Immunogenic components

Abstract

The present invention relates to immunogenic compositions comprising antigens derived from Salmonella enterica serotype tiphimurium (S. Typhimurium), Salmonella enterica serotype enteritidis (S. Enteritidis), and Salmonella enterica serotype typhi (S. Typhi). The present invention further relates to methods and compositions comprising GMMA for boosting the immune response to the S. Typhi antigen, vaccines and methods comprising immunogenic compositions, and the use of immunogenic compositions.

Description

[Technical Field]

[0001] The present invention relates to immunogenic compositions comprising antigens derived from Salmonella enterica serotype tiphimurium (S. Typhimurium), Salmonella enterica serotype enteritidis (S. Enteritidis), and Salmonella enterica serotype typhi (S. Typhi). The present invention further relates to methods and compositions comprising GMMA for boosting the immune response to the S. Typhi antigen, vaccines and methods comprising immunogenic compositions, and the use of immunogenic compositions. [Background technology]

[0002] Typhoid fever is a bacterial disease caused by Salmonella enterica subspecies enterica serotype tiffi (Salmonella tiffi or S. typhi), a human host-limiting organism [Crump, 2019]. The disease is found worldwide, primarily affecting children and young adults, but is endemic in developing countries in Africa and Asia, while in developed countries it is occasionally reported in travelers recently returning from endemic areas [Smith, 2016]. The accurate burden of typhoid fever is said to be significantly underestimated due to the difficulty in establishing its diagnosis in endemic areas. In 2017, an estimated 10.9 million cases of typhoid fever and 116,800 deaths attributed to Salmonella tiffi were reported. Similarly, the yearly life loss (YLL) due to typhoid fever was 8.3 million, and the disability-adjusted life year (DALY) was 8.4 million. Despite a reduction in the disease burden due to improvements in water and public health, significant public health problems remain [Global Burden of Disease, 2017]. The burden of typhoid fever is highest in school-age children and those under 5 years of age. Recent studies have shown that the adjusted incidence of typhoid fever confirmed by blood cultures per 100,000 people-years of observation, including 95% confidence intervals, ranges from 861 (599-1203) in Malawi to 3228 (2276-4757) in Bangladesh for the 5-9 year age group, while for the 0-4 year age group it was 632 (398-965) and 2625 (1764-4244) in Malawi and Bangladesh, respectively [Meiring, 2021]. Without prompt diagnosis and treatment, typhoid fever requires hospitalization and can potentially lead to fatal complications such as typhoid-induced intestinal perforation (TIP). In developing countries where typhoid fever is endemic, surgical intervention is often delayed, thus further worsening disease outcomes [Contini, 2017].

[0003] Antibiotic treatment for typhoid fever has been hampered by the emergence of multidrug-resistant (MDR) typhoid Salmonella strains, first identified in 1980 and defined as resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. While the emergence of bacterial resistance has been overcome to some extent by newer antibiotics, challenges remain and hinder effective control of the disease [Radhakrishnan, 2018]. Similarly, S. typhi clones carrying resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole), as well as fluoroquinolones and third-generation cephalosporins, classified as extremely drug-resistant (XDR), have been reported in Asia [Klemm, 2018].

[0004] S. paratyphi A (Salmonella 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, or paratyphoid fever, caused by S. paratyphi A 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 been rising 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).

[0005] Therefore, improvements to the Salmonella vaccine are necessary. [Overview of the project]

[0006] Examples of the present invention demonstrate that a trivalent vaccine containing antigens (GMMA) derived from S. Typhimurium, S. Enteritidis, and S. Typhi is safe and highly immunogenic. Similarly, examples of the present invention demonstrate that a tetravalent vaccine containing a trivalent vaccine and an antigen (GMMA or O antigen conjugate) derived from S. Paratyphi A is also highly immunogenic, and no antigenic interference is observed among the four antigens. Furthermore, examples demonstrate that GMMA (derived from S. Enteritidis and / or S. Typhimurium) is immunogenic, and that antigen (CRM) derived from S. Typhi is immunogenic. 197 This demonstrates that the immune response to fVi conjugated to can be boosted.

[0007] In a first aspect of the present invention, (a) Salmonella enterica serotype tiphimurium antigen; (b) Salmonella enterica serotype enteritidis antigen; and (c) Salmonella enterica serotype Typhi antigen An immunogenic composition containing the following is provided.

[0008] A second aspect of the present invention provides a method for boosting an immune response to S. Typhi or S. Paratyphi A antigen, comprising administering a composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA.

[0009] A third aspect of the present invention provides a method for preventing infection by S. Typhi or S. Paratyphi A, comprising administering an immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA, wherein GMMA boosts the immune response to the S. Typhi antigen or S. Paratyphi A antigen.

[0010] A fourth aspect of the present invention provides an immunogenic composition comprising GMMA for use in a method for boosting an immune response to S. Typhi or S. Paratyphi A antigen, comprising administering the immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA.

[0011] A fifth aspect of the present invention provides an immunogenic composition for use in a method of preventing infection by S. Typhi or S. Paratyphi A, comprising administering the immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA, wherein the immunogenic composition boosts the immune response to S. Typhi antigen or S. Paratyphi A antigen.

[0012] A sixth aspect of the present invention provides a vaccine comprising the immunogenic composition of the present invention.

[0013] A seventh aspect of the present invention provides a method for preventing infection, comprising administering an effective amount of the immunogenic composition or vaccine of the present invention.

[0014] An eighth aspect of the present invention provides the use of an immunogenic composition or vaccine of the present invention for the manufacture of a pharmaceutical for use in a method of preventing infection. [Brief explanation of the drawing]

[0015] [Figure 1-1] The iNTS-TCV vaccine induces a specific serum IgG response to the target antigen, and the antibodies are bactericidal in mice. Study design: iNTS-TCV formulation; 8 mice / group; immunization IP: days 0 and 28; blood collection: days 27 and 42; toxicity lot at time 0. [Figure 1-2]The iNTS-TCV vaccine induces a specific serum IgG response to the target antigen, and the antibodies are bactericidal in mice. Study design: iNTS-TCV formulation; 8 mice / group; immunization IP: days 0 and 28; blood collection: days 27 and 42; toxicity lot at time 0. [Figure 2-1] Anti-STm OAg IgG response in mice treated with STmGMMA / Alhydrogel versus iNTS-TCV2 on day 27 (Figure 2(a)) and day 42 (Figure 2(b)). Anti-SEn OAg IgG response in mice treated with SEnGMMA / Alhydrogel versus iNTS-TCV2 on day 27 (Figure 2(c)) and day 42 (Figure 2(d)). Anti-Vi IgG response in mice treated with Vi-CRM197 versus iNTS-TCV2 on day 27 (Figure 2(e)) and day 42 (Figure 2(f)). [Figure 2-2] Anti-STm OAg IgG response in mice treated with STmGMMA / Alhydrogel versus iNTS-TCV2 on day 27 (Figure 2(a)) and day 42 (Figure 2(b)). Anti-SEn OAg IgG response in mice treated with SEnGMMA / Alhydrogel versus iNTS-TCV2 on day 27 (Figure 2(c)) and day 42 (Figure 2(d)). Anti-Vi IgG response in mice treated with Vi-CRM197 versus iNTS-TCV2 on day 27 (Figure 2(e)) and day 42 (Figure 2(f)). [Figure 2-3] Anti-STm OAg IgG response in mice treated with STmGMMA / Alhydrogel versus iNTS-TCV2 on day 27 (Figure 2(a)) and day 42 (Figure 2(b)). Anti-SEn OAg IgG response in mice treated with SEnGMMA / Alhydrogel versus iNTS-TCV2 on day 27 (Figure 2(c)) and day 42 (Figure 2(d)). Anti-Vi IgG response in mice treated with Vi-CRM197 versus iNTS-TCV2 on day 27 (Figure 2(e)) and day 42 (Figure 2(f)). [Figure 3] Anti-STm OAg and SEn OAg IgG antibody units detected in individual rabbit serum in response to bivalent iNTS-GMMA (total dose of 40 μg). [Figure 4-1]Reaction scheme for the conjugation of S. paratyphi AO antigen to CRM197 using a random CDAP chemical method. [Figure 4-2] Reaction scheme for the conjugation of S. paratyphi AO antigen to CRM197 using a random CDAP chemical method. [Figure 5-1] Both pan-salmonella formulations induced specific serum IgG responses to four antigens, and the antibodies were bactericidal in mice. Figures 5(a)–(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 5(e)–(g) show the SBA results. In Figure 5(e), the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. In Figures 5(f) and (g), each bar represents 42 days after immunization. [Figure 5-2] Both pan-salmonella formulations induced specific serum IgG responses to four antigens, and the antibodies were bactericidal in mice. Figures 5(a)–(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 5(e)–(g) show the SBA results. In Figure 5(e), the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. In Figures 5(f) and (g), each bar represents 42 days after immunization. [Figure 5-3]Both pan-salmonella formulations induced specific serum IgG responses to four antigens, and the antibodies were bactericidal in mice. Figures 5(a)–(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 5(e)–(g) show the SBA results. In Figure 5(e), the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. In Figures 5(f) and (g), each bar represents 42 days after immunization. [Figure 5-4] Both pan-salmonella formulations induced specific serum IgG responses to four antigens, and the antibodies were bactericidal in mice. Figures 5(a)–(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 5(e)–(g) show the SBA results. In Figure 5(e), the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. In Figures 5(f) and (g), each bar represents 42 days after immunization. [Figure 6-1] Evaluation of immune interference between vaccine components in pan-Salmonella formulations. Figures 6(a), (c), (e), and (g) show IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 6(b), (d), and (f) show SBA results. In Figure 6(b), the left-hand column is one day before immunization, and the right-hand column is 42 days after immunization. In Figures 6(d) and (f), each bar represents 42 days after immunization. [Figure 6-2]Evaluation of immune interference between vaccine components in pan-Salmonella formulations. Figures 6(a), (c), (e), and (g) show IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 6(b), (d), and (f) show SBA results. In Figure 6(b), the left-hand column is one day before immunization, and the right-hand column is 42 days after immunization. In Figures 6(d) and (f), each bar represents 42 days after immunization. [Figure 6-3] Evaluation of immune interference between vaccine components in pan-Salmonella formulations. Figures 6(a), (c), (e), and (g) show IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 6(b), (d), and (f) show SBA results. In Figure 6(b), the left-hand column is one day before immunization, and the right-hand column is 42 days after immunization. In Figures 6(d) and (f), each bar represents 42 days after immunization. [Figure 6-4] Evaluation of immune interference between vaccine components in pan-Salmonella formulations. Figures 6(a), (c), (e), and (g) show IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 6(b), (d), and (f) show SBA results. In Figure 6(b), the left-hand column is one day before immunization, and the right-hand column is 42 days after immunization. In Figures 6(d) and (f), each bar represents 42 days after immunization. [Figure 6-5] Evaluation of immune interference between vaccine components in pan-Salmonella formulations. Figures 6(a), (c), (e), and (g) show IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 6(b), (d), and (f) show SBA results. In Figure 6(b), the left-hand column is one day before immunization, and the right-hand column is 42 days after immunization. In Figures 6(d) and (f), each bar represents 42 days after immunization. [Figure 6-6]Evaluation of immune interference between vaccine components in pan-Salmonella formulations. Figures 6(a), (c), (e), and (g) show IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 6(b), (d), and (f) show SBA results. In Figure 6(b), the left-hand column is one day before immunization, and the right-hand column is 42 days after immunization. In Figures 6(d) and (f), each bar represents 42 days after immunization. [Figure 6-7] Evaluation of immune interference between vaccine components in pan-Salmonella formulations. Figures 6(a), (c), (e), and (g) show IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 6(b), (d), and (f) show SBA results. In Figure 6(b), the left-hand column is one day before immunization, and the right-hand column is 42 days after immunization. In Figures 6(d) and (f), each bar represents 42 days after immunization. [Figure 7] The relative abundance percentage of each subclass, calculated as subclass / total subclass percentage. The top segment is IgG3, the next segment is IgG2b, the next segment is IgG2a, and the bottom segment is IgG1. [Figure 8-1] Both tetravalent pansalmonella preparations induce specific serum IgG responses to four antigens, and the antibodies are bactericidal in rabbits. Figures 8(a)-(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 8(e)-(g) show the SBA results, where the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. [Figure 8-2]Both tetravalent pansalmonella preparations induce specific serum IgG responses to four antigens, and the antibodies are bactericidal in rabbits. Figures 8(a)-(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 8(e)-(g) show the SBA results, where the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. [Figure 8-3] Both tetravalent pansalmonella preparations induce specific serum IgG responses to four antigens, and the antibodies are bactericidal in rabbits. Figures 8(a)-(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 8(e)-(g) show the SBA results, where the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. [Figure 8-4] Both tetravalent pansalmonella preparations induce specific serum IgG responses to four antigens, and the antibodies are bactericidal in rabbits. Figures 8(a)-(d) show the IgG responses one day before immunization (left-hand column), 27 days after immunization (center column), and 42 days after immunization (right-hand column). Figures 8(e)-(g) show the SBA results, where the left-hand column (for O:2-CRM197 and ParA GMMA respectively) is one day before immunization, and the right-hand column (for O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization. [Figure 9-1]SBA on heterogeneous panels of mouse serum induced by monovalent components (STm or SEn GMMA) (Figure 9(a) and (b)). SBA on heterogeneous panels of mouse serum induced by bivalent vaccine (STm and SEn GMMA) (Figure 9(c)) and trivalent vaccine iNTS-TCV (STm and SEn GMMA and fVi polysaccharide derived from S. Typhi) (Figure 9(d)). SBA on heterogeneous panels of rabbit serum induced by bivalent vaccine (STm and SEn GMMA) (Figure 9(e)) and trivalent vaccine iNTS-TCV (STm and SEn GMMA and fVi polysaccharide derived from S. Typhi) (Figure 9(f)). [Figure 9-2] SBA on heterogeneous panels of mouse serum induced by monovalent components (STm or SEn GMMA) (Figure 9(a) and (b)). SBA on heterogeneous panels of mouse serum induced by bivalent vaccine (STm and SEn GMMA) (Figure 9(c)) and trivalent vaccine iNTS-TCV (STm and SEn GMMA and fVi polysaccharide derived from S. Typhi) (Figure 9(d)). SBA on heterogeneous panels of rabbit serum induced by bivalent vaccine (STm and SEn GMMA) (Figure 9(e)) and trivalent vaccine iNTS-TCV (STm and SEn GMMA and fVi polysaccharide derived from S. Typhi) (Figure 9(f)). [Figure 9-3] SBA on heterogeneous panels of mouse serum induced by monovalent components (STm or SEn GMMA) (Figure 9(a) and (b)). SBA on heterogeneous panels of mouse serum induced by bivalent vaccine (STm and SEn GMMA) (Figure 9(c)) and trivalent vaccine iNTS-TCV (STm and SEn GMMA and fVi polysaccharide derived from S. Typhi) (Figure 9(d)). SBA on heterogeneous panels of rabbit serum induced by bivalent vaccine (STm and SEn GMMA) (Figure 9(e)) and trivalent vaccine iNTS-TCV (STm and SEn GMMA and fVi polysaccharide derived from S. Typhi) (Figure 9(f)). [Figure 10]The tetravalent pansalmonella formulation induces bactericidal antibodies against a broad panel of Salmonella strains. The panel includes invasive STm isolates from Africa and Southeast Asia, as well as S. enterica serotypes other than STm, SEn, ParA, and Thphi. [Figure 11] The published structure of the O antigen (including the core domain) derived from S. paratyphi A. [Figure 12] The sequence of CRM197. [Modes for carrying out the invention]

[0016] general definition Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention pertains.

[0017] Generally, the term "comprising" is intended to mean "including but not limited to." For example, the phrase "An immunogenic composition comprising a Salmonella Typhimurium antigen" should be interpreted as meaning that the immunogenic composition contains a Salmonella Typhimurium antigen, but the immunogenic composition may also contain further components.

[0018] In some embodiments of the present invention, the word “comprising” is replaced by the phrase “consisting of.” The term “consisting of” is intended to be restrictive. For example, the phrase “An immunogenic composition consisting of a Salmonella Typhimurium antigen” should be understood to mean that the immunogenic composition has a Salmonella Typhimurium antigen and no further components.

[0019] 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 certain further components may be present, i.e., those that do not substantially affect the essential characteristics of the subject.

[0020] The terms "about" or "around" refer to a value that falls within a reasonable range of scientific error. In some cases, a value is "about x" or "around x" if it is within 10%, 5%, or 1% of x.

[0021] The singular forms "a," "an," and "the" refer to multiple objects unless otherwise explicitly indicated in the text. Thus, for example, a reference to "the GMMA" includes two or more examples or versions of such GMMAs.

[0022] All publications, patents, and patent applications cited herein, whether listed above or below, are incorporated herein by reference in their entirety.

[0023] Detailed explanation Salmonella tiphymuria antigen, Salmonella enteritidis antigen, and Salmonella paratiphyta A antigen In some embodiments, the immunogenic composition of the present invention comprises Salmonella tiphimurium antigen. In some embodiments, the immunogenic composition of the present invention comprises Salmonella enteritidis antigen. In some embodiments, the immunogenic composition of the present invention comprises Salmonella paratyphi A antigen.

[0024] O antigen Various S. Typhimurium, S. Enteritidis, and S. Paratyphi A antigens are known to those skilled in the art. In particular, all S. Typhimurium, S. Enteritidis, and S. Paratyphi A bacteria have an outer membrane containing the O antigen, and S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A antigens may also contain the O antigen.

[0025] For the purposes of this invention, the terms O antigen, OAg, and O:2 are interchangeable. The outer membrane of Gram-negative bacteria contains lipopolysaccharide. This lipopolysaccharide contains O antigen linked to the lipid A domain via a core domain. The terms "O antigen," "OAg," and "O:2" refer to a polysaccharide composed solely of O antigen, or more preferably, O antigen linked to the core domain of a lipopolysaccharide.

[0026] As discussed above, the S. Typhimurium antigen, S. Enteritidis antigen, and / or S. Paratyphi A may also be the O antigen. A typical process for purifying these O antigens is based on the Westphal and Jann phenol-water method (Westphal and Jann (1965) Methods Carbohydr. Chem. 5:83-91), first described in the 1960s, followed by detoxification of lipopolysaccharides with acetic acid or anhydrous hydrazine. The O antigen is modified to remove lipid A. For example, polysaccharide extraction and purification can be carried out by acetic acid hydrolysis as described in Watson et al. (1992) Infect Immun. 60(11):4679-86; Konadu et al. (1996) Infect Immun. (7):2709-15; Konadu et al. (1994) Infect Immun. 62(11):5048-54; Ahmed et al. (2006) J Infect Dis. 193(4):515-21; Cox et al. (2011) Glycoconj J 28:165-182; Chu et al. (1991) Infect Immun. 59(12):4450-58; and Micoli et al., 2012 PlosOne, 7(11): e47039.

[0027] 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) (1→3) linked to 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). The published structure of the O antigen derived from S. Paratyphi A, which contains a KDO subunit and a primary amine group (within a pyrophosphoethanolamine group) in the core domain, is shown in Figure 11.

[0028] O antigen conjugate In embodiments in which the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A antigens include the O antigen, the O antigen derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A may be part of a conjugate. The term “conjugate” refers to a molecule formed by a covalent linkage between an antigen (such as the O antigen) and a carrier. The carrier may be a carrier protein. Generally, the conjugation of a polysaccharide to a carrier protein enhances the immunogenicity of the polysaccharide because it converts them from T cell-independent antigens to T cell-dependent antigens, thus enabling the priming of immunological memory.

[0029] Examples of carrier proteins include bacterial toxins such as diphtheria or tetanus toxin, or their toxoids or variants. In some embodiments, the carrier protein is CRM 197 This is CRM. 197 The sequence is provided in Figure 12 (Sequence ID 1).

[0030] In certain embodiments, the S. Paratyphi A antigen comprises the S. Paratyphi AO antigen conjugated to a carrier protein. The carrier protein is diphtheria toxoid or CRM. 197 It may also be the carrier protein, CRM 197 That is the case.

[0031] O antigens derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A can be conjugated to carrier proteins by a method that includes introducing more than one activation site into the O antigen. In some cases, O antigens derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A contain more than one activation site.

[0032] The term “activation site” refers to a site or functional group on a polysaccharide that is activated by a step in a conjugation chemistry process such that it is primed to be conjugated to a carrier protein. For example, when a P antigen is conjugated by a method using CDAP chemistry, the polysaccharide is “activated” by the addition of CDAP if the addition of CDAP introduces cyanoester groups. CDAP activation introduces cyanoester groups to one or more sites, and the locations of these introduced cyanoester groups are considered “activation sites.” Once the O antigen is activated, it can be conjugated to a carrier protein at one or more (in some examples, all) activation sites. For the purposes of this invention, the term “activation site” includes both activated sites not conjugated to a carrier protein and activated sites conjugated to a carrier protein.

[0033] Depending on the circumstances, O antigens derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A may contain 1.5 or more, 2.0 or more, or 2.5 or more activated sites. Assuming that the O antigen is part of a composition containing multiple O antigenic sugars, the O antigen will contain 1.5 or more activated sites if the average number of activated sites on each O antigen molecule in the composition is 1.5 or more.

[0034] In some cases, the S. Paratyphi A antigen is an O antigen conjugated to a carrier protein, and the O antigen is conjugated to a carrier protein by a method comprising introducing one or more activation sites into the S. Paratyphi O antigen and / or S. Paratyphi AO antigen, which contain one or more activation sites.

[0035] O antigens derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A can be conjugated to carrier proteins by CDAP chemistry, possibly via a linker.

[0036] A linker is a compound that can be used to link proteins and polysaccharides. Any suitable linker can be used in the conjugate and method of the present invention. Suitable linkers include the following structures:

[0037] [ka] Examples of compounds possessing this feature include adipic acid dihydrazide (ADH) linkers.

[0038] Other suitable linkers include adipic acid, glutaric acid, carbonyl, β-propionamide (WO00 / 10599), bis(N-hydroxysuccinimide) adipate, dihydrazides similar to ADH but with different chain lengths, hexamethylenediamine (or similar diamines with different chain lengths), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halogens (US Patent No. 4,057,685), and glycosidic bonds (US Patent No. 4,673,574; No. 4,761,283; Examples include (and patents No. 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), and the C4-C12 portion (US Patent No. 4,663,160).

[0039] In embodiments in which O antigens derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A are conjugated to a carrier protein by CDAP chemistry, the O antigens can be conjugated to a carrier protein by a method comprising the step of activating the O antigen by CDAP chemistry to provide an activated O antigen.

[0040] Activation of the O antigen by CDAP chemistry involves mixing the O antigen with CDAP such that cyanoester groups are introduced into the polysaccharide or O antigen. For example, Example 9 discloses a preferred method for activating the O antigen by CDAP chemistry. Activation of the O antigen by CDAP chemistry results in an activated O antigen. Activation of the O antigen by CDAP chemistry introduces cyanoester groups, and therefore, the method includes the step of activating the O antigen by CDAP chemistry if the number of cyanoester groups present on the O antigen after the step of mixing with CDAP is higher than the number of cyanoester groups present on the O antigen before that step. The number of cyanoester groups present can be measured using the ADH quenching / TNBS colorimetric analysis method reported in Lees A., Vaccines (Basel), 2020; 8(4):777.

[0041] In some cases, activation of the O antigen by CDAP chemistry involves mixing the O antigen with CDAP in a w / w ratio (ratio of CDAP to O antigen) of 0.05:1 to 5:1, 0.1:1 to 5:1, 0.2:1 to 2:1, or approximately 0.3:1.

[0042] The step of activating the O antigen by CDAP chemistry, which may include mixing the O antigen with CDAP, is carried out in a salt solution such as a solution of NaCl or KCl. The step of activating the O antigen by CDAP chemistry, which may include mixing the O antigen with CDAP, is carried out in a solution of NaCl or KCl at concentrations of 50 mM to 1 M, 100 mM to 250 mM, 125 mM to 200 mM, or approximately 150 mM.

[0043] If necessary, after mixing the O antigen with CDAP, adjust the pH to pH 6-10, 7-9, or 9-10. If necessary, adjust the pH by adding a base such as triethylamine, sodium hydroxide, or pyridine. If necessary, adjust the pH by adding 5%-15%, 8%-12%, or approximately 10% (v / v) triethylamine. If necessary, after mixing the O antigen with CDAP, incubate the mixture at a temperature of 18°C-30°C, 20°C-28°C, room temperature, or approximately 25°C. If necessary, incubate the solution with stirring before conjugation of the activated O antigen to the carrier protein.

[0044] A preferred method for activating polysaccharides by CDAP chemistry is described in Example 9.

[0045] As described above, O antigens activated using CDAP chemistry (activated polysaccharides or activated O antigens) contain cyanoester groups (at the activation site), and these cyanoester groups can be covalently linked to hydrazide or amino groups. Therefore, O antigens activated using CDAP chemistry can be directly linked to a carrier protein (via amino groups) or conjugated to a carrier protein via a linker containing a hydrazide or amino group. A suitable linker is the ADH linker mentioned above. Thus, activated O antigens can be conjugated to a carrier protein by a method comprising reacting the activated O antigen with a hydrazide / amino group on the carrier protein or a carrier protein-linker compound. Thus, the method may further include a step of preparing a carrier protein-linker compound. For example, if the linker is an ADH linker, the method may include a step of preparing an ADH-carrier protein compound (such as the ADH-CRM197 compound) as reported, for example, Micoli et al., Vaccine 2011, 29, (4), 712-20.

[0046] The reaction between the activated O antigen and the hydrazide / amino group on the carrier protein or carrier protein-linker compound may involve mixing the carrier protein or carrier protein-linker compound with the activated O antigen under conditions suitable for forming a covalent bond between the cyanoester group (activation site) on the activated O antigen and the hydrazide / amino group on the carrier protein or carrier protein-linker compound. For example, this may simply involve mixing the activated O antigen with the carrier protein or carrier protein-linker compound.

[0047] The reaction of the activated O antigen with the hydrazide / amino group on the carrier protein or carrier protein-linker compound may include mixing the activated O antigen with the carrier protein or carrier protein-linker compound in a w / w ratio (ratio of O antigen to carrier protein or carrier protein-linker) of 0.1:1 to 5:1, 0.2:1 to 3:1, 0.5:1 to 2:1, or approximately 1:1. Optionally, the step of mixing the activated O antigen with the carrier protein or carrier protein-linker provides a conjugation mixture. Optionally, the mixing of the activated O antigen with the carrier protein or carrier protein-linker compound is carried out at a pH of 8 to 11, 9 to 10, or approximately 9.5. Optionally, the pH is maintained at 8 to 11, 9 to 10, or approximately 9.5 for at least 1 hour, at least 2 hours, at least 30 minutes to 10 hours, 1 hour to 5 hours, or 2 hours to 3 hours. If necessary, maintain the pH using a base such as triethylamine, sodium hydroxide, or pyridine. If necessary, maintain the pH using triethylamine.

[0048] If applicable, after mixing the activated O antigen and, if applicable, maintaining the mixture at pH 8–11 for at least 1 hour, the method may further include a step of adding a glycine solution (to quench the cyanoester group). If applicable, the glycine solution is added at a concentration of 0.5 M–5 M, 0.5 M–2 M, or approximately 1 M. If applicable, the glycine solution is added to a volume of the conjugation mixture substantially equal to the volume of the glycine solution. One volume is substantially equal to another if the other is within 10%. If applicable, there is a further step of adjusting the pH using a base such as triethylamine, sodium hydroxide, or pyridine. If applicable, this further step is performed after the step of adding the glycine solution. If applicable, the pH is adjusted to pH 7–9, or approximately 8. If applicable, there is an incubation step after the further step of adjusting the pH using a base. Depending on the circumstances, the incubation step may include incubation at temperatures below 15°C, between 12°C and 15°C, below 10°C, between 0°C and 10°C, or between 2°C and 8°C. Depending on the circumstances, the incubation step may be carried out over a period of 10 to 30 hours, or 10 to 20 hours.

[0049] When an activated O antigen is mixed with a carrier protein or carrier protein-linker compound under conditions suitable for conjugation (such as those described in the previous two paragraphs), a conjugate will be formed. The method may further include a chromatographic step to remove any unconjugated O antigens. The chromatographic step may include hydrophobic interaction chromatography or anion exchange chromatography.

[0050] A preferred method for conjugating polysaccharides activated using CDAP to a carrier protein or linker is described in Example 9.

[0051] Outer membrane vesicles In some embodiments, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A antigens contain or consist of outer membrane vesicles such as GMMA.

[0052] For the purposes of this invention, the terms “outer membrane vesicles” or “OMV” are interchangeable 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 the turgor pressure of their cell envelope, and these are natural OMVs. OMVs are immunogenic, cell surface-bound, periplasmic and secretory antigen-rich, and have been used as vaccines.

[0053] 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)), detergent-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)).

[0054] 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 important aspects. First, the membrane structure is modified by the deletion of genes encoding key structural components such as tolR to induce GMMA formation (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 Salmonella bacteria modified to lack the gene encoding a functional TolR).

[0055] The Gram-negative bacteria from which the OMV (GMMA, etc.) of the present invention is purified may be one or more of the group consisting of Salmonella enterica subspecies enterica serotype tiphimulium (Salmonella tiphimulium), Salmonella enterica subspecies enteritidis (Salmonella enteritidis), and Salmonella enterica subspecies enterica serotype paratifi A (Salmonella paratifi A). Suitable purification methods are known in the art and include various filtration and chromatographic methods. A preferred two-step filtration purification process is described in WO 2011 / 036562, which is incorporated herein by reference.

[0056] Modification of Lipid A In some embodiments, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A antigen comprises or consists of GMMA, i.e., GMMA of S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A. The GMMA of S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A may contain modified lipid A, or may be derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria containing modified lipid A. The modified lipid A is lipid A having a different structure compared to the corresponding wild-type lipid A.

[0057] The structure of lipid A can be determined using MALDI-TOF analysis of lipid A isolated from GMMA. For the assay, lipid A is separated after treatment of GMMA with acetic acid and then assayed by MALDI-TOF. GMMA with a protein concentration of approximately 1 mg / mL (micro-BCA 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). 2 μl of the mixture is loaded onto a target plate, the spots are 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 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.

[0058] In some cases, lipid A is 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. The 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. In some cases, modified lipid A is 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 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.

[0059] "Corresponding wild-type lipid A" refers to the lipid A found in the corresponding wild-type bacteria and strains. For example, in the context of GMMA for S. Typhimurium, modified lipid A compared to "corresponding wild-type lipid A" is interpreted as meaning lipid A that has been modified (e.g., so that it is less toxic) compared to the lipid A found in wild-type S. Typhimurium.

[0060] In some cases, the modified lipid A is 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.

[0061] GMMA from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A may be derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria, including any preferred modifications that result in the production of GMMA containing lipid A that is less toxic than wild-type lipid A.

[0062] HtrB, MsbB, and PagP are proteins involved in the production of lipid A 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 do not produce native lipid A, but rather modified, detoxified lipid A. Thus, GMMA of S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A may originate from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria that do not express functional versions of MsbB and / or PagP. In some cases, GMMA from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria may be derived from S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria that do not contain genes encoding functional MsbB and / or PagP proteins.

[0063] By isolating lipid A from GMMA and analyzing its structure using the MALDI-TOF method described above, it is possible to determine whether the bacteria from which GMMA originates express functional versions of MsbB and / or PagP, or whether they contain genes encoding functional MsbB and / or PagP proteins. If lipid A is detoxified, the bacteria from which GMMA originates will not express functional versions of Msb and / or PagP, or will not contain genes encoding functional MsbB and / or PagP proteins.

[0064] In some cases, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates contain mutations in the genes encoding functional proteins (such as htrB, msbB, and / or pagP), and therefore these bacteria do not contain those genes. In some cases, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates do not contain the genes encoding the functional HtrB, MsbB, and / or PagP proteins. In some cases, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates contain the genes encoding at least some of the HtrB, MsbB, and / or PagP proteins, but one of the genes is mutated such that the encoded HtrB, MsbB, and / or PagP proteins are missing one or more important amino acids, or a portion of the gene is deleted. For example, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may contain substitution or deletion mutations in the htrB, msbB, and / or pagP genes. Alternatively, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may have additive mutations in the htrB, msbB, and / or PagP genes, such as additive mutations that cause a frameshift. In some cases, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates 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, with at least 10%, at least 20%, at least 25%, at least 50%, or at least 75% of the htrB, msbB, and / or pagP genes being deleted.In some cases, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates lack the htrB, msbB, and / or pagP genes (e.g., complete deletion of the htrB, msbB, and / or pagP genes (ΔhtrB, ΔmsbB, and / or ΔpagP mutations)).

[0065] In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A in which at least a portion of the msbB and / or pagP genes are replaced by different genes. In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A in which at least a portion of the msbB and / or pagP genes are replaced by the tetracycline (tet) or kanamycin (kan) gene, respectively. In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A in which at least a portion of the msbB and / or pagP genes are replaced by the tetracycline (tet) or kanamycin (kan) gene, respectively. In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A where pagP::kan and / or msbB::tet. "::" indicates that the gene before "::" is replaced by the gene after "::". Therefore, "pagP::kan" means that the pagP gene is replaced by kanamycin.

[0066] Excessive bleb formation The S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may be modified (e.g., genetically modified) to excessively form blebs, i.e., have a greater amount of outer membrane "budding" compared to the corresponding Gram-negative bacteria that do not have the genetic mutation.

[0067] The S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may contain any preferred modifications resulting in excessive bleb formation. In some cases, the modifications are mutations, and for example, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may contain a mutation in a gene encoding a functional protein (such as tolR), and therefore the bacteria may not contain that gene. In some cases, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may not contain the gene encoding the functional TolR protein. In some cases, the S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may contain a gene encoding at least a portion of the TolR protein, but one of the genes is mutated such that the encoded TolR protein is missing one or more important amino acids, or a portion of the gene is deleted. For example, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may contain substitution or deletion mutations in the tolR gene. Alternatively, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may have additive mutations in the tolR gene, such as additive mutations that cause a frameshift. In some cases, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates may contain deletion mutations in the tolR gene. In some cases, the tolR gene contains deletion mutations, with at least 10%, at least 20%, at least 25%, at least 50%, or at least 75% of the tolR gene being deleted. In some cases, S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates lack the tolR gene (e.g., due to a complete deletion of the tolR gene (ΔtolR mutation)).

[0068] In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A in which at least a portion of the tolR gene is replaced by a different gene. In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A in which at least a portion of the tolR gene is replaced by the chloramphenicol acetyltransferase (cat) gene. In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A in which the tolR gene is replaced by the chloramphenicol acetyltransferase (cat) gene. In some cases, the OMV or GMMA of S. Paratyphi A originates from S. Paratyphi A where tolR::cat.

[0069] Whether a genetic modification causes excessive bleb formation in S. Typhimurium, S. Enteritidis, and / or S. Paratyphi A bacteria from which GMMA originates can be tested using the following excessive bleb formation assay. The user should prepare two bacterial cultures. The first culture should contain bacteria with the genetic modification to be tested (test culture), and the second culture should 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 identical conditions and determine 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 into the test culture is greater than the amount released into the reference culture, the genetic modification causes excessive bleb formation in the bacteria. The level of released outer membrane vesicles can be determined, for example, by O antigen quantification according to Example 3.

[0070] KK In some cases, the immunogenic composition may contain GMMA of S. Typhimurium derived from S. Typhimurium strain 2192 (see, for example, De Benedetto et al., 2017, Multiple Techniques for Size Determination of Generalized Modules for Membrane Antigens from Salmonella typhimurium and Salmonella enteritidis. ACS Omega. 2017 Nov 30;2(11):8282-8289). In some cases, the immunogenic composition may contain GMMA of S. Enteritidis derived from S. Enteritidis strain 618 (see, for example, Lanzilao L, Stefanetti G, Saul A, MacLennan CA, Micoli F, Rondini S. Strain Selection for Generation of O-Antigen-Based Glycoconjugate Vaccines against Invasive Nontyphoidal Salmonella Disease. PLoS One. 2015 Oct 7;10(10):e0139847). In some cases, the immunogenic composition may contain GMMA of S. Paratyphi A derived from S. Paratyphi A 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). For example, the S. Paratyphi A strain may be tolR::cat pagP::kan msbB::tet.

[0071] Even if modifications have been made to strain 2192 (e.g., mutations in the msbB, pagP, or tolR genes), if the strain used was based on S. Typhimurium strain 2192, the immunogenic composition will contain GMMA of S. Typhimurium derived from S. Typhimurium strain 2192.

[0072] dose The immunogenic composition may contain S. Paratyphi A O antigen in doses of 1-100 μg, 1-50 μg, 15-50 μg, 20-30 μg, 1-20 μg, 1-10 μg, approximately 25 μg, or approximately 5 μg. The dose of S. Paratyphi A O antigen in the composition can be determined by mild hydrolysis of the O antigen in the immunogenic composition and detection of the amount of paratose by HPAEC-PAD. Paratose is a monosaccharide present in S. Paratyphi A O antigen but not in O antigens derived from S. Enteritidis or S. Typhimurium. A preferred method for determining the amount of S. Paratyphi A O antigen by HPAEC-PAD is described in Example 10. If the S. Paratyphi A O antigen is part of a conjugate (including a carrier protein), the amount of carrier protein may vary. For example, if the ratio of carrier protein to O antigen in the conjugate is greater than 2, the amount of carrier protein present to achieve a 1 μg dose of O antigen is greater than if the ratio of carrier protein to O antigen in the conjugate is less than 2.

[0073] The immunogenic composition of the present invention may contain S. Typhimurium antigen or GMMA of S. Typhimurium in doses 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 of (O antigen). The GMMA used in the immunogenic composition contains the O antigen. The dose of GMMA can be quantified as the dose of O antigen; that is, if the immunogenic composition contains a dose of GMMA of 1 μg (O antigen), then the immunogenic composition contains enough GMMA to yield 1 μg of O antigen to bind to that GMMA (for example, if the GMMA is GMMA of S. Typhimurium, then the immunogenic composition contains GMMA containing a total of 1 μg of S. Typhimurium O antigen). This means that if the immunogenic composition contains GMMA rich in O antigen, the actual amount of GMMA present to achieve a dose of 1 μg of (O antigen) may be less than the amount of GMMA required when the O antigen is low. The amount of S. Typhimurium O antigen present in the immunogenic composition can be determined by mild hydrolysis of the O antigen in the immunogenic composition (to provide monosaccharide avequoise) and detection of the amount of avequoise using HPAEC-PAD. Assuming that no "free" S. Typhimurium O antigen is added, the amount of O antigen in the S. Typhimurium GMMA composition will coincide with the O antigen dose of S. Typhimurium GMMA.

[0074] The immunogenic compositions of the present invention may contain S. Enteritidis antigen or S. Enteritidis GMMA in doses (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. The amount of S. Enteritidis O antigen present in the immunogenic composition can be determined by mild hydrolysis of the O antigen in the immunogenic composition (to provide the monosaccharide tiberose) and detection of the amount of tiberose using HPAEC-PAD. Assuming that no "free" S. Enteritidis O antigen is added, the amount of O antigen in the S. Enteritidis GMMA composition will be equivalent to the O antigen dose of S. Enteritidis GMMA.

[0075] The immunogenic compositions of the present invention may contain GMMA of S. Paratyphi A in doses (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. The amount of S. Paratyphi A O antigen present in the immunogenic composition can be determined by mild hydrolysis of the O antigen in the immunogenic composition (to provide monosaccharide paratose) and detection of the amount of paratose using HPAEC-PAD. Assuming that no "free" S. Paratyphi A O antigen is added, the amount of O antigen in the GMMA of S. Paratyphi A will be equivalent to the O antigen dose of GMMA of S. Paratyphi A.

[0076] The O antigen / protein ratio of S. Paratyphi A OMV or GMMA 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 at most 0.8, 0.9, 1.0, or 2.0. Optionally, the O antigen content can be quantified by HPAEC-PAD, for example, as described in Example 5. Optionally, the protein concentration can be quantified by micro-BCA, for example, as described in PCT / EP2022 / 073501.

[0077] The immunogenic compositions of the present invention may contain doses of fVi polysaccharide in amounts of 1-100 μg, 1-50 μg, 15-50 μg, 20-30 μg, 1-20 μg, 1-10 μg, approximately 25 μg, or approximately 5 μg. The dose of fVi polysaccharide in the composition can be determined by hydrolyzing the fVi polysaccharide in the immunogenic composition (by acid hydrolysis) and detecting the monomeric sugars of the repeating units using HPAEC-PAD. A preferred method for determining the amount of fVi polysaccharide by HPAEC-PAD is described in Example 5. If the fVi polysaccharide is part of a conjugate (containing a carrier protein), the amount of carrier protein may vary. For example, if the ratio of carrier protein to fVi polysaccharide in the conjugate is greater than 2, the amount of carrier protein present to achieve a dose of 1 μg of fVi polysaccharide is greater than if the ratio of carrier protein to fVi polysaccharide in the conjugate is less than 2.

[0078] Salmonella Tiffin Antigen The immunogenic composition may further contain an antigen derived from Salmonella typhi (S. Typhi). In some cases, the antigen derived from S. Typhi is a Vi polysaccharide.

[0079] The term "Vi" or "Vi polysaccharide" refers to the capsular polysaccharide of Salmonella enterica serotype Tiffi purified from Citrobacter (Rondini et al., J. Infect. Dev. Ctries, 2012).

[0080] In some cases, Vi polysaccharides are fragmented Vi polysaccharides (fVi). The term "fragmented" referring to Vi polysaccharides means that they have undergone size reduction, thus reducing the number of repeating units in the polysaccharide. Therefore, fragmented Vi have 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.

[0081] [ka]

[0082] 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 vary and may decrease to about 65% O-acetylation. O-acetylation can be determined by standard measurements such as 1H NMR or colorimetric analysis of hestrin.

[0083] 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. In some cases, fVi polysaccharide has 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.

[0084] Typically, the average molecular weight is calculated by electrophoresis of the sample on a TSK Gel 3000 PWXL column (30 cm x 7.8 mm; particle size 7 μm; cod. 808021) with a TSK Gel PWXL Guard column (4.0 cm x 6.0 mm; particle size 12 μm; cod. 808033) (Tosoh Bioscience) using dextran as a standard (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 (30 min isocratic method). 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] The narrower the molecular weight distribution, the closer the PDI value will be to 1.

[0089] The fVi polysaccharide 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. The fVi polysaccharide 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. The fVi polysaccharide 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.

[0090] 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.

[0091] 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 having a lower average molecular weight than when mechanical methods are used. A preferred method for fragmenting Vi polysaccharide is described in Example 2.

[0092] The fVi polysaccharide may be part of an fVi conjugate containing fVi and a carrier protein. In some cases, the carrier protein in the fVi conjugate may be tetanus toxoid, CRM 197 , or diphtheria toxoid. In some cases, the carrier protein is CRM 197 That is the case.

[0093] fVi polysaccharides can be conjugated to carrier proteins by any suitable conjugation chemistry.

[0094] The conjugation of fVi polysaccharide to the carrier protein may be via an -NH2 group, for example, via the side chain of a lysine or arginine residue 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 of a cysteine ​​residue in the carrier polypeptide. Or, the fVi polysaccharide can be conjugated to the carrier protein via a linker molecule.

[0095] 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).

[0096] 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. Linker-mediated conjugation 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).

[0097] 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.

[0098] In some cases, the fVi conjugate may 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 (or a derivatized carrier protein, if applicable) to produce an fVi conjugate. It can be obtained by a method comprising (i.e., a method for preparing an fVi conjugate), or can be obtained by this method.

[0099] Such a method is described in more detail in WO2015068129.

[0100] The carrier protein can be derivatized by reacting it with a carbodiimide and a linker. In some cases, the carbodiimide is 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. In some cases, derivatization of the carrier protein results in a derivatized carrier protein. In some cases, the carrier protein is CRM197, and the derivatization of the carrier protein comprises one or more of the following steps: (i) Preparing CRM in a suitable buffer, in some cases, a MES buffer 197 ; (ii) Mixing CRM 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 (ratio of CRM to EDAC by weight / weight); 197 197 ; (iii) Mixing CRM and ADH at a ratio of 1:1 to 1:6, 1:2 to 1:4, or approximately 1:3.5 (ratio of CRM to ADH by weight / weight); 197 197 ; (iv) Incubating the mixture of CRM, EDAC, and optionally ADH for at least 30 minutes or for 30 minutes to 2 hours, optionally with stirring; and 197 (v) Purifying the derivatized CRM by tangential flow filtration (crossflow filtration), optionally 197 ; which comprises.

[0101] 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.

[0102] fVi conjugates can be obtained, or may be obtained, by a method comprising the step of reacting fVi polysaccharides with carbodiimide and N-hydroxysuccinimide at a pH of 5-6 to form N-hydroxysuccinimide ester fVi derivatives. In some cases, the carbodiimide is EDC (N-3-dimethylaminopropyl(-N-ethylcarbodiimide)). In some cases, the reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide involves mixing fVi with a carbodiimide such as EDC in the presence of N-hydroxysuccinimide (NHS). In some cases, the reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide involves mixing fVi polysaccharide with NHS. In some cases, the reaction of fVi polysaccharide with carbodiimide and N-hydroxysuccinimide results in an NHS concentration of 0.1 M to 0.5 M, or approximately 0.33 M, and an fVi polysaccharide concentration of 1 mg / mL to 100 mg / mL, or The reaction may involve mixing the fVi polysaccharide with NHS to a concentration of approximately 50 mg / ml. Optionally, the reaction of the fVi polysaccharide with carbodiimide and N-hydroxysuccinimide may involve mixing the fVi polysaccharide with EDC to have a molar ratio of EDC to fVi repeating units of 1:1 to 20:1, 1:1 to 10:0, 2:1 to 7:1, or approximately 5:1. Optionally, the mixing of the fVi polysaccharide with EDC may be performed after mixing the fVi polysaccharide with NHS. Optionally, the reaction of the fVi polysaccharide with carbodiimide and N-hydroxysuccinimide may involve incubating the mixture of the fVi polysaccharide, NHS, and EDC at room temperature for at least 30 minutes, or approximately 1 hour.

[0103] In some cases, the reaction between an N-hydroxysuccinimide ester fVi derivative and a carrier protein (or a derivatized carrier protein) involves mixing the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative). In some cases, the reaction between an N-hydroxysuccinimide ester fVi derivative and a carrier protein (or a derivatized carrier protein) involves mixing the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative) in a ratio of (w / w) 1:0.1 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:2, or approximately 1:1. In some cases, the mixing of the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative) is carried out in a buffer with a pH of 5 to 7, or approximately 6. In some cases, the mixing of the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or a carrier protein derivative) is carried out in MES buffer. Depending on the circumstances, 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.

[0104] 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) Depending on the case, a step to purify the fVi conjugate using hydrophobic interaction chromatography; (iv) If applicable, a step of concentrating the fVi conjugate by tangential flow filtration; and (v) Depending on the case, filter the conjugate using one or more 0.2 μm filters. It may include.

[0105] Depending on the circumstances, a method for preparing an fVi conjugate may include two or more, three or more, four or more, or all five of the steps (i) to (v) above. Depending on the circumstances, the method may include step (i) above. Depending on the circumstances, the method may include steps (i) to (iii) above. Depending on the circumstances, the method may include steps (i) to (v) above. Depending on the circumstances, the method may include steps (i) to (iii) above in the order listed above. Depending on the circumstances, the method may include steps (i) to (v) above in the order listed above.

[0106] EDAC Chemistry uses ADH linker to convert fVi polysaccharide to CRM 197 A preferred method for conjugating is described in Example 2.

[0107] immunogenic composition The immunogenic compositions of the present invention, or used in the present invention, may include additional components such as pharmaceutically acceptable excipients, adjuvants, and / or further antigens.

[0108] The immunogenic composition may further contain pharmaceutically acceptable excipients. A typical “pharmaceutically acceptable excipient” includes any carrier that does not itself induce the production of antibodies harmful to the organism receiving the composition. Preferred carriers are typically large, slowly metabolized polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those skilled in the art. The pharmaceutically acceptable excipient may also contain diluents such as water, saline, or glycerol. Furthermore, auxiliary substances such as wetting agents or emulsifiers and pH buffers may be present. Since phosphates in phosphate-buffered saline can inhibit outer membrane vesicles that bind to aluminum, sterile, pyrogenic Tris-buffered saline is a preferred carrier, especially when using aluminum adjuvants. However, in certain embodiments, the immunogenic composition includes phosphate-buffered saline (and optionally, aluminum adjuvants as further described below). In some cases, the immunogenic composition may contain a pH of 6-7, for example, phosphate-buffered saline with a pH of 6.5.

[0109] Immunogenic compositions can be prepared as injectable liquid solutions or suspensions (suspensions). A solid form suitable for the solution or suspension in a liquid vehicle can also be prepared before injection (e.g., lyophilized compositions or spray-lyophilized compositions). Immunogenic compositions can be prepared for topical administration, for example, as ointments, creams, or powders. Immunogenic compositions can be prepared for oral administration, for example, as tablets or capsules, sprays, or syrups (optionally flavored). Immunogenic compositions can be prepared for pulmonary administration, for example, as inhalants using fine powders or sprays. Compositions can be prepared as suppositories or pessaries. Immunogenic compositions can be prepared for nasal, ocular, or ocular administration, for example, as drops. Immunogenic compositions may also be in kit form, designed so that the combined compositions are reconstituted immediately before administration to a mammal. Such kits may contain one or more antigens in liquid form and one or more lyophilized antigens. The immunogenic composition may be supplied in vials or in pre-filled syringes. Syringes with or without needles may be supplied. The syringes will contain a single dose of the composition, while the vials may contain single or multiple doses.

[0110] The immunogenic compositions of the present invention, or those used in the present invention, can be packaged in unit dose form or multiple dose form. For multiple dose forms, vials are preferred over pre-filled syringes. The effective dose volume can be routinely established, but a typical human dose of the composition has a volume of 0.5 ml, for example, for intramuscular injection.

[0111] The composition is sterile. The immunogenic composition of the present invention, or used in the present invention, may be isotonic with respect to humans.

[0112] Thus, the immunogenic compositions of the present invention, or used in the present invention, may be useful as vaccines. The vaccines according to the present invention may be prophylactic (i.e., prevent infection) or therapeutic (i.e., treat infection), but are typically prophylactic.

[0113] An immunogenic composition used as a vaccine contains an effective amount of antigen, and optionally any other components. “Effective amount” (i.e., immunological 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 to be treated, age, the taxonomic group of the individual to be treated (e.g., non-human primates, primates, etc.), the individual’s ability to synthesize antibodies, the desired level of protection, the vaccine formulation, the medical condition assessment by the treating physician, and other relevant factors. The immunogenic compositions of the present invention may also contain antimicrobial agents, particularly when packaged in multi-dose forms.

[0114] 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 a mineral salt, such as an aluminum salt or a calcium salt. Suitable mineral salts include hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, or mixtures of various mineral compounds in any suitable form (e.g., gel, crystal, amorphous, etc.) with compounds that are preferably adsorbed. Mineral-containing compositions can also be formulated as particles of metal salts.

[0115] 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.

[0116] 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 be composed of, any compound containing ions.

[0117] In some cases, the aluminum adjuvant contains or consists of aluminum hydroxide. The aluminum hydroxide adjuvant may contain or consist of an aluminum hydroxide salt. The aluminum hydroxide adjuvant may contain or consist of an aluminum hydroxide salt that is at least partially crystalline. The aluminum hydroxide salt, which can be represented by the formula AlO(OH), is determined 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 ​​have been seen as having a higher ability for antigen adsorption. Fibrous morphology (e.g., as seen in transmission electron microscopy) is typical for aluminum hydroxide adjuvants.

[0118] Suitable examples of aluminum hydroxide adjuvants will be obvious to those skilled in the art, and include, for example, ALHYDROGEL®.

[0119] 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.

[0120] immunogenicity The examples demonstrate that the immunogenic composition of the present invention exhibits good immunogenicity. Immunogenicity can be measured according to the assay described in Example 6.

[0121] 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; and (b) Step 42: Measure the levels of anti-S. Typhimurium O antigen antibody and / or anti-S. Enteritidis O antigen antibody by ELISA. In an immunogenicity assay including at least 10 3 EU / ml anti-S. Typhimurium O antigen antibody and / or at least 10 3 It can induce EU / ml anti-S. Enteritidis O antigen antibodies.

[0122] When the immunogenic composition is used to immunize mice, at least 10 3 If an immunogenic composition can induce an anti-S. Typhimurium O antigen antibody at a level of EU / ml, it is considered to "induce" this level of antibody. Whether an immunogenic composition can induce this level of antibody when used to immunize mice can be determined by testing a sample of the immunogenic composition using an immunogenicity assay.

[0123] The dosages of GMMA and sugar can be determined as discussed in the section titled "Dosage" above.

[0124] A suitable ELISA is: - Coat the ELISA plate with S. typhimurium or S. enteritidis O antigen; - 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 did not bind to the S. Typhimurium or S. Enteritidis O antigen; and - Detect the amount of anti-S. Typhimurium or S. Enteritidis O antigen antibody bound to the S. Typhimurium or S. Enteritidis O antigen on an ELISA plate using an anti-IgG antibody conjugated to a detection portion such as alkaline phosphatase. It may include.

[0125] 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) Step 42: Measure the level of anti-fVi conjugate antibody by ELISA. In an immunogenicity assay including at least 10 3 It can induce EU / ml anti-fVi conjugate antibodies.

[0126] The dosages of GMMA and sugar can be determined as discussed in the section titled "Dosage" above.

[0127] A suitable ELISA is: - Coat the ELISA plate with fVi ​​polysaccharide; - 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 did not bind to the fVi polysaccharide; and - Detect the amount of anti-fVi conjugate antibody bound to fVi polysaccharide on an ELISA plate using an anti-IgG antibody conjugated to a detection portion such as alkaline phosphatase. It may include.

[0128] 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) Step 42: Measure the anti-S. Paratyphi AO antigen antibody level by ELISA. In an immunogenicity assay including at least 10 3 EU / ml or 10 3.5 It can induce anti-S. paratyphi AO antigen antibodies.

[0129] The dosages of GMMA and sugar can be determined as discussed in the section titled "Dosage" above.

[0130] A suitable ELISA is: - Coat the ELISA plate with S. paratyphi AO antigen; - 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 did not bind to the S. Paratyphi AO antigen; and - Detect the amount of anti-S. Paratyphi AO antigen antibody bound to the S. Paratyphi AO antigen on an ELISA plate using an anti-IgG antibody conjugated to a detection portion such as alkaline phosphatase. It may include.

[0131] In the immunogenic composition of the present invention, the level of anti-S. Typhimurium O antigen antibody induced may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the level of anti-S. Typhimurium O antigen antibody induced by the corresponding monovalent S. Typhimurium immunogenic composition. The levels of antibodies in the immunogenic composition and the corresponding monovalent immunogenic composition can be determined using the "preferred ELISA" described above. The "corresponding monovalent S. Typhimurium immunogenic composition" is identical to the immunogenic composition except that the only antigen present is the S. Typhimurium antigen. For example, if the immunogenic composition of the present invention contains 5 μg of O antigen derived from S. Typhimurium, O antigen derived from S. Enteritidis, fVi polysaccharide conjugate, aluminum adjuvant, and phosphate-buffered saline, the corresponding monovalent S. Typhimurium immunogenic composition contains 5 μg of O antigen derived from S. Typhimurium, aluminum adjuvant, and phosphate-buffered saline.

[0132] In the immunogenic composition of the present invention, the level of anti-S. Enteritidis O antigen antibody induced may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the level of anti-S. Enteritidis O antigen antibody induced by the corresponding monovalent S. Enteritidis immunogenic composition. The levels of antibodies in the immunogenic composition and the corresponding monovalent immunogenic composition can be determined using the "preferred ELISA" described above. The "corresponding monovalent S. Enteritidis immunogenic composition" is identical to the immunogenic composition except that the only antigen present is the S. Enteritidis antigen. For example, if the immunogenic composition of the present invention contains S. Typhimurium-derived O antigen, 5 μg of S. Enteritidis-derived O antigen, fVi polysaccharide conjugate, aluminum adjuvant, and phosphate-buffered saline, the corresponding monovalent S. Enteritidis immunogenic composition contains 5 μg of S. Enteritidis-derived O antigen, aluminum adjuvant, and phosphate-buffered saline.

[0133] In the immunogenic composition of the present invention, the level of anti-fVi conjugate antibody induced may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the level of anti-fVi conjugate antibody induced by the corresponding monovalent fVi polysaccharide immunogenic composition. The levels of antibody in the immunogenic composition and the corresponding monovalent immunogenic composition can be determined using the "preferred ELISA" described above. The "corresponding monovalent fVi polysaccharide immunogenic composition" is identical to the immunogenic composition except that the only antigen present is the fVi polysaccharide antigen. For example, if the immunogenic composition of the present invention contains O antigen derived from S. Typhimurium, O antigen derived from S. Enteritidis, 5 μg of fVi polysaccharide conjugate, aluminum adjuvant, and phosphate-buffered saline, the corresponding monovalent fVi polysaccharide immunogenic composition contains 5 μg of fVi polysaccharide, aluminum adjuvant, and phosphate-buffered saline.

[0134] In the immunogenic composition of the present invention, the level of anti-S. Paratyphi AO antigen antibody induced may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the level of anti-S. Paratyphi AO antigen antibody induced by the corresponding monovalent S. Paratyphi A immunogenic composition. The levels of antibodies in the immunogenic composition and the corresponding monovalent immunogenic composition can be determined using the "preferred ELISA" described above. The "corresponding monovalent S. Paratyphi A immunogenic composition" is identical to the immunogenic composition except that the only antigen present is the S. Paratyphi A antigen. For example, if the immunogenic composition of the present invention contains O antigen derived from S. Typhimurium, 5 μg of O antigen derived from S. Paratyphi A, fVi polysaccharide conjugate, aluminum adjuvant, and phosphate-buffered saline, the corresponding monovalent S. Paratyphi A immunogenic composition contains 5 μg of O antigen derived from S. Paratyphi A, aluminum adjuvant, and phosphate-buffered saline.

[0135] In the immunogenic composition or method of the present invention, the levels of anti-S. Typhimurium O antigen antibody, anti-S. Enteritidis O antigen antibody, anti-fVi conjugate antibody and / or anti-S. Paratyphi AO antigen antibody are as follows: (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) Step 42: Measure the levels of anti-S. Typhimurium, anti-S. Enteritidis, anti-fVi conjugate antibody and / or anti-S. Paratyphi AO antigen antibody by ELISA. It is measured in immunogenicity assays that include [specific component].

[0136] 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) S. Typhimurium (Salmonella enterica Serovar 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) S. Typhimurium (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) S. Typhimurium (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) S. Typhimurium (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) S. Typhimurium (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) S. enterica serotype Derby (Salmonella porcine) (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) S. enterica serotype Dublin (bovine salmonella) (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) S. 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) S. Enteritidis (Enterococcus enterica) 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) S. 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.

[0137] The immunogenic composition of the present invention is (i) S. Enteritidis and S. Dublin; (ii) S. Typhimurium, S. Derby, and S. Dublin; (iii) S. Typhimurium, S. Enteritidis, S. Derby and S. Dublin; or (iv) S. Typhimurium, S. Enteritidis, S. Derby, S. Dublin and S. Paratyphi A Antibodies against it may be induced.

[0138] An immunogenic composition "induces" antibodies against (e.g.) S. Typhimurium ST34 if it can induce these antibodies when used to immunize mice. Whether an immunogenic composition can induce these antibodies when used to immunize mice can be determined by testing a sample of the immunogenic composition using a cross-protection assay.

[0139] Whether an immunogenic composition induces antibodies against one or more of the above-mentioned strains can be determined by performing a cross-protection assay. Specifically, the user can: - Immunize mice intraperitoneally with 500 μL of immunogenic composition; and - The level of bactericidal antibodies produced against the target strain can be measured using a serum bactericidal assay (SBA).

[0140] The SBA assay may be based on the assay described in Example 6, except that the user should measure bactericidal activity against the target strains listed above (e.g., Salmonella paratifi A NVGH308).

[0141] In some cases, the IC50 (serum dilution that gives 50% inhibition of ATP levels) obtained in the SBA assay is 10 2 If the threshold exceeds this, the immunogenic composition induces antibodies against the other strain mentioned above.

[0142] 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) Step 42: Measuring the level of the anti-S. Paratyphi AO antigen antibody subtype by ELISA. When determined using an antibody class assay including [specific antibody class], anti-S. Paratyphi A antibodies can be induced in each of the IgG3, IgG2b, IgG2a, and IgG1 classes, respectively.

[0143] An immunogenic composition "induces" anti-S. Paratyphi A antibodies in the respective 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-S. Paratyphi AO antigen antibody subtypes described below.

[0144] A suitable ELISA for measuring the level of S. paratyphi AO antigen antibody subtypes is: - Coat the ELISA plate with S. paratyphi AO antigen; - 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 did not bind to the S. Paratyphi AO antigen; and - Detecting the amount of anti-S. Paratyphi AO antigen IgG3 antibody conjugated to a detection portion such as alkaline phosphatase on an ELISA plate; - Repeat the first three steps, using an anti-IgG2b antibody instead of an anti-IgG3 antibody, then an anti-IgG2a antibody instead of an anti-IgG3 antibody, and finally an anti-IgG1 antibody instead of an anti-IgG3 antibody. It may include.

[0145] Tolerance "Tolerogenic" or "tolerogenic" refers to an immunogenic composition that does not induce a significant negative response in the recipient. In particular, immunogenic compositions containing outer membrane vesicles are known to induce fever in patients to whom they are administered. Therefore, an immunogenic composition may be judged to be tolerogenic if it does not induce a significant fever.

[0146] An immunogenic composition is considered to "induce" fever if it induces fever when used to immunize mice. Whether or not it induces fever when used to immunize mice can be determined by testing a sample of the immunogenic composition using the toxicity assay described below.

[0147] The immunogenic composition is prepared in the following steps; (a) A step of measuring the rabbit's initial body temperature; (b) A step of administering an immunogenic composition to rabbits in doses of 20 μg of (O antigen) per GMMA and 25 μg of sugar per sugar conjugate; (c) A step of monitoring the rabbit's body temperature for 5 hours; and (d) Step to record the rabbit's maximum body temperature Includes, In a toxicity assay where the body temperature rise is calculated as equal to the rabbit's maximum body temperature minus its initial body temperature, an immunogenic composition may be tolerogenic if it induces a body temperature rise of less than 1.8°C, less than 1.7°C, less than 1.6°C, or less than 1.5°C.

[0148] The immunogenic composition is prepared in the following steps; (a) A step of administering an immunogenic composition to rabbits in doses of 20 μg of (O antigen) per GMMA and 25 μg per sugar; (b) A step of monitoring the rabbit's body temperature for 5 hours; and (c) Steps to record the rabbit's maximum body temperature In toxicity assays involving immunogenic compositions, if an immunogenic composition induces a maximum body temperature of 41°C or lower, 40.9°C or lower, or 40.8°C or lower, it may be tolerogenic.

[0149] How to boost your immune response A further aspect of the present invention provides a method for boosting an immune response to an antigen, comprising administering a composition comprising the antigen and GMMA. Also provided is a method for preventing infection by Salmonella enterica bacteria, comprising administering an immunogenic composition comprising the antigen of Salmonella enterica and GMMA, wherein the administration of GMMA boosts an immune response to Salmonella enterica bacteria.

[0150] For example, the method of the present invention may include boosting an immune response to S. Typhi or S. Paratyphi A antigen by administering a composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA. The method of the present invention may also include a method for preventing infection by S. Typhi or S. Paratyphi A by administering an immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA, wherein GMMA boosts an immune response to S. Typhi antigen or S. Paratyphi A antigen.

[0151] The present invention also provides an immunogenic composition comprising GMMA for use in a method of boosting an immune response to S. Typhi or S. Paratyphi A antigen, comprising administering an immunogenic composition comprising S. Typhi or S. Paratyphi A antigen and GMMA. The present invention also provides an immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA for use in a method of preventing infection by S. Typhi or S. Paratyphi A, wherein GMMA boosts an immune response to S. Typhi or S. Paratyphi A antigen.

[0152] In a method or immunogenic composition for use of the present invention, the method may be a method for boosting an immune response to S. Typhi antigen, and the immunogenic composition may contain S. Typhi antigen. In a method or immunogenic composition for use of the present invention, the method may be a method for boosting an immune response to S. Paratyphi A antigen, and the immunogenic composition may contain S. Paratyphi A antigen.

[0153] In a method or immunogenic composition for use of the present invention, the method may be a method for preventing infection by S. Typhi, the immunogenic composition may contain S. Typhi antigen, and GMMA may boost the immune response to S. Typhi antigen. In a method or immunogenic composition for use of the present invention, the method may be a method for preventing infection by S. Paratyphi A, the immunogenic composition may contain S. Paratyphi A antigen, and GMMA may boost the immune response to S. Paratyphi A antigen.

[0154] A method or immunogenic composition for use of the present invention, wherein GMMA comprises at least one selected from the group consisting of GMMA of S. Typhimurium, GMMA of S. Enteritidis, and GMMA of S. Paratyphi A. GMMA of S. Typhimurium may boost the immune response to S. Typhi or S. Paratyphi A antigen. GMMA of S. Paratyphi A may boost the immune response to S. Typhi antigen. GMMA of S. Enteritidis may boost the immune response to S. Typhi or S. Paratyphi A antigen. GMMA of S. Paratyphi may boost the immune response to S. Typhi antigen.

[0155] If the immune response to S. Typhi or S. Paratyphi A antigen is higher when S. Typhi or S. Paratyphi A antigen is part of an immunogenic composition containing GMMA compared to when S. Typhi or S. Paratyphi A antigen is not part of an immunogenic composition containing GMMA, then the method is a method that "boosts the immune response" to S. Typhi or S. Paratyphi A antigen. This is due to the adjuvant effect of GMMA. In other words, when GMMA and S. Typhi or S. Paratyphi A antigen are in the same composition, GMMA increases the immune response to S. Typhi or S. Paratyphi A antigen compared to S. Typhi or S. Paratyphi A antigen alone.

[0156] To boost the immune response to S. typhi antigen or S. paratyphi A antigen, the S. typhi antigen and S. paratyphi A antigen may be polysaccharides. In some cases, the S. typhi antigen may contain fVi polysaccharide, and in some cases, the fVi polysaccharide is part of an fVi conjugate containing fVi and a carrier protein, and further in some cases, the carrier protein is CRM 197 In some cases, the S. Paratyphi A antigen may also contain the S. Paratyphi A O antigen, and in some cases, the S. Paratyphi A O antigen is conjugated to a carrier protein, and in some cases, the carrier protein is CRM 197 That is the case.

[0157] If the immune response to S. Typhi antigen or S. Paratyphi A antigen is higher when S. Typhi antigen or S. Paratyphi A antigen is part of an immunogenic composition containing GMMA compared to when S. Typhi antigen or S. Paratyphi A antigen is not part of an immunogenic composition containing GMMA, then GMMA boosts the immune response to S. Typhi antigen or S. Paratyphi A antigen.

[0158] If the immune response to S. Typhi antigen or S. Paratyphi A antigen is at least 5-fold, at least 10-fold, or at least 20-fold higher when S. Typhi antigen or S. Paratyphi A antigen is part of an immunogenic composition containing GMMA compared to the immune response to S. Typhi antigen or S. Paratyphi A antigen when S. Typhi antigen or S. Paratyphi A antigen is not part of an immunogenic composition containing GMMA, then the method is a method for boosting the immune response to S. Typhi antigen or S. Paratyphi A antigen, or GMMA boosts the immune response to S. Typhi antigen or S. Paratyphi A antigen.

[0159] The immune response to S. Typhi antigen or S. Paratyphi A antigen produced by the immunogenic composition of the present invention or by the method of the present invention may be the number of antibodies produced when determined by ELISA 42 days after administration of an immunogenic composition containing S. Typhi antigen or S. Paratyphi A antigen and GMMA at a dose of 0.78 μg of S. Typhi antigen or S. Paratyphi A antigen and 0.63 μg (O antigen) of GMMA.

[0160] A suitable ELISA is: - Coat the ELISA plate with S. typhi antigen or S. paratyphi AO antigen; - 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 did not bind to the S. Typhi antigen or S. Paratyphi AO antigen; and - Detect the amount of anti-S. Typhi antigen or S. Paratyphi AO antigen antibody bound to the S. Typhi antigen or S. Paratyphi AO antigen on an ELISA plate using an anti-IgG antibody conjugated to a detection portion such as alkaline phosphatase. It may include.

[0161] Medical use and treatment methods Further aspects of the present invention provide an immunogenic composition of the present invention for use in a method for preventing infection. Further aspects of the present invention provide a method 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 a pharmacopoeia for use in a method for preventing infection. The prevention method of the present invention may comprise administering an effective amount of the immunogenic composition or vaccine of the present invention to a subject.

[0162] Methods of preventing infection may also be methods of preventing Salmonella infection. In some cases, methods of preventing infection may be methods of preventing infection by S. Typhimurium, S. Enteritidis, S. Typhi and / or S. Paratyphi A.

[0163] 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.

[0164] The subject of this invention is mammals, and optionally humans. If the vaccine is for prophylactic use, the human may be an adult, i.e., the subject is 18 years of age or older. 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.

[0165] If the vaccine is for preventative use, children may be approximately 9 months of age.

[0166] If the vaccine is for therapeutic use, the human subject is preferably a child.

[0167] Vaccines intended for children can also be administered to adults to evaluate, for example, their safety, dosage, or immunogenicity.

[0168] (Examples) [Example 1] GMMA production of S. typhimurium and S. enteritidis Salmonella enterica serotype tiphimurum 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).

[0169] Salmonella enterica serotype enteritidis WT strain 618 was provided by Quotient Bioresearch Limited, UK. The animal-derived strain was isolated by European Antimicrobial Susceptibility Surveillance in Animals (EASSA).

[0170] From the above Salmonella strains, recombinant mutants of Salmonella tiphimurium ΔtolRΔpagPΔmsbB and Salmonella enteritidis ΔtolRΔpagPΔmsbB were generated for each strain, 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).

[0171] GMMAs derived from the above Salmonella strains (GMMA from Salmonella tiphimuria (STmGMMA) and GMMA from Salmonella enteritidis (SEnGMMA)) were purified and isolated. The GMMAs were purified using a method similar to that previously reported for GMMA from S. sonnei (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.

[0172] [Example 2] Production of fVi-CRM197 conjugate for immunization against S. typhi Using the following protocol, divalent compositions were produced containing the S. Paratyphi A OAg-CRM197 conjugate prepared as described above and a conjugate of fragmented Vi polysaccharide (fVi) derived from S. Typhi conjugated to CRM197.

[0173] 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 min. 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.

[0174] 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.

[0175] Step 3: Stabilization of fVi polysaccharide: After fragmentation, stabilize the 30kDa retained product by incubation at 80±5℃ for 120±15min.

[0176] 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 molecular size (kDa) distribution.

[0177] Step 5: Desalting: The pooled Capto-Q fraction is concentrated by tangential flow filtration (TFF) using a cassette with a 10 kDa cutoff, and then dialyzed using WFI until the conductivity of the filtration product reaches 30 μS / cm or less.

[0178] Step 6: Filtration of fVi polysaccharide through a 0.2 μm filter: Filter the fVi polysaccharide through a 0.22 μm filter. Store the purified fVi polysaccharide in a PETG bottle.

[0179] 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.

[0180] Step 2: Buffer exchange with 100mM MES buffer: CRM 197 After thawing, buffer replacement is performed using TFF with 100 mM MES buffer (pH 6.0 ± 0.2) in a 10 kDa cassette.

[0181] Step 3: CRM 197 Derivatization: CRM at the required concentration197 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 under mixed conditions for 60 ± 15 minutes. At the end of the reaction, an equal volume of 5 mM MES buffer (pH 7.0 ± 0.2) is added.

[0182] Step 4: CRM 197 Purification: After the reaction, CRM197 is purified by TFF using a 10kDa cassette containing 5mM MES buffer.

[0183] Step 5: Dialysis-filtered CRM 197 Filtration: Dialysis-filtered CRM 197 After filtering the solution through a 0.2 μm filter, store it in a glass bottle at 2-8°C.

[0184] 3) Conjugation of fragmented Vi polysaccharide with derivatized CRM197: Stabilization 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.

[0185] 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. 197This 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.

[0186] 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.

[0187] 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.

[0188] Step 3: Filtration of the conjugation mixture: fVi-CRM 197 Filter the crude conjugate through a 0.65 filter.

[0189] Step 4: fVi-CRM 197Purification 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.

[0190] 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.

[0191] Step 6: Use a 0.2 μm filter with fVi-CRM 197 Pre-filtration of conjugate: Filter the fVi-CRM197 conjugate through a 0.2 μm filter to reduce bioburden.

[0192] 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.

[0193] [Example 3] Formulation of a bivalent (iNTS-GMMA) vaccine against S. typhimurium and S. enteritidis adsorbed onto aluminum hydroxide adjuvant. Aluminum hydroxide (Alum) in phosphate-buffered saline (pH 6.5) 3+ STmGMMA and SEnGMMA were separately adsorbed to a final concentration of 0.7 mg / mL, and two different pharmaceutical formulations were obtained by vialing each in a 3 mL Type I 2R vial. The bulk GMMA solution was diluted to obtain a GMMA concentration of 80 μg / ml of O antigen and a 0.7 mg / ml aluminum hydroxide (Al3+) concentration, based on the amount of O-Ag measured in bulk GMMAx. Before administration, the contents of the STmGMMA / Alhydrogel pharmaceutical vial were mixed with the contents of the SEnGMMA / Alhydrogel vial to obtain an iNTS-GMMA vaccine containing each O antigen at a final concentration of 40 μg / mL adsorbed onto aluminum hydroxide (final concentration 0.7 mg / mL) in phosphate-buffered saline, thereby forming the final mixed pharmaceutical product.

[0194] The amount of OAg in the final mixed drug was also determined. The assay protocol used to evaluate the amount of OAg is described in the following paragraph.

[0195] Quantification of Salmonella enteritidis O antigen on bulk GMMA or final mixed pharmaceutical formulations using HPAEC-PAD: Quantification of OAg in S. enteritidis. GMMA is performed by high-speed anion exchange chromatography (HPAEC-PAD) analysis using pulsed current detection after acid hydrolysis of the sample. Quantification determines the concentrations of rhamnose, galactose, glucose, and mannose present in the sample.

[0196] OAg is quantified based on the known sugar ratios present in the OAg repeating units 1xRha (rhamnose); 1xGal (galactose); 1xMan (mannose); and 1xTyv (tiverose). Glucose is calculated from the glucose measured in the analysis after subtracting the glucose attributable to the core.

[0197] Standard dilution series of each sugar in the range of 0.5 - 10 μg / mL are electrophoresed in each HPAEC-PAD analysis, and the μg / mL of the corresponding sugar present in the OAg of the sample is interpolated using the peak area.

[0198] 450 μL of the dilution of each of the standard and the sample are simultaneously treated with 150 μL of 8M TFA at 100 °C for 4 hours. Then the sample is cooled at 2 °C - 8 °C for about 30 minutes, dried in a centrifugal evaporator, resuspended in 450 μL of water, filtered and analyzed. HPAEC-PAD is performed using a Dionex ICS3000 (or 5000) equipped with a CarboPac PA10 column connected to a PA10 guard column. The separation is carried out at 25 °C using the following conditions: · 20 min, 18 mM NaOH, flow rate 1 mL / min (separation step) · 10 min, 28 mM NaOH, 100 mM sodium acetate, flow rate 1 mL / min (column washing step) · 20 min, 18 mM NaOH, flow rate 1 mL / min (column equilibration step). The effluent is monitored using an electrochemical detector.

[0199] Quantification of Salmonella Typhimurium O antigen on bulk GMMA by HPAEC-PAD Quantification of OAg in GMMA of S. Typhimurium is performed using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) after GMMA acid hydrolysis. Standard dilution series of solutions containing rhamnose, galactose, glucose and mannose are electrophoresed in each HPAEC-PAD analysis series and used as calibration curves to quantify the sugars present in the samples.

[0200] The quantification of OAg is performed based on the known sugar ratios present in the OAg repeating unit 1xRha (rhamnose); 1xGal (galactose); 1x(mannose); 1xAbe (abequose), and glucose is calculated from the glucose measured in the analysis after subtracting the glucose present in the core.

[0201] Quantification of OAg in the final mixed pharmaceutical (SEn-GMMA / STm GMMA) by HPAEC-PAD The quantification of OAg of S. Typhimurium or S. Enteritidis on GMMA adsorbed on aluminum hydroxide is performed using HPAEC-PAD after GMMA acid hydrolysis. Standard dilution series of solutions containing fucose, rhamnose, N-acetylglucosamine, glucose, galactose, and mannose are electrophoresed in each HPAEC-PAD analysis series and used as calibration curves to quantify rhamnose, glucose, galactose, and mannose in the samples. The procedure is the same as that used for the quantification of the raw materials of S. Typhimurium and S. Enteritidis.

[0202] Add 150 μL of 8M TFA to each 450 μL dilution of the standard and the sample. Heat all vials in parallel at 100 °C for 4 hours. Then cool the samples at 2 °C - 8 °C for about 30 minutes, dry in a centrifugal evaporator, resuspend in 450 μL of water, filter, and analyze. Perform HPAEC-PAD using a Dionex ICS3000 (or 5000) equipped with a CarboPac PA10 column connected to a PA10 guard column.

[0203] Separation is performed at 25 °C using the following conditions: · 20 min, 18 mM NaOH, flow rate 1 mL / min (separation step) · 10 min, 28 mM NaOH, 100 mM sodium acetate, flow rate 1 mL / min (column washing step) · 20 min, 18 mM NaOH, flow rate 1 mL / min (column equilibration step).

[0204] The effluent is monitored using an electrochemical detector.

[0205] OAg quantification is performed based on the known sugar ratios present in the OAg repeating units in the formulation (Rha; Glc; Gal; Man and tiberose (SEn) or avequoise (STm) equal to Rha). After quantifying rhamnose, glucose, galactose, and mannose directly as nmol / mL from the analysis, the amount of OAg is calculated considering the molecular weight of each sugar and its content in the repeating units (OAg = 600.61). * Rha+162.15 * Glc total -162.15 * Gal total +162.15 * Man).

[0206] [Example 4] Adsorption of SEnGMMA and STmGMMA to aluminum hydroxide enhanced in vivo tolerability in rabbits. The effects of adsorption of SEnGMMA and STmGMMA onto aluminum hydroxide were investigated, and it was determined that adsorption onto aluminum hydroxide reduces the exothermic properties of GMMA.

[0207] A group of three New Zealand white rabbits were compared with saline control and Alhydrogel control (Alhydrogel (0.7 mg / ml Al). 3+Immunization in muscle was performed using one of the following vaccines: (1) STmGMMA adsorbed to Alhydrogel (20 μg of O antigen in a 0.5 mL dose) (STmGMMA / hydrogel), (2) SEnGMMA adsorbed to Alhydrogel (20 μg of O antigen in a 0.5 mL dose) (SEnGMMA / hydrogel), (3) iNTS-GMMA vaccine (20 μg of O antigen in a 0.5 mL dose), or (4) bivalent STmGMMA and SEnGMMA vaccines with a dose of 2 μg of O antigen per 0.5 mL of GMMA that is not adsorbed to Alhydrogel. These four types of vaccines were prepared as described in Examples 1-3.

[0208] The rabbits' body temperature was monitored for 5 hours after vaccination.

[0209] The initial body temperature of each rabbit was within the range of 38.0–39.8°C, and the temperature difference between rabbits within the group was less than 1.0°C. In rabbits treated with Alhydrogel control or saline, the absolute body temperature after administration did not exceed 39.2°C. In one rabbit treated with STmGMMA / Alhydrogel, the peak absolute body temperature was 40.6°C; the temperatures of the other two rabbits in this group did not exceed 39.6°C. In three rabbits treated with SEnGMMA / Alhydrogel, the measured peak body temperatures were 39.3°C, 39.8°C, and 40.2°C. In the two-component iNTS-GMMA vaccine group (the third group above), the measured peak body temperature did not exceed 40.4°C, and the maximum temperature increase in individual rabbits was 0.9°C, 1.2°C, and 1.5°C. On the other hand, in rabbits treated with a mixture of unadsorbed STmGMMA and SEnGMMA (Group 4 above), the measured maximum body temperature was 41.3°C, which was similar in all three rabbits. Peak body temperature was reached in all rabbits during a 5-hour period (typically by 180–270 minutes). The rabbits that received the Alhydrogel formulation (Groups 1–3 above) showed a mean maximum body temperature increase of 1°C–1.3°C, while the rabbits that received unformulated GMMA at one-tenth the dose (Group 4 above) showed a mean maximum body temperature increase of 1.9°C. The group that received unformulated GMMA and reached peak earlier (typically by 120–180 minutes) had a steeper, higher rise and a steeper, more rapid decline.

[0210] [Example 5] Formulation of a trivalent (iNTS-TCV) vaccine against Salmonella tiphimulium, S. enteritidis, and S. typhi. STmGMMA and SEnGMMA were successively adsorbed into aluminum hydroxide phosphate-buffered saline at pH 6.5. The adsorbed STmGMMA and SEnGMMA were then mixed together, and after the addition of a phosphate quencher, fVi-CRM197 suspended in sodium phosphate-buffered saline was subsequently added.

[0211] To prepare solutions of the precise concentrations of STmGMMA and SEnGMMA shown above, the amount of O antigen (OAg) in the bulk GMMA solution was determined, and then a method for diluting the bulk GMMA to obtain the precise GMMA concentration was determined using this determination. The amounts of OAg and Vi polysaccharide in the final mixed pharmaceutical product were also determined. The protocol for determining the amount of O antigen in STmGMMA and SEnGMMA is described in Example 3. Similarly, the following paragraphs describe fVi-CRM 197 Summarize the protocol used to add the precise amount of conjugate.

[0212] Identity and quantification of STm and SEn OAg in the final mixed drug iNTS-TCV by FAcE The identity and quantification of the O antigen are evaluated by a FAcE (Factorized Alhydrogel Competitive ELISA) assay, designed to detect single antigenic components of S. Enteritidis and S. Typhimurium OAg polysaccharides in the final formulation. The FAcE assay is a competitive ELISA method in which serotype-specific anti-Salmonella OAg monoclonal antibodies (mAbs) bind to the respective OAg in the OAg coated on the ELISA plate and the OAg in the formulated GMMA suspension present in the ELISA wells. The more serotype-specific OAg present in the formulated GMMA suspension, the more mAbs bind to them, and the fewer mAbs are available to bind to the coated antigen. The ELISA signal is given by the binding of mouse mAbs to the coated OAg; therefore, the lowest signal is obtained at the highest GMMA concentration, and vice versa. After detecting mAb binding using an enzyme-labeled anti-mouse antibody, the substrate solution is added, and the formation of yellow is detected by absorbance at 405 and 490 nm. The results are calculated as the OD difference between 405 nm and 490 nm. The quantification of the target antigen in the test sample by FAcE assay is obtained using a reference standard curve constructed with serial dilutions of freshly formulated serotype-specific GMMA of aluminum hydroxide, starting from a known concentration of OA g μg / mL. The test sample is assayed with different dilutions selected to fit within the linear and central portion of the standard curve. The amount of OA g in the test sample is calculated by 4-parameter logistic regression analysis and interpolation of absorbance readings into the fitted standard curve. Since the quantification of OA g by FAcE is performed using an anti-OA g specific mAb, this also confirms the identity of the OA g.

[0213] Identity and quantification of Vi in the final mixed drug iNTS-TCV using HPAEC-PAD The identity and quantification of Vi polysaccharides are performed by HPAEC-PAD analysis after acid hydrolysis of the sample using the following protocol.

[0214] Hydrolyze the Vi polysaccharide into monomeric sugars of the repeating unit corresponding to the chromatographic peak. Prepare a dilution series of 0.16 - 5 μg / mL of the Vi standard and electrophorese it in each HPAEC-PAD analysis. Use the area of the resulting peak to construct a standard curve, interpolate the peak area of the unknown sample, and quantify the corresponding μg / mL.

[0215] Prepare the standard calibration curve by acid hydrolysis; perform the acid hydrolysis of the sample and the standard simultaneously, and using this method, the concentration of the polysaccharide present in the sample can be determined.

[0216] Add 1000 μL of the TFA / HCl mixture (1:6.7 v / v) to 300 μL of each dilution of the standard curve and the sample. Heat all vials simultaneously at 80 °C for 4.5 hours (final concentration: 8 M HCl, 10% TFA). Then cool the sample at 2 - 8 °C for about 15 minutes, dry it under a nitrogen stream and finally in a centrifugal evaporator for at least 1.5 hours, resuspend it in 300 μL of water, filter it, and analyze it. Perform HPAEC-PAD using a Dionex ICS3000 (or 5000) equipped with a CarboPac PA1 column connected to a PA1 guard column. Perform the separation at 25 °C using the following conditions: 15 min, 400 mM NaOH, flow rate 1.5 mL / min (the washing step is not required during injection). Monitor the effluent using an electrochemical detector.

[0217] [Example 6] Immunogenicity assay [[ID=十六]]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.

[0218] SBA Evaluation of serum bactericidal activity by SBA. Individual mouse serums collected on day 42 were heat-inactivated (HI) at 56°C for 30 min and then tested in a serum bactericidal assay based on luminescence readings against Salmonella paratyphi A NVGH308, Salmonella enteritidis 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). As previously described, dilutions of HI test serum were incubated for 3 hours in the presence of exogenous complement (rabbit complement [RBC]) and bacteria (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 min, 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 min, 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.

[0219] IgG subclass Individual serum samples isolated from blood samples taken from immunized mice as described in the section titled "Example 12 - 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 %.

[0220] [Example 7] Immunogenicity of trivalent (iNTS-TCV) vaccine in mice Eight CD1 mice were immunized with iNTS-TCV vaccine produced as described in Example 5, at doses of 0.01 μg to 0.63 μg (O antigen) of GMMA and 0.012 μg to 0.78 μg of Vi polysaccharide (total polysaccharide dose of 0.032 to 2.04 μg). Immunization included intraperitoneal immunization with 0.032 to 2.04 μg in 500 μl at days 0 and 28. Blood samples were collected at days 27 and 42, and the resulting antibodies were measured using the ELISA assay described above in Example 6. The results are shown in Figure 1.

[0221] The iNTS-TCV vaccine candidate was well-tolerated at all doses. Two antigenic GMMA components (SEnGMMA and STmGMMA) and the antigenic Vi component induced an antibody dose response. Three antigenic components (SEn OAg, STm OAg, and Vi) induced an increased SBA response at four lower doses on day 42.

[0222] In the second experiment, a group of 8 CD1 mice was used. - iNTS-TCV vaccine produced as described in Example 5, in doses of 0.032, 0.13, 0.51, and 2.04 μg (total polysaccharides). - SEnGMMA adsorbed onto Alhydrogel in doses of 0.01, 0.04, 0.16, and 0.63 μg (O antigen) (produced as described in Example 1) - STmGMMA adsorbed onto Alhydrogel in doses of 0.01, 0.04, 0.16, and 0.63 μg (O antigen) (produced as described in Example 1) - fVi-CRM197 conjugates in doses of 0.012, 0.05, 0.19, and 0.78 μg (Vi polysaccharide) (produced as described in Example 2) And then it became immune.

[0223] Immunization involved intraperitoneal immunization at corresponding doses for each product in 500 μl at days 0 and 28. Blood samples were collected at days 27 and 42, and the resulting antibodies were measured using the ELISA and SBA assays described in Example 6. The results are shown in Figure 2.

[0224] Overall, no immunoassay was performed on the iNTS GMMA component in iNTS-TCV by ELISA and SBA measurements. However, when combined with GMMA in iNTS-TCV, the fVi-CRM197 component exhibited a higher anti-Vi IgG response and serum bactericidal activity.

[0225] In the third experiment, a group of three New Zealand white rabbits was immunized with the bivalent iNTS-GMMA vaccine described in Example 3. Immunization included intramuscular immunization with 40 μg (O antigen) of total GMMA on day 0 and day 28. Blood samples were collected on days 21, 28, and 42, and the resulting antibodies were measured using the ELISA assay described in Example 6. The results are shown in Figure 3.

[0226] The vaccine was well tolerated, and all rabbits immunized with the two-component iNTS-GMMA candidate vaccine responded with high levels of anti-SEn OAg and anti-STm OAg serum IgG.

[0227] [Example 8] Production of GMMA from S. Paratyphi A. S. Paratyphi strain ED199 containing the ΔtolRΔpagPΔmsbB mutation was prepared using a protocol based on that described for S. Enteritidis and S. Thphimurium in Example 1, except that the specific mutations used to delete tolR, pagP, and msbB were tolR::cat pagP::kan msbB::tet. GMMA was isolated from the bacteria as described in Example 1.

[0228] [Example 9] S. paratyphi AO antigen and CRM 197 Production of conjugates OAg CRM using CDAP Chemistry 197 Conjugation to (with or without ADH linker) Paratyphi A OAg (O:2) was converted to CRM using random CDAP chemical methods, either via an ADH linker or without an ADH linker. 197 It was conjugated. As expected, the random CDAP chemical method introduces multiple bonds between OAg and CRM197. The resulting chemistry is shown in Figure 4.

[0229] Specifically, the OH groups of O:2 are activated with CDAP using a w / w ratio of 1:0.3 for O:2 in a 150 mM NaCl solution. The pH is adjusted to 9-10 using 10% v / v triethylamine, and the solution is incubated at room temperature for 3 ± 0.5 minutes with stirring.

[0230] The activated cyanoester group of O:2 is used in CRM 197 ADH / CRM 197 By covalently bonding the hydrazide / amino group to O:2-CDAP-ADH-CRM 197 / O:2-CDAP-CRM 197 To form.

[0231] CRM197 ADH / CRM 197 Add to O:2 at a concentration of 10 mg / mL in an equal w / w ratio (1:1) (O:2 and CRM 197 ADH / CRM 197 The final concentration is 5 mg / mL). Maintain the pH at 9.5 ± 0.5 with 10% triethylamine and mix the solution at room temperature for 2-3 hours. Then, add an equal volume of 1 M glycine solution to the conjugation mixture and adjust the pH to 8.0 ± 0.2 with 10% triethylamine; incubate the solution at 2-8°C for 15 ± 5 hours. Then, exchange the buffer of the crude conjugate and remove unbound and unreacted O:2 using HIC phenyl HP resin.

[0232] Stability tests based on the test method described in Example 3 (however, free OAg derived from samples taken on days 0, 8, 14, and 28 were measured) were performed on OAg-CRM197 conjugates produced using CDAP chemistry with or without an ADH linker. The results (percentage of free OAg) are shown in the table below.

[0233] [Table 1] Compared to OAg-CRM197 prepared using the chemistry described in Examples 2-5, significantly less free OAg was observed in the stability test.

[0234] [Example 10] Formulation of two quadrivalent (pan-salmonella) vaccines against S. typhimurium, S. enteritidis, S. typhi, and S. paratyphi for preclinical trials. Two quadrivalent vaccines were formulated. The first is (Pansalmonella O:2-CRM) 197 ) are GMMA (described in Example 1) and CRM derived from S. Enteritidis and S. Typhimurium. 197S. Paratyphi AO antigen conjugated to (O:2-CRM described in Example 9) 197 ) and fVi-CRM 197 It contained a conjugate (described in Example 2). The second (Pan-Salmonella_ParA GMMA) was similar, except that the S. Paratyphi AO antigen conjugate was replaced with S. Paratyphi A GMMA (as described in Example 8).

[0235] STmGMMA and SEnGMMA were adsorbed onto Alhydrogel. After mixing (1-2 hours), S. Paratyphi GMMA (or O:2-CRM) was added. 197 Add ). Subsequently, after a quenching step using phosphoric acid and osmotic pressure adjustment using sodium chloride, add fVi-CRM197.

[0236] The concentration of the phosphate buffer was optimized while quenching to ensure the optimal particle size, resulting in the highest adsorption.

[0237] The final formulation contains GMMA sufficient to provide 40 μg / ml of STm, SEn, and S. Paratyphi AO antigen (sPa), and 50 μg / ml of Vi polysaccharide in a phosphate-buffered saline matrix containing 0.7 mg / mL of aluminum hydroxide.

[0238] The protocols for determining the amount of O antigen in STmGMMA, SEnGMMA, and Paratyphi A are based on those described in Example 5. The methods used to determine the O antigen levels for S. Enteritidis, S. Typhimurium, and S. Paratyphi A are described below.

[0239] Determination of OAg for Salmonella enteritidis, tiphimurum, and paratifi A Prior to analysis with HPAEC-PAD, each individual OAg is hydrolyzed to release its relative dideoxy monosaccharide corresponding to the chromatographic peak (SEn OAg for tyberose; STm OAg for avequoise; SPa OAg for paratose).

[0240] In fact, dideoxy is the only sugar that differs from the others among the components of the SEn, STm, and SPa OAg repeating unit (RU).

[0241] Dilute the sample with milliQ water by volume (450 μL) or weight on an analytical balance to bring each OAg within the calibration curve range. Add 120 μL of 1 M TFA to the vial containing the standard or sample and incubate at 75°C for 1.5 hours. After hydrolysis, cool the vial in the refrigerator at 2–8°C for 15 minutes. Remove the solvent / TFA by drying the sample and standard overnight on a centrifugal evaporator at room temperature (RT). Dissolve the pellet in 450 μL of milliQ water. Filter the sample and standard over a 1 mL well of AcroPrep Advance 96 Filter Plate 0.2 μm Supor and load the plate onto an HPAEC-PAD.

[0242] [Example 11] Immunogenicity of tetravalent pansalmonella vaccine in mice A group of 10 CD1 mice was immunized with one of the pan-salmonella vaccines described in Example 10 at the following doses:

[0243] [Table 2]

[0244] Immunization included intraperitoneal immunization of 200 μl of each formulation at days 0 and 28. Blood samples were collected at days 1, 27, and 42, and the resulting antibodies were tested using the assay described in Example 6 above. The results are shown in Figure 5. In summary, both pan-salmonella vaccines induced specific serum IgG responses to S. Paratyphi AO antigen, S. Typhimurium O antigen, S. Enteritidis O antigen, and Vi polysaccharide, and the antibodies were bactericidal in mice.

[0245] In the second experiment, a group of 10 CD1 mice was used. - Tetravalent pansalmonella O:2-CRM (STm GMMA + SEn GMMA + O:2-CRM) produced as described in Example 10, in doses of 1.0 (μg O antigen), 1.0 (μg O antigen), 1.25 (μg O antigen), and 1.25 (μg Vi polysaccharide), respectively. 197 + fVi-CRM 197 ) - Tetravalent pansalmonella_ParAGMMA (STm GMMA + SEn GMMA + ParA GMMA + fVi-CRM) produced as described in Example 10, in doses of 1.0 (μg O antigen), 1.0 (μg O antigen), 1.17 (μg O antigen), and 1.25 (μg Vi polysaccharide), respectively. 197 ) - Trivalent iNTS-TCV (STm GMMA + SEn GMMA + fVi-CRM) produced as described in Example 5, in doses of 1.0 (μg O antigen), 1.0 (μg O antigen), and 1.25 (μg Vi polysaccharide), respectively. 197 ) - Divalent O:2-CRM produced as described in Example 3, in doses of 1.25 (μg O antigen) and 1.25 (μg Vi polysaccharide), respectively. 197 + fVi-CRM 197 - 1.25 μg (Vi polysaccharide) dose, O:2-CRM 197 Conjugate (produced as described in Example 9) - ParA GMMA adsorbed onto Alhydrogel at a dose of 1.17 μg (Vi polysaccharide) (produced as described in Example 8) - 1.25 μg (Vi polysaccharide) dose of fVi-CRM 197 Conjugate (produced as described in Example 2) And then it became immune.

[0246] Immunization involved intraperitoneal immunization at corresponding doses for each product in 200 μl at days 0 and 28. Blood samples were collected at days 27 and 42, and the resulting antibodies were measured using the ELISA and SBA assays described in Example 6. The results are shown in Figure 6.

[0247] 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, compared to trivalent iNTS-TCV, a significantly higher anti-SEn IgG response was induced by the pan-Salmonella formulation containing O:2-CRM on both days 27 and 42, and also by the pan-Salmonella formulation containing ParA GMMA on day 27 only. Furthermore, compared to trivalent iNTS-TCV, a significantly higher anti-STm IgG response was induced by the pan-Salmonella formulation containing O:2-CRM on day 27 only. It is also important to consider that the O:2-OAg dose used in pan-Salmonella_ParAGMMA is slightly different (approximately 7%) from the O:2 dose used in pan-Salmonella_O:2-CRM.

[0248] The presence of iNTS GMMA in the pan-Salmonella formulation containing O:2-CRM had an overall positive impact on the anti-O:2 IgG response elicited: a significantly higher response 1 week later was induced by the tetravalent formulation containing O:2-CRM, compared to both the bivalent O:2-CRM + fVi-CRM and the monovalent O:2-CRM, and a significantly higher response 2 weeks later was elicited compared to the bivalent O:2-CRM + fVi-CRM formulation. Also, the SBA titers elicited by the pan-Salmonella formulations containing O:2-CRM were significantly higher compared to the corresponding bivalent formulations. Conversely, no significant differences were demonstrated either in the anti-O:2 IgG response elicited or in the function of the antibodies against ParA induced by pan-Salmonella containing ParA GMMA and by the monovalent formulation ParA GMMA.

[0249] [Example 12] Antibody subclasses generated by the tetravalent pan-Salmonella vaccine in mice A group of 10 CD1 mice was immunized with one of the pan-Salmonella vaccines described in Example 10 at the following doses:

[0250]

Table 3

[0251] Immunization included intraperitoneal immunization of 200 μl of each formulation on day 0 and day 28. Blood samples were collected on days -1, 27, and 42. The class of antibodies produced against Vi antigen polysaccharide or S. Paratyphi AO:2 O antigen was determined using the assay described in Example 6 under the heading "IgG subclass". The results are shown in Figure 7. The absolute values ​​for Figure 7 are shown in the table below:

[0252] Antibody titer against S. Paratyphi AO:2 O antigen [Table 4]

[0253] Antibody titer against Vi antigen polysaccharide [Table 5]

[0254] [Example 13] Immunogenicity of the quadrivalent pan-salmonella vaccine in rabbits A group of eight New Zealand white rabbits was immunized with one of the pan-salmonella vaccines described in Example 10 at the following doses:

[0255] [Table 6]

[0256] Immunization included intramuscular immunization of 500 μl of each formulation on day 0 and day 28. Blood samples were collected on days -1, 27, and 42, and the resulting antibodies were tested using the assay described in Example 6 above. The results are shown in Figure 8. In summary, both pan-salmonella vaccines induced specific serum IgG responses to S. Paratyphi AO antigen, S. Typhimurium O antigen, S. Enteritidis O antigen, and fVi polysaccharide, and the antibodies were bactericidal in rabbits.

[0257] [Example 14] Administration of iNTS-TCV vaccine to human subjects The safety, reactiongenicity, and immune response of the trivalent iNTS-TCV vaccine against invasive non-typhoid salmonella (iNTS) and typhoid fever, administered intramuscularly on days 1, 57, and 169 to healthy European and African adults, will be evaluated in a nine-group, phase 1 / 2a, observer-blind, randomized, dose-escalation, controlled, multinational, two-stage, and alternating trial.

[0258] The entire study (both Stage 1 and Stage 2) will be conducted on approximately 155 healthy adult participants (18-50 years old). Healthy European adults will be randomly assigned to one of the groups indicated for Stage 1. Healthy African-American adults will be randomly assigned to one of the groups indicated for Stage 2. Each group will receive two of the eleven study interventions at each dose, with the exception of the control Stage 2 group, which will receive four study interventions (with saline and different active comparators at each dosing time).

[0259] Each participant receives one randomly selected intramuscular trial intervention per group on days 1, 57, and 169.

[0260] Stage 1 Stage 1 (Europe) will use a two-step alternating design with dose escalation, delivering all trial interventions at low doses. A sentinel technique will be used for the first 10 participants in both Step 1 and Step 2, treating only one participant daily. This is done to ensure maximum participant safety.

[0261] In Step 1, 10 healthy European adults were randomized in a ratio of 2:2:1. • Low doses of candidate iNTS-TCV vaccine and accompanying saline administered in different groups (arms), or • Low doses of iNTS-GMMA and TCV (fVi-CRM) in different groups (arms) 197 and S. Paratyphi AO antigen-CRM 197 Separate administration of vaccines (containing conjugates), or • Placebo and saline in different groups To receive.

[0262] In Step 2, 40 healthy European adults will be randomized in a 2:2:1 ratio. An alternating approach will be used for the first 10 sentinel participants, followed by a safety follow-up call to these participants the day after administration of the study intervention. The remaining 30 participants will receive the study intervention sequentially (at least at 60-minute intervals). Participants in Step 2 will be: • The total dose of candidate iNTS-TCV vaccine and accompanying saline administered in different groups (arms), or • Separate administration of the full doses of iNTS-GMMA and TCV vaccine in different groups (arms), or • Placebo and saline in different groups (arms) To receive.

[0263] In Stage 2 (African American), a total of 105 healthy African American adults were randomized in a ratio of 3:3:1. • The total dose of candidate iNTS-TCV vaccine and accompanying saline administered in different groups (arms), or • Separate administration of the full doses of iNTS-GMMA and TCV vaccine in different groups (arms), or In different arm groups, the first dose consists of MenACWY (Menveo) and saline, the second dose consists of TdaP (Boostrix) and saline, and the third dose consists of Typhoid Vi polysaccharide vaccine (Typhim Vi) and saline. This is the control stage 2 group.

[0264] The first 21 participants in Stage 2 will be recruited sequentially at least 60-minute intervals as the administration progresses. Subsequently, these participants will be contacted by phone for a safety follow-up the day after the administration of the study intervention. Recruitment of the remaining 84 participants in Stage 2 will be initiated only if there are positive assessments of all safety data derived from these participants up to 7 days after the first administration of the study intervention. The study intervention will be administered to the remaining 84 participants simultaneously.

[0265] The composition of the 0.5 mL total dose of iNTS-TCV vaccine is as follows:

[0266] [Table 7] The composition of a 0.5 mL low-dose iNTS-TCV vaccine is as follows:

[0267] [Table 8]

[0268] [Example 15] Antibodies induced by GMMA-based vaccine candidates show cross-protection. CD1 mice were immunized intraperitoneally on days 0 and 28 using 500 μL of monovalent STm GMMA (per injection, per mouse dose in μg 2.5 STm OAg) or monovalent SEn GMMA (per injection, per mouse dose in μg 2.5 SEn OAg). SBA was performed on mouse serum obtained on day 42.

[0269] Mouse serum induced by STm GMMA showed bactericidal activity against S. Typhimurium, S. Derby, and S. Dublin (Figure 9(a)). Mouse serum induced by SEm GMMA showed bactericidal activity against S. Enteritidis and S. Dublin (Figure 9(b)).

[0270] CD1 mice were immunized intraperitoneally on days 0 and 28 with either a 200 μL injection volume of bivalent iNTS GMMA vaccine (STm and SEn GMMA) (per injection, per mouse dose, in μg 1.0 STm OAg + 1.0 SEn OAg) or a trivalent iNTS-TCV GMMA vaccine (STm and SEn GMMA and S. Typhi fVi polysaccharide) (per injection, per mouse dose, in μg 1.0 STm OAg + 1.0 SEn OAg + 0.125 fVi). SBA was performed on mouse serum obtained on day 42.

[0271] Mouse serum induced by the bivalent iNTS GMMA vaccine showed bactericidal activity against S. Typhimurium, S. Enteritidis, S. Derby, and S. Dublin (Figure 9(c)). Mouse serum induced by the trivalent iNTS-TCV vaccine also showed bactericidal activity against S. Typhimurium, S. Enteritidis, S. Derby, and S. Dublin (Figure 9(d)).

[0272] New Zealand female rabbits were intramuscularly immunized on days 0 and 28 with either a 500 μL injectable volume of bivalent iNTS GMMA vaccine (STm and SEn GMMA) (dose per rabbit per injection: μg 20 STm OAg + 20 SEn OAg) or trivalent iNTS-TCV GMMA vaccine (STm and SEn GMMA and S. Typhi fVi polysaccharide) (dose per rabbit per injection: μg 20 STm OAg + 20 SEn OAg + 25 fVi). SBA was performed on rabbit serum obtained on day 42.

[0273] Rabbit serum induced by the bivalent iNTS GMMA vaccine showed bactericidal activity against S. Typhimurium, S. Enteritidis, S. Derby, S. Dublin, and S. Paratyphi A (Figure 9(e)). Mouse serum induced by the trivalent iNTS-TCV vaccine also showed bactericidal activity against S. Typhimurium, S. Enteritidis, S. Derby, S. Dublin, and S. Paratyphi A (Figure 9(f)).

[0274] CD1 mice were immunized intraperitoneally on day 0 and day 28 with two quadrivalent pansalmonella vaccines: (i) STm and SEn GMMA, S. Typhi fVi polysaccharide and S. Paratyphi A OAg conjugate (per injection, per mouse dose in μg 1 STm + 1 SEn + 1.25 ParA + 1.25 fVi); and (ii) STm and SEn GMMA, S. Typhi fVi polysaccharide and S. Paratyphi A GMMA (per injection, per mouse dose in μg 1 STm + 1 SEn + 1.17 ParA + 1.25 fVi). SBA was performed on mouse serum obtained on day 42.

[0275] (i) Mouse serum induced by STm and SEn GMMA, S. Typhi fVi polysaccharide, and S. Paratyphi A OAg conjugate; and (ii) Mouse serum induced by STm and SEn GMMA, S. Typhi fVi polysaccharide, and S. Paratyphi A GMMA both showed bactericidal activity against S. Typhimurium, S. Enteritidis, S. Derby, S. Dublin, and S. Paratyphi A (Figure 10).

[0276] Embodiments of the present invention 1. (a) Salmonella enterica serotype tiphimurium antigen; (b) Salmonella enterica serotype enteritidis antigen; and (c) Salmonella enterica serotype Typhi antigen An immunogenic composition containing [a specific substance].

[0277] 2. A method for boosting an immune response to S. Typhi or S. Paratyphi A antigen, comprising administering a composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA.

[0278] 3. A method for preventing infection by S. Typhi or S. Paratyphi A, comprising administering an immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA, wherein GMMA boosts the immune response to S. Typhi antigen or S. Paratyphi A antigen.

[0279] 4. An immunogenic composition containing GMMA for use in a method of boosting an immune response to S. Typhi or S. Paratyphi A antigen, comprising administering the immunogenic composition containing S. Typhi or S. Paratyphi A antigen and GMMA.

[0280] 5. An immunogenic composition for use in a method of preventing infection by S. Typhi or S. Paratyphi A, comprising administering an immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA, wherein the GMMA boosts the immune response to S. Typhi antigen or S. Paratyphi A antigen.

[0281] 6. The method or immunogenic composition for use according to Embodiment 2 or 4, wherein the method is a method for boosting an immune response to the S. Typhi antigen, and the immunogenic composition comprises the S. Typhi antigen.

[0282] 7. A method or immunogenic composition for use according to Embodiment 2 or 4, wherein the method is a method for boosting an immune response to the S. Paratyphi A antigen, and the immunogenic composition comprises the S. Paratyphi A antigen.

[0283] 8. A method for preventing infection by S. typhi, wherein the immunogenic composition comprises S. typhi antigen, and GMMA boosts the immune response to S. typhi antigen, the method or immunogenic composition for use according to Embodiment 3 or 5.

[0284] 9. A method for preventing infection by S. Paratyphi A, wherein the immunogenic composition comprises S. Paratyphi A antigen, and GMMA boosts the immune response to S. Paratyphi A antigen, according to Embodiment 3 or 5, or an immunogenic composition for use.

[0285] 10. A method for use or immunogenic composition according to any one of Embodiments 2 to 9, wherein the GMMA comprises at least one selected from the group consisting of GMMA of S. Typhimurium, GMMA of S. Enteritidis, and GMMA of S. Paratyphi A.

[0286] 11. The immunogenic composition is (d) Salmonella enterica serotype paratyphi A antigen An immunogenic composition or method according to any one of Embodiments 1 to 10, further comprising the above.

[0287] 12. An immunogenic composition or method according to Embodiment 1 or 11, wherein the S. Typhimurium antigen comprises the O antigen of S. Typhimurium.

[0288] 13. An immunogenic composition or method according to any one of Embodiments 1, 11, or 12, wherein the S. Typhimurium antigen comprises or consists of outer membrane vesicles derived from S. Typhimurium.

[0289] 14. An immunogenic composition or method according to any one of Embodiments 1 or 11-13, wherein the S. Typhimurium antigen comprises or consists of GMMA of S. Typhimurium.

[0290] 15. An immunogenic composition or method according to Embodiment 10 or 14, wherein GMMA of S. Typhimurium contains a modified lipid A.

[0291] 16. The immunogenic composition or method according to Embodiment 15, wherein the modified lipid A is detoxified lipid A.

[0292] 17. The immunogenic composition or method according to Embodiment 15 or 16, wherein the modified lipid A is penta-acylated lipid A.

[0293] 18. An immunogenic composition or method according to any one of Embodiments 15 to 17, wherein the GMMA of S. Typhimurium is derived from S. Typhimurium that does not contain a gene encoding a functional MsbB protein.

[0294] 19. An immunogenic composition or method according to any one of Embodiments 15 to 18, wherein the GMMA of S. Typhimurium is derived from S. Typhimurium, which is ΔmsbB.

[0295] 20. An immunogenic composition or method according to any one of Embodiments 10 or 14-19, wherein the GMMA of S. Typhimurium is derived from S. Typhimurium that does not contain a gene encoding a functional PagP protein.

[0296] 21. An immunogenic composition or method according to any one of Embodiments 10 or 14-20, wherein the GMMA of S. Typhimurium is derived from S. Typhimurium, which is ΔpagP.

[0297] 22. An immunogenic composition or method according to any one of Embodiments 10 or 14-21, wherein the GMMA of S. Typhimurium is derived from S. Typhimurium that does not contain a gene encoding a functional TolR protein.

[0298] 23. An immunogenic composition or method according to any one of Embodiments 10 or 14-22, wherein the GMMA of S. Typhimurium is derived from S. Typhimurium strain 2192.

[0299] 24. An immunogenic composition or method according to any one of Embodiments 10 or 14-23, wherein the GMMA of S. Typhimurium is derived from S. Typhimurium, which is ΔtolR.

[0300] 25. An immunogenic composition or method according to Embodiment 1 or any one of Embodiments 10 to 24, wherein the immunogenic composition comprises S. Typhimurium antigen or GMMA of S. Typhimurium in doses (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.

[0301] 26. An immunogenic composition or method according to any one of Embodiments 10 or 14-25, wherein GMMA of S. Typhimurium boosts the immune response to S. Typhi or S. Paratyphi A antigen.

[0302] 27. An immunogenic composition or method according to Embodiment 1 or any one of Embodiments 11 to 26, wherein the S. Enteritidis antigen comprises the O antigen of S. Enteritidis.

[0303] 28. An immunogenic composition or method according to Embodiment 1 or any one of Embodiments 11 to 24, wherein the S. Enteritidis antigen comprises or consists of outer membrane vesicles derived from S. Enteritidis.

[0304] 29. An immunogenic composition or method according to Embodiment 1 or any one of Embodiments 11 to 28, wherein the S. Enteritidis antigen comprises or consists of GMMA of S. Enteritidis.

[0305] 30. An immunogenic composition or method according to Embodiment 10 or 29, wherein GMMA of S. Enteritidis comprises modified lipid A.

[0306] 31. The immunogenic composition or method according to Embodiment 30, wherein the modified lipid A is detoxified lipid A.

[0307] 32. The immunogenic composition or method according to Embodiment 30 or 31, wherein the modified lipid A is penta-acylated lipid A.

[0308] 33. An immunogenic composition or method according to any one of Embodiments 10 or 29-32, wherein the GMMA of S. Enteritidis is derived from S. Enteritidis that does not contain a gene encoding a functional MsbB protein.

[0309] 34. An immunogenic composition or method according to Embodiment 10 or any one of 29-33, wherein the GMMA of S. Enteritidis is derived from S. Enteritidis, which is ΔmsbB.

[0310] 35. An immunogenic composition or method according to any one of Embodiments 10 or 29-34, wherein the GMMA of S. Enteritidis is derived from S. Enteritidis that does not contain a gene encoding a functional PagP protein.

[0311] 36. An immunogenic composition or method according to any one of Embodiments 10 or 29-35, wherein the GMMA of S. Enteritidis is derived from S. Enteritidis, which is ΔpagP.

[0312] 37. An immunogenic composition or method according to any one of Embodiments 10 or 29-36, wherein the GMMA of S. Enteritidis is derived from S. Enteritidis that does not contain a gene encoding a functional TolR protein.

[0313] 38. An immunogenic composition or method according to any one of Embodiments 10 or 29-37, wherein the GMMA of S. Enteritidis is derived from S. Enteritidis strain 618.

[0314] 39. An immunogenic composition or method according to any one of Embodiments 10 or 29-38, wherein the GMMA of S. Enteritidis is derived from S. Enteritidis with ΔtolR.

[0315] 40. An immunogenic composition or method according to Embodiment 1 or any one of Claims 10 to 39, wherein the immunogenic composition comprises S. Enteritidis antigen or GMMA of S. Enteritidis in doses (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.

[0316] 41. An immunogenic composition or method according to Embodiment 10 or any one of Embodiments 29-40, wherein GMMA of S. Enteritidis boosts the immune response to S. Typhi or S. Paratyphi A antigen.

[0317] 42. An immunogenic composition or method according to any one of Embodiments 1 to 6, 8, or 10 to 41, wherein the S. Typhi antigen comprises a Vi polysaccharide.

[0318] 43. An immunogenic composition or method according to any one of Embodiments 1 to 6, 8, or 10 to 42, wherein the S. Typhi antigen comprises a fragmented Vi(fVi) polysaccharide.

[0319] 44. The immunogenic composition or method according to Embodiment 43, wherein the fVi polysaccharide has an average molecular weight of 40 kDa to 55 kDa, 41 kDa to 49 kDa, or 51 kDa to 55 kDa.

[0320] 45. The immunogenic composition or method according to Embodiment 43 or 44, wherein the fVi polysaccharide has an average molecular weight of 51 kDa to 55 kDa.

[0321] 46. ​​An immunogenic composition or method according to any one of Embodiments 43 to 45, wherein the fVi polysaccharide is part of an fVi conjugate comprising fVi and a carrier protein.

[0322] 47. The immunogenic composition or method according to Embodiment 46, wherein the carrier protein is CRM197 or diphtheria toxoid.

[0323] 48. The immunogenic composition or method according to Embodiment 47, wherein the carrier protein is CRM197.

[0324] 49. An immunogenic composition or method according to any one of Embodiments 46 to 48, wherein the fVi polysaccharide is optionally conjugated to a carrier protein by carbodiimide chemistry via a linker.

[0325] 50. The fVi conjugate is a. A step of fragmenting Vi polysaccharides to obtain fragmented Vi(fVi) polysaccharides having an average molecular weight of 40kDa-55kDa, 41kDa-49kDa, or 51kDa-55kDa; b. A step to activate the fVi polysaccharide by reacting the fVi polysaccharide obtained in step a 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 to produce an fVi conjugate. An immunogenic composition or method according to any one of embodiments 46 to 49, which is obtained or can be obtained by a method comprising the above.

[0326] 51. An immunogenic composition or method according to any one of Embodiments 46 to 50, wherein the carbodiimide is EDC.

[0327] 52. An immunogenic composition or method according to Embodiment 50 or 51, wherein the carrier protein is derivatized by reacting it with a carbodiimide and a linker.

[0328] 53. The immunogenic composition or method according to Embodiment 52, wherein the linker is an adipic acid dihydrazide (ADH) linker.

[0329] 54. The immunogenic composition or method according to Embodiment 52 or 53, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and the carbodiimide chemistry is EDAC chemistry.

[0330] 55. An immunogenic composition or method according to any one of Embodiments 1 to 6, 8, or 10 to 54, wherein the immunogenic composition comprises an amount of fVi polysaccharide of 1 to 100 μg, 1 to 50 μg, 15 to 50 μg, 20 to 30 μg, 1 to 20 μg, 1 to 10 μg, approximately 25 μg, or approximately 5 μg.

[0331] 56. An immunogenic composition or method according to any one of Embodiments 2 to 50, wherein the S. Paratyphi A antigen comprises the O antigen of S. Paratyphi A.

[0332] 57. The immunogenic composition or method according to Embodiment 56, wherein the immunogenic composition comprises the O antigen of S. Paratyphi A conjugated to a carrier protein.

[0333] 58. The immunogenic composition or method according to Embodiment 57, wherein the carrier protein is diphtheria toxoid or CRM197.

[0334] 59. The immunogenic composition or method according to Embodiment 58, wherein the carrier protein is CRM197.

[0335] 60. An immunogenic composition or method according to any one of Embodiments 57 to 59, wherein the O antigen of S. Paratyphi A is conjugated to a carrier protein by a method comprising introducing one or more activation sites into an S. Paratyphi AO antigen and / or an S. Paratyphi AO antigen containing one or more activation sites.

[0336] 61. An immunogenic composition or method according to any one of Embodiments 57 to 60, wherein the O antigen of S. Paratyphi A is conjugated to a carrier protein by CDAP chemistry, optionally via a linker.

[0337] 62. The immunogenic composition or method according to Embodiment 61, wherein the O antigen of S. Paratyphi A is conjugated to a carrier protein via a linker.

[0338] 63. The immunogenic composition or method according to Embodiment 62, wherein the linker is adipic acid dihydrazide (ADH).

[0339] 64. An immunogenic composition or method according to any one of embodiments 56 to 63, wherein the immunogenic composition contains S. Paratyphi A O antigen in doses of 1 to 100 μg, 1 to 50 μg, 15 to 50 μg, 20 to 30 μg, 1 to 20 μg, 1 to 10 μg, approximately 25 μg, or approximately 5 μg.

[0340] 65. An immunogenic composition or method according to any one of Embodiments 2 to 64, wherein the S. Paratyphi A antigen comprises GMMA of S. Paratyphi A.

[0341] 66. An immunogenic composition or method according to Embodiment 10 or 65, wherein GMMA of S. Paratyphi A contains a modified lipid A.

[0342] 67. The immunogenic composition or method according to Embodiment 66, wherein the modified lipid A is detoxified lipid A.

[0343] 68. The immunogenic composition or method according to Embodiment 66 or 67, wherein the modified lipid A is penta-acylated lipid A.

[0344] 69. An immunogenic composition or method according to any one of Embodiments 10 or 65-68, wherein the GMMA of S. Paratyphi A is derived from S. Paratyphi A that does not contain a gene encoding a functional MsbB protein.

[0345] 70. An immunogenic composition or method according to any one of Embodiments 10 or 65-69, wherein the GMMA of S. Paratyphi A is derived from S. Paratyphi A, which is ΔmsbB.

[0346] 71. The immunogenic composition or method according to Embodiment 69 or 70, wherein at least a portion of the msbB gene is replaced by at least a portion of the tetracycline (tet) gene.

[0347] 72. The immunogenic composition according to Embodiment 69, 70, or 71, wherein GMMA is derived from S. Paratyphi A, which is msbB::tet.

[0348] 73. An immunogenic composition or method according to any one of Embodiments 10 or 65-72, wherein the GMMA of S. Paratyphi A is derived from S. Paratyphi A that does not contain a gene encoding a functional PagP protein.

[0349] 74. An immunogenic composition or method according to any one of Embodiments 10 or 65-73, wherein the GMMA of S. Paratyphi A is derived from S. Paratyphi A, which is ΔpagP.

[0350] 75. The immunogenic composition according to Embodiment 73 or 74, wherein at least a portion of the pagP gene is replaced by at least a portion of the kanamycin (kan) gene.

[0351] 76. An immunogenic composition according to any one of embodiments 73 to 75, wherein rGMMA is derived from S. Paratyphi A, which is pagP::kan.

[0352] 77. An immunogenic composition or method according to any one of Embodiments 10 or 65-76, wherein the GMMA of S. Paratyphi A is derived from S. Paratyphi A that does not contain the gene encoding a functional TolR protein.

[0353] 78. An immunogenic composition or method according to any one of Embodiments 10 or 65-77, wherein the GMMA of S. Paratyphi A is derived from S. Paratyphi A strain ED199.

[0354] 79. An immunogenic composition or method according to any one of Embodiments 10 or 65-78, wherein the GMMA of S. Paratyphi A is derived from S. Paratyphi A with ΔtolR.

[0355] 80. An immunogenic composition or method according to any one of Embodiments 10 or 65-79, wherein the immunogenic composition comprises GMMA of S. Paratyphi A in doses 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 (O antigen).

[0356] 81. The immunogenic composition according to any one of Embodiments 77, 79, or 80, wherein at least a portion of the tolR gene is replaced by at least a portion of the chloramphenicol acetyltransferase (cat) gene.

[0357] 82. An immunogenic composition according to any one of Embodiments 77 or 79-81, wherein GMMA is derived from S. Paratyphi A, which is tolR::cat.

[0358] 83. An immunogenic composition or method according to Embodiment 10 or any one of 65-82, wherein GMMA of S. Paratyphi A boosts the immune response to the S. Typhi antigen.

[0359] 84. An immunogenic composition or method according to any one of Embodiments 2 to 83, wherein the immune response to S. Typhi antigen is higher when S. Typhi antigen or S. Paratyphi A antigen is part of an immunogenic composition containing GMMA, compared to the immune response to S. Typhi antigen when S. Typhi antigen or S. Paratyphi A antigen is not part of an immunogenic composition containing GMMA, the method is a method for boosting the immune response to S. Typhi antigen or S. Paratyphi A antigen.

[0360] 85. An immunogenic composition or method according to any one of Embodiments 2 to 84, wherein GMMA boosts the immune response to the S. Typhi antigen or S. Paratyphi A antigen when the S. Typhi antigen or S. Paratyphi A antigen is part of an immunogenic composition containing GMMA, compared to the immune response to the S. Typhi antigen or S. Paratyphi A antigen when the S. Typhi antigen or S. Paratyphi A antigen is not part of an immunogenic composition containing GMMA.

[0361] 86. The immunogenic composition or method according to Embodiment 84 or 85, wherein the method is a method for boosting the immune response to the S. Typhi antigen or S. Paratyphi A antigen, or GMMA boosts the immune response to the S. Typhi antigen or S. Paratyphi A antigen, when the S. Typhi antigen or S. Paratyphi A antigen is part of an immunogenic composition containing GMMA, compared to the immune response to the S. Typhi antigen or S. Paratyphi A antigen when the S. Typhi antigen or S. Paratyphi A antigen is not part of an immunogenic composition containing GMMA, and the immune response to the S. Typhi antigen or S. Paratyphi A antigen is at least 5 times, at least 10 times, or at least 20 times higher when the S. Typhi antigen or S. Paratyphi A antigen is part of an immunogenic composition containing GMMA.

[0362] 87. An immunogenic composition or method according to any one of embodiments 84 to 86, wherein the immune response to S. Typhi antigen or S. Paratyphi A antigen is the number of antibodies produced when determined by ELISA 42 days after administration of an immunogenic composition containing 0.78 μg of S. Typhi antigen or S. Paratyphi A antigen and 0.63 μg (O antigen) of GMMA.

[0363] 88. An immunogenic composition or method according to any one of Embodiments 1 to 87, wherein the immunogenic composition is tolerogenic.

[0364] 89. The following steps: (a) A step of measuring the rabbit's initial body temperature; (b) A step of administering an immunogenic composition to rabbits in doses of 20 μg of (O antigen) per GMMA and 25 μg of sugar per sugar conjugate; (c) A step of monitoring the rabbit's body temperature for 5 hours; and (d) Step to record the rabbit's maximum body temperature Includes, An immunogenic composition or method according to Embodiment 88, wherein, in a toxicity assay where the body temperature rise is calculated to be equal to the rabbit's maximum body temperature minus the rabbit's initial body temperature, the immunogenic composition is tolerogenic if it induces a body temperature rise of less than 1.8°C, less than 1.7°C, less than 1.6°C, or less than 1.5°C.

[0365] 90. The following steps: (a) A step of administering an immunogenic composition to rabbits in doses of 20 μg (O antigen) per GMMA and 25 μg per sugar; (b) A step of monitoring the rabbit's body temperature for 5 hours; and (c) Steps to record the rabbit's maximum body temperature An immunogenic composition or method according to Embodiment 2 or 88, wherein in a toxicity assay including the immunogenic composition, if the immunogenic composition induces a maximum body temperature of 41°C or lower, 40.9°C or lower, or 40.8°C or lower, it is tolerogenic.

[0366] 91. 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; and (b) Step of measuring the level of anti-S. Typhimurium O antigen antibody and / or anti-S. Enteritidis O antigen antibody by ELISA on the 42nd day In an immunogenic assay comprising 3 at least 10 3 EU / ml of anti-S. Typhimurium O antigen antibody and / or at least 10

[0367] 92. The following steps: (a) Step of immunizing mice intraperitoneally on days 0 and 28 with an immunogenic composition at a dose of 1 μg of (O antigen) per GMMA and 1.25 μg of sugar per sugar conjugate; and (b) Step of measuring the level of anti-fVi conjugate antibody by ELISA on the 42nd day In an immunogenic assay comprising 3 at least 10

[0368] 93. The following steps: (a) Step of immunizing mice intraperitoneally on days 0 and 28 with an immunogenic composition at a dose of 1 μg of (O antigen) per GMMA and 1.25 μg of sugar per sugar conjugate; and (b) Step of measuring the level of anti-S. Paratyphi A O antigen antibody by ELISA on the 42nd day In an immunogenic assay comprising 3 at least 10

[0369] 94. 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) Step 42: Measure the anti-S. Paratyphi AO antigen antibody level by ELISA. In an immunogenicity assay including the immunogenicity composition, the immunogenicity composition is at least 10 3.5 An immunogenic composition or method according to any one of Embodiments 1 to 93 for inducing an EU / ml anti-S. Paratyphi AO antigen antibody.

[0370] 95. An immunogenic composition or method according to Embodiment 1 or any one of Claims 10 to 94, wherein the level of induced anti-S. Typhimurium O antigen antibody is at least 90%, at least 95%, or at least 98% of the level of anti-S. Typhimurium O antigen antibody induced by the corresponding monovalent S. Typhimurium immunogenic composition.

[0371] 96. An immunogenic composition or method according to Embodiment 1 or any one of Claims 10 to 95, wherein the level of induced anti-S. Enteritidis O antigen antibody is at least 90%, at least 95%, or at least 98% of the level of anti-S. Enteritidis O antigen antibody induced by the corresponding monovalent S. Enteritidis immunogenic composition.

[0372] 97. An immunogenic composition or method according to any one of Embodiments 1-6, 8, or 10-96, wherein the level of the induced anti-fVi conjugate antibody is at least 90%, at least 95%, or at least 98% of the level of the anti-fVi conjugate antibody induced by the corresponding monovalent fVi capsular polysaccharide immunogenic composition.

[0373] 98. An immunogenic composition or method according to any one of Embodiments 2-5, 7, or 11-97, wherein the level of induced anti-S. Paratyphi AO antigen antibody is at least 90%, at least 95%, or at least 98% of the level of anti-S. Paratyphi AO antigen antibody induced by the corresponding monovalent S. Paratyphi A immunogenic composition.

[0374] 99. The levels of anti-S. Typhimurium O antigen antibody, anti-S. Enteritidis O antigen antibody, anti-fVi conjugate antibody and / or anti-S. Paratyphi O antigen antibody are as follows: (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) Step 42: Measure the levels of anti-S. Typhimurium antibody, anti-S. Enteritidis antibody, anti-fVi conjugate antibody, and / or anti-S. Paratyphi AO antigen antibody by ELISA. An immunogenicity composition or method according to any one of embodiments 95 to 98, measured in an immunogenicity assay comprising [the specified element].

[0375] 100. The immunogenic composition is the following strain: (a) S. Typhimurium ST34; (b) S. Typhimurium 10433_3; (c) S. Typhimurium D23580; (d) S. Typhimurium ST4 / 74; (e) S. Typhimurium A130; (f) S. enterica serotype Derby; (g) S. enterica serovar Dublin; (h) S. Enteritidis A1636; (i) S. Enteritidis CP255; and (j) S. Enteritidis D7795 An immunogenic composition or method according to any one of Embodiments 1 to 99, which induces antibodies against three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten of the following.

[0376] 101. 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) Step 42: Measure the level of the anti-S. Paratyphi AO antigen antibody subtype by ELISA. An immunogenic composition or method according to any one of Embodiments 2-5, 7, or 11-100, wherein the immunogenic composition induces anti-S. Paratyphi A antibodies in each of the IgG3, IgG2b, IgG2a, and IgG1 classes, as determined using an antibody class assay comprising the above.

[0377] 102. An immunogenic composition or method according to any one of Embodiments 1 to 101, wherein the immunogenic composition further comprises an adjuvant.

[0378] 103. The immunogenic composition or method according to Embodiment 102, wherein the adjuvant is an aluminum adjuvant.

[0379] 104. An immunogenic composition or method according to Embodiment 102 or 103, wherein the adjuvant comprises aquatic aluminum and / or aluminum phosphate.

[0380] 105. The immunogenic composition or method according to Embodiment 104, wherein the adjuvant is aluminum hydroxide.

[0381] 106. The adjuvant is 0.1 mg to 10 mg of Al3+ , 0.1mg~5mg Al 3+ , 0.3mg~0.4mg Al 3+ , or approximately 0.35 mg of Al 3+ An immunogenic composition or method according to any one of embodiments 103 to 105, including the above.

[0382] 107. An immunogenic composition or method according to any one of Embodiments 1 to 106, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient.

[0383] 108. The immunogenic composition or method according to Embodiment 107, wherein the pharmaceutically acceptable excipient comprises phosphate-buffered saline.

[0384] 109. The immunogenic composition or method according to Embodiment 108, wherein the pH of the phosphate-buffered saline is 6-7, or approximately 6.5.

[0385] 110. A vaccine comprising the immunogenic composition described in Embodiment 1 or any one of Claims 11 to 109.

[0386] 111. An immunogenic composition or vaccine according to Embodiment 1 or any one of Embodiments 11 to 110 for use in a method of preventing infection.

[0387] 112. A method for preventing infection, comprising administering an effective amount of an immunogenic composition or vaccine described in Embodiment 1 or any one of Embodiments 11 to 110 to a target.

[0388] 113. Use of an immunogenic composition or vaccine according to Embodiment 1 or any one of 11 to 110 for the manufacture of a pharmaceutical product for use in a method of preventing infection.

[0389] 114. An immunogenic composition or vaccine for use according to Embodiment 111, or the use according to Embodiment 113, wherein a method for preventing infection comprises administering an effective amount of the immunogenic composition or vaccine according to Embodiment 1 or any one of Embodiments 11 to 110 to the target.

[0390] 115. An immunogenic composition or vaccine, method, or use for use according to any one of Embodiments 111 to 114, wherein the method for preventing infection is a method for preventing Salmonella infection.

[0391] 116. An immunogenic composition or vaccine, method, or use for use according to any one of Embodiments 111 to 115, wherein the method for preventing infection is a method for preventing invasive unclassifiable Salmonella infection.

[0392] 117. An immunogenic composition or vaccine, method or use according to any one of Embodiments 111 to 116, wherein the method for preventing infection is a method for preventing infection by S. Typhimurium, S. Enteritidis, S. Typhi and / or S. Paratyphi A.

[0393] 118. An immunogenic composition, immunogenic composition or vaccine for use, method or use, according to Embodiment 10 or any one of 65-117, wherein the GMMA O antigen / protein ratio of S. Paratyphi A is at least 0.4.

Claims

1. (a) S. Typhimurium antigens, which are Salmonella enterica serotype tiphimurium antigens containing or consisting of outer membrane vesicles derived from S. Typhimurium; (b) S. Enteritidis antigen, wherein the Salmonella Enterica serotype Enteritidis (S. Enteritidis) antigen contains or consists of outer membrane vesicles derived from S. Enteritidis; and (c) Salmonella enterica serotype Typhi antigen contains Vi polysaccharide, S. Typhi antigen An immunogenic composition containing [a specific substance].

2. A method for boosting an immune response to S. Typhi or S. Paratyphi A antigen, comprising administering a composition containing S. Typhi antigen or S. Paratyphi A antigen and GMMA.

3. A method for preventing infection by S. Typhi or S. Paratyphi A, comprising administering an immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA, wherein GMMA boosts the immune response to S. Typhi antigen or S. Paratyphi A antigen.

4. An immunogenic composition comprising GMMA for use in a method for boosting an immune response to S. Typhi or S. Paratyphi A antigen, wherein the method comprises administering the immunogenic composition comprising S. Typhi or S. Paratyphi A antigen and GMMA.

5. An immunogenic composition for use in a method of preventing infection by S. Typhi or S. Paratyphi A, comprising administering an immunogenic composition comprising S. Typhi antigen or S. Paratyphi A antigen and GMMA, wherein the GMMA boosts the immune response to S. Typhi antigen or S. Paratyphi A antigen.

6. A method for use or immunogenic composition according to any one of claims 2 to 5, wherein the GMMA comprises at least one selected from the group consisting of GMMA of S. Typhimurium, GMMA of S. Enteritidis, and GMMA of S. Paratyphi A.

7. The immunogenic composition (d) Salmonella enterica serotype paratyphi A antigen The immunogenic composition or method according to any one of claims 1 to 6, further comprising:

8. The immunogenic composition or method according to claim 1 or 7, wherein the S. Typhimurium antigen comprises or consists of GMMA of S. Typhimurium.

9. The immunogenic composition or method according to claim 6 or 8, wherein the GMMA of S. Typhimurium comprises modified lipid A, and optionally the modified lipid A is detoxified lipid A.

10. The immunogenic composition or method according to any one of claims 1 or 7 to 9, wherein the S. Enteritidis antigen comprises or consists of GMMA of S. Enteritidis.

11. The immunogenic composition or method according to claim 6 or 10, wherein GMMA of S. Enteritidis comprises modified lipid A, and optionally the modified lipid A is detoxified lipid A.

12. The immunogenic composition or method according to any one of claims 1 to 11, wherein the S. typhi antigen comprises a fragmented Vi(fVi) polysaccharide.

13. The immunogenic composition or method according to claim 12, wherein the fVi polysaccharide is part of an fVi conjugate comprising fVi and a carrier protein.

14. Carrier protein CRM 197 The immunogenic composition or method according to claim 13, or diphtheria toxoid.

15. The immunogenic composition or method according to any one of claims 2 to 14, wherein the S. Paratyphi A antigen comprises the O antigen of S. Paratyphi A.

16. The immunogenic composition or method according to any one of claims 2 to 15, wherein the S. Paratyphi A antigen comprises GMMA of S. Paratyphi A.

17. The immunogenic composition or method according to claim 6 or 16, wherein the GMMA of S. Paratyphi A comprises a modified lipid A.

18. The immunogenic composition or method according to claim 17, wherein the modified lipid A is detoxified lipid A.

19. The immunogenic composition or method according to any one of claims 1 to 18, wherein the immunogenic composition further comprises an adjuvant.

20. The immunogenic composition or method according to claim 19, wherein the adjuvant is an aluminum adjuvant.

21. A method for preventing infection, comprising administering an effective amount of the immunogenic composition according to any one of claims 1 or 7 to 20 to a target.

22. Use of the immunogenic composition according to any one of claims 1 or 7 to 20 for the manufacture of a pharmaceutical for use in a method of preventing infection.

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