Method of diagnosing a subpopulation of subjects with a post-covid-19 condition and of treating the subjects using BAFF inhibition

Abstract

The present disclosure provides a method for treating long CO VID ( e.g., severe long CO VID) or at least one symptom thereof in a subpopulation of subjects displaying a combination of biomarkers namely a blood level of secreted B-cell activating factor and of membrane BAFF in white blood cells higher than a corresponding reference level; and at least one of blood level of precursor- like marginal zone B-cell populations, zonulin, anti-Mi-2 nuclear antigen autoantibodies, anti-SmD autoantibodies, anti-Ul-snRNP-A autoantibodies, anti-RCN2 autoantibodies, lipopolysaccharide-binding protein (LBP), and p-D-glucan higher than a corresponding reference level, and APRIL lower than a corresponding reference level, comprising administering a therapeutically effective amount of a BAFF inhibitor to the subject. It also provides a method of diagnosing a subject as having long CO VID using the combination of biomarkers and a diagnosis kit comprising ligands for the biomarkers.

Description

[0001] TITLE

[0002] METHOD OF DIAGNOSING A SUBPOPULATION OF SUBJECTS WITH A POST-COVID-19 CONDITION AND OF TREATING THE SUBJECTS USING BAFF INHIBITION CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application is a PCT application Serial No PCT / CA2026 / filed on February 9, 2026, and published in English under PCT Article 21(2), which itself claims benefit of U.S. provisional application Serial No. 63 / 756,367, filed on February 10th, 2025. All documents above are incorporated herein in their entirety by reference.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0005] N.A.

[0006] FIELD OF THE DISCLOSURE

[0007] The present disclosure relates to the use of a combination of biomarkers for the diagnosis of a subpopulation of subjects as suffering from a post-COVID-19 condition such as long COVID (e.g., severe long COVID) and to a method of treating the subpopulation of subjects using BAFF inhibition.

[0008] REFERENCE TO SEQUENCE LISTING

[0009] Pursuant to 37 C.F.R. 1.821(c), a sequence listing is submitted herewith as an ASCII compliant xml file named G10992-00077-Sequence listing, that was created on February 9, 2026, and has a size of 11 kilobytes. The content of the aforementioned file named G10992-00077-Sequence listing is hereby incorporated by reference in its entirety. BACKGROUND OF THE DISCLOSURE

[0010] Widespread acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has left in its wake an epidemic of postcoronavirus disease 2019 (COVID-19) condition or post-acute sequelae of COVID-19 (PASC), commonly known as long-COVID (LC). LC is an infection-associated chronic condition with one or more symptoms that are present for at least 3 months, can be continuous, relapsing and remitting or progressive and affect one or multiples organ systems1'4and can be extremely debilitating for years. It impacts 5-19% of individuals previously infected with SARS-CoV-2 and is now considered a chronic disease which can be severe and significantly impact functional status and quality of life1 5. In addition to the burden of disease impacting several organ systems that can be extremely debilitating36, SARS-CoV-2 infection is associated with increased long-term risk of neurological7, mental health8, cardiovascular9, renal10, metabolic11 12and gastrointestinal disorders13. Although LC is a complex multisystemic disease that has been associated with more than 200 symptoms14, it is now thought to be a disease with distinct phenotypic and biological subtypes. The most severe (almost prototypic) form of LC, which especially impacts women, includes symptoms such as post-exertional malaise, fatigue, brain fog, trouble with memory and concentration, and dysautonomia1 15. While the risk of LC increases with the severity of the acute infection, more than 90% of individuals with LC had a mild SARS-CoV-2 infection due to the much higher prevalence of mild cases3. While studies elucidating the pathogenic underpinnings of LC are ongoing, LC remains a clinical diagnosis which is becoming increasingly more challenging tomake with decreasing COVID-19 testing and the inclusion of the exacerbation of past medical conditions as part of the LC definition1. Moreover, treatment for LC is currently symptom-based as there are currently no specific pharmacological treatments for LC or other post-COVID-19 conditions.

[0011] There is therefore a need for reliable diagnostic biomarkers of LC and effective treatments thereof.

[0012] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0013] SUMMARY OF THE DISCLOSURE

[0014] The present invention demonstrates that non-hospitalized individuals who developed LC, especially severe LC, have intestinal barrier dysfunction associated with increased levels of B-cell activating factor (BAFF), dysregulation of the B-cell compartment and autoimmunity which persist for at least 24 months post-COVID-19 infection. The present invention demonstrates that transferring fecal microbiota alone from individuals with severe LC into wild-type germ-free mice is sufficient to produce the LC phenotype observed in patients with LC, including intestinal barrier dysfunction, immune dysregulation associated with systemic autoimmunity, and neurobehavioral changes associated with neuroinflammation. The present invention further demonstrates that this phenotype significantly improves with treatment using an anti-BAFF monoclonal antibody. Together, these data demonstrate that intestinal barrier dysfunction may be a key factor driving autoimmunity and end-organ complications in LC, and BAFF may represent a new treatment target for LC.

[0015] More specifically, in accordance with the present disclosure, there are provided the following items:

[0016] Item 1. Method for treating long COVID (LC) or at least one symptom thereof in a subject displaying a blood (i) level of secreted B-cell activating factor (BAFF) higher than a corresponding reference level; and (ii) level of membrane BAFF in white blood cells higher than a corresponding reference level; and at least one of blood (iii) level of precursorlike marginal zone B-cell (MZp) populations higher than higher than a corresponding reference level; (iv) level of zonulin higher than a corresponding reference level; (v) detectable level of anti-Mi-2 nuclear antigen autoantibodies; (vi) detectable level of anti-SmD autoantibodies; (vii) detectable level of anti-U1-snRNP-A autoantibodies; (viii) detectable level of anti-RCN2 autoantibodies; (ix) level of lipopolysaccharide-binding protein (LBP) higher than a corresponding reference level; (x) level of [3-D-glucan higher than a corresponding reference level; and (xi) level of A Proliferation Inducing Factor (APRIL) lower than a corresponding reference level, comprising administering a therapeutically effective amount of a BAFF inhibitor to the subject.

[0017] Item 2. The method of item 1, wherein the BAFF inhibitor is an anti-BAFF monoclonal antibody.

[0018] Item 3. The method of item 1 or 2, wherein the at least one symptom thereof includes at least one of a neurological dysfunction, improvement of intestinal barrier dysfunction, and improvement of immune dysregulation associated with systemic autoimmunity.

[0019] Item 4. A method of diagnosing a long COVID (LC) in a subject comprising detecting in a blood sample from the subject: (i) a level of secreted B-cell activating factor (BAFF) higher than a corresponding reference level; and (ii) alevel of membrane BAFF on white blood cells higher than a corresponding reference level; and at least one of (iii) a level of precursor-like marginal zone B-cell (MZp) populations higher than higher than a corresponding reference level; (iv) a level of zonulin higher than a corresponding reference level; (v) a detectable level of anti-Mi-2 nuclear antigen autoantibodies; (vi) a detectable level of anti-SmD autoantibodies; (vii) a detectable level of anti-U1-snRNP-A autoantibodies; (viii) a detectable level of anti-RCN2 autoantibodies; (ix) a level of lipopolysaccharide-binding protein (LBP) higher than a corresponding reference level; (x) a level of [3-D-glucan higher than a corresponding reference level; and (xi) a level of A Proliferation Inducing Factor (APRIL) lower than a corresponding reference level, wherein detecting a level in (i) and (ii) higher than their corresponding reference level, and detecting at least one of (iii) to (x) higher than their corresponding reference level, and (xi) lower than its corresponding reference level is an indication that the subject suffers from a LC.

[0020] Item 5. The method of any one of items 1 to 4, wherein:

[0021] (I) the LC is severe LC; and / or

[0022] (II) the subject displays:

[0023] (a) at least two of (iii) to (vii);

[0024] (b) at least three of (iii) to (vii);

[0025] (c) at least four of (iii) to (vii); or

[0026] (d) all five of (iii) to (vii).

[0027] Item 6. The method of any one of items 1 to 5, wherein the subject is a human.

[0028] Item 7. A diagnostic kit comprising (a) (i) a B-cell activating factor (BAFF) ligand; (ii) a ligand for each of CD3, CD19, CD1c, CD27, IgM, and CD10; (iii) a zonulin ligand; and at least one of (iv) an anti-Mi-2 nuclear antigen autoantibody ligand; (v) an anti-SmD autoantibody ligand; (vi) an anti-U1-snRNP-A autoantibody ligand; and (vii) an anti-RCN2 autoantibody ligand; and (b) optionally instructions to use the kit for diagnosing long COVID (LC).

[0029] Item 8. The kit of the item 7, wherein one or more of the ligands of (i) to (vii) is an antibody.

[0030] Item 9. The kit of the item 7, wherein LC is severe LC.

[0031] Item 10. The kit of any one of items 7 to 9, wherein the kit comprises:

[0032] (a) at least two of (iv) to (vii);

[0033] (b) at least three of (iv) to (vii);

[0034] (c) at least four of (iv) to (vii); or

[0035] (d) all five of (iv) to (vii).

[0036] Item 11. The kit of any one of items 7 to 10, wherein the kit further comprises one or more of (viii) a lipopolysaccharide-binding protein (LBP) ligand; (ix) a [3-D-glucan ligand; (x) an A Proliferation Inducing Factor (APRIL) ligand; (xi) one or more ligands for total memory B-cells populations; (xii) one or more ligands for resting switched memory B-cells; (xiii) one or more ligands for naive B-cells; and (xiv) one or more ligands for transitional immature B-cells.

[0037] Item 12. The kit of item 11 , wherein one or more of the ligands of (vii) to (xiv) is an antibody.Item 13. Method of generating a mouse model of long COVID comprising administering fecal microbiota from a human subject diagnosed with long COVID into a germ-free mouse by oral gavage and allowing microbiota engraftment in the mouse for a least three weeks.

[0038] Item 14. Mouse generated by the method as defined in item 13.

[0039] Other objects, advantages and features of the present disclosure will become more apparent upon reading the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0040] BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In the appended drawings:

[0042] FIG. 1A: Levels of serum zonulin in Long COVID (LC) participants. Serum zonulin concentrations were measured in pandemic controls, and LC participants at 3-6, 12- and 24- months post-COVID-19 infection (FIG. 1A). * p < 0.05. FIG. 1 B: Sustained levels of serum zonulin levels in LC participants. Serum zonulin concentrations were measured in pandemic controls, recovered individuals, and participants with LC at 3-6 months, 12 months, and 24 months post-COVID-19 infection. Individuals with LC exhibited increased serum zonulin levels compared to pandemic controls and recovered participants at all examined timepoints, with elevations persisting up to 24 months post-infection.* p < 0.05; **p < 0.01.

[0043] FIGs. 1C-H: Correlations between levels of two markers of microbial translocation, lipopolysaccharide (LPS) Binding Protein (LBP) (FIGs. 1C-E) and [3-D-glucan (FIGs. 1F-H), and zonulin levels at 3-6, 12- and 24- months post-COVID-19 infection. * p < 0.05; ** p < 0.01.

[0044] FIGs. 2A-F: Relative percentage (%) of membrane B-cell activating factor (mBAFF) expression (left) and geometric mean fluorescence intensity (GeoMFI) (right) measured by multicolor flow-cytometry in total peripheral blood mononuclear cells (PBMCs) (FIG. 2A), T-cells (FIG. 2B), B-cells (FIG. 2C), and classic (FIG. 2D), intermediate (FIG.

[0045] 2E) and non-classic (FIG. 2F) monocytes at 3-6, 12 and 24 months post-COVID-19 infection in patients with LC and pandemic controls. * p < 0.05; ** p < 0.01 ; *** p < 0.001.

[0046] FIGs. 3A-I: Correlations between blood levels of soluble B-cell Activating Factor (BAFF) and zonulin (FIGs. 3A, 3D, 3G) or A Proliferation Inducing Factor (APRIL) (FIGs. 3B, 3E, 3H) in patients with LC at 3-6, 12- and 24- months post-COVID-19 infection. Correlations between B-cell Maturation Antigen (BCMA) and (3-D-glucan are shown at 3-6, 12-and 24-months post-COVID-19 infection (FIGs. 3C, 3F, 3I).

[0047] FIGs. 4A-J: Comparison of circulating frequencies of total (CD3 CD19+) (FIG. 4A), total memory (CD3 CD19+CD1c CD27+CD20+) (FIG. 4B), resting switched memory (CD3CD19+CD1cCD27+CD20+CD21+lgM ) (FIG. 4C), naive (CD3-CD19+CD20+CD27-CD1c-CD10-CD21hi9h) (FIG. 4D), transitional immature (CD3CD19+CD20+CD10+CD1cCD27-CD21high) (FIG. 4E), Marginal Zone (MZ) (CD3-CD19+CD1c+CD27+lgMbri9htCD10-) (FIG. 4F), and Precursor-like MZ (MZp) (CD3’CD19+CD1c+CD27+lgMbri9htCD10+) (FIG. 4G) B-cells, lgA+blasts cells (CD3-CD19+CD20-CD38+lgA+) (FIG.

[0048] 4H), lgM+blasts cells (CD3 CD19+CD20 CD38+lgM+) (FIG. 4I) and lgG+blasts cells (CD3 CD19+CD20 CD38+lgG+)(FIG. 4J) in patients with LC at 3-6, 12 and 24 months post-COVID-19 infection * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

[0049] FIG. 4K: Increased frequencies of precursor marginal zone B cells in Long COVID. The frequency of circulating precursor marginal zone B cells (MZp) was assessed in pandemic controls and participants with LC at 12 months, and 24 months post-COVID-19. Participants with LC demonstrated increased frequencies of MZp cells compared to pandemic controls, with sustained elevation observed through 24 months post-infection.

[0050] FIGs. 5A-C: Correlations between circulating precursor-like Marginal Zone (MZp B-cell) frequencies and soluble B-Cell Activating Factor (BAFF) levels at 3-6, 12- and 24-months post-COVID-19 infection. * p < 0.05.

[0051] FIGs. 6A-G: Tbet expression in circulating precursor-like Marginal Zone (MZp) B-cells in blood from patients with LC at 3-6, 12- and 24-months post COVID-19 infection (FIG. 6A) according to levels of soluble B-Cell Activating Factor (BAFF) (FIGs. 6B-D) or serum zonulin (FIGs. 6E-G). * p < 0.05; ** p < 0.01; *** p < 0.001.

[0052] FIGs. 7A-G: NR4A3 expression in circulating precursor-like Marginal Zone (MZp) B-cells in blood from patients with LC at 3-6, 12- and 24-months post COVID-19 infection (FIG. 7 A) according to levels of soluble B-Cell Activating Factor (BAFF) (FIGs. 7B-D) or serum zonulin (FIGs. 7E-G). * p < 0.05; ** p < 0.01; *** p < 0.001.

[0053] FIGs. 8A-F: Total immunoglobulin (Ig) levels in sera from patients with LC compared to pandemic controls at 3-6 and 12-months post-COVID-19 infection (FIGs. 8A-B). Serum IgM measurements according to low, intermediate or high levels of soluble B-Cell Activating Factor (BAFF) (FIGs. 8C) or zonulin (FIGs. 8D) and corresponding correlation analyses at 12 months post-COVID-19 infection (FIGs. 8E, 8F). * p < 0.05; *** p < 0.001.

[0054] FIGs. 9A-B: Correlations between precursor-like marginal zone (pMZ) B-cells and serum IgA (FIG. 9A) or IgM (FIG.

[0055] 9B) levels at 12 months post-COVID-19 infection.

[0056] FIGs. 10A-C: Number of participants with positive vs. negative autoantibodies to DNA Topoisomerase (FIG. 10A), Mi-2 (FIG. 10B) and SmD (FIG. 10C) based on timepoint (3-6, 12- and 24- months post-COVID-19 infection), compared to pandemic controls.

[0057] FIGs. 11A-J: Number of participants with positive vs. negative autoantibodies to dsDNA (FIG. 11 A), DNA Topoisomerase (FIG. 11 B), Histidyl-tRNA synthase (FIG. 11C), Mi-2 (FIG. 11D), SmD (FIG. 11E), LHsnRNP (FIG.

[0058] 11F), LHsnRNP A (FIG. 11G), Ro / SS-A (FIG. 11 H), LA / SS-B (FIG. 111) and Ro / SS-AA (FIG. 11 J) based on serum zonulin levels (low, intermediate, high) compared to pandemic controls.

[0059] FIG. 12. Elevated serum anti-RCN2 autoantibody levels in Long COVID. Serum levels of autoantibodies targeting reticulocalbin-2 (RCN2) were measured in pandemic controls and participants with LC at 12 months, and 24 months post-COVID-19. Individuals with LC exhibited increased anti-RCN2 autoantibody levels relative to pandemic controls across all examined timepoints.

[0060] FIGs. 13A-C. Correlation of anti-RCN2 autoantibodies with markers of intestinal permeability and immune activation. Serum anti-RCN2 autoantibody levels were correlated with serum zonulin concentrations (FIG. 13A), frequencies of intermediate monocytes expressing membrane-bound BAFF (mBAFF) (FIG. 13B), and frequencies of precursormarginal zone B cells (MZp) (FIG. 13C). Positive correlations were observed, supporting an association between intestinal barrier dysfunction, BAFF-related immune activation, and autoantibody production in Long COVID.

[0061] FIG. 14: Schematic representation of approach used for the establishment of the gnotobiotic long COVID (LC) mouse model.

[0062] FIG. 15: The microbiota from patients with severe LC transfers increased intestinal barrier permeability. Serum lipopolysaccharide binding protein (LBP) in gnotobiotic mice colonized with the fecal microbiota from severe compared to mild LC. **p < 0.01.

[0063] FIGs. 16A-F: The microbiota from patients with severe LC transfers immune dysregulation. Serum B-cell activating factor (BAFF) (FIG. 16A) and relative percentage (%) of membrane BAFF (mBAFF) expression in lamina propria (FIG.

[0064] 16B) or splenic (FIG. 16C) cell suspensions from gnotobiotic mice colonized with fecal microbiota from patients with mild or severe LC. Relative frequencies (%) of CD 19+CD1 d+lgMbri9htB-cells (FIG. 16D), IgA blasts (FIG. 16E) and IgM blasts (FIG. 16F) in the spleen of gnotobiotic mice colonized with fecal microbiota from patients with mild or severe were calculated relative to total CD19+B-cells, and total CD19+CD138+blasts respectively. Data are representative of 3 independent experiments each evaluating the impact of the fecal microbiota from distinct patients with LC.

[0065] FIG. 17: Schematic representation of approach used for the establishment of the gnotobiotic long COVID (LC) mouse model and treatment with a B-cell activating factor (BAFF) antagonist compared to an isotype control.

[0066] FIGs. 18A-C: Treatment with a B-cell activating factor (BAFF) antagonist significantly decreases blood levels of soluble BAFF (FIG. 18A) and relative percentage (%) of membrane BAFF (mBAFF) expression (FIG. 18B) and geometric mean fluorescence intensity (GeoMFI) (FIG. 18C) measured by multicolor flow-cytometry in spleen cell suspensions, * p < 0.05; *** p < 0.001; **** P < 0.0001.

[0067] FIGs. 19A-E: Evaluation of B-cell compartment in splenic cell suspensions in gnotobiotic mice with LC phenotype treated with BAFF antagonist. Relative frequencies (%) of total CD19+B-cells were calculated relatively to total CD45+cells (FIG. 19A), GeoMFI of CD73 expression by CD19+CD1d+lgMbri9htB-cells (FIG. 19B), total CD19+CD138+blasts calculated relative to total CD45+cells (FIG. 19C), IgA blasts (FIG. 19D) and IgM blasts (FIG. 19E) were calculated relative to total blasts in spleen.

[0068] FIG. 20: Treatment with B-cell activating factor (BAFF) antagonist decreases autoimmunity in gnotobiotic mice with severe LC. Serum anti-nuclear antibodies (ANA; U / ml) measurements in gnotobiotic mice colonized with the fecal microbiota from patients with mild or severe LC compared to mice with severe LC phenotype treated with anti-BAFF monoclonal or isotype control antibody.

[0069] FIGs. 21A-B: Treatment with B-cell activating factor (BAFF) antagonist decreases neuroinflammation in gnotobiotic mice with severe LC phenotype. Quantification of fluorescence intensity of scanned brain sections from gnotobiotic mice colonized with the fecal microbiota from a patient with severe LC either untreated or treated with an anti-BAFF monoclonal antibody compared to mice colonized with the fecal microbiota from a patient with mild LC. Fluorescence was quantified in 5 selected regions of the hippocampus for glial fibrillary acidic protein (GFAP) (n=2 mice) (FIG. 21 A),and the hind brain for the microglia marker ionized calcium-binding adaptor molecule 1 (IBA1) (n=2 mice) (FIG. 21B). Data are shown as integrated density values (i.e., integration of the area x mean of fluorescence).

[0070] FIGs. 22A-D: Nucleotide (cDNA) (FIGs. 22A and 220) and amino acid (FIGs. 22B and 22D) sequences of human BAFF isoforms 1 (FIGs. 22A-B) (SEQ ID NOs: 1-2) and 2 (FIGs. 22C-D) (SEQ ID NOs: 3-4). Without being bound by this predicted structure16, BAFF’s cytoplasmic domain is italicized, its transmembrane domain is underlined, and its soluble form is bolded in FIGs. 22B and 22D.

[0071] DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS DEFINITIONS

[0072] For clarity, definitions of the following terms in the context of the present invention are provided.

[0073] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0074] The terms "comprising", "having", "including", and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0075] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0076] The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0077] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0078] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

[0079] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0080] Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in stem cell biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0081] Unless otherwise indicated, the techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al.

[0082] In the studies described herein, the present inventors have shown that subjects displaying severe LC symptoms (symptoms including fatigue and at least one of the following symptoms: brain fog, trouble with memory and / or concentration and / or conditions highly associated with LC (i.e., post-exertional malaise [PEM] / post-exertional symptom exacerbation [PESE] and / or cardiac dysautonomia)), with the symptoms lasting for at least 2 months, and being present 12 weeks post-acute COVID-19 infection (i.e., firstday of symptoms or date of COVID-19 diagnosis) and not explained by an alternate diagnosis) present a specific combination of biomarkers. This characterization of a subgroup of subjects confers the advantages associated with personalized medicine, by e.g., ensuring that available treatments can be more precisely directed to patient subgroups that are more likely to respond thereto, minimizing the risk of adverse effects, and ultimately reducing healthcare costs by improving outcomes. The present inventors have also created an LC mouse model. The present inventors have finally also shown that the inhibition of BAFF in these murine subjects reduces LC symptoms. By transferring the microbiome of subjects displaying severe LC, to germ-free (GF) wild-type (WT) mice, the inventors observed that the mice displayed a severe LC phenotype including intestinal barrier compromise, immune dysregulation, autoimmunity and neuroinflammation. The inventors have finally demonstrated that administering a BAFF inhibitor to these severe LC mice resulted in a marked improvement in the severe LC phenotype including reduced serum BAFF levels and mBAFF expression, increased intestinal barrier integrity and decreased neuroinflammation. Overall, the results presented herein provide compelling evidence that inhibition of BAFF is a suitable approach for improving severe LC symptoms in a severe LC subjects subgroup.

[0083] In one aspect the present disclosure provides a method of diagnosing post-COVID-19 condition (e.g., severe LC) comprising detecting in a blood sample from the subject a: (i) level of secreted B-cell activating factor (BAFF) higher than a corresponding reference level; and (ii) level of membrane BAFF in white blood cells (e.g., myeloid cells) higher than a corresponding reference level; and at least one of (iii) level of precursor-like marginal zone B-cell (MZp) populations higher than a corresponding reference level; (iv) level of zonulin higher than a corresponding reference level; (v) presence of anti-Mi-2 nuclear antigen autoantibodies; (vi) presence of anti-SmD autoantibodies; (vii) presence of anti-U1-snRNP-A autoantibodies; (viii) presence of anti-RCN2 autoantibodies; (ix) level of lipopolysaccharide-binding protein (LBP) higher than a corresponding reference level; (x) level of [3-D-glucan higher than a corresponding reference level; and (xi) level of A Proliferation Inducing Factor (APRIL) lower than a corresponding reference level. In another aspect, the present disclosure provides a method for treating a post-COVID condition (e.g., severe LC) or a symptom thereof in a subject displaying the following biomarkers (hereinafter referred to as “LC biomarkers”): (i) level of secreted B-cell activating factor (BAFF) in plasma or serum higher than a corresponding reference level; and (ii) level of membrane BAFF in white blood cells (e.g., myeloid cells) higher than a corresponding reference level; and at least one of (iii) blood level of precursor-like marginal zone B-cell (MZp) populations higher than a corresponding reference level; (iv) level of plasma or serum zonulin higher than a corresponding reference level; (v) presence of anti-Mi-2 nuclear antigen autoantibodies; (vi) presence of anti-SmD autoantibodies; (vii) presence of anti-U1-snRNP-A autoantibodies; (viii) presence of anti-RCN2 autoantibodies; (ix) level of lipopolysaccharide-binding protein (LBP) higher than a corresponding reference level; (x) level of [3-D-glucan higher than a corresponding reference level; and (xi) level of A Proliferation Inducing Factor (APRIL) lower than a corresponding reference level, comprising administering a therapeutically effective amount of a BAFF inhibitor to the subject. In specific embodiments, the method comprises a measure of the LC biomarkers, prior to administering the therapeutically effective amount of a BAFF inhibitor to the subject.

[0084] In specific embodiments, wherein e.g., the subject is suspected of having been infected by COVID-19, 24 months prior (i.e. prior to applying a diagnosis or treatment method of the present disclosure), one or more of the following biomarkers can further be detected in a blood sample from the subject: (xi) level of total memory B-cells populations higher than a corresponding reference level; (xii) level of resting switched memory B-cells higher than a corresponding reference level. In other specific embodiments, wherein e.g., the subject is suspected of having been infected by COVID-19, 3 to 12 months prior i.e. prior to applying a diagnosis or treatment method of the present disclosure), one or more of the following biomarkers can further be detected in a blood sample from the subject: (xiii) level of naive B-cells higher than a corresponding reference level; and (xiv) level of transitional immature B-cells higher than a corresponding reference level.

[0085] In another aspect, the present disclosure provides a diagnostic panel comprising (i) at least one reagent (e.g., ligand such as an anti-BAFF antibody) to measure secreted B-cell activating factor (BAFF) in blood; (ii) at least one reagent (e.g., at least one ligand such as an anti-BAFF antibody) to measure membrane BAFF expression by e.g., flow cytometry (e.g., anti-BAFF antibody); and at least one of (iii) at least one reagent to measure precursor marginal zone B cell (MZp) cell frequencies by e.g., flow cytometry (e.g., at least one ligand such as anti-MZp antibodies) (e.g., anti-CD3, anti-CD19, anti-CD1c, anti-CD27, anti-IgM, and anti-CD10 antibodies); (iv) at least one reagent (e.g., at least one ligand such as an anti-zonulin antibody to measure serum or plasma zonulin levels; (v) an antibody to detect anti-Mi-2 nuclear antigen autoantibodies; (vi) an antibody to detect anti-SmD autoantibodies; (vii) an antibody to detect anti-U1-snRNP-A autoantibodies; and (viii) an antibody to detect anti-RCN2 autoantibodies. In specific embodiments, the kit may further comprise one of more of the following biomarkers: (ix) at least one reagent (e.g., at least one ligand such as an antibody) to measure LBP; (x) at least one reagent (e.g., at least one ligand such as an antibody) to measure P-D-glucan; (xi) at least one reagent (e.g., at least one ligand such as an antibody) to measure APRIL; (xii) at leastone reagent to measure total memory B-cells populations (e.g., at least one ligand such as antibodies) (e.g., anti-CD3, anti-CD19, anti-CD1c, anti-CD27, anti-CD20); (xiii) at least one reagent to measure resting switched memory B-cells e.g., at least one ligand such as antibodies) (e.g., anti-CD3-CD19, anti-CD1c, anti-CD27, anti-CD20, anti-CD21, and anti-l gM); (xiv) at least one reagent to measure naive B-cells (e.g., at least one ligand such as antibodies) (e.g., anti-CD3, anti-CD19, anti-CD20, anti-CD27, anti-CD1c, anti-CDW, anti-CD21); and (xv) at least one reagent to measure transitional immature B-cells (e.g., at least one ligand such as antibodies) (e.g., anti-CD3, anti-CD19, anti-CD20, anti-CD27, anti-CD1c, anti-CDW, anti-CD21).

[0086] In specific embodiments of the methods (diagnosis and / or method of treatment) of the disclosure, the levels of the biomarkers can be determined using antibodies specific to the biomarkers. Hence (i) the level of secreted BAFF can be measured with an anti-BAFF antibody; (ii) the level of membrane BAFF can be measured with an anti-BAFF antibody; (iii) the level of MZp B-cell populations can be measured with anti-CD3, anti-CDW, anti-CD1c, anti-CD27, anti-l gM, and anti-CD 10 antibodies; (iv) level of zonulin can be measured with an anti-zonulin antibody; (v) the presence of anti-Mi-2 nuclear antigen autoantibodies can be measured with an antibody specific to anti-Mi-2 nuclear antigen autoantibodies; (vi) the presence of anti-SmD autoantibodies can be measured with an antibody specific to anti-SmD autoantibodies; (vii) the presence of anti-U1-snRNP-A autoantibodies can be measured with an antibody specific to anti-U1-snRNP-A autoantibodies; (viii) the presence of anti-RCN2 autoantibodies can be measured with an antibody specific to anti-RCN2 autoantibodies; (ix) the level of LBP can be measured with an anti-LBP antibody; (x) the level of P-D-glucan can be measured with an anti-|3-D-glucan; (xi) the level of A Proliferation Inducing Factor (APRIL) can be measured with an anti- APRIL antibody; (xii) the level of total memory B-cells can be measured with anti-CD3, anti-CDW, anti-CD1c, anti-CD27, and anti-CD20 antibodies; (xiii) the level of resting switched memory B-cells can be measured with anti-CD3-CDW, anti-CD1c, anti-CD27, anti-CD20, anti-CD21, and anti-IgM antibodies; (xiv) the level of naive B-cells can be measured with anti-CD3, anti-CDW, anti-CD20, anti-CD27, anti-CD1c, anti-CDW, anti-CD21 antibodies; and (xv) the level of transitional immature B-cells can be measured with anti-CD3, anti-CDW, anti-CD20, anti-CD27, anti-CD1c, anti-CDW, and anti-CD21 antibodies.

[0087] In another aspect, the inventors provide an LC mouse model and a method of generating it.

[0088] As used herein, the term “ long COVID “ or “LC” is used herein to refer to a continuation or development, in subjects diagnosed with a SARS-CoV-2 infection, of new or continued symptoms 3 months after the initial SARS-CoV-2 infection, with symptoms lasting for at least 2 months, without other explanation4. An “LC symptom “refers to any of the foregoing common symptoms, but not limited to : fatigue, brain fog, trouble with memory, trouble with concentration, shortness of breath or breathlessness, palpitations, trouble with sleep, and muscle aches. Conditions highly associated with LC include post-exertional malaise (PEM / post-exertional symptom exacerbation (PESE), and / or dysautonomia. As used herein the term “severe LC” refers to an LC wherein the subject has at least the following symptoms 12 weeks post-acute COVID-19 infection, the symptoms lasting at least two months: (i) fatigue; and (ii) at least one of the following neurological dysfunctions: (i) brain fog, (ii) trouble with memory, and (iii) trouble with concentration. The subject with severe LC may further present post-exertional malaise [PEM] / post-exertional symptom exacerbation [PESE] and / orcardiac dysautonomia. Without being so limited, neurological dysfunction can be associated with (result from) neuroinflammation (i.e., neuroinflammation can lead to neurological dysfunction).

[0089] As used herein the term “treating” or “treatment” in reference to LC or a symptom thereof (e.g., severe LC) in a symptomatic subject is meant to refer to a reduction or improvement (e.g., reduction of intensity, duration and / or frequency) of one or more symptoms as defined above in a subject as compared to the one or more symptoms before the start of the treatment, namely e.g., fatigue; neurological dysfunction such as neuroinflammation, brain fog, trouble with memory, and / or trouble with concentration; shortness of breath or breathlessness; palpitations; trouble with sleep; muscle aches;, post-exertional malaise; and / or dysautonomia as compared to the intensity, duration and / or frequency of that or these symptoms prior to the start of the treatment. In specific embodiments, the comparison can be made with corresponding symptoms in untreated subjects. It may also refer to any one of a reduction in blood or cell membrane levels of BAFF, a reduction of blood levels of MZp B-cells, a reduction of blood levels of zonulin, a reduction in blood levels of anti-Mi-2 nuclear antigen autoantibodies, anti-SmD autoantibodies, anti-U1-snRNP-A autoantibodies and / or anti-RCN2 autoantibodies as compared to the corresponding levels prior to the start of the treatment of the subject. In specific embodiments, the comparison can be made with corresponding reference levels. In specific embodiments, treatment refers to at least one of a reduced neurological dysfunction (e.g., reduced neuroinflammation), improvement of intestinal barrier dysfunction (e.g., detectable by e.g., reduced or normalized zonulin levels), improvement of immune dysregulation associated with systemic autoimmunity (e.g., detectable by at least one of a reduced blood BAFF level, reduced white blood cell BAFF membrane level, reduced B-cell frequencies, increased CD73 levels, reduced lgA+ blast frequency, reduced anti-nuclear antibodies levels as compared to the corresponding levels of the subject prior to the treatment).

[0090] As used herein the term "therapeutically effective amount" or “effective amount” or "therapeutically effective dosage" of a BAFF inhibitor of the present disclosure provided herein results in a treatment of Long COVID or of at least one symptom thereof in a subject (e.g., a subject in need thereof).

[0091] As used herein the term “corresponding reference level” e.g., in the context of the LC biomarkers refers, unless otherwise specified, to a level range or a level considered normal in the art or the level in one or more corresponding control subjects i.e. a control subject or a control subject population.

[0092] As used herein the term “corresponding control subject” refers to a corresponding healthy subject or healthy subject population of the same species as the subject assessed or treated by a method of the present disclosure, i.e. a same species subject or subject population that does not have LC or another post-COVID-19 condition. In specific embodiments, the corresponding control subject is age-matched and / or gender-matched and / or ethnicity-matched with the subject assessed or treated by a method of the present disclosure.

[0093] In specific embodiments, the subject to which the diagnostic method and / or method of treatment of the present disclosure will be applied is a subject suspected of having a LC based on a prior diagnostic or the presence of one or more symptoms characterizing LC. In other specific embodiments, prior to applying the diagnostic method and / or method of treatment of the present disclosure to a subject, the subject is tested to determine whether he has or hashad in the past a SARS-CoV2 infection that may cause LC (e.g., severe LC), by methods known in the art e.g., tests to determine presence of viral antigen or viral load in blood or tissue, preferably blood, with methods known in the art (e.g., anti-viral antigen antibody to detect antigen, PCR etc. to detect nucleic acid).

[0094] As used herein the term “higher” in reference to a level of a biomarker of LC (e.g., secreted B-cell activating factor (BAFF), membrane BAFF, precursor-like marginal zone B-cell (MZp) population, plasma or serum zonulin) in a subject refers to level that is higher as compared to a corresponding reference level of the biomarker. In specific embodiments it refers to level at least 10% higher than the corresponding reference level; at least 15% higher; at least 20% higher; at least 25% higher; at least 30% higher; at least 35% higher; at least 40% higher; at least 45% higher; at least 50% higher; at least 55% higher; at least 65% higher; at least 70% higher; at least 75% higher; at least 80% higher; at least 85% higher; at least 90% higher; at least 95% higher; at least 100% higher; at least 110% higher; at least 120% higher; at least 130% higher; at least 140% higher; at least 150% higher; at least 160% higher; at least 170% higher; at least 180% higher; at least 190% higher; at least 200% higher; at least 210% higher; at least 220% higher; at least 230% higher or more than the reference level. In other specific embodiments, the term “higher” it refers to a as least 1 standard deviation higher than the mean of the corresponding control population.

[0095] As used herein the term “lower” or “reduced” in reference to a level of a biomarker of LC (e.g., in a diagnosed subject blood level, prior to treatment: APRIL; or in a subject treated with a BAFF inhibitor: BAFF blood and membrane level, zonulin blood level, MZp B-cells blood levels, etc.) in a subject refers to level that is lower as compared to a corresponding reference level of the biomarker. In specific embodiments it refers to level at least 10% lower than the corresponding reference level; at least 15% lower; at least 20% lower; at least 25% lower; at least 30% lower; at least 35% lower; at least 40% lower; at least 45% lower; at least 50% lower; at least 55% lower; at least 65% lower; at least 70% lower; at least 75% lower; at least 80% lower; at least 85% lower; at least 90% lower; at least 95% lower; at least 100% lower; at least 110% lower; at least 120% lower; at least 130% lower; at least 140% lower; at least 150% lower; at least 160% lower; at least 170% lower; at least 180% lower; at least 190% lower; at least 200% lower; at least 210% lower; at least 220% lower; at least 230% lower or more than the reference level. In other specific embodiments, the term “lower'1it refers to a as least 1 standard deviation lower than the mean of the corresponding control population. The term “sample” as used herein is any type of biological sample (from a tested subject or from (a) control subject(s) or subject population (e.g., used to assess a reference level)) which may be used in the methods of the present disclosure to measure the level of one or more of the biomarkers of the present disclosure in a subject. As used herein, the term sample encompasses “cell sample” referring to a sample which originally comprised cells (e.g., white blood cells such as myeloid cells such as monocytes, lymphoid cells such as T-cells and B-cells) from the subject. The term “blood sample” refers to whole blood sample, or to plasma or serum. The sample as used herein may be a crude sample or a purified sample, it may be processed to a nucleic acid sample.

[0096] Without being so limited, methods of determining whether a subject has had COVID-19 include: subject providing molecular test results (i.e., PCR, SARS-CoV2 serologies, rapid COVID-19 test results) performed during the acute COVID-19 infection, the detection of antibodies specific to SARS-CoV-2, such as through detection of SARS-CoV-2 Spike (S1) Protein (in COVID-19 unvaccinated subjects) or Nucleocapsid (can distinguish COVID-19 infection fromvaccination). Antibody detection assays can be of low sensitivity and antibodies are only detected for a limited time. As such, negative COVID-19 antibody detection results do not exclude the possibility that a subject had COVID-19. B-cell activating factor (BAFF), also referred to as BLyS, TALL-1, THANK, and TNFSF13B, is a ligand that is part of the TNF superfamily and is crucial for the development and survival of B lineage cells. It can exist as a homotrimer or as a heteromer when associated with the related protein APRIL. It is produced by various hematopoietic cell types, and activated neutrophils can release a soluble form of it. Both BAFF and APRIL transmit signals through the receptors BCMA and TACI, with BAFF also signaling through BAFF R. There are secreted and membrane forms of BAFF. Membrane forms of BAFF can be found on myeloid cells.

[0097] As used herein plasma or serum levels of secreted BAFF can be measured by anti-BAFF antibodies. As used herein the term “ligand for plasma or serum BAFF” refers to anti-BAFF antibodies such as those from R&D systems (AF124; MAB1357; AF2106; MAB124; MAB1241; BAF2106, etc.). In a specific embodiment, it is measured by BAFF / BLyS / TNFSF13B Quantikine™ ELISA Kit (R&D systems, Minneapolis, MN, USA).

[0098] As used herein, membrane BAFF in white blood (e.g., myeloid cells) cells can be measured by anti-BAFF antibodies on cells analyzed using e.g., flow cytometry. As used herein the term “ligand for membrane BAFF” refers to anti-BAFF antibodies such as those from R&D systems (e.g., MAB1241).

[0099] As used herein the term precursor-like Marginal Zone B-cells (MZp) refers to a population sharing features of both transitional immature (Tl) and marginal zone (MZ) B-cells, expressing CD3'CD19+CD1c+CD27+lgMbr'9htCD10+at their surfaces and termed herein Marginal Zone precursor-like (MZp) cells.

[0100] As used herein, levels of precursor-like Marginal Zone B-cells (MZp) cell populations can be measured by antibodies directed to their signature cluster of differentiation proteins (CD) cell surface markers and level of expression of IgM (anti-CD19, anti-CD1c, anti-CD27, anti-CD10, and anti-IgM on cells analyzed using e.g., flow cytometry. As used herein the term “ligand for precursor marginal zone B cell (MZp) cell” refers inter alia to these antibodies. In a specific embodiment, levels of MZp populations can be measured with BV650-anti-CD19, PerCP-eFluor710-anti-CD1c, AlexaFluor700-anti-CD27, BB515-anti-lgM, BV421-anti-CD3 and BV421 -anti-CD10.

[0101] Increased intestinal permeability (i.e., leaky gut) refers to breached epithelial barrier and microbial translocation, as has been demonstrated in chronic inflammatory conditions such as Human Immunodeficiency Virus (HI V)-1 infection. One marker for this condition is plasma or serum levels of the protein zonulin. Zonulin (haptoglobin 2 precursor) is a 406-amino acid protein member of the Peptidase S1 family protein that increases the permeability of tight junctions between cells of the wall of the digestive tract.

[0102] As used herein plasma or serum levels of zonulin can be detected by anti-zonulin antibodies. As used herein the term “ligand for zonulin” refers to, without being so limited, anti-zonulin antibodies such as MyBiosource, Inc., (MBS622563, MBS2003934, MBS9411531, MBS9410566, MBS2005921 etc.); Antibodies Online (ABIN165305, etc.); Immundiagnostik AG (A 1105.1, A 1105.2); Abbexa Ltd. (abx104827), etc. In a specific embodiment, Human Zonulin ELISA Kit (Elabscience) is used to detect plasma or serum levels of zonulin.Mi-2 nuclear antigen is a protein found in the nucleus that is a component of the nucleosome remodeling-deacetylase (NuRD) and is involved in transcription regulation. SmD is a small nuclear ribonucleoprotein that is a target of autoantibodies in systemic lupus erythematosus (SLE) and is the most. Specific Sm antigen for SLE. U1-snRNP-A is a small nuclear ribonucleoprotein particle that is involved in RNA processing. It is also a key regulator of mRNA length and is a target of autoreactive B and T cells in some rheumatic diseases. Reticulocalbin 2 (RCN2) is a 39 kDa calcium-binding protein located in the endoplasmic reticulum and implicated in endothelial cell function and cellular stress responses. Anti-Mi-2 nuclear antigen autoantibodies, Anti-SmD autoantibodies, Anti-U1-snRNP-A autoantibodies and Anti-RCN2 autoantibodies are antibodies used to detect antibodies against Mi-2 nuclear antigen, SmD, U1-snRNP-A, and RCN2 respectively.

[0103] As used herein the term “ligand for anti-Mi-2 nuclear antigen autoantibody” refers to an antibody that specifically binds to anti-Mi-2 nuclear antigen autoantibody. In a specific embodiment, it refers to Anti-Nuclear Antibodies (ANA) BioAssay ELISA Kit (US Biological Life Sciences).

[0104] As used herein the term “ligand for anti-SmD autoantibody” refers to an antibody that specifically binds to anti-SmD autoantibody. In a specific embodiment, it refers to Anti-Nuclear Antibodies (ANA) BioAssay ELISA Kit (US Biological Life Sciences).

[0105] As used herein the term “ligand for anti-U1-snRNP-A autoantibody” refers to an antibody that specifically binds to anti-U1-snRNP-A autoantibody. In a specific embodiment, it refers to Anti-Nuclear Antibodies (ANA) BioAssay ELISA Kit (US Biological Life Sciences).

[0106] As used herein the term “ligand for anti-RCN2 autoantibody” refers to an antibody that specifically binds to anti-RCN2 autoantibody. In a specific embodiment, it refers to Anti-RCN2 antibody Abeam™ ab104516, or home-made antibody. As used herein a “BAFF inhibitor” refers to an agent that decreases BAFF expression (e.g., protein levels) and / or activity, and includes, without being so limited, small molecule, a peptide, a polypeptide, a BAFF-binding molecule including but not limited to an antibody or an antigen-binding fragment thereof, or an RNA interference agent.

[0107] Without being so limited, BAFF small molecules antagonists include OSX-100 (Ossianix, Inc.), KR-32592 (Sejong University), and small molecules designed by Psivant Therapeutics.

[0108] Anti-BAFF peptides and polypeptides

[0109] Without being so limited anti-BAFF peptides and polypeptides include peptides and proteins including as fusion proteins such as BAFF receptor-IgG Fc fusion proteins, Blisibimod (Amgen, Inc.), Briobacept (Biogen (Denmark) A / S), Mitumprotimut T (MMRGIobal, Inc.). Examples of anti-BAFF peptides and polypeptides that reduce or inhibit the expression of BAFF are described, e.g., in publications Nos. WO / 2017 / 223534, W02001058949A2, W02004074511A1, US20050048626A1, W02004089982A3 or US7709220B2.

[0110] Antibodies or an antigen-binding fragments thereof

[0111] The term “antibodies or antigen-binding fragments thereof” encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies),humanized antibodies, CDR-grafted antibodies, chimeric antibodies, multispecific antibodies (e.g., bispecific antibodies), and fragments thereof so long as they exhibit the desired antigenic specificity / binding activity. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain FV, scFV), single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VH regions (VH, VH-VH), anticalins, antibody-T-cell epitope fusions (troybodies) or peptibodies.

[0112] The term "monoclonal antibody" as used herein refers to an antibody derived from a population of substantially homogeneous antibodies. This means that the individual antibodies within the population are substantially similar, and bind to the same epitope(s), with the exception of minor variants that may occur during the production of the monoclonal antibody. Typically, such a monoclonal antibody includes an antibody with a variable region that specifically binds to a target, and it is obtained through a selection process involving the identification of the antibody from a larger group of antibodies. For instance, this selection process may involve isolating a unique clone from a collection of clones, such as those from hybridoma, phage display, or recombinant DNA techniques. It is important to note that the selected antibody can undergo further modifications to enhance its affinity for the target, humanize the antibody, improve its production in cell culture, reduce its immunogenicity in vivo, or create a multispecific antibody. An antibody that possesses an altered variable region sequence also qualifies as a monoclonal antibody within the scope of this invention. In addition to their specificity, monoclonal antibody preparations are typically free from contamination by other immunoglobulins. The term "monoclonal" denotes that the antibody originates from a largely homogeneous population and should not be interpreted as necessitating a specific production method. Indeed, the monoclonal antibodies utilized in this invention may be generated through various techniques, including the hybridoma method and methods for producing human or human-like antibodies from animals possessing part or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences known in the field.

[0113] The monoclonal antibodies herein specifically include "chimeric" or “recombinant” antibodies in which a portion of the light and / or heavy chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. Chimeric antibodies of interest herein include "humanized" antibodies.

[0114] In some embodiments, the antibodies and antigen binding fragments thereof specifically recognize an antigen of a full-length antibody. In some embodiments, the heavy and light chains of an antibody can be full-length or can be an antigen-binding portion (a Fab, F(ab')2, Fv or a single chain Fv fragment (scFv)). In other embodiments, the antibody heavy chain constant region is chosen from, e.g., lgG1 , lgG2, lgG3, lgG4, IgM, lgA1, lgA2, IgD, and IgE, particularly chosen from, e.g., lgG1, lgG2, lgG3, and lgG4, more particularly, lgG1 (e.g., human lgG1). In another embodiment,the antibody light chain constant region is chosen from, e.g., kappa or lambda, particularly kappa.

[0115] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0116] Single-domain antibodies (sdAbs) are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, the singledomain antibody is a human single-domain antibody.

[0117] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that may not be produced by enzyme digestion of a naturally occurring intact antibody. In some embodiments, the antibody fragment is a scFv.

[0118] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization, typically to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0119] Without being so limited, antibodies and an antigen-binding fragments thereof encompassed by the term “BAFF inhibitor” include anti-BAFF antibodies and anti-BAFF antigen binding fragments. Examples of antibodies or antigenbinding fragments thereof that inhibit the activity of BAFF include Belimumab (GlaxoSmithKline PLC), UBP-1213 (Shanghai Junshi Biosciences Co., Ltd.), atacicept, Tabalumab (Eli Lilly & Co.), BAFF / IL-17 bispecific antibody, ardenermin. Sandy-2 is directed to mouse BAFF. Other examples of antibodies or antigen-binding fragments thereof that inhibit the activity of BAFF are described, e.g., in publications Nos. US20240254247A1, US7709220B2 WO2024182541 A1, US20190135923, WO2024182541A1, WO2022263551A1, W02020210670A1, WO2019136311 A2, WO2019210168A1 , WO2017015433, US20030095967A1, W02004011611A2.In addition to antibodies and antigen-binding fragments thereof, the term “BAFF-binding molecule” also encompasses adnectins; affibodies; affilins; affimers; affitins; alphabodies; antibody mimetics; anticalins; aptamers; armadillo repeat protein-based scaffolds; atrimers; avimers; DARPins; fynomers; knottins; Kunitz domain peptides; monobodies; and nanofitins.

[0120] RNA interference agent

[0121] Without being so limited, anti-BAFF RNA interference agents include an RNA interference (RNAi) agent targeting an mRNA encoding BAFF. In specific embodiments, it is a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or an antisense oligonucleotide (ASO). Examples of anti-BAFF RNA interference agents that inhibit the activity of BAFF include Gene Therapy (Gsk).

[0122] In specific embodiments, the nucleic acid BAFF inhibitor is a single-stranded antisense oligonucleotide (ASO), a microRNA (miRNA) (non-coding RNA), or a dsRNA (e.g., siRNA, miRNA (e.g.., precursor or mimic, eventually processed into single stranded miRNA)) specific to BAFF mRNA, or a CRISPR-Cas base editor (cytosine, adenine or prime editor) or CRISPR-Cas prime editors, with guide RNAs (gRNAs) or prime editing guide RNAs (pegRNAs) targeting specific regions of the BAFF DNA locus. While the present disclosure is not limited by any particular mechanism of action, in some embodiments, the nucleic acid enters a cell and causes the degradation, blocks the translation, blocks the interaction with another factor or affects the splicing of an RNA of complementary or identical sequences, including endogenous RNAs (mRNA or non-coding).

[0123] In an embodiment, the BAFF inhibitor is an antisense oligonucleotide (ASO) specific for BAFF mRNA (e.g., human). ASOs are synthetic single stranded strings of nucleic acids (natural or modified (e.g., Locked Nucleic Acid (LNA), phosphorothioate, 2’0-Methyl, 2’0-Methoxy, phosphoramidite, etc.), between 8 and 50 nucleotides in length, preferably between 10 and 35, between 15 and 25, or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 in length. In some embodiments, the ASO is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 single-stranded nucleotides in length. They bind to RNA through standard Watson-Crick base pairing. ASOs encompassed with the present disclosure interfere with BAFF RNA and result in its degradation; in preventing BAFF translation; in affecting splicing of the BAFF mRNA in a way that affects the resulting protein, and leading to a loss of function (e.g., ASO targeting BAFF splicing site); or in inhibiting the binding of a protein important for the processing of the mRNA (e.g., by targeting a binding partner’s site on BAFF).

[0124] ASOs may target exons or introns. In specific embodiments, ASO comprises a subsequence of a nucleic acid encoding BAFF (e.g., a subsequence of the sequence of FIGs. 20A and C (SEQ ID NOs: 1 or 3). In other specific embodiments, the inhibitors are gapmers, i.e., ASOs that contains a central block of deoxynucleotide monomers sufficiently long to induce ribonuclease cleavage. In a specific embodiment, the ASOs of the present disclosure target exons. In specific embodiments, the ASOs of the present disclosure results in BAFF (e.g., >50% knock down of BAFF) RNA degradation. In certain embodiments, the above-mentioned BAFF inhibitors (e.g., BAFF ASOs, siRNAs, or miRNA mimics) are chemically modified to increase their stability and / or help them evade immune response (e.g., phosphorothioate (PS)(e.g., increases stability), 2’0-Methyl (2’OMe) (e.g., increases stability and reduces immune response), 2’0-Methoxy (2’MOE) e.g., (increases stability and reduces immune response), phosphoramidite (NP) (e.g., increases stability), locked nucleic acid or phosphoramidate morpholino (PMO), and peptide nucleic acid (PNA) groups. Such modifications may also assist in loading in the RISC complex and in excluding the passenger strand.

[0125] In other embodiments, the BAFF inhibitor is a double-stranded RNA (dsRNA) molecule (or a molecule comprising region of double-strandedness). In some embodiments, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length. In some embodiments, the dsRNA is a small interfering RNA (siRNA), a shorthairpin RNA (shRNA), or a microRNA (miRNA) (miRNA mimics).

[0126] Also provided herein are double-stranded RNA (dsRNA) molecules, comprising a portion of the mature polypeptide coding sequence of any one of the coding sequences of the polypeptides disclosed herein of inhibiting expression of that polypeptide in a cell.

[0127] When a cell is exposed to a dsRNA, RNAs containing complementary sequences are selectively degraded by a process called RNA interference (RNAi). In some embodiments, dsRNAs provided herein are used in gene-silencing methods. In one aspect, methods are provided to selectively degrade RNA using the dsRNAis disclosed herein. In some embodiments, the BAFF inhibitor is a shRNA expressed by a DNA vector transfected or transduced into a target cell. In some embodiments, the BAFF inhibitor is a virus encoding a shRNA. In some embodiments, the BAFF inhibitor is a vector encoding a shRNA. The process is alternatively practiced in vitro, ex vivo or in vivo. In one aspect, the dsRNA molecules are used to generate a loss-of-function mutation in a cell, an organ or an organism. Methods for making and using dsRNA molecules to selectively degrade RNA are described in the art, see, for example, U.S. Patent No.

[0128] 6,506,559; U.S. Patent No. 6,511,824; U.S. Patent No. 6,515,109; and U.S. Patent No. 6,489,127.

[0129] In some embodiments, a nucleic acid BAFF inhibitor (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) comprises a circular nucleic acid molecule, wherein the nucleic acid BAFF inhibitor (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is about 38 to about 70 (e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in length having about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23) base pairs wherein the circular oligonucleotide forms a dumbbell shaped structure having about 19 base pairs and 2 loops. In some embodiments, a circular a nucleic acid BAFF inhibitor (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) contains two loop motifs, wherein one or both loop portions of the dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) is biodegradable. In some embodiments, degradation of the loop portions of a circular a nucleic acid BAFF inhibitor (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) generates a double-stranded nucleic acid BAFF inhibitor (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) with 3'-terminal overhangs, such as 3'-terminal nucleotide overhangs comprising about 2 nucleotides. The sense strand of a double stranded dsRNA molecule (e.g., RNAi molecules, siRNA molecules, miRNA molecules, and analogues thereof) may have a terminal cap moiety such as an inverted deoxybasic moiety, at the 3'-end, 5'-end, or both 3' and 5'-ends of the sense strand.

[0130] In some embodiments, the 3'-terminal nucleotide overhangs of an anti-BAFF dsRNA molecule (e.g., RNAi molecules,siRNA molecules, miRNA molecules, and analogues thereof) comprise ribonucleotides or deoxyribonucleotides that are chemically modified at a nucleic acid sugar, base, or backbone. In some embodiments, the 3'-terminal nucleotide overhangs comprise one or more universal base ribonucleotides. In some embodiments, the 3'-terminal nucleotide overhangs comprise one or more acyclic nucleotides.

[0131] In other specific embodiments, the BAFF inhibitor is an oligonucleotide with the nucleic acid sequence of a miRNA (regulatory) specific for BAFF RNA expression (e.g., human) (miRNA mimics). miRNAs are double-stranded RNAs (e.g., 18-22-nucleotide-long) that regulate gene expression post-transcriptionally by base-pairing with the 3' untranslated region of target messenger RNAs and inhibiting their expression. The miRNA mimic technology (miR-Mimic) is innovative gene silencing approach to generate nonnatural double-stranded miRNA-like RNA fragments. Such an RNA fragment is designed to have its 5'-end bearing a partially complementary motif to the selected sequence in the 3'UTR unique to the target gene. Once introduced into cells, this RNA fragment, mimicking an endogenous miRNA, can bind specifically to its target gene and produce posttranscriptional repression, more specifically translational inhibition, of the gene. Unlike endogenous miRNAs, miR-Mimics act in a gene-specific fashion.

[0132] In the present disclosure, miRNAs (or miRNA mimics) are natural or modified (e.g., Locked Nucleic Acid, phosphorothioate, 2’0-Methyl, 2’0-Methoxy, phosphoramidite, etc.) double stranded strings of nucleic acids between 8 and 50 nucleotides in length, preferably between 10 and 35, between 15 and 25, between 18 and 22 or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 in length that target the BAFF transcript. In some embodiments, the miRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more nucleotides in length targeting one or more regions of the BAFF mRNA. In specific embodiments, the oligonucleotide comprises or consists of the nucleic acids of miRNA (miRNA mimics) targeting one ormoreof (ortwoormore of orall three of) exon 14, exon 15 and 3’UTR of BAFF. In some embodiments, the miRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more single-stranded nucleotides in length.

[0133] The structures and sequences of the BAFF inhibitors described in the above-noted patents and patent application are incorporated herein by reference.

[0134] As used herein, the term “ligand” refers to any molecule capable of binding the designated LC biomarker as defined herein. Without being so limited, it includes binding-molecules such as antibodies and antigen binding fragments thereof, adnectins; affibodies; affilins; affimers; affitins; alphabodies; antibody mimetics; anticalins; aptamers; armadillo repeat protein-based scaffolds; atrimers; avimers; DARPins; fynomers; knottins; Kunitz domain peptides; monobodies; and nanofitins. Antibodies and antigen binding fragments thereof are as defined above in so far as they can be used for the detection of the LC biomarkers as defined herein. It further includes peptides (i.e. short chains of amino acids designed or selected to bind specifically to the target biomarkers. Peptide ligands can be engineered for high specificity and affinity), small molecules (i.e. low molecular weight compounds (e.g., less than 1000 Daltons) that can be designed or screened for their ability to bind to specific target biomarkers), lectins (i.e. proteins that bind specifically to carbohydrate moieties on glycoproteins or glycolipids. They can be used to detect biomarkers with specific glycosylation patterns (e.g., BAFF is glycosylatedat residue e.g., 124)), nanoparticles (i.e. functionalized nanoparticles can be engineered to bind specific biomarkers. These can include gold nanoparticles, quantum dots, or magnetic nanoparticles, often used in conjunction with other ligands like peptides or aptamers for enhanced selectivity, molecularly Imprinted Polymers (MIPs) (i.e. synthetic polymers that have been structured to have selective binding sites for target molecules, functioning similarly to natural receptors). Without being so limited, in specific embodiments, ligands used to detect LC biomarkers include antibodies specific to LC biomarkers such as BV650-anti-CD19 (e.g., BD Biosciences), BB515-anti-lgM (e.g., BD Biosciences), BV421 -anti-CD10 (e.g., BD Biosciences), AlexaFluor / OO-anti-CD27 (e.g., BD Biosciences), BUV395-anti-CD20 (e.g., BD Biosciences), PE-anti-CD21 (e.g., BD Biosciences), PerCP-eFluor710-anti-CD1c (e.g., eBioscience), Anti-Human Zonulin (e.g., Elabscience) ELISA kits, PE-anti-BAFF (e.g., Biolegend), BV421-anti-CD3 (e.g., Biolegend), anti-APRIL Human (e.g., ThermoFisher Scientific), anti-Mi-2 nuclear antigen autoantibodies (e.g., U-plex MSD Kit (MSD)), anti-SmD autoantibodies (e.g., U-plex MSD Kit (MSD)), anti-U1-snRNP-A autoantibodies (e.g., U-plex MSD Kit (MSD)), and anti-RCN2 autoantibodies (e.g., Abeam™ ab104516).

[0135] Routes of administration

[0136] In some embodiments, compounds and compositions provided herein are administered by one or more routes of administration using one or more of a variety of suitable methods.

[0137] As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. Routes of administration for BAFFs inhibitors for uses and methods herein include, but are not limited to, intravenous, intraperitoneal, subcutaneous, intramuscular, intradermal, spinal or other parenteral routes of administration, for example by injection or infusion. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion. Alternatively, BAFF inhibitors provided herein are administered by a non-parenteral route, such as a topical, epidermal or mucosal route of administration, for example, oral, intranasal, vaginal, rectal, sublingual or topical.

[0138] When BAFF inhibitors of the present disclosure are ASOs, they can be administered parenterally or orally to subjects in need thereof in the form of naked nucleic acid or encapsulated nucleic acid (e.g., lipid-based particles such as liposomes or lipid nanoparticles (LNPs)) As indicated above, in specific embodiments, ASOs of the present disclosure are chemically modified to increase their nuclease resistance and avoid triggering an immune response and enable their administration as naked nucleic acids.

[0139] Parenteral routes appropriate for ASOs of the present disclosure include intravenous, intraperitoneal, or subcutaneous administrations.

[0140] The present disclosure also encompasses vectors (plasmids) comprising the above-mentioned dRNAs (e.g., shRNA).The vectors are contemplated to be of any type suitable, e.g., for expression of said polypeptides or propagation of genes encoding said polypeptides in a particular organism. In some embodiments, the organism is of eukaryotic or prokaryotic origin. The specific choice of vector depends on the host organism and is known to a person skilled in the art. In an embodiment, the vector comprises transcriptional regulatory sequences or a promoter operably linked to a nucleic acid comprising a sequence encoding BAFF. A first nucleic acid sequence is “operably linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in reading frame. However, since enhancers for example generally function when separated from the promoters by several kilobases and intronic sequences are often of variable lengths, some polynucleotide elements are operably linked but not contiguous. “Transcriptional regulatory sequences” or “transcriptional regulatory elements” are generic terms that refer to DNA sequences, such as initiation and termination signals, enhancers, and promoters, splicing signals, polyadenylation signals, etc., which induce or control transcription of protein coding sequences with which they are operably linked.

[0141] A recombinant expression vector comprising a double stranded nucleic acid sequence provided herein, in some embodiments is introduced into a cell, e.g., a host cell, which includes living cells capable of expressing a BAFF inhibitor provided herein encoded by a recombinant expression vector. Accordingly, also provided herein are cells, such as host cells, comprising the nucleic acid and / or vector as described above. The suitable host cell is any cell of eukaryotic or prokaryotic (bacterial) origin that is suitable, e.g., for expression of the nucleic acid. In some embodiments, the eukaryotic cell line is of mammalian, of yeast, or invertebrate origin. The specific choice of cell line is known to a person skilled in the art. Choice of bacterial strain will depend on the task at hand and is known to a person skilled in the art. The terms “host cell” and “recombinant host cell” are used interchangeably herein. Such terms refer not only to the particular subject cell, but also to the progeny or potential progeny of such a cell. Because certain modifications often occur in succeeding generations due to either mutation or environmental influences, such progeny are often not, in fact, identical to the parent cell, but are still included within the scope of the term as used herein. Vectors are introduced into cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection” refer to techniques for introducing foreign nucleic acid into a host cell, including calcium phosphate or calcium chloride coprecipitation, DEAE-dextran-mediated transfection, lipofection, electroporation, microinjection, and viral-mediated transfection. Suitable methods for transforming or transfecting host cells is for example found in Sambrook et al. (supra), Sambrook and Russell (supra) and other laboratory manuals. Methods for introducing nucleic acids into mammalian cells in vivo are also known and are often used to deliver the vector DNA of a BAFF inhibitor provided herein to a subject for gene therapy.

[0142] The above-mentioned nucleic acid or vector, in some embodiments, is delivered to cells in vivo using methods well known in the art (such as direct injection of nucleic acid, receptor-mediated nucleic acid uptake, viral-mediated transfection or non-viral transfection and lipid-based transfection), all of which often involve the use of gene therapy vectors. Direct injections have been used to introduce naked or chemically modified nucleic acid into cells in vivo. Insome embodiments, a delivery apparatus (e.g., a "gene gun") for injecting nucleic acid into cells in vivo is used. In some embodiments, such an apparatus is commercially available (e.g., from BioRad). In some embodiments, naked or chemically modified nucleic acid is introduced into cells by complexing the nucleic acid to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor. Binding of the nucleic acid-ligand complex to the receptor, in some embodiments, facilitates uptake of the nucleic by receptor-mediated endocytosis. A nucleic acidligand complex linked to adenovirus capsids which disrupt endosomes, thereby releasing material into the cytoplasm, in some embodiments, is used to avoid degradation of the complex by intracellular lysosomes.

[0143] Defective retroviruses are well characterized for use as gene therapy vectors (for a review see Miller, A. D., Blood 76:271 (1990)). Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses are found in Current Protocols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art. Examples of suitable packaging virus lines include psiCrip, psiCre, psi2 and psiAm. Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and / or in vivo.

[0144] For use as a gene therapy vector, the genome of an adenovirus, in some embodiments, is manipulated so that it encodes and expresses a nucleic acid of a BAFF inhibitor provided herein (e.g., a nucleic acid encoding an anti-BAFF targeting BAFF) but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art. Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and are often used to infect a wide variety of cell types, including airway epithelium, endothelial cells, hepatocytes, and muscle cells.

[0145] In some embodiments, adeno-associated virus (AAV) is used as a gene therapy vector for delivery of DNA for gene therapy purposes. AAV is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. In some embodiments, AAV is used to integrate DNA into non-dividing cells. In some embodiments, lentiviral gene therapy vectors are adapted for use in methods provided herein.

[0146] In certain embodiments, the BAFF inhibitors (e.g., anti-BAFF antibody, siRNA or ASO) provided herein are formulated to ensure proper distribution in vivo.

[0147] For example, the therapeutic compounds provided herein, in some embodiments, are formulated in lipid-based nanoparticles or liposomes. In some embodiments, the nanoparticles or liposomes comprise one or more moieties which are selectively transported into specific cells (e.g., immune cells such asT cells), tissues or organs, thus enhance targeted drug delivery (see, e.g., Ranade V.V., J. Clin. Pharmacol. 29:685 (1989)). In an embodiment, the BAFF inhibitors provided herein are formulated to be delivered to immune cells, such as T cells (e.g., CD4 and / or CD8 T cells). Biodegradable, biocompatible polymers used in some embodiments, such as lipids, ethylene vinyl acetate,polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. In some embodiments, therapeutic compositions are administered with medical devices known in the art.

[0148] In another aspect, compositions are provided, e.g., a pharmaceutical composition, comprising one or a combination of BAFF inhibitors (e.g., anti-BAFF antibody, siRNA or ASO) provided herein, formulated togetherwith a pharmaceutically acceptable carrier and / or excipient.

[0149] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the BAFF inhibitor coated in a material to protect the compound from the action of acids and other natural conditions that, in some embodiments, inactivate the compound.

[0150] Pharmaceutical compositions provided herein, in some embodiments, include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0151] Examples of suitable aqueous and non-aqueous carriers that are employed in the pharmaceutical compositions of provided herein include, but are not limited to, water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity is maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0152] In some embodiments, compositions herein contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of presence of microorganisms is ensured, in some embodiments, both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. In some embodiments, it is desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form, in some embodiments, is brought about by the inclusion of agents which delay absorption such as, aluminum monostearate and gelatin.

[0153] Pharmaceutically acceptable carriers or excipients include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions provided herein iscontemplated. In some embodiments, supplementary active compounds are incorporated into the compositions. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. In some embodiments, the composition is formulated as a solution, microemulsion, liposome, lipid nanoparticles, or other ordered structure suitable to high drug concentration. In some embodiments, the carrier is a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, isotonic agents are included, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

[0154] Prolonged absorption of the injectable compositions is brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

[0155] Sterile injectable solutions are prepared, in some embodiments, by incorporating the BAFF inhibitor (e.g., anti-BAFF antibody, siRNA or ASO), in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-fi Itered solution thereof.

[0156] The amount of BAFF inhibitor (e.g., anti-BAFF antibody, siRNA or ASO), to be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated, and the particular mode of administration. The amount of active ingredient combined with a carrier material to produce a single dosage form will generally be that amount of the composition which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01 per cent to about ninety-nine percent of active ingredients, from about 0.1 per cent to about 70 per cent, or from about 1 percent to about 30 percent of active ingredient in combination with a pharmaceutically acceptable carrier.

[0157] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, in some embodiments, a single bolus is administered. In some embodiments, several divided doses are administered over time. In some embodiments, the dose is proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of BAFF inhibitors provided herein are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.In some embodiments, for administration of a BAFF inhibitor (e.g., anti-BAFF antibody, siRNA or ASO), exemplary dosage ranges include but are not limited to from about 0.0001 to about 100 mg / kg of the host body weight. In some embodiments, dosage ranges are from about 0.01 to about 5 mg / kg of the host body weight. In some embodiments, dosages are about 0.3 mg / kg body weight, about 1 mg / kg body weight, about 3 mg / kg body weight, about 5 mg / kg body weight, or about 10 mg / kg body weight or within the range of about 1-10 mg / kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Exemplary dosage regimens BAFF inhibitor (e.g., anti-BAFF antibody, siRNA or ASO) provided herein include, but are not limited to about 1 mg / kg body weight or about 3 mg / kg body weight by intravenous administration.

[0158] The BAFF inhibitor (e.g., anti-BAFF antibody, siRNA or ASO) is usually administered on multiple occasions. Intervals between single dosages are, for example, weekly, monthly, every three months or yearly. In some embodiments, intervals are irregular as indicated by measuring blood levels of BAFF inhibitor (e.g., anti-BAFF antibody, siRNA or ASO), in the patient. In some methods, dosage is adjusted to achieve a plasma concentration of the BAFF inhibitor, of about 1-1000 pg / ml and in some methods about 25-300 pg / ml.

[0159] Alternatively, BAFF inhibitors are administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the inhibitor in the patient. The dosage and frequency of administration vary, in some embodiments, depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, in some embodiments, the patient is administered a prophylactic regime.

[0160] Actual dosage levels of the active ingredients in the pharmaceutical compositions provided herein, in some embodiments, are varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response (e.g., decreased of at least one LC symptom) for a particular individual, composition, and mode of administration, without being toxic to the individual. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0161] As used herein the term “subject” refers to an animal, a mammal and to a human in a specific embodiment. The compositions of the present disclosure may also be used for veterinary applications and be used in pets or other animals (e.g., pets such as cats, dogs, horses, etc.; and cattle, fishes, swine, poultry, etc.). In a specific embodiment, the subject is 18 years old or older. As used herein a “subject in need thereof’ is a subject presenting the symptoms of LC as defined herein. In specific embodiments, the subject is pre-diagnosed as having been infected with SARS-CoV-2.

[0162] The present disclosure is illustrated in further detail by the following non-limiting examples.

[0163] EXAMPLE 1: Materials and methods

[0164] Study design and population

[0165] The Institut de Recherches Cliniques de Montreal (IRCM) post-COVID-19 (IPCO) research clinic, the first clinic of its kind in Quebec, integrates clinical care into a prospective observational cohort study with biobank (IPCO protocol #2021-1092, ClinicalTrials.gov Identifier: NCT04736732). 228 male and female Quebec residents aged 18 to 100 years were enrolled between February 12th, 2021, and July 25th, 2022, onto the IPCO protocol, which was conducted with participant informed consent and full approval from the IRCM Research Ethics Board. The cohort included, among others, participants meeting the WHO clinical case definition of post COVID-19 condition (LC group), and people recruited during the pandemic who never tested positive for COVID-19 infection and never had symptoms consistent with acute COVID-19 infection (Pandemic Control group). All participants in the LC group had a COVID-19 diagnosis confirmed by PCR at least 3 months prior to recruitment and while they could enroll onto the study at any time following their COVID-19 infection, in-person study visits with biobanking were offered at 3-6 months, 12 months and 24 months post-infection. Among the 228 recruited participants, the study focused on the 125 participants, including males and females, with LC and pandemic controls, who opted into providing stool samples.

[0166] Further validation data was generated using samples from additional participants enrolled in the IRCM post-COVID-19 (IPCO) cohort. For this validation, analyses focused on individuals with Long COVID (LC) for whom plasma and peripheral blood samples were available at three post-infection timepoints: 3-6 months, 12 months, and 24 months following acute SARS-CoV-2 infection. A recovered group (participants who had COVID-19 but did not have any COVID-19 associated symptoms beyond 12 weeks post-COVID) were included. Data measured in these additional participants are presented in FIGs. 1 B, 4K, 12 and 13. Comparator groups used in FIG. 1 B were as follows

[0167] 1. Pandemic ctrl= Pandemic controls; n=24;

[0168] 2. Recovered 3-6m= Recovered, visit was at 3-6 months post-COVID-19; n=12;

[0169] 3. Recovered 12m= Recovered, visit was at 12 months post-COVID-19; n=9;

[0170] 4. LC 3-6m= patient with Long COVID 3-6 months post-COVID-19; n=13;

[0171] 5. LC 12m= patient with Long COVID 12 months post-COVID-19; n=12; and

[0172] 6. LC 24m= patient with Long COVID 24 months post-COVID-19; n=12.

[0173] Sample collection and processing

[0174] Blood and stool samples were collected at 3-6-month, 12-month and 24-month study visits. Plasma and serum were collected after centrifugation of whole blood at 400g for 10min at room temperature (RT). Undiluted plasma and serum were aliquoted and stored at -80°C. Peripheral blood mononuclear cells (PBMCs) were isolated using SepMate™ PBMC isolation tubes (Stemcell Technologies) as per the manufacturer’s protocol and cryopreserved in liquid nitrogen.Stool was self-collected in sterile, dry tubes and immediately stored at -20°C and transferred to -80°C upon receipt at the clinic.

[0175] Measurement of soluble biomarkers in blood

[0176] Human participants

[0177] Plasma B-cell activating factor (BAFF), a proliferation inducing ligand (APRIL), lipopolysaccharide-binding protein (LBP) and [3-D-glucan were quantified by enzyme-linked immunosorbent assay (ELISA) using commercial kits (Human BAFF / BLyS, R&D systems; human APRIL, Thermo Fisher Scientific; human LBP, Abeam; and [3-D-glucan, Glucatell™, Associates of Cape Cod) according to the manufacturers’ instructions. Serum zonulin was measured by ELISA using a commercial kit (human Zonulin, Elabscience). Serum soluble transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA) were measured using Meso Scale Discovery (MSD) assays (R-PLEX™ human TACI; U-PLEX™ human BCMA) according to the manufacturer’s instructions. MSD data were analysed using Discovery Workbench software (MSD).

[0178] Mice

[0179] Soluble BAFF and LBP levels were measured in mouse serum using the mouse BAFF / BLyS / TNFSF13B (R&D Systems) and the mouse LBP (Abeam) ELISA kits, respectively, as per the manufacturer’s instructions.

[0180] Ig isotype and autoantibody measurements in serum

[0181] Human participants

[0182] Serum IgG autoantibodies were measured using an electrochemiluminescence-based multiplex assay (MSD U-PLEX™). Briefly, pooled biotinylated antigens (double stranded DNA (dsDNA), DNA topoisomerase I, histidyl-tRNA-synthetase, Mi-2, SmD, U1-snRNP, U1-snRNP A, Ro / SS-A, La / SS-B, and Ro / SS-A52; Surmodics IVD) were prepared according to the MSD Biotinylation QuickGuide and coupled to U-PLEX™ linkers (final coating concentration, 66 nM per antigen). A fixed positivity threshold of 1,000 electrochemiluminescence (ECL) counts was used, selected a priori as threefold the blank (background) signal to prioritize specificity. Data were analysed in Discovery Workbench (MSD). Plasma autoantibodies against human RCN2 were quantified by an in-house (homemade) ELISA. Briefly, 96-well high-binding microplates were coated overnight with recombinant human RCN2 (1 pig / mL) diluted in bicarbonate coating buffer (pH 9.6). Plates were washed three times with wash buffer consisting of 1 x PBS containing 0.05% (v / v) Tween™-20 (prepared by combining Tween™-20 (0.5 mL), 10x PBS (100 mL), and deionized water (900 mL) to a final volume of 1 L). Non-specific binding was blocked with 10% (v / v) heat-inactivated fetal bovine serum in PBS for 2 h, followed by three washes. Plasma samples were diluted 1:2000 in assay diluent and added to the plate. A standard curve was generated in parallel using a rabbit polyclonal anti-RCN2 antibody starting at 350 ng / mL followed by seven serial 1:2 dilutions. Samples and standards were incubated for 2 h, and plates were then washed five times with wash buffer. For detection, an HRP-conjugated anti-human IgG antibody (1:10,000) was used for plasma samples, and an HRP-conjugated anti-rabbit IgG antibody (1:5,000) was used for the standard curve, followed by a 1-h incubation. Plates were washed five times, and color was developed using the substrate and stop solutions supplied in a SARS-CoV-2spike IgG ELISA kit (Thermo Fisher Scientific, cat. BMS2325). Absorbance was measured at 450 nm, and anti-RCN2 IgG levels in plasma were interpolated from the standard curve.

[0183] Mice

[0184] Anti-nuclear antibodies (ANA) were detected in mouse sera using the BioAssay mouse ELISA kit (USBiological Life Sciences) according to the manufacturer’s instructions.

[0185] Flow cytometry

[0186] Human participants

[0187] Cryopreserved PBMCs were thawed, washed with Iscove's Modified Dulbecco's Medium (IMDM; Gibco) and phosphate-buffered saline (PBS), and stained for viability using LIVE / Dead™ Fixable Aqua (Invitrogen). Non-specific binding was blocked in FACS buffer (PBS, 2% heat-inactivated foetal bovine serum (FBSi; Gibco) and 0.1% sodium azide) supplemented with 20% FBSi, mouse IgG (50pig; Sigma-Aldrich) and Human BD Fc Block (7 L per 106cells; BD Biosciences).

[0188] Surface staining was performed using the following fluorochrome-conjugated mouse anti-human monoclonal antibodies: BV650 anti-CD19, BB515 anti-IgM, BV42 anti-CD10, PE-Cy7 anti-CD83, AlexaFluor700 anti-CD27, APC-H7 anti-IgG, BV711 anti-CD14, BUV395 anti-CD20 and PE anti-CD21 (BD Biosciences); PerCP-eFluor710 anti-CD1c, APC anti-CD38 (eBioscience); BV785 anti-CD16, BV605 anti-CD11c, PE anti-BAFF, BV421 anti-CD3 and PE-Cy7 anti-CD66b (BioLegend); FITC anti-lgA (Millipore).

[0189] For intranuclear staining, cells were processed with the eBioscience™ FoxP3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) and stained with PE anti-NR4A3 (Santa Cruz Biotechnology) and PE anti-T-bet (Biolegend). Fluorescence-minus-one (FMO) controls were used to define gates, and anti-mouse IgK compensation beads (Thermo Fisher Scientific) were used for compensation.

[0190] Mice

[0191] Live / dead exclusion was performed using Aqua-LIVE / Dead™ Fixable Stain (Invitrogen Life Technologies). Nonspecific binding was blocked using FACS buffer (PBS 1x + 2% FBSi and 0.1% sodium azide supplemented with 20% FBSi and 2 piL / 106cells of anti-Mouse CD16 / CD32 (eBioscience), as well as normal goat and normal rat serum (2piL of each per well)). The following conjugated rat, hamster or goat anti-mouse antibodies were used to detect extracellular markers: PE-Cy7-rat anti-CD45, BV711-rat anti-CD19, BV605-rat anti-CD138, Pacific Blue-rat anti-CD11b, Alexa-Fluor 488-ratanti-Ly6C, APC-Cy7-ratanti-Ly6G, BV605-rat anti-CD3, PE-rat anti-IgM (Biolegend); APC-rat anti-CD1d, PerCP-eFluor™ 710-rat anti-CD73, Alexa-Fluor 700-rat anti-CD3, PE-rat anti-lgA (eBioscience); Alexa-Fluor 488-goat anti-IgG (Invitrogen); PE-Cy5.5-hamster anti-CD11c, Alexa-Fluor 700-rat anti-MHC II (eBioscience); PE / Dazzle 594-rat anti-F4 / 80 and PE-rat anti-BAFF (R&D Systems). Intranuclear labeling was performed using the FoxP3 / Transcription Factor Staining Buffer Set (Invitrogen). The following conjugated rat or mouse anti-mouse antibodies were used to detect intranuclear markers in staining cocktails: FITC-rat anti-FoxP3 (eBioscience); PE / Dazzle 594-mouse anti-Tbet (Biolegend); and BV650-mouse anti-RORyT (BD Biosciences). All stained sampleswere compared to an unstained sample to control for background fluorescence. OneComp eBeads™ (Invitrogen Life Technologies) were used to optimize fluorescence compensation settings.

[0192] For all flow cytometry experiments, cells were kept at 4°C in 1.25% paraformaldehyde prior to data acquisition with a FACSFortessa™ (BD Biosciences). Analyses were done with FlowJo10.8.1 software and GraphPad™ Prism (10.3.1).

[0193] Fecal microbiota transplantation into germ-free mice

[0194] Mice

[0195] Female C57BL / 6 mice aged 5-7 weeks were purchased from the Germ-Free & Gnotobiotic Platform of the University of Calgary and maintained in the IRCM Germ-Free and Gnotobiotic Facility in sterile cages inside Flexible Film Isolators (Class Biologically Clean) and subjected to ambient temperature of 22 ± 1 °C under a 12h light / dark cycle. Mice were provided with sterile food and water ad libitum and tested regularly for contaminants. Experiments were conducted in a biosafety cabinet under sterile conditions according to the standards of the Canadian Council of Animal Care and approved by the IRCM Animal Care Committee.

[0196] Human stool aliquots were resuspended into a sterile brain-heart-infusion (BHI) broth with 30% glycerol solution (200mg of stool / 1 mL of solution) under anaerobic conditions. Stool suspensions were filtered through a 70pim strainer. After a 2-week acclimation period, germ-free mice were administered 200piL of fecal suspension by oral gavage 3 times per week (every other day) for 1 week and allowed 3 weeks for microbiota engraftment prior to further testing. 3 sets of experiments were performed in germ-free mice (5-10 female mice per experimental group). In each experiment, stool from a distinct female human donor was examined and included 2 experimental groups (i.e., stool from a participant with severe LC compared to stool from a participant with mild LC).

[0197] Evaluation ofanti-BAFF treatment in LC gnotobiotic mouse model

[0198] Female C57BL / 6 gnotobiotic mice previously colonized with stool from a patient with severe LC were treated with a single administration of anti-BAFF (mouse) mAb (Sandy-2; n=5) or a mouse lgG1 isotype control (n=5; Adipogen Life Sciences) i.p. on day 1 and tissue was harvested for downstream analysis on day 18.

[0199] Mouse tissue harvest and cell suspensions

[0200] After sedation with ketamine, mice were sacrificed by cardiac puncture. Serum was isolated by clotting and stored in dry sterile tubes at -80°C until further use. Colon, Peyer's patches, spleen and brain were harvested in Hank’s balanced salt solution (HBSS; Multicell) and kept on ice until further processing. Colons were measured for length and sectioned transversely with one half fixed in 10% formalin and the other half cut into 2cm sections and processed for colon epithelial cell (CEC) isolation and lamina propria (LP) cell suspension. CECs were isolated by washing colons in HBSS lacking Ca2+ and Mg2+with 25mM HEPES (ThermoFisher Scientific) and incubated in pre-warmed HBSS with 15mM HEPES, 10% FBS, 5mM EDTA, and 1mM DTT for 15min at37°C with shaking every 5min. Samples were then filtered through a 100pm cell strainer and LP cells were isolated by incubation with pre-warmed 5mL IMDM with 1% Penicillin-Streptomycin-Glutamine (Gibco), 340pL Liberase™ (2mg / mL sterile water) and 15pL of DNase I (Roche) for 1 hour at 37°C with shaking every 15min, filtering through 100pm and 40pm strainers and washing with PBS 1x before flowcytometry analysis. Half of the spleen was reserved for immunofluorescent imaging and half was passed through a 70pim strainer and kept on ice in IMDM + 2% FBSi until flow cytometry analysis.

[0201] Mouse tissue fixation and immunofluorescence microscopy

[0202] Spleens and Peyer’s patches were embedded in Tissue-Plus™ OCT Compound (Scigen) and stored at -80°C. Serial 10pm spleen and Peyer’s patch cryosections were fixed in acetone (-20°C), air dried and stored at -80°C until immunolabelling and imaging. Formalin-fixed mouse colons were embedded in paraffin and 10pm sections prepared and stored at room temperature (RT) (IRCM Histology core). Brain hemispheres were fixed in 4% paraformaldehyde, transferred into 30% sucrose, embedded in OCT compound, cut into 14pm serial cryosections and stored at -80°C (Institut de Recherche en Immunologie et en Cancerologie Histology core).

[0203] Immunolabelling was performed by the Centre de Recherche du Centre Hospitalier de I’Universite de Montreal (CRCHUM) Molecular Pathology core. All sections were stained using the Discovery Ultra automated stainer (Ventana Medical Systems, Roche). Antigen retrieval was performed on paraffin-embedded sections with Cell Conditioning #1 solution (Ventana Medical System; Tris-EDTA buffer, pH 7.8) for 60 min at95°C. Slides were incubated for 30 minutes at RT in PBS-BSA 1%, followed by incubations with primary and then secondary antibodies for 2 hours each at RT, and a 10-minute incubation with DAPI (1 / 3000) at RT. Slides were then washed in PBS, incubated for 15 minutes in 0.1% Suddan black / 70% ethanol solution, washed again and mounted on cover slips with Fluormount™ (Sigma). All slides were scanned using an Aperio Verso 200 slide scanner microscope equipped with a 20x0.8 NA objective and a resolution of 0.275 MPP (Leica Biosystems). Image visualization was performed using the Aperio™ Image Scope software (Leica Biosystems).

[0204] The following conjugated / unconjugated reagents / antibodies were used for immunolabelling: biotinylated peanut agglutinin (PNA) (MJS Biolynx), Texas Red (TXRD) Goat anti-mouse IgM and Rat anti-mouse CD45R(B220), biotinylated Goat anti-mouse IgA (Southern Biotech), Alexa Fluor 488 Streptavidin, Rabbit anti-mouse ZO-1, Alexa Fluor 488 Goat anti-rabbit (ThermoFisher Scientific), Alexa Fluor 488 glial fibrillary acidic protein (GFAP) Monoclonal Antibody (GA5) (eBioscience) and Rabbit anti-mouse IBA1 (ThermoFisher Scientific).

[0205] Statistical analyses

[0206] For clustering of BAFF and zonulin measurements in human plasma, outliers were removed using the Z-score approach and the maximum number of clusters per analytes data frame was determined using the Silhouette method. The k-means algorithm was used to reconstitute clusters with a random number for the center set to 25 (center method) and a maximum iteration set to 50. All determined clusters were aggregated, and a function was appended to determine the lowest (minimum) and the highest (maximum) value for each cluster. The lowest cluster is labelled as “Low”, the highest as “High” and the middle as “Intermediate” if any.

[0207] Statistical significance of differences between groups was assessed with a one-way ANOVA with post-hoc Tukey test for normally distributed data or a Kruskal-Wallis test with post-hoc Dunn test for non-normally distributed data. For 2-group comparisons, a paired t-test was used for normally distributed data. Otherwise, a Wilcoxon test was used. Normality was assessed with the Shapiro-Wilk test. Pearson and Spearman correlations were performed on normallyand non-normally distributed data, respectively. Analyses were performed using GraphPad Prism 9.1.0 and p values SO.05 were considered statistically significant.

[0208] EXAMPLE 2: Increased intestinal barrier permeability in adults with LC for at least 24 months

[0209] The inventors measured markers of intestinal barrier integrity in the subjects from the IPCO cohort (people with LC and pandemic controls). First, they measured levels of zonulin, a marker of intestinal permeability, in the serum of LC participants at 3-6, 12- and 24-months post-infection (FIG. 1A). The inventors found that serum zonulin levels are increased in people with LC when compared to pandemic controls and that this persists over time.

[0210] In a further validation group described in Example 1, individuals with LC exhibited increased serum zonulin levels relative to pandemic controls and recovered participants. Elevated zonulin levels were observed at all examined LC timepoints and remained increased up to 24 months post-COVID-19, indicating sustained intestinal barrier dysfunction (FIG. 1B).

[0211] The inventors then assessed microbial translocation by measuring blood levels of LPS-binding protein (LBP) and |3-D-glucan, which are markers of bacterial and fungal translocation, respectively. The inventors found significant positive correlations between markers of microbial translocation and zonulin levels (FIGs. 1C-H). The most significant correlations are observed at 12 months post-infection, but some persist for up to 24 months. These observations suggest that intestinal permeability is increased in people with LC compared to pandemic controls.

[0212] EXAMPLE 3: Increased intestinal permeability is closely associated with modulation of the BAFF / APRIL system in adults with LC

[0213] The inventors next evaluated the BAFF / APRIL system in subjects from the IPCO cohort at all 3 timepoints (3-6, 12-24-months). They first observed that membrane BAFF (mBAFF) expression was significantly increased in total PBMCs, T-cells, B-cells and in all 3 monocyte subsets from people with LC up to 24 months post-COVID-19 (FIGs. 2A-2F). The inventors also found that increased circulating BAFF levels significantly positively correlated with serum zonulin levels and negatively correlated with plasma APRIL levels up to 12 months post-COVID-19. Soluble levels of B-cell Maturation Antigen (BCMA), a receptor for BAFF and / or APRIL that is shed with B-cell dysregulation, was also found by the inventors to be significantly positively correlated with (3-D-glucan levels up to 24 months post-infection (FIGs.

[0214] 3A-3I). Together, these data suggest that the phenotype of increased intestinal permeability observed in people with LC is strongly associated with modulation of the BAFF / APRIL system.

[0215] EXAMPLE 4: B-cell compartment dysregulation in adults with LC

[0216] Based on the inventors’ observed alterations in the BAFF / APRIL system in LC, they evaluated the circulating B-cell compartment in people with LC compared to pandemic controls at 3-6, 12- and 24- months post-COVID-19 (FIGs. 4A-4K). Based on their previously established protocol to analyze human blood CD1c+ MZ and MZp B-cell populations, the inventors compared the frequencies of circulating total (CD3-CD19+), total memory (CD3-CD19+CD1c-CD27+CD20+), resting switched memory (CD3-CD19+CD1c-CD27+CD20+CD21+lgM-), naive (CD3-CD19+CD20+CD27-CD1c-CD10-CD21h'9h), transitional immature (CD3-CD19+CD20+CD10+CD1c-CD27-CD21hi9h), MZ (CD3-CD19+CD1c+CD27+lgMbr'9htCD10-) and MZp (CD3-CD19+CD1c+CD27+lgMbri9htCD10+) B-cells (FIGs. 4A-4G), as well as total B-cell blasts (CD3-CD19CD20-CD38+lg+) (FIG. 4G-4I). There were no differences in the frequencies of total B-cells and MZ B-cells between people with LC and pandemic controls at all time points. However, an increase in total memory and resting switched memory B-cells was observed at 24 months compared to the earlier timepoints in people with LC. Correspondingly, frequencies of naive and transitional immature B-cells were increased at 3-6 and 12-months post-infection in people with LC but returned to levels similar to pandemic controls by 24 months. Interestingly, MZp B-cell frequencies were significantly increased in people with LC compared to pandemic controls and remained elevated for at least 24 months (FIGs. 4G and 4K), while frequencies of IgG-expressing blasts increased specifically at 24 months (FIG. 4J). Since excess BAFF decreases the Breg potential of MZp B-cells, the inventors correlated MZp B-cell frequencies with soluble BAFF levels and observed positive correlations, especially at 12 months (FIGs. 5A-5C). Together, these results suggest that people with LC have important dysregulation of the circulating B-cell compartment that shifts over time but does not fully resolve by 24 months post-COVID-19.

[0217] EXAMPLE 5: Blood MZp B-cell Breg potential is reduced in adults with LC and involves high levels of zonulin and BAFF

[0218] The inventors measured Tbet and NR4A3 expression in blood MZp B-cells from people with LC at 3-6, 12- and 24-months post-COVID-19 infection (FIGs. 6A and 7A). The inventors observed that Tbet expression in MZp B-cells is increased in people with LC at 3-6 and 12-months post-infection compared to pandemic controls but decreases to levels similar to controls at 24 months post-infection. Correspondingly, NR4A3 expression in blood MZp B-cells was significantly decreased at 3-6 and 12 months and recovered at 24 months post-infection in people with LC compared to pandemic controls. The inventors also demonstrated that increased Tbet expression in MZp B-cells is associated with increased plasma BAFF levels at 3-6 and 12 months and increased serum zonulin levels at 3-6 months post-COVID-19 infection (FIGs. 6B-G). The inventors also show a significant downregulation of NR4A3 expression in blood MZp B-cells from people with LC that is associated with increased BAFF and zonulin levels in blood, especially at 3-6 and 12-months post-infection (FIGs. 7B-G). These results suggest that up to 12 months post-infection, in the context of elevated BAFF and zonulin levels, MZp Breg potential is decreased in exchange for an activation / exhaustion profile which seems to return to a certain level of homeostasis at 24 months despite MZp B-cell frequencies remaining elevated.

[0219] EXAMPLE 6 Elevated BAFF and zonulin levels are associated with increased autoimmunity in people with LC Given the above observations, the inventors then assessed whether patients with LC had evidence of increased autoimmunity. Since B-cell dysregulation appeared to peak at 12 months post-infection in people with LC, evaluations were done at the 3-6- and 12-month time points. Total Ig levels in blood significantly increased between the 3-6- and 12- month time points in LC participants (FIGs. 8A-B). The inventors also found that increased levels of soluble BAFFand zonulin were associated with increased concentrations of IgM in blood compared to pandemic controls at 12 months post-infection (FIGs. 8C-F). Correspondingly, increased MZp B-cell frequencies were associated with increased serum IgM levels (FIG. 9B) and decreased serum IgA levels (FIG. 9A). Next, the inventors developed an assay to evaluate for the presence of 10 different autoantibodies in the blood of LC participants. The inventors observed significantly increased detection of autoreactivity against 3 targets, DNA topoisomerase, Mi-2 and SmD, in people with LC compared to pandemic controls, especially at 3-6 and 12-months post-COVID-19 infection (FIGs. 10A-B). Interestingly, there was no reactivity towards these targets in pandemic controls. Furthermore, at 12 months postinfection, people with LC and high zonulin levels were found to have the most autoreactivity (FIGs. 11 A-J). Altogether, these results suggest that people with LC and increased intestinal barrier compromise and / or dysregulation of the B-cell compartment have increased circulating levels of IgM, at the expense of IgA, and evidence of increased autoimmunity.

[0220] EXAMPLE 7: Identification of autoantibody associated with Long COVID

[0221] To further evaluate immune dysregulation associated with LC, serum samples were screened for autoantibodies targeting reticulocalbin-2 (RCN2), a protein implicated in endothelial cell function and cellular stress responses. Individuals with LC demonstrated increased serum levels of anti-RCN2 autoantibodies compared to pandemic controls, with elevated levels detected at 12 months, and 24 months post-COVID-19.

[0222] Serum anti-RCN2 autoantibody levels positively correlated with serum zonulin concentrations (FIG. 13A), frequencies of intermediate monocytes expressing elevated membrane-bound B-cell activating factor (mBAFF) (FIG. 13B), and frequencies of circulating MZp cells (FIG. 13C). These correlations further support an association between intestinal barrier dysfunction, BAFF-related immune activation, and autoantibody production in LC.

[0223] EXAMPLE 8 T ransfer of the microbiota from patients with LC can reproduce the LC phenotype in gnotobiotic mice

[0224] To demonstrate a causal relationship between intestinal dysbiosis, increased intestinal barrier permeability and autoimmunity, the inventors developed a unique mouse model where they colonized germ-free (GF) wild-type (WT) mice with the fecal microbiota from patients with severe or mild LC. After allowing for sufficient time for adequate engraftment of the human microbiota into GF mice, mice were evaluated for intestinal barrier permeability and immune dysregulation (FIG. 14).

[0225] The inventors found that GF mice colonized with the severe LC microbiota had increased intestinal barrier permeability as evidenced by increased levels of serum lipopolysaccharide (LPS) binding protein (LBP) (FIG. 15) and a lack of a continuous pattern (i.e., a punctuated pattern) of immune fluorescent (IF) staining for the tight junction protein zonula occludens 1 (ZO-1) in the colon epithelium (data not shown). In contrast, GF mice colonized with the mild LC microbiota had colons sections showing smooth and continuous ZO-1 staining along the colon epithelium (data not shown). Mice colonized with severe LC microbiota had higher serum BAFF (FIG. 16A) and increased frequency of BAFF-expressing CD45+ cells in both colonic LP and spleen compared with mice colonized with mild LC microbiota (FIGs.

[0226] 16B-C). Since IgA expression is important for gut homeostasis and decreased IgA levels were found in patients with LC and increased MZp B-cell frequencies (FIG. 9A), the inventors analyzed IgA expression in cryosections of Peyer’s Patches and found that IgA expression was significantly more expressed in Peyer’s Patches from GF mice colonized with the mild LC microbiota compared to those colonized with the severe LC microbiota (data not shown).

[0227] Subsequent evaluation of B-cell subpopulations in the spleen by flow-cytometry revealed a significant increase in innate MZ-like, (CD3'CD19+CD1d+lgMbr'9ht) B-cells in GF mice colonized with the severe LC microbiota compared to mice colonized with mild LC microbiota (FIG. 16D). This was accompanied by an increase in blasts (CD3CD138+) expressing IgM (FIG. 16F) and a trend towards decreased frequencies of lgA+blasts (FIG. 16E).

[0228] Together, these data demonstrate that the dysbiotic microbiota from patients with severe LC alone can transfer a severe LC phenotype to GF mice which includes increased intestinal barrier permeability, as well as excess BAFF and a B-cell dysregulation profile.

[0229] EXAMPLE 9: Treatment with a BAFF-inhibitor improves intestinal barrier compromise, immune dysregulation and neuroinflammation in gnotobiotic mice with severe LC

[0230] The inventors next evaluated whether treatment with a BAFF antagonist could improve key features of the severe LC phenotype. After colonization of GF mice with the microbiota from a patient with severe LC, mice were treated with a single dose of the lgG1 anti-mouse BAFF monoclonal antibody Sandy-2 (anti-BAFF) or with an isotype control (2 mg / kg dose administered via intra-peritoneal injection) and tissue was harvested for further examination 18 days later (FIG.

[0231] 17).

[0232] To evaluate treatment efficacy, serum BAFF levels and mBAFF expression in the spleen were measured in mice with severe LC treated with anti-BAFF or isotype control (FIGs. 18A-C). As expected, soluble BAFF and mBAFF expression in splenocytes were both significantly decreased in mice treated with anti-BAFF compared to those who received the isotype control, thereby confirming that the anti-BAFF treatment can effectively reduce systemic BAFF levels.

[0233] The inventors next showed that treatment with anti-BAFF improves intestinal barrier integrity as demonstrated by the continuous ZO-1 IF staining in the colon epithelium of mice with severe LC treated with anti-BAFF compared to those treated with the isotype control (data not shown).

[0234] Evaluation of immune responses showed that B-cell frequencies were decreased in the spleen of mice treated with anti-BAFF (FIG. 19A). This was expected since BAFF is a key factor for B-cell selection and survival. Anti-BAFF treatment also significantly increased the expression of CD73, a known regulatory marker on CD 1 d+l gMbri9htcells (which are likely innate B-cells) in the spleen (FIG. 19B) and decreased blast cell frequencies (FIG. 19C). Moreover, treatment with anti-BAFF significantly increased lgA+blast frequencies which was associated with a trend towards decreased lgG+blasts (FIGs. 19D-E). Finally, treatment with anti-BAFF also significantly decreased production of anti-nuclear antibodies (ANA) compared to the isotype control (FIG. 20).

[0235] Since almost all people with severe LC have neurological symptoms, the inventors also evaluated neuroinflammationin GF mice colonized with the microbiota from patients with severe or mild LC. As anticipated, the mice colonized with the severe LC microbiota had significantly increased inflammation in the brain as demonstrated by significantly increased detection by IF staining of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes in the hippocampus compared to mice with mild LC (image not shown, quantification shown in FIG. 21A). Increased neuroinflammation in severe LC mice was also demonstrated by increased IF staining of the microglia marker ionized calcium-binding adaptor molecule 1 (IBA1) in the hindbrain compared to mice with mild LC (image not shown, quantification shown in FIG. 21 B). The IBA1 IF staining in brains from severe LC mice also reveals the presence of bushy and amoeboid-like activated microglia. Interestingly, treatment with anti-BAFF significantly decreased neuroinflammation as demonstrated by decreased staining of both GFAP and IBA1 in severe LC mice compared to untreated or isotype control-treated mice.

[0236] The scope of the claims should not be limited by the embodiments set forth in the examples but should be given the broadest interpretation consistent with the description as a whole.

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[0255] 16 Schneider P, et al. BAFF, a novel ligand of the tumor necrosis factor family, stimulates B cell growth. J Exp Med. 1999; 189:1747-1756.

Claims

CLAIMS:

1. Method for treating long COVID (LC) or at least one symptom thereof in a subject displaying a blood (i) level of secreted B-cell activating factor (BAFF) higher than a corresponding reference level; and (ii) level of membrane BAFF in white blood cells higher than a corresponding reference level; and at least one of blood (iii) level of precursorlike marginal zone B-cell (MZp) populations higher than higher than a corresponding reference level; (iv) level of zonulin higher than a corresponding reference level; (v) detectable level of anti-Mi-2 nuclear antigen autoantibodies; (vi) detectable level of anti-SmD autoantibodies; (vii) detectable level of anti-U1-snRNP-A autoantibodies; (viii) detectable level of anti-RCN2 autoantibodies; (ix) level of lipopolysaccharide-binding protein (LBP) higher than a corresponding reference level; (x) level of [3-D-glucan higher than a corresponding reference level; and (xi) level of A Proliferation Inducing Factor (APRIL) lower than a corresponding reference level, comprising administering a therapeutically effective amount of a BAFF inhibitor to the subject.

2. The method of claim 1 , wherein the BAFF inhibitor is an anti-BAFF monoclonal antibody.

3. The method of claim 1 or 2, wherein the at least one symptom thereof includes at least one of a neurological dysfunction, improvement of intestinal barrier dysfunction, and improvement of immune dysregulation associated with systemic autoimmunity.

4. A method of diagnosing a long COVID (LC) in a subject comprising detecting in a blood sample from the subject: (i) a level of secreted B-cell activating factor (BAFF) higher than a corresponding reference level; and (ii) a level of membrane BAFF on white blood cells higher than a corresponding reference level; and at least one of (iii) a level of precursor-like marginal zone B-cell (MZp) populations higher than higher than a corresponding reference level; (iv) a level of zonulin higher than a corresponding reference level; (v) a detectable level of anti-Mi-2 nuclear antigen autoantibodies; (vi) a detectable level of anti-SmD autoantibodies; (vii) a detectable level of anti-U1-snRNP-A autoantibodies; (viii) a detectable level of anti-RCN2 autoantibodies; (ix) a level of lipopolysaccharide-binding protein (LBP) higher than a corresponding reference level; (x) a level of [3-D-glucan higher than a corresponding reference level; and (xi) a level of A Proliferation Inducing Factor (APRIL) lower than a corresponding reference level, wherein detecting a level in (i) and (ii) higher than their corresponding reference level, and detecting at least one of (iii) to (x) higher than their corresponding reference level, and (xi) lower than its corresponding reference level is an indication that the subject suffers from a LC.

5. The method of any one of claims 1 to 4, wherein:(I) the LC is severe LC; and / or(II) the subject displays:(a) at least two of (iii) to (vii);(b) at least three of (iii) to (vii);(c) at least four of (iii) to (vii); or(d) all five of (iii) to (vii).

6. The method of any one of claims 1 to 5, wherein the subject is a human.

7. A diagnostic kit comprising (a) (i) a B-cell activating factor (BAFF) ligand; (ii) a ligand for each of CD3, CD19, CD1c, CD27, IgM, and CD10; (iii) a zonulin ligand; and at least one of (iv) an anti-Mi-2 nuclear antigen autoantibody ligand; (v) an anti-SmD autoantibody ligand; (vi) an anti-U1-snRNP-A autoantibody ligand; and (vii) an anti-RCN2 autoantibody ligand; and (b) optionally instructions to use the kit for diagnosing long COVID (LC).

8. The kit of the claim 7, wherein one or more of the ligands of (i) to (vii) is an antibody.

9. The kit of the claim 7, wherein LC is severe LC.

10. The kit of any one of claims 7 to 9, wherein the kit comprises:(a) at least two of (iv) to (vii);(b) at least three of (iv) to (vii);(c) at least four of (iv) to (vii); or(d) all five of (iv) to (vii).

11. The kit of any one of claims 7 to 10, wherein the kit further comprises one or more of (viii) a lipopolysaccharide-binding protein (LBP) ligand; (ix) a [3-D-glucan ligand; (x) an A Proliferation Inducing Factor (APRIL) ligand; (xi) one or more ligands for total memory B-cells populations; (xii) one or more ligands for resting switched memory B-cells; (xiii) one or more ligands for naive B-cells; and (xiv) one or more ligands for transitional immature B-cells.

12. The kit of claim 11, wherein one or more of the ligands of (vii) to (xiv) is an antibody.

13. Method of generating a mouse model of long COVID comprising administering fecal microbiota from a human subject diagnosed with long COVID into a germ-free mouse by oral gavage and allowing microbiota engraftment in the mouse for a least three weeks.

14. Mouse generated by the method as defined in claim 13.

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