WO2022117823A1

WO2022117823A1 - Methods and compositions for preventing and/or treating autoimmune diseases

Info

Publication number: WO2022117823A1
Application number: PCT/EP2021/084190
Authority: WO
Inventors: Melissa HANSON; Marine MEUNIER; Klaus Schwamborn
Original assignee: Valneva Se
Priority date: 2020-12-04
Filing date: 2021-12-03
Publication date: 2022-06-09

Abstract

Described herein are compositions and methods for prevention and/or treating autoimmune diseases, such as autoimmune diseases like Inflammatory bowel disease, e.g. Crohn's disease or Ulcerative Colitis.

Description

METHODS AND COMPOSITIONS FOR PREVENTING AND/OR TREATING AUTOIMMUNE DISEASES

FIELD

The disclosure relates to compositions and methods for prevention and/or treating autoimmune diseases, such as autoimmune diseases like inflammatory bowel disease (herein also referred to as “IBD”), e.g. Crohn’s disease or Ulcerative Colitis.

BACKGROUND

An autoimmune disease is a condition arising from an abnormal immune response to a functioning body part (“Autoimmune diseases fact sheet". Office on Women's Health. U.S. Department of Health and Human Services. 16 July 2012). There are at least 80 types of autoimmune diseases. Nearly any body part can be involved (Borgelt LM (2010). Women's Health Across the Lifespan: A Pharmacotherapeutic Approach. ASHP. p. 579. ISBN 978-1- 58528-194-7). Common symptoms include low grade fever and feeling tired. Often symptoms come and go.

The cause is generally unknown. Some autoimmune diseases such as lupus run in families, and certain cases may be triggered by infections or other environmental factors. Some common diseases that are generally considered autoimmune include celiac disease, diabetes mellitus type 1, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus (Hohlfeld R, Dornmair K, Meinl E, Wekerle H (February 2016). "The search for the target antigens of multiple sclerosis, part 1 : autoreactive CD4+ T lymphocytes as pathogenic effectors and therapeutic targets". The Lancet. Neurology. 15 (2): 198-209. doi: 10.1016/S1474-4422(15)00334-8. PMID 26724103. S2CID 20082472). The diagnosis can be difficult to determine.

Treatment depends on the type and severity of the condition. Nonsteroidal antiinflammatory drugs (NSAIDs) and immunosuppressants are often used. Intravenous immunoglobulin may also occasionally be used. While treatment usually improves symptoms, they do not typically cure the disease.

About 24 million (7%) people in the United States are affected by an autoimmune disease. Women are more commonly affected than men.

Inflammatory bowel disease (IBD) can be further categorized as Crohn's disease or ulcerative colitis. In both cases, individuals with IBD lose immune tolerance for normal bacteria present in the gut microbiome. In this case, the immune system attacks the bacteria and induces chronic inflammation, which has been linked to increased cancer risk (Franks AL, Slansky JE (April 2012). "Multiple associations between a broad spectrum of autoimmune diseases, chronic inflammatory diseases and cancer". Anticancer Research. 32 (4): 1119-36. PMC 3349285. PMID 22493341).

The global prevalence of inflammatory bowel disease (IBD) has been increasing since 2000, and IBD now affects up to 1 in 200 individuals in Western countries (Ng, S. C. et al. Worldwide incidence and prevalence of inflammatory bowel disease in the 21st century: a systematic review of population- based studies. Lancet 390, 2769-2778 (2018)). IBD encompasses two distinct disorders, Crohn’s disease (CD) and ulcerative colitis (UC), which differ in pathophysiology, affected parts of the gastrointestinal (GI) tract, symptoms, complications, disease course and management. The cause of CD is still unclear but genetic, immunological and environmental factors contribute to risk of disease onset and progression (Torres, J., Mehandru, S., Colombel, J.- F. & Peyrin- Biroulet, L. Crohn’s disease. Lancet 389,1741-1755 (2017)). CD is characterized by skip intestinal lesions (that is, areas of inflammation interposed between normal- appearing mucosa) anywhere in the GI tract, and involves chronic, relapsing transmural inflammation that can lead to chronic abdominal pain, diarrhea, obstruction and/or perianal lesions. UC affects only the colon, the lesions are continuous and inflammation is superficial, which can lead to erosions, ulcers and bloody diarrhea. CD is progressive and destructive — 21-47% of patients also present with systemic, extraintestinal manifestations (EIMs), which strongly affect patients in a multitude of ways, such as their quality of life (QOL) and long- term outcomes, including risk of hospitalization, complications and surgery. Furthermore, half of all patients with CD develop intestinal complications, such as strictures or fistulae, within 10 years of diagnosis. Population- based cohort studies have demonstrated that up to 30% of patients with CD have evidence of bowel damage at diagnosis, and half of these patients require surgery in the 20 years following the diagnosis (Thia, K. T., Sandborn, W. J., Harmsen, W. S., Zinsmeister, A. R. & Loftus, E. V. Risk factors associated with progression to intestinal complications of Crohn’s disease in a population- based cohort. Gastroenterology 139, 1147-1155 (2010)) and (Fiorino, G., Bonifacio, C., Peyrin- Biroulet, L. & Danese, S. Preventing collateral damage in Crohn’s disease: the Lemann index. J. Crohns Colitis 10, 495-500 (2016)). CD most often presents in patients younger than 30 years, although the incidence is increasing in older individuals. Higher incidence has been reported for Ashkenazi Jews, urban populations and those in northern latitudes, with a peak between the second decade and the fourth decade of life. Many studies have failed to find any sex difference in incidence in Western countries, whereas the incidence of CD is higher in men than in women in Asian populations.

A number of approaches in this field are in development such as targeting of regulatory T cells, interfering with immune trafficking, providing cytokines or activating pathways to increase or reduce cytokines, modulation of immune cells, regenerative approaches such as wound healing, alteration of the microbiome, and activating or modulating the innate immune system. Although many attempts have been made and or in the development in order to improve the outcome for these subjects, there is a large unmet need to improve the prevention but also the treatment in the different stages of IBD or other autoimmune diseases.

Vibrio cholerae is a Gram-negative, comma-shaped bacterium. The bacterium's natural habitat is brackish water or saltwater where they attach themselves easily to the chitin-containing shells of crabs, shrimps, and other shellfish. Some strains of V. cholerae cause the disease cholera, which can be derived from the consumption of undercooked or raw marine life species. V. cholerae is a facultative anaerobe and has a flagellum at one cell pole as well as pili. V. cholerae can undergo respiratory and fermentative metabolism. When ingested, V. cholerae can cause diarrhea and vomiting in a host within several hours to 2-3 days of ingestion. Cholera toxin (also known as choleragen and sometimes abbreviated to CTX, Ctx or CT) is AB5 multimeric protein complex secreted by the bacterium V. cholerae. CTX is responsible for the massive, watery diarrhea characteristic of cholera infection. It is a member of the Heat-labile enterotoxin family. The complete toxin is a hexamer made up of a single copy of the A subunit (part A, enzymatic, P01555), and five copies of the B subunit (part B, receptor binding, P01556), denoted as AB5. Subunit B binds while subunit A activates the G protein which activates adenylate cyclase. The three-dimensional structure of the toxin was determined using X-ray crystallography by Zhang et al. in 1995 (Zhang R, Scott D, Westbrook M, Nance S, Spangler B, Shipley G, Westbrook E (1995). "The three- dimensional crystal structure of cholera toxin". J Mol Biol. 251 (4): 563-73. doi: 10.1006/jmbi.1995.0456. PMID 7658473). The five B subunits — each weighing 11 kDa, form a five-membered ring. The A subunit, which is 28 kDa, has two important segments. The Al portion of the chain (CTA1) is a globular enzyme payload that ADP-ribosylates G proteins, while the A2 chain (CTA2) forms an extended alpha helix which sits snugly in the central pore of the B subunit ring. This structure is similar in shape, mechanism, and sequence to the heat-labile enterotoxin secreted by some strains of the Escherichia coli bacterium. The oral cholera vaccines are generally of two forms: inactivated and attenuated. Two variants of the inactivated oral vaccine currently are in use: WC-rBS and BivWC. WC- rBS (marketed as "Dukoral®") is a monovalent inactivated vaccine containing killed whole cells of V. cholerae 01 plus additional recombinant cholera toxin B subunit. BivWC (marketed as "Shanchol®" and "mORCVAX®") is a bivalent inactivated vaccine containing killed whole cells of V. cholerae 01 and V. cholerae 0139. mORCVAX is only available in Vietnam. ORCVAX, i.e. bacterial strains of both Inaba and Ogawa serotypes and of El Tor and Classical biotypes are included in the vaccine. Dukoral® is taken orally with bicarbonate buffer, which protects the antigens from the gastric acid. The vaccine acts by inducing antibodies against both the bacterial components and CTB. The antibacterial intestinal antibodies prevent the bacteria from attaching to the intestinal wall, thereby impeding colonization of V. cholerae 01. The anti -toxin intestinal antibodies prevent the cholera toxin from binding to the intestinal mucosal surface, thereby preventing the toxin-mediated diarrhoeal symptoms.

A live, attenuated oral vaccine (CVD 103-HgR or Vaxchora), derived from a serogroup 01 classical Inaba strain, was approved by the US FDA in 2016 and by the EMA in 2020.

Recombinant CTB was tested in an open-label, multicentre, nonrandomized trial including 15 patients with mild/moderate CD. Patients received an oral solution of 5 mg recombinant cholera toxin B subunit three times weekly for 2 weeks. Reduction in CD Activity Index (CD Al) with >100 between baseline and days 15, 29, 42 and 70 defined clinical response. Patients with CD Al score <150 were defined as being in remission. A significant decrease in CD Al score was observed. Response rates were 40% in the full analysis set and 42% in the per protocol analysis. Two patients receiving adjuvant treatment after day 29 were excluded, after which 40% were in remission at 4 weeks and 30% at 8 weeks post-treatment. Mild side effects (arthralgia, headache and pruritus) were seen in 33% of patients (Clinical trial: the safety and short-term efficacy of recombinant cholera toxin B subunit in the treatment of active Crohn’s disease. Aliment Pharmacol & Ther 31(3): 387- 395P. Stal, R. Befrits, A. Ronnblom, A. Danielsson, O. Suhr, D. Stahlberg, A. Brinkberg, R. Lofberg. 03 January 2010 https://doi.Org/10. l l l l/j.1365-2036.2009.04185.x).

SUMMARY

Autoimmune disease therapeutics and preventatives for use in humans is a field where improved clinical options and solutions are in high need. Despite recent progress with various immunomodulatory agents, alternative solutions and compositions, e.g. with agents as described herein are very important. Accordingly, new methods of treating and/or preventing autoimmune diseases or enhancing survival of patients with autoimmune diseases such as Crohn’s diseases (herein also referred to simply as “CD”) are desired.

The present disclosure provides methods of treating and/or preventing autoimmune diseases such as CD involving administering compositions comprising at least one strain of Vibrio choleras, or compositions comprising at least one strain of Vibrio choleras and cholera toxin. Also provided herein are methods of enhancing survival of subjects having an autoimmune disease such as CD involving administering compositions comprising at least one strain of Vibrio choleras, compositions comprising cholera toxin, or compositions comprising a combination of at least one strain of Vibrio cholerae and cholera toxin. We herein postulate that administration of at least one strain of Vibrio cholerae in the context of autoimmune disease such as CD i.e., through stimulation of regulatory T cells and/or tolerogenic monocytes, macrophages and dendritic cells in a subject in need thereof, or administration of at least one strain of Vibrio cholerae and cholera toxin (e.g. cholera toxin subunit B)), results in a reduction of inflammation and/or induction of tolerance and is a major process that is able to provide an improvement to a subject who is at risk of developing or already has an autoimmune disease. Alternative mechanisms are improvement of the microbiome, regeneration of the epithelial cell wall and/or rebalancing the innate immune system. One of the ways to measure this improvement is by measuring the amount of TNF- alpha and/or IL- 10 of a subject within a group of such subjects who are at risk of developing or already have autoimmune disease (e.g., have been diagnosed with an autoimmune disease).

Also the present disclosure provides methods of treating and/or preventing autoimmune diseases such as CD involving administering compositions comprising lipopolysaccharide (LPS) from at least one strain of Vibrio cholerae, or compositions comprising lipopolysaccharide (LPS) from at least one strain of Vibrio cholerae and cholera toxin. Furthermore, herein provided are methods of enhancing survival of subjects having an autoimmune disease such as CD involving administering compositions comprising lipopolysaccharide (LPS) from at least one strain of Vibrio cholerae, compositions comprising cholera toxin, or compositions comprising a combination of LPS from at least one strain of Vibrio cholerae and cholera toxin. We herein postulate that administration of the LPS in the context of autoimmune disease such as CD i.e., through stimulation of regulatory T cells and/or promotion of anti-inflammatory monocytes, macrophages and dendritic cells in a subject in need thereof, and/or cholera toxin (e.g. cholera toxin subunit B)), results in a reduction of inflammation and/or induction of tolerance and is a major process that is able to provide an improvement to a subject who is at risk of developing or already has an autoimmune disease. Alternative mechanisms are improvement of the microbiome, regeneration of the epithelial cell wall and/or rebalancing the innate immune system. One of the ways to measure this improvement is by measuring the amount of TNF-alpha and/or IL- 10 of a subject within a group of such subjects who are at risk of developing or already have autoimmune disease (e.g., have been diagnosed with an autoimmune disease).

Compositions for use in accordance with the methods described herein comprise a sufficient amount of Vibrio choleras, LPS from Vibrio choleras, cholera toxin, or a combination of Vibrio cholerae and cholera toxin, or a combination of LPS from Vibrio cholerae and cholera toxin, wherein the LPS from Vibrio cholerae is associated with inactivated or live-attenuated variants of a strain of Vibrio cholerae. Commercial products that may be used in subjects in need thereof are products with the tradenames Dukoral®, Shanchol®, Euvichol®, and/or Vaxchora®. Dosage strength may be a strength used for the vaccine but preferably may be higher or may be administered more frequently than currently done, i.e., may be administered three times or more (e.g., 3-, 4-, 5-, 6- or 7- times or more).

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. The figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

Figure 1 Comparison of cytokine secretion profile from PBMCs pretreated with media alone, purified LPS from E. coli, or inactivated V. cholerae of Dukoral®, and subsequently challenged with A. coli LPS. A) TNFa cytokine levels in cell supernatants 6 hrs post-LPS challenge. B) IL- 10 cytokine levels in cell supernatant 24 hrs post-LPS challenge, n = 3 per group. Data shown are representative of two independent experiments with different human PBMC donors.

Figure 2 Comparison of cytokine secretion profile from THP-1 monocytes pretreated with media alone, purified LPS from E. coH, or inactivated V. choleras of Dukoral®, and subsequently challenged with A", coll LPS. A) TNFa cytokine levels in cell supernatants 24 hrs post-LPS challenge. 1-way ANOVA and Sidak comparisons were used to compare each group to all other groups. B) IL- 10 cytokine levels in cell supernatant 24 hrs post-LPS challenge, n = 3 per group. Data shown are representative of two independent experiments.

Figure 3 Comparison of cytokine secretion profile from PBMCs pretreated with media alone, inactivated E. coH, or purified LPS from E. coli at high (10 ng/mL) or low (0.9 ng/mL) doses; PBMCs were subsequently challenged with 1 pg/mL E. coli LPS. PBMCs were from two different donors (filled bars = Donor 1, empty bars = Donor 2). A) TNFa cytokine levels in cell supernatants 6 hrs post-LPS challenge. B) IL- 10 cytokine levels in cell supernatant 24 hrs post-LPS challenge, n = 3 per group.

Figure 4 Comparison of viability of THP-1 macrophages pretreated with media alone, purified LPS from E. coli, inactivated V. choleras of Dukoral® or inactivated E. coli, and subsequently challenged with E. coli LPS for 24 hours, n = 3 per group. Data is representative of two independent experiments.

Figure 5 Comparison of cytokine secretion profile from PBMCs pretreated with media alone, purified LPS from E. coli, recombinant CTB and E. coli LPS, or inactivated Dukoral®, and subsequently challenged with E. coli LPS. A) Pro-inflammatory TNFa cytokine levels 6 hrs post-LPS challenge. B) Anti-inflammatory IL-10 cytokine levels 24 hrs post-LPS challenge, n = 3 per group. Data shown are representative of two independent experiments with different human PBMC donors.

Figure 6 Evaluation of anti-inflammatory properties of V. cholerae strains in a DSS mouse model. C57BL/6 mice orally received DSS (Dextran sulfate sodium) from day 0 to day 5 in parallel to the administration of V. cholerae strains of Dukoral®; or control (PBS, vehicle); on days -6, -3, 0, 3 & 6. At the end of the experiment (day 12), mice were monitored for weight loss, clinical score and stool consistency to determine the Disease Activity Index (DAI). An inflammation negative control group didn’t receive the DSS treatment neither the

V. cholerae strains, n = 10 per group, statistics: permutation test

DETAILED DESCRIPTION

Aspects of the present disclosure provide methods of treating and/or preventing autoimmune diseases such as CD involving administering to a subject in need thereof a therapeutically effective amount of a composition comprising lipopolysaccharide from at least one strain of Vibrio cholerae or a composition comprising a combination of at least one strain of Vibrio cholerae and cholera toxin. Also provided herein are methods of enhancing survival of subjects having an autoimmune disease such as CD by administering compositions comprising at least one strain of Vibrio cholerae or compositions comprising a combination at least one strain of Vibrio cholerae and cholera toxin.

Another aspect of the present disclosure provides methods of treating and/or preventing autoimmune diseases such as CD involving administering to a subject in need thereof a therapeutically effective amount of a composition comprising lipopolysaccharide from at least one strain of Vibrio cholerae or a composition comprising a combination of lipopolysaccharide from at least one strain of Vibrio cholerae and cholera toxin. Also provided herein are methods of enhancing survival of subjects having an autoimmune disease such as CD by administering compositions comprising lipopolysaccharide from at least one strain of Vibrio cholerae or compositions comprising a combination of lipopolysaccharide from at least one strain of Vibrio cholerae and cholera toxin.

Vibrio cholerae

V. cholerae is a Gram-negative, curved rod-shaped bacterium with a polar flagellum. It is a facultative anaerobe and tends to tolerate alkaline media but is sensitive to acid (Finkelstein, Medical Microbiology “Cholera, Vibrio cholerae 01 and 0139, and other Pathogenic Vibrios; 4^th Edition U.T. Medical Branch at Galveston (1996)). V. cholerae may be classified into distinct groups based on the structure of the O antigen of the LPS. In general, V. cholerae strains are classified as serogroup 01, serogroup 0139, or non-01/non- 0139 based on agglutination of the bacterial cells (or lack thereof) in 01 and/or 0139 antiserum. The non-Ol/non-O139 strains have been divided into groups 02 through 0138 based on the lipopolysaccharide (LPS) somatic (O) antigen. The majority of non-01/non- 0139 strains are not associated with cholera disease.

In some embodiments, the V. cholerae strain is V. cholerae 01. In some embodiments, the V. cholerae strain is V. cholerae 0139. In some embodiments, the V. cholerae belongs to a non-01 serogroup. Examples of non-01 serogroups include the 02, 03, 04, 05, 06, 07, 08, 09, 010, Oi l, 012, 013, 014, 015, 016, 017, 018, 019, 020, 021, 022, 023, 024, 025, 026, 027, 028, 029, 030, 031, 032, 033, 034, 035, 036, 037,

038, 039, 040, 041, 042, 043, 044, 045, 046, 047, 048, 049, 050, 051, 052, 053, 054,

055, 056, 057, 058, 059, 060, 061, 062, 063, 064, 065, 066, 067, 068, 069, 070, 071,

072, 073, 074, 075, 076, 077, 078, 079, 080, 081, 082, 083, 084, 085, 086, 087, 088,

089, 090, 091, 092, 093, 094, 095, 096, 097, 098, 099, 0100, 0101, 0102, 0103, 0104, 0105, 0106, 0107, 0108, 0109, OHO, 0111, 0112, 0113, 0114, 0115, 0116, 0117, 0118, 0119, 0120, 0121, 0122, 0123, 0124, 0125, 0126, 0127, 0128, 0129, 0130, 0131, 0132, 0133, 0134, 0135, 0136, 0137, and 0138 groups.

As will be evident to one of ordinary skill in the art, in some embodiments, the compositions described herein may contain V. cholerae belonging to different O groups. In some embodiments, the compositions comprise V. cholerae 01 and one or more strains of V. cholerae belonging another O group.

The V. cholerae 01 group contains two major biotypes, El Tor and classical, each of which can be further distinguished into three serotypes based on the composition of the O antigen: Inaba, Ogawa, and Hikojima. Bacterial cells of each of the serotypes express the common “A” antigen; cells of the Ogawa serotype also express the “B” antigen i.e. express A+B antigens; cells of the Inaba serotype also express the “C” antigen, i.e. express A+C antigens; and cells of the Hikojima serotype express also the “B” and “C” antigens, i.e. express A+B+C antigens.

In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 El Tor biotype and at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype. In some embodiments, the compositions described herein comprise at least one (e.g, 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor biotype. In some embodiments, the compositions described herein comprise at least one e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical Hikojima biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 El Tor Hikojima biotype.

Regulatory T cells (Tregs), characterized as CD4+, CD25 high and Foxp3+, downregulate immune responses to both foreign and self-antigens. Tregs play important roles in autoimmune disorders and allergies including the ability of transferred Tregs to clear inflammatory lesions in IBD models (Yamada et al. (2016) “Role of regulatory T cell in the pathogenesis of inflammatory bowel disease.” World Journal of Gastroenterology 22: 2195- 2205). Despite that bacteria with its LPS are typically associated with an acute pro- inflammatory effect, it was reported also that exposure of bacteria to particular cell types, such as CD4+ CD25+ cells (e.g., Tregs), promotes survival and proliferation of these cells (Caramalho et al. (2003) “Regulatory T cells selectively express toll-like receptors and are activated by lipopolysaccharide.” J. Exp. Med. 197(4): 403-11; Lavelle et al. (2004) “Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent.” J. Leukocyte Biology 75: 756-763). Furthermore, both in vitro and in vivo, exposure to low doses of LPS can promote tolerance and prevent toxicity from endotoxin challenge (Lopez-Collazo E. and del Fresno C. (2013) “Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences.” Critical Care 17: 242-253). Without wishing to be bound by any particular theory, we herein postulate that administration of at least one strain of V. cholerae (possibly in conjunction with the cholera toxin (e.g., cholera toxin subunit B)), in the context of autoimmune disease i.e., through stimulation of regulatory T cells and/or promotion of tolerogenic monocytes, macrophages and dendritic cells in the subject results in a reduction of inflammation and is a major process providing an improvement in the autoimmune disease setting and thus overall improves the life conditions and/or provides numerous benefits for a subject in need thereof, i.e. having (or is at risk of developing) au autoimmune disease such as CD.

The phrase “at least one strain of V. cholerae" refers to V. cholerae, which may be in the form of whole V. cholerae bacteria (inactivated strains (heat and/or formalin inactivated) and/or live attenuated strains) or outer membrane vesicles (OMVs) (Chatterjee D et al. Genetic Engineering & Biotechnology News (2011): 1357-1362). A preferred embodiment is a composition comprising V. cholerae bacterial cells, e.g., in the form of whole V. cholerae bacteria (inactivated strains (heat and/or formalin inactivated) and/or live attenuated strains) that comprise LPS and/or cholera toxin such as subunit B (CTB) (see e.g., Chatteijee D et al. Genetic Engineering & Biotechnology News (2011): 1357-1362). More preferred embodiments are methods of treatment of (or uses in) autoimmune disease subjects in need thereof with compositions comprising inactivated 01 strains (of biotypes El Tor and/or classical; and subtypes Inaba and/or Ogawa inactivated by formalin and/or heat). Most preferred embodiments are methods of treatment of (or uses in) autoimmune disease subjects in need thereof with Cholera vaccines known in certain jurisdictions such as the US and the EU under the trademarks Dukoral®, Shanchol®, Euvichol® and/or Vaxchora®.In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. choleras 01 El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. choleras 01 classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 El Tor biotype and at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical Hikojima biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 El Tor Hikojima biotype and cholera toxin.

In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 classical biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and/or classical biotype and at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139.

In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba El Tor biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa El Tor biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba classical biotype. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa classical biotype.

In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae Olclassical biotype and cholera toxin. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139. In some embodiments, the compositions described herein comprise at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and/or classical biotype and at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139 and cholera toxin.

In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa classical biotype and cholera toxin.

In some embodiments, the compositions described herein comprise at least two strains, wherein at least one of the strains belongs to V. cholerae El Tor biotype and at least one of the strains belongs to V. cholerae classical biotype. In some embodiments, the compositions described herein comprise at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Ogawa classical biotype. In some embodiments, the compositions described herein comprise at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise at least two strains, wherein at least one of the strains belongs to V. cholerae Inaba classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise a combination of at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae El Tor biotype and at least one of the strains belongs to V. cholerae classical biotype. In some embodiments, the compositions described herein comprise a combination of at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Ogawa classical biotype. In some embodiments, the compositions described herein comprise a combination of at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise a combination of at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise a combination of at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise a combination of at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise a combination of at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Inaba classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise three strains of V. cholerae. In some embodiments, the compositions described herein comprise at least three strains, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Ogawa classical biotype, and at least one strain belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise at least three strains, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise at least three strains, wherein at least one strain belongs to V. cholerae Ogawa classical biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise a combination of three strains of V. cholerae and cholera toxin. In some embodiments, the compositions described herein comprise a combination of at least three strains and cholera toxin, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Ogawa classical biotype, and at least one strain belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise a combination of at least three strains and cholera toxin, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise a combination of at least three strains and cholera toxin, wherein at least one strain belongs to V. cholerae Ogawa classical biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise four strains of V. cholerae. In some embodiments, the compositions described herein comprise five strains of V. cholerae. In some embodiments, the compositions described herein comprise six or more strains of V. cholerae.

In some embodiments, the compositions described herein comprise a combination of four strains of V. cholerae and cholera toxin. In some embodiments, the compositions described herein comprise a combination of five strains of V. cholerae and cholera toxin. In some embodiments, the compositions described herein comprise a combination of six or more strains of V. cholerae and cholera toxin. In some embodiments, the compositions described herein comprise LPS that is the form of whole V. choleras bacteria. As used herein, the term “whole V. choleras bacteria” refers to a population of bacteria that are substantially intact bacteria. In some embodiments, the whole V. cholerae bacteria have not been subjected to a process of bacteriolysis or have not been separated into distinct fractions or components. As will be appreciated by one of ordinary skill in the art, whole V. cholerae bacteria may include a portion of bacteria that are not in whole bacterial form, such as a portion of bacteria that have lysed. In some embodiments, the whole V. cholerae bacteria does not contain a substantial amount of lysed bacteria. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or up to 100% of the bacteria of the whole V. cholerae bacteria are in whole bacterial form (e.g., not lysed or fractionated).

Methods for quantifying the amount of whole bacteria in a composition are known in the art and include microscopy methods and assays for detecting bacterial components (e.g., nucleic acid, cytoplasmic components) indicative that the bacteria are not in whole bacterial form.

In some embodiments, the whole V. cholerae bacteria are killed or inactivated bacteria. In some embodiments, the whole V. cholerae bacteria are subjected to a process by which the bacteria is rendered dead or metabolically inactive. A variety of methods of killing or inactivating bacteria are known in the art. In some embodiments, the bacteria are inactivated by chemical inactivation, thermal inactivation, pH inactivation, ionizing radiation inactivation, or UV inactivation. The viability or activity of the bacteria following the process of killing or inactivation may be assessed, for example by viability staining or plating on growth medium.

In some embodiments, the whole V. cholerae bacteria are killed or inactivated by thermal inactivation. In some embodiments, the whole V. cholerae bacteria are heat inactivated. In general, heat inactivation of bacteria involves subjecting the bacteria to elevated temperatures for a duration of time sufficient to inactivate the bacteria or eliminate viability.

In some embodiments, the whole V. cholerae bacteria are killed or inactivated by chemical inactivation. Examples of chemical agents for use in chemically inactivating bacteria include, without limitation, formalin, alcohols, salt, antibiotics, and detergents. In some embodiments, the whole V. cholerae bacteria are chemically inactivated. In general, chemical inactivation of bacteria involves subjecting the bacteria to a chemical agent (e.g., formalin) under conditions for a period of time sufficient to inactivate the bacteria or eliminate viability. In some embodiments, the whole V. cholerae bacteria are formalin- inactivated.

As will be appreciated by one of ordinary skill in the art, in some embodiments, each of the V. cholerae strains of a composition may be inactivated by the same or different method. For example, in some embodiments, the composition may comprise V. cholerae bacteria that have been thermally-inactivated. In some embodiments, the composition may comprise V. cholerae bacteria that have been heat-inactivated. In some embodiments, the composition may comprise at least one V. cholerae strain that has been heat-inactivated. In some embodiments, each of the V. cholerae strains of the composition have been heat- inactivated. In some embodiments, the composition may comprise V. cholerae bacteria that have been chemically inactivated. In some embodiments, the composition may comprise V. cholerae bacteria that have been formalin inactivated. In some embodiments, the composition may comprise at least one V. cholerae strain that has been formalin-inactivated. In some embodiment, each of the V. cholerae strains of the composition have been formalin- inactivated.

In some embodiments, the composition may comprise bacteria that have been heat- inactivated and bacteria that have been formalin-inactivated. In some embodiments, the composition may comprise bacteria of a V. cholerae strain that has been heat-inactivated and bacteria of the same V. cholerae strain that has been formalin-inactivated. In some embodiments, each of the V. cholerae strains have been inactivated using the same method.

In some embodiments, the whole V. cholerae bacteria are live, attenuated V. cholerae bacteria, such as lyophilized V. cholerae CVD 103-HgR (Chen et al. Clinical Infectious Diseases (2016) 62: 1329f).

Lipopolysaccharide or LPS

Lipopolysaccharide, also referred to as LPS, is a major component of the outer membrane of Gram-negative bacteria. Each LPS molecule is composed of a hydrophobic lipid section (lipid A), which is responsible for the toxic properties of the molecule; a hydrophilic core polysaccharide chain; and a repeating hydrophilic O-antigenic oligosaccharide side chain (O antigen) that is specific to the bacterial serotype.

LPS is generally considered to be a pro-inflammatory molecule, as the lipid A portion of LPS is the biologically active moiety that causes septic shock when present in the bloodstream. LPS binds the toll-like receptor 4 on immune cells such as macrophages and dendritic cells, resulting in the release of pro-inflammatory cytokines such as TNFa, IL-6, and IL-1.

Regulatory T cells (Tregs), characterized as CD4+, CD25 high and Foxp3+, downregulate immune responses to both foreign and self-antigens. Tregs play important roles in autoimmune disorders and allergies including the ability of transferred Tregs to clear inflammatory lesions in IBD models (Yamada et al. (2016) “Role of regulatory T cell in the pathogenesis of inflammatory bowel disease.” World Journal of Gastroenterology 22: 2195- 2205). Despite that LPS is typically associated with an acute pro-inflammatory effect and reports that LPS promotes liver metastasis in colorectal cancer by stimulating TLR4 signaling (Hsu YC et al. (2011) “LPS-induced TLR4 signaling in human colorectal cancer cells increases betal integrin-mediated cell adhesion and liver metastasis.” Cancer Research 71(5): 1989f), it was reported also that exposure of LPS to particular cell types, such as CD4+ CD25+ cells (e.g., Tregs), promotes survival and proliferation of these cells (Caramalho et al. (2003) “Regulatory T Cells Selectively Express Toll-like Receptors and Are Activated by Lipopolysaccharide.” J. Exp. Med. 197(4): 403-11; Lavelle et al. (2004) “Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent.” J. Leukocyte Biology 75: 756-763). Furthermore, both in vitro and in vivo, exposure to low doses of LPS can promote tolerance and prevent toxicity from endotoxin challenge (Lopez- Collazo E. and del Fresno C. (2013) “Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences.” Critical Care 17: 242-253). Without wishing to be bound by any particular theory, we herein postulate that administration of the LPS (possibly in conjunction with the cholera toxin (e.g., cholera toxin subunit B), in the context of autoimmune diseases i.e., through direct stimulation of regulatory T cells and/or the induction of tolerogenic intestinal monocytes, dendritic cells and macrophages in the subject results in a reduction of inflammation and is a major process providing an improvement in the cancer disease setting and thus overall improves the probability of survival for a subject in need thereof, i.e. having (or is at risk of developing) autoimmune disease such as CD.

The phrase “LPS from at least one strain of V. cholerae" refers to LPS produced by V. cholerae, which may be in the form of LPS isolated from bacterial cells or LPS associated with V. cholerae cells, e.g., in the form of whole V. cholerae bacteria (inactivated strains (heat and/or formalin inactivated) and/or live attenuated strains) or outer membrane vesicles (OMVs) released by V. cholerae (Chatteijee and Chauhuri. (2013) “Vibrio cholerae 0395 Outer Membrane Vesicles Modulate Intestinal Epithelial Cells in a NODI Protein-dependent Manner and Induce Dendritic Cell-mediated Th2/Thl7 Cell Responses.” J Biol Chem 288(6): 4299-4309). However, any type of LPS might be beneficial for the uses as disclosed herein, i.e. use in the prevention and/or treatment of autoimmune diseases such as CD. Also within the definition of LPS from at least one strain of V. choleras are mimitopes of LPS (i.e., LPS- mimicking peptides) as described by Ghazi (Ghazi and Gargari. (2017) “Synthetic peptides mimicking lipopolysaccharide as a potential vaccine candidates against Vibrio cholerae serogroup 01.” Iran J Microbiol 9(4): 244-250J. Pept. Sci. (2016): 22(11-12): 682-688) and others.

In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 El Tor biotype and at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical Hikojima biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 El Tor Hikojima biotype and cholera toxin.

In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor biotype. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 classical biotype. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and/or classical biotype and at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139.

In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba El Tor biotype. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. choleras 01 Ogawa El Tor biotype. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. choleras 01 Inaba classical biotype. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2,

3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa classical biotype.

In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strains belonging to V. cholerae 01 classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and at least one e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise LPS from at least one e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139. In some embodiments, the compositions described herein comprise LPS from at least one e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 El Tor and/or classical biotype and at least one e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 0139 and cholera toxin.

In some embodiments, the compositions described herein comprise a combination of LPS from at least one e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa El Tor biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3,

4, 5, or more) strain belonging to V. cholerae 01 Inaba classical biotype and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa classical biotype and cholera toxin.

In some embodiments, the compositions described herein comprise LPS from at least two strains, wherein at least one of the strains belongs to V. cholerae El Tor biotype and at least one of the strains belongs to V. cholerae classical biotype. In some embodiments, the compositions described herein comprise LPS from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Ogawa classical biotype. In some embodiments, the compositions described herein comprise LPS from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise LPS from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise LPS from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise LPS from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise LPS from at least two strains, wherein at least one of the strains belongs to V. cholerae Inaba classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise a combination of LPS from at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae El Tor biotype and at least one of the strains belongs to V. cholerae classical biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Ogawa classical biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least two strains and cholera toxin, wherein at least one of the strains belongs to V. cholerae Inaba classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise LPS from three strains of V. cholerae. In some embodiments, the compositions described herein comprise LPS from at least three strains, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Ogawa classical biotype, and at least one strain belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise LPS from at least three strains, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise LPS from at least three strains, wherein at least one strain belongs to V. cholerae Ogawa classical biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise a combination of LPS from three strains of V. cholerae and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from at least three strains and cholera toxin, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Ogawa classical biotype, and at least one strain belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least three strains and cholera toxin, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise a combination of LPS from at least three strains and cholera toxin, wherein at least one strain belongs to V. cholerae Ogawa classical biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise LPS from four strains of V. cholerae. In some embodiments, the compositions described herein comprise LPS from five strains of V. cholerae. In some embodiments, the compositions described herein comprise LPS from six or more strains of V. cholerae. In some embodiments, the compositions described herein comprise a combination of LPS from four strains of V. choleras and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from five strains of V. choleras and cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS from six or more strains of V. cholerae and cholera toxin.

In some embodiments, the compositions comprise LPS derived from or obtained from V. cholerae. For example, in some embodiments, the LPS may be isolated or separated from the V. cholerae bacteria. Methods of obtaining LPS from bacteria are known in the art, for example, the rapid isolation method, which involves gas chromatography and mass spectroscopy (see, e.g, Yi et al. Analyst. 2000 Apr;125(4):651-6), and hot aqueous-phenol extraction (see, e.g, Davis et al. J Vis Exp. (2012)(63): 3916). In some embodiments, the compositions comprise LPS derived from or obtained from V. cholerae as described previously and cholera toxin derived from or obtained from V. cholerae.

In some embodiments, the compositions described herein comprise LPS from more than one (e.g., 2, 3, 4, 5, or more) V. cholerae strain. In some embodiments, the compositions described herein comprise a combination of LPS from more than one (e.g., 2, 3, 4, 5, or more) V. cholerae strain and cholera toxin. In such embodiments, as will be appreciated by one of ordinary skill in the art, the LPS from each of the V. cholerae strains may be in the same or different form. For example, in some embodiments, the composition may comprise LPS from more than one V. cholerae strain, in which the LPS from each of the V. cholerae strains has been isolated from the V. cholerae bacteria. In some embodiments, the composition may comprise LPS from more than one V. cholerae strain, in which the LPS from each of the V. cholerae strains is present in the form of whole V. cholerae bacteria, meaning the composition comprises whole bacteria of more than one V. cholerae strain. In some embodiments, the composition may comprise LPS from more than one V. cholerae strain, in which the LPS from one (or more) of the V. cholerae strains has been isolated from the V. cholerae bacteria and the LPS from one (or more) of the V. cholerae strains is present in the form of whole V. cholerae bacteria.

Examples of compositions

Examples of compositions comprising LPS and/or cholera toxin are known in the art, e.g., Dukoral®, Vaxchora®, Shanchol® and/or Euvichol®. In some embodiment, the composition is the cholera vaccine Dukoral®, as e.g. described in WO 2011/034495A1 and summary of product characteristics of Dukoral® provided by EMA. In some embodiments, the composition is the cholera vaccine Vaxchora®. Examples of compositions comprising LPS and cholera toxin are known in the art, as e.g. described in summary of product characteristics of Vaxchora® provided by the FDA. In some embodiment, the composition is the cholera vaccine Shanchol®, as e.g. described in the officially approved packet inserts in licensed countries. In some embodiment, the compositions is the cholera vaccine Euvichol®, as e.g. described in the officially approved packet inserts in licensed countries.

In short, the above-mentioned four cholera vaccines contain the active ingredients as listed in Tables A-C.

Table A: Active ingredients and quantity of Dukoral®:

Table B: Active ingredients and quantity of Vachora®:

Table C: Active ingredients and quantity of Shanchol® and Euvichol®:

Cholera toxin

Aspects of the present disclosure provide methods of prevention and/or treating autoimmune diseases such as CD comprising administering compositions comprising a combination of at least one strain of Vibrio cholerae and cholera toxin, whereas various embodiments as outlined above are also included. Also provided are methods of enhancing survival of a subject having an autoimmune disease such as CD comprising administering compositions comprising a combination of at least one strain of Vibrio cholerae and cholera toxin, whereas various embodiments as outlined above are also included.

Cholera toxin is the main virulence factor produced by the CTX(|) bacteriophage residing in V. cholerae. Cholera toxin is composed of six protein subunits: a single copy of the A subunit and five copies of the B subunit. During infection with V. cholerae, the B subunit ring of the cholera toxin binds to target cells and the entire toxin complex is endocytosed by the cell, leading to release of the cholera toxin A subunit. Subunit B of cholera toxins is not toxic alone. Cholera toxin binds to human cells through interaction between the cholera toxin B subunit with GM1 ganglioside receptors on the cell surface.

Cholera toxin subunit B has adjuvant activity for mucosal vaccines; this may be due to the enhanced antigen presentation by various types of antigen-presenting cells, such as macrophages and dendritic cells (Bharati et al. Indian J. Med. Res. (2011) 133: 179-187; Baldauf et al. Toxins (2015) 7: 974-996). In addition to its adjuvant properties, cholera toxin subunit B may function as an anti-inflammatory agent by modulating specific signal transduction pathways and may function as an immunomodulatory agent when used for treatment of various autoimmune disorders (Bharati et al. Indian J. Med. Res. (2011) 133: 179-187; Baldauf et al. Toxins (2015) 7: 974-996). Oral administration of cholera toxin can upregulate the accumulation of macrophages, natural killer (NK) cells, and the regulatory T cells, as well as IL-10 production, and can downregulate the accumulation of neutrophils (Doulberis et al. Carcinogenesis (2015) 36(2): 280-290).

Administration of cholera toxin in the context of inflammation-associated carcinogenesis models has been shown to reduce tumor formation (see, e.g., Poutahidis et al. Oncolmmunology (2015) 4: 10, el027474 and Doulberis et al. Carcinogenesis (2015) 36(2): 280-290). Additionally, use of cholera toxin as an anti-inflammatory agent to treat inflammatory disease has been proposed (see, e.g., Royal et al. Toxins (2017) 9: 379). Recombinant CTB was tested in an open-label, multicentre, nonrandomized trial including 15 patients with mild/moderate CD. Patients received an oral solution of 5 mg recombinant cholera toxin B subunit three times weekly for 2 weeks. Reduction in CD Activity Index (CD Al) with >100 between baseline and days 15, 29, 42 and 70 defined clinical response. Patients with CD Al score <150 were defined as being in remission. A significant decrease in CD Al score was observed. Response rates were 40% in the full analysis set and 42% in the per protocol analysis. Two patients receiving adjuvant treatment after day 29 were excluded, after which 40% were in remission at 4 weeks and 30% at 8 weeks post-treatment. Mild side effects (arthralgia, headache and pruritus) were seen in 33% of patients (supra). The immunomodulatory function of CTB may be due to its specific properties, such as the ability of binding to specific GM1 ganglioside receptors present in the gut mucosa, and facilitating antigen uptake and presentation. It might be because of its ability to upregulate the accumulation of macrophages, natural killer (NK) cells, and the regulatory T cells, as well as IL- 10 production, and can downregulate the accumulation of neutrophils (Doulberis et al. Carcinogenesis (2015) 36(2): 280-290). Furthermore, previous studies have found that MAPK phosphatase- 1 expression can be induced by CTB alone and can subsequently inhibit the activation of Janus kinase and p38, thus leading to a substantial attenuation of TNFa and IL-6 production from macrophages (Chen et al. J. Immunol. (2002) 169:6408-6416).

In some embodiments, the compositions described herein comprise a subunit of cholera toxin. In some embodiments, the compositions described herein comprise subunit B of cholera toxin. In some embodiments, the compositions described herein comprise a subunit B variant of cholera toxin such as e.g. EPICERTIN - a recombinant variant of cholera toxin B subunit containing an ER retention motif (Hamorsky et al. PLoS Negl Trop Dis, (2013) 7(3):e2046). It was shown that oral administration of EPICERTIN promotes mucosal healing in the colon (Baldauf et al. (2017) Mucosal Immunol. 10(4):887-900, W02017/004168). Moreover, non-toxic variant of subunit A of the cholera toxin is also within the reach of this invention (Lebens et al. (2016) “Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae ” Vaccine 34(18): 2121-8. doi: 10.1016/j. vaccine.2016.03.002. Epub 2016 Mar 11 - Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae).

In some embodiments, the compositions described herein comprise a combination of LPS and a subunit of cholera toxin. In some embodiments, the compositions described herein comprise a combination of LPS and subunit B of cholera toxin.

As discussed in Baldauf et al. (Toxins (2015) 7: 974-996) the presence of cholera toxin or the presence of subunit A of cholera toxin in a preparation of subunit B of cholera toxin may affect one or more effect (e.g., stimulation of TNFa) of subunit B of cholera toxin. In some embodiments, the compositions described herein do not comprise subunit A of cholera toxin. In some embodiments, the compositions described herein do not comprise a detectable amount of subunit A of cholera toxin. In some embodiments, the compositions described herein do not comprise a substantial amount of subunit A of cholera toxin.

In some embodiments, any of the compositions described herein comprise subunit B of cholera toxin that is at least 95.0%, 95.5%, 96.0%, 96.5%, 97.0%, 97.5%, 98.0%, 98.5%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% pure. In some embodiments, any of the compositions described herein comprise subunit B of cholera toxin and less than 0.50%, 0.045%, 0.40%, 0.35%, 0.30%, 0.25%, 0.20%, 0.15%, 0.10%, 0.05%, 0.01% cholera toxin and/or subunit A of cholera toxin.

Cholera toxin, including subunits of cholera toxin, can be obtained by any method known in the art. For example, cholera toxin or subunits thereof may be isolated from V. cholerae strains that produce the toxin. In some embodiments, cholera toxin or subunits thereof may be recombinantly produced, for example by expressing the toxin or toxin subunit in a cell or expression system. In some embodiments, the cholera toxin subunit B is recombinantly produced. In some embodiments, the cholera toxin or subunits thereof may be recombinantly produced and added to a composition comprising LPS. In some embodiments, the cholera toxin or subunits thereof may be present in the LPS, for example in LPS obtained from V. cholerae. In some embodiments, the LPS is in the form of whole V. cholerae bacteria.

In some embodiments, as will be appreciated by one of ordinary skill in the art, the LPS and cholera toxin may be obtained from or derived from the same V. cholerae strain or different V. cholerae strains.

Also with the scope of the present disclosure are cholera toxin subunit B variants and cholera toxin subunit A variants. As used herein, the term “cholera toxin subunit B variant” or “cholera toxin subunit A variant” refers to a cholera toxin subunit B or cholera toxin subunit A having at least one amino acid mutation (e.g., insertion, deletion, substitution) relative to the amino acid sequence of a wild type or naturally occurring cholera toxin subunit B or cholera toxin subunit A. Examples of such variants are Epicertin, a recombinant variant of cholera toxin B subunit containing an ER retention motif (Hamorsky et al. PLoS Negl Trop Dis, 2013;7(3):e2046) and a non toxic variant of subunit A of the cholera toxin, mmCT (Lebens et al. “Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae ' Vaccine 2016 Apr 19;34(18):2121-8. doi: 10.1016/j.vaccine.2016.03.002. Epub 2016 Mar 11 - Construction and preclinical evaluation of mmCT, a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae).

As discussed herein, V. cholerae may be classified into distinct groups based on the structure of the O antigen of the LPS. In some embodiments, the cholera toxin is from a V. cholerae strain that is of serogroup V. cholerae 01. In some embodiments, the cholera toxin is from a V. cholerae strain that is of serogroup V. cholerae 0139. In some embodiments, the cholera toxin is from a V. cholerae strain that belongs to a non-01 serogroup. Examples of non-01 serogroups include the 02, 03, 04, 05, 06, 07, 08, 09, 010, Oi l, 012, 013, 014, 015, 016, 017, 018, 019, 020, 021, 022, 023, 024, 025, 026, 027, 028, 029, 030, 031,

032, 033, 034, 035, 036, 037, 038, 039, 040, 041, 042, 043, 044, 045, 046, 047, 048,

049, 050, 051, 052, 053, 054, 055, 056, 057, 058, 059, 060, 061, 062, 063, 064, 065,

066, 067, 068, 069, 070, 071, 072, 073, 074, 075, 076, 077, 078, 079, 080, 081, 082,

083, 084, 085, 086, 087, 088, 089, 090, 091, 092, 093, 094, 095, 096, 097, 098, 099,

0100, 0101, 0102, 0103, 0104, 0105, 0106, 0107, 0108, 0109, OHO, 0111, 0112,

0113, 0114, 0115, 0116, 0117, 0118, 0119, 0120, 0121, 0122, 0123, 0124, 0125,

0126, 0127, 0128, 0129, 0130, 0131, 0132, 0133, 0134, 0135, 0136, 0137, and 0138 groups.

As will be evident to one of ordinary skill in the art, in some embodiments, the compositions described herein may contain cholera toxin from strains of V. cholerae belonging to different O groups. In some embodiments, the compositions comprise cholera toxin from one or more strains of V. cholerae 01 and one or more strains of V. cholerae belonging another O group.

In some embodiments, the compositions described herein comprise cholera toxin from more than one (e.g., 2, 3, 4, 5, or more) V. cholerae strain. In some embodiments, the compositions described herein comprise cholera toxin from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Inaba El Tor biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Ogawa El Tor biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Hikojima classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least one (e.g., 1, 2, 3, 4, 5, or more) strain belonging to V. cholerae 01 Hikojima El Tor biotype.

In some embodiments, the compositions described herein comprise cholera toxin from at least two strains, wherein at least one of the strains belongs to V. cholerae El Tor biotype and at least one of the strains belongs to V. cholerae classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Ogawa classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa El Tor biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least two strains, wherein at least one of the strains belongs to V. cholerae Ogawa classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least two strains, wherein at least one of the strains belongs to V. cholerae Inaba classical biotype and at least one of the strains belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise cholera toxin from three strains of V. cholerae. In some embodiments, the compositions described herein comprise cholera toxin from at least three strains, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Ogawa classical biotype, and at least one strain belongs to V. cholerae Inaba classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least three strains, wherein at least one strain belongs to V. cholerae Ogawa El Tor biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype. In some embodiments, the compositions described herein comprise cholera toxin from at least three strains, wherein at least one strain belongs to V. cholerae Ogawa classical biotype, at least one strain belongs to V. cholerae Inaba classical biotype, and at least one strain belongs to V. cholerae Inaba El Tor biotype.

In some embodiments, the compositions described herein comprise cholera toxin from a strain belonging to V. cholerae 01 Inaba classical biotype, a strain belonging to V. cholerae 01 Inaba El Tor biotype, and/or a strain belonging to V. cholerae 01 Ogawa classical biotype. In some embodiments, the compositions described herein comprise cholera toxin from four strains of V. choleras. In some embodiments, the compositions described herein comprise cholera toxin from five strains of V. choleras. In some embodiments, the compositions described herein comprise cholera toxin from six or more strains of V. cholerae.

Methods of obtaining cholera toxin from bacteria are known in the art, for example, utilizing crossflow microfiltration followed by ion exchange chromatography (see, e.g., Jang et al, J Microbiol Biotechnol. 2009 Jan; 19(1): 108-112), and fractionation onto two successive phosphocellulose columns (see, e.g., Mekalanos et al. Infect Immun. 1978 May;20(2): 552-558).

In some embodiments, the compositions described herein comprise cholera toxin that is associated with whole V. cholerae bacteria. As used herein, the term “whole V. cholerae bacteria” refers to a population of bacteria that are substantially intact bacteria. In some embodiments, the whole V. cholerae bacteria have not been subjected to a process of bacteriolysis or have not been separated into distinct fractions or components.

In some embodiments, the composition comprises cholera toxin from strains belonging to V. cholerae 01 Inaba classical biotype; V. cholerae 01 Inaba, El Tor biotype; and V. cholerae 01 Ogawa classical biotype. In some embodiments, the composition comprises subunit B of cholera toxin from strains belonging to V. cholerae 01 Inaba classical biotype; V. cholerae 01 Inaba, El Tor biotype; and V. cholerae 01 Ogawa classical biotype. In some embodiments, the composition comprises recombinant cholera toxin having the same amino acid sequence as cholera toxin from strains belonging to V. cholerae 01 Inaba classical biotype; V. cholerae 01 Inaba, El Tor biotype; and V. cholerae 01 Ogawa classical biotype. In some embodiments, the composition is the cholera vaccine, Dukoral®. Examples of compositions comprising cholera toxin are known in the art, e.g., Dukoral®, Vaxchora®, Shanchol® and/or Euvichol®. In some embodiment, the composition is the cholera vaccine, Dukoral® as e.g. described in PCT Publication WO 2011/034495A1, summary of product characteristics of Dukoral® provided by EMA. In some embodiments, the composition is the cholera vaccine, Vaxchora®. Examples of compositions comprising cholera toxin are known in the art, e.g., summary of product characteristics of Vaxchora® provided by the FDA. In some embodiment, the composition is the cholera vaccine Shanchol®, as e.g. described in the officially approved packet inserts in licensed countries. In some embodiment, the composition is the cholera vaccine Euvichol®, as e.g. described in the officially approved packet inserts in licensed countries.

Methods of administration

Provided herein are methods for administering compositions comprising at least one V. choleras strain or compositions comprising a combination of at least one V. choleras strain and cholera toxin from at least one V. choleras strain to a subject in need thereof. As used herein, a “subject,” “individual,” and “patient” are used interchangeably, and refer to a vertebrate, preferably a mammal. In some embodiments, the subject is a mammalian subject, such as a human, non-human primate, rodent, rabbit, sheep, dog, cat, horse, or cow. In some embodiments, the subject is a human subject, such as a patient.

The presence of another disease or disorder may predispose a subject to having or being at risk of having an autoimmune disease, such as an autoimmune disease characterized by chronic inflammation such as CD. In some embodiments, a subject may be at risk of having an autoimmune disease if the subject has a chronic inflammatory disorder. In some embodiments, a subject may be at risk of having an autoimmune disease if the subject has a dysbalanced immune system.

In some embodiments, the subject has an autoimmune disease or is predisposed to (at risk of) developing an autoimmune disease. Examples of an autoimmune disease include, without limitation, inflammatory bowel disease such as CD or ulcerative colitis, rheumatoid arthritis juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, gastric ulcer, seronegative arthropathies, osteoarthritis, systemic lupus erythematosus, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/wegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, food intolerance, hypersensitivity pneumonitis, transplants, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, gram positive sepsis, gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage, bums, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory pathologies, sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis, atopic diseases, hypersensitivity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphylaxis, dermatitis, pernicious anemia, hemolytic disease, thrombocytopenia, graft rejection of any organ or tissue, kidney transplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant (BMT) rejection, skin allograft rejection, cartilage transplant rejection, bone graft rejection, small bowel transplant rejection, fetal thymus implant rejection, parathyroid transplant rejection, xenograft rejection of any organ or tissue, allograft rejection, anti-receptor hypersensitivity reactions, Graves disease, Raynaud's disease, type B insulin-resistant diabetes, asthma, myasthenia gravis, antibody- meditated cytotoxicity, type DI hypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary biliary cirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IN hypersensitivity , contact dermatitis, hypersensitivity pneumonitis, allograft rejection, granulomas due to intracellular organisms, drug sensitivity, metabolic/idiopathic, Wilson's disease, hemochromatosis, alpha- 1 -antitrypsin deficiency, diabetic retinopathy, hashimoto's thyroiditis, osteoporosis, hypothalamic-pituitary-adrenal axis evaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, chronic obstructive pulmonary disease (COPD), familial hemophagocytic lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerular ' nephritis, acute renal failure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy, anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy (e.g., including but not limited to asthenia, anemia, cachexia, and the like), chronic salicylate intoxication, sleep apnea, obesity, heart failure, sinusitis, , and the like. See, e.g., the Merck Manual, 12th-17th Editions, Merck & Company, Rahway, NJ (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al., eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each entirely incorporated by reference. A preferred use of the compositions of the invention is in the prevention and/or treatment of CD.

In some embodiments, the autoimmune disease is associated with or characterized by chronic inflammation. Inflammation is a condition caused by the body’s immune system as a response to injury or infection. Non-limiting symptoms associated inflammation include increased blood flow to the injury or infection site, leading redness and swelling, increased white blood cell production, and increased body temperature in the subject, in particular e.g. for CD it is believed to be driven by an imbalance of pro & anti-inflammatory responses in the intestinal epithelium. Inflammation can be categorized as either acute or chronic. Acute inflammation typically persists for only a few days to a few weeks. However, chronic inflammation may be long-term or reoccurring, and can last from several months or longer. Chronic inflammation may result, for example, from failure to eliminate the cause of acute inflammation, an autoimmune response to a self-antigen, or a chronic irritant of low intensity that persists. Chronic inflammation has been linked to numerous illnesses, including autoimmune diseases like CD.

Chronic inflammation is a critical component in progression of the autoimmune disease. The microenvironment of the affected area, which is largely controlled by inflammatory cells, is an indispensable participant in the process, fostering proliferation, survival, and migration of an autoimmune disease. Furthermore, autoimmune diseases, such as inflammatory bowel disease and Crohn’s disease, which may lead to colorectal cancer; and inflammatory conditions of uncertain etiology, such as prostatitis, which may lead to prostate cancer (Colotta et al, Carcinogenesis (2009) 30(70): 1073-81).

Whether a subject has an autoimmune disease or is at risk of having an autoimmune disease will be evident to one of skill in the art and may be determined by performing an assessment, such as, but not limited to a colonoscopy or a computed tomography (CT) scan. In some embodiments, the subject has one or more symptoms associated with an autoimmune disease, such as fatigue, joint pain and swelling, skin problems, abdominal pain or digestive issues, recurring fever, and swollen glands.

In some embodiments, the autoimmune disease is CD. In general, CD is herein considered an autoimmune disease and is a type of inflammatory bowel disease (IBD) that may affect any segment of the gastrointestinal tract from the mouth to the anus (National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). Archived from the original on December 8, 2019. Retrieved December 8, 2019). Symptoms often include abdominal pain, diarrhea (which may be bloody if inflammation is severe), fever, abdominal distension, and weight loss.

Other complications outside the gastrointestinal tract may include anemia, skin rashes, arthritis, inflammation of the eye, and fatigue. The skin rashes may be due to infections as well as pyoderma gangrenosum or erythema nodosum. Bowel obstruction may occur as a complication of chronic inflammation, and those with the disease are at greater risk of colon cancer and small bowel cancer. While the causes of Crohn's disease are unknown, it is believed to be caused by a combination of environmental, immune, and bacterial factors in genetically susceptible individuals. It results in a chronic inflammatory disorder, in which the body's immune system defends the gastrointestinal tract, possibly targeting microbial antigens (Marks DJ, Rahman FZ, Sewell GW, Segal AW (2010). "Crohn's disease: an immune deficiency state". Clinical Reviews in Allergy & Immunology. 38 (1): 20-31. doi: 10.1007/sl2016-009-8133-2. PMC 4568313. PMID 19437144). About half of the overall risk is related to genetics, with more than 70 genes having been found to be involved (Barrett JC, Hansoul S, Nicolae DL, Cho JH, Duerr RH, Rioux JD, et al. (2008). "Genome-wide association defines more than 30 distinct susceptibility loci for Crohn's disease". Nature Genetics. 40 (8): 955-62. doi: 10.1038/ng, 175. PMC 2574810. PMID 18587394). Tobacco smokers are twice as likely to develop Crohn's disease as nonsmokers. It also often begins after gastroenteritis (Baumgart DC, Sandborn WJ (August 2012). "Crohn's disease". Lancet.

380 (9853): 1590-605. doi: 10.1016/S0140-6736(12)60026-9. PMID 22914295). Diagnosis is based on a number of findings, including biopsy and appearance of the bowel wall, medical imaging, and description of the disease. Other conditions that can present similarly include irritable bowel syndrome and Behcet's disease.

There are no medications or surgical procedures that can cure Crohn's disease. Treatment options are intended to help with symptoms, maintain remission, and prevent relapse. In those newly diagnosed, a corticosteroid may be used for a brief period of time to rapidly improve symptoms, alongside another medication such as either methotrexate or a thiopurine used to prevent recurrence. Stopping smoking is recommended in people with Crohn's disease. One in five people with the disease is admitted to the hospital each year, and half of those with the disease will require surgery for the disease at some point over a ten- year period. While surgery should be used as little as possible, it is necessary to address some abscesses, certain bowel obstructions, and cancers. Checking for bowel cancer via colonoscopy is recommended every few years, starting eight years after the disease has begun.

In some embodiments, the subject has CD or is at risk of having CD. Whether a subject has CD or at risk of CD will be evident to one of skill in the art and may be determined by performing an assessment, such as a colonoscopy or biopsy. In some embodiments, the subject has one or more symptoms associated with CD, such as abdominal pain, diarrhea (which may be bloody if inflammation is severe), fever, abdominal distension, and weight loss. The presence of another disease or disorder may predispose a subject to having or being at risk of having CD. In some embodiments, a subject may be at risk of having CD if the subject has a chronic inflammatory disorder.

In some embodiments, the autoimmune disease is ulcerative colitis (herein also referred to simply as “UC”). Ulcerative colitis (UC) is a long-term condition that results in inflammation and ulcers of the colon and rectum. The primary symptoms of active disease are abdominal pain and diarrhea mixed with blood. Weight loss, fever, and anemia may also occur. Often, symptoms come on slowly and can range from mild to severe. Symptoms typically occur intermittently with periods of no symptoms between flares. Complications may include abnormal dilation of the colon (megacolon), inflammation of the eye, joints, or liver, and colon cancer. The cause of UC is unknown. Theories involve immune system dysfunction, genetics, changes in the normal gut bacteria, and environmental factors. Rates tend to be higher in the developed world with some proposing this to be the result of less exposure to intestinal infections, or to a Western diet and lifestyle. The removal of the appendix at an early age may be protective. Diagnosis is typically by colonoscopy with tissue biopsies. It is a kind of inflammatory bowel disease (IBD) along with Crohn's disease and microscopic colitis. Dietary changes, such as maintaining a high-calorie diet or lactose-free diet, may improve symptoms. Several medications are used to treat symptoms and bring about and maintain remission, including aminosalicylates such as mesalazine or sulfasalazine, steroids, immunosuppressants such as azathioprine, and biologic therapy. Removal of the colon by surgery may be necessary if the disease is severe, does not respond to treatment, or if complications such as colon cancer develop. Removal of the colon and rectum generally cures the condition.

The presence of another disease or disorder may predispose a subject to having or being at risk of having UC. In some embodiments, a subject may be at risk of having UC if the subject has a chronic inflammatory disorder.

In some embodiments, the methods include assessing whether a subject has cancer. In some embodiments, the subject may be assessed for cancer prior to administration of the compositions comprising LPS, compositions comprising cholera toxin, or the compositions comprising a combination of LPS and cholera toxin described herein. In some embodiments, if the subject is assessed as having cancer, any of the compositions comprising LPS, compositions comprising cholera toxin, or compositions comprising a combination of LPS and cholera toxin described herein may be administered to the subject. In some embodiments, the methods include determining whether a subject is predisposed to developing an autoimmune disease, such as a characterized by chronic inflammation. In some embodiments, determining whether a subject is predisposed to developing an autoimmune disease involves assessing the subject for an inflammatory condition associated with an autoimmune disease. In some embodiments, if the subject is determined to be predisposed to an autoimmune disease characterized by chronic inflammation, the subject is administered any of the compositions described herein. In some embodiments, determining whether a subject is predisposed to developing an autoimmune disease involves assessing the subject immune balance or dysbalance.

In some embodiments, the methods include determining the stage of the autoimmune disease. In some embodiments, the stage of the autoimmune disease may be determined prior to administration of any of the compositions described herein. In some embodiments, if the autoimmune disease is determined to be a particular stage, any of the compositions comprising at least a strain of Vibrio choleras, LPS derived from a strain of Vibrio choleras, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein may be administered to the subject. In some embodiments, a composition comprising at least a strain of Vibrio cholerae, LPS derived from a strain of Vibrio cholerae, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein is administered, if the autoimmune disease is determined to be of mild stage. In some embodiments, a composition comprising at least a strain of Vibrio cholerae, LPS derived from a strain of Vibrio cholerae, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein is administered, if the autoimmune disease is determined to be of moderate stage. In some embodiments, a composition comprising at least a strain of Vibrio cholerae, LPS derived from a strain of Vibrio cholerae, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein is administered, if the autoimmune disease is determined to be of severe stage.

In some embodiments, the methods include determining the stage of the CD or UC. In some embodiments, the stage of the CD or UC may be determined prior to administration of any of the compositions described herein. In some embodiments, if the CD or UC is determined to be a particular stage, any of the composition comprising at least a strain of Vibrio cholerae, LPS derived from a strain of Vibrio cholerae, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein may be administered to the subject. In some embodiments, a composition comprising at least a strain of Vibrio cholerae, LPS derived from a strain of Vibrio cholerae, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein is administered, if the autoimmune disease is determined to be mild stage CD or mild stage UC. In some embodiments, a composition comprising at least a strain of Vibrio cholerae, LPS derived from a strain of Vibrio cholerae, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein is administered, if the CD or UC is determined to be of moderate stage. In some embodiments, a composition comprising at least a strain of Vibrio cholerae, LPS derived from a strain of Vibrio cholerae, or compositions comprising a combination of at least a strain of Vibrio cholerae, or LPS derived from a strain of Vibrio cholerae and cholera toxin described herein is administered, if the CD or UC is determined to be of severe stage.

In some embodiments, the subject has undergone or is currently undergoing another therapy to treat the autoimmune disease (e.g., for CD is treated with an anti-TGF-alpha antibody). In some embodiments, the subject has undergone a surgical procedure to remove diseased tissue or tissue suspected of being diseased. In some embodiments, the subject has undergone or is currently undergoing an autoimmune disease therapy to treat the autoimmune disease. In some embodiments, this therapy is an immunotherapy or immune suppression therapy. In some embodiments, the subject has undergone one or more rounds of immunotherapy to treat the autoimmune disease.

Examples of immunotherapeutic agents e.g. for CD include, without limitation, prednisolone, methylprednisolone, IV hydrocortisone, budesonide, aminosalicylate: mesalamine, olsalazine, azathioprine, anti-IL-12, anti-IL-23, anti-TNF-alpha, anti-integrin such as an alpha 4 beta 7 integrin inhibitor, SIP receptor modulator, JAK1 inhibitor, antibiotic combinations and the like.

Any of the composition comprising at least a strain of Vibrio cholerae described herein may be administered to the subject twice or more (e.g., as a triple or quadruple dose). In some embodiments, any of the compositions comprising at least a strain of Vibrio cholerae described herein may be administered to the subject more than once (e.g., as multiple doses). In some embodiments, the composition comprising at least a strain of Vibrio cholerae

31 described herein is administered to the subject on at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more occasions.

Any of the composition comprising LPS derived from a strain of Vibrio choleras described herein may be administered to the subject twice or more (e.g, as a triple or quadruple dose). In some embodiments, any of the composition comprising LPS derived from a strain of Vibrio choleras may be administered to the subject more than once (e.g, as multiple doses). In some embodiments, composition comprising LPS derived from a strain of Vibrio cholerae described herein is administered to the subject on at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more occasions.

Any of compositions comprising a combination of LPS from at least one strain of V. cholerae and cholera toxin may be administered to the subject twice or more (e.g., as a triple or quadruple dose). In some embodiments, any of the compositions comprising a combination of LPS from at least one strain of V. cholerae and cholera toxin may be administered to the subject more than once (e.g., as multiple doses). In some embodiments, the composition comprising a combination of LPS from at least one strain of V. cholerae and cholera toxin is administered to the subject on at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more occasions.

Any of compositions comprising a combination of at least one strain of V. cholerae and cholera toxin may be administered to the subject twice or more (e.g., as a triple or quadruple dose). In some embodiments, any of the compositions comprising a combination of at least one strain of V. cholerae and cholera toxin may be administered to the subject more than once (e.g., as multiple doses). In some embodiments, the composition comprising a combination of at least one strain of V. cholerae and cholera toxin is administered to the subject on at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more occasions.

Each administration of any of the compositions described herein may be in the form of a “dose.” Any administration of the compositions described herein that is administered after the first administration may be referred to as a “booster.”

In some embodiments, the more than one administration of any of the compositions described herein are administered sequentially to the subject. In some embodiments, a subsequent administration of any of the compositions described herein is administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or at least 60 days after the first administration. In some embodiments, a subsequent administration of any of the compositions described herein is administered at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, or longer after the first administration. Determining whether a subject is in need of one or more additional administrations of any of the compositions described herein will be evident to one of ordinary skill in the art.

The compositions described herein may be administered by any route known in the art. Routes of administration include, but are not limited to, oral, intravenous, subcutaneous, parenteral, intratumoral, intramuscular, intranasal, intracranial, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal. In some embodiments, the compositions are orally administered to the subject.

Any of the methods or uses described herein may be for the prevention and/or treatment of an autoimmune disease in a subject. As used herein, the terms “treat” and “treating,” include the administration of a composition comprising at least one V. cholerae strain (or LPS from at least one V. cholerae strain), or a composition comprising a combination of at least one V. cholerae strain (or LPS from at least one V. cholerae strain) and cholera toxin to a subject to cure, ameliorate, prevent, reduce, or delay the onset of the symptoms, complications, pathologies or biochemical indicia of the disease (e.g., CD or UC, preferably CD), alleviating the symptoms or arresting or inhibiting further development of the disease, and/or reduce complications of the disease. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent or reduce the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease.

In some embodiments, administration of any of the compositions described herein to a subject reduces the burden, and/or complications of an autoimmune disease (e.g., CD or UC) in the subject by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to the burden, and/or complications of the autoimmune disease in the subject prior to administration with the composition (e.g. for CD measured by the CD Al (Crohn's Disease Activity Index). In some embodiments, administration of any of the compositions described herein to a subject reduces the burden, and/or complication of the autoimmune disease (e.g, CD) in the subject by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to the burden, and/or complication of the autoimmune disease in subjects that did not receive the compositions.

In some embodiments, administration of any of the compositions described herein to a subject reduces the severity or incidence of one or more symptoms associated with the autoimmune disease (e.g., CD or UC) in the subject by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to the severity or incidence of the symptoms in the subject prior to administration with the composition. In some embodiments, administration of any of the compositions described herein to a subject reduces the severity or incidence of one or more symptoms associated with the autoimmune disease such as CD or UC in the subject by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more as compared to the severity or incidence of the symptoms in subjects that did not receive the compositions.

The methods described herein may result in an improvement of the CD Al in the subject being administered the compositions as described herein.

Any of the compositions comprising LPS from at least one V. choleras strain, compositions comprising cholera toxin, or compositions comprising a combination of LPS from at least one V. choleras strain and cholera toxin described herein may be administered to a subject with, prior to, or after administration of an acid neutralizing agent. An acid neutralizing agent is any agent that reduces acidity (z.e., increases the pH, neutralizes). Examples of acid neutralizing agents include, without limitation, sodium bicarbonate (sodium hydrogen carbonate), sodium dihydrogen phosphate dihydrate, disodium hydrogen phosphate dihydrate, algeldrate (aluminium hydroxide), magnesium carbonate, calcium carbonate, magnesium hydroxide, and simethicone.

Determining whether subject who administered the compositions comprising at least one V. cholerae strain, or compositions comprising a combination of at least one V. cholerae strain and cholera toxin from at least one V. cholerae strain described herein are also administered an acid neutralizing agent may depend on factors, such as the route of administration and/or components of the compositions. For example, a composition that is administered by an oral route may encounter the acid environment of the stomach. In some embodiments, oral administration of an acid neutralizing agent reduces the acidity (e.g., increases the pH, neutralizes) of the stomach.

In some embodiments, any of the compositions comprising at least one V. cholerae strains also comprises an acid neutralizing agent. In some embodiments, any of the compositions comprising at least one V. cholerae strains may be resuspended in an acid neutralizing agent and the composition and acid neutralizing agent are administered simultaneously to the subject. In some embodiments, any of the compositions comprising a combination of at least one V. cholerae strains and cholera toxin also comprises an acid neutralizing agent. In some embodiments, any of the compositions comprising a combination of at least one V. cholerae strains and cholera toxin may be resuspended in an acid neutralizing agent and the composition and acid neutralizing agent are administered simultaneously to the subject.

Any of the compositions comprising at least one V. cholerae strain, compositions comprising cholera toxin, or compositions comprising a combination of at least one V. cholerae strain and cholera toxin described herein may be administered to a subject with, prior to, or after administration of one or more additional therapeutic agents. In some embodiments, compositions comprising at least one V. cholerae strain, or compositions comprising a combination of at least one V. cholerae strain and cholera toxin described herein may be administered to a subject with, prior to, or after administration of an immune suppressor.

Any of the compositions comprising at least one V. cholerae strain (e.g. LPS from at least one V. cholerae strain), or compositions comprising a combination of LPS from at least one V. cholerae strain and cholera toxin described herein may be administered to a subject in a therapeutically effective amount. As used herein, a “therapeutically effective amount” or an “effective amount” of composition is any amount that results in a desired response or outcome in a subject, such as those described herein, including but not limited to preventing or treating an autoimmune disease such as CD or UC. In some embodiments, the compositions comprising at least one V. cholerae strain, or compositions comprising a combination of at least one V. cholerae strain and cholera toxin may be formulated for administration in a pharmaceutical composition. The term “pharmaceutical composition” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g. at least one V. cholerae strain or a combination of at least one V. cholerae strain and cholera toxin, and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g. at least one V. cholerae strain, or a combination of at least one V. cholerae strain and cholera toxin, and a co-agent, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the 2 compounds in the body of the patient.

Pharmaceutical compositions of the disclosure, including vaccines, can be prepared in accordance with methods well known and routinely practiced in the art (see e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co. 20th ed. 2000; and Ingredients of Vaccines - Fact Sheet from the Centers for Disease Control and Prevention, e.g., adjuvants, enhancers, preservatives, and stabilizers). Pharmaceutical compositions are preferably manufactured under GMP conditions. The compositions described herein may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art.

The compositions comprising at least one strain of V. cholerae or compositions comprising a combination of at least one strain of V. cholerae and cholera toxins are typically administered to subjects as pharmaceutical compositions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. The nature of the pharmaceutical carrier and other components of the pharmaceutical composition will depend on the mode of administration. The pharmaceutical compositions of the disclosure may be administered by any means and route known to the skilled artisan in carrying out the treatment methods described herein. In some embodiments, the compositions are formulated for oral administration.

Formulations may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. In some embodiments, the compositions are present in a power or lyophilized form for resuspension prior to administration.

The compositions may be formulated for parenteral administration by injection. As used herein, “parenteral” administration includes, without limitation, subcutaneous, intracutaneous, intravenous, intratumoral, intramuscular, intraarticular, intrathecal, or by infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compositions in water soluble form. Additionally, suspensions of the active compositions may be prepared as appropriate oily injection suspensions. Alternatively, the active compositions may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. For oral administration, the compositions can be formulated readily by combining the compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the disclosure to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated. Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl- cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally, the oral formulations may also be formulated in saline or buffers, e.g., EDTA for neutralizing internal acid conditions, or may be administered without any carriers.

For oral delivery, the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films. A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic powder; for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

Dosage regimens are adjusted to provide the optimum desired response. Dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired pharmaceutical response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, 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 subject being treated, and like factors.

A physician, veterinarian or other trained practitioner, can start doses of compositions comprising at least one V. cholerae strain, or compositions comprising a combination of at least one V. cholerae strain and cholera toxin employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions disclosed herein, for the prophylactic and therapeutic treatment of groups of people as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy.

In some embodiments, the dosage of at least one V. cholerae strain, cholera toxin, or combination of at least one V. cholerae strain and cholera toxin in a composition refers to the amount of V. cholerae strain, or combination of V. cholerae strain and cholera toxin that is administered to the subject in the composition. It will be appreciated the amount of V. cholerae strain, or combination of V. cholerae strain and cholera toxin may be presented as the direct weight of V. cholerae strain, or combination of V. cholerae strain and cholera toxin molecules, respectively; the weight of bacterial cells as an indirect amount of V. cholerae strain, or combination of V. cholerae strain and cholera toxin, respectively; or as the quantity of bacterial cells in the composition, referring to the amount of V. cholerae strain, or combination of V. cholerae strain and cholera toxin, respectively, associated with the quantity of bacterial cells. In a preferred embodiment, the dosage of a V. cholerae strain and/or cholera toxin composition is that of Shanchol®, Euvichol®, Vaxchora® or Dukoral®. In a more preferred embodiment, the dosage of a V. cholerae strain and/or cholera toxin composition is that of Dukoral® (see Table A).

Table A: Active ingredients of Dukoral® vaccine (suspension)

By “Vibrio choleras of Dukoral®”, it is meant within the context of this invention, the following equal mix of bacteria: Vibrio choleras 01 Inaba, classical biotype (heat- inactivated), Vibrio cholerae 01 Inaba, El Tor biotype (formalin- inactivated), Vibrio cholerae 01 Ogawa, classical biotype (heat- inactivated), and Vibrio cholerae 01 Ogawa, classical biotype (formalin- inactivated).

In some embodiments, the compositions comprise between about 0.1 pg/mL - 10 mg cholera toxin such as e.g. the compositions comprise between about 0.1 pg/mL - 10 mg recombinant cholera toxin subunit B per dosage. In some embodiments, the compositions comprise 0.1 pg - 5 mg, 0.1 pg - 7 mg, 0.1 pg/mL - 3 mg, 0.2 pg - 4 mg cholera toxin such as e.g. recombinant cholera toxin subunit B per dosage. In some embodiments, the compositions contain about 1 mg cholera toxin such as e.g. recombinant cholera toxin subunit B per dosage.

In some embodiments, the compositions contain between 10⁵ and 10¹⁵ of each V. cholerae strain per dosage. In some embodiments, the compositions contain between 10⁵ and 10¹⁵, between 10⁶ and 10¹⁴, between 10⁷ and 10°, between 10⁸ and 10¹², between 10⁹ and

10¹¹ of each V. cholerae strain per dosage. In some embodiments, the compositions contain between IO¹⁰ and 10¹¹ bacterial cells per dosage. In some embodiments, the compositions contain approximately 3xlO¹⁰ cells of each V. cholerae strain per dosage.

In some embodiments, the compositions contain between 10⁵ and 10¹⁵ total V. cholerae cells per dosage. In some embodiments, the compositions contain between 10⁵ and 10¹⁵, between 10⁶ and 10¹⁴, between 10⁷ and 10¹³, between 10⁹ and 10¹², between IO¹⁰ and

10¹² total V. cholerae cells per dosage. In some embodiments, the compositions contain between 10¹¹ and 10¹² bacterial cells per dosage. In some embodiments, the compositions contain approximately 1.25xlOⁿ total V. cholerae cells per dosage.

In some embodiments, the compositions contain between 10⁵ and 10¹⁵ colonyforming units (CFUs) of live-attenuated V. cholerae per dosage. In some embodiments, the compositions contain between 10⁵ and 10¹⁵, between 10⁶ and 10¹⁴, between 10⁷ and 10°, between 10⁶ and 10⁷, between 10⁸ and 10⁹ total CFUs of live-attenuated V. cholerae per dosage. In some embodiments, the compositions contain between 10⁸ and 10⁹ bacterial cells per dosage. In some embodiments, the compositions contain approximately 5xl0⁸ total CFUs of V. cholerae per dosage.

The invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of “including,” “comprising,” or “having,” “containing,” “involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms hall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well- known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, microbiology, virology, cell or tissue culture, genetics and protein and nucleic chemistry described herein are those well- known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. However, the citation of any reference is not intended to be an admission that the reference is prior art. EXAMPLES

Materials and Methods

Cell lines, bacteria, vaccines & reagents

THP-1 cells were obtained from ATCC® (TIB-202™) and cultured following ATCC recommendations at 37°C and 5% CO2 in RPMI 1640 medium (Life Technologies, A10491- 01) containing 0.05 mM 2-mercaptoethanol (Sigma, M3148), 10% fetal bovine serum (SAFC, 12003C) and penicillin and streptomycin antibiotics (Lonza, DE17-602E). According to bibliography (Daigneault M, Preston JA, Marriott HM, Whyte MK, Dockrell DH (2010) “The Identification of Markers of Macrophage Differentiation in PMA-Stimulated; THP-1 Cells and Monocyte-Derived Macrophages.” PLoS ONE 5(1): e8668 doi: 10.1371/journal. pone.0008668), THP-1 monocytes were differentiated into THP-1 macrophages via incubation of cells for 3 days at 37°C and 5% CO2 in a 200 nM Phorbol 12- myristate 13 -acetate (PMA, Sigma, P8139) solution and then for rested for 4 additional days in a fresh complete growth medium.

Inactivated Vibrio cholerae strains ( cholerae strain Inaba 6973 El Tor and V. cholerae strain Ogawa 50 classical serotype 01, formalin inactivated; and V. cholerae strain Inaba 48 classical serotype 01 and V. cholerae strain Ogawa 50 classical serotype 01, heat inactivated) and Dukoral® were obtained from Valneva Soina entity. Inactivated E. coli (MBD0017) and A. coli LPS (L4391) were both purchased at Sigma. Bacterial number of inactivated E. coli was determined by regression after OD readings at 600 nm.

Isolation of PBMCs from healthy human donors

Human PBMCs were isolated from freshly sampled blood from normal healthy donors. Whole blood was centrifuged on a lymphocytes separation medium gradient (Eurobio - CMSMSL01-01) at 900 g for 25 min, followed by washing of the enriched cells in a PBS solution (Gibco, 10010-015) containing 1 mM EDTA (Gibco, 15575-038) and 2% FBS: one at 400 g for 8 minutes and two at 200 for 10 minutes. Cells were resuspended in RPMI 1640 (Sigma, R0883) supplemented with 10% FBS, 2 mM glutamine and penicillin and streptomycin antibiotics. In stimulation assays, isolated PBMCs were cultured at 37°C and 5% CO2 at a final concentration at IxlO⁶ cells/mL

Cytokine analyses

Following cells stimulation assays; TNFa and IL-10 cytokines concentrations in supernatants were determined by ELISA, using human DuoSet ELISA kits (R&D systems, DY210 and DY217B respectively), according to manufacturer recommendations and adapted to use high binding half-well plates (Greiner, 675061). Briefly, after an overnight coating with capture antibody and a saturation step, appropriately diluted samples and standards were incubated on plates for 2 hours at room temperature. After washing, detection antibody and then a streptavidin solution were incubated in wells before a revelation step using a TMB solution (SeraCare, 5120-0047) and H2SO4 (LCH Chimie, MC3067691000) as a stop solution. OD reading was performed at 450 and 570 nm and cytokines concentrations determined by regression from standard curves.

Endotoxin assay

Endotoxin levels of inactivated E. coli were determined using E-TOXATE™ semi- quantitative kit (Sigma, ET0200), according to manufacturer instructions. Briefly, appropriately diluted controls, samples and standards were mixed 1 : 1 with E-TOXATE reagent working solution in pyrogen free glass culture tubes (Lonza, N205). After 1 hour incubation at 37°C, evidence of gelation was observed for each control, standards and samples tubes. Endotoxin level was calculated by multiplying the inverse of the highest dilution of sample found positive by the lowest concentration of Endotoxin Standard found positive. The LPS concentration of inactivated Vibrio cholerae of Dukoral® was provided by the manufacturer, Valneva Soina.

Statistics

Statisctical analyses were performed using GraphPad Prism software. All values and error bars are mean ± standard deviation. Comparisons of experimental groups versus control group at a single time point were performed using 1-way ANOVA and Dunnet’s tests, unless otherwise noted.

Table 1: PBMC endotoxin challenge protocol

Table 2: THP1 Monocytes endotoxin challenge protocol

Table 3: THP1 Macrophages differentiation and endotoxin challenge protocol

Background Autoimmune diseases occur in the absence of immune system tolerance, when the immune system generates an inflammatory response to the body itself. Mucosal tolerance, in particular, is of critical importance as it occurs at the interface of self and non-self (e.g. the gut epithelium) where the immune system must simultaneously be tolerogenic to innocuous antigens yet recognize and effectively respond to dangerous pathogens (Rezende, R.M., Cox, L.M., & Weiner, H.L. “Mucosal tolerance therapy in humans: Past and future.” Clinical &

Experimental Neuroimmunology 10 (Suppl. 1), 20-31, (2019)). Both innate and adaptive immune cells play critical roles in the development and maintenance of tolerance: monocytes differentiate into tolerogenic macrophages and dendritic cells, which in turn, via signaling through IL- 10, IDO1, retinoic acid and TGF^_,-P induce naive T cells to a Treg phenotype. Treg cells are key effector cells to suppress inflammation and maintain a tolerogenic state (Wambre, E., Jeong, D. “Oral Tolerance Development and Maintenance.” Immunology and Allergy Clinics of North America. 38, 27 - 37, (2018)). Incomplete oral tolerance can lead to autoimmune diseases (such as IBD), IgE-mediated food allergies, celiac disease and infections (Wambre, E., Jeong, D. “Oral Tolerance Development and Maintenance.” Immunology and Allergy Clinics of North America. 38, 27 - 37, (2018)).

Lipopolysaccharides (LPS aka endotoxin) are large molecules of both lipid and polysaccharide which are ubiquitous in the membrane of gram-negative bacteria, however each bacterium has its own unique LPS structures. The lipid A portion of LPS binds to the TLR4/MD-2 complex of innate immune cells and is key for initiating inflammatory responses to gram-negative bacteria. One hallmark of oral tolerance is a hyporesponsivess of intestinal epithelial cells, macrophages and dendritic cells to LPS via down-regulation of TLR4 signaling (Cerovic C. et al. “Hyporesponsiveness of Intestinal Dendritic Cells to TLR Stimulation is Limited to TLR4.” Journal of Immunology 182, 2405-2415 (2009) and Lotz M., Giitle D., Walther S., Menard S., Bogdan C., Hornef M. “Postnatal acquisition of endotoxin tolerance in intestinal epithelial cells.” Journal of Experimental Medicine 203, 973-984 (2006)). In contrast, TLR4 expression and sensitivity is increased in active human Crohn’s disease and ulcerative colitis as well as in the presence of inflammatory cytokines such as TNFa and IFN-y (Cario, E. “Toll-like receptors in inflammatory bowel diseases: A decade later.” Inflammatory Bowel Disease 16, 1583-1597, (2010)).

Lack of response to LPS, termed endotoxin tolerance, can be induced both in vitro and in vivo via initial exposure to low doses of LPS prior to challenge with large LPS quantities (Mendes M. et al. “Differential expression of toll-like receptor signaling cascades in LPS-tol erant human peripheral blood mononuclear cells.” Immunobiology 216, 285-295, (2011) and Lopez-Collazo E. and del Fresno C. “Pathophysiology of endotoxin tolerance: mechanisms and clinical consequences.” Critical Care 17, 242-253 (2013)). Typically in vitro endotoxin tolerance models employ peripheral blood mononuclear cells (PBMCs) or a monocyte cell line (such as human THP-1 monocytes) to demonstrate induction of innate immune cell tolerance. In addition to low dose LPS, cholera toxin B (CTB) can also induce tolerance in these endotoxin challenge models (Burkart V. et al. “Cholera Toxin B Pretreatment of Macrophages and Monocytes Diminishes Their Proinflammatory Responsiveness to Lipopolysaccharide.” Journal of Immunology 168, 1730-1737, (2002), and Chen P., Li J., Barnes J., Kokkonen G., Lee J., Liu Y. “Restraint of Proinflammatory Cytokine Biosynthesis by Mitogen- Activated Protein Kinase Phosphatase- 1 in Lipopolysaccharide-Stimulated Macrophages.” Journal of Immunology 169, 6408-6416, (2002)).

Example 1: Analysis of immune response to inactivated V. cholerae pretreatment of peripheral blood mononuclear cells (PBMCs) upon endotoxin challenge

To investigate the tolerogenic capacity of the inactivated bacteria component of Dukoral®, an in vitro endotoxin tolerance models was implemented using PBMCs (Table 1). Briefly, PBMCs were isolated from healthy human donors and subjected either to LPS isolated from E. coH, or inactivated Vibrio cholerae of Dukoral®. Both groups contained 0.9 ng/mL endotoxin. After 16 hours of pretreatment, cells were challenged with 1 pg/mL of E. coli LPS. Supernatants were collected at 6 and 24 hours for analysis of cytokine secretion via ELISA. In previous experiments, 6 hours and 24 hours were established as the timepoints of peak expression levels for TNFa and IL-10, respectively.

As shown in Figure 1A, as compared to untreated samples, low doses of both E. coli LPS and V. cholerae of Dukoral® significantly suppressed TNFa secretion in PBMCs upon LPS challenge, demonstrating an ability to limit the inflammatory response to LPS challenge. However, only the V. cholerae of Dukoral® were capable of promoting anti-inflammatory responses, as demonstrated by the enhanced secretion of IL- 10 in Figure IB. Conversely, E. coli LPS actually suppressed IL- 10 secretion, thus low dose E. coli LPS pretreatment generates an anergic state rather than a functionally tolerogenic in this model.

Example 2: Analysis of immune response to inactivated V. cholerae pretreatment of THP-1 monocytes upon endotoxin challenge

As PBMCs constitute a mixture of T cells, B cells, monocytes, and other leukocytes, we sought to discern the tolerogenic properties of Dukoral® in a single cell type, using the human cell line THP-1 monocytes. As described in Table 2, THP-1 cells were pretreated with LPS isolated from E. coli, or inactivated Vibrio cholera of Dukoral®. Both groups contained 0.9 ng/mL endotoxin. After 16 hours of pretreatment, cells were challenged with 1 pg/mL of E. coli LPS. Supernatants were collected at 24 hours for cytokine quantification. As shown in Figure 2A, as compared to untreated samples, low doses of both E. coli LPS and V. cholerae of Dukoral® significantly suppressed TNFa secretion in THP-1 monocytes upon LPS challenge, demonstrating an ability to limit the inflammatory response to LPS challenge. However, the V. cholerae of Dukoral® suppressed TNFa secretion to a less than half the amount generated in the E. coli LPS group. Furthermore, V. cholerae of Dukoral® generate modest amounts of IL-10 in THP-1 cells, in contrast to the absence of IL- 10 signaling in cells pretreated with media alone or with E. coli LPS (Figure 2B).

Example 3: Analysis of immune response to inactivated E. coli pretreatment of PBMCs after endotoxin challenge

To determine if the tolerogenic properties of inactivated V. cholerae of Dukoral® were ubiquitous to all gram-negative bacteria, inactivated E. coli was tested in the LPS challenge mode; see Table 1 for protocol details. PBMCs were pretreated with inactivated A. coli containing 3.4 ng/mL LPS, or purified E. coli LPS at 0.9 ng/mL or 10 ng/mL (summarized in Table 4). After 16 hours of pretreatment, cells were challenged with 1 pg/mL of E. coli LPS. Supernatants were collected at 6 and 24 hours for analysis of cytokine secretion via ELISA. As shown in Figure 3A, all three treatment groups significantly inhibited TNFa secretion and in the inactivated E. coli group, TNFa secretion was almost completely silenced. Although inactivated V. cholerae from Dukoral® is capable of inducing anti-inflammatory IL-10, this was not observed for inactivated E. coli (Figure 3B). Rather, inactivated E. coli appears to function as LPS from E. coli, as it suppressed rather than promoted IL- 10 signaling. These results suggest that inactivated E. coli is immune-suppressive, not immune tolerogenic and thus the tolerogenic properties of V. cholerae of Dukoral® are unique. Previous work has demonstrated that LPS from E. coli is a substantially stronger stimulator of TLR4 than V. cholerae LPS (Hankins J.V., Madsen J. A., Giles D. K., Brodbelt J.S., Trent M.S. “Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in grampositive and gram-negative bacteria,” PNAS 109, 8722-8727, (2012)); likely this weakened innate immune activation via TLR4 contributes to the tolerogenic phenotype induced by V. cholerae of Dukoral®.

Table 4: LPS concentrations of experimental groups of Example 3

Example 4: Pretreatment with inactivated V. choleras of Dukoral® protects THP-1 macrophages from LPS toxic shock

To investigate the impact of inactivated bacteria on professional APCs, THP-1 monocytes were differentiated into THP-1 macrophages according to the methods outlined in Table 3. THP-1 macrophages were pretreated for 24 hours with either purified LPS from E. coli, or inactivated V. cholerae of Dukoral®, or inactivated E. coli. The inactivated bacteria concentrations were standardized to via OD regression at 600 nm. The endotoxin concentration in each group is reported in Table 5. THP-1 macrophages were then challenged with 1 pg/mL E. coli LPS and analyzed via flow cytometry at 24 hours post-challenge for viability. As shown in Figure 4, cells which did not receive any pretreatment has substantial mortality (average viability: 15%), which was significantly reduced via pretreatment with V. cholerae of Dukoral® (average viability: 64%). In contrast, inactivated A. coli only slightly increased viability to 25%. These results suggest the V.cholerae of Dukoral® effectively promotes anti-inflammatory responses, which protect macrophages from LPS-induced toxic shock (Sant-Anna Melo E. et al. Gene expression reprogramming protects macrophage from septic-induced cell death. Molecular Immunology 47, 2587-2593, (2010)).

Table 5: LPS concentrations of experimental groups of Example 4

Example 5: Investigation of V. choleras' therapeutic effect in TNBS mouse colitis model

2,4,6-trinitrobenzene sulfonic acid (TNBS) is a haptenizing molecule which generates colitis when delivered rectally to mice. The TNBS model is characterized by THl-mediated transmural colitis and is widely used to study immunologic aspects of Crohn’s Disease (Kiesler P., Fuss I., Strober I, “Experimental Models of Inflammatory Bowel Disease.” Cellular and Molecular Gastroenterology and Hepatology 1, 154-170, (2015)). Furthermore, TNBS mice enabled the discovery that oral tolerance results in development of T regs which control gut inflammation (Kiesler P., Fuss I., Strober I, “Experimental Models of Inflammatory Bowel Disease.” Cellular and Molecular Gastroenterology and Hepatology 1, 154-170, (2015)). Therefore, the TNBS model is utilized to characterize the ability of Dukoral® to treat mouse colitis. Mice receive TNBS intrarectally, and then are treated either with recombinant CTB (as in Coccia E.M. et al. “Cholera toxin subunit B inhibits IL- 12 and IFN-y production and signaling in experimental colitis and Crohn’s disease.” Gut 11, 1558- 1564, (2005)), with the V. choleras of Dukoral®, or the full Dukoral® vaccine, as shown in Table 6. Mice are monitored for weight loss, clinical score, stool consistency, Disease Activity Index (DAI), colon length and weight, histology and immunohistochemistry, myeloperoxidase (MPO) activity and analysis of lamina propria mononuclear cells for cytokine and cell surface marker expression.

Table 6: Experimental groups of TNBS mouse colitis model

Example 6: Investigation of V. cholerae' therapeutic effect in DSS mouse colitis model

Dextran sulfate sodium (DSS) is a water-soluble sulfated polysaccharide which causes epithelial cell injury, resulting in entry of luminal organisms/molecules into the lamina propria, which in turn causes intestinal inflammation. The DSS model is widely used to study innate immune aspects of IBD as well as to study mucosal healing dynamics (Kiesler P., Fuss I., Strober I, “Experimental Models of Inflammatory Bowel Disease.” Cellular and Molecular Gastroenterology and Hepatology 1, 154-170, (2015)). Therefore, the DSS model is utilized to characterize the ability of V. cholerae to initiate epithelial repair and alleviate colitis. In short, adult C57BL/6J female mice received 2.5% oral DSS in their drinking water from day 0 to day 5 and in parallel were treated via oral gavage with either the V. cholerae strains of Dukoral® at regular intervals (i.e. at D-6, D-3, DO, D3 & D6 wherein DO is the day of colitis induced DSS and D-6, day minus 6, is the experimental start and first V. cholerae administration), or the control vehicle (PBS) following the same schedule, as shown in Table 7. Mice were monitored for weight loss, clinical score, stool consistency, to determine the Disease Activity Index (DAI) at day 12 at which point the experiment was stopped. Statistical comparisons were performed using the permutation test for two independent samples.

Table 7: Experimental groups of DSS mouse colitis model

* V. cholerae strains dose corresponds to a mix of the following 4 different strains included in the Dukoral vaccine (each bacterial strain in an amount of 7.21xl0⁸ bacteria; for a total of 2.88xl0⁹ bacteria): Cholera Inaba 6973 El Tor formalin inactivated, Cholera Inaba 48 classical serotype 01 heat inactivated, Cholera Ogawa 50 classical serotype 01 formalin inactivated and Cholera Ogawa 50 classical serotype 01 heat inactivated. The DAI score was recorded at Day 12 corresponding to the wound- healing/beginning of chronic colitis phase. As expected, the DAI score for the DSS + Vehicle (PBS) group was significantly increased compared to healthy control group (CTRL + Vehicle) (3.90±0.31 vs 0.00±0.00, p=0.00033) indicating that colitis remains at sustained level in the C57bl6 mice even 7 days after the last DSS administration colitis (Figure 6).

A significant decrease of the DAI score was recorded at Day 12 in colitic mice receiving the mix of inactivated of Vibrio cholera (2.10±0.35 vs 3.90±0.31, p=0.0035) inducing a 46% of improvement of the DAI score compared to colitic mice receiving the vehicle (Figure 6).

These results confirm the anti-inflammatory properties of the mix of strains of tested Vibrio cholera in the DSS model and thus suggests also an anti-inflammatory property in humans and thus also a potential beneficial effect in inflammatory bowel disease, such as e.g. Crohn’s disease or Ulcerative Colitis.

Example 7: Pretreatment with Dukoral® protects PBMCs from endotoxin challenge

To investigate the tolerogenic capacity of the inactivated bacteria component of Dukoral®, an in vitro endotoxin tolerance models was implemented using PBMCs (Lopez- Collazo and Del Fresno. Critical Care (2013)). As shown in Figure 5 A, as compared to untreated samples, low doses of E. coli LPS, or A. coli LPS and recombinant CTB, or Dukoral® significantly suppressed pro-inflammatory TNFa secretion in PBMCs upon LPS challenge. However, only the Dukoral® was able to increase the secretion of the key antiinflammatory cytokine IL-10 in Figure 5B. Conversely, E. coli LPS actually suppressed IL- 10 secretion, either when used alone or in combination with CTB. These results suggest that Dukoral® - as the combination of rCTB and inactivated V. cholerae bacteria - is a unique driver of a strongly tolerogenic state, both suppressing inflammation and promoting antiinflammatory responses.

Preferred Aspects

Al . A method of treating or preventing an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of

(1) a composition comprising at least one strain of inactivated or attenuated Vibrio cholerae, (2) a composition comprising a combination of i) at least one strain of inactivated or attenuated Vibrio cholerae bacteria, and ii) a cholera toxin or a modified version thereof.

A2. The method of aspect 1, wherein the cholera toxin is a subunit of cholera toxin.

A3. The method of aspect 2, wherein the subunit of cholera toxin is cholera toxin subunit B or a variant thereof such as rCTB or a non-toxic variant of cholera toxin subunit A such as mmCT.

A4. The method of any one of aspects 2-3, wherein the cholera toxin is recombinantly produced.

A5. The method of any one of aspects 1-4, wherein the cholera toxin is from strains belonging to V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and V. cholerae Ogawa classical biotype.

A6. The method of any one of aspects 1-5, wherein the at least one strain of V. cholerae is of serotype 01.

A7. The method of aspect 6, wherein the at least one strain of V. cholerae is selected from the group consisting of V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and Ogawa classical biotype.

A8. The method of aspect 6 or 7, wherein the composition comprises LPS from a strain of V. cholerae instead of the whole V. cholerae bacteria.

A9. The method of aspect 8, wherein the LPS is from V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and V. cholerae Ogawa classical biotype.

A10. The method of any one of aspects 1-7, wherein the V. cholerae bacteria is in the form of whole V. cholerae bacteria.

Al l. The method of aspect 10, wherein the whole V. cholerae bacteria are killed/ inactivated and/or heat-inactivated. A12. The method of aspect 10, wherein the whole V. cholerae bacteria are attenuated by deletion of at least a portion of a nucleic acid sequence encoding the A subunit of cholera toxin.

Al 3. The method of any one of aspects 8-9, wherein the LPS is in the form of whole V. cholerae bacteria of three strains of V. cholerae.

A14. The method of aspect 13, wherein the bacteria of at least one of the strains of V. cholerae are killed/inactivated.

A15. The method of aspect 14, wherein the bacteria of at least one of the strains of V. cholerae are heat-inactivated.

A16. The method of aspect 14 or 15, wherein the bacteria of at least one of the strains of V. cholerae are formalin-inactivated.

Al 7. The method of any one of aspects 1-16, further comprising administering an acidneutralizing agent.

A18. The method of aspect 17, wherein the acid-neutralizing agent is a bicarbonate buffer.

Al 9. The method of any one of aspects 1-18, wherein the composition is administered to the subject i) in an induction phase up to 6 doses in 2 weeks; and ii) in a maintenance phase once about every 3 months, 6 months, 12 months or 24 months, preferably once every 3 months at least twice.

A20. The method of any one of aspects 1-19, wherein the administration is oral administration or parenteral administration.

A21. The method of any one of aspects 1-20, wherein the subject has or is predisposed to have an autoimmune disease characterized by chronic inflammation. A22. The method of any one of aspects 1-21, wherein the subject has or is predisposed to have an autoimmune disease such as e.g. inflammatory bowel disease, Crohn’s disease, ulcerative colitis, rheumatoid arthritis, preferably Crohn’s disease.

A23. The method of any one of aspects 1-22, further comprising administering one or more additional therapeutic agent.

A24. The method of any one of aspects 1-23, further comprising administering an agent selected from the group of agent consisting of i) glucocorticosteroid such as e.g. prednisolone, methylprednisolone, hydrocortisone, ii) budesonide, iii) aminosalicylate such as e.g. mesalamine, olsalazine, iv) anti-TNF alpha such as e.g. Infliximab, adalimumab, v) immunosuppressants such as azathioprine, methotrexate, vi) anti-IL- 12/23: ustekinumab, and vii) anti-integrin monoclonal antibodies such as e.g. vedolizumab.

A25. The method of any one of aspects 1-24, further comprising administering an anti- TNF alpha such as e.g. Infliximab, adalimumab.

A26. The method of any one of aspects 1-25, wherein the autoimmune disease is Crohn’s disease or ulcerative colitis, preferably Crohn’s disease.

A27. A method of enhancing survival of a subject having an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of

(1) a composition comprising lipopolysaccharide (LPS) from at least one strain of Vibrio choleras, or

(2) a composition comprising lipopolysaccharide (LPS) from at least one strain of Vibrio choleras and cholera toxin.

A28. The method of aspect 27, wherein the cholera toxin is a subunit of cholera toxin.

A29. The method of aspect 28, wherein the subunit of cholera toxin is cholera toxin subunit B.

A30. The method of any one of aspects 27-29, wherein the cholera toxin is recombinantly produced. A31. The method of any one of aspects 27-30, wherein the cholera toxin is from strains belonging to V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and V. cholerae Ogawa classical biotype.

A32. The method of any one of aspects 27-31, wherein the at least one strain of V. cholerae is of serotype 01.

A33. The method of aspect 32, wherein the at least one strain of V. cholerae is selected from the group consisting of V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and Ogawa classical biotype.

A34. The method of aspect 32 or 33, wherein the composition comprises LPS from three strains of V. cholerae.

A35. The method of aspect 34, wherein the LPS is from V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and Ogawa classical biotype.

A36. The method of any one of aspects 27-35, wherein the LPS is in the form of whole V. cholerae bacteria.

A37. The method of aspect 36, wherein the whole V. cholerae bacteria are killed/inactivated and/or heat-inactivated.

A38. The method of aspect 37, wherein the whole V. cholerae bacteria are attenuated by deletion of at least a portion of a nucleic acid sequence encoding the A subunit of cholera toxin.

A39. The method of any one of aspects 36-38, wherein the LPS is in the form of whole V. cholerae bacteria of three strains of V. cholerae or LPS is in the form of an outer membrane vesicle.

A40. The method of aspect 39, wherein the bacteria of at least one of the strains of V. cholerae are killed/inactivated. A41. The method of aspect 40, wherein the bacteria of at least one of the strains of V. cholerae are heat-inactivated.

A42. The method of aspect 40 or 41, wherein the bacteria of at least one of the strains of V. cholerae are formalin-inactivated.

A43. The method of any one of aspects 27-42, further comprising administering an acidneutralizing agent.

A44. The method of aspect 43, wherein the acid-neutralizing agent is a bicarbonate buffer.

A45. The method of any one of aspects 26-44, wherein the composition is administered to the subject at least twice.

A46. The method of any one of aspects 27-45, wherein the administration is oral administration or parenteral administration.

A47. The method of any one of aspects 27-46, wherein the subject has or is predisposed to have an autoimmune disease characterized by chronic inflammation or by expression of GM1 ganglioside receptors.

A48. The method of any one of aspects 27-47, wherein the subject has or is predisposed to have Crohn’s disease or ulcerative colitis.

A49. The method of any one of aspects 27-48, wherein the subject has undergone a surgical procedure to remove inflamed tissue.

A50. The method of any one of aspects 27-49, further comprising administering one or more additional therapeutic agent.

A51. The method of any one of aspects 27-50, further comprising administering an agent selected from the group of agent consisting of i) glucocorticosteroid such as e.g. prednisolone, methylprednisolone, hydrocortisone, ii) budesonide, iii) aminosalicylate such as e.g. mesalamine, olsalazine, iv) anti-TNF alpha such as e.g. infliximab, adalimumab, v) immunosuppressants such as azathioprine, methotrexate, vi) anti-IL- 12/23: ustekinumab, and vii) anti-integrin monoclonal antibodies such as e.g. vedolizumab.

A52. The method of any one of aspects 27-51, further comprising administering an anti- TNF alpha such as e.g. Infliximab, adalimumab.

Bl. A pharmaceutical composition comprising

(1) at least one strain of Vibrio choleras, or

(2) a combination of i) at least one strain of Vibrio choleras, and ii) a cholera toxin or a modified version thereof, for use as set out in A1-A52 such as e.g. in the prevention and/or treatment of autoimmune disease such as CD.

Cl . Use of a composition comprising

(1) at least one strain of Vibrio cholerae, or

(2) a combination of i) at least one strain of Vibrio cholerae, and ii) a cholera toxin or a modified version thereof, for use as set out in A1-A52 such as e.g. in the prevention and/or treatment of autoimmune disease such as CD.

Claims

CLAIMS What is claimed is:

1. A method of treating or preventing an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of

(1) a composition comprising at least one strain of inactivated or attenuated Vibrio choleras, or

(2) a composition comprising a combination of i) at least one strain of inactivated or attenuated Vibrio choleras bacteria, and ii) a cholera toxin or a modified version thereof.

2. The method of claim 1, wherein the cholera toxin is a subunit of cholera toxin.

3. The method of claim 2, wherein the subunit of cholera toxin is cholera toxin subunit B or a variant thereof such as rCTB or a non-toxic variant of cholera toxin subunit A such as mmCT.

4. The method of any one of claims 2-3, wherein the cholera toxin is recombinantly produced.

5. The method of any one of claims 1-4, wherein the cholera toxin is from strains belonging to V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and V. cholerae Ogawa classical biotype.

6. The method of any one of claims 1-5, wherein the at least one strain of V. cholerae is of serotype 01.

7. The method of claim 6, wherein the at least one strain of V. cholerae is selected from the group consisting of V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and V. cholerae Ogawa classical biotype.

8. The method of claim 6 or 7, wherein the composition comprises LPS from a strain of V. cholerae instead of the whole V. cholerae bacteria.

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9. The method of claim 8, wherein the LPS is from V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and V. cholerae Ogawa classical biotype.

10. The method of any one of claims 1-7, wherein the V. cholerae bacteria is in the form of whole V. cholerae bacteria.

11. The method of claim 10, wherein the whole V. cholerae bacteria are killed/ inactivated and/or heat-inactivated.

12. The method of claim 10, wherein the whole V. cholerae bacteria are attenuated by deletion of at least a portion of a nucleic acid sequence encoding the A subunit of cholera toxin.

13. The method of any one of claims 8-9, wherein the LPS is in the form of whole V. cholerae bacteria of three strains of V. cholerae.

14. The method of claim 13, wherein the bacteria of at least one of the strains of V. cholerae are killed/inactivated.

15. The method of claim 14, wherein the bacteria of at least one of the strains of V. cholerae are heat-inactivated.

16. The method of claim 14 or 15, wherein the bacteria of at least one of the strains of V. cholerae are formalin-inactivated.

17. The method of any one of claims 1-16, further comprising administering an acidneutralizing agent.

18. The method of claim 17, wherein the acid-neutralizing agent is a bicarbonate buffer.

19. The method of any one of claims 1-18, wherein the composition is administered to the subject i) in an induction phase up to 6 doses in 2 weeks; and ii) in a maintenance phase once about every 3 months, 6 months, 12 months or 24 months, preferably once every 3 months.

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20. The method of any one of claims 1-19, wherein the administration is oral administration or parenteral administration.

21. The method of any one of claims 1-20, wherein the subject has or is predisposed to have an autoimmune disease characterized by chronic inflammation.

22. The method of any one of claims 1-21, wherein the subject has or is predisposed to have an autoimmune disease such as e.g. inflammatory bowel disease, Crohn’s disease, ulcerative colitis, rheumatoid arthritis, preferably Crohn’s disease.

23. The method of any one of claims 1-22, further comprising administering one or more additional therapeutic agent.

24. The method of any one of claims 1-23, further comprising administering an agent selected from the group of agent consisting of i) glucocorticosteroid such as e.g. prednisolone, methylprednisolone, hydrocortisone, ii) budesonide, iii) aminosalicylate such as e.g. mesalamine, olsalazine, iv) anti-TNF alpha such as e.g. Infliximab, adalimumab, v) immunosuppressants such as azathioprine, methotrexate, vi) anti-IL- 12/23: ustekinumab, and vii) anti-integrin monoclonal antibodies such as e.g. vedolizumab.

25. The method of any one of claims 1-24, further comprising administering an anti- TNF alpha such as e.g. Infliximab, adalimumab.

26. The method of any one of claims 1-25, wherein the autoimmune disease is Crohn’s disease or ulcerative colitis, preferably Crohn’s disease.

27. A method of enhancing survival of a subject having an autoimmune disease, comprising administering to a subject in need thereof a therapeutically effective amount of

(2) a composition comprising i) a lipopolysaccharide (LPS) from at least one strain of Vibrio choleras, and ii) a cholera toxin or a modified version thereof.

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28. The method of claim 27, wherein the cholera toxin is a subunit of cholera toxin.

29. The method of claim 28, wherein the subunit of cholera toxin is cholera toxin subunit B.

30. The method of any one of claims 27-29, wherein the cholera toxin is recombinantly produced.

31 The method of any one of claims 27-30, wherein the cholera toxin is from strains belonging to V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and V. cholerae Ogawa classical biotype.

32. The method of any one of claims 27-31, wherein the at least one strain of V. cholerae is of serotype 01.

33. The method of claim 32, wherein the at least one strain of V. cholerae is selected from the group consisting of V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and Ogawa classical biotype.

34. The method of claim 32 or 33, wherein the composition comprises LPS from three strains of V. cholerae.

35. The method of claim 34, wherein the LPS is from V. cholerae Inaba classical biotype, V. cholerae Inaba El Tor biotype, and Ogawa classical biotype.

36. The method of any one of claims 27-35, wherein the LPS is in the form of whole V. cholerae bacteria.

37. The method of claim 36, wherein the whole V. cholerae bacteria are killed/inactivated and/or heat-inactivated.

38. The method of claim 37, wherein the whole V. cholerae bacteria are attenuated by deletion of at least a portion of a nucleic acid sequence encoding the A subunit of cholera toxin.

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39. The method of any one of claims 36-38, wherein the LPS is in the form of whole V. choleras bacteria of three strains of V. choleras or LPS is in the form of an outer membrane vesicle.

40. The method of claim 39, wherein the bacteria of at least one of the strains of V. cholerae are killed/inactivated.

41. The method of claim 40, wherein the bacteria of at least one of the strains of V. cholerae are heat-inactivated.

42. The method of claim 40 or 41, wherein the bacteria of at least one of the strains of V. cholerae are formalin-inactivated.

43. The method of any one of claims 27-42, further comprising administering an acidneutralizing agent.

44. The method of claim 43, wherein the acid-neutralizing agent is a bicarbonate buffer.

45. The method of any one of claims 26-44, wherein the composition is administered to the subject at least twice.

46. The method of any one of claims 27-45, wherein the administration is oral administration or parenteral administration.

47. The method of any one of claims 27-46, wherein the subject has or is predisposed to have an autoimmune disease characterized by chronic inflammation.

48. The method of any one of claims 27-47, wherein the subject has or is predisposed to have Crohn’s disease or ulcerative colitis.

49. The method of any one of claims 27-48, wherein the subject has undergone a surgical procedure to remove inflamed tissue.

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50. The method of any one of claims 27-49, further comprising administering one or more additional therapeutic agent.

51. The method of any one of claims 27-50, further comprising administering an agent selected from the group of agent consisting of i) glucocorticosteroid such as e.g. prednisolone, methylprednisolone, hydrocortisone, ii) budesonide, iii) aminosalicylate such as e.g. mesalamine, olsalazine, iv) anti-TNF alpha such as e.g. infliximab, adalimumab, v) immunosuppressants such as azathioprine, methotrexate, vi) anti-IL- 12/23: ustekinumab, and vii) anti-integrin monoclonal antibodies such as e.g. vedolizumab.

52. The method of any one of claims 27-51, further comprising administering an anti- TNF alpha such as e.g. Infliximab, adalimumab.

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