A vaccine for the treatment or prevention of burkholderia species infection in a subject.

Abstract

A composition comprising BpPA26 is provided. The composition is for use in vaccine therapy to treat or prevent pathogenic Burkholderia species infection in a mammal, typically a human or an equine mammal. The species include Burkholderia cepacia complex, Burkholderia mallei, and Burkholderia pseudomallei.

Description

[0001] Title of the Invention

[0002] A vaccine for the treatment or prevention of Burkholderia species infection in a subject.

[0003] Field of the Invention

[0004] The invention relates to a vaccine for the treatment or prevention of Burkholderia infection in a subject. The infection is caused by a pathogenic member of the Burkholderia genus selected from Burkholderia cepacia complex, Burkholderia mallei and Burkholderia pseudomallei. In an embodiment, the vaccine is for treatment or prevention of melioidosis.

[0005] Background of the invention

[0006] Burkholderia is a genus whose pathogenic members include Burkholderia cepacia complex, Burkholderia mallei, and Burkholderia pseudomallei.

[0007] Burkholderia mallei is responsible for glanders, a disease that occurs mostly in horses and related animals, such as mules and donkeys. It is usually caused by ingestion of contaminated feed or water. Signs of glanders include the formation of nodular lesions in the lungs and ulceration of the mucous membranes in the upper respiratory tract. B. mallei can also infect humans.

[0008] Burkholderia cepacia complex (BCC) is a group of catalase-producing gram-negative bacteria with at least 20 different species, including B. cepacia, B. multivorans, B. cenocepacia, B. vietnamiensis, B. stabilis, B. ambifaria, B. dolosa, B. anthina, B. pyrrocinia and B. ubonensis. BCC causes pneumonia in immunocompromised individuals with underlying lung diseases, such as cystic fibrosis or chronic granulomatous disease and it is an emerging pathogen in nosocomial infections among chemotherapy patients and other immunosuppressed individuals. Once a patient is colonized with BCC, it is rarely eradicated due to the resistance of BCC to antibiotics and its inherent resistance to antimicrobial peptides.

[0009] Burkholderia pseudomallei (Bp) is the causative agent of melioidosis. Acute infection is characterized by localized abscesses in different organs, ulceration, and sepsis with or without pneumonia. This gram-negative bacterium is estimated to cause around 165,000 cases per year worldwide, of which 89,000 are expected to be fatal. Although most of the cases are concentrated in endemic areas of Southeast Asia, such as Thailand, and northern Australia, B. pseudomallei is emerging in non-endemic areas like Brazil, China, or India. It is estimated that melioidosis is severely underreported in the 45 countries in which it is endemic and is likely to be endemic in a further 34 countries. A recent modelling framework analysis predicted that B. pseudomallei is ubiquitous throughout the tropics. The bacterium grows in soils and waters, and thus the main route of infection in some countries is by inoculation whilst working in flooded fields such as rice paddies. Therefore, the increase seen is exacerbated by monsoon rains every year. Climate change is making monsoons more robust and a higher prevalence of B. pseudomallei is detected at soil depths greater than 30 cm. Monsoon rainfalls may release bacteria from deeper soils and make rice paddies riskier for farmers. Diabetes is a major risk factor for severe disease and the growing prevalence of diabetes in these regions, particularly Asia, may give rise to a substantial increase in melioidosis disease severity.

[0010] The main therapeutic options for melioidosis at present include p-lactams, (e.g., ceftazidime, certain p-lactam-p-lactamase inhibitor combinations), carbapenems, trimethoprimsulfamethoxazole (TMP-SMX), and doxycycline, depending on the phase of treatment. Antibiotic treatment is for extensive periods of time and in many cases (up to 50%) ventilation in the intensive care unit (ICU) is required for several weeks.

[0011] Although the protective immune responses to this infection are poorly understood, IFN-y responses in CD4+and CD8+T cells appear to be essential to repress the pathogen, and IFN- y expressing NK and NKT cell subsets may also be protective.

[0012] There is no approved vaccine available to prevent melioidosis, although many potential vaccine candidates have been evaluated in preclinical studies (Casey & McClean, Exploiting Molecular Virulence Determinants in Burkholderia to Develop Vaccine Antigens, Current Medicinal Chemistry 2015; 22(14) .2015). The inventors’ group previously showed that B. pseudomallei homologue OmpW (BpOmpW) protected mice from B. pseudomallei challenge (Casey et al., Identification of an OmpW homologue in Burkholderia pseudomallei, a protective vaccine antigen against melioidosis. Vaccine. 2016 May 17;34(23):2616-21). This work is disclosed in WO2014 / 096070. The cytoplasmic Alkyl hydroperoxide reductase (AhpC) (Reynolds et al., J Immunol 194, 4814-4824 (2015)) or the hemolysin-coregulated protein (Hcp1) (Burtnick et al., Infect Immun 86 (2018)) are also potential vaccine antigens.

[0013] Acetoacetyl-CoA reductase (BpPA26) is a mainly cytosolic enzyme with essential functions inside cells. It belongs to the family oxidoreductases and catalyzes the oxidation of 3-hydroxy acyl-CoA with the production of NADPH (Rodarte, J.V. et al. Crystal structure of acetoacetyl- CoA reductase from Rickettsia felis. Acta Crystallogr F Struct Biol Commun 77, 54-60 (2021)). BpPA26 has also been described as a moonlighting protein with different functions in secondary metabolites' biosynthesis, transport, and metabolism (Mariappan, V., et al., Adaptation of Burkholderia pseudomallei Alters Metabolism and Virulence: a Global Proteome Analysis. Sci Rep 7, 9015 (2017)). There is an urgent unmet clinical need for a vaccine to protect populations, such populations of subtropical and tropical countries, from infection with pathogenic Burkholderia species, in particular infection caused by Burkholderia pseudomallei. The current invention provides a solution to this problem.

[0014] Summary of the Invention

[0015] The inventors have surprisingly found that putative Acetoacetyl-CoA reductase (herein “BpPA26”) provides a protective immune responses against pathogenic Burkholderia species, such as Burkholderia pseudomallei, infection in subjects. It can also have a therapeutic effect in subjects infected with a pathogenic Burkholderia species. This has not been previously disclosed.

[0016] As shown by the inventors in the examples described herein, immunization of mice with BpPA26 recombinant antigen stimulated both humoral and IFN-y responses in both non- and insulin-resistant mice, which correlate with protective immune responses against melioidosis (Figure 1 , 3, and 5). The inventors have also shown that Th2 responses, determined by significantly increased amounts of IL-4 cytokine, were promoted by the immunisation of noninsulin resistant mice with recombinant BpPA26 antigen (Figure 4). Regulatory T cells, which are essential to prevent the excessive inflammatory responses to bacterial endotoxins, were elevated in BpPA26 immunised mice group relative to the control.

[0017] Overall, the elevated NKT cell cytokine responses in BpPA26 immunised insulin-resistant mice, including IFN-y, is significant given that IFN-y expressing NKT cells showed a role in survival to melioidosis in patients with diabetes mellitus (Figure 5). It is also noteworthy that BpPA26 also promoted the stimulation of IFN-y responses in NK and CD4+T cells in both non- and insulin-resistant mice, as the strongest correlations of IFN-y responses against B. pseudomallei in melioidosis patients were in CD4+ T cells, followed by NK cells.

[0018] Accordingly, in an aspect of the invention, there is provided a composition comprising BpPA26 or an immunogenic portion thereof (or a nucleotide or mRNA encoding the same) (herein “composition of the invention”).

[0019] In an embodiment, BpPA26 is a protein.

[0020] In an embodiment, the immunogenic portion is a polypeptide or a peptide.

[0021] In an embodiment, the composition is an immunogenic composition.

[0022] In an embodiment, the immunogenic composition is a vaccine.

[0023] In an embodiment, the vaccine is an adenoviral vector vaccine. In an embodiment, the vaccine is a mRNA vaccine.

[0024] In an embodiment, the vaccine is a liposome-based vaccine.

[0025] The composition may optionally include one or more adjuvants.

[0026] The composition may optionally include one or more additional antigens.

[0027] The composition may be a pharmaceutical composition and optionally comprising a pharmaceutically acceptable excipient.

[0028] Preferably, the BpPA26 or immunogenic portion is present in a therapeutically effective amount.

[0029] An aspect of the invention provides BpPA26 or an immunogenic portion thereof (or a nucleotide or mRNA encoding the same), for use in a vaccine therapy to prevent or treat infection by a pathogenic Burkholderia species in a subject.

[0030] An aspect of the invention provides the composition of the invention for use in a vaccine therapy to prevent or treat infection by a pathogenic Burkholderia species in a subject.

[0031] In an embodiment, the infection is melioidosis.

[0032] An aspect of the invention provides BpPA26 or an immunogenic portion thereof (or a nucleotide or mRNA encoding the same), for use as a therapy to induce an immune response against a pathogenic Burkholderia species in a subject.

[0033] The invention also relates to a method of treating or preventing infection by a pathogenic Burkholderia species in a subject, comprising administering a therapeutically effective amount of BpPA26 or an immunogenic portion (or a nucleotide or mRNA encoding the same) to the subject.

[0034] The invention also relates to a method of treating or preventing infection by a pathogenic Burkholderia species in a subject, comprising administering the composition of the invention to the subject.

[0035] In an embodiment of any aspect of the invention, the pathogenic Burkholderia species is one or more of Burkholderia cepacia complex, Burkholderia mallei, and Burkholderia pseudomallei. In an embodiment, the species is Burkholderia pseudomallei.

[0036] The invention also relates to a method of treating or preventing melioidosis in a subject, comprising administering a therapeutically effective amount of BpPA26 or an immunogenic portion (or a nucleotide or mRNA encoding the same) to the subject. The invention also relates to a method of treating or preventing melioidosis in a subject, comprising a step of administering the composition of the invention to the subject.

[0037] In an embodiment of any aspect of the invention, the subject is a mammal, such as a human or an animal.

[0038] Definitions and general preferences

[0039] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0040] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

[0041] As used herein, the term "comprise," or variations thereof such as "comprises" or "comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method / process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method / process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method / process steps.

[0042] As used herein, the term “disease” is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired irrespective of the nature of the aetiology (or indeed whether the aetiological basis for the disease is established). It therefore encompasses conditions arising from infection, trauma, injury, surgery, radiological ablation, poisoning, or nutritional deficiencies.

[0043] As used herein, the term "treatment" or "treating" refer to an intervention (e.g. the administration of an agent to a subject) which cures, ameliorates or lessens the symptoms of a disease or removes (or lessens the impact of) its cause(s). In this case, the term is used synonymously with the term “therapy”. It can be manifested by a permanent or temporary improvement in the subject's condition. In this context, it includes limiting and / or reversing disease progression. As used herein the terms "prevention" or "preventing" refers to an intervention (e.g., the administration of an agent or vaccine to a subject), which prevents or delays the onset or progression of a disease or infection, e.g., Burkholderia pseudomallei infection, or the severity of a disease in a subject, or reduces (or eradicates) its incidence within a treated population.

[0044] When used herein, the term “composition” should be understood to mean something made by the hand of man, and not including naturally occurring compositions. Compositions may be formulated in unit dosage form, i.e., in the form of discrete portions containing a unit dose, or a multiple or sub-unit of a unit dose.

[0045] When used herein, the term “pharmaceutical composition” may comprise one or more pharmaceutically acceptable diluents, excipients or carriers. Even though the proteins, peptides and compositions of the present invention can be administered alone, they will generally be administered in admixture with a pharmaceutical carrier, excipient or diluent, particularly for human therapy. The pharmaceutical compositions may be for human or animal usage in human and veterinary medicine. Examples of such suitable excipients for the different forms of pharmaceutical compositions described herein may be found in the “Handbook of Pharmaceutical Excipients”, 2ndEdition, (1994), edited by A Wade and PJ Weller. In particular, formulations for topical delivery are described in “Topical Drug Delivery Formulations” edited by David Osborne and Antonio Aman, Taylor & Francis, the complete contents of which are incorporated herein by reference. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).

[0046] In this specification, the term “sequence identity” should be understood to mean the amount of nucleic acids or amino acids which match between different sequences. For example, a sequence, such as a polypeptide or a polynucleotide sequence, that shares 90% sequence identity with a sequence is one in which any 90% of aligned residues are identical to the corresponding nucleic acids or amino acids in the reference sequence across the entire length of the sequence. Sequence homology can be calculated by a BLAST program (www.ncbi.nlm.nih.gov / cgi-bin Blast). Hereby, gaps are not counted.

[0047] The term subject (which is to be read to include "individual", "animal", "patient" or "mammal" where the context permits) defines any subject, particularly a mammalian subject, for whom treatment or prevention is indicated. Mammal is a human or an animal. In many cases, the subject will be a mammal suspected of having a pathogenic Burkholderia infection, or at risk of having a pathogenic Burkholderia infection In this specification, the term “infection by a pathogenic Burkholderia species”, should be understood to mean infections of mammals that are caused by a pathogenic species of Burkholderia. The term “pathogenic Burkholderia species” includes Burkholderia cepacia complex, Burkholderia mallei, and Burkholderia pseudomallei ( Eberl L, et al., Members of the genus Burkholderia: good and bad guys. FIOOORes. 2016 May 26;5:F1000 Faculty Rev- 1007). In this context, the species is one having an Acetoacetyl-CoA reductase with an amino acid sequence having at least 60% sequence identity, to BpPA26 of the current invention. In an embodiment, the sequence identity is 64% or more, 74% or more, 80% or more, 90% or more, 95% or more, or 97%.

[0048] As used herein the term “infection” by Burkholderia pseudomallei, or “Burkholderia pseudomallei infection” should be understood to mean an infection of a subject, such as a human, that is caused by a pathogenic strain of Burkholderia pseudomallei.

[0049] As used herein the term “infection by BCC” in a subject should be understood to mean infections caused by one or more members of B. cepacia complex. Members include catalaseproducing gram-negative bacteria species B. cepacia, B. multivorans, B. cenocepacia, B. vietnamiensis, B. stabilis, B. ambifaria, B. dolosa, B. anthina, B. pyrrocinia and B. ubonensis.

[0050] As used herein the term “BpPA26” refers to putative Acetoacetyl-CoA reductase that is expressed by strains of B. pseudomallei. The protein has a molecular weight of 26906.69 daltons and an approximate PI of 6.30. The amino acid sequence of BpPA26 is:

[0051] MGMQAKRVAFVTGGMGGLGAAI SRRLHDAGMAVAVSHSERNDHVSTWLMHERDAGRDFKAYAVDVADF ESCERCAEKVLADFGKVDVLINNAGITRDATFMKMTKGDWDAVMRTDLDAMFNVTKQFIAGMVERRFG RIVNIGSVNGSRGAFGQANYASAKAGIHGFTKTLALETAKRGITVNTVSPGYLATAMVEAVPQDVLEA KILPQI PVGRLGQPDEVAALIAFLCSDDAGSVTGADLAINGRDAHVLNS (SEQUENCE ID NO.1)

[0052] The nucleic acid sequence is:

[0053] ATGCAAGCAAAACGCGTGGCATTCGTGACGGGCGGTATGGGCGGACTGGGTGCGGCGATCAGCCGGCGGC TGCACGACGCCGGCATGGCTGTGGCGGTGTCGCATTCGGAGCGCAACGATCATGTGTCGACGTGGCTGAT GCACGAGCGCGACGCCGGACGCGATTTCAAGGCGTATGCGGTCGACGTCGCGGATTTCGAATCGTGCGAG CGGTGCGCGGAGAAGGTGCTCGCCGATTTCGGCAAGGTCGACGTGCTGATCAACAACGCGGGGATCACGC GCGATGCGACGTTCATGAAGATGACGAAGGGCGACTGGGACGCGGTGATGCGCACCGATCTCGATGCGAT GTTCAACGTGACGAAACAGTTCATCGCGGGCATGGTCGAGCGGCGCTTCGGGCGCATCGTCAACATCGGT TCGGTGAACGGCTCGCGCGGCGCGTTCGGTCAGGCGAATTATGCGTCGGCGAAGGCGGGCATCCACGGTT TCACGAAGACGCTCGCGCTCGAGACGGCGAAGCGCGGGATCACGGTCAACACGGTGTCGCCCGGCTATCT CGCGACCGCGATGGTCGAAGCGGTGCCGCAGGACGTGCTCGAGGCGAAGATCCTGCCGCAGATTCCGGTT GGCCGGCTCGGCCAGCCCGACGAAGTGGCCGCGCTCATCGCGTTTCTGTGCTCGGACGACGCGGGGTTCG TCACCGGCGCCGATCTCGCGATCAACGGCGGGATGCACATGTCCTGA (SEQUENCE ID NO. 2) The term BpPA26 should be understood to include therapeutically effective variants that share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQUENCE ID NO: 1.

[0054] In this specification, the term “effective variant” or “functional variant” as applied to BpPA26 means a protein or polypeptide that shares at least 90% sequence identity with SEQUENCE ID NO: 1 and that is capable of eliciting a protective immune response in a mammal immunised with the variant against subsequent challenge by pathogenic Burkholderia, e.g. B. pseudomallei as described below. The term can include peptides or proteins having an amino acid sequence that is substantially identical to the reference, and which is therapeutically effective as defined herein. Thus, for example, the term should be taken to include variants that are altered in respect of one or more amino acid residues. Preferably, such alterations involve the insertion, addition, deletion and / or substitution of 6 or fewer amino acids, preferably 5 or fewer, 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged. It should be noted that any variant will have principally the same therapeutic effect, or may have an enhanced effect when tested in in vitro or in vivo models of the disease.

[0055] The term “effective variant” or “functional variant” may also be applied to the immunogenic portions, mRNA and nucleotide sequences of the invention. For instance, the “effective variant”, or “functional variant” of the nucleotide is one that encodes a protein or immunogenic portion thereof that is capable of eliciting a protective immune response in a mammal immunised with the protein or immunogenic portion thereof against subsequent challenge by pathogenic Burkholderia species as described below.

[0056] The term “immunogen” is a substance that is capable of generating a B-cell and / or T-cell immune response in a host organism. In this context, the immunogen may be a protein or an immunogenic portion of the protein such as an isolated polypeptide, or peptide. One immunogen is BpPA26 immunogen. The immunogens may be purified, recombinant or synthetic.

[0057] In this specification, the term “immunogenic portion thereof” as applied to BpPA26 should be understood to mean a portion of the protein that when administered to a mammal using the methods described below elicits an immune response that is specific to the portion that is administered. In this context, immune response includes IFN-y response in natural killer cells, CD4+ and CD8+ T cells and humoral responses (B-cells). In an embodiment, the immunogenic portion is a peptide. In an embodiment, the peptide is a short fragment of SEQ ID NO. 1 . The peptide may have a length of up to 50 amino acids, or up to 20 amino acids. It may have from 10 to 25 amino acids in length from any portion of SEQ ID NO. 1. It may be a peptide fragment derived from the first 50 amino acids of SEQUENCE ID NO. 1. It may be a peptide derived from amino acid 50 to 150 of SEQUENCE ID NO. 1. It may be a peptide fragment derived from amino acid 150 to 253 amino acids of SEQUENCE ID NO. 1. Functional variants of the immunogenic portion are also part of the current invention.

[0058] The term “vaccine therapy” should be understood to mean the administration of an immunogen, e.g., a protein or immunogenic portion thereof or an mRNA or viral vector encoding the protein to a mammal with a view to eliciting a response by the host immune system. Typically, it results in the immunogen being destroyed and subsequently recognised by the host immune system.

[0059] The term “immune response” should be understood to mean induced humoral or cellular response in a host subject. In this context immune response includes IFN-y response in natural killer cells, CD4+ and CD8+ T cells and humoral responses (B-cells).

[0060] As used herein, the term “active pharmaceutical ingredient” or “API” refers to an immunogen that may be used in vaccine therapy such as a protein, protein subunit, polypeptide, botanical (e.g., a plant extract) a fraction of a cell such as a bacterial cell, viral cell, or yeast cell (e.g. a cell wall fraction), or a nucleic acid encoding an immunogen. The nucleic acid may be an RNA (e.g. mRNA) or DNA molecule.

[0061] In this specification, the term “vaccine” should be understood to mean a composition comprising at least one immunogen, e.g., a protein of the invention, and optionally a suitable adjuvant and / or carrier. The preparation of vaccines comprising a protein or peptide as an active agent is well known in the art, for example US4599230 and US4601903 as examples. In an embodiment, the vaccine is a subunit vaccine. A “subunit” vaccine contains one or more specific antigens from a pathogen. It is acellular, i.e. it does not contain whole bacteria.

[0062] The term “DNA vaccine” should be understood to mean a composition that comprises a nucleic acid, typically on a nucleic acid construct that is capable of being delivered to a subject and encodes one or more proteins or immunogenic portions of the invention. The nucleic acid construct expresses said one or more protein or immunogenic portions in said subject. The protein or immunogenic portion thereof may be expressed as separate entities or in the form of a fusion protein. The vaccine may include one or more suitable adjuvants and / or one or more suitable pharmaceutically acceptable carriers. The construct may be an expression vector. Expression vectors suitable for this purpose are known in the art, for example a plasmid or a virus. In an embodiment, the vector is an adenovirus vector. The term “adjuvant” should be understood to mean an agent that enhances the subject, i.e. , the recipient’s, immune response to an immunogenic protein or portion thereof. Details of a suitable adjuvant composition are well known to a person skilled in the art.

[0063] The term “immunogenic composition” refers to a composition comprising one or more proteins of the invention, or an immunogenic portion thereof, (or nucleotide or mRNA encoding same) that is capable of inducing an immune response in an individual.

[0064] Proteins, peptides, and polypeptides (and their variants, including fragments) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid in DNA or mRNA format. The proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984). They may be recombinant.

[0065] In this specification, the term “amount effective” or “therapeutically effective amount” should be taken to mean an amount which results in a clinically significant reduction or prevention of pathogenic Burkholderia infection, e.g. Burkholderia pseudomallei infection.

[0066] Suitably, the immunogenic component of the vaccine is administered at a dose of from 1 pg to 1OOpg. In one embodiment, it is administered at a dose of from 1Opg to 70pg, or from 1Opg to 50pg, or from 20pg to 40pg, or from 25pg to 30pg.

[0067] Typically, it is given as a bolus dose.

[0068] In the context of the therapeutic aspects of the present invention, the term “individual in need thereof’ shall be taken to mean a mammal, preferably a human, who has an infection caused by a pathogenic Burkholderia species, such as Burkholderia pseudomallei. In therapeutic aspects which include prevention, the term “individual in need thereof” can also include a horse or camel animal who is at risk of developing an infection caused by a B. mallei strain.

[0069] The term "symptom" is defined as an indication of disease, illness, injury, or that something is not right in the body.

[0070] The term "excipient" refers to a diluent, adjuvant, carrier or vehicle with which the therapeutic or active is administered. Such pharmaceutical carriers or excipients can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous buffered solution, are the preferred carriers when the pharmaceutical composition is administered intramuscularly. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

[0071] The vaccine or composition of the invention can be formulated in any suitable means for the desired route of administration. Formulation should suit the mode of administration. Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g. , intra-nasally. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intranasal, intracerebral, and oral routes. Typically, the mode is intramuscularly, subcutaneous, oral or transdermal, such as by microneedle patches. In a typical embodiment, the mode is intramuscularly. The vaccine or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). The vaccine or composition may be administered together with adjuvants and or other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the vaccine or composition of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[0072] In this specification, the term “vaccine therapy” should be understood to mean the administration of an immunogenic peptide or protein (or a nucleotide or mRNA) to a subject with a view to eliciting an immune response by the host immune system that results in the peptide or protein been recognised and processed by the innate immune system and subsequently recognised by the components of the host adaptive immune system to destroy infectious microorganisms.

[0073] An “mRNA vaccine” is a vaccine that uses a copy of messenger RNA (mRNA) to produce an immune response. The vaccine delivers the immunogen encoding mRNA into the subject’s immune cells. This uses the mRNA to provide the protein, in this case the B. pseudomallei protein BpPA26, or one or more immunogenic portions. It is translated in the cell cytosol. This protein then stimulates the immune response which trains the body to identify, retain a memory for and destroy the pathogen. Typically, the mRNA is delivered by a co-formulation of the RNA encapsulated in a lipid nanoparticle that protects the RNA strand and aids absorption into the cells. Methods to make mRNA and mRNA vaccines are known in the art. mRNA vaccines are described in Kowalski PS, Rudra A, Miao L, Anderson DG (April 2019). "Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery". Mol Ther. 27 (4): 710- 28; and Verbeke R, Lentacker I, De Smedt SC, Dewitte H (October 2019). "Three decades of messenger RNA vaccine development". Nano Today. 28: 100766. The protein or immunogenic portion thereof may be expressed as separate entities or in the form of a fusion protein. The vaccine may include one or more suitable adjuvants and / or one or more suitable pharmaceutically acceptable carriers. The construct may be a delivery vector. Delivery vectors suitable for this purpose are known in the art, for example a plasmid a lipid nanoparticle. In an embodiment, the vector is a Lipid Nanoparticle

[0074] The term “peptide” used herein refers to a polymer composed of up to 50 amino acids, for example 5 to 50 amino acid monomers typically linked via peptide bond linkage. The peptide may have up to 20 amino acids. Peptides may be generated wholly or partly by chemical synthesis or by expression from nucleic acid. For example, the peptides can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984).

[0075] The term “soliped” refers to mammal having a single hoof on each foot. It includes but is not limited to a horse, ass, donkey, and mule.

[0076] In this specification, the term “sample” or “fluid” is a biological sample. The sample is one obtained from a subject, such as, for example, urine or urine cell-sediment, blood, saliva, or a tissue sample.

[0077] The term “assaying” should be understood to mean quantitative detection of BpPA26 or a gene encoding BpPA26 in the sample. Suitable methods will be known to a person skilled in the art.

[0078] Brief Description of the Figures

[0079] The current invention will now be described with reference to the following Figures in which;

[0080] Figure 1. Immunization of non-insulin resistant mice with recombinant BpPA26 induced humoral and IFN responses. A) Purified recombinant PA26 from B. pseudomallei (BpPA26). B) Schematic illustration of the experimental timeline of non-insulin-resistant mouse study. C-E) Total IgG, lgG1 , and lgG2a specific responses against BpPA26 in the non-insulin resistant mice. SAS adjuvanted control mice (blue circles). BpPA26-SAS immunized mice (red circles.) Figure 2. Analysis of splenocytes from adjuvanted PA26 immunised mice re-exposed in vitro to BpPA26 (red circles) and splenocytes from SAS only adjuvanted treated mice exposed to BpPA26 in vitro (blue circles) analysed by flow cytometry. A) Percentages of T cells expressing CD45RB. B-E) Percentages of CD4 and CD8 T cells expressing CD25 (B and D) and CD44 (C and E) activation markers. F-G) Percentages of CD4 (F) and CD8 (G) T cells expressing IL-2 cytokine. H-J) Percentages of CD4 T cells expressing IFN-y (H), IL-4 (I), and IL-17 (J). K) Percentages of CD8 T cells expressing IFN-y cytokine. L) Percentages of CD4 T cells expressing TNF cytokine. M) Percentages of CD8 T cells expressing TNF cytokine. O) Percentages of CD4+CD25+Foxp3+Regulatory T cells (Tregs). Asterisks denote statistically significant differences according to the two-tailed t-test. The significant levels are represented as follows: (p < 0.05, *); (p < 0.01 , **); (p < 0.001 , ***).

[0081] Figure 3. Immunization of non-insulin resistant mice with recombinant BpPA26 induced IFN-y responses in NK cells. Analysis of splenocytes from adjuvant only immunised mice exposed in vitro to BpPA26 (blue circles) and splenocytes from SAS adjuvanted BpPA26 immunized mice re-exposed to BpPA26 in vitro (red circles) analysed by flow cytometry. A-B) Percentages of Natural Killer (NK) cells expressing IFN-y (A) and TNF (B). C-H) Percentages of Natural Killer T cells expressing CD44 (C), IL-2 (D), IFN-y (E), IL-4 (F), IL-17 (G), and TNF (H); (I) percentages of Double negative cells representing NKT cells SAS: splenocytes from adjuvant only immunised mice exposed to BpPA26 (blue circles). BpPA26-SAS: splenocytes from SAS adjuvanted BpPA26 immunized mice re-exposed with BpPA26 in vitro (red circles). Asterisks denote statistically significant differences according to the two-tailed t-test. The significant levels are represented as follows: (p < 0.05, *); (p < 0.01 , **).

[0082] Figure 4. Murine HFD-lnduced Insulin Resistant mouse model and Characterization. A) Schematic illustration of the experimental timeline of the development of insulin-resistant mouse model. B) Weight gain of 20 C57BL6 / j mice during 98 days of HFD (red circles) or normal diet (ND) (black circles) feeding. C) Glucose level of 70 HFD-fed C57BL6 / j mice at 14, 28, 56, and 84 weeks (red circles) and the corresponding control of 10 ND fed C57BL6 / j mice (black circles). D) Insulin tolerant test (ITT) of HFD (red circles) and ND (black circles) fed mice at 8 weeks and E) 12 weeks of feeding. Lines represent the average of each group throughout the experiment. Asterisks mark significant differences according to the two-tailed Student's t-test. The significant levels are represented as follows: (p < 0.0001 , ****).

[0083] Figure 5. Immunization of a mouse model of T2D with BpPACR produced IFN-y responses as well. Analysis of splenocytes from adjuvant only immunised mice exposed in vitro to BpPACR (blue circles) and splenocytes from SAS adjuvanted BpPACR immunized mice re-exposed to BpPACR in vitro (red circles) analysed by flow cytometry. A) Percentages of T cells expressing CD45RB. B-E) Percentages of CD4 and CD8 T cells expressing CD25 (B and D) and CD44 (C and E) activation markers. F-G) Percentages of CD4 (F) and CD8 (G) T cells expressing IL-2 cytokine. H-J) Percentages of CD4 T cells expressing IFN-y (H), IL-4 (I), and IL-17 (J). K) Percentages of CD4 T cells expressing TNF cytokine. L) Percentages of CD8 T cells expressing IFN-y cytokine. M) Percentages of CD8 T cells expressing TNF cytokine. N-O) Percentages of NK cells expressing IFN-y (N) and TNF (O). P-T) Percentages of Natural Killer T cells expressing IL-17 (P), TNF (Q), IL-2 (R), IFN-y (S), IL-4 (T). U) Percentages of CD4+CD25+Foxp3+Regulatory T cells (Tregs). Asterisks denote statistically significant differences according to the two-tailed t-test. The significant levels are represented as follows: (p < 0.05, *); (p < 0.01 , “); (p < 0.001 , ***).

[0084] Figure 6. Total IgG, lgG1, and lgG2a specific responses against BpPA26 in the insulin resistant mice after a single immunisation with BpPA26. SAS adjuvanted control mice (blue circles). BpPA26-SAS immunized mice (red circles.).

[0085] Detailed Description of the Invention

[0086] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

[0087] There is a need to provide a vaccine to protect and / or treat infections of pathogenic Burkholderia species.

[0088] For B. pseudomallei, the search for a vaccine that elicits protective responses in subjects with and without diabetes is of vital importance. There are currently no vaccines. An effective vaccine would protect the population from B. pseudomallei infection, and the vaccine would be particularly beneficial to those subjects with Diabetes mellitus due to the severity of the illness that it causes in that population.

[0089] Typically, the subject is one at risk of an infection with B. pseudomallei.

[0090] In an embodiment, then the infection is by B. pseudomallei the subject is one with diabetes. This may be type 1 or type 2 diabetes.

[0091] The subject may be immunocompromised. The subject may be one undergoing chemotherapy. The subject may be an alcoholic. The subject may be affected by chronic lung disease. The subject may be affected by cystic fibrosis. The subject may be affected by kidney disease. The subject may be one in an endemic area. The subject may be one with two or more of these criteria. In an embodiment, the subject is a child less than 18 years of age, but typically less 16 years of age or less. The child may be 12 years of age or less, or 10 years of age or less. Children are a target group for acute potentially fatal infections. Other risk groups and thus subjects in relation to B. pseudomallei infection include but are not limited to subjects over 45 years of age, excessive alcohol users, a subject with chronic kidney disease, a subject with chronic lung disease, e.g. chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF), a subject with a malignancy, a subject that is immune compromised, a subject that has non-HIV immune suppression, and a subject with thalassemia.

[0092] When the infection is caused by BCC, in an embodiment the subject has CF or chronic granulomatous disease. The subject may be immunocompromised. The subject may be one undergoing chemotherapy. The subject may be one with a malignancy. The subject may be one with a nosocomial infection.

[0093] When the infection relates to B. mallei typically the subject is an equine mammal such as a solipeds, e.g. a horse, donkey or a mule. Typically, the infection causes glanders in the mammal. In an embodiment, the subject is a human who works with, or is otherwise in contact with, solipeds.

[0094] In an embodiment, the immunogen BpPA26 is used in a composition as the sole immunogen. Typically, BpPA26 immunogen or antigen used in the current invention is a protein or polypeptide. However, it will be appreciated that in the aspects and embodiments disclosed above, the composition or vaccine may comprise a nucleotide encoding BpPA26, or an mRNA encoding BpPA26 instead of the protein or polypeptide itself.

[0095] Notably, in an embodiment, BpPA26 can be combined with one or more additional immunogens or antigens that are immunogenic against B. pseudomallei. Examples include but are not limited to one or more of Hcp1 (Burtnick et al., infect Immun. 2018; 86(1)), BipD (Druar et al., FEMS Immunol Med Microbiol, 2008; 52(1):78-87), LolC (Champion et al., Vaccine, 2016; 34(14): 1665-71), FliC (Koosakulnirand et al., PLoS One. 2018; 13(6)), BpOmpW (Tomas-Cortazar et al., Front. Immunol. 2021 Dec 13; 12; Casey et al..Vaccine 2016 May 17; 34(23):2616-21 ), OmpA (Petersen et al., Proc Vaccinol, 2014; 8:38-42; Nieves et al., Vaccine, 2011 ; 29(46): 8381-9; Nieves et al., Clin Vaccine Immunol. 2014; 21(5): 747-54), Omp85 (Su et al..Vaccine, 2010 Jul 12 ; 28(31) : 5005-11), and Hcp2 (Burtnick et al., Infect. Immun. 2011 ; 79 :1512-1525). In an embodiment, BpPA26 and BpOmpW are combined in a vaccine composition. There may be 1 additional antigen only, there may be 2 or more additional antigens. In an embodiment, there is from 1 to 9 additional antigens in the vaccine composition. In such an embodiment, the vaccine composition may be a DNA vaccine. It may be an adenoviral vector vaccine which comprises one or more nucleotide sequences encoding BpPA26 of the invention.

[0096] The invention relates to a DNA vaccine comprising an active agent, optionally a pharmaceutically acceptable carrier and optionally an adjuvant. In an embodiment, the active agent is a nucleic acid sequence encoding BpPA26 or an immunogenic portion thereof. In an embodiment, the nucleotide acid sequence comprises (or consists of) SEQUENCE ID NO. 2 or a therapeutically effective variant thereof, typically having at least 90% sequence identity with SEQUENCE ID NO. 2.

[0097] The nucleotide sequence is one that encodes the sequence of SEQUENCE ID NO. 1.

[0098] In an embodiment, the vaccine is an mRNA vaccine. Thus, in an aspect, a vaccine is provided that comprises an mRNA that encodes BpPA26 or an immunogenic portion thereof.

[0099] The vaccine may have one or more adjuvants. Typically, the adjuvant is one that tailors the effect of the antigen to induce T cell and B cell response, e.g., the CAFseries, such as CAF- 01 or CAF-09 or CpG ODNS (ODN 2006). It may be manno-heptose capsular polysaccharide (CPS) (Scott et al., Infect Immun. 2014 Aug;82(8):3206-13). It will be appreciated that such adjuvants are known in the art.

[0100] The vaccine or composition of the invention may comprise a combination of immunogenic portions.

[0101] The invention also relates to a diagnostic agent comprising or consisting of, BpPA26, or an immunogenic portion thereof, or an antibody that binds specifically thereto.

[0102] In an aspect, the invention also provides a method for diagnosing pathogenic Burkholderia infection in a subject, the method comprising assaying a sample from the subject for the presence of BpPA26 (or a gene encoding the protein), wherein presence of BpPA26 (or the gene) in the sample is indicative of infection.

[0103] Suitably, the assay method may be selected from an immunohistochemical detection a Western Blot, Enzyme-linked Immunosorbent Assay (ELISA) a Northern Blot, a Southern Blot, quantitative polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), quantitative real time RT-PCR (qRTPCR), an enzyme-linked immunosorbent assay (ELISA), protein determination on polyacrylamide gels, and such methods known to those skilled in the art.

[0104] An aspect of the invention BpPA26 immunogen of the invention or the composition of the invention comprising BpPA26 immunogen for use in immunotherapy. Thus also provided by the invention is an antibody (or a composition comprising the same) directed to BpPA26 and its use in immunotherapy. The composition may contain multiple antibodies directed to BpPA26 and one or more additional Burkholderia immunogens or antigens. Antibodies to said immunogens or antigens can be obtained with routine methods known in the art, for example hybridoma technology.

[0105] An embodiment of the treatment aspect of the current invention includes the use of an antibody directed or raised against, to one or more immunogens of the invention, which are suitable for immunotherapy and administered to the subject. The antibody is typically a monoclonal antibody specific to the immunogen of the invention

[0106] As noted, an aspect of the invention relates to the treatment of pathogenic Burkholderia infection in a subject, comprising administration of the composition of the invention to the subject.

[0107] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0108] EXAMPLES

[0109] MATERIAL AND METHODS

[0110] BpPA26 expression and purification

[0111] Sequence-confirmed BpPA26 clones in E. coli BL21(DE3) were grown in Luria-Bertani medium supplemented with ampicillin and induced with 1 mM IPTG for 16 h at 20 °C. The His- tag fusion protein cloned in pHisParallel 2 vector was then lysed in 20mM Tris-HCI pH 8.8; 150mM NaCI; 5% (v / v) glycerol; 0.5mM TCEP; 10mM imidazole purified by nickel affinity chromatography (Ni-NTA) (Cytiva, Uppsala, Sweden) washed with 20mM Tris-HCI pH 8.8; 150mM NaCI; 5% (v / v) glycerol; 0.5mM TCEP; 0.5% (v / v) Triton X-114; 10mM imidazole to remove bacterial endotoxins from the protein sample followed by Triton X-114-free wash buffer to eliminate the remaining Triton X114 from the Ni-NTA column. The sample was then eluted in 20mM Tris-HCI pH 8.8; 150mM NaCI; 5%(v / v) glycerol; 0.5mM TCEP; 250 mM Imidazole. An additional purification step using gel filtration chromatography was performed by loading BpPA26 onto a HiLoad 16 / 600 Superdex 75 column (Cytiva, Uppsala, Sweden) preequilibrated in 50mM Tris-HCI pH 7.5; 150mM NaCI; 5% (v / v) glycerol; 0,5mM TCEP, using an AKTA chromatography system (Cytiva, Uppsala, Sweden). The fractions with the protein of interest were pooled and the protein was concentrated and stored at - 80 °C until use. The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific). Endotoxin quantification was performed using the Pierce™ Chromogenic Endotoxin Quant Kit (Thermo Fisher Scientific). Protein identity was confirmed with bands excised from SDS-PAGE gels and in-gel trypsin digested, analysed by MALDI-TOF Mass spectrometry (Bruker, Coventry, UK) and compared to known peptides via the MASCOT database (Supplemental Excel Putative acetyl coA reductase MS data).

[0112] Ethics statement

[0113] All work involving animals was approved by the University College Dublin Ethics Committee (AREC-19-13-McClean), and mice were maintained according to the regulations of the Health Products Regulatory Authority (Directive 2010 / 63 / EU and Irish Statutory Instrument 543 of 2012) with the Authorisation number AE18982 / P166.

[0114] Immunisation of mice with BpPA26

[0115] Male C57BL / 6J mice were used in these studies and were given free access to food and water and subjected to a 12h light / dark cycle. Groups of C57BL / 6J male mice were immunized subcutaneously with 50ug BpPA26 in Sigma Adjuvant System (SAS, Sigma) or SAS adjuvant alone as adjuvant control. Two weeks later, mice were humanely killed by sedation with isoflurane followed by CO2 exposure. Then, blood were extracted by cardiac puncture for the serological analysis of antigen-specific immunoglobulins. Spleens were also processed for splenocyte restimulation with the vaccine antigen.

[0116] Determination of BpPA26-specific IgG isotypes by ELISA

[0117] Microtiter plates were coated with purified BpPA26 in sodium bicarbonate buffer (pH 9.4) at 4°C overnight. The coating solution was removed, and the plates were blocked with 10% (v / v) FBS solution in PBS at room temperature for 1 h. The wells were washed three times with 0.05% (v / v) Tween 20 in PBS using a plate washer. The serum samples were serially diluted (5-fold) in PBS containing 10% (v / v) FBS and 100ul added to wells in triplicate at RT for 2h and the plates washed again three times before the addition of anti-mouse IgG, lgG1 , or lgG2a-HRP conjugated antibodies (ab97023, ab97240, ab97245, respectively from Abeam) at RT for one hour. Then, TMB substrate was added and incubated until the colour developed. The reactions were stopped with 2M sulfuric acid and the plates read at 450nm. For the detection of BpPA26-specific IgG in human plasma, the same protocol was applied using an anti-human IgG antibody (ab6858, abeam) in place of anti-mouse IgG antibodies.

[0118] Ex-vivo Splenocyte Restimulation with BpPA26 antigen.

[0119] Splenocytes were extracted from the spleens using Ammonium-Chloride-Potassium (ACK) lysing buffer to remove red blood cells. Cells from BpPA26 immunized or adjuvant only control mice were then counted automatically (Invitrogen™ Countess™), two million plated per well and stimulated for 60h with 50ug / mL BpPA26 in 96-well-plate using 10% (v / v) FBS RPMI medium and Penicillin / Streptomycin. Between five and six hours before harvesting the cells, 5ug / mL Brefeldin A was added to block cell trafficking in order to increase the accumulation of intracellular cytokines. Cells were collected by centrifugation and stained for flow cytometry.

[0120] Flow Cytometry

[0121] The cells were incubated with Fc Block (anti-CD16 / CD32; BD BioSciences) for 5 minutes on ice and labeled with Viakrome 808 (Beckman Coulter) and fluorochrome-labeled antibodies against CD4, CD49b, CD45RB, CD8a, CD25, CD44, and CD3 surface markers (BD Biosciences) for 30 minutes on ice. Then, intracellular IL-2, IL-4, IL-17, IFN-y, IL-9, TNF, and Foxp3 (BD Bioscience) were also analysed with a BD Cytofix / CytoPerm™ / Fixation / Permeabilization Solution Kit (BD Biosciences) according to the manufacturer instructions. The antibodies were all titrated for optimal performance and Beckman Coulter Versacomp beads were used for compensation. A Beckman Coulter CytoFLEX LX (NUV full configuration) was used for the analysis of the samples with the gains for each one of the markers optimised by performing a gaintration. The Quality Control of the instrument was performed using the Beckman Coulter Daily QC beads and IR Daily QC as per manufacturer specifications. Fluorescence Minus One controls (FMOs) were used for every single marker in order to generate the gates and several FMOs were used daily for verification purposes. The data analysis and reanalysis were performed with Beckman Coulter CytExpert v. 2.4. Supplemental Excel file shows the full MIFIowCyt: The Minimum Information about a Flow Cytometry Experiment.

[0122] Insulin Resistant Mouse model

[0123] C57BL / 6J male mice were fed with a D12492i rodent diet comprised of 60% kcal from fat (Research Diets, Inc) or regular chow starting at 6-8 weeks of age until they were humanely killed. Insulin resistance was determined at 8 and 12 weeks by fasting the mice for 6h at which time 0.5 units / Kg of insulin was administered subcutaneously. In order to alleviate any pain, EM LA cream was applied to the whole tail 10 minutes before blood was sampled from the tail vein using a 27-gauge needle, a drop of blood was extracted at 15, 30, 45 & 60 minutes, and glucose levels measured by AlphaTRAK® 2 Blood Glucose Monitor. The time required for individual mice to develop insulin resistance varied and only mice that showed insulin resistance were considered eligible to be randomly selected for immunization studies. Statistical Analysis

[0124] The data are presented as means ± SE, unless otherwise stated. The differences in the means between groups were tested using a two-tailed T-test using GraphPad Prism, version 7. A p- value < 0.05 was considered statistically significant.

[0125] RESULTS

[0126] Immunization of non-insulin resistant mice with recombinant BpPA26 induced humoral and IFN responses

[0127] As BpPA26 antigen candidate was predicted to have immunogenic epitopes for both B and T cells, the inventors then examined the immune responses to BpPA26 in immunised mice to investigate whether it might elicit strong humoral and T cell responses. The inventors expressed and purified BpPA26 in its tetramer form (eluted around 52.5mL in a HiLoad Superdex 75 16 / 600) (Figure 1A,B), confirmed its identity by Mass Spectrometry and removed endotoxin (0.2 EU / dose) to the recombinant BpPA26. The band at lower molecular weight is due to degradation of the whole protein (Fig. 1 B). Groups of 11 mice were immunized in the presence or absence of adjuvant and, humoral and T cell responses were evaluated in sera and splenocytes, respectively after two weeks (Fig. 1C). Mice immunized with BpPA26 showed moderate humoral antibody responses as determined by specific total IgG, lgG1 , and lgG2a against the antigen (Fig. 1 , D-F). Conversely, when splenocytes from immunized mice were re-exposed in vitro to antigen and interrogated for T cell markers and cytokines, the CD45RB marker was significantly decreased compared to the control group (Fig. 2A; p = 0.0343) and the autocrine growth factor IL-2 in both T type of cells (CD4+and CD8+T cells) was elevated relative to the control group (Fig. 2, B-C; p = 0.0123, p = 0.0054, respectively), both outcomes indicative of T cell activation. The activation of T cells by BpPA26 produced a mixed Th1 / Th2 response, as determined by concomitant increases in IFN-y and IL-4, respectively, in CD4+T cells with respect to control splenocytes (Fig.2, D-E; p = 0.0035, p = 0.0002). In turn, Th17 cells (Fig. 2, F), together with CD25 and CD44 levels in both CD4+and CD8+T cells (Fig. 2, G-J), as well as IFN-y expressing CD8+T cells (Fig. 2, K) remained unchanged relative to the control group. Finally, several studies have observed increased levels of TNF during melioidosis infection in both humans and animal models and TNF is required for optimal control of B. pseudomallei infection. In line with this, TNF was also elevated in CD4+T cells (Fig. 2, L; p = 0.0234) whereas TNF-expressing CD8+T cells remained comparable to the control group (Fig. 2, M). The inventors also evaluated CD4+CD25+Foxp3+regulatory T cells (Tregs) as they can suppress pro-inflammatory damage. Tregs were also elevated in the presence of BpPA26 antigen with respect to the control group (Fig. 3, O; p = 0.0004). It has been recently reported that NK and DN T cell effector functions correlate with survival in patients with melioidosis. In this study, the number of IFN-y producing NK cells was higher in the BpPA26 immunised group relative to the control group (Fig. 3, A; p = 0.0141), whereas TNF expressing NK cells were comparable to the control group (Fig. 2, B). Expression of CD49b+ demonstrated that NKT cells represented virtually all these DN cells (Fig. 3I) In addition, the activation marker CD44 and the cytokines levels elicited following BpPA26 immunisation in NKT cells were comparable to the control group (Fig. 3, C-H). TNF-expressing NKT cells were the only cells elevated relative to the control group (Fig. 3, H)

[0128] Immunization of a mouse model of T2D with BpPA26 produced IFN-y responses

[0129] Due to the capacity of BpPA26 to activate T cells and stimulate IFN-y responses and the fact that T2D increases susceptibility to melioidosis, we developed a polygenic insulin-resistant mouse model of T2D by feeding C57BL / 6J male mice for 17 weeks with a high-fat diet (HFD) (Fig. 4, A). (Tomas Cortazar et al (2021). Frontiers in Immunology, 12, 5235.). Mice on the HFD gained more weight (Fig. 4, B; p < 0.0001) and developed insulin resistance (Fig. 4, C, D; p < 0.0001) from week 8 of HFD feeding. The inventors immunized groups of nine insulinresistant mice with recombinant BpPA26 (Fig. 1 , B) plus adjuvant to examine the vaccine response in the insulin-resistant context. BpPA26 elicited similar humoral responses to those observed in non-insulin resistant study (Fig. 5). In turn, immunisation with the antigen did not alter CD45RB, CD25 or CD44 on T cells (Fig. 4, A-E) but augmented IL-2 in both CD4+and CD8+T cells relative to the control groups (Fig. 5, F-G; p = 0.041 and p = 0.0423). As a consequence of T cell activation, IFN-y in CD4+T cells was elevated in the BpPA26 immunised group compared to the control group (Fig. 5, H; p = 0.0035). However, both IL-4 (Fig. 4, I; p = 0.0794) and IL-17 (Fig. 5, J; p = 0.5316) expressing CD4+T cells showed no significant differences following BpPA26 immunisation compared with the control group. No differences were observed in TNF-producing CD4+T cells between both groups either (Fig. 5, K). The CD8+T cells subset in immunised mice showed unchanged IFN-y levels (Fig. 5, L) but elevated levels of TNF (Fig. 5, M; p = 0.0500) relative to the control group. Furthermore, NK cells elicited higher amounts of IFN-y (Fig. 5, N; p = 0.0316) and comparable values of TNF (Fig. 5, O) relative to the control group following BpPA26 immunisation. While IL-17 and TNF in the BpPA26 group were comparable to the control groups in NKT cells (Fig. 4, P-Q), elevated IL- 2 (p = 0.0037), IFN-y (p = 0.0159) and IL-4 (p = 0.0002) were observed in the BpPA26 immunised group compared to the control group (Fig. 5, R-T). Finally, CD4+CD25+Foxp3+regulatory T cells remained unchanged in the BpPA26 group relative to the control group (Fig. 5, U).

[0130] DISCUSSION Acetoacetyl-CoA reductase was suggested to be a moonlighting protein in B. pseudomallei. Prior to evaluating BpPA26 preclinically the inventors used a structural vaccinology-based B and T cell epitopes predictive method as a tool to avoid unnecessary preclinical studies in the absence of immunogenic epitopes. The inventors discovered that the antigen had potential B and T cell immunoreactivity and therefore allowed them to proceed to preclinical studies in mice with more confidence.

[0131] The immunisation of mice with BpPA26 recombinant antigen stimulated humoral and IFN-y responses in both non- and insulin-resistant mice, which correlate with protective immune responses against melioidosis. The moderate humoral responses induced by BpPA26 were achieved despite the animals receiving only one single immunisation. Th2 responses, determined by significantly increased amounts of IL-4 cytokine, were promoted by the immunisation of non-insulin resistant mice with recombinant BpPA26 antigen, but not in insulin resistant mice probably because C57BL / 6j mice preferentially differentiate to Th1 phenotype in response to HFD (Jovicic et al., 2015). Regulatory T cells which are essential to prevent the excessive inflammatory responses to bacterial endotoxins were elevated in BpPA26 immunised mice group relative to the control but remained comparable to control in insulin resistance mice. Regulatory T cells are described to be downregulated in diabetes (Wagner et al., 2013; Cipolleta, 2014. Overall the elevated NKT cell cytokine responses in BpPA26 immunised insulin resistant mice, including IFN-y, is significant given that IFN-y expressing NKT cells showed a role in survival to melioidosis in patients with diabetes mellitus. It is also noteworthy that BpPA26 also promoted the stimulation of IFN-y responses in NK and CD4+T cells in both non- and insulin-resistant mice, as the strongest correlations of IFN-y responses against B. pseudomallei in melioidosis patients were in CD4+ T cells, followed by NK cells.

[0132] Although BpPA26 antigen stimulated moderate humoral responses in mice, a single immunisation of whole protein was able to elicit the predominant IFN-y responses observed against B. pseudomallei (from NK and CD4+T cells) in both non- and insulin-resistant mice, suggesting that, even for people with diabetes as well, it could be used as an antigen in a vaccine or an additional antigen for a subunit vaccine to increase the efficacy and enhance its breadth of coverage.

[0133] Equivalents

[0134] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

Claims1. A vaccine composition comprising BpPA26, or an immunogenic portion thereof (or a nucleotide, or mRNA encoding same).

2. The vaccine composition of Claim 1 , wherein BpPA26 is a protein or polypeptide.

3. The vaccine composition of Claim 1 , wherein BpPA26 has the amino acid sequence of SEQUENCE ID NO. 1 , or an effective variant thereof.

4. The vaccine composition of Claim 3, wherein the effective variant has 1 to 5 amino acid alterations compared with SEQUENCE ID NO. 1 , wherein each alteration is independently selected from an insertion, addition, deletion or substitution of an amino acid.

5. The vaccine composition of Claim 1 , which comprises an immunogenic portion of BpPA26 having an amino acid sequence that is a fragment of SEQUENCE ID NO. 1 of from 10 to 25 amino acids in length, or an effective variant of the immunogenic portion having from 1 to 3 amino acid alterations compared with the sequence of the immunogenic portion, wherein each alteration is independently selected from an insertion, addition, deletion or substitution of an amino acid.

6. The vaccine composition of any one of the preceding claims, which is an adenoviral vector vaccine and comprising a nucleotide encoding BpPA26 or the immunogenic portion thereof.

7. The vaccine composition of any one of Claims 1 to 5, which is an mRNA vaccine and comprising an mRNA encoding BpPA26 or the immunogenic portion thereof.

8. The vaccine composition of any one of the preceding claims, further comprising one or more additional B. pseudomallei antigens.

9. The vaccine composition of Claim 8, wherein the additional antigen is one or more of Hcp1 , BipD, LolC, FliC, BpOmpW, OmpA, Omp85, and Hcp2 (or a nucleotide or mRNA encoding same).

10. The vaccine composition of Claim 9, wherein the additional antigen is BpOmpA.

11. The vaccine composition of any one of the preceding claims, further comprising one or more adjuvants.

12. The vaccine composition of Claim 11 , wherein the one or more adjuvants are selected from the group comprising a CAF series adjuvant and manno-heptose capsular polysaccharide (CPS).

13. The vaccine composition of any one of the preceding claims, for use in vaccine therapy to prevent or treat a pathogenic Burkholderia species infection in a subject.

14. The vaccine composition for use of Claim 13, wherein the pathogenic Burkholderia species is selected from Burkholderia cepacia complex, Burkholderia mallei, and Burkholderia pseudomallei.

15. The vaccine composition for use of Claim 14, wherein the species is Burkholderia cepacia complex.

16. The vaccine composition for use of Claim 14, wherein the species is Burkholderia pseudomallei and the infection causes melioidosis.

17. The vaccine composition for use of any one of Claims 13 to 16, wherein the use is to prevent infection and the subject is one at risk of pathogenic Burkholderia species infection.

18. The vaccine composition for use of any one of Claims 13 to 17, wherein the subject is one with diabetes.

19. The vaccine composition for use of any one of Claims 13 to 17, wherein the subject is an immunocompromised subject with an underlying lung condition.

20. The vaccine composition for use of Claim 19, wherein the lung condition is chronic lung disease.

21. The vaccine composition for use of Claim 20, wherein the lung disease is chronic obstructive pulmonary disease (COPD) or cystic fibrosis (CF).

22. The vaccine composition for use of any one of Claims 13 to 17, wherein the subject is an excessive alcohol user, a subject with chronic kidney disease, a subject with a malignancy, a subject that is immune compromised, a subject that has non-HIV immune suppression, and a subject with thalassemia.

23. The vaccine composition for use of any one of Claims 13 to 22 wherein the subject is 45 years or older.

24. The vaccine composition for use of any one of Claims 13 to 22, wherein the subject is a child of 16 years of age or less.

25. The vaccine composition for use of Claim 13, wherein the species is Burkholderia mallei and the subject is a horse or a camel.

26. A method of treating or preventing Burkholderia species infection in a subject, said method comprising the step of administration of a vaccine of any one of Claims 1 to 12 to the subject.

27. A method of treating or preventing melioidosis in a subject, comprising administering a vaccine of any one of Claims 1 to 12 to the subject.

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