vaccine

Abstract

The present invention relates to a conjugate comprising a polysaccharide containing a 3-deoxy-D-manno-octulosonic acid (KDO) moiety, in particular a conjugate produced using a random conjugation method, a method for preparing such a conjugate, an immunogenic composition and vaccine comprising the conjugate, and a method of treatment or medical use using the composition and vaccine.

Description

[Technical Field]

[0001] The present invention relates to a conjugate containing a polysaccharide comprising a 3-deoxy-D-mannoocturosonic acid (KDO) moiety, particularly a conjugate produced using a random conjugation method, a method for preparing such a conjugate, an immunogenic composition and vaccine comprising the conjugate, and a method of treatment or medical use using the composition and vaccine. [Background technology]

[0002] Typhoid fever is a bacterial disease caused by Salmonella enterica subspecies enterica serotype chyfi (Salmonella chyfi), a human host-limited microorganism [Crump, 2019]. The disease occurs globally, primarily affecting children and young adults, but is endemic in developing countries in Africa and Asia, while in developed countries it is occasionally reported in travelers returning from recently endemic countries [Smith, 2016]. Due to difficulties in establishing diagnosis in endemic regions, the accurate burden of typhoid fever is believed to be significantly underestimated. In 2017, there were an estimated 10.9 million cases of typhoid fever and 116,800 deaths due to Salmonella chyfi. Similarly, there were 8.3 million lost life years (YLLs) and 8.4 million disability-adjusted life years (DALYs) due to typhoid fever. Although improvements in water and sanitation have reduced the disease burden, it remains a significant public health issue [Global Burden of Disease, 2017]. The burden of typhoid fever is highest in school-aged children and those under 5 years of age. Recent studies have shown that the adjusted typhoid fever incidence per 100,000 people-years observed, as confirmed by blood cultures, ranged from 861 (599-1203) in Malawi to 3228 (2276-4757) in Bangladesh for the 5-9 year group, while for the 0-4 year group, it was 632 (398-965) and 2625 (1764-4244) in Malawi and Bangladesh, respectively [Meiring, 2021]. Without rapid diagnosis and treatment, typhoid fever can require hospitalization and lead to fatal complications such as typhoid ileus (TIP). In developing countries where typhoid fever is endemic, surgical intervention is often delayed, resulting in worse disease outcomes [Contini, 2017].

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

[0004] S. paratifi A resides in the human gut, and its clinical symptoms are indistinguishable from typhoid fever. S. paratifi A is ranked as the second leading cause of typhoid fever, preceded only by Salmonella enterica serotype tifi (S. tifi). Typhoid fever or paratyphoid fever caused by S. paratifi A was thought to account for a relatively small proportion of typhoid fever cases. However, since the 1980s, the incidence and relative frequency of paratyphoid fever have been increasing in Nepal, Pakistan, and Thailand. Furthermore, India and China, with their large populations, have reported a considerable number of S. paratifi A cases. Countries that do not consider typhoid fever endemic, such as the United States, have reported an increasing trend in typhoid fever, particularly among travelers from South Asia (Irfan et al, Ceftriaxone resistant Salmonella enterica serovar Paratyphi A identified in a case of enteric fever: first case report from Pakistan. BMC Infect Dis. 2023 Apr 26;23(1):267. doi: 10.1186 / s12879-023-08152-9. Erratum in: BMC Infect Dis. 2023 May 23;23(1):346. PMID: 37101111;PMCID: PMC10132421.).

[0005] Bacterial sugars (or polysaccharides) have been used in vaccines for many years. Polysaccharides are T-cell independent antigens, but they have poor immunogenicity. Conjugation of carriers can convert T-cell independent antigens into T-cell dependent antigens, thereby enhancing the memory response and enabling the exertion of protective immunity. Therefore, the most influential polysaccharide vaccines are based on sugar conjugates, and the prototype conjugate vaccine is against Haemophilus influenzae type b (Hib) [see, for example, Chapter 14 of Vaccines (2004) eds. Plotkin & Orenstein. ISBN 0-7216-9688-0].

[0006] Gram-negative bacteria are surrounded by an outer membrane containing lipopolysaccharides. Lipopolysaccharides are a diverse group of molecules that act as endotoxins, inducing a potent immune response in mammals. Each lipopolysaccharide contains three parts: the O-antigen (referred to as O-specific polysaccharide or O-polysaccharide), the core domain, and the lipid A domain. Antibodies against the O-antigen from a particular Gram-negative bacterium may provide protection against infection by that bacterium. Therefore, vaccines have been envisioned to contain the O-antigen conjugated to a carrier protein. For example, O-antigen-based conjugated vaccines have been proposed against various Salmonella species (e.g., serotypes of Salmonella enterica, Salmonella tiphimurium and Salmonella paratifei A), Sigella species, and Escherichia coli. In these vaccines, the O-antigen is conjugated to the core domain of a full-length lipopolysaccharide (i.e., the conjugated polysaccharide is lipopolysaccharide without the lipid A domain). Polysaccharides are conjugated to the carrier via the core domain.

[0007] Various methods have been investigated for conjugating O-antigens from bacteria such as Salmonella paratifida (S. paratifida). For example, WO 2013 / 038375 proposes the use of a selective method to link the KDO moiety to a linker or carrier protein using direct (selective) reductive amination. [Overview of the project] [Problems that the invention aims to solve]

[0008] The examples herein demonstrate that conjugates produced by the direct (selective) reductive amination method of WO2013 / 038375 are not sufficiently stable. In particular, S. paratifida O-antigen-CRM 197 Conjugate Salmonella chifi (S. chifi) Vi-CRM 197 When formulated into a bivalent vaccine further containing a conjugate, S. paratifida A O-antigen-CRM prepared using the selective reductive amination method of WO2013 / 038375 197The conjugate was unstable. This is particularly concerning because infections caused by S. paratifi A and S. chifi are especially prevalent in developing countries, where vaccine stability is crucial for supply chains. [Means for solving the problem]

[0009] However, the examples herein demonstrate that when S. paratifida O-antigen is conjugated using a random conjugation method that introduces multiple active sites, the conjugate is remarkably stable and therefore highly suitable for use in vaccines.

[0010] Therefore, the first embodiment provides a polysaccharide conjugate comprising a 3-deoxy-D-manno-octulosonic acid (KDO) moiety conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide conjugate comprises two or more activation sites.

[0011] In a second embodiment, a conjugate comprising an O-antigen conjugated to a carrier protein by a random conjugation method is provided, wherein the polysaccharide conjugate contains two or more activation sites.

[0012] In the third aspect, the following steps are taken: (i) Activating polysaccharides by 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) chemistry to provide activated polysaccharides; or (ii) To provide oxidized polysaccharides by oxidizing polysaccharides, We provide a conjugate containing a polysaccharide with a KDO moiety, which is conjugated to a carrier protein using a method that includes [a specific method].

[0013] In the fourth aspect, the following steps are taken: (i) Activating O-antigens by CDAP chemistry to provide activated O-antigens; or (ii) To provide an oxidized O-antigen by oxidizing the O-antigen, We provide a conjugate containing an O-antigen conjugated to a carrier protein using a conjugation method that includes [specific method / method].

[0014] The fifth aspect provides a method for generating a conjugate comprising a polysaccharide containing a KDO moiety conjugated to a carrier protein, the method comprising the step of introducing a plurality of activation sites into the polysaccharide.

[0015] The sixth aspect provides a method for generating a conjugate containing an O-antigen conjugated to a carrier protein, the method comprising the step of introducing a plurality of activation sites into a polysaccharide.

[0016] A seventh aspect provides a method for generating a conjugate comprising a polysaccharide containing a KDO moiety conjugated to a carrier protein, comprising the following steps: (i) Activating polysaccharides by CDAP chemistry to provide activated polysaccharides; or (ii) To provide oxidized polysaccharides by oxidizing polysaccharides, This provides a method that includes [something].

[0017] In the eighth aspect, a method for generating a conjugate containing an O-antigen conjugated to a carrier protein, comprising the following steps: (i) Activating O-antigens by CDAP chemistry to provide activated O-antigens; or (ii) To provide an oxidized O-antigen by oxidizing the O-antigen, This provides a method that includes [something].

[0018] In the ninth aspect, a conjugate obtainable by the method of the present invention is provided.

[0019] In the tenth aspect, a conjugate obtained by the method of the present invention is provided.

[0020] In the eleventh aspect, an immunogenic composition comprising the conjugate of the present invention is provided.

[0021] In the twelfth aspect, a vaccine comprising the immunogenic composition of the present invention is provided.

[0022] In the thirteenth aspect, a method for preventing infection is provided, comprising administering an effective amount of the immunogenic composition or vaccine of the present invention to a subject.

[0023] In the fourteenth aspect, the use of the immunogenic composition or vaccine of the present invention is provided for the manufacture of a pharmaceutical product used in a method for preventing infection. [Brief explanation of the drawing]

[0024] [Figure 1] Reaction scheme for the selective reductive amination method described in WO2013 / 038375. [Figure 2] A reaction scheme for conjugating S. paratifida A O-antigen to CRM197-ADH using NHS-EDAC chemistry. [Figure 3] A reaction scheme for conjugating S. paratifida O-antigen to CRM197 by reductive amination with a random oxidation step using periodate. [Figure 4] A reaction scheme for conjugating S. paratifida A O-antigen to CRM197 using a random CDAP chemistry approach. [Figure 5] A graph showing the immunogenicity of various S. paratifida O-antigen conjugate vaccines. Mice were immunized as described in Example 8, and the immunogenicity of the vaccines was measured by ELISA. Hatched bars represent antibody levels 42 days after initial immunization, and unfilled bars represent antibody levels 28 days after initial immunization. [Figure 6] Previously reported structure of the O-antigen (including core domain) from S. paratifei A. [Figure 7] Structure of the Vi monomer repeating unit. [Figure 8] Array list. [Figure 9] A graph showing the geometric mean concentration (GMC) of anti-Vi antibodies measured by ELISA. The administered vaccine (and the dose used) is as described in Example 9. [Figure 10] A graph showing the geometric mean concentration (GMC) of anti-O:2 (S. paratifida O-antigen) antibodies measured by ELISA. The administered vaccine (and the dose used) is as described in Example 9. [Figure 11] A graph showing the geometric mean titer (GMT) of anti-S. paratifida O-antigen bactericidal antibodies measured by SBA. The administered vaccine (and the dose used) is as described in Example 9. [Figure 12-1] A) HPLC SEC of O:2[16 kDa + 100 kDa], O:2[16 kDa], O:2[100 kDa]. [Figure 12-2] B) Analytical characterization of the O:2 population used for conjugation with CRM197. [Figure 13] This report presents schematic graphs of the geometric mean units (bars) and individual antibody levels (dots) of anti-O:2 IgG. The bars on the left represent 27 days, and the bars on the right represent 42 days. A) Effect of S. paratifida AO:2 size on the immune response of mice: O:2-CRM197 conjugates synthesized using similar O:2 / CRM197 w / w ratios were compared using O:2 of different sizes (16 kDa, 100 kDa, 16 kDa + 100 kDa). B) Effect of O:2 / CRM197 w / w ratio: O:2-CRM197 conjugates synthesized from O:2 of specific sizes but exhibiting different O:2 / CRM197 w / w ratios were compared. [Figure 14-1] A) A table showing the percentage of O-acetylation of O:2[16 kDa + 100 kDa] after treatment with increasing ammonia concentrations from 5 mM to 1 M. [Figure 14-2] B) Treatment of O:2 with increasing ammonia concentration allowed for the acquisition of partially de-acetylated O:2. The graph reports a mathematical function correlating the OAc level of residual O:2 with ammonia concentration. [Figure 15] A) Effects of O:2-CRM197 conjugates with different O-acetylation levels on anti-O:2 IGG in mice. The left bar represents 27 days, and the right bar represents 42 days. Five different levels of O-acetylation were compared for O:2[16 kDa + 100 kDa], and two different levels for O:2[16 kDa]. Schematic graphs of geometric mean units (bars with 95% CI) and individual antibody levels (dots) for anti-O:2 IgG are reported; B) Linear regression analysis with 95% CI for log EU (42 days) vs. OAc%. [Modes for carrying out the invention]

[0025] General definition Unless otherwise specified, technical and scientific terms used herein have the same meanings as those ordinarily understood by those skilled in the art.

[0026] Generally, the term "comprising" means that something includes, but is not limited to. For example, the phrase "an immunogenic composition containing a conjugate" is interpreted as meaning that the immunogenic composition contains a conjugate, but may also contain further components.

[0027] In some embodiments of the present invention, the word “comprising” is replaced with the phrase “consisting of.” The term “consisting of” is intended to be restrictive. For example, the phrase “an immunogenic composition comprising a conjugate” is understood to mean that the immunogenic composition comprises a conjugate but does not contain any further components.

[0028] In some embodiments of the present invention, the word “comprising” is replaced with the phrase “consisting essentially of.” The term “consisting essentially of” means that certain additional components may be present, which, so to speak, do not substantially affect the essential properties of the subject.

[0029] When referring to a value, the terms "approximately" or "about" mean that the value is within a reasonable range of scientific error. Optionally, a value is "approximately x" or "about x" if it is within 10%, 5%, or 1% of x.

[0030] The singular forms "a," "an," and "the" refer to multiple objects unless the context clearly indicates otherwise. Therefore, a reference to, for example, "the GMMA" includes one or more instances or versions of such GMMAs.

[0031] All publications, patents, and patent applications cited herein are incorporated in their entirety, either above or below.

[0032] Conjugate In some embodiments, the present invention relates to conjugates. The term “conjugate” refers to a molecule formed by a covalent bond between an antigen and a carrier. The conjugates of the present invention include polysaccharides. The term “polysaccharide” refers to a linear or branched polymer consisting of monosaccharide residues linked by glycosidic bonds, and therefore includes oligosaccharides. The carrier is generally a carrier protein. “Protein” means, or includes, a linear or branched molecule consisting of amino acid residues.

[0033] Polysaccharide containing the KDO moiety and / or O-antigen The conjugate of the present invention comprises a polysaccharide containing a KDO moiety and / or an O-antigen. Similarly, the method of the present invention is a method for producing a conjugate comprising a polysaccharide containing a KDO moiety and / or an O-antigen.

[0034] 3. The KDO section has the following structure: [ka] Generally, polysaccharides contained within a conjugate have a KDO moiety at their reducing end. The KDO moiety can be used to directly and selectively couple the polysaccharide to a carrier protein. However, as described above, the examples herein demonstrate that when polysaccharides containing a KDO moiety are conjugated using such a selective approach, the conjugate is not sufficiently stable.

[0035] The conjugate may contain an O-antigen (also called OAg or O:2). A polysaccharide containing a KDO moiety may also be an O-antigen. The outer membrane of Gram-negative bacteria contains lipopolysaccharide. This lipopolysaccharide contains an O-antigen, which is linked to the lipid A domain via a core domain. The terms "O-antigen," "OAg," and "O:2" refer to a polysaccharide composed solely of an O-antigen, or an O-antigen linked to the core domain of a lipopolysaccharide. Purification of these O-antigen cores can be carried out using a method based on the phenol-water method first described in the 1960s by Westphal and Jann (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 polysaccharides can be carried out by acetic acid hydrolysis, as described below: Watson et al., (1992) Infect Immun. 60(11):4679-86; Konadu et al. (1996) Infect Immun. (7):2709-15; Konadu et al. (1994) Infect Immun. 62(11):5048-54; Ahmed et al. (2006) J Infect Dis. 193(4):515-21; Cox et al. (2011) Glycoconj J 28:165-182; Chu et al. (1991) Infect Immun. 59(12):4450-58; and Micoli et al., 2012 PlosOne, 7(11): e47039.

[0036] Typically, O-antigens are derived from the lipopolysaccharides of Salmonella bacteria, such as Salmonella serogroups A, B, or D, and especially Salmonella paratifida A. The O-antigens of Salmonella serogroups A, B, and D have been described and are thought to share the following common skeleton: →2-α-D-Manp-(1→4)-α-L-Rhap-(1→3)-α-D-Galp-(1→). The serogroup specificity of Salmonella paratifida A is given by α-3,6-dideoxyglucose (α-D-paratose) linked (1→3) to mannose in the skeleton. The α-L-rhamnose in the skeleton is partially O-acetylated at C-3 (Konadu et al. (1996) Infect Immun.). (7):2709-15). The previously reported structures of the O-antigen and core domain from S. parathifolia A are shown in Figure 6, which include the KDO subunit and primary amine group (within the pyrophosphoethanolamine group) within the core domain. The O-antigen may also be from S. tiphimurium. The O-antigen may also be from S. enteritidis. Naturally occurring O-antigens may contain structural variations compared to the previously reported structures for these O-antigens.

[0037] The O-antigen may be from the genus Sigella, for example, S. flexinella. Other Sigella species that provide the O-antigen used in this invention include S. sonne, S. dicentellie, and S. boyd [Knirel et al. (2011) Glycobiology. (10): 1362-72]. The O-antigen and core domain may also be from Escherichia coli, such as E. coli O157. Other lipopolysaccharide-containing Gram-negative bacteria that can provide the O-antigen used in the present invention include Klebsiella pneumoniae [Chhibber et al. (2005) Indian JExp Biol. 43(1):40-5], Vibrio cholera [Gupta et al. (1992) Infect Immun. 60(8):3201-8], Haemophilus influenzae, and Neisseria meningitidis [Cox et al. (2005) Vaccine. 23(43):5045-54].

[0038] Polysaccharides and / or O-antigens may be chemically modified compared to naturally occurring polysaccharides and / or O-antigens. For example, polysaccharides and / or O-antigens may be (partially or completely) de-acetylated, but it is preferable that the O-antigen is not de-acetylated. If de-acetylation occurs, it may occur before, during, or after other processing steps, but typically before the coupling step. The effects of de-acetylation and other modifications can be evaluated by conventional assays. For example, the relationship of O-acetylation in the O-antigen of S. paratifida A is discussed in Konadu et al. (1996) Infect Immun. (7):2709-15. The natural O-antigen of S. paratifida A is said to have approximately 80% O-acetylation in this document. Conjugated de-acetylated O-antigens did not induce anti-lipopolysaccharide antibodies with bactericidal activity. Therefore, when the O-antigen used in the present invention is the S. paratifi A O-antigen, the O-antigen may have 0-100% O-acetylation, but it is preferable that the O-antigen is O-acetylated. The level of O-acetylation may depend on the bacterial strain providing the O-antigen. For example, the degree of O-acetylation of the S. paratifi A O-antigen is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 55-95%, 55-85%, 60-80%, or 65-75%. Typically, the degree of O-acetylation of the S. paratifi A O-antigen is 55-85%, particularly 65-75%, or 45-80%, particularly 40-50% or 60-70%. However, higher degrees of O-acetylation, such as 70-100%, and especially 85-95%, are also typical in some strains. Therefore, the degree of O-acetylation in S. paratifida A O-antigen may be 60-100%, 70-100%, 80-100%, or 90-100%. Optionally, the degree of O-acetylation may also be determined based on the amount of rhamnose (Rha).

[0039] The degree of O-acetylation of polysaccharides can be determined by any method known in the art, such as proton NMR (e.g., Ravenscroft et al. Carbohydr Res. 2015;404:108-16), the hestrin method

[24] , or HPAEC-CD

[25] . The O-acetyl group can be removed by hydrolysis, for example, treatment with a base such as sodium hydroxide or anhydrous hydrazine [Konadu et al. (1996) Infect Immun. (7):2709-15]. To maintain a high level of O-acetylation on polysaccharides, treatments that result in hydrolysis of the O-acetyl group, such as treatments at extreme pH levels, should be minimized.

[0040] In some embodiments, when the O-antigen used in the present invention is S. paratiphii A O-antigen, the average molecular weight of the O-antigen is in the range of 5 kDa to 120 kDa, 10 kDa to 100 kDa, 10 kDa to 80 kDa, 10 kDa to 70 kDa, 10 kDa to 60 kDa, 10 kDa to 50 kDa, 10 kDa to 40 kDa, 10 kDa to 30 kDa, 10 kDa to 25 kDa, 10 kDa to 20 kDa, 11 kDa to 19 kDa, 12 kDa to 19 kDa, 13 kDa to 19 kDa, 14 kDa to 18 kDa, 15 kDa to 18 kDa, or 16 kDa to 18 kDa. Optionally, the O-antigen has a target molecular weight of 16 kDa–18 kDa (for example, if prepared by a typical method that produces an O-antigen with a molecular weight within this range). The molecular weight of the O-antigen may be determined by HPLC-SEC.

[0041] Conjugation of carrier proteins The conjugate of the present invention comprises a carrier protein. Similarly, the method of the present invention is a method for producing a conjugate comprising a carrier protein. Generally, conjugation of polysaccharides to carrier proteins enhances the immunogenicity of polysaccharides by converting them from T cell-independent antigens to T cell-dependent antigens, thereby enabling priming of immunological memory.

[0042] The carrier protein includes bacterial toxins such as diphtheria toxin and tetanus toxin, or their toxoids and variants. In some embodiments, the carrier protein is CRM 197 This is CRM. 197 The sequence is provided as SEQ ID NO:1.

[0043] In some embodiments, the polysaccharide of the present invention is S paratiphyte A O-antigen, and the carrier protein is CRM 197 And O:2 / CRM 197 The w / w ratio may be 0.1-3.5, 0.2-3.0, 0.25-3.0, 0.25-0.9, 0.25-0.8, 0.35-0.8, 0.4-0.8, 0.5-0.8, 0.6-0.7, 0.61-0.67, or 0.62-0.66. The ratio may also be 0.5-3.0, 0.7-3.0, 1.0-3.0, 1.5-3.0, 2.0-3.0, or 2.5-3.0.

[0044] Random conjugation and activation sites In some embodiments, the conjugate of the present invention is a conjugate comprising a polysaccharide and / or O-antigen conjugated to a carrier protein by a random conjugation method. In some embodiments, the method of the present invention is a method for generating a conjugate by a random conjugation method.

[0045] The term "random conjugation method" refers to a non-selective method of conjugating polysaccharides to carrier proteins, where either (i) the number of linkages (i.e., the number of positions on the polysaccharide to which the carrier protein links) and (ii) the linkage site (i.e., the position on the polysaccharide where conjugation occurs) or both are not predetermined by the chemistry used and may differ among conjugates produced using the same conjugation chemistry. For example, in a composition containing multiple conjugates, some conjugates may contain two linkages (i.e., two linkages between the polysaccharide and the carrier protein, or two carrier protein molecules linking to the same polysaccharide), while others may contain only one. Similarly, some conjugates may conjugate at a first reaction site on the polysaccharide, while others may conjugate at a second reaction site on the polysaccharide. Appropriate random conjugation methods include conjugation using 1-cyano-4-dimethylaminopyridine (CDAP) chemistry, and conjugation using reductive amination reactions with random oxidation steps, such as using a drug like sodium periodate.

[0046] If a polysaccharide or O-antigen contains two or more activation sites, or if the method includes a step of introducing multiple activation sites to a polysaccharide or O-antigen, the term "activation site" refers to a site or functional group on the polysaccharide that is activated by a step in conjugation chemistry and prepared for conjugation to a carrier protein. For example, if a random conjugation method uses CDAP chemistry, and CDAP addition introduces cyanoester groups, the polysaccharide is "activated" by CDAP addition. CDAP activation introduces cyanoester groups to one or more sites, and the locations of these introduced cyanoester groups are considered "activation sites." Once activated, the polysaccharide may be conjugated to a carrier protein at one or more (and possibly all) activation sites. In this invention, the term "activation site" includes both activated sites that are not conjugated to a carrier protein and activated sites that are conjugated to a carrier protein. Optionally, a random conjugation method may include the following steps: (i) To provide activated polysaccharides or activated O-antigens by activating polysaccharides or O-antigens using CDAP chemistry; or (ii) To provide an oxidized polysaccharide or oxidized O-antigen by oxidizing a polysaccharide or O-antigen, Includes.

[0047] It is possible to determine the average number of activated sites in polysaccharides. If activated sites are introduced using CDAP chemistry, the number of activated sites can be measured using the ADH inactivation / TNBS colorimetric method, as reported in Lees A, Vaccines (Basel). 2020;8(4):777. If activated sites are introduced by oxidation, the number of activated sites can be measured using a micro-BCA reagent to measure the formed aldehyde, or HPAEC-PAD to detect the oxidized sugar ring. Both options are reported in Stefanetti et al. Vaccine. 2014;32(46):6122-9.

[0048] Selectively, the polysaccharide or O-antigen contains 1.5 or more, 2.0 or more, or 2.5 or more activation sites. Assuming that the polysaccharide or O-antigen is part of a composition containing multiple polysaccharides or O-antigen sugars, if the average number of activation sites on the polysaccharide or O-antigen molecule is 1.5 or more, then the polysaccharide or O-antigen will contain 1.5 or more activation sites.

[0049] Optionally, the conjugate of the present invention is part of a composition comprising multiple conjugates, and / or a composition comprising multiple conjugates is produced by the method of the present invention, and the average number of active sites on the conjugates in the composition is greater than 1, and optionally 1.5 or more, 2 or more, or 2.5 or more active sites. In such cases, different O-antigen molecules in the composition may have different numbers of active sites.

[0050] The conjugate also includes a linker. The conjugate may further contain a linker. The method of the present invention may include conjugating an activated or oxidized polysaccharide to a linker and / or carrier protein-linker compound.

[0051] A linker is a compound used to link proteins and polysaccharides. Any suitable linker can be used in the conjugate and method of the present invention. A suitable linker is the adipic acid dihydrazide (ADH) linker, which is a compound having the following structure: [ka]

[0052] Other suitable linkers include adipic acid, glutaric acid, carbonyl, β-propionamide (WO00 / 10599), bis(N-hydroxysuccinimide) adipate, dihydrazides (analogs of ADH but with different chain lengths), hexamethylenediamine (or diamine analogs with different chain lengths), and nitrophenylethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165). Examples include 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-aminocaproic acid (US Patent No. 4,459,286), N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) (US Patent No. 5,204,098), and C4-C12 moieties (US Patent No. 4,663,160).

[0053] Activation of polysaccharides by CDAP chemistry In some embodiments, the conjugate comprises a polysaccharide containing a KDO moiety and / or an O-antigen, conjugated to a carrier protein by a conjugation method comprising the step of activating the polysaccharide and / or O-antigen by CDAP chemistry to provide an activated polysaccharide. In some embodiments, the method comprises the step of activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide.

[0054] Activation of a polysaccharide or O-antigen by CDAP chemistry involves mixing the polysaccharide and / or O-antigen with CDAP, thereby introducing cyanoester groups into the polysaccharide or O-antigen. For example, Example 7 discloses a suitable method for activating an O-antigen by CDAP chemistry. Activation of a polysaccharide and / or O-antigen by CDAP chemistry results in an activated polysaccharide or activated O-antigen. Activation of a polysaccharide and / or O-antigen by CDAP chemistry introduces cyanoester groups, thereby the activated polysaccharide and / or activated O-antigen contains cyanoester groups introduced using CDAP. Similarly, if the method involves mixing the polysaccharide and / or O-antigen with CDAP, and the number of cyanoester groups present on the polysaccharide and / or O-antigen after the mixing step with CDAP is greater than the number of cyanoester groups present on the polysaccharide and / or O-antigen before that step, the method includes the step of activating the polysaccharide and / or O-antigen by CDAP chemistry. The number of cyanoester groups present can be measured using the ADH inactivation / TNBS colorimetric method, as reported in Lees A., Vaccines (Basel), 2020;8(4):777.

[0055] Selectively, activation of O-antigens and / or polysaccharides by CDAP chemistry includes mixing the polysaccharide or O-antigen with CDAP in the following w / w ratios: 0.05:1 to 5:1, 0.1:1 to 5:1, 0.2:1 to 2:1, or approximately 0.3:1 (ratio of CDAP to polysaccharide or O-antigen).

[0056] The activation step of the O-antigen and / or polysaccharide by CDAP chemistry, which optionally involves mixing the polysaccharide or O-antigen with CDAP, is carried out in a salt solution, such as a NaCl solution or a KCl solution. Optionally, the activation step of the O-antigen and / or polysaccharide by CDAP chemistry is carried out in a NaCl solution or KCl solution of the following concentrations: 50 mM to 1 M, 100 mM to 250 mM, 125 mM to 200 mM, or approximately 150 mM.

[0057] Optionally, after mixing the polysaccharide and / or O-antigen with CDAP, the pH is optionally adjusted to pH 6-10, 7-9, or 9-10. Optionally, the pH is adjusted by adding a base, such as triethylamine, sodium hydroxide, or pyridine. Optionally, the pH is adjusted by adding 5%-15%, 8%-12%, or approximately 10% (v / v) triethylamine. Optionally, after mixing the polysaccharide and / or O-antigen with CDAP, the mixture is incubated at 18°C-30°C, 20°C-28°C, room temperature, or approximately 25°C. Optionally, the solution is incubated with agitation before conjugating the activated polysaccharide or activated O-antigen to the carrier protein.

[0058] A suitable method for activating polysaccharides by CDAP chemistry is described in Example 7.

[0059] Conjugation of activated polysaccharides to carrier proteins or linkers using CDAP As described above, polysaccharides and / or O-antigens activated using CDAP chemistry (activated polysaccharides or activated O-antigens) have cyanoester groups (at the activation site), and these cyanoester groups may be covalently linked to hydrazide or amino groups. Therefore, polysaccharides and / or O-antigens activated using CDAP chemistry can be directly linked to a carrier protein (via amino groups) or conjugated to a carrier protein via a linker containing a hydrazide or amino group. Suitable linkers include the ADH linkers described above. Therefore, this method may include a step of reacting the activated polysaccharide or O-antigen with a hydrazide / amino group on a carrier protein or carrier protein linker compound.

[0060] The method may include a step of reacting an activated polysaccharide or activated O-antigen with a hydrazide / amino group on a carrier protein linker compound. Therefore, the method may further include a step of preparing a carrier protein linker compound. For example, if the linker is an ADH linker, the method may include an ADH-carrier protein compound (e.g., ADH-CRM) as reported, for example, in Micoli et al. Vaccine 2011, 29, (4), 712-20. 197 This may include a step of preparing compounds, etc.

[0061] Reacting an activated polysaccharide or activated O-antigen with a hydrazide / amino group involves mixing the activated polysaccharide or activated O-antigen with a carrier protein or carrier protein linker compound, the mixing being carried out under conditions suitable for the formation of a covalent bond between the cyanoester group (activation site) on the activated polysaccharide or activated O-antigen and the hydrazide / amino group on the carrier protein or carrier protein linker compound. For example, the activated polysaccharide or activated O-antigen may simply be mixed with the carrier protein or carrier protein linker compound.

[0062] Reacting an activated polysaccharide or activated O-antigen with a hydrazide / amino group on a carrier protein or carrier protein-linker compound involves mixing the activated polysaccharide or O-antigen with the carrier protein or carrier protein-linker compound in a w / w ratio (ratio of polysaccharide or O-antigen to carrier protein or carrier protein-linker) of 0.1:1 to 5:1, 0.2:1 to 3:1, 0.5:1 to 2:1, or approximately 1:1. Optionally, the step of mixing the activated polysaccharide or activated O-antigen with the carrier protein or carrier protein-linker compound provides a conjugation mixture. Optionally, the mixing of the activated polysaccharide or activated O-antigen with the carrier protein or carrier protein-linker compound is carried out at a pH of 8 to 11, 9 to 10, or approximately 9.5. Optionally, the pH is maintained at 8–11, 9–10, or approximately 9.5 for at least 1 hour, at least 2 hours, 30 minutes–10 hours, 1 hour–5 hours, or 2 hours–3 hours. Optionally, the pH is maintained using a base, such as triethylamine, sodium hydroxide, or pyridine. Optionally, the pH is maintained using triethylamine.

[0063] Optionally, the method further includes the step of adding a glycine solution after mixing an activated polysaccharide or activated O-antigen, and optionally maintaining the mixture at pH 8–11 for at least 1 hour (to eliminate the cyanoester group). Optionally, the glycine solution is added at a concentration of 0.5 M–5 M, 0.5 M–2 M, or approximately 1 M. Optionally, the glycine solution is added to a conjugate mixture in a volume substantially equal to the volume of the glycine solution. If the volume is within 10%, the volume is considered substantially equal to the other volume. Optionally, there is a further step of adjusting the pH using a base, such as triethylamine, sodium hydroxide, or pyridine. Optionally, this further step is performed after the glycine solution addition step. Optionally, the pH is adjusted to 7–9, or approximately 8. Optionally, there is an incubation step after the further step of adjusting the pH using a base. Optionally, the incubation step may include incubation at a temperature below 15°C, below 12°C, below 10°C, between 0°C and 10°C, or between 2°C and 8°C. Optionally, the incubation step may be performed for 10 to 30 hours, or between 10 to 20 hours.

[0064] A conjugate is formed when an activated polysaccharide or activated O-antigen is mixed with a carrier protein or carrier protein-linker compound under conditions suitable for conjugation to occur (e.g., as disclosed in the previous paragraph). The method may further include a chromatographic step to remove unconjugated polysaccharide and / or unconjugated O-antigen. Optionally, the chromatographic step includes hydrophobic interaction chromatography or anion exchange chromatography.

[0065] A suitable method for conjugating activated polysaccharides to a carrier protein or linker using CDAP is described in Example 7.

[0066] Oxidizing polysaccharides provides oxidized polysaccharides. In some embodiments, the conjugate comprises a polysaccharide and / or O-antigen containing a KDO moiety, conjugated to a carrier protein using a conjugation method that includes the step of oxidizing the polysaccharide and / or O-antigen to provide an oxidized polysaccharide. In some embodiments, the method includes the step of oxidizing the polysaccharide to provide an oxidized polysaccharide.

[0067] The oxidation of polysaccharides and / or O-antigens may be used as part of a random conjugation method, such that the oxidation step may be used to introduce one or more activation (oxidation) sites at various positions on the polysaccharide chain. Optionally, the oxidation step of a polysaccharide or O-antigen may include mixing the polysaccharide or O-antigen with an oxidizing agent. Suitable oxidizing agents include periodates, such as sodium periodate.

[0068] Optionally, the mixing of a polysaccharide or O-antigen with an oxidizing agent includes mixing the polysaccharide or O-antigen with the oxidizing agent in a ratio of 1 mg / mL:10 mM to 100 mg / mL:10 mM, 1 mg / mL:10 mM to 50 mg / mL:10 mM, 1 mg / mL:10 mM to 25 mg / mL:10 mM, or approximately 10 mg / mL:10 mM (ratio of polysaccharide or O-antigen to oxidizing agent). Optionally, the mixing of the polysaccharide or O-antigen with the oxidizing agent is carried out at pH 3 to 7, pH 4 to 6, or approximately pH 5. Optionally, the mixing of the polysaccharide or O-antigen with the oxidizing agent is carried out in a buffer, such as acetate buffer. Preferably, the buffer is sodium acetate. Optionally, the step of oxidizing the polysaccharide or O-antigen includes incubation of the mixture of the polysaccharide or O-antigen with the oxidizing agent. Optionally, incubation of the mixture of the polysaccharide or O-antigen with the oxidizing agent is carried out in the dark. The step of incubating the polysaccharide or O-antigen mixture with the oxidizing agent may be performed for 1 to 10 hours, 1 to 5 hours, 1 to 3 hours, or approximately 2 hours. The incubation of the polysaccharide or O-antigen mixture with the oxidizing agent may be performed at 20°C to 30°C, 22°C to 28°C, approximately 25°C, or room temperature.

[0069] The step of oxidizing the polysaccharide or O-antigen may include a step of deactivating excess oxidizing agent. The step of deactivating excess oxidizing agent is optionally carried out in water after the step of mixing the polysaccharide or O-antigen with the oxidizing agent and / or incubation the mixture of the polysaccharide or O-antigen and oxidizing agent. The step of deactivating excess oxidizing agent may include adding a deactivator. If the oxidizing agent is a periodate, a suitable deactivator is sodium sulfite (Na2SO3). Optionally, deactivating excess oxidizing agent may involve adding sodium sulfite to the mixture of the polysaccharide or O-antigen and oxidizing agent and incubating the resulting mixture at a temperature of 15°C to 30°C, 20°C to 28°C, room temperature, or approximately 25°C for at least 10 minutes, 5 to 30 minutes, or approximately 15 minutes.

[0070] The step of oxidizing the polysaccharide or O-antigen may further include a step of purifying the oxidized polysaccharide or oxidized O-antigen. The step of purifying the oxidized polysaccharide or oxidized O-antigen may include desalting the mixture of the polysaccharide or O-antigen and the oxidizing agent, for example, using a PD10 column.

[0071] The method may also include the step of freeze-drying an oxidized polysaccharide or oxidized O-antigen to provide a freeze-dried oxidized polysaccharide or freeze-dried oxidized O-antigen.

[0072] A suitable method for oxidizing polysaccharides is described in Example 6.

[0073] Conjugation of oxidized polysaccharides to carrier proteins with or without a linker. The method may include a step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein or a carrier protein-linker compound containing a carrier protein.

[0074] Oxidized polysaccharides or oxidized O-antigens can be reacted with carrier proteins or carrier protein-linker compounds using reductive amination. For example, the step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein includes mixing the oxidized polysaccharide or oxidized O-antigen and the carrier protein with a reducing agent. Suitable reducing agents include cyanoborohydride salts (e.g., sodium cyanoborohydride), borohydride salts (e.g., sodium borohydride), or boranes (e.g., aminoborane, pyridineborane, dimethylborane, trimethylamineborane). In some embodiments, the reducing agent is a cyanoborohydride salt. In some embodiments, the reducing agent is sodium cyanoborohydride.

[0075] The step of optionally reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein or carrier protein-linker compound includes mixing the oxidized polysaccharide or oxidized O-antigen with the carrier protein or carrier protein-linker compound in a w / w ratio (ratio of polysaccharide or O-antigen to carrier protein) of 0.5:1 to 20:1, 1:1 to 10:1, 1.5:1 to 5:1, or approximately 2:1. The step of optionally reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein or carrier protein-linker compound includes mixing the carrier protein or carrier protein-linker compound with a reducing agent in a w / w ratio (ratio of carrier protein or carrier protein-linker compound to reducing agent) of 0.25:1 to 20:1, 0.5:1 to 10:1, 0.75:1 to 5:1, or approximately 1:1. The optional step of mixing an oxidized polysaccharide or oxidized O-antigen with a carrier protein or carrier protein-linker compound yields a reaction mixture containing the oxidized polysaccharide or oxidized O-antigen and the carrier protein or carrier protein-linker compound.

[0076] The method may include incubating the reaction mixture for 4 hours to 20 hours, 6 hours to 15 hours, 7 hours to 10 hours, or overnight. The method may include incubating the reaction mixture at 30°C to 42°C, 33°C to 40°C, 35°C to 39°C, or approximately 37°C.

[0077] The method may further include deactivating. Optionally, deactivating deactivates residual oxidation sites. Optionally, deactivating includes adding a deactivating agent. Suitable deactivating agents include sodium borohydride and lithium aluminum hydride. Optionally, the deactivating agent is sodium borohydride, and the w / w ratio of the oxidized polysaccharide or oxidized O-antigen to sodium borohydride is 10:1 to 0.1:1, 5:1 to 0.5:1, or approximately 1:1 (w / w ratio of the polysaccharide or O-antigen to sodium borohydride).

[0078] Optionally, the conjugate is purified by chromatography, such as hydrophobic interaction chromatography.

[0079] Suitable methods for conjugating the oxidized polysaccharide to a carrier protein or linker are described in Example 6.

[0080] Conjugates obtained / obtainable by the method In some embodiments of the invention, there is provided a conjugate obtained / obtainable by the method of the invention. Such a conjugate may include any of the characteristics of the conjugates described herein. For example, such a conjugate may include S. paratyphi A O-antigen and CRM as a carrier protein 197 and may be included.

[0081] immunogenic composition The conjugate of the invention may be included in an immunogenic composition. The immunogenic composition may include the conjugate of the invention and additional components, such as pharmaceutically acceptable excipients, adjuvants, and / or additional antigens.

[0082] The immunogenic composition may further contain pharmaceutically acceptable excipients. Typical “pharmaceutically acceptable excipients” include any carrier that does not itself induce the production of antibodies harmful to the organism receiving the composition. Suitable carriers typically include large, slowly metabolized polymers such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymerized amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (e.g., oil droplets or liposomes). Such carriers are well known in the art. Diluents may also include diluents such as water, saline, and glycerol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffers, and similar substances may be present. Particularly when using aluminum adjuvants, Tris-buffered saline is a suitable carrier because the phosphate in phosphate-buffered saline may interfere with the binding of outer membrane vesicles to aluminum. However, in certain embodiments, the immunogenic composition includes phosphate-buffered saline (and optionally aluminum adjuvants as described later). Optionally, the immunogenic composition may include phosphate-buffered saline with a pH of 6-7, for example, pH 6.5.

[0083] Immunogenic compositions may be prepared as liquid solutions or suspensions for injection. Solid forms suitable for solution or suspension in a liquid vehicle before injection can also be prepared (e.g., lyophilized compositions or spray-lyophilized compositions). Immunogenic compositions may be prepared for topical administration, e.g., as ointments, creams, or powders. Immunogenic compositions may be prepared for oral administration, e.g., as tablets or capsules, as sprays, or as syrups (optionally flavored). Immunogenic compositions may be prepared for pulmonary administration, e.g., as inhalants using fine powders or sprays. Compositions may be prepared as suppositories or vaginal suppositories. Immunogenic compositions may be prepared for nasal, ear, or ocular administration, e.g., as droplets. Immunogenic compositions may be provided in kit form, designed to reconstitute the mixed composition immediately before administration to a mammal. Such kits may contain one or more antigens in liquid form and one or more lyophilized antigens. Immunogenic compositions may be present in vials or in pre-filled syringes. Syringes are supplied with or without needles. Syringes contain a single dose of the composition, while vials may contain a single dose or multiple doses.

[0084] The immunogenic composition of the present invention may be packaged in single-dose or multi-dose forms. For multi-dose forms, vials are preferred over pre-filled syringes. Effective doses can be established by convention, but a typical human dose, for example, for intramuscular injection, is 0.5 ml in volume.

[0085] The composition is sterile. The immunogenic composition of the present invention may be isotonic to humans.

[0086] Therefore, the immunogenic compositions of the present invention are useful as vaccines. Vaccines according to the present invention may be either prophylactic (i.e., prevent infection) or therapeutic (i.e., treat infection), but are typically prophylactic.

[0087] An immunogenic composition used as a vaccine contains an effective amount of antigen(s) and any other components as appropriate. “Effective amount” (i.e., immunologically effective amount) means that administering that amount to an individual, either as a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending on health and physical condition, age, taxonomic group of the individual being treated (e.g., non-human primates, primates, etc.), the individual’s immune system’s ability to synthesize antibodies, the desired level of protection, vaccine formulation, assessment of the medical condition by the administering physician, and other relevant factors. The amount is expected to fall within a relatively wide range, which can be determined through conventional testing. The immunogenic compositions of the present invention may also contain antimicrobial agents, particularly when packaged in a multi-dose form.

[0088] In some embodiments, the immunogenic composition comprises the conjugate (polysaccharide or O-antigen) of the present invention in doses of 1 μg to 50 μg, 10 μg to 50 μg, 20 μg to 30 μg, or approximately 25 μg. In some embodiments, the immunogenic composition comprises the fVi conjugate in doses of 1 μg to 50 μg, 10 μg to 50 μg, 20 μg to 30 μg, or approximately 25 μg (fVi).

[0089] Adjuvant The immunogenic compositions of the present invention may contain an adjuvant. 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, or mixtures of different mineral compounds, the compounds can take any suitable form (e.g., gel-like, crystalline, amorphous, etc.), and adsorbents are preferred. The mineral-containing composition may also be formulated as particles of metal salts.

[0090] Adjuvants known as "aluminum hydroxide" are typically aluminum oxyhydroxide salts, usually at least partially crystalline. Aluminum oxyhydroxide can be represented by the formula AlO(OH) and is obscured by infrared (IR) spectroscopy, particularly at 1070 cm⁻¹. -1 Adhesion band and 3090~3100 cm -1 The presence of a strong shoulder distinguishes it from other aluminum compounds, such as Al(OH)3 [Reference Chapter 9, Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X)]. The degree of crystallinity of aluminum hydroxide adjuvants is reflected by the diffraction bandwidth (WHH) at 20 half-value, with less crystalline particles showing a broader linewidth due to smaller crystallite size. As WHH increases, so does the surface area, and adjuvants with high WHH values ​​have been shown to have high antigen adsorption capacity. Fibrous morphology (e.g., as seen in transmission electron microscopy) is typical of aluminum hydroxide adjuvants. The pl of aluminum hydroxide adjuvants is typically about 11, meaning that the adjuvant itself has a positive surface charge at physiological pH. For aluminum hydroxide adjuvants, Al at pH 7.4 +++ An adsorption capacity of 1.8–2.6 mg of protein per 1 mg has been reported.

[0091] Optionally, the aluminum salt contains or consists of aluminum phosphate and / or aluminum hydroxide. Optionally, the aluminum salt contains or consists of aluminum hydroxide.

[0092] In some embodiments, the immunogenic composition is 0.05 mg to 1.00 mg, 0.05 mg to 0.50 mg, 0.30 mg to 0.40 mg, or approximately 0.375 mg (Al 3+ Contains aluminum hydroxide in the dose of ).

[0093] Salmonella cyfii antigen The immunogenic composition may further contain antigens from Salmonella chifi (S. chifi). Optionally, the antigen from S. chifi is the Vi polysaccharide.

[0094] The terms "Vi" or "Vi polysaccharide" refer to the capsular polysaccharide of Salmonella enterica serotype cifii purified from the genus Citrobacter (Rondini et al., J. Infect. Dev. Ctries, 2012).

[0095] Selectively, Vi polysaccharides are fragmented Vi polysaccharides (fVi). The term "fragmented" in reference to Vi polysaccharides refers to Vi polysaccharides in which the number of repeating units within the polysaccharide has been reduced through size reduction. Therefore, fragmented Vi has a lower average molecular weight compared to natural Vi. For example, compared to more than 600 repeating units in natural Vi, fragmented Vi may contain 30 to 300 repeating units. The structure of the Vi monomer repeating units is shown in Figure 7. In fragmented Vi, preferably no change in the structure of the repeating units is observed compared to natural Vi. This can be confirmed by 1H NMR analysis (see WO2015 / 068129). In addition, the percentage of O-acetyl groups in fragmented Vi is preferably similar to that of natural Vi (i.e., about 95% O-acetylation), but may change and decrease to about 65% O-acetylation. O-acetylation can be determined by standard measurements, such as 1H NMR or hestrin colorimetric methods.

[0096] In its natural state, Vi polysaccharides have an average molecular weight of approximately 165 kDa, as measured by HPLC-size exclusion chromatography (HPLC-SEC). In some embodiments, fVi polysaccharides have average molecular weights of 10 kDa–90 kDa, 25 kDa–70 kDa, 40 kDa–55 kDa, 41 kDa–49 kDa, or 51 kDa–55 kDa. Optionally, fVi polysaccharides have a target molecular weight of 51 kDa–55 kDa (e.g., those produced by methods typically generating fVi with molecular weights within this range). The molecular weight of Vi polysaccharides may be determined by HPLC-SEC.

[0097] Typically, the average molecular weight is measured by running the sample on a TSK Gel 3000 PWXL column (30 cm × 7.8 mm; particle size 7 μm; code 808021) (Tosoh Bioscience) using a TSK Gel PWXL Guard column (4.0 cm × 6.0 mm; particle size 12 μm; code 808033) with dextran as a standard substance (5, 25, 50, 80, 150 kDa). The mobile phase is 0.1 M NaCl, 0.1 M NaH2PO4, 5% CH3CN, pH 7.2 at a flow rate of 0.5 mL / min (single elution method for 30 minutes). Void volume and bed volume calibration are performed using λ-DNA (λ-DNA Molecular Weight Marker III 0.12~21.2 kb; Roche) and sodium azide (NaN3; Merck), respectively.

[0098] The fragmented Vi polysaccharide 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.

[0099] The fVi polysaccharide used in this invention has a specific average molecular weight (avMW) range distribution, which is further characterized in terms of polydispersity index (PDI).

[0100] The polyvariance index is calculated as shown by the following formula: PDI = Mw / Mn In the formula, Mw is the weight-average molecular weight and Mn is the number-average molecular weight.

[0101] The narrower the molecular weight distribution, the closer the PDI value approaches 1. fVi polysaccharides may have an avMW distribution characterized by at least 80% of the pool having avMW between 25 kDa and 70 kDa. fVi polysaccharides may have an avMW distribution characterized by at least 50% of the pool having avMW between 35 kDa and 60 kDa. fVi polysaccharides may have an avMW distribution characterized by at least 30% of the pool having avMW between 41 kDa and 55 kDa.

[0102] Fragmentation of Vi polysaccharides can be carried out by several methods known in the art, such as chemical hydrolysis of natural polysaccharides, enzymatic fragmentation of natural polysaccharides, gamma irradiation of natural polysaccharides, or by mechanical methods, such as sonication of natural polysaccharides, or by high-pressure homogenizers / microfluidizers / HPCDS (high-pressure cell disruption systems). The fragmentation method used in the present invention is selected to obtain fVi polysaccharides having avMW of less than 90 kDa, less than 80 kDa, less than 60 kDa, or 40-55 kDa. The method may also be selected so as not to change the structure of the repeating units.

[0103] Preferably, fragmentation is not carried out by mechanical means. Preferably, fragmentation is not carried out by alkaline hydrolysis. fVi polysaccharides may 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 changing the repeating unit structure. Furthermore, hydrolysis with hydrogen peroxide was able to enable the formation of fragmented Vi with a lower average molecular weight than when using mechanical methods. A suitable method for fragmenting Vi polysaccharides is described in Example 2.

[0104] fVi polysaccharides may be part of an fVi conjugate containing fVi and a carrier protein. Optionally, the carrier protein in the fVi conjugate may be tetanus toxoid, CRM 197 , or diphtheria toxoid. Optionally, the carrier protein is CRM 197 That is the case.

[0105] fVi polysaccharides can be conjugated to carrier proteins through any suitable conjugation chemistry.

[0106] Conjugation of fVi polysaccharides to carrier proteins may occur via an -NH2 group, for example, through the side chains of lysine or arginine residues in the carrier polypeptide. If the fVi polysaccharide has a free aldehyde group, this group reacts with amines in the protein, forming a conjugate by reductive amination. Conjugation to the carrier may also occur via an -SH group, for example, through the side chains of cysteine ​​residues in the carrier polypeptide. Alternatively, fVi polysaccharides may be conjugated to carrier proteins by linker molecules.

[0107] fVi polysaccharides are typically activated or functionalized before conjugation. Activation requires, for example, cyanating reagents such as CDAP (1-cyano-4-dimethylaminopyridinium tetrafluoroborate). Other suitable techniques include the use of carbodiimides, hydrazides, activated esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, and TSTU (see, for example, the preface to WO 98 / 42721).

[0108] Direct conjugation to carrier proteins may involve oxidation of the fVi polysaccharide followed by reductive amination with the protein, as described, for example, in U.S. Patents 4,761,283 and 4,356,170. Conjugation via linker groups may be carried out using any known method, e.g., the methods described in U.S. Patents 4,882,317 and 4,695,624. Typically, the linker is attached via the anomeric carbon of the polysaccharide. A preferred type of linker is the adipic acid linker, which may be formed by coupling a free -NH2 group (e.g., introduced into the polysaccharide by amination) with adipic acid (e.g., using diimide activation), after which the protein is coupled to the resulting sugar-adipic acid intermediate (see, e.g., EP-B-0477508, Mol. Immunol, (1985) 22, 907-919, and EP-A-0208375). A similarly preferred type of linker is the glutaric acid linker, which can be formed by coupling a free -NH group with glutaric acid. Adipic acid linkers and glutaric acid linkers may also be formed by direct coupling to polysaccharides, i.e., without introducing a free group (e.g., a free -NH group) to the polysaccharide beforehand, and then coupling the protein to the resulting sugar-adipic acid / glutaric acid intermediate. Another preferred type of linker is the carbonyl linker, which may be formed by the reaction of a free hydroxyl group of a modified polysaccharide with CDI (Bethell GS et al. (1979) J. Biol. Chem. 254, 2572-4 and Hearn MTW (1981) J. Chromatogr. 218, 509-18), and then react with the protein to form a carbamate bond.Other linkers include β-propionylamide (WO00 / 10599), nitrophenylethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (US Patent No. 4,057,685), glycosidic bonds (US Patents No. 4,673,574; No. 4,761,283; and No. 4,808,700), 6-aminocaproic acid (US Patent No. 4,459,286), N-succinimidyl-3-(2-pyridyldithio)-propionate (SPDP) (US Patent No. 5,204,098), adipic acid dihydrazide (ADH) (US Patent No. 4,965,338), and C4-C12 moieties (US Patent No. 4,663,160). Carbodiimide condensation can also be used (WO2007 / 000343).

[0109] A bifunctional linker can be used to provide a first group for coupling with an amine group in a polysaccharide (e.g., introduced into the polysaccharide by amination) and a second group for coupling with a carrier (typically for coupling with an amine in the carrier). Alternatively, the first group can be directly coupled to the polysaccharide, i.e., without prior introduction of a group (e.g., an amine group) into the polysaccharide.

[0110] Optionally, the fVi conjugate may be obtained or obtainable by 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 having average molecular weights of 10 kDa-90 kDa, 25 kDa-70 kDa, 40 kDa-55 kDa, 41 kDa-49 kDa, or 51 kDa-55 kDa; b. The fVi polysaccharide obtained in step a is reacted with carbodiimide and N-hydroxysuccinimide at pH 5-6 to form an N-hydroxysuccinimide ester fVi derivative; c. React the N-hydroxysuccinimide ester fVi derivative obtained in step b with a carrier protein (a carrier protein that has been selectively derivatized) to produce an fVi conjugate.

[0111] This method is described in more detail in WO2015068129.

[0112] The carrier protein may be derivatized by reacting it with a carbodiimide and a linker (i.e., derivatization). Optionally, the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC). Any suitable linker (such as those discussed above) may be used. In some embodiments, the linker is an ADH linker. Optionally, the derivatization of the carrier protein produces a derivatized carrier protein. Optionally, the carrier protein is CRM 197 The derivatization of the carrier protein involves one or more of the following steps: (i) Appropriate buffer, optionally CRM in MES buffer 197 To provide; (ii) CRM 197 The ratio of EDAC to (CRM) is 1:0.05~1:0.5, 1:0.1~1:0.3, or approximately 1:0.15. 197 Mixing with EDAC at a w / w ratio; (iii) CRM 197 The ratio of ADH to 1:1-1:6, 1:2-1:4, or approximately 1:3.5 (CRM 197 Mix with ADH in a w / w ratio; (iv) CRM 197 And EDAC, and optionally ADH, are incubated with stirring for at least 30 minutes, or optionally 30 minutes to 2 hours; and (v) Derivatized CRM 197 The product is optionally purified by tangential flow filtration.

[0113] In some embodiments, the carrier protein is derivatized by a method comprising steps (i), (ii), and (iv). In some embodiments, the carrier protein is derivatized by a method comprising steps (i), (ii), (iii), and (iv). In some embodiments, the carrier protein is derivatized by a method comprising steps (i), (ii), (iv), and (v). In some embodiments, the carrier protein is derivatized by a method comprising all of the above steps (i) to (v). In some embodiments, the above steps (i) to (v) are carried out in the above order, except that steps (ii) and (iii) may be carried out simultaneously.

[0114] fVi conjugates are obtainable or have been obtained by a method comprising the step of reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide at pH 5-6 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 involves mixing fVi with carbodiimide (e.g., EDC) in the presence of N-hydroxysuccinimide (NHS). Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide involves mixing fVi polysaccharide with NHS. Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide involves mixing the fVi polysaccharide with NHS such that the NHS concentration is 0.1 M to 0.5 M, or approximately 0.33 M, and the fVi polysaccharide concentration is 1 mg / mL to 100 mg / mL, or approximately 50 mg / mL. Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide involves mixing the 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 approximately 5:1. Optionally, mixing the fVi polysaccharide with EDC is performed after mixing the fVi polysaccharide with NHS. Optionally, reacting fVi polysaccharide with carbodiimide and N-hydroxysuccinimide involves the step of incubating a mixture of fVi polysaccharide, NHS and EDC at room temperature for at least 30 minutes, or approximately 1 hour.

[0115] Selectively reacting an N-hydroxysuccinimide fVi derivative with a carrier protein (selectively derivatized carrier protein) includes mixing the N-hydroxysuccinimide fVi derivative with the carrier protein (or carrier protein derivative). Selectively reacting an N-hydroxysuccinimide fVi derivative with a carrier protein (selectively derivatized carrier protein) includes mixing the N-hydroxysuccinimide fVi derivative with the carrier protein (or carrier protein derivative) in a ratio (w / w) of 1:0.1 to 1:10, 1:0.5 to 1:5, 1:0.75 to 1:2, or approximately 1:1. Selectively mixing the N-hydroxysuccinimide fVi derivative with a carrier protein (or carrier protein derivative) is carried out in a buffer at pH 5 to 7, or approximately 6. Selectively mixing the N-hydroxysuccinimide fVi derivative with a carrier protein (or carrier protein derivative) is carried out in MES buffer. Selectively mixing an N-hydroxysuccinimide ester fVi derivative with a carrier protein (or carrier protein derivative) is performed at a temperature of 20°C to 30°C, or approximately room temperature, with selective mixing.

[0116] A method for preparing an fVi conjugate may include, after the step of reacting an N-hydroxysuccinimide fVi derivative with a carrier protein (or carrier protein derivative), one or more of the following additional steps: (i) Inactivating by adding an inactivating agent (e.g., phenyl HP buffer); (ii) Filtering the fVi conjugate; (iii) Optionally purify the fVi conjugate using hydrophobic interaction chromatography; (iv) Selectively concentrate the fVi conjugate by tangential flow filtration; (v) Filter the conjugate using one or more 0.2 μm filters as optional.

[0117] Optionally, a method for preparing an fVi conjugate includes two or more, three or more, four or more, or all five of the above steps (i) to (v). Optionally, a method includes step (i). Optionally, a method includes steps (i) to (iii). Optionally, a method includes steps (i) to (v). Optionally, a method includes steps (i) to (iii) in the above order. Optionally, a method includes steps (i) to (v) in the above order.

[0118] ADH linker is used to convert fVi polysaccharides to CRM using EDAC chemistry. 197 A suitable method for conjugating is described in Example 2.

[0119] Selectively, the immunogenic composition may include (i) O-antigen and CRM from S. paratifida A. 197 Conjugates including (ii) fVi polysaccharide and CRM 197 It contains conjugates with a (w / w) ratio (ratio of O-antigen to fVi) of 1:0.1-1:10, 1:0.5-1:5, 1:0.75-1:3, or approximately 1:1.

[0120] Stability assay The stability of the conjugate of the present invention (optionally selected within the immunogenic composition of the present invention) (e.g., the amount of polysaccharide or O-antigen released from the conjugate after 4 weeks) may be determined using the following stability assay: (i) Incubate the immunogenic composition at 37°C for 4 weeks; (ii) Prepare a post-incubation sample of the incubated immunogenic composition and remove the conjugated polysaccharide or conjugated O-antigen by deoxycholic acid precipitation; (iii) The amount of free polysaccharides or free O-antigen in the incubated sample is determined as a percentage of the total polysaccharide amount in the incubated sample.

[0121] Incubating an immunogenic composition at 37°C for 4 weeks is simply a matter of maintaining the immunogenic composition at 37°C for 4 weeks. For example, if the immunogenic composition is CRM 197 If the composition contains a conjugate containing the O-antigen and an fVi conjugate, the user should transfer a sample of the immunogenic composition to an appropriate container (e.g., a single-dose syringe) and store it at 37°C.

[0122] The incubated sample may be of any appropriate size (i.e., any size that allows for the accurate detection of the amount of free polysaccharides or free O-antigen in the immunogenic composition).

[0123] The amount of free polysaccharide or O-antigen in the incubation sample is determined by removing the intact conjugate and measuring the amount of remaining polysaccharide or O-antigen. This requires deoxycholic acid precipitation. Deoxycholic acid precipitates the carrier protein, and any remaining polysaccharide or O-antigen as part of the conjugate precipitates together with the carrier protein. The precipitated protein can be removed by centrifugation.

[0124] As an alternative to deoxycholic acid precipitation, the conjugate (carrier protein and any polysaccharide or O-antigen that is part of the conjugate) can be captured in a C4 solid-phase extraction disposable column, and the free polysaccharide can be eluted with 20% acetonitrile containing a 0.05% TFA solution.

[0125] Subsequently, it is desirable to remove the polysaccharide or O-antigen that is part of the conjugate and for the user to confirm the amount of polysaccharide or O-antigen remaining in the sample. This can be achieved using a phenol-sulfuric acid assay, or preferably high-performance anion exchange chromatography (HPAED-PAD) with pulsed amperometric detection.

[0126] The phenol-sulfuric acid assay quantifies the total amount of detectable sugars in a sample as a hexose weight equivalent (glucose calibration curve). The assay uses concentrated sulfuric acid and phenol. With the addition of sulfuric acid, the temperature rises due to acid hydration, and polysaccharides are hydrolyzed to their corresponding monosaccharides. Under these conditions, hexose monosaccharides (derived from glucose standards or O:2 sample hydrolysis) react with phenol to form hydroxymethylfurfural, resulting in the formation of a chromophore (Mollisch reaction). The absorbance of the sample and glucose standard at 490 nm is read using a spectrophotometer. Multiple dilutions (0, 25, 50, 75, 100 μg / mL) of glucose standard solutions are treated as samples in the assay and used as calibration curves.

[0127] The HPAED-PAD method can be used to detect the amount of detectable sugars in the S. paratifida O-antigen repeat unit, such as rhamnose (rha), galactose (gal), glucose (glc), and mannose (man). Calibration curves may be created using commercially available monomeric sugars.

[0128] A suitable HPAED-PAD method is described in detail in PLoS One (2012): 7(11):e47039. For example, the amount of S. paratifida O-antigen may be detected using the following method.

[0129] To detect the amounts of Rha, Gal, and Glc in S. paratifida OAg samples diluted to contain sugar monomers in the range of 0.5 to 10 μg / mL, hydrolysis is performed in 2 M TFA at 100°C for 4 hours.

[0130] After hydrolysis, the sample is cooled at 2–8°C for approximately 30 minutes, dried overnight using SpeedVac, and reconstituted with water and filtered using a 0.45 μm Acrodisc (PALL) filter before chromatographic analysis.

[0131] HPAEC-PAD is performed using a Dionex ICS3000 equipped with a CarboPac PA10 column (4 × 250 mm) and a PA10 guard column (4 × 50 mm) linked together. Sugar separation is performed by eluting with 18 mM NaOH at a flow rate of 1 mL / min for 20 minutes. Alternatively, the solution may be washed with 100 mM AcONa in 28 mM NaOH for 20 minutes, followed by re-equilibrium with 18 mM NaOH for 20 minutes.

[0132] The eluate may be monitored using a pulsed amperometric electrochemical detector with a gold working electrode and an Ag / AgCl reference electrode. A Dionex quadruple potential waveform for standard carbohydrates can be used. The obtained chromatographic data can be processed using Chromeleon software 6.8. Calibration curves may be prepared for each sugar monomer (0.5–10 μg / mL). Standards can be hydrolyzed and analyzed in the same manner as the samples.

[0133] Once the amount of free polysaccharide or O-antigen is determined, the user can calculate the amount of free polysaccharide or free O-antigen in the sample as a percentage of the total amount of polysaccharide in the sample. An experienced user can easily calculate the amount of conjugate in the sample by knowing the conjugate concentration in the immunogenic composition, determining the volume of conjugate in the sample, and multiplying that volume by the concentration.

[0134] stability The conjugates of the present invention exhibit improved stability compared to conjugates prepared using a non-random conjugation method.

[0135] The conjugate is stable in the immunogenic composition for at least 4 weeks (37°C). For example, the immunogenic composition of the present invention may include the conjugate of the present invention (containing a polysaccharide or O-antigen with a KDO moiety) and the fVi conjugate, in which case the conjugate of the present invention is stable in the immunogenic composition in the presence of the fVi conjugate. For example, the immunogenic composition may include S. paratifida O-antigen and CRM197 Conjugates including and fVi conjugates containing S. paratifi O-antigen and CRM 197 The conjugate containing is stable in the composition for at least 4 weeks.

[0136] If, optionally, less than 20%, less than 18%, less than 17%, less than 15%, 0% to 20%, or 1% to 15% of the polysaccharide or O-antigen is released from the conjugate within 4 weeks, the conjugate (containing the polysaccharide or O-antigen with the KDO moiety) is stable in the immunogenic composition for at least 4 weeks. The amount of polysaccharide or O-antigen released from the conjugate within 4 weeks may be determined using a stability assay as described above.

[0137] Selectively, the amount of polysaccharide or O-antigen released from the conjugate over 4 weeks is determined by whether the conjugate uses direct reductive amination via the ADH-SIDEA linker. 197 When substantially less than that of an equivalent immunogenic composition containing the conjugated S. paratifida O-antigen, the conjugate (containing a polysaccharide with a KDO moiety or the O-antigen) is stable in the immunogenic composition for at least 4 weeks. "Equivalent" means otherwise identical. For example, an equivalent immunogenic composition should preferably contain the same excipients as the immunogenic composition, with the only difference being the properties of the conjugate in the immunogenic composition.

[0138] Selectively, the amount of polysaccharide or O-antigen released from the conjugate over 4 weeks is determined by the amount of the conjugate used in CRM using CDAP chemistry. 197 The conjugate is stable in the immunogenic composition for at least 4 weeks if it is similar in amount to that released in an equivalent reference immunogenic composition containing the conjugated S. paratifi O-antigen. Optionally, "similar" means within 25%, 15%, or 10%.

[0139] immunogenicity The examples demonstrate that the conjugate of the present invention is not only stable but also possesses good immunogenicity. Therefore, in some embodiments, the immunogenic composition induces similar levels of anti-polysaccharide or anti-O-antigen antibodies compared to equivalent immunogenic compositions in which the conjugate comprises a polysaccharide or O-antigen conjugated to a carrier protein using a selective (non-random) conjugation method. In some embodiments, the immunogenic composition induces similar levels of anti-polysaccharide or anti-O-antigen antibodies compared to equivalent immunogenic compositions in which the conjugate comprises a polysaccharide or O-antigen conjugated to a carrier protein using selective reductive amination, such as the reductive amination method described in Example 1.

[0140] In some embodiments, the immunogenic composition is a conjugate that uses direct reductive amination via an ADH-SIDEA linker to CRM 197 It induces similar levels of anti-S. paratifida O-antigen antibodies compared to equivalent immunogenic compositions containing S. paratifida O-antigen conjugated to it.

[0141] An immunogenic composition should be considered to "induce" antibodies if it has the ability to induce antibodies, that is, if it possesses structural characteristics that enable it to induce antibodies when administered to mammals such as mice. Whether an immunogenic composition "induces" antibodies can be determined by administering a sample of the immunogenic composition to mice and determining whether the relevant antibodies are produced.

[0142] Selectively, similar levels are defined as levels within 25%, 15%, or 10%.

[0143] Optionally, the user may determine whether an immunogenic composition induces anti-polysaccharide antibodies or anti-O-antigen antibodies by measuring the level of anti-polysaccharide antibodies or anti-O-antigen antibodies induced using the following immunogenicity assay: (i) Immunize mice on day 0 and day 28 with a subcutaneous conjugate of 25 μg (polysaccharide); (ii) On day 42, measure the anti-polysaccharide antibody level by ELISA.

[0144] If the immunogenic composition contains S. paratifida A O-antigen, the level of induced anti-O-antigen antibodies may then be measured using the following immunogenicity assay: (i) Immunize mice on day 0 and day 28 with a subcutaneous conjugate of 25 μg (O-antigen); (ii) On day 42, measure the anti-O antigen antibody level by ELISA.

[0145] Appropriate ELISA assays include: - Coat the ELISA plate with a polysaccharide or O-antigen; - On day 42 (42 days after initial immunization), obtain a serum sample from the immunized mouse and spread it on a plate; and - Measure the antibody titer by adding an anti-mouse Fc antibody conjugated with an enzyme and a chromogenic substrate.

[0146] The appropriate ELISA assay is described in WO213 / 038375.

[0147] Further antigens The immunogenic composition may contain additional antigens. Optionally, the additional antigens may include Salmonella antigens. Optionally, the additional antigens may include Salmonella tiphimurium antigen and / or Salmonella enteritidis antigen. Optionally, the Salmonella tiphimurium antigen and / or Salmonella enteritidis antigen are outer membrane vesicles.

[0148] Optionally, the immunogenic composition contains S. tiphimurium GMMA and / or S. enteritidis GMMA. Optionally, S. tiphimurium GMMA and / or S. enteritidis GMMA contain detoxification lipid A. Optionally, S. tiphimurium GMMA and / or S. enteritidis GMMA are derived from bacteria that do not contain genes encoding functional MsbB and / or PagP proteins. Optionally, S. tiphimurium GMMA and / or S. enteritidis GMMA are derived from bacteria modified to at least partially delete the msbB and / or pagP genes. Optionally, S. tiphimurium GMMA and / or S. enteritidis GMMA are ΔmsbB and / or ΔpagP. Optionally, S. tiphimurium GMMA and / or S. enteritidis GMMA are derived from hyperbraving bacteria. Selectively, S. tiphimurium GMMA and / or S. enteritidis GMMA are derived from bacteria that do not contain the gene encoding the functional TolR protein. Selectively, S. tiphimurium GMMA and / or S. enteritidis GMMA are derived from bacteria that have been modified to at least partially delete the tolR gene. Selectively, S. tiphimurium GMMA and / or S. enteritidis GMMA are ΔtolR.

[0149] Medical uses and treatment methods Further embodiments of the present invention provide an immunogenic composition for use in a method for preventing infection. Further embodiments of the present invention provide a method for preventing infection, comprising administering an effective amount of the immunogenic composition or vaccine of the present invention to a subject. Further embodiments of the present invention provide the use of the immunogenic composition or vaccine of the present invention for the manufacture of a pharmaceutical for use in a method for preventing infection. The method for preventing infection may include administering an effective amount of the immunogenic composition or vaccine of the present invention to a subject.

[0150] Methods of preventing infection may include preventing Salmonella infection, or, optionally, preventing invasive atypical Salmonella infection. Optional methods of preventing infection include preventing infection by S. tiphimulium, S. enteritidis, S. tiphi and / or S. paratiphi A.

[0151] In the methods / uses of immunogenic compositions / pharmaceuticals of the present invention, the term "prevents Salmonella infection" includes inducing an immune response in the subject. The immune response is protective and may produce antibodies, such as IgG antibodies.

[0152] The subject of this invention is mammals, optionally humans. When the vaccine is for prophylactic use, humans are adults, i.e., the subject is 18 years of age or older. When the vaccine is for prophylactic use, humans may be children, i.e., under 18 years of age. When the vaccine is for prophylactic use, children may be 12 to 72 months of age, preferably 24 to 59 months of age, more preferably 6 to 12 months of age.

[0153] If the vaccine is for preventative use, the child may be approximately 9 months old.

[0154] If the vaccine is for therapeutic use, the human subject is preferably a child.

[0155] Children's vaccines may also be administered to adults, for example, to evaluate their safety, dosage, and immunogenicity. [Examples]

[0156] Example 1 - S. paratifida OAg-CRM via reductive amination using an ADH-SIDEA linker 197 Conjugate generation S. paratifida A O-antigen (also called O:2) and CRM 197 The conjugate (via the ADH-SIDEA linker) was generated using the following protocol.

[0157] S. paratifida O-antigen-CRM via reductive amination using the ADH-SIDEA linker. 197 The method used for generating the conjugate is described in detail in WO2013 / 038375.

[0158] In short, S. paratifida O-antigen (OAg) is dissolved in 100 mM AcONa pH 4.5 at a concentration of 20-40 mg / mL, and ADH and then NaBH3CN are sequentially added as solids to achieve an OAg / ADH / NaBH3CN ratio of 1:2:2 w / w / w. After mixing at 30°C for 1 hour (h), the reaction mixture is desalted with water using a G-25 column. The resulting OAg-ADH is dried and dissolved in 1:9 (v / v) water / DMSO at a concentration of approximately 50 mg / mL (O-Ag). Triethylamine (TEA) and SIDEA (see Figure 1 for SIDEA structure) are added to achieve a molar ratio of NH2(OAg):TEA:SIDEA of 1:5:12, and the reaction is maintained at room temperature for 3 hours. By adding twice the volume of 100 mM citrate buffer pH 3, unreacted SIDEA precipitates and is discarded after centrifugation. OAg-ADH-SIDEA is separated from the reaction mixture by precipitation with the addition of ethanol up to 80% (v / v), and recovered after centrifugation. The pellet is washed twice with 100% ethanol and dried under vacuum. OAg-ADH-SIDEA is then processed using CRM. 197 Dissolve in phosphate buffer pH 7.2 containing [the substance], and set the protein concentration to 20 mg / mL and the active ester group on OAg-ADH-SIDEA and CRM 197 This yields a molar ratio of 30:1. The reaction mixture is mixed at room temperature for 2 hours, and the conjugate is purified by size exclusion chromatography (SEC) or hydrophobic interaction chromatography (HIC).

[0159] Example 2 - Salmonella citrate Vi-CRM 197 Conjugate formation and formulation of divalent compositions (S. thifi and S. parathifi A) The following protocol was used to generate S. paratifida A OAg-CRM as described above. 197Fragmented Vi polysaccharide (fVi) from conjugates and S. thifi is processed by CRM. 197 A divalent composition containing a conjugate was prepared by conjugating it.

[0160] 1) Fragmentation of Vi polysaccharide: Step 1: Fragmentation and Inactivation: Vi polysaccharide fragmentation is achieved by oxidation using hydrogen peroxide in the presence of iron sulfate. The reaction is inactivated with EDTA (ethylenediaminetetraacetic acid). The natural Vi polysaccharide is diluted by WFI. Calculated volumes of 10 mM FeSO4 and H2O2 are added to give final concentrations of 0.5 mM FeSO4 and 0.5% v / v H2O2, respectively, in the reaction mixture. The reaction mixture is incubated at 15±5°C for 120±10 minutes. The reaction is stopped by adding an equivalent volume of 250 mM EDTA to give a final EDTA concentration of 10 mM, and then stirred.

[0161] Step 2: Buffer exchange: To remove residual H2O2, perform buffer exchange using tangential flow filtration (TFF) with 100 mM sodium phosphate buffer (pH: 7.2 ± 0.2) using a 30 kDa cassette. Concentrate the fragmented Vi (fVi) polysaccharide.

[0162] Step 3: Stabilization of fVi polysaccharide: After fragmentation, stabilize by incubating the 30 kDa retention solution at 80±5°C for 120±15 minutes.

[0163] Step 4: Purification of fVi by anion exchange (resin: Capto-Q): Separation of fVi polysaccharides (25-70 kDa) of the desired molecular weight is performed using the AA chromatography step. This is carried out using linear gradient elution with Capto-Q Buffer A and Capto-Q Buffer B, using Capto-Q resin with a binding capacity of 13 mg fVi / mL. Elution fractions are collected based on conductivity at 1 mS / cm intervals, i.e., 35-50 mS / cm, and the molecular weight distribution is estimated by SEC / HPLC. The Capto-Q fractions are pooled based on molecular weight (kDa) distribution.

[0164] Step 5: Desalting: The pooled Capto-Q fraction is concentrated by tangential flow filtration (TFF) using a 10 kDa cutoff cassette, and then diafiltration is performed using WFI until the conductivity of the permeate reaches ≤30 μS / cm.

[0165] Step 6: 0.2 μm filtration of fVi polysaccharide: Filter the fVi polysaccharide through a 0.22 μm filter. Store the purified fVi polysaccharide in a PETG bottle.

[0166] 2) CRM 197 Derivatization: Step 1: CRM 197 Defrosting: Purified CRM 197 Thaw at 2-8°C before buffer exchange with 100 mM MES (morpholinoethanesulfonic acid) buffer. After thawing, CRM 197 The mixture is filtered using a 0.5 μm filter.

[0167] Step 2: Buffer exchange with 100 mM MES buffer: CRM 197 After thawing, buffer exchange is performed using TFF with 100 mM MES buffer (pH 6.0±0.2) using a 10 kDa cassette.

[0168] Step 3: CRM 197 Derivatization: Required concentration of CRM197 Dilute with 100 mM MES buffer, then add the calculated amounts of ADH (adipate dihydrazide) and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) to achieve a CRM:ADH:EDAC ratio of 1:3.5:0.15 w / w / w. After adding ADH and EDAC, incubate the reaction mixture at room temperature for 60 ± 15 minutes under mixed conditions. At the end of the reaction, add an equivalent volume of 5 mM MES buffer (pH 7.0 ± 0.2).

[0169] Step 4: CRM 197 Purification: After the reaction, CRM 197 It is purified by TFF using a 10 kDa cassette with 5 mM MES buffer.

[0170] Step 5: Diafiltration-processed CRM 197 Filtration: Diafiltration-prepared CRM 197 The solution is filtered through a 0.2 micron filter, and then stored in a glass bottle at 2-8°C.

[0171] 3) Fragmented Vi polysaccharides and derivatized CRM 197 Conjugation with: Activation of fVi polysaccharide: Step 1: Drying of fVi polysaccharide by Rota Vapor: Further concentrate fVi by drying it at 30°C using a Rota Vapor. The concentrated fVi polysaccharide is reconstituted using 100 mM MES buffer (pH: 6.0) to obtain a concentration of 50 mg / mL.

[0172] Step 2: Activation of fVi polysaccharide by NHS: The carboxyl group (-COOH) of fVi is activated by EDC (N-3-dimethylaminopropyl-N-ethylcarbodiimide) in the presence of N-hydroxysuccinimide (NHS) to form an active ester intermediate, and the CRM is pre-activated by ADH. 197The conjugation efficiency with is improved. The dried fVi polysaccharide is reconstituted to the desired concentration (50 mg / mL) with 100 mM MES buffer (pH: 6.2 ± 0.2); activated in the presence of NHS (concentration 0.33 M), and then EDAC is added to achieve a molar ratio of EDAC / fVi RU of 5:1. The EDAC solution is added after the addition of NHS to ensure complete dissolution. The reaction is incubated at room temperature for 1 hour with slow mixing.

[0173] Conjugation: Step 1: fVi and CRM 197 -Conjugation with ADH: The conjugation reaction involves activated fVi and CRM 197 -Forms a covalent bond with ADH. The activated and derivatized reaction mixture is diluted with 100 mM MES pH:6.0 and CRM 197 -ADH in a 1:1 w / w ratio (fVi:CRM 197 ) is added to activate fVi and CRM 197 - The final concentration of ADH is brought to 5 mg / mL. The conjugation reaction is carried out slowly at room temperature with mixing until protein consumption by HPLC-SEC at 280 nm absorbance is ≥70%.

[0174] Step 2: Inactivation and preparation of the conjugation reaction: Inactivate the conjugation reaction by adding an equivalent volume of Phenyl HP Buffer-B Tris 50 mM pH:8.0. Add NaCl in powder form to reach a final salt concentration of 3 M.

[0175] Step 3: Filtration of the conjugation mixture: fVi-CRM 197 The coarse conjugate is filtered through a 0.65 filter.

[0176] Step 4: fVi -CRM 197Purification of Crude Conjugate: Purification of the conjugate from the prepared reaction mixture is performed through a HIC phenyl Sepharose high-performance (HP) column. Column integrity is checked every 5–10 cycles according to standard procedure. The column is equilibrated with phenyl Sepharose HP buffer A, Tris 50 mM, NaCl 3M, pH 8.0. After adding the preparation buffer and NaCl, the crude conjugate is packed into the column. Column washing is performed with phenyl Sepharose HP buffer A, followed by elution of the product with phenyl Sepharose HP buffer B, Tris 50 mM, pH 8. The fractions are collected and stored at 2–8°C until further use. All fractions from multiple runs are pooled.

[0177] Step 5: Concentration and Buffer Exchange with PBS: The purified conjugate is concentrated by TFF using a 50 kDa cutoff cassette, and then the buffer is exchanged with PBS buffer until the permeate conductivity matches the conductivity of the PBS buffer.

[0178] Step 6: fVi-CRM using a 0.2 μm filter 197 Conjugate pre-filtration: fVi-CRM for bioburden reduction 197 Filter the conjugate using a 0.2 μm filter.

[0179] Step 7: fVi-CRM using a 0.2 μm cellulose acetate filter 197 Aseptic filtration of conjugates: fVi-CRM 197 The conjugate is filtered through a 0.2 μm cellulose acetate filter. Purified fVi-CRM 197 Collect the conjugate and store it at 2-8°C.

[0180] Preparation of divalent compositions Divalent formulations with and without aluminum adjuvant were prepared according to the following protocol.

[0181] Non-adsorbent formulation: (25 μg fVi, 25 μg O:2 (each dose contains O-antigen from S. paratifida A and 4 mg of 2-PE)) Aseptically inject 2-PE-containing physiological saline into the preparation container, add an appropriate volume of fVi conjugate antigen (prepared in Example 2) (refer to the header for dosage, depending on the required number of doses), and stir the solution for 15-20 minutes. Add the calculated volume of S. paratifi O-antigen (prepared in Example 1) (refer to the header for dosage, depending on the required number of doses), and stir the solution for 15-20 minutes. Check the pH and adjust it to 6.5 ± 0.2. Continue stirring for 15-20 minutes.

[0182] The formulation solution is aseptically filtered into another sterile glass vial using a sterile 0.22 μm filter.

[0183] Adsorbent preparation: (per dose: fVi 25 μg, O:2 25 μg, Al 3+ (Contains 0.375 mg and 4 mg of 2-PE) Aseptically inject physiological saline containing 2-PE into the preparation container. Add the specified volume of Vi antigen and stir the solution for 15-20 minutes. Add the calculated volume of O:2 antigen and stir the solution for 15-20 minutes. Check the pH and adjust it to 6.5 ± 0.2. Continue stirring for 15-20 minutes.

[0184] The preliminary mixture is aseptically filtered into a separate sterile glass vial using a sterile 0.22 μm filter. Aluminum hydroxide gel is added to the filtered intermediate bulk. The container is stirred for 60–90 minutes.

[0185] Example 3 - Stability study of the divalent composition produced and prepared in Example 2 A divalent non-adsorptive preparation, a sample composed of fVi-CRM and O:2-CRM (O:2 ADH-SIDEA-CRM according to Example 1) was stored in a refrigerator at 2-8°C for 12 months, and the presence of free parathyroid OAg was tested at 0 months, 3 months, 6 months, 9 months and 12 months using the following assays. To determine the percentage of free OAg, a phenol-sulfuric acid colorimetric assay was used and performed after precipitation of proteins (conjugates) with deoxycholic acid under acidic conditions.

[0186] The phenol-sulfuric acid assay quantifies the total detectable sugars in a sample as a hexose weight equivalent (glucose calibration curve). The assay uses concentrated sulfuric acid and phenol. When sulfuric acid is added, the temperature rises due to the acid hydration reaction and the polysaccharide is hydrolyzed to the corresponding sugar monomers. Under these conditions, hexose monosaccharides (from glucose standard solution or from hydrolysis of O:2 sample) react with phenol to form hydroxymethylfurfural which results in a chromophore (Molisch reaction). The absorbance at 490 nm of the sample and glucose standard solution is read using a spectrophotometer. Glucose standard solutions at multiple dilution rates (0, 25, 50, 75, 100 μg / mL) are treated as samples in the assay and used as the calibration curve.

[0187] Table 1 shows the change in free OAg (percentage relative to total OAg) over 12 months.

[0188]

Table 1

[0189] Example 4 - Investigation of the origin of instability The various steps of the reductive amination conjugation chemistry (see Figure 1) were investigated to confirm whether they were the origin of conjugate instability, as summarized below.

[0190] 1. The stability of paraffin A OAg was evaluated using an accelerated stability study conducted over a period of 4 weeks at 37°C. 1 1H NMR and HPLC-SEC data indicated that the sugar composition and chain length of paraffin A OAg did not change over time.

[0191] 2. The purity of sodium cyanoborohydride reagent and its degradation kinetics under reaction conditions were evaluated using NMR. The NMR spectrum indicated that the sodium cyanoborohydride reagent did not contain significant impurities and an appropriate amount of residue still remained after 5 hours under reaction conditions. Therefore, it is considered that the impurities of sodium cyanoborohydride reagent and its degradation were not the origin of instability (due to incomplete reduction of the imine bond in the reaction intermediate).

[0192] 3. The efficiency of the step of linking ADH to paraffin OAg was investigated by the TNBS colorimetric method, and it was shown that OAg-ADH linkage existed after this step (92% of the composition was OAg-ADH).

[0193] 4. In the sodium cyanoborohydride reduction step, whether the C=N bond (see Figure 1) was completely reduced was evaluated using the HPLC-SEC method that measures absorbance at 252 nm. More specifically, incomplete reduction of the C=N bond leaves a chromophore on OAg that has a characteristic absorbance at 252 nm formed by the α-keto acid (KDO) group and the hydrazide (ADH) group, and can be easily confirmed by HPLC-SEC. Incomplete reduction of the C=N bond is not considered to be the origin of instability.

[0194] 5. The stability of paraffin OAg-ADH conjugate was evaluated. Specifically, OAg-ADH samples in Falcon tubes at different pH and different buffers (see Table 2) were incubated at 37°C for 4 weeks, and samples were taken at 0 week, 2 weeks, and 4 weeks. The level of free ADH was evaluated using the following assay.

[0195] Unbound (free) ADH was quantified by HPLC-SEC using a TOSOH TSKGEL-3000PW-XL column (eluate: 100 mM sodium phosphate, 100 mM sodium chloride, pH 7.2, containing 5% acetonitrile). The ADH peak in the sample was quantified by comparison with the ABS 214 nm and the ADH standard calibration curve.

[0196] [Table 2]

[0197] The data in Table 2 shows that paratifide OAg-ADH binding decomposes over time, and OAg-CRM 197 This suggests that it is likely the origin of the conjugate instability.

[0198] Example 5 - An attempt to improve stability by changing the chemistry used in conjugation. More stable OAg-CRM 197 To determine if they could be used to generate conjugates, we investigated various alternative conjugation chemistry, as briefly summarized below.

[0199] 1. The ADH linker was replaced with a DAH (1,6-diaminehexane) linker. This modification to the original chemistry allows for the determination of whether the instability is due to the hydrazide bond or also present in the aliphatic amine bond. A stability study based on the research method described in Example 3 (where free paratifida OAg is measured from samples obtained at 37°C on days 0, 8, 14, and 28) was performed on the OAg-DAH-SIDEA-CRM 197 The test was conducted using a conjugate. The results (percentage of free OAg) are shown in Table 6 below.

[0200] [Table 3]

[0201] 2. Remove the ADH linker and convert paratifide OAg to SIDEA-CRM via the PPetN portion in the core region of OAg. 197 The stability of the linkage via the pyrophosphate group was determined by direct conjugation. A stability study based on the research method described in Example 3 (however, free OAg was measured from samples obtained at day 0, week 2, and week 4, and free OAg was separated from the conjugate by solid-phase extraction using a C4 disposable column) was performed on OAg-SIDEA-CRM. 197 The test was conducted using a conjugate. The results (percentage of free OAg) are shown in Table 7 below.

[0202] [Table 4]

[0203] 3. To determine whether the linkage to ADH via the KDO carboxyl moiety is more stable, OAg was converted to CRM via NHS-EDC chemistry (see Figure 2 for a description of the chemistry used). 197 - Conjugated with ADH (CRM protein was previously derivatized with ADH by EDAC chemistry). Stability studies based on the research method described in Example 3 (however, free OAg was measured from samples obtained on days 0, 8, 14, and 28) were performed on OAg-EDC-NHS-ADH-CRM. 197 The test was conducted using a conjugate. The results (percentage of free OAg) are shown in Table 8 below.

[0204] [Table 5]

[0205] However, in all the cases described above, a significant amount of free OAg was released during the stability assay, which means that none of these chemistrys have adequate stability for OAg-CRM. 197 It was shown that it cannot be used to prepare conjugates.

[0206] Example 6 - CRM conjugation of OAg using a random oxidation followed by reductive amination approach 197 Conjugation to As described in the following paragraphs, paratif OAg (O:2) was conjugated to CRM using a random oxidation with periodic acid followed by a reductive amination approach to introduce multiple bonds between the OAg chain and the CRM 197 protein molecule. The chemistry is described in Figure 3 197 The chemistry is described in Figure 3

[0207] Oxidation O:2 at 10 mg / mL was oxidized (to O:2ox) with 10 mM NaIO4 in buffer AcONa 10 mM pH 5. The solution was maintained at 25 °C in the dark for 2 hours. Subsequently, the excess NaIO4 was deactivated with 20 mM Na2SO3 in H2O. The mixture was gently stirred at room temperature for 15 minutes. The oxidation mixture was purified by desalting on a PD10 column against water and lyophilized

[0208] Conjugation The final O:2ox concentration was 10 mg / mL, and the CRM 197 / NaCNBH3 concentration was adjusted to 5 mg / mL (O:2ox:CRM 197 :NaBH3CN = 2:1:1 w / w) by resuspending O:2ox in NaPi 100 pH 7.2 and adding CRM 197 the carrier protein and NaCNBH3. The reaction mixture was incubated overnight at 37 °C. To deactivate the remaining oxidized free radicals, NaBH4 was added to the conjugation mixture to reach an O:2ox:NaBH4 of 1:1 w / w ratio. The mixture was maintained at 37 °C for 2 hours

[0209] The conjugate was purified through HIC chromatography

[0210] Stability studies based on the research method described in Example 3 (however, free OAg was measured from samples obtained on days 0, 14, and 28; free OAg was separated from the conjugate by solid-phase extraction using a C4 disposable column and quantified using HPAEC-PAD analysis of sugars) were performed on OAg-CRM produced using random oxidation followed by reductive amination using the method described in Micoli F et al. PLoS One. 2012;7(11):e47039. 197 The test was performed on a conjugate. The results (percentage of free OAg) are shown in Table 9 below.

[0211] [Table 6]

[0212] In stability studies, OAg-CRM was prepared using different conjugate chemistry. 197 Significantly lower levels of free OAg were observed compared to the conjugate.

[0213] Example 7 - CRM of OAg using CDAP chemistry 197 Conjugation to (with or without ADH linker) Paratifide A OAg(O:2) was conjugated to CRM197 using a random CDAP chemistry approach, either via an ADH linker or without an ADH linker. Again, the random CDAP chemistry approach conjugated OAg and CRM 197 Multiple bonds are introduced between them. The chemistry is shown in Figure 4.

[0214] Specifically, the OH groups of O:2 are activated by CDAP using a w / w ratio of 1:0.3 between O:2 and CDAP in a 150 mM NaCl solution. The pH is adjusted to 9-10 with 10% v / v triethylamine, and the solution is incubated at room temperature for 3 ± 0.5 minutes with stirring.

[0215] The activated cyanoester group of O:2 is CRM 197ADH / CRM 197 covalently binds to the hydrazide group / amino group of O:2-CDAP-ADH-CRM 197 / O:2-CDAP-CRM 197 is formed. CRM 197 ADH / CRM 197 is added at a w / w ratio (1:1) equivalent to O:2 at a concentration of 10 mg / mL (the final concentration of O:2 and CRM 197 ADH / CRM 197 is 5 mg / mL). The pH is maintained at 9.5 ± 0.5 with 10% triethylamine, and the solution is mixed at room temperature for 2 - 3 hours. Then, a 1M glycine solution is added to an equal amount of the conjugation mixture, the pH is adjusted to 8.0 ± 0.2 with 10% triethylamine, and the solution is incubated at 2 - 8°C for 15 ± 5 hours. Subsequently, the crude conjugate is subjected to buffer exchange, and unbound and unreacted O:2 is removed using HIC phenyl HP resin.

[0216] Stability studies based on the research methods described in Example 3 (however, free OAg was measured from samples obtained on days 0, 8, 14, and 28) were performed on OAg-CRM 197 conjugates generated using CDAP chemistry with or without an ADH linker. The results (free OAg percentage) are shown in Table 10 below.

[0217]

Table 7

[0218] In the stability studies, significantly less free OAg was observed compared to the OAg-CRM 197 conjugates prepared using the chemistries described in Examples 2 - 5.

[0219] Example 8 - Comparison of immunogenicity of bivalent formulations by O:2-CRM 197 generated through ADH-SIDEA chemistry or CDAP chemistry. To compare the ability of conjugates generated using different conjugation chemistries to induce an immune response, a group of 10 BALB / c mice was immunized subcutaneously on days 0 and 28 with a 50 μl dose of 2.5 μg (each component (fVi or S. paratyphi A O-antigen)) without aluminum hydroxide, or a bivalent formulation with a dose of 1.25 μg (each component) with 50 μl of 75 μg of aluminum hydroxide.

[0220] Mice were bled on days 28 and 42, and the levels (GMT) of O:2 and Vi IgG were measured by ELISA. For O:2, serum functionality was also confirmed by SBA. The ELISA protocol used was the same as that described in WO2013 / 038375. The SBA protocol used is described in Necchi F et al. “Setup of luminescence-based serum bactericidal assay against Salmonella Paratyphi A” J Immunol Methods. 2018;461:117-121.

[0221] Groups of mice were immunized with or without aluminum hydroxide as follows: - O:2-ADH-SIDEA-CRM 197 (Generated by reductive amination by selective reduction as in Example 1) - O:2(CDAP)-CRM 197 (Generated as in Example 7) In each case, formulated as a bivalent formulation with fVi-CRM 197 (Generated as in Example 2 at the doses indicated above).

[0222] The results obtained are shown in Figure 5. As can be judged from Figure 5, the conjugates involving CDAP conjugation chemistry induced an antibody response and functionality to O:2 similar to those of the conjugates using ADH-SIDEA chemistry. However, as demonstrated in the previous examples, the CDAP-based conjugates were more stable.

[0223] Example 9 - O:2-CRM produced through CDAP chemistry 197 Human clinical trials demonstrating the immunogenicity of a bivalent formulation including [specific compound] O:2(CDAP)-CRM 197 The conjugate (generated in Example 7) is used in fVi-CRM 197 To characterize the safety and immunogenicity profile of the vaccine (produced as in Example 2), a Phase I, observer-blinded, randomized, controlled, single-center human clinical trial was conducted. The study number for the clinical trial is NCT05613205.

[0224] Ninety-six healthy European adults (18-50 years old) were administered one of the formulations listed in Table 11, or the TYPHIM Vi control described below.

[0225] [Table 8]

[0226] The above vaccine formulations were compared to a control dose of 0.5 mL of TYPHIM Vi (containing 25 μg of Salmonella cyphi Vi capsular polysaccharide).

[0227] Blood samples were obtained on day 1 (the same day as vaccine administration) and day 29 (i.e., 28 days after vaccine administration). Antibody levels in the samples were tested using ELISA and SBA assays.

[0228] For the estimation of anti-Vi IgG, a robust and qualified ELISA method was used. In this method, a 96-well ELISA plate was coated with Vi PS diluted in coating buffer (1X PBS) and maintained overnight at 2–8°C. The coated plate was washed with washing buffer and blocked with blocking solution (5% skim milk powder in PBS). After further washing, optimally diluted serum samples (in dilution buffer) were added to the designated wells and incubated at 25°C for 2 hours. After three further washes, AP conjugate anti-mouse IgG secondary antibody (in dilution buffer) at an optimized dilution was added and incubated for 1 hour. After washing, the substrate was added to the plate and incubated. After 1 hour, absorbance was measured using an ELISA reader. A standard curve was plotted against the concentration of the anti-Vi IgG reference standard substance for the obtained absorbance or optical density (OD), and fitted using a polynomial regression line. Subsequently, the anti-Vi IgG concentration in each serum sample was calculated using the same standard curve.

[0229] To estimate anti-O:2 IgG in animal or human serum, a conventional method similar to that used for anti-Vi IgG was employed, by coating ELISA plates with O:2-PS. In short, a 96-well ELISA plate was coated with O:2 PS (in 1X carbonate) and maintained overnight at 2–8°C. The coated plate was washed once with washing buffer and blocked with blocking solution (5% non-fat milk in PBS). After further washing, optimally diluted serum samples (in dilution buffer) were added to the designated wells and incubated at 25°C for 2 hours. After three further washes, an optimized dilution of AP conjugate anti-mouse IgG secondary antibody (in dilution buffer) was added and incubated for 1 hour. After washing, the substrate was added to the plate and incubated. After 1 hour, absorbance was measured using an ELISA reader. Standard curves were plotted against the acquired absorbance or optical density (OD) versus the anti-O:2 IgG reference standard and fitted using polynomial regression lines. Subsequently, the ELISA units of anti-O:2 IgG in each serum sample were calculated using the same standard curve.

[0230] Serum bactericidal activity against Salmonella paratifida A was measured by L-SBA based on previously reported methods [Necchi, 2017; Rossi, 2020]. The results are expressed as serum titers and defined as the serum dilution ratio (IC50) that inhibits bacterial growth by 50%.

[0231] The results are shown in Figures 9-11.

[0232] Using the SBA and ELISA results, the percentage of patients with more than 2 μg / ml of anti-Vi IgG GMC (at 29 days) and the percentage of patients with more than 4.3 μg / ml of anti-Vi IgG GMC (at 29 days) were calculated, and these data are shown in Tables 12 and 13 below. Similarly, the percentage of patients with more than a fourfold increase in anti-O:2 GMC (by ELISA or SBA assay) was calculated, and these data are shown in Tables 14 and 15 below.

[0233] [Table 9]

[0234] [Table 10]

[0235] [Table 11]

[0236] [Table 12]

[0237] Example 10 - O:2 size and O:2 / CRM in mice 197 Ratio O:2-CRM 197 Effects of glycoconjugates on immunogenicity Conjugate synthesis and characterization O:2 size and O:2 / CRM 197 To evaluate the effect of the w / w ratio on immunogenicity, we synthesized a panel of conjugates with different characteristics and thoroughly characterized them.

[0238] S. paratifi strain A ED199ΔtolR was used as the source of O:2 to generate a bimodal O-antigen population with molecular weight (MW), where the two main peaks were located at 16 kDa and 100 kDa, and the w / w ratio was 65:35 (Figure 12A). Purified O:2 [16 kDa + 100 kDa] Starting with a mixed population, size exclusion chromatography (SEC) was performed to determine the O:2 ratio. [16 kDa] and O:2 [100 kDa] Two populations, named as [Population A] and [Population B], were separated. The two populations showed similar characteristics in glucosylation% and O-acetylation% (Figure 12B). During SEC, the glucosylation level remained unchanged, but the O-acetylation level was partially affected, and O:2 [16 kDa + 100 kDa] From 60% of the mixed population, O:2 [16 kDa] and O:2 [100 kDa] Then it decreased to about 45% (Figure 12B).

[0239] Different O:2-CRM 197 Conjugate, O:2 [16 kDa] (Conjugate 1-3, Table 13), O:2 [100 kDa] (Conjugate 4-6, Table 13), or O:2 [16 kDa + 100 kDa] Synthesized using (Conjugates 7-9, Table 13). Different O:2 / CRM were used in the conjugation process. 197 / CDAP weight ratio is used, which is not only the size of O:2 but also O:2 and CRM 197 This resulted in a panel of conjugates with different ratios (Table 13). After purification, all conjugates had a residual free O:2 of less than 5%.

[0240] [Table 13]

[0241] Mouse immunogenicity research Mice underwent two intraperitoneal (IP) immunizations with a 2.5 μg conjugate (O:2 dose) at 28-day intervals. The anti-O:2 IgG response was determined by ELISA, and serum functional activity was determined by its ability to kill S. paratifi A bacteria in the presence of complement.

[0242] In general, all conjugates tested (Table 13) were immunogenic regardless of specific structural differences and exhibited a significant booster effect after a second immunization (Figure 13A).

[0243] O:2 size influence O:2 / CRM 197 The w / w ratio is similar, but the O:2 MW is different for O:2 / CRM. 197 Conjugates (16 kDa, 100 kDa, or 100 kDa + 16 kDa) were compared. After one injection, no significant difference was observed between the conjugates. Two weeks after the second injection, O:2 [16 kDa] -CRM 197 O:2 [100 kDa] It induced a significantly higher anti-O:2 IgG response than the similar conjugate synthesized by (p = 0.0040). Both conjugates induced a response of 0.37 (O:2 [16 kDa] -CRM 197 ) and 0.39(O:2 [100 kDa] -CRM 197 ) and similar O:2 / CRM 197 It showed a w / w ratio (Figure 13A). Similarly, it had a high O:2 / CRM ratio. 197 w / w ratio (O:2 [16 kDa] -CRM 197 0.62, O:2 [16 kDa + 100 kDa] -CRM 197 The same applies to the conjugate of 0.53), O:2 [16 kDa] -CRM 197 This induced the highest anti-O:2 IgG response (Figure 13A).

[0244] O:2 and CRM 197 The effect of the ratio O:2 load CRM 197 The impact on O:2 was also investigated.[16 kDa] , O:2 [100 kDa] Starting with a mixed population of O:2[16 kDa + 100 kDa] and carried out under different reaction conditions, different O:2 / CRM 197 We were able to synthesize w / w ratio conjugates (Table 13). O:2 / CRM 197 O:2 with w / w ratios of 0.39 and 0.79 [100 kDa] Conjugates 4 and 5 (Table 13) induced similar anti-O:2 IgG responses (Figure 13B) and serum bactericidal titers. [16 kDa + 100 kDa] Obtained from a mixed population, O:2 / CRM 197 Conjugates 7 and 8 (Table 13), with a w / w ratio of 0.28, also induced similar responses. On the other hand, O:2 [16 kDa] Regarding O:2 / CRM 197 Conjugate 3, with a w / w ratio of 0.62, induced a significantly higher total anti-O:2 IgG response than conjugates 1 and 2, which had lower ratios of 0.25 (p = 0.0051) and 0.37 (p = 0.0494), respectively (Table 13) (Figure 13B).

[0245] O:2-CRM 197 Effect of O-acetylation level on the immunogenicity of glycoconjugates Partial deoxyacetylation of O:2 by ammonia treatment Bacterial polysaccharides often contain O-acetyl esters (OAc), which may constitute an important part of immune dominance epitopes. To evaluate the impact of O-acetylation on immune responses, we developed O:2-CRM with different levels of O-acetylation. 197 Panels of conjugates were synthesized (Table 14). Partial removal of the OAc group from O:2 was achieved by treating O:2 with a weak base such as ammonia, and CRM 197 This was performed before the conjugation. O:2 [16 kDa + 100 kDa]The mixed population had 60% initial O-acetylation. Panels of partially or completely de-O-acetylated O:2 were obtained by treating the polysaccharides with increasing ammonia concentration (in the range of 5 ÷ 1000 mM) at 25°C. Correlation functions were established by plotting the obtained O-acetylation levels against ammonia (NH4OH) concentration (Figure 14). De-O-acetylation of total O:2 was obtained by using 1 M ammonia.

[0246] Using the correlation function, O:2 [16 kDa] The material was treated with an appropriate ammonia concentration to gradually obtain de-O-acetylated polysaccharides.

[0247] O:2-CRM with different O-acetylation levels 197 Conjugate generation Start O:2 [16 kDa + 100 kDa] , as well as completely deoxyacetylated O:2 [16 kDa + 100 kDa] CRM 197 Conjugation was performed, and conjugates with 54% or 0% OAc were obtained (Conjugates 1 and 5, Table 14). In addition, selections of partially de-acetylated O:2 were conjugated, resulting in O:2-CRM with OAc content of 45.2%, 35.3%, and 18.5%, respectively. 197 A panel of conjugates was provided (Conjugates 2, 3, 4, Table 14). Similarly, O:2 [16 kDa] and partially deoxyacetylated O:2 [16 kDa] CRM 197 Conjugation resulted in two conjugates with OAc concentrations of 58.5% and 25.2% (Conjugates 6 and 7, Table 14).

[0248] O:2 activation by CDAP followed by CRM 197 Conjugation to did not affect the initial O:2 OAc level.

[0249] All conjugates have been thoroughly characterized and are similar to O:2 / CRM conjugates, as indicated by their HPLC-SEC fluorescence emission profiles.197 The w / w ratio (Table 14) and similar cross-linking levels were shown.

[0250] [Table 14]

[0251] Immunogenicity of conjugates at different O-acetylation levels To investigate the effect of O-acetylation on immunogenicity, the conjugates reported in Table 14 were tested in mice. Mice received two intraperitoneal (IP) immunizations with 2.5 μg of conjugate (O:2 dose) at 28-day intervals. [16 kDa + 100 kDa] In this case, five different conjugates with varying O-acetylation levels (0% to 54%) were tested. Results were analyzed by performing regression analysis of ELISA results (logarithmically transformed) versus OAc. All conjugates were immunogenic and showed a booster response after a second immunization (Figure 15A). The 42-day analysis (Figure 15B) showed a significant slope (p = 0.027) in the O-acetylation range of 0–45%, with no significant lack of fit (p = 0.303), indicating an increase in anti-O:2 IgG antibody with increasing O-acetylation levels. A significant trend was observed in the OAc levels from 0% to 54%, with a total increase factor of 8.8 (CI). 95% The geometric mean ratio was 1.3-59 EU / mL. On day 27, regression analysis did not demonstrate a significant trend with respect to the slope.

[0252] O:2 at two different O-acetylation levels (58.5% and 25.2%, conjugates 6 and 7, Table 14) [16 kDa] Similar results were obtained using conjugates: at both 27 days (p = 0.0016, Mann-Whitney study) and 42 days (p = 0.0063, Mann-Whitney study), the 58.5% O-acetylated conjugate showed a higher response than the 25.2% OAc conjugate (Figure 15A).

[0253] manner 1. A conjugate comprising a polysaccharide containing a 3-deoxy-D-manno-octulosonic acid (KDO) moiety and conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide contains two or more activation sites.

[0254] 2. A conjugate comprising an O-antigen conjugated to a carrier protein by a random conjugation method, wherein the O-antigen contains two or more activation sites.

[0255] 3. A conjugate comprising a polysaccharide containing a KDO moiety and conjugated to a carrier protein using a conjugation method, The conjugation method involves the following steps: (i) Activating polysaccharides by 1-cyano-4-dimethylaminopyridinetetrafluoroborate (CDAP) chemistry to provide activated polysaccharides; or (ii) To provide oxidized polysaccharides by oxidizing polysaccharides, A conjugate that includes this.

[0256] 4. A conjugate containing an O-antigen conjugated to a carrier protein using a conjugation method, wherein the conjugation method consists of the following steps: (i) Activating O-antigens by CDAP chemistry to provide activated O-antigens; or (ii) To provide an oxidized O-antigen by oxidizing the O-antigen, A conjugate that includes this.

[0257] 5. A method for generating a conjugate comprising a polysaccharide containing a KDO moiety and conjugated to a carrier protein, comprising the step of introducing multiple activation sites into the polysaccharide.

[0258] 6. A method for generating a conjugate comprising an O-antigen conjugated to a carrier protein, comprising the step of introducing multiple activation sites to the O-antigen.

[0259] 7. A method for producing a conjugate containing a KDO moiety and a polysaccharide conjugated to a carrier protein, The following steps: (i) Activating polysaccharides by CDAP chemistry to provide activated polysaccharides; or (ii) To provide oxidized polysaccharides by oxidizing polysaccharides, Methods that include...

[0260] 8. A method for generating a conjugate containing an O-antigen conjugated to a carrier protein, comprising the following steps: (i) Activate the O-antigen by CDAP chemistry to provide an activated O-antigen; or (ii) Oxidize the O-antigen to provide an oxidized O-antigen. Methods that include...

[0261] 9. A conjugate or method according to any one of embodiments 1, 3, 5, or 7, wherein the polysaccharide is an O-antigen.

[0262] 10. A conjugate or method according to any one of embodiments 2, 4, 6, 8, or 9, wherein the O-antigen is an O-antigen from Salmonella paratifida (S. paratifida).

[0263] 11. Carrier protein is CRM 197 A conjugate or method according to any one of the preceding embodiments, selected from the group consisting of tetanus toxoid (TT) or diphtheria toxoid (DT).

[0264] 12. Carrier protein is CRM 197 The conjugate or method described in embodiment 11.

[0265] 13. A conjugate or method according to any one of the preceding embodiments, further comprising a linker.

[0266] 14. A conjugate or method according to any one of the preceding embodiments, wherein the linker is an adipic acid dihydrazide (ADH) linker.

[0267] 15. The conjugate or method according to embodiment 13 or 14, wherein the linker is located between the sugar or O-antigen and the carrier protein.

[0268] 16. A conjugate or method according to any one of embodiments 3, 4, or 7-15, wherein the activation or oxidation step introduces multiple activation sites into a polysaccharide or O-antigen.

[0269] 17. A conjugate or method according to any one of the preceding embodiments, wherein the polysaccharide or O-antigen contains 1.5 or more, 2.0 or more, or 2.5 or more activation sites, or the activation or oxidation step introduces 1.5 or more, 2.0 or more, or 2.5 or more activation sites.

[0270] 18. The step of introducing a random conjugation method or multiple activation sites is (i) Activating polysaccharides or O-antigens by CDAP chemistry to obtain activated polysaccharides or activated O-antigens; or (ii) To obtain oxidized polysaccharides or oxidized O-antigens by oxidizing polysaccharides or O-antigens, A conjugate according to any one of embodiments 1, 2, 5, 6, or 9-17, including the above.

[0271] 19. A conjugate or method according to any one of embodiments 3, 4, or 7-18, wherein the activation of an O-antigen and / or polysaccharide by CDAP chemistry comprises mixing the O-antigen or polysaccharide with CDAP in a w / w ratio (ratio of CDAP to polysaccharide or O-antigen) of 0.05:1-5:1, 0.1:1-5:1, 0.2:1-2:1, or approximately 0.3:1.

[0272] 20. The conjugate or method according to any one of embodiments 3, 4, or 7-19, wherein the activation of the O-antigen and / or polysaccharide by CDAP chemistry comprises mixing the O-antigen and / or polysaccharide containing the KDO moiety in a NaCl solution or KCl solution of 50 mM to 1 M, 100 mM to 250 mM, 125 mM to 200 mM, or approximately 150 mM.

[0273] 21. A conjugate or method according to any one of embodiments 3, 4, or 7-20, wherein the activation of an O-antigen and / or polysaccharide by CDAP chemistry comprises adjusting the pH to 9-10 using triethylamine and optionally incubating the solution at room temperature for 1-5 minutes with stirring.

[0274] 22. A conjugate or method according to any one of embodiments 3, 4, or 7-21, further comprising reacting an activated polysaccharide or activated O-antigen with a hydrazide group / amino group on a carrier protein or a carrier protein-linker compound containing a carrier protein.

[0275] 23. The conjugate or method according to embodiment 22, wherein reacting an activated polysaccharide or activated O-antigen with a hydrazide group / amino group on a carrier protein or a carrier protein-linker compound containing a carrier protein is mixed with the activated polysaccharide or activated O-antigen in a w / w ratio (ratio of polysaccharide or O-antigen to carrier protein or carrier protein-linker) of 0.1:1 to 5:1, 0.2:1 to 3:1, 0.5:1 to 2:1, or approximately 1:1.

[0276] 24. Carrier protein is CRM 197 The conjugate or method according to embodiment 22 or 23.

[0277] 25. Carrier protein-linker compound is CRM 197 -ADH, the conjugate or method according to any one of embodiments 22 to 24.

[0278] 26. The conjugation method is as follows: (i) After mixing the activated polysaccharide or activated O-antigen with the carrier protein or carrier protein-linker compound, adjust the pH to 9-10 using triethylamine and incubate at room temperature for 1-10 hours with stirring, and / or (ii) Add glycine solution, adjust the pH to 7-9 using triethylamine, and incubate at 2-8°C for 5-50 hours or 10-20 hours, and / or (iii) Step to remove unreacted polysaccharides or O-antigens. A conjugate or method according to any one of embodiments 22 to 25, including the method described herein.

[0279] 27. The conjugate or method according to embodiment 26, wherein the step of removing unreacted polysaccharide or O-antigen comprises a chromatography step (which may optionally include hydrophobic interaction chromatography or anion exchange chromatography).

[0280] 28. A conjugate or method according to any one of embodiments 3, 4, or 7-18, wherein the step of oxidizing a polysaccharide or O-antigen comprises a step of optionally mixing the polysaccharide or O-antigen with an oxidizing agent at a pH of 4-6.

[0281] 29. The conjugate or method according to embodiment 28, wherein the oxidizing agent is a periodate (optionally sodium periodate).

[0282] 30. The conjugate or method according to embodiment 28 or 29, wherein mixing a polysaccharide or O-antigen with an oxidizing agent comprises mixing the polysaccharide or O-antigen with the oxidizing agent in a ratio of 1 mg / mL:10 mM to 100 mg / mL:10 mM, 1 mg / mL:10 mM to 50 mg / mL:10 mM, 1 mg / mL:10 mM to 25 mg / mL:10 mM, or approximately 10 mg / mL:10 mM (ratio of polysaccharide or O-antigen to oxidizing agent).

[0283] 31. The step of oxidizing the polysaccharide or O-antigen is as follows: (i) Allow the polysaccharide or O-antigen and oxidizing agent solution to stand in the dark at a temperature of 20°C to 30°C for 1 to 5 hours, and / or (ii) Deactivating excess oxidizing agents using Na2SO3 as an option, and / or (iii) Selectively desalting oxidized polysaccharides or oxidized O-antigens using a PD10 column. A conjugate or method according to any one of embodiments 28 to 30, including the method described above.

[0284] 32. A conjugate or method according to any one of embodiments 3, 4, 7-18 or 28-31, wherein the conjugation method further comprises the step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein.

[0285] 33. The conjugate or method according to embodiment 32, wherein the step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein comprises mixing the oxidized polysaccharide or oxidized O-antigen and the carrier protein with a reducing agent.

[0286] 34. The conjugate or method according to embodiment 33, wherein the reducing agent is sodium borohydride.

[0287] 35. The conjugate or method according to embodiment 33 or 34, wherein the step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein comprises mixing the oxidized polysaccharide or oxidized O-antigen and the carrier protein in a w / w ratio (ratio of polysaccharide or O-antigen to carrier protein) of 0.5:1 to 20:1, 1:1 to 10:1, 1.5:1 to 5:1, or approximately 2:1.

[0288] 36. A conjugate or method according to any one of embodiments 33 to 35, wherein the step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein comprises mixing the carrier protein and a reducing agent in a w / w ratio (ratio of carrier protein to reducing agent) of 0.25:1 to 20:1, 0.5:1 to 10:1, 0.75:1 to 5:1, or approximately 1:1.

[0289] 37. The conjugation method is as follows: (i) Incubate a reaction mixture containing an oxidized polysaccharide or oxidized O-antigen and a carrier protein at a temperature of 35°C to 39°C for 4 to 20 hours; and (ii) Inactivation by adding sodium borohydride, wherein the w / w ratio of oxidized polysaccharide or oxidized O-antigen to sodium boride is optionally 10:1 to 0.1:1, 5:1 to 0.5:1, or approximately 1:1 (ratio of polysaccharide to O-antigen). A conjugate or method according to any one of embodiments 32 to 36, including the method described above.

[0290] 38. A conjugate obtainable by any one of the methods described in embodiments 5 to 37.

[0291] 39. A conjugate obtained by any one of the methods described in aspects 5 to 37.

[0292] 40. An immunogenic composition comprising a conjugate according to any one of embodiments 1 to 4 or 9 to 39.

[0293] 41. The immunogenic composition according to embodiment 40, further comprising a pharmaceutically acceptable excipient and / or adjuvant.

[0294] 42. An immunogenic composition according to embodiment 40 or 41, further comprising an antigen from Salmonella cifida (S. cifida), optionally selected Vi polysaccharide.

[0295] 43. The immunogenic composition according to embodiment 42, wherein the Vi polysaccharide is a fragmented Vi polysaccharide (fVi).

[0296] 44. The immunogenic composition according to embodiment 43, wherein the average molecular weight of the fVi polysaccharide is 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.

[0297] 45. The immunogenic composition according to embodiment 43 or 44, wherein the target average molecular weight of the fVi polysaccharide is 51 kDa to 55 kDa.

[0298] 46. ​​The immunogenic composition according to any one of embodiments 43 to 45, wherein the fVi polysaccharide is part of an fVi conjugate containing fVi and a carrier protein.

[0299] 47. Carrier protein in fVi conjugate is CRM 197 The immunogenic composition according to embodiment 46, which is a diphtheria toxoid.

[0300] 48. Carrier protein in fVi conjugate is CRM 197 The immunogenic composition according to embodiment 47.

[0301] 49. An immunogenic composition according to any one of embodiments 43 to 48, wherein an fVi polysaccharide is optionally conjugated to a carrier protein via a linker by carbodiimide chemistry.

[0302] 50. The fVi conjugate is performed in the following steps: a. Fragmenting Vi polysaccharide to obtain fragmented Vi polysaccharide (fVi) having an average molecular weight of 10 kDa-90 kDa, 25 kDa-70 kDa, 40 kDa-55 kDa, 41 kDa-49 kDa, or 51 kDa-55 kDa; b. Activating the fVi polysaccharide by reacting the fVi polysaccharide obtained in step a. with carbodiimide and N-hydroxysuccinimide at pH 5-6 to form an N-hydroxysuccinimide ester fVi derivative; and c. The N-hydroxysuccinimide ester fVi derivative obtained in step b. is reacted with a carrier protein to produce an fVi conjugate. An immunogenic composition according to any one of embodiments 43 to 49, which is obtained or can be obtained by a method including the above.

[0303] 51. The immunogenic composition according to embodiment 49 or 50, wherein the carbodiimide is EDC.

[0304] 52. The immunogenic composition according to embodiment 51, wherein the carrier protein is derivatized by reacting it with a carbodiimide and a linker.

[0305] 53. The immunogenic composition according to embodiment 52, wherein the linker is an adipic acid dihydrazide (ADH) linker.

[0306] 54. The immunogenic composition according to embodiment 52 or 53, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), or the carbodiimide chemistry is EDAC chemistry.

[0307] 55. The immunogenic composition according to any one of embodiments 40 to 54, wherein the conjugate is stable in the immunogenic composition for at least 4 weeks.

[0308] 56. The immunogenic composition according to embodiment 55, wherein the conjugate in the immunogenic composition is stable for at least 4 weeks when the proportion of polysaccharides or O-antigens released from the conjugate in 4 weeks is less than 20%, less than 18%, less than 17%, less than 15%, 0% to 20%, or 1% to 15%.

[0309] 57. The amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is determined by direct reductive amination via the ADH-SIDEA linker. 197 The immunogenic composition according to embodiment 55 or 56, wherein the conjugate in the immunogenic composition is stable for at least 4 weeks when the amount released is substantially less than the amount released from an equivalent reference immunogenic composition containing S. paratifi O-antigen conjugated to it.

[0310] 58. The immunogenic composition according to embodiment 57, wherein substantially less means at least 10% less, 15% less, 20% less, or 25% less.

[0311] 59. The amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is determined using CDAP chemistry in CRM. 197 The immunogenic composition according to any one of embodiments 55 to 58, wherein the conjugate in the immunogenic composition is stable for at least 4 weeks when the amount released is similar to that released from an equivalent reference immunogenic composition containing S. paratifi O-antigen conjugated to it.

[0312] 60. The immunogenic composition according to embodiment 59, wherein being similar means within ±25%, within ±15%, or within ±10%.

[0313] 61. The amount of polysaccharide or O-antigen released from the conjugate after 4 weeks is determined by the following steps: (i) Incubate the immunogenic composition at 37°C for 4 weeks; (ii) Prepare a post-incubation sample of the incubated immunogenic composition and remove conjugated polysaccharides or O-antigens by deoxycholic acid precipitation; and (iii) Determine the amount of free polysaccharides or free O-antigen in the incubated sample as a percentage of the total polysaccharide or total O-antigen amount. An immunogenic composition according to any one of embodiments 56 to 60, determined by using a stability assay including [a specific component].

[0314] 62. Immunogenic compositions undergo CRM by direct reductive amination via ADH-SIDEA linker. 197 A conjugate, method, or immunogenic composition according to any one of the preceding embodiments, which induces a similar level of anti-S. paratifida O-antigen antibody compared to an equivalent immunogenic composition containing S. paratifida O-antigen conjugated with the conjugated S. paratifida O-antigen.

[0315] 63. The conjugate, method, or immunogenic composition according to embodiment 62, wherein the similar level is within ±25%, ±15%, or ±10%.

[0316] 64. Anti-S. paratificentide O-antigen antibody levels are measured in the following steps: (i) Immunize mice by subcutaneous administration of a 25 μg (O-antigen) conjugate on days 0 and 28; (ii) On day 42, measure the level of anti-S. paratifi O-antigen antibody by ELISA. A conjugate, method, or immunogenic composition according to embodiment 62 or 63, which is measured using an immunogenicity assay comprising the above.

[0317] 65. (i) Salmonella tiphimurium (S. tiphimurium) antigen; and / or (ii) Salmonella enteritidis (S. enteritidis) antigen, An immunogenic composition according to any one of embodiments 40 to 64, further comprising the above.

[0318] 66. The immunogenic composition according to embodiment 65, further comprising both S. tiphimurium antigen and S. enteritidis antigen.

[0319] 67. The immunogenic composition according to embodiment 65 or 66, wherein the S. tiphimurium antigen comprises or is derived from S. tiphimurium GMMA.

[0320] 68. An immunogenic composition according to any one of embodiments 65 to 67, wherein the S. enteritidis antigen comprises or is derived from S. enteritidis GMMA.

[0321] 69. The immunogenic composition according to embodiment 67 or 68, comprising S. tiphimurium GMMA and / or S. enteritidis GMMA, and containing detoxification lipid A.

[0322] 70. S. ciphyllum GMMA and / or S. enteritidis GMMA, as follows: (i) a gene encoding a functional MSbB protein; and / or (ii) The gene encoding the functional PagP protein, An immunogenic composition according to embodiment 69, which is derived from S. tiphimulium and / or S. enteritidis, and does not contain any of the above.

[0323] 71. An immunogenic composition according to any one of embodiments 67 to 70, wherein S. tiphimulium GMMA and / or S. enteritidis GMMA are derived from S. tiphimulium and / or S. enteritidis that do not contain a gene encoding a functional tolR protein.

[0324] 72. A vaccine comprising the immunogenic composition described in any one of embodiments 40 to 71.

[0325] 73. An immunogenic composition or vaccine according to any one of embodiments 40 to 72 for use in a method for preventing infection.

[0326] 74. A method for preventing infection, comprising administering an effective amount of an immunogenic composition or vaccine described in any one of embodiments 40 to 72 to a target.

[0327] 75. Use of an immunogenic composition or vaccine according to any one of embodiments 40 to 72 for the manufacture of a pharmaceutical product for use in a method of preventing infection.

[0328] 76. An immunogenic composition or vaccine for use according to embodiment 73, or use according to embodiment 75, wherein a method for preventing infection includes administering an effective amount of the immunogenic composition or vaccine according to any one of embodiments 40 to 72 to a target.

[0329] 77. An immunogenic composition or vaccine for use, method, or use according to any one of embodiments 73 to 76, wherein the method for preventing infection is a method for preventing Salmonella infection.

[0330] 78. An immunogenic composition or vaccine for use, method, or use according to any one of embodiments 73 to 77, wherein the method for preventing infection is a method for preventing invasive atypical Salmonella infection.

[0331] 79. An immunogenic composition or vaccine for use, method or use according to any one of embodiments 73 to 78, wherein the method for preventing infection is a method for preventing infection by S. tiphimulium, S. enteritidis, S. tif and / or S. paratif A.

[0332] 80. A conjugate, method, immunogenic composition, vaccine, immunogenic composition or vaccine for use, or use according to any one of embodiments 2, 4, 6 or 8-79, wherein the O-antigen is an O-antigen from Salmonella paratifida (S. paratifida), and the average molecular weight of the O-antigen is 10-25 kDa, 10-20 kDa, 11-19 kDa, 12-19 kDa, 13-19 kDa, 14-18 kDa, 15-18 kDa, or 16-18 kDa.

[0333] 81. A conjugate, method, immunogenic composition, vaccine, immunogenic composition or vaccine for use, or use according to any one of embodiments 2, 4, 6 or 8-80, wherein the O-antigen is an O-antigen from Salmonella paratifida (S. paratifida A), and the degree of O-acetylation of the S. paratifida A O-antigen is 10-100%, 20-100%, 30-100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%.

[0334] 82. The O-antigen is derived from Salmonella paratifida (S. paratifida), and the carrier protein is CRM. 197 O:2 / CRM 197 A conjugate, method, immunogenic composition, vaccine, immunogenic composition or vaccine for use, or use according to any one of embodiments 2, 4, 6 or 8 to 81, wherein the w / w ratio is 0.25 to 3.0, 0.4 to 0.8, 0.5 to 0.8, 0.6 to 0.7, 0.61 to 0.67, or 0.62 to 0.66.

Claims

1. below: (a) 3-deoxy-D-manno-octulosonic acid (KDO) moiety; and / or (b) O-antigen, A conjugate comprising a polysaccharide conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide contains two or more activation sites.

2. below: (a) KDO portion; and / or (b) O-antigen, A conjugate comprising a polysaccharide conjugated to a carrier protein using a conjugation method, The conjugation method involves the following steps: (i) A step of activating a polysaccharide by 1-cyano-4-dimethylaminopyridinetetrafluoroborate (CDAP) chemistry to provide an activated polysaccharide; or (ii) A step of oxidizing a polysaccharide to provide an oxidized polysaccharide. A conjugate that includes this.

3. below: (a) KDO portion; and / or (b) O-antigen, A method for generating a conjugate comprising a polysaccharide conjugated to a carrier protein, the method comprising the step of introducing multiple activation sites into the polysaccharide.

4. below: (a) KDO portion; and / or (b) O-antigen, A method for generating a conjugate containing a polysaccharide conjugated to a carrier protein, The following steps: (i) A step of activating a polysaccharide by CDAP chemistry to provide an activated polysaccharide; or (ii) A step of oxidizing a polysaccharide to provide an oxidized polysaccharide, Methods that include...

5. (a) Polysaccharides are O-antigens; (b) The O-antigen is the O-antigen from Salmonella paratifida (S. paratifida); (c) The carrier protein is CRM 197 Selected from the group consisting of tetanus toxoid (TT) or diphtheria toxoid (DT); (d) Carrier protein is CRM 197 It is; (e) further comprising a conjugate a linker, optionally an adipic acid dihydrazide (ADH) linker; (f) The conjugate further comprises a linker between the sugar or O-antigen and the carrier protein, optionally an ADH linker; and / or (g) The polysaccharide or O-antigen contains 1.5 or more, 2.0 or more, or 2.5 or more activation sites, or the activation or oxidation step introduces 1.5 or more, 2.0 or more, or 2.5 or more activation sites. The conjugate or method according to any one of claims 1 to 4.

6. The conjugate or method according to any one of claims 2 or 4 to 5, wherein the activation step or oxidation step introduces a plurality of activation sites into a polysaccharide or O-antigen.

7. Random conjugation or introduction of multiple activation sites follows these steps: (i) A step of activating a polysaccharide or O-antigen by CDAP chemistry to provide an activated polysaccharide or activated O-antigen; or (ii) A step of oxidizing a polysaccharide or O-antigen to provide an oxidized polysaccharide or oxidized O-antigen, A conjugate according to any one of claims 1, 3, or 5 to 6, including the following:

8. (a) The step of activating an O-antigen and / or polysaccharide by CDAP chemistry includes mixing the polysaccharide or O-antigen with CDAP in a w / w ratio (ratio of CDAP to polysaccharide or O-antigen) of 0.05:1 to 5:1, 0.1:1 to 5:1, 0.2:1 to 2:1, or approximately 0.3:1; (b) The step of activating the O-antigen and / or polysaccharide by CDAP chemistry includes mixing the O-antigen and / or polysaccharide containing the KDO moiety in a NaCl or KCl solution at concentrations of 50 mM to 1 M, 100 mM to 250 mM, 125 mM to 200 mM, or approximately 150 mM; (c) The step of activating the O-antigen and / or polysaccharide by CDAP chemistry includes adjusting the pH to 9–10 using triethylamine and optionally incubating the solution at room temperature for 1–5 minutes with stirring; (d) The method further comprises the step of reacting an activated polysaccharide or activated O-antigen with a hydrazide / amino group on a carrier protein or a carrier protein-linker compound containing a carrier protein, wherein the carrier protein is CRM 197 The carrier protein-linker compound is CRM 197 -ADH; and / or (e) The method further comprises the step of reacting an activated polysaccharide or activated O-antigen with a hydrazide group / amino group on a carrier protein or a carrier protein-linker compound containing a carrier protein, the step of reacting an activated polysaccharide or activated O-antigen with a hydrazide group / amino group on a carrier protein or a carrier protein-linker compound comprising mixing the activated polysaccharide or activated O-antigen with the carrier protein or carrier protein-linker compound in a w / w ratio of 0.1:1 to 5:1, 0.2:1 to 3:1, 0.5:1 to 2:1, or approximately 1:1 (ratio of polysaccharide or O-antigen to carrier protein or carrier protein-linker), wherein the carrier protein is optionally CRM 197 and / or carrier protein-linker compound CRM 197 -ADH The conjugate or method according to any one of claims 2 or 4 to 7.

9. The conjugation method follows these steps: (i) After mixing the activated polysaccharide or activated O-antigen with the carrier protein or carrier protein-linker compound, adjust the pH to 9-10 using triethylamine and incubate the solution at room temperature for 1-10 hours with stirring; and / or (ii) Add glycine solution, adjust the pH to 7–9 using triethylamine, and incubate the solution at 2–8°C for 5–50 hours or 10–20 hours; and / or (iii) a step of removing unreacted polysaccharide or unreacted O-antigen, the step of removing unreacted polysaccharide or unreacted O-antigen comprising a step of chromatography, optionally hydrophobic interaction chromatography or anion exchange chromatography. The conjugate or method according to any one of claims 2 or 4 to 8, further comprising:

10. The conjugate or method according to any one of claims 2 or 4 to 7, wherein the step of oxidizing a polysaccharide or O-antigen comprises optionally mixing the polysaccharide or O-antigen with an oxidizing agent at a pH of 4 to 6.

11. (a) The oxidizing agent is a periodate, optionally sodium periodate; (b) Mixing of polysaccharides or O-antigens with oxidizing agents includes mixing the polysaccharides or O-antigens with oxidizing agents in ratios of 1 mg / mL:10 mM to 100 mg / mL:10 mM, 1 mg / mL:10 mM to 50 mg / mL:10 mM, 1 mg / mL:10 mM to 25 mg / mL:10 mM, or approximately 10 mg / mL:10 mM (ratio of polysaccharides or O-antigens to oxidizing agents); and / or (c) The step of oxidizing the polysaccharide or O-antigen is as follows: (i) Leave a solution of polysaccharide or O-antigen and oxidizing agent to stand in the dark at a temperature of 20°C to 30°C for 1 to 5 hours; and / or (ii) Deactivating excess oxidizing agents using Na2SO3 in an optional manner; and / or (iii) Selectively desalting oxidized polysaccharides or oxidized O-antigens using a PD10 column. including, The conjugate or method according to claim 10.

12. The conjugate or method according to any one of claims 2, 4-7, or 10-11, further comprising the step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein.

13. (a) The step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein comprises mixing the oxidized polysaccharide or oxidized O-antigen and the carrier protein with a reducing agent, optionally the reducing agent being sodium cyanoborohydride; (b) The step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein includes mixing the oxidized polysaccharide or oxidized O-antigen with the carrier protein in a w / w ratio (ratio of polysaccharide or O-antigen to carrier protein) of 0.5:1 to 20:1, 1:1 to 10:1, 1.5:1 to 5:1, or approximately 2:1; (c) The step of reacting an oxidized polysaccharide or oxidized O-antigen with a carrier protein includes mixing the carrier protein and the reducing agent in a w / w ratio (ratio of carrier protein to reducing agent) of 0.25:1 to 20:1, 0.5:1 to 10:1, 0.75:1 to 5:1, or approximately 1:1; and / or (d) The conjugation method involves the following steps: (i) A reaction mixture containing an oxidized polysaccharide or oxidized O-antigen and a carrier protein is incubated at a temperature of 35°C to 39°C for 4 to 20 hours; (ii) A step of inactivating by adding sodium borohydride, wherein optionally the w / w ratio of the oxidized polysaccharide or oxidized O-antigen to sodium borohydride is 10:1 to 0.1:1, 5:1 to 0.5:1, or approximately 1:1 (w / w ratio of the polysaccharide or O-antigen to sodium borohydride), The conjugate or method according to claim 12, further comprising:

14. A conjugate obtainable by the method described in any one of claims 3 to 13.

15. A conjugate obtained by the method described in any one of claims 3 to 13.

16. An immunogenic composition comprising the conjugate described in any one of claims 1, 2, or 5 to 15, and optionally further comprising a pharmaceutically acceptable excipient and / or adjuvant.

17. The immunogenic composition according to claim 16, further comprising an antigen from Salmonella cifii (S. cifii), optionally further comprising a Vi polysaccharide.

18. The immunogenic composition according to claim 17, wherein the Vi polysaccharide is fragmented Vi polysaccharide (fVi).

19. fVi polysaccharide: (a) Having an average molecular weight 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) Having a target average molecular weight of 51 kDa to 55 kDa; (c) Part of an fVi conjugate including fVi and a carrier protein; (d) fVi and CRM 197 Alternatively, it is part of an fVi conjugate containing a carrier protein that is a diphtheria toxoid; (e) fVi, and CRM 197 It is part of an fVi conjugate that includes a carrier protein; (f) A part of an fVi conjugate comprising fVi and a carrier protein, wherein the fVi polysaccharide is optionally conjugated to the carrier protein via a linker by carbodiimide chemistry; (g) A part of an fVi conjugate comprising fVi and a carrier protein, wherein the fVi conjugate is involved in the following steps: (i) Fragmenting Vi polysaccharide to obtain fragmented Vi(fVi) polysaccharide having an average molecular weight of 10 kDa–90 kDa, 25 kDa–70 kDa, 40 kDa–55 kDa, 41 kDa–49 kDa, or 51 kDa–55 kDa; (ii) A step of activating the fVi polysaccharide by reacting the fVi polysaccharide obtained in step a. with carbodiimide and N-hydroxysuccinimide at pH 5-6 to form an N-hydroxysuccinimide ester fVi derivative; and (iii) A step of reacting the N-hydroxysuccinimide ester fVi derivative obtained in step b. with a carrier protein to produce an fVi conjugate, Those obtained, or that can be obtained, by means of a method including; (h) Part of an fVi conjugate containing fVi and a carrier protein, where the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, and the carbodiimide is EDC; (i) part of an fVi conjugate comprising fVi and a carrier protein, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, the carbodiimide is EDC, and the carrier protein is derivatized by reacting the carbodiimide with a linker, optionally with an adipic acid dihydrazide (ADH) linker; and / or (j) Part of an fVi conjugate containing fVi and a carrier protein, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, and the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), or the carbodiimide chemistry is EDAC chemistry. The immunogenic composition according to claim 18.

20. (a) The conjugate is stable in the immunogenic composition for at least 4 weeks and can be optionally used. (i) If the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is less than 20%, less than 18%, less than 17%, less than 15%, 0% to 20%, or 1% to 15%, the conjugate is considered stable in the immunogenic composition for at least 4 weeks; (ii) If the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is substantially less than the amount released in an equivalent immunogenic composition that is the S. paratyphi O-antigen conjugated to CRM 197 via an ADH-SIDEA linker by direct reductive amination, the conjugate is said to be stable in the immunogenic composition for at least 4 weeks, and optionally, substantially less means at least 10% less, at least 15% less, at least 20% less, or at least 25% less; (iii) The amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is determined by the conjugate using CDAP chemistry CRM 197 The amount of S. paratifi O-antigen conjugated to is similar to that released in an equivalent reference immunogenic composition, the conjugate is considered stable in the immunogenic composition for at least 4 weeks, and optionally, similar means within 25%, 15%, or 10%; and / or (iv) The amount of polysaccharide or O-antigen released from the conjugate after 4 weeks is determined by the following steps: (A) Incubate the immunogenic composition at 37°C for 4 weeks; (B) A step of preparing a post-incubation sample of the incubated immunogenic composition and removing conjugated polysaccharides or O-antigens by deoxycholic acid precipitation; and (C) A step of determining the amount of free polysaccharides or O-antigen in the incubated sample as a percentage of the total amount of polysaccharides or total O-antigen in the incubated sample. Determined using a stability assay that includes; and / or (b) The immunogenic composition is a conjugate that uses direct reductive amination via the ADH-SIDEA linker to CRM 197 Compared to equivalent immunogenic compositions containing S. paratifi O-antigen conjugated to it, it induces similar levels of anti-S. paratifi O-antigen antibodies, and selectively: (i) Similar levels are those within 25%, 15%, or 10%; and / or (ii) The level of anti-S. paratificent O-antigen antibody is determined in the following steps: (A) A step in which mice are immunized on day 0 and day 28 by subcutaneous administration of a conjugate with a dose of 25 μg (O-antigen); (B) Step 42: Measure the anti-S. paratifi O-antigen antibody level by ELISA. Measured using an immunogenicity assay that includes, The immunogenic composition according to any one of claims 16 to 19.

21. below: (i) Salmonella tiphimurium (S. tiphimurium) antigens in which, optionally, the S. tiphimurium antigen contains or consists of S. tiphimurium GMMA; and / or (ii) Salmonella enteritidis (S. enteritidis) antigen, wherein the S. enteritidis antigen optionally contains S. enteritidis GMMA or consists of S. enteritidis GMMA. The immunogenic composition according to any one of claims 16 to 20, further comprising:

22. (a) S. tiphimurium GMMA and / or S. enteritidis GMMA contain detoxification lipid A; (b) S. ciphyllum GMMA and / or S. enteritidis GMMA are as follows: (i) a gene encoding a functional MsbB protein; and / or (ii) Genes encoding functional PagP protein Derived from S. tiphimulium and / or S. enteritidis, which do not contain; and / or (c) S. tiphimulium GMMA and / or S. enteritidis GMMA are derived from S. tiphimulium and / or S. enteritidis that do not contain the gene encoding the functional tolR protein. The immunogenic composition according to claim 21.

23. A vaccine comprising the immunogenic composition according to any one of claims 16 to 22.

24. An immunogenic composition or vaccine according to any one of claims 16 to 23, for use in a method for preventing infection.

25. Methods to prevent infection: (a) comprising administering an effective amount of the immunogenic composition or vaccine described in any one of claims 16 to 23 to a target; (b) Methods to prevent Salmonella infection; (c) A method for preventing invasive atypical Salmonella infection; and / or (d) Methods for preventing infection by S. thiphyllum, S. enteritidis, S. thiphyllum and / or S. parathiphyllum A, An immunogenic composition for use according to claim 24.

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