Immunogenic components

Abstract

The present invention relates to an immunogenic composition containing outer membrane vesicles (OMVs) of Bacillus paratyphi A, a vaccine containing the immunogenic composition, and a method and use of the immunogenic composition. The present invention also relates to Bacillus paratyphi A containing modified lipid A.

Description

[Technical Field]

[0001] The present invention relates to an immunogenic composition comprising outer membrane vesicles (OMVs) of *S. enterica serovar Paratyphi A*, a vaccine comprising the immunogenic composition, and methods and uses of the immunogenic composition. The present invention also relates to *S. Paratyphi A* bacterium comprising modified lipid A. [Background technology]

[0002] S. paratyphi A is a commensal bacterium in the human gut, and its clinical findings are indistinguishable from those of typhoid fever. S. paratyphi A is the second leading cause of typhoid fever, preceded only by Salmonella enterica serotype tiphi (S. thphi). Typhoid fever caused by S. paratyphi A, or paratyphoid fever itself, was thought to account for a relatively small proportion of typhoid cases. However, since the 1980s, the incidence and relative frequency of paratyphoid fever have increased in Nepal, Pakistan, and Thailand. Furthermore, populous countries such as India and China have reported a considerable number of S. paratyphi A cases. Non-endemic countries like the United States have reported an increasing trend in paratyphoid fever, particularly among travelers from South Asia (Irfan et al., Ceftriaxone resistant Salmonella enterica serovar Paratyphi A identified in a case of enteric fever: first case report from Pakistan. BMC Infect Dis. 2023 Apr 26;23(1):267. doi: 10.1186 / s12879-023-08152-9. Erratum in: BMC Infect Dis. 2023 May 23;23(1):346. PMID: 37101111; PMCID: PMC10132421).

[0003] Therefore, an effective vaccine is needed to protect against paratyphi A bacteria.

Summary of the Invention

[0004] This example demonstrates that an immunogenic composition containing Salmonella paratyphi A outer membrane vesicles (OMV) exhibits high immunogenicity.

[0005] In a first aspect of the present invention, an immunogenic composition containing Salmonella paratyphi A membrane vesicles is provided.

[0006] In a second aspect of the present invention, Salmonella paratyphi A bacteria containing modified lipid A are provided.

[0007] In a third aspect of the present invention, outer membrane vesicles or GMMA obtainable from the Salmonella paratyphi A bacteria of the present invention are provided.

[0008] In a fourth aspect of the present invention, outer membrane vesicles or GMMA obtained from the Salmonella paratyphi A bacteria of the present invention are provided.

[0009] In a fifth aspect of the present invention, an immunogenic composition containing the outer membrane vesicles or GMMA of the present invention is provided.

[0010] In a sixth aspect of the present invention, a vaccine containing the immunogenic composition of the present invention is provided.

[0011] In a seventh aspect of the present invention, the immunogenic composition or vaccine of the present invention for use in a method of preventing infection is provided.

[0012] In an eighth aspect of the present invention, a method of preventing infection is provided, which includes administering an effective amount of the immunogenic composition or vaccine of the present invention.

[0013] In a ninth aspect of the present invention, the present invention provides the use of the immunogenic composition or vaccine of the present invention for the manufacture of a medicament for use in a method of preventing infection.

Brief Description of the Drawings

[0014] [Figure 1-1]Figure 1 illustrates that the Pan-Salmonella vaccine induces a specific serum IgG response against Salmonella paratyphi A OAg and Salmonella typhi (S. Typhi) Vi, and that the antibodies exhibit bactericidal activity in mice. Figures 1(a) and (c) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figure 1(b) shows the SBA results. The left bar represents one day before immunization, and the right bar represents 42 days after immunization. [Figure 1-2] Figure 1 illustrates that the Pan-Salmonella vaccine induces a specific serum IgG response against Salmonella paratyphi A OAg and Salmonella typhi (S. Typhi) Vi, and that the antibodies exhibit bactericidal activity in mice. Figures 1(a) and (c) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figure 1(b) shows the SBA results. The left bar represents one day before immunization, and the right bar represents 42 days after immunization. [Figure 2-1] Figure 2 shows the evaluation of immune interference between vaccine components in Pan-Salmonella formulations. Figures 2(a), (c), (e), and (g) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figures 2(b), (d), and (f) show the SBA results. In Figure 2(b), the left bar represents one day before immunization, and the right bar represents 42 days after immunization. In Figures 2(d) and (f), each bar represents 42 days after immunization. [Figure 2-2]Figure 2 shows the evaluation of immune interference between vaccine components in Pan-Salmonella formulations. Figures 2(a), (c), (e), and (g) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figures 2(b), (d), and (f) show the SBA results. In Figure 2(b), the left bar represents one day before immunization, and the right bar represents 42 days after immunization. In Figures 2(d) and (f), each bar represents 42 days after immunization. [Figure 2-3] Figure 2 shows the evaluation of immune interference between vaccine components in Pan-Salmonella formulations. Figures 2(a), (c), (e), and (g) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figures 2(b), (d), and (f) show the SBA results. In Figure 2(b), the left bar represents one day before immunization, and the right bar represents 42 days after immunization. In Figures 2(d) and (f), each bar represents 42 days after immunization. [Figure 2-4] Figure 2 shows the evaluation of immune interference between vaccine components in Pan-Salmonella formulations. Figures 2(a), (c), (e), and (g) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figures 2(b), (d), and (f) show the SBA results. In Figure 2(b), the left bar represents one day before immunization, and the right bar represents 42 days after immunization. In Figures 2(d) and (f), each bar represents 42 days after immunization. [Figure 3] Figure 3 shows the relative abundance percentage of each subclass, calculated as subclass / total subclass percentage. The top segment is IgG3, the next is IgG2b, the following is IgG2a, and the bottom segment is IgG1. [Figure 4-1]Figure 4 illustrates that the quadrivalent Pan-Salmonella vaccine induces a specific serum IgG response against Salmonella paratyphi A OAg and Salmonella typhi Vi, and that the antibodies exhibit bactericidal activity in rabbits. Figures 4(a) and (c) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figure 4(b) shows the SBA results, with the left bar representing one day before immunization and the right bar representing 42 days after immunization. [Figure 4-2] Figure 4 illustrates that the quadrivalent Pan-Salmonella vaccine induces a specific serum IgG response against Salmonella paratyphi A OAg and Salmonella typhi Vi, and that the antibodies exhibit bactericidal activity in rabbits. Figures 4(a) and (c) show the IgG response one day before immunization (left bar), 27 days after immunization (center bar), and 42 days after immunization (right bar). Figure 4(b) shows the SBA results, with the left bar representing one day before immunization and the right bar representing 42 days after immunization. [Figure 5] Figure 5 shows that the quadrivalent Pan-Salmonella vaccine induces bactericidal antibodies against a broad panel of Salmonella strains. The panel includes invasive STm isolates from Africa and Southeast Asia, as well as Salmonella (S. enterica) serotypes other than STm, SEn, ParA, and Typhi. [Figure 6] Figure 6 shows the published structure (including the core region) of the O-antigen from *Salvelinus paratyphi* A. [Figure 7] Figure 7 shows the sequence list. [Modes for carrying out the invention]

[0015] General definition Unless otherwise defined, the technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this invention pertains.

[0016] In general, the term "comprising" means that it is not limited to including. For example, the expression "an immunogenic composition comprising paratyphi a outer membrane vesicles" should be interpreted as meaning that the immunogenic composition contains paratyphi a outer membrane vesicles, but the immunogenic composition may contain further components.

[0017] In some embodiments of the present invention, the word “comprising” is replaced with the expression “consisting of.” This term “consisting of” is intended to be restrictive. For example, the expression “an immunogenic composition comprising paratyphi a outer membrane vesicles” should be understood to mean that the immunogenic composition has paratyphi a outer membrane vesicles and no further components.

[0018] In some embodiments of the present invention, the word “comprising” is replaced by the phrase “consisting essentially of.” The term “consisting essentially of” means that there may be certain further components, i.e., those that do not substantially affect the essential characteristics of the subject.

[0019] The terms "about" or "around" refer to a value that falls within a reasonable range of scientific error. A value may be "about x" or "approximately x" if it is within 10%, 5%, or 1% of x.

[0020] The singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. Therefore, for example, a reference to "the GMMA" includes two or more examples or versions of such GMMAs.

[0021] All publications, patents, and patent applications cited herein, whether listed above or below, are incorporated herein by reference in their entirety.

[0022] Detailed explanation O-antigen All Bacteria paratyphi A contain an outer membrane containing the O-antigen. The Bacteria paratyphi A OMV (e.g., GMMA) in the immunogenic composition of the present invention contains the O-antigen.

[0023] For the purposes of this invention, the terms O-antigen, O-Ag, and O:2 are considered interchangeable.

[0024] The outer membrane of Gram-negative bacteria contains lipopolysaccharide. This lipopolysaccharide contains an O-antigen linked to the lipid A domain via a core domain. The terms "O-antigen," "O-Ag," and "O:2" refer to a polysaccharide composed solely of the O-antigen, or more preferably, an O-antigen linked to the core domain of a lipopolysaccharide.

[0025] The O-antigens of Salmonella serogroups A, B, and D have been described and are thought to share a common backbone: →2-α-D-Manp-(1→4)-α-L-Rhap-(1→3)-α-D-Galp-(1→). The serogroup specificity of Salmonella paratyphi A is conferred by α-3,6-dideoxyglucose (α-D-paratose) linked (1→3) to the mannose in the backbone. The α-L-rhamnose in the backbone is partially O-acetylated at C-3 (Konadu et al. (1996) Infect Immun. (7):2709-15). α-D-paratose has also been reported to have various degrees of O-acetylation. The published structure of the O-antigen derived from Salmonella paratyphi A, which contains a KDO subunit and a primary amine group (within a pyrophosphoethanolamine group) in the core domain, is shown in Figure 6.

[0026] Outer membrane vesicles The immunogenic composition of the present invention contains outer membrane vesicles (e.g., GMMA) of Salmonella enterica subspecies enterica serotype paratyphi A (Bacillus paratyphi A).

[0027] For the purposes of this invention, the terms “outer membrane vesicles” or “OMV” are considered interchangeable herein and refer to any type of outer membrane vesicle. Preferred OMVs include natural OMVs. Gram-negative bacteria can spontaneously release outer membrane vesicles (OMVs) during growth due to turgor pressure of the cell cortex, and these are natural OMVs. OMVs are rich in immunogenic periplasmic and secretory antigens attached to the cell surface and are used as vaccines.

[0028] The OMVs of the present invention include a generalized module for membrane antigens (GMMA), natural OMVs ("NOMV" (see Katial et al., 2002, Infect Immun, 70: 702-707), microvesicles (MV (see WO 02 / 09643)), surfactant-extracted OMVs (DOMV), mutant-derived OMVs (m-OMV), and blebs, which are outer membrane projections that remain bound to bacteria before release as MVs (see Beveridge, 1999, J. Bacteriol. 181: 4725-4733)).

[0029] A generalized module for membrane antigens (GMMA) is a type of OMV. GMMAs differ from natural outer membrane vesicles (NOMVs) spontaneously released from Gram-negative bacteria in two crucial aspects. First, to induce GMMA formation, the membrane structure is modified by the deletion of genes encoding key structural components such as tolR (resulting in hyperblebbing). Second, as a result of the genetic modification, a large amount of outer membrane "budding" (or "hyperblebbing") provides a practical source of membrane material for vaccine production. Therefore, for the purposes of this invention, the term "GMMA" refers to an OMV spontaneously released from bacteria modified to hyperblebify (such as *Bacteria paratyphia* modified to lack the gene encoding functional TolR).

[0030] The OMV in the immunogenic composition of the present invention is derived from Bacteria paratyphi A. “Derived from” may mean “purified,” i.e., the OMV in the immunogenic composition of the present invention is purified from Bacteria paratyphi A. Suitable purification methods are known in the art, and various filtration and chromatographic methods are cited as examples. A suitable two-step filtration purification process is described in WO 2011 / 036562, which is incorporated herein by reference.

[0031] Modification of Lipid A In some embodiments, the paratyphi a bacterium OMV contains or consists of GMMA, i.e., paratyphi a bacterium GMMA. The paratyphi a bacterium GMMA may be derived from a paratyphi a bacterium containing modified lipid A. The paratyphi a bacterium of the present invention may contain modified lipid A.

[0032] Modified lipid A is lipid A that has a different structure compared to the corresponding wild-type lipid A. The structure of lipid A can be determined using MALDI-TOF analysis of lipid A isolated from GMMA. For the assay, lipid A is isolated after treatment of GMMA with acetic acid and then assayed by MALDI-TOF. GMMA with a protein concentration of approximately 1 mg / mL (microBCA calibration curve) or a cell bank suspension with an OD600 of approximately 3 (4 mL sample) is treated with 1% acetic acid (final concentration) at 100°C for 2 or 6 hours, respectively, to obtain a precipitate containing lipid A. The precipitate is then collected, washed with water, and lipid A is extracted in chloroform / methanol 4:1. The final solution containing lipid A is mixed 1:1 with a saturated Super DHB (Fluka, 50862) solution (acetonitrile / water 1:1). A 2 μl mixture is loaded onto a target plate, the spot is allowed to dry at room temperature, and then the plate is inserted into a mass spectrometer. The spectrum (negative reflectron mode) generally shows peaks corresponding to the lipid A molecular species and includes several peaks attributable to lipid A fragmentation (i.e., loss of one or more fatty acid chains), sodium adducts (+22 m / z), and lipid A dephosphorylation (-80 m / z). The lipid A species is identified by comparing the molecular peak mass m / z with that expected for the sample in the analysis.

[0033] Lipid A may be modified to be detoxified (i.e., modified lipid A is detoxified lipid A). "Detoxified" means that lipid A is less toxic than wild-type lipid A. Wild-type lipid A used in comparison is the corresponding wild-type lipid A. In this context, "toxic" or "toxic" refers to the degree to which the innate immune system is activated by lipid A, particularly via the Toll-like receptor IV pathway. Highly toxic lipid A can lead to uncontrolled inflammation, apoptosis, and, in extreme cases, septic shock, among other effects. Modified lipid A may be less toxic if it is less reactive than the corresponding wild-type lipid A. For example, a person skilled in the art can determine whether a modified lipid A is less toxic by administering it to an animal such as a rabbit and using a monocyte activation test to determine whether it activated more monocytes compared to the corresponding wild-type lipid A.

[0034] "Corresponding wild-type lipid A" refers to the lipid A found in the corresponding wild-type bacterium and strain. For example, in the context of GMMA for *Cardiomycosis paratyphi* A, modified lipid A relative to "corresponding wild-type lipid A" is interpreted as lipid A that has been modified relative to the lipid A found in wild-type *Cardiomycosis paratyphi* A (for example, so that it is less virulent).

[0035] The modified lipid A may be penta-acylated lipid A. Those skilled in the art can determine whether GMMA contains penta-acylated lipid A by determining the structure of lipid A using the MALDI-TOF analysis described above.

[0036] The Paratyphi A bacterial GMMA may be derived from Bacteria paratyphi A, including any preferred modification that results in the production of GMMA containing lipid A that is less toxic than wild-type lipid A. Similarly, the Paratyphi A bacteria may include any preferred modification that results in the production of GMMA containing lipid A that is less toxic than wild-type lipid A.

[0037] HtrB, MsbB, and PagP are proteins involved in lipid A production in Gram-negative bacteria. Of these, MsbB and PagP are important in Salmonella. Salmonella bacteria that do not express functional versions of MsbB and / or PagP will not produce true lipid A, but rather a modified and detoxified lipid A. Therefore, the Paratyphi A GMMA may be derived from Paratyphi A bacteria that do not express functional versions of MsbB and / or PagP. Similarly, the Paratyphi A bacteria may not express functional versions of MsbB and / or PagP. In some cases, the Paratyphi A GMMA may be derived from Paratyphi A bacteria that do not contain genes encoding functional MsbB and / or PagP proteins. Similarly, the Paratyphi A bacteria may not contain genes encoding functional MsbB and / or PagP proteins.

[0038] Whether the Paratyphi A bacteria from which GMMA is derived, or whether the Paratyphi A bacteria express functional versions of MsbB and / or PagP, or whether they contain genes encoding functional MsbB and / or PagP proteins, may be determined by isolating lipid A from the GMMA as described above and analyzing its structure by MALDI-TOF. If lipid A is detoxified, then the Paratyphi A bacteria from which GMMA is derived, or the Paratyphi A bacteria, do not express functional versions of Msb and / or PagP, or do not contain genes encoding functional MsbB and / or PagP proteins.

[0039] In some cases, the Paratyphi A bacteria from which the GMMA is derived, or the Paratyphi A bacteria, do not contain genes encoding functional proteins (e.g., htrB, msbB, and / or pagP) because they contain mutations within those genes. In some cases, the Paratyphi A bacteria from which the GMMA is derived, or the Paratyphi A bacteria, do not contain genes encoding functional HtrB, MsbB, and / or PagP proteins. In some cases, the Paratyphi A bacteria from which the GMMA is derived, or the Paratyphi A bacteria, contain genes encoding at least a portion of the HtrB, MsbB, and / or PagP proteins, but the genes are mutated such that the encoded HtrB, MsbB, and / or PagP proteins lack one or more important amino acids, or a portion of the gene is deleted. For example, the Paratyphi A bacteria from which the GMMA is derived, or the Paratyphi A bacteria, may contain substitution or deletion mutations in the htrB, msbB, and / or pagP genes. Alternatively, the Paratyphi A bacteria from which the GMMA is derived, or the Paratyphi A bacteria, may have additive mutations in the htrB, msbB, and / or PagP genes, such as additive mutations that cause a frameshift. In some cases, the Paratyphi A bacteria from which the GMMA is derived, or the Paratyphi A bacteria, may contain deletion mutations in the htrB, msbB, and / or pagP genes. In some cases, the htrB, msbB, and / or pagP genes contain deletion mutations, and at least 10%, at least 20%, at least 25%, at least 30%, at least 50%, or at least 75% of the htrB, msbB, and / or pagP genes are deleted. In some cases, the Bacterium paratyphi A from which the GMMA is derived, or the Bacterium paratyphi A itself, is deficient in the htrB, msbB, and / or pagP genes (e.g., due to a complete deletion of the htrB, msbB, and / or pagP genes (ΔhtrB, ΔmsbB, and / or ΔpagP mutation)). In some cases, the Bacterium paratyphi A OMV or GMMA is derived from Bacterium paratyphi A in which at least a portion of the msbB and / or pagP genes are replaced by different genes.In some cases, the Paratyphi A OMV or GMMA is derived from a strain of Paratyphi A in which at least a portion of the msbB and / or pagP genes are replaced with tetracycline (tet) or kanamycin (kan) genes, respectively. In some cases, the Paratyphi A OMV or GMMA is derived from a strain of Paratyphi A in which at least a portion of the msbB and / or pagP genes are replaced with tetracycline (tet) or kanamycin (kan) genes, respectively. In some cases, the Paratyphi A OMV or GMMA is derived from a strain of Paratyphi A in which pagP::kan and / or msbB::tet. "::" indicates that the gene before "::" is replaced with the gene after "::". Therefore, "pagP::kan" means that the pagP gene is replaced with kanamycin.

[0040] In one embodiment, the Salmonella paratyphi A GMMA of the present invention is derived from a Salmonella paratyphi A GMMA bacterium containing one or more mutations resulting in the deletion of pagP and / or msbB. Similarly, the Salmonella paratyphi A bacterium of the present invention may contain one or more mutations resulting in the deletion of pagP and / or msbB. As a non-limiting example, a suitable strain of Salmonella paratyphi A may be selected from the group consisting of ΔpagP and ΔmsbB (ΔpagP refers to a Salmonella strain in which the pagP gene is deleted and / or replaced by a different gene such as an antibiotic resistance gene).

[0041] Hyperblebbing The Paratyphi A bacterium, or Paratyphi bacterium (S. Paratyphi bacterium), from which the GMMA is derived may be modified (e.g., genetically modified) to hyperbleb, that is, to "bud off" a larger amount of outer membrane compared to the corresponding Gram-negative bacterium that does not have the genetic mutation.

[0042] The Paratyphi A bacteria, or Paratyphi bacterium, from which the GMMA is derived may contain any preferred modification that results in hyperbraving. In some cases, the modification is a mutation, and for example, the Paratyphi A bacteria, or Paratyphi bacterium, from which the GMMA is derived may not contain a gene encoding a functional protein (e.g., tolR) because it contains a mutation. In some cases, the Paratyphi A bacteria, or Paratyphi bacterium, from which the GMMA is derived may not contain a gene encoding the functional TolR protein. In some cases, the Paratyphi A bacteria, or Paratyphi bacterium, from which the GMMA is derived may contain a gene encoding at least a portion of the TolR protein, but the gene is mutated such that the encoded TolR protein lacks one or more important amino acids, or a portion of the gene is deleted. For example, the Paratyphi A bacteria, or Paratyphi bacterium, from which the GMMA is derived may contain substitution or deletion mutations in the tolR gene. Alternatively, the Bacteria paratyphi A from which the GMMA is derived, or the Bacteria paratyphi, may have an additive mutation in the tolR gene, such as an additive mutation causing a frameshift. In some cases, the Bacteria paratyphi A from which the GMMA is derived, or the Bacteria paratyphi, may contain a deletion mutation in the tolR gene. In some cases, the tolR gene may contain a deletion mutation, and at least 10%, at least 20%, at least 25%, at least 50%, or at least 75% of the tolR gene may be deleted. In some cases, the Bacteria paratyphi A from which the GMMA is derived, or the Bacteria paratyphi, may lack the tolR gene (for example, due to a complete deletion of the tolR gene (ΔtolR mutation)). In some cases, Bacteria paratyphi A OMV or GMMA may be derived from Bacteria paratyphi A in which at least a portion of the tolR gene is replaced by a different gene. In some cases, the paratyphi A OMV or GMMA strains originate from a strain of paratyphi A in which at least a portion of the tolR gene is replaced by the chloramphenicol acetyltransferase (cat) gene.In some cases, the paratyphi A OMV or GMMA is derived from a strain of paratyphi A in which the tolR gene is replaced by chloramphenicol acetyltransferase (cat). In some cases, the paratyphi A OMV or GMMA is derived from a strain of paratyphi A in which tolR::cat.

[0043] Whether a certain genetic modification causes bleb over-formation in Paratyphi A bacteria from which the GMMA originates, or in Paratyphi bacteria, may be tested using the following hyperblebing assay. The user should prepare two bacterial cultures. The first culture must contain bacteria with the genetic modification to be tested (test culture), and the second culture must contain equivalent bacteria that are identical except for the genetic modification to be tested (reference culture). The user should grow the test culture and the reference culture under the same conditions and then measure the number of outer membrane vesicles released from the bacteria in the test culture and the bacteria in the reference culture. If the amount of outer membrane vesicles released in the test culture is greater than the amount released in the reference culture, then the genetic modification causes bleb over-formation in the bacteria. The level of released outer membrane vesicles may be measured by O-antigen quantification, for example by HPAEC-PAD, as described in Example 5.

[0044] In some cases, the immunogenic composition contains Salmonella paratyphi A GMMA derived from strain ED199 (see, for example, Mylona E, Sanchez-Garrido J, Hoang Thu TN, Dongol S, Karkey A, Baker S, Shenoy AR, Frankel G. Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death. Cell Microbiol. 2021 May;23(5):e13306). Similarly, the Salmonella paratyphi A bacterium may be Salmonella paratyphi A ED199.

[0045] If the strain used is based on the Paratyphi A strain ED199, then even if modifications have been made to strain ED199 (e.g., mutations in the msbB, pagP, or tolR genes), the immunogenic composition will contain Paratyphi A GMMA derived from Paratyphi A strain ED199, or the Paratyphi A bacterium will be ED199 Paratyphi A bacterium. For example, the Paratyphi A strain may be tolR::cat pagP::kan mabB::tet.

[0046] dose The immunogenic composition of the present invention may contain doses of GMMA of *Streptococcus paratyphia* (O-antigen) of 1 μg to 50 μg, 2 μg to 25 μg, 2 μg to 10 μg, 15 μg to 25 μg, approximately 20 μg, or approximately 4 μg.

[0047] The dose of GMMA may be quantified as an O-antigen dose; that is, if the immunogenic composition contains a 1 μg (O-antigen) dose of GMMA, then the immunogenic composition contains enough GMMA to provide 1 μg of the O-antigen associated with that GMMA (for example, the immunogenic composition contains GMMA containing a total of 1 μg of Salmonella typhimurium O-antigen). This means that when an immunogenic composition contains O-antigen-rich GMMA, the actual amount of GMMA present to obtain a 1 μg (O-antigen) dose may be less than the amount required when the GMMA is poor in O-antigen.

[0048] The amount of *Salvelinus paratyphi* O-antigen present in an immunogenic composition may be measured by mild hydrolysis of the O-antigen in the immunogenic composition (to provide the monosaccharide paratose) and detection of the amount of paratose using HPAEC-PAD. Assuming that "free" *Salvelinus paratyphi* O-antigen is not added, the amount of O-antigen in the *Salvelinus paratyphi* GMMA composition corresponds to the O-antigen dose of the *Salvelinus paratyphi* GMMA.

[0049] The O-antigen / protein ratio of OMV or GMMA of *Salvelinus paratyphi* A present in the immunogenic composition may be at least 0.2, 0.3, 0.4, 0.5, or at least 0.6, typically at least 0.4. The O-antigen / total protein ratio may be up to 0.8, 0.9, 1.0, or 2.0. The O-antigen content may be quantified by HPAEC-PAD, for example, as described in Example 5. The protein concentration may be quantified by micro-BCA, for example, as described in PCT / EP2022 / 073501.

[0050] Outer membrane vesicles or GMMA obtainable from or acquired from the aforementioned Paratyphi A bacteria In some aspects of the present invention, an outer membrane vesicle or GMMA obtained or obtainable by the method of the present invention is provided. Such an outer membrane vesicle or GMMA may contain any of the features of the outer membrane vesicle or GMMA described herein. For example, such an outer membrane vesicle or GMMA may contain penta-acylated lipid A.

[0051] immunogenic composition The immunogenic composition of the present invention or the immunogenic composition used in the present invention may contain additional components, such as pharmaceutically acceptable excipients, adjuvants, and / or further antigens.

[0052] The immunogenic composition may further contain pharmaceutically acceptable excipients. Typical "pharmaceutically acceptable excipients" include any carrier that does not itself induce the production of antibodies harmful to the individual to whom the composition is administered. Preferred carriers are typically large, slowly metabolized polymers, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such carriers are well known to those skilled in the art. Pharmaceutically acceptable excipients may also contain diluents, such as water, saline, glycerol, etc. Furthermore, auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc. Sterilized, pyrogenic-free Tris-buffered saline is a particularly preferred carrier when using aluminum adjuvants, because phosphates in phosphate-buffered saline may interfere with the binding of outer membrane vesicles to aluminum. However, in certain embodiments, the immunogenic composition comprises phosphate-buffered saline (and optionally an aluminum adjuvant as further described below). In some cases, the immunogenic composition comprises phosphate-buffered saline with a pH of 6-7, for example, pH 6.5.

[0053] The immunogenic composition may be prepared as an injectable liquid solution (liquid solution) or suspension (suspension). A solid form suitable for solution or suspension in a liquid vehicle may also be prepared before injection (e.g., lyophilized composition or spray lyophilized composition). The immunogenic composition may be prepared for topical administration, for example, as an ointment, emulsion, or powder. The immunogenic composition may be prepared for oral administration, for example, as a tablet or capsule, as a spray, or as a syrup (possibly flavored). The immunogenic composition may be prepared for pulmonary administration, for example, as an inhaler, using a fine powder or spray. The composition may be prepared as a suppository or vaginal suppository. The immunogenic composition may be prepared for nasal, ocular, or ocular administration, for example, as a drop. The immunogenic composition may also be in the form of a kit, designed so that the combined composition is reconstituted immediately before administration to a mammal. Such a kit may contain one or more antigens in liquid form and one or more lyophilized antigens. The immunogenic compositions may be provided in vials or in pre-filled syringes. The syringes may or may not have needles. The syringes may contain a single dose of the composition, while the vials may contain single or multiple doses.

[0054] The immunogenic composition of the present invention, or the immunogenic composition used in the present invention, may be packaged in unit dose form or in multiple dose form. In the case of multiple dose form, vials are preferred over pre-filled syringes. An effective dosage can be conventionally established, but a typical human dose of the composition is, for example, 0.5 mL for intramuscular injection.

[0055] The aforementioned composition will be sterile. The immunogenic composition of the present invention or the immunogenic composition used in the present invention may be isotonic in humans.

[0056] Therefore, the immunogenic composition of the present invention or the immunogenic composition used in the present invention may be useful as a vaccine. A vaccine according to the present invention may be prophylactic (i.e., for preventing infection) or therapeutic (i.e., for treating infection), but will typically be prophylactic.

[0057] The immunogenic composition used as a vaccine comprises an effective amount(s) of antigen(s), and any other components as needed. “Effective amount” (i.e., immunologically effective amount) means that the amount administered to an individual, either as a single dose or as part of a series of doses, is effective for treatment or prevention. This amount varies depending on the health and physical condition of the individual being treated, age, taxonomy of the individual being treated (e.g., non-human primates, primates, etc.), the individual's ability to synthesize antibodies, the desired degree of protection, the formulation of the vaccine, the physician's assessment of the medical situation, and other relevant factors. The immunogenic composition of the present invention may also contain antimicrobial agents, particularly when packaged in multiple doses.

[0058] Salmonella Typhi antigen The immunogenic composition may further contain an antigen derived from Salmonella typhi (Salmonella typhi antigen). The Salmonella typhi antigen may be a Vi polysaccharide.

[0059] The term "Vi" or "Vi polysaccharide" refers to the capsular polysaccharide of Salmonella enterica serovar Typhi purified from Citrobacter (Rondini et al., J. Infect. Dev. Ctries, 2012).

[0060] Vi polysaccharides may also be fragmented Vi polysaccharides (fVi). The term "fragmented" in relation to Vi polysaccharides refers to Vi polysaccharides that have undergone size reduction, resulting in a decrease in the number of repeating units in the polysaccharide. Therefore, fragmented Vi has a lower average molecular weight compared to natural Vi. For example, fragmented Vi may contain 30 to 300 repeating units, compared to more than 600 repeating units in natural Vi. The structure of the Vi monomer repeating unit is shown below.

[0061] [ka]

[0062] In fragmented Vi, preferably, no structural changes in the repeating units are observed compared to natural Vi. This can be confirmed by 1H NMR analysis (see WO2015 / 068129). Furthermore, the percentage of O-acetyl groups in fragmented Vi is preferably the same as in natural Vi (i.e., about 95% O-acetylation), but may differ and may be reduced to about 65% O-acetylation. O-acetylation can be determined by standard measurements such as 1H NMR or colorimetric analysis of hestrin.

[0063] In its natural size, fVi polysaccharide has an average molecular weight of approximately 165 kDa, as measured by HPLC-size exclusion chromatography (HPLC-SEC). In some embodiments, fVi polysaccharide has an average molecular weight of 10 kDa–90 kDa, 25 kDa–70 kDa, 40 kDa–55 kDa, 41 kDa–49 kDa, or 51 kDa–55 kDa. fVi polysaccharide may have a target molecular weight of 51 kDa–55 kDa (for example, it is prepared by a method that typically produces fVi with a molecular weight within this range). The molecular weight of Vi polysaccharide can be determined by HPLC-SEC.

[0064] Typically, the average molecular weight is calculated by electrophoresis on a TSK Gel 3000 PWXL column (30 cm x 7.8 mm; particle size 7 μm; cod. 808021) (Tosoh Bioscience) with a TSK Gel PWXL guard column (4.0 cm x 6.0 mm; particle size 12 μm; cod. 808033) using dextran as a standard substance (5, 25, 50, 80, 150 kDa). The mobile phase is 0.1 M NaCl, 0.1 M NaH2PO4, 5% CH3CN, pH 7.2 at a flow rate of 0.5 mL / min (isocratic method for 30 minutes). Void and bed volume calibration are performed using λ-DNA (λ-DNA molecular weight marker III 0.12~21.2 kb; Roche) and sodium azide (NaN3; Merck), respectively.

[0065] The fragmented Vi polysaccharides can be further separated into pools of different average molecular weight ranges. This can be achieved by methods known in the art, such as anion exchange chromatography, size exclusion chromatography, and tangential flow filtration.

[0066] The fVi polysaccharide used in the present invention has a specific average molecular weight (avMW) range distribution, which can be further characterized using the polydispersity index (PDI) as a unit.

[0067] The polyvariance index is given by the following formula: PDI = Mw / Mn (In the formula, Mw is the weight-average molecular weight and Mn is the number-average molecular weight.) It is calculated as shown.

[0068] The narrower the molecular weight distribution, the closer the PDI value will be to 1.

[0069] fVi polysaccharides may have an avMW distribution characterized in that at least 80% of the pool has avMW in the range of 25 kDa to 70 kDa. fVi polysaccharides may have an avMW distribution characterized in that at least 50% of the pool has avMW in the range of 35 kDa to 60 kDa. fVi polysaccharides may have an avMW distribution characterized in that at least 30% of the pool has avMW in the range of 41 kDa to 55 kDa.

[0070] Fragmentation of Vi polysaccharides can be carried out by several methods known in the art, such as chemical hydrolysis of natural polysaccharides, enzymatic fragmentation of natural polysaccharides, gamma irradiation of natural polysaccharides, sonication of natural polysaccharides, or mechanical methods such as high-pressure homogenizers / microfluidizers / HPCDS (high-pressure cell disruption systems). The fragmentation method used in the present invention is selected so that it can yield fVi polysaccharides having avMW of less than 90 kDa, less than 80 kDa, less than 60 kDa, or 40-55 kDa. The method may also be selected so as not to change the structure of the repeating units.

[0071] Preferably, fragmentation is not carried out by mechanical means. Preferably, fragmentation is not carried out by alkaline hydrolysis. fVi polysaccharide can be obtained by chemical hydrolysis using hydrogen peroxide. It has been found that this method can reduce the size of Vi polysaccharide without changing the structure of the repeating units. Furthermore, hydrolysis using hydrogen peroxide can enable the formation of fragmented Vi with a lower average molecular weight than when mechanical methods are used.

[0072] The fVi polysaccharide may be part of an fVi conjugate containing fVi and a carrier protein. The carrier protein in the fVi conjugate may be tetanus toxoid or CRM. 197 , or diphtheria toxoid. The carrier protein is CRM 197 That's fine.

[0073] fVi polysaccharides can be conjugated to carrier proteins by any suitable conjugation chemistry.

[0074] The conjugation of fVi polysaccharide to the carrier protein may be via an -NH2 group, for example, via the side chain(s) of lysine residue(s) or arginine residue(s) in the carrier polypeptide. If the fVi polysaccharide has a free aldehyde group, this group can react with an amine in the protein to form a conjugate by reductive amination. Alternatively, the conjugation to the carrier may be via an -SH group, for example, via the side chain(s) of cysteine ​​residue(s) in the carrier polypeptide. Or, the fVi polysaccharide can be conjugated to the carrier protein via a linker molecule.

[0075] fVi polysaccharides are typically activated or functionalized before conjugation. Activation may involve cyanylating reagents such as CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroboric acid). Other preferred techniques include the use of carbodiimides, hydrazides, activated esters, norboranes, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU (see, for example, the introduction to WO 98 / 42721).

[0076] Direct conjugation to a carrier protein may include, for example, oxidation of the fVi polysaccharide followed by reductive amination using the protein, as described in U.S. Patents 4,761,283 and 4,356,170. Conjugation via a linker group can be carried out using any known procedure, for example, the procedure described in U.S. Patents 4,882,317 and 4,695,624. Typically, the linker is bonded via the anomeric carbon of the polysaccharide. A preferred type of linker is the adipic acid linker, which can be formed by coupling a free -NH2 group (e.g., introduced into the polysaccharide by amination) with adipic acid (e.g., using diimide activation), and then coupling the protein to the resulting sugar-adipic acid intermediate (see, e.g., EP-B-0477508, Mol. Immunol, (1985) 22, 907-919, and EP-A-0208375). A similar preferred type of linker is the glutaric acid linker, which can be formed in the same way by coupling a free -NH group with glutaric acid. Adipic acid and glutaric acid linkers can also be formed directly, i.e., by coupling a polysaccharide with a free group, e.g., a free -NH group, without prior introduction of the free group into the polysaccharide, and then coupling the protein to the resulting sugar-adipic acid / glutaric acid intermediate. Another preferred type of linker is a carbonyl linker, which can be formed by the reaction of the free hydroxyl group of a modified polysaccharide with CDI (Bethell GS et al. (1979) J. Biol. Chem. 254, 2572-4 and Hearn MTW (1981) J. Chromatogr. 218, 509-18), followed by a reaction with a protein to form a carbamate bond.Other linkers include β-propionamide (WO00 / 10599), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), halogenated haloacyl (US Patent No. 4,057,685), glycosidic bonds (US Patents No. 4,673,574, 4,761,283, and 4,808,700), 6-aminocaproic acid (US Patent No. 4,459,286), N-succinimidyl-3-(2-pyridyldithio)-propionic acid (SPDP) (US Patent No. 5,204,098), adipic acid dihydrazide (ADH) (US Patent No. 4,965,338), and C4-C12 moieties (US Patent No. 4,663,160). Carbodiimide condensation can also be used (WO2007 / 000343).

[0077] A bifunctional linker can be used to provide a first group for coupling to an amine group in a polysaccharide (e.g., introduced into the polysaccharide by amination) and a second group for coupling to a carrier (typically for coupling to an amine in the carrier). Alternatively, the first group can be directly coupled to the polysaccharide, i.e., without prior introduction of the group, e.g., an amine group, into the polysaccharide.

[0078] fVi conjugate is a. A step of fragmenting Vi polysaccharides to obtain fragmented Vi(fVi) polysaccharides having an average molecular weight of 10kDa-90kDa, 25kDa-70kDa, 40kDa-55kDa, 41kDa-49kDa, or 51kDa-55kDa; b. A step in which the fVi polysaccharide obtained in step a. is reacted with carbodiimide and N-hydroxysuccinimide at a pH of 5-6 to form an N-hydroxysuccinimide ester fVi derivative; and c. The N-hydroxysuccinimide ester fVi derivative obtained in step b. is reacted with a carrier protein (which may be a derivatized carrier protein) to produce an fVi conjugate. It may be obtained by a method comprising (i.e., a method for preparing an fVi conjugate), or it may be possible to obtain it.

[0079] Such methods are described in more detail in WO2015068129.

[0080] The carrier protein can be derivatized by reacting it with a carbodiimide and a linker. The carbodiimide may be 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). Any suitable linker (such as those discussed above) can be used. In some embodiments, the linker is an ADH linker. Derivatization of the carrier protein may result in a derivatized carrier protein. The carrier protein may be CRM 197 and the derivatization of the carrier protein comprises one or more of the following steps: (i) preparing CRM 197 in a suitable buffer, optionally a MES buffer; (ii) mixing CRM 197 and EDAC at a ratio of 1:0.05 to 1:0.5, 1:0.1 to 1:0.3, or approximately 1:0.15 (CRM 197 [[ID=z0]]to EDAC (w / w)); (iii) mixing CRM 197 and ADH at a ratio of 1:1 to 1:6, 1:2 to 1:4, or approximately l:3.5 (CRM 197 to ADH (w / w)); (iv) incubating the mixture of CRM 197 with EDAC, and optionally ADH, for at least 30 minutes or for 30 minutes to 2 hours, optionally with stirring; and (v) purifying the derivatized CRM 197 by tangential flow filtration, optionally. [[ID=z6]]This includes.

[0081] In some embodiments, the carrier protein is derivatized by a method comprising steps (i), (ii), and (iv). In some embodiments, the carrier protein is derivatized by a method comprising steps (i), (ii), (iii), and (iv). In some embodiments, the carrier protein is derivatized by a method comprising steps (i), (ii), (iv), and (v). In some embodiments, the carrier protein is derivatized by a method comprising all of the above steps (i) to (v). In some embodiments, the above steps (i) to (v) are carried out in the above order, except that steps (ii) and (iii) can be carried out simultaneously.

[0082] fVi conjugates can be obtained by a method comprising the step of reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide at a pH of 5-6 to form N-hydroxysuccinimide ester fVi derivatives. The carbodiimide may be EDC (N-3-dimethylaminopropyl(-N-ethylcarbodiimide)). The reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide may involve mixing fVi with a carbodiimide such as EDC in the presence of N-hydroxysuccinimide (NHS). The reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide may involve mixing fVi polysaccharide with NHS. The reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide results in an NHS concentration of 0.1 M-0.5 M, or approximately 0.33 M, and an fVi polysaccharide concentration of 1 mg / mL-100 mg / mL, or approximately 50 mg / mL. The reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide may include mixing fVi polysaccharide with EDC to have a molar ratio of 1:1 to 20:1, 1:1 to 10:0, 2:1 to 7:1, or approximately 5:1 for repeating units from EDC to fVi. The mixing of fVi polysaccharide with EDC may be carried out after mixing fVi polysaccharide with NHS. The reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide may include the step of incubating the mixture of fVi polysaccharide, NHS, and EDC at room temperature for at least 30 minutes, or approximately 1 hour.

[0083] The reaction between an N-hydroxysuccinimide ester fVi derivative and a carrier protein (which may be a derivatized carrier protein) may involve mixing the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative). The reaction between an N-hydroxysuccinimide ester fVi derivative and a carrier protein (which may be a derivatized carrier protein) may involve mixing the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative) in a ratio (w / w) of 1:0.1 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:2, or approximately 1:1. The mixing of the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative) may be carried out in a buffer with a pH of 5 to 7, or approximately 6. The mixing of the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative) may be carried out in MES buffer. The mixing of the N-hydroxysuccinimide fVi derivative with the carrier protein (or carrier protein derivative) may be carried out at a temperature of 20°C to 30°C, or around room temperature, while mixing is performed as needed.

[0084] A method for preparing an fVi conjugate involves reacting an N-hydroxysuccinimide fVi derivative with a carrier protein (or carrier protein derivative), followed by one or more of the following additional steps: (i) Quenching by adding a quencher such as phenyl HP buffer; (ii) A step of filtering the fVi conjugate; (iii) A step to purify the fVi conjugate, possibly using hydrophobic interaction chromatography; (iv) a step of concentrating the fVi conjugate by tangential flow filtration if applicable; and (v) The step of filtering the conjugate using one or more 0.2 μm filters, if applicable. It may include.

[0085] A method for preparing an fVi conjugate may include two or more, three or more, four or more, or all five of the above steps (i) to (v). A method may include step (i). A method may include steps (i) to (iii). A method may include steps (i) to (v). A method may include steps (i) to (iii) in the order listed above. A method may include steps (i) to (v) in the order listed above.

[0086] Adjuvant The immunogenic compositions of the present invention, or used in the present invention, may contain an adjuvant. Any suitable adjuvant can be used. However, in some embodiments, the adjuvant is an inorganic salt such as an aluminum salt or a calcium salt. Suitable inorganic salts include hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, or mixtures of various inorganic compounds (mineral compounds) in any suitable form (e.g., gels, crystals, amorphous materials, etc.) with compounds that are preferred for adsorption. The mineral-containing composition can also be formulated as particles of metal salts.

[0087] The immunogenic compositions of the present invention, or used in the present invention, are aluminum adjuvants, i.e., Al 3+ It may contain any compound that includes ions.

[0088] Aluminum adjuvants are aluminum phosphate (Al 3+ and PO4 3- Any compound containing ions) and / or aluminum hydroxide (Al 3+ and OH - It may contain, or may consist of, any compound containing ions.

[0089] Aluminum adjuvants may contain or consist of aluminum hydroxide. Aluminum hydroxide adjuvants may contain or may consist of aluminum hydroxide salts. Aluminum hydroxide adjuvants may contain or may consist of aluminum hydroxide salts that are at least partially crystalline. Aluminum hydroxide salts that can be represented by the formula AlO(OH) are analyzed by infrared (IR) spectroscopy, particularly at 1070 cm⁻¹. -1 Adhesive band and 3090~3100cm -1 The presence of a strong shoulder distinguishes it from other aluminum compounds such as aluminum hydroxide salts (Al(OH)3) (see Chapter 9 of Vaccine Design: The Subunit and Adjuvant Approach (Powell & Newman (eds.)) Plenum Press 1995 (ISBN 0-306-44867-X)). The crystallinity of aluminum hydroxide adjuvants is reflected in the width of the diffraction bands (WHH) at 20 half-high, with less crystalline particles showing greater line broadening due to smaller microcrystalline sizes. As WHH increases, the surface area increases, and adjuvants with higher WHH values ​​appeared to have a higher ability for antigen adsorption. Fibrous morphology (e.g., as seen in transmission electron micrographs) is typical for aluminum hydroxide adjuvants.

[0090] Suitable examples of aluminum hydroxide adjuvants will be obvious to those skilled in the art, and include, for example, ALHYDROGEL®.

[0091] Aluminum adjuvants are 0.1 mg to 10 mg of Al 3+ , 0.1mg~5mg Al 3+ , 0.3mg~0.4mg Al 3+ , or approximately 0.35 mg of Al 3+ It may include, or may consist of.

[0092] immunogenicity This example demonstrates that the immunogenic composition of the present invention exhibits good immunogenicity. Immunogenicity can be measured according to the assay described in Example 4.

[0093] The immunogenic composition of the present invention is obtained by following the steps (a) and (b), namely, (a) Immunizing mice intraperitoneally with the immunogenic composition at doses of 1 μg (O-antigen) / GMMA and 1.25 μg sugar / sugar conjugate on days 0 and 28, and (b) A step in which the anti-paratyphimurium O-antigen antibody level is measured by ELISA on day 42. In an immunogenicity assay including at least 10 3 It may induce EU / mL of O-antigen polysaccharide antibodies against *Salvia paratyphi A*. The doses of the GMMA and the sugar may be determined as described in the preceding chapter titled "Dosage".

[0094] A suitable ELISA is as follows: - Coat the ELISA plate with the paratyphi A bacterium O-antigen. - After applying a blood sample collected from a mouse on day 42 to a coated ELISA plate, the plate is washed to remove antibodies that did not bind to the Paratyphi A O-antigen, and - The amount of anti-paratyphimurium A O-antigen antibody bound to the paratyphimurium A O-antigen on the ELISA plate is detected using an anti-IgG antibody conjugated to a detection portion such as alkaline phosphatase. It may include.

[0095] Cross-defense The immunogenic compositions of the present invention may exhibit cross-protection. For example, the immunogenic compositions of the present invention may be effective against the following strains: (a) Salmonella Typhimurium ST34 (Mather et al., New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease in Immunocompromised Patients in Vietnam. mBio. 2018 Sep 4;9(5):e01056-18); (b) Salmonella typhimurium 10433_3 (Van Puyvelde et al., An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation. Nat Commun. 2019 Sep 19;10(1):4280); (c) Salmonella typhimurium D23580 (Van Puyvelde et al., An African Salmonella Typhimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation. Nat Commun. 2019 Sep 19;10(1):4280); (d) Salmonella typhimurium ST4 / 74 (Hurley et al., Atypical Salmonella enterica Serovars in Murine and Human Macrophage Infection Models. Infect Immun. 2020 Mar 23;88(4):e00353-19); (e) Salmonella Typhimurium A130 (Okoro et al., Intracontinental spread of human invasive Salmonella Typhimurium pathovariants in sub-Saharan Africa. Nat Genet. 2012 Nov;44(11):1215-21); (f) Salmonella enterica serovar Derby (Pullinger et al., Identification of Salmonella enterica serovar Dublin-specific sequences by subtractive hybridization and analysis of their role in intestinal colonization and systemic translocation in cattle. Infect Immun. 2008 Nov;76(11):5310-21); (g) Salmonella enterica serovar Dublin (Pullinger et al., Identification of Salmonella enterica serovar Dublin-specific sequences by subtractive hybridization and analysis of their role in intestinal colonization and systemic translocation in cattle. Infect Immun. 2008 Nov;76(11):5310-21); (h) Salmonella Enteritidis A1636 (Perez-Sepulveda et al., Complete Genome Sequences of African Salmonella enterica Serovar Enteritidis Clinical Isolates Associated with Bloodstream Infection. Microbiol Resour Announc. 2021 Mar 25;10(12):e01452-20); (i) Enterococcus CP255 (Perez-Sepulveda et al., Complete Genome Sequences of African Salmonella enterica Serovar Enteritidis Clinical Isolates Associated with Bloodstream Infection. Microbiol Resour Announc. 2021 Mar 25;10(12):e01452-20); and (j) Enteritidis D7795 (Perez-Sepulveda et al., Complete Genome Sequences of African Salmonella enterica Serovar Enteritidis Clinical Isolates Associated with Bloodstream Infection. Microbiol Resour Announc. 2021 Mar 25;10(12):e01452-20) Antibodies may be induced against 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or all 10 of the following.

[0096] The immunogenic composition of the present invention is (i) Enterococcus and Salmonella bovine (S. Dublin); (ii) Salmonella typhimurium, Salmonella porcine (S. derby), and Salmonella bovine; (iii) Salmonella typhimurium, Salmonella enteritis, Salmonella porcine and Salmonella bovine; or (iv) Salmonella typhi, Salmonella enteritis, Salmonella porcine, Salmonella bovine, and Salmonella paratyphi A Antibodies against it may be induced.

[0097] An immunogenic composition "induces" antibodies against (e.g.) Salmonella typhimurium ST34 if it can induce these antibodies when used to immunize mice. Whether it can induce these antibodies when used to immunize mice can be determined by testing a sample of the immunogenic composition using the following cross-protection assay.

[0098] Whether or not an immunogenic composition induces antibodies against one or more of the above-mentioned strains can be determined by performing a cross-protection assay.

[0099] Specifically, users - It is possible to immunize mice in the peritoneal cavity using 500 μL of immunogenic composition; and - It is possible to measure the level of bactericidal antibodies produced against the relevant strain using a serum bactericidal assay (SBA). Here, the related stock is one of the stocks (a) to (j) listed above.

[0100] The SBA assay may be based on the assay described in Example 4, except that the user should measure bactericidal activity against the relevant strains listed above (for example, *Salvin paratyphi* NVGH308).

[0101] The IC50 (serum dilution that causes 50% inhibition of ATP levels) obtained in the SBA assay is 10 2 If the threshold exceeds [value], the immunogenic composition may induce antibodies against the other strain mentioned above.

[0102] The immunogenic composition of the present invention is prepared in the following steps: (a) Immunizing mice intraperitoneally on days 0 and 28 with an immunogenic composition containing 1 μg of (O-antigen) per GMMA and 1.25 μg of sugar per sugar conjugate; and (b) A step on day 42 to measure the level of the anti-paratyphi A bacterium O-antigen antibody subtype by ELISA. When determined using an antibody class assay including the following, anti-paratyphimurium A antibodies can be induced in each of the IgG3, IgG2b, IgG2a, and IgG1 classes.

[0103] An immunogenic composition "induces" anti-paratyphi A antibodies in each of the IgG3, IgG2b, IgG2a, and IgG1 classes, if it can induce these antibodies when used to immunize mice. Whether it can induce these antibodies when used to immunize mice can be determined by testing a sample of the immunogenic composition using an ELISA assay to measure the levels of the anti-paratyphi A O-antigen antibody subtypes described below.

[0104] A suitable ELISA for measuring the level of O-antigen antibody subtypes of Bacillus subparatyphi A is: - Coat the ELISA plate with the O-antigen of *Salvelinus paratyphi*; - After adding a blood sample taken from a 42-day-old mouse to a coated ELISA plate, the plate is washed to remove antibodies that are not bound to the Paratyphi A O-antigen; and - Detecting the amount of anti-paratyphimurium A O-antigen IgG3 antibody bound to the paratyphimurium A O-antigen on an ELISA plate using an anti-IgG3 antibody conjugated to a detection portion such as alkaline phosphatase; - Repeat the first three steps: first using an anti-IgG2b antibody instead of an anti-IgG3 antibody, then using an anti-IgG2a antibody instead of an anti-IgG3 antibody again, and finally using an anti-IgG1 antibody instead of an anti-IgG3 antibody. It may include.

[0105] Medical use and treatment methods Further aspects of the present invention provide immunogenic compositions of the present invention for use in methods for preventing infection. Further aspects of the present invention provide methods for preventing infection, comprising administering an effective amount of the immunogenic composition or vaccine of the present invention to a subject. Further aspects of the present invention provide the use of the immunogenic composition or vaccine of the present invention for the manufacture of pharmaceuticals for use in methods for preventing infection. Methods for preventing infection of the present invention may comprise administering an effective amount of the immunogenic composition or vaccine of the present invention to a subject.

[0106] A method of preventing infection may be a method of preventing Salmonella infection. A method of preventing infection may also be a method of preventing invasive unclassified Salmonella infection. A method of preventing infection may also be a method of preventing Salmonella typhimurium, Salmonella enteritis, Salmonella typhimurium and / or Salmonella paratyphi A infection.

[0107] The method of use of the immunogenic composition in the manufacture of the pharmaceutical of the present invention / immunogenic composition for use / term in use "preventing Salmonella infection" includes inducing an immune response in the subject. The immune response may be protective and may produce antibodies such as IgG antibodies.

[0108] The subject of this invention is a mammal, which may be a human. If the vaccine is for prophylactic use, the human may be an adult, i.e., 18 years of age or over 18 years of age. If the vaccine is for prophylactic use, the human may be a child, i.e., under 18 years of age. If the vaccine is for prophylactic use, the child may be 12 to 72 months of age, preferably 24 to 59 months of age, more preferably 6 to 12 months of age.

[0109] If the vaccine is for preventative use, children may be approximately 9 months of age.

[0110] If the vaccine is for therapeutic use, the human subject is preferably a child.

[0111] Vaccines intended for children can also be administered to adults to evaluate, for example, their safety, dosage, or immunogenicity. [Examples]

[0112] Example 1 - Production of GMMA from Salmonella typhimurium and Enterococcus pallidum Salmonella typhimurium wild-type (WT) strain 2192 was provided by the Salmonella Genetic Stock Center (SGSC) at the University of Calgary, Canada, and belongs to the global Salmonella reference collection A (SARA12).

[0113] Enterococcal WT strain 618 was provided by Quotient Bioresearch Limited, UK. The animal-derived strain was isolated by European Antimicrobial Susceptibility Surveillance in Animals (EASSA).

[0114] From the above Salmonella strains, recombinant mutants of Salmonella typhimurium ΔtolRΔpagPΔmsbB and Salmonella enteritidis ΔtolRΔpagPΔmsbB 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).

[0115] GMMAs derived from the above Salmonella strains (GMMA from Salmonella typhimurium (STmGMMA) and GMMA from Salmonella enteritis (SEnGMMA)) were purified and isolated. The GMMAs were purified using a method similar to that previously reported for Shigella 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: 10.1371 / journal.pone.0134478 [doi];PONE-D-15-08654). In short, GMMA released into the fermentation broth was purified using two consecutive tangential flow filtration (TFF) steps: microfiltration to separate the culture supernatant containing GMMA from the bacteria, and ultrafiltration to separate GMMA from soluble proteins and nucleic acids.

[0116] Example 2 - fVi-CRM for immunization against Salmonella typhi 197 Conjugate Production Paratyphi A OAag-CRM was prepared using the following protocol as described above. 197 Conjugate and CRM 197 A divalent composition was produced containing a conjugate of fragmented Vi polysaccharide (fVi) derived from Salmonella typhi, which was conjugated to [the compound].

[0117] 1) Fragmentation of Vi polysaccharide: Step 1: Fragmentation and Quenching: Vi polysaccharide fragmentation is achieved by oxidation using hydrogen peroxide in the presence of iron sulfate. The reaction is quenched with EDTA (ethylenediaminetetraacetic acid). The natural Vi polysaccharide is diluted with WFI. Calculated volumes of 10 mM FeSO4 and H2O2 are added to obtain final concentrations of 0.5 mM FeSO4 and 0.5% v / v H2O2, respectively, in the reaction mixture. The reaction mixture is incubated at 15±5°C for 120±10 minutes. The reaction is stopped by adding an equal volume of 250 mM EDTA to obtain a final EDTA concentration of 10 mM, and the mixture is stirred.

[0118] Step 2: Buffer exchange: Remove residual H2O2 by tangential flow filtration (TFF) using 100 mM sodium phosphate (pH: 7.2 ± 0.2) with a 30 kDa cassette. Concentrate the fragmented Vi(fVi) polysaccharide.

[0119] Step 3: Stabilization of fVi polysaccharide: After fragmentation, stabilize the 30kDa retained product by incubation at 80±5°C for 120±15 minutes.

[0120] Step 4: Purification of fVi by anion exchange (resin: Capto-Q): Separate fVi polysaccharides of the desired molecular size (25-70 kDa) using the AA chromatography step. This is done using linear gradient elution with Capto-Q buffer A and Capto-Q buffer B, using Capto-Q resin with a binding capacity of 13 mg fVi / mL. Eluten fractions are collected based on conductivity of 1 mS / cm, i.e., 35-50 mS / cm, and the molecular size distribution is estimated by SEC / HPLC. The Capto-Q fractions are pooled based on the molecular size (kDa) distribution.

[0121] Step 5: Desalting: The pooled Capto-Q fraction is concentrated by tangential flow filtration (TFF) using a 10 kDa cutoff cassette, and then dialyzed using WFI until the conductivity of the filtration product reaches 30 μS / cm or less.

[0122] Step 6: 0.2 μm filtration of fVi polysaccharide: Filter the fVi polysaccharide through a 0.22 μm filter. Store the purified fVi polysaccharide in a PETG bottle.

[0123] 2) CRM 197 Derivatization of: Step 1: CRM 197 Unpacking: Refined CRM 197 Thaw at 2-8°C before buffer exchange with 100 mM MES (morpholinoethanesulfonic acid) buffer. After thawing, CRM 197 This is filtered using a 0.5 μm filter.

[0124] Step 2: Buffer exchange with 100mM MES buffer: CRM 197 After thawing, the buffer is replaced using TFF with 100mM MES buffer (pH 6.0 ± 0.2) in a 10kDa cassette.

[0125] Step 3: CRM 197 Derivatization: CRM at the required concentration 197 After diluting with 100 mM MES buffer, the calculated amounts of ADH (adipate dihydrazide) and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) are added to achieve a CRM:ADH:EDAC ratio of 1:3.5:0.15 w / w / w. After the addition of ADH and EDAC, CRM 197 The reaction mixture is incubated at room temperature for 60 ± 15 minutes under mixed conditions. At the end of the reaction, an equal volume of 5 mM MES buffer (pH 7.0 ± 0.2) is added.

[0126] Step 4: CRM 197 Purification: After the reaction, CRM 197 This is purified by TFF using a 10kDa cassette containing 5mM MES buffer.

[0127] Step 5: Dialysis-filtered CRM 197Filtration: Dialysis-filtered CRM 197 After filtering the solution through a 0.2 μm filter, store it in a glass bottle at 2-8°C.

[0128] 3) Derivatized CRM of fragmented Vi polysaccharides 197 Conjugation with: Activation of fVi polysaccharide: Step 1: Drying of fVi polysaccharide by Rota Vapor: Further concentrate fVi by drying it at 30°C using a rotavapor. Reconstitute the concentrated fVi polysaccharide using 100 mM MES buffer (pH: 6.0) to obtain a concentration of 50 mg / mL.

[0129] Step 2: Activation of fVi polysaccharide with NHS: The fVi carboxylate (-COOH) is activated with EDC (N-3-dimethylaminopropyl-N-ethylcarbodiimide) in the presence of N-hydroxysuccinimide (NHS) by forming an active ester intermediate, and then pre-activated CRM with ADH. 197 This increases the efficiency of conjugation. The dried fVi polysaccharide is reconstituted to the desired concentration (50 mg / mL) using 100 mM MES buffer (pH: 6.2 ± 0.2), activated in the presence of NHS (0.33 M concentration), and then EDAC is added so that the EDAC / fVi RU molar ratio is 5:1. The EDAC solution is added after the addition of NHS to ensure complete dissolution. The reaction mixture is incubated at room temperature for 1 hour with slow mixing.

[0130] Conjugation: Step 1: fVi, CRM 197 -Conjugation with ADH: The conjugation reaction involves activated fVi and CRM 197 -A covalent bond is formed with ADH. The activated and derivatized reaction mixture is diluted with 100 mM MES pH:6.0 and 5 mg / mL of activated fVi and CRM 197 -CRM to reach the final fVi concentration of ADH 197- Add ADH in a 1:1 (fVi:CRM197) w / w ratio. Perform the conjugation reaction at room temperature while slowly mixing until protein consumption reaches 70% or more, as measured by HPLC-SEC at 280 nM absorbance.

[0131] Step 2: Quenching and conditioning of the conjugation reaction: Quench the conjugation reaction by adding an equal volume of phenyl HP buffer B Tris 50mM pH:8.0. Add NaCl as a powder until a final salt concentration of 3M is reached.

[0132] Step 3: Filtration of the conjugation mixture: fVi-CRM 197 Filter the crude conjugate through a 0.65 filter.

[0133] Step 4: fVi-CRM 197 Purification of Crude Conjugate: Purify the conjugate from the conditioned reaction mixture by passing it through a HIC Phenyles Sepharose High Performance (HP) column. Perform column integration every 5–10 cycles per standard procedure. Equilibrate the column using Phenyles Sepharose HP Buffer A Tris 50mM NaCl 3M pH 8. After adding the conditioning buffer and NaCl, place the crude conjugate on the column. Wash the column with Phenyles Sepharose HP Buffer A, then elute the product using Phenyles Sepharose HP Buffer B Tris 50mM pH 8. Collect the fractions and store them at 2–8°C until further use. Pool all fractions derived from multiple electrophoresis runs.

[0134] Step 5: Concentration and Buffer Exchange using PBS: After concentrating the conjugate purified by TFF using a 50kDa cutoff cassette, perform a buffer exchange using PBS buffer until the conductivity of the filtered product matches the conductivity of the PBS buffer.

[0135] Step 6: Use a 0.2 μm filter with fVi-CRM 197 Conjugate pre-filtration: fVi-CRM 197 The conjugate is filtered through a 0.2 μm filter to reduce bioburden.

[0136] Step 7: Use a 0.2 μm cellulose acetate filter with fVi-CRM 197 Sterile filtration of conjugates: fVi-CRM 197 The conjugate is filtered through a 0.2 μm cellulose acetate filter. The purified fVi-CRM197 conjugate is sampled and stored at 2–8°C.

[0137] Example 3 - Production of GMMA derived from Paratyphi A bacteria Paratyphimurium strain ED199 containing the ΔtolR ΔpagP ΔmsbB mutant was prepared using a protocol based on the protocol described for Enterococcus and Salmonella mucinous in Example 1, except that the specific mutants used to delete tolR, pagP, and msbB were tolR::cat pagP::kan msbB::tet. GMMA was isolated from Paratyphimurium strain A as described in Example 1.

[0138] Example 4 - Immunogenicity Assay ELISA Evaluation of anti-Vi and anti-OAg specific total IgG by ELISA. As previously reported, anti-OAg and anti-Vi antigen-specific IgG levels were measured by ELISA two weeks after the second immunization (day 42) (Rondini et al., Evaluation of the immunogenicity and biological activity of the Citrobacter freundii Vi-CRM197 conjugate as a vaccine for Salmonella enterica serovar Typhi. Clin Vaccine Immunol. 2011 Mar;18(3):460-8). Briefly, 96-well round-bottom MaxiSorp microtiter plates (Nunc, Roskilde, Denmark) were coated with 100 ml / well antigen overnight at 4°C. OAg purified from S. Paratyphi A (O:2) or S. Enteritidis (O:9) and Vi purified from C. freundii s.1 were used at concentrations of 15 mg / ml and 2 mg / ml in carbonate buffer or 1 mg / ml in phosphate buffer, respectively (Micoli et al., A scalable method for O-antigen purification applied to various Salmonella serovars. Anal Biochem. 2013 Mar 1;434(1):136-45; Micoli et al., Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi. Vaccine. 2012 Jan 20;30(5):853-61). Plates were blocked with PBS + 5% nonfat milk (Sigma) for 1 hour at room temperature (RT), and then washed three times with PBS + 0.05% Tween 20 (PBS-T). Serum samples were diluted to 1:100 and 1:4,000 in PBS-T (dilution buffer) with 0.1% BSA added, and both dilutions were assayed in three replicates.After incubation at RT for 2 hours, the plates were washed three times with PBS-T and incubated at 25°C for 1 hour with anti-mouse goat IgG alkaline phosphatase (Sigma) diluted in dilution buffer at 1:6,000, 1:8,800, and 1:2,600 (for Vi, O:2, or O:9, respectively). After washing three times with PBS-T, the plates were colored by adding alkaline phosphatase substrate (SIGMAFAST N2770; Sigma) and read at 405 nm and 490 nm using an ELx 800 reader (BioTek). ELISA units were expressed against a mouse antigen-specific antibody standard serum curve consisting of 10 standard points and 2 blank wells (performed twice on each plate), and the best 5-parameter fit was determined by a modified Hill plot. One ELISA unit is defined as the reciprocal of the dilution ratio of the standard serum that gives an absorbance value equal to 1 in this assay.

[0139] SBA Evaluation of serum bactericidal activity by SBA. Individual mouse serums collected on day 42 were heat-inactivated (HI) at 56°C for 30 minutes and then tested in a serum bactericidal assay based on luminescence readings against Salmonella paratyphi A NVGH308, Salmonella enteritis CMCC3014, and Vi-positive Citrobacter freundii broad strain 3056 (Necchi et al., Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout. PLoS One. 2017 Feb 13;12(2):e0172163; Necchi et al., Setup of luminescence-based serum bactericidal assay against Salmonella Paratyphi A. J Immunol Methods. 2018 Oct;461:117-121). SBA was performed in 96-well round-bottom sterile plates (Corning). Dilutions of HI test serum were incubated for 3 hours in the presence of exogenous complement (rabbit complement [RBC]) and bacteria, as previously described (Necchi et al., Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout. PLoS One. 2017 Feb 13;12(2):e0172163). Briefly, a sufficient volume of reaction mixture containing target bacterial cells (approximately 100,000 CFU / ml), BRC (50% for S. Enteritidis, 20% for S. paratyphi A, and 5% for C. freundii s.1.) and buffer (PBS) was added to an SBA plate containing HI serum dilution and incubated at 37°C for 3 hours.At the end of incubation, the plate was centrifuged at 4,000 xg for 10 minutes, the supernatant was discarded to remove ATP from dead bacteria, and the live bacterial pellet, resuspended in PBS, was transferred to a white round-bottom 96-well plate (Greiner) and mixed with BacTiter-Glo reagent (Promega) in a 1:1 (vol / vol) ratio. The reaction mixture was incubated in an orbital shaker at RT for 5 minutes, and the luminescence signal was measured using a luminometer (Viktor). As previously described, a four-parameter nonlinear regression was applied to the raw luminescence for all serum dilutions tested (Rossi et al., Intra-Laboratory Evaluation of Luminescence Based High-Throughput Serum Bactericidal Assay (L-SBA) to Determine Bactericidal Activity of Human Sera against Shigella. High Throughput. 2020 Jun 8;9(2):14). SBA titers are reported as IC50, defined as the serum dilution that results in a 50% inhibition of ATP levels in the negative control well. An IC50 of 50 was arbitrarily assigned to titers below the minimum measurable level of luminescence, representing half of the initial dilution of the tested serum (i.e., 100). GraphPad Prism 7 software (GraphPad Software) was used for fitting and IC50 determination.

[0140] IgG subclass Individual serum samples isolated from blood samples taken from immunized mice as described in the section titled "Example 7 - Antibody Subclasses Produced by Quadrivalent Pan-Salmonella Vaccine in Mice" were tested, and the isotypes of the antibodies produced were determined using an ELISA-based assay operating on the same principle as described under the heading "ELISA". The assay was repeated to determine the EU / mL for each individual serum sample at the doses tested as secondary antibodies (anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, and anti-mouse IgG3) in the standard assay. The results are shown in Example 12 and are expressed as subclass / total subclass %.

[0141] Example 5 Example 5 - Formulation of a quadrivalent (Pan-Salmonella) vaccine against Salmonella typhi, Salmonella enteritis, Salmonella typhi, and Salmonella paratyphi for preclinical trials. A quadrivalent vaccine called Pan-Salmonella ParaA GMMA was formulated. This vaccine contains GMMA obtained from Enterococcus and Salmonella typhimurium (described in Example 1), Salmonella paratyphi A GMMA (described in Example 3), and fVi-CRM. 197 It contained a conjugate (described in Example 2).

[0142] STmGMMA and SEnGMMA were adsorbed onto an alpha hydrogel. After mixing (1-2 hours), the paratyphi GMMA was added. Subsequently, after a quenching step using phosphate and osmotic pressure adjustment using sodium chloride, the fVi-CRM was added.

[0143] Maximum adsorption was achieved by optimizing the concentration of the phosphate buffer, while quenching was performed to ensure optimal particle size.

[0144] The final formulation contained GMMA in a phosphate-buffered saline matrix containing 0.7 mg / mL of aluminum hydroxide, providing 40 μg / mL of STm, SEn, and *Salvelinus paratyphi* O-antigen (sPa), and 50 μg / mL of Vi polysaccharide.

[0145] The methods used to determine the O-antigen levels of the GMMA of the aforementioned enteric bacteria, Salmonella typhimurium, and Salmonella paratyphi A are described below.

[0146] OAg determination for enteric bacteria, typhimurium, and paratyphi A. Each single OAg was hydrolyzed before HPAEC-PAD analysis to release its associated di-deoxy monosaccharide corresponding to the chromatographic peak (tiberose from SEn OAg, avequoise from STm OAg, and paratose from SPa OAg). In fact, di-deoxy is the only sugar that makes up the repeating unit (RU) of SEn, STm, and SPa OAg that is different from the others.

[0147] The samples were diluted by volume (450 μL) or weight on a chemical balance with Milli-Q water to fall within the calibration curve range for each OAg. 120 μL of 1 M TFA was added to the vial containing the standard or sample, and incubated at 75°C for 1.5 hours. After hydrolysis, the vial was cooled in a refrigerator at 2–8°C for 15 minutes. The samples and standards were dried overnight at room temperature (RT) on a centrifugal evaporator to remove the solvent / TFA. The pellets were dissolved in 450 μL of Milli-Q water. The samples and standards were filtered on an AcroPrep Advance 96 Filter Plate 0.2 μm Supor 1 mL well, and the plate was loaded onto an HPAEC-PAD.

[0148] Example 6 Example 6 - Immunogenicity of the quadrivalent pan-salmonella vaccine in mice Multiple groups of 10 CD1 mice were administered either the Pan-Salmonella vaccine described in Example 5 (Pan-Salmonella-ParAGMMA) or an alternative vaccine containing a conjugate of the Paratyphi A O-antigen instead of the Paratyphi A GMMA (Pan-Salmonella-O:2-CRM). 197 Using the following dosages,

[0149] [Table 1] And I became immunized.

[0150] The aforementioned immunizations included intraperitoneal immunization with 200 μL of each formulation on day 0 and day 28. Blood samples were collected on days -1, 27, and 42, and the produced antibodies were tested using the assay described in Example 4 above. The results are shown in Figure 1. In summary, both pan-salmonella vaccines induced a specific serum IgG response to the paratyphi A O-antigen, and the antibodies showed bactericidal activity in mice. Both pan-salmonella vaccines also induced a specific serum IgG response to Salmonella typhi Vi.

[0151] In the second experiment, multiple groups of 10 CD1 mice were used, as follows: - Tetravalent Pan-Salmonella O:2-CRM (STm GMMA + SEn GMMA + O:2-CRM) 197 + fVi-CRM 197 Using ), the values ​​were 1.0 (μg O-antigen), 1.0 (μg O-antigen), 1.25 (μg O-antigen), and 1.25 (μg Vi polysaccharide), respectively. - Tetravalent Pan-Salmonella_ParAGMMA (STm GMMA + SEn GMMA + ParA GMMA + fVi-CRM) prepared as described in Example 5 197 Using ), the doses were 1.0 (μg O-antigen), 1.0 (μg O-antigen), 1.17 (μg O-antigen), and 1.25 (μg Vi polysaccharide), respectively. - Trivalent iNTS-TCV (STm GMMA + SEn GMMA + fVi-CRM 197 Using ), at doses of 1.0 (μg O-antigen), 1.0 (μg O-antigen), and 1.25 (μg Vi polysaccharide), - Divalent O:2-CRM 197 + fViCRM 197 Using the following methods, at doses of 1.25 (μg O-antigen) and 1.25 (μg Vi polysaccharide), - O:2-CRM 197 Using a conjugate, a dose of 1.25 μg (Vi polysaccharide) was administered. - Using ParA GMMA adsorbed onto an alpha hydrogel (prepared as described in Example 3), a dose of 1.17 μg (Vi polysaccharide) was administered. - fVi-CRM 197 Using a conjugate (prepared as described in Example 2), a dose of 1.25 μg (Vi polysaccharide) was administered. I became immune.

[0152] The aforementioned immunization included intraperitoneal immunization at corresponding doses for each product in 200 μL on days 0 and 28. After blood samples were collected on days 27 and 42, the produced antibodies were measured using the ELISA assay and SBA assay described in Example 4. The results are shown in Figure 2.

[0153] No negative immune interference was detected in the induced anti-SEn and anti-STm functional antibody responses from the combination of iNTS-TCV and the ParA component. However, a significantly higher anti-SEn IgG response was elicited by the Pan-Salmonella formulation containing O:2-CRM on both days 27 and 42, and similarly by the Pan-Salmonella formulation containing ParA GMMA on day 27, compared to trivalent iNTS-TCV. Furthermore, a significantly higher anti-STm IgG response was elicited by the Pan-Salmonella formulation containing O:2-CRM on day 27, compared to trivalent iNTS-TCV. It is also important to consider that the O:2-OAg dose used with Pan-Salmonella_ParAGMMA differed slightly (approximately 7%) from the O:2 dose used with Pan-Salmonella_O:2-CRM.

[0154] Example 7 Example 7 - Subclasses of antibodies produced in mice by the quadrivalent Pan-Salmonella vaccine Multiple groups, each consisting of 10 CD1 mice, were administered the following doses of the Pan-Salmonella vaccine described in Example 6, i.e.,

[0155] [Table 2] And I became immunized.

[0156] The additional mouse group was compared to the following comparators, namely: - 1 μg (O-antigen) of STmGMMA, 1 μg (O-antigen) of SEnGMMA, and 1.25 μg of fVi-CRM 197 iNTS-TCV, including - fVi-CRM described in Example 2 197 (1.25 μg) and ParA O:2-CRM 197 A divalent composition containing (1.25 μg), - fVi-CRM described in Example 2 197 A divalent composition comprising (1.25 μg) and ParA GMMA (1.17 μg O-Ag) as described in Example 3, - ParA O:2-CRM 197 Conjugate (1.25 μg), - ParA GMMA (1.17 μg O-Ag) as described in Example 3, - fVi-CRM described in Example 2 197 (1.25 μg) And I became immunized.

[0157] The aforementioned immunization included intraperitoneal immunization with 200 μL of each preparation on day 0 and day 28. Blood samples were collected on days -1, 27, and 42. The class of antibodies produced against the *Salmonella paratyphi A* O:2 O-antigen and *Salmonella typhi Vi* was determined using the assay described in Example 4 under the section "IgG subclass". The results are shown in Figures 3(a) and (b). The absolute values ​​for Figures 3(a) and (b) are shown in the table below:

[0158] [Table 3]

[0159] [Table 4]

[0160] Example 8 Example 8 - Immunogenicity of the quadrivalent Pan-Salmonella vaccine in rabbits Multiple groups, each consisting of 8 New Zealand white rabbits, were administered the following doses of the Pan-Salmonella vaccine described in Example 6, i.e.,

[0161] [Table 5] And I became immunized.

[0162] The immunization included intramuscular immunization with 500 μL of the formulation on day 0 and day 28. Blood samples were collected on days -1, 27, and 42, and the produced antibodies were tested using the assay described in Example 4. The results are shown in Figure 4. In summary, the Pan-Salmonella vaccine induced a specific serum IgG response to the O-antigen of Salmonella paratyphi, and its antibodies showed bactericidal activity in rabbits. The Pan-Salmonella vaccine also induced a specific serum IgG response to Salmonella typhi Vi.

[0163] Example 9

[0164] Example 9 - Antibodies induced by GMMA-based vaccines show cross-protection. CD1 mice were given the following doses of the quadrivalent Pan-Salmonella vaccine described in Example 5 on days 0 and 28, i.e.,

[0165] [Table 6] Then, intraperitoneal immunization was performed.

[0166] SBA was performed on mouse serum obtained on day 42.

[0167] Mouse serum induced by the aforementioned quadrivalent Pan-Salmonella vaccine showed bactericidal activity against Salmonella typhimurium, Salmonella enteritis, Salmonella derby, Salmonella dublin, and Salmonella paratyphi A (Figure 5).

[0168] Embodiments of the present invention

[0169] 1. An immunogenic composition containing outer membrane vesicles of *Cyperus paratyphi*.

[0170] 2. The immunogenic composition of Embodiment 1, wherein the outer membrane vesicles of *Cyprinus paratyphi* are *Cyprinus paratyphi* GMMA.

[0171] 3. Paratyphi A bacteria containing modified lipid A.

[0172] 4. The immunogenic composition of Embodiment 1 or 2, wherein the outer membrane vesicles of Bacillus paratyphi A or GMMA comprises modified lipid A.

[0173] 5. The immunogenic composition of Embodiment 3 or 4 or the bacterium Paratyphi A, wherein the modified lipid A is detoxified lipid A.

[0174] 6. An immunogenic composition or bacterium paratyphi A according to any one of embodiments 3 to 5, wherein the modified lipid A is penta-acylated lipid A.

[0175] 7. An immunogenic composition according to any one of Embodiments 1, 2, or 4-6, wherein the paratyphi a outer membrane vesicles or GMMA are derived from paratyphi a bacteria that do not contain a gene encoding a functional MsbB protein.

[0176] 8. The immunogenic composition of Embodiment 7, wherein the paratyphi a outer membrane vesicles or GMMA are derived from paratyphi a bacteria in which the msbB gene is at least partially deleted.

[0177] 9. A Paratyphi A bacterium according to any one of Embodiments 3, 5, or 6, wherein the Paratyphi A bacterium does not contain a gene encoding a functional MsbB protein.

[0178] 10. The Paratyphi A bacterium of Embodiment 9, wherein the msbB gene in the Paratyphi A bacterium is at least partially deleted.

[0179] 11. The immunogenic composition of Embodiment 8, or the Bacteria paratyphi A of Embodiment 10, wherein at least 20%, at least 30%, at least 50%, or at least 75% of the msbB gene is deleted.

[0180] 12. The immunogenic composition of Embodiment 11 or Bacteria paratyphi A, wherein the entire msbB gene is deleted.

[0181] 13. The immunogenic composition of Embodiment 7, wherein the paratyphi a outer membrane vesicle or GMMA is derived from paratyphi a bacterium in which ΔmsbB is present.

[0182] 14. The immunogenic composition of Embodiment 7 or 13, wherein at least a portion of the msbB gene is replaced with at least a portion of the tetracycline (tet) gene.

[0183] 15. The immunogenic composition of Embodiments 7, 13, or 14, wherein the OMV or GMMA is derived from *Salvelinus paratyphi* A, which is msbB::tet.

[0184] 16. The Paratyphi A bacterium of Embodiment 9, wherein the Paratyphi A bacterium is ΔmsbB.

[0185] 17. Paratyphi A bacterium of embodiment 9 or 16, wherein at least a portion of the msbB gene is replaced with at least a portion of the tetracycline (tet) gene.

[0186] 18. Paratyphi A bacterium of embodiment 9, 16, or 17, which is msbB::tet.

[0187] 19. An immunogenic composition according to any one of Embodiments 1, 2, 4-8, or 11-13, wherein the paratyphi a outer membrane vesicles or GMMA are derived from paratyphi a bacteria that do not contain a gene encoding a functional PagP protein.

[0188] 20. The immunogenic composition of Embodiment 19, wherein the paratyphi a outer membrane vesicles or GMMA are derived from paratyphi a bacteria in which the pagP gene is at least partially deleted.

[0189] 21. The Paratyphi A bacterium according to any one of Embodiments 3, 5, 6, 9-12, or 16, wherein the Paratyphi A bacterium does not contain a gene encoding a functional PagP protein.

[0190] 22. The Paratyphi A bacterium of Embodiment 21, wherein the pagP gene in the Paratyphi A bacterium is at least partially deleted.

[0191] 23. The immunogenic composition of Embodiment 20, or the Bacteria paratyphi A of Embodiment 22, wherein at least 20%, at least 30%, at least 50%, or at least 75% of the pagP gene is deleted.

[0192] 24. The immunogenic composition of Embodiment 23 or the bacterium *Paratyphi a*, wherein the entire PagP gene is deleted.

[0193] 25. The immunogenic composition of Embodiment 19, wherein the paratyphi a outer membrane vesicle or GMMA is derived from paratyphi a bacterium in which ΔpagP is present.

[0194] 26. The immunogenic composition of Embodiment 19 or 25, wherein at least a portion of the pagP gene is replaced with at least a portion of the kanamycin (kan) gene.

[0195] 27. The immunogenic composition of Embodiments 19, 25, or 26, wherein the OMV or GMMA is derived from *Typhi a paratyphi*, which is pagP::kan.

[0196] 28. The Paratyphi A bacterium of Embodiment 21, wherein the Paratyphi A bacterium is ΔpagP.

[0197] 29. Paratyphi A bacterium of embodiment 21 or 28, wherein at least a portion of the pagP gene is replaced with at least a portion of the kanamycin (kan) gene.

[0198] 30. Paratyphi A bacteria of embodiment 21, 28, or 29, wherein pagP::kan.

[0199] 31. An immunogenic composition or bacterium paratyphi A of any one of the above embodiments, wherein the outer membrane vesicles or GMMA are derived from bacterium paratyphi A that excessively forms blebs, or the bacterium paratyphi A is such that it excessively forms blebs.

[0200] 32. An immunogenic composition according to any one of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, or 31, wherein the paratyphi a outer membrane vesicles or GMMA are derived from paratyphi a bacteria that do not contain a gene encoding a functional TolR protein.

[0201] 33. The immunogenic composition of Embodiment 32, wherein the paratyphi a outer membrane vesicles or GMMA are derived from paratyphi a bacteria in which the tolR gene is at least partially deleted.

[0202] 34. A paratyphi A bacterium according to any one of Embodiments 3, 5, 6, 9-12, 16, 21-25, 28, or 29, wherein the paratyphi A bacterium does not contain a gene encoding a functional tolR protein.

[0203] 35. The Paratyphi A bacterium of Embodiment 34, wherein the tolR gene in the Paratyphi A bacterium is at least partially deleted.

[0204] 36. An immunogenic composition of Embodiment 33, or a Bacteria paratyphi A of Embodiment 27, wherein at least 20%, at least 30%, at least 50%, or at least 75% of the tolR gene is deleted.

[0205] 37. The immunogenic composition of Embodiment 36 or Bacteria paratyphi A, wherein the entire tolR gene is deleted.

[0206] 38. The immunogenic composition of Embodiment 32, wherein the paratyphi a outer membrane vesicle or GMMA is derived from paratyphi a bacterium in which ΔtolR is present.

[0207] 39. Paratyphi A bacteria of embodiment 34, wherein the Paratyphi A bacteria is ΔtolR.

[0208] 40. The immunogenic composition of Embodiment 32 or 38, wherein at least a portion of the tolR gene is replaced with at least a portion of the chloramphenicol acetyltransferase (cat) gene.

[0209] 41. The immunogenic composition of Embodiments 32, 38, or 40, wherein the OMV or GMMA is derived from *Typhi a paratyphi*, which is tolR::cat.

[0210] 42. Paratyphi A bacterium of embodiment 34 or 39, wherein at least a portion of the tolR gene is replaced by at least a portion of the chloramphenicol acetyltransferase (cat) gene.

[0211] 43. Bacteria paratyphi a, of embodiment 34, 39, or 42, which is tolR::cat.

[0212] 44. Outer membrane vesicles or GMMA obtainable from any one of the Paratyphi A bacteria of Embodiments 3, 5, 6, 9-12, 16, 21-25, 28, or 29, 34-37, or 39.

[0213] 45. Outer membrane vesicles or GMMA obtained from any one of the Bacteria paratyphi A species of Embodiments 3, 5, 6, 9-12, 16, 21-25, 28, or 29, 34-37, or 39.

[0214] 46. ​​An immunogenic composition comprising outer membrane vesicles or GMMA as of Embodiment 44 or 45.

[0215] 47. The immunogenic composition is subjected to the following steps (a) and (b), namely, (a) Immunizing mice intraperitoneally with the immunogenic composition at doses of 1 μg (O-antigen) / GMMA and 1.25 μg sugar / sugar conjugate on days 0 and 28, and (b) Step of measuring anti - Salmonella paratyphi O - antigen antibody level by ELISA on the 42nd day In an immunogenic assay comprising 3 an immunogenic composition according to any one of embodiments 1, 2, 4 - 8, 11 - 13, 19, 20, 23 - 25, 31 - 33, 36 - 38, or 46 that induces Salmonella paratyphi A O - antigen polysaccharide antibody at least 10

[0216] 48. The immunogenic composition, which comprises the following steps (a) and (b), namely, (a) Step of intraperitoneally immunizing mice with the immunogenic composition at doses of 1 μg (O - antigen) / GMMA and 1.25 μg of sugar / sugar conjugate on days 0 and 28, and (b) Step of measuring anti - Salmonella paratyphi A O - antigen antibody subtype level by ELISA on the 42nd day an immunogenic composition or method according to any one of embodiments 1, 2, 4 - 8, 11 - 13, 19, 20, 23 - 25, 31 - 33, 36 - 38, 46, or 47 that induces anti - Salmonella paratyphi A O - antigen antibodies of each of classes IgG3, IgG2b, IgG2a, and IgG1 determined using an antibody class assay comprising

[0217] 49. An immunogenic composition according to any one of embodiments 1, 2, 4 - 8, 11 - 13, 19, 20, 23 - 25, 31 - 33, 36 - 38, or 46 - 48, wherein the immunogenic composition further comprises an adjuvant.

[0218] 50. The immunogenic composition of embodiment 49, wherein the adjuvant is an aluminum adjuvant.

[0219] 51. The immunogenic composition of embodiment 49 or 50, wherein the adjuvant comprises aluminum hydroxide and / or aluminum phosphate.

[0220] 52. The immunogenic composition of embodiment 51, wherein the adjuvant comprises aluminum hydroxide.

[0221] 53. The adjuvant is 0.1 mg to 10 mg of Al 3+ , 0.1 mg to 5 mg of Al 3+ , 0.3 mg to 0.4 mg of Al 3+ , or approximately 0.35 mg of Al 3+ An immunogenic composition comprising any one of embodiments 49 to 52.

[0222] 54. An immunogenic composition according to any one of Embodiments 19, 20, 23-25, 31-33, 36-38, or 46-53, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient.

[0223] 55. The immunogenic composition of Embodiment 54, wherein the pharmaceutically acceptable excipient comprises phosphate-buffered saline.

[0224] 56. The immunogenic composition of Embodiment 55, wherein the phosphate buffered saline has a pH of 6 to 7, or approximately 6.5.

[0225] 57. A vaccine comprising any one immunogenic composition of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, 31-33, 36-38, or 46-56.

[0226] 58. Any one immunogenic composition or vaccine of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, 31-33, 36-38, or 46-57 for use in methods of preventing infection.

[0227] 59. A method for preventing infection, comprising administering an effective amount of any one of the immunogenic compositions or vaccines of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, 31-33, 36-38, or 46-57 to a target.

[0228] 60. Use of any one immunogenic composition or vaccine of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, 31-33, 36-38, or 46-57 for the manufacture of a pharmaceutical for use in a method of preventing infection.

[0229] 61. An immunogenic composition or vaccine for use in Embodiment 58, or use in Embodiment 60, wherein the method for preventing the aforementioned infection includes administering an effective amount of any one of the immunogenic compositions or vaccines of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, 31-33, 36-38, or 46-57 to a target.

[0230] 62. An immunogenic composition or vaccine, method, or use for any one of Embodiments 58 to 61, wherein the method for preventing the aforementioned infection is a method for preventing Salmonella infection.

[0231] 63. An immunogenic composition or vaccine, method, or use for any one of Embodiments 58 to 62, wherein the method for preventing the aforementioned infection is a method for preventing invasive, non-typeable Salmonella infection.

[0232] 64. An immunogenic composition or vaccine, method, or use for any one of Embodiments 58 to 63, wherein the method for preventing the aforementioned infection is a method for preventing infection by Platyphi a bacillus.

[0233] 65. Any one immunogenic composition, immunogenic composition or vaccine for use, method or use, of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, 31-33, 36-38, or 46-64, wherein the O-antigen / protein ratio of the paratyphi A outer membrane vesicles or GMMA is at least 0.4.

[0234] 66. Any one immunogenic composition, immunogenic composition or vaccine for use, method, or use of Embodiments 1, 2, 4-8, 11-13, 19, 20, 23-25, 31-33, 36-38, or 46-65, wherein the immunogenic composition or vaccine further comprises Salmonella typhi antigen.

[0235] 67. The immunogenic composition of Embodiment 66, an immunogenic composition or vaccine for use, a method or use, wherein the typhoid antigen comprises a fragmented Vi(fVi) polysaccharide.

[0236] 68. The immunogenic composition of Embodiment 67, an immunogenic composition or vaccine for use, a method or use, wherein the fVi polysaccharide is part of an fVi conjugate comprising fVi and a carrier protein.

[0237] 69. The carrier protein is CRM 197 Alternatively, an immunogenic composition of Embodiment 69, an immunogenic composition or vaccine for use, a method, or a use, which is a diphtheria toxoid.

Claims

1. An immunogenic composition comprising a paratyphi a outer membrane vesicle, wherein the paratyphi a outer membrane vesicle contains modified lipid A, and the modified lipid A is detoxified lipid A.

2. The immunogenic composition according to claim 1, wherein the outer membrane vesicles of *Salvelinus paratyphi* are *Salvelinus paratyphi* GMMA.

3. Paratyphi A bacteria containing modified lipid A.

4. The immunogenic composition or Bacteria paratyphi A according to any one of claims 1 to 3, wherein the modified lipid A is penta-acylated lipid A.

5. The immunogenic composition according to any one of claims 1, 2, or 4, wherein the outer membrane vesicles or GMMA of the paratyphi a bacilli are derived from a bacilli paratyphi a bacilli that does not contain a gene encoding a functional MsbB protein, and in some cases, the outer membrane vesicles or GMMA of the paratyphi a bacilli are derived from a bacilli paratyphi a bacilli in which msbB::tet.

6. The paratyphi A bacterium according to claim 3 or 4, wherein the paratyphi A bacterium does not contain a gene encoding a functional MsbB protein, and in some cases the paratyphi A bacterium is msbB::tet.

7. The immunogenic composition according to any one of claims 1, 2, 4, or 5, wherein the outer membrane vesicles or GMMA of the paratyphi a bacilli are derived from a paratyphi a bacilli that does not contain a gene encoding a functional PagP protein, and optionally, the outer membrane vesicles or GMMA of the paratyphi a bacilli are derived from a paratyphi a bacilli that is pagP::kan.

8. The paratyphi A bacterium according to any one of claims 3, 4, 6, or 7, wherein the paratyphi A bacterium does not contain a gene encoding a functional PagP protein, and optionally the paratyphi bacterium is pagP::kan.

9. The immunogenic composition or bacterium paratyphi A according to any one of claims 1 to 8, wherein the outer membrane vesicles or GMMA of bacterium paratyphi A are derived from bacterium paratyphi A that excessively forms blebs, or the bacterium paratyphi A is such that it excessively forms blebs.

10. The immunogenic composition according to any one of claims 1, 2, 4, 5, 7, or 9, wherein the outer membrane vesicles or GMMA of the paratyphi A bacteria are derived from a strain of paratyphi A that does not contain a gene encoding a functional TolR protein, and optionally, the outer membrane vesicles or GMMA of the paratyphi A bacteria are derived from a strain of paratyphi A that is tolR::cat.

11. The paratyphi A bacterium according to any one of claims 3, 4, 6, 7, 8, or 9, wherein the paratyphi A bacterium does not contain a gene encoding a functional tolR protein, and optionally the paratyphi A bacterium is tolR::cat.

12. Outer membrane vesicles or GMMA obtainable from or obtained from Bacteria paratyphi A according to any one of claims 3, 4, 6, 7, 8, 9, or 11.

13. An immunogenic composition comprising outer membrane vesicles or GMMA as described in claim 12.

14. The immunogenic composition is then subjected to the following steps (a) and (b), namely, (a) Immunizing mice intraperitoneally with the immunogenic composition at doses of 1 μg (O-antigen) / GMMA and 1.25 μg sugar / sugar conjugate on days 0 and 28, and (b) A step in which the anti-paratyphimurium O-antigen antibody level is measured by ELISA on day 42. In an immunogenicity assay including at least 10 3 An immunogenic composition according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13, which induces EU / mL of O-antigen polysaccharide antibody against *Streptococcus paratyphia*.

15. The immunogenic composition is then subjected to the following steps (a) and (b), namely, (a) Immunizing mice intraperitoneally with the immunogenic composition at doses of 1 μg (O-antigen) / GMMA and 1.25 μg sugar / sugar conjugate on days 0 and 28, and (b) A step in which the anti-paratyphi A bacterium O-antigen antibody subtype level is measured by ELISA on day 42. An immunogenic composition according to any one of claims 1, 2, 4, 5, 7, 9, 10, 13, or 14, which induces anti-paratyphi A antibodies of class IgG3, IgG2b, IgG2a, and IgG1, respectively, as determined by an antibody class assay comprising the above.

16. The immunogenic composition according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13-15, wherein the immunogenic composition further comprises an aluminum adjuvant.

17. The immunogenic composition according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13-16, further comprising a pharmaceutically acceptable excipient.

18. An immunogenic composition according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13-17, for use in a method for preventing infection.

19. A method for preventing infection, comprising administering an effective amount of an immunogenic composition according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13-15 to a target.

20. Use of the immunogenic composition according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13-19 for the manufacture of a pharmaceutical for use in a method of preventing infection.

21. The immunogenic composition, method, or use according to any one of claims 18 to 20, wherein the method for preventing the aforementioned infection is a method for preventing Salmonella infection.

22. An immunogenic composition, method, or use for use according to any one of claims 18 to 21, wherein the method for preventing the aforementioned infection is a method for preventing an invasive, untyped Salmonella infection.

23. An immunogenic composition, method, or use for use according to any one of claims 18 to 22, wherein the method for preventing the aforementioned infection is a method for preventing infection by Salmonella paratyphi A.

24. An immunogenic composition, method, use, or immunogenic composition for use according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13-23, wherein the O-antigen / protein ratio of the paratyphi a outer membrane vesicle or GMMA is at least 0.

4.

25. The immunogenic composition or vaccine further comprises Salmonella typhi antigen, which may be fVi polysaccharide, and which is fVi and CRM 197 An immunogenic composition, method, use, or immunogenic composition for use according to any one of claims 1, 2, 4, 5, 7, 9, 10, or 13-24, which is part of an fVi conjugate comprising a carrier protein.

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