Vaccine

Abstract

The present invention relates to conjugates comprising polysaccharides comprising 3- deoxy-D-manno-actulosonic acid (KDO) moieties, particularly conjugates produced using random conjugation methods, methods for preparing such conjugates, immunogenic compositions and vaccines comprising the conjugates, and methods of treatment or medical uses using the compositions and vaccines.

Description

[0001] Vaccine Field of the invention The present invention relates to conjugates comprising polysaccharides comprising 3- deoxy-D-manno-actulosonic acid (KDO) moieties, particularly conjugates produced using random conjugation methods, methods for preparing such conjugates, immunogenic compositions and vaccines comprising the conjugates, and methods of treatment or medical uses using the compositions and vaccines. Background to the invention Typhoid fever is a bacterial disease caused by Salmonella enterica subspecies enterica serovar Typhi (Salmonella Typhi), a human host–restricted organism [Crump, 2019]. The disease occurs globally, affecting predominantly children and young adults but is endemic in the developing countries of Africa and Asia, while in the developed countries it is reported occasionally in travellers that recently returned from endemic countries [Smith, 2016]. The exact burden of typhoid fever is said to be grossly underestimated due to difficulties in establishing its diagnosis in endemic areas. In 2017, there were estimated 10.9 million cases of typhoid fever and 116.8 thousand deaths due to Salmonella Typhi. Similarly, the years of life lost (YLLs) attributed to typhoid fever were 8.3 million and disability-adjusted life-years (DALYs) were 8.4 million. Despite a decline in the disease burden due to improvement in water and sanitation, it still remains a significant public health problem [Global Burden of Disease, 2017]. The burden of typhoid fever is highest in school aged children and in the under-5 age group. Recent studies show that in the 5-9 years age group, the adjusted incidence of blood-culture-confirmed typhoid fever / 100,000 person-years of observation with 95% confidence interval ranged from 861 (599-1203) in Malawi to 3228 (2276-4757) in Bangladesh while in the 0-4 years ago group, it was 632 (398–965) and 2625 (1764–4244) in Malawi and Bangladesh respectively [Meiring,2021]. In the absence of prompt diagnosis and treatment, intervention, typhoid fever may require hospitalization and potentially fatal complications such as Typhoid Intestinal Perforation (TIP). In developing countries where typhoid fever is endemic, surgical intervention is often delayed thus further worsening the outcome of the disease [Contini, 2017]. Antimicrobial treatment of typhoid fever is hampered by the emergence of multidrug- resistant (MDR) typhoidal Salmonella, first identified in 1980 and defined as strains resistant to ampicillin, chloramphenicol and trimethoprim sulfamethoxazole. The emergence of resistant strains of the bacteria has been somewhat overcome with newer antimicrobials, but the challenge remains and hampers effective control of the disease [Radhakrishnan, 2018]. Similarly, S. Typhi clones harboring resistance to three first- line drugs (chloramphenicol, ampicillin, and trimethoprim-sulfamethoxazole) as well as fluoroquinolones and third generation cephalosporins, which are classified as Extremely Drug Resistant (XDR) has been reported in Asia [Klemm, 2018]. S. Paratyphi A resides in the human gut and its clinical manifestations are indistinguishable from Typhoid fever. S. Paratyphi A is ranked second as a causative agent of enteric fever, preceded only by Salmonella enterica serovar Typhi (S. Typhi). Enteric fever caused by S. Paratyphi A, or Paratyphoid fever was thought to be responsible for a comparatively smaller proportion of enteric fever cases. However, since the 1980s both the incidence and relative frequency of Paratyphoid fever have risen in Nepal, Pakistan, and Thailand. Moreover, the populous nations of India and China have reported substantial numbers of S. Paratyphi A cases. Non-endemic countries like the United States report an increasing trend of Paratyphoid fever especially, amongst 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.) Saccharides (or polysaccharides) from bacteria have been used for many years in vaccines. As polysaccharides are T-independent antigens, however, they are poorly immunogenic. Conjugation to a carrier can convert T-independent antigens into T- dependent antigens, thereby enhancing memory responses and allowing protective immunity to develop. The most effective polysaccharide vaccines are therefore based on glycoconjugates, and the prototype conjugate vaccine was against Haemophilus influenzae type b ('Hib') [e.g. see chapter 14 of Vaccines (2004) eds. Plotkin & Orenstein. ISBN 0-7216-9688-0]. Gram-negative bacteria are surrounded by an outer membrane that contains lipopolysaccharide. Lipopolysaccharides are a diverse group of molecules that act as endotoxins and elicit strong immune responses in mammals. Each lipopolysaccharide comprises three parts: an O-antigen (also referred to as the O-specific polysaccharide or O-polysaccharide), a core domain, and a lipid A domain. Antibodies against the O- antigen from a particular Gram-negative bacterium may confer protection against infection by that bacterium. Vaccines have therefore been envisaged that contain O- antigens conjugated to carrier proteins. For example, O-antigen-based conjugate vaccines have been proposed for various Salmonellae (e.g. serovars Salmonella Typhimurium and Salmonella Paratyphi A of Salmonella enterica); Shigella species; and Escherichia coli. In these vaccines, the O-antigen is linked to the core domain from the full- length lipopolysaccharide (i.e. the conjugated polysaccharide is a lipopolysaccharide without its lipid A domain). The polysaccharide is conjugated to the carrier via its core domain. Various methods for conjugating O-antigens from bacteria such as Salmonella Paratyphi A (S. Paratyphi A) have been considered. For example WO 2013 / 038375 proposes using a selective method in which the KDO moiety is linked to a linker or a carrier protein using direct (selective) reductive amination. Summary of the invention The present Examples demonstrate that conjugates produced by the direct (selective) reductive amination method of WO2013 / 038375 are not very stable. In particular, the Examples demonstrate that, when a S. Paratyphi A O-antigen-CRM197conjugate was formulated into a bivalent vaccine further comprising a Salmonella Typhi (S. Typhi) Vi- CRM197conjugate, the S. Paratyphi A O-antigen-CRM197conjugate made using the selective reductive amination method of WO2013 / 038375 was not stable. This is particularly concerning as infections by S. Paratyphi A and S. Typhi are particularly prevalent in developing countries, and in such countries it is particularly important for the supply chain that a vaccine be stable. The present Examples demonstrate, however, that if the S. Paratyphi A O-antigen is conjugated using a random conjugation method introducing multiple activated sites, the conjugate is significantly more stable and therefore much more suitable for use in a vaccine. Accordingly, a first aspect provides a conjugate comprising a polysaccharide comprising a 3-deoxy-D-manno-octulosonic acid (KDO) moiety conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide comprises more than one activated site. In a second aspect, there is provided a conjugate comprising an O-antigen conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide comprises more than one activated site. In a third aspect, there is provided a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the polysaccharide by 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide. In a fourth aspect, there is provided a conjugate comprising an O-antigen conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O- antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen. In a fifth aspect, there is provided a method for producing a conjugate comprising a polysaccharide comprising KDO moiety conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide. In a sixth aspect, there is provided a method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide. In a seventh aspect, there is provided a method for producing a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein comprising a step of: (i) activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide. In an eighth aspect, there is provided a method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O- antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen. In a ninth aspect, there is provided a conjugate obtainable by the method of the invention. In a tenth aspect, there is provided a conjugate obtained by the method of the invention. In an eleventh aspect, there is provided an immunogenic composition comprising the conjugate of the invention. In a twelfth aspect, there is provided a vaccine comprising the immunogenic composition of the invention. In a thirteenth aspect, there is provided a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of the invention to a subject. In a fourteenth aspect, there is provided a use of the immunogenic composition or vaccine of the invention for the manufacture of a medicament for use in a method of preventing an infection. Brief description of the Figures Figure 1 - Reaction scheme for the selective reductive amination method of WO2013 / 038375. Figure 2 - Reaction scheme for conjugation of S. Paratyphi A O-antigen to CRM197- ADH by NHS-EDAC chemistry. Figure 3 - Reaction scheme for conjugation of S. Paratyphi A O-antigen to CRM197by reductive amination with a step of random oxidation using periodate. Figure 4 - Reaction scheme for conjugation of S. Paratyphi A O-antigen to CRM197by a random CDAP chemistry approach. Figure 5 - Graphs showing the immunogenicity of various S. Paratyphi O-antigen conjugate vaccines. Mice were immunised as described in Example 8, and the immunogenicity of the vaccines measured by ELISA. Bars with hatching represent the antibody level 42 days after first immunisation and bars with no fill represent the antibody levels 28 days after first immunisation. Figure 6 - Published structure of the O-antigen (including the core domain) from S. Paratyphi A. Figure 7 - Structure of the Vi monomeric repeating unit. Figure 8 - Sequence listing. Figure 9 - Graph showing geometric mean concentrations (GMCs) of anti-Vi antibodies as measured by ELISA. The vaccines administered (and doses used) are as described in Example 9. Figure 10 - Graph showing geometric mean concentrations (GMCs) of anti-O:2 (S. Paratyphi O-antigen) antibodies as measured by ELISA. The vaccines administered (and doses used) are as described in Example 9. Figure 11 - Graph showing geometric mean titres (GMTs) of anti- S. Paratyphi O- antigen bactericidal antibodies as measured by SBA. The vaccines administered (and doses used) are as described in Example 9. Figure 12 - A) HPLC SEC of O:2[16kDa + 100kDa], O:2[16kDa], O:2[100kDa]; B) analytical characterization of the O:2 populations used for the conjugation with CRM197. Figure 13 - Summary graphs of anti-O:2 IgG geometric mean units (bars) and individual antibody levels (dots) are reported. Left-hand bars represent Day 27, and right-hand bars represent Day 42. A) Impact on the immune response in mice of S. Paratyphi A O:2 size: O:2-CRM197conjugates synthesized using O:2 with different sizes (16 kDa, 100 kDa and 16kDa + 100kDa) and with a similar O:2 / CRM197w / w ratio were compared. B) Impact of O:2 / CRM197w / w ratio: O:2-CRM197conjugates synthesized from O:2 of a certain size, but showing different O:2 / CRM197w / w ratio were compared. Figure 14 - A) Table showing O:2[16 kDa + 100 kDa]O-acetylation % after treatment with increasing concentration of ammonia from 5 mM up to 1 M . B) Treatment of O:2 with increasing concentration of ammonia allowed to obtain partially de-O-acetylated O:2. The graph reports the mathematical function, correlating the residual O:2 OAc level and ammonia concentration. Figure 15 - A) Impact of O:2-CRM197conjugates, differing for O-acetylation levels, on anti-O:2 IgG in mice. Left-hand bars represent Day 27, and right-hand bars represent Day 42. Five different levels of O-acetylation were compared for O:2 [16 kDa + 100 kDa] and two different levels for O:2[16 kDa]. Summary graphs of anti-O:2 IgG geometric mean units (bars with 95% CI) and individual antibody levels (dots) are reported; B) Linear regression analysis for Log EU(Day 42) vs OAc% with 95% CI. General Definitions Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs. In general, the term “comprising” is intended to mean including but not limited to. For example, the phrase “An immunogenic composition comprising a conjugate” should be interpreted to mean that the immunogenic composition comprises the conjugate, but the immunogenic composition may comprise further components. In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting of”. The term “consisting of” is intended to be limiting. For example, the phrase “An immunogenic composition consisting of a conjugate” should be understood to mean that the immunogenic composition has a conjugate and no further components. In some embodiments of the invention, the word “comprising” is replaced with the phrase “consisting essentially of”. The term “consisting essentially of” means that specific further components can be present, namely those not materially affecting the essential characteristics of the subject matter. The term “about” or “around” when referring to a value refers to that value but within a reasonable degree of scientific error. Optionally, a value is “about x” or “around x” if it is within 10%, within 5%, or within 1% of x. The singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “the GMMA” includes two or more instances or versions of such GMMA. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. A conjugate In some aspects, the invention relates to conjugates. The term “conjugate” refers to a molecule formed by a covalent linkage between an antigen and a carrier. The conjugates of the present invention comprise a polysaccharide. The term “polysaccharide” refers to any linear or branched polymer consisting of monosaccharide residues, usually linked by glycosidic linkages, and thus includes oligosaccharides. The carrier is generally a carrier protein. By “protein” we mean or include any linear or branched molecule consisting of amino acid residues. Polysaccharide comprising a KDO moiety and / or an O-antigen The conjugates of the invention comprise a polysaccharide comprising a KDO moiety and / or an O-antigen. Similarly, the methods of the invention are methods for producing conjugates comprising a polysaccharide comprising a KDO moiety and / or an O-antigen. A 3 KDO moiety has the following structure: Generally, the polysaccharides comprised within the conjugates comprise a KDO moiety at the reducing terminus. The KDO moiety may be used for direct selective coupling of the polysaccharide to a carrier protein. However, as discussed above, the present Examples demonstrate that when polysaccharides comprising a KDO moiety are conjugated using such a selective approach then the conjugates are not very stable. The conjugates may comprise an O-antigen (also referred to as OAg and O:2). The polysaccharide comprising a KDO moiety may be an O-antigen. The outer membrane of gram-negative bacteria comprises a lipopolysaccharide. This lipopolysaccharide comprises an O-antigen, which is linked via to the core domain to a lipid A domain. The terms “O-antigen” “OAg” and “O:2” refer to a polysaccharide made up of the O- antigen alone, or the O-antigen linked to the core domain of the lipopolysaccharide. Purification of these O-antigen-cores may be carried out using a method based on the phenol-water method of Westphal and Jann, first described in the 1960s (Westphal and Jann (1965) Methods Carbohydr. Chem. 5:83-91), followed by detoxification of the lipopolysaccharide with acetic acid or anhydrous hydrazine. The O-antigen is modified to remove the lipid A. For example, extraction and purification of polysaccharide can be performed by acetic acid hydrolysis as described in for example Watson et al., (1992) Infect Immun. 60(11):4679-86; Konadu et al. (1996) Infect Immun. (7):2709-l5; 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. Typically, the O-antigen is from the lipopolysaccharide of a Salmonella bacterium, e g. from Salmonella serogroups A, B or D, and particularly from Salmonella Paratyphi A. The O-antigens of Salmonella serogroups A, B and D have been described and are thought to share a common backbone: →2-α-D-Manp-(l →4)-α-L-Rhap-(l→3)-α-D- Galp-(1→. The serogroup specificity of Salmonella Paratyphi A is conferred by an α- 3,6-dideoxyglucose (α-D-paratose) linked (1→3) to the mannose of the backbone. The α-L-rhamnose of the backbone is partially O-acetylated at C-3 (Konadu et al. (1996) Infect Immun. (7):2709-l5). The published structure of the O-antigen and core domain from S. Paratyphi A is shown in Figure 6, including the KDO subunit and primary amine group (within a pyrophosphoethanolamine group) in the core domain. The O- antigen may also be from S. Typhimurium. The O-antigen may also be from S. Enteriditis. Naturally-derived O-antigens may contain structural variations compared to the published structures for these O-antigens. The O-antigen may also be from Shigella species, e.g. from S. flexneri. Other Shigella species that may provide the O-antigen used in the invention are S. sonnei, S. dysenteriae and S. boydii [Knirel et al. (2011) Glycobiology. (10): 1362-72]. The O- antigen and core domain may also be from E. coli, such as E. coli 0157. Other lipopolysaccharide-containing Gram-negative bacteria that may provide the O-antigen used in the invention are Klebsiella pneumonia [Chhibber et al. (2005) Indian JExp Biol. 43(l):40-5], Vibrio cholerae [Gupta et al. (1992) Infect Immun. 60(8):3201-8], Haemophilus influenzae and Neisseria meningitidis [Cox et al. (2005) Vaccine. 23(43):5045-54]. The polysaccharide and / or O-antigen may be chemically modified relative to the polysaccharide and / or O-antigen as found in nature. For example, the polysaccharide and / or O-antigen may be de-O-acetylated (partially or fully), but it is preferred for O- antigens not to be de-O-acetylated. If it takes place, then de-acetylation may occur before, during or after other processing steps, but typically occurs before any coupling step. The effect of de-acetylation etc. can be assessed by routine assays. For example, the relevance of O-acetylation on S. Paratyphi A O-antigen is discussed in Konadu et al. (1996) Infect Immun. (7):2709-l5. The native O-antigen of S. Paratyphi A is said in this document to have about 80% O-acetylation. Conjugated de-O-acetylated O-antigen did not elicit anti-lipopolysaccharide antibodies with bactericidal activity. Accordingly, when the O-antigen used in the present invention is S. Paratyphi A O-antigen, the O- antigen may have between 0 and 100% O-acetylation, but it is preferred for the O- antigen to be O-acetylated. The level of O-acetylation may depend on the bacterial strain that provided the O-antigen. For example, the degree of O-acetylation of the S. Paratyphi A O-antigen may be 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. Paratyphi A O-antigen is 55-85%, particularly 65-75%, or 45-80%, particularly 40-50% or 60-70%. However, higher degrees of O-acetylation, e.g. 70-100%, particularly 85- 95% are also typical in some strains. Therefore, the degree of O-acetylation of the S. Paratyphi A O-antigen may also be 60-100%, 70-100%, 80-100%, or 90-100%. Optionally, the degree of O-acetylation is based on the amount of Rhamnose (Rha). The degree of O-acetylation of the polysaccharide can be determined by any method known in the art, for example, by proton NMR {e.g. as described in Ravenscroft et al. Carbohydr Res. 2015 ;404:108-16, the Hestrin method

[0024] or HPAEC-CD

[0025] . O- acetyl groups may be removed by hydrolysis, for example by treatment with a base such as sodium hydroxide or anhydrous hydrazine [Konadu et al. (1996) Infect Immun. (7):2709-l5]. To maintain high levels of O-acetylation on the polysaccharide, treatments that lead to hydrolysis of the O-acetyl groups are minimised, e.g. treatments at extremes of pH. In some aspects, when the O-antigen used in the present invention is S. Paratyphi A O- antigen, the average molecular weight of the O-antigen is between 5 kDa and 120 kDa, between 10 kDa and 100 kDa, between 10 kDa and 80 kDa, between 10 kDa and 70 kDa, between 10 kDa and 60 kDa, between 10 kDa and 50, between 10 kDa and 40 kDa, between 10 kDa and 30 kDa, between 10 and 25 kDa, between 10 and 20 kDa, 11 kDa and 19 kDa, 12 kDa and 19 kDa, 13 kDa and 19 kDa, 14 kDa and 18 kDa, 15 and 18 kDa, or between 16 kDa and 18 kDa . Optionally, the O-antigen has a target molecular weight of between 16 and 18 kDa (e.g. it has been made by a method that typically generates O-antigen having a molecular weight within this range). The molecular weight of the O-antigen may be determined by HPLC-SEC. Conjugated to a carrier protein The conjugates of the invention comprise a carrier protein. Similarly, the methods of the invention are methods for producing conjugates comprising a carrier protein. In general, conjugation of polysaccharides to carrier proteins enhances the immunogenicity of the polysaccharides as it converts them from T-independent antigens to T-dependent antigens, thus allowing priming for immunological memory. Carrier proteins include bacterial toxins, such as diphtheria or tetanus toxins, or toxoids or mutants thereof. In some embodiments, the carrier protein is CRM197. The sequence of CRM197is provided in SEQ ID NO: 1. In some aspects, the polysaccharide of the invention is S. Paratyphi A O-antigen and the carrier protein is CRM197,and the O:2 / CRM197w / w ratio may be between 0.1 and 3.5, between 0.2 and 3.0, between 0.25 and 3.0, between 0.25 and 0.9, between 0.25 and 0.8, between 0.35 and 0.8, between 0.4 and 0.8, between 0.5 and 0.8, between 0.6 and 0.7, between 0.61 and 0.67 or between 0.62 and 0.66. The ratio may also be between 0.5 and 3.0, 0.7 and 3.0, 1.0 and 3.0, 1.5 and 3.0, 2.0 and 3.0, or 2.5 and 3.0. Random conjugation and activated sites In some aspects, the conjugates of the invention are conjugates comprising a polysaccharide and / or an O-antigen conjugated to a carrier protein by a random conjugation method. In some aspects, the methods of the invention are methods for producing a conjugate by a random conjugation method. The term “random conjugation method” refers to a method of conjugating a polysaccharide to a carrier protein which is not selective, i.e. a conjugation method in which one or both of (i) the number of linkages (i.e. the number of positions on the polysaccharide to which a carrier protein is linked) and (ii) the position of the linkages (i.e. where on the polysaccharide the linkages occur) is not predetermined by the chemistry used and may differ between conjugates produced using the same conjugation chemistry. For example, in a composition comprising multiple conjugate compounds, some of the conjugates may comprise 2 linkages (i.e. two linkages between the polysaccharide and the carrier protein or two carrier protein molecules being linked to the same polysaccharide) and other conjugates may comprise only 1. Similarly, in some cases some of the conjugates are conjugated at a first reaction site on the polysaccharide, but some of the conjugates are conjugated at a second reaction site on the polysaccharide. Suitable random conjugation methods include conjugation using 1- cyano-4-dimethylaminopyridine (CDAP) chemistry and using reductive amination using a step of random oxidation, for example using an agent such as sodium periodate. The polysaccharide or O-antigen may comprise more than one activated site or the method may comprise a step of introducing multiple activated sites into the polysaccharide or O-antigen. The term “activated site” is intended to refer to a site or functional group on the polysaccharide that has been activated by a step in a conjugation chemistry method such that it is primed to be conjugated to a carrier protein. For example, if the random conjugation method uses CDAP chemistry, the polysaccharide is “activated” by the addition of CDAP if the addition of CDAP introduces cyanoester groups. The CDAP activation introduces cyanoester groups at one or more sites, and the positions of these introduced cyanoester groups would be considered to be “activated sites”. Once the polysaccharide has been activated, it may be linked (conjugated) to a carrier protein at one or more (in some cases all) of the activated sites. For the purposes of the present invention, the term “activated sites” includes sites that have been activated and not linked to carrier protein and also sites that have been activated and are linked to a carrier protein. Optionally, the random conjugation method comprising a step of: (i) activating the polysaccharide or the O-antigen by CDAP chemistry to provide an activated polysaccharide or O-antigen; or (ii) oxidising the polysaccharide or the O-antigen to provide an oxidised polysaccharide or O-antigen. It is possible to determine the average number of activated sites of a polysaccharide. If the activated sites are introduced using CDAP chemistry, the number of activated sites may be measured using the ADH quenching / TNBS colorimetric method as reported in Lees A, Vaccines (Basel). 2020;8(4):777. If the activated sites are introduced by oxidation, the number of activated sites may be measured using microBCA reagents to measure the formed aldehydes or by HPAEC-PAD to detect the oxidized sugar ring; both options are reported in Stefanetti et al. Vaccine. 2014;32(46):6122-9. Optionally, the polysaccharide or the O-antigen comprises 1.5 or more, 2.0 or more, or 2.5 or more activated sites. Assuming that the polysaccharide or the O-antigen is part of a composition comprising multiple polysaccharide or O-antigen saccharides, the polysaccharide or O-antigen will comprise 1.5 or more activated sites if the average number of activated sites on each polysaccharide or O-antigen molecule is 1.5 or more. Optionally, the conjugates of the invention are part of a composition comprising multiple conjugates, and / or the methods of the invention produce a composition comprising multiple conjugates, and the average number of activated sites on the conjugates in the composition is greater than 1, optionally 1.5 or more, 2 or more, or 2.5 or more activated sites. In such cases different O-antigen molecules within the composition may have different numbers of activated sites. Conjugate further comprises a linker The conjugate may further comprise a linker. The methods of the invention may comprise conjugating an activated or oxidised polysaccharide to a linker and / or a carrier protein-linker compound. A linker is a compound that can be used to link a protein and a polysaccharide. Any suitable linker may be used in the conjugates and methods of the invention. Suitable linkers include an adipic acid dihydrazide (ADH) linker, which is a compound having the following structure: . Other suitable linkers include adipic acid, glutaric acid, carbonyl, β-propionamido (WO00 / 10599), adipic acid bis(N-hydroxysuccinimmide), dihydrazides analogous to ADH but with different chain lengths, hexamethylenediamine (or analogous diamines with different chain lengths), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat. Nos. 4,673,574; 4,761,283; and 4,808,700), 6- aminocaproic acid (U.S. Pat. No. 4,459,286), N- succinimidyl-3-(2-pyridyldithio)- propionate (SPDP) (U.S. Pat. No. 5,204,098), C4 to C12 moieties (U.S. Pat. No. 4,663, 160), etc. Activating the polysaccharide by CDAP chemistry In some aspects, the conjugate comprises a polysaccharide comprising a KDO moiety and / or an O-antigen that was conjugated to a carrier protein using a conjugation method comprising a step of activating the polysaccharide and / or the O-antigen by CDAP chemistry to provide an activated polysaccharide. In some aspects, the methods comprise a step of activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide. Activating the polysaccharide or O-antigen by CDAP chemistry comprises mixing the polysaccharide and / or the O-antigen with CDAP such that cyanoester groups are introduced into the polysaccharide or O-antigen. For example, Example 7 discloses a suitable method of activating an O-antigen by CDAP chemistry. Activating the polysaccharide and / or the O-antigen by CDAP chemistry results in an activated polysaccharide or O-antigen. Activating the polysaccharide and / or the O-antigen by CDAP chemistry introduces cyanoester groups, and so an activated polysaccharide and / or O-antigen comprises cyanoester groups that were introduced using CDAP. Similarly, a method comprises a step of activating a polysaccharide and / or an O-antigen by CDAP chemistry if the method comprises mixing the polysaccharide and / or the O- antigen with CDAP and the number of cyanoester groups present on the polysaccharide and / or the O-antigen present after the step of mixing with CDAP is higher than the number of cyanoester groups present on the polysaccharide and / or the O-antigen prior to that step. The number of cyanoester groups present may be measured using the ADH quenching / TNBS colourimetric method as reported in Lees A., Vaccines (Basel), 2020; 8(4):777. Optionally, activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises mixing the polysaccharide or the O-antigen with CDAP at a w / w ratio of between 0.05:1 and 5:1, between 0.1:1 and 5:1, between 0.2:1 and 2:1, or around 0.3:1 (CDAP to polysaccharide or O-antigen). Optionally, the step of activating the O-antigen and / or the polysaccharide by CDAP chemistry comprising mixing the polysaccharide or the O-antigen with CDAP takes place in a salt solution, such as a solution of NaCl or KCl. Optionally, the step of activating the O-antigen and / or the polysaccharide by CDAP chemistry comprising mixing the polysaccharide or the O-antigen with CDAP takes place in a solution of NaCl or KCl at a concentration between 50 mM and 1 M, between 100 mM and 250 mM, between 125 mM and 200 mM, or around 150 mM. Optionally, after the polysaccharide and / or the O-antigen has been mixed with the CDAP the pH is adjusted, optionally to a pH between 6 and 10, between 7 and 9, or between 9 and 10. Optionally, the pH is adjusted by adding a base, such as triethylamine, sodium hydroxide, or pyridine. Optionally, the pH is adjusted by adding between 5 % and 15%, between 8% and 12%, or around 10% (v / v) triethylamine. Optionally, after the polysaccharide and / or the O-antigen has been mixed with the CDAP, the mixture is incubated at a temperature between 18ºC and 30ºC, between 20ºC and 28ºC, room temperature, or around 25ºC. Optionally, the solution is incubated with stirring prior to conjugation of the activated polysaccharide or O-antigen to the carrier protein. A suitable method for activating the polysaccharide by CDAP chemistry is described in Example 7. Conjugating a polysaccharide activated using CDAP to a carrier protein or a linker As set out above, polysaccharides and / or O-antigens that have been activated using CDAP chemistry (activated polysaccharides or activated O-antigens) comprise cyanoester groups (at activated sites), and these cyanoester groups may be covalently linked to hydrazide or amino groups. Accordingly, polysaccharides and / or O-antigens that have been activated using CDAP chemistry may be linked directly to carrier proteins (via amino groups), or may be conjugated to a carrier protein via a linker comprising a hydrazide or amino group. Suitable linkers include the ADH linker described above. Accordingly, the methods may comprise reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on a carrier protein or a carrier-protein linker compound. The methods may comprise a step of reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on a carrier protein-linker compound. Thus, the method may further comprise steps to prepare the carrier protein-linker compound. For example, if the linker is an ADH linker, the method may comprise a step of preparing an ADH-carrier protein compound (such as an ADH-CRM197compound), for example as reported in Micoli et al. Vaccine 2011, 29, (4), 712-20. Reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on a carrier protein or a carrier-protein linker compound may comprise mixing the carrier protein or the carrier-protein linker compound with the activated polysaccharide or O- antigen under conditions suitable for a covalent bond to be formed between the cyanoester groups (activated sites) on the activated polysaccharide or O-antigen and the hydrazide / amino groups on the carrier protein or the carrier protein-linker compound. For example, it may be simply a case of mixing the activated polysaccharide or O- antigen with the carrier protein or the carrier protein-linker compound. Reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on the carrier protein or a carrier protein-linker compound may comprises mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound at a w / w ratio of between 0.1:1 and 5:1, between 0.2:1 and 3:1, between 0.5:1 and 2:1, or around 1:1, (polysaccharide or O-antigen to carrier protein or carrier protein-linker). Optionally, the step of mixing the activated polysaccharide or O- antigen with the carrier protein or the carrier protein-linker provides a conjugation mixture. Optionally, mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound takes place at a pH between 8 and 11, between 9 and 10, or around 9.5. Optionally, the pH is maintained at between 8 and 11, between 9 and 10, or around 9.5 for at least 1 hour, at least 2 hours, between 30 minutes and 10 hours, between 1 hour and 5 hours, or between 2 hours and 3 hours. Optionally, the pH is maintained using a base, such as triethylamine, sodium hydroxide, or pyridine. Optionally, the pH is maintained using triethylamine. Optionally, after the activated polysaccharide or O-antigen has been mixed and optionally after the mixture has been maintained at a pH between 8 to 11 for at least 1 hour, the method may further comprise a step of adding glycine solution (to quench the cyanoester groups). Optionally, the glycine solution is added in a concentration of 0.5M to 5M, 0.5M to 2M, or around 1M. Optionally, the glycine solution is added to a volume of the conjugation mixture with is substantially equal to the volume of the glycine solution. A volume is substantially equal to another volume if it is within 10%. Optionally, there is a further step of adjusting the pH using a base, such as triethylamine, sodium hydroxide, or pyridine. Optionally, this further step occurs after a step of adding a glycine solution. Optionally, the pH is adjusted to a pH between 7 and 9, or around 8. Optionally, there is an incubation step after the further step of adjusting the pH using a base. Optionally, the incubation step comprises incubating at a temperature below 15ºC, between 12ºC, below 10ºC, between 0ºC and 10ºC, or between 2ºC and 8ºC. Optionally, the incubation step takes place for between 10 and 30 hours, or between 10 and 20 hours. Once the activated polysaccharide or O-antigen has been mixed with the carrier protein or the carrier protein-linker compound under suitable conditions for conjugation to occur (such as those set out in the two preceding paragraphs), a conjugate will be formed. The method may further comprise a chromatography step to remove any unconjugated polysaccharide and / or O-antigen. Optionally, the chromatography step comprises hydrophobic interaction chromatography or anion exchange chromatography. A suitable method for conjugating a polysaccharide activated using CDAP to a carrier protein or a linker is described in Example 7. Oxidising the polysaccharide to provide an oxidised polysaccharide In some aspects, the conjugate comprises a polysaccharide comprising a KDO moiety and / or an O-antigen that was conjugated to a carrier protein using a conjugation method comprising a step of oxidising the polysaccharide and / or the O-antigen to provide an oxidised polysaccharide. In some aspects, the methods comprise a step of oxidising the polysaccharide to provide an oxidised polysaccharide. Oxidising the polysaccharide and / or O-antigen may be used as part of a random conjugation method, as an oxidation step may be used to introduce one or more activated (oxidised) sites at a variety of positions on the polysaccharide chain. Optionally, the step of oxidising the polysaccharide or O-antigen comprises mixing the polysaccharide or O-antigen with an oxidising agent. Suitable oxidising agents include periodate, for example sodium periodate. Optionally, mixing the polysaccharide or O-antigen with an oxidising agent comprises mixing the polysaccharide or O-antigen with an oxidising agent at a ratio of between 1 mg / ml: 10 mM and 100 mg / mL: 10 mM, between 1 mg / mL :10 mM and 50 mg / mL: 10 mM, between 1 mg / mL: 10 mM and 25 mg / mL: 10 mM, or around 10mg / mL: 10 mM (polysaccharide or O-antigen to oxidising agent). Optionally, mixing the polysaccharide or O-antigen with an oxidising agent occurs at a pH between 3 and 7, between 4 and 6, or around 5. Optionally, mixing the polysaccharide or O-antigen with an oxidising agent occurs in a buffer such as an acetate buffer. Suitably, the buffer is sodium acetate. Optionally, the step of oxidising the polysaccharide or O-antigen comprises incubating a mixture of the polysaccharide or O-antigen and the oxidising agent. Optionally, incubating the mixture of the polysaccharide or O-antigen and the oxidising agent takes place in the dark. Optionally, incubating the mixture of the polysaccharide or O-antigen and the oxidising agent takes place for between 1 and 10 hours, between 1 and 5 hours, between 1 and 3 hours, or around 2 hours. Optionally, incubating the mixture of the polysaccharide or O-antigen and the oxidising agent takes place at a temperature between 20ºC and 30ºC, between 22ºC and 28ºC, or around 25ºC, or at room temperature. The step of oxidising the polysaccharide or O-antigen may also comprise a step of quenching excess oxidising agent. The step of quenching excess oxidising agent may take place after the step of mixing the polysaccharide or O-antigen with an oxidising agent and / or the step of incubating the mixture of the polysaccharide or O-antigen and the oxidising agent, optionally in water. The step of quenching excess oxidising agent may comprise adding a quenching agent. If the oxidising agent is periodate, a suitable quenching agent is sodium sulphite (Na2SO3). Optionally, quenching excess oxidising agent comprises adding sodium sulphite to the mixture of the polysaccharide or O- antigen and oxidising agent and incubating the resulting mixture for at least 10 minutes, between 5 minutes and 30 minutes, or around 15 minutes at a temperature between 15ºC and 30ºC, between 20ºC and 28ºC, room temperature, or around 25ºC. The step of oxidising the polysaccharide or O-antigen may also comprise a step of purifying the oxidised polysaccharide or O-antigen. The step of purifying the oxidised polysaccharide or O-antigen may comprise desalting the mixture of the polysaccharide or O-antigen and the oxidising agent, for example using a PD10 column. The methods may also comprise a step of lyophilising the oxidised polysaccharide or O- antigen to provide a lyophilised oxidised polysaccharide or O-antigen. A suitable method for oxidising the polysaccharide is described in Example 6. Conjugating an oxidised polysaccharide to a carrier protein with or without a linker The methods may comprise a step of reacting the oxidised polysaccharide or O-antigen with the carrier protein or a carrier protein-linker compound comprising the carrier protein. The oxidised polysaccharide or O-antigen may be reacted with the carrier protein or the carrier protein-linker compound using reductive amination. For example, the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein with a reducing agent. Suitable reducing agents include cyanoborohydrides such as sodium cyanoborohydride, borohydrides such as sodium borohydride, or boranes such as amino borane, pyridine borane, dimethylborane or trimethylamine borane. In some embodiments, the reducing agent is a cyanoborohydride. In some embodiments, the reducing agent is sodium cyanoborohydride. Optionally, the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein or carrier protein-linker compound comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein or carrier protein-linker compound at a w / w ratio of between 0.5: 1 and 20: 1, between 1:1 and 10: 1, between 1.5:1 and 5:1, or around 2:1 (polysaccharide or O-antigen to carrier protein). Optionally, the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein or carrier protein-linker compound comprises mixing the carrier protein or the carrier protein- linker compound and the reducing agent at a w / w ratio of between 0.25:1 and 20: 1, between 0.5:1 and 10: 1, between 0.75:1 and 5:1, or around 1:1 (carrier protein or carrier protein-linker compound to reducing agent). Optionally, the step of mixing the oxidised polysaccharide or O-antigen with the carrier protein or carrier protein-linker compound results in a reaction mixture comprising the oxidised polysaccharide or O- antigen and the carrier protein or carrier protein-linker compound. The method may comprise incubating the reaction mixture for between 4 and 20 hours, between 6 and 15 hours, between 7 and 10 hours, or overnight. The method may comprise incubating the reaction mixture at a temperature between 30ºC and 42ºC, between 33ºC and 40ºC, between 35ºC and 39ºC, or around 37ºC. The method may further comprise quenching. Optionally, the quenching quenches residual oxidised sites. Optionally, the quenching comprises adding a quenching agent. Suitable quenching agents include sodium borohydride and lithium aluminium hydride. Optionally, the quenching agent is sodium borohydride and the w / w ratio of oxidised polysaccharide or O-antigen to sodium borohydride is between 10:1 and 0.1: 1, between 5:1 and 0.5: 1, or around 1:1 (w / w polysaccharide or O-antigen to sodium borohydride). Optionally, the conjugate is purified by chromatography such as hydrophobic interaction chromatography. A suitable method for conjugating an oxidised polysaccharide to a carrier protein or a linker is described in Example 6. Conjugates obtained / obtainable by the methods In some aspects of the invention there is provided a conjugate obtained by or obtainable by the methods of the invention. Such conjugates may comprise any of the features of the conjugates described here. For example, such conjugates may comprise S. Paratyphi A O-antigen and CRM197as the carrier protein. Immunogenic compositions The conjugates of the invention may be comprised within an immunogenic composition. The immunogenic composition may comprise a conjugate of the invention and additional components, such as a pharmaceutically acceptable excipient, an adjuvant, and / or further antigens. The immunogenic composition may further comprise a pharmaceutically acceptable excipient. Typical ‘pharmaceutically acceptable excipients’ include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers are typically large, slowly metabolised macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, sucrose, trehalose, lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable excipients may also contain diluents, such as water, saline, glycerol, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free, Tris-buffered physiologic saline is a suitable carrier particularly when using aluminium adjuvants since the phosphate in phosphate buffered saline may interfere with outer membrane vesicle binding to aluminium. However, in a particular embodiment, the immunogenic composition comprises phosphate buffered saline (and optionally an aluminium adjuvant as described further below). Optionally, the immunogenic composition comprises phosphate buffered saline at a pH between 6 and 7, for example pH 6.5. Immunogenic compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g. a lyophilised composition or a spray-freeze dried composition). The immunogenic composition may be prepared for topical administration e.g. as an ointment, cream or powder. The immunogenic composition may be prepared for oral administration e.g. as a tablet or capsule, as a spray, or as a syrup (optionally flavoured). The immunogenic composition may be prepared for pulmonary administration e.g. as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The immunogenic composition may be prepared for nasal, aural or ocular administration e.g. as drops. The immunogenic composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a mammal. Such kits may comprise one or more antigens in liquid form and one or more lyophilised antigens. Immunogenic compositions may be presented in vials, or they may be presented in pre-filled syringes. The syringes may be supplied with or without needles. A syringe will include a single dose of the composition, whereas a vial may include a single dose or multiple doses. Immunogenic compositions of the invention may be packaged in unit dose form or in multiple dose form. For multiple dose forms, vials are preferred to pre-filled syringes. Effective dosage volumes can be routinely established, but a typical human dose of the composition has a volume of 0.5ml e.g. for intramuscular injection. The composition will be sterile. Immunogenic compositions of the invention may be isotonic with respect to humans. Thus, immunogenic compositions of the invention may be useful as vaccines. Vaccines according to the invention may either be prophylactic (i.e. to prevent infection) or therapeutic (i.e. to treat infection), but will typically be prophylactic. Immunogenic compositions used as vaccines comprise an effective amount of antigen(s), as well as any other components, as needed. By “effective amount” (i.e. an immunologically effective amount), it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g. non-human primate, primate, etc.), the capacity of the individual's immune system to synthesise antibodies, the degree of protection desired, the formulation of the vaccine, the treating doctor's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials. Immunogenic compositions of the invention may include an antimicrobial, particularly when packaged in multiple dose formats. In some embodiments, the immunogenic composition comprises a conjugate of the invention at a dose of 1μg to 50μg, 10μg to 50μg, 20μg to 30μg, or around 25μg (polysaccharide or O-antigen). In some embodiments, the immunogenic composition comprises an fVi conjugate at a dose of 1μg to 50μg, 10μg to 50μg, 20μg to 30μg, or around 25μg (fVi). Adjuvants The immunogenic compositions of the invention may comprise an adjuvant. Any suitable adjuvant may be used. However, in some embodiments the adjuvant is a mineral salt, such as an aluminium salt or a calcium salt. Suitable mineral salts include hydroxides (e.g. oxyhydroxides), phosphates (e.g. hydroxyphosphates, orthophosphates), sulphates, etc. or mixtures of different mineral compounds, with the compounds taking any suitable form (e.g. gel, crystalline, amorphous, etc.), and with adsorption being preferred. The mineral containing compositions may also be formulated as a particle of metal salt. The adjuvants known as “aluminium hydroxide” are typically aluminium oxyhydroxide salts, which are usually at least partially crystalline. Aluminium oxyhydroxide, which can be represented by the formula AlO(OH), can be distinguished from other aluminium compounds, such as aluminium hydroxide Al(OH)3, by infrared (IR) spectroscopy, in particular by the presence of an adsorption band at 1070cm-1and a strong shoulder at 3090-3100cm-1[chapter 9 of ref. Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell & Newman) Plenum Press 1995 (ISBN 0-306-44867-X). The degree of crystallinity of an aluminium hydroxide adjuvant is reflected by the width of the diffraction band at 20 half height (WHH), with poorly-crystalline particles showing greater line broadening due to smaller crystallite sizes. The surface area increases as WHH increases, and adjuvants with higher WHH values have been seen to have greater capacity for antigen adsorption. A fibrous morphology (e.g. as seen in transmission electron micrographs) is typical for aluminium hydroxide adjuvants. The pI of aluminium hydroxide adjuvants is typically about 11 i.e. the adjuvant itself has a positive surface charge at physiological pH. Adsorptive capacities of between 1.8-2.6 mg protein per mg Al+++at pH 7.4 have been reported for aluminium hydroxide adjuvants. Optionally, the aluminium salt comprises or consists of aluminium phosphate and / or aluminium hydroxide. Optionally, the aluminium salt comprises or consists of aluminium hydroxide. In some embodiments, the immunogenic compositions comprise aluminium hydroxide at a dose of 0.05 mg to 1.00 mg, 0.05 mg to 0.50 mg, 0.30 mg to 0.40 mg, or around 0.375 mg (Al3+). Salmonella Typhi antigen The immunogenic composition may further comprise an antigen from Salmonella typhi (S. typhi). Optionally the antigen from S. Typhi is a Vi polysaccharide. The term “Vi” or “Vi polysaccharide” relates to the capsular polysaccharide of Salmonella enterica serovar Typhi purified from Citrobacter (Rondini et al., J. Infect. Dev. Ctries, 2012). Optionally, the Vi polysaccharide is a fragmented Vi polysaccharide (fVi). The term “fragmented” in reference to the Vi polysaccharide refers to the Vi polysaccharide having undergone size reduction thus reducing the number of repeating units in the polysaccharide. Fragmented Vi therefore has a lower average molecular weight compared to native Vi. For example, fragmented Vi may comprise 30 to 300 repeating units, compared to over 600 repeating units for native Vi. A structure of Vi monomeric repeating unit is shown in Figure 7. In the fragmented Vi preferably no changes in the structure of the repeating unit is observed compared to native Vi. This can be confirmed by 1H NMR analysis (see WO2015 / 068129). In addition, the percentage of O-acetyl groups in the fragmented Vi is preferably the same as the native Vi (i.e. about 95% O-acetylation) but may vary and decrease to about 65% O-acetylation. O- acetylation can be determined by standard measurements such as 1H NMR or the Hestrin colorimetric method. In its native size, the Vi polysaccharide has an average molecular weight measured by HPLC size exclusion chromatography (HPLC-SEC) of about 165kDa. In some embodiments, the fVi polysaccharide has an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa. Optionally, the fVi polysaccharide has a target molecular weight of between 51 kDa and 55 kDa (e.g. it has been made by a method that typically generates fVi having a molecular weight within this range). The molecular weight of the Vi polysaccharide may be determined by HPLC-SEC. Typically, the average molecular weight is calculated by running the sample on a TSK gel 3000 PWXL column, (30 cm x 7.8 mm; particle size 7 μm; cod. 808021) with a TSK gel PWXL guard column (4.0 cm x 6.0 mm; particle size 12 μm; cod. 808033) (Tosoh Bioscience) using dextrans as standards (5, 25, 50, 80, 150 kDa).The mobile phase is 0.1 M NaCI, 0.1 M NaH2PO4, 5% CH3 CN, pH 7.2, at the flow rate of 0.5 mL / min (isocratic method for 30 min). Void and bed volume calibration is performed with λ-DNA (λ-DNA Molecular Weight Marker III 0.12-21.2 kb; Roche) and sodium azide (NaN3; Merck), respectively. Fragmented Vi polysaccharide can further be 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. The fVi polysaccharide used in the present invention have certain average molecular weight (avMW) range distributions which can be further characterized in terms of polydispersity index (PDI). The polydispersity index is calculated as shown in the equation below: PDI = Mw / Mn where Mw is the weight average molecular weight and Mn is the number average molecular weight. The narrower the molecular weight distribution, the closer the PDI value is to 1. The fVi polysaccharide may have an avMW distribution characterised in that at least 80% of the pool has an avMW between 25 kDa and 70 kDa. It may have an avMW distribution characterised in that at least 50% of the pool has an avMW between 35 kDa and 60 kDa. It may have an avMW distribution characterised in that at least 30% of the pool has an avMW between 41 kDa and 55 kDa. Fragmentation of the Vi polysaccharide may be carried out by a number of methods known in the art such as chemical hydrolysis of the native polysaccharide, enzymatic fragmentation of the native polysaccharide, gamma irradiation of the native polysaccharide, or mechanical methods such as sonication, or high pressure homogenizer / microfluidizer / HPCDS (High pressure cell disruption system) of the native polysaccharide. The fragmentation method used in the present invention is selected such that it can yield fVi polysaccharide having an avMW of less than 90 kDa, less than 80 kDa, less than 60 kDa, or between 40 and 55 kDa. The method may also be selected such that there are no alterations to the repeating units' structure. Preferably, fragmentation is not by mechanical methods. Preferably, fragmentation is not by alkaline hydrolysis. The fVi polysaccharide may be obtained by chemical hydrolysis with hydrogen peroxide. Using this method, it was found that the Vi polysaccharide could be reduced in size without altering the repeating units' structure. Also, hydrolysis with hydrogen peroxide could enable the formation of fragmented Vi having a lower average molecular weight than when using mechanical methods. A suitable method for fragmenting Vi polysaccharide is set out in Example 2. The fVi polysaccharide may be part of an fVi conjugate comprising fVi and a carrier protein. Optionally, the carrier protein in the fVi conjugate is tetanus toxoid, CRM197, or diphtheria toxoid. Optionally, the carrier protein is CRM197. The fVi polysaccharide may be conjugated to the carrier protein via any suitable conjugation chemistry. Conjugation of the fVi polysaccharide to the carrier protein may be via a -NH2 group, e.g., through the side chain(s) of a lysine residue(s) or arginine residue(s) in the carrier polypeptide. Where the fVi polysaccharide has a free aldehyde group, this group can react with an amine in the protein to form a conjugate by reductive amination. Conjugation to the carrier may also be via a -SH group, e.g., through the side chain(s) of a cysteine residue(s) in the carrier polypeptide. Alternatively, the fVi polysaccharide may be conjugated to the carrier protein via a linker molecule. The fVi polysaccharide will typically be activated or functionalised prior to conjugation. Activation may involve, for example, cyanylating reagents such as CDAP (l-cyano-4-dimethylamino pyridinium tetrafluoro borate). Other suitable techniques use carbodiimides, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, TSTU (see, e.g., the introduction to WO 98 / 42721). Direct conjugation to the carrier protein may comprise oxidation of the fVi polysaccharide followed by reductive amination with the protein, as described in, for example, U.S. Pat No. 4,761,283 and U.S. Pat No. 4,356,170. Conjugation via a linker group may be made using any known procedure, for example, the procedures described in U.S. Pat No. 4,882,317 and U.S. Pat No. 4,695,624. Typically, the linker is attached via an anomeric carbon of the polysaccharide. A preferred type of linker is an adipic acid linker, which may be formed by coupling a free -NH2 group (e.g., introduced to a polysaccharide by amination) with adipic acid (using, for example, diimide activation), and then coupling a protein to the resulting saccharide-adipic acid intermediate (see, e.g., EP-B-0477508, Mol. Immunol, (1985) 22, 907-919, and EP-A-0208375). A similar preferred type of linker is a glutaric acid linker, which may be formed by coupling a free -NH group with glutaric acid in the same way. Adipic and glutaric acid linkers may also be formed by direct coupling to the polysaccharide, i.e., without prior introduction of a free group, e.g., a free -NH group, to the polysaccharide, followed by coupling a protein to the resulting saccharide-adipic / glutaric acid intermediate. Another preferred type of linker is a carbonyl linker, which may be formed by reaction of a free hydroxyl group of a modified polysaccharide with CDI (Bethell G.S. et al. (1979) J. Biol. Chem. 254, 2572-4 and Hearn M.T.W. (1981) J. Chromatogr. 218, 509-18); followed by reaction with a protein to form a carbamate linkage. Other linkers include β-propionamido (WO00 / 10599), nitrophenyl-ethylamine (Gever et al. (1979) Med. Microbiol. Immunol. 165, 171-288), haloacyl halides (U.S. Pat. No. 4,057,685), glycosidic linkages (U.S. Pat. Nos. 4,673,574; 4,761,283; and 4,808,700), 6- aminocaproic acid (U.S. Pat. No. 4,459,286), N- succinimidyl-3-(2-pyridyldithio)- propionate (SPDP) (U.S. Pat. No. 5,204,098), adipic acid dihydrazide (ADH) (U.S. Pat. No. 4,965,338), C4 to C12 moieties (U.S. Pat. No. 4,663, 160), etc. Carbodiimide condensation can also be used (WO2007 / 000343). A bifunctional linker may be used to provide a first group for coupling to an amine group in the polysaccharide (e.g., introduced to the polysaccharide by amination) and a second group for coupling to the carrier (typically for coupling to an amine in the carrier). Alternatively, the first group is capable of direct coupling to the polysaccharide, i.e., without prior introduction of a group, e.g., an amine group, to the polysaccharide. Optionally, the fVi conjugate is obtained by or obtainable by a method (i.e. a method for preparing an fVi conjugate) comprising the steps of: a. fragmenting Vi polysaccharide to obtain a fragmented Vi (fVi) polysaccharide having an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; b. reacting the fVi polysaccharide obtained in step a. with a carbodiimide and N- hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative; and c. reacting the N-hydroxysuccinimide ester fVi derivative obtained in step b. with the carrier protein (optionally derivatised carrier protein) to produce the fVi conjugate. Such a method is described in more detail in WO2015068129. The carrier protein may be derivatised by reacting it with a carbodiimide and a linker. 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, derivatising the carrier protein produces a derivatised carrier protein. Optionally, the carrier protein is CRM197and derivatising the carrier protein comprises one or more of the following steps: (i) providing CRM197is an appropriate buffer, optionally MES buffer; (ii) mixing the CRM197with EDAC at a ratio between 1:0.05 and 1:0.5, between 1:0.1 and 1:0.3, or around 1:0.15 (w / w CRM197to EDAC); (iii) mixing the CRM197with ADH at a ratio of between 1:1 and 1:6, between 1:2 and 1:4, or around 1:3.5 (w / w CRM197to ADH); (iv) incubating a mixture of CRM197and EDAC and optionally ADH for at least 30 minutes, or between 30 minutes and 2 hours, optionally with stirring; and (v) purifying derivatised CRM197, optionally by tangential flow filtration. In some embodiments, the carrier protein is derivatised by a method that comprises steps (i), (ii), and (iv). In some embodiments, the carrier protein is derivatised by a method that comprises steps (i), (ii), (iii), and (iv). In some embodiments, the carrier protein is derivatised by a method that comprises steps (i), (ii), (iv), and (v). In some embodiments, the carrier protein is derivatised by a method that comprises all of steps (i) to (v) above. In some embodiments, steps (i) to (v) above are performed in the order set out above, except that steps (ii) and (iii) may be performed simultaneously. The fVi conjugate may be obtainable or obtained by a method comprising a step of reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative. Optionally, the carbodiimide is EDC (N-3-dimethylamino propyl(-N-ethyl carbodiimide). Optionally, reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide comprises mixing the fVi with a carbodiimide such as EDC in the presence of N- hydroxysuccinimide (NHS). Optionally, reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide comprises mixing the fVi polysaccharide with NHS. Optionally, reacting the fVi polysaccharide with a carbodiimide and N- hydroxysuccinimide comprises mixing the fVi polysaccharide with NHS such that the NHS concentration is between 0.1 M and 0.5M, or around 0.33M, and the fVi polysaccharide concentration is between 1 mg / mL and 100 mg / ml, or around 50 mg / ml. Optionally, reacting the fVi polysaccharide with a carbodiimide and N- hydroxysuccinimide comprises mixing the fVi polysaccharide with EDC to have a molar ratio of EDC to fVi repeating unit or between 1:1 and 20:1, between 1:1 and 10:0, between 2:1 and 7:1, or around 5:1. Optionally, mixing the fVi polysaccharide with EDC is carried out after mixing the fVi polysaccharide with NHS. Optionally, reacting the fVi polysaccharide with a carbodiimide and N-hydroxysuccinimide comprises a step of incubating a mixture of fVi polysaccharide, NHS and EDC for at least 30 minutes, or around 1 hour at room temperature. Optionally, reacting the N-hydroxysuccinimide ester fVi derivative with the carrier protein (optionally the derivatised carrier protein) comprises mixing the N- hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative). Optionally, reacting the N-hydroxysuccinimide ester fVi derivative with the carrier protein (optionally the derivatised carrier protein) comprises mixing the N- hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) at a ratio of between (w / w) 1:0.1 and 1:10, between 1:0.5 and 1:5, between 1:0.75 and 1:2, or around 1:1. Optionally, mixing the the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) is carried out in a buffer at a pH between 5 and 7, or around 6. Optionally, mixing the N- hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) is carried out in MES buffer. Optionally, mixing the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative) is carried out at a temperature between 20ºC and 30ºC or around room temperature, optionally with mixing. The method for preparing an fVi conjugate may comprise one or more of the following additional steps, after the step of reacting the N-hydroxysuccinimide ester fVi derivative with the carrier protein (or carrier protein derivative): (i) quenching by addition of a quencher such as Phenyl HP buffer; (ii) filtering the fVi conjugate; (iii) purifying the fVi conjugate, optionally using hydrophobic interaction chromatography; (iv) concentrating the fVi conjugate, optionally by tangential flow filtration; and (v) filtering the conjugate, optionally using one or more 0.2μm filters. Optionally, the method for preparing an fVi conjugate comprises 2 or more, 3 or more, 4 or more, or all 5 of steps (i) to (v) above. Optionally, the method comprises step (i) above. Optionally, the method comprises steps (i) to (iii) above. Optionally, the method comprises steps (i) to (v) above. Optionally, the method comprises steps (i) to (iii) above in the order recited above. Optionally, the method comprises steps (i) to (v) above in the order recited above. A suitable method for conjugating the fVi polysaccharide to CRM197using EDAC chemistry via an ADH linker is set out in Example 2. Optionally, the immunogenic composition comprises (i) a conjugate comprising O- antigen from S. Paratyphi A and CRM197and (ii) a conjugate comprising fVi polysaccharide and CRM197at a (w / w) ratio of between 1:0.1 and 1:10, between 1:0.5 and 1:5, between 1:0.75 and 1:3, or around 1:1 (O-antigen to fVi). Stability assay The stability of the conjugates of the invention (optionally within an immunogenic composition of the 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 for 4 weeks at 37ºC; (ii) prepare a post-incubation sample of the incubated immunogenic composition and remove the conjugated polysaccharide or O-antigen by deoxycholate precipitation; and (iii) determine the amount of free polysaccharide or O-antigen in the post- incubation sample as a percentage of the amount of total polysaccharide in the post-incubation sample. Incubating the immunogenic composition for 4 weeks at 37ºC is a simple step in which the immunogenic composition is simply maintained at 37ºC for 4 weeks. For example, if the immunogenic composition comprises a conjugate comprising O-antigen conjugated to CRM197and an fVi conjugate, the user will transfer a sample of that immunogenic composition to an appropriate vessel (such as a syringe with a single dosage) and store it at 37ºC. The post-incubation sample(s) may be a sample of any suitable size (i.e. any size that allows the user to accurately detect the amount of free polysaccharide or O-antigen in the immunogenic composition). The amount of free polysaccharide or O-antigen in the post-incubation sample may be determined by removing the intact conjugate and measuring the amount of polysaccharide or O-antigen remaining. This involves deoxycholate precipitation. The deoxycholate will precipitate out the carrier protein, and any polysaccharide or O- antigen that is still part of a conjugate will be precipitated out with the carrier protein. The precipitated protein may be removed by centrifugation. In an alternative to the deoxycholate precipitation, the conjugate (carrier protein, and any polysaccharide or O-antigen that is still part of a conjugate) can be trapped in a C4 solid phase extraction disposable column eluting the free polysaccharide with 20% acetonitrile with 0.05% TFA solution. The user should then examine the amount of polysaccharide or O-antigen that is remaining in the sample (post-incubation) once the polysaccharide or O-antigen that is still part of a conjugate has been removed. This may be achieved using a Phenol- sulphuric assay or preferably high performance anion exchange chromatography with pulsed amperometric detection (HPAED-PAD). The Phenol-Sulphuric Assay quantifies the total amount of detectable sugar in the sample as hexose weight equivalent (glucose calibration curve). The assay uses concentrated sulphuric acid and phenol. With the addition of sulphuric acid the temperature raises due to acid hydratation and the polysaccharide is hydrolysed to corresponding sugar monomers. In these conditions hexose monosaccharides (glucose standard or the ones coming from O:2 sample hydrolysis) form hydroxymethyl furfurals that react with phenol resulting in the production of chromophores (Molisch reaction). Sample and glucose standard solutions absorbance at 490 nm is read using a spectrophotometer. Glucose standard solution at several dilutions (0, 25, 50, 75, 100 μg / mL) is treated as the sample in the assay and used as a calibration curve. HPAED-PAD can be used to detect the amount of detectable sugars, such as the amounts of the rhamnose (rha), galactose (gal), glucose (glc) and mannose (man) of the S. Paratyphi A O-antigen repeating unit. Commercial monomer sugars may be used to build calibration curves. A suitable HPAED-PAD method is described in more detail in PLoS One. (2012): 7(11):e47039. For example, the following method may be used to detect amounts of the S. Paratyphi O-antigen. For detecting amounts of Rha, Gal, Glc in the S. Paratyphi A OAg samples, diluted to have the sugar monomer in the range 0.5–10 μg / mL, are hydrolyzed at 100°C for 4 h in 2 M TFA. After the hydrolysis, samples may be chilled at 2–8°C for about 30 min, dried by SpeedVac overnight, reconstituted in water and filtered using 0.45 μm Acrodisc (PALL) filters before chromatographic analysis. HPAEC-PAD may be performed with a Dionex ICS3000 equipped with a CarboPac PA10 column (4×250 mm) coupled with PA10 guard column (4×50 mm). Separation of the sugars may be performed with a flow rate of 1 mL / min eluting in 18 mM NaOH over 20 min. After washing for 20 min with 100 mM AcONa in 28 mM NaOH, the column may be re-equilibrated with 18 mM NaOH for 20 min. The effluent may be monitored using an electrochemical detector in the pulse amperometric mode with a gold working electrode and an Ag / AgCl reference electrode. The Dionex standard quadruple-potential waveform for carbohydrates may be used. The resulting chromatographic data may be processed using Chromeleon software 6.8. Calibration curves may be built for each sugar monomer (0.5–10 μg / mL). The standards may be hydrolysed and analysed in the same way as samples. Once the amount of free polysaccharide or O-antigen has been determined, the user can then calculate the amount of free polysaccharide or O-antigen in the sample as a percentage of the total amount of polysaccharide in the sample. The skilled person should know the concentration of conjugate in the immunogenic composition, and can readily calculate the amount of conjugate in the sample by determining the volume of the conjugate in the sample and multiplying the volume by the concentration. Stability The conjugates of the invention have increased stability compared to conjugates of the invention prepared using non-random conjugation methods. The conjugate may be stable in the immunogenic composition for at least 4 weeks (at 37ºC). For example, an immunogenic composition of the invention may comprise a conjugate of the invention (comprising a polysaccharide comprising a KDO moiety or an O-antigen) and an fVi conjugate, and in these embodiments the conjugate of the invention will be stable in the immunogenic composition in the presence of the fVi conjugate. For example, the immunogenic composition may comprise a conjugate comprising an S. Paratyphi A O-antigen and CRM197and an fVi conjugate, and the conjugate comprising an S. Paratyphi O-antigen and CRM197is stable in the composition for at least 4 weeks. Optionally, the conjugate (comprising the polysaccharide comprising a KDO moiety or the O-antigen) is stable in the immunogenic composition for at least 4 weeks if less than 20%, less than 18%, less than 17%, less than 15%, between 0% and 20%, or between 1% and 15% of the polysaccharide or O-antigen is released from the conjugate in 4 weeks. The amount of polysaccharide or O-antigen released from the conjugate in 4 weeks may be determined using a stability assay as set out above. Optionally, the conjugate (comprising the polysaccharide comprising a KDO moiety or the O-antigen) is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is substantially less than an equivalent immunogenic composition in which the conjugate comprises an S. Paratyphi O-antigen conjugated to CRM197 using direct reductive amination via an ADH-SIDEA linker. By “equivalent” we mean otherwise identical. For example, the equivalent immunogenic composition should contain the same excipients etc. as the immunogenic composition, and the only difference should be the nature of the conjugate in the immunogenic composition. Optionally, the conjugate is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is similar to the amount released in an equivalent reference immunogenic composition in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM197using CDAP chemistry. Optionally, similar to is within 25%, within 15%, or within 10% of. Immunogenicity The Examples demonstrate that the conjugates of the invention are not only stable, but also have good immunogenicity. Thus, in some embodiments, the immunogenic composition induces a similar level of anti-polysaccharide or anti-O-antigen antibodies compared to an equivalent immunogenic composition 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 a similar level of anti-polysaccharide or anti-O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises the polysaccharide or O-antigen conjugated to a carrier protein using selective reductive amination, such as the reductive amination method described in Example 1. In some embodiments, the immunogenic composition induces a similar level of anti-S. Paratyphi O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises an S. Paratyphi O-antigen conjugated to CRM197using direct reductive amination via an ADH-SIDEA linker. An immunogenic composition should be considered to be one that “induces” antibodies if it is capable of inducing antibodies, i.e. if the immunogenic composition has the structural features of an immunogenic composition that can induce antibodies if it is administered to a mammal such as a mouse. Whether an immunogenic composition “induces” antibodies may be determined by administering a sample of the immunogenic composition to a mouse and determining whether the relevant antibodies are raised. Optionally, a similar level is a level within 25%, within 15%, or within 10%. Optionally, a user may determine whether the immunogenic composition is one that induces anti-polysaccharide or anti-O-antigen antibodies by measuring the level of anti- polysaccharide or anti-O-antigen antibodies induced using the following immunogenic assay: (i) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 μg (polysaccharide); (ii) measure the anti-polysaccharide antibody level by ELISA at day 42. If the immunogenic composition comprises an S. Paratyphi A O-antigen, then the level of anti-O-antigen antibodies induced may be measured using the following immunogenic assay: (i) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 μg (O-antigen); (ii) measure the anti-O-antigen antibody level by ELISA at day 42. A suitable ELISA assay involves: - coating ELISA plates with the polysaccharide or O-antigen; - taking serum samples from the immunised mice at day 42 (42 days after first immunisation), and applying them to the plates; and - measuring the antibody titers by adding an anti-mouse Fc antibody conjugated with an enzyme and the color substrate. A suitable ELISA assay is described in WO213 / 038375. Further antigens The immunogenic composition may comprise further antigens. Optionally, the further antigens comprise Salmonella antigens. Optionally, the further antigens comprise a Salmonella Typhimurium antigen, and / or a Salmonella Enteritidis antigen. Optionally, the Salmonella Typhimurium antigen, and / or the Salmonella Enteritidis antigen are outer membrane vesicles. Optionally, the immunogenic composition comprises S. Typhimurium GMMA and / or S. Enteritidis GMMA. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA comprise detoxified lipid A. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from bacteria that do not comprise a gene encoding a functional MsbB and / or PagP protein. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from bacteria that are modified to at least partially delete the msbB and / or pagP gene. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are ∆msbB and / or ∆pagP. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from bacteria that are hyperblebbing. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from bacteria that do not comprise a gene encoding a functional TolR protein. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from bacteria that are modified to at least partially delete the tolR gene. Optionally, the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are ∆tolR. Medical uses and methods of treatment In a further aspect of the invention, there is provided an immunogenic composition of the invention for use in a method of preventing an infection. In a further aspect of the invention, there is provided a method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of the invention to a subject. In a further aspect of the invention, there is provided a use of the immunogenic composition or vaccine of the invention for the manufacture of a medicament for use in a method of preventing an infection. The method of preventing an infection may comprise administering an effective amount of the immunogenic composition or vaccine of the invention to a subject. The method of preventing an infection may be a method of preventing Salmonella infection, optionally an invasive non-typeable Salmonella infection. Optionally the method of preventing an infection is a method of preventing infection by S. Typhimurium, S. Enteritidis, S. Typhi and / or S. Paratyphi A. The term “preventing Salmonella infection” in the method / immunogenic composition for use / use of the immunogenic composition in the manufacture of a medicament of the invention comprises raising an immune response in a subject. The immune response may be protective and may raises antibodies, such as IgG antibodies. The subject of the invention is a mammal, optionally a human. Where the vaccine is for prophylactic use, the human may be an adult i.e. subject is 18 years old or above 18 years old. Where the vaccine is for prophylactic use, the human may be a child i.e. below 18 years old. Where the vaccine is for prophylactic use, the child may be between 12 to 72 months, preferably between 24 to 59 months, more preferably between 6 to 12 months. Where the vaccine is for prophylactic use, the child may be around 9 months. Where the vaccine is for therapeutic use, the human is preferably a child. A vaccine intended for children may also be administered to adults e.g. to assess safety, dosage, or immunogenicity. Examples Example 1 - Production of S. Paratyphi OAg – CRM197conjugates via an ADH-SIDEA linker through reductive amination Conjugates of the S. Paratyphi A O-antigen (also referred to as O:2) and CRM197(via an ADH-SIDEA linker) were produced using the following protocol. The method used for producing S. Paratyphi A O antigen-CRM197 conjugates via an ADH-SIDEA linker through reductive amination is described in detail in WO2013 / 038375. In brief, S. Paratyphi O-antigen (OAg) is solubilized in 100 mM AcONa pH 4.5 at a concentration of 20–40 mg / mL, ADH and then NaBH3CN are added in sequence as solids to have a ratio of OAg / ADH / NaBH3CN 1 : 2 : 2 w / w / w. After mixing the solution at 30 °C for 1 hour (h), the reaction mixture is desalted against water using a G- 25 column. Resulting OAg-ADH is dried, solubilized in water / DMSO 1:9 (v / v) at a concentration of about 50 mg / mL (O-Ag), triethylamine (TEA) and SIDEA (see Figure 1 for the SIDEA structure) are added to have an NH2(OAg) : TEA : SIDEA molar ratio of 1 : 5 :12, and the reaction is kept at room temperature for 3h. Adding twice the reaction volume of 100 mM citrate buffer pH 3, unreacted SIDEA precipitates and is discarded after centrifugation. OAg-ADH-SIDEA is isolated from the reaction mixture by precipitation adding ethanol up to 80% (v / v) and is recovered after centrifugation. The pellet is washed twice with 100% EtOH and dried under vacuum. OAg-ADH- SIDEA is solubilized in phosphate buffer pH 7.2 with CRM197to give a protein concentration of 20 mg / mL and a molar ratio of active ester groups on OAg-ADH- SIDEA to CRM197of 30 to 1. The reaction mixture is mixed at room teemperature for 2h and the conjugate is purified by Size Exclusion Chromatography (SEC) or by Hydrophobic Interaction Chromatography (HIC). Example 2 - Production of Salmonella Typhi Vi-CRM197conjugate and formulation of bivalent composition (S. Typhi and S. Paratyphi A) The following protocol was used to produce a bivalent composition comprising an S. Paratyphi A OAg-CRM197conjugate made as described above and a conjugate of fragmented Vi polysaccharide from S. Typhi (fVi) conjugated to CRM197. 1) Fragmentation of Vi Polysaccharide: Step 1: Fragmentation Reaction and Quenching: Vi-Polysaccharide fragmentation is achieved by an Oxidation-reaction using hydrogen peroxide in the presence of iron sulphate. The reaction is quenched with EDTA (Ethylenediaminetetraacetic acid). Native Vi polysaccharide is diluted with WFI. A calculated volume of 10 mM FeSO4 and H2O2is added to get a final concentration of 0.5 mM FeSO4and 0.5% v / v H2O2in the reaction mixture respectively. The reaction mixture is incubated at 15±5°C for 120 + / - 10 min. The reaction is stopped by adding equal volumes of 250 mM EDTA to get a final EDTA concentration of 10 mM and is stirred. Step 2: Buffer Exchange: Buffer exchange is performed by Tangential Flow Filtration (TFF) with 100mM Sodium phosphate (pH: 7.2 ± 0.2) using 30 kDa cassettes to remove residual H2O2. Fragmented Vi (fVi) polysaccharide is concentrated. Step 3: Stabilization of fVi polysaccharide: Post fragmentation, 30 kDa retentate is stabilized by incubating at 80±5° C for 120 + / - 15 min. Step 4: fVi Purification by Anion Exchange (Resin: Capto-Q): AA chromatography step is used to separate the desired molecular size of fVi polysaccharide (25-70 KDa). This is performed using a linear gradient elution, with Capto-Q Buffer A and Capto-Q Buffer B using a Capto-Q Resin which has the binding capacity of 13 mg of fVi / mL. The eluted fractions are collected based on the conductivity for every 1 mS / cm; i.e., from 35 to 50 mS / cm and estimating the Molecular size distribution by SEC / HPLC. The Capto- Q fractions are pooled based on the Molecular Size (kDa) distribution. Step 5: Desalting: Pooled Capto-Q fractions are concentrated by Tangential Flow Filtraion (TFF) using a 10 kDa Cut-off cassette and then dia-filtered using WFI until permeate conductivity reaches ≤ 30 μS / cm. Step 6: 0.2 μm Filtration of fVi polysaccharide: The fVi polysaccharide is filtered through a 0.22 μm filter. The purified fVi polysaccharide is stored in PETG bottles. 2) CRM197 Derivatization: Step1: Thawing of CRM197: Purified CRM197 is thawed at 2-8°C prior to buffer exchange with 100 mM MES (Morpholino Ethanesulfonic acid) buffer. Post thawing, CRM197is filtered using 0.5 μm filter. Step 2: Buffer Exchange with 100 mM MES Buffer: Buffer exchange is performed by TFF with 100 mM MES buffer (pH 6.0±0.2) using 10 KDa cassette after CRM197thawing. Step 3: CRM197Derivatization: The required concentration of CRM197is diluted with 100 mM MES buffer followed by addition of calculated quantity of ADH (Adipic Acid Dihydrazide) and EDAC (1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide) to make a CRM : ADH : EDAC 1 : 3.5 : 0.15 w / w / w ratio. After addition of ADH and EDAC, the CRM197the reaction mixture is incubated at room temperature under mixing conditions for 60 + / - 15 min. At the end of the reaction an equal volume of 5 mM MES buffer (pH 7.0±0.2) is added. Step 4: Purification of CRM197: Post reaction, CRM197is purified by TFF using a 10 KDa cassette with 5 mM MES buffer. Step 5: Filtration of Dia-Filtered CRM197: 0.2-micron filtration is performed for dia- filtered CRM197solution followed by storage at 2-8°C in glass bottle. 3) Conjugation of Fragmented Vi Polysaccharide with Derivatized CRM197: Activation of fVi polysaccharide: Step 1: fVi polysaccharide drying by Rota Vapor: fVi is further concentrated by drying at 30°C using rotavapor. Concentrated fVi polysaccharide is reconstituted by using 100 mM MES buffer (pH: 6.0) in order to get a 50 mg / mL concentration. Step 2: Activation of fVi polysaccharide with NHS: fVi carboxylates (-COOH) are activated with EDC (N-3-Dimethylamino propyl-N Ethyl Carbodimide) in the presence of N-hydroxysuccinimide (NHS), by forming an active ester intermediate, to increase the efficiency of conjugation with CRM197previously activated with ADH. The dried fVi polysaccharide is re-constituted to a desired concentration (50 mg / mL) with 100 mM MES buffer (pH: 6.2±0.2); and activated in the presence of NHS (concentration of 0.33 M) followed by EDAC addition to have an EDAC / fVi RU molar ratio of 5 :1. EDAC solution is added after addition of NHS to ensure complete dissolution. The reaction is incubated at room temperature with slow mixing for 1 h. Conjugation: Step 1: Conjugation of fVi with CRM197-ADH: The conjugation reaction creates a covalent bond between the activated fVi and CRM197-ADH. The activated and derivatized reaction mixture is diluted with 100mM MES pH: 6.0 and CRM197-ADH is added in a w / w ratio of 1 : 1 (fVi : CRM197) to reach the final fVi concentration of the activated fVi and CRM197-ADH of 5 mg / mL. The conjugation reaction is performed at room temperature with slow mixing until protein consumption is ≥ 70% measured by HPLC-SEC at 280 nm absorbance. Step 2: Quenching and conditioning of Conjugation Reaction: The conjugation reaction is quenched by adding equal volume of Phenyl HP Buffer-B Tris 50 mM pH: 8.0. NaCl as powder is added to reach a final salt concentration of 3 M. Step 3: Conjugation Mixture Filtration: The fVi-CRM197crude conjugate is 0.65 filtered. Step 4: Purification of fVi-CRM197Crude Conjugate: Purification of the conjugate from the conditioned reaction mixture is performed through a HIC Phenyl Sepharose High Performance (HP) column. Column integrity is performed for every 5-10 cycles as per standard procedure. The column is equilibrated by using Phenyl Sepharose HP Buffer A Tris 50 mM NaCl 3M pH 8. After addition of conditioning buffer and NaCl, crude conjugate is loaded on to the column. Column washing is done using Phenyl Sepharose HP Buffer-A followed by product elution using Phenyl Sepharose HP Buffer-B Tris 50mM pH: 8. Fractions are collected and stored at 2-8ºC till further usage. All the fractions from multiple runs are pooled. Step 5: Concentration and Buffer Exchange using PBS: Purified conjugate is concentrated by TFF using a 50 kDa Cut-off cassette and then buffer exchanged using PBS buffer until the permeate conductivity meets PBS buffer conductivity. Step 6: Pre Filtration of fVi-CRM197 Conjugate using 0.2 μm filter: fVi-CRM197 conjugate is filtered through a 0.2 μm filter for bioburden reduction. Step 7: Sterile Filtration of fVi-CRM197Conjugate using 0.2 μ cellulose acetate filter: The fVi-CRM197conjugate is filtered through a 0.2 μm cellulose acetate filter. The purified fVi-CRM197conjugate is sampled and stored at 2-8°C. Preparation of the bivalent composition Bivalent formulations with or without an aluminium adjuvant were prepared according to the following protocols. Not adsorbed formulation: (containing 25 ^g fVi, 25 ^g O:2 (O-antigen from S. Paratyphi A, 4 mg 2-PE per dose) Saline solution containing 2-PE is dispensed aseptically into the formulation vessel, the appropriate volume of fVi conjugate antigen (as prepared in Example 2) is added (depending on the number of doses required – see header for dosage) and the solution is stirred for 15-20 minutes. A calculated volume of S. Paratyphi O-Antigen (as prepared in Example 1) is added (depending on the number of doses required – see header for dosage) and the solution is stirred for 15-20 minutes. pH is checked and adjusted to 6.5 ± 0.2. Stirring is continued for 15-20 minutes. The formulated solution is filtered using sterilized 0.22 μm filter into another sterilized glass bottle aseptically. Adsorbed formulation: (containing 25 ^g fVi, 25 ^g O:2, 0.375 mg Al3+, 4 mg 2-PE per dose) Saline solution with 2-PE is dispensed aseptically into the formulation vessel, the volume of Vi Antigen is added and the solution is stirred for 15-20 minutes. Calculated volume of O:2 Antigen is added and the solution is stirred for 15-20 minutes. The pH is checked and adjusted to 6.5 ± 0.2. Stirring is continued for 15-20 minutes. The pre blend is filtered using sterilized 0.22 μm filter into another sterilized glass bottle aseptically. The Aluminium hydroxide gel is added to filtered intermediate bulk. The container is stirred for 60 to 90 minutes. Example 3 - Stability study on the bivalent composition produced prepared in Example 2 Samples of a bivalent not adsorbed formulation, composed of fVi-CRM and O:2-CRM (O:2ADH-SIDEA-CRM as per example 1) of the bivalent composition were stored in a fridge at 2-8ºC for 12 months, and the presence of free Paratyphi OAg was tested at 0 months, 3 months, 6 months, 9 months and 12 months using the following assay. To determine the percentage of free OAg, a phenol sulfuric colorimetric assay is used, performed after protein (conjugate) precipitation with Deoxycholate in acidic conditions. The phenol-Sulfuric Assay quantifies the total amount of detectable sugar in the 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 raises due to acid hydration and the polysaccharide is hydrolized to corresponding sugar monomers. In these conditions hexose monosaccharides (glucose standard or the ones coming from O:2 sample hydrolysis) form hydroxymethyl furfurals that react with phenol resulting in chromophors (Molisch reaction). Sample and glucose standard solutions absorbance at 490 nm is read using a spectrophotometer. Glucose standard solution at several dilutions (0, 25, 50, 75, 100 μg / mL) is treated as the sample in the assay and used as a calibration curve. Table 1 shows the change in free OAg (as a percentage to total OAg) over 12 months. Table 1 0 months 3 months 6 months 9 months 12 months 6.59 22.67 21.69 30.05 32.27 Example 4 - Investigations into the source of the instability Various steps of the reductive amination conjugation chemistry (see Figure 1) were investigated to see whether they were the source of the conjugate instability as summarised briefly below. 1. The stability of the Paratyphi A OAg was assessed using an accelerated stability study performed at 37ºC over 4 weeks of time.1H NMR and HPLC-SEC data indicated that Paratyphi A OAg sugar composition and chain length did not change over time. 2. The purity of the sodium cyanoborohydride reagent as well as its decomposition kinetic in reaction conditions were assessed using NMR. The NMR spectra indicated that the sodium cyanoborohydride reagent did not contain any significant impurities and a suitable residual quantity is still present after 5h in reaction conditions, and so it seems that impurity of the sodium cyanoborohydride reagent and its decomposition was not the source of the instability (due to incomplete reduction of imine bond in the intermediate of reaction). 3. The efficiency of the step of linking the ADH to the Paratyphi OAg was investigated by TNBS colorimetric method, which indicated that that the OAg-ADH link was present after this step (92% of the composition was OAg-ADH). 4. Whether the C=N bond (see Figure 1) was fully reduced in the sodium cyanoborohydride reduction step was assessed using a HPLC-SEC method measuring absorption at 252 nm. More specifically, the incomplete reduction of the C=N bond leaves on the OAg a chromophore that has a characteristic absorbance at 252 nm formed by alpha-keto-acid (KDO) and hydrazide (ADH) groups and can be easily seen by HPLC-SEC. It seems that incomplete reduction of the C=N bond was not the source of the instability. 5. The stability of the Paratyphi OAg-ADH linkage was assessed. Specifically, OAg- ADH samples in Falcon tubes at different pHs and in different buffers (see Table 2) were incubated at 37ºC for 4 weeks, and samples were taken at 0 weeks, 2 weeks and 4 weeks. The level of free ADH was assessed using the following assay. The unbound (free) ADH was quantified by HPLC-SEC (eluent: sodium phosphate 100 mM, sodium chloride 100 mM pH 7.2 with 5% Acetonitrile) using a TOSOH TSKGEL- 3000PW-XL column. The ADH peak in the sample is quantified by comparison of the ABS 214 nm with a ADH standard calibration curve. Table 2: free ADH percentage in OAg-ADH samples put in accelerated stabilities at 37°C at different pHs Time OAg- OAg- OAg- OAg- OAg- OAg- ADH in ADH in ADH in ADH in ADH in ADH in 20 mM 20 mM 100 mM 100 mM 20 mM 20 mM AcONa, NaPi, 100 HEPES HEPES NaPi, 100 AcONH4, 100 mM mM NaCl (pH 7.2) (pH 7.2) mM NaCl 100 mM NaCl (pH 6.5) and CaCl2(pH 8) NaCl (pH 5) (10 mM) (pH 9) 0 weeks 2.8 1.3 4.2 3.3 1.8 4.3 2 weeks 8.4 5.5 11.3 10.9 11.4 28.6 4 weeks 7.6 14.0 13.0 17.4 The data in Table 2 indicates that the Paratyphi OAg-ADH link does break down over time, and is likely a source of the instability of the OAg-CRM197conjugate. Example 5 - attempts to improve stability by altering the chemistry used in the conjugation Various alternative conjugation chemistries were explored to see whether they could be used to create a more stable OAg-CRM197conjugate, as summarised briefly below. 1. The ADH linker was replaced with a DAH (1,6-Diaminehexane) linker. This modification to the original chemistry allows one to determine if the instability is due to the hydrazide linkage or it is present also with an alifatic amine linkage. A stability study based on the study method described in Example 3 (but measuring free Paratyphi A OAg from samples taken at 0 days, 8 days, 14 days and 28 days at 37°C) was performed on the OAg-DAH-SIDEA-CRM197conjugate. The results (percentage free OAg) are shown in Table 6 below. Table 6 0 days 8 days 14 days 28 days 10 24 26 36 2. The ADH linker was removed, and the Paratyphi OAg was conjugated directly to SIDEA-CRM197via a PPetN moiety in the core region of the OAg to understand the stability of linkage through pyrophosphate groups. A stability study based on the study method described in Example 3 (but measuring free OAg from samples taken at 0 days, 2 weeks and 4 weeks and separating free OAg from conjugate with a solid phase extraction method using a C4 disposable column) was performed on the OAg-SIDEA- CRM197conjugate. The results (percentage free OAg) are shown in Table 7 below. Table 7 0 days 2 weeks 4 weeks 13 29 30 3. The OAg was conjugated to CRM197-ADH (CRM protein is previously derivatized with ADH by EDAC chemistry) though NHS- EDC chemistry (see Figure 2 for a description of the chemistry used) to understand if a linkage through a KDO- carboxylate moiety to ADH is more stable. A stability study based on the study method described in Example 3 (but measuring free OAg from samples taken at 0 days, 8 days, 14 days and 28 days) was performed on the OAg-EDC-NHS-ADH-CRM197conjugate. The results (percentage free OAg) are shown in Table 8 below. Table 8 0 days 8 days 14 days 28 days 5 7 14 23 However, in all cases described above a significant amount of free OAg was released during the stability assay, indicating that none of these chemistries could be used to prepare a suitably stable OAg-CRM197conjugate. Example 6 - Conjugating OAg to CRM197using a random oxidation followed by reductive amination approach Paratyphi OAg (O:2) was conjugated to CRM197using a random oxidation with periodate followed by reductive amination approach, which introduces multiple linkages between OAg chains and CRM197protein molecules, as described in the following paragraphs. The chemistry is described in Figure 3. Oxidation O:210 mg / mL was oxidized (to O:2ox)with 10 mM NaIO4in buffer AcONa 10mM pH 5. The solution was kept at 25 °C in the dark, for 2 hours. After that, NaIO4excess was quenched with 20 mM Na2SO3 in H2O. The mixture was gently stirred at room temperature for 15 minutes. The oxidation mixture was purified by desalting with PD10 column against water and lyophilized. Conjugation O:2ox was resuspended with NaPi 100 pH 7.2 and added to CRM197carrier protein and NaCNBH3in order to reach the final O:2ox concentration of 10 mg / mL and CRM197 / NaCNBH3concentration of 5 mg / mL (O:2ox : CRM197: NaBH3CN = 2 :1 : 1 w / w). The reaction mixture was incubated overnight at 37°C. Conjugation mixture was added with NaBH4to reach O:2ox : NaBH41 : 1 w / w ratio, to quench residual oxidized free groups. The mixture was maintained at 37 °C for 2 hours. The conjugate was purified through HIC chromatography. A stability study based on the study method described in Example 3 (but measuring free OAg from samples taken at 0 days, 14 days and 28 days; separating free OAg from conjugate with solid phase extraction method with C4 disposable column and quantifying it using HPAEC-PAD analysis on sugar) was performed on the OAg- CRM197conjugate produced using random oxidation followed by reductive amination using the method described in Micoli F et al. PLoS One. 2012;7(11):e47039. The results (percentage free OAg) are shown in Table 9 below. Table 9 0 days 2 weeks 4 weeks 0 4 7 Significantly less free OAg was seen in the stability study compared to OAg-CRM197conjugates made using different conjugated chemistries. Example 7 - Conjugating OAg to CRM197using CDAP chemistry (with or without an ADH linker) Paratyphi A OAg (O:2) was conjugated to CRM197 using a random CDAP chemistry approach either via an ADH linker or without the ADH linker. Again, the random CDAP chemistry approach introduces multiple linkages between the OAg and the CRM197. The chemistry is described in Figure 4. Specifically, O:2 OH groups are activated with CDAP, using O:2 to CDAP w / w ratio of 1:0.3 in 150 mM NaCl solution. pH is adjusted to 9-10 with 10% v / v triethylamine and the solution is incubated at room temperature for 3 ± 0.5 minutes on stirring. Activated cyanoester groups of O:2 are covalently bound with hydrazide / amino groups of CRM197ADH / CRM197 to form O:2-CDAP-ADH-CRM197 / O:2-CDAP-CRM197. CRM197ADH / CRM197is added in the equal w / w ratio (1:1) of O:2 at a concentration of 10 mg / mL (final concentration of O:2 and CRM197ADH / CRM197is 5 mg / mL). The pH is maintained to 9.5± 0.5 with 10% triethylamine and the solution is mixed for 2-3 hours at room temperature. 1M glycine solution is then added to an equal volume of conjugation mixture and the pH is adjusted to 8.0 ± 0.2 with 10% triethylamine; the solution is incubated at 2-8°C for 15±5 hours. The crude conjugate is then buffer exchanged and unbound and unreacted O:2 is removed using HIC Phenyl HP resin. A stability study based on the study method described in Example 3 (but measuring free OAg from samples taken at 0 days, 8 days, 14 days and 28 days) was performed on the OAg-CRM197conjugate produced using CDAP chemistry with or without an ADH linker. The results (percentage free OAg) are shown in Table 10 below. Table 10 Conjugate 0 days 8 days 14 days 28 days OAg-CDAP- <5 9 10 14 CRM OAg-CDAP- <5 7 9 11 ADH-CRM Significantly less free OAg was seen in the stability study compared to OAg-CRM197conjugates made using the chemistries described in Examples 2-5. Example 8 - Comparison of immunogenicity for bivalent formulations with O:2-CRM197produced through ADH-SIDEA or CDAP chemistry. In order to compare the ability of conjugates generated using different conjugation chemistries to elicit an immune response, groups of 10 BALB / c mice were immunised subcutaneously at days 0 and 28 with bivalent formulations with a dose of 2.5 μg (each component (fVi or S. Paratyphi A O-antigen) in 50μl without aluminium hydroxide or a dose of 1.25 μg (each component) with 75 μg aluminium hydroxide in 50 μl. The mice were bled at days 28 and 42 and the level of O:2 and Vi IgG (GMT) measured by ELISA. For O:2 also sera functionality by SBA was checked. The ELISA protocol used was the same as that described in WO2013 / 038375. The SBA protocol used was that described in Necchi F et al. “Setup of luminescence-based serum bactericidal assay against Salmonella Paratyphi A” J Immunol Methods. 2018; 461:117-121. Groups of mice were immunized with or without aluminium hydroxide with: - O:2-ADH-SIDEA-CRM197(produced by selective reductive amination as in Example 1) - O:2(CDAP)-CRM197 (produced as in Example 7) in each case formulated in bivalent formulations with fVi-CRM197(produced as in Example 2 at the doses indicated above). The results obtained are shown in Figure 5. As can be seen from Figure 5, the conjugate involving CDAP conjugation chemistry elicited similar antibody responses and functionality for O:2 with respect to the conjugate employing the ADH-SIDEA chemistry. As demonstrated in earlier Examples though, the CDAP based conjugates were more stable. Example 9 – Human clinical trial showing immunogenicity of bivalent formulations with O:2-CRM197produced through CDAP chemistry A Phase I, observer-blind, randomised, controlled, single-centre human clinical trial was performed to characterise the safety and immunogenicity profile of a vaccine containing the O:2(CDAP)-CRM197conjugate (produced in Example 7) combined with fVi-CRM197(produced as in Example 2). The clinical trial has the study number NCT05613205. 96 healthy adults (18-50 years of age) in Europe were administered one of the formulations in Table 11 or the TYPHIM Vi control described below. Table 11 Formulation name Contents Low Dose (5 / 5) O:2(CDAP)-CRM197(5μg O:2) and fVi- CRM197(5μg fVi) in water for injection (0.1 mL total volume) Low Dose + Alum (5 / 5 with aluminium O:2(CDAP)-CRM197(5μg O:2) and fVi- hydroxide) CRM197(5μg fVi) adsorbed on aluminium hydroxide (0.075 mg Al3+) in water for injection (0.1 mL total volume) High Dose (25 / 25) O:2(CDAP)-CRM197(25μg O:2) and fVi- CRM197(25μg fVi) in water for injection (0.5 mL total volume) High Dose + Alum (25 / 25 with O:2(CDAP)-CRM197(25μg O:2) and fVi- aluminium hydroxide) CRM197(25μg fVi) adsorbed on aluminium hydroxide (0.375 mg Al3+) in water for injection (0.5 mL total volume) The above vaccine formulations were compared with TYPHIM Vi at a 0.5 mL dose (containing 25 μg of the Vi capsular polysaccharide of Salmonella Typhi) which served as a control. Blood samples were taken at day 1 (the same day as vaccine administration) and day 29 (i.e. 28 days after the day of vaccine administration). The antibody levels in the samples were examined using ELISA and SBA assays. For the estimation of anti-Vi IgG, a robust and qualified ELISA method was used. In this method, 96-well ELISA plates were coated with Vi PS diluted in coating buffer (1X PBS) and kept overnight at 2 – 8 °C. The coated plate was washed with wash-buffer and blocked with blocking solution (5% skimmed milk in PBS). After another round of washing, optimally diluted sera samples (in dilution buffer) were added in the designated wells and incubated at 25°C for 2 hours. After another round of 3 washes, optimized dilution of AP-conjugated anti-mouse IgG secondary antibody was added (in dilution buffer) and incubated for 1 hour. After washing, the substrate was added to the plate and incubated. Absorbance was measured using an ELISA reader after 1 hour. A standard curve was plotted with obtained absorbance or Optical Density (OD) versus concentration of anti-Vi IgG reference standard and fitted with a polynomial regression line. The concentration of anti-Vi IgG in each sera sample was then calculated using the same standard curve. For the estimation of anti-O:2 IgG in animal or human sera, a traditional method similar to that for the anti-Vi IgG, was used by coating O:2-PS on to ELISA plate. Briefly, 96- well ELISA plates were coated with O:2 PS (in 1X Carbonate buffer) and kept overnight at 2 – 8 °C. The coated plate was washed one time with wash-buffer and blocked with blocking solution (5% fat-free milk in PBS). After another round of washing, optimally diluted sera samples (in dilution buffer) was added in the designated wells and incubated at 25°C for 2 hours. After another round of 3 washes, optimized dilution of AP-conjugated anti-mouse IgG secondary antibody was added (in dilution buffer) and incubated for 1 hour. After washing, the substrate was added to the plate and incubated. Absorbance was measured using an ELISA reader after 1 hour. A standard curve was plotted with obtained absorbance or Optical Density (OD) versus anti-O:2 IgG reference standard and fitted with a polynomial regression line. The Elisa Units of anti-O:2 IgG in each sera sample were then calculated using the same standard curve. Serum bactericidal activity against Salmonella Paratyphi A was measured by L-SBA, based on a method previously reported [Necchi, 2017; Rossi, 2020]. Results are expressed in serum titres, defined as serum dilutions giving 50% inhibition of bacterial growth (IC50). The results are provided in Figure 9 to 11. The SBA and ELISA results were used to calculate the percentage of patients having an anti-Vi IgG GMC greater than or equal to 2μg / ml (at day 29) and the percentage of patients having an anti-Vi IgG GMC greater than or equal to 4.3 μg / ml (at day 29), and these data are set out in Tables 12 and 13 below. Similarly, the percentage of patients having an anti-O:2 GMC increased by more than 4-fold (according to the ELISA assay or the SBA assay) was calculated, and these data are set out in Tables 14 and 15 below. Table 12 Low dose Low dose Full dose Full dose TYPHIM (%) + alum (%) + alum Vi (%) (%) (%) Percentage of 0 18.2 4.8 4.8 0 patients with a GMC of anti-Vi antibodies greater than 2μg / ml at day 1 (baseline) as determined by ELISA Percentage of 100 100 100 95.7 83.3 patients with a GMC of anti-Vi antibodies greater than 2μg / ml at day 29 (post- vaccination) as determined by ELISA Table 13 Low dose Low dose Full dose Full dose TYPHIM (%) + alum (%) + alum Vi (%) (%) (%) Percentage of 0 18.2 0 4.2 0 patients with a GMC of anti-Vi antibodies greater than 4.3μg / ml at day 1 (baseline) as determined by ELISA Percentage of 100 88.9 100 95.7 54.2 patients with a GMC of anti-Vi antibodies greater than 4.3μg / ml at day 29 (post-vaccination) as determined by ELISA Table 14 Low dose Low dose Full dose Full dose TYPHIM (%) + alum (%) + alum Vi (%) (%) (%) Percentage of 100 81.8 100 83.3 4.2 patients having a 4- fold increase in anti- O:2 antibodies at day 29 (post- vaccination) as determined by ELISA Table 15 Low dose Low dose Full dose Full dose TYPHIM (%) + alum (%) + alum Vi (%) (%) (%) Percentage of 91.7 72.7 90.5 75 0 patients having a 4- fold increase in anti- O:2 antibodies at day 29 (post- vaccination) as determined by SBA Example 10 – Impact of O:2 size and O:2 / CRM197 ratio on the immunogenicity of O:2- CRM197 glycoconjugate in mice Synthesis and characterization of the conjugates Aiming to evaluate the impact of O:2 size and O:2 / CRM197w / w ratio on immunogenicity, a panel of conjugates differing for these attributes was synthesized and fully characterized. S. Paratyphi A strain ED199 ΔtolR used as source of O:2, produces a bimodal molecular weight (MW) O-antigen population, with two main peaks at 16 kDa and 100 kDa with a w / w ratio of 65:35 (Figure 12A). The two populations, named as O:2[16 kDa]and O:2[100kDa], were separated by size exclusion chromatography (SEC), starting from the purified O:2[16 kDa + 100 kDa]mixed population. The two populations showed similar characteristics in terms of glucosylation and O-acetylation % (Figure 12B). Glucosylation level was not altered during SEC, while O-acetylation level was partially impacted, decreasing from 60% in the O:2[16 kDa + 100 kDa]mixed population, to ~45% in O:2[16 kDa]and O:2[100kDa] (Figure 12B). Different O:2-CRM197conjugates were synthesized using either O:2[16 kDa](Conjugates 1-3, Table 13), O:2[100 kDa] (Conjugates 4-6, Table 13), or O:2[16 kDa + 100 kDa] (Conjugates 7-9, Table 13). Different O:2 / CRM197 / CDAP weight ratios were used during conjugation, resulting in a panel of conjugates differing not only for O:2 size, but also O:2 to CRM197ratio (Table 13). After purification, all conjugates had a residual free O:2 below 5%. Table 13. O:2-CRM197conjugates differing for O:2 size and O:2 / CRM197w / w ratio. Conjugates were synthesized using O:2 populations of different size and changing the weight proportion of O:2, CRM197and CDAP. Cojugation conditions s Conjugate ize Nr Conjugate (weight proportion) 1 O:2[16 kDa]-CRM19716 1 2 0.2 0.25 2 O:2[16 kDa]-CRM19716 1 0.5 0.2 0.37 3 O:2[16 kDa]-CRM19716 1 0.5 0.5 0.62 4 O:2[100 kDa]-CRM197 100 1 2 0.2 0.39 5 O:2[100 kDa]-CRM197100 1 0.5 0.2 0.79 6 O:2[100 kDa]-CRM197100 1 0.5 0.5 0.65 O:2[16 kDa + 100 kDa]-CRM19716 + 100 1 0.5 0.2 0.53 O:2[16 kDa + 100 kDa]-CRM19716 + 100 1 2 0.2 0.28 O:2[16 kDa + 100 kDa]-CRM19716 + 100 1 2 0.5 0.22 * Expressed as w / w ratio between O:2 and conjugated CRM197Mouse immunogenicity study Mice received two intraperitoneal (IP) immunizations, with a 28 day interval, of 2.5 μg conjugates (O:2 dose). Anti-O:2 IgG response was determined by ELISA and sera functional activity was determined by their ability to kill S. Paratyphi A bacteria in the presence of complement. In general, all tested conjugates (Table 13), regardless of their specific structural differences, were immunogenic, with a significant booster effect after the second immunization (Figure 13A). Effect of O:2 size O:2-CRM197conjugates with a similar O:2 / CRM197w / w ratio, but differing for O:2 MW (16 kDa, 100 kDa or 100 kDa + 16 kDa) were compared. No significant differences were found among the conjugates after 1 injection. Two weeks after the second injection, O:2[16 kDa]-CRM197induced a significantly higher anti-O:2 IgG response than the analogous conjugate synthesized with O:2[100 kDa](p = 0.0040). Both conjugates presented a similar O:2 / CRM197w / w ratio of 0.37 (O:2[16 kDa]-CRM197) and 0.39 (O:2[100kDa]-CRM197) (Figure 13A). Same was valid for conjugates at a similarly higher O:2 / CRM197w / w ratio (0.62 for O:2[16 kDa]-CRM197; 0.65 for O:2[16 kDa + 100 kDa]-CRM197and 0.53 for O:2[16 kDa + 100 kDa]-CRM197), with O:2[16 kDa]-CRM197inducing the highest anti-O:2 IgG response (Figure 13A). Effect of O:2 to CRM197ratio Impact of the O:2 loading on CRM197was also investigated. Starting from O:2[16 kDa]; O:2[100 kDa]and O:2[16k Da + 100 kDa]mixed population, by working with different reaction conditions, it was possible to synthesize conjugates with different O:2 / CRM197w / w ratios (Table 13). O:2[100 kDa]conjugates 4 and 5 (Table 13) with O:2 / CRM197w / w ratio of 0.39 and 0.79 elicited similar anti-O:2 IgG responses (Figure 13B) and serum bactericidal titers. Also the conjugates 7 and 8 (Table 13), obtained from O:2[16k Da + 100kDa]mixed population with an O:2 / CRM197w / w ratio of 0.28 and 0.53 induced similar responses. Instead, for O:2[16 kDa], the conjugate 3 with O:2 / CRM197w / w of 0.62 induced significantly higher total anti-O:2 IgG response than the conjugates 1 and 2 (Table 13) with lower ratios of 0.25 (p = 0.0051) and 0.37 (p = 0.0494), respectively (Figure 13B). Impact of O-acetylation level on immunogenicity of O:2-CRM197glycoconjugate in mice Partial de-O-acetylation of O:2 through ammonia treatment Bacterial polysaccharides often contain O-acetyl esters (OAc) which may constitute an important part of the immunodominant epitopes. To evaluate the impact of O- acetylation on the immune response, a panel of O:2-CRM197conjugates with different O-acetylation levels was synthesized (Table 14). Partial removal of the OAc groups from O:2 was performed before conjugation to CRM197by treating O:2 with a weak base as ammonia. The O:2[16 kDa + 100 kDa] mixed population had a starting 60% of O- acetylation. By treating the polysaccharide with increasing ammonia concentrations (range 5 ÷ 1000 mM) at 25°C, we obtained a panel of partially or totally de-O- acetylated O:2. By plotting the resulting O-acetylation level against ammonia (NH4OH) concentration, a correlation function was established (Figure 14). Total O:2 de-O- acetylation was obtained by using 1M ammonia. Using the correlation function, O:2[16 kDa]was treated with the suitable ammonia concentration, to obtain a gradually de-O-acetylated polysaccharide. Generation of O:2-CRM197conjugates with different O-acetylation levels The starting O:2[16 kDa + 100 kDa],as well as the completely de-O-acetylated O:2[16 kDa + 100kDa],were conjugated to CRM197, obtaining conjugates with of 54% or 0% OAc (conjugates 1 and 5, Table 14). In addition, a selection of partially de-O-acetylated O:2 were conjugated, resulting in a panel of O:2-CRM197conjugates with 45.2%, 35.3% and 18.5 % OAc (conjugates 2, 3 and 4, Table 14). Similarly, O:2[16 kDa]and partially de-O- acetylated O:2[16 kDa]were conjugated to CRM197, resulting in two conjugates with 58.5% and 25.2% OAc (conjugates 6 and 7, Table 14). O:2 activation by CDAP, followed by conjugation to CRM197, did not impact the initial O:2 OAc levels. All conjugates were fully characterized and showed similar O:2 / CRM197w / w ratio (Table 14) and similar level of cross-linking, as showed by HPLC SEC fluorescence emission profiles. Table 14. Analytical characterization of O:2-CRM197 conjugates synthesized through CDAP chemistry starting from O:2[16 kDa +100 kDa] and O:2[16 kDa] at different O- acetylation levels. O:2 size Conjugate OAc Free Nr Conjugate O:2 / CRM (kDa) 197 level % O:2 % w / w ratio 416 kDa + 100O:2[18.5% OAc]- 0.41 18.5 2.9 kDa CRM197516 kDa + 100O:2[0-CRM 0.38 0 2.5 kDa% OAc] 1976 16 kDaO:2[58.5% OAc]-CRM1970.39 58.5 6.1 716 kDaO:2[25.2% OAc]-CRM1970.36 25.2 5.6 Immunogenicity of conjugates with different O-acetylation levels To investigate the impact of O-acetylation on immunogenicity, the conjugates reported in Table 14 were tested in mice. Mice received two intraperitoneal (IP) immunizations, with a 28 day interval, of 2.5 μg conjugates (O:2 dose). In the case of O:2[16 kDa + 100 kDa], conjugates at five different O-acetylation levels (from 0% to 54%) were tested. Results were analyzed by performing a regression analysis of ELISA results (log transformed) vs OAc degree. All conjugates were immunogenic, with a booster response after the second immunization (Figure 15A). Analysis at day 42 (Figure 15B) showed a significant slope (p = 0.027) in the O-acetylation range 0 - 54%, with a non-significant lack of fit (p = 0.303), indicating that anti-O:2 IgG antibodies increase with increasing O-acetylation levels. There was a significant trend, with a total fold increase of 8.8 (CI95%1.3 – 59) EU / mL Geomean ratio, from 0% to 54% OAc levels. At day 27 the regression analysis did not evidence any significant trend in terms of slope. Same result was obtained using O:2[16 kDa]conjugates at two different O-acetylation levels (58.5% and 25.2%, conjugates 6 and 7, Table 14): both at day 27 (p = 0.0016, Mann Whitney test) and 42 (p = 0.0063, Mann Whitney test), the conjugate with 58.5% O-acetylation elicited higher response that the conjugate at 25.2% OAc (Figure 15A).

[0002] ASPECTS 1. A conjugate comprising a polysaccharide comprising a 3-deoxy-D-manno- octulosonic acid (KDO) moiety conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide comprises more than one activated site. 2. A conjugate comprising an O-antigen conjugated to a carrier protein by a random conjugation method, wherein the O-antigen comprises more than one activated site. 3. A conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the polysaccharide by 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide. 4. A conjugate comprising an O-antigen conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O-antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen. 5. A method for producing a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide. 6. A method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of introducing multiple activated sites into the O- antigen. 7. A method for producing a conjugate comprising a polysaccharide comprising a KDO moiety conjugated to a carrier protein comprising a step of: (i) activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide. 8. A method for producing a conjugate comprising an O-antigen conjugated to a carrier protein comprising a step of: (i) activating the O-antigen by CDAP chemistry to provide an activated O-antigen; or (ii) oxidising the O-antigen to provide an oxidised O-antigen. 9. The conjugate or method of any one of aspects 1, 3, 5 or 7, wherein the polysaccharide is an O-antigen. 10. The conjugate or method of any one of aspects 2, 4, 6, 8 or 9, wherein the O- antigen is an O-antigen from Salmonella Paratyphi A (S. Paratyphi A). 11. The conjugate or method of any one of the preceding aspects, wherein the carrier protein is selected from the group consisting of CRM197, tetanus toxoid (TT) or diphtheria toxoid (DT). 12. The conjugate or method of aspect 11, wherein the carrier protein is CRM197. 13. The conjugate or method of any one of the preceding aspects, wherein the conjugate further comprises a linker. 14. The conjugate or method of any one of the preceding aspects, wherein the linker is an adipic acid dihydrazide (ADH) linker. 15. The conjugate or method of aspect 13 or 14, wherein the linker is between the saccharide or O-antigen and the carrier protein. 16. The conjugate or method of any one of aspects 3, 4 or 7 to 15, wherein the activating or oxidising step introduces multiple activated sites into the polysaccharide or O-antigen. 17. The conjugate or method of any one of the preceding aspects, wherein the polysaccharide or O-antigen comprises 1.5 or more, 2.0 or more, or 2.5 or more activated sites or the activating or oxidising step introduces 1.5 or more, 2.0 or more, or 2.5 or more activated sites. 18. The conjugate of any one of aspects 1, 2, 5, 6 or 9 to 17, wherein the random conjugation method or introducing multiple activated sites comprises a step of: (i) activating the polysaccharide or the O-antigen by CDAP chemistry to provide an activated polysaccharide or O-antigen; or (ii) oxidising the polysaccharide or the O-antigen to provide an oxidised polysaccharide or O-antigen. 19. The conjugate or method of any one of aspects 3, 4, or 7 to 18, wherein activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises mixing the polysaccharide or the O-antigen with CDAP at a w / w ratio of between 0.05:1 and 5:1, between 0.1:1 and 5:1, between 0.2:1 and 2:1, or around 0.3:1 (CDAP to polysaccharide or O-antigen). 20. The conjugate or method of any one of aspects 3, 4, or 7 to 19, wherein activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises mixing the O-antigen and / or the polysaccharide comprising a KDO moiety in a solution of NaCl or KCl at a concentration between 50 mM and 1 M, between 100 mM and 250 mM, between 125 mM and 200 mM, or around 150 mM. 21. The conjugate or method of any one of aspects 3, 4, or 7 to 20, wherein activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises adjusting the pH to between 9 and 10 using triethylamine and optionally incubating the solution at room temperature for 1 to 5 minutes with stirring. 22. The conjugate or method of any one of aspects 3, 4, or 7 to 21, wherein the method further comprises reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on the carrier protein or a carrier protein-linker compound comprising the carrier protein. 23. The conjugate or method of aspect 22, wherein reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on the carrier protein or the carrier protein-linker compound comprises mixing the activated polysaccharide or O- antigen with the carrier protein or the carrier protein-linker compound at a w / w ratio of between 0.1:1 and 5:1, between 0.2:1 and 3:1, between 0.5:1 and 2:1, or around 1:1 (polysaccharide or O-antigen to carrier protein or carrier protein-linker). 24. The conjugate or method of aspect 22 or 23, wherein the carrier protein is CRM197. 25. The conjugate or method of any one of aspects 22 to 24, wherein the carrier protein-linker compound is CRM197-ADH. 26. The conjugate or method of any one of aspects 22 to 25, wherein the conjugation method further comprises: (i) a step of adjusting the pH to between 9 and 10 using triethylamine and incubating the solution at room temperature for 1 to 10 hours with stirring after the step of mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound; and / or (ii) a step of adding glycine solution and adjusting the pH to pH 7 to 9 using triethylamine and incubating the solution at 2-8ºC for between 5 and 50, or between 10 and 20 hours; and / or (iii) a step of removing unreacted polysaccharide or O-antigen. 27. The conjugate or method of aspect 26, wherein the step of removing unreacted polysaccharide or O-antigen comprises a step of chromatography, optionally hydrophobic interaction chromatography or anion exchange chromatography. 28. The conjugate or method of any one of aspects 3, 4, or 7 to 18, wherein the step of oxidising the polysaccharide or O-antigen comprises mixing the polysaccharide or O- antigen with an oxidising agent, optionally at a pH between 4 and 6. 29. The conjugate or method of aspect 28, wherein the oxidising agent is periodate, optionally sodium periodate. 30. The conjugate or method of aspect 28 or 29, wherein mixing the polysaccharide or O-antigen with an oxidising agent comprises mixing the polysaccharide or O-antigen with an oxidising agent at a ratio of between 1 mg / ml: 10 mM and 100 mg / mL : 10 mM, between 1 mg / mL :10 mM and 50 mg / mL: 10 mM, between 1 mg / mL: 10 mM and 25 mg / mL: 10 mM, or around 10mg / mL:10 mM (polysaccharide or O-antigen to oxidising agent). 31. The conjugate or method of any one of aspects 28 to 30, wherein the step of oxidising the polysaccharide or O-antigen comprises: (i) leaving a solution of the polysaccharide or O-antigen and the oxidising agent in the dark at a temperature between 20ºC and 30ºC for between 1 and 5 hours; and / or (ii) quenching excess oxidising agent, optionally using Na2SO3; and / or (iii) desalting the oxidised polysaccharide or O-antigen, optionally with a PD10 column. 32. The conjugate or method of any one of aspects 3, 4, 7 to 18 or 28 to 31, wherein the conjugation method further comprises a step of reacting the oxidised polysaccharide or O-antigen with the carrier protein. 33. The conjugate or method of aspect 32, wherein the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein with a reducing agent. 34. The conjugate or method of aspect 33, wherein the reducing agent is sodium cyanoborohydride. 35. The conjugate or method of aspect 33 or 34, wherein the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein at a w / w ratio of between 0.5: 1 and 20: 1, between 1:1 and 10: 1, between 1.5:1 and 5:1, or around 2:1 (polysaccharide or O-antigen to carrier protein). 36. The conjugate or method of any one of aspects 33 to 35, wherein the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the carrier protein and the reducing agent at a w / w ratio of between 0.25:1 and 20: 1, between 0.5:1 and 10: 1, between 0.75:1 and 5:1, or around 1:1 (carrier protein to reducing agent). 37. The conjugate or method of any one of aspects 32 to 36, wherein the conjugation method further comprises: (i) incubating a reaction mixture comprising the oxidised polysaccharide or O- antigen and the carrier protein at a temperature between 35ºC and 39ºC for between 4 and 20 hours; (ii) quenching by adding sodium borohydride, optionally wherein the w / w ratio of oxidised polysaccharide or O-antigen to sodium borohydride is between 10:1 and 0.1: 1, between 5:1 and 0.5: 1, or around 1:1 (w / w polysaccharide or O-antigen to sodium borohydride). 38. A conjugate obtainable by the method of any one of aspects 5 to 37. 39. A conjugate obtained by the method of any one of aspects 5 to 37. 40. An immunogenic composition comprising the conjugate of any one of aspects 1 to 4 or 9 to 39. 41. The immunogenic composition of aspect 40, further comprising a pharmaceutically acceptable excipient and / or an adjuvant. 42. The immunogenic composition of aspect 40 or 41, wherein the immunogenic composition further comprises an antigen from Salmonella Typhi (S. Typhi), optionally a Vi polysaccharide. 43. The immunogenic composition of aspect 42, wherein the Vi polysaccharide is a fragmented Vi polysaccharide (fVi). 44. The immunogenic composition of aspect 43, wherein the fVi polysaccharide has an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa. 45. The immunogenic composition of aspect 43 or 44, wherein the fVi polysaccharide has a target average molecular weight of between 51 kDa and 55 kDa. 46. The immunogenic composition of any one of aspects 43 to 45, wherein the fVi polysaccharide is part of an fVi conjugate comprising fVi and a carrier protein. 47. The immunogenic composition of aspect 46, wherein the carrier protein in the fVi conjugate is CRM197or diphtheria toxoid. 48. The immunogenic composition of aspect 47, wherein the carrier protein in the fVi conjugate is CRM197. 49. The immunogenic composition of any one of aspects 43 to 48, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, optionally via a linker. 50. The immunogenic composition of any one of aspects 43 to 49, wherein the fVi conjugate is obtained by or obtainable by a method comprising the steps of: a. fragmenting Vi polysaccharide to obtain a fragmented Vi (fVi) polysaccharide having an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; b. activating the fVi polysaccharide by reacting the fVi polysaccharide obtained in step a. with a carbodiimide and N-hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative; and c. reacting the N-hydroxysuccinimide ester fVi derivative obtained in step b. with the carrier protein to produce the fVi conjugate. 51. The immunogenic composition of aspect 49 or 50, wherein the carbodiimide is EDC. 52. The immunogenic composition of aspect 51, wherein the carrier protein is derivatised by reacting it with a carbodiimide and a linker. 53. The immunogenic composition of aspect 52, wherein the linker is an adipic acid dihydrazide (ADH) linker. 54. The immunogenic composition of aspect 52 or 53, wherein the carbodiimide is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) or the carbodiimide chemistry is EDAC chemistry. 55. The immunogenic composition of any one of aspects 40 to 54, wherein the conjugate is stable in the immunogenic composition for at least 4 weeks. 56. The immunogenic composition of aspect 55, wherein the conjugate is stable in the immunogenic composition for at least 4 weeks if less than 20%, less than 18%, less than 17%, less than 15%, between 0% and 20%, or between 1% and 15% of the polysaccharide or O-antigen is released from the conjugate in 4 weeks. 57. The immunogenic composition of aspect 55 or 56, wherein the conjugate is stable in the immunogenic composition for at least 4 weeks, 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 in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM197using direct reductive amination via an ADH-SIDEA linker. 58. The immunogenic composition of aspect 57, wherein substantially less is at least 10% less, at least 15% less, at least 20% less, or at least 25% less. 59. The immunogenic composition of any one of aspects 55 to 58, wherein the conjugate is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is similar to the amount released in an equivalent reference immunogenic composition in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM197using CDAP chemistry. 60. The immunogenic composition of aspect 59, wherein similar to is within 25%, within 15%, or within 10% of. 61. The immunogenic composition of any one of aspects 56 to 60, wherein the amount of the polysaccharide or O-antigen released from the conjugate after 4 weeks is determined using a stability assay comprising the following steps: (i) incubate the immunogenic composition for 4 weeks at 37ºC; (ii) prepare a post-incubation sample of the incubated immunogenic composition and remove the conjugated polysaccharide or O-antigen by deoxycholate precipitation; and (iii) determine the amount of free polysaccharide or O-antigen in the post-incubation sample as a percentage of the amount of total polysaccharide or O-antigen in the post- incubation sample. 62. The conjugate, method or immunogenic composition of any one of the preceding aspects, wherein the immunogenic composition induces a similar level of anti-S. Paratyphi O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises an S. Paratyphi O-antigen conjugated to CRM197using direct reductive amination via an ADH-SIDEA linker. 63. The conjugate, method or immunogenic composition of aspect 62, wherein a similar level is a level within 25%, within 15%, or within 10%. 64. The conjugate, method or immunogenic composition of aspect 62 or 63, wherein the level of anti-S. Paratyphi O-antigen antibodies is measured using an immunogenic assay comprising the following steps: (i) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 μg (O-antigen); (ii) measure the anti-S. Paratyphi O-antigen antibody level by ELISA at day 42. 65. The immunogenic composition of any one of aspects 40 to 64 further comprising: (i) a Salmonella Typhimurium (S. Typhimurium) antigen; and / or (ii) a Salmonella Enteritidis (S. Enteritidis) antigen. 66. The immunogenic composition of aspect 65, wherein the immunogenic composition further comprises a S. Typhimurium antigen and an S. Enteritidis antigen. 67. The immunogenic composition of aspect 65 or 66, wherein the S. Typhimurium antigen comprises or consists of S. Typhimurium GMMA. 68. The immunogenic composition of any one of aspects 65 to 67, wherein the S. Enteritidis antigen comprises or consists of S. Enteritidis GMMA. 69. The immunogenic composition of aspect 67 or 68, wherein the S. Typhimurium GMMA and / or the S. Enteritidis GMMA comprises detoxified lipid A. 70. The immunogenic composition of aspect 69, wherein the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from S. Typhimurium and / or S. Enteritidis that does not comprise: (i) a gene encoding a functional MsbB protein; and / or (ii) a gene encoding a functional PagP protein. 71. The immunogenic composition of any one of aspects 67 to 70, wherein the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from S. Typhimurium and / or S. Enteritidis that does not comprise gene encoding a functional tolR protein. 72. A vaccine comprising the immunogenic composition of any one of aspects 40 to 71. 73. The immunogenic composition or vaccine of any one of aspects 40 to 72, for use in a method of preventing an infection. 74. A method of preventing an infection comprising administering an effective amount of the immunogenic composition or vaccine of any one of aspects 40 to 72 to a subject. 75. Use of the immunogenic composition or vaccine of any one of aspects 40 to 72, for the manufacture of a medicament for use in a method of preventing an infection. 76. The immunogenic composition or vaccine for use of aspect 73, or the use of aspect 75, wherein the method of preventing an infection comprises administering an effective amount of the immunogenic composition or vaccine of any one of aspects 40 to 72 to a subject. 77. The immunogenic composition or vaccine for use, method, or use of any one of aspects 73 to 76, wherein the method of preventing an infection is a method of preventing Salmonella infection. 78. The immunogenic composition or vaccine for use, method, or use of any one of aspects 73 to 77, wherein the method of preventing an infection is a method of preventing invasive non-typeable Salmonella infection. 79. The immunogenic composition or vaccine for use, method, or use of any one of aspects 73 to 78, wherein the method of preventing an infection is a method of preventing infection by S. Typhimurium, S. Enteritidis, S. Typhi and / or S. Paratyphi A. 80. The conjugate, method, immunogenic composition, vaccine, immunogenic composition or vaccine for use, or use of any one of aspects 2, 4, 6 or 8 to 79, wherein the O-antigen is an O-antigen from Salmonella Paratyphi A (S. Paratyphi A), and the average molecular weight of the O-antigen is between 10 and 25 kDa, between 10 and 20 kDa, 11 kDa and 19 kDa, 12 kDa and 19 kDa, 13 kDa and 19 kDa, 14 kDa and 18 kDa, 15 and 18 kDa, or between 16 kDa and 18 kDa. 81. The conjugate, method, immunogenic composition, vaccine, immunogenic composition or vaccine for use, or use of any one of aspects 2, 4, 6 or 8 to 80, wherein the O-antigen is an O-antigen from Salmonella Paratyphi A (S. Paratyphi A), and the degree of O-acetylation of the S. Paratyphi A O-antigen is 10-100%, 20-100%, 30- 100%, 40-100%, 50-100%, 60-100%, 70-100%, 80-100%, or 90-100%. 82. The conjugate, method, immunogenic composition, vaccine, immunogenic composition or vaccine for use, or use of any one of aspects 2, 4, 6 or 8 to 81, wherein the O-antigen is an O-antigen from Salmonella Paratyphi A (S. Paratyphi A) and the carrier protein is CRM197,and the O:2 / CRM197w / w ratio is between 0.25 and 3.0, between 0.4 and 0.8, between 0.5 and 0.8, between 0.6 and 0.7, between 0.61 and 0.67 or between 0.62 and 0.66.

Claims

CLAIMS 1. A conjugate comprising a polysaccharide comprising (a) a 3-deoxy-D-manno-octulosonic acid (KDO) moiety, and / or (b) an O-antigen conjugated to a carrier protein by a random conjugation method, wherein the polysaccharide comprises more than one activated site.

2. A conjugate comprising a polysaccharide comprising (a) a KDO moiety, and / or (b) an O-antigen conjugated to a carrier protein using a conjugation method comprising a step of: (i) activating the polysaccharide by 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP) chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.

3. A method for producing a conjugate comprising a polysaccharide comprising (a) a KDO moiety, and / or (b) an O-antigen conjugated to a carrier protein comprising a step of introducing multiple activated sites into the polysaccharide.

4. A method for producing a conjugate comprising a polysaccharide comprising (a) a KDO moiety, and / or (b) an O-antigen conjugated to a carrier protein comprising a step of: (i) activating the polysaccharide by CDAP chemistry to provide an activated polysaccharide; or (ii) oxidising the polysaccharide to provide an oxidised polysaccharide.

5. The conjugate or method of any one of the preceding claims, wherein: (a) the polysaccharide is an O-antigen;(b) the O-antigen is an O-antigen from Salmonella Paratyphi A (S. Paratyphi A); (c) the carrier protein is selected from the group consisting of CRM197, tetanus toxoid (TT) or diphtheria toxoid (DT); (d) the carrier protein is CRM197;(e) the conjugate further comprises a linker, optionally an adipic acid dihydrazide (ADH) linker; (f) the conjugate further comprises a linker between the saccharide or O- antigen and the carrier protein which is optionally an ADH linker; and / or (g) the polysaccharide or O-antigen comprises 1.5 or more, 2.0 or more, or 2.5 or more activated sites or the activating or oxidising step introduces 1.5 or more, 2.0 or more, or 2.5 or more activated sites.

6. The conjugate or method of any one of claims 2 or 4 to 5, wherein the activating or oxidising step introduces multiple activated sites into the polysaccharide or O- antigen.

7. The conjugate of any one of claims 1, 3 or 5 to 6, wherein the random conjugation method or introducing multiple activated sites comprises a step of: (i) activating the polysaccharide or the O-antigen by CDAP chemistry to provide an activated polysaccharide or O-antigen; or (ii) oxidising the polysaccharide or the O-antigen to provide an oxidised polysaccharide or O-antigen.

8. The conjugate or method of any one of claims 2 or 4 to 7, wherein: (a) activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises mixing the polysaccharide or the O-antigen with CDAP at a w / w ratio of between 0.05:1 and 5:1, between 0.1:1 and 5:1, between 0.2:1 and 2:1, or around 0.3:1 (CDAP to polysaccharide or O-antigen); (b) activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises mixing the O-antigen and / or the polysaccharide comprising a KDO moiety in a solution of NaCl or KCl at a concentration between 50 mM and 1 M,between 100 mM and 250 mM, between 125 mM and 200 mM, or around 150 mM; (c) activating the O-antigen and / or the polysaccharide by CDAP chemistry comprises adjusting the pH to between 9 and 10 using triethylamine and optionally incubating the solution at room temperature for 1 to 5 minutes with stirring; (d) the method further comprises reacting the activated polysaccharide or O- antigen with hydrazide / amino groups on the carrier protein or a carrier protein- linker compound comprising the carrier protein, optionally wherein the carrier protein is CRM197and / or the carrier protein-linker compound is CRM197-ADH; and / or (e) the method further comprises reacting the activated polysaccharide or O- antigen with hydrazide / amino groups on the carrier protein or a carrier protein- linker compound comprising the carrier protein and reacting the activated polysaccharide or O-antigen with hydrazide / amino groups on the carrier protein or the carrier protein-linker compound comprises mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound at a w / w ratio of between 0.1:1 and 5:1, between 0.2:1 and 3:1, between 0.5:1 and 2:1, or around 1:1 (polysaccharide or O-antigen to carrier protein or carrier protein-linker), optionally wherein the carrier protein is CRM197and / or the carrier protein-linker compound is CRM197-ADH.

9. The conjugate or method of any one of claims 2 or 4 to 8, wherein the conjugation method further comprises: (i) a step of adjusting the pH to between 9 and 10 using triethylamine and incubating the solution at room temperature for 1 to 10 hours with stirring after the step of mixing the activated polysaccharide or O-antigen with the carrier protein or the carrier protein-linker compound; and / or (ii) a step of adding glycine solution and adjusting the pH to pH 7 to 9 using triethylamine and incubating the solution at 2-8ºC for between 5 and 50, or between 10 and 20 hours; and / or (iii) a step of removing unreacted polysaccharide or O-antigen,optionally wherein the step of removing unreacted polysaccharide or O-antigen comprises a step of chromatography, optionally hydrophobic interaction chromatography or anion exchange chromatography.

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

11. The conjugate or method of claim 10, wherein: (a) the oxidising agent is periodate, optionally sodium periodate; (b) mixing the polysaccharide or O-antigen with an oxidising agent comprises mixing the polysaccharide or O-antigen with an oxidising agent at a ratio of between 1 mg / mL: 10 mM and 100 mg / mL: 10 mM, between 1 mg / mL :10 mM and 50 mg / mL: 10 mM, between 1 mg / mL: 10 mM and 25 mg / mL: 10 mM, or around 10mg / mL:10 mM (polysaccharide or O-antigen to oxidising agent; and / or (c) the step of oxidising the polysaccharide or O-antigen comprises: (i) leaving a solution of the polysaccharide or O-antigen and the oxidising agent in the dark at a temperature between 20ºC and 30ºC for between 1 and 5 hours; and / or (ii) quenching excess oxidising agent, optionally using Na2SO3; and / or (iii) desalting the oxidised polysaccharide or O-antigen, optionally with a PD10 column.

12. The conjugate or method of any one of claims 2, 4 to 7, or 10 to 11, wherein the conjugation method further comprises a step of reacting the oxidised polysaccharide or O-antigen with the carrier protein.

13. The conjugate or method of claim 12, wherein: (a) the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen andthe carrier protein with a reducing agent, optionally wherein the reducing agent is sodium cyanoborohydride; (b) the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the oxidised polysaccharide or O-antigen and the carrier protein at a w / w ratio of between 0.5: 1 and 20: 1, between 1:1 and 10: 1, between 1.5:1 and 5:1, or around 2:1 (polysaccharide or O-antigen to carrier protein); (c) the step of reacting the oxidised polysaccharide or O-antigen with the carrier protein comprises mixing the carrier protein and the reducing agent at a w / w ratio of between 0.25:1 and 20: 1, between 0.5:1 and 10: 1, between 0.75:1 and 5:1, or around 1:1 (carrier protein to reducing agent); and / or (d) the conjugation method further comprises: (i) incubating a reaction mixture comprising the oxidised polysaccharide or O-antigen and the carrier protein at a temperature between 35ºC and 39ºC for between 4 and 20 hours; (ii) quenching by adding sodium borohydride, optionally wherein the w / w ratio of oxidised polysaccharide or O-antigen to sodium borohydride is between 10:1 and 0.1: 1, between 5:1 and 0.5: 1, or around 1:1 (w / w polysaccharide or O-antigen to sodium borohydride).

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

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

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

17. The immunogenic composition of claim 16, wherein the immunogenic composition further comprises an antigen from Salmonella Typhi (S. Typhi), optionally a Vi polysaccharide.

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

19. The immunogenic composition of claim 18, wherein the fVi polysaccharide: (a) has an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; (b) has a target average molecular weight of between 51 kDa and 55 kDa; (c) is part of an fVi conjugate comprising fVi and a carrier protein; (d) is part of an fVi conjugate comprising fVi and a carrier protein which is CRM197or diphtheria toxoid; (e) is part of an fVi conjugate comprising fVi and a carrier protein which is CRM197; (f) is part of an fVi conjugate comprising fVi and a carrier protein, and wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry, optionally via a linker; (g) is part of an fVi conjugate comprising fVi and a carrier protein, and wherein the fVi conjugate is obtained by or obtainable by a method comprising the steps of: (i) fragmenting Vi polysaccharide to obtain a fragmented Vi (fVi) polysaccharide having an average molecular weight of between 10 kDa and 90 kDa, between 25 kDa and 70 kDa, between 40 kDa and 55 kDa, between 41 kDa and 49 kDa, or between 51 kDa and 55 kDa; (ii) activating the fVi polysaccharide by reacting the fVi polysaccharide obtained in step a. with a carbodiimide and N- hydroxysuccinimide at a pH of 5 to 6 to form an N-hydroxysuccinimide ester fVi derivative; and (iii) reacting the N-hydroxysuccinimide ester fVi derivative obtained in step b. with the carrier protein to produce the fVi conjugate; (h) is part of an fVi conjugate comprising fVi and a carrier protein, wherein the fVi polysaccharide is conjugated to the carrier protein by carbodiimide chemistry and the carbodiimide is EDC;(i) is 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 derivatised by reacting it with a carbodiimide and a linker, optionally an adipic acid dihydrazide (ADH) linker; and / or (j) is part of an fVi conjugate comprising 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.

20. The immunogenic composition of any one of claims 16 to 19, wherein: (a) the conjugate is stable in the immunogenic composition for at least 4 weeks, optionally wherein (i) the conjugate is stable in the immunogenic composition for at least 4 weeks if less than 20%, less than 18%, less than 17%, less than 15%, between 0% and 20%, or between 1% and 15% of the polysaccharide or O-antigen is released from the conjugate in 4 weeks; (ii) the conjugate is stable in the immunogenic composition for at least 4 weeks, 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 in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM197using direct reductive amination via an ADH-SIDEA linker, optionally wherein substantially less is at least 10% less, at least 15% less, at least 20% less, or at least 25% less; (iii) the conjugate is stable in the immunogenic composition for at least 4 weeks, if the amount of polysaccharide or O-antigen released from the conjugate in 4 weeks is similar to the amount released in an equivalent reference immunogenic composition in which the conjugate is an S. Paratyphi O-antigen conjugated to CRM197using CDAP chemistry, optionally wherein similar to is within 25%, within 15%, or within 10% of; and / or(iv) the amount of the polysaccharide or O-antigen released from the conjugate after 4 weeks is determined using a stability assay comprising the following steps: (A) incubate the immunogenic composition for 4 weeks at 37ºC; (B) prepare a post-incubation sample of the incubated immunogenic composition and remove the conjugated polysaccharide or O-antigen by deoxycholate precipitation; and (C) determine the amount of free polysaccharide or O-antigen in the post-incubation sample as a percentage of the amount of total polysaccharide or O-antigen in the post-incubation sample; and / or (b) the immunogenic composition induces a similar level of anti-S. Paratyphi O-antigen antibodies compared to an equivalent immunogenic composition in which the conjugate comprises an S. Paratyphi O-antigen conjugated to CRM197using direct reductive amination via an ADH-SIDEA linker, optionally wherein: (i) a similar level is a level within 25%, within 15%, or within 10%; and / or (ii) the level of anti-S. Paratyphi O-antigen antibodies is measured using an immunogenic assay comprising the following steps: (A) immunise mice at days 0 and 28 with the conjugate subcutaneously at a dose of 25 μg (O-antigen); (B) measure the anti-S. Paratyphi O-antigen antibody level by ELISA at day 42.

21. The immunogenic composition of any one of claims 16 to 20 further comprising: (i) a Salmonella Typhimurium (S. Typhimurium) antigen, optionally wherein the S. Typhimurium antigen comprises or consists of S. Typhimurium GMMA; and / or (ii) a Salmonella Enteritidis (S. Enteritidis) antigen, optionally wherein the S. Enteritidis antigen comprises or consists of S. Enteritidis GMMA.

22. The immunogenic composition of claim 21, wherein: (a) the S. Typhimurium GMMA and / or the S. Enteritidis GMMA comprises detoxified lipid A; (b) the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from S. Typhimurium and / or S. Enteritidis that does not comprise: (i) a gene encoding a functional MsbB protein; and / or (ii) a gene encoding a functional PagP protein; and / or (c) the S. Typhimurium GMMA and / or the S. Enteritidis GMMA are derived from S. Typhimurium and / or S. Enteritidis that does not comprise gene encoding a functional tolR protein.

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

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

25. The immunogenic composition for use of claim 24, wherein the method of preventing an infection: (a) comprises administering an effective amount of the immunogenic composition or vaccine of any one of claims 16 to 23 to a subject; (b) is a method of preventing Salmonella infection; (c) is a method of preventing invasive non-typeable Salmonella infection; and / or (d) is a method of preventing infection by S. Typhimurium, S. Enteritidis, S. Typhi and / or S. Paratyphi A.

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