Immunogenic composition

Abstract

The present invention relates to immunogenic compositions comprising outer membrane vesicles (OMVs) adsorbed to an aluminium hydroxide adjuvant, whereby the aluminium hydroxide adjuvant is quenched. The present invention also relates to the use of the immunogenic compositions for providing protection against diseases caused by Gram-negative bacteria. The present invention also relates to a method for reducing particle aggregation and / or average particle diameter in compositions comprising OMVs and an aluminium hydroxide adjuvant.

Description

[0001] IMMUNOGENIC COMPOSITION

[0002] Field of the Invention

[0003] The present invention relates to immunogenic compositions comprising outer membrane vesicles (OMVs) adsorbed to an aluminium hydroxide adjuvant, whereby the aluminium hydroxide adjuvant is quenched. The present invention also relates to the use of the immunogenic compositions for providing protection against diseases caused by Gram-negative bacteria. The present invention also relates to a method for reducing particle aggregation and / or average particle diameter in compositions comprising OMVs and an aluminium hydroxide adjuvant.

[0004] Background to the Invention

[0005] Aluminium hydroxide is an adjuvant onto which antigens can be adsorbed. The major mechanisms by which aluminium-containing adjuvants adsorb antigens are electrostatic attraction, hydrophobic forces and ligand exchange. Electrostatic attraction in particular seems to play a large role in these interactions: the positive charge of aluminium hydroxide is able to attract negatively charged molecules, leading to adsorption.

[0006] A type of negatively charged molecule is GMMA (generalised modules for membrane antigens). GMMA are particles derived from the outer membrane of Gram-negative bacteria that have high levels of LPS, lipoproteins, proteins and other antigens that activate the innate immune response. GMMA from bacteria such as Shigella and Salmonella are useful as components for vaccines - see, for example, Gerke et al. Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB. PLoS One. 2015;10(8):e0134478, and Rossi et al. Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis. Clin Vaccine Immunol. 2016;23(4):304-14). GMMA are generally well-adsorbed onto aluminium hydroxide. However, when GMMA are adsorbed onto aluminium hydroxide, they may form particles that are large and even visible to the naked eye. The presence of particles that are visible to the naked eye can be problematic in, for example, vaccine compositions, as such large particles may cause pain when injected and injection site reactions. Moreover, if mechanically stressed, foam formation can occur and there may be an increase of the crystalline forms of aluminium hydroxide. This phenomenon breaks the amorphous status of the adjuvant, leading to a loss of homogeneity of the suspension and a possible different presentation of GMMA to the immune system once injected.

[0007] Therefore, there is a need to provide a way of preventing aggregation of, and therefore reduce the particle diameter of, aluminium hydroxide-antigen complexes.

[0008] Summary of the Invention

[0009] As discussed above, there is a need to provide a way of preventing aggregation of, and therefore reduce the particle diameter of, aluminium hydroxide antigen complexes. The present Examples demonstrate that this may be achieved by adding a quencher to a composition comprising an antigen and aluminium hydroxide. Accordingly, the present invention provides the following,

[0010] In a first aspect of the invention, there is provided an immunogenic composition comprising outer membrane vesicles (OMVs) adsorbed to a quenched aluminium hydroxide adjuvant.

[0011] In a second aspect of the invention, there is provided an immunogenic composition comprising OMVs adsorbed to an aluminium hydroxide adjuvant and further comprising a quencher. In a third aspect of the invention, there is provided a method for reducing particle aggregation, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition.

[0012] In a fourth aspect of the invention, there is provided a method of producing an immunogenic composition with reduced average particle diameter, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition.

[0013] In a fifth aspect of the invention, there is provided a method of preventing a disease comprising administering an effective amount of the immunogenic composition of the invention.

[0014] In a sixth aspect of the invention, the invention provides a use of the immunogenic composition of the invention in the manufacture of a medicament for use in a method of preventing a disease.

[0015] Brief description of the Figures

[0016] Figure 1. Comparison in particle size of a composition comprising STmGMMA adsorbed onto aluminium hydroxide that is quenched with phosphate ions (continuous line) or not quenched (dotted line).

[0017] Figures 2a and 2b. Comparison in particle size of a composition comprising STmGMMA (Figure 2a) or SEnGMMA (Figure 2b) adsorbed onto aluminium hydroxide that is quenched with 20mM phosphate ions (continuous line) or lOmM phosphate ions (dotted line). Detailed Description

[0018] General Definitions

[0019] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[0020] In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “An immunogenic composition comprising outer membrane vesicles” should be interpreted to mean that the immunogenic composition contains outer membrane vesicles, but the immunogenic composition may comprise further components.

[0021] In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of'. The term “consisting of' is intended to be limiting. For example, the phrase “An immunogenic composition consisting of outer membrane vesicles” should be understood to mean that the immunogenic composition has outer membrane vesicles and no other components.

[0022] In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting essentially of”. The term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter.

[0023] The term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error. Optionally, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x. For example, if the concentration of ions is “about” or “around” 20mM, then the concentration can be within 10%, within 5%, or within 1% of20mM. The singular forms “a”, “an ”, and “the ” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “the GtVltVlA” includes two or more instances or versions of such GMMA.

[0024] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0025] Immunogenic compositions

[0026] The present invention includes immunogenic compositions comprising outer membrane vesicles (OMVs) adsorbed to a quenched aluminium hydroxide adjuvant. The present invention also includes immunogenic compositions comprising OMVs adsorbed to an aluminium hydroxide adjuvant and further comprising a quencher.

[0027] Methods for reducing particle aggregation / producing an immunogenic composition with reduced average particle diameter

[0028] The present invention also includes a method for reducing particle aggregation, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition.

[0029] The present invention also includes a method of producing an immunogenic composition with reduced average particle diameter, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition. The step of obtaining OMVs may be obtaining OMVs from one or more Gram-negative bacteria. The Gram-negative bacteria may be from any species in any of genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia, Haemophilus, Klebsiella, Legionella, Porphyromonas, Pseudomonas, Yersinia, Helicobacter, Salmonella, or Vibrio. For example, the bacterium may be Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Moraxella catarrhalis, Escherichia coli, Haemophilus influenzae (including non- typeable stains), Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, Porphyromonas gingivalis, Pseudomonas aeruginosa, Yersinia enterocolitica, Helicobacter pylori, Salmonella enterica (including serovars Typhi and Typhimurium, as well as serovars Paratyphi and Enteritidis), Shigella (such as S. dysenteriae, S.flexneri, S. boydii or S. sonnei), Vibrio cholerae, etc.

[0030] The Gram-negative bacteria may be Salmonella and / or Shigella and / or or Escherichia coli. The Gram-negative bacteria may be one or more of the subspecies of Salmonella enterica, and / or Shigella sonnei and / or one or more of the serotypes of Shigella flexneri and / or Escherichia coli. For example, the Gram-negative bacteria may be one or more of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium), Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis), Salmonella enterica subspecies enterica serovar Typhi (Salmonella Typhi), Salmonella enterica subspecies enterica serovar Paratyphi A (Salmonella Paratyphi A), Shigella sonnei, and / or Shigella flexneri serotype la, lb, 2a, 2b, 3a, 3b, 4a, 5b, 6 and / or X and / or Enterotoxigenic Escherichia coli (ETEC).

[0031] Obtaining OMVs from one or more Gram-negative bacteria may comprise growing the Gram-negative bacteria and isolating the OMVs. Growing the Gram-negative bacteria can be carried out by fermentation under suitable pH, temperature and oxygen conditions, for example conditions set out under the “Bacterial culture” section under the “ MODES FOR CARRYING OUT THE INVENTION' section of WO 2011 / 036562 herein incorporated by reference, the “ Fermentation" section under the “MODES FOR CARRYING OUT THE INVENTION' section of WO 2016 / 202872 herein incorporated by reference, and the citations set out in Example 1. The isolating the OMVs may comprise a step of ultra-filtration and / or a step of dia-filtration, for example those described in the “The first filtration" and “The second filtration" sections in WO 2011 / 036562 herein incorporated by reference.

[0032] The step of adding a quencher may be immediately followed by stirring for about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. The step of adding a quencher may be immediately followed by stirring for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, and / or at most 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0033] The step of adding a quencher may alternatively comprise adding the quencher and stirring for a duration of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, i.e. the quencher may be added gradually (for example in small aliquots) whilst being continuously stirred over a period of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours. The step of adding a quencher may comprise adding the quencher and stirring for a duration of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, and / or at most 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, i.e. the quencher may be added gradually (for example in small aliquots) whilst being continuously stirred over a period of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, and / or at most 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0034] The method may further comprise adding one or more additional antigens to the immunogenic composition. As a result, the immunogenic composition may be bivalent, trivalent, quadrivalent, pentavalent, hexavalent, heptavalent, octavalent, nonavalent or decavalent. The one or more additional antigens may be any antigen purified from a Gram-negative bacterium that raises an immune response in a subject. The immune response may be protective and may raises antibodies, such as IgG antibodies.

[0035] The one or more additional antigens may be one or more antigens purified from one or more Gram-negative bacteria of the group consisting of: Salmonella enterica (for example Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium), Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis), Salmonella enterica subspecies enterica serovar Typhi (Salmonella Typhi), and / or Salmonella enterica subspecies enterica serovar Paratyphi A (Salmonella Paratyphi A)), Shigella sonnei, Shigella flexneri (for example Shigella flexneri serotypes la, lb, 2a, 2b, 3a, 3b, 4a, 5b, 6 and / or X), or Escherichia coli (for example Enterotoxigenic Escherichia coli (ETEC)).

[0036] The one or more additional antigens may be one or more OMVs from the same bacterium but different serotype as (one of) the antigens already present in the immunogenic composition, or from a different bacterium.

[0037] The one or more additional antigens may be GMMA. For example, the one or more additional antigens may be Salmonella Typhimurium GMMA, Salmonella Enteritidis GMMA, Shigella sonnei GMMA, and / or Shigella flexneri la, lb, 2a, 2b, 3a, 3b, 4a, 5b, 6 and / or X GMMA, or a combination of these GMMA.

[0038] The one or more additional antigens may be antigens that are not OMVs. For example, the one or more additional antigens may be an S. Typhi Vi-CRM197 saccharide conjugate, S. Paratyphi A OAg-CRM197 saccharide conjugate and / or an Enterotoxigenic Escherichia coli (ETEC) colonisation factor.

[0039] The one or more additional antigens may be one or more OMVs (for example GMMA) and one or more additional antigens that are not OMVs. For example, the one or more additional antigens may be Salmonella Typhimurium GMMA, Salmonella Enteritidis GMMA, Salmonella Typhi Vi-CRM197 saccharide conjugate, Salmonella Paratyphi A GMMA and / or Salmonella Paratyphi A OAg-CRM197 saccharide conjugate.

[0040] The present invention also includes immunogenic compositions obtainable or obtained from the methods of the invention.

[0041] Outer membrane vesicles (OMVs)

[0042] For the purpose of the present invention, the term “OMV” refers to any type of outer membrane vesicle. Suitable OMVs include native OMVs. Gram-negative bacteria can spontaneously release outer membrane vesicles (OMVs) during growth due to the turgor pressure of the cell envelope, and these are native OMVs. OMVs are rich in immunogenic cell surface-associated, periplasmic and secreted antigens and have been used as vaccines.

[0043] OMVs of the invention include Generalised Modules for Membrane Antigens (GMMA), native OMVs (‘NOMVs’ (see Katial et al. 2002, Infect Irnmun, 70: 702- 707), microvesicles (MVs (see WO 02 / 09643)), detergent-extracted OMVs (DOMVs), mutant-derived OMVs (m-OMV), and blebs, which are outer-membrane protrusions that remain attached to bacteria prior to release as MVs (see Beveridge, 1999, J.

[0044] Bacteriol. 181: 4725-4733)).

[0045] Generalised Modules for Membrane Antigens (GMMA) are a type of OMV. GMMA are distinct from native outer membrane vesicles (NOMV), which are released spontaneously from Gram-negative bacteria, in two crucial aspects. First, to induce GMMA formation, the membrane structure has been modified by the deletion of genes encoding key structural components, such as tolR (leading to hyperblebbing). Second, as a consequence of the genetic modification, large quantities of outer membrane “bud off' (or “hyperb lei '’) to provide a practical source of membrane material for vaccine production. Accordingly, for the purpose of the present invention, the term “GMMA ” refers to OMVs which are released spontaneously from bacteria modified to hyperbleb (such as Salmonella or Shigella bacteria which are modified such that they do not comprise a gene encoding functional TolR).

[0046] OMVs of the invention are purified from Gram-negative bacteria

[0047] The OMVs (such as GMMA) of the invention can be purified from any Gram-negative bacteria. Suitable purification methods are known in the art, and include a variety of filtration and chromatography methods. A preferred two-step filtration purification process is described in WO 2011 / 036562 herein incorporated by reference.

[0048] The OMVs (such as GMMA) of the invention may be purified from Gram-negative bacteria that have high surface O-antigen on the OMVs (such as GMMA). For example, the w / w ratio between protein and O-antigen displayed by the OMVs (such as GMMA) may be at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0, or at most 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.

[0049] The amount of OAg in OMVs (such as GMMA) can be quantified by using HPAEC- PAD.

[0050] The total amount of protein in OMVs (such as GMMA) can be quantified using the micro BCA assay (Micro BCA assay kit 23225, ThermoFischer scientific). This assay makes use of the protein reactivity that in alkaline conditions reduces copper (II) ions which are revealed through the formation of a purple complex with bicinchoninic acid. The amount of protein in the sample is determined by reading the 562 nm absorbance of the resulting product against a standard curve. The standard curve for the measurements is generated with bovine serum albumin (BSA) in the range of 1.8-20 pg / mL. To quantify the soluble protein in OMVs (such as GMMA), the assay is applied on the sample supernatant following OMV (such as GMMA) purification by ultracentrifugation (110000 rpm, 30 minutes 4°C, using a rotor with K factor 15).

[0051] In some aspects, the OMVs are obtained from Gram negative bacteria. In some embodiments, the OMVs are obtained from any Gram negative bacteria genera, such as from species in any of genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia, Haemophilus, Klebsiella, Legionella, Porphyromonas, Pseudomonas, Yersinia, Helicobacter, Salmonella, or Vibrio.

[0052] For example, the bacterium may be Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Moraxella catarrhalis, Escherichia coli, Haemophilus influenzae (including non-typeable stains), Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, Porphyromonas gingivalis, Pseudomonas aeruginosa, Yersinia enterocolitica, Helicobacter pylori, Salmonella enterica (including serovars Typhi and Typhimurium, as well as serovars Paratyphi and Enteritidis), Shigella (such as S. dysenteriae, S.flexneri, S. boydii or S. sonnei), Vibrio cholerae, etc.

[0053] The OMVs (such as GMMA) of the invention may be purified from one or more of the serotypes of Salmonella enterica, Shigella sonnei, or one or more of the serotypes of Shigella flexneri and / or Escherichia coli. For example, the OMVs (such as GMMA) of the invention may be purified from one or more of the group consisting of: Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium), Salmonella enterica subspecies enterica serovar Enteritidis (Salmonella Enteritidis), Salmonella enterica subspecies enterica serovar Typhi (Salmonella Typhi), Salmonella enterica subspecies enterica serovar Paratyphi A (Salmonella Paratyphi A), Shigella sonnei, Shigella flexneri serotypes la, lb, 2a, 2b, 3a, 3b, 4a, 5b, 6 and / or X (for example Shigella flexneri serotypes lb, 2a, and / or 3a) and / or Enterotoxigenic Escherichia coli (ETEC)). Hyperblebbing

[0054] The Gram-negative bacteria from which the OMVs (such as GMMA) of the invention are purified may have been modified (for example genetically modified) to hyperbleb i.e. more quantities of outer membrane “bud off' compared to a corresponding Gramnegative bacterium that does not have the genetic mutation.

[0055] The Gram-negative bacteria may comprise any suitable modification that leads to hyperblebbing. Optionally, the modification is a mutation, for example the Gramnegative bacterium may not comprise a gene (such as tolR) encoding a functional protein because it comprises a mutation in that gene. Optionally, the Gram-negative bacteria do not comprise a gene encoding a functional TolR protein. Optionally, the Gram-negative bacteria comprise a gene encoding at least a portion of the TolR protein, but either the gene is mutated such that the TolR protein encoded is missing one or more important amino acids or a portion of the gene is deleted. For example, the Gramnegative bacterium may comprise a substitution or deletion mutation in the tolR gene. Alternatively, the Gram-negative bacterium may have an addition mutation in the tolR gene, for example an addition mutation causing a frame shift. Optionally, the Gramnegative bacterium comprises a deletion mutation in the tolR gene. Optionally, the tolR gene comprises a deletion mutation, and at least 10%, at least 20%, at least 25%, at least 50% or at least 75% of the tolR gene is deleted. Optionally, the Gram-negative bacterium lacks a tolR gene (for example because the complete tolR gene has been deleted (a AtolR mutation).

[0056] Whether or not a genetic modification causes a Gram-negative bacterium to hyperbleb may be tested using the following hyperblebbing assay. The user should prepare two cultures of bacterium. The first culture should comprise the bacterium having the genetic modification to be tested (the test culture), and the second culture should comprise an equivalent bacterium which is identical but for the genetic modification to be tested (the reference culture). The user should grow the test culture and the reference culture under identical conditions and determine the number of outer membrane vesicles released from the bacteria in the test culture and bacteria in the reference culture. If the amount of outer membrane vesicles released in the test culture is higher than the amount of outer membrane vesicles released in the reference culture, then the genetic modification causes the bacterium to hyperbleb. The level of outer membrane vesicles released may be determined by O-Antigen quantification, for example, for example by using the methods set out in the “O-antigerT section.

[0057] Modification of lipid A

[0058] The GMMA of the invention may comprise modified lipid A. A modified lipid A is a lipid A that has a different structure compared to a corresponding wild type lipid A.

[0059] The structure of lipid A may be determined using MALDI-TOF analysis of lipid A isolated from the GMMA. For the assay, the lipid A is separated after treatment of GMMA with acetic acid and then assayed by MALDI-TOF. GMMA with a protein concentration of about 1 mg / mL (micro BCA calibration curve) or a cell bank suspension with an OD600 of about 3 (4 mL sample) are treated with 1% acetic acid (final concentration) for 2 or 6 hours, respectively, at 100°C to obtain a precipitate containing the lipid A. The precipitate is then collected, washed with water and the lipid A is extracted in chloroform / methanol 4:1. The final solution, which contains the lipid A, is mixed 1:1 with Super DHB (Fluka, 50862) saturated solution (acetonitrile / water 1:1). Two microliters of the mixture are loaded onto the target plate and after the spot is dried at room temperature, the plate is inserted in the mass spectrometer. The spectra (negative reflectron mode) generally show peaks corresponding to the lipid A molecular species and contain several peaks due to fragmentation of the lipid A (i.e. loss of one or more fatty acid chains), sodium adduct (+22 m / z) and lipid A dephosphorylation (- 80 m / z). The species of lipid A is identified by comparison of the molecular peak mass m / z to what is expected for the sample in analysis.

[0060] Optionally, the modified lipid A is modified to be less toxic than wildtype lipid A. The wildtype lipid A used in the comparison is a corresponding wildtype lipid A. “Toxicity” or “toxic” in this context refers to the extent to which the innate immune system is activated by lipid A, particularly through the Toll-like receptor 4 pathway. Highly toxic lipid A can lead to uncontrolled inflammation, apoptosis, and in extreme cases septic shock, among other effects. Optionally, a modified lipid A is less toxic if it is less reactogenic than a corresponding wildtype lipid A. For example, one can determine whether a modified lipid A is less toxic by administering it to an animal such as a rabbit, and determining whether it activates more monocytes compared to a corresponding wildtype lipid A using a monocyte activation test. Optionally, a modified lipid A is less toxic than wildtype lipid A if the monocyte activation test shows that the cells with the modified lipid A has a reduced IL-6 release compared to cells with wildtype lipid A.

[0061] “Corresponding wildtype lipid A” refers to lipid A that can be found in the corresponding wildtype bacterium and strain. For example, lipid A that is modified relative to a “corresponding wildtype lipid A” in the context of Shigella flexneri serotype lb GMMA is interpreted to mean a modified lipid A (e.g. such that it is less toxic) relative to lipid A found in wildtype Shigella flexneri serotype lb.

[0062] The Gram-negative bacteria may comprise any suitable modification that leads to production of GMMA comprising lipid A that is less toxic than wildtype lipid A. Optionally, the modification is a mutation, for example the Gram-negative bacterium may not comprise a gene (such as htrB, msbB, msbBl, msbB2 and / or pagP) encoding a functional protein because it comprises a mutation in that gene. Optionally, the Gramnegative bacteria do not comprise a gene encoding a functional HtrB, MsbB, MsbBl, MsbB2 and / or PagP protein. Optionally, the Gram-negative bacteria comprise a gene encoding at least a portion of the HtrB, MsbB, MsbBl, MsbB2 and / or PagP protein, but either the gene is mutated such that the HtrB, MsbB, MsbBl, MsbB2 and / or PagP protein encoded is missing one or more important amino acids or a portion of the gene is deleted. For example, the Gram-negative bacterium may comprise a substitution or deletion mutation in the htrB, msbB, msbBl, msbB2 and / or pagP gene. Alternatively, the Gram-negative bacterium may have an addition mutation in the tolR gene, for example an addition mutation causing a frame shift. Optionally, the Gram-negative bacterium comprises a deletion mutation in the htrB, msbB, msbBl, msbB2 and / or pagP gene. Optionally, the htrB, msbB, msbBl, msbB2 and / or pagP gene comprises a deletion mutation, and at least 10%, at least 20%, at least 25%, at least 50% or at least 75% of the htrB, msbB, msbBl, msbB 2 and / or pagP gene is deleted. Optionally, the Gram-negative bacterium lacks a htrB, msbB, msbBl, msbB2 and / or pagP gene (for example because the complete htrB, msbB, msbBl, msbB2 and / or pagP gene has been deleted (a ShtrB, SmsbB, SmsbBl, SmsbB2 and / or SpagP mutation).

[0063] ""Inactivation” in the context of a gene refers to mutating or deleting the gene such that the protein to be transcribed can no longer carry out the function of the corresponding wildtype protein, or carries out the function to a lesser extent. For example, "''inactivation of htrB” refers to deleting the gene, or mutating the gene, in a Shigella bacterium such that acylation of lipid A in the Shigella bacterium is reduced.

[0064] Optionally, whether or not Shigella bacteria include one or mutations resulting in “ inactivation ” of htrB or an msbB protein may be determined by isolating GMMA from the Shigella bacteria and analysing the lipid A in the GMMA using MALDI-TOF analysis. For example, one can compare a spectrum generated by MALDI-TOF analysis for a certain lipid A sample with a spectrum produced by analysing lipid A from Shigella that comprise wild type htrB and msbB genes, and if the amount of hexaacylation is reduced, then the Shigella bacteria includes one or mutations resulting in inactivation of htrB and / or msbB. A similar analysis can be applied to Salmonella enterica bacteria, with pagP and / or msbB. Inactivation of pagP and / or msbB results in Salmonella enterica that produces GMMA comprising lipid A that is less toxic than wildtype lipid A. Example 1 provides an example of how such Salmonella enterica GMMA can be produced.

[0065] Inactivation of htrB, msbBl and / or msbB2 results in Shigella that produces GMMA comprising lipid A that is less toxic than wildtype lipid A (see WO 2023 / 025815 for example).

[0066] In an embodiment, the Salmonella enterica GMMA of the invention are produced from Salmonella enterica strains that include one or more mutations resulting in inactivation of pagP and / or msbB. By way of non-limiting example, suitable Salmonella enterica strains may be selected from the group consisting of ApagP and AmsbB (ApagP refers to a Salmonella enterica strain which has the pagP gene deleted and / or replaced with a different gene such as an antibiotic resistance gene).

[0067] In an embodiment, the Shigella GMMA of the invention are produced from Shigella strains that include one or more mutations resulting in inactivation of htrB, msbBl and / or msbB2. Inactivation of htrB, msbBl and / or msbB2 results in Shigella that produces GMMA comprising lipid A that is less toxic than wildtype lipid A. The Shigella sonnei and Shigella flexneri GMMA may be produced as described in WO 2023 / 025815. By way of non-limiting example, suitable Shigella strains may be selected from the group consisting of AhtrB, AmsbB 1 and AmsbB2 (AhtrB refers to a Shigella strain which has the htrB gene deleted). For simplicity, double deletions of both msbBl and msbB2 may also be referred to as ADmsbB. Inactivation of htrB or msbBl and msbB2 reduces acylation in lipid A. In the invention, inactivation of msbBl and / or msbB 2 is preferred.

[0068] In an embodiment, the Salmonella enterica and / or Shigella GMMA of the invention may comprise penta-acylated lipid A and / or hexa-acylated lipid A wherein the lauroyl - chain is replaced by a palmitoleoyl chain or wherein C14 comprises a myristoyl group. O-antigen

[0069] The OMVs and GMMA of the invention may be from Gram-negative bacteria that produce LPS comprising the O-antigen (OAg). The O-antigen is a polysaccharide moiety of the LPS. O-antigen is exposed on the outer surface of Gram-negative bacteria, and thus can be recognised by host antibodies. O-antigen can be quantified by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) analysis after acid hydrolysis of the sample.

[0070] For example, for Salmonella Enteritidis O-antigen quantification on bulk GMMA or final mixed drug product formulation, the quantification of OAg is performed on the basis of the known sugar ratios present in the OAg repeating unit IxRha (Rhamnose); IxGal (Galactose); IxMan (Mannose) IxTyv (Tyvelose) and the glucose is calculated from the glucose measured in the analysis after subtraction of the glucose due to the core.

[0071] For example, for Salmonella Typhimurium O-antigen quantification on bulk GMMA or final mixed drug product formulation, the quantification of OAg is performed on the basis of the known sugar ratios present in the OAg repeating unit IxRha (rhamnose); IxGal (galactose); lx Man (mannose); IxAbe (Abequose) and the glucose is calculated from the glucose measured in the analysis after subtraction of the glucose present in the core.

[0072] A standard dilution series of each sugar in the range of 0.5-10 pg / mL is run in each HPAEC-PAD analysis and the peak areas are used to interpolate the pg / mL of the corresponding sugar present in the sample OAg.

[0073] 450 pL each of the dilutions of the standard and sample are treated in parallel with

[0074] 150 pL of 8 M TFA for 4 hours at 100°C. Samples are then chilled at 2°C to 8°C for approximately 30 minutes, dried in a centrifugal evaporator, resuspended in 450 pL of water, filtered and analysed. HPAEC-PAD is performed with a Dionex ICS3000 (or 5000) equipped with a CarboPac PA10 column coupled with PA10 guard column. Separation is performed at 25°C using the following conditions:

[0075] • 20 min, 18 mM NaOH, flow rate 1 mL / min (separation step)

[0076] • 10 min 28 mM NaOH, Sodium acetate 100 mM, flow rate 1 mL / min (column washing step)

[0077] • 20 min 18 mM NaOH, flow rate 1 mL / min (column equilibration step) The effluent is monitored using an electrochemical detector.

[0078] Adjuvant

[0079] The adjuvant of the invention is or comprises an aluminium hydroxide adjuvant, i.e. any compound comprising Al3+and OH" ions (though other ions may also form part of the adjuvant of the invention). The aluminium hydroxide adjuvant may comprise or be an aluminium oxyhydroxide salt. The aluminium hydroxide adjuvant may comprise or be an aluminium oxyhydroxide salt that is at least partially crystalline. Aluminium oxyhydroxide salt, which can be represented by the formula AIO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide salt (A1(OH)3), by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070cm'1and a strong shoulder at 3090-3100cm'1(chapter 9 of ref. Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)). The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at 20 half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. Suitable examples of aluminium hydroxide adjuvants will be apparent to one of skill in the art, for example ALHYDROGEL®.

[0080] The PZC of the aluminium hydroxide adjuvant may be about 7, 8, 9, 10, or 11. The PZC of the aluminium hydroxide adjuvant of the invention may be greater than 7, greater than 8 or greater than 9, greater than 10, at most 8, at most 9, at most 10, or at most 11. The PZC can be measured by methods known to one of skill in the art, for example by salt acid-base titration methods.

[0081] The concentration of the aluminium hydroxide may be about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 mg Al3+1 ml. The concentration of the aluminium hydroxide may be at least 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, or 1.7 mg Al3+ / ml, and / or at most 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, or 1.8 mg Al3+ / ml.

[0082] Quenching / quencher

[0083] The quencher of the invention is a compound that quenches aluminium hydroxide as determined using the following quenching assay: a) obtain an assay immunogenic composition comprising S. Enteritidis GMMA adsorbed to the aluminium hydroxide adjuvant; b) partition the assay immunogenic composition into a first test aliquot and a second reference aliquot; c) incubate the reference aliquot for 2 hours, to provide an incubated reference aliquot, then measure average particle diameter in the incubated reference aliquot using static light scattering; d) add the compound at a concentration that is in excess compared to the concentration of aluminium hydroxide to the test aliquot and incubate the test aliquot comprising the quencher for two hours with stirring to provide an incubated test aliquot; e) measure the average particle diameter (as measured by either D90 and D4;3) in the incubated test aliquot by static light scattering and values; and f) compare the average particle diameter in the incubated test aliquot with the average particle diameter in the incubated reference aliquot, and if the average particle diameter in the incubated test aliquot is reduced by at least 10% (as measured by either D90 or D4;3), then the compound is a compound that quenches aluminium hydroxide.

[0084] The quenched aluminium hydroxide adjuvant of the invention refers to an aluminium hydroxide that has been mixed with a quencher under conditions suitable for quenching to occur. Optionally, the quenched aluminium hydroxide of the invention refers to an aluminium hydroxide to which OMVs have been pre-adsorbed that has subsequently been mixed with a quencher under conditions suitable for quenching to occur.

[0085] The OMVs (such as GMMA) and aluminium hydroxide of the immunogenic composition of the invention are preferably mixed with a quencher under conditions suitable for quenching to occur. Optionally, the OMVs (such as GMMA) are preadsorbed to the aluminium hydroxide before being mixed with a quencher under conditions suitable for quenching to occur.

[0086] “ Conditions suitable for quenching to occur” refer to conditions that allow the quencher to reduce the average particle diameter of the immunogenic composition comprising OMVs (such as GMMA) and aluminium hydroxide by at least 10% (as measured by either D90 or D4;3) using the quenching assay above. The conditions may comprise mixing the immunogenic composition comprising the aluminium hydroxide adjuvant with a quencher and stirring the mixture for at least 30 mins, for at least 1 hour, for at least 2 hours, at least 4 hours, at least 6 hours, between 1 hour and 100 hours, between 2 hours and 50 hours, or between 6 hours and 25 hours. Optionally, the OMVs (such as GMMA) are pre-adsorbed to the aluminium hydroxide adjuvant in the immunogenic composition before mixing the immunogenic composition with a quencher and stirring the mixture for at least 30 mins, for at least 1 hour, for at least 2 hours, at least 4 hours, at least 6 hours, between 1 hour and 100 hours, between 2 hours and 50 hours, or between 6 hours and 25 hours.

[0087] The quencher of the invention may comprise an anionic compound, in other words a compound that possesses a negatively charged ion. The anion of the anionic compound may be a stronger anion than the hydroxide anion of aluminium hydroxide. The anion of the anionic compound may be able to displace the hydroxide anion of aluminium hydroxide.

[0088] The quencher of the invention may comprise an anionic compound selected from the group consisting of phosphate ions, tricarboxylic acid ions and dicarboxylic ions. The tricarboxylic acid ions may be citrate ions. The dicarboxylic ions may be succinate ions.

[0089] Optionally, the quencher may comprise an anionic compound selected from the group consisting of phosphate ions, tricarboxylic acid ions (for example citrate ions) and dicarboxylic ions (for example succinate ions), and further comprise suitable counterions, for example sodium and / or potassium ions. Optionally, the quencher may comprise sodium phosphate and / or potassium phosphate.

[0090] The final concentration of the quencher in the immunogenic composition of the invention may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mM. The concentration may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mM. The concentration may be at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mM. The concentration may be between about 5 and 30mM, 6 and 28mM, 7 and 26mM, 8 and 24mM, 9 and 22mM, or 10 and 20mM. The concentration may be at least about 30, 40, 50, 60, 60, 80, 90, or 100 mM. The concentration may be at most about 50, 60, 70, 80, 90, 100 or 1 lOmM. The concentration may be between about 50 and 110, 60 and 110, 70 and 100, 80 and 100, or 90 and lOOmM. The final concentration of the quencher is the concentration of the quencher in the immunogenic composition of the invention once no more quencher is to be added.

[0091] The concentration of the quencher of the invention may be in excess of the concentration of the aluminium hydroxide. The concentration of the quencher of the invention may be low enough that the quencher does not cause significant desorption of the OMVs (such as GMMA) from the aluminium hydroxide adjuvant. Specifically, increasing concentrations of quencher can be tested to determine concentrations that do not cause significant desorption of OMVs or GMMA from the aluminium hydroxide whilst retaining a desired average particle diameter.

[0092] ‘M desired average particle diameter” is one that where the particles are not visible to any naked eye. The desired average particle diameter of the immunogenic compositions of the present invention may be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, and / or at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, compared to an equivalent composition lacking the quencher. The desired average particle diameter of the invention may be less than about 100pm, less than about 95pm, less than about 90pm, less than about 85pm, less than about 80pm, less than about 75pm, less than about 70pm, less than about 65pm, less than about 60pm, less than about 55pm, less than about 50pm, less than about 45pm, less than about 40pm, less than about 35pm, less than about 30pm, less than about 25pm, less than about 20pm, less than about 15pm, less than about 10pm, or less than about 5pm.

[0093] “ Average particle diameter” refers to the diameter of the particles in a composition as measured by static light scattering, for example by using a Beckman Coulter static light scattering machine. Average particle diameter can refer to either D90 or D4;3 values. D90 means that 90% of the total particles are smaller than the size reported. D4;3 is the volume-weighted mean particle diameter. “Reduced average particle diameter" refers to an average particle diameter in an immunogenic composition of the present invention that is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, and / or at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, compared to an equivalent composition lacking the quencher. Reduced average particle diameter may refer to reduced D90, D4;3 or both.

[0094] “Reducing particle aggregation''’ refers to reducing the average particle diameter in an immunogenic composition of the present invention by at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, and / or at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, compared to an equivalent composition lacking the quencher. Reducing particle aggregation may refer to reducing the D90, the D4;3 or both.

[0095] The average particle diameter of the invention may be such that the particles are not visible to any naked eye. The average particle diameter of the immunogenic compositions of the present invention may be at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, and / or at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, compared to an equivalent composition lacking the quencher. The average particle diameter of the invention may be less than about 100pm, less than about 90pm, less than about 80pm, less than about 70pm, less than about 60pm, less than about 50pm, less than about 40pm, less than about 35pm less than about 30pm, less than about 20pm, less than about 10pm, or less than about 5 pm.

[0096] Formulation of the immunogenic compositions

[0097] The immunogenic compositions of the invention may further comprise a pharmaceutically acceptable excipient. Typical "pharmaceutically acceptable excipients ’ include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable excipients may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, Tris-buffered physiologic saline is a suitable carrier particularly when using aluminium adjuvants since the phosphate in phosphate buffered saline may interfere with outer membrane vesicle binding to aluminium. However, in a particular embodiment, the immunogenic composition comprises phosphate buffered saline (and optionally an aluminium adjuvant as described further below). Optionally, the immunogenic composition comprises phosphate buffered saline at a pH between 6 and 7, for example pH 6.5.

[0098] Immunogenic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition). The immunogenic composition may be prepared for topical administration e.g. as an ointment, cream or powder. The immunogenic composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The immunogenic composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The immunogenic composition may be prepared for nasal, aural or ocular administration e.g. as drops. The immunogenic composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a mammal. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens. Immunogenic compositions may be presented in vials, or they may be presented in pre-fdled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses.

[0099] Immunogenic compositions of the invention may be packaged in unit dose form or in multiple dose form. For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition has a volume of 0.5ml e.g. for intramuscular injection.

[0100] The pH of the immunogenic composition is preferably between 6 and 8, for example, 6, 6.5, 7, 7.5 or 8. For compositions comprising acetylated O-antigens particularly the pH of the composition is less than 7, preferably about 6 (to slow the rate of deesterification). Stable pH may be maintained by the use of a buffer. The immunogenic compositions of the invention may comprise a Tris [Tris(hydroxymethyl)aminomethane] buffer. The Tris buffer may comprise about 1- 20mM [Tris(hydroxymethyl)aminomethane], e.g. 1.25 mM, 2.5 mM, 5.0 mM or 10.0 mM. For immunogenic compositions comprising acetylated O-antigens, in particular, the buffer is not a Tris buffer. The immunogenic compositions of the invention may comprise a 5-20mM succinate buffer, e.g. 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM or 20 mM. The immunogenic compositions of the invention may comprise a 5-20mM histidine buffer, e.g. 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM or 20 mM. The immunogenic compositions of the invention preferably comprise a 5- 20mM sodium phosphate buffer e.g. 5 mM, 7.5 mM, 10 mM, 12.5 mM, 15 mM, 17.5 mM or 20 mM. The composition will be sterile. Immunogenic compositions of the invention may be isotonic with respect to humans.

[0101] Thus, immunogenic compositions of the invention may be useful as vaccines. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s), as well as any other components, as needed. By

[0102] “ immunologically effective amount”, it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individuals to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

[0103] Immunogenic compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose formats.

[0104] Medical uses and methods of treatment

[0105] In a further aspect of the invention, there is provided an immunogenic composition of the invention for use in a method of preventing an infection. In a further aspect of the invention, there is provided a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of the invention to a subject. In a further aspect of the invention, there is provided a use of the immunogenic composition or vaccine of the invention for the manufacture of a medicament for use in a method of preventing an infection. The method of preventing an infection may comprise administering an effective amount of the immunogenic composition or vaccine of the invention to a subject.

[0106] The method of preventing an infection may be a method of preventing infection by any species in any of genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia, Haemophilus, Klebsiella, Legionella, Porphyromonas, Pseudomonas, Yersinia, Helicobacter, Salmonella, or Vibrio. For example, the method of preventing an infection may be a method of preventing infection by Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis, Moraxella catarrhalis, Escherichia coli, Haemophilus influenzae (including non- typeable stains), Klebsiella pneumoniae, Legionella pneumophila, Neisseria gonorrhoeae, Neisseria meningitidis, Neisseria lactamica, Porphyromonas gingivalis, Pseudomonas aeruginosa, Yersinia enterocolitica, Helicobacter pylori, Salmonella enterica (including serovars Typhi and Typhimurium, as well as serovars Paratyphi and Enteritidis), Shigella (such as S. dysenteriae, S.flexneri, S. boydii or S. sonnei), Vibrio cholerae, etc.

[0107] The method of preventing an infection may be a method of preventing Salmonella infection. Optionally the method of preventing an infection is a method of preventing infection by Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Typhi, and / or Salmonella Paratyphi A.

[0108] The method of preventing an infection may be a method of preventing Shigella infection. Optionally the method of preventing an infection is a method of preventing infection by Shigella sonnei, or Shigella flexneri serotype la, lb, 2a, 2b, 3a, 3b, 4a, 5b, 6 and / or X.

[0109] The method of preventing an infection may be a method of preventing E. coli infection. Optionally the method of preventing an infection is a method of preventing infection by Enterotoxigenic Escherichia coli (ETEC).

[0110] The term “preventing an infection"’ in the method / immunogenic composition for use / use of the immunogenic composition in the manufacture of a medicament of the invention comprises raising an immune response in a subject. The immune response may be protective and may raises antibodies, such as IgG antibodies. The subject of the invention is a mammal, optionally a human. Where the vaccine is for prophylactic use, the human may be an adult i.e. subject is 18 years old or above 18 years old. Where the vaccine is for prophylactic use, the human may be a child i.e. below 18 years old. Where the vaccine is for prophylactic use, the child may be between 12 to 72 months, preferably between 24 to 59 months, more preferably between 6 to 12 months.

[0111] Where the vaccine is for prophylactic use, the child may be around 9 months. Where the vaccine is for therapeutic use, the human is preferably a child.

[0112] A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, or immunogenicity. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, or immunogenicity.

[0113] Examples

[0114] Example 1 - Quenching aluminium hydroxide with phosphate ions prevents aggregation formation

[0115] GMMA production

[0116] Salmonella enterica serovar Typhimurium wild-type (WT) strain 2192 was provided by the Salmonella Genetic Stock Center (SGSC) at the University of Calgary, Canada, which belongs to the global Salmonella reference collection A (SARA 12).

[0117] Salmonella enterica serovar Enteritidis WT strain 618 was provided by Quotient Bioresearch Limited, UK. The strain of animal origin was isolated by the European Antimicrobial Susceptibility Surveillance in Animals (EASSA).

[0118] From the Salmonella strains above, Salmonella Typhimurium Ato / RApagP MnsbB and Salmonella Enteritidis Ato / RApagP MnsbB recombinant mutants for each strain were generated as previously reported (Rossi O, Caboni M, Negrea A, Necchi F, Alfini R, Micoli F, et al. Toll-Like Receptor Activation by Generalized Modules for Membrane Antigens from Lipid A Mutants of Salmonella enterica Serovars Typhimurium and Enteritidis. Clin Vaccine Immunol. 2016;23(4):304-14.)

[0119] GMMA derived from the Salmonella strains above (Salmonella Typhimurium GMMA (STmGMMA) and Salmonella Enteritidis GMMA (SEnGMMA)) were purified and isolated. GMMA were purified using similar methods previously reported for S. sonnei GMMA (Gerke C, Colucci AM, Giannelli C, Sanzone S, Vitali CG, Sollai L, et al. Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB. PLoS One. 2015;10(8):e0134478. doi:

[0120] 10.1371 / journal.pone.0134478 [ doi] ;PONE-D-15-08654). Briefly, GMMA released into the fermentation broth were purified using two consecutive Tangential Flow Filtration (TFF) steps: a microfiltration in which the culture supernatant containing the GMMA is separated from the bacteria, and an ultrafiltration, in which the GMMA are separated from soluble proteins and nucleic acids.

[0121] Adsorption of GMMA to aluminium hydroxide

[0122] STmGMMA and SEnGMMA drug product formulations were prepared at IL scale starting from a sterile filtered (using a Sartobran Pl 50) suspension of either SEnGMMA or STmGMMA at a concentration of >1000 pg / mL OAg in 154 mM NaCl. This solution was added to aluminium hydroxide (ALHYDROGEL®, 2% Brenntag, wet gel suspension) in water to the final OAg concentration of 80 pg / mL and stirred (90-180 rpm) for 2 hours at room temperature.

[0123] The particle size was then analysed by static light scattering (Beckman Coulter).

[0124] Quenching

[0125] To quench the aluminium hydroxide, sodium phosphate (pH 6.5) was added to the composition to have a final phosphate concentration of 20mM and the resulting composition was stirred for 2 hours using a magnetic stirrer at 180 rpm. The particle size was then analysed by static light scattering (Beckman Coulter) again.

[0126] The final osmolarity was adjusted with 1.5 M NaCl to a final concentration of 154 mM NaCl, the formulation was mixed for a further 1 hour and aliquoted at 0.7 mL in 3 mL glass vials.

[0127] Quenching the aluminium hydroxide with phosphate ions leads to lower particle size as shown in Figure 1. Example 2 - Quenching aluminium hydroxide using different concentrations of phosphate

[0128] The experiment in Example 1 was repeated. The experiment in Example 1 was then carried out to take the final phosphate concentration to lOmM (instead of 20mM). The particle sizes for the 20mM and lOmM concentrations were compared (Figure 2).

[0129] Figure 2 shows that 20mM sodium phosphate concentration leads to better quenching than lOmM.

[0130] Example 3 - Quenching aluminium hydroxide using other ions

[0131] The adsorption step in Example 1 was carried out again using STmGMMA. The quenching step in Example 1 was then carried out in the same way but instead of adding phosphate ions, lOmM citrate or succinate ions were added.

[0132] Aluminium hydroxide was shown to be quenched using citrate and succinate ions, leading to lower particle size.

[0133] Example 4 - Aggregation is prevented for other types of GMMA

[0134] After the results obtained on STmGMMA and SEnGMMA, the formulation as in Example 1 was developed again but this time using Shigella GMMA instead of STmGMMA and SEnGMMA. Specifically, Shigella sonnei and Shigella flexneri lb, 2a and 3a GMMA were used, each strain of which was made up to a final OAg concentration of 60 pg / mL. The Shigella sonnei and Shigella flexneri GMMA were produced, purified and isolated according to WO 2016 / 202872.

[0135] Shigella GMMA was also shown to be quenched using succinate ions, leading to lower particle size. Embodiments of the Invention

[0136] 1. An immunogenic composition comprising outer membrane vesicles (OMVs) adsorbed to a quenched aluminium hydroxide adjuvant.

[0137] 2. An immunogenic composition comprising OMVs adsorbed to an aluminium hydroxide adjuvant and further comprising a quencher.

[0138] 3. A method for reducing particle aggregation, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition.

[0139] 4. A method of producing an immunogenic composition with reduced average particle diameter, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition.

[0140] 5. The immunogenic composition of embodiment 1 or 2, or the method of embodiment 3 or 4, wherein the OMVs are generalised modules for membrane antigens (GMMA), native OMVs, microvesicles, detergent-extracted OMVs, mutant-derived OMVs, or blebs.

[0141] 6. The immunogenic composition or method of any one of the preceding embodiments, wherein the OMVs and / or the GMMA are obtained from species in any of genera Escherichia, Shigella, Neisseria, Moraxella, Bordetella, Borrelia, Brucella, Chlamydia, Haemophilus, Klebsiella, Legionella, Porphyromonas, Pseudomonas, Yersinia, Helicobacter, Salmonella, or Vibrio. 7. The immunogenic composition or method of any one of the preceding embodiments, wherein the OMVs and / or the GMMA are obtained from Shigella and / or Salmonella bacteria.

[0142] 8. The immunogenic composition or method of embodiment 6 or 7, wherein the OMVs and / or the GMMA are obtained from at least one of the group consisting of: Shigella sonnei, Shigella flexneri lb, Shigella flexneri 2a and Shigella flexneri 3a.

[0143] 9. The immunogenic composition or method of embodiment 6, 7 or 8, wherein the OMVs and / or GMMA are obtained from at least one of Salmonella Typhimurium and / or Salmonella Enteritidis.

[0144] 10. The immunogenic composition of any one of embodiments 1 or 5 to 9, wherein a quenched aluminium hydroxide adjuvant is an aluminium hydroxide adjuvant that has been mixed with a quencher under conditions suitable for quenching to occur.

[0145] 11. The immunogenic composition of embodiment 10, wherein mixing the aluminium hydroxide adjuvant with a quencher under conditions suitable for quenching to occur comprises mixing the immunogenic composition comprising the aluminium hydroxide adjuvant with a quencher and stirring the mixture for at least 30 mins, for at least 1 hour, for at least 2 hours, at least 4 hours, at least 6 hours, between 1 hour and 100 hours, between 2 hours and 50 hours, or between 6 hours and 25 hours.

[0146] 12. The immunogenic composition of embodiment 10 or 11, wherein the aluminium hydroxide adjuvant is mixed with a quencher under conditions suitable for quenching to occur after adsorption of OMVs to the aluminium hydroxide.

[0147] 13. The immunogenic composition of embodiment 12, wherein mixing the aluminium hydroxide adjuvant with a quencher under conditions suitable for quenching to occur comprises mixing the immunogenic composition comprising the aluminium hydroxide adjuvant to which OMVs have been pre-adsorbed with a quencher and stirring the mixture for at least 30 mins, for at least 1 hour, for at least 2 hours, at least 4 hours, at least 6 hours, between 1 hour and 100 hours, between 2 hours and 50 hours, or between 6 hours and 25 hours.

[0148] 14. The immunogenic composition of embodiment 11, wherein the mixture is stirred for at most 3 hours, at most 4 hours, at most 5 hours, at most 6 hours, at most 7 hours, or at most 8 hours.

[0149] 15. The immunogenic composition or method of any one of embodiments 2 to 14, wherein, a quencher is a compound that quenches aluminium hydroxide as determined using the following quenching assay: a) obtain an assay immunogenic composition comprising S. Enteritidis GMMA adsorbed to the aluminium hydroxide adjuvant; b) partition the assay immunogenic composition into a first test aliquot and a second reference aliquot; c) incubate the reference aliquot for 2 hours, to provide an incubated reference aliquot, then measure the average particle diameter in the incubated reference aliquot using static light scattering; d) add the compound at a concentration that is in excess compared to the concentration of aluminium hydroxide to the test aliquot and incubate the test aliquot comprising the quencher for two hours with stirring to provide an incubated test aliquot; e) measure the average particle diameter (D90 and D4;3) in the incubated test aliquot by static light scattering and values; and f) compare the average particle diameter in the incubated test aliquot with the average particle diameter in the incubated reference aliquot, and if the average particle diameter in the incubated test aliquot is reduced by at least 10% (for either D90 or D4;3), then the compound is a compound that quenches aluminium hydroxide. 16. The immunogenic composition or method of any one of embodiments 2 to 15, wherein the quencher comprises an anionic compound.

[0150] 17. The immunogenic composition or method of any one of embodiments 1 to 16, wherein the aluminium hydroxide has a PZC that is greater than 7, greater than 8, or greater than 9, at most 8, at most 9 or at most 10.

[0151] 18. The immunogenic composition or method of embodiment 16 or 17, wherein the anionic compound is an anionic compound that is stronger anion than the hydroxide anion.

[0152] 19. The immunogenic composition or method of any one of embodiments 16 to 18, wherein the anionic compound is able to displace the hydroxide anion of the aluminium hydroxide.

[0153] 20. The immunogenic composition or method of any one of embodiments 2 to 19, wherein the quencher comprises an anionic compound selected from the group consisting of phosphate ions, tricarboxylic acid ions and dicarboxylic ions.

[0154] 21. The immunogenic composition or method of any one of embodiments 2 to 20, wherein the quencher comprises phosphate ions.

[0155] 22. The immunogenic composition or method of embodiment 20, wherein the tricarboxylic acid ions are citrate ions.

[0156] 23. The immunogenic composition or method of embodiment 20, wherein the dicarboxylic acid ions are succinate ions. 24. The immunogenic composition or method of embodiment 21, wherein the final concentration of phosphate ions is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mM.

[0157] 25. The immunogenic composition or method of embodiment 21, wherein the final concentration of phosphate ions is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 mM.

[0158] 26. The immunogenic composition or method of embodiment 21 or 15, wherein the final concentration of phosphate ions is at most 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 60, 70, 80, 90, 100 or 110 mM.

[0159] 27. The immunogenic composition or method of any one of embodiments 21 or 24 to 26, wherein the final concentration of phosphate ions is between 5 and 30mM.

[0160] 28. The immunogenic composition or method of any one of embodiments 21 or 24 to 27, wherein the final concentration of phosphate ions is between 10 and 20mM, 50 and 1 lOmM, 60 and 110 mM, 70 and 100 mM, 80 and 100 mM, or 90 and lOOmM.

[0161] 29. The immunogenic composition or method of any one of the preceding embodiments, wherein the average particle diameter in the immunogenic composition is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, and / or at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, compared to an equivalent immunogenic composition lacking the quencher. 30. The immunogenic composition or method of any one of the preceding embodiments, wherein the particle diameter (or average particle diameter) in the immunogenic composition is less than 100, 90, 80, 70, 60, or 50 pm, or less than 45pm.

[0162] 31. The immunogenic composition or method of any one of the preceding embodiments, wherein the immunogenic composition does not comprise visible particles.

[0163] 32. The immunogenic composition or method of any one of embodiments 5 to 31, wherein the GMMA are purified from Gram-negative bacteria that have been modified to hyperbleb.

[0164] 33. The immunogenic composition or method of any one of embodiments 5 to 32, wherein the GMMA are purified from Gram-negative bacteria that do not comprise a gene encoding a functional TolR protein.

[0165] 34. The immunogenic composition or method of any one of embodiments 5 to 33, wherein the GMMA are purified from Gram-negative bacteria that have a AtolR mutation.

[0166] 35. The immunogenic composition or method of any one of embodiments 5 to 34, wherein the GMMA comprise modified lipid A.

[0167] 36. The immunogenic composition or method of any one of embodiments 5 to 35, wherein the GMMA comprise lipid A that has been modified to be less toxic than wildtype lipid A, optionally wherein the lipid A is penta-acylated.

[0168] 37. The immunogenic composition or method of embodiment 36, wherein the GMMA comprise lipid A that is penta-acylated and / or hexa-acylated. 38. The immunogenic composition or method of any one of embodiments 5 to 37, wherein the w / w ratio between protein and O-antigen displayed by each GMMA is at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0,

[0169] 2.1, 2.2, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0, or at most 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1,

[0170] 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0.

[0171] 39. The immunogenic composition or method of any one of embodiments 5 to 38, wherein the GMMA comprise purified Salmonella Typhimurium GMMA and / or purified Salmonella Enteritidis GMMA.

[0172] 40. The immunogenic composition or method of embodiment 39, wherein the purified Salmonella Typhimurium GMMA are purified from Salmonella Typhimurium with a AtolR mutation and / or the purified Salmonella Enteritidis GMMA are purified from Salmonella Enteritidis with a AtolR mutation.

[0173] 41. The immunogenic composition or method of embodiment 39 or 40, wherein the purified Salmonella Typhimurium GMMA are purified from Salmonella Typhimurium with a AmsbB mutation and / or the purified Salmonella Enteritidis GMMA are purified from Salmonella Enteritidis with a AmsbB mutation.

[0174] 42. The immunogenic composition or method of any one of embodiments 39 to 41, wherein the purified Salmonella Typhimurium GMMA are purified from Salmonella Typhimurium with a APagP mutation and / or the purified Salmonella Enteritidis GMMA are purified from Salmonella Enteritidis with a APagP mutation.

[0175] 43. The immunogenic composition or method of any one of embodiments 5 to 42, wherein the GMMA comprises purified Shigella sonnei GMMA and / or purified Shigella flexneri GMMA. 44. The immunogenic composition or method of embodiment 43, wherein the purified Shigella flexneri GMMA are purified from at least one strain selected from the group consisting of Shigella flexneri lb, 2a and 3a.

[0176] 45. The immunogenic composition or method of embodiment 43 or 44, wherein the purified Shigella sonnei GMMA are purified from Shigella sonnei with a AtolR mutation and / or the purified Shigella flexneri GMMA are purified from Shigella flexneri with a AtolR mutation.

[0177] 46. The immunogenic composition or method of any one of embodiments 43 to 45, wherein the purified Shigella sonnei GMMA are purified from Shigella sonnei with a AmsbB mutation and / or the purified Shigella flexneri GMMA are purified from Shigella flexneri with a AmsbB mutation.

[0178] 47. The immunogenic composition or method of any one of embodiments 43 to 46, wherein the AmsbB mutation is a AmsbB 1 and / or AmsbB2 mutation.

[0179] 48. The method of any one of embodiments 3 to 47, wherein the step of adding a quencher is followed by stirring for about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0180] 49. The method of any one of embodiments 3 to 48, wherein the step of adding a quencher is followed by stirring for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours, and / or at most 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, or 24 hours.

[0181] 50. The method of any one of embodiments 3 to 49, wherein obtaining OMVs from at least one Gram-negative bacterium comprises growing a Gram-negative bacterium and isolating the OMVs. 51. The method of embodiment 50, wherein isolating the OMVs comprises a step of ultra-filtration and a step of dia-filtration.

[0182] 52. The method of any one of embodiments 3 to 51 , wherein the method further comprises adding one or more additional antigens to the immunogenic composition.

[0183] 53. An immunogenic composition obtainable from the method of any one of embodiments 3 to 52.

[0184] 54. An immunogenic composition obtained from the method of any one of embodiments 3 to 53.

[0185] 55. The immunogenic composition of any one of embodiments 1, 2, 5 to 47, 53 or 54 for use in a method of preventing a disease.

[0186] 56. A method of preventing a disease comprising administering an effective amount of the immunogenic composition of any one of embodiments 1, 2, 5 to 47, 43 or 54 to a subject.

[0187] 57. Use of the immunogenic composition of any one of embodiments 1, 2, 5 to 47, 53 or 54 in the manufacture of a medicament for use in a method of preventing a disease.

[0188] 58. The immunogenic composition for use of embodiment 55 or the use of embodiment 57, wherein the method of preventing a disease comprises administering an effective amount of the immunogenic composition to a subject.

[0189] 59. The immunogenic composition for use, method of treatment, or use of any one of embodiments 55 to 58, wherein the method of preventing a disease is a method of preventing a disease caused by Salmonella or Shigella. 60. The immunogenic composition for use, method of treatment, or use of any one of embodiments 55 to 59, wherein the method of preventing a disease is a method of preventing a disease caused by Salmonella Enteritidis, Salmonella Typhimurium, Shigella flexneri and / or Shigella sonnei.

Claims

Claims1. An immunogenic composition comprising outer membrane vesicles (OMVs) adsorbed to a quenched aluminium hydroxide adjuvant.

2. An immunogenic composition comprising OMVs adsorbed to an aluminium hydroxide adjuvant and further comprising a quencher.

3. A method for reducing particle aggregation, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition.

4. A method of producing an immunogenic composition with reduced average particle diameter, the method comprising: a) obtaining OMVs; b) adsorbing the OMVs onto aluminium hydroxide; and c) adding a quencher to the OMV and aluminium hydroxide composition.

5. The immunogenic composition of claim 1 or 2, or the method of claim 3 or 4, wherein the OMVs are generalised modules for membrane antigens (GMMA).

6. The immunogenic composition or method of any one of the preceding claims, wherein the OMVs and / or the GMMA are obtained from Shigella and / or Salmonella bacteria.

7. The immunogenic composition or method of claim 6, wherein the OMVs and / or the GMMA are obtained from at least one of the group consisting of: Shigella sonnei, Shigella flexneri lb, Shigella flexneri 2a and Shigella flexneri 3a.

8. The immunogenic composition or method of claim 6 or 7, wherein the OMVs and / or GMMA are obtained from at least one of Salmonella Typhimurium and / or Salmonella Enteritidis.

9. The immunogenic composition of any one of claims 1 or 5 to 8, wherein a quenched aluminium hydroxide adjuvant is an aluminium hydroxide adjuvant that has been mixed with a quencher under conditions suitable for quenching to occur.

10. The immunogenic composition or method of any one of claims 2 to 9, wherein the quencher comprises an anionic compound.

11. The immunogenic composition or method of claim 10, wherein the anionic compound is an anionic compound that is stronger anion than the hydroxide anion.

12. The immunogenic composition or method of any one of claims 2 to 11, wherein the quencher comprises an anionic compound selected from the group consisting of phosphate ions, tricarboxylic acid ions and dicarboxylic ions.

13. The immunogenic composition or method of any one of claims 2 to 12, wherein the quencher comprises phosphate ions.

14. The immunogenic composition or method of claim 13, wherein the final concentration of phosphate ions is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mM.

15. The immunogenic composition or method of claim 13 or 14, wherein the final concentration of phosphate ions is between 10 and 20mM.

16. The immunogenic composition or method of any one of the preceding claims, wherein the average particle diameter in the immunogenic composition is at least 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, and / or at most 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 or 95% smaller, compared to an equivalent immunogenic composition lacking the quencher.

17. The immunogenic composition or method of any one of the preceding claims, wherein the particle diameter (or average particle diameter) in the immunogenic composition is less than 100, 90, 80, 70, 60, or 50 pm, or less than 45pm.

18. The immunogenic composition or method of any one of the preceding claims, wherein the immunogenic composition does not comprise visible particles.

19. The immunogenic composition or method of any one of claims 5 to 18, wherein the GMMA are purified from Gram-negative bacteria that have been modified to hyperbleb.

20. The immunogenic composition or method of any one of claims 5 to 19, wherein the GMMA are purified from Gram-negative bacteria that do not comprise a gene encoding a functional TolR protein.

21. The immunogenic composition or method of any one of claims 5 to 20, wherein the GMMA are purified from Gram-negative bacteria that have a AtolR mutation.

22. The immunogenic composition or method of any one of claims 5 to 21, wherein the GMMA comprise lipid A that has been modified to be less toxic than wildtype lipid A, optionally wherein the lipid A is penta-acylated.

23. The immunogenic composition of any one of claims 1, 2, or 5 to 22 for use in a method of preventing a disease.

24. The immunogenic composition for use of claim 23, wherein the method of preventing a disease is a method of preventing a disease caused by Salmonella or Shigella.

25. The immunogenic composition for use of claims 23 or 24, wherein the method of preventing a disease is a method of preventing a disease caused by Salmonella Enteritidis, Salmonella Typhimurium, Shigella flexneri and / or Shigella sonnei.

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