Treatment of crohn's disease by mycobacterium avium subsp. paratuberculosis agent

Abstract

An immunotherapeutic agent against Mycobacterium avium subspecies paratuberculosis (MAP) for use in a method of treating or preventing Crohn's disease.

Description

[0001] THERAPY

[0002] Field of the Invention

[0003] The present invention relates to the treatment or prevention of Crohn’s disease. to the Invention

[0004] Crohn’s disease is a chronic, incurable and progressive inflammatory bowel disease characterised by a variety of intestinal and extra-intestinal manifestations that impair an individual’s quality of life. Although much has been learned in recent years about the disease mechanisms, there has been little progress in the recognition of disease causation. Indeed, current treatments for Crohn’s disease do not target the cause of the disease but rather aim to reduce or control symptoms using, for example, immunomodulating, immunosuppressive and anti-inflammatory medications.

[0005] Although the exact aetiology of Crohn’s disease remains unclear, it is thought to arise from a combination of genetic predisposition, altered gut microbiota and environmental factors leading to dysregulated immune responses (Honap et al. (2021) Frontline Gastroenterol. 12(5): 397-403). For over a century, researchers have explored the association between Mycobacterium avium subspecies paratuberculosis (MAP) and the development of Crohn’s disease. However, the suggestion that MAP is the causative agent of Crohn’s disease, and a potential cause for immune dysregulation, has been strongly debated.

[0006] MAP is an atypical, non-tuberculous, obligate intracellular pathogen part of the Mycobacterium avium complex. It is very slow-growing and can cause systemic infection and chronic inflammation of the intestines in many animal species including primates. In 1912, MAP was identified as the cause of Johne’s disease, a chronic granulomatous enteritis affecting domestic livestock. The following year, the clinicopathological features of Johne’s disease were found to be similar to the clinicopathological features of a stricturing and fistulising enteritis affecting humans, later identified as Crohn’s disease. Hypotheses surrounding the causal association of MAP with Crohn’s disease present today stem from here. However, despite studies investigating the link between MAP and Crohn’s disease over the past century, evidence is circumstantial and the link unproven (Honap et al. (2021) Frontline Gastroenterol. 12(5): 397-403). Due to the paucity of data, anti-MAP therapy is not presently featured in international guidelines as a treatment for Crohn’s disease.

[0007] HAV Vaccines Limited has developed two virally vectored anti-MAP vaccines: a simian adenovirus vectored vaccine, ChAdOx2 HAV, and poxvirus Modified Vaccinia Ankara vectored (MVA) vaccine, MVA HAV. The vectors each contain the vaccine insert, Havilah (HAV), as described in International Publication No. WO 2007 / 017635, which is incorporated herein by reference. The HAV insert comprises a 95-kDa fusion construct from four MAP genes present in all MAP strains, 1589c (ahpC), 1234 (gsd), 2444c (p 12) and 1235 (mpa). AhpC is a secreted virulence factor in MAP shared by other pathogenic mycobacteria. Gsd is directly involved in the synthesis and transport of fucose, contributing to the relatively inert and highly chemical and enzymic resistance characteristics of MAP. P12 consists mostly of the extracellular carboxy-terminal portion of the IS 900 protein released from the mycobacterial cell and involved in pathogenicity. Mpa is a cell-surface acyltransferase and may have a pore function which contributes to MAP’s intrinsic resistance pattern. Sequences similar to those of all four of the MAP genes in the HAV insert can be found in secondary co-pathogens in Crohn’s disease, including some E. coli and other M. avium sp. The DNA of the selected MAP genes was codon-optimised for mammalian cell expression and strung together to express the single HAV vaccine antigen. For patient safety, this was further edited to remove any genetic sequences with homology to a mammalian sequence.

[0008] A phase la clinical trial has demonstrated that the ChAdOx2 HAV and the MVA HAV vaccines are safe, well-tolerated, and immunogenic in healthy subjects (Folegatti et al. (2021) Vaccines (Basel). 9(3): 262).

[0009] Summary of the Invention

[0010] The present application describes a phase lb clinical trial conducted to investigate the safety, immunogenicity, and clinical response of the ChAdOx2 HAV and MVA HAV vaccines, as a single dose or in a heterologous prime-boost regime, in subjects with active Crohn’s disease. The phase lb clinical trial demonstrates that the ChAdOx2 HAV and MVA HAV vaccines are safe and well-tolerated in patients with active Crohn’s disease. In addition, significant clinical improvement was observed in subjects, particularly in those who received a ChAdOx2 HAV vaccine prime followed by an MVA HAV vaccine boost. This is the first demonstration that inducing an immune response against MAP is effective in the treatment of Crohn’s disease. Thus, immunotherapeutic agents (e.g. vaccines) which generate and / or enhance an immune response against MAP are effective in the treatment of Crohn’s disease.

[0011] Accordingly, the invention provides an immunotherapeutic agent against MAP for use in a method of treating or preventing Crohn’s disease.

[0012] The invention further provides: use of an immunotherapeutic agent against MAP in the manufacture of a medicament for treating or preventing Crohn’s disease; and a method of treating or preventing Crohn’s disease comprising administering to a subject in need thereof an effective amount of an immunotherapeutic agent against MAP.

[0013] Brief Description of the Figures

[0014] Figure 1 is a Consolidated Standards of Reporting Trials (CONSORT) diagram, which summarises the design of the phase lb clinical trial investigating the safety, immunogenicity and clinical response of ChAdOx2 HAV and MVA HAV vaccines when administered to patients with active Crohn’s disease as single doses or in a prime-boost regime.

[0015] Figure 2 shows the T cell response following vaccination in patients with active Crohn’s disease. (A) shows interferon-y (IFN-y) enzyme-linked immunospot (ELISpot) assays responses to the entire HAV vaccine insert according to vaccine regimen. Lines represent medians with IQR. (B) shows individual IFN-y ELISpot responses by time point and vaccine regime. Lines represent medians. (C) shows responses to individual antigens at post-prime (day 28) and post-boost (day 84) time points.

[0016] Figure 3 shows the changes in the clinical markers of Crohn’s disease severity following vaccination. (A) shows the Crohn’s Disease Activity Index (CD Al) at baseline and after vaccination in each vaccine group. A score below the dotted line represents clinical remission. (B) shows the change in CD Al after either priming alone or primeboost vaccination. A change above the dotted line represents clinical response. (C) shows the Simple Endoscopic Score for Crohn’s Disease (SES-CD) at screening and post-prime- boo st- vaccination . Brief Description of the Sequences

[0017] SEQ ID NOs: 1 and 2 are the amino acid and nucleic acid sequences, respectively, for the MAP ahpC gene.

[0018] SEQ ID NOs: 3 and 4 are the amino acid and nucleic acid sequences, respectively, for the MAP gsd gene.

[0019] SEQ ID NOs: 5 and 6 are the amino acid and nucleic acid sequences, respectively, for the MAP pl2 gene.

[0020] SEQ ID NOs: 7 and 8 are the amino acid and nucleic acid sequences, respectively, for the MAP mpa gene.

[0021] SEQ ID NOs: 9 and 10 are the amino acid and nucleic acid sequences, respectively, of a modified version of the MAP ahpC gene, in which the nucleic acid sequence has been codon-optimised for human use.

[0022] SEQ ID NOs: 11 and 12 are the amino acid and nucleic acid sequences, respectively, of a modified version of the MAP gsd gene, in which the nucleic acid sequence has been codon-optimised for human use and which is truncated at the N- terminus in order to remove the cysteine residue at position 22.

[0023] SEQ ID NOs: 13 and 14 are the amino acid and nucleic acid sequences, respectively, of a modified version of the MAP pl2 gene, in which the nucleic acid sequence has been codon-optimised for human use.

[0024] SEQ ID NOs: 15 and 16 are the amino acid and nucleic acid sequences, respectively, of a modified version of the MAP mpa gene, in which the nucleic acid sequence has been codon-optimised for human use and a number of transmembrane regions have been removed.

[0025] SEQ ID NOs: 17 and 18 are the amino acid and nucleic acid sequences, respectively, of the N-terminal region of the MAP P900 polypeptide.

[0026] SEQ ID NOs: 19 to 27 are the amino acid sequences of the immunogenic regions of the MAP P900 polypeptide.

[0027] SEQ ID NOs: 28 and 29 are the amino acid and nucleic acid sequences, respectively, of the HAV vaccine construct.

[0028] SEQ ID NO: 30 is the amino acid sequence of the HAVX1 vaccine construct.

[0029] SEQ ID NO: 31 is the amino acid sequence of the HAVX2 vaccine construct. SEQ ID NOs: 32 to 35 are the amino acid sequences of linker peptides.

[0030] SEQ ID NO: 36 is a consensus amino acid sequence for 2A peptides.

[0031] SEQ ID NO: 37 is the amino acid sequence of a 2A peptide.

[0032] Detailed Description of the Invention

[0033] Definitions

[0034] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field of this application.

[0035] The singular forms “a”, “an”, and “the” used herein include plural referents unless the content clearly dictates otherwise. For example, reference to “a polypeptide” includes “two or more polypeptides” , reference to “a polynucleotide” includes “two or more polynucleotides” , and reference to “a vector” includes “two or more vectors”.

[0036] The term “and / or" used herein includes any and all combinations of one or more related listed items.

[0037] The term “comprising” used herein is intended to mean “including but not limited to”. For example, the phrase “the immunotherapeutic agent comprises a polypeptide” should be interpreted to mean that the immunotherapeutic agent includes a polypeptide, but the immunotherapeutic agent may include further components.

[0038] The term “consisting of’ used herein is intended to be limiting. For example, the phrase “the immunotherapeutic agent consists of a polypeptide” should be understood to mean that the immunotherapeutic agent includes a polypeptide and no further components.

[0039] The term “consisting essentially of’ used herein means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.

[0040] The term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error. For example, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x.

[0041] Numeric ranges are inclusive of the numbers defining the range. Thus, every numerical range described herein is intended to encompass every narrower numerical range that falls within such broader numerical range as if such narrower numerical ranges were all expressly written herein. It is also intended that every maximum or minimum numerical limitation disclosed herein includes every lower or higher numerical limitation as if such lower or higher numerical limitations were expressly written herein.

[0042] The term “antigen” used herein refers to any agent that can elicit an immunological response in an individual. The term “antigen” is generally used herein to refer to a polypeptide which contains one or more epitopes. An “antigen” includes a polypeptide having modifications, such as deletions, additions and substitutions (generally conservative in nature) to the native sequence, so long as the polypeptide maintains sufficient immunogenicity. These modifications may be deliberate, for example through site- directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the antigens. An “immune response” against an antigen is the development in an individual of a humoral and / or a cellular immune response to that antigen. A “humoral immune response” refers to an immune response mediated by antibody molecules, while a “cellular immune response” is one mediated by T-lymphocytes and / or other white blood cells.

[0043] The term “epitope” used herein generally refers to the site on a target antigen which is recognised by an immune receptor such as a T-cell receptor and / or an antibody. Preferably, it is a short peptide derived from or as part of a protein. However, the term is also intended to include peptides with glycopeptides and carbohydrate epitopes. A single antigenic molecule may comprise several different epitopes. The term “epitope” used herein also includes modified sequences of amino acids or carbohydrates which stimulate responses which recognise the whole organism.

[0044] The term “polypeptide” used herein refers to a compound of subunit amino acids. The terms “polypeptide” and “peptide” are used interchangeably herein. The term “polypeptide” includes short peptide sequences and also longer polypeptides and proteins.

[0045] The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include a gene fragment, messenger RNA (mRNA), cDNA. A polynucleotide may be a recombinant polynucleotide. A polynucleotide may be provided in isolated or purified form.

[0046] The term “adjuvant” used herein refers to any material or composition capable of specifically or non- specifically altering, enhancing, directing, redirecting, potentiating, or initiating an antigen-specific immune response. The term “therapeutic” or “treatment” used herein includes any of following: prevention, alleviation, reduction, cure or at least partial arrest of symptoms of, and / or complications associated with, Crohn’s disease.

[0047] The term “therapeutically or prophylactically effective dose” used herein refers to a dose in an amount sufficient to prevent, alleviate, reduce, cure or at least partially arrest symptoms of, and / or complications associated with, Crohn’s disease.

[0048] Therapeutic Methods

[0049] The invention provides an immunotherapeutic agent against MAP for use in a method of treating or preventing Crohn’s disease. The immunotherapeutic agents described herein generate and / or enhance an immune response against MAP, and effectively treat or prevent Crohn’s disease in a subject.

[0050] Also provided is the use of the immunotherapeutic agent in the manufacture of a medicament for treating or preventing Crohn’s disease. The invention additionally provides a method of treating or preventing Crohn’s disease comprising administering to a subject in need thereof an effective amount of the immunotherapeutic agent.

[0051] The invention is broadly applicable to vaccination methods and the immunotherapeutic agent may be used as a therapeutic and / or prophylactic vaccine. Thus, references herein to treatment include curative, palliative, and prophylactic treatment. Treatment may be effected therapeutically (i.e. following the initiation or development of active Crohn’s disease) or prophylactically (i.e. prior to the initiation or development of active Crohn’s disease).

[0052] The immunotherapeutic agent may be administered as part of a composition, such as a pharmaceutical composition. The pharmaceutical composition may be a vaccine composition, for example a vaccine composition comprising one or more adjuvants.

[0053] The treatment typically results in the generation and / or enhancement of an immune response against MAP. The immune response against MAP may prevent an increase in the amount of MAP present in the subject being treated, reduce the amount of MAP present in the subject being treated, or eliminate MAP from the subject being treated. The immune response against MAP may prevent an increase in the amount of MAP present in intestinal cells of the subject being treated, reduce the amount of MAP present in intestinal cells of the subject being treated, or eliminate MAP from intestinal cells of the subject being treated.

[0054] The reduction or elimination of MAP may result in the treatment alleviating, reducing, curing or at least partially arresting symptoms of, and / or complications associated with, Crohn’s disease. Treatment with the immunotherapeutic agent may result in remission of Crohn’s disease. The alleviation, reduction, cure or at least partial arrest of symptoms of, and / or complications associated with, Crohn’s disease may be indicated by a decrease in the subject’s Crohn’s Disease Activity Index (CD Al) score following the treatment. For example, the reduction or elimination of MAP may result in a decrease in the subject’s CD Al score by more than about 80 points, for example more than about 90 points, more than about 110 points, more than about 120 points, more than about 130 points, more than about 140 points, or more than about 150 points. Typically, the reduction or elimination of MAP results in a decrease in the subject’s CD Al score by more than about 100 points. A decrease in the CD Al score to a score of less than about 150 typically indicates that the treatment has caused the subject to go into clinical remission. Alternatively, or additionally, the alleviation, reduction, cure or at least partial arrest of symptoms of, and / or complications associated with, Crohn’s disease may be indicated by a decrease in the subject’s Simple Endoscopic Score for Crohn’s Disease (SES-CD) following the treatment. For example, the reduction or elimination of MAP may result in a decrease in the subject’s SES-CD by about 35% or more, for example about 40% or more, about 45% or more, about 50% or more, or about 55%. Typically, the reduction or elimination of MAP may result in a decrease in the subject’s SES-CD by more than about 50%. A decrease in the SES-CD to a score of about 0 to 2, or about 0 to 3, typically indicates that the treatment has caused the subject to go into clinical remission.

[0055] Alternatively, the reduction or elimination of MAP may result in the treatment preventing the initiation or development of active Crohn’s disease. The immmunotherapeutic agent may be provided to prevent the initiation or development of active Crohn’s disease in a subject who has previously had active Crohn’s disease (e.g. a subject in clinical remission) or in a subject who has not previously had active Crohn’s disease. Where the subject is in clinical remission, the immunotherapeutic agent may be provided to prevent the subject going into relapse. The prevention of the initiation or development of active Crohn’s disease may comprise the prevention of the onset of symptoms of, and / or complications associated with, active Crohn’s disease. The prevention of the initiation or development of active Crohn’s disease may be indicated by a decrease in the subject’s CD Al score following the treatment, and / or by a decrease in the subject’s SES-CD following the treatment as described above.

[0056] Subject to be Treated

[0057] The invention relates to the methods of treating or preventing Crohn’s disease in a subject using an immunotherapeutic agent against MAP. The subject may have Crohn’s disease, be suspected to have Crohn’s disease, or be susceptible to developing Crohn’s disease.

[0058] The subject may have quiescent Crohn’s disease (i.e. the subject may be in remission for Crohn’s disease). The subject may have active Crohn’s disease. The active Crohn’s disease may be mildly active Crohn’s disease, moderately active Crohn’s disease, or severe Crohn’s disease. The subject typically has mildly to moderately active Crohn’s disease. The subject may exhibit one or more symptoms of, and / or complications associated with, Crohn’s disease at the time of treatment. The subject may be susceptible to developing symptoms of, and / or complications associated with, Crohn’s disease. A subject susceptible to developing symptoms of, and / or complications associated with, Crohn’s disease may be a subject who has previously exhibited symptoms of, and / or complications associated with, Crohn’s disease but who is not presenting with such symptoms and / or complications at the time of treatment.

[0059] A subject susceptible to developing symptoms of Crohn’s disease may be genetically predisposed to Crohn’s disease. A subject susceptible to developing symptoms of Crohn’s disease may have a family member who has Crohn’s disease, such as a grandparent, parent, sibling or child with Crohn’s disease. A subject susceptible to developing symptoms of Crohn’s disease may be a smoker or an ex-smoker.

[0060] Methods for determining whether a subject has Crohn’s disease are known in the art. For example, Crohn’s disease may be diagnosed according to standard clinical, endoscopic, radiological, and / or histological criteria. Such diagnosis may involve carrying out a colonoscopy, MRI scan and / or CT scan, and / or obtaining and analysing a biopsy. Methods for determining the severity of Crohn’s disease in a subject are also known in the art. For example, the severity of Crohn’s disease may be determined by measuring the CRP level, faecal calprotectin level, CD Al, and / or SES-CD of the subject, and comparing the measured values against standards.

[0061] Accordingly, the subject may have a CRP level of more than about 1 mg / L, such as a CRP level of from about 1 mg / L to 228 mg / L. Lor example, the subject may have a CRP level of more than about 2 mg / L, more than about 3 mg / L, more than about 4 mg / L, more than about 5 mg / L, more than about 10 mg / L, more than about 15 mg / L, more than about 20 mg / L, more than about 25 mg / L, more than about 30 mg / L, more than about 35 mg / L, more than about 40 mg / L, more than about 45 mg / L, more than about 50 mg / L, more than about 60 mg / L, more than about 70 mg / L, more than about 80 mg / L, more than about 90 mg / L, more than about 100 mg / L, more than about 150 mg / L, or more than about 200 mg / L, or more than about 250 mg / L. Where the method is a therapeutic method (e.g. where the subject has active Crohn’s disease), the subject typically has a CRP level of more than about 10 mg / L.

[0062] The subject may have a faecal calprotectin level of from about 100 mcg / g (quiescent disease) to 500 mcg / g or more (very severe disease). The subject may have a faecal calprotectin level of more than about 30 mcg / g, for example more than about 40 mcg / g, more than about 50 mcg / g, more than about 60 mcg / g, more than about 70 mcg / g, more than about 80 mcg / g, more than about 90 mcg / g, more than about 100 mcg / g, more than about 110 mcg / g, more than about 120 mcg / g, more than about 130 mcg / g, more than about 140 mcg / g, more than about 150 mcg / g, more than about 200 mcg / g, more than about 250 mcg / g, more than about 300 mcg / g, more than about 350 mcg / g, more than about 400 mcg / g, more than about 450 mcg / g, more than about 500 mcg / g, more than about 550 mcg / g, more than about 600 mcg / g, or more than about 650 mcg / g. Where the method is a therapeutic method (e.g. where the subject has active Crohn’s disease), the subject typically has a faecal calprotectin level of more than about 150 mcg / g.

[0063] The subject may have a CD Al score of between about 100 and 600, for example between about 150 and 450, between about 150 and 200, or between about 180 and 300, between about 220 and 450, such as about 240 to 300. Typically, a CD Al score of less than about 150 indicates inactive or quiescent disease, a CD Al score of between about 150 and 220 indicates mildly active disease, a CD Al score of between about 220 and 450 indicates moderately active disease, and a CD Al score of more than about 450 indicates severe disease. Where the method is a therapeutic method (e.g. where the subject has active Crohn’s disease), the subject typically has a CD Al score of between about 150 and 320.

[0064] The subject may have an SES-CD of more than about 1, for example more than about 2, more than about 3, more than about 4, more than about 5, more than about 6, more than about 7, more than about 8, more than about 9, more than about 10, more than about 11, more than about 12, more than about 13, more than about 14, more than about 15, more than about 16, more than about 17, more than about 18, more than about 19, more than about 20, more than about 25, more than about 30, more than about 40, more than about 45, more than about 50, more than about 55, or about 60. Typically, an SES-CD of between about 0 and 2 or about 0 to 3 indicates inactive or quiescent disease, an SES-CD of between about 3 and 6 indicates mildly active disease, an SES-CD of between about 7 and 15 indicates moderately active disease, and an SES-CD of about 16 or more indicates severe disease. Where the method is a therapeutic method (e.g. where the subject has active Crohn’s disease), the subject typically has an SES-CD of more than about 3.

[0065] The subject may have one or more symptoms of, and / or complications associated with, Crohn’s disease, such as abdominal pain, diarrhoea, bowel obstruction, fever, abdominal distension, weight loss, anaemia, skin rash, arthritis, inflammation of the eye, and fatigue. The subject may have active Crohn’s inflammation in at least one segment of the gastrointestinal tract, for example in at least one segment of the ileum or colon. Inflammation in at least one segment of the ileum or colon may be determined by colonoscopy and / or flexible sigmoidoscopy.

[0066] The subject typically has a MAP infection. The MAP infection may be diagnosed or undiagnosed. A MAP infection may be diagnosed by any suitable clinical diagnostic. The MAP infection may, for example, be detected by a diagnostic for MAP infection using monoclonal antibodies as described in International Publication No. WO 2018 / 130836, which is incorporated herein by reference. Other suitable diagnostic techniques include fluorescence in situ hybridisation (FISH) and PCR.

[0067] The subject may or may not have been tested for a MAP infection. The subject may have been tested for MAP infection and shown a negative result. The negative result may be a false negative result, such as a negative PCR result. The subject may have been tested for MAP infection and shown a positive result. The subject is preferably not immunocompromised. For example, the subject may not be receiving, or has not received, for example within 3 months of being administered the treatment, an immunoinhibitory treatment. For example, the subject may not be receiving, or have received, for example within 3 months of being administered the treatment, an oral corticosteroid, a thiopurine, methotrexate, tacrolimus, anti-TNF-a antibody, anti-a4p7 antibody, or anti-p40 antibody treatment.

[0068] A therapeutically effective treatment amount of the immunotherapeutic agent results in the treatment alleviation, reduction, cure or at least partially arrest of symptoms of, and / or complications associated with, Crohn’s disease. The subject typically has alleviated, reduced, cured or at least partially arrested symptoms of, and / or complications associated with, Crohn’s disease as a result of a reduction or elimination of MAP following administration of the immunotherapeutic agent. The alleviation, reduction, curing or at least partial arrest of symptoms of, and / or complications associated with, Crohn’s disease may be indicated by a decrease in the subject’s CD Al score following the treatment described herein. Typically, a therapeutically effective treatment results in a decrease in the subject’s CD Al score by more than about 100 points. A therapeutically effective treatment may result in a decrease in the subject’s CD Al score to less than about 150 points, which indicates inactive disease or remission. Alternatively, or in addition, the alleviation, reduction, cure or at least partial arrest of symptoms of, and / or complications associated with, Crohn’s disease may be indicated by a decrease in the subject’s SED-CD following administration of the immunotherapeutic agent. Typically, a therapeutically effective treatment results in a decrease in the subject’s SES-CD by more than about 50%. A therapeutically effective treatment may result in a decrease in the subject’s SES-CD to about 0 to 2 or about 0 to 3, which indicates inactive disease or remission.

[0069] The subject is typically a human. The subject may be any age. For example, the subject may be an adult subject or a paediatric subject. The adult subject is typically at least 18 years old, such as 18 to 80 years old, 18 to 70 years old, 18 to 60 years old, or 18 to 50 years old. The paediatric subject is typically under 18 years old, such as 0 to 17 years old or 12 to 17 years old. Immunotherapeutic Agent

[0070] The invention provides an immunotherapeutic agent against MAP for use in a method of treating or preventing Crohn’s disease. The immunotherapeutic agent against MAP may comprise a molecule which is capable of modifying an immune response against MAP, such as generating and / or enhancing an immune response against MAP.

[0071] The immunotherapeutic agent may be a MAP antigen, such as an immunogenic polypeptide comprising an amino acid sequence found in a MAP polypeptide. The amino acid sequence found in a MAP polypeptide is referred to herein as a MAP amino acid sequence.

[0072] The MAP amino acid sequence typically comprises an epitope which can be recognised by the host immune system, such as by a host immune receptor. The host immune receptor may be an MHC Class I receptor or an MHC Class II receptor.

[0073] The MAP amino acid sequence may be any amino acid sequence, so long as it comprises at least one epitope of the MAP polypeptide. The amino acid sequence may comprise one or more epitopes, such as two or more epitopes, three or more epitopes, four or more epitopes, or five or more epitopes from the same MAP polypeptide and / or different MAP polypeptides.

[0074] The epitope may be specific to MAP. An epitope specific to MAP is an epitope found in a MAP polypeptide but not in polypeptides from other organisms, such as other microorganisms or unrelated organisms.

[0075] The epitope may be present in a polypeptide from a microorganism other than MAP, such as another mycobacterium, as well as in a MAP polypeptide. The epitope may be related to equivalent epitopes in other mycobacteria, such as M. avium, which may be a secondary co-pathogen in Crohn’s disease.

[0076] Epitopes can be identified based on knowledge of the amino acid sequence of the MAP polypeptide and from the nature of particular amino acids in the amino acid sequence, such as size or charge. Suitable epitopes may be identified by routine methods, such as those described in International Publication No. WO 2020 / 012177, which is incorporated herein by reference. For example, a library of short peptides which are fragments of a MAP polypeptide may be generated and each of these peptides may be assessed separately for their ability to generate an immune response against the full-length polypeptide. Members of the library may be screened in groups or pools. Alternatively, individual members of the library, such as individual members of a single pool, may be assessed separately. In a further example, epitope scanning of individual proteins may reveal a number of predicted MHC class I and MHC class II epitopes.

[0077] The amino acid sequence of an MHC class I epitope is typically at least 8 or 9 amino acids long to fit into an MHC class I complex. The amino acid sequence of an MHC class II epitope is typically at least 8 to 25 amino acids long, such as at least 13 to 25 amino acids long, to fit into an MHC class II complex. Therefore, the MAP amino acid sequence is preferably at least about 8 amino acids long. Where the MAP amino acid sequence comprises an MHC class I epitope, the MAP amino acid sequence is preferably at least about 8 or 9 amino acids long. Where the MAP amino acid sequence comprises an MHC class II epitope, the MAP amino acid sequence may be at least about 8, at least about 10, at least about 12, at least about 13, at least about 15, at least about 20 or at least about 25 amino acids long.

[0078] As polypeptides may be cut into fragments within a host cell, the MAP amino acid sequence is preferably longer than these lengths, such as at least about 30, at least about 35, at least about 40, at least about 45, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, or at least about 300 amino acids long. Hence, a MAP amino acid sequence may comprise, for example, from about 8 to about 300 amino acids, such as from 9 to 200, 10 to 100, 12 to 70, 15 to 60, 18 to 55, 20 to 50, 25 to 45, or 30 to 40 contiguous amino acids as found in a MAP polypeptide. Preferably, the amino acid sequence comprises an appropriate anchor motif. An anchor motif facilitates binding of a peptide to an MHC class I or MHC class II molecule.

[0079] The MAP amino acid sequence may comprise one or more epitopes from any MAP polypeptide. A suitable MAP polypeptide may be a secreted component of MAP, may be expressed on the cell surface of MAP, or may be an internal MAP polypeptide. The MAP polypeptide may be involved in the pathogenicity of MAP. The MAP polypeptide may be, for example, an AhpC polypeptide, a Gsd polypeptide, a P900 polypeptide, such as a P 12 polypeptide, or an Mpa polypeptide.

[0080] AhpC is a secreted component shared by many pathogenic mycobacteria. It is involved in the ability of MAP to survive within macrophages and is upregulated on entry into a state of microbial dormancy. The amino acid sequence of the MAP AhpC polypeptide is shown in SEQ ID NO: 1.

[0081] Gsd is a glycosyl transferase encoded by the GS pathogenicity element, with a predicted signal sequence and lipid acylation site. Microarray analysis shows that it is upregulated in the intracellular environment. It is expressed on the microbial cell surface and is predicted to transfer GDP-fucose to sub-terminal rhamnose to cap surface glycopeptidolipid on MAP with derivatised fucose, thus giving the pathogen in its ZN- negative state an inert, hydrophobic, and highly resistant cell surface. The amino acid sequence of the MAP Gsd polypeptide is shown in SEQ ID NO: 3.

[0082] P12 is the carboxy-terminal 17-kDa fragment of P900 (also known as P43) encoded by IS 900 which is also upregulated intracellularly. It is strongly predicted on the cell surface. Both in MAP and in p43.rec.E.coli, it is the substrate for specific proteolytic cleavage and exodomain release. The amino acid sequence of the MAP P12 polypeptide is shown in SEQ ID NO: 5.

[0083] Mpa is also expressed on the surface of MAP and is believed to be unique to the pathogen. It is both an acetylase and a predicted pore molecule with 10 transmembrane regions and a large extracellular peptide loop. The amino acid sequence of the MAP Mpa polypeptide is shown in SEQ ID NO: 7.

[0084] Thus, a suitable AhpC polypeptide may have the amino acid sequence of SEQ ID NO: 1. A suitable Gsd polypeptide may have the amino acid sequence of SEQ ID NO: 3. A suitable P12 polypeptide may have the amino acid sequence of SEQ ID NO: 5. A suitable Mpa polypeptide may have the amino acid sequence of SEQ ID NO: 7.

[0085] The immunogenic polypeptides described herein may comprise two or more MAP amino acid sequences, such as three or more MAP amino acid sequences, four or more MAP amino acid sequences, or five or more MAP amino acid sequences. The two or more, three or more, four or more, or five or more MAP amino acid sequences may be present in the same MAP polypeptide. The amino acid sequences may, for example, be non-contiguous stretches of the same MAP polypeptide. The two or more, three or more, four or more, or five or more MAP amino acid sequences may be found in different MAP polypeptides, such as in two or more, three or more, four or more, or five or more separate MAP polypeptides. Two or more of the three or more, four or more, or five or more MAP amino acid sequences may be found in the same MAP polypeptide whilst one or more of the three or more, four or more, or five or more MAP amino acid sequences may be found in one or more different MAP polypeptides. The two or more, three or more, four or more, or five or more MAP amino acid sequences may each be found in a different MAP polypeptide. For example, the immunogenic polypeptide may comprise MAP amino acid sequences from two or more, such as three or all of the MAP AhpC, Gsd, P900, e.g. P12, and Mpa polypeptides.

[0086] The two or more, three or more, four or more, or five or more amino acid sequences may be comprised in the same immunogenic polypeptide, for example as a fusion protein. The immunotherapeutic agent may comprise two or more immunogenic polypeptides. Hence, the two or more, three or more, four or more, or five or more amino acid sequences may be comprised in different immunogenic polypeptides, such as in two or more, three or more, four or more, or five or more separate immunogenic polypeptides. The amino acid sequences may each be comprised in a different immunogenic polypeptide. The separate immunogenic polypeptides may or may not be linked by non- covalent linkages. For example, the amino acid sequences present in the AhpC, Gsd, P12, and Mpa polypeptides may be comprised in different immunogenic polypeptides or in the same immunogenic polypeptide as a fusion protein. The amino acid sequences may be joined in any order. In the fusion protein, linker sequences may separate the MAP amino acid sequences from different MAP polypeptides, such as from the AhpC, Gsd, P900, and Mpa polypeptides, preferably from the AhpC, Gsd, Pl 2, and Mpa polypeptides or the AhpC, Gsd, P900, and Mpa polypeptides, including both a P12 amino acid sequence and an additional P900 sequence. Thus, in the fusion protein, 1, 2, 3, or all of the MAP amino acid sequences found in the AhpC, Gsd, P12, and Mpa polypeptides may be contiguous with each other or may be separated from each other, for example by such linkers. There may or may not be additional sequences present at the amino-terminal or carboxy- terminal of the fusion protein. A suitable fusion protein may comprise the amino acid sequences of SEQ ID NOs: 9, 11, 13, and 15. A suitable fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 28, 30, or 31.

[0087] Where the two or more, three or more, four or more, or five or more amino acid sequences are comprised in the same immunogenic polypeptide, the amino acid sequences may be joined directly to each other, or the immunogenic polypeptide may comprise one or more additional linking amino acids, such as from 1 to 20, 2 to 15, 3 to 10, 4 to 8 or 5 to 6 amino acids. The fusion protein may, for example, comprise 0, 1, 2, 3, or more such linkers. The linkers are typically 1, 2, 3, 4 or more amino acids in length, but may be longer, such as up to 20, 22 or 25 amino acids. For example, A or AA may be used as a linker. Other examples of linkers include: GGG, GG, SGSG (SEQ ID NO: 32), AG, GGGS (SEQ ID NO: 33), AAY, a dilysine linker (KK), EAAAK (SEQ ID NO: 34), AAY and HEYGAEALERAG (SEQ ID NO: 35). Preferably, the peptide linker is non- immunogenic.

[0088] The two or more, three or more, four or more, or five or more amino acid sequences may be joined by a “self-cleaving” peptide. A “self-cleaving” peptide is a peptide that mediates cleavage of the polypeptide it is contained in during translation. One example of a self-cleaving peptide is a 2A polypeptide. 2A polypeptides are 18 to 22 amino acid long viral polypeptides that mediate cleavage of polypeptides during translation in eukaryotic cells. The 2A polypeptide may be derived from any virus that includes a 2A polypeptide in its viral genome. The 2A polypeptide typically comprises the conserved sequence GDVEXNPGP (SEQ ID NO: 36). One example of a 2A polypeptide has the sequence APVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 37).

[0089] The MAP amino acid sequence found in the MAP P900 polypeptide may comprise an amino acid sequence of at least 8 contiguous amino acids from the region of MAP P900 set out in SEQ ID NO: 17. The immunogenic polypeptides described herein may comprise the amino acid sequence MVINDDAQRLLSQR (SEQ ID NO: 19) and any one of or any combination of an amino acid sequence present in an AhpC polypeptide, a Gsd polypeptide, a P12 polypeptide, and an Mpa polypeptide described above. The immunogenic polypeptide may be phosphorylated at the serine residue in the amino acid sequence MVINDDAQRLLSQR (SEQ ID NO: 19).

[0090] The amino acid sequence of exemplary immunogenic regions of MAP P900 are MVINDDAQRLLSQR (SEQ ID NO: 19), which may be phosphorylated to form MVINDDAQRLL[pS]QR (SEQ ID NO: 20); VTTLADGGEVTWAID (SEQ ID NO: 21); VSIRTDPSSR (SEQ ID NO: 22); YLSALVSIRTDPSSR (SEQ ID NO: 23), which may be phosphorylated to form YLSALVSIRTDPS[pS]R (SEQ ID NO: 24), YLSALVSIRTDP[pS]SR (SEQ ID NO: 25) or YLSALVSIRTDP[pS][pS]R (SEQ ID NO: 26); and EVVVAQPVWAGVDAGKADHY (SEQ ID NO: 27). The immunotherapeutic agent may comprise an amino acid sequence found in the amino-terminal of a P900 polypeptide and an amino acid sequence found in any one or more of the AhpC, Gsd, Pl 2, and Mpa polypeptides in the same immunogenic polypeptide as a fusion protein. The P900 amino acid sequence and the AhpC, Gsd, P12, and / or Mpa amino acid sequences may be provided in any order in the fusion protein. The amino acid sequences may, for example, be provided in the order amino-terminal P900 - AhpC - Gsd - P12 - Mpa. As described above, the amino acid sequence present in the AhpC, Gsd, P12, and Mpa polypeptides may be present in a fusion protein, and a suitable fusion protein may comprise the amino acid sequences of SEQ ID NOs: 9, 11, 13, and 15. A suitable fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 28, 30, or 31.

[0091] In the fusion protein, linker sequences may separate the amino acid sequences. There may or may not be additional sequences present at the amino-terminal or carboxyterminal of the fusion protein. Typically, the fusion protein comprises 1, 2, 3, or more such linkers. The linkers are typically 1, 2, 3, 4 or more amino acids in length. Thus, in the fusion protein, 1, 2, 3, 4, or all of the amino acid sequences may be contiguous with each other or may be separated from each other, for example by such linkers.

[0092] The MAP amino acid sequences may be present in two or more separate immunogenic polypeptides, which may or may not be linked by non-covalent linkages. For example, the MAP amino acid sequences may be provided separately, or may be provided in two, three or more separate fusion proteins. For example, the amino acid sequence found in the amino-terminal of the P900 polypeptide may be comprised in one immunogenic polypeptide, and the amino acid sequences found in the AhpC, Gsd, P12, and Mpa polypeptides may be comprised in another immunogenic polypeptide, such as the fusion protein described above.

[0093] Exemplary immunogenic polypeptides include those described in International Publication Nos. WO 2007 / 017635 and WO 2020 / 012177, both of which are incorporated herein by reference. For example, the immunogenic polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO: 28, 30, or 31.

[0094] Particular modifications can be made to any of the polypeptides described herein. For example, modification can be made to try to improve the overall properties of the polypeptide as an immunogen. The MAP amino acid sequence may, for example, be modified by deletion or substitution to remove an acylation site. Such an acylation site might affect the overall conformation of the polypeptide. By omitting acylation sites, for example by excluding or substituting a cysteine residue, the presentation of effective epitopes within the protein may be optimised. For example, the wild-type MAP Gsd polypeptide sequence of SEQ ID NO: 3 includes a cysteine residue at position 22. In the modified amino acid sequence of SEQ ID NO: 3 shown in SEQ ID NO: 11, the amino acid sequence has been modified by truncation at the amino-terminal such that this cysteine residue is no longer present. Similarly, the amino acid sequence of the amino-terminal fragment of the P900 polypeptide shown in SEQ ID NO: 17 comprises a cysteine residue at position 25. This cysteine residue may be deleted, or substituted by one or more amino acid, such as by a peptide linker.

[0095] The MAP amino acid sequence may be modified to disable or remove potential cross -reacting epitopes. For example, the amino acid sequence may be modified to disable or remove potential cross -reacting human epitopes, such as sequences which generate antibodies in human patients which may cross-react with similar sequences in human proteins. Modifications may thus be made to the MAP sequences to avoid such crossreactivity but to maintain the ability to generate an anti-MAP immune response.

[0096] For example, within the wild-type MAP Gsd polypeptide sequence, the lysine residues at positions 239 and 241 of SEQ ID NO: 3 may each be substituted with asparagine. An equivalent substitution may be made in any of the Gsd sequences described herein. For example, in the amino acid sequence of SEQ ID NO: 11, the lysine residues at positions 216 and 218 may be replaced with asparagines. This may be achieved by modifying the nucleic acid sequence which encodes the Gsd polypeptide. For example, in the gsd polynucleotide sequence of SEQ ID NO: 4, the AAG codons at positions 646 to 648 and 651 to 654 may be replaced by AAT. This maintains the optimised human codon usage of SEQ ID NO: 4 and further removes potentially crossreacting human epitopes.

[0097] Similarly, modifications may be made to the MAP AhpC polypeptide sequence. In the wild-type AhpC polypeptide sequence of SEQ ID NO: 1, the lysine at position 29 may be replaced with threonine and the proline at position 31 may be replaced with leucine. An equivalent substitution may be made in any of the AhpC sequences described herein. For example, in the modified variant amino acid sequence of SEQ ID NO: 9, the same substitutions may be made at the position 28 lysine and the position 30 proline. This may be achieved by modifying the nucleic acid sequence which encodes the AhpC polypeptide. For example, in the ahpC polynucleotide sequence of SEQ ID NO: 10, the AAA codon at positions 82 to 84 may be replaced by ACA and the CCC codon at positions 88 to 90 may be replaced by CTC. This maintains the optimised human codon usage of SEQ ID NO: 10 and further removes potentially cross -reacting human epitopes.

[0098] Similarly, modifications may be made to reduce the hydrophobicity of the polypeptide and thus to help optimise the surface presentation of epitopes. For example, the wild-type Mpa polypeptide sequence of SEQ ID NO: 7 includes ten transmembrane regions. In order to reduce the hydrophobicity of the polypeptide, one or more of these regions, or parts of these regions, may be omitted or substituted. For example, one or more, or all, of the transmembrane regions may be deleted. Such regions may be deleted totally or partially, optionally leaving none, one, two or more amino acid residues from the ends of the transmembrane sequence in the polypeptide. Thus, one modification may be the deletion or substitution of one or more hydrophobic amino acids. An example of this is SEQ ID NO: 15, in which most of the transmembrane sequences of the MAP Mpa polypeptide have been deleted, leaving only one or two amino acids from the transmembrane regions in the variant polypeptide. Another example is deletion of the four amino-terminal amino acid residues, MTVT, of SEQ ID NO: 17.

[0099] The MAP amino acid sequence may be a fragment of a MAP polypeptide, such as a truncated MAP polypeptide, for example a MAP polypeptide that is truncated by removal of one or more amino acids from the amino- and / or carboxy-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the amino- and / or carboxy-terminal in this way. Fragments may also be generated by one or more internal deletions. For example, a fragment may comprise two or more epitope regions from a full-length polypeptide of the region in the absence of non-epitope amino acids. Preferably, a fragment of an amino-terminal AhpC, Gsd, P12, Mpa, or P900 polypeptide comprises at least one epitope capable of inducing an immune response against the unmodified MAP polypeptide. Such fragments may be derived from an amino acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, or 17, or may be derived from a variant polypeptide described herein. Preferably, such fragments are between 8 and 150 residues in length, for example 8 to 50 or 8 to 30 residues. Alternatively, fragments may be longer sequences, for example comprising at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the full-length polypeptide.

[0100] In the AhpC variant sequence of SEQ ID NO: 9, predicted strong class II epitopes have been identified at amino acids 48 to 56, 90 to 101 and 161 to 169. The immunogenic polypeptide may comprise an amino acid sequence from the AhpC polypeptide which comprises at least one, for example one, two or all three of these epitopes.

[0101] In the Gsd variant sequence of SEQ ID NO: 11, predicted class I epitopes have been identified at amino acids 1 to 32, 58 to 68, 99 to 119, 123 to 147, 159 to 169, 180 to 194 and 200 to 231, and predicted strong class II epitopes have been identified at amino acids 64 to 76, 95 to 110, 192 to 206 and 223 to 240. The immunogenic polypeptide may comprise an amino acid sequence from the Gsd polypeptide which comprises at least one, for example one, two, three, four, five, six, seven, eight, nine, ten or all of these epitopes.

[0102] In the P12 variant sequence of SEQ ID NO: 13, predicted class I epitopes have been identified at amino acids 33 to 56 and 98 to 117 and a predicted strong class II epitope has been identified at amino acids 3 to 10. The immunogenic polypeptide may comprise an amino acid sequence from the P12 polypeptide which comprises at least one, for example one, two or all three of these epitopes.

[0103] In the Mpa variant sequence of SEQ ID NO: 15, a predicted class I epitope has been identified at amino acids 130 to 160, and predicted strong class II epitopes have been identified at amino acids 56 to 64 and 150 to 160. The immunogenic polypeptide may comprise an amino acid sequence from the Mpa polypeptide which comprises at least one, for example one, two or all three of these epitopes. A particularly strong T cell epitope has also been identified at amino acids 357 to 365 of SEQ ID NO: 7 and amino acids 177 to 185 of SEQ ID NO: 15. This epitope is found in the construct of SEQ ID NO: 28 at amino acids 761 to 769. A preferred Mpa polypeptide sequence is a sequence which forms this strong T cell epitope, optionally in addition to one, two or all three of the predicted class I and class II epitopes mentioned above.

[0104] The strong T cell epitope is believed to be located in the fifth extracellular loop of Mpa. A preferred Mpa polypeptide may therefore maintain the sequence of the fifth extracellular loop. An immunogenic polypeptide may therefore comprise the amino acid sequence which forms this epitope and also adjacent amino acids from the fifth extracellular loop of Mpa. Preferably, this fifth extracellular loop will be present in a polypeptide in a suitable form and conformation for it to be recognised by the immune system.

[0105] The immunogenic polypeptides may comprise further additional sequences, such as those encoded by the polynucleotides and vectors described below. For example, an immunogenic polypeptide may comprise additional epitopes, therapeutic polypeptides, adjuvants, or immunomodulatory molecules. A suitable adjuvant may be an ADP- ribosylating bacterial toxin, such as diphtheria toxin (DT), pertussis toxin (PT), cholera toxin (CT), the Escherichia coli heat labile toxins (LT1 and LT2), Pseudomonas endotoxin A, Pseudomonas exotoxin S, Bacillus cereus exoenzyme, Bacillus sphaericus toxin, Clostridium botulinum C2 and C3 toxins, Clostridium limosum exoenzyme, toxins from Clostridium perfringens, Clostridium spiriforma and Clostridium, difficile, and Staphylococcus aureus EDIN. Most ADP-ribosylating bacterial toxins contain A and B subunits.

[0106] The immunogenic polypeptides may comprise a leader or signal peptide sequence, i.e. an amino acid sequence at or near the amino-terminus of the polypeptide that functions in targeting or regulating the polypeptide. For example, an amino acid sequence may be included in the polypeptide which targets it to particular tissues in the body, or which helps the processing or folding of the polypeptide upon expression. Such sequences are well known in the art and could be selected by the skilled person depending upon, for example, the desired properties and production method of the polypeptide. Typically, a signal peptide sequence encodes a peptide of 10 to 30 amino acids, for example 15 to 20 amino acids. Typically, the amino acids of the signal peptide sequence are predominantly hydrophobic.

[0107] The immunogenic polypeptides may be chemically modified, such as post- translationally modified. For example, an immunogenic polypeptide may be modified by phosphorylation, for example 3-amino phosphorylation, and by glycosylation, for example mannosylation.

[0108] Chemically modified polypeptides also include those having one or more residues chemically derivatized by reaction of a functional side group. Such derivatized side groups include those which have been derivatized to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups, and formyl groups. Free carboxyl groups may be derivatized to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine may be derivatized to form N-im-benzylhistidine.

[0109] Also included as chemically modified peptides are those which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline or homoserine may be substituted for serine.

[0110] The immunogenic polypeptides may be modified at the amino-terminus and / or at the carboxy-terminus, and / or may be conjugated or coupled to a carrier molecule. Polypeptides may, for example, be conjugated to a bacterial saccharide or a carrier protein, such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA), human serum albumin (HSA), ovalbumin (OVA). The polypeptide may be biotinylated at the aminoterminus or carboxy- terminus, may be amidated at the amino-terminus or carboxyterminus, and / or may have a peptide tag added at the amino-terminus or the carboxyterminus. The peptide tag may be, for example, a polylysine, such as a branched polylysine octamer, or a cell penetrating peptide, such as an oligo-arginine (e.g. a polyarginine octamer or nonomer). The polypeptide may be biotinylated at the aminoterminus and have an amide group or a branched polylysine octamer at the carboxyterminus. One or more additional amino acid residues may be added at the aminoterminus and / or the carboxy-terminus, optionally in addition to other terminal modifications. For example, one or more, such as two, alanine residues may be added at the amino-terminus to increase immunogenicity and specificity and / or charged residues. For example, GKK may be added at the amino-terminus, or preferably at the carboxyterminus, to reduce hydrophobicity. Where residues, such as GKK, are added at one terminus, the mirror image residues, such as KKG, may be added at the other terminus.

[0111] Any of the immunogenic polypeptides described herein may be produced from, or delivered to the subject in the form of, a polynucleotide which encodes and is capable of expressing the polypeptide. Accordingly, the immunotherapeutic agent against MAP for use according to the invention may be a polynucleotide encoding an immunogenic polypeptide described herein. The immunotherapeutic agent may comprise a polynucleotide encoding any one or more of the immunogenic polypeptides, such as the fusion proteins, described above. The immunotherapeutic agent may comprise a polynucleotide comprising two or more copies, such as three, four, five or more copies of a nucleic acid sequence encoding any one or more of the immunogenic polypeptides described above. The immunotherapeutic agent may comprise one or more polynucleotides, wherein each encodes a different immunogenic polypeptide described above.

[0112] A nucleic acid sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5'-terminus (corresponding to the amino-terminus) and a translation stop codon at the 3'- terminus (corresponding to the carboxy- terminus). Such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, and synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0113] Therefore, a polynucleotide may comprise a nucleic acid sequence encoding one or more of the immunogenic polypeptides described above.

[0114] A suitable polynucleotide encoding the AhpC polypeptide described above may have the nucleic acid sequence of SEQ ID NO: 2. A suitable polynucleotide encoding the Gsd polypeptide described above may have the nucleic acid sequence of SEQ ID NO: 4. A suitable polynucleotide encoding the P12 polypeptide described above may have the nucleic acid sequence of SEQ ID NO: 6. A suitable polynucleotide encoding the Mpa polypeptide may have the nucleic acid sequence of SEQ ID NO: 8. A suitable polynucleotide encoding the fusion protein described above may have the nucleic acid sequence of SEQ ID NO: 29.

[0115] The polynucleotide may comprise mRNA. The immunotherapeutic agent may be a mRNA vaccine. The mRNA comprises one or more protein-encoding sequences, either each or collectively encoding one or more of the polypeptides described herein. Preferably, the mRNA does not substantially induce an immune response (e.g. an innate immune response) of a host cell into which the mRNA is introduced. The skilled person can readily determine the nucleic acid sequence of an mRNA from the amino acid sequences described herein and / or from the DNA sequences described herein. The nucleic acid sequence of the polynucleotide may be a variant of any of the nucleic acid sequences described above. For example, a variant may be a substitution, deletion or addition variant of any of the nucleic acid sequences described above. A variant of one of the nucleic acid sequences encoding the one or more amino acid sequences present in the AhpC, Gsd, P12, Mpa, and / or P900 polypeptides described above may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or more nucleic acid substitutions and / or deletions from the nucleic acid sequences described above.

[0116] Suitable variants may be at least 70% homologous to one of the nucleic acid sequences described above, preferably at least 80%, 85% or 90%, and more preferably at least 95%, 96%, 97%, or 99% homologous to the nucleic acid sequences described above. Methods of measuring homology are well known in the art. It will be understood by the skilled person that, in the present context, homology is calculated on the basis of nucleic acid identity. Such homology may exist over the entire length of the polynucleotide sequence.

[0117] A variant polynucleotide may encode the same polypeptide sequence as another polynucleotide but may have a different nucleic acid sequence due to the use of different codons to encode the same amino acids.

[0118] The coding sequence of the polynucleotides described above may be optimised to more closely resemble the codon usage of highly expressed genes in mammalian cells, for example human cells. Where more than one codon is available to code for a given amino acid, it has been observed that the codon usage patterns of organisms are highly nonrandom. Different species show a different bias in their codon selection and utilisation of codons may be markedly different in a single species between genes which are expressed at high and low levels. For example, humans are less strongly biased than certain bacteria or viruses. Therefore, it is possible that, for example, a mycobacterial gene expressed in human cells will have an inappropriate distribution of codons for efficient expression. It is believed that the presence in a heterologous DNA sequence of clusters of codons which are rarely observed in the host in which expression is to occur is predictive of low heterologous expression levels in that host.

[0119] Accordingly, in the polynucleotide described above, the codon usage pattern may be altered from that present in MAP to more closely represent the codon bias of humans. Thus, the particular polynucleotide sequence which encodes an immunogenic polypeptide described above may be altered to optimise the codons for human use. As an example, the MAP polynucleotide sequences given in SEQ ID NOs: 10, 12, 14, and 16 have been codon-optimised for human use. Such modifications may improve the ability of polynucleotides to express their encoded polypeptides in a human cell.

[0120] As explained above in relation to polypeptides, the polynucleotides described herein may also be modified to disable or remove potential cross -reacting epitopes in the encoded polypeptide.

[0121] A polynucleotide variant may be a fragment of a polynucleotide described above. Polynucleotide fragments may be made by truncation, for example by removal of one or more nucleotides from one or both ends of a polynucleotide. Up to 10, up to 20, up to 30, up to 40, up to 50, up to 75, up to 100, up to 200 or more nucleic acids may be removed from the 3' and / or 5' end of the polynucleotide in this way. Fragments may also be generated by one or more internal deletions. For example, a variant may encode a polypeptide which comprises two or more epitope regions from a full-length polypeptide described above in the absence of non-epitope amino acids. Preferably, a fragment of a polynucleotide sequence encoding an AhpC, Gsd, Pl 2, Mpa, or P900 polypeptide sequence comprises at least one region encoding an epitope capable of generating and / or enhancing an immune response against the unmodified MAP polypeptide. Such fragments may be derived from a polynucleotide described above. Preferably, such fragments are between 24 and 500 residues in length, for example 24 to 400, 24 to 300, 24 to 100, 100 to 200, or 200 to 400 residues. Alternatively, fragments may be longer sequences, for example comprising at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of a full-length polynucleotide described above.

[0122] Polynucleotides may be synthesised according to methods well known in the art. Polynucleotides encoding an immunogenic polypeptide described above can be obtained using recombinant methods, such as by screening cDNA and genomic libraries from cells expressing an antigen. Furthermore, the desired nucleic acid sequences can be isolated directly from cells containing the same, using standard techniques, such as phenol extraction and PCR of cDNA or genomic DNA. Polynucleotide sequences can also be produced synthetically rather than cloned.

[0123] Methods of synthesising RNA are known in the art. Once nucleic acid sequences have been obtained, they may be linked together to provide a nucleic acid molecule using standard cloning or molecular biology techniques. Alternatively, the sequences can be produced synthetically rather than cloned. The polynucleotide sequence can be designed with the appropriate codons for the particular amino acid sequence desired.

[0124] A polynucleotide described above may also be modified. It may be desirable to modify the polynucleotide such that the rate of degradation of the modified polynucleotide in a host cell into which the polynucleotide is introduced is lower than the rate of degradation of the unmodified polynucleotide. For example, the polynucleotide may be modified to modulate (e.g. suppress) the immune response of the host cell into which the polynucleotide is introduced. The modification may modulate the innate immune response of the host cell. An innate immune response comprises a cellular response to exogenous polynucleotides, which involves the induction of cytokine expression and release, and cell death. Protein synthesis is also reduced during the innate cellular immune response. Thus, suppressing the innate immune response may enhance the viability of host cells, intracellular retention of polynucleotides, and / or the efficiency of the production of the polypeptides encoded by the polynucleotides.

[0125] Modifications which modulate the immune response (e.g. the innate immune response) may comprise one or more modifications to the polynucleotide. One or more of the nucleosides or nucleotides of the polynucleotide may be modified. In cases where the polynucleotide is an mRNA, the modification may decrease the interaction of the mRNA with major groove-binding partners. Major groove-binding partners include RNA recognition receptors which detect and respond to RNA ligands through interactions with the major groove face of a nucleotide. Examples of major groove-binding partners include Toll-like Receptors (TLR) 3, 7, and 8, which can respond to single- and double- stranded RNAs within membranes, and members of the superfamily 2 class of DEX(D / H) helicases and ATPases (e.g. retinoic acid-inducible gene I (RIG-I), melanoma differentiation- associated gene 5 (MDA5), laboratory of genetics and physiology 2 (LGP2), HIN-200 domain-containing proteins, and helicase domain-containing proteins), which can sense RNAs within the cytoplasm. Such modifications may hence decrease the immunogenicity of the mRNA, and / or expression and secretion of pro-inflammatory cytokines, relative to the unmodified mRNA by reducing the interaction of the mRNA with one or more of the major groove-binding partners described above. Nucleoside or nucleotide modifications which suppress immune responses are known in the art.

[0126] Alternatively, it may be desirable to increase the rate of intracellular degradation of a polynucleotide introduced into a host cell if the precise timing of polypeptide production is desired. Thus, the polynucleotide may comprise a degradation domain, which is capable of being acted on in a directed manner within a host cell. Suitable polynucleotide degradation domains are known in the art.

[0127] In cases where the polynucleotide comprises an mRNA, the mRNA may be modified to comprise a further sequence in addition to the protein-encoding sequence. The further sequence may be a coding sequence. The further sequence may be a non-coding sequence. For example, the mRNA may comprise a 5' untranslated region (UTR) and / or a 3' UTR. Either or both of the UTRs may independently comprise one or more nucleoside modifications. Nucleoside modifications may be present in the translatable region of the mRNA. As a further example, the mRNA may comprise one or more intronic nucleotide sequences which can be excised from the mRNA. As a further example, the mRNA may comprise a Kozak sequence. As a further example, the mRNA may comprise an internal ribosome entry site (IRES). The IRES may act as the sole ribosome-binding site or may serve as one of multiple ribosome binding sites of the mRNA. An mRNA comprising more than one functional ribosome-binding site may encode multiple polypeptides which are translated independently by ribosomes. In cases where the mRNA is provided with an IRES, a second translatable protein-coding region may be provided. Examples of IRES sequences that can be used include those from picornaviruses (e.g. foot-and-mouth disease viruses), pest viruses (e.g. classical swine fever viruses), polioviruses, encephalomyocardititis viruses, hepatitis C viruses, murine leukaemia viruses, simian immune deficiency viruses, and cricket paralysis viruses.

[0128] Any of the polynucleotides described above may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the immunogenic polypeptide described above in vivo humans. These expression cassettes, in turn, are typically provided within vectors, which are suitable for use as reagents for nucleic acid immunisation. Such an expression cassette may be administered directly to a subject. Alternatively, a vector comprising a polynucleotide described above may be administered to a subject. Preferably, the polynucleotide is administered using a vector. A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information and allowing expression of a polypeptide described above.

[0129] Accordingly, any of the polynucleotides described above may be comprised in a vector. The immunotherapeutic agent may comprise a vector comprising two or more copies, such as three, four, five or more copies of any one or more of the polynucleotides described above.

[0130] Vectors are routinely constructed in the art of molecular biology and may, for example, involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as polyadenylation signals, which may be necessary and which are positioned in the correct orientation in order to allow for expression of a polypeptide.

[0131] Thus, an immunogenic polypeptide described above may be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, a polynucleotide comprised in a vector is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.

[0132] A number of expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleic acid sequence of interest operably linked to expression control sequences. These control sequences include transcriptional promoter sequences and transcriptional start and termination sequences. A suitable vector may be, for example, a plasmid, virus, or phage vector provided with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally a regulator of the promoter. A plasmid is a vector in the form of an extrachromosomal genetic element.

[0133] Promoters and other expression regulation signals may be selected to be compatible with the host cell for which expression is designed. Mammalian promoters, such as 0- actin promoters, may be used. Tissue- specific promoters are especially preferred. Mammalian promoters include the metallo thionein promoter which can be induced in response to heavy metals such as cadmium.

[0134] A viral promoter may be used to drive expression from the polynucleotide. Typical viral promoters for mammalian cell expression include the SV40 large T antigen promoter, adenovirus promoters, the Moloney murine leukaemia virus (MMLV) long terminal repeat (LTR) promoter, the mouse mammary tumour virus LTR promoter, the rous sarcoma virus (RSV) LTR promoter, the simian virus 40 (SV40) early promoter, the human cytomegalovirus (CMV) immediate early (IE) promoter, adenovirus promoters, including the adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoters, such as HSV IE promoters, or human papillomavirus (HPV) promoters, such as the HPV upstream regulatory region (URR). All these promoters are readily available in the art.

[0135] The promoter may be a cytomegalovirus (CMV) promoter. A preferred promoter element is the CMV IE promoter devoid of intron A but including exon 1. Thus, the expression from the polynucleotide may be under the control of human CMV IE early promoter. Expression vectors using the human CMV IE promoter include, for example, pWRG7128, pBC12 / CMV, and pJW4303. A human CMV IE promoter sequence can be obtained using known methods. A native human CMV IE promoter can be isolated directly from a sample of the virus using standard techniques. The sequence of a human CMV IE promoter is available at Genbank #M60321 (human CMV Towne strain) and X17403 (human CMV Adl69 strain). A native sequence could therefore be isolated by PCR using PCR primers based on the known sequence. A suitable human CMV promoter sequence could also be isolated from an existing plasmid vector. Promoter sequences can also be produced synthetically.

[0136] Typically, transcription termination and polyadenylation sequences will also be present in the vector, located 3' to the translation stop codon. Preferably, a sequence for optimisation of initiation of translation, located 5' to the coding sequence, is also present. Examples of transcription terminator or polyadenylation signals include those derived from SV40 as well as a bovine growth hormone terminator sequence. Introns, containing splice donor and acceptor sites, may also be designed into the vector.

[0137] Expression systems often include transcriptional modulator elements, such as enhancers. Examples of suitable enhancers include the SV40 early gene enhancer, the enhancer / promoter derived from the LTR of the RSV, and elements derived from human or murine CMV, for example elements included in the CMV intron A sequence. Nucleic acid immunisation involves the introduction of a nucleic acid molecule encoding one or more selected antigens into a host cell for the in vivo expression of the one or more antigens. Efficient expression of the polynucleotide in the transfected cell is required to provide a sufficient amount of the antigenic gene product. Several factors are known to affect the levels of expression obtained, including transfection efficiency, and the efficiency with which the sequence of interest is transcribed and the mRNA translated.

[0138] A vector described above may be administered directly as “a naked nucleic acid construct", preferably further comprising flanking sequences homologous to the host cell genome. A “naked vector" is a vector, such as a viral vector or plasmid comprising the polynucleotide described herein. A “naked vector" is not carried in any delivery vehicle. When such a vector enters a host cell, the polynucleotide(s) it comprises are transcribed and the polypeptide(s) encoded by the polynucleotide(s) are translated within the cell.

[0139] The vector described herein may be introduced into suitable host cells using a variety of viral techniques which are known in the art, such as infection with a recombinant viral vector, such as a DNA viral vector or an RNA viral vector. For example, the vector itself may be a recombinant viral vector, such as a DNA viral vector or an RNA viral vector. Suitable recombinant viral vectors include but are not limited to adenovirus vectors, adeno-associated viral (AAV) vectors, herpes simplex virus (HSV) vectors, retroviral vectors, lentiviral vectors, baculoviral vectors, poxvirus vectors, and parvovirus vectors. In the case of viral vectors, administration of the polynucleotide is mediated by viral infection of a target cell.

[0140] A number of virus-based systems have been developed for transfecting mammalian cells. For example, the polynucleotide described herein may be inserted into an adenovirus vector. A number of adenovirus vectors are known. Adenovirus subgroup C serotypes 2 and 5 are commonly used as vectors. A simian adenovirus derived from the AdC68 strain may be used. The wild-type adenovirus genome is approximately 35 kb of which up to 30 kb can be replaced with foreign DNA. There are four early transcriptional units (El, E2, E3, and E4), which have regulatory functions, and a late transcript, which codes for structural proteins. Adenovirus vectors may have the El and / or E3 gene inactivated, for example deleted. The missing gene(s) may then be supplied in trans by a helper virus or plasmid, or integrated into a helper cell genome. Adenovirus vectors may use an E2a temperature-sensitive mutant or an E4 deletion. Minimal adenovirus vectors may contain only the inverted terminal repeats (ITRs) and a packaging sequence around the transgene, with all the necessary viral genes being provided in trans by a helper virus. Suitable adenoviral vectors thus include Ad5 vectors and simian adenovirus vectors. Viral vectors may also be derived from the pox family of viruses, including vaccinia viruses and avian poxviruses, such as fowlpox virus. For example, MVA is a highly attenuated poxvirus. MVA is a strain of vaccinia virus which does not replicate in most cell types, including normal human tissues. A recombinant MVA vector may therefore be used to deliver the polynucleotide described above.

[0141] Preferred immunotherapeutic agents include ChAdOx2 HAV and MVA HAV.

[0142] RNA virus-based systems have also been developed. For example, the polynucleotides described above may be inserted into a vector and packaged as retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. Retroviral vectors may be based upon the MMLV. In a retroviral vector, one or more of the viral genes (gag, pol and env) are preferably replaced with a nucleic acid sequence described above.

[0143] Alternatively, the vector may be a plasmid, which is an autonomously replicating, extrachromosomal circular or linear DNA molecule. The plasmid may include additional elements, such as an origin of replication. Such elements are known in the art and can be included using standard techniques. Numerous suitable expression plasmids are known in the art. For example, one suitable plasmid is pSG2. This plasmid was originally isolated from Streptomyces ghanaensis. Single restriction sites of 13.8 kb in length for Hindlll, EcoRV and PvuII, and the possibility of deleting non-essential regions of the plasmid, make pSG2 a suitable basic replicon for vector development.

[0144] As an alternative to viral vector systems, liposomal preparations can be used to deliver the polynucleotides described above. Useful liposomal preparations include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred. Cationic liposomes may mediate intracellular delivery of plasmid DNA and mRNA.

[0145] As another alternative to viral vector systems, the polynucleotides described above may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). Other particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyomithine, spermine, spermidine, as well as conjugates of these molecules. The vector described above may be a targeted vector. A targeted vector is a vector whose ability to infect, transfect or transduce a cell, or to be expressed in a host and / or target cell, is restricted to certain cell types within the host subject, usually cells having a common or similar phenotype. The vector may target intestinal cells, such as MAP- infected intestinal cells.

[0146] The vector described above may comprise a single expression cassette from which a single immunogenic polypeptide described above can be expressed. Alternatively, the vector may comprise two or more expression cassettes each capable of expressing a different immunogenic polypeptide described above, such that the vector as a whole is capable of expressing all required immunogenic polypeptides. In cases where the polypeptides are expressed from more than one locus in the vector, or are expressed as multiple separate polypeptides, the expression of the multiple sequences is preferably coordinated such that all polypeptides are expressed together. For example, the same or similar promoters may be used to control expression of the various components. Inducible promoters may be used so that expression of the various polypeptides can be coordinated.

[0147] In some cases, it may be preferable to administer an antibody which binds to MAP, for example an antibody that binds to the immunogenic polypeptide described herein. Accordingly, the immunotherapeutic agent may comprise an antibody which binds to MAP, for example an antibody which binds to the immunogenic polypeptide described herein. The antibody may be specific to one or more of the amino acid sequences of any one or more of the MAP polypeptides described herein. The antibody preferably binds to native MAP. Exemplary antibodies which bind to native MAP are described in International Publication No. WO 2018 / 130836.

[0148] In some cases, it may be preferably to administer a cell which is capable of generating and / or enhancing an immune response against MAP. The cell may be modified to express the immunogenic polypeptide described herein or a receptor which binds to the immunogenic polypeptide described herein. Accordingly, the immunotherapeutic agent may comprise a cell which expresses the immunogenic polypeptide described herein or a receptor which binds to the immunogenic polypeptide described herein.

[0149] Whereas the polynucleotide described herein may be introduced directly into the subject, the polynucleotide alternatively may be introduced ex vivo into cells which have been removed from a subject. In the latter case, cells containing the polynucleotide are re- introduced into the subject such that an immune response can be mounted against the immunogenic polypeptide encoded by the polynucleotide. Thus, the cells may be used therapeutically or prophylactically to deliver the polypeptides described herein to a subject. Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known, including dextran-mediated transfection, calcium phosphate precipitation, electroporation, and direct microinjection into nuclei.

[0150] Modified cells expressing a receptor which binds to the polypeptide described above include immune cells. The immune cells may be of the lymphoid lineage, including T and B cells. T cells are lymphocytes that mature in the thymus and are chiefly responsible for cell-mediated immunity. T cells are also involved in the adaptive immune system. T cells may include, but are not limited to, helper T cells, cytotoxic T cells, central memory T cells, effector memory T cells, regulatory T cells, natural killer T cells, mucosal-associated invariant T cells, and gamma-delta T cells. Cytotoxic T cells are capable of inducing the death of infected somatic or tumour cells. Preferably, the modified cell is a T cell, optionally a CD4+T cell or a CD8+T cell. Thus, the modified cell may be a T cell, optionally a CD4+T cell or a CD8+T cell. The modified cell may be a population of modified cells comprising CD4+T cells and / or CD8+T cells.

[0151] A subject’s T cells may be genetically modified to target specific antigens through the introduction of a heterologous TCR or a heterologous CAR. That is, the subjects T cells may be transduced with, or engineered to comprise, a nucleic acid sequence encoding a heterologous TCR or CAR, for example by gene knock-in. Thus, the modified cells comprising the heterologous TCR or CAR may comprise a polynucleotide, expression cassette, or vector encoding the heterologous TCR or CAR. The modification and subsequent expansion of the modified cells may be performed in vitro and / or ex vivo.

[0152] Preferably, the heterologous TCR or CAR binds to an amino acid sequence comprised in an immunogenic polypeptide described above. As the amino acid sequence is also present in a MAP polypeptide, the heterologous TCR or CAR is capable of binding the MAP polypeptide. Upon binding to a MAP polypeptide in a MAP-infected subject, the modified cell expressing the heterologous TCR and / or CAR preferably exhibits T cell effector functions and / or cytolytic effects towards MAP, and / or undergo proliferation and / or cell division. Activated modified cells comprising the heterologous TCR or CAR may secrete anti-tumour cytokines which can include, but are not limited to, TNF-a, IFN- y, and IL2.

[0153] Pharmaceutical Compositions

[0154] The immunotherapeutic agent may be provided in a composition. Any of the immunogenic polypeptides, polynucleotides, vectors, antibodies, and cells described herein may be provided in a composition. The composition may be formulated using standard pharmaceutical formulation chemistries and methodologies, all of which are readily available to the skilled person. For example, compositions comprising the immunotherapeutic agent described herein may comprise one or more pharmaceutically acceptable excipients or vehicles. The compositions may comprise one or more adjuvant. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient or vehicle. These excipients, vehicles, and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the subject receiving the composition, and which may be administered without undue toxicity. Pharmaceutically acceptable excipients or vehicles include, but are not limited to, liquids, such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts can also be included therein. Suitable pharmaceutically acceptable salts include mineral acid salts, such as hydrochlorides, hydrobromides, phosphates, sulphates, and the like, and the salts of organic acids, such as acetates, propionates, malonates, benzoates, and the like.

[0155] Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions may further comprise one or more additional ingredients including, but not limited to, suspending, stabilising, or dispersing agents. In cases where the composition is for parenteral administration, the active ingredient may be provided in dry (e.g. a powder or granule) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. The compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the prior art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally- acceptable diluent or solvent, such as water or 1,3-butane diol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono- or diglycerides.

[0156] Other parentally-administrable compositions which are useful include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

[0157] In cases where the composition comprises a nucleic acid molecule, certain facilitators of nucleic acid uptake and / or expression (i.e. transfection-facilitating agents) may also be included in the composition. Suitable transfection-facilitating agents include bupivacaine, cardiotoxin and sucrose, and transfection-facilitating vehicles, such as liposomal or lipid preparations, which are routinely used to deliver nucleic acid molecules. Anionic and neutral liposomes are widely available and well-known for delivering nucleic acid molecules. Cationic lipid preparations are also well-known vehicles for use in the delivery of nucleic acid molecules. Suitable lipid preparations include DOTMA (N-[l- (2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride) and DOTAP (1,2- bis(oleyloxy)-3-(trimethylammonio)propane). These cationic lipids may preferably be used in association with a neutral lipid, for example DOPE (dioleyl phosphatidylethanolamine). Further transfection-facilitating agents that can be added to the above lipid or liposome preparations include spermine derivatives and membrane- permeabilising compounds, such as GALA, Gramicidine S, and cationic bile salts.

[0158] Alternatively, the nucleic acid molecules described herein may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides). Other particulate systems and polymers can also be used. Suitable polymers include polylysine, polyarginine, polyomithine, spermine, spermidine, as well as conjugates of these molecules.

[0159] The formulated compositions will include a therapeutically or prophylactically effective dose or amount of the immunotherapeutic agent described herein. An appropriate effective amount can be readily determined by the skilled person.

[0160] In some cases, the immunotherapeutic agent against MAP for use according to the invention may be a vaccine composition comprising one or more of the immunogenic polypeptides or polynucleotides described herein. The immunotherapeutic agent (e.g. polypeptide or polynucleotide) in the vaccine composition may be delivered using any suitable delivery system, for example an emulsion-based delivery system, a liposomebased delivery system, a virosome-based delivery system, a transfersome-based delivery system, an archeosome-based delivery system, a niosome-based delivery system, a cochleate-based delivery system, and / or a particulate delivery system.

[0161] The vaccine composition may include, or the immunotherapeutic agent may be administered with, an adjuvant, or the vaccine composition may include an adjuvant. The adjuvant may, for example, be selected from Freund’s complete adjuvant (CFA or FCA), Freund’s incomplete adjuvant (IFA or FIA), Montanide™ ISA 720, ISCOMs, or ISCOMATRIX™. Particulate delivery systems can also serve as adjuvants. The particulate delivery system may comprise nanoparticles. The nanoparticles may, for example, be made of a natural polymer, such as albumin, collagen, starch, chitosan or dextran, or of a synthetic polymer, such as polylactides (PLA), polyglycolides or polyglocolic acid (PGA) and their copolymers poly(lactide-co-glycolide) PLGA, poly(e- caprolactone) (PCL), poly(hydroxybutyrate) (PHB) and their copolymers. Alternatively, the nanoparticles may be carbon nanotubes, silicon dioxide nanoparticles, dendrimers, ferritin nanoparticles, peptide nanocarriers, gold nanoparticles, liposome-polycation-DNA (LPD) complex, oligosaccharide ester derivative (OED) microparticles, and combination systems.

[0162] Delivery Methods

[0163] Once formulated, a composition (e.g. a vaccine composition) described herein may be delivered to a subject in vivo using a variety of known routes and techniques. For example, a composition can be provided as an injectable solution, suspension or emulsion, and administered via parenteral, subcutaneous, epidermal, intradermal, intramuscular, intraarterial, intraperitoneal, intravenous injection using a conventional needle and syringe, or using a liquid jet injection system. Typically, the composition described herein is administered intramuscularly. Compositions can also be administered topically to skin or mucosal tissue, such as nasally, intratracheally, intestinal, rectally or vaginally, or provided as a finely divided spray suitable for respiratory or pulmonary administration. Other modes of administration include oral administration, suppositories, and active or passive transdermal delivery techniques. Particularly in relation to the invention, compositions may be administered directly to the gastrointestinal tract.

[0164] Alternatively, the composition described herein may be administered ex vivo, for example by delivery and reimplantation of transformed cells into a subject.

[0165] Delivery Regimes

[0166] The immunotherapeutic agents, or compositions comprising a therapeutic agent, may be administered to a subject in an amount that is compatible with the dosage formulation and that will be therapeutically and / or prophylactically effective. An appropriate therapeutically or prophylactically effective dose will fall in a relatively broad range but can be readily determined the skilled person by routine trials. Therapy or prophylaxis may be accomplished by a single direct administration at a single time point or by multiple administrations, for example at multiple time points. Administration can also be delivered to a single or to multiple sites. The skilled person can adjust the dosage and concentration to suit the particular route of delivery. A single dose may be administered on a single occasion. Alternatively, multiple doses may be administered on the same occasion but, for example, at different sites. Alternatively, multiple doses may be administered on multiple occasions. Such multiple doses may be administered in batches (i.e. with multiple administrations at different sites on the same occasion) or may be administered individually, with one administration on each of multiple occasions, optionally at multiple sites. Any combination of such administration regimes may be used. Thus, a first dose of the immunotherapeutic agent described herein may be administered to a subject followed by a second dose of the immunotherapeutic agent described herein.

[0167] Different compositions may be administered at different sites or on different occasions as part of the same treatment regime. It is known that improved immune responses may be generated to an antigen by varying the vectors used to deliver the antigen. There is evidence that, in some instances, antibody and / or cellular immune responses may be improved by using two different vectors administered sequentially as a "prime" and a "boost" .

[0168] Thus, the immunotherapeutic agent may be administered as a prime, and then subsequently administered as a boost. The prime is equivalent to a first dose and the boost is equivalent to a second dose.

[0169] The immunotherapeutic agent administered as the first dose may be identical to or different from the immunotherapeutic agent administered as the second dose. For example, the immunotherapeutic agent used for the first dose may comprise or encode the same MAP epitopes as the immunotherapeutic agent used for the second dose, but the vector used to deliver the active component and / or the composition comprising the active component may be different. For example, the immunotherapeutic agent used for the first dose may comprise the same active component, such as the same polynucleotide encoding the immunogenic polypeptide, as the second dose, but the vector used to deliver the active component and / or the composition comprising the active component may be different. The immunotherapeutic agents administered in the first and second doses may comprise polynucleotides encoding the same immunogenic polypeptide, wherein the immunogenic polypeptide in the first dose is encoded by a first vector and the immunogenic polypeptide in the second dose is encoded a second, different vector. For example, the first and second vectors may be different viral vectors.

[0170] In one example, the immunotherapeutic agent used for the first dose may comprise an immunogenic polypeptide described herein and the immunotherapeutic agent used for the second dose may comprise a polynucleotide encoding the immunogenic polypeptide described herein. The immunotherapeutic agent of the first dose may comprise an immunogenic polypeptide and the immunotherapeutic agent of the second dose may be a polynucleotide which encodes the same immunogenic polypeptide. Alternatively, the immunotherapeutic agent used for the first dose may comprise a polynucleotide encoding the immunogenic polypeptide described herein and the immunotherapeutic agent used for the second dose may comprise the immunogenic polypeptide described herein. The immunogenic agent may be a polynucleotide, wherein the polynucleotide of the first dose and the polynucleotide of the second dose encode, different immunogenic polypeptides. The polynucleotide of the first dose may encode, and the polynucleotide of the second dose may comprise, the same immunogenic polypeptide. The polynucleotide of the first dose may encode, and the second dose may comprise, different immunogenic polypeptides.

[0171] The first dose and the second dose may differ in the choice of vector comprising the polynucleotide. Different vectors each selected from plasmid vectors, viral vectors (e.g. adenovirus vectors or poxvirus vectors), or other vectors described herein may be administered sequentially. Thus, the method of treating or preventing Crohn’s disease may comprise administering a first dose and a second dose of the immunotherapeutic agent against MAP, wherein the first dose comprises a polynucleotide encoding the immunogenic polypeptide described herein, wherein the polynucleotide of the first dose is comprised in a first vector, and the second dose comprises a polynucleotide encoding the immunogenic polypeptide described herein, wherein the polynucleotide of the second dose is comprised in a second vector, wherein the first and second vectors are different. The first vector may be an adenovirus vector (e.g. a simian adenovirus vector, such as a simian adenovirus vector derived from the AdC68 strain) and the second vector may be a poxvirus vector (e.g. an MVA vector). Alternatively, the first vector may be a poxvirus vector (e.g. an MVA vector) and the second vector may be an adenovirus vector (e.g. a simian adenovirus vector, such as a simian adenovirus vector derived from the AdC68 strain). Preferably, the immunotherapeutic agent of the first dose (prime) is ChAdOx2 HAV and the immunotherapeutic agent of the second dose (boost) is MVA HAV. As a further example, the first vector may be a plasmid vector (e.g. pSG2) and the second vector may be a viral vector (e.g. a poxvirus vector, such as MVA). Alternatively, the first vector may be a viral vector (e.g. a poxvirus vector, such as MVA) and the second vector may be a plasmid vector (e.g. pSG2). The polynucleotide comprised in the first vector and the polynucleotide comprised in the second vector may encode the same immunogenic polypeptide or a variant thereof. Alternatively, the polynucleotide comprised in the first vector and the polynucleotide comprised in the second vector may encode different immunogenic polypeptides. In these cases, a prime may be effected by administering the first dose and a boost may be effected by administering the second dose at a later time.

[0172] Further doses of the immunotherapeutic agent may be administered to further boost the immune response. The immunotherapeutic agent administered in a further dose may be the same or different to the immunotherapeutic agents used in the first and second doses. For example, the further dose of the immunotherapeutic agent may comprise a polynucleotide encoding the immunogenic polypeptide in a third vector, such as a third vaccine vector, or may be an immunogenic polypeptide.

[0173] In a prime-boost regime, one or more administrations of the prime and / or the boost may be performed. For example, the prime and / or boost step may be achieved using a single administration, or using two or more administrations at different sites and / or on different occasions. Two administrations on different occasions may be given for the prime step and a single administration on a later occasion may be given for the boost step. Thus, the method of treating or preventing Crohn’s disease described herein may comprise administering more than two doses of the immunotherapeutic agent against MAP described herein, such as three of more doses, four or more doses, five or more doses, or six or more doses.

[0174] Different doses may be administered on the same occasion, on the same day, one, two, three, four, five, six, seven, eight, nine, or ten days apart, or one, two, three, four, five, six, seven, eight, nine, ten or more weeks apart. Preferably, administrations are about 5 to 10 weeks apart, more preferably about 7 to 9 weeks apart, such as about 7 weeks, about 8 weeks or about 9 weeks apart. More preferably, administrations are about 8 weeks apart. For example, the first dose and the second dose described above may be administered about 8 weeks apart. The schedule and timing of such multiple administrations can be optimised for a particular composition by the skilled person by routine trials.

[0175] Dosages for administration will depend upon a number of factors including the nature of the composition, the route of administration, and the schedule and timing of the administration regime. Suitable doses of an immunotherapeutic agent described herein may be in the order of up to about 15 pg, up to about 20 pg, up to about 25 pg, up to about 30 pg, up to about 50 pg, up to about 100 pg, up to about 500 pg or more per administration. For some immunotherapeutic agents described herein, such as plasmids, the dose used may be higher, for example up to about 1 mg, up to about 2 mg, up to about 3 mg, up to about 4 mg, up to about 5 mg or higher. Such doses may be provided in a liquid formulation at a concentration suitable to allow an appropriate volume for administration by the selected route. In the case of a viral vector, a dose of about 106, 107, 108, IO9, IO10or more pfu may be given per administration. Alternatively, a dose of about

[0176] 109, IO10, 1011or more vp may be given per administration.

[0177] For example, the MVA vector described herein may be administered at a dose of 5 x 107pfu to 2 x 108pfu on a single occasion or multiple occasions. As a further example, the simian adenovirus vector (e.g. the simian adenovirus vector derived from the AdC68 strain) described herein may be administered at a dose of 2.5 x IO10vp to 5 x IO10vp on a single occasion. Each of the MVA vector and the adenovirus vector may be administered at a single site or at multiple sites. The MVA vector may be administered at multiple sites on a single occasion or multiple occasions. The adenovirus vector may be administered at multiple sites on a single occasion or multiple occasions.

[0178] As described above, the dosages, the nature of the composition, the route of administration, and the schedule and timing of the administration regime may vary. For example, the method of treating or preventing Crohn’s disease may comprise administering a first dose and a second dose of an immunotherapeutic agent against MAP, wherein the first dose comprises a polynucleotide encoding an immunogenic polypeptide, wherein the polynucleotide of the first dose is comprised in a first vector, and the second dose comprises a polynucleotide encoding an immunogenic polypeptide, wherein the polynucleotide of the second dose is comprised in a second vector, wherein the first and second vectors are different. The first vector may be an adenovirus vector (e.g. a simian adenovirus vector, such as a simian adenovirus vector derived from the AdC68 strain) and the second vector may be a poxvirus vector (e.g. an MVA vector). In this example, the first dose and the second dose may be administered about 8 weeks apart. The first dose and the second dose may be administered intramuscularly. The first vector may be administered at a dose of 5 x IO10vp, and the second vector may be administered at a dose of 2 x 108pfu.

[0179] Combined Therapies

[0180] The method of treating or preventing Crohn’s disease described herein may comprise administering one or more further therapeutic agents or therapies used in the treatment or prevention of Crohn’s disease, and / or one or more further therapeutic agents having activity against MAP. The further therapeutic agent may be one or more antimicrobial agents, such as a combination including rifabutin and clarithromycin. The further therapeutic agent may be a therapeutic or prophylactic MAP vaccine. The further therapeutic agent may be an aminosalicylate, corticosteroid, immunomodulator, anti-TNF-a antibody, anti-integrin antibody, anti-IL12 antibody, anti-IL23 antibody, acetaminophen, antibiotic, and / or loperamide.

[0181] The immunotherapeutic agent may be administered in an amount which is sufficient to augment the therapeutic effects against Crohn’s disease and / or anti-MAP effects of the further therapeutic agent or therapy, or vice versa. Numerous further therapeutic agents may be used in the treatment or prevention of Crohn’s disease. These include aminosalicylates, such as balsalazide, mesalamine, olsalazine and sulfasalazine, corticosteroids, such as budesonide, hydrocortisone, methylprednisolone and prednisone, immunomodulators, such as 6-mercaptopurine, azathioprine, cyclosporine and methotrexate, anti-TNF-a antibodies, such as adalimumab, certolizumab and infliximab, anti-integrin antibodies, such as natalizumab and vedolizumab, anti-IL12 and anti-IL23 antibodies, such as ustekinumab, acetaminophen, antibiotics, and loperamide. Other therapies for the treatment of Crohn’s disease include surgery, such as small bowel resection, subtotal colectomy, proctocolectomy, and ileostomy. Numerous further therapeutic agents may be used in the treatment of MAP infection. These include the rifamycins, such as rifabutin and rifaximin, clarithromycin and other macrolides, azathioprine, methotrexate, Humira, 6-mercaptopurine, and / or Infliximab. Various antituberculosis drugs may also be used.

[0182] The method of treating or preventing Crohn’s disease described herein may comprise administering one or more further therapeutic agents which potentiate the effect of the immunotherapeutic agent. For example, the further therapeutic agent may be an immunomodulatory molecule or an adjuvant which enhances the immune response to an immunotherapeutic agent. Alternatively, the further therapeutic agent may increase the susceptibility of MAP present in the subject to the immune system.

[0183] The immunotherapeutic agent may be administered separately, simultaneously, or sequentially with any of the further therapeutic agents described herein. The immunotherapeutic agent may be administered in the same or different compositions as any of the further therapeutic agents described herein. A composition may therefore be formulated which comprises the immunotherapeutic agent and one or more other further therapeutic agents.

[0184] The method of treating or preventing Crohn’s disease described herein may comprise administering one or more of the immunotherapeutic agents described herein and one or more further therapeutic agents in any order and in any combination. The invention also encompasses the use of the immunotherapeutic agent in combination with one or more further therapeutic agents in the manufacture of a medicament for treating or preventing Crohn’s disease.

[0185] Kits

[0186] An immunotherapeutic agent suitable for use in the method according to the invention may be packaged in the form of a kit. Such kits may comprise one or more immunotherapeutic agents described herein, such as an immunogenic polypeptide and a polynucleotide encoding an immunogenic polypeptide, or two such polynucleotides each in a different vector, such as MVA HAV and ChAdOx2 HAV. The kit may further comprise one or more further components. For example, the kit may comprise: (a) the immunotherapeutic agent, e.g. an immunogenic polypeptide and / or a polynucleotide encoding an immunogenic polypeptide, such as one or more vectors comprising a polynucleotide encoding an immunogenic polypeptide; and (b) at least one further therapeutic agent described herein, for simultaneous, sequential or separate use. The kit may comprise one or more vectors comprising the polynucleotide encoding the immunogenic polypeptide, such as two or more different vectors, and optionally one or more further therapeutic agents. The inclusion of multiple immunotherapeutic agents described herein, such as two or more such agents, facilitates the use of the kit in primeboost treatment regimes. The kit may optionally contain other suitable reagent(s), control(s) or instructions, and the like. The instructions may be instructions for using the kit in the method of treating or preventing Crohn’s disease described herein. Examples

[0187] Example 1: Methods

[0188] ChAdOx2 HAV and MVA HAV Vaccines

[0189] ChAdOx2 is a simian adenovirus vector derived from the AdC68 strain. The vector is replication-deficient, as the El and E3 genes are deleted. MVA is a highly attenuated poxvirus vector which has been extensively used as a vaccine since the 1970s and, more recently, in vaccine clinical trials for multiple diseases.

[0190] Both the ChAdOx2 and MVA vectors were modified to include the vaccine insert, HAV. The HAV insert comprises a 95-kDa fusion construct from four MAP genes present in all MAP strains, 1589c (ahpC), 1234 (gsd), 2444c (p 12) and 1235 (mpa).

[0191] The ChAdOx2 HAV and MVA HAV vaccines were manufactured to current good manufacturing practices by the Clinical Biomanufacturing Facility (University of Oxford, Oxford, UK) and IDT Biologika GmbH (Dessau-Rosslau, Germany), respectively.

[0192] Study Design and Participants

[0193] Enrolment into the phase lb clinical trial commenced in September 2021 and was undertaken at the Clinical Research Facility at Guy’s Hospital (Guy’s & St. Thomas’ Hospitals NHS Foundation Trust), London (Figure 1).

[0194] 28 participants were enrolled according to the eligibility criteria described in Table

[0195] 1. Participant sex was self-reported by individuals recruited from clinical practice.

[0196] Sample size and trial design were chosen to match the phase la clinical trial in healthy adults (Folegatti et al. (2021) Vaccines (Basel). 9(3): 262).

[0197] Table 1. Eligibility criteria for enrolment.

[0198] Accepted participants were randomly assigned to one of five vaccine groups as set out in Table 2 and dose-escalated. Initially, recruited patients were assigned according to time of recruitment. The only alteration to this process was ensuring that any patient in Group 5 would have disease assessable for flexible sigmoidoscopy. A staggered- enrolment approach was used for the participants in each group and interim safety reviews conducted prior to dose escalation. Table 2. Summary of study group numbers, dosage, and route of administration.

[0199] The primary objective was to assess the safety of ChAd0x2 HAV and MVA HAV vaccines administered alone and in a prime -boost regime in adult volunteers with active Crohn’s disease. Safety was assessed by recording solicited adverse events (AEs) for 7 days, unsolicited AEs for 28 days post-vaccine administration, and the occurrence of any serious AEs throughout the trial. The primary outcome measure was to define the Maximum Tolerated Dose (MTD) of ChAdOx2 HAV and MVA HAV vaccines in patients with active Crohn’s disease not receiving immunosuppressive therapy.

[0200] The following parameters were assessed for all study groups.

[0201] Occurrence of local reactogenicity signs and symptoms for 7 days following the vaccination.

[0202] Occurrence of systemic reactogenicity signs and symptoms for 7 days following the vaccination.

[0203] Occurrence of adverse events for 28 days following the vaccination. Change from baseline for safety laboratory measures.

[0204] Occurrence of serious adverse events during the whole study duration. The secondary objectives were to assess the immunogenicity and clinical response in individuals with active Crohn’s disease of ChAdOx2 HAV vaccine administered alone and in a prime-boost regimen with MVA HAV vaccine. To assess cellular immunogenicity, ex vivo interferon-y (IFN-y) enzyme-linked immunospot (ELISpot) assays were completed. This required freshly isolated peripheral blood mononuclear cells (PBMC) stimulated with pools of peptides spanning the HAV vaccine construct. Methodology and calculation of results have been described previously elsewhere (Folegatti et al. (2021) Vaccines (Basel). 9(3): 262).

[0205] Clinical response for Groups 1, 2, and 5 was assessed using the Crohn’s Disease Activity Index (CD Al). CD Al is a weighted index comprising eight clinical and laboratory variables that estimate disease activity in Crohn’s disease and is the most used tool in clinical trials that assess efficacy of Crohn’s disease treatments. A clinical response is generally considered to be a decrease of at least 100 points in the CD Al score, with an absolute score of less than 150 being often defined as clinical remission.

[0206] For Group 5, scoring by endoscopy was included to obtain a Simple Endoscopic Score for Crohn’s Disease (SES-CD) at screening and at Day 112. It has been argued that CD Al is poorly associated with intestinal inflammation, and current practice in clinical trials is to include SES-CD as an adjunct to assess disease response and a marker for mucosal healing. Endoscopic response in Crohn’s disease clinical trials is currently widely accepted as a reduction of 50% in baseline SES-CD, with inactive disease or remission being defined as an SES-CD score of 0 to 2 or 0 to 3.

[0207] Participants in Groups 1 and 2 underwent clinical follow-up for 20 weeks following completion of the vaccination regimen. For participants in Groups 3 and 4, who received the MVA HAV vaccine only, a shorter follow-up period of 12 weeks was considered appropriate, as MVA vectored vaccines have been used extensively with no significant safety concerns reported to date.

[0208] Participants in Group 5 were only vaccinated with the prime-boost regime after data generated from the individual vaccine Groups 1 to 4 had confirmed it was safe to proceed with the proposed higher doses in combination.

[0209] Participants in Group 5 underwent clinical follow-up for a further 20 weeks following completion of the boost vaccination regimen. The duration of follow-up reflects the desire to obtain sufficient safety data with the use of the ChAdOx2 HAV vaccine, the MVA HAV vaccine, and the prime-boost regime with ChAdOx2 HAV and MVA HAV vaccines in humans with Crohn’s disease.

[0210] All participants were excluded from taking any treatments that might affect Crohn’s disease during the follow-up period following vaccination, including immunomodulating, immunosuppressive, anti-inflammatory, and antimicrobial medication.

[0211] Procedures

[0212] All vaccines were administered intramuscularly given the favourable safety and immunogenicity profile of this route of administration with viral vector vaccines. Safety and immunogenicity data generated from participants receiving a single dose of ChAdOx2 HAV vaccine in Groups 1 and 2 were used to inform the dose to be used in the primeboost Group 5. The doses of MVA HAV vaccine used in the phase lb clinical trial (i.e. 5 x 107and 2 x 108pfu) were chosen in light of reassuring safety and immunogenicity data generated by hundreds of individuals who have safely received MVA vectored vaccines following priming with simian adenovirus vectored vaccines. The optimal dose of MVA has been shown consistently to be 1 x 108to 2 x 108pfu. Higher doses of MVA, in particular 2.5 x 108to 5 x 108pfu, have been associated with marked reactogenicity, with severe ‘flu-like’ systemic AEs recorded in a previous study. Lower doses of MVA enable an acceptable reactogenicity profile without significantly compromising vaccine immunogenicity.

[0213] Participants were asked to record any AEs using paper diaries and were regularly reviewed at follow-up visits up to 140 days post- vaccination. Investigators assessed the severity of AEs using the following criteria: (a) Grade 1, mild (short-lived or mild symptoms with no limitation to usual activity); (b) Grade 2, moderate (mild to moderate limitation in usual activity); and (c) Grade 3, severe (considerable limitation in activity, medication, or medical attention required). Unsolicited AEs were reviewed for causality by an independent clinician, and events were categorised as having a possible, probable, or definite relation to the vaccines. Laboratory AEs were graded using site- specific toxicity tables which were adapted from the US Food and Drug Administration toxicity grading scale. A timetable of assessments, assays, and procedures for each Group are set out in Table 3.

[0214] Table 3. Schedule of procedures and assessments. Vital signs included pulse, blood pressure, and temperature. Physical examination, other than at screening and vaccination, only took place if considered necessary. Biochemistry included sodium, potassium, urea, creatinine, albumin, and liver function tests. Immunology assay included ex vivo ELISpot responses to IFN-y. CD Al is Crohn's Disease Activity Index score. SES-CD is Simple Endoscopic Score for Crohn's Disease.

[0215] Statistical Analysis

[0216] The trial design, including eligibility criteria and definitions of active disease, was reviewed and approved by the GSTT R&D Gastroenterology Group and Clinical Research Facility Review Board. As a phase 1 clinical trial, the study's primary focus was on safety and tolerability, rather than efficacy. The sample size was determined based on practical considerations, such as the number of participants needed to adequately assess safety and tolerability. It is also consistent with the previous HAV001 phase la clinical trial in healthy volunteers, ensuring adequate numbers for safety and immunogenicity assessments. Safety endpoints are described as frequencies with their respective percentages alongside their exact 95% confidence intervals (CI). Statistical analysis of clinical and immunogenicity data was conducted using GraphPad Prism version 9.5.1 for Mac (GraphPad Software Inc., California, USA). Comparisons of responses between paired immunogenicity timepoints were made using Wilcoxon matched pairs signed rank test or a Mann- Whitney test for unpaired data. For comparison of data from more than two timepoints (in Group 5), a Friedman test was using with Dunn’s multiple comparisons test. Correlations were performed using a Spearman’s test. Alpha levels of <0.05 were considered significant and all -valucs are reported as two-tailed.

[0217] Ethics

[0218] The trial was registered with EudraCT (reference number 2018-003462-14) and ISRCTN (identifier ISRCTN36126048). The Medicines and Healthcare products Regulatory Agency (MHRA) granted Clinical Trial Authorisation (CTA) as an Acceptance of the Amended Request CTA 51689 / 0001 / 001-0001 on 5thSeptember 2019. Ethical approval was received from the London Westminster Research Ethics Committee (reference number 19 / LO / 1738) on 19thDecember 2019. However, trial activation was delayed due to the COVID- 19 pandemic. Initial Research and Development (R&D) approval was provided by Guy's and St Thomas' (GSTT) R&D on 25thMay 2021, allowing investigators to share participant information sheets with potential participants. Written informed consent was obtained from all participants before their involvement in the study, in compliance with the Declaration of Helsinki and Good Clinical Practice (GCP) guidelines. Recruitment commenced following the GSTT R&D ‘green light’ on 16thAugust 2021, with the first participant consented and screened during the week of 20thSeptember 2021. The first vaccination was administered during the week of 27thSeptember 2021. This trial adhered to the UK Policy Framework for Health and Social Care Research and the Medicines for Human Use (Clinical Trials) Regulations 2004 and its amendments. For further details on the trial approvals and to view the clinical trial protocol, please refer to the ISRCTN registry.

[0219] Example 2: Results

[0220] Study Population

[0221] Between 20thSeptember 2021 and 23rdAugust 2022, 28 adult participants with active Crohn’s disease were screened and received either a single dose of ChAdOx2 HAV vaccine (n=12), a single dose of MVA HAV vaccine (n=6), or a prime dose of ChAdOx2 HAV vaccine (n=10) followed by a boost dose of MVA HAV vaccine (n=9) eight weeks apart. The baseline characteristics of these five groups are summarised in Table 4. One volunteer received a prime dose of ChAdOx2 HAV vaccine but withdrew consent before their booster appointment and left the study.

[0222] Table 4. Demographics of trial participants.

[0223] Primary Endpoints: Vaccine Safety and Tolerability

[0224] The ChAdOx2 HAV and MVA HAV vaccines were safe and well-tolerated in all groups. The total number of solicited AEs was 196 in all 28 (100%) participants. Of all solicited AEs, 149 / 196 (76.0%, 95% CI 69.6%— 81.5%) were graded as mild and 45 / 196 (23.3%, 95% CI 17.2%— 28.8%) were graded as moderate. Three AEs were graded as severe (grade 3) in Group 1 (Table 5).

[0225] Table 5. Grade 3 adverse drug reactions (AEs that are possibly / probably / definitely related to study drug).

[0226] Most of the solicited AEs completely resolved within 7 days (187 / 196, 95.4%) and the majority (111 / 196, 56.6%) had their onset within the first 72 hours post-vaccination (64 / 196, 32.6% at Day 0, 27 / 196, 13.8% at Day 1, and 20 / 196, 10.2% at Day 2). In Group 5, vaccination arm pain was the most common local AE, reported by 6 / 10 (60%) participants after ChAdOx2 HAV vaccine administration and 8 / 9 (89%) after MVA HAV vaccine administration. The reported vaccination arm pain was predominantly mild in severity and, in majority, resolved after 48 hours.

[0227] A total of 164 local and systemic solicited AEs was reported by 28 / 28 (100%) participants within 7 days post-vaccination. Most of these solicited AEs were mild (129 / 165, 78.2%; 95% CI 71.3%-83.8%) or moderate (35 / 165, 21.2%; 95% CI 15.7%- 28.1%). One solicited AE was graded as severe and lasted for 1 day (severe migraine attack in a Group 1 participant). There were two serious AEs recorded, one from a participant of Group 4 and the other from a participant of Group 5, being a Crohn’s flare thought to be secondary to gastroenteritis and a flare of a chronic Crohn’s -related perianal abscess. These were categorised as unlikely or unrelated to the vaccination (Table 6).

[0228] Table 6. Serious adverse events.

[0229] Secondary Endpoints: Cellular Immunogenicity

[0230] Prior to vaccination, ELISpot responses to the HAV vaccine insert were low with a median of 53 SFC / 106PBMC. One month after vaccination with ChAdOx2 HAV vaccine alone (Groups 1 and 2), response to vaccination with doses of 2.5xlO10vp and 5xlO10vp had increased to a median of 87 SFC / 106PBMC and 131 SFC / 106PBMC, respectively. In Groups 3 and 4 (MVA HAV vaccine only), ELISpot assays were not performed, as T cell responses were expected to be low or undetectable. For the nine participants in Group 5 who received the higher ChAdOx2 HAV vaccine dose and boosted eight weeks later with the higher dose of the MVA HAV vaccine, responses 28 days after boosting (Day 84) increased significantly above that at Day 28 (p<0.01), reaching a median of 1335 SFC / 106PBMC. This remained high at a median of 544 SFC / 106PBMC two months after boosting (Day 112) (Figures 2A and B).

[0231] Immune responses to individual antigens in the HAV vaccine construct were also assessed in the nine participants that were primed and boosted. Immune responses to all four antigens increased significantly after boosting with the MVA HAV vaccine (Figure 2C).

[0232] Secondary Endpoints: Clinical Responses

[0233] There was a marked decrease in CD Al in all vaccine groups, and this was statistically significant in Groups 2 and 5. For all six Group 2 participants who received a single higher dose of the ChAd0x2 HAV vaccine, a significant CD Al response was evident with a median score of 205.5 having decreased to 47.5 two months after vaccination (Figure 3B). The individual scores of all six participants decreased by more than 100 points and were below 150 after two months, compared with their Crohn’s disease activity at screening (Figures 3B and 3 A, respectively). This response contrasts with the scores of the participants in Group 1, who received half the dose received by the participants in Group 2, where only two participants’ scores decreased by more than 100 points (Figure 3B), suggesting a possible dose effect. For Groups 3 and 4, individually, group sizes were too small to undertake statistical analyses, although one participant in Group 4 showed a substantial reduction of 204 points (Figure 3B). One participant in Group 5 showed minimal Crohn’s disease activity at screening based on CD Al score. For the remaining eight participants in Group 5 who, based on their CD Al scores, exhibited mildly to moderately active Crohn’s disease at screening, clinical response was similar to that seen in Group 2. Their median CD Al score declined from 225 to 76 after two months and remained low at 66 two months after booster vaccination with the MVA HAV vaccine (Figure 3B). The individual CD Al scores of all eight participants decreased by more than 100 points with six of the eight remaining in that state two months after boosting (Figure 3B). Seven of the eight had a score of below 150 two months following the priming vaccination with six of the eight remaining below 150 two months after boosting (Figure 3A).

[0234] As regards SES-CD, of the eight participants in Group 5 exhibiting mild to moderate Crohn’s disease activity at screening (SES-CD>3), four showed a decrease of >50% and a further two marginally less than 50% (Table 7 and Figure 3C) with an overall decrease in median score from 9.5 to 5.

[0235] Table 7. Crohn's Disease Activity Index score (CDAI) and Simple Endoscopic Score for Crohn's Disease (SES-CD) for participants in Group 5 at baseline (Day 0) and Day 112. PID is the participant identifier number for the trial.

[0236] Example 3: Discussion

[0237] The phase lb clinical trial demonstrated that the candidate MAP vaccines, ChAdOx2 HAV and MVA HAV, are safe, well-tolerated, and immunogenic in patients with active Crohn’s disease when given alone and / or as part of a heterologous prime-boost regime. Most AEs were mild or moderate in severity, and all were self-limiting. The profile of adverse events reported here is similar to that for other simian adenovirus and MVA vectored vaccines expressing different antigens.

[0238] Modest T cell responses were observed following ChAdOx2 HAV single-dose vaccinations. However, T cell responses were significantly boosted by MVA HAV vaccination following ChAdOx2 HAV priming. The boosted T cell responses persisted above baseline levels for at least two months post-boost. This is consistent with results seen in previous reports (Vuitton et al. (2016) Gut. 65(9): 1447-55; and Berthoud et al. (2011) Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 52(1): 1-7). As such, a heterologous prime-boost approach is preferred over a prime-only strategy with ChAdOx2 HAV and MVA HAV vaccines in patients with active Crohn’s disease. This is in keeping with T cell, rather than antibody, responses being considered responsible for protection against intracellular agents, such as those in the Mycobacterium avium complex.

[0239] The immunogenicity results show that ChAdOx2-HAV prime with MVA-HAV boost regime is equally immunogenic in both healthy adults and patients with active Crohn’s disease. Within the HAV insert, none of the individual antigens drive the immune response more than the others, and boosting does not affect this (Figure 2). There is a signal of clinical improvement in disease markers. CD Al score decreased in all participants who received the heterologous prime -boost regime. At a mucosal level, over the 4 months, all eight participants in Group 5 with endoscopic evidence of active Crohn’s disease at screening (SES-CD >3) showed a decrease in the SES-CD score. Four participants showed a defined clinical response of >50% and a further two between 40% and 50%. It should be noted that CD Al and SES-CD do not always correlate and patients may report symptoms that differ from the level of active mucosal inflammation at endoscopy. However, the results indicate that, for the participants in Group 5, a mucosal response also occurred in accordance with the CD Al improvement.

[0240] In conclusion, the ChAdOx2 HAV and MVA HAV vaccines were found to be safe and well-tolerated in patients with active Crohn’s disease when given alone or in a heterologous prime-boost regime. T cell responses significantly improved and were sustained for at least 2 months post-boost when given as part of the heterologous primeboost regime. Similarly, immune responses to all four antigens in the HAV construct increased significantly after boosting with the MVA HAV vaccine. A clinical response was evident in all Group 5 participants as measured by CD Al and in half of the Group 5 participants as measured by SES-CD.

Claims

CLAIMS1. An immunotherapeutic agent against Mycobacterium avium subspecies paratuberculosis (MAP) for use in a method of treating or preventing Crohn’s disease.

2. The immunotherapeutic agent for use according to claim 1, wherein the immunotherapeutic agent comprises an immunogenic polypeptide comprising a first amino acid sequence present in a MAP polypeptide or a polynucleotide encoding the immunogenic polypeptide.

3. The immunotherapeutic agent for use according to claim 2, wherein the polynucleotide is comprised in a vector.

4. The immunotherapeutic agent for use according to claim 3, wherein the vector is a viral vector.

5. The immunotherapeutic agent for use according to claim 4, wherein the viral vector is an adenovirus vector or a poxvirus vector.

6. The immunotherapeutic agent for use according to claim 5, wherein the adenovirus vector is a simian adenovirus vector or the poxvirus vector is a Modified Vaccinia Ankara (MV A) vector.

7. The immunotherapeutic agent for use according to claim 2, wherein the polynucleotide comprises an mRNA.

8. The immunotherapeutic agent for use according to any one of claims 2 to 7, wherein the immunogenic polypeptide further comprises a second amino acid sequence present in a MAP polypeptide, or the polynucleotide further encodes a second amino acid sequence present in a MAP polypeptide.

9. The immunotherapeutic agent for use according to claim 8, wherein the first and second amino acid sequences are present in different MAP polypeptides.

10. The immunotherapeutic agent for use according to claim 8 or 9, wherein the polypeptide further comprises a third amino acid sequence present in a MAP polypeptide and a fourth amino acid sequence present in a MAP polypeptide, or the polynucleotide further encodes a third amino acid sequence present in a MAP polypeptide and a fourth amino acid sequence present in a MAP polypeptide.

11. The immunotherapeutic agent for use according to claim 10, wherein the first, second, third and fourth amino acid sequences are each present in a different MAP polypeptide.

12. The immunotherapeutic agent for use according to claim 11, wherein the first, second, third and fourth amino acid sequences are present in the MAP polypeptides AhpC, Gsd, P12 and Mpa.

13. The immunotherapeutic agent for use according to any one of the preceding claims, wherein the first amino acid sequence is, and optionally the second amino acid sequence is or the second, third and fourth amino acid sequences are, at least 8 amino acids in length.

14. The immunotherapeutic agent according to any one of the preceding claims, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 28, or the polynucleotide encodes the amino acid sequence of SEQ ID NO: 29.

15. The immunotherapeutic agent for use according to any one of the preceding claims, wherein the method comprises administering a first dose and a second dose of the immunotherapeutic agent.

16. The immunotherapeutic agent for use according to claim 15, wherein the first dose comprises the immunogenic polypeptide and the second dose comprises thepolynucleotide encoding the immunogenic polypeptide, or the first dose comprises the polynucleotide encoding the immunogenic polypeptide and the second dose comprises the immunogenic polypeptide.

17. The immunotherapeutic agent for use according to claim 15, wherein the first dose comprises the polynucleotide encoding the immunogenic polypeptide, wherein the polynucleotide of the first dose is comprised in a first vector, and the second dose comprises the polynucleotide encoding the immunogenic polypeptide, wherein the polynucleotide of the second dose is comprised in a second vector, wherein the first and second vectors are different.

18. The immunotherapeutic agent for use according to claim 17, wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 28, the first vector is a simian adenovirus vector and the second vector is a Modified Vaccinia Ankara (MVA) vector.

19. The immunotherapeutic agent for use according to any one of claims 15 to 19, wherein the second dose is administered up to 8 weeks after administering the first dose.

20. The immunotherapeutic agent for use according to claim 18 or 19, wherein the simian adenovirus vector is administered at a dose of 5 x IO10vp and the MVA vector is administered at a dose of 2 x 108pfu.

21. Use of an immunotherapeutic agent against MAP in the manufacture of a medicament for treating or preventing Crohn’s disease.

22. The use according to claim 21, wherein the immunotherapeutic agent is as defined in any one of claims 2 to 14, and / or the medicament is for treating or preventing Crohn’s disease by a method as defined in any one of claims 15 to 20.

23. A method of treating or preventing Crohn’s disease comprising administering to a subject in need thereof an effective amount of an immunotherapeutic agent against MAP.

24. The method according to claim 23, wherein the immunotherapeutic agent is as defined in any one of claims 2 to 14, and / or the method is as defined in any one of claims 15 to 20.

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