be induced by herpesviruses such as EBV and HHV-6 (Sutkowski, 2001 ; Tai, 2009).

Consistent with that work and with the data presented here, EBV and HHV-6 have been observed in the duodenum of ME/CFS subjects (Fremont, 2009).

The expression of HERV proteins in autoimmune diseases such as SLE, MS and Sjogren's syndrome is also evident by reports of antibodies to retroviral proteins in subjects that are not found to be infected with an exogenous retrovirus (Balada, 2010). If the expression of HERV proteins in pDCs (an antigen-presenting cell) is found to be associated with these autoimmune diseases, it may help explain the presence of such antibodies. Inflammation can be known to increase HERV expression (Rolland, 2006; Kelleher, 1996);

therefore, if pDC-associated inflammation is driving the expression of

endogenous retroviruses, it's also conceivable that unregulated expression of other proteins in pDCs may occur. Consequently, the antigen-presenting abilities of pDCs may contribute to the production of auto-reactive antibodies, as is observed in ME/CFS subjects (von Mikecz, 1997; Hokama, 2008; Klein, 1995; Tanaka, 2003; Vernon, 2005; Wheatland, 2005). The etiopathology associated with autoimmune disease, HERV expression, and pDCs is not a simple relationship. Nevertheless, the prospect of a HERV/pDC involvement in ME/CFS pathology raises the possibility of a specific target for disease treatment. A case study conducted by Dreyfus (Dreyfus, 201 1 ) suggests that antiviral drugs, such as Acyclovir and Raltegravir, may be effective against certain autoimmune diseases characterized by HERV expression and pDC involvement. However, rigorously controlled clinical trials will be required before any claims can be supported regarding efficacy and safety.

The expression of HERV proteins in pDCs may lead to ME/CFS-related pathology. Conversely, their expression might merely be the result of the inflammation associated with the disease or perhaps a combination of both. Nevertheless, the presence of these proteins in the pDCs of ME/CFS subjects but not in control subjects does support a pDC involvement in ME/CFS. EXAMPLE 10: TLR INHIBITION AND HERV EXPRESSION BYPDCS

The following example demonstrates that HERV expression by gut- associated pDCs in ME/CFS subjects is associated with TLR inhibition.

Purified pDCs from healthy donors were cultured for 5 days in the presence of small interfering RNA (siRNA) to TLR-7 and TLR-9 or with the TLR inhibitors C661 and IRS-954. Non-treated cells and cells treated with an irrelevant siRNA were used as controls. HERV expression was established by immunohistochemistry and total RNA was collected on Trizol reagent, depleted of ribosomal RNA, and used to conduct transcriptional analysis by unbiased Next Generation Sequencing (NGS). Transcripts expressed by TLR-inhibited pDCs were determined significant when their expression was four-fold greater than that of the control cells. Lastly, a monoclonal antibody specific for the most

significantly upregulated transcript was used to screen duodenum biopsies of ME/CFS subjects for the presence of the respective protein to verify in vivo expression.

Results showed that cultured pDCs expressed HERV proteins in the presence of TLR-7 and TLR-9 siRNA as well as TLR inhibitors, but not control cells. NGS analysis identified several significantly upregulated transcripts, the greatest of which (500-fold increase) was phospholipase C-like 2 (PLCL2), a protein previously associated with multiple sclerosis. Monoclonal antibodies to PLCL2 were uniquely immunoreactive to pDCs in biopsies of ME/CFS subjects, but not in controls. As such, PLCL2 upregulation can be a biomarker of TLR-7 or TLR-9 inhibition.

These data are consistent with an inhibition of TLR-7 or TLR-9 in the gut- associated pDCs of subjects with ME/CFS.

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Claims

CLAIMS What is claimed is:

1 . A method of diagnosing an autoimmune disease, an autoimmune- associated disease, a neuroimmune disease, or a neuroimmune-associated disease in a subject, the method comprising:

(a) determining in a biological sample of a subject one or more of

(i) a level of plasmacytoid dendritic cells (pDCs) cell-localized immunoreactivity to an antibody specific for human endogenous retrovirus (HERV); (ii) a level of pDCs;

(iii) a level of pDC-localized activity of Toll Like Receptor (TLR)-7 or TLR-9; or

(iv) a level of pDC-localized phospholipase C-like 2 (PLCL2) expression; (b) comparing one or more of

(i) the level of pDC-localized immunoreactivity to the anti-HERV antibody with a first control,

(ii) the level of pDCs with a second control,

(iii) the level of pDC-localized activity of TLR-7 or TLR-9 with a third control; or

(iv) the level of pDC-localized expression of PLCL2 with a fourth control; and

(c) diagnosing the subject with an autoimmune disease, an autoimmune- associated disease, a neuroimmune disease, or a neuroimmune-associated disease when at least one of the following is satisfied

(i) the level of pDC-localized immunoreactivity to the anti-HERV antibody is detected or is higher than the first control;

(ii) the level of pDCs is higher than the second control; (iii) the level of pDC-localized activity of TLR-7 or TLR-9 is inhibited compared to the third control; or

(iv) the level of pDC-localized PLCL2 expression is upregulated compared to a fourth control.

2. The method of claim 1 , wherein the disease comprises myalgic encephalomyelitis/chronic fatigue syndrome ("ME/CFS").

3. The method of any one of claims 1 -2, wherein the biological sample comprises gut-associated lymphoid tissue.

4. The method of any one of claims 1 -3, wherein the biological sample comprises a duodenum biopsy sample.

5. The method of any one of claims 1 -4, wherein the subject is diagnosed with the autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimmune-associated disease when at least one of the following are satisfied:

(i) the level of pDC-localized immunoreactivity to the anti-HERV antibody is about 1 % higher than the first control;

(ii) the level of pDCs is about two times higher than the second control;

(iii) the level of pDC-localized activity of TLR-7 or TLR-9 is about 1 % lower than the third control; or

(iv) the level of pDC-localized PLCL2 expression is about 10 times higher than the fourth control.

6. The method of any one of claims 1 -5, wherein the subject is diagnosed with the autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimnnune-associated disease when at least two of the following are satisfied:

(i) the level of pDC-localized immunoreactivity to the anti-HERV antibody is about 1 % higher than the first control;

(ii) the level of pDCs is about two times higher than the second control;

(iii) the level of pDC-localized activity of TLR-7 or TLR-9 is about 1 % lower than the third control; or

(iv) the level of pDC-localized PLCL2 expression is about 10 times higher than the fourth control.

7. The method of any one of claims 1 -6, wherein the subject is diagnosed with the autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimmune-associated disease when at least three of the following are satisfied:

(i) the level of pDC-localized immunoreactivity to the anti-HERV antibody is about 1 % higher than the first control;

(ii) the level of pDCs is about two times higher than the second control;

(iii) the level of pDC-localized activity of TLR-7 or TLR-9 is about 1 % lower than the third control; or

(iv) the level of pDC-localized PLCL2 expression is about 10 times higher than the fourth control.

8. The method of any one of claims 1 -7, wherein the first control, the second control, the third control, or the fourth control comprise a sample from a subject not having the autoimmune-associated disease.

9. The method of any one of claims 1 -8, wherein the first control, the second control, the third control, or the fourth control are comprised in a single sample from a subject not having the autoimmune-associated disease.

10. The method of any one of claims 1 -9, wherein the first control, the second control, the third control, and the fourth control comprise a sample from a subject not having the autoimmune-associated disease.

1 1 . The method of any one of claims 1 -10, wherein the subject is diagnosed with the autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimmune-associated disease when the level of cells immunoreactive to anti-HERV antibodies in the sample is substantially the number of cells in the sample.

12. A method of treating an autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimmune-associated disease in a subject comprising: diagnosing an autoimmune disease, autoimmune-associated disease, neuroimmune disease, or neuroimmune-associated disease according to the method of any one of claims 1 -1 1 ; and administering an antiviral agent to a subject in need thereof.

13. The method of any one of claims 1 -12, wherein the disease is selected from the group consisting of chronic fatigue syndrome, fibromyalgia, myalgic encephalitis, atypical multiple sclerosis, autism, non-epileptic seizures, and Gulf War Syndrome.