Immunogenic composition

Abstract

The present invention relates to an immunogenic composition comprising antigens from Salmonella enterica mouse typhoid serotype (Salmonella typhoid), Salmonella enterica enteritis serotype (Salmonella enteritidis), and Salmonella enterica typhoid serotype (Salmonella typhoid). The invention also relates to methods and uses of compositions comprising GMMA for enhancing an immune response against Salmonella typhi antigens, vaccines comprising said immunogenic compositions, and methods and uses of said immunogenic compositions.

Description

Technical Field

[0001] This invention relates to immunogenic compositions comprising antigens from enteric Salmonella typhimurium serotype (Salmonella typhimurium), enteric Salmonella enteritidis serotype (Salmonella enteritidis), and enteric Salmonella typhimurium serotype (Salmonella typhimurium). The invention also relates to methods and uses of compositions comprising GMMA for enhancing immune responses against Salmonella typhimurium antigens, vaccines comprising said immunogenic compositions, and methods and uses of said immunogenic compositions. Background Technology

[0002] Typhoid fever is a bacterial disease caused by the human host-restricted organism *Salmonella enterica* serotype *Salmonella typhi* (*Salmonella typhi*) [Crump, 2019]. This disease occurs globally, primarily affecting children and young adults, but is prevalent in developing countries in Africa and Asia, and occasionally in developed countries among travelers who have recently returned from endemic countries [Smith, 2016]. The exact burden of typhoid fever is believed to be severely underestimated due to the difficulty in diagnosing it in endemic areas. In 2017, an estimated 10.9 million cases of typhoid fever and 116,800 deaths due to *Salmonella typhi* were reported. Similarly, the estimated life years lost (YLL) and disability-adjusted life years (DALY) due to typhoid fever were 8.3 million and 8.4 million, respectively. Although the disease burden has decreased due to improvements in water and sanitation, it remains a significant public health problem [Global Burden of Disease, 2017]. The burden of typhoid fever is most severe among school-aged children and those under 5 years of age. Recent studies indicate that in the 5-9 year age group, the adjusted incidence (observed values ​​per 100,000 person-years, 95% confidence interval) of typhoid fever diagnosed by blood culture ranged from 861 cases (599-1203) in Malawi to 3228 cases (2276-4757) in Bangladesh; while in the 0-4 year age group, the incidence rates in Malawi and Bangladesh were 632 cases (398-965) and 2625 cases (1764-4244), respectively [Meiring, 2021]. Without timely diagnosis, treatment, and intervention, typhoid fever can lead to hospitalization and fatal complications such as typhoid intestinal perforation (TIP). In developing countries where typhoid fever is prevalent, surgical intervention is often delayed, further worsening disease outcomes [Contini, 2017].

[0003] Antimicrobial treatment of typhoid fever has been hampered by the emergence of multidrug-resistant (MDR) Salmonella typhi, strains first discovered in 1980 and defined as resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. While the threat of resistant strains has been mitigated with the advent of newer antimicrobials, this challenge remains and hinders effective disease control [Radhakrishnan, 2018]. Similarly, Salmonella typhi clones carrying resistance to three first-line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole), as well as fluoroquinolones and third-generation cephalosporins have been reported in Asia; these strains are classified as extensively drug-resistant (XDR) [Klemm, 2018].

[0004] Salmonella paratyphi A is present in the human intestine, and its clinical manifestations are indistinguishable from typhoid fever. Salmonella paratyphi A is the second leading cause of enteric fever, after Salmonella typhi serotype (Salmonella typhi). Enteric fever caused by Salmonella paratyphi A or paratyphoid fever was once thought to account for a small proportion of enteric fever cases. However, since the 1980s, the incidence and relative prevalence of paratyphoid fever have increased in Nepal, Pakistan, and Thailand. Furthermore, large numbers of cases of Salmonella paratyphi A have been reported in populous countries such as India and China. Reports of paratyphoid fever are also on the rise in non-endemic countries (such as the United States), particularly among travelers from South Asia (Irfan et al.). people 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 May23; 23(1):346.PMID: 37101111; PMCID: PMC10132421).

[0005] Therefore, improved vaccines against Salmonella are needed. Summary of the Invention

[0006] Examples of the present invention demonstrate that trivalent vaccines comprising Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Typhimurium antigens (GMMA) are safe and highly immunogenic. Similarly, examples of the present invention demonstrate that quadrivalent vaccines comprising the aforementioned trivalent vaccine and Salmonella Paratyphi A antigen (GMMA or O-antigen conjugate) are also highly immunogenic, and no antigenic interference was observed among the four antigens. Furthermore, these examples demonstrate that GMMA (from Salmonella Enteritidis and / or Salmonella Typhimurium) can enhance protection against Salmonella Typhimurium antigens (fVi and CRM). 197 (Fuse) immune response.

[0007] In a first aspect of the invention, an immunogenic composition is provided, comprising: (a) Enteric Salmonella Typhimurium serotype (Salmonella Typhimurium) antigen; (b) Salmonella enteritis serotype (Salmonella enteritidis) antigen; and (c) Enteric Salmonella typhi serotype (Salmonella enterica) cold Salmonella antigen.

[0008] In a second aspect of the invention, a method for enhancing an immune response to Salmonella typhi or Salmonella paratyphi A antigen is provided, the method comprising administering a composition comprising the Salmonella typhi antigen or the Salmonella paratyphi A antigen and GMMA.

[0009] In a third aspect of the invention, a method for preventing infection with Salmonella Typhi or Salmonella Paratyphi A is provided, the method comprising administering an immunogenic composition comprising Salmonella Typhi antigen or Salmonella Paratyphi A antigen and GMMA, wherein the GMMA enhances the immune response to the Salmonella Typhi antigen or Salmonella Paratyphi A antigen.

[0010] In a fourth aspect of the invention, an immunogenic composition comprising GMMA is provided for use in a method of enhancing an immune response to an antigen of Salmonella Typhi or Salmonella Paratyphi A, wherein the method comprises administering an immunogenic composition comprising the Salmonella Typhi antigen or the Salmonella Paratyphi A antigen and GMMA.

[0011] In a fifth aspect of the invention, an immunogenic composition is provided for use in a method of preventing infection with Salmonella Typhi or Salmonella Paratyphi A, the method comprising administering an immunogenic composition comprising Salmonella Typhi antigen or Salmonella Paratyphi A antigen and GMMA, wherein the GMMA enhances the immune response to the Salmonella Typhi antigen or Salmonella Paratyphi A antigen.

[0012] In a sixth aspect of the invention, a vaccine comprising the immunogenic composition described herein is provided.

[0013] In a seventh aspect of the invention, a method for preventing infection is provided, the method comprising administering an effective amount of the immunogenic composition or vaccine described in this invention.

[0014] In an eighth aspect of the invention, the use of the immunogenic composition or vaccine described herein in the preparation of a medicament for the prevention of infection is provided. Attached Figure Description

[0015] Figure 1 The iNTS-TCV vaccine induced a specific serum IgG response against the target antigen, and the antibody exhibited bactericidal activity in mice. Study design: iNTS-TCV drug product; 8 mice per group; Immunization IP: Day 0 and Day 28; Blood collection: Day 27 and Day 42; Zero-time toxicology batch.

[0016] Figure 2 STmGMMA / alhydrogel compared to iNTS-TCV2 in anti-STm OAg IgG response in mice, at day 27 ( Figure 2 (a) and day 42 ( Figure 2 (b)).

[0017] SEnGMMA / aluminum gel compared to iNTS-TCV2 in anti-SEn OAg IgG response in mice, at day 27 ( Figure 2 (c) and day 42 ( Figure 2 (d)).

[0018] Vi-CRM197 compared to iNTS-TCV2 in anti-Vi IgG response in mice, at day 27 ( Figure 2 (e) and day 42 ( Figure 2 (f)).

[0019] Figure 3 Anti-STm OAg and SEn OAg IgG antibody units against bivalent iNTS-GMMA (full dose 40 ug) were detected in individual rabbit serum.

[0020] Figure 4 Salmonella paratyphi A O-antigen was combined with CRM using a randomized CDAP chemistry method. 197 Combination reaction schemes.

[0021] Figure 5 Both pan-Salmonella preparations induced specific serum IgG responses against four antigens, and the antibodies exhibited bactericidal activity in mice.

[0022] Figure 5 (a)-(d) show the IgG response at the following time points: one day before immunization (left column), 27 days after immunization (middle column), and 42 days after immunization (right column).

[0023] Figure 5 (e)-(g) Display SBA results. Figure 5 In (e), the left column (corresponding to O:2-CRM197 and ParA GMMA, respectively) represents the day before immunization, and the right column (corresponding to O:2-CRM197 and ParA GMMA, respectively) represents 42 days after immunization. Figure 5 In (f) and (g), each column represents 42 days after immunization.

[0024] Figure 6 Evaluation of immune interference between components in pan-Salmonella vaccine formulations.

[0025] Figure 6 (a), (c), (e) and (g) show IgG responses at the following time points: one day before immunization (left column), 27 days after immunization (middle column), and 42 days after immunization (right column).

[0026] Figure 6 (b), (d), and (f) show the SBA results. Figure 6 In (b), the left column represents the day before immunization, and the right column represents 42 days after immunization. Figure 6 In (d) and (f), each column represents 42 days after immunization.

[0027] Figure 7 The relative abundance of subclasses (%) is calculated as subclass / total abundance of all subclasses (%). The top segment is IgG3, the next segment is IgG2b, the next segment after that is IgG2a, and the bottom segment is IgG1.

[0028] Figure 8 Two tetravalent pansalmonella preparations induced specific serum IgG responses against four antigens, and the antibodies also had bactericidal effects in rabbits.

[0029] Figure 8 (a)-(d) show the IgG response at the following time points: one day before immunization (left column), 27 days after immunization (middle column), and 42 days after immunization (right column).

[0030] Figure 8(e)-(g) show the SBA results, where the left column (corresponding to O:2-CRM197 and ParA GMMA respectively) is the day before immunization, and the right column (corresponding to O:2-CRM197 and ParA GMMA respectively) is 42 days after immunization.

[0031] Figure 9 Monovalent component (STm or SEn GMMA) induced SBA in mouse serum heterologous groups ( Figure 9 (a) and (b)).

[0032] Bivalent vaccines (STm and SEn GMMA) Figure 9 (c) and the trivalent vaccine iNTS-TCV (STm and SEn GMMA and fVi polysaccharide from Salmonella typhi) Figure 9 (d) SBA induced in the mouse serum heterologous group.

[0033] Bivalent vaccines (STm and SEn GMMA) Figure 9 (e) and the trivalent vaccine iNTS-TCV (from Salmonella typhi STm and SEn GMMA and fVi polysaccharide) Figure 9 (f) SBA induced by heterologous rabbit serum.

[0034] Figure 10 The tetravalent pan-Salmonella preparation elicits bactericidal antibodies against a range of Salmonella strains. This range includes invasive STm isolates from Africa and Southeast Asia, as well as other Salmonella enteritidis serotypes besides STm, SEn, ParA, and Typhi.

[0035] Figure 11 The disclosed structure of the O-antigen (containing the core domain) from Salmonella paratyphi A.

[0036] Figure 12 CRM 197 sequence.

[0037] General definition Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0038] Generally speaking, the term " Include "Intended to indicate, but not limited to, this. For example, the phrase " Including Salmonella typhimurium Immunogenic compositions of bacterial antigens "This should be interpreted as meaning that the immunogenic composition contains Salmonella typhimurium antigen, but the immunogenic composition may contain other components."

[0039] In some embodiments of the present invention, the term " Include"Replaced with phrase" Composed of... ".the term" Composed of... "Intended to indicate a limiting meaning. For example, the phrase " Immunogenic group composed of Salmonella typhimurium antigen Compounds "It should be understood that the immunogenic composition contains only Salmonella typhimurium antigen and no other components."

[0040] In some embodiments of the present invention, words "Include" Replaced with phrase "Basically composed of..." .the term "Basically composed of..." This means that there may be specific further components, namely those components that do not substantially affect the basic characteristics of the subject.

[0041] the term" about "or" About "When referring to a numerical value, it indicates that the value but allows for a reasonable range of scientific error. Optionally, if a value falls within 10%, 5%, or 1% of x, it can be expressed as " Approximately x "or" About x”.

[0042] singular form "a" or "an" and "the" This includes the plural referents, unless the context explicitly specifies otherwise. Therefore, for example, mentioning " The GMMA "When it includes two or more instances or variants of this type of GMMA.

[0043] All publications, patents, and patent applications cited in this article, whether above or below, are incorporated herein by reference in their entirety. Detailed Implementation

[0044] Salmonella typhimurium antigen, Salmonella enteritidis antigen, and Salmonella paratyphi A antigen In some aspects, the immunogenic compositions of the present invention comprise Salmonella typhimurium (Salmonella typhimurium). S. Typhimurium antigen. In some aspects, the immunogenic compositions of the present invention contain Salmonella enteritidis (Typhimurium) antigen. S. Enteritidis) antigen. In some aspects, the immunogenic compositions of the present invention contain Salmonella paratyphi A (Enteritidis) antigen. S. Paratyphi A antigen.

[0045] O-antigen Various Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Paratyphi A antigens are known to those skilled in the art. In particular, all Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Paratyphi A bacteria contain an outer membrane including the O-antigen, and the Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A antigens may include the O-antigen.

[0046] For the purposes of this invention, the terms O-antigen, OAg, and O:2 are considered to be used interchangeably. The outer membrane of Gram-negative bacteria contains lipopolysaccharide. The lipopolysaccharide contains O-antigen, which is linked to a lipid A domain via a core domain. The term "..." O-antigen "", OAg” and" O:2” It refers to a polysaccharide composed solely of OAg, or more preferably, to an O-antigen linked to the core domain of lipopolysaccharide.

[0047] As mentioned above, Salmonella typhimurium antigen, Salmonella enteritidis antigen, and / or Salmonella paratyphi A antigen can be O-antigens. Typical purification methods for these O-antigens are based on the phenol-water method first proposed by Westphal and Jann in the 1960s (Westphal and Jann (1965)). Methods Carbohydr . Chem. 5:83-91), followed by detoxification of the lipopolysaccharide with acetic acid or anhydrous hydrazine. The O-antigen is modified to remove lipid A. For example, the extraction and purification of the polysaccharide can be carried out by acetic acid hydrolysis, as described in the following literature: Watson et al. (1992) Infect Immun. 60(11):4679-86; - Konadu et al. (1996) Infect Immun. (7):2709-l5; Konadu et al. people (1994) InfectImmun. 62(11):5048-54; Ahmed et al. people (2006) J Infect Dis.193(4):515-21; Cox et al. people (2011) Glycoconj J 28:165-182; Chu et al. people (1991) Infect Immun. 59(12):4450-58; and Micoli et al. people, 2012 PlosOne, 7(11): e47039.

[0048] The O-antigen of Salmonella serogroups A, B, and D has been described, and they are considered to share a common backbone: →2-α-D-Man p -(l→4)-α-L-Rha p -(l→3)-α-D-Gal p-(1→. The serospecificity of Salmonella paratyphi A is conferred by α-3,6-dideoxyglucose (α-D-porase) linked to the mannose (1→3) in the main chain. The α-L-rhamnose in the main chain is partially O-acetylated at the C-3 position (Konadu et al. (1996) Infect Immun. (7):2709-l5). The disclosed structure of the O-antigen of Salmonella paratyphi A is as follows: Figure 11 As shown, it includes the KDO subunit and primary amine group (located within the ethanolamine pyrophosphate group) in the core structural domain.

[0049] O-antigen conjugate In the implementation scheme, when the antigens of *Salmonella typhimurium*, *Salmonella enteritidis*, and / or *Salmonella paratyphi A* contain O-antigens, the O-antigens from *Salmonella typhimurium*, *Salmonella enteritidis*, and / or *Salmonella paratyphi A* can be part of the conjugate. The term "..." Conjugate A "carrier" is a molecule formed by a covalent link between an antigen (such as an O-antigen) and a carrier. The carrier can be a carrier protein. Typically, the conjugation of a polysaccharide to a carrier protein enhances the immunogenicity of the polysaccharide because it transforms it from a T-cell-independent antigen to a T-cell-dependent antigen, thereby allowing the induction of immune memory.

[0050] Carrier proteins include bacterial toxins, such as diphtheria toxin or tetanus toxin, or toxoids or mutants thereof. In some embodiments, the carrier protein is a CRM. 197 CRM 197 The sequence in Figure 12 Provided in (SEQ ID NO: 1).

[0051] In a specific implementation, the Salmonella paratyphi A antigen comprises a Salmonella paratyphi A O-antigen conjugated to a carrier protein. The carrier protein may be a diphtheria toxoid or CRM. 197 Optionally, the carrier protein is CRM. 197 .

[0052] O-antigens from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A can be conjugated to a carrier protein by a method comprising introducing multiple activation sites into the O-antigen. Optionally, the O-antigen from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A contains multiple activation sites.

[0053] the term" Activation site"" refers to a site or functional group on the polysaccharide that has been activated through steps in a conjugation chemistry process, making it ready to conjugate with a carrier protein. For example, if P-antigen conjugation is performed using CDAP chemistry, the polysaccharide is activated when the addition of CDAP introduces a cyano ester group. "activation" CDAP activation introduces cyanoester groups at one or more sites; these sites where cyanoester groups are introduced are considered... "Activation site" Once activated, the O-antigen can bind (conjugate) to a carrier protein at one or more (in some cases all) activation sites. For the purposes of this invention, the term "..." Excitement live site "This includes sites that have been activated but are not linked to carrier proteins, as well as sites that have been activated and are linked to carrier proteins."

[0054] Optionally, the O-antigen from *Salmonella typhimurium*, *Salmonella enteritidis*, and / or *Salmonella paratyphi A* contains 1.5 or more, 2.0 or more, or 2.5 or more activation sites. Assuming the O-antigen is part of a composition containing multiple O-antigen sugars, if the average number of activation sites on each O-antigen molecule in the composition is 1.5 or more, then the activated O-antigen will contain 1.5 or more sites.

[0055] Optionally, the Salmonella paratyphi A antigen is an O-antigen conjugated to a carrier protein, and the O-antigen is conjugated to the carrier protein by introducing more than one activation site into the Salmonella paratyphi A O-antigen and / or the Salmonella paratyphi A O-antigen containing more than one activation site.

[0056] O-antigens from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A can be optionally conjugated to a carrier protein via a linker through CDAP chemistry.

[0057] Linkers are compounds that can be used to connect proteins and polysaccharides. Any suitable linker can be used in the conjugates and methods of this invention. Suitable linkers include adipic acid dihydrazide (ADH) linkers, which are compounds having the following structure: .

[0058] Other suitable linkers include adipic acid, glutaric acid, carbonyl, β-propionamidyl (WO00 / 10599), adipic acid bis(N-hydroxysuccinimide), dihydrazides similar to ADH but with different chain lengths, hexamethylenediamine (or similar diamines with different chain lengths), and nitrophenylethylamine (Gever et al.(1979) Med. Microbiol. Immunol. 165,171-288), haloacyl halides (US Patent No. 4,057,685), glycosidic bonds (US Patent Nos. 4,673,574; 4,761,283; and 4,808,700), 6-aminohexanoic acid (US Patent No. 4,459,286), N-succinimide-3-(2-pyridinedithio)propionate (SPDP) (US Patent No. 5,204,098), C4 to C12 moieties (US Patent No. 4,663,160), etc.

[0059] In the implementation scheme, when the O-antigen from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A is conjugated to the carrier protein via CDAP chemistry, the O-antigen can be conjugated to the carrier protein via a method that includes a CDAP chemistry method to activate the O-antigen to provide an activated O-antigen step.

[0060] Activating an O-antigen via CDAP chemistry involves mixing the O-antigen with CDAP, thereby introducing cyano ester groups into the polysaccharide or O-antigen. For example, Example 9 discloses a suitable method for activating an O-antigen via CDAP chemistry. Activated O-antigen is obtained by CDAP chemistry activation. Introducing cyano ester groups into the O-antigen via CDAP chemistry involves a step of activating the O-antigen via CDAP chemistry, provided that the method includes mixing the O-antigen with CDAP, and that the number of cyano ester groups present on the O-antigen after the mixing step is higher than the number of cyano ester groups present on the O-antigen before the step. The number of cyano ester groups present can be determined using an ADH quenching / TNBS colorimetric method, as described in the following literature: Lees A, Vaccines (Basel), 2020; 8(4):777.

[0061] Optionally, chemically activating the O-antigen via CDAP includes mixing the O-antigen with CDAP at a weight ratio of 0.05:1 to 5:1, 0.1:1 to 5:1, 0.2:1 to 2:1, or about 0.3:1 (CDAP to O-antigen).

[0062] Optionally, the step of chemically activating the O-antigen by CDAP includes mixing the O-antigen with CDAP in a saline solution (e.g., NaCl or KCl solution). Alternatively, the step of chemically activating the O-antigen by CDAP includes mixing the O-antigen with CDAP in a NaCl or KCl solution with a concentration of 50 mM to 1 M, 100 mM to 250 mM, 125 mM to 200 mM, or about 150 mM.

[0063] Optionally, after mixing the O-antigen with CDAP, the pH is adjusted to 6 to 10, 7 to 9, or 9 to 10. Optionally, the pH is adjusted by adding a base (e.g., triethylamine, sodium hydroxide, or pyridine). Optionally, the pH is adjusted by adding 5% to 15%, 8% to 12%, or about 10% (v / v) triethylamine. Optionally, after mixing the O-antigen with CDAP, the mixture is incubated at 18°C ​​to 30°C, 20°C to 28°C, room temperature, or about 25°C. Optionally, the solution is incubated with stirring before conjugating the activated O-antigen to the carrier protein.

[0064] Example 9 describes a suitable method for chemically activating polysaccharides via CDAP.

[0065] As described above, O-antigens activated by CDAP chemistry (activated polysaccharides or activated O-antigens) contain cyano ester groups (located at the activation site), which can be covalently linked to hydrazide or amino groups. Therefore, O-antigens activated by CDAP chemistry can be directly linked to carrier proteins (via amino groups) or conjugated to carrier proteins via hydrazide / amino group-containing linkers. Suitable linkers include the ADH linkers described above. Therefore, activated O-antigens can be conjugated to carrier proteins by reacting the activated O-antigen with hydrazide / amino groups on the carrier protein or a carrier protein-linker compound. Therefore, the method may further include the step of preparing a carrier protein-linker compound. For example, if the linker is an ADH linker, the method may include the preparation of an ADH-carrier protein compound (such as ADH-CRM). 197 The steps of (compounds), for example, such as Micoli et al. As reported in Vaccine 2011, 29, (4), 712-20.

[0066] The reaction of the activated O-antigen with the acylhydrazine / amino group on the carrier protein or carrier protein-promoter compound may include mixing the carrier protein or carrier protein-promoter compound with the activated O-antigen under suitable conditions where a covalent bond is formed between the cyano ester group (activation site) on the activated O-antigen and the acylhydrazine / amino group on the carrier protein or carrier protein-promoter compound. For example, the activated O-antigen can be simply mixed with the carrier protein or carrier protein-promoter compound.

[0067] Reaction of the activated O-antigen with the hydrazide / amino group on the carrier protein or carrier protein-promoter compound may include mixing the activated O-antigen with the carrier protein or carrier protein-promoter compound in a ratio of 0.1:1 to 5:1, 0.2:1 to 3:1, 0.5:1 to 2:1, or about 1:1 (weight ratio of O-antigen to carrier protein or carrier protein-promoter compound). Optionally, the step of mixing the activated O-antigen with the carrier protein or carrier protein-promoter compound provides a conjugated mixture. Optionally, mixing the activated O-antigen with the carrier protein or carrier protein-promoter compound is carried out at pH 8 to 11, 9 to 10, or about 9.5. Optionally, the pH is maintained at 8 to 11, 9 to 10, or about 9.5 for at least 1 hour, at least 2 hours, 30 minutes to 10 hours, 1 hour to 5 hours, or 2 hours to 3 hours. Optionally, a base (e.g., triethylamine, sodium hydroxide, or pyridine) is used to maintain the pH. Optionally, triethylamine is used to maintain the pH.

[0068] Optionally, after mixing the activated O-antigen and optionally maintaining the mixture at pH 8 to 11 for at least 1 hour, the method may further include the step of adding a glycine solution (to quench cyano ester groups). Optionally, the glycine solution is added at a concentration of 0.5M to 5M, 0.5M to 2M, or about 1M. Optionally, the glycine solution is added to a conjugated mixture with a volume substantially equal to that of the glycine solution. If the volume difference is within 10%, it is considered substantially equal. Optionally, a further step of adjusting the pH using a base (e.g., triethylamine, sodium hydroxide, or pyridine) is included. Optionally, the further step is performed after the step of adding the glycine solution. Optionally, the pH is adjusted to 7 to 9 or about 8. Optionally, an incubation step follows the further step of pH adjustment using a base. Optionally, the incubation step includes incubation at below 15°C, 12°C, below 10°C, 0°C to 10°C, or 2°C to 8°C. Optionally, the incubation process may last 10 to 30 hours or 10 to 20 hours.

[0069] The activated O-antigen is mixed with a carrier protein or a carrier protein-connector compound under conditions suitable for conjugation (e.g., those described in the first two paragraphs) to form a conjugate. The method may further include a chromatographic step for removing unconjugated O-antigen. Optionally, the chromatographic step includes hydrophobic interaction chromatography or anion exchange chromatography.

[0070] A method suitable for conjugating CDAP-activated polysaccharides to carrier proteins or linkers is described in Example 9.

[0071] outer membrane vesicles In some respects, Salmonella typhimurium, Salmonella enteritidis, and / or Salmonella paratyphi A antigens contain or are composed of outer membrane vesicles (such as GMMA).

[0072] For the purposes of this invention, the term " outer membrane vesicles "or" OMV "" is considered interchangeable and refers to any type of outer membrane vesicle. Appropriate OMVs include natural OMVs. Gram-negative bacteria spontaneously release outer membrane vesicles (OMVs) during growth due to cell membrane turgor pressure; these are natural OMVs. OMVs are rich in immunogenic cell surface-associated antigens, periplasmic antigens, and secretory antigens and have been used in vaccines.

[0073] The OMV of this invention includes a universal membrane antigen module (GMMA), a natural OMV (NOMV, see Katial et al.) people 2002, Infect Immun, 70: 702-707), microvesicles (MV (see WO 02 / 09643)), detergent-extracted OMV (DOMV), mutant-derived OMV (m-OMV), and bleb, which are outer membrane protrusions that remain attached to bacteria before being released as MV (see Beveridge, 1999, J. Bacteriol. 181: 4725-4733)).

[0074] Universal membrane antigen modules (GMMAs) are a type of OMV. GMMAs differ from natural outer membrane vesicles (NOMVs) in two key aspects, the latter being products spontaneously released by Gram-negative bacteria. First, to induce GMMA formation, the membrane structure is modified by the deletion of genes encoding key structural components (such as tolR, which can lead to supervesicles). Second, the genetic modification results in a large number of outer membrane "..." budding (or "excessive foam"), thus providing a practical source of membrane material for vaccine production. Therefore, for the purposes of this invention, the term " GMMA "OMV" refers to spontaneously released OMV from bacteria that have been modified to produce superfoaming (e.g., Salmonella that has been modified to no longer contain the gene encoding a functional TolR).

[0075] The Gram-negative bacteria that yield the OMV (such as GMMA) of this invention after purification can be one or more of the following groups: *Salmonella enterica* subsp. *enterotyphimurium* serotype (*Salmonella typhimurium*), *Salmonella enterica* subsp. *enterotyphimurium* serotype (*Salmonella enteritidis*), and *Salmonella enterica* subsp. *enterotyphimurium* serotype (*Salmonella paratyphimurium*). Suitable purification methods are known in the art, including various filtration and chromatographic methods. A preferred two-step filtration purification method is described in WO 2011 / 036562, which is incorporated herein by reference. Modification of lipid A In some respects, Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A antigens contain or consist of the following: GMMA, namely Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A GMMA. The Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A GMMA may contain modified lipid A, or may be derived from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A containing modified lipid A. Modified lipid A refers to lipid A that has a different structure compared to the corresponding wild-type lipid A.

[0077] The structure of lipid A isolated from GMMA can be determined by MALDI-TOF analysis. In the above assay, lipid A was isolated after GMMA treatment with acetic acid, and then measured by MALDI-TOF. GMMA with a protein concentration of approximately 1 mg / mL (micro BCA calibration curve) or a cell bank suspension (4 mL sample) with an OD600 of approximately 3 was treated with 1% acetic acid (final concentration) at 100°C for 2 or 6 hours to obtain a precipitate containing lipid A. The precipitate was then collected, washed with water, and lipid A was extracted with a chloroform / methanol (4:1) mixture. The final solution containing lipid A was mixed with a saturated Super DHB (Fluka, 50862) solution (acetonitrile / water = 1:1) at a 1:1 ratio. 2 μL of the mixture was spotted onto a target plate, and after the spots dried at room temperature, the target plate was inserted into the mass spectrometer. The spectra (negative reflectance mode) typically show peaks corresponding to the type of lipid A molecule, and include several peaks resulting from lipid A cleavage (i.e., loss of one or more fatty acid chains), sodium adducts (+22 m / z), and lipid A dephosphorylation (-80 m / z). The type of lipid A is identified by comparing the molecular peak mass m / z with the expected value for the analyzed sample.

[0078] Optionally, lipid A is modified to be detoxified (i.e., the modified lipid A is detoxified lipid A). Detoxified This means that the toxicity of lipid A is lower than that of wild-type lipid A. The wild-type lipid A used for comparison is the corresponding wild-type lipid A. In the context of this article, toxicity "or" Toxic "Activation" refers to the degree to which lipid A activates the innate immune system, particularly through the Toll-like receptor 4 pathway. Highly toxic lipid A can lead to uncontrolled inflammation, apoptosis, and, in extreme cases, septic shock. Optionally, modified lipid A is less toxic if it has lower reactivity than the corresponding wild-type lipid A. For example, its low toxicity can be determined by administering modified lipid A to animals (such as rabbits) and using a monocyte activation assay to determine whether the modified lipid A activates more monocytes than the corresponding wild-type lipid A.

[0079] “ Corresponding wild-type lipid A "Refers to lipid A present in the corresponding wild-type bacteria and strains. For example, in the context of Salmonella Typhimurium GMMA, relative to " Corresponding wild-type lipid A "Modified lipid A should be understood as lipid A that has been modified (e.g., to reduce its toxicity) relative to lipid A in wild-type Salmonella typhimurium."

[0080] Optionally, the modified lipid A is a pentacylated lipid A. Whether GMMA contains pentacylated lipid A can be determined by measuring the structure of lipid A using the MALDI-TOF analysis described above.

[0081] Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A GMMA can be derived from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A bacteria containing any suitable modifications that result in the production of GMMA containing lipid A with lower toxicity than wild-type lipid A.

[0082] HtrB, MsbB, and PagP are proteins involved in lipid A production in Gram-negative bacteria. MsbB and PagP play important roles in Salmonella. Salmonella strains that do not express functional forms of MsbB and / or PagP will not produce native lipid A, but instead will produce modified, detoxified lipid A. Therefore, GMMA from *Salmonella typhimurium*, *Salmonella enteritidis*, and / or *Salmonella paratyphi A* can be derived from functional forms of *Salmonella typhimurium*, *Salmonella enteritidis*, and / or *Salmonella paratyphi A* that do not express MsbB and / or PagP. Optionally, GMMA from *Salmonella typhimurium*, *Salmonella enteritidis*, and / or *Salmonella paratyphi A* derives from *Salmonella typhimurium*, *Salmonella enteritidis*, and / or *Salmonella paratyphi A* that do not contain genes encoding functional MsbB and / or PagP proteins.

[0083] Whether bacteria derived from GMMA express the functional forms of MsbB and / or PagP, or whether they contain genes encoding functional MsbB and / or PagP proteins, can be determined by isolating lipid A from GMMA and analyzing its structure using MALDI-TOF technology as described above. If lipid A is detoxified, it indicates that bacteria derived from GMMA do not express the functional forms of MsbB and / or PagP, or do not carry genes encoding functional MsbB and / or PagP proteins.

[0084] Optionally, GMMA may be derived from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A due to gene mutations (such as...). htrB、msbB and / or pagPTherefore, it does not contain genes encoding the corresponding functional proteins. Optionally, Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A derived from GMMA do not contain genes encoding functional HtrB, MsbB, and / or PagP proteins. Optionally, Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A derived from GMMA contain at least a portion of genes encoding HtrB, MsbB, and / or PagP proteins, but said genes are mutated to the point that the encoded HtrB, MsbB, and / or PagP proteins lose one or more key amino acids, or a portion of the gene sequence is missing. For example, Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A derived from GMMA may... htrB msbB and / or pagP The gene contains substitution or deletion mutations. Alternatively, GMMA derived from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A may have addition mutations in the htrB, msbB, and / or PagP genes, such as frameshift addition mutations. Optionally, GMMA derived from Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A contains htrB、msbB and / or pagP Gene deletion mutations. Optional, htrB、msbB and / or pagP The gene contains deletion mutations, and at least 10%, at least 20%, at least 25%, at least 50%, or at least 75% of them are deletions. htrB、msbB and / or pagP Gene deletion. Optionally, GMMA is deleted from its derivatives in Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A. htrB、msbB and / or pagP Genes (e.g., due to complete) htrB、msbB and / or pagP Gene missing (existing) htrB , msbB and / or pagP mutation)).

[0085] Optionally, *Salmonella paratyphi A* strains whose OMV or GMMA are derived from the msbB and / or pagP genes have at least a portion replaced by different genes. Optionally, *Salmonella paratyphi A* strains whose OMV or GMMA are derived from the msbB and / or pagP genes have at least a portion replaced by tetracycline (tet) or kanamycin (kan) genes, respectively. Optionally, *Salmonella paratyphi A* strains whose OMV or GMMA are derived from the msbB and / or pagP genes have at least a portion replaced by tetracycline (tet) or kanamycin (kan) genes, respectively. Optionally, *Salmonella paratyphi A* strains whose OMV or GMMA are derived from the msbB and / or pagP genes are pagP::kan and / or msbB::tet. "::" indicates that the gene before "::" is replaced by the gene after "::". Therefore, "pagP::kan" indicates that the pagP gene has been replaced by kanamycin.

[0086] Excess foam GMMA can be modified (e.g., genetically modified) from its derivatives, such as Salmonella typhimurium, Salmonella enteritidis, and / or Salmonella paratyphi A, to achieve superfoaming, i.e., more outer membrane “budding” compared to the corresponding Gram-negative bacteria without the aforementioned gene mutation.

[0087] GMMA-derived Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A may contain any suitable modifications that result in vesicle extrusion. Optionally, the modification is a mutation; for example, GMMA-derived Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A may not contain a gene encoding a functional protein (such as tolR) due to a mutation in said gene. Optionally, GMMA-derived Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A may not contain a gene encoding a functional TolR protein. Optionally, GMMA-derived Salmonella Typhimurium, Salmonella Enteritidis, and / or Salmonella Paratyphi A may contain at least a portion of a gene encoding a TolR protein, but said gene may be mutated to result in the loss of one or more essential amino acids in the encoded TolR protein, or a partial deletion of said gene. For example, *Salmonella typhimurium*, *Salmonella enterica*, and / or *Salmonella paratyphi A* derived from GMMA may have substitution or deletion mutations in the tolR gene. Alternatively, *Salmonella typhimurium*, *Salmonella enterica*, and / or *Salmonella paratyphi A* derived from GMMA may have addition mutations in the tolR gene, such as addition mutations causing frameshifts. Optionally, *Salmonella typhimurium*, *Salmonella enterica*, and / or *Salmonella paratyphi A* bacteria derived from GMMA contain deletion mutations in the tolR gene. Optionally, the tolR gene contains deletion mutations, and at least 10%, at least 20%, at least 25%, at least 50%, or at least 75% of the tolR gene is deleted. Optionally, *Salmonella typhimurium*, *Salmonella enterica*, and / or *Salmonella paratyphi A* bacteria derived from GMMA lack the tolR gene (e.g., due to the complete tolR gene being deleted). tolR mutation).

[0088] Optionally, the *Salmonella paratyphi A* OMV or GMMA is derived from *Salmonella paratyphi A*, wherein at least a portion of the tolR gene has been replaced by a different gene. Optionally, the *Salmonella paratyphi A* OMV or GMMA is derived from *Salmonella paratyphi A*, wherein at least a portion of the tolR gene has been replaced by the chloramphenicol acetyltransferase (cat) gene. Optionally, the *Salmonella paratyphi A* OMV or GMMA is derived from *Salmonella paratyphi A*, wherein the tolR gene has been replaced by the chloramphenicol acetyltransferase (cat) gene. Optionally, the *Salmonella paratyphi A* OMV or GMMA is derived from *Salmonella paratyphi A*, and the strain is tolR::cat.

[0089] Whether a genetic modification causes vesicle ...

[0090] strain Optionally, the immunogenic composition comprises Salmonella typhimurium GMMA derived from Salmonella typhimurium strain 2192 (see, for example, De Benedetto et al.) people , 2017, Multiple Techniques for SizeDetermination of Generalized Modules for Membrane Antigens from Salmonella typhimurium and Salmonella enteritidisACS Omega. 2017 Nov 30; 2(11):8282-8289). Optionally, the immunogenic composition comprises Salmonella GMMA derived from Salmonella enterica 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). Optionally, the immunogenic composition comprises Salmonella paratyphi A GMMA derived from Salmonella 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 Salmonella paratyphi A strain could be tolR::cat pagP::kan msbB::tet.

[0091] The immunogenic composition contains Salmonella typhimurium GMMA derived from Salmonella typhimurium strain 2192, provided that the strain used is based on Salmonella typhimurium strain 2192, even if strain 2192 has been modified (e.g., mutations in the msbB, pagP, or tolR genes).

[0092] dose The immunogenic composition may contain 1 to 100 µg, 1 to 50 µg, 15 to 50 µg, 20 to 30 µg, 1 to 20 µg, 1 to 10 µg, about 25 µg, or about 5 µg of Salmonella paratyphi A O-antigen. The dosage of Salmonella paratyphi A O-antigen in the composition can be determined by mild hydrolysis of the O-antigen in the immunogenic composition (to provide the monosaccharide porase) and by detecting the porase content using HPAEC-PAD. Porase is a monosaccharide present in Salmonella paratyphi A O-antigen but not in O-antigens from Salmonella enteritidis or Salmonella typhimurium. Example 10 illustrates a suitable method for determining the content of Salmonella paratyphi A O-antigen by HPAEC-PAD. If the Salmonella paratyphi A O-antigen is part of a conjugate (containing a carrier protein), the content of the carrier protein may vary. For example, if the ratio of carrier protein to O-antigen in the conjugate is greater than 2, then the amount of carrier protein required to achieve a dose of 1 µg O-antigen will be higher than when the ratio of carrier protein to O-antigen is less than 2.

[0093] The immunogenic compositions of the present invention may comprise Salmonella Typhimurium antigen or Salmonella Typhimurium 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, about 20 µg, or about 4 µg. The GMMA used in the immunogenic compositions comprises O-antigen. The dose of GMMA can be quantified as an O-antigen dose, i.e., if the immunogenic composition contains a dose of 1 µg (O-antigen) of GMMA, then the immunogenic composition contains sufficient GMMA to provide 1 µg of O-antigen associated with the GMMA (e.g., if the GMMA is Salmonella Typhimurium GMMA, then the immunogenic composition contains GMMA containing a total of 1 µg of Salmonella Typhimurium O-antigen). This means that if the immunogenic composition contains O-antigen-rich GMMA, the actual GMMA content required to achieve a 1 µg (O-antigen) dose may be lower than the amount required with GMMA that is not O-antigen-rich. The content of Salmonella Typhimurium O-antigen in the immunogenic composition can be determined by mild hydrolysis of the O-antigen in the composition (to provide the monosaccharide abigolose) and detection of the abigolose content using HPAEC-PAD. Assuming no addition of… free "The amount of O-antigen in the Salmonella Typhimurium GMMA composition will correspond to the O-antigen dose of Salmonella Typhimurium GMMA." 。

[0094] The immunogenic compositions of the present invention may contain doses of Salmonella enterica antigen or Salmonella enterica GMMA (O-antigen) ranging from 1 µg to 50 µg, 2 µg to 25 µg, 2 µg to 10 µg, 15 µg to 25 µg, about 20 µg, or about 4 µg. The amount of Salmonella enterica 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 tivasugar) and by detecting the tivasugar content using HPAEC-PAD. Assuming no " free "The amount of O-antigen in the Salmonella enteritidis GMMA composition will correspond to the O-antigen dose of Salmonella enteritidis GMMA." 。

[0095] The immunogenic compositions of the present invention may contain doses of 1 µg to 50 µg, 2 µg to 25 µg, 2 µg to 10 µg, 15 µg to 25 µg, about 20 µg, or about 4 µg of Salmonella paratyphi A GMMA (O-antigen). The amount of Salmonella 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 the monosaccharide porase) and detection of the amount of porase using HPAEC-PAD. Assuming no addition of “free” Salmonella paratyphi A O-antigen, the amount of O-antigen in the Salmonella paratyphi A GMMA composition will correspond to the dose of O-antigen in Salmonella paratyphi A GMMA.

[0096] The O-antigen / protein ratio of Salmonella 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 may be quantified by HPAEC-PAD, for example as described in Example 5. Optionally, the protein concentration may be quantified by micro-BCA method, for example as described in PCT / EP2022 / 073501.

[0097] The immunogenic compositions of the present invention may contain 1 to 100 µg, 1 to 50 µg, 15 to 50 µg, 20 to 30 µg, 1 to 20 µg, 1 to 10 µg, about 25 µg, or about 5 µg of fVi polysaccharide. The dosage of fVi polysaccharide in the composition can be determined by hydrolyzing (by acid hydrolysis) the fVi polysaccharide in the immunogenic composition and measuring the monomeric sugars of the repeating unit by HPAEC-PAD determination. A suitable method for determining the fVi polysaccharide content by HPAEC-PAD is described in Example 5. If the fVi polysaccharide is a component of a conjugate (containing a carrier protein), the amount of carrier protein can be varied. For example, if the ratio of carrier protein to fVi polysaccharide in the conjugate is greater than 2, then the amount of carrier protein required to achieve a dose of 1 µg of fVi polysaccharide will be higher than when the ratio of carrier protein to fVi polysaccharide is less than 2.

[0098] Salmonella typhi antigen The immunogenic composition may further contain an antigen from Salmonella Typhi. Optionally, the antigen from Salmonella Typhi is Vi polysaccharide.

[0099] the term" Vi "or" Vi polysaccharide "Involves capsular polysaccharides of Salmonella typhi purified from Citrobacter (Rondini et al.)" people , J. Infect. Dev. Ctries, 2012).

[0100] Optionally, Vi polysaccharides are fragmented Vi polysaccharides (fVi). The term "..." Fragmented "When referring to Vi polysaccharides, it means that the size of the Vi polysaccharide has been reduced, thereby reducing the number of repeating units in the polysaccharide. Therefore, fragmented Vi has a lower average molecular weight compared to natural Vi. For example, fragmented Vi may contain 30 to 300 repeating units, while natural Vi contains more than 600 repeating units. The structure of the repeating unit of Vi monomer is shown below."

[0101] In fragmented Vi, preferably, no change in the repeating unit structure is observed compared to native Vi. This can be confirmed by 1H NMR analysis (see WO2015 / 068129). Furthermore, the percentage of O-acetylated groups in fragmented Vi is preferably the same as in native Vi (i.e., about 95% O-acetylation), but it may vary and decrease to about 65% O-acetylation. O-acetylation can be determined by standard methods, such as 1H NMR or the Hestrin colorimetric method.

[0102] At its natural size, fVi polysaccharides have an average molecular weight of approximately 165 kDa, as determined by HPLC size exclusion chromatography (HPLC-SEC). In some embodiments, fVi polysaccharides have average molecular weights of 10 kDa to 90 kDa, 25 kDa to 70 kDa, 40 kDa to 55 kDa, 41 kDa to 49 kDa, or 51 kDa to 55 kDa. Optionally, fVi polysaccharides have a target molecular weight of 51 kDa to 55 kDa (e.g., prepared by methods typically producing fVi with molecular weights in this range). The molecular weight of Vi polysaccharides can be determined by HPLC-SEC.

[0103] Typically, the average molecular weight was calculated by running samples on a TSK Gel 3000 PWXL column (30 cm x 7.8 mm; particle size 7 μm; No. 808021) along with a TSK Gel PWXL guard column (4.0 cm x 6.0 mm; particle size 12 μm; No. 808033) (Tosoh Bioscience) using dextran as standards (5, 25, 50, 80, 150 kDa). The mobile phase was 0.1 M NaCl, 0.1 M NaH₂PO₄, 5% CH₃CN, pH 7.2, and the flow rate was 0.5 mL / min (isocratic method, 30 min). Void volume and bed volume were calibrated using λ-DNA (λ-DNA molecular weight marker III 0.12–21.2 kb; Roche) and sodium azide (NaN₃; Merck), respectively.

[0104] 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.

[0105] The fVi polysaccharides used in this invention have a specific range of average molecular weights (avMW), which can be further represented by the polydispersity index (PDI).

[0106] The polydispersity index is calculated according to the following equation: PDI = Mw / Mn Where Mw is the weight-average molecular weight and Mn is the number-average molecular weight.

[0107] The narrower the molecular weight distribution, the closer the PDI value is to 1.

[0108] fVi polysaccharides may have an avMW distribution characteristic, wherein at least 80% of the mixture has an avMW of 25 kDa to 70 kDa. Alternatively, they may have an avMW distribution characteristic, wherein at least 50% of the mixture has an avMW of 35 kDa to 60 kDa. Or, they may have an avMW distribution characteristic, wherein at least 30% of the mixture has an avMW of 41 kDa to 55 kDa.

[0109] Fragmentation of Vi polysaccharides can be performed using various methods known in the art, such as chemical hydrolysis of natural polysaccharides, enzymatic fragmentation of natural polysaccharides, gamma irradiation of natural polysaccharides, or mechanical methods such as ultrasonic treatment, or high-pressure homogenizers / microfluidizers / HPCDS (high-pressure cell disruption systems) of natural polysaccharides. The fragmentation method used in this invention needs to produce fVi polysaccharides with average molecular weights of less than 90 kDa, less than 80 kDa, less than 60 kDa, or 40 to 55 kDa. The selected method can also be operated without altering the repeating unit structure.

[0110] Preferably, fragmentation is not performed by mechanical means. Preferably, fragmentation is not performed by alkaline hydrolysis. fVi polysaccharides can be obtained by chemical hydrolysis with hydrogen peroxide. Using this method, it has been found that the size of Vi polysaccharides can be reduced without altering the structure of the repeating units. Furthermore, hydrogen peroxide hydrolysis can form fragmented Vi with a lower average molecular weight than the products obtained by mechanical methods. A method suitable for fragmenting Vi polysaccharides is described in Example 2.

[0111] fVi polysaccharides can be part of an fVi conjugate containing fVi and a carrier protein. Optionally, the carrier protein in the fVi conjugate is tetanus toxoid, CRM, etc. 197 Or diphtheria toxoid. Optionally, the carrier protein is CRM. 197 .

[0112] fVi polysaccharides can be conjugated to carrier proteins using any suitable conjugation chemistry method.

[0113] The conjugation of fVi polysaccharides to carrier proteins can be achieved via -NH2 groups, for example, through the side chains of lysine or arginine residues in the carrier polypeptide. When fVi polysaccharides have free aldehyde groups, these groups can form conjugates with amines in the protein through reductive amination. Conjugation with the carrier can also be achieved via -SH groups, for example, through the side chains of cysteine ​​residues in the carrier polypeptide. Alternatively, fVi polysaccharides can be conjugated to carrier proteins via adaptor molecules.

[0114] fVi polysaccharides are typically activated or functionalized prior to conjugation. Activation may involve, for example, cyaniding agents such as CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate). Other applicable techniques use carbodiimides, hydrazides, active esters, norbornane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU (see, for example, WO 98 / 42721 for details).

[0115] Direct conjugation with a carrier protein may include oxidation of the fVi polysaccharide followed by reductive amination of the protein, as described in U.S. Patents 4,761,283 and 4,356,170. Conjugation via a linker group can be performed by any known procedure, such as those described in U.S. Patents 4,882,317 and 4,695,624. Typically, the linker is connected via the anomeric carbon of the polysaccharide. A preferred type of linker is an adipic acid linker, which can be formed by conjugating a free -NH2 group (e.g., introduced into the polysaccharide via amination) with adipic acid (e.g., activated using a diimide), and then conjugating the protein with the resulting sugar-adipic acid intermediate (see, for example, 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 by conjugating a free -NH group to glutaric acid in the same manner. Adipic acid and glutaric acid linkers can also be formed by direct conjugation with polysaccharides. Right now No prior introduction of free groups (e.g., free -NH groups) onto the polysaccharide is required. The protein is then conjugated with the resulting sugar-adipic acid / glutaric acid intermediate. Another preferred type of linker is the carbonyl linker, which can be formed by reacting the free hydroxyl groups of the modified polysaccharide with CDI (Bethell GS). et al.(1979) J. Biol. Chem. 254, 2572-4 and Hearn MTW (1981) J. Chromatogr. 218, 509-18); subsequently reacts with proteins to form carbamate bonds. Other linkers include β-propionamidyl (WO00 / 10599), nitrophenylethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (US Patent No. 4,057,685), glycosidic linkages (US Patent Nos. 4,673,574, 4,761,283; and 4,808,700), 6-aminohexanoic acid (US Patent No. 4,459,286), N-succinimide-3-(2-pyridinedithio)-propionate (SPDP) (US Patent No. 5,204,098), adipic acid dihydrazide (ADH) (US Patent No. 4,965,338), and C4 to C12 moieties (US Patent No. 4,663,160). Carbodiimide condensation (WO2007 / 000343) can also be used.

[0116] A bifunctional linker can be used to provide a first group for conjugation with an amino group in the polysaccharide (e.g., introduced into the polysaccharide via amination) and a second group for conjugation with a support (typically used for coupling with an amine in the support). Alternatively, the first group can be conjugated directly with the polysaccharide, i.e., without the need to pre-introduce a group (e.g., an amine group) into the polysaccharide.

[0117] Optionally, the fVi conjugate can be obtained by or through a method comprising the following steps (i.e., a method for preparing the fVi conjugate): a. Fragmenting Vi polysaccharides to obtain fragmented Vi (fVi) polysaccharides with average molecular weights of 10 kDa to 90 kDa, 25 kDa to 70 kDa, 40 kDa to 55 kDa, 41 kDa to 49 kDa, or 51 kDa to 55 kDa; b. The fVi polysaccharide obtained in step a is reacted with carbodiimide and N-hydroxysuccinimide at pH 5 to 6 to form N-hydroxysuccinimide ester fVi derivatives; and c. The N-hydroxysuccinimide ester fVi derivative obtained in step b is reacted with a carrier protein (optionally derivatized carrier protein) to produce an fVi conjugate.

[0118] This method is described in more detail in WO2015068129.

[0119] The carrier protein can be derivatized by reacting it with a carbodiimide and a linker. Optionally, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). Any suitable linker (e.g., those discussed above) can be used. In some embodiments, the linker is an ADH linker. Optionally, derivatizing the carrier protein produces a derivatized carrier protein. Optionally, the carrier protein is a CRM. 197 Furthermore, the derivatization of the carrier protein includes one or more of the following steps: (i) Provide CRM in an appropriate buffer solution 197 Optional MES buffer; (ii) CRM 197 Compared to EDAC, the ratios are 1:0.05 to 1:0.5, 1:0.1 to 1:0.3, or approximately 1:0.15 (CRM). 197 Mixed in proportion to the weight ratio of EDAC; (iii) CRM 197 Compared to ADH, the ratio is 1:1 to 1:6, 1:2 to 1:4, or approximately 1:3.5 (CRM). 197 Mix in a ratio of (weight ratio of ADH); (iv) CRM 197 Incubate with a mixture of EDAC and optionally ADH for at least 30 minutes or from 30 minutes to 2 hours, optionally with stirring; and (v) Purified and derivatized CRM 197 Optionally filtered by tangential flow.

[0120] In some embodiments, the carrier protein is derived by a method comprising steps (i), (ii), and (iv). In some embodiments, the carrier protein is derived by a method comprising steps (i), (ii), (iii), and (iv). In some embodiments, the carrier protein is derived by a method comprising steps (i), (ii), (iv), and (v). In some embodiments, the carrier protein is derived by a method comprising all of the above steps (i) to (v). In some embodiments, steps (i) to (v) are performed in the order listed above, but steps (ii) and (iii) may be performed simultaneously.

[0121] fVi conjugates can be obtained or prepared by a method comprising the following steps: reacting fVi polysaccharides with carbodiimide and N-hydroxysuccinimide at pH 5 to 6 to form N-hydroxysuccinimide ester fVi derivatives. Optionally, the carbodiimide is EDC (N-3-dimethylaminopropyl(-N-ethylcarbodiimide). Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide includes mixing fVi with a carbodiimide such as EDC in the presence of N-hydroxysuccinimide (NHS). Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide includes mixing fVi polysaccharide with NHS. Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide includes mixing fVi polysaccharide with NHS such that the NHS concentration is from 0.1 M to 0.5 M, or about 0.33 M, and the fVi polysaccharide concentration is from 1 mg / mL to 100 mg / mL, or about 50 mg / mL. mg / ml. Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide includes mixing fVi polysaccharide with EDC such that the molar ratio of EDC to fVi repeating units is 1:1 to 20:1, 1:1 to 10:0, 2:1 to 7:1, or about 5:1. Optionally, mixing fVi polysaccharide with EDC is performed after mixing fVi polysaccharide with NHS. Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide includes incubating the mixture of fVi polysaccharide, NHS, and EDC at room temperature for at least 30 minutes or about 1 hour.

[0122] Optionally, reacting the N-hydroxysuccinimide fVi derivative with a carrier protein (optionally derivatized carrier protein) comprises mixing the N-hydroxysuccinimide fVi derivative with the carrier protein (or a carrier protein derivative). Optionally, reacting the N-hydroxysuccinimide fVi derivative with the carrier protein (optionally derivatized carrier protein) comprises mixing the N-hydroxysuccinimide fVi derivative with the carrier protein (or a carrier protein derivative) at a (w / w) ratio of 1:0.1 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:2, or about 1:1. Optionally, the mixing of the N-hydroxysuccinimide fVi derivative with the carrier protein (or a carrier protein derivative) is carried out in a buffer solution at pH 5 to 7 or about 6. Optionally, the mixing of the N-hydroxysuccinimide fVi derivative with the carrier protein (or a carrier protein derivative) is carried out in a MES buffer solution. Optionally, the N-hydroxysuccinimide fVi derivative is mixed with the carrier protein (or carrier protein derivative) at a temperature of 20°C to 30°C or about room temperature, optionally accompanied by mixing.

[0123] Following the step of reacting the N-hydroxysuccinimide ester fVi derivative with a carrier protein (or a carrier protein derivative), the method for preparing the fVi conjugate may include one or more additional steps: (i) Quenching by adding a quencher (e.g., Phenyl HP buffer); (ii) Filter fVi conjugates; (iii) Purify the fVi conjugate, optionally using hydrophobic interaction chromatography; (iv) Concentrate the fVi conjugate, optionally by tangential flow filtration; and (v) Filter the conjugate, optionally using one or more 0.2µm filters.

[0124] Optionally, the method for preparing the fVi conjugate includes two or more, three or more, four or more, or all five steps of steps (i) to (v) above. Optionally, the method includes step (i) above. Optionally, the method includes steps (i) to (iii) above. Optionally, the method includes steps (i) to (v) above. Optionally, the method includes steps (i) to (iii) above in the order described above. Optionally, the method includes steps (i) to (v) above in the order described above.

[0125] Example 2 illustrates the use of EDAC chemistry to combine fVi polysaccharides with CRM via an ADH linker. 197 Appropriate methods for joining.

[0126] The immunogenic compositions of the present invention, or the immunogenic compositions used in the present invention, may contain additional components, such as pharmaceutically acceptable excipients, adjuvants, and / or further antigens.

[0127] The immunogenic composition may also contain pharmaceutically acceptable excipients. Typical " Pharmaceutically acceptable excipient "This includes any carrier that does not itself induce the production of antibodies harmful to individuals receiving the composition. Suitable carriers are typically large, slowly metabolized macromolecules, 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. Pharmaceutically acceptable excipients may also contain diluents such as water, physiological saline, glycerol." waitAdditionally, auxiliary substances may be present, such as wetting agents or emulsifiers, pH buffers, etc. Sterile, pyrogen-free Tris-buffered saline is particularly suitable as a carrier for use with aluminum adjuvants because phosphate in phosphate-buffered saline may interfere with the binding of outer membrane vesicles to aluminum. However, in a particular embodiment, the immunogenic composition comprises phosphate-buffered saline (and optionally aluminum adjuvant as described below). Optionally, the immunogenic composition comprises phosphate-buffered saline with a pH of 6 to 7 (e.g., pH 6.5).

[0128] Immunogenic compositions can be formulated as injections, liquid solutions, or suspensions. They can also be formulated in solid forms (e.g., lyophilized compositions or spray-dried compositions) suitable for dissolution or suspension in a liquid carrier prior to injection. The immunogenic compositions can be formulated for topical application, such as as ointments, creams, or powders. They can be formulated for oral administration, such as as tablets or capsules, sprays, or syrups (optionally flavored). They can be formulated for pulmonary administration, such as using inhalers in the form of fine powders or sprays. They can be formulated as suppositories or vaginal suppositories. They can be formulated for nasal, ear, or ocular administration, such as as drops. Immunogenic compositions can be designed as kits that allow for immediate reconstitution into a combination composition prior to administration to mammals. Such kits may contain one or more liquid antigens and one or more lyophilized antigens. Immunogenic compositions may be contained in vials or in pre-filled syringes. These syringes may or may not include needles. The syringe will contain a single dose of the composition, while the vial may contain a single or multiple doses.

[0129] The immunogenic compositions of the present invention, or the immunogenic compositions used in the present invention, can be packaged in single-dose or multi-dose forms. For multi-dose forms, vials are preferred over pre-filled syringes. The effective dose can be determined by conventional methods, but a typical human dose volume of the composition is 0.5 ml, for example, for intramuscular injection.

[0130] The composition will be sterile. The immunogenic composition of the present invention, or the immunogenic composition used in the present invention, may be isotonic relative to humans.

[0131] Therefore, the immunogenic compositions of the present invention, or the immunogenic compositions used in the present invention, can be used as vaccines. Vaccines according to the present invention can be prophylactic (i.e., for the prevention of infection) or therapeutic (i.e., for the treatment of infection), but are generally prophylactic.

[0132] An immunogenic composition used as a vaccine contains an effective amount of antigen and any other components as needed. The term "immunogenic composition" Effective dose"(Immune effective dose)" refers to the amount that is effective for treatment or prevention when administered to an individual as a single dose or as part of a series of doses. The dose varies depending on the individual's health status and physiological condition, age, taxonomic class (e.g., non-human primate, primate, etc.), the individual's immune system's ability to synthesize antibodies, the required level of protection, the vaccine formulation, the treating physician's assessment of the medical condition, and other relevant factors. The immunogenic compositions of the present invention may contain antimicrobial agents, particularly when packaged in multiple doses.

[0133] adjuvant The immunogenic compositions of the present invention, or the immunogenic compositions used in the present invention, may contain adjuvants. Any suitable adjuvant may 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, and mixtures of different mineral compounds, which may be in any suitable form (e.g., gels, crystals, amorphous, etc.) and are preferably in an adsorbed form. Mineral-containing compositions may also be formulated as metal salt particles.

[0134] The immunogenic compositions of the present invention, or the immunogenic compositions used in the present invention, may contain aluminum adjuvants, i.e., any composition containing Al. 3+ Compounds containing ions.

[0135] The aluminum adjuvant may contain aluminum phosphate (any substance containing Al). 3+ and PO4 3- (Ionic compounds) and / or aluminum hydroxide (any compound containing Al) 3+ and OH - (A compound of ions) or composed of them.

[0136] Optionally, the aluminum adjuvant comprises or is composed of aluminum hydroxide. The aluminum hydroxide adjuvant may comprise or be an aluminum oxyhydroxide salt. The aluminum hydroxide adjuvant may comprise or be at least partially crystalline aluminum oxyhydroxide salt. Aluminum oxyhydroxide salts (which may be represented by the formula AlO(OH)) can be distinguished from other aluminum compounds (such as aluminum hydroxide salts (Al(OH)3)) by infrared (IR) spectroscopy, specifically at 1070 cm⁻¹. -1 There is an adsorption zone at 3090-3100 cm. -1A strong shoulder peak is observed at the following reference (see Chapter 9: Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)). The crystallinity of aluminum hydroxide adjuvants is reflected by the width of the diffraction band at half-height (WHH). Poorly crystallized particles exhibit greater spectral broadening due to their smaller grain size. Surface area increases with increasing WHH, and adjuvants with higher WHH values ​​have been observed to have stronger antigen adsorption capacity. Fibrous morphology (as shown in transmission electron microscopy images) is a typical characteristic of aluminum hydroxide adjuvants.

[0137] Suitable embodiments of aluminum hydroxide adjuvants will be apparent to those skilled in the art, such as aluminum gel (ALHYDROGEL®).

[0138] Aluminum adjuvants may contain or consist of the following: 0.1 mg to 10 mg Al 3+ 0.1 mg to 5 mg Al 3+ 0.3 mg to 0.4 mg Al 3+ Or approximately 0.35 mg Al 3+ .

[0139] Immunogenicity The embodiments of this invention demonstrate that the immunogenic compositions of this invention exhibit good immunogenicity. Immunogenicity can be measured according to the assay methods listed in Example 6.

[0140] The immunogenic composition of the present invention can induce at least 10 in an immunogenicity assay including the following steps. 3 EU / ml of anti-Salmonella typhimurium O-antigen antibody and / or at least 10 3 EU / ml of anti-Salmonella O-antigen antibody: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg glucose / glucose on days 0 and 28; and (b) On day 42, the antibody levels against Salmonella Typhimurium O antigen and / or Salmonella Enteritidis O antigen were measured by ELISA.

[0141] An immunogenic composition that induces the antibody level when used to immunize mice, then " Induction "At least 10" 3EU / ml of anti-Salmonella typhimurium O-antigen antibody. Whether this level of antibody can be induced when used to immunize mice can be determined by testing a sample of the immunogenic composition using an immunogenicity assay.

[0142] The dosage of GMMA and carbohydrates can be determined as described in the "Dosage" section above.

[0143] A suitable ELISA may involve: - Coat ELISA plates with Salmonella typhimurium or Salmonella enteritidis O-antigen; - Blood samples collected from mice on day 42 were applied to coated ELISA plates, which were then washed to remove antibodies that did not bind to the O-antigen of Salmonella Typhimurium or Salmonella Enteritidis; and - Use anti-IgG antibodies conjugated to the detection portion (such as alkaline phosphatase) to detect the amount of anti-Salmonella typhimurium or Salmonella enteritidis O-antigen antibodies bound to the ELISA plate.

[0144] The immunogenic composition of the present invention can induce at least 10 in an immunogenicity assay comprising the following steps. 3 EU / ml of anti-fVi conjugate antibody: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) The level of the anti-fVi conjugate antibody was measured by ELISA on day 42.

[0145] The dosage of GMMA and carbohydrates can be determined as described in the "Dosage" section above.

[0146] A suitable ELISA may involve: - Coat ELISA plates with fVi ​​polysaccharide; - Blood samples collected from mice on day 42 were applied to coated ELISA plates, which were then washed to remove antibodies that did not bind to the fVi polysaccharide; and - The amount of anti-fVi conjugate antibodies bound to fVi polysaccharides on an ELISA plate is detected using anti-IgG antibodies that are partially conjugated with alkaline phosphatase and other detection modalities.

[0147] The immunogenic composition of the present invention can induce at least 10 in an immunogenicity assay comprising the following steps. 3 EU / ml or 10 3.5 Salmonella paratyphi A O-antigen antibody: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) The level of anti-Salmonella paratyphi A O-antigen antibody was measured by ELISA on day 42.

[0148] The dosage of GMMA and carbohydrates can be determined as described in the "Dosage" section above.

[0149] A suitable ELISA may involve: - Coat ELISA plates with Salmonella paratyphi A O-antigen; - Blood samples collected from mice on day 42 were applied to coated ELISA plates, which were then washed to remove antibodies that did not bind to the Salmonella paratyphi A O-antigen; and - The amount of anti-Salmonella paratyphi A O-antigen antibody that binds to Salmonella paratyphi A O-antigen on the ELISA plate is detected using anti-IgG antibodies that are conjugated to alkaline phosphatase and other detection components.

[0150] In the immunogenic composition of the present invention, the induced level of anti-Salmonella typhimurium O-antigen antibody can reach at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the level of anti-Salmonella typhimurium O-antigen antibody induced by the corresponding monovalent Salmonella typhimurium immunogenic composition. The antibody levels of the immunogenic composition and the corresponding monovalent immunogenic composition can be determined by the above-described… Suitable ELISA The method was used for determination. The corresponding unit price of typhus Salmonella immunogenic composition "This is completely identical to the immunogenic composition, except that the only antigen present is Salmonella typhimurium antigen. For example, if the immunogenic composition of the present invention contains 5 µg of O-antigen from Salmonella typhimurium, O-antigen from Salmonella enteritidis, fVi polysaccharide conjugate, aluminum adjuvant, and phosphate-buffered saline, then the corresponding monovalent Salmonella typhimurium immunogenic composition should contain 5 µg of O-antigen from Salmonella typhimurium, aluminum adjuvant, and phosphate-buffered saline."

[0151] In the immunogenic composition of the present invention, the level of anti-Salmonella O-antigen antibodies induced can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the level of anti-Salmonella O-antigen antibodies induced by the corresponding monovalent Salmonella immunogenic composition. The antibody levels of the immunogenic composition and the corresponding monovalent immunogenic composition can be determined by the above-described… Suitable ELISA The method was used for determination. The corresponding unit price of sausage Salmonella immunogenic composition "Similar to the immunogenic composition, the difference is that the only antigen present is Salmonella enterica antigen. For example, if the immunogenic composition of the present invention contains O antigen from Salmonella typhimurium, 5 µg of O antigen from Salmonella enterica, fVi polysaccharide conjugate, aluminum adjuvant, and phosphate-buffered saline, then the corresponding monovalent Salmonella enterica immunogenic composition will contain 5 µg of O antigen from Salmonella enterica, aluminum adjuvant, and phosphate-buffered saline."

[0152] In the immunogenic compositions of the present invention, the induced anti-fVi conjugate antibody level can reach at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the anti-fVi conjugate antibody level induced by the corresponding monovalent fVi polysaccharide immunogenic compositions. The antibody levels of the immunogenic compositions and the corresponding monovalent immunogenic compositions can be determined by the above-described… Suitable ELISA The method was used for determination. Corresponding monovalent fVi polysaccharide immunogenic compositions "Similar to the immunogenic composition, the difference being that the only antigen present is fVi." many Sugar antigens. For example, if the immunogenic composition of the present invention comprises O-antigen from Salmonella Typhimurium, O-antigen from Salmonella Enteritidis, 5 µg fVi polysaccharide, aluminum adjuvant, and phosphate-buffered saline, then the corresponding monovalent fVi polysaccharide immunogenic composition shall comprise 5 µg fVi polysaccharide, aluminum adjuvant, and phosphate-buffered saline.

[0153] In the immunogenic compositions of the present invention, the induced antibody level against Salmonella paratyphi A O-antigen can reach at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or at least 98% of the antibody level induced by the corresponding monovalent Salmonella paratyphi A immunogenic composition. The antibody levels of the immunogenic composition and the corresponding monovalent immunogenic composition can be determined by the above-described… Suitable ELISA The method was used for determination. Corresponding Monovalent Paratyphoid A Immunogenic Composition "This is completely identical to the immunogenic composition, except that the only antigen present is Salmonella paratyphi A antigen. For example, if the immunogenic composition of the present invention contains O antigen from Salmonella typhimurium, 5 µg O antigen from Salmonella paratyphi A, fVi polysaccharide conjugate, aluminum adjuvant, and phosphate-buffered saline, then the corresponding monovalent Salmonella paratyphi A immunogenic composition will contain 5 µg O antigen from Salmonella paratyphi A, aluminum adjuvant, and phosphate-buffered saline."

[0154] In the immunogenic compositions or methods of the present invention, the levels of anti-Salmonella typhimurium O-antigen antibody, anti-Salmonella enteritidis O-antigen antibody, anti-fVi conjugate antibody and / or anti-Salmonella paratyphi A O-antigen antibody are measured by an immunogenicity assay comprising the following steps: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) On day 42, the antibody levels against Salmonella typhimurium, Salmonella enteritidis, fVi conjugate and / or Salmonella paratyphi A O-antigen were measured by ELISA.

[0155] Cross protection The immunogenic compositions of the present invention can exhibit cross-protection. For example, the immunogenic compositions of the present invention can induce 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 strains: (a) Mouse Typhoid fever Salmonella ST34 (Mather et al.) people , New Variant of Multidrug-Resistant Salmonella enterica Serovar Typhimurium Associated with Invasive Disease inImmunocompromised Patients in Vietnam. mBio.2018 Sep 4; 9(5):e01056-18); (b) Salmonella Typhimurium 10433_3 (Van Puyvelde et al.) people , An African SalmonellaTyphimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation. Nat Commun. 2019 Sep 19; 10(1):4280); (c) Salmonella Typhimurium D23580 (Van Puyvelde et al.) people, An African SalmonellaTyphimurium ST313 sublineage with extensive drug-resistance and signatures of host adaptation. Nat Commun. 2019 Sep 19; 10(1):4280); (d) Salmonella typhimurium ST4 / 74 (Hurley et al.) people , Atypical Salmonella entericaSerovars in Murine and Human Macrophage Infection Models. Infect Immun. 2020Mar 23; 88(4):e00353-19); (e) Salmonella Typhimurium A130 (Okoro et al.) people , Intracontinental spread of humaninvasive Salmonella Typhimurium pathovariants in sub-Saharan Africa. NatGenet.2012 Nov; 44(11):1215-21); (f) Intestine road Salmonella Derby serotype (Pullinger et al.) people , Identification of Salmonella enterica serovar Dublin-specific sequences by subtractivehybridization and analysis of their role in intestinal colonization and systemic translocation in cattle. Infect Immun. 2008 Nov; 76(11):5310-21); (g) Intestine road Salmonella Dublin serotype (Pullinger, etc.) people, Identification of Salmonella enterica serovar Dublin-specific sequences by subtractive hybridization and analysis of their role in intestinal colonization and systemic translocation in cattle. Infect Immun. 2008 Nov; 76(11):5310-21); (h) Salmonella enterica A1636 (Perez-Sepulveda et al people , 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) Salmonella enterica CP255 (Perez-Sepulveda et al people , 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) Salmonella enterica D7795 (Perez-Sepulveda et al people , 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).

[0156] The immunogenic composition of the present invention can induce antibodies against the following: (i) Salmonella enteritidis and Salmonella Dublinii; (ii) Salmonella typhimurium, Salmonella debby, and Salmonella Dublin; (iii) Salmonella typhimurium, Salmonella enteritidis, Salmonella debilis, and Salmonella Dublin; or (iv) Salmonella typhimurium, Salmonella enteritidis, Salmonella debye, Salmonella Dublin, and Salmonella paratyphi A.

[0157] Immunogenic composition " "Inducement" Antibodies against, for example, Salmonella typhimurium ST34, are provided that they are able to 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.

[0158] Whether the 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 with 500 µL of the immunogenic composition via the intraperitoneal route; and - The level of bactericidal antibodies produced against the relevant strains was determined using the serum bactericidal assay (SBA).

[0159] The SBA test can be based on the test described in Example 6, except that the user should determine the bactericidal activity against the relevant strains listed above (rather than, for example, Salmonella paratyphi A NVGH308).

[0160] Optionally, if the IC50 (serum dilution with 50% inhibition of ATP levels) obtained in the SBA trial is greater than 10 2 The immunogenic composition can induce antibodies against one or more of the strains described above.

[0161] The immunogenic compositions of the present invention can induce antibodies against Salmonella paratyphi A in each of the classes IgG3, IgG2b, IgG2a, and IgG1, as determined by antibody class assay comprising the following steps: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) The level of the anti-Salmonella paratyphi A O-antigen antibody subtype was measured by ELISA on day 42.

[0162] The immunogenic composition “induces” antibodies against each of the classes of IgG3, IgG2b, IgG2a, and IgG1 against Salmonella paratyphi A, provided that it is able to induce these antibodies when used to immunize mice. Whether the immunogenic composition can induce these antibodies when used to immunize mice can be determined by measuring the level of the anti-Salmonella paratyphi A O-antigen antibody subtype using an ELISA assay, as described below.

[0163] Suitable ELISA methods for measuring the O-antigen antibody subtype levels against Salmonella paratyphi A may include: - Coat ELISA plates with Salmonella paratyphi A O-antigen; - Blood samples collected from mice on day 42 were applied to coated ELISA plates, which were then washed to remove antibodies that did not bind to the Salmonella paratyphi A O-antigen; and - The amount of anti-Salmonella paratyphi A O-antigen IgG3 antibody that binds to the Salmonella paratyphi A O-antigen on the ELISA plate was detected using an anti-IgG3 antibody that is partially conjugated to alkaline phosphatase and other detection components. - Repeat the first three steps, but use anti-IgG2b antibody instead of anti-IgG3 antibody, then use anti-IgG2a antibody instead of anti-IgG3 antibody, and finally use anti-IgG1 antibody instead of anti-IgG3 antibody.

[0164] Tolerability "Tolerogenic" or "tolerable" refers to an immunogenic composition that does not elicit a significant negative reaction in the subject to which it is administered. In particular, immunogenic compositions comprising outer membrane vesicles are known to cause fever in patients to which they are administered. Therefore, optionally, an immunogenic composition may be considered tolerable if it does not elicit a significant fever.

[0165] If an immunogenic composition causes fever when used to immunize mice, it will be considered " Induction "Fever. Whether the immunogenic composition induces fever when used to immunize mice can be determined by testing the immunogenic composition sample using the following toxicity assay."

[0166] An immunogenic composition may be tolerable if it induces a temperature increase of less than 1.8°C, less than 1.7°C, less than 1.6°C, or less than 1.5°C in a toxicity assay including the following steps: (a) Measure the rabbit's initial body temperature; (b) The immunogenic composition was administered to the rabbits at a dose of 20 µg (O-antigen) / GMMA and 25 µg sugar / sugar conjugate; (c) Monitor the rabbit's body temperature for 5 hours; and (d) Record the highest body temperature of the rabbit. The temperature rise is calculated as equal to the rabbit's highest body temperature minus the rabbit's initial body temperature.

[0167] In a toxicity assay that includes the following steps, if the highest body temperature induced by the immunogenic composition is 41°C or lower, 40.9°C or lower, or 40.8°C or lower, then it may be tolerable: (a) The immunogenic composition was administered to rabbits at doses of 20 µg (O-antigen) / GMMA and 25 µg / carbohydrate; (b) Monitor the rabbit's body temperature for 5 hours; and (c) Record the highest body temperature of the rabbit.

[0168] Methods for enhancing immune responses In a further aspect of the invention, a method for enhancing an immune response to an antigen is provided, the method comprising administering a composition comprising an antigen and GMMA. A method for preventing enteric Salmonella infection is also provided, the method comprising administering an immunogenic composition comprising enteric Salmonella antigen and GMMA, wherein the administration of GMMA enhances the immune response to enteric Salmonella.

[0169] For example, the method of the present invention may include enhancing the immune response to Salmonella typhi or Salmonella paratyphi A antigens by administering a composition comprising the Salmonella typhi or Salmonella paratyphi A antigens and GMMA. The method of the present invention may also include preventing Salmonella typhi or Salmonella paratyphi A infection by administering an immunogenic composition comprising Salmonella typhi or Salmonella paratyphi A antigens and GMMA, wherein the GMMA enhances the immune response to the Salmonella typhi or Salmonella paratyphi A antigens.

[0170] The present invention also provides an immunogenic composition comprising GMMA for use in a method of enhancing an immune response to Salmonella typhi or Salmonella paratyphi A antigen, wherein the method comprises administering an immunogenic composition comprising the Salmonella typhi antigen or the Salmonella paratyphi A antigen and GMMA. The present invention also provides an immunogenic composition for use in a method of preventing Salmonella typhi or Salmonella paratyphi A infection, wherein the immunogenic composition comprises the Salmonella typhi antigen or the Salmonella paratyphi A antigen and GMMA, wherein the GMMA enhances the immune response to the Salmonella typhi antigen or the Salmonella paratyphi A antigen.

[0171] In the method or immunogenic composition used in this invention, the method may be a method for enhancing the immune response to Salmonella typhi antigen, and the immunogenic composition contains Salmonella typhi antigen. In the method or immunogenic composition used in this invention, the method may be a method for enhancing the immune response to Salmonella paratyphi A antigen, and the immunogenic composition may contain Salmonella paratyphi A antigen.

[0172] In the methods or immunogenic compositions used in this invention, the method may be a method for preventing Salmonella typhi infection, the immunogenic composition may contain Salmonella typhi antigen, and the GMMA may enhance the immune response to the Salmonella typhi antigen. In the methods or immunogenic compositions used in this invention, the method may be a method for preventing Salmonella paratyphi A infection, the immunogenic composition may contain Salmonella paratyphi A antigen, and the GMMA may enhance the immune response to the Salmonella paratyphi A antigen.

[0173] The method or immunogenic composition used in this invention, wherein the GMMA comprises at least one selected from the group consisting of: *Salmonella typhimurium* GMMA, *Salmonella enteritidis* GMMA, and *Salmonella paratyphi A* GMMA. *Salmonella typhimurium* GMMA can enhance the immune response to *Salmonella typhimurium* or *Salmonella paratyphi A* antigens. *Salmonella paratyphi A* GMMA can enhance the immune response to *Salmonella typhimurium* antigens. *Salmonella enteritidis* GMMA can enhance the immune response to *Salmonella typhimurium* or *Salmonella paratyphi A* antigens. The *Salmonella paratyphimurium* GMMA can enhance the immune response to *Salmonella typhimurium* antigens.

[0174] One approach is to target the Salmonella typhi antigen or the Salmonella paratyphi A antigen. Enhance immune response The method, provided that the immune response induced when *Salmonella typhi* or *Salmonella paratyphi A* antigens are used as components of an immunogenic composition containing GMMA is higher than the immune response induced when these antigens are not used as components of the GMMA-containing immunogenic composition. This is due to the adjuvant effect of GMMA. In other words, when GMMA is in the same composition as *Salmonella typhi* or *Salmonella paratyphi A* antigens, the GMMA enhances the immune response to these antigens compared to using them alone.

[0175] For the purpose of enhancing the immune response to Salmonella typhi antigen or Salmonella paratyphi A antigen, the Salmonella typhi antigen and Salmonella paratyphi A antigen may be polysaccharides. Optionally, the Salmonella typhi antigen may contain fVi polysaccharide, optionally wherein the fVi polysaccharide is part of an fVi conjugate comprising fVi and a carrier protein, and further optionally wherein the carrier protein is CRM. 197 Optionally, the Salmonella paratyphi A antigen may include Salmonella paratyphi A O-antigen, optionally wherein the Salmonella paratyphi A O-antigen is conjugated to a carrier protein, and further optionally wherein the carrier protein is a CRM. 197 .

[0176] GMMA enhances the immune response to Salmonella typhi or Salmonella paratyphi A antigens when the Salmonella typhi or Salmonella paratyphi A antigens are part of an immunogenic composition containing GMMA, and the resulting immune response is higher when the Salmonella typhi or Salmonella paratyphi A antigens are not part of an immunogenic composition containing GMMA.

[0177] One approach is a method of enhancing the immune response to Salmonella typhi antigen or Salmonella paratyphi A antigen, or GMMA enhancing the immune response to Salmonella typhi antigen or Salmonella paratyphi A antigen, provided that the immune response induced when the Salmonella typhi antigen or Salmonella paratyphi A antigen is part of an immunogenic composition containing GMMA is at least 5, at least 10, or at least 20 times higher than the immune response induced when the Salmonella typhi antigen or Salmonella paratyphi A antigen is not part of an immunogenic composition containing GMMA.

[0178] The immune response against Salmonella typhi antigen or Salmonella paratyphi A antigen induced by the immunogenic composition or method of the present invention can be measured by ELISA as the number of antibodies produced 42 days after administration of an immunogenic composition containing Salmonella typhi antigen or Salmonella paratyphi A antigen and GMMA, wherein the immunogenic composition contains 0.78 µg of Salmonella typhi antigen or Salmonella paratyphi A antigen and GMMA. cold Salmonella antigen or Salmonella paratyphi A antigen and 0.63µg (O-antigen) of GMMA.

[0179] A suitable ELISA may involve: - Coat ELISA plates with Salmonella Typhi antigen or Salmonella Paratyphi A O-antigen; - Blood samples collected from mice on day 42 were applied to coated ELISA plates, which were then washed to remove antibodies that did not bind to Salmonella typhi antigen or Salmonella paratyphi A O-antigen; and - Using anti-IgG antibodies conjugated to alkaline phosphatase and other detection components, the amount of anti-Salmonella antigen or Salmonella paratyphi A O-antigen antibodies bound to the Salmonella typhi antigen or Salmonella paratyphi A O-antigen on the ELISA plate is detected.

[0180] Medical uses and treatments In another aspect of the invention, an immunogenic composition of the invention is provided for use in methods of preventing infection. In another aspect of the invention, a method of preventing infection is provided, comprising administering an effective amount of the immunogenic composition or vaccine of the invention to a subject. In another aspect of the invention, use of the immunogenic composition or vaccine of the invention in the preparation of a medicament for preventing infection is provided. The method of preventing infection may include administering an effective amount of the immunogenic composition or vaccine of the invention to a subject.

[0181] The method for preventing infection may be a method for preventing Salmonella infection. Optionally, the method for preventing infection is a method for preventing infection with Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Typhimurium, and / or Salmonella Paratyphi A.

[0182] In the context of the methods of the present invention / the use of the immunogenic compositions of the present invention in the preparation of the pharmaceuticals of the present invention, the term " prevention salmonella Infect "This includes inducing an immune response in the subject. The immune response can be a protective immune response and can produce antibodies, such as IgG antibodies."

[0183] The subjects of this invention are mammals, optionally humans. When the vaccine is used for the prevention of disease, the human may be an adult, i.e., the subject is 18 years of age or older. When the vaccine is used for the prevention of disease, the human may be a child, i.e., under 18 years of age. When the vaccine is used for the prevention of disease, the child may be 12 to 72 months old, preferably 24 to 59 months old, and more preferably 6 to 12 months old.

[0184] When vaccines are used to prevent disease, children can be around 9 months old.

[0185] When vaccines are used to treat diseases, children are preferred as the recipients.

[0186] Vaccines intended for children may also be administered to adults, for example, to assess safety, dosage, or immunogenicity.

[0187] Example Example 1 - Production of GMMA from Salmonella Typhimurium and Salmonella Enteritidis The wild-type (WT) 2192 strain of Salmonella typhimurium was provided by the Salmonella Genetic Stock Center (SGSC) at the University of Calgary, Canada. This strain belongs to the global Salmonella reference collection A (SARA 12).

[0188] The enterocolitis serotype WT strain 618 of *Salmonella enterica* was provided by Quotient Bioresearch Limited, UK. The animal-derived strain was isolated from *European Antimicrobial Susceptibility Surveillance in Animals* (EASSA).

[0189] Based on the above Salmonella strains, Salmonella Typhimurium was generated for each strain. tolR ΔpagPΔmsbB and Salmonella enteritidis The tolR ΔpagP ΔmsbB recombinant mutant, the relevant research results are as described above (Rossi O, Caboni M, Negrea A, Necchi F, Alfini R, Micoli F, et al. Toll-LikeReceptor Activation by Generalized Modules for Membrane Antigens from Lipid AMutants of Salmonella enterica Serovars Typhimurium and Enteritidis. ClinVaccine Immunol. 2016; 23(4):304-14.).

[0190] GMMA derived from the aforementioned Salmonella strains (Salmonella typhimurium GMMA (STmGMMA) and Salmonella enteritidis GMMA (SEnGMMA)) was purified and isolated. The GMMA was purified using a method similar to that previously reported for Shigella sonnei GMMA. Gerke C, Colucci AM, Giannelli C, Sanzone S, Vitali CG, Sollai L, et al. Production of a Shigella sonnei Vaccine Based on Generalized Modules for Membrane Antigens (GMMA), 1790GAHB.PLoS One.2015; 10 (8):e0134478. doi: 10.1371 / journal.pone.0134478 [doi]; PONE-D-15-08654In short, the GMMA released into the fermentation broth is purified through two consecutive tangential flow filtration (TFF) steps: microfiltration, which separates the culture supernatant containing GMMA from the bacteria; and ultrafiltration, which separates the GMMA from soluble proteins and nucleic acids.

[0191] Example 2 - Production fVi-CRM for immunization against Salmonella typhi. 197 Conjugate The following method is used to produce a divalent composition comprising the above-prepared Salmonella paratyphi A OAg-CRM. 197 Conjugates and fragmented Vi polysaccharides (fVi) from Salmonella Typhi with CRM 197 A conjugated compound.

[0192] 1) Fragmentation of Vi polysaccharides: Step 1: Fragmentation reaction and quenching Vi polysaccharide fragmentation was achieved through oxidation using hydrogen peroxide in the presence of ferrous sulfate. The reaction was quenched with EDTA (ethylenediaminetetraacetic acid). Natural Vi polysaccharide was diluted with WFI. Calculated amounts of 10 mM FeSO4 and H2O2 were added to the reaction mixture to obtain final concentrations of 0.5 mM FeSO4 and 0.5% v / v H2O2, respectively. The reaction mixture was incubated at 15 ± 5 °C for 120 ± 10 min. The reaction was terminated by adding an equal volume of 250 mM EDTA to achieve a final EDTA concentration of 10 mM and stirring.

[0193] Step 2: Buffer exchange Residual H2O2 was removed by tangential flow filtration (TFF) using a 30 kDa cassette membrane and buffer exchange with 100 mM sodium phosphate buffer (pH: 7.2 ± 0.2). The fragmented Vi (fVi) polysaccharide was then concentrated.

[0194] Step 3: Stabilization of fVi polysaccharides After fragmentation, the 30 kDa cutoff was stabilized by incubating at 80±5℃ for 120+ / -15 min.

[0195] Step 4: fVi anion exchange purification (resin: Capto-Q) fVi polysaccharides with the desired molecular weight (25-70 kDa) were separated using chromatographic procedures. This process employed Capto-Q resin (binding capacity 13 mg fVi / mL) with a linear gradient elution using Capto-Q buffer A and Capto-Q buffer B. Elution fractions were collected based on conductivity per 1 mS / cm, i.e., from 35 to 50 mS / cm, and the molecular weight distribution was estimated by SEC / HPLC. Capto-Q fractions were combined based on their molecular weight (kDa) distribution.

[0196] Step 5: Desalination The Capto-Q combined fraction was concentrated by tangential flow filtration (TFF) using a 10 kDa molecular weight cutoff membrane, followed by percolation with WFI until the percolate conductivity was ≤30 µS / cm.

[0197] Step 6: 0.2 µm filtration of fVi polysaccharides The fVi polysaccharide was filtered through a 0.22 µm filter. The purified fVi polysaccharide was stored in PETG bottles.

[0198] 2) CRM 197 Derivatization: Step 1: CRM 197 Thawing: Purified CRM 197 Thaw at 2-8°C before buffer exchange with 100 mM MES (morpholinoethanesulfonic acid) buffer. After thawing, CRM 197 Use a 0.5 µm filter for filtration.

[0199] Step 2: Perform buffer exchange using 100 mM MES buffer. In CRM 197 After thawing, a 10 kDa membrane was used to exchange buffer with 100 mM MES buffer (pH 6.0 ± 0.2) via TFF.

[0200] Step 3: CRM 197 Derivatization : Apply the required concentration of CRM 197 Dilute with 100 mM MES buffer, then add calculated amounts of ADH (adipic acid dihydrazide) and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to prepare a CRM:ADH:EDAC ratio of 1:3.5:0.15 w / w / w. After adding ADH and EDAC, CRM... 197 The reaction mixture was incubated at room temperature for 60 ± 15 min under mixed conditions. At the end of the reaction, an equal volume of 5 mM MES buffer (pH 7.0 ± 0.2) was added.

[0201] Step 4: CRM 197 Purification After the reaction, CRM was purified via TFF using a 10 kDa membrane pack with 5 mM MES buffer. 197 .

[0202] Step 5: Post-percolation CRM 197 Filtering: CRM after percolation 197The solution was filtered through a 0.2-micron filter and then stored in glass bottles at 2-8°C.

[0203] 3) Fragmented Vi polysaccharides and derived CRM 197 Adhesion: Activation of fVi polysaccharides: Step 1: Dry fVi polysaccharides using a rotary evaporator fVi was further concentrated and dried using a rotary evaporator at 30°C. The concentrated fVi polysaccharide was reconstituted using 100 mM MES buffer (pH: 6.0) to obtain a concentration of 50 mg / mL.

[0204] Step 2: Activation of fVi polysaccharides and NHS fVi carboxylate (-COOH) is activated with EDC (N-3-dimethylaminopropyl-N-ethylcarbodiimide) in the presence of N-hydroxysuccinimide (NHS) via the formation of an active ester intermediate to enhance its reactivity with CRM previously activated with ADH. 197 The conjugation efficiency was determined. Dry fVi polysaccharides were reconstituted to the desired concentration (50 mg / mL) using 100 mM MES buffer (pH: 6.2 ± 0.2) and activated in the presence of NHS (0.33 M). EDAC was then added to achieve an EDAC / fVi RU molar ratio of 5:1. The EDAC solution was added after the NHS addition to ensure complete dissolution. The reaction was incubated at room temperature with slow mixing for 1 h.

[0205] Adhesion: Step 1: fVi and CRM 197 -ADH conjugation : Conjugation reaction in activated fVi with CRM 197 Covalent bonds are formed between -ADH groups. The activated and derivatized reaction mixture was diluted with 100 mM MES at pH 6.0 and mixed at a 1:1 ratio (fVi : CRM). 197 The weight ratio of CRM added 197 -ADH to achieve activation of fVi and CRM 197 The final fVi concentration of -ADH was 5 mg / mL. The conjugation reaction was carried out at room temperature, with slow mixing until protein consumption was ≥70% as measured by HPLC-SEC at 280 nm absorbance.

[0206] Step 2: Quenching and Regulation of the Conjugation Reaction The conjugation reaction was initiated by adding an equal volume of Phenyl HP buffer-B Tris 50mM pH: 8.0. Powdered NaCl was added until the final salt concentration reached 3 M.

[0207] Step 3: Filtering the conjugated mixture fVi-CRM 197 The coarse conjugate was filtered through a 0.65 filter.

[0208] Step 4: fVi-CRM 197 Purification of crude conjugate Purification of the conjugate from the conditioned reaction mixture was performed using a HICPhenyl Sepharose High Performance (HP) column. Column integrity was checked every 5–10 cycles according to standard procedures. The column was equilibrated with Phenyl Sepharose HP buffer A (Tris 50 mM NaCl 3 M pH 8). After adding conditioned buffer and NaCl, the crude conjugate was loaded onto the column. The column was washed with Phenyl Sepharose HP buffer A, followed by elution with Phenyl Sepharose HP buffer B (Tris 50 mM pH 8). The fraction was collected and stored at 2–8°C until subsequent use. All fractions from multiple runs were combined.

[0209] Step 5: Concentrate and exchange buffer using PBS The purified conjugate was concentrated using a TFF membrane with a molecular weight cutoff of 50 kDa, and then buffered with PBS buffer until the conductivity of the permeate was consistent with that of the PBS buffer.

[0210] Step 6: Use a 0.2 µm filter for fVi-CRM 197 Pre-filtration of conjugates fVi-CRM 197 The conjugate was filtered through a 0.2 µm filter to reduce bioburden.

[0211] Step 7: Perform fVi-CRM using a 0.2 µ cellulose acetate filter. 197 Aseptic filtration of conjugates: fVi-CRM 197 The conjugate was filtered through a 0.2 µm cellulose acetate membrane. The purified fVi-CRM was then collected. 197 Conjugate samples should be stored at 2-8℃.

[0212] Example 3 - Formulation of a bivalent (iNTS-GMMA) vaccine against Salmonella Typhimurium and Salmonella Enteritidis adsorbed onto aluminum hydroxide adjuvant STmGMMA and SEnGMMA were adsorbed onto aluminum hydroxide (Alum) in phosphate buffered saline (pH 6.5), respectively. 3+The final concentration was 0.7 mg / mL, yielding two different drug product formulations, each aliquoted into 3 mL Type I 2R vials. The GMMA stock solution was diluted to obtain a GMMA concentration of 80 µg / mL O-antigen (based on the O-Ag content measured in GMMAx) and an aluminum hydroxide concentration of 0.7 mg / mL (Al). 3+ Prior to administration, the contents of the STmGMMA / aluminum gel vial are mixed with the contents of the SEnGMMA / aluminum gel vial to obtain the iNTS-GMMA vaccine, wherein the final concentration of each O-antigen adsorbed on aluminum hydroxide (final concentration 0.7 mg / mL) in phosphate buffered saline is 40 µg / ml, forming the final mixed drug product.

[0213] The OAG content in the final mixed drug product was also determined. The assay protocol for assessing OAG content is described below.

[0214] HPAEC-PAD was used to analyze Salmonella O-antigen in GMMA stock solution or final mixed pharmaceutical product formulation. Quantitative: Quantification of OAG in Salmonella enteritidis. GMMA quantification was performed using high-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD) after acidic hydrolysis of the sample. The quantification determined the concentrations of rhamnose, galactose, glucose, and mannose present in the sample.

[0215] The quantification of OAg was based on the known sugar ratios in the OAg repeating unit 1xRha (rhamnose); 1xGal (galactose); 1xMan (mannose); 1xTyv (Tyvose), and glucose was calculated by subtracting the contribution of the core component from the glucose measured in the analysis.

[0216] Each HPAEC-PAD analysis was performed using standard dilution series of sugars in the range of 0.5–10 µg / mL, and the µg / mL concentration of the corresponding sugar in the sample OAg was calculated using peak area interpolation.

[0217] 450 µL each of the standard and sample diluent were treated in parallel with 150 µL of 8 M TFA at 100 °C for 4 hours. The samples were then cooled at 2 °C to 8 °C for approximately 30 minutes, centrifuged, evaporated to dryness, resuspended in 450 µL of water, filtered, and analyzed. HPAEC-PAD was performed using a Dionex ICS3000 (or 5000) system equipped with a CarboPac PA10 column and a PA10 guard column. Separation was performed at 25 °C under 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 was monitored using an electrochemical detector.

[0218] Quantification of Salmonella Typhimurium O-antigen in GMMA stock solution using HPAEC-PAD Following acidic hydrolysis of GMMA, high-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD) was used to quantify OAg in Salmonella typhimurium GMMA. Each HPAEC-PAD analytical series included a standard dilution series containing rhamnose, galactose, glucose, and mannose solutions, which were used as calibration curves to quantify the sugars present in the samples.

[0219] The quantification of OAg is based on the known sugar ratios in the OAg repeating unit 1xRha (rhamnose); 1xGal (galactose); 1xMan (mannose); 1xAbe (abicosylose), and the glucose calculation is derived from the glucose value measured in the analysis minus the glucose content of the core portion.

[0220] Quantification of OAG in the final mixed drug product (SEn-GMMA / STm GMMA) using HPAEC-PAD The quantification of OAG in *Salmonella typhimurium* or *Salmonella enteritidis* GMMA adsorbed on aluminum hydroxide was performed using the HPAEC-PAD method after acidic hydrolysis of GMMA. Each HPAEC-PAD analysis series included a series of dilutions of standard solutions containing fucose, rhamnose, N-acetylglucosamine, glucose, galactose, and mannose, which were used as calibration curves to quantify rhamnose, glucose, galactose, and mannose in the sample. The procedure was identical to that used for quantifying *Salmonella typhimurium* and *Salmonella enteritidis* active pharmaceutical ingredients.

[0221] Add 150 µL of 8 M TFA to 450 µL of each dilution of standard and sample. Heat all vials in parallel at 100 °C for 4 hours. Then cool the samples at 2 °C to 8 °C for approximately 30 minutes, dry them using a centrifugal evaporator, resuspend them in 450 µL of water, filter, and proceed with analysis. Perform HPAEC-PAD using a Dionex ICS3000 (or 5000) system equipped with a CarboPac PA10 column and an external PA10 guard column.

[0222] The separation was operated at 25°C under 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 was monitored using an electrochemical detector.

[0223] OAg quantification was based on the known sugar proportions in the OAG repeating units (Rha; Glc; Gal; Man and Tavicanose (SEn) or Abicosose (STm)) present in the formulation. Rhamnose, glucose, galactose, and mannose were directly quantified as nmol / mL by analysis, and the amount of OAG was subsequently calculated based on the molecular weight of each sugar and its content in the repeating unit (OAg = 600.61). Rha+ 162.15 Glc 总量 – 162.15 Gal 总量 +162.15 Man).

[0224] Example 4 - The adsorption of SEnGMMA and STmGMMA onto aluminum hydroxide enhanced tolerance in rabbits. The effects of SEnGMMA and STmGMMA adsorption to aluminum hydroxide were investigated, and it was determined that adsorption to aluminum hydroxide can reduce the pyrogenicity of GMMA.

[0225] Three groups of New Zealand white rabbits were immunized via intramuscular immunization: a saline control, an aluminum glue control (0.5 ml dose), and an aluminum glue control (0.7 mg / ml Al). 3+ (20 mM phosphate and 154 mM NaCl), or one of the following vaccines: (1) STmGMMA adsorbed on aluminum gel (0.5 mL dose, containing 20 µg O-antigen, STmGMMA / hydrogel); (2) SEnGMMA adsorbed on aluminum gel (0.5 mL dose, containing 20 µg O-antigen, SEnGMMA / hydrogel); (3) iNTS-GMMA vaccine (0.5 mL dose, containing 20 µg O-antigen); (4) bivalent STmGMMA and SEnGMMA vaccine not adsorbed on aluminum gel (0.5 mL dose, 2 µg O-antigen / GMMA). These four vaccines were prepared according to the methods described in Examples 1-3.

[0226] Monitor the rabbits' body temperature within 5 hours after vaccination.

[0227] The initial body temperature of each rabbit ranged from 38.0 to 39.8°C, and the temperature difference between rabbits in the same group was <1.0°C. For rabbits treated with aluminum gel control or saline, the absolute body temperature after administration did not exceed 39.2°C. In one rabbit treated with STmGMMA / aluminum gel, the highest absolute body temperature was 40.6°C; the body temperatures of the other two rabbits in the same group did not exceed 39.6°C. In the three rabbits treated with SEnGMMA / aluminum gel, the highest measured body temperatures were 39.3°C, 39.8°C, and 40.2°C, respectively. In the 2-component iNTS-GMMA vaccine group (group (3) above), the highest measured body temperature did not exceed 40.4°C, and the maximum temperature rise of individual rabbits was 0.9°C, 1.2°C, and 1.5°C, respectively. For rabbits treated with unadsorbed STmGMMA and SEnGMMA mixture (group (4) above), the highest measured body temperature was 41.3°C, and the results of the three rabbits were similar. Peak body temperature was reached in all rabbits within a five-hour period (typically 180–270 minutes). Rabbits receiving the aluminum gel formulation (groups (1) to (3) above) showed an average maximum temperature rise of 1°C to 1.3°C, while rabbits receiving unformulated GMMA (at a dose 10 times lower) (group (4) above) showed an average maximum temperature rise of 1.9°C. The group receiving unformulated GMMA reached peak temperature earlier (typically 120–180 minutes), with a steeper and higher temperature rise and a steeper and faster temperature drop.

[0228] Example 5 - Trivalent vaccine formulation against Salmonella Typhimurium, Salmonella Enteritidis, and Salmonella Typhimurium (iNTS-TCV) STmGMMA and SEnGMMA were sequentially adsorbed into an aluminum hydroxide phosphate buffer solution at pH 6.5. The adsorbed STmGMMA and SEnGMMA were then mixed, and a phosphate quencher was added, followed by the addition of fVi-CRM suspended in a sodium phosphate buffer solution. 197 .

[0229] To prepare STmGMMA and SEnGMMA solutions of the specified concentrations, the content of O-antigen (OAg) in the GMMA stock solution was first determined to determine how to dilute the GMMA stock solution to obtain the correct GMMA concentration. The contents of OAg and Vi polysaccharides in the final mixed pharmaceutical product were also determined. The protocol for determining the O-antigen content of STm GMMA and SEn GMMA is as described in Example 3. Similarly, the following paragraphs summarize the steps for adding the correct amount of fVi-CRM. 197 Operational procedures for conjugates.

[0230] Identification and quantification of STm and SEn OAg in the final mixed drug product iNTS-TCV using FAcE The identification and quantification of the O-antigen were evaluated using a FAcE (prepared aluminum gel competitive ELISA) assay, designed to detect the monoantigen components of Salmonella enteritidis and Salmonella 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 OAg coated on the ELISA plate and the corresponding OAg contained in the GMMA suspension prepared in the ELISA wells. The more serotype-specific OAg present in the prepared GMMA suspension, the more mAbs bind to it, and the fewer mAbs available for binding to the coating antigen. The ELISA signal is generated by the binding of mouse mAbs to the coated OAg; therefore, the lowest signal is obtained at the highest GMMA concentration, and vice versa. mAb binding was detected using enzyme-labeled anti-mouse antibodies, followed by the addition of substrate solution and detection of the formation of a yellow product by absorbance at 405 nm and 490 nm. The results are the calculated OD difference from 405 nm to 490 nm. Quantitative analysis of the target antigen in test samples by FAcE assay employed a reference standard curve (starting from a known concentration of OAG µg / mL) established by serial dilutions of freshly prepared aluminum hydroxide adjuvant serum-type-specific GMMA. Test samples were measured at different dilutions, with each dilution falling within the linear center of the standard curve. The OAG content in the test sample was calculated by interpolating absorbance readings to a standard curve fitted using a 4-parameter logistic regression analysis. Since FAcE uses anti-OAg specific mAbs for OAG quantification, this method simultaneously verifies the identity of OAG.

[0231] Vi identification and quantification of the final mixed drug product iNTS-TCV according to HPAEC-PAD The following method was used to perform acidic hydrolysis on the samples, followed by HPAEC-PAD analysis to identify and quantify Vi polysaccharides.

[0232] Vi polysaccharide is hydrolyzed into repeating monomeric sugars, corresponding to chromatographic peaks. In each HPAEC-PAD analysis, a series of Vi standard dilutions of 0.16–5 µg / mL were run. A standard curve was established using the resulting peak areas, and the peak areas of unknown samples were interpolated to quantify the corresponding µg / mL.

[0233] The standard calibration curve is generated by acidic hydrolysis; the acidic hydrolysis of the sample and the standard is carried out simultaneously, and the concentration of polysaccharides present in the sample can be determined using the method described above.

[0234] Add 1000 µL of TFA / HCl mixture (1:6.7 v / v) to each 300 µL of the standard curve and each dilution of the sample. Heat all vials in parallel at 80 °C for 4.5 h (final concentration: HCl 8M TFA 10%). Then cool the samples at 2 °C to 8 °C for approximately 15 min, dry them under a nitrogen stream for at least 1.5 h, and finally dry them in a centrifugal evaporator. Resuspend the samples in 300 µL of water, filter, and analyze. Perform HPAEC-PAD using a Dionex ICS3000 (or 5000) system equipped with a CarboPac PA1 column and a PA1 guard column. Separation was performed at 25 °C under the following conditions: 15 min, 400 mM NaOH, flow rate 1.5 mL / min (no washing step required at the injection site). Monitor the eluent using an electrochemical detector.

[0235] Example 6 - Immunogenicity Assay ELISA Anti-Vi and anti-OAg-specific total IgG were assessed by ELISA. Anti-OAg and anti-Vi antigen-specific IgG levels were measured by ELISA two weeks after the second immunization (day 42), as previously reported (Rondini et al.). people Evaluation of the immunogenicity and biological activity of the Citrobacter freundii Vi-CRM197 conjugate as a vaccine for Salmonella enterica serovar Typhi. ClinVaccine Immunol. 2011 Mar; 18(3):460-8). In short, 96-well round-bottom MaxiSorp microplates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with 100 ml / well of antigen. OAg purified from Salmonella paratyphi A (O:2) or Salmonella enteritidis (O:9) and from the broad strain of Citrobacter freundii (O:9) were administered at 15 mg / ml and 2 mg / ml (in carbonate buffer) or 1 mg / ml (in phosphate buffer saline), respectively. C. freundii sl) purified Vi antigen (Micoli et al.) people, A scalable method for O-antigen purification applied tovarious Salmonella serovars. Anal Biochem. 2013 Mar 1; 434(1):136-45; Micoli et al. people Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi. Vaccine. 2012 Jan 20; 30(5):853-61). The plate was blocked with PBS containing 5% skim milk (Sigma) at room temperature (RT) for 1 h, and then washed three times with PBS containing 0.05% Tween 20 (PBS-T). Serum samples were diluted 1:100 and 1:4,000 in PBS-T (dilution buffer) supplemented with 0.1% BSA, and each dilution was performed in triplicate. After incubation at room temperature for 2 h, the plate was washed three times with PBS-T and incubated at 25°C for 1 h with anti-mouse goat IgG-alkaline phosphatase (Sigma) (diluted in dilution buffer at 1:6,000, 1:8,800 and 1:2,600 (corresponding to Vi, O:2 or O:9, respectively). After washing three times with PBS-T, the assay was developed by adding alkaline phosphatase substrate (SIGMAFAST N2770; Sigma) and readouts were obtained at 405 nm and 490 nm using an ELx 800 reader (BioTek). ELISA units were expressed relative to a mouse antigen-specific antibody standard serum curve consisting of 10 standard spots and 2 blank wells (run twice on each plate), where the optimal five-parameter fit was determined by a modified Hill plot. One ELISA unit was defined as the reciprocal of the standard serum dilution that produced an absorbance value equal to 1 in this assay.

[0236] SBA Serum bactericidal activity was assessed using SBA. Individual mouse serum collected on day 42 was heat-inactivated (HI) at 56°C for 30 minutes, followed by SBA testing against Salmonella paratyphi A NVGH308, Salmonella enteritidis CMCC3014, and Vi-positive Citrobacter freundii strain 3056 (Necchi et al.). people, Development of a high-throughputmethod to evaluate serum bactericidal activity using bacterial ATPmeasurement as survival readout. PLoS One.2017 Feb 13; 12(2):e0172163; Necchi et al. people The luminescence-based serum bactericidal assay (SBA) was performed in a 96-well round-bottom sterile plate (Corning). The luminescence readout of the HI test serum was detected in the serum bactericidal assay. The assay was performed using a luminescence-based serum bactericidal assay (SBA) in a 96-well round-bottom sterile plate (Corning). The dilution of the HI test serum was incubated for 3 hours in the presence of exogenous complement (rabbit complement [BRC]) and bacteria, as previously described (Necchi et al., Development of a high-throughput method to evaluate serum bactericidal activity using bacterial ATP measurement as survival readout. PLoS One. 2017 Feb 13; 12(2):e0172163). In short, an appropriate amount of reaction mixture containing target bacterial cells (approximately 100,000 CFU / ml), BRC (50% Salmonella enteritidis, 20% Salmonella paratyphi A, and 5% broad-spectrum Citrobacter freundii), and PBS was added to an SBA plate containing HI serum dilution and incubated at 37°C for 3 h. After incubation, the plate was centrifuged at 4,000 × g for 10 min, the supernatant was discarded to remove ATP derived from dead bacteria, and the viable bacterial pellet resuspended in PBS was transferred to a white round-bottom 96-well plate (Greiner) and mixed with BacTiter-Glo reagent (Promega) at a 1:1 (v / v) ratio. The reaction mixture was incubated in an orbital shaker at room temperature for 5 min, and the luminescence signal was measured using a luminescence detector (Viktor). The raw luminescence was analyzed using a 4-parameter nonlinear regression method for all tested serum dilutions, as previously described (Rossi et al.). peopleIntra-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 inhibits ATP levels in the negative control wells by 50%. For titers below the lowest measurable luminescence level, we artificially set their IC50 value to 50, representing half of the initial serum dilution tested (i.e., 100). Curve fitting and IC50 values ​​were performed using GraphPad Prism 7 software (GraphPad Software).

[0237] IgG subclass Serum from individuals immunized with a second immunization, isolated from blood samples collected from mice immunized according to the section titled "Example 12 – Antibody Subtypes Produced in Mice with a Quadrivalent Pan-Salmonella Vaccine," was tested using an ELISA-based assay of the same principle as described under the heading "ELISA" to determine the isotypes of the produced antibodies. Anti-mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, and anti-mouse IgG3 antibodies were used as secondary antibodies in the standard assay, which was repeated to determine the EU / mL of serum from each individual at the tested dose. Results are as shown in Example 12 and are expressed as subtype / total subtype %

[0238] Example 7 - Immunogenicity of trivalent (iNTS-TCV) vaccine in mice Eight CD1 mice in each group were immunized with the iNTS-TCV vaccine prepared as described in Example 5, at doses ranging from 0.01 µg to 0.63 µg of each GMMA (O-antigen) and 0.012 µg to 0.78 µg of Vi polysaccharide (total polysaccharide dose 0.032–2.04 µg). Immunization consisted of intraperitoneal immunization at 0.032–2.04 µg per 500 µl on days 0 and 28. Blood samples were collected on days 27 and 42, and antibodies produced were measured using the ELISA assay described in Example 6. Results are as follows. Figure 1 As shown.

[0239] The iNTS-TCV candidate vaccine demonstrated good tolerability at all doses. Both antigen GMMA components (SEnGMMA and STmGMMA) and the antigen Vi component induced antibody dose responses. All three antigen components (SEnOAg, STmOAg, and Vi) generated increased SBA responses in the four lower dose groups on day 42.

[0240] In the second experiment, the eight CD1 mice were immunized with the following: - The iNTS-TCV vaccine produced as described in Example 5 was administered at doses of 0.032, 0.13, 0.51, and 2.04 µg (total polysaccharides). - SEnGMMA (produced as described in Example 1) adsorbed onto aluminum gel at doses of 0.01, 0.04, 0.16, and 0.63 µg (O-antigen). - STmGMMA (produced as described in Example 1) adsorbed onto aluminum gel at doses of 0.01, 0.04, 0.16, and 0.63 µg (O-antigen). - fVi-CRM 197 The conjugates (produced according to the method described in Example 2) were administered at doses of 0.012, 0.05, 0.19, and 0.78 µg (Vi polysaccharide).

[0241] Immunization consisted of intraperitoneal immunization with the appropriate dose (500 µl) of each product on days 0 and 28. Blood samples were collected on days 27 and 42, and antibodies produced were measured using the ELISA and SBA assays described in Example 6. Results are as follows: Figure 2 As shown.

[0242] Overall, no immunological interference was observed in the iNTS GMMA component of iNTS-TCV as measured by ELISA and SBA, but when fVi-CRM... 197 When the components were combined with GMMA in iNTS-TCV, a higher anti-Vi IgG response and serum bactericidal activity were observed.

[0243] In the third experiment, three groups of New Zealand white rabbits were immunized with the bivalent iNTS-GMMA vaccine as described in Example 3. The immunizations involved intramuscular administration of 40 µg (O-antigen) total GMMA on days 0 and 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. Results are as follows... Figure 3 As shown.

[0244] The vaccine was well tolerated, and all rabbits vaccinated with the two-component iNTS-GMMA candidate vaccine produced high levels of anti-SEn OAg and anti-STm OAg serum IgG responses.

[0245] Example 8 - Production of GMMA from Salmonella Paratyphi A Using the scheme described in Example 1 for Salmonella enteritidis and Salmonella typhimurium (the difference being that the specific mutations used to delete tolR, pagP, and msbB are tolR::cat pagP::kan msbB::tet), a product containing Δ was prepared. tolR Δ pagP Δ msbB A mutant strain of Salmonella paratyphi A, ED199. GMMA was isolated from the bacteria as described in Example 1.

[0246] Example 9 - Salmonella Paratyphi A O-antigen and CRM 197 Production of conjugates Using CDAP chemistry (with or without ADH connectors) to mix OAG with CRM 197 Perform a splicing Salmonella paratyphi A OAg(O:2) was conjugated to CRM197 using a randomized CDAP chemistry method with or without an ADH linker. It should be noted that the randomized CDAP chemistry method will conjugate OAG and CRM... 197 Multiple bonds are formed between them. Chemical processes such as Figure 4 As shown.

[0247] Specifically, the O:2 OH groups were activated in 150 mM NaCl solution using an O:2 to CDAP w / w ratio of 1:0.3. The pH was adjusted to 9-10 with 10% TEA (v / v), and the solution was incubated at room temperature with stirring for 3 ± 0.5 minutes.

[0248] O:2 activates the cyano ester group and CRM 197 ADH / CRM 197 The hydrazide / amino group is covalently bonded to form O:2-CDAP-ADH-CRM 197 / O:2-CDAP-CRM 197 Add CRM at a w / w ratio of 0:2 (1:1). 197 ADH / CRM 197 The concentration was 10 mg / mL (O:2 and CRM). 197 ADH / CRM 197The final concentration was 5 mg / mL. The pH was maintained at 9.5 ± 0.5 with 10% triethylamine, and the solution was mixed at room temperature for 2–3 hours. Then, 1 M glycine solution was added to an equal volume of the conjugated mixture, and the pH was adjusted to 8.0 ± 0.2 with 10% triethylamine; the solution was incubated at 2–8 °C for 15 ± 5 hours. The crude conjugate was then subjected to buffer exchange, and unbound and unreacted O:2 was removed using HIC Phenyl HP resin.

[0249] Based on the research method described in Example 3 (but measuring the free OAg content in samples obtained on days 0, 8, 14, and 28), the OAg-CRM produced using CDAP chemistry with or without an ADH connector was analyzed. 197 The stability of the conjugate was studied. The results (percentage of free OAG) are shown in the table below.

[0250] Stability studies showed that, compared with OAg-CRM prepared using the chemical methods described in Examples 2-5, [the results were positive]. 197 Compared to conjugates, free OAG is significantly reduced.

[0251] Example 10 - Formulations of two tetravalent (pan-Salmonella) vaccines against Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Paratyphimurium for preclinical studies. Two types of quadrivalent vaccines were formulated. The first type (Salmonella Pancreaticobiliaria O:2-CRM) 197 ) contains GMMA (as described in Example 1) from Salmonella enteritidis and Salmonella typhimurium, and CRM 197 Conjugated Salmonella paratyphi A O-antigen (such as O:2-CRM as described in Example 9) 197 ) and fVi-CRM 197 The first type is a conjugate (as described in Example 2). The second type (Pan-Salmonella_ParA GMMA) is similar, except that the Salmonella Paratyphi A O-antigen conjugate is replaced with Salmonella Paratyphi A GMMA (as described in Example 8).

[0252] STmGMMA and SEnGMMA are adsorbed onto the aluminum gel. After mixing (1-2 hours), Salmonella paratyphi GMMA (or O:2-CRM) is added. 197 Subsequently, after a quenching step with phosphate and adjustment of osmotic pressure with sodium chloride, fVi-CRM was added. 197 .

[0253] Optimize the phosphate buffer solution concentration to achieve the highest adsorption rate, while quenching is performed to ensure the optimal particle size.

[0254] The final formulation contains sufficient GMMA to provide 40 µg / ml STm, SEn, and Salmonella paratyphi A O-antigen (sPa) and 50 µg / ml Vi polysaccharide in a phosphate-buffered saline matrix containing 0.7 mg / mL aluminum hydroxide.

[0255] The protocol for determining STm GMMA, SEnGMMA, and Salmonella paratyphi A O-antigen levels is based on the method described in Example 5. The method for determining the O-antigen levels of Salmonella enteritidis, Salmonella typhimurium, and Salmonella paratyphi A is described below.

[0256] OAG assay for Salmonella enteritidis, Salmonella typhimurium, and Salmonella paratyphi A Each individual OAg was hydrolyzed to release its corresponding dideoxymonosaccharide (SEn OAg releases tivesaccharide; STm OAg releases apicoose; SPa OAg releases porase), and these monosaccharides corresponded to chromatographic peaks, which were then analyzed by HPAEC-PAD.

[0257] In fact, among the sugars that make up the repeating units (RU) of SEn, STm, and SPa OAg, dideoxy sugars are the only type that is different from the others.

[0258] Dilute the sample with milliQ water by volume (450 µL) or by weighing using an analytical balance to ensure that the concentration of each OAG is 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 h. After hydrolysis, cool the vial in a refrigerator at 2–8 °C for 15 min. Dry the sample and standard overnight at room temperature (RT) in a centrifugal evaporator to remove the solvent / TFA. Dissolve the precipitate in 450 µL of milliQ water. Filter the sample and standard through the 0.2 µm Supor 1 mL well of an AcroPrep Advance 96-well plate and load the plate onto an HPAEC-PAD.

[0259] Example 11 - Immunogenicity of tetravalent pan-Salmonella vaccine in mice Each group, consisting of 10 CD1 mice, was immunized with the pan-Salmonella vaccine described in Example 10 at the following dosages:

[0260] The immunizations involved included intraperitoneal injections of 200 µl of each formulation on day 0 and day 28, respectively. Blood samples were collected on day -1, day 27, and day 42, and the resulting antibodies were studied using the assays described in Example 6 above. The results are as follows: Figure 5As shown. In summary, both pan-Salmonella vaccines induced specific serum IgG responses against Salmonella paratyphi A O-antigen, Salmonella typhimurium O-antigen, Salmonella enteritidis O-antigen, and Vi polysaccharide, and the antibodies exhibited bactericidal activity in mice.

[0261] In the second experiment, 10 CD1 mice were immunized with the following: - Tetravalent Pansalmonella_O:2-CRM (STm GMMA + SEn GMMA + O:2-CRM) 197 + fVi-CRM 197 Produced according to the method described in Example 10, with dosages of 1.0 (µg O-antigen), 1.0 (µg O-antigen), 1.25 (µg O-antigen), and 1.25 (µg Vi polysaccharide). - Tetravalent Pansalmonella_ParAGMMA (STm GMMA + SEn GMMA + ParA GMMA + fVi-CRM) 197 Produced according to Example 10, with dosages of 1.0 (µg O-antigen), 1.0 (µg O-antigen), 1.17 (µg O-antigen), and 1.25 (µg Vi polysaccharide). - Trivalent iNTS-TCV (STm GMMA + SEn GMMA + fVi-CRM) 197 Produced according to Example 5, with dosages of 1.0 (µg O-antigen), 1.0 (µg O-antigen), and 1.25 (µg Vi polysaccharide). - Divalent O:2-CRM 197 + fVi-CRM 197 Produced according to Example 3, with dosages of 1.25 (µg O-antigen) and 1.25 (µg Vi polysaccharide), respectively. - O:2-CRM 197 The conjugate (produced according to the method described in Example 9) was prepared at a dosage of 1.25 µg (Vi polysaccharide). - ParA GMMA (produced according to the method described in Example 8) adsorbed onto aluminum gel, at a dosage of 1.17 µg (Vi polysaccharide). - fVi-CRM 197 The conjugate (produced according to the method described in Example 2) was prepared at a dose of 1.25 µg (Vi polysaccharide).

[0262] Immunization consisted of intraperitoneal immunization with the appropriate dose (200 µl) of each product on days 0 and 28. Blood samples were collected on days 27 and 42, and antibodies produced were measured using the ELISA and SBA assays described in Example 6. Results are as follows: Figure 6 As shown.

[0263] In the induced anti-SEn and anti-STm functional antibody responses, no negative immune interference was detected when iNTS-TCV was used in combination with the ParA component. However, compared to trivalent iNTS-TCV, the Pansalmonella preparation containing O:2-CRM elicited significantly higher anti-SEn IgG responses on both days 27 and 42, while the Pansalmonella preparation containing ParA GMMA also elicited significantly higher anti-SEn IgG responses only on day 27. Furthermore, compared to trivalent iNTS-TCV, the Pansalmonella preparation containing O:2-CRM elicited significantly higher anti-STm IgG responses only on day 27. It should also be noted that the O:2-OAg dose used in Pansalmonella_ParAGMMA differs slightly (approximately 7%) from the O:2 dose used in Pansalmonella_O:2-CRM.

[0264] The presence of iNTS GMMA in Pansalmonella formulations containing O:2-CRM generally had a positive impact on the induced anti-O:2 IgG response: the tetravalent formulation with O:2-CRM induced a significantly higher response after primary immunization compared to the bivalent O:2-CRM + fVi-CRM and monovalent O:2-CRM; and it induced a significantly higher response after secondary immunization compared to the bivalent O:2-CRM + fVi-CRM formulation. The SBA titer induced by the O:2-CRM-containing Pansalmonella formulation was also significantly higher than that of the corresponding bivalent formulation. Conversely, no significant differences were shown in the induced anti-O:2 IgG response, or in the anti-ParA antibody function induced by the ParaA GMMA-containing Pansalmonella formulation versus the monovalent ParaA GMMA formulation.

[0265] Example 12 - Antibody subclasses produced by the tetravalent pan-Salmonella vaccine in mice Each group, consisting of 10 CD1 mice, was immunized with the pan-Salmonella vaccine described in Example 10 at the following dosages:

[0266] The other mouse groups were inoculated with the following control formulation: • iNTS-TCV (as described in Example 5), with a dosage of 1 µg (O-antigen) STmGMMA, 1 µg (O-antigen) SEnGMMA and 1.25 µg fVi-CRM 197 • A divalent composition comprising the fVi-CRM described in Example 2 197 (1.25 µg) and the ParAO:2-CRM described in Example 9 197 Conjugate (1.25 µg) • A divalent composition comprising the fVi-CRM described in Example 2 197 (1.25 µg) and ParAGMMA (1.17 µg O-antigen) as described in Example 8 • ParaA O:2-CRM as described in Example 9 197 Conjugate (1.25 µg) • ParA GMMA (1.17 µg O-Ag) as described in Example 8 • fVi-CRM as described in Example 2 197 (1.25 µg) The immunization involved intraperitoneal injection of 200 µl of each formulation on day 0 and day 28, respectively. Blood samples were collected on day -1, day 27, and day 42. The antibody classes generated against Vi antigen polysaccharide or Salmonella paratyphi A O:2 O-antigen were as described in Example 6. IgG subclass The determination was performed using the method described in the heading. The results are as follows: Figure 7 As shown. Figure 7 The absolute values ​​are shown in the table below: Antibody titer against Salmonella paratyphi A O:2 O- antigen

[0267] Antibody titer against Vi antigen polysaccharide

[0268] Example 13 - Immunogenicity of tetravalent pan-Salmonella vaccine in rabbits Each group, consisting of 8 New Zealand white rabbits, was immunized with one of the Pan Salmonella vaccines described in Example 10 at the following dosages:

[0269] The immunizations involved included intramuscular injections of 500 µl of each formulation on day 0 and day 28, respectively. Blood samples were collected on day -1, day 27, and day 42, and the resulting antibodies were studied using the assays described in Example 6 above. The results are as follows: Figure 8As shown. In summary, both pan-Salmonella vaccines induced specific serum IgG responses against Salmonella paratyphi A O-antigen, Salmonella typhimurium O-antigen, Salmonella enteritidis O-antigen, and fVi polysaccharide, and the antibodies exhibited bactericidal activity in rabbits.

[0270] Example 14 - Administration of iNTS-TCV vaccine to human subjects A nine-group, phase 1 / 2a, observer-blinded, randomized, dose-escalation, controlled, multinational, interleaved phase 2 study will be conducted to evaluate the efficacy of trivalent iNTS-TCV vaccine versus placebo in healthy European and African adults administered intramuscularly on days 1, 57, and 169 against invasive nontyphoidal Salmonella. Salmonella Safety, reactivity and immune response of (iNTS) and typhoid fever.

[0271] The study will be conducted in approximately 155 healthy adult participants (aged 18 to 50 years) across both phases 1 and 2. Healthy European adults will be randomly assigned to one of the designated groups in phase 1. Healthy African adults will be randomly assigned to one of the designated groups in phase 2. Each group will receive two of the 11 study interventions at each administration, except for the "Control_Stage 2" group, which will receive four study interventions (using a different active counterpart at each administration time point, along with saline).

[0272] Each participant will be randomly assigned to one intramuscular study intervention on days 1, 57, and 169, in each group.

[0273] Phase 1 Phase 1 (Europe) will employ a two-step staggered design, with all study interventions starting at a low dose in an escalating manner. A sentinel approach will be used for the first 10 participants in both Step 1 and Step 2, treating only one participant per day. This is to ensure maximum participant safety.

[0274] In step 1, 10 healthy European adults will be randomly assigned in a 2:2:1 ratio to receive: • Administer low-dose candidate iNTS-TCV vaccine with accompanying saline solution in different branches, or • Low-dose iNTS-GMMA vaccine and TCV vaccine (containing fVi-CRM) were administered to different groups, respectively. 197 Salmonella paratyphi A O-antigen-CRM 197 (conjugate), or • Placebo and saline were administered in different groups.

[0275] In step 2, 40 healthy European adults will be randomly assigned in a 2:2:1 ratio. The first 10 sentinel participants will be staggered and followed up by a safety phone call the day after the study intervention is administered. The remaining 30 participants will receive the study intervention sequentially (at least 60 minutes apart). Participants in step 2 will receive: • Full doses of candidate iNTS-TCV vaccine administered in different groups with accompanying saline, or • Full-dose iNTS-GMMA and TCV vaccines administered separately in different groups, or • Placebo and saline were administered in different groups.

[0276] In Phase 2 (Africa), a total of 105 healthy African adults will be randomly assigned in a 3:3:1 ratio to receive: • Full doses of candidate iNTS-TCV vaccine administered in different groups with accompanying saline, or • Full-dose iNTS-GMMA and TCV vaccines administered separately in different groups, or • The first administration in each group was MenACWY (Menveo) and saline, the second administration was TdaP (Boostrix) and saline, and the third administration was Typhim Vi polysaccharide vaccine (Typhim Vi) and saline. This is the control group (Phase 2).

[0277] The first 21 participants in Phase 2 will be recruited, and the intervention will be administered sequentially with at least 60-minute intervals. These participants will receive a safety follow-up phone call the day after the intervention is administered. Recruitment of the remaining 84 participants in Phase 2 will only begin after all participants have received positive safety data assessments within 7 days of the first administration of the intervention. The remaining 84 participants will receive the intervention concurrently.

[0278] The composition of a 0.5 mL full dose of iNTS-TCV vaccine is as follows:

[0279] The composition of a 0.5 mL low-dose iNTS-TCV vaccine is as follows:

[0280] Example 15 - Antibodies induced by GMMA-based candidate vaccines exhibit cross-protective effects CD1 mice were intraperitoneally immunized on days 0 and 28 with either a 500 µL injection volume of monovalent STm GMMA (2.5 μg STm OAg per mouse per injection) or monovalent SEn GMMA (2.5 μg SEn OAg per mouse per injection). SBA was measured on mouse serum obtained on day 42.

[0281] Mouse serum induced by STm GMMA showed bactericidal activity against Salmonella Typhimurium, Salmonella Debeni, and Salmonella Dublin. Figure 9 (a)). SEm GMMA-induced bactericidal activity against Salmonella enteritidis and Salmonella Dublin was observed in mouse serum. Figure 9 (b)).

[0282] CD1 mice were intraperitoneally immunized on days 0 and 28 with either a 200 µL injection volume of bivalent iNTS GMMA vaccine (STm and SEn GMMA) (1.0 μg STm OAg + 1.0 μg SEn OAg per mouse per injection) or a trivalent iNTS-TCV GMMA vaccine (STm and SEn GMMA and Salmonella typhi fVi polysaccharide) (1.0 μg STm OAg + 1.0 μg SEn OAg + 0.125 μg fVi per mouse per injection). SBA assays were performed on mouse serum obtained on day 42.

[0283] Bivalent iNTS GMMA vaccine-induced mouse serum showed bactericidal activity against Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Debye, and Salmonella Dublin. Figure 9 (c)). Mouse serum induced by the trivalent iNTS-TCV vaccine also showed bactericidal activity against Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Debye, and Salmonella Dublin. Figure 9 (d)).

[0284] New Zealand female rabbits were intramuscularly immunized on days 0 and 28 with either a 500 µL dose of bivalent iNTS GMMA vaccine (STm and SEn GMMA) (20 μg STm OAg + 20 μg SEn OAg per rabbit per injection) or a trivalent iNTS-TCV GMMA vaccine (STm and SEn GMMA and Salmonella Typhi fVi polysaccharide) (20 μg STm OAg + 20 μg SEn OAg + 25 μg fVi per rabbit per injection). SBA assays were performed on rabbit serum obtained on day 42.

[0285] Rabbit serum induced by the bivalent iNTS GMMA vaccine showed bactericidal activity against Salmonella typhimurium, Salmonella enteritidis, Salmonella debilis, Salmonella Dublin, and Salmonella paratyphi A. Figure 9 (e)). Serum from mice induced by the trivalent iNTS-TCV vaccine also showed bactericidal activity against *Salmonella typhimurium*, *Salmonella enteritidis*, *Salmonella debilis*, *Salmonella dublinii*, and *Salmonella paratyphi A*. Figure 9 (f)).

[0286] CD1 mice were intraperitoneally immunized on days 0 and 28 with two tetravalent pan-Salmonella vaccines: (i) STm and SEn GMMA, Salmonella typhi fVi polysaccharide, and Salmonella paratyphi A OAG conjugate (each injection was 1 μg STm + 1 μg SEn + 1.25 μg ParA + 1.25 μg fVi per mouse); and (ii) STm and SEn GMMA, Salmonella typhi fVi polysaccharide, and Salmonella paratyphi A GMMA (each injection was 1 μg STm + 1 μg SEn + 1.17 μg ParA + 1.25 μg fVi per mouse). SBA was measured in mouse serum obtained on day 42.

[0287] The bactericidal activities against Salmonella typhimurium, Salmonella enteritidis, Salmonella debilis, Salmonella paratyphi A OAg conjugates were shown in mouse serum induced by (i) STm and SEn GMMA, Salmonella typhimurium fVi polysaccharide and Salmonella paratyphi A GMMA; (ii) STm and SEn GMMA, Salmonella typhimurium fVi polysaccharide and Salmonella paratyphi A GMMA. Figure 10 ).

[0288] Embodiments of the present invention 1. An immunogenic composition comprising: (a) Enteric Salmonella Typhimurium serotype (Salmonella Typhimurium) antigen; (b) Salmonella enteritis serotype (Salmonella enteritidis) antigen; and (c) Enteric Salmonella typhi serotype (Salmonella typhi) antigen.

[0289] 2. A method for enhancing an immune response to Salmonella typhi or Salmonella paratyphi A antigen, the method comprising administering a composition comprising the Salmonella typhi antigen or the Salmonella paratyphi A antigen and GMMA.

[0290] 3. A method for preventing infection with Salmonella Typhi or Salmonella Paratyphi A, the method comprising administering an immunogenic composition comprising Salmonella Typhi antigen or Salmonella Paratyphi A antigen and GMMA, wherein the GMMA enhances the immune response to the Salmonella Typhi antigen or Salmonella Paratyphi A antigen.

[0291] 4. An immunogenic composition comprising GMMA for use in a method of enhancing an immune response to an antigen of Salmonella Typhi or Salmonella Paratyphi A, wherein the method comprises administering an immunogenic composition comprising the Salmonella Typhi antigen or the Salmonella Paratyphi A antigen and GMMA.

[0292] 5. An immunogenic composition for use in a method of preventing infection with Salmonella Typhi or Salmonella Paratyphi A, the method comprising administering an immunogenic composition comprising Salmonella Typhi antigen or Salmonella Paratyphi A antigen and GMMA, wherein the GMMA enhances the immune response to the Salmonella Typhi antigen or Salmonella Paratyphi A antigen.

[0293] 6. The method or immunogenic composition used as described in embodiment 2 or 4, wherein the method is a method for enhancing an immune response to Salmonella typhi antigen, and the immunogenic composition comprises the Salmonella typhi antigen.

[0294] 7. The method or immunogenic composition used as described in embodiment 2 or 4, wherein the method is a method for enhancing an immune response to Salmonella paratyphi A antigen, and the immunogenic composition comprises the Salmonella paratyphi A antigen.

[0295] 8. The method or immunogenic composition used as described in embodiment 3 or 5, wherein the method is a method for preventing Salmonella typhi infection, the immunogenic composition comprising the Salmonella typhi antigen, and the GMMA enhancing the immune response to the Salmonella typhi antigen.

[0296] 9. The method or immunogenic composition used as described in embodiment 3 or 5, wherein the method is a method for preventing Salmonella paratyphi A infection, the immunogenic composition comprising the Salmonella paratyphi A antigen, and the GMMA enhancing the immune response to the Salmonella paratyphi A antigen.

[0297] 10. The method or immunogenic composition for use as described in any one of embodiments 2 to 9, wherein the GMMA comprises at least one selected from the group consisting of: Salmonella typhimurium GMMA, Salmonella enteritidis GMMA, and Salmonella paratyphi A GMMA.

[0298] 11. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition further comprises: (d) Enteric Salmonella paratyphoid serotype (Salmonella paratyphoid) antigen.

[0299] 12. The immunogenic composition or method as described in embodiment 1 or 11, wherein the Salmonella typhimurium antigen comprises Salmonella typhimurium O-antigen.

[0300] 13. The immunogenic composition or method as described in any one of embodiments 1, 11 or 12, wherein the Salmonella typhimurium antigen comprises or is composed of outer membrane vesicles derived from Salmonella typhimurium.

[0301] 14. An immunogenic composition or method as described in any one of embodiments 1 or 11 to 13, wherein the Salmonella typhimurium antigen comprises or is composed of Salmonella typhimurium GMMA.

[0302] 15. The immunogenic composition or method as described in embodiment 10 or 14, wherein the Salmonella typhimurium GMMA comprises modified lipid A.

[0303] 16. The immunogenic composition or method as described in embodiment 15, wherein the modified lipid A is a detoxified lipid A.

[0304] 17. The immunogenic composition or method as described in embodiment 15 or 16, wherein the modified lipid A is pentacylated lipid A.

[0305] 18. The immunogenic composition or method as described in any one of embodiments 15 to 17, wherein the Salmonella typhimurium GMMA is derived from Salmonella typhimurium that does not contain a gene encoding a functional MsbB protein.

[0306] 19. The immunogenic composition or method according to any one of embodiments 15 to 18, wherein the Salmonella typhimurium GMMA is derived from... Salmonella typhimurium msbB.

[0307] 20. The immunogenic composition or method as described in any one of embodiments 10 or 14 to 19, wherein the Salmonella typhimurium GMMA is derived from Salmonella typhimurium that does not contain a gene encoding a functional PagP protein.

[0308] 21. The immunogenic composition or method as described in any one of embodiments 10 or 14 to 20, wherein the Salmonella typhimurium GMMA is derived from... pagP Salmonella Typhimurium.

[0309] 22. The immunogenic composition or method as described in any one of embodiments 10 or 14 to 21, wherein the Salmonella typhimurium GMMA is derived from Salmonella typhimurium that does not contain a gene encoding a functional TolR protein.

[0310] 23. The immunogenic composition or method as described in any one of embodiments 10 or 14 to 22, wherein the Salmonella Typhimurium GMMA is derived from Salmonella Typhimurium strain 2192.

[0311] 24. The immunogenic composition or method as described in any one of embodiments 10 or 14 to 23, wherein the Salmonella typhimurium GMMA is derived from... Salmonella typhimurium from tolR.

[0312] 25. An immunogenic composition or method as described in any one of embodiments 1 or 10 to 24, wherein the immunogenic composition comprises a dose (O-antigen) of 1 µg to 50 µg, 2 µg to 25 µg, 2 µg to 10 µg, 15 µg to 25 µg, about 20 µg, or about 4 µg of Salmonella typhimurium antigen or Salmonella typhimurium GMMA.

[0313] 26. The immunogenic composition or method as described in any one of embodiments 10 or 14 to 25, wherein the Salmonella typhimurium GMMA enhances the immune response to Salmonella typhimurium or Salmonella paratyphimurium antigens.

[0314] 27. An immunogenic composition or method as described in any one of embodiments 1 or 11 to 26, wherein the Salmonella enteritidis antigen comprises Salmonella enteritidis O-antigen.

[0315] 28. An immunogenic composition or method as described in any one of embodiments 1 or 11 to 24, wherein the Salmonella enterica antigen comprises or is composed of: outer membrane vesicles derived from Salmonella enterica. 。

[0316] 29. An immunogenic composition or method as described in any one of embodiments 1 or 11 to 28, wherein the Salmonella enteritidis antigen comprises or is composed of Salmonella enteritidis GMMA.

[0317] 30. The immunogenic composition or method as described in embodiment 10 or 29, wherein the Salmonella enteritidis GMMA comprises modified lipid A.

[0318] 31. The immunogenic composition or method as described in embodiment 30, wherein the modified lipid A is a detoxified lipid A.

[0319] 32. The immunogenic composition or method as described in embodiment 30 or 31, wherein the modified lipid A is pentacylated lipid A.

[0320] 33. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 32, wherein the Salmonella enterica GMMA is derived from Salmonella enterica that does not contain a gene encoding a functional MsbB protein.

[0321] 34. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 33, wherein the Salmonella enteritidis GMMA is derived from... Salmonella enteritidis msbB.

[0322] 35. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 34, wherein the Salmonella enterica GMMA is derived from Salmonella enterica that does not contain a gene encoding a functional PagP protein.

[0323] 36. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 35, wherein the Salmonella enteritidis GMMA is derived from... pagP is a type of Salmonella enteritidis.

[0324] 37. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 36, wherein the Salmonella enterica GMMA is derived from Salmonella enterica that does not contain a gene encoding a functional TolR protein.

[0325] 38. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 37, wherein the Salmonella enterica GMMA is derived from Salmonella enterica strain 618.

[0326] 39. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 38, wherein the Salmonella enteritidis GMMA is derived from... Salmonella enteritidis from tolR.

[0327] 40. An immunogenic composition or method as described in any one of embodiments 1 or 10 to 39, wherein the immunogenic composition comprises a dose (O-antigen) of 1 µg to 50 µg, 2 µg to 25 µg, 2 µg to 10 µg, 15 µg to 25 µg, about 20 µg, or about 4 µg of Salmonella enteritidis antigen or Salmonella enteritidis GMMA.

[0328] 41. The immunogenic composition or method as described in any one of embodiments 10 or 29 to 40, wherein the Salmonella enteritidis GMMA enhances the immune response to Salmonella typhi or Salmonella paratyphi A antigens.

[0329] 42. The immunogenic composition or method as described in any one of embodiments 1 to 6, 8 or 10 to 41, wherein the Salmonella typhi antigen comprises Vi polysaccharide.

[0330] 43. The immunogenic composition or method as described in any one of embodiments 1 to 6, 8 or 10 to 42, wherein the Salmonella typhi antigen comprises fragmented Vi (fVi) polysaccharide.

[0331] 44. The immunogenic composition or method as described in embodiment 43, wherein the average molecular weight of the fVi polysaccharide is 40 kDa to 55 kDa, 41 kDa to 49 kDa, or 51 kDa to 55 kDa.

[0332] 45. The immunogenic composition or method as described in embodiment 43 or 44, wherein the average molecular weight of the fVi polysaccharide is 51 kDa to 55 kDa.

[0333] 46. ​​The immunogenic composition or method as described in any one of embodiments 43 to 45, wherein the fVi polysaccharide is part of an fVi conjugate comprising fVi and a carrier protein.

[0334] 47. The immunogenic composition or method as described in embodiment 46, wherein the carrier protein is CRM. 197 Or diphtheria toxoid.

[0335] 48. The immunogenic composition or method as described in embodiment 47, wherein the carrier protein is CRM. 197 .

[0336] 49. The immunogenic composition or method of any one of embodiments 46 to 48, wherein the fVi polysaccharide is conjugated to the carrier protein via carbodiimide chemistry, optionally via a linker. 50. The immunogenic composition or method as described in any one of embodiments 46 to 49, wherein the fVi conjugate is obtained by or through a method comprising the following steps: a. Fragmenting Vi polysaccharide to obtain fragmented Vi (fVi) polysaccharide, said fragmented Vi polysaccharide having an average molecular weight of 40 kDa to 55 kDa, 41 kDa to 49 kDa or 51 kDa to 55 kDa; b. Activating the fVi polysaccharide obtained in step a by reacting it with carbodiimide and N-hydroxysuccinimide at pH 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative; and c. The N-hydroxysuccinimide ester fVi derivative obtained in step b is reacted with the carrier protein to prepare the fVi conjugate.

[0338] 51. The immunogenic composition or method as described in any one of embodiments 46 to 50, wherein the carbodiimide is EDC.

[0339] 52. The immunogenic composition or method as described in embodiment 50 or 51, wherein the carrier protein is derivatized by reacting it with carbodiimide and a linker.

[0340] 53. The immunogenic composition or method as described in embodiment 52, wherein the adapter is an adipic acid dihydrazide (ADH) adapter.

[0341] 54. The immunogenic composition or method as described in embodiment 52 or 53, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or the carbodiimide chemistry is an EDAC chemistry.

[0342] 55. An immunogenic composition or method as described in any one of embodiments 1 to 6, 8, or 10 to 54, wherein the immunogenic composition comprises 1 to 100 µg, 1 to 50 µg, 15 to 50 µg, 20 to 30 µg, 1 to 20 µg, 1 to 10 µg, about 25 µg, or about 5 µg of fVi polysaccharide.

[0343] 56. The immunogenic composition or method as described in any one of embodiments 2 to 50, wherein the Salmonella paratyphi A antigen comprises Salmonella paratyphi A O-antigen.

[0344] 57. The immunogenic composition or method as described in embodiment 56, wherein the immunogenic composition comprises Salmonella paratyphi A O-antigen conjugated to a carrier protein.

[0345] 58. The immunogenic composition or method as described in embodiment 57, wherein the carrier protein is diphtheria toxoid or CRM. 197 .

[0346] 59. The immunogenic composition or method as described in embodiment 58, wherein the carrier protein is CRM. 197 .

[0347] 60. The immunogenic composition or method of any one of embodiments 57 to 59, wherein the Salmonella paratyphi A O-antigen is conjugated to the carrier protein by means of introducing more than one activation site into the Salmonella paratyphi A O-antigen and / or the Salmonella paratyphi A O-antigen contains more than one activation site.

[0348] 61. The immunogenic composition or method as described in any one of embodiments 57 to 60, wherein the Salmonella paratyphi A O-antigen is optionally conjugated to the carrier protein via a linker through CDAP chemistry.

[0349] 62. The immunogenic composition or method as described in embodiment 61, wherein the Salmonella paratyphi A O-antigen is conjugated to the carrier protein via a linker.

[0350] 63. The immunogenic composition or method as described in embodiment 62, wherein the linker is adipic dihydrazide (ADH).

[0351] 64. The immunogenic composition or method according to any one of embodiments 56 to 63, wherein the immunogenic composition comprises 1 to 100 µg, 1 to 50 µg, 15 to 50 µg, 20 to 30 µg, 1 to 20 µg, 1 to 10 µg, about 25 µg, or about 5 µg of Salmonella paratyphi A O-antigen.

[0352] 65. The immunogenic composition or method as described in any one of embodiments 2 to 64, wherein the Salmonella paratyphi A antigen comprises Salmonella paratyphi A GMMA.

[0353] 66. The immunogenic composition or method as described in embodiment 10 or 65, wherein the Salmonella paratyphi A GMMA comprises modified lipid A.

[0354] 67. The immunogenic composition or method as described in embodiment 66, wherein the modified lipid A is a detoxified lipid A.

[0355] 68. The immunogenic composition or method as described in embodiment 66 or 67, wherein the modified lipid A is pentacylated lipid A.

[0356] 69. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 68, wherein the Salmonella paratyphi A GMMA is derived from Salmonella paratyphi A that does not contain a gene encoding a functional MsbB protein.

[0357] 70. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 69, wherein the Salmonella paratyphi A GMMA is derived from... Salmonella paratyphi A of msbB.

[0358] 71. The immunogenic composition or method as described in 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.

[0359] 72. The immunogenic composition as described in embodiments 69, 70 or 71, wherein the GMMA is derived from Salmonella paratyphi A of msbB::tet.

[0360] 73. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 72, wherein the Salmonella paratyphi A GMMA is derived from Salmonella paratyphi A that does not contain a gene encoding a functional PagP protein.

[0361] 74. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 73, wherein the Salmonella paratyphi A GMMA is derived from... pagP Salmonella paratyphi A.

[0362] 75. The immunogenic composition as described in 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.

[0363] 76. The immunogenic composition according to any one of embodiments 73 to 75, wherein the rGMMA is derived from Salmonella paratyphi A of pagP::kan.

[0364] 77. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 76, wherein the Salmonella paratyphi A GMMA is derived from Salmonella paratyphi A that does not contain a gene encoding a functional TolR protein.

[0365] 78. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 77, wherein the Salmonella paratyphi A GMMA is derived from Salmonella paratyphi A strain ED199.

[0366] 79. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 78, wherein the Salmonella paratyphi A GMMA is derived from Salmonella paratyphi A with the denomination ΔtolR.

[0367] 80. An immunogenic composition or method as described in any one of embodiments 10 or 65 to 79, wherein the immunogenic composition comprises a dose (O-antigen) of 1 µg to 50 µg, 2 µg to 25 µg, 2 µg to 10 µg, 15 µg to 25 µg, about 20 µg, or about 4 µg of Salmonella paratyphi A GMMA.

[0368] 81. The immunogenic composition as described in 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.

[0369] 82. The immunogenic composition as described in any one of embodiments 77 or 79 to 81, wherein the GMMA is derived from Salmonella paratyphi A of tolR::cat.

[0370] 83. The immunogenic composition or method as described in any one of embodiments 10 or 65 to 82, wherein the Salmonella paratyphi A GMMA enhances the immune response to the Salmonella typhi antigen.

[0371] 84. An immunogenic composition or method as described in any one of embodiments 2 to 83, wherein one method is for enhancing an immune response to a Salmonella typhi antigen or a Salmonella paratyphi A antigen, provided that the immune response to the Salmonella typhi antigen or the Salmonella paratyphi A antigen induced when the Salmonella typhi antigen or the Salmonella paratyphi A antigen is a component of an immunogenic composition comprising GMMA is higher than the immune response induced when the Salmonella typhi antigen or the Salmonella paratyphi A antigen is not a component of an immunogenic composition comprising GMMA.

[0372] 85. An immunogenic composition or method as described in any one of embodiments 2 to 84, wherein the GMMA enhances the immune response to the Salmonella typhi antigen or the Salmonella paratyphi A antigen, provided that the immune response to the Salmonella typhi antigen or the Salmonella paratyphi A antigen induced when the Salmonella typhi antigen or the Salmonella paratyphi A antigen is a component of the immunogenic composition comprising the GMMA is higher than the immune response induced when the Salmonella typhi antigen or the Salmonella paratyphi A antigen is not a component of the immunogenic composition comprising the GMMA.

[0373] 86. An immunogenic composition or method as described in embodiment 84 or 85, wherein one method is a method or GMMA-enhanced immune response against Salmonella typhi or Salmonella paratyphi A antigen, wherein the immune response against the Salmonella typhi or Salmonella paratyphi A antigen induced when the Salmonella typhi or Salmonella paratyphi A antigen is part of an immunogenic composition containing GMMA is at least 5, at least 10, or at least 20 times higher than the immune response induced when the Salmonella typhi or Salmonella paratyphi A antigen is not part of an immunogenic composition containing GMMA.

[0374] 87. The immunogenic composition or method as described in any one of embodiments 84 to 86, wherein the immune response induced against Salmonella typhi or Salmonella paratyphi A antigen is determined by ELISA as the number of antibodies produced 42 days after administration of the immunogenic composition comprising the Salmonella typhi or Salmonella paratyphi A antigen and the GMMA at a dose of 0.78 µg of Salmonella typhi or Salmonella paratyphi A antigen and 0.63 µg of (O-antigen) of the GMMA.

[0375] 88. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition is tolerogenic.

[0376] 89. The immunogenic composition or method as described in embodiment 88, wherein the immunogenic composition is tolerable when it induces a body temperature increase of less than 1.8°C, less than 1.7°C, less than 1.6°C, or less than 1.5°C in a toxicity assay comprising the following steps: (a) Measure the rabbit's initial body temperature; (b) The immunogenic composition was administered to the rabbits at a dose of 20 µg (O-antigen) / GMMA and 25 µg sugar / sugar conjugate; (c) Monitor the rabbit's body temperature for 5 hours; and (d) Record the highest body temperature of the rabbit. The temperature rise is calculated as equal to the rabbit's highest body temperature minus the rabbit's initial body temperature.

[0377] 90. The immunogenic composition or method as described in embodiment 2 or 88, wherein the immunogenic composition is tolerable when it induces a maximum body temperature of 41°C or lower, 40.9°C or lower, or 40.8°C or lower in a toxicity assay comprising the following steps: (a) The immunogenic composition was administered to the rabbits at doses of 20 µg (O-antigen) / GMMA and 25 µg / carbohydrate; (b) Monitor the rabbit's body temperature for 5 hours; and (c) Record the highest body temperature of the rabbit. 。

[0378] 91. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition induces at least 10 [units of measurement] in an immunogenicity assay comprising the following steps. 3 EU / ml of anti-Salmonella typhimurium O-antigen antibody and / or at least 10 3 EU / ml of anti-Salmonella O-antigen antibody: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg glucose / glucose on days 0 and 28; and (b) On day 42, the antibody levels against Salmonella Typhimurium O antigen and / or Salmonella Enteritidis O antigen were measured by ELISA.

[0379] 92. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition induces at least 10 [units of measurement] in an immunogenicity assay comprising the following steps. 3 EU / ml of anti-fVi conjugate antibody: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) The level of the anti-fVi conjugate antibody was measured by ELISA on day 42.

[0380] 93. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition induces at least 10 [units of measurement] in an immunogenicity assay comprising the following steps. 3 EU / ml Salmonella paratyphi A O-antigen antibody: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) On day 42, the level of anti-Salmonella paratyphi A O-antigen antibody was measured by ELISA.

[0381] 94. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition induces at least 10 [units of measurement] in an immunogenicity assay comprising the following steps. 3.5 EU / ml Salmonella paratyphi A O-antigen antibody: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) On day 42, the level of anti-Salmonella paratyphi A O-antigen antibody was measured by ELISA.

[0382] 95. The immunogenic composition or method as described in any one of embodiments 1 or 10 to 94, wherein the induced level of anti-Salmonella typhimurium O-antigen antibody is at least 90%, 95%, or 98% of the level of anti-Salmonella typhimurium O-antigen antibody induced by the corresponding monovalent Salmonella typhimurium immunogenic composition.

[0383] 96. The immunogenic composition or method as described in any one of embodiments 1 or 10 to 95, wherein the induced level of anti-Salmonella enteritidis O-antigen antibody is at least 90%, 95%, or 98% of the level of anti-Salmonella enteritidis O-antigen antibody induced by the corresponding monovalent Salmonella enteritidis immunogenic composition.

[0384] 97. The immunogenic composition or method as described in any one of embodiments 1 to 6, 8 or 10 to 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.

[0385] 98. The immunogenic composition or method as described in any one of embodiments 2 to 5, 7 or 11 to 97, wherein the level of the induced anti-Salmonella paratyphi A O-antigen antibody is at least 90%, at least 95% or at least 98% of the level of the anti-Salmonella paratyphi A O-antigen antibody induced by the corresponding monovalent Salmonella paratyphi A immunogenic composition.

[0386] 99. The immunogenic composition or method according to any one of embodiments 95 to 98, wherein the levels of the anti-Salmonella typhimurium O-antigen antibody, the anti-Salmonella enteritidis O-antigen antibody, the anti-fVi conjugate antibody, and / or the anti-Salmonella paratyphi A O-antigen antibody are measured in an immunogenicity assay comprising the following steps: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) On day 42, the antibody levels against Salmonella typhimurium, Salmonella enteritidis, fVi conjugate and / or Salmonella paratyphi A O-antigen were measured by ELISA.

[0387] 100. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition 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 strains: (a) Salmonella typhimurium ST34; (b) Salmonella typhimurium 10433_3; (c) Salmonella Typhimurium D23580; (d) Salmonella typhimurium ST4 / 74; (e) Salmonella typhimurium A130; (f) Enteric Salmonella Derby serotype; (g) Enteric Salmonella Dublin serotype; (h) Salmonella enteritidis A1636; (i) Salmonella enteritidis CP255; and (j) Salmonella enteritidis D7795.

[0388] 101. An immunogenic composition or method as described in any one of embodiments 2 to 5, 7, or 11 to 100, wherein the immunogenic composition induces antibodies against Salmonella paratyphi A O-antigen of each class of IgG3, IgG2b, IgG2a, and IgG1, as determined by an antibody class assay including the following steps: (a) Mice were intraperitoneally immunized with the immunogenic composition at doses of 1 µg (O-antigen) / GMMA and 1.25 µg sugar / sugar conjugate on days 0 and 28; and (b) The level of the anti-Salmonella paratyphi A O-antigen antibody subtype was measured by ELISA on day 42.

[0389] 102. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition further comprises an adjuvant.

[0390] 103. The immunogenic composition or method as described in embodiment 102, wherein the adjuvant is an aluminum adjuvant.

[0391] 104. The immunogenic composition or method as described in embodiment 102 or 103, wherein the adjuvant comprises aluminum hydroxide and / or aluminum phosphate.

[0392] 105. The immunogenic composition or method as described in embodiment 104, wherein the adjuvant comprises aluminum hydroxide.

[0393] 106. The immunogenic composition or method according to any one of embodiments 103 to 105, wherein the adjuvant comprises 0.1 mg to 10 mg of Al. 3+ Al from 0.1 mg to 5 mg 3+ Al, 0.3 mg to 0.4 mg 3+ Or approximately 0.35 mg Al 3+ .

[0394] 107. The immunogenic composition or method as described in any of the foregoing embodiments, wherein the immunogenic composition further comprises a pharmaceutically acceptable excipient.

[0395] 108. The immunogenic composition or method as described in embodiment 107, wherein the pharmaceutically acceptable excipient comprises phosphate-buffered saline.

[0396] 109. The immunogenic composition or method as described in embodiment 108, wherein the pH of the phosphate buffered saline is 6 to 7, or about 6.5.

[0397] 110. A vaccine comprising an immunogenic composition as described in any one of embodiments 1 or 11 to 109.

[0398] 111. The immunogenic composition or vaccine as described in any one of embodiments 1 or 11 to 110, used in a method for preventing infection.

[0399] 112. A method for preventing infection, the method comprising administering to a subject an effective amount of an immunogenic composition or vaccine as described in any one of embodiments 1 or 11 to 110.

[0400] 113. Use of the immunogenic composition or vaccine as described in any one of embodiments 1 or 11 to 110 in the preparation of a medicament for use in a method of preventing infection.

[0401] 114. The immunogenic composition or vaccine as described in embodiment 111 or the use as described in embodiment 113, wherein the method of preventing infection comprises administering to a subject an effective amount of the immunogenic composition or vaccine as described in embodiment 1 or any one of 11 to 110.

[0402] 115. An immunogenic composition or vaccine, method or use as described in any one of embodiments 111 to 114, wherein the method for preventing infection is a method for preventing Salmonella infection.

[0403] 116. An immunogenic composition or vaccine, method or use as described in any one of embodiments 111 to 115, wherein the method for preventing infection is a method for preventing infection with invasive undifferentiated Salmonella.

[0404] 117. An immunogenic composition or vaccine, method or use as described in any one of embodiments 111 to 116, wherein the method for preventing infection is a method for preventing infection with Salmonella Typhimurium, Salmonella Enteritidis, Salmonella Typhimurium and / or Salmonella Paratyphi A.

[0405] 118. An immunogenic composition, immunogenic composition or vaccine, method or use as described in any one of embodiments 10 or 65 to 117, wherein the O-antigen / protein ratio of said Salmonella paratyphi A GMMA is at least 0.4.

Claims

1. An immunogenic composition comprising: (a) a Salmonella enterica serovar typhimurium (S. typhimurium) antigen, wherein the S. typhimurium antigen comprises or consists of outer membrane vesicles from S. typhimurium; (b) a Salmonella enterica serovar enteritidis (S. enteritidis) antigen, wherein the S. enteritidis antigen comprises or consists of outer membrane vesicles from S. enteritidis; and (c) a Salmonella enterica serovar typhi (S. typhi) antigen, wherein the S. typhi antigen comprises Vi polysaccharide.

2. A method for enhancing an immune response to a S. typhi or S. paratyphi A antigen, the method comprising administering a composition comprising the S. typhi or S. paratyphi A antigen and a GMMA.

3. A method for preventing S. typhi or S. paratyphi A infection, the method comprising administering an immunogenic composition comprising a S. typhi or S. paratyphi A antigen and a GMMA, wherein the GMMA enhances an immune response to the S. typhi or S. paratyphi A antigen.

4. An immunogenic composition comprising a GMMA for use in a method of enhancing an immune response to a S. typhi or S. paratyphi A antigen, wherein the method comprises administering an immunogenic composition comprising the S. typhi or S. paratyphi A antigen and a GMMA.

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

6. The method or immunogenic composition for use of any one of claims 2 to 5, wherein the GMMA comprises at least one selected from the group consisting of: a S. typhimurium GMMA, a S. enteritidis GMMA, and a S. paratyphi A GMMA.

7. The immunogenic composition or method of any one of the preceding claims, wherein the immunogenic composition further comprises: (d) a Salmonella enterica serovar paratyphi A (S. paratyphi A) antigen.

8. The immunogenic composition or method of claim 1 or 7, wherein the S. typhimurium antigen comprises or consists of a S. typhimurium GMMA.

9. The immunogenic composition or method of claim 6 or 8, wherein the S. typhimurium GMMA comprises a modified lipid A, optionally wherein the modified lipid A is a detoxified lipid A.

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

11. The immunogenic composition or method of claim 6 or 10, wherein the S. enteritidis GMMA comprises a modified lipid A, optionally wherein the modified lipid A is a detoxified lipid A.

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

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

14. The immunogenic composition or method of claim 13, wherein the carrier protein is CRM 197 or diphtheria toxoid.

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

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

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

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

19. The immunogenic composition or method of any one of the preceding claims, wherein the immunogenic composition further comprises an adjuvant.

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

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

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

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