Immunogenic compositions against enteric diseases and methods for preparation thereof

Abstract

To provide immunogenic compositions for prophylaxis against infections caused by Salmonella and non-typhoidal Salmonella infections, and methods for preparation thereof.SOLUTION: The present disclosure relates to novel immunogenic monovalent and multivalent polysaccharide-protein conjugate vaccine compositions comprising a polysaccharide selected from Salmonella serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis. Salmonella typhi and non-typhi related diseases in subjects are reduced or prevented by inducing an immune response in subjects against Salmonella typhi and non-typhi related diseases by using the disclosed compositions. The vaccine elicits bactericidal antibodies and is useful for prevention of gastroenteritis, enteric fever and typhoid fever.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present disclosure relates to the field of vaccine production, and more particularly, the present disclosure relates to immunogenic compositions for the prevention against infections caused by Salmonella and non-typhoidal Salmonella infections, and methods for the preparation thereof. [Background technology]

[0002] The following background information herein is relevant to the present disclosure but is not necessarily prior art. Salmonella infections remain a major health problem affecting millions of people annually throughout the world, especially in developing countries. Salmonella infections can cause enteritis, which can be complicated by bacteremia (enteric fever) and gastroenteritis in both normal and immunocompromised individuals.

[0003] The genus Salmonella belongs to the family Enterobacteriaceae and includes gram-negative nonspore-forming facultative anaerobic rods. Salmonella enterica serovar typhi (S.typhi), as well as Salmonella enterica serovar paratyphi (S.paratyphi) A and B, cause enteric fever, a systemic febrile illness occurring exclusively in humans that is distinct from the more common self-limited acute gastroenteritis caused by numerous other Salmonella serotypes. Enteric fever caused by members of the genus Salmonella, including typhoid and paratyphoid, continues to constitute a significant morbidity and mortality burden in developing country populations (Lancet 2005;366:749 762) and represents a notable risk for travelers (Lancet Infect Dis.2005:5(10):623-628). Typhoid fever remains endemic in low- and middle-income countries (LMICs). 12.5-20.6 million cases of enteric fever occur annually in LMICs, especially in South Asia and sub-Saharan Africa (Lancet Glob Heal. 2014;2:e570-e580 and J Glob Health. 2012;2:10401). Salmonella paratyphi is responsible for an increasing proportion of enteric fever in parts of Asia, including Nepal, Cambodia and China. Salmonella paratyphi has the highest burden in the Indian subcontinent and Southeast Asia. One study in Nepal, where typhoid fever is highly endemic, found that municipal water was contaminated with both S. typhi and Salmonella enterica serovar paratyphi A (PLoS Negl Trop Dis. 2016 Jan;10(1):e0004346 and PLoS Negl Trop Dis. 2013;7(8):e2391).

[0004] Nontyphoidal Salmonella enterica (NTS) serotypes are important causes of invasive Salmonella disease worldwide. Of the more than 2,500 NTS serotypes, NTS serotypes typhimurium and enteritidis account for nearly 80 percent of all human isolates of NTS reported worldwide. Furthermore, invasive nontyphoidal Salmonella (iNTS) infections caused by serotypes enteritidis (SE) and typhimurium (STm) are a major pediatric health problem in sub-Saharan Africa. NTS has been increasingly recognized in recent years as a major cause of invasive bacterial infections in young children and immunocompromised individuals, as well as the elderly, worldwide. These two serotypes are also the leading causes of gastroenteritis in healthy children and adults in industrialized countries. NTS can also cause severe extraintestinal invasive bacteremia, which is referred to as iNTS. iNTS usually manifests as a febrile illness. Indeed, iNTS often occurs without gastrointestinal symptoms in both adults and children.

[0005] Of the more than 2,500 nontyphoid serotypes, Salmonella enterica subspecies enterica serovar typhimurium (S. typhimurium) and Salmonella enterica serovar enteritidis (S. enteritidis) account for nearly 80 percent of all human isolates of NTS reported worldwide. NTS has been increasingly recognized in recent years as a major cause of invasive bacterial infections in young children and HIV-infected individuals in sub-Saharan Africa, as well as in the elderly and immunocompromised individuals worldwide.

[0006] The global incidence of NTS gastroenteritis in 2010 was estimated at 93 million cases, of which approximately 80.3 million were food-borne transmissions, with 155,000 deaths. The economic burden of NTS is enormous in developed countries. In the United States alone, NTS costs US$3.3 billion annually, with 17,000 quality-adjusted life years lost, mostly due to some food-borne pathogen. As already mentioned, NTS can also cause severe extraintestinal invasive bacteremia, which is called iNTS. Invasive infections with Salmonella are more common throughout the developing world and are the most common cause of bacteremia in tropical Africa, especially in young children and individuals with HIV. It usually manifests as a febrile illness. In fact, iNTS often occurs without gastrointestinal symptoms in both adults and children. The symptoms of iNTS are similar to malaria and include fever and sweats (over 90 percent), and splenomegaly (40 percent). It is not clear why iNTS is such a problem in Africa, but this may be related to distinct monophyletic groups of increasingly invasive iNTS bacteria found in Africa and not elsewhere (e.g., S. typhimurium ST313), weakened host immunity associated with HIV infection, malaria and malnutrition, and increased opportunities for person-to-person transmission, for example through contaminated water supplies; NTS bacteremia in HIV-infected African adults is associated with high mortality (up to 47 percent) and recurrence rates (43 percent).

[0007] Antibiotics are used to treat typhoidal and nontyphoidal Salmonella-associated infections, with the choice of antimicrobial agent and length of treatment being determined by the cost and effectiveness of the antibiotic, local patterns of resistance and the patient's response to treatment. It is increasingly recognized in both developed and developing countries that multiple antibiotic-resistant strains are emerging as important causes of invasive bacteremia and gastroenteritis complications, leading to hospitalization and mortality.

[0008] As the effectiveness of available treatment tools decreases, the problem of typhoidal and nontyphoidal Salmonella-associated infections is likely to continue to increase, making vaccine development a key priority for disease control efforts. Vaccines are also an important protective tool for people traveling to areas where typhoidal and nontyphoidal Salmonella-associated infections are endemic.

[0009] Currently, three typhoid vaccines are licensed for use: i) typhoid conjugate vaccine (TCV), ii) non-conjugated Vi polysaccharide (ViPS) vaccine, and iii) live attenuated Ty21a vaccine. The World Health Organization (WHO) has recommended the increased use of typhoid vaccines, with typhoid conjugate vaccine (TCV) being preferred (WHO position paper. Wkly Epidemiol Rec 2018;93:153-72).

[0010] No vaccine is currently available for S. paratyphi. Similarly, there is a need for immunogenic compositions / vaccines that can simultaneously confer immunity against typhoidal and non-typhoidal Salmonella.

[0011] A drawback of currently available vaccines is that they are all directed only at S. typhi. S. paratyphi A causes enteric fever in the same geographic distribution as S. typhi, and the diseases are clinically indistinguishable. Therefore, in South and Southeast Asia, a vaccine that could protect against both serotypes would be more valuable than a vaccine that is restricted to one. The same is also true for S. typhimurium and S. enteritidis in sub-Saharan Africa, indicating the importance of a vaccine that could additionally protect against NTS serotypes for this region.

[0012] It is unclear whether vaccine candidates in the pipeline are protective against both gastroenteritis and the invasive manifestations of iNTS. While our understanding of the disease burden of typhoid in LMICs is improving, the global medical need for conjugate vaccines that protect against typhoidal and nontyphoidal Salmonella infections is growing in importance. Peak demand for typhoid conjugate vaccines is likely to occur in 2023-2026, approaching 300 million doses per year in 133 countries (Clin Infect Dis. 2019 Mar 15;68(Suppl 2):S154-S160).

[0013] Furthermore, upstream, downstream, conjugation and formulation development can often be rate-limiting steps in the early introduction of biologics to the market and matching population demand. Upstream includes the entire process from initial cell isolation and culture, cell banking, culture growth and final harvest of the bacterial fermentation process. Cell culture is scaled up from 100-500 milliliters to 3-20,000 liter bioreactors. Further steps include primary recovery of Salmonella polysaccharides, as well as removal of cells and debris. Furthermore, to facilitate cost-effective Salmonella polysaccharide-based conjugate vaccine development, structurally intact polysaccharides need to be obtained in high yield and purity. Yields of less than 40% have been previously reported for Salmonella spp. polysaccharides. Increasing the capsular polysaccharide (CPS) “fermentation harvest stage yield” by using novel feeding strategies and improved fermentation media is one of the preferred approaches to achieve the above objective. Merritt et al. (2000) showed that fed-batch culture in a 500 liter production-scale bioreactor increased cell density and capsular polysaccharide yield by approximately four-fold when compared to batch culture.

[0014] Studies of capsular polysaccharide production by other pathogens, e.g., Haemophilus influenzae type B and Neisseria meningitidis, have shown that production depends on fermentation conditions (temperature, pH, DO, osmolality) and medium components, and that these optima differed for each bacterium. Zhan et al. (2002) similarly showed that pH control and modifications to fed-batch fermentation increased cell yield and capsular polysaccharide production.

[0015] The expression of capsular polysaccharides is highly regulated in relation to specific fermentation conditions such as osmolality, and a decrease in polysaccharide synthesis has been reported at high osmolality. Polysaccharides were produced under various concentrations of glucose, casamino acids and phosphate ions.

[0016] Baruque-Ramos et al., 2005 showed that higher yields of capsular polysaccharide were obtained when N. meningitidis (serogroup C) was cultivated in a medium where the glucose concentration was maintained below 1.0 g / l, and that low oxygen tension favored higher polysaccharide production. Furthermore, it was observed that the final cell density was limited by concentrated casamino acids in the feed solution. Furthermore, growth and polysaccharide yield in a particular medium also depend on the ratio of carbohydrate to nitrogen source. In one of the fed-batch fermentation methods for S. typhi, ammonia was supplied as a nitrogen source along with the feed medium. However, in Aureobasidium pullulan Polysaccharide formation by S. was affected by the ammonia nitrogen source in the medium, with the yield being observed to be reduced in the presence of excess ammonium ions, even under conditions that otherwise supported its synthesis (Appl Microbiol Biotechnol (1990) 32:637-644).

[0017] Growth and polysaccharide synthesis in defined media was greatest when amino acids replaced ammonia as the nitrogen source. The use of casamino acids as a nitrogen source in defined media has been reported. However, animal-derived casamino acids, most likely derived from bovine casein, have been reported as allergens, limiting their use. Additionally, the use of casein digest / tryptone media as a nitrogen source in defined media has been reported, but this may not support the growth of fastidious organisms.

[0018] The addition of animal component-free hydrolysates (Bacto TC Yeastolate, Phytone Peptone) to chemically defined media is one of the techniques to increase cell density, culture viability and productivity in a timely manner. Hydrolysates are protein digests composed of amino acids, small peptides, carbohydrates, vitamins and minerals that provide nutritional support to the medium. Non-animal derived hydrolysates from soybean, wheat and yeast are commonly used in cell culture media to provide improved polysaccharide yields (see US9284371). However, due to their complex composition, yeast extracts and hydrolysates can be a significant source of medium variability due to lot-to-lot variability and the undesirable effect of thickening the medium. Foam formation during large-scale fermentation can i) reduce capsular polysaccharide yields due to loss of cells and culture medium into the foam phase, ii) be detrimental to cells due to shear forces generated when the bubbles burst, iii) result in loss of sterility when the foam is cleared, and iv) lead to overpressure if the outlet filter is clogged due to foaming.

[0019] The fermentation cell supernatant is subjected to various steps of purification to isolate the purified polysaccharides and remove host cell impurities such as proteins, nucleic acids and lipopolysaccharides. Filtration techniques play an important role in downstream processing or purification of bacterial polysaccharides from host cell impurities. Downstream includes inactivation of the bacterial culture, separation of cells from the medium, isolation, concentration and purification of the product. Downstream processing is the most challenging part of the process due to its complexity.

[0020] Each bacterium has a different capsular polysaccharide and different serotypes of the same bacterium, further differing in the chemical structure of the bacterial capsular polysaccharide. A case in point is when S. typhi expresses the Vi polysaccharide capsule, which is a linear homopolymer of α(1-4)-D-GalpA, N-acetylated at C-2 and O-acetylated at C-3. The N- and O-acetyls occupy the surface and are essential for both antigenicity and immunogenicity of Vi, whereas, on the contrary, S. paratyphi A and B, as well as NTS (with rare exceptions), do not express a capsular polysaccharide. Rather, their surface polysaccharide is the O polysaccharide (OPS) of lipopolysaccharide. They share a common trisaccharide backbone →2)-α-D-Manp-(1→4)-α-L-Rhap-(1→3)-α-D-Galp-(1→), which constitutes epitope 12 serologically. However, dideoxyhexose sugars linked α-(3→6) to the mannose of the repeating trisaccharide result in the immunodominant epitope that confers the Salmonella group specificity. In the case of S. typhimurium, the galactose of the trisaccharide backbone epitope 12 becomes α-(1→6) glucosylated. (See Lindberg AA, Le Minor L. Serology of Salmonella: In Bergan T (ed.), Methods in Microbiology: Academic Press, 1984:1-141.)

[0021] This diversity of bacterial polysaccharide structures makes the purification of these polysaccharides more challenging and difficult. Vaccines containing polysaccharides must meet certain quality standards. Previous purification methods that can be performed in large volumes include dissolution and precipitation of impurities such as nucleic acids and lipids using solvents, pH manipulation, and detergents such as sodium deoxycholate and Triton-X.

[0022] Sodium deoxycholate (DOC) is a mild detergent and one of the most commonly used detergents for polysaccharide purification. Sodium deoxycholate, with a core steroid structure, is not easily denatured and its solubilizing strength is limited, which destroys endotoxins without affecting their chemical structure, and therefore, upon removal of sodium deoxycholate, endotoxins regain their biological activity. Also, DOC-based procedures do not work effectively in removing contaminants from polysaccharides, especially sialic acid-containing polysaccharides. This may be due to the weak detergent activity of DOC on lipopolysaccharide-protein bonds formed during downstream processing, leading to high levels of endotoxin and protein content in the final isolated polysaccharides. Moreover, sodium deoxycholate is an animal-derived product, and even the presence of its residual amounts in the final product may result in a product that is not acceptable by regulatory authorities and certain religious communities.

[0023] A disadvantage of using Triton-X is that residual detergent remains in the extraction phase and removal requires extensive washing to remove all residues. For some CPS types, precipitation with zinc acetate / ammonium sulfate / sodium citrate for removal of protein contaminants is also included. However, to reach a high level of purity, repeated ammonium sulfate precipitations must be performed, making the process more cumbersome and labor intensive. Furthermore, sometimes this also precipitates capsular polysaccharides, leading to a loss of total polysaccharides.

[0024] Some other methods use enzymes, which aid in the degradation of protein and nucleic acid contaminants; however, the removal of enzymes and hydrolyzed materials can be an overwhelming task, leading to loss of the desired product. Furthermore, regulatory agencies have restricted the use of animal enzymes in human products due to the risk of prion contamination. Besides being expensive, the use of enzymes introduces many regulatory issues into the cGMP framework, such as the origin of the enzyme (animal or recombinant), variability in enzyme activity between different vendors and lots, etc.

[0025] Some other methods use Benzonase, Proteinase K or Nargase for degradation of residual protein and / or nucleic acid material followed by chromatographic purification, resulting in high costs and processes that are not easily scalable.

[0026] Some other methods use toxic organic solvents such as phenol, butanol, toluene and chloroform for the isolation of endotoxins from bacterial polysaccharides. This method is expensive and time consuming. Moreover, working with toxic organic solvents produces toxic waste products, which is undesirable.

[0027] The need for high purity of vaccine-specific polysaccharides has led to the development of new purification methods based on differential precipitation, ion exchange chromatography, gel filtration and affinity chromatography. Chromatographic techniques such as size-exclusion chromatography, ion exchange chromatography and hydrophobic interaction chromatography have been successfully used for the isolation of bacterial polysaccharides with effective removal of protein and nucleic acid contaminants. Despite the success of the isolation of bacterial polysaccharides according to the WHO standard, the use of chromatographic techniques involves multiple steps of laborious and time-consuming sample preparation, involves problems of scale-up and requires extensive purification of capsular polysaccharides. The recovery of the contaminants is dramatically compromised and therefore not suitable as a low-cost option for downstream processing on an industrial scale. Moreover, the addition of new purification steps to remove these contaminants increases the process complexity, reduces the final yield and increases the economic cost.

[0028] Salmonella typhi purified polysaccharide obtained by a previously reported purification process has an endotoxin content of 25-50 EU / μg. Therefore, there is a need for alternative purification methodologies aimed at maximizing recovery of Salmonella polysaccharide while removing impurities to acceptable levels.

[0029] Various methods such as acid hydrolysis, alkaline degradation, oxidation with periodate, ozonolysis (Wang et al., Carb. Res. 1999, 319, 1-4, 141-147), enzymatic hydrolysis, sonication (Pawlowski et al., Vaccine, 2000, 18.18, 1873-1885), and electron beam fragmentation (Pawlowski et al., Micro Lett, 1999, 174.2, 255-263) have been described for the depolymerization (sizing) of bacterial and non-bacterial polysaccharides. However, acid hydrolysis and alkaline degradation are time consuming and the resulting size-reduced samples have high polydispersity. Also, oxidation with periodate has a detrimental effect on labile antigenic epitopes of some polysaccharides. Moreover, ozonolysis can only be used with polysaccharides containing β-D-aldoside bonds, and only few endoglycanases have been isolated to date. US20090041802 discloses fragmentation of meningococcal polysaccharide with an Emulsiflex C-50 (conventional homogenizer) (Avestin). However, conventional homogenizers operate at peak pressure for only a small fraction of each cycle (approximately 7%), resulting in wider variability and less stable product, and requiring more runs or the use of higher pressures than would otherwise be required.

[0030] Ultrasonic treatment has been used to depolymerize polysaccharides (see, for example, WO2010 / 055250). However, ultrasonic depolymerization methods are not suitable for industrial depolymerization of large amounts of polysaccharides due to their low efficiency.

[0031] US20090234108 describes a method for partial deacetylation of pneumococcal serotype 1 polysaccharide by chemical treatment with sodium carbonate buffer (pH 9.0). This method is time consuming and prone to destroy immunogenic moieties, which may affect the immunogenicity of the conjugate.

[0032] For all the reasons stated above, a simple, low-cost method for the depolymerization of polysaccharides or polysaccharide derivatives remains desirable in the art. Producing a polysaccharide-protein conjugate vaccine is specific to the particular carrier protein and natural polysaccharide involved in the conjugation process.

[0033] A variety of conjugation techniques are known in the art. Conjugates can be prepared using TSTU, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) conjugation chemistry, by direct reductive amination methods, carbodiimide conjugation chemistry, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC.

[0034] The activated polysaccharide is coupled to the amino group of the carrier protein directly or via a spacer (linker) group. The linker used for conjugation disclosed in the prior art is N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (SZU et al., 1987). Another linker, B-propionamide (WO0 0 / 10599), nitrophenyl-ethylamine (Gever et al. (1979) Med Microbiol Immunol 165;171-288), haloalkyl halides (U.S. Pat. No. 4,057,685), glycosidic bonds (U.S. Pat. No. 4,673,574), hexanediamine and 6-aminocaproic acid (U.S. Pat. No. 4,459,286). Marburg et al., J. Am. Chem. Soc., 108, 5282 (1986) disclosed one method of conjugation of polysaccharides and immunogenic proteins by bigenic spacers. Proteins (PRO) are derivatized to display pendant nucleophilic or electrophilic groups (PRO * ), while the partner polysaccharide (Ps) was functionalized to display pendant groups of opposite reactivity (Ps * ). Ps * PRO *Upon combination with , a bigenic spacer was formed that covalently linked Ps to PRO (Ps-PRO). Upon acid hydrolysis, the bigenic spacer (linker) was released as an unusual amino acid and quantified by amino acid analysis, thereby providing a means of demonstrating covalent binding capacity.

[0035] The polysaccharide component of polysaccharide-protein conjugate vaccines undergoes gradual depolymerization at a rate that depends on the type of conjugate, formulation components and storage conditions. This leads to an increase in free polysaccharides that may adversely affect the stability of the product. Polysaccharide-carrier protein conjugates are known to release free polysaccharides after conjugation during further processing, lyophilization or storage in liquid and solid formulations. Only Salmonella polysaccharides covalently bound to the carrier protein (i.e., conjugated polysaccharides) are immunologically important for clinical protection, and excessive levels of unbound polysaccharides may potentially result in a reduced immune response to the polysaccharide (see WHO / TRS / 924, p. no. 14, A.3.3.5). In particular, Salmonella typhi conjugates reported in the literature have a high free polysaccharide content of up to 34% and a high free protein content of more than 5%, indicating a lower conjugation efficiency and a lower stability of the conjugate, which is undesirable. Therefore, there is a need for a vaccine that exhibits a free polysaccharide content of less than 10%.

[0036] Indeed, if it were possible to have a generic process that could be used to manufacture and formulate all vaccine candidates, the time and resources required for process development would be significantly reduced. This could have a significant impact on the number of clinical candidates that could be introduced into clinical trials. Also, processes developed for early-phase clinical trials, including those developed using platforms, may not be optimal in terms of process economics, yield, pool volume and throughput and may not be suitable for manufacturing the quantities required for later-stage or commercial campaigns. Another important consideration is the speed of process development, given that process development needs to occur prior to the introduction of therapeutic candidates into clinical trials. See Abhinav A. Shukla et al., Journal of Chromatography B, 848 (2007) 28-39.

[0037] Furthermore, Salmonella disease burden is high in developing countries where availability of electricity and refrigeration is often inadequate and therefore vaccine stability across temperature excursions is presumed to be more relevant in these regions.

[0038] Thus, an efficient platform process for producing an effective vaccine against Salmonella serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis that meets multiple criteria including good immunogenicity, safety and affordability, in particular i) improved polysaccharide yield over the fermentation and purification process, ii) an improved purification process showing optimal percentage recovery and minimum impurity levels, and iii) an improved ratio of polysaccharide-protein conjugates in the vaccine, iv) low viscosity, avoidance of aggregation and long term viability over a wide temperature range is provided. There is a need for a platform process that provides improved formulations that exhibit stability.

[0039] In order to overcome the aforementioned limitations of the prior art and to solve a global medical need that has been felt to be unmet for a long time, the Applicant proposes improved and alternative fermentation, purification, conjugation processes, formulations for the preparation of monovalent Salmonella typhoid conjugates, as well as multivalent vaccines comprising at least one additional conjugate derived from Salmonella paratyphi (S. paratyphi A, B, C) and non-typhoidal Salmonella enterica serovars typhimurium (S. typhimurium) and enteritidis (S. enteritidis). Summary of the Invention [Problem to be solved by the invention]

[0040] It is an object of the present disclosure to ameliorate one or more of the problems of the prior art, or at least to provide a useful alternative. The objective of the present disclosure is to develop an effective vaccine formulation for preventing and treating infections caused by Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis in humans.

[0041] Another object of the present disclosure is to provide an improved process for the production of polysaccharide-protein conjugate vaccines comprising polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis that can be used on an industrial scale.

[0042] Yet another object of the present disclosure is to provide a monovalent polysaccharide-protein conjugate vaccine comprising polysaccharides from any of the Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis.

[0043] Yet another object of the present disclosure is to provide a bivalent polysaccharide-protein conjugate vaccine comprising polysaccharides from any of the Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis in any combination thereof.

[0044] Yet another object of the present disclosure is to provide a multivalent polysaccharide-protein conjugate vaccine comprising polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis in any combination thereof.

[0045] Yet another object of the present disclosure is to provide an improved fed-batch process for producing polysaccharides of Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis.

[0046] Yet another object of the present disclosure is to provide an improved method for the purification of polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis.

[0047] Yet another object of the present disclosure is to provide a method for the transfection of Salmonella enterica serovar S. typhi, The objective of the present invention is to provide an improved method for the conjugation of polysaccharides (with or without size reduction) from S. paratyphi A, S. typhimurium and S. enteritidis.

[0048] Yet another object of the present disclosure is to provide a method for conjugation of polysaccharides (with or without size reduction) from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis to carrier proteins with or without linker (spacer) molecules.

[0049] Yet another object of the present disclosure is to provide immunogenic vaccine formulations comprising polysaccharide-protein conjugates in suitable single dose and multi-dose vials to be administered to infants and adults in suitable concentrations effective to confer protection or treatment of infection against Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis, or to prevent, ameliorate or delay the onset or progression of clinical symptoms thereof.

[0050] Other objects and advantages of the present disclosure will become more apparent from the following description, which is not intended to limit the scope of the disclosure. [Means for solving the problem]

[0051] The present disclosure relates to a) an immunogenic composition comprising one or more of the polysaccharide-protein conjugates, wherein the polysaccharides are from Salmonella serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis; b) Fed-batch methods for cultivating and processing polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis; c) downstream processing steps to obtain polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis; d) Methods for conjugation of polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis (with or without size reduction) to carrier proteins, with or without linker molecules; and e) A method for inducing an immune response in a subject by administering to the subject a therapeutically effective amount of an immunogenic composition to confer protection or treatment against infection with, or prevent, ameliorate or delay the onset or progression of clinical symptoms of, Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis. to provide. [Brief description of the drawings]

[0052] [Figure 1] See Figure 1: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP 1:0.8:1.3). [Diagram 2] Figure 2: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP is 1:0.8:1.3). [Diagram 3] See Figure 3: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP 1:0.8:1.1). [Figure 4] See Figure 4: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP 1:0.8:1.1). [Diagram 5] See Figure 5: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP is 1:1:1.3). [Figure 6] See Figure 6: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP is 1:1:1.3). [Figure 7] Figure 7: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP is 1:1:1.3). [Figure 8] See Figure 8: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP 1:0.8:1.25). [Figure 9] Figure 9: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP is 1:0.8:1.25). [Figure 10]See Figure 10: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP1:0.9:1.3). [Figure 11] See Figure 11: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP 1:0.9:1.3). [Figure 12] See Figure 12: Chromatogram showing the progress of the conjugation reaction (reaction quenched at 23 hours). [Figure 13] See Figure 13: Chromatogram showing the progress of the conjugation reaction. [Figure 14] See Figure 14: Chromatogram showing the progress of the conjugation reaction (reaction quenched at 20 hours). [Figure 15] See Figure 15: chromatogram showing purified conjugate (pooled fractions). [Figure 16] See Figure 16: Chromatogram showing the progress of the conjugation reaction (reaction quenched at 4 hours). [Figure 17] See Figure 17: chromatogram showing the purified conjugate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] While the present disclosure may be capable of various embodiments, certain specific embodiments are set forth in the following detailed discussion, which can be considered as an exemplification of the principles of the disclosure, with the understanding that it is not intended to limit the scope of the disclosure to what is illustrated and disclosed herein.

[0054] The embodiments are provided to fully and completely convey the scope of the present disclosure to those skilled in the art. Numerous details regarding specific components and processes are provided to provide a thorough understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that the details provided in the embodiments should not be interpreted as limiting the scope of the present disclosure. In some embodiments, well-known compositions, well-known processes and well-known techniques are not described in detail.

[0055] The terminology used in this disclosure is for the purpose of describing particular embodiments only, and such terms are not to be construed as limiting the scope of the disclosure. As used in this disclosure, the forms "a", "an" and "the" may be intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0056] The terms "comprises," "comprising," "including," and "having" are open-ended transitional phrases and thus specify the presence of stated features, integers, steps, operations, elements, modules, units, and / or components, but do not prohibit the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. The particular order of steps disclosed in the methods of the present disclosure is not necessarily required to perform them in the exact order described or illustrated. Additional It is also understood that alternative steps may be used.

[0057] The terms such as first, second, third, etc. should not be interpreted as limiting the scope of the present disclosure, and the aforementioned terms may only be used to distinguish one element, component, region, layer or section from another component, region, layer or section. The terms such as first, second, third, etc., when used herein, do not imply a specific sequence or order, unless clearly indicated by the present disclosure. The present disclosure provides an immunogenic composition and a method for preparing the same.

[0058] The term "vaccine" is optionally interchangeable with the term "immunogenic composition" and vice versa. "D antigen unit" (also called "International Unit" or IU): The D antigen form of poliovirus induces protective neutralizing antibodies. The D antigen unit referred to herein (e.g. in the vaccine of the invention) is the total D antigen unit measurement of each unadsorbed bulk IPV antigen type before formulation of the final vaccine, which is added to each human dose of the formulated vaccine (typically 0.5 mL final volume). Reliable methods for measuring D antigen units are well known in the art and published, for example, by the European Pharmacopoeia. For example, D antigen units can be measured using an ELISA test as described in Example 1 below ("D antigen quantification by ELISA"). The European Pharmacopoeia provides test samples (available from the European Pharmacopoeia Biological Reference Preparation-Ph.Eur.Secretariat, e.g. code P 216 0000) to standardize such methods between manufacturers (Pharmeuropa Special Issue, Bio 96-2). Thus, D antigen unit values ​​are well understood in the art.

[0059] The term "dose" as used herein typically refers to one administration of the vaccine of the present invention, which is typically one injection. A typical human dose is 0.5 mL. Of course, different doses may be administered in a vaccine administration schedule.

[0060] The term "IPV" or immunogenic compositions comprising these components is intended herein to mean inactivated poliovirus type 1 (e.g., Mahoney, which is preferably used), type 2 (e.g., MEF-1) or type 3 (e.g., Saukett), or a Sabin serotype 1, 2, 3 combination of any two or all three of these types. An example of a full (or standard) dose (40-8-32 D antigen units of IPV types 1, 2 and 3, respectively, based on Salk) IPV immunogenic composition for the purposes of the present invention may be Poliovac® (Serum Institute of India Pvt. Ltd.).

[0061] The term "saccharide" may refer to polysaccharides or oligosaccharides throughout this specification and includes both. Capsular saccharide antigens may be full-length polysaccharides or they may be extended to bacterial "sized saccharides" and "oligosaccharides" (which are polysaccharides that naturally have fewer repeating units or are reduced in size for handleability but are still capable of inducing a protective immune response in the host).

[0062] According to a first embodiment of the present disclosure, the immunogenic composition may comprise one or more of the polysaccharide-protein conjugates, wherein the polysaccharides are derived from Salmonella serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis.

[0063] According to a second embodiment of the present disclosure, a fed-batch process is used to culture Salmonella serovar St The method for obtaining polysaccharides from S. yphi, S. paratyphi A, S. typhimurium and S. enteritidis may comprise any subset or all of the following steps: 1. Inoculation, cultivation and harvesting of bacteria in a fermentation medium composition; 2. Inactivation; 3. Separation; 4. Clarification, and 5. Sterile filtration.

[0064] According to a first aspect of the second embodiment, the fermentation medium composition may comprise a carbon source, a magnesium salt, a phosphate source, yeast extract and a soy hydrolysate. The carbon source may be selected from the group consisting of glucose, mannitol, sucrose, lactose, fructose and trehalose, preferably glucose. The magnesium salt may be selected from magnesium chloride, magnesium sulfate, preferably magnesium sulfate heptahydrate. The potassium source may be selected from disodium hydrogen phosphate heptahydrate, sodium dihydrogen phosphate monohydrate, potassium phosphate and dipotassium phosphate. Preferably, the potassium source is a combination of disodium hydrogen phosphate heptahydrate, sodium dihydrogen phosphate. Preferably, the soy hydrolysate is hysoy.

[0065] Further according to this, the fermentation medium was selected from Antifoam 204, Antifoam C, SE-15, Y-30, Antifoam EX-Cell, S184 (pure silicone oil), SLM54474 (polypropylene glycol: PPG), VP1133 (silicone oil / PPG mixture), BREOX (polyalkylene glycol), J673 STRUKTOL (plant-based alkoxylated fatty acid ester) and Wacker-Chemie Co., Ltd. SE9 (aqueous emulsion with 10% silicon oil component). The combination of the antifoam agent with the soy hydrolysate and yeast extract may help improve the yield of polysaccharides. More preferably, the antifoam agent may be Antifoam C or J673 STRUKTOL.

[0066] According to an embodiment of the present disclosure, the yeast extract may be yeast autolysate, ultrafiltered yeast extract or synthetic yeast extract. The yeast extract may be selected from BD BBL™, BD BACTO™, Difco™, and the like. In a preferred embodiment, the yeast extract may be ultrafiltered yeast extract, such as Difco™ Yeast Extract, UF. The soy hydrolysate may be selected from, but is not limited to, soy meal, soy peptone, and soy flour. In one embodiment, the soy hydrolysate may be Difco™ Select Phytone™ UF. In another embodiment, the soy hydrolysate may be hysoy.

[0067] The combination of antifoam, soy peptone and yeast extract improves harvest yields compared to other media. According to a second aspect of the second embodiment, the method may follow a two-stage strategy by incorporating the feed at a fixed rate at specific fixed time intervals when the fermentation is already underway and / or allowing continuous feeding in a fed-batch mode of fermentation, including multiple stages with proportionally increasing batch size for each stage.

[0068] According to a third aspect of the second embodiment, the fermentation parameters are: Temperature: 36.0±2℃ Mixing: 150-600 rpm pH: 7.0±0.5 Dissolved oxygen: 30%~90% Air (nl / min): 2-10 Gas flow: 60~600nl / min Osmolality: 400-600mOsm / kg It may be composed of:

[0069] According to a fourth aspect of the second embodiment, the inactivation of the bacterial culture may be carried out using formalin. More preferably, the inactivation of the bacterial culture may be carried out using formaldehyde in the range of 0.1-2% v / v, preferably 0.5% v / v, and incubating at 34-38°C, preferably 36°C, for 5-12 hours, preferably 8-12 hours.

[0070] According to a fifth aspect of the second embodiment, the separation may be performed by centrifugation, more preferably with parameters set as follows: temperature 2-8° C., RPM 7000-8000, and centrifugation time 40-60 minutes.

[0071] According to a sixth aspect of the second embodiment, the clarification may be performed by depth filtration. According to a seventh aspect of the second embodiment, the clarified harvest may be sterilized by filtration using a 0.2 μM sterile filter.

[0072] According to an eighth aspect of the second embodiment, the crude Salmonella enterica serovar typhi Vi polysaccharide (ViPs) yield during the fermentation stage may be at least 40% and the average Vi polysaccharide yield may be in the range of 100 mg / L to 5000 mg / L, more preferably 100 to 700 mg / L.

[0073] According to a third embodiment of the present disclosure, the fermentation harvest may be subjected to any subset, or in any order, or all of the following downstream purification steps to obtain Vi polysaccharides (ViPs) of the desired quality: a) clarification of the bacterial capsular polysaccharide harvest by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.2 micrometers; b) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using membranes with molecular weight cut-offs (MWCO) between 10 and 300 kDa or kDa; c) treatment with anionic or cationic detergents, ethylenediaminetetraacetic acid (EDTA) (4-10 mM) and sodium acetate (5%-10%) for denaturation of proteins, nucleic acids and lipopolysaccharides; d) Alcohol precipitation (40%-70%), e) centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 μM; f) treatment with an alkaline salt to remove excess detergent, followed by centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 μM; g) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using membranes with molecular weight cut-offs (MWCO) between 10 and 300 kDa or kDa; h) treatment with anionic or cationic detergents; i) centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.45 micrometers to about 0.2 micrometers; j) removal of protein and nucleic acid impurities by washing the pellet with alcohol (50%-70%) in the presence of sodium chloride (0.1M-2M); k) selective precipitation of polysaccharides by using alcohol (<75% OR >95%); l) dissolving the polysaccharide in WFI and subjecting it to concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a molecular weight cut-off (MWCO) of 10 to 300 kDa or kD; m) sterile filtration under sterile conditions through at least one sterile filter having a pore size of about 0.2 micrometers.

[0074] According to a first aspect of the third embodiment, the purification method may not include a chromatography step. According to the second aspect of the third embodiment, the purification method can result in a large recovery of about 40%-65% with the desired O-acetyl level (>2.0 mmol / g polysaccharide), the purified Vi polysaccharide yield can be in the range of 1000-4000 mg / L, the average molecular weight can be in the range of 40-400 kDa, contains less than 1% protein / peptide, less than 2% nucleic acid, less than 100 EU endotoxin per μg polysaccharide (PS), and molecular size distribution (>50% of PS elutes before a distribution coefficient (KD) of 0.25 is reached).

[0075] More preferably, the average molecular weight of the purified Vi polysaccharide may be in the range of 40 to 400 kDa. According to a third aspect of the third embodiment, the anionic detergent may be selected from the group comprising alkyl sulfates, sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfate, sodium oleyl sulfate, sodium N-oleoyl poly(amino acid), sodium alkylbenzene sulfonate, sodium alpha olefin sulfonate, sodium alkyl sulfonate, alpha-sulfomonocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonates, alkyl naphthalene sulfonates, sodium alkanesulfonates, sodium lignosulfonate and sodium alkyl glyceryl ether sulfonates.

[0076] More preferably, the anionic detergent may be an alkyl sulfate, more preferably sodium dodecyl sulfate, at a final concentration in the range of 0.1% to 20%, more preferably 1 to 20%, added to the retentate and stirred at 25°C to 30°C for 2 hours.

[0077] According to a fourth aspect of the third embodiment, the alcohol precipitation may be carried out using methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone or t-butyl alcohol, or a combination thereof.

[0078] More preferably, the alcohol may be ethanol. According to a fifth aspect of the third embodiment, the alkali salt may be selected from the group of sodium, potassium, calcium and magnesium salts. More preferably, the alkali salt may be a potassium salt selected from the group consisting of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate and other potassium salts, or a combination of two or more thereof.

[0079] More preferably still, the potassium salt may be potassium chloride mixed with the supernatant at a final concentration ranging from 0.1 M to 2 M, and upon dissolution, the mixture was incubated at 2-8° C. for >3 hours.

[0080] According to a sixth aspect of the third embodiment, the cationic detergent may be selected from the group comprising cetyltrimethylammonium salts, tetrabutylammonium salts, myristyltrimethylammonium salts and hexadimethrine bromide, or a combination thereof.

[0081] More preferably, the cationic detergent may be cetyltrimethylammonium bromide (CTAB) at a final concentration ranging from 0.1% to 12%, preferably 2% to 3%, which may be added to the retentate and stirred at 25°C to 30°C for 1 to 2 hours.

[0082] According to a seventh aspect of the third embodiment, the final purified polysaccharide bulk may be stored at -20°C or below. According to a fourth embodiment of the present disclosure, the fermentation harvest may be subjected to any subset, or in any order, or to all of the following downstream purification steps to obtain O-specific polysaccharide of the desired quality from the Salmonella Paratyphi A lipopolysaccharide (LPS): a) Centrifugation and separation; b) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using membranes with molecular weight cut-offs (MWCO) between 10 and 300 kDa; c) acid hydrolysis of LPS; d) centrifugation and separation; e) neutralization; f) clarification of the LPS by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and 0.2 micrometers; g) treatment with anionic or cationic detergents; h) Centrifugation and separation; i) direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 to 0.2 micrometers; j) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using membranes with molecular weight cut-offs (MWCO) between 10 and 300 kDa; k) Alcohol precipitation (40%-70%), l) centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 μM; m) treatment with an alkaline salt to remove excess detergent, followed by centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 μM; n) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using membranes with molecular weight cutoffs (MWCO) between 10 and 300 kDa; o) removal of protein and nucleic acid impurities by washing the pellet with alcohol (50%-70%) in the presence of sodium chloride (0.1M-2M); p) dissolving the polysaccharide in WFI and subjecting it to concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a molecular weight cutoff (MWCO) of 10 to 300 kDa; and q) sterile filtration under sterile conditions through at least one sterile filter having a pore size of about 0.2 micrometers.

[0083] According to a first aspect of the fourth embodiment, acid hydrolysis of LPS may be carried out preferably using acetic acid (final concentration 0.5-5%) at a pH of about 2.0-3.0, at a temperature of 30-90°C and for a time of about 100-200 minutes.

[0084] According to a second aspect of the fourth embodiment, acid hydrolysis neutralization may be carried out, preferably using aqueous ammonia to achieve a final pH of 7.0. According to a third aspect of the fourth embodiment, the anionic detergent is selected from the group consisting of alkyl sulfates, sodium dodecyl sulfate, sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, sodium N-oleoyl poly(amino acid), alkylbenzene sulfonate, and the like. The sulfonate may be selected from the group including sodium sulfonate, sodium alpha olefin sulfonate, sodium alkyl sulfonate, alpha-sulfomonocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonates, alkyl naphthalene sulfonates, sodium alkanesulfonates, sodium lignosulfonate and sodium alkyl glyceryl ether sulfonates.

[0085] More preferably, the anionic detergent may be sodium deoxycholate at a final concentration in the range of 0.1% to 20%, more preferably in the range of 1 to 2%, which may be added to the retentate and stirred at 25°C to 30°C for 10 to 120 minutes.

[0086] According to a fourth aspect of the fourth embodiment, the alcohol precipitation may be carried out using methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone or t-butyl alcohol, or a combination thereof.

[0087] More preferably, the alcohol may be ethanol. According to a fifth aspect of the fourth embodiment, the alkali salt may be selected from the group of sodium, potassium, calcium and magnesium salts. More preferably, the alkali salt may be a potassium salt selected from the group consisting of potassium chloride, potassium acetate, potassium sulfate, potassium carbonate, potassium bicarbonate, potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, potassium nitrate and other potassium salts, or a combination of two or more thereof.

[0088] More preferably still, the potassium salt may be potassium chloride mixed with the supernatant at a final concentration ranging from 0.1 M to 2 M, and upon dissolution, the mixture was incubated at 2-8° C. for >3 hours.

[0089] According to a sixth aspect of the fourth embodiment, the cationic detergent may be selected from the group comprising cetyltrimethylammonium salts, tetrabutylammonium salts, myristyltrimethylammonium salts and hexadimethrine bromide, or a combination thereof.

[0090] According to a seventh aspect of the fourth embodiment, the purification method may not include a chromatography step. According to an eighth aspect of the fourth embodiment, a purification method may result.

[0091] According to a ninth aspect of the fourth embodiment, the method results in desirable O-acetyl levels (>2.0 mmol / g polysaccharide) together with significant reduction in endotoxin (<100 EU endotoxin / μg PS), protein (<1%) and nucleic acid (<2%) impurities, preferably higher capsular polysaccharide recovery in the range of 40%-65%, molecular size distribution (>50% of PS elutes before a distribution coefficient (KD) of 0.25 is reached) and average molecular weight of purified O-specific polysaccharide may be in the range of 40-200 kDa.

[0092] According to a tenth aspect of the fourth embodiment, the final purified polysaccharide bulk may be stored at -20°C or below. According to a fifth embodiment of the present disclosure, purified Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharides may be covalently attached to a carrier protein (CP) using carbodiimide, reductive amination or cyanylation conjugation reactions.

[0093] According to a first aspect of the fifth embodiment, purified Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and and S. enteritidis polysaccharides, tetanus toxin, tetanus toxoid (TT), diphtheria toxoid (DT), CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, E. coli LT, E. coli ST, Escherichia coli heat-labile toxin-B subunit, Neisseria meningitidis outer membrane complex, rEPA, Protein D of H. influenzae, Flagellin FliC, Limulus hemocyanin, Exotoxin A from Pseudomonas aeruginosa, Outer Membrane Complex c (OMPC), Porin, Transferrin-binding protein, Pneumolysin, Pneumococcal surface protein A (PspA), Pneumococcal surface adhesin A (PsaA), Pneumococcal PhtD, Pneumococcal surface proteins BVH-3 and BVH-11, Bacillus anthracis protective antigen (PA), and Bacillus The antibody may be covalently linked to a carrier protein (CP) selected from the group including detoxified edema factor (EF) and lethal factor (LF) of C. anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins containing multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N19, iron uptake proteins, toxin A or B from C. difficile and S. agalactiae proteins with or without linkers, and fragments, derivatives, and variants thereof.

[0094] Still preferably, the carrier protein used for conjugation with Salmonella typhi Vi polysaccharide may be tetanus toxoid. The polysaccharides from S. paratyphi A, S. typhimurium and S. enteritidis may be individually conjugated to a carrier protein, preferably selected from tetanus toxoid, diphtheria toxoid or CRM197. Immunogenic compositions comprising the following polysaccharide-carrier protein conjugates are envisaged according to the present disclosure: S. typhi conjugated to tetanus toxoid, S. paratyphi A conjugated to TT or DT or CRM197, S. typhimurium conjugated to TT or DT or CRM197 and S. enteritidis conjugated to TT or DT or CRM197; S. typhi conjugated to tetanus toxoid, S. paratyphi conjugated to tetanus toxoid A; S. typhimurium conjugated to CRM197 and S. enteritidis conjugated to CRM197; and S. typhi conjugated to tetanus toxoid, S. paratyphi conjugated to DT A; S. typhimurium conjugated to CRM197 and S. enteritidis conjugated to tetanus toxoid, S. typhi conjugated to tetanus toxoid, S. paratyphi conjugated to CRM197 A; S. typhimurium conjugated to CRM197 and S. enteritidis conjugated to tetanus toxoid.

[0095] According to the present disclosure, CRM197 is obtained from recombinant strain CS463-003 (MB101) of Pseudomonas fluorescens from Pfenex USA.

[0096] According to the present disclosure, TT is obtained from Clostridium Tetani (Harvard No. 49205) obtained from the Central research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. The Central research Institute (CRI) has acquired this strain from the NVI, Netherlands. Obtained from.

[0097] According to the present disclosure, DT is produced from a culture of Cornynebacterium diphtheriae Park-Williams Number 8 strain, a strain obtained from the Central Research Institute (CRI), Kasauli, Himachal Pradesh, India.

[0098] According to a second aspect of the fifth embodiment, prior to conjugation, the purified Salmonella enterica serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharides may be subjected to depolymerization / sizing by chemical means selected from the group of FeCl3, H2O2, sodium metaperiodate and sodium acetate, or mechanical means selected from the group of high pressure cell disruptors and homogenizers.

[0099] Even more preferably, purified Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharides may be subjected to depolymerisation / sizing by high pressure cell disruption.

[0100] More preferably, the purified Salmonella enterica serovar typhi polysaccharides (ViPs) may be subjected to depolymerization / sizing with sodium acetate (5%-10%), wherein the average molecular weight of the ViPs may range from 40-400 kDa.

[0101] More preferably, the purified Salmonella enterica serovar typhi polysaccharides (ViPs) may be subjected to depolymerization / sizing with sodium acetate (5%-10%), wherein the average molecular weight of the ViPs may be in the range of 100-250 kDa.

[0102] Applicants have found that conjugates prepared using partially size-reduced polysaccharides have higher conjugation efficiency / conjugate yield and immunogenicity compared to conjugates prepared from full-length polysaccharides. Size reduction of polysaccharides also reduces the viscosity of the solution and increases the number of reactive end groups, both factors that contribute to an increased frequency of covalent bond formation.

[0103] Still alternatively, the purified Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharides may not be subjected to depolymerization / sizing.

[0104] According to a third aspect of the fifth embodiment, prior to conjugation, the carrier protein (CP) may be derivatized to contain amino and / or carboxyl groups using carbodiimide, reductive amination or cyanylation reactions via a hetero- or homobifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethylamine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propinamido, nitrophenylethylamine, haloalkyl halide, 6-aminocaproic acid, and combinations thereof.

[0105] Even more preferably, the hetero- or homobifunctional linker may be a dihydrazide, more preferably adipic dihydrazide. Hydrazide groups can be introduced into proteins through the carboxyl groups of aspartic acid and glutamic acid residues of proteins using carbodiimide, reductive amination, cyanylation reactions, for example, by reaction with hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride) or any other dihydrazide in the presence of a carbodiimide such as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC). EDC is used as a catalyst to activate and modify protein reactants with hydrazine or dihydrazide.

[0106] More preferably, the reaction of the carrier protein (CP) with adipic dihydrazide (ADH) in the presence of a carbodiimide such as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) may be carried out at a pH of 5 to 7, more preferably 6.

[0107] Thus, the reaction of a carrier protein (CP) with adipic acid dihydrazide (ADH) in the presence of a carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), may be stopped by raising the pH from about 6 to about 7-8.

[0108] Alternatively, after derivatization of the carrier protein with ADH, the method may further comprise the steps of buffer exchange by diafiltration (DF) using membranes of either 10 kDa or 30 kDa or 50 kDa molecular weight cut-off (MWCO) and sterile filtration using a 0.2 u filter, whereby the ADH-derivatized carrier protein is buffer exchanged for at least 10 volumes, or passing it through a suitable gel filtration column to remove substantially all unreacted compounds, residual ADH and residual EDC to obtain a purified ADH-derivatized carrier protein.

[0109] Still alternatively, prior to conjugation, the carrier protein may not be derivatized to contain amino and / or carboxyl groups via a hetero- or homobifunctional linker.

[0110] According to a third aspect of the fifth embodiment, prior to conjugation, purified Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharides may be derivatized to contain amino and / or carboxyl groups using carbodiimide, reductive amination or cyanylation reactions via a hetero- or homobifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, dihydrazide, mixtures thereof, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethylamine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propionamide, nitrophenylethylamine, haloalkyl halides, 6-aminocaproic acid, and combinations thereof.

[0111] Even more preferably, the hetero- or homobifunctional linker may be a dihydrazide, more preferably adipic dihydrazide. Hydrazide groups can be introduced into the Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharides by using carbodiimide, reductive amination, cyanylation reactions, for example, the hydrazide derivative of the Vi polysaccharide can be formed by reaction of the polysaccharide with hydrazine, carbohydrazide, succinyl dihydrazide, adipic dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride) or any other dihydrazide in the presence of cyanogen bromide (CNBr).

[0112] Still preferably, the Salmonella enterica serovar Paratyphi A OSP is derivatized with an adipic acid dihydrazide (ADH) linker using cyanylation conjugation chemistry, the cyanylation reagent being selected from the group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ("CDAP"), p-nitrophenyl cyanate, and N-cyanotriethylammonium tetrafluoroborate ("CTEA") or 2-cyanopyridazin-3(2H)one (2-CPO).

[0113] Still preferably, Salmonella enterica serovar Paratyphi A OSP is derivatized with adipic acid dihydrazide (ADH) linker using cyanylation conjugation chemistry, the cyanylation reagent is 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) mixed in a weight ratio of OSP:ADH of 1:1 to 1:10, the ratio of OSP:CPPT may be 0.5 to 1.5, and the reaction is carried out at a pH ranging from 7 to 10, for a reaction duration of 1 to 2 hours, and at a temperature ranging from 2°C to 30°C.

[0114] According to yet another aspect of the fifth embodiment, prior to conjugation, the polysaccharide may not be derivatized to contain amino and / or carboxyl groups via a hetero- or homobifunctional linker.

[0115] In a preferred aspect of the fifth embodiment, Salmonella enterica serovar typhi polysaccharides (ViPs) may be covalently attached to a carrier protein using carbodiimide conjugation chemistry, and any water-soluble carbodiimide, more preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), may be used as a catalyst.

[0116] More preferably, the Vi polysaccharide may be covalently attached to the ADH-derivatized carrier protein using carbodiimide conjugation chemistry, where any water-soluble carbodiimide, more preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), may be used as a catalyst.

[0117] More preferably, the Vi polysaccharide may be covalently attached to the ADH-derivatized tetanus toxoid using carbodiimide conjugation chemistry, where any water-soluble carbodiimide, more preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), may be used as a catalyst.

[0118] Alternatively, the ADH-derivatized Vi polysaccharide may be covalently attached to the ADH-derivatized tetanus toxoid using carbodiimide conjugation chemistry, where any water-soluble carbodiimide, more preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), may be used as a catalyst.

[0119] Alternatively, the ADH-derivatized Vi polysaccharide may be covalently attached to tetanus toxoid using carbodiimide conjugation chemistry, where any water-soluble carbodiimide, more preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), may be used as a catalyst.

[0120] More preferably, the purified ViPs is purified in a ViPs:EDAC weight ratio of 1:0.5 to 1:2. It is covalently attached to a carrier protein (CP) using a carbodiimide conjugation reaction in the presence of mixed 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC), the ratio of Vi polysaccharide and carrier protein may be 0.5-1.5, and the reaction is carried out at a pH ranging from 5-7 and a temperature ranging from 2°C to 30°C.

[0121] According to yet another aspect of the fifth embodiment, the Vi polysaccharide (ViPs) may be covalently attached to the ADH-derivatized tetanus toxoid (TT) in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), the weight ratio of ViPs:TT:EDC may be 1:1:2, the concentrations of the Vi polysaccharide and tetanus toxoid may be 0.1 mg / mL to 10.0 mg / mL, and the ratio of the Vi polysaccharide and tetanus toxoid may be 0.5 to 1.5.

[0122] According to yet another aspect of the fifth embodiment, the reaction of Vi polysaccharides (ViPs) with derivatized tetanus toxoid (TT) in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) may be carried out at a pH in the range of 5 to 7, more preferably 6, at a temperature in the range of 2°C to 30°C, more preferably 10 to 25°C, the conjugation conversion efficiency is ≧70%, more preferably ≧90%, and the molecular size of the conjugate is preferably 1000 to 1600 kDa.

[0123] Additionally, the reaction of derivatized tetanus toxoid (TT) and Vi polysaccharides (ViPs) in the presence of a carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), may be stopped by raising the pH from about 6 to about 7-8.

[0124] According to one aspect of the fifth embodiment, S. paratyphi A polysaccharide (OSP) may be conjugated to a carrier protein using cyanylation conjugation chemistry, where the cyanylation reagent is selected from the group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ("CDAP"), p-nitrophenyl cyanate, and N-cyanotriethylammonium tetrafluoroborate ("CTEA") or 2-cyanopyridazin-3(2H)one (2-CPO).

[0125] More preferably, the S. paratyphi A OSP may be conjugated to the carrier protein using cyanylation conjugation chemistry, the cyanylation reagent being 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) or (CPIP).

[0126] Still preferably, the purified S. paratyphi A OSP is covalently conjugated to a carrier protein (CP) using cyanylation conjugation chemistry, the cyanylation reagent is 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) mixed in a weight ratio of OSP:CPPT of 1:0.5 to 1:2, the ratio of OSP and carrier protein may be 0.5 to 1.5, and the reaction is carried out at a pH ranging from 5 to 7, at a temperature ranging from 2°C to 30°C.

[0127] Even more preferably, the carrier protein used for conjugation with the S. paratyphi A polysaccharide OSP may be tetanus toxoid. Even more preferably, the carrier protein used for conjugation with the S. paratyphi A polysaccharide OSP may be diphtheria toxoid.

[0128] Even more preferably, the carrier protein used for conjugation with S. paratyphi A polysaccharide OSP may be CRM197. According to yet another aspect of the fifth embodiment, the S. typhimurium polysaccharide may be conjugated to the carrier protein using cyanylation conjugation chemistry, the cyanylation reagent being selected from the group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ("CDAP"), p-nitrophenyl cyanate and N-cyanotriethylammonium tetrafluoroborate ("CTEA") or 2-cyanopyridazin-3(2H)one (2-CPO).

[0129] More preferably, the S. typhimurium polysaccharide may be conjugated to the carrier protein using cyanylation conjugation chemistry, the cyanylation reagent being 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT).

[0130] More preferably, the carrier protein used for conjugation with the S. typhimurium polysaccharide may be tetanus toxoid. More preferably, the carrier protein used for conjugation with the S. typhimurium polysaccharide may be diphtheria toxoid.

[0131] Even more preferably, the carrier protein used for conjugation with S. typhimurium polysaccharides may be CRM197. According to yet another aspect of the fifth embodiment, the S. enteritidis polysaccharide may be conjugated to the carrier protein using cyanylation conjugation chemistry, the cyanylation reagent being selected from the group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ("CDAP"), p-nitrophenyl cyanate and N-cyanotriethylammonium tetrafluoroborate ("CTEA") or 2-cyanopyridazin-3(2H)one (2-CPO).

[0132] More preferably, the S. enteritidis polysaccharide may be conjugated to the carrier protein using cyanylation conjugation chemistry, the cyanylation reagent being 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT).

[0133] Even more preferably, the carrier protein used for conjugation with the S. enteritidis polysaccharide may be tetanus toxoid. Even more preferably, the carrier protein used for conjugation with the S. enteritidis polysaccharide may be diphtheria toxoid.

[0134] Even more preferably, the carrier protein used for conjugation with the S. enteritidis polysaccharide may be CRM197. Applicants have found a method to stabilize the final polysaccharide-protein conjugate by using appropriate linkers, appropriately sized polysaccharides, utilizing alternative methods of conjugation, polysaccharide to protein ratios, and polysaccharide to coupling agent ratios. This method can result in improved ratios of polysaccharide-protein conjugates in the vaccine, which in turn can reduce the number of free sugars and free proteins in the conjugate, leading indirectly to better immune responses through reduced carrier protein inhibition, improved sterile filterability of the conjugate, better control of conjugation, and increased intra-moiety cross-linking.

[0135] Applicants have found that various factors affect the coupling of polysaccharides and proteins, which depend on the molecular weight of the ViPs, the type of carrier protein selected, the ratio of the amount of polysaccharide:carrier protein used, activation of functional groups, the use of spacers and the conjugation chemistry.

[0136] According to yet another aspect of the fifth embodiment, after the conjugation reaction, the method may include steps of concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using membranes having either 100 kDa or 300 kDa molecular weight cut-off (MWCO), which concentrates the conjugate bulk at least three-fold and removes substantially all unreacted compounds, unconjugated polysaccharide, unconjugated proteins and residual EDC to obtain a purified Vi polysaccharide conjugate vaccine.

[0137] Additionally, the method may include gel filtration chromatography, which concentrates the conjugate bulk at least three-fold and removes substantially all unreacted compounds, unconjugated polysaccharide, unconjugated protein and residual EDC to obtain purified S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharide conjugate vaccines, with a conjugate yield of ≧50%.

[0138] Additionally, purification methods may include a combination of ultrafiltration and gel filtration chromatography. According to a sixth embodiment, the immunogenic composition may be a monovalent vaccine comprising either S. typhi Vi polysaccharide conjugated to a carrier protein or S. paratyphi A polysaccharide conjugated to a carrier protein or S. typhimurium conjugated to a carrier protein or S. enteritidis conjugated to a carrier protein.

[0139] According to a seventh embodiment of the present disclosure, the immunogenic composition may comprise at least one bivalent combination of: a) Salmonella enterica serovar typhi glyco-carrier protein (CP) conjugate antigen; b)Salmonella enterica serovar Paratyphi A sugar-carrier protein (CP) conjugate antigen, or a) Salmonella enterica serovar typhi glyco-carrier protein (CP) conjugate antigen; b) Salmonella enterica serovar typhimurium carbohydrate-carrier protein (CP) conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; or a)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, b) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; or a)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen.

[0140] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen.

[0141] Still preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0142] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0143] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0144] According to an eighth embodiment of the present disclosure, the immunogenic composition may comprise at least one of the following trivalent combinations: a) Salmonella enterica serovar typhi glyco-carrier protein (CP) conjugate antigen; b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; c) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; or a)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen.

[0145] More preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella. Nella enterica serovar typhi ViPs-TT conjugate antigen.

[0146] Still preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0147] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0148] Still preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0149] According to a ninth embodiment of the present disclosure, the tetravalent immunogenic composition may comprise i) S. typhi Vi polysaccharide conjugated to a carrier protein (CP), ii) S. paratyphi A polysaccharide conjugated to a carrier protein, iii) S. enteritidis polysaccharide conjugated to a carrier protein, and iv) S. typhimurium polysaccharide conjugated to a carrier protein.

[0150] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen.

[0151] Still preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0152] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0153] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0154] According to a tenth embodiment of the present disclosure, the immunogenic composition includes, but is not limited to, Salmonella paratyphi B, Salmonella paratyphi C, Salmonella antigens, such as outer membrane vesicles, outer membrane proteins (e.g., OmpC, OmpD, OmpF), siderophores (enterobactin), type III secretion system proteins (e.g., SipB, SipD, SseB, SseC, and PrgI), flagellin, non-typhoidal Salmonella species, Shigella, Shigella sonnei, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Escherichia coli, Enterobacter species, Yersinia species, Pseudomonas species, Pseudomonas aeruginosa, Haemophilus influenzae (serotypes a, c, d, e, f and non-encapsulated strains), hepatitis (strains A, C, D, E, F and G), influenza, Staphylococcus species, Staph ylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8, Streptococcus species, Streptococcus pneumoniae (1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E, 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B, 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48), group A Streptococcus, group B Streptococcus (groups Ia, Ib, II, III, IV, V, Vl, VII VII, VIII and IX), Neisseria meningitidis, Haemophilus pneumonia, Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans, Enterococcus faecalis, Enterococcus faecium, Enterococcus faecalis Neisseria gonorrhoeae, Bacillus anthracis, Vibrio cholerae, Pasteurella pestis, Campylobacter sp., Campylobacter jejuni, Clostridium sp., Clostridium tetani, Clostridium difficile, Mycobacterium sp., Mycobacterium tuberculosis, M.catarrhalis, Klebsiella pneumoniae, Treponema sp., Borrelia sp., Borrelia burgdorferi, Leptospira sp., Hemophilus ducreyi, Corynebacterium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Shigella spp., Ehrlichia spp., Rickettsia spp. and N. meningitidis polysaccharides (A, B, C, D, W135, X, Y, Z and 29E) acellular pertussis antigens, modified adenylate cyclase, malaria antigens (RTS, S), splenic encephalitis, dengue, malaria, measles, mumps, rubella, BCG, human papillomavirus, Japanese encephalitis, dengue, Zika, Ebola, Chikungunya, poliovirus, rotavirus, smallpox, yellow fever, flavivirus, varicella zoster and varicella virus antigens.

[0155] According to an eleventh embodiment of the present disclosure, the composition comprises a combination of at least one of the following: a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis W-135 carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 carbohydrate-carrier protein conjugate antigen; g) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; g) Neisseria meningitidis W-135 carbohydrate-carrier protein conjugate antigen; h) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; g) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; h) Neisseria meningitidis W-135 glyco-carrier protein Conjugated antigens, i) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen, or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 carbohydrate-carrier protein conjugate antigen; g) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis W-135 carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C sugar-carrier protein conjugate Gate antigen, e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen.

[0156] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen.

[0157] Still preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0158] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0159] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0160] Even more preferably, 0.5ml of the composition contains a dose of 5μg of Neisseria meningitidis A saccharide-TT conjugate antigen. Even more preferably, 0.5ml of the composition contains a dose of 5μg of Neisseria meningitidis C saccharide-CRM197 conjugate antigen.

[0161] Even more preferably, 0.5ml of the composition contains a dose of 5μg of Neisseria meningitidis Y saccharide-CRM197 conjugate antigen. Even more preferably, 0.5ml of the composition contains a dose of 5μg of Neisseria meningitidis W saccharide-CRM197 conjugate antigen.

[0162] Even more preferably, 0.5ml of the composition contains a dose of 5μg of Neisseria meningitidis X saccharide-TT conjugate antigen. According to a twelfth embodiment of the present disclosure, the immunogenic composition comprises a combination of at least one of the following: a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; c) diphtheria toxoid (D) antigen, d) tetanus toxoid (T) antigen; e) whole cell pertussis (wP) antigen or acellular pertussis (aP), f) Hepatitis B surface antigen (HBsAg), and g) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; c) rotavirus antigens, d) diphtheria toxoid (D) antigen; e) tetanus toxoid (T) antigen, f) whole cell pertussis (wP) antigen or acellular pertussis (aP), g) Hepatitis B surface antigen (HBsAg), and h) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; d) diphtheria toxoid (D) antigen; e) tetanus toxoid (T) antigen, f) whole cell pertussis (wP) antigen or acellular pertussis (aP), g) Hepatitis B surface antigen (HBsAg), and h) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; e) diphtheria toxoid (D) antigen, f) tetanus toxoid (T) antigen, g) whole cell pertussis (wP) antigen or acellular pertussis (aP), h) Hepatitis B surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; e) diphtheria toxoid (D) antigen, f) tetanus toxoid (T) antigen, g) whole cell pertussis (wP) antigen or acellular pertussis (aP), h) Hepatitis B surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) rotavirus antigen, e) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; f) diphtheria toxoid (D) antigen, g) tetanus toxoid (T) antigen, h) whole cell pertussis (wP) antigen or acellular pertussis (aP), i) Hepatitis B surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; e) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; f) diphtheria toxoid (D) antigen, g) tetanus toxoid (T) antigen, h) whole cell pertussis (wP) antigen or acellular pertussis (aP), i) Hepatitis B surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; e) rotavirus antigens, f) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strains; g) diphtheria toxoid (D) antigen, h) tetanus toxoid (T) antigen, i) whole cell pertussis (wP) antigen or acellular pertussis (aP), j) Hepatitis B surface antigen (HBsAg), and k) Haemophilus influenzae type b antigen (Ηib).

[0163] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen.

[0164] Still preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0165] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0166] Still preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0167] More preferably, the composition contains diphtheria toxoid (D) antigen in an amount of 1-50 Lf per 0.5 ml. Even more preferably, the composition contains tetanus toxoid (T) in an amount of 1-30 Lf per 0.5 ml.

[0168] Still preferably, the composition comprises whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen including one or more modified adenylate cyclases, 1-50 μg pertussis toxin (PT), 1-50 μg filamentous hemagglutinin (FHA), 1-20 μg pertactin (P69 or PRN), or 2-25 μg fimbrial proteins (FIM1, 2 and 3) per 0.5 ml.

[0169] More preferably, the composition contains Hepatitis B virus surface antigen (HBsAg) in an amount of 1-20 μg per 0.5 ml. Even more preferably, the composition comprises Haemophilus influenzae type b antigen (Hib) in an amount of 1-20 μg per 0.5 ml.

[0170] Even more preferably, the composition comprises an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml.

[0171] More preferably, the composition comprises an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strain, in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3, per 0.5 ml.

[0172] According to a thirteenth embodiment of the present disclosure, the immunogenic composition comprises a combination of at least one of the following: a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b) rotavirus antigens, c) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; d) Shigella species antigen; e) Campylobacter jejuni antigen, f) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen, f) Campylobacter jejuni antigen, g) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen, f) Campylobacter jejuni antigen, or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) Diarrhea progenitor Escherichia coli species (enterotoxigenic and enterohemorrhagic )antigen, e) Shigella species antigen, or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) Shigella species antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) rotavirus antigens, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen, f) Campylobacter jejuni antigen, g) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) rotavirus antigen, e) diarrheagenic Escherichia coli spp. (enterotoxigenic and enterohaemorrhagic) antigens; f) Shigella species antigen; g) Campylobacter jejuni antigen, h) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi glyco-carrier protein conjugate antigen, b)Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; e) rotavirus antigens, f) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; g) Shigella species antigen; h) Campylobacter jejuni antigen, i) Vibrio cholerae antigen.

[0173] More preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella. Nella enterica serovar typhi ViPs-TT conjugate antigen.

[0174] Still preferably, 0.5 ml of the composition contains a dose range of 1.25 to 50 μg of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0175] Even more preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0176] Still preferably, 0.5 ml of the composition contains a dose range of 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen, where the CP is either TT or DT or CRM197.

[0177] Even more preferably, the composition comprises an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml.

[0178] According to one aspect of the embodiment, the immunogenic composition may further comprise a buffer selected from the group consisting of carbonate, phosphate, acetate, HEPES, succinate, histidine, Tris, borate, citrate, butyrate, gluconate and tartrate, as well as more complex organic buffers including phosphate buffers containing sodium phosphate and / or potassium phosphate in a ratio selected to reach a desired pH. In another example, the buffer contains tris(hydroxymethyl)aminomethane or "Tris" formulated to reach a desired pH. In yet another example, the buffer may be a minimum essential medium containing Hank's salt. Other buffers such as HEPES, piperazine-N,N'-bis (PIPES) and 2-ethanesulfonic acid (MES) are also contemplated by the present disclosure. The buffer helps stabilize the immunogenic composition of the present disclosure. The amount of the buffer may be in the range of 0.1 mM to 100 mM, preferably selected from 5 mM, 6 mM, 7 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM and 30 mM. The amount of the buffer may be in an amount of 0.1 mg to 2.0 mg.

[0179] Citrate buffer may be prepared by dissolving citric acid monohydrate (CAM) in the range of 1.05-2.63 mg and trisodium citrate anhydrous (TCD) in the range of 1.47-3.68 mg.

[0180] Preferably, the immunogenic composition may further comprise a Tris or citrate buffer or a histidine buffer or a succinate buffer in the range of 0.1 mg to 2.0 mg. Preferably, the immunogenic composition may further comprise Tris buffer in the range of 0.61 mg to 1.52 mg.

[0181] Preferably, the immunogenic composition may further comprise a citrate buffer in the range of 10 mM to 25 mM. Preferably, the immunogenic composition may further comprise a histidine buffer in the range of 0.78 to 1.94 mg.

[0182] Preferably, the immunogenic composition may further comprise a succinate buffer in the range of 0.59 to 1.48 mg. According to yet another aspect of the embodiment, the immunogenic composition may further comprise a pharma- ceutically acceptable excipient selected from the group consisting of a sugar, a surfactant, a polymer, a salt, an amino acid, or a pH adjuster.

[0183] Examples of surfactants may include ionic and non-ionic surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethanol, t-octylphenoxypolyethoxyethanol, oxtoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin, and poloxamer.

[0184] Preferably, the immunogenic composition may include polysorbate 20 as a pharma- ceutically acceptable excipient. Examples of polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, and the like.

[0185] Examples of salts may include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaCl2, MgCl2, etc. Preferably, the salt may be NaCl. Typically, the amount of salt may range from 100 mM to 200 mM.

[0186] Preferably, the immunogenic composition may contain sodium chloride in the range of 1-10 mg. Examples of amino acids as excipients are selected from the group of L-histidine, lysine, isoleucine, methionine, glycine, aspartic acid, tricine, arginine, leucine, glutamine, alanine, peptides, hydrolyzed proteins or proteins such as serum albumin.

[0187] Preferably, the immunogenic composition may include histidine. Examples of sugars as excipients are selected from the group of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol or combinations thereof.

[0188] Preferably, the immunogenic composition may further comprise sucrose. Examples of polymers as excipients are selected from the group consisting of dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin.

[0189] Even more preferably, the single dose composition is preservative-free. Still preferably, the multi-dose immunogenic composition may further comprise a preservative selected from the group consisting of 2-phenoxyethanol, benzethonium chloride (femerol), phenol, m-cresol, thiomersal, formaldehyde, paraben esters (e.g., methyl-, ethyl-, propyl- or butyl-paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-m-cresol or benzyl alcohol, or combinations thereof. The vaccine composition may contain material for a single immunization or may contain material for multiple immunizations (i.e., a "multi-dose" kit). Inclusion of a preservative is preferred in a multi-dose configuration. Alternatively (or in addition) to including a preservative in the multi-dose composition, the composition may be contained in a container having a sterile adaptor for removal of material. Preferably, the preservative may be 2-phenoxyethanol in the range of 0.1 mg to 50 mg, more preferably 1 to 10 mg.

[0190] According to yet another aspect of the embodiment, the immunogenic composition may further comprise auxiliary substances, such as wetting or emulsifying agents, diluent pH buffering agents, gelling or thickening additives, preservatives, flavoring agents, coloring agents, and the like, depending on the desired route of administration and preparation.

[0191] According to yet a preferred aspect of the embodiment, the immunogenic composition may further comprise water for injection as a diluent. According to yet another aspect of the embodiment, the immunogenic composition may further comprise an adjuvant selected from the group of aluminum salts, aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate and potassium aluminum sulfate.

[0192] According to yet another aspect of the embodiment, the immunogenic composition is selected from the group consisting of an oil-water emulsion, MF-59, liposomes, lipopolysaccharides, saponins, lipid A, lipid A derivatives, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, oligonucleotides, oligonucleotides containing at least one unmethylated CpG and / or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers including block copolymers, polymer p 1005, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonist, flagellin, flagellin from gram-negative bacteria, TLR-5 agonist, fragments of flagellin capable of binding to the TLR-5 receptor, alpha-C-galactosylceramide, chitosan, interleukin-2, QS-21, ISCOMS, squalene mixture (SAF-1), Quil A, cholera toxin B subunit, polyphosphazenes and derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, non-ionic block copolymer surfactants, OMV, fHbp, saponin combinations with sterols and lipids.

[0193] According to yet another aspect of the embodiment, the immunogenic composition may be entirely liquid. Suitable forms of liquid preparations may include solutions buffered to a selected pH, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions and elixirs.

[0194] More preferably, the immunogenic composition may be completely liquid and stable at 2-8°C, 25°C and 40°C over a period of 6 months, with the free polysaccharide after 6 months being 7.5% or less for the 180-220 kDa polysaccharide and 10.5% or less for the 388 / 80 / 45 kDa polysaccharide.

[0195] The immunogenic composition of the present disclosure may be in the form of a transdermal preparation, including lotion, gel, spray, ointment or other suitable technology. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), the composition is in a form and can be administered by a squeeze spray dispenser, pump dispenser or aerosol dispenser. Aerosols are usually under pressure with a hydrocarbon. Pump dispensers can preferably administer metered doses or doses with a specific particle size. When in the form of a solution, suspension and gel, in some embodiments, the immunogenic composition contains a large amount of water (preferably purified water) in addition to the active ingredient. According to yet an alternative aspect of the embodiment, the immunogenic composition may be a lyophilized or freeze-dried composition.

[0196] As used herein, the term "freeze-drying" or "lyophilizing" or "lyophilization" includes lyophilization and refers to the process of freezing a suspension / solution followed by removal of water by sublimation at low pressure. As used herein, the term "sublimation" refers to the physical process of a composition in which the composition changes directly from a solid state to a gaseous state without becoming a liquid. Refers to a change in characteristics.

[0197] Thus, the lyophilized immunogenic composition may be stable for 12-36 months at 2-8°C, 2-6 months at 25°C, 1-4 weeks at 37°C, 2-7 days at 42°C, and 2-7 days at 55°C.

[0198] According to one aspect of the embodiment, the method for reconstituting the lyophilized immunogenic composition may comprise the step of reconstituting the lyophilized immunogenic composition with an aqueous solution, optionally saline or water for injection (WFI), wherein the final pH of the immunogenic composition after reconstitution is in the range of pH 6.0 to pH 8.0, more preferably in the range of pH 7.0 to pH 8.0, even more preferably in the range of pH 7.2 to pH 7.9, and most preferably in the range of pH 7.5 to pH 7.9.

[0199] According to a ninth embodiment of the present disclosure, the immunogenic composition may be formulated for use in a method for reducing or preventing the onset of a health condition, including Salmonella serotypes S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis infection, comprising administering an immunologically effective amount of the immunogenic composition to a human subject via parenteral or subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration, or injectable administration, or sustained release from an implant, or ophthalmic or nasal or rectal or buccal or vaginal, oral or intragastric or mucosal or sublingual, alveolar or gingival or olfactory or respiratory mucosal administration, or any other immunization route.

[0200] According to a preferred aspect of the embodiment, the immunogenic composition may be administered to a human subject via the intramuscular route or subcutaneously. According to a preferred aspect of the embodiment, an immunologically effective amount of an immunogenic composition comprising a polysaccharide-protein conjugate for vaccination against bacterial infection with the Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis infections can be about 1 μg / 0.5 ml or less of polysaccharide conjugate of the Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium or S. enteritidis to about 100 μg / 0.5 ml or more of polysaccharide conjugate of the Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium or S. enteritidis. In some other aspects, this may be from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 μg to about 55, 60, 65, 70, 75, 80, 85, 90, or 95 μg per single 0.5 ml dose. Immunologically effective amounts for vaccination against bacterial infections are between 1 μg / 0.5 ml and 50 μg / 0.5 ml, more preferably the Salmonella enterica serovar Typhi A sugar-carrier protein conjugate antigen, Salmonella enterica serovar Typhimurium sugar-carrier protein conjugate antigen, Salmonella enterica serovar Enteritidis sugar-carrier protein conjugate antigen are present in a dosage range of about 5 ug / 0.5 ml to about 30 ug / 0.5 ml, even more preferably the Salmonella enterica serovar Typhi sugar-carrier protein conjugate antigen, Salmonella enterica serovar Enteritidis sugar-carrier protein conjugate antigen are present in a dosage range of about 5 ug / 0.5 ml to about 30 ug / 0.5 ml, even more preferably the Salmonella enterica serovar Typhi sugar-carrier protein conjugate antigen, Salmonella enterica serovar Enteritidis sugar-carrier protein conjugate antigen are present in a dosage range of about 5 ug / 0.5 ml to about 30 ug / 0.5 ml. Paratyphi A carbohydrate-carrier protein conjugate antigen, Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen, Salmonella enterica serovar ent The C. eritidis saccharide-carrier protein conjugate antigen is present at a dose of approximately 25ug / 0.5ml.

[0201] According to the tenth embodiment of the present disclosure, the immunogenic composition may be administered intramuscularly or subcutaneously in a dose effective for producing neutralizing antibodies and protection. The vaccine is administered in a manner compatible with the dosage formulation and in an amount that is prophylactically and / or therapeutically effective against typhoid and NTS infection. The immunogenic composition of the present disclosure may be administered as a primary preventative agent in the elderly, teenagers, adults or children at risk of infection, or may be used as a secondary agent to treat infected patients. For example, the immunogenic composition disclosed herein may be used in the elderly, teenagers, adults or children under or over 2 years of age at risk of infection with Salmonella serotypes S.typhi, S.paratyphi A, S.typhimurium and S.enteritidis, or may be used as a secondary agent to treat patients infected with Salmonella serotypes S.typhi, S.paratyphi A, S.typhimurium and S.enteritidis.

[0202] Alternatively, the NTS vaccine may be administered to infants aged 2-4 months, prior to the peak incidence occurring at approximately 12 months of age. In addition, it is believed that vaccine rollouts may also include HIV-infected populations, as they are at higher risk of NTS infection. In developed countries, NTS vaccines may target the elderly, who experience a very high case fatality rate (up to 50 percent). In children, it has been proposed that planned field rollouts could be directly integrated into existing immunization expansion program schedules, possibly at 6, 10, and 14 weeks.

[0203] More preferably, the immunogenic composition may be administered intramuscularly or subcutaneously in a dosage volume of about 0.5 ml or 1 ml. According to an eleventh embodiment of the present disclosure, the immunogenic composition may be formulated as a single or multi-dose vial (2-dose or 5-dose or 10-dose vial), or as a multi-dose kit, or as a pre-filled syringe, and said immunogenic composition may be administered in a single dose schedule, or preferably in a multiple dose schedule, with a primary course of vaccination followed, if necessary, by 1-3 divided doses administered at subsequent time intervals of 1-3 years later. The dosage regimen is also determined, at least in part, according to the need for booster doses required to confer protective immunity.

[0204] Even more preferably, the immunogenic composition may be formulated for administration to a human subject, be it an elderly, a teenager, an adult or a child under or over 2 years of age, according to a one or two dose regimen consisting of a first dose and / or a second dose administered 3 months to 2 years after the first dose and / or a third dose administered 3 months to 2 years after the second dose, or a three dose regimen.

[0205] According to an eleventh embodiment of the present disclosure, the immunogenic composition may be administered simultaneously with other drugs or any other vaccine. According to one aspect of the eleventh embodiment, the single-dose vaccine kit comprises: Lyophilized (freeze-dried) immunogenic composition: a) 5 μg Neisseria meningitidis A saccharide-TT conjugate antigen per 0.5 ml; b) 5 μg Neisseria meningitidis C saccharide-CRM197 conjugate antigen per 0.5 ml; c) 5 μg Neisseria meningitidis Y glyco-CRM197 conjugate antigen per 0.5 ml; d) 5 μg Neisseria meningitidis W-135 saccharide-CRM197 conjugate antigen per 0.5 ml; e) 5 μg Neisseria meningitidis X-glyco-TT conjugate antigen per 0.5 ml; f) 1 to 12 mg of sucrose per 0.5 ml; g) 0.1-2 mg of sodium citrate (dihydrate) per 0.5 ml; h) 0.05 to 0.5 mg of Tris buffer per 0.5 ml a first container containing; and a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride per 0.5 ml; c) Water for injection (WFI) in appropriate volume A second container containing a liquid composition for the reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising For protection against typhoid fever caused by Salmonella typhi and Neisseria meningitidis antigens, there is no antigenic interference of ViPs-TT with Neisseria meningitidis antigens, and the vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi in children under 2 years of age, teenagers, adults and the elderly with only one injection comprising a complete vaccination schedule.

[0206] Still according to a second aspect of the eleventh embodiment, the single dose vaccine kit comprises: Lyophilized (freeze-dried) immunogenic composition: a) 5 μg Neisseria meningitidis A saccharide-TT conjugate antigen per 0.5 ml; b) 5 μg Neisseria meningitidis C saccharide-CRM197 conjugate antigen per 0.5 ml; c) 5 μg Neisseria meningitidis Y glyco-CRM197 conjugate antigen per 0.5 ml; d) 5 μg Neisseria meningitidis W-135 saccharide-CRM197 conjugate antigen per 0.5 ml; e) 5 μg Neisseria meningitidis X-glyco-TT conjugate antigen per 0.5 ml; f) 1 to 12 mg of sucrose per 0.5 ml; g) 0.1-2 mg of sodium citrate (dihydrate) per 0.5 ml; h) 0.05 to 0.5 mg of Tris buffer per 0.5 ml a first container containing; and a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197); c) 1 to 10 mg of sodium chloride per 0.5 ml; d) Water for injection (WFI) in appropriate volume A second container containing a liquid composition for the reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising and typhoid fever caused by Salmonella typhi, S. paratyphi and Neisseria meningitidis antigens. For protection against paratyphoid fever, there is no antigenic interference of the ViPs-TT;OSP antigen with Neisseria meningitidis antigens, and the vaccine formulation is sufficient to induce the necessary T-dependent immune responses against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly with only one injection comprising the complete vaccination schedule.

[0207] Still according to a third aspect of the eleventh embodiment, the single dose vaccine kit comprises: A fully liquid hexavalent immunogenic composition comprising: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1-50 D antigenic units (DU) for IPV type 1, 1-50 D antigenic units (DU) for IPV type 2, or 1-50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; c) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; d) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen comprising one or more of the following modified adenylate cyclases selected from pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (P69 or PRN), 1-20 μg, or fimbrial proteins (FIM1, 2, and 3), per 0.5 ml; e) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; f) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; And a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride per 0.5 ml, and / or c) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer per 0.5 ml, and / or d) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25 to 500 μg per 0.5 ml, and / or e) 2-phenoxyethanol, from 1 to 10 mg per 0.5 ml, and / or f) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising and for protection against Salmonella typhi and typhoid fever caused by hexavalent antigens, there is no antigenic interference of ViPs-TT with the hexavalent immune composition, and said vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi, including in children under 2 years of age, teenagers, adults and the elderly, with only one injection comprising a complete vaccination schedule.

[0208] Still according to a fourth aspect of the eleventh embodiment, the single dose vaccine kit comprises: Complete liquid hexavalent immunogenic composition: a) inactivated poliovirus (IPV) selected from the Sabin or Salk strains in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3 per 0.5 ml; antigen, b) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; c) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; d) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen comprising one or more of the following modified adenylate cyclases selected from pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (P69 or PRN), 1-20 μg, or fimbrial proteins (FIM1, 2, and 3), per 0.5 ml; e) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; f) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; And a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197); c) 1 to 10 mg of sodium chloride, and / or d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer per 0.5 ml, and / or g) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25 to 500 μg per 0.5 ml, and / or h) 1 to 10 mg of 2-phenoxyethanol per 0.5 ml, and / or e) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising and for protection against Salmonella typhi, S. paratyphi and typhoid and paratyphoid caused by the hexavalent antigen, there is no antigenic interference of the ViPs-TT;OSP antigen with the hexavalent antigen, and said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly, with only one injection comprising a complete vaccination schedule.

[0209] Still according to a fifth aspect of the eleventh embodiment, the single dose vaccine kit comprises: Complete liquid heptavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml; c) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; d) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; e) Whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or 1-50 μg pertussis toxin (PT) or 1-50 μg filamentous hemagglutinin (FHA) per 0.5 ml. acellular pertussis (aP) antigens comprising one or more of the following: 1-20 μg of modified adenylate cyclase selected from 1-20 μg of pertactin (P69 or PRN) or 2-25 μg of fimbrial proteins (FIM1, 2 and 3); f) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; g) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; and a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride per 0.5 ml; c) Water for injection (WFI) in appropriate volume A second container containing a liquid composition for the reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising and for protection against Salmonella typhi and typhoid fever caused by the heptavalent antigen, there is no antigenic interference of ViPs-TT with the heptavalent immune composition, and the vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi, including in children under 2 years of age, teenagers, adults and the elderly, with only one injection comprising a complete vaccination schedule.

[0210] Still according to a sixth aspect of the eleventh embodiment, the single dose vaccine kit comprises: Complete liquid heptavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml; c) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; d) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; e) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen containing one or more of the following modified adenylate cyclases selected from pertussis toxin (PT), filamentous hemagglutinin (FHA), pertactin (P69 or PRN), 1-20 μg, or fimbrial proteins (FIM1, 2, and 3), per 0.5 ml; f) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; g) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; And a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197); c) 1 to 10 mg of sodium chloride per 0.5 ml, and / or d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer per 0.5 ml, and / or g) Polysorbate 20, Polysorbate 40, Polysorbate 60, Polysorbate 65, Polysorbate 80, Polysorbate 8 in amounts of 25-500 μg per 0.5 ml 5, polysorbates selected from nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer, and / or h) 1 to 10 mg of 2-phenoxyethanol per 0.5 ml, and / or e) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising for protection against Salmonella typhi, S. paratyphi and typhoid and paratyphoid caused by the heptavalent antigen, there is no antigenic interference of the ViPs-TT;OSP antigen with the heptavalent antigen, and said vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly with only one injection comprising a complete vaccination schedule.

[0211] Salmonella serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis used to develop the immunogenic compositions of the present disclosure include Salmonella enterica serovar typhi TY2 strain harboring the Vi polysaccharide-specific "tviB" gene (identified by Geneombio Technologies Private Limited, Pune and isolated from a stool sample of a patient with confirmed typhoid fever at Villoo Poonawalla Memorial Hospital, Pune); S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150, CMCC50073, CMCC50973 obtained from Chromachemie Laboratory Private Limited, Bangalore; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219; S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC19585; ATCC 700408; (LT2 / SL134(ST19)); S. typhimurium 177(ST19)CDC 6516-60; ATCC 700720. Additionally, any attenuated Salmonella serovar strain (S. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used in the preparation of the immunogenic compositions of the present disclosure.

[0212] Salmonella enterica serovar typhi deposited at the international depository authority NCMR-NCCS, Pune and assigned accession number MCC 0193; strain designation PDL-1.

[0213] Other embodiments disclosed herein also include vaccine kits comprising a first container containing a lyophilized (freeze-dried) immunogenic composition and a second container containing an aqueous solution, optionally saline or WFI (water for injection), for reconstituting the lyophilized (freeze-dried) immunogenic composition.

[0214] The foregoing description of specific embodiments fully reveals the general nature of the embodiments herein, such that anyone, by applying current knowledge, may easily modify and / or adapt such specific embodiments to various applications without departing from the general concept, and therefore such adaptations and modifications are to be understood as falling within the meaning and scope of equivalents of the embodiments of the present disclosure. It is to be understood, and is intended, that the phraseology or terminology used herein is for the purpose of description and not limitation. Thus, although the embodiments herein have been described with reference to preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modifications within the spirit and scope described herein.

[0215] Throughout this specification the word "comprise" or variations such as "comprises" or "comprising" imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but does not imply the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0216] The use of the phrase "one or more" or "at least one" connotes the use of one or more elements or ingredients or amounts by which one or more desired objects or results can be achieved in the embodiments of the invention.

[0217] Any discussion of documents, works, materials, devices, articles or the like which has been included in this specification is solely for the purpose of providing a context for the present disclosure. The existence of any prior to the priority date of this application is not to be construed as an admission that any or all of such matters form part of the prior art body or are common general knowledge in the field relevant to this disclosure.

[0218] The numerical values ​​given for various physical parameters, dimensions and amounts are merely approximations, which means that, unless stated to the contrary herein, values ​​higher than the numerical values ​​assigned to the physical parameters, dimensions and amounts are assumed to be within the scope of the invention.

[0219] Although certain features of the preferred embodiments have been emphasized herein, it will be recognized that many additional features may be added and that many modifications may be made in the preferred embodiments without departing from the principles of the present disclosure. These and other modifications in the preferred embodiments of the present disclosure will be apparent to those skilled in the art from the present disclosure, and it will be clearly understood that the foregoing description is intended to be merely illustrative of the present disclosure and not limiting. Technical advantages: 1. The present disclosure provides monovalent and multivalent polysaccharide-protein conjugate vaccines comprising polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis, in any combination thereof, effective to confer protection against or treatment of infection against, or to prevent, ameliorate or delay the onset or progression of clinical symptoms thereof.

[0220] 2. Improved upstream, downstream and conjugation processes. 3. The upstream fermentation medium does not contain casein digest / tryptone or casamino acids. 4. Use of i) a combination of antifoam, soy peptone and yeast extract, ii) a specific DO of 36-39%, and iii) an osmolality of 400-600 mOsmol / kg, results in an improvement in harvest stage polysaccharide yield of >50%.

[0221] 5. An improved purification method with low endotoxin <10EU / μg, low protein <1% and low nucleic acid content <2%, wherein the purification process is i) free of sodium deoxycholate (DOC), which is an animal-derived product and even its residual presence in the final product can result in a product that is unacceptable by regulatory authorities and certain religious communities, ii) free of Triton X, iii) free of ammonium sulfate, iv) free of any adsorbents (hydophors, etc.) The purification method does not include any of the above-mentioned compounds (e.g., hydroxyapatite, calcium phosphate or apatite).

[0222] 6. Improved conjugation efficiency (>60%), improved conjugate yield (≧50%) and conjugate stability with i) Ps:Pr:EDAC ratio of 1:1:2 and ii) stable free polysaccharide <5% (preferably <3%) and free protein <5% (preferably <4%).

[0223] 7. Improved immunogenicity of the conjugates resulting from i) the Vi polysaccharide-protein conjugates having a characteristic size of 1200 kDa to 1600 kDa, and ii) the polysaccharide used for conjugation having an average molecular weight of 150 to 300 kDa (preferably 150 to 250 kDa).

[0224] 8. The immunogenic composition is stable at 2-8°C, 25°C and 40°C over a period of 6 months with free polysaccharides after 6 months being less than or equal to 7.5% for the 180-220 kDa polysaccharide and free polysaccharides after 6 months being less than or equal to 10.5% for the 388 / 80 / 45 kDa polysaccharide.

[0225] 9. Groups of mice injected with bivalent (typhoid and paratyphoid) SIIPL vaccines such as a) SIIPL Vi PS-TT+SIIPL O-SP A DT, b) SIIPL Vi PS-TT+SIIPL O-SP A TT and c) SIIPL Vi PS-TT+SIIPL O-SP A CRM Vi TT showed more than 4-fold higher induction of IgG (compared to mice receiving unconjugated Vi PS or unconjugated SIIPL O-SP A), thus indicating the immunogenic potential of the bivalent SIIPL vaccine containing the combination of Vi TT and SIIPL O-SP A (DT / TT / CRM).

[0226] 10. Minimal components are included in the vaccine composition. EXAMPLES

[0227] The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will recognize that the compositions and techniques disclosed in the following examples represent techniques discovered by the inventors to work well in the practice of the present invention, and therefore can be considered to constitute preferred modes of its practice. However, those skilled in the art should recognize in light of this disclosure that many changes can be made in the specific embodiments disclosed and still obtain similar or similar results without departing from the spirit and scope of the present invention.

[0228] Example 1: A) Stocks: The Salmonella serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis used to develop the immunogenic compositions of the present disclosure include Salmonella enterica serovar typhi strain TY2 (identified by Geneombio Technologies Private Limited, Pune and isolated from a stool sample of a patient with confirmed typhoid fever at Villoo Poonawalla Memorial Hospital, Pune) carrying the Vi polysaccharide-specific "tviB" gene; Salmonella enterica serovar typhi deposited with the international depository authority NCMR-NCCS, Pune and assigned the accession number MCC 0193; strain designation PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: Chromachemie Obtained from Laboratory Private Limited, Bangalore: ATCC 9150, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219; S. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC19585; ATCC 700408; (LT2 / SL134(ST19)); S. typhimurium 177(ST19)CDC 6516-60; ATCC 700720. Additionally, any attenuated Salmonella serovar strain (S. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used in the preparation of the immunogenic compositions of the present disclosure.

[0229] WO2018037365, WO2019016654 and WO2020075184 are incorporated by reference for the preparation of diphtheria toxoid (DT), tetanus toxoid (TT), inactivated whole cell pertussis (wP), acellular pertussis (aP), Hepatitis B surface antigen (HBsAg), Haemophilus influenzae type b antigen (Hib) and inactivated poliovirus (standard dose and reduced dose poliovirus).

[0230] WO2018037365 is incorporated by reference for the preparation of inactivated poliovirus and inactivated rotavirus. WO2013114268 is incorporated by reference for the preparation of meningococcal polysaccharide antigens of serotypes A, C, W, X and Y. B) A method for obtaining polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis by fed-batch process, comprising the following steps: (upstream fermentation) 1) S1 stage of inoculation and harvest: Dilute a 0.5 mL culture from a cell bank vial to 5 mL with medium, and streak a loopful of this culture onto an SCDA plate and incubate at 36 ± 0.5 °C for 28-32 h.

[0231] 2) S2 stage of inoculation and harvest: Inoculate 10 well-isolated colonies from the S1 stage plate into a conical flask containing 40 mL medium. Incubate the flask at 36 ± 0.5 °C and 150 ± 10 RPM until the optical density (OD) of the culture reaches the 2.5 - 3.5 range.

[0232] 3) S3 stage of inoculation and harvest: When the optical density (OD) of the S2 stage culture reaches the 2.5-3.5 range, increase the 40 ml culture to 800 ml and distribute 160 ml contents into five 1 L flasks each. Incubate the flasks at 36 ± 0.5 °C and 150 ± 10 rpm until the OD of the culture reaches the 2.5-3.5 range.

[0233] 4) S4 stage of inoculation and harvest (20L fermenter): Once the OD of the S3 stage culture reaches the 2.5-3.5 range, pool the cultures from all five flasks into the seed bottle assembly and inoculate the 20 L fermenter. Continue the fermentation using the following parameters:

[0234] Fermentation process parameters:

[0235] [Table 1]

[0236] After the third hour, start feeding the medium and allow it to increase proportionally to the OD. Continue the fermentation for 13-15 hours. 5) Formalin inactivation at the S4 stage: After the S4 stage is complete, add formaldehyde solution to the fermenter to a final concentration of 0.5-1.0% and incubate the culture at 36 °C ± 2 °C for 8-10 h.

[0237] 6) Cell separation by centrifugation: After the inactivation step is complete, the broth is clarified by centrifugation and the supernatant is collected. Centrifugation is carried out using the following parameters:

[0238] [Table 2]

[0239] 7) Clarification by depth filtration: After centrifugation is complete, the supernatant is filtered using a depth filter. 8) 0.2μ filtration: After depth filtration, the filtrate is subjected to 0.2μ filtration. B1. Medium optimization for the growth of Salmonella typhi, S. paratyphi A, S. typhimurium and S. enteritidis.

[0240] The following experiments were performed for medium optimization and batch parameters. Experiment No: 01 - Frantz medium was selected for the experiment as it is widely used for the growth of Gram-negative microorganisms and was evaluated for the growth of Salmonella Typhi. Frantz medium was used for the growth and fermentation of the original species. Glucose was constituted in the feed medium as the carbon source.

[0241] [Table 3]

[0242] [Table 4]

[0243] The starter seed was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi microorganisms. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

[0244] observation:

[0245] [Table 5]

[0246] Conclusion: Frantz medium was found to support the growth of Salmonella typhi organisms to a limited extent. Further optimization of the medium was required. Experiment No: 02 - Yeast extract was added to the fermentation and feed media to support cell growth.

[0247] [Table 6]

[0248] [Table 7]

[0249] procedure: The starter seed was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi microorganisms. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

[0250] observation:

[0251] [Table 8]

[0252] Conclusion: The addition of yeast extract was found to support the growth of Salmonella typhi organisms to a limited extent. Further optimization of the medium was required. Experiment No: 03 - Soy peptone was added to the fermentation and feed media to support cell growth.

[0253] [Table 9]

[0254] [Table 10]

[0255] procedure: The starter seed was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi microorganisms. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

[0256] observation:

[0257] [Table 11]

[0258] Conclusion: The addition of soy peptone was found to support the growth of Salmonella typhi organisms. Optimization of batch parameters for production was required. Experiment No: 04 - Antifoam Ctobe was replaced with antifoam Struktol for improved cell growth.

[0259] [Table 12]

[0260] [Table 13]

[0261] procedure: The starter seed was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi microorganisms. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

[0262] observation:

[0263] [Table 14]

[0264] Conclusion: Replacement of Antifoam C with Antifoam Struktol was found to improve the growth of Salmonella typhi organisms. Experiment No: 05 - Growth pattern of Salmonella typhi was studied in 20L scale batches with the following fermentation parameters. Suitable parameters were defined for Salmonella typhi batches. Dissolved oxygen and osmolality were controlled near the experimental set points.

[0265] [Table 15]

[0266] [Table 16]

[0267] [Table 17]

[0268] Procedure: Seed was grown in disposable flasks (250ml and 1L) and inoculated into a 20L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi microorganisms. The batch was operated at a temperature of 36°C, pH 7.00 and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during fermentation.

[0269] observation:

[0270] [Table 18]

[0271] Conclusion: From the observations, it was concluded that for the growth of Salmonella typhi microorganism, optimization of the parameters mentioned in the experimental set points table of this experiment was required.

[0272] Experiment No: 06 - Growth pattern of Salmonella typhi was studied in 20L scale batches with the following fermentation parameters. Suitable parameters were defined for Salmonella typhi batches. Dissolved oxygen and osmolality were controlled near the experimental set points.

[0273] [Table 19]

[0274] [Table 20]

[0275] [Table 21]

[0276] Procedure: The starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was incubated at a temperature of 36° C., pH 7. The fermentation was operated at .00 and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during fermentation.

[0277] observation:

[0278] [Table 22]

[0279] Conclusion: From the observations, it was concluded that for the growth of Salmonella typhi microorganism, optimization of the parameters mentioned in the experimental set points table of this experiment was required.

[0280] Experiment No: 07 - Growth pattern of Salmonella typhi was studied in 20L scale batches with the following fermentation parameters. Suitable parameters were defined for Salmonella typhi batches. Dissolved oxygen and osmolality were controlled near the experimental set points.

[0281] [Table 23]

[0282] [Table 24]

[0283] [Table 25]

[0284] Procedure: The starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was incubated at a temperature of 36° C., pH 7. The fermentation was operated at .00 and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during fermentation.

[0285] observation:

[0286] [Table 26]

[0287] Conclusion: From the observations, it was concluded that for the growth of Salmonella typhi microorganism, optimization of the parameters mentioned in the experimental set points table of this experiment was required.

[0288] Experiment No: 08 - Growth pattern of Salmonella typhi was studied in 20L scale batches with the following fermentation parameters. Suitable parameters were defined for Salmonella typhi batches. Dissolved oxygen and osmolality were controlled near the experimental set points.

[0289] [Table 27]

[0290] [Table 28]

[0291] [Table 29]

[0292] Procedure: The starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermentor. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermentor to support the growth of Salmonella Typhi microorganisms. The batch was operated at a temperature of 36°C, pH 7.00 and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during fermentation.

[0293] observation:

[0294] [Table 30]

[0295] Conclusion: From the observations, it was concluded that for the growth of Salmonella typhi microorganism, optimization of the parameters mentioned in the experimental set points table of this experiment was required.

[0296] Experiment No: 09 - Growth pattern of Salmonella typhi was studied in 20L scale batches with the following fermentation parameters. Suitable parameters were defined for Salmonella typhi batches. Dissolved oxygen and osmolality were controlled near the experimental set points.

[0297] [Table 31]

[0298] [Table 32]

[0299] [Table 33]

[0300] Procedure: The starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was incubated at a temperature of 36° C., pH 7. The fermentation was operated at .00 and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during fermentation.

[0301] observation:

[0302] [Table 34]

[0303] Conclusion: From the observations, it was concluded that for the growth of Salmonella typhi microorganism, optimization of the parameters mentioned in the experimental set points table of this experiment was required.

[0304] Experiment Number: The growth pattern of Salmonella typhi was studied with the following fermentation parameters in 10-20L scale batches. Suitable parameters were defined for the Salmonella typhi batches. Dissolved oxygen and osmolality were controlled near the experimental set points.

[0305] [Table 35]

[0306] [Table 36]

[0307] [Table 37]

[0308] Procedure: The starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermenter. After inoculation, the fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was incubated at a temperature of 36° C., pH 7. The fermentation was operated at .00 and 150-500 RPM agitation (cascade mode to maintain DO). Antifoam was added intermittently to control foaming during fermentation.

[0309] observation:

[0310] [Table 38]

[0311] Conclusion:

[0312] [Table 39]

[0313] Fed-batch mode of cultivation to obtain high yield harvest of polysaccharides from Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis by fed-batch process involving use of a combination of antifoam J673 STRUKTOL, soy peptone Difco™ Select Phytone™ UF in the range of 40-70 g / L and yeast extract Difco™ Yeast Extract, UF in the range of 40-70 g / L during cultivation resulted in improved yield (100-700 mg / L) and fermentation parameters consisted of pH maintained in the range of 6.7-7.1, temperature maintained in the range of 34.0-38.0°C, dissolved oxygen level maintained at 36-39%, agitation (rpm) maintained at 150-500 and osmolality of 400-600 mOsmol / kg.

[0314] Example 2: C) Methods for purifying polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis (downstream purification) C1) The ViPs fermentation harvest was subjected to the following downstream purification steps to obtain Vi polysaccharide (ViPs) of desired quality: n) clarification of the bacterial capsular polysaccharide harvest by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.2 micrometers; o) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a 100 kDa molecular weight cut-off (MWCO) (20 mM Tris buffer pH 7.5 containing 5 mM EDTA); p) treatment with SDS (20% SDS stock input), ethylenediaminetetraacetic acid (EDTA) (4-10 mM) and sodium acetate (5%-10%) for denaturation of proteins, nucleic acids and lipopolysaccharides; q) Ethanol precipitation (40%-70%), r) centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 μM; s) treatment with 2 M potassium chloride (KCl) to remove excess detergent, followed by centrifugation and filtration by direct flow filtration (DFF) through at least one clarifying filter having a pore size of about 0.2 μM; t) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a molecular weight cut-off (MWCO) of 30 kDa; u) Selective precipitation of PS with CTAB (12% CTAB stock input); v) centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.45 micrometers to about 0.2 micrometers; w) removal of protein and nucleic acid impurities by washing the pellet with 60% ethanol (50%-70%) in the presence of 1M NaCl (0.1M-2M); x) selective precipitation of polysaccharides by utilizing 60% ethanol (<75% OR >95%); y) dissolving the polysaccharide in WFI or 1M NaCl and subjecting it to concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a molecular weight cut-off (MWCO) of 30 kDa; and z) sterile filtration through at least one sterile filter having a pore size of about 0.2 micrometers under sterile conditions and storage at ≦−20° C. C2) Lipopolysaccharide (LPS) Fermentation The harvested Salmonella paratyphi O-specific polysaccharide (OSP) was subjected to the following downstream purification steps to obtain the desired quality of O-specific polysaccharide from Salmonella Paratyphi A lipopolysaccharide (LPS): r) centrifugation at 7000 rpm, 4° C. for 30 minutes, collecting the cell pellet (approximately 1 kg), suspending the supernatant and cell pellet in 15 L of 1M NaCl, and stirring at room temperature for 1 hour; s) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using 0.45 μm Prostak cassettes and membranes with a 30 kDa molecular weight cut-off (MWCO); t) acid hydrolysis of LPS with 1% acetic acid (pH about 2.8-3.0) at a temperature of 90°C for 180 minutes; u) cooling the mixture to room temperature (RT) and removing impurity precipitates by centrifugation at 7000 rpm at 25° C. for 45 minutes; v) neutralizing the supernatant in a glass bottle and neutralizing it to pH 7.0 with aqueous ammonia; w) clarification by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and about 0.2 micrometers; x) adding sodium deoxycholate stock solution to a final concentration of 1% and incubating for 30 minutes with stirring at a temperature of 30° C., adjusting the pH to 2.0 with acetic acid and incubating for 15 minutes with stirring at 30° C.; y) Centrifuge at 7000 rpm, 25°C for 45 minutes; z) direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and about 0.2 micrometers; aa) Concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a 10 kDa molecular weight cut-off (MWCO); bb) obtaining purified O-specific polysaccharide (OSP) by sterile filtration under sterile conditions through at least one sterile filter having a pore size of about 0.2 micrometers.

[0315] [Table 40-1]

[0316] [Table 40-2]

[0317] Results and Interpretation:

[0318] [Table 41]

[0319] The improved method of Vi PS purification has clear advantages both in terms of ease of operation and several other advantages which are as follows. Improved polysaccharide purification (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, and viscosity) when using sodium acetate (6%) versus sodium acetate (2%) Values ​​when sodium acetate (6%) is used DSP % Recovery Rate - 50% Ps Viscosity - No data available Ps polydispersity - no data available O-Acetyl - 2.9mmol / gmPS Values ​​when sodium acetate (2%) is used DSP % Recovery Rate - 40% Ps Viscosity - No data available Ps polydispersity - no data available O-Acetyl - 2.6mmol / gmPS Sodium acetate reacts with nucleic acids. It dissociates into Na+ and (CH3COO)-. The positively charged sodium ions neutralize the negative charge PO3- of nucleic acids, thus aiding in the precipitation of nucleic acids. Therefore, higher concentrations of sodium acetate, i.e. 6%, effectively precipitate host cell impurities such as nucleic acids.

[0320] Improved polysaccharide purification (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, viscosity) with CTAB (3%) versus CTAB (0.5, 1%). CTAB is an amine-based cationic quaternary surfactant. CTAB interacts with anionic polysaccharides (ionic interaction) and then its solubility is reduced, so CTAB precipitates the polysaccharides from the solution. Therefore, a higher concentration of CTAB (2%) always precipitates a higher amount of polysaccharides, which results in a higher yield and the removal of protein impurities is an added advantage.

[0321] · Improved polysaccharide purification (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, viscosity) in current processes without DOC versus DOC-based processes.

[0322] Advantages of the improved method without the DOC process 1. Regulatory Issues: DOC is an animal-derived ingredient and is not HALAL compliant. DOC is manufactured and supplied by a single vendor worldwide, whereas SDS is a synthetic detergent, is HALAL certified and is available from several suppliers worldwide.

[0323] 2. Functional issues: DOC degrades endotoxins without affecting their chemical composition and once the detergent is removed, endotoxins regain their biological activity, whereas SDS, due to its amphiphilic nature and higher aggregation number, denatures and solubilizes proteins well and also irreversibly destroys endotoxins into their monomeric units.

[0324] A new and improved method for Vi PS purification developed without the use of animal-derived components sodium deoxycholate (DOC) or phenol. The improved method based on reverse purification of polysaccharides where host cell impurities like proteins, nucleic acids and lipopolysaccharides are removed in an initial step by using sodium acetate (6%), sodium dodecyl sulfate (2%) and ethanol (40%) as well as concentration in various steps and diafiltration using a 30 kDa cut-off membrane, then polysaccharides are precipitated by cationic detergent CTAB and a polishing purification step is performed to achieve a higher yield of purified Vi polysaccharide meets WHO specifications.

[0325] The purification process results in high recovery rates of about 40%-65%, purified Vi polysaccharide yields in the range of 400-4000mg / L with the desired O-acetyl levels (>2.0mmol / g polysaccharide), average molecular weights found to be in the range of 40-400kDa, containing less than 1% protein / peptide, less than 2% nucleic acid, less than 100EU endotoxin per μg polysaccharide (PS), and molecular size distribution (>50% of PS elutes before reaching a distribution coefficient (KD) of 0.25).

[0326] The O-specific polysaccharide (OSP) purification method results in a significant reduction in endotoxin (<100 EU endotoxin / μg PS), protein (<1%) and nucleic acid (<2%) impurities, and a higher capsular polysaccharide recovery, preferably in the range of 40%-65%, with the desired O-acetyl levels (>2.0 mmol / g polysaccharide), molecular size distribution (>50% of PS elutes before a distribution coefficient (KD) of 0.25 is reached), resulting in purified O-specific The average molecular weight of the polysaccharides (OSPs) was found to be in the range of 40-200 kDa.

[0327] Example 3: D) A method for conjugating polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis to a carrier protein.

[0328] The carrier protein used to conjugate with the Salmonella typhi Vi polysaccharide is tetanus toxoid. The polysaccharides from S. paratyphi A, S. typhimurium and S. enteritidis are individually conjugated to a carrier protein selected from tetanus toxoid (TT), diphtheria toxoid (DT) or CRM197.

[0329] According to the present disclosure, CRM197 is obtained from recombinant strain CS463-003 (MB101) of Pseudomonas fluorescens from Pfenex USA.

[0330] According to the present disclosure, TT is obtained from Clostridium Tetani (Harvard No 49205) obtained from the Central research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. The Central research Institute (CRI) obtained this strain from NVI, Netherland.

[0331] According to the present disclosure, DT is produced from a culture of Cornynebacterium diphtheriae Park-Williams Number 8 strain obtained from the Central Research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. The Central research Institute (CRI) acquired this strain from Wellcome Research Laboratories. D1) Preparation of monovalent Salmonella typhi conjugates using carbodiimide chemistry: Step 1: Concentration and derivatization of TT procedure: a) GFC purified TT (>90% monomer content) was concentrated using a 10 kDa membrane to reach a protein concentration of ≥12 mg / ml (Lowry assay).

[0332] b) Derivatisation ratios were used as shown below and the amounts required were calculated accordingly. Pr:ADH:EDC is 1:6.0:1 Derivatization scale: 13gm c) Concentrated TT (in 0.9% NaCl), 1M MES pH 6.0, ADH solution (dissolved in 0.1M MES pH 6.0) and EDC solution (dissolved in 0.1M MES pH 6.0) were added sequentially and brought to the final required volume by addition of 0.1M MES buffer pH 6.0. The final Pr concentration in the reaction was approximately 4.5mg / ml.

[0333] d) The reaction mixture was stirred and continued at pH 5.90 for approximately 1 hour. The derivatization reaction was then quenched by adjusting the pH to above 7.5 using 0.1 M phosphate buffer containing EDTA pH 8.0.

[0334] e) The quenched reaction mixture was diafiltered using 22 volumes of 10 mM phosphate buffer, followed by at least 12 volumes of 0.1 M MES buffer pH 6.0 using a 10 kDa cut-off membrane to remove unreacted moieties and other residues.

[0335] f) Derivatized samples were analyzed for total Pr concentration by Lowry assay and degree of derivatization value by colorimetric (TNBS) assay. result: Derivatized Pr concentration by Lowry assay: 15mg / ml Degree of Derivatization (DOA): 19 Percentage recovery rate: Approximately 75% Step 2: Conjugation of ViPs to derivatization of TT procedure: Vi PS-derivatized TT conjugation was carried out using carbodiimide chemistry.

[0336] The following PS and Pr parameters were used for conjugation: A ratio of conjugation reactions of Ps:Pr:EDC::1:0.9:1.75 (+0.5 for Pr and EDC) was used. Conjugation scale 10 gm.

[0337] b. The required batch volume of PS was added to a sterile glass bottle. c. 1M MES buffer pH 6.0 (original buffer) was added to a measured volume of PS to reach a concentration of 0.1M MES.

[0338] d. The mixture was stirred at about 100 rpm for homogeneous mixing. e. A measured volume of derivatized TT was then added to the Ps-containing bottle. f. Freshly prepared EDC (dissolved in 0.1 M MES buffer pH 6.0) was added to the above reaction immediately after the addition of TT.

[0339] g. The pH was observed and recorded (pH 6.0 + 0.5). h. Samples were analyzed at different time intervals using SE-HPLC to monitor the conjugation conversion percentage and protein consumption.

[0340] i. When <90% protein was consumed, the reaction was quenched after 1.45 hours by raising the pH to 7.5 using 0.1 M phosphate buffer containing EDTA.

[0341] j. The quenched reaction mixture was stored at 2-8 °C until further use. Step 3: Purification of the quenched Vi-TT conjugate Purification of the quenched conjugate was performed by two methods implemented to remove unreacted Ps, unreacted Pr, and other residues using the following parameters. 1. Ultrafiltration: The following parameters were used for diafiltration:

[0342] Diafiltration scale: 8.5gm Ps concentration during D / F: approx. 2mg / ml Fractionation membrane: 300kDa Membrane manufacturing: Pall Membrane area: 2.5m 2 Buffer A used: 10 mM PBS minimum 25 volumes Buffer B used: 0.9% NaCl 30 vol.

[0343] Final volume: 6.5L The purified conjugate was filtered using a 0.2 μM filter. 2. Gel filtration chromatography: Approximately 1.5 gm of the quenched conjugate was concentrated to approximately 2-4 mg / ml using a 0.1 m2 300 kDa cut-off membrane. Conjugate purification was performed using gel filtration chromatography to remove unbound PS, unbound TT and residual EDC.

[0344] Two separate GFC runs were performed with the same purification parameters. The following chromatographic conditions were used for purification:

[0345] [Table 42]

[0346] 1 A total of 27 fractions (100 ml each) were collected, analyzed by SE-HPLC and pooled together based on the chromatographic profile. The two fractions were analyzed for Ps content by the HPAD method, for Pr content by the Lowry method, and for % free Ps by the DOC-HPAD method.

[0347] 3 Based on the analysis, fractions were pooled (1–23) and filtered using a 0.22 μM filter. 4 Samples were analyzed for Ps content, Pr content and free Ps analysis as well as endotoxin, O-acetyl content, sterility, pH value and endotoxin analysis.

[0348] Results and Interpretation:

[0349] [Table 43]

[0350] Comparative conjugation process data - improved conjugation efficiency, improved conjugate yield and stability (free Ps, free Pr) of SIIPL Typhi conjugate (Ps:Pr:EDAC ratio is 1:1:2 compared to other Ps:Pr:EDAC ratios).

[0351] Improved conjugation efficiency: Conjugation conversion percentages of over 90% were observed. Improved conjugate yield: Purification was performed using different methods, i.e., ultrafiltration and gel filtration, at various conjugation purification scales, with recoveries ranging from 40 to 87%.

[0352] For Vi-TT conjugation, it was found that optimal conjugation conversion (%) could be obtained using ratios of 1:1:2 versus 1:1.5:1.2, 1:0.9:2, 1:0.9:1.75, 1:0.8:1.8:, 1:0.7:1.8, 1:0.9:0.6, 1:0.9:1 and 1:0.7:1.5, with lower protein of 1.5 EDC.

[0353] It was found that ViPs of various sizes could be conjugated with ADH-activated TT. Different ratios such as Ps:Pr:EDC::1:0.7:1.8, 1:0.8:1.8, 1:0.9:2 and 1:1:2 used at lower pH with different purification techniques resulted in good recovery of the conjugate with PS / Pr ratios above 0.5 and lower free ps. D2) Preparation of monovalent Salmonella paratyphi conjugates: Two types of conjugation chemistries (cyanylation and carbodiimide chemistry) were applied for the conjugation of Paratyphoid OSP to the carrier proteins diphtheria toxoid (DT), CRM197 and tetanus toxoid (TT). 1) Conjugation of Paratyphoid OSP to the carrier proteins diphtheria toxoid (DT), CRM197, and tetanus toxoid (TT) based on cyanylation chemistry A) Derivatization of diphtheria toxoid (DT) using carbodiimide chemistry (attachment of the linker ADH) and conjugation with concentrated OSP using cyanylation chemistry. B) Derivatization of CRM197, tetanus toxoid (TT) using carbodiimide chemistry (addition of the linker ADH) and conjugation with concentrated OSP using cyanylation chemistry. Gasion C) Derivatization of tetanus toxoid (TT) using carbodiimide chemistry (attachment of the linker ADH) and conjugation with concentrated OSP using cyanylation chemistry 1A) Preparation of S. Paratyphi conjugates using diphtheria toxoid as the carrier protein. 1) Protein derivatization: Highly monomeric diphtheria toxoid (DT) was concentrated on a 10 kDa membrane (10-20 mg / ml) and analyzed for protein content.

[0354] [Table 44]

[0355] To the concentrated DT was added freshly prepared 1M MES buffer, adipic acid dihydrazide (ADH) (dissolved in 100 mM MES buffer pH:5.8 at 75-100 mg / ml) in a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved in 100 mM MES buffer pH:5.8 at 30-40 mg / ml) in a weight ratio of 1:1. The reaction was continued for about 1 h at pH 5.8, and then the reaction mixture was diafiltered with a 10 kDa TFF in 50 mM borate buffer pH 9.0 to remove residual and unreacted components. The final sample was analyzed for protein content and degree of derivatization.

[0356] [Table 45]

[0357] 2) Conjugation of S. Paratyphi A polysaccharide (OSP) and ADH-derivatized DT: Two experiments were carried out by varying the 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) ratio.

[0358] Experiment No.: 1 - OSP was concentrated with a 10 kDa membrane to reach a concentration of (10-15 mg / ml).

[0359] [Table 46]

[0360] To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP was 1:0.8:1.3).

[0361] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS.

[0362] See Figure 1: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP 1:0.8:1.3) 3) Conjugate purification by GFC: Column XK16 / 70 was prepared with GFC resin (Tyopearl HW65F) at a bed height of 40 cm. The column was packed and stabilized at a flow rate of 100 cm / hr, and 1% column volume of 1M NaCl was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm / hr, the conjugate was loaded onto the column, and fractions were collected at 1 min intervals. Fractions 2-9 were pooled according to their HPLC profile. The final pooled fraction was filtered and sent for analysis.

[0363] [Table 47]

[0364] Figure 2: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP is 1:0.8:1.3). Test Number: 2 - To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.1. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP was 1:0.8:1.1).

[0365] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS.

[0366] See Figure 3: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP 1:0.8:1.1). Conjugate purification by GFC: Column XK16 / 70 was prepared with GFC resin (Tyopearl HW65F) at 40 cm bed height. The column was packed and stabilized at a flow rate of 100 cm / hr, and 1% column volume of 1M NaCL was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCL at 30 cm / hr, the conjugate was loaded onto the column, and fractions were collected at 1 min intervals. Fractions 2-8 were pooled according to their HPLC profile. The final pooled fraction was filtered and sent for analysis.

[0367] [Table 48]

[0368] See Figure 4: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP 1:0.8:1.1). Conclusion: The conjugation of Paratyphi A PS with ADH-derivatized DT was successful, and the PS / PR ratio was satisfactory. 1B. Preparation of S. Paratyphi conjugates using CRM197 as a carrier protein 1) Protein derivatization: A cross-reacting mutant (CRM197) received from the manufacturing department (SIIPL) was used for derivatization.

[0369] [Table 49]

[0370] To the concentrated CRM197, add freshly prepared 1M MES buffer, adipic acid dihydrazide (ADH) (dissolved in 100 mM MES buffer pH:6.5 at 75-100 mg / ml) at a weight ratio of 1:3.5, and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved in 100 mM MES buffer pH:6.5 at 30-40 mg / ml) at a weight ratio of 1:0.25. Add 5% Tween 80. The final reaction volume was brought to reach a final concentration of 3-4 mg / ml using MES buffer, the reaction was continued for approximately 3 hours at pH 6.5, and the reaction mixture was then diafiltered with a 10 kDa TFF in 50 mM borate buffer and 0.005% Tween 80 pH 9.0 to remove residual and unreacted components. The final sample was analyzed for protein content and degree of derivatization.

[0371] Brief description or reference of the assay: Protein content was measured by BCA (bicinchoninic acid) assay.

[0372] [Table 50]

[0373] 2) Conjugation of S. Paratyphi A polysaccharide (OSP) and ADH-derivatized CRM197: The OSP received from the DSP was concentrated with a 10 kDa membrane to reach a concentration of (10–15 mg / ml).

[0374] [Table 51]

[0375] To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:1 (PS:PR:CPIP was 1:1:1.3).

[0376] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS.

[0377] See Figures 5 and 6: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP is 1:1:1.3). 3) Conjugate purification by GFC: Column XK16 / 70 was prepared with GFC resin (Tyoperal HW65F) at a bed height of 40 cm. The column was packed and stabilized at a flow rate of 100 cm / hr, and 1% column volume of 1M NaCl was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm / hr, the conjugate was loaded onto the column, fractions were collected at 1 min intervals, and fractions 2-7 were pooled according to their HPLC profile. The final pooled fraction was filtered and sent for analysis.

[0378] [Table 52]

[0379] Figure 7: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP is 1:1:1.3). Conclusion: The conjugation of Paratyphi A PS using ADH-derivatized CRM197 was successful, and the PS / PR ratio was satisfactory. 1C) Preparation of S. Paratyphi conjugates using tetanus toxoid as a carrier protein: 1) Protein Derivatization: High monomeric tetanus toxoid (TT) received from the manufacturing unit (SIIPL) was concentrated (15-20mg / ml) on a 30kDa membrane and analyzed for protein content.

[0380] [Table 53]

[0381] To the concentrated TT was added freshly prepared 1M MES buffer, adipic acid dihydrazide (ADH) (dissolved in 100 mM MES buffer pH:6.0 at 75-100 mg / ml) in a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved in 100 mM MES buffer pH:6.0 at 30-40 mg / ml) in a weight ratio of 1:1. The reaction was continued for about 1 h at pH 6.0, and then the reaction mixture was diafiltered with a 30 kDa TFF in 10 mM phosphate buffer pH 7.2 to remove residual and unreacted components. The final sample was analyzed for protein content and degree of derivatization.

[0382] Brief description or reference of the assay: Protein content was measured by the Lowry assay.

[0383] [Table 54]

[0384] 2) Conjugation of S. Paratyphi A polysaccharide (OSP) and ADH-derivatized TT: OSP received from the DSP team was concentrated using a 10 kDa membrane to reach a concentration of (10-13 mg / ml).

[0385] [Table 55]

[0386] 1-Cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CP Two experiments were performed by varying the IP ratio. Test Number: 1 To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.25. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP was 1:0.8:1.25).

[0387] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS.

[0388] See Figure 8: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP 1:0.8:1.25). 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration with a 300 kDa TFF membrane in 10 mM PBS pH 7.2, followed by 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis.

[0389] [Table 56]

[0390] Figure 9: Chromatogram of GFC-purified OSP-DT ADH conjugate (PS:PR:CPIP is 1:0.8:1.25) Test Number: 2 To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.9 (PS:PR:CPIP 1:0.9:1.3).

[0391] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS.

[0392] See Figure 10: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP1:0.9:1.3). 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration with 300 kDa TFF membrane in 10 mM PBS pH 7.2 followed by diafiltration in 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis.

[0393] [Table 57]

[0394] See Figure 11: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP 1:0.9:1.3). Conclusion: The conjugation of Paratyphi A PS with ADH-derivatized TT was successful, applying two different types of diafiltration strategies, and both processes yielded satisfactory PS / Pr ratios. 2) Conjugation of Paratyphoid OSP to the carrier proteins diphtheria toxoid (DT), CRM197, and tetanus toxoid (TT) based on carbodiimide chemistry A) Derivatization of concentrated OSP (attachment of the linker ADH) using cyanylation chemistry and conjugation with tetanus toxoid (TT) using carbodiimide chemistry.

[0395] B) Derivatization of concentrated OSP (attachment of the linker ADH) using cyanylation chemistry and conjugation with diphtheria toxoid (DT) using carbodiimide chemistry. C) Derivatization of concentrated OSP using cyanylation chemistry (addition of the linker ADH) and conjugation with CRM197 using carbodiimide chemistry. 2A) Preparation of S. Paratyphi conjugates using tetanus toxoid as the carrier protein. 1) Polysaccharide derivatization: OSP received from the DSP team was concentrated using a 10 kDa membrane to reach a concentration of (10-13 mg / ml).

[0396] [Table 58]

[0397] To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:0.7. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for a maximum of 3 min. Adipic acid dihydrazide (ADH) (dissolved at 75-100 mg / ml in 0.5 M sodium bicarbonate buffer pH:8.0) was then added in a weight ratio of 1:10 (PS:ADH:CPIP 1:10:0.7). The reaction was continued for approximately 2 h at pH 9.5 and quenched using 2 M glycine 10, and the reaction mixture was then diafiltered with a 10 kDa TFF in 100 mM MES buffer pH 6.0 to remove residual and unreacted components. The final sample was analyzed for polysaccharide content.

[0398] [Table 59]

[0399] 2) Protein Preparation: High monomeric tetanus toxoid (TT) received from the manufacturing unit (SIIPL) was concentrated (10-15mg / ml) using a 30kDa membrane and analyzed for protein content.

[0400] Brief description or reference of the assay: Protein content was measured by the Lowry assay.

[0401] [Table 60]

[0402] 3) Conjugation of ADH-derivatized S. Paratyphi A polysaccharide (OSP) and concentrated TT: Two experiments were performed at different temperatures to confirm Pr conversion. Test Number: 1 To the ADH-derivatized PS, protein was added at a weight ratio of 1:0.9 and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 3040 mg / ml in 100 mM MES buffer) at a weight ratio of 1:0.86. The reaction was continued at pH 6.0 and a temperature of 6° C. (PS:PR:EDC ratio was 1:0.9:0.86).

[0403] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 23 h, the reaction was quenched by adding 100 mM phosphate buffer containing EDTA.

[0404] See Figure 12: Chromatogram showing the progress of the conjugation reaction (reaction quenched at 23 hours). 4) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration with 300 kDa TFF membrane in 10 mM PBS pH 7.2 followed by diafiltration in 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis.

[0405] [Table 61]

[0406] Test Number: 2 To the ADH-derivatized PS, protein was added at a ratio of 1:0.9 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0 and temperature of 10 °C (PS:PR:EDC ratio was 1:0.9:0.86).

[0407] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 4 h, the reaction was quenched by adding 100 mM phosphate buffer containing EDTA.

[0408] See Figure 13: Chromatogram showing the progress of the conjugation reaction. 4) Conjugate purification by GFC: Column XK16 / 70 was prepared with GFC resin (Tyoperal HW65F) at a bed height of 40 cm. The column was packed and stabilized at a flow rate of 100 cm / hr, and 1% column volume of 1M NaCl was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm / hr, the conjugate was loaded onto the column, fractions were collected at 1 min intervals, and fractions 2-10 were pooled according to their HPLC profile. The final pooled fraction was filtered and sent for analysis.

[0409] [Table 62]

[0410] Conclusion: Using reverse conjugation chemistry (to activate PS), conjugation of ADH-derivatized Paratyphi A PS and concentrated TT was successful by increasing the temperature from 6° C. to 10° C. The conjugation rate of the reaction increased and the PS / Pr ratio was very low in both reactions. 2B) Preparation of S. Paratyphi conjugates using diphtheria toxoid as the carrier protein. 1) Polysaccharide derivatization: OSP received from the DSP team was concentrated using a 10 kDa membrane to reach a concentration of (10–13 mg / ml).

[0411] [Table 63]

[0412] To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (0.114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:0.7. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Then, dihydroadipate was added. Hydrazide (ADH) (dissolved at 75-100 mg / ml in 0.5 M sodium bicarbonate buffer pH:8.0) was added at a weight ratio of 1:10 (PS:ADH:CPIP was 1:10:0.7). The reaction was continued for approximately 2 hours at pH 9.5, quenched using 2 M glycine 10, and the reaction mixture was then diafiltered with a 10 kDa TFF in 100 mM MES buffer pH 6.0 to remove residuals and unreacted components. The final sample was analyzed for polysaccharide content.

[0413] [Table 64]

[0414] 2) Protein Preparation: High monomeric diphtheria toxoid (DT) received from the manufacturing unit (SIIPL) was concentrated (10-20mg / ml) using a 10kDa membrane and analyzed for protein content.

[0415] Brief description or reference of the assay: Protein content was measured by the Lowry assay.

[0416] [Table 65]

[0417] 3) Conjugation of ADH-derivatized S. Paratyphi A polysaccharide (OSP) and concentrated DT: The same conditions as for the TT conjugate were applied to DT to confirm Pr conversion.

[0418] Test Number: 1 To the ADH-derivatized PS, protein was added at a ratio of 1:0.8 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0 and 6 degrees (PS:PR:EDC ratio was 1:0.8:0.86).

[0419] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 20 h, the reaction was quenched by adding 100 mM phosphate buffer containing EDTA.

[0420] See Figure 14: Chromatogram showing the progress of the conjugation reaction (reaction quenched at 20 hours). 4) Conjugate purification by GFC: Column XK16 / 70 was prepared with a bed height of 40 cm using GFC resin (Tyoperal HW65F). The column was packed and stabilized at a flow rate of 1000 x 100 cm / hr and the integrity of the packed column was assessed by passing 1% column volume of 1M NaCl. The column was equilibrated using 0.9% NaCl at 30 cm / hr and the conjugate was loaded onto the column, fractions were collected at 1 min intervals and fractions 2-12 were pooled according to their HPLC profile. The final pooled fraction was filtered and sent for analysis.

[0421] [Table 66]

[0422] See Figure 15: chromatogram showing purified conjugate (pooled fractions). Test Number: 2 To the ADH-derivatized PS, protein was added at a weight ratio of 1:1.0 and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) at a weight ratio of 1:0.9. The reaction was continued at pH 6.0 and temperature of 10°C (PS:PR:EDC ratio was 1:1.0:0.9).

[0423] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 4 h, the reaction was quenched by adding 100 mM phosphate buffer containing EDTA.

[0424] See Figure 16: Chromatogram showing the progress of the conjugation reaction (reaction quenched at 4 hours). 4) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration with 300 kDa TFF membrane in 10 mM PBS pH 7.2 followed by diafiltration in 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis.

[0425] See Figure 17: chromatogram showing the purified conjugate.

[0426] [Table 67]

[0427] Conclusion: Conjugation of ADH-derivatized Paratyphi A PS using carbodiimide chemistry was successful using concentrated DT. The rate of the conjugation reaction increased with increasing temperature, but the PS / Pr ratio was low in both experiments. D3) A method to conjugate polysaccharides from Salmonella enterica serovars S. typhimurium and S. enteritidis to carrier proteins selected from tetanus toxoid (TT), diphtheria toxoid (DT) or CRM197. 1) Diphtheria toxoid (DT), a carrier protein based on cyanylation chemistry, CRM19 7. Conjugation of S. typhimurium and S. enteritidis polysaccharides to tetanus toxoid (TT) A) Derivatization of diphtheria toxoid (DT) (attachment of the linker ADH) using carbodiimide chemistry and conjugation to concentrated S. typhimurium and S. enteritidis polysaccharides using cyanylation chemistry.

[0428] B) Derivatization of CRM197 (addition of the linker ADH) using carbodiimide chemistry and conjugation with concentrated S. typhimurium and S. enteritidis using cyanylation chemistry.

[0429] C) Derivatization of tetanus toxoid (TT) (attachment of the linker ADH) using carbodiimide chemistry and conjugation to concentrated S. typhimurium and S. enteritidis using cyanylation chemistry.

[0430] D) Conjugation of Paratyphoid OSP to carrier proteins diphtheria toxoid (DT), CRM197 and tetanus toxoid (TT) based on cyanylation chemistry without derivatization of polysaccharide or carrier protein (CPPT).

[0431] Steps followed: A) Preparation of S. typhimurium and S. enteritidis polysaccharide conjugates using diphtheria toxoid (DT) as the carrier protein. 1) Protein derivatization: Highly monomeric diphtheria toxoid (DT) was concentrated on a 10 kDa membrane (10-20 mg / ml) and analyzed for protein content.

[0432] To the concentrated DT was added freshly prepared 1M MES buffer, adipic acid dihydrazide (ADH) (dissolved in 100 mM MES buffer pH:5.8 at 75-100 mg / ml) in a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved in 100 mM MES buffer pH:5.8 at 30-40 mg / ml) in a weight ratio of 1:1. The reaction was continued for about 1 h at pH 5.8, and then the reaction mixture was diafiltered with a 10 kDa TFF in 50 mM borate buffer pH 9.0 to remove residual and unreacted components. The final sample was analyzed for protein content and degree of derivatization. 2) Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) and ADH-derivatized DT: Two experiments were carried out by varying the 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) ratio.

[0433] Experiment No.: 1 - S. typhimurium and S. enteritidis polysaccharides were concentrated on a 10 kDa membrane to reach a concentration of (10-15 mg / ml). To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP was 1:0.8:1.3).

[0434] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS. 3) Conjugate purification by GFC: GFC resin (Tyopearl HW65F) A Column XK16 / 70 was prepared with a bed height of 40 cm using a 50% ethanol centrifuge. The column was packed and stabilized at a flow rate of 100 cm / hr and 1% column volume of 1M NaCl was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm / hr and the conjugate was loaded onto the column and fractions were collected at 1 min intervals. The final pooled fraction was filtered and sent for analysis.

[0435] Test Number: 2 To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.1. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP was 1:0.8:1.1).

[0436] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS.

[0437] Conjugate purification by GFC: Column XK16 / 70 was prepared with GFC resin (Tyoperal HW65F) at 40 cm bed height. The column was packed and stabilized at a flow rate of 100 cm / hr and 1% column volume of 1M NaCL was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCL at 30 cm / hr, the conjugate was loaded onto the column and fractions were collected at 1 min intervals. The final pooled fraction was filtered and sent for analysis.

[0438] Conclusion: The conjugation of S. typhimurium and S. enteritidis polysaccharides using ADH-derivatized DT was successful, and the PS / PR ratio was satisfactory. B. Preparation of S. typhimurium and S. enteritidis polysaccharide conjugates using CRM197 as a carrier protein 1) Protein derivatization: A cross-reacting mutant (CRM197) received from the manufacturing department (SIIPL) was utilized for derivatization.

[0439] To the concentrated CRM197, add freshly prepared 1M MES buffer, adipic acid dihydrazide (ADH) (dissolved in 100 mM MES buffer pH:6.5 at 75-100 mg / ml) at a weight ratio of 1:3.5, and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved in 100 mM MES buffer pH:6.5 at 30-40 mg / ml) at a weight ratio of 1:0.25. Add 5% Tween 80. The final reaction volume was brought to a final concentration of 3-4 mg / ml using MES buffer, the reaction was continued for approximately 3 hours at pH 6.5, and the reaction mixture was then diafiltered with a 10 kDa TFF in 50 mM borate buffer and 0.005% Tween 80 pH 9.0 to remove residual and unreacted components. The final sample was analyzed for protein content and degree of derivatization. 2) Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) and ADH-derivatized CRM197: The PS received from the DSP was concentrated with a 10 kDa membrane to reach a concentration of (10–15 mg / ml).

[0440] To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. The protein was then diluted by weight. The addition ratio was 1:1 (PS:PR:CPIP was 1:1:1.3).

[0441] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS. 3) Conjugate purification by GFC: Column XK16 / 70 was prepared with GFC resin (Tyoperal HW65F) at a bed height of 40 cm. The column was packed and stabilized at a flow rate of 100 cm / hr, and 1% column volume of 1M NaCL was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCL at 30 cm / hr, the conjugate was loaded onto the column, fractions were collected at 1 min intervals, and the final pooled fraction was filtered and sent for analysis.

[0442] Conclusion: Conjugation of S. typhimurium and S. enteritidis PS using ADH-derivatized CRM197 was successful, and the PS / PR ratio was satisfactory. C) Preparation of S. Paratyphi conjugates using tetanus toxoid as a carrier protein: 1) Protein Derivatization: High monomeric tetanus toxoid (TT) received from the manufacturing unit (SIIPL) was concentrated to (15-20mg / ml) with a 30kDa membrane and analyzed for protein content. To the concentrated TT, freshly prepared 1M MES buffer, adipic acid dihydrazide (ADH) (dissolved in 100mM MES buffer pH:6.0 at 75-100mg / ml) at a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved in 100mM MES buffer pH:6.0 at 30-40mg / ml) at a weight ratio of 1:1 were added. The reaction was continued at pH 6.0 for about 1 hour, and then the reaction mixture was subjected to diafiltration with a 30kDa TFF in 10mM phosphate buffer pH 7.2 to remove residual and unreacted components. The final sample was analyzed for protein content and degree of derivatization. 2) Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) and ADH-derivatized TT: OSP received from the DSP team was concentrated using a 10 kDa membrane to reach a concentration of (10–13 mg / ml).

[0443] Two experiments were carried out by varying the 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) ratio. Test Number: 1 To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.25. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP was 1:0.8:1.25).

[0444] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS. 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration with 300 kDa TFF membrane in 10 mM PBS pH 7.2 followed by diafiltration in 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis.

[0445] Test Number: 2 To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for up to 3 min. Protein was then added in a weight ratio of 1:0.9 (PS:PR:CPIP 1:0.9:1.3).

[0446] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 3-4 h, the reaction was quenched by adding 2 M glycine at a weight ratio 10 times that of PS. 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration with 300 kDa TFF membrane in 10 mM PBS pH 7.2 followed by diafiltration in 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis.

[0447] Conclusion: Conjugation of S. typhimurium and S. enteritidis polysaccharides (PS) using ADH-derivatized TT was successful, applying two different types of diafiltration strategies, both processes yielding satisfactory PS / Pr ratios. 2) Conjugation of S. typhimurium and S. enteritidis polysaccharides to the carrier proteins diphtheria toxoid (DT), CRM197, and tetanus toxoid (TT) based on carbodiimide chemistry A) Derivatization of concentrated S. typhimurium and S. enteritidis polysaccharides (attachment of the linker ADH) using cyanylation chemistry and conjugation with tetanus toxoid (TT) using carbodiimide chemistry.

[0448] B) Derivatization of concentrated S. typhimurium and S. enteritidis polysaccharides (addition of the linker ADH) using cyanylation chemistry and conjugation with diphtheria toxoid (DT) using carbodiimide chemistry.

[0449] C) Derivatization of concentrated S. typhimurium and S. enteritidis polysaccharides using cyanylation chemistry (addition of the linker ADH) and conjugation with CRM197 using carbodiimide chemistry.

[0450] D) Conjugation of Paratyphoid OSP to the carrier proteins diphtheria toxoid (DT), CRM197 and tetanus toxoid (TT) based on carbodiimide chemistry (EDAC) without derivatization of the polysaccharide or carrier protein.

[0451] Steps followed: 1) Polysaccharide (PS) derivatization: Received S. typhimurium and S. enteritidis polysaccharides were concentrated with a 10 kDa membrane to reach a concentration of (10-13 mg / ml). To the concentrated PS, 0.9% NaCl was added and freshly prepared CPIP solution (114 mg / ml in acetonitrile) was added to the polysaccharides in a weight ratio of 1:0.5-1:2 (1:0.7). The pH was immediately shifted to 9.5 by 2.5 M NaOH and held for up to 3 min. Adipic acid dihydrazide (ADH) (dissolved at 75-100 mg / ml in 0.5 M sodium bicarbonate buffer pH:8.0) was then added in a weight ratio of 1:10 (PS:ADH:CPIP was 1:2:0.7-1:10:0.7). The reaction was continued for approximately 2 hours at pH 9.5 and quenched using 2M glycine 10, and the reaction mixture was then diafiltered with a 10 kDa TFF in 100 mM MES buffer pH 6.0 to remove residual and unreacted components. The final sample was analyzed for polysaccharide content. 2) Protein preparation: 2A) Protein preparation: High monomeric tetanus toxoid (TT) received from the manufacturing division (SIIPL) was concentrated to (10-15mg / ml) with a 30kDa membrane to obtain protein content The quantity was analyzed.

[0452] 2B) Protein Preparation: High monomeric diphtheria toxoid (DT) received from the manufacturing unit (SIIPL) was concentrated (10-20mg / ml) on a 30kDa membrane and analyzed for protein content.

[0453] 2C) Protein Preparation: High monomer CRM197 received from the manufacturing unit (SIIPL) was concentrated (10-20mg / ml) on a 30kDa membrane and analyzed for protein content. 3) Conjugation 3A) Conjugation of ADH-derivatized S. typhimurium and S. enteritidis polysaccharides and concentrated TT: To the ADH-derivatized PS, protein was added at a ratio of 1:0.9 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio was 1:0.9:0.86).

[0454] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 23 h, the reaction was quenched by adding 100 mM phosphate buffer containing EDTA.

[0455] 3B) Conjugation of ADH-derivatized S. typhimurium and S. enteritidis polysaccharides and concentrated DT: The same conditions as for the TT conjugates were applied to DT to confirm Pr conversion.

[0456] To the ADH-derivatized PS, protein was added at a ratio of 1:0.8 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio was 1:0.8:0.86).

[0457] Protein conversion was monitored using successive Shodex columns SB-804 HQ and SB-805 HQ at a flow rate of 1 ml / min with PBS as the mobile phase. After 20 h, the reaction was quenched by adding 100 mM phosphate buffer containing EDTA.

[0458] 3C) Conjugation of ADH-derivatized S. typhimurium and S. enteritidis polysaccharides and concentrated CRM197: The same conditions as for the TT and DT conjugates were applied to CRM197 to confirm Pr conversion.

[0459] To the ADH-derivatized PS, protein was added at a ratio of 1:0.8 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio was 1:0.8:0.86).

[0460] Protein conversion was monitored using Shodex columns SB-804 HQ and SB-805 HQ in succession at a flow rate of 1 ml / min with PBS as the mobile phase. The reaction was then quenched by adding 100 mM phosphate buffer containing EDTA. 4) Conjugate purification 4A) Conjugate purification by ultrafiltration: The quenched conjugate was diluted with 10 mM Purification was performed by 300 kDa TFF membrane diafiltration in PBS pH 7.2 followed by 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis.

[0461] 4B) Conjugate purification by GFC: Column XK16 / 70 was prepared with GFC resin (Tyoperal HW65F) at 40 cm bed height. The column was packed and stabilized at a flow rate of 100 cm / hr, and 1% column volume of 1M NaCl was passed through to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm / hr, the conjugate was loaded onto the column, fractions were collected at 1 min intervals, and fractions 2-12 were pooled according to their HPLC profile. The final pooled fraction was filtered and sent for analysis.

[0462] Conclusion: Using reverse conjugation chemistry (to activate PS) and increasing the temperature from 6 to 10° C., the conjugation of ADH-derivatized S. typhimurium and S. enteritidis polysaccharides and concentrated TT, DT, and CRM197 was successful. The conjugation rates of the reactions increased and the PS / Pr ratios were very low in all reactions.

[0463] Conjugation of ADH-derivatized S. typhimurium and S. enteritidis polysaccharides and concentrated TT, DT, and CRM197 using carbodiimide chemistry was successful. The rate of the conjugation reaction increased with increasing temperature, but the PS / Pr ratio was low in both experiments.

[0464] Example 4: Immunogenic composition

[0465] [Table 68]

[0466] The single dose does not contain the preservative 2-phenoxyethanol. The multi-dose composition may further comprise 2-phenoxyethanol - 5 mg (1-10 mg).

[0467] [Table 69]

[0468] [Table 70]

[0469] [Table 71]

[0470] [Table 72]

[0471] [Table 73]

[0472] [Table 74]

[0473] Further, the pH of the composition disclosed above is adjusted to about 6 with sodium hydroxide / sodium carbonate. Adjust to 0.0-7.0 and make up to volume by adding saline (0.9%). It may further comprise one of the following combinations of preservatives:

[0474] i. 2-phenoxyethanol in an amount of 1-10 mg per 0.5 ml (v / v); ii. 2-phenoxyethanol in an amount between 1 and 10 mg (v / v) per 0.5 ml and methylparaben in an amount between 0.1 and 1.5 mg (w / v) per 0.5 ml; or iii. 2-phenoxyethanol in an amount between 1 and 10 mg (v / v) per 0.5 ml and propylparaben in an amount between 0.05 and 0.2 mg (w / v) per 0.5 ml, or iv. 2-phenoxyethanol in an amount of 1 to 10 mg (v / v) per 0.5 ml, methylparaben in an amount of 0.1 to 1.5 mg (w / v) per 0.5 ml, and propylparaben in an amount of 0.05 to 0.2 mg (w / v) per 0.5 ml.

[0475] A combination vaccine composition comprising reduced dose IPV, D, T, HepB, ViPs-TT, OSP conjugate, acellular pertussis and Hib antigens may comprise a detoxified (particularly genetically or chemically detoxified) form of Bordetella toxin, particularly an acellular pertussis antigen selected from pertussis toxoid, filamentous hemagglutinin, pertactin or fimbriae, in particular 1 to 50 micrograms (more particularly 8 μg) pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) pertactin and optionally 2 to 25 micrograms of fimbriae per 0.5 ml.

[0476] [Table 75]

[0477] [Table 76]

[0478] Additionally, the pH of the composition disclosed above is adjusted to about 6.0-7.0 with sodium hydroxide / sodium carbonate and made up to volume with saline (0.9%). It may further comprise one of the following combinations of preservatives:

[0479] i. 2-phenoxyethanol in an amount of 1-10 mg per 0.5 ml (v / v); ii. 2-phenoxyethanol in an amount between 1 and 10 mg (v / v) per 0.5 ml and methylparaben in an amount between 0.1 and 1.5 mg (w / v) per 0.5 ml; or iii. 2-phenoxyethanol in an amount between 1 and 10 mg (v / v) per 0.5 ml and propylparaben in an amount between 0.05 and 0.2 mg (w / v) per 0.5 ml, or iv. 2-phenoxyethanol in an amount of 1 to 10 mg (v / v) per 0.5 ml, methylparaben in an amount of 0.1 to 1.5 mg (w / v) per 0.5 ml, and propylparaben in an amount of 0.05 to 0.2 mg (w / v) per 0.5 ml.

[0480] A combination vaccine composition comprising reduced dose IPV, D, T, HepB, ViPs-TT, OSP conjugate, acellular pertussis and Hib antigens may comprise a detoxified (particularly genetically or chemically detoxified) form of Bordetella toxin, particularly an acellular pertussis antigen selected from pertussis toxoid, filamentous hemagglutinin, pertactin or fimbriae, in particular 1 to 50 micrograms (more particularly 8 μg) pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) pertactin and optionally 2 to 25 micrograms of fimbriae per 0.5 ml.

[0481] [Table 77]

[0482] [Table 78]

[0483] Further, the pH of the composition disclosed above is adjusted to about 6 with sodium hydroxide / sodium carbonate. Adjust to 0.0-7.0 and make up to volume by adding saline (0.9%). It may further comprise one of the following combinations of preservatives:

[0484] v. 2-phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v / v); vi. 2-phenoxyethanol in an amount of 1-10 mg (v / v) per 0.5 ml and methylparaben in an amount of 0.1-1.5 mg (w / v) per 0.5 ml, or vii. 2-phenoxyethanol in an amount of 1-10 mg (v / v) per 0.5 ml and propylparaben in an amount of 0.05-0.2 mg (w / v) per 0.5 ml, or viii. 2-phenoxyethanol in an amount of 1 to 10 mg (v / v) per 0.5 ml, methylparaben in an amount of 0.1 to 1.5 mg (w / v) per 0.5 ml, and propylparaben in an amount of 0.05 to 0.2 mg (w / v) per 0.5 ml.

[0485] A combination vaccine composition comprising reduced dose TT, IPV, IRV, D, T, HepB, ViPs-TT, acellular pertussis and Hib antigens may comprise a detoxified (particularly genetically or chemically detoxified) form of Bordetella toxin, particularly an acellular pertussis antigen selected from pertussis toxoid, filamentous hemagglutinin, pertactin or fimbriae, in particular 1 to 50 micrograms (more particularly 8 μg) pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) pertactin and optionally 2 to 25 micrograms of fimbriae per 0.5 ml.

[0486] [Table 79]

[0487] [Table 80]

[0488] Additionally, the pH of the above disclosed composition is adjusted to about 6.0-7.0 with sodium hydroxide / sodium carbonate and made up to volume with saline (0.9%). It may further comprise one of the following combinations of preservatives: v. 2-phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v / v); vi. 2-phenoxyethanol in an amount of 1-10 mg per 0.5 ml (v / v) and methylparaben in an amount of 0.1 to 1.5 mg (w / v) per 0.5 ml, or vii. 2-phenoxyethanol in an amount of 1-10 mg (v / v) per 0.5 ml and propylparaben in an amount of 0.05-0.2 mg (w / v) per 0.5 ml, or viii. 2-phenoxyethanol in an amount of 1 to 10 mg (v / v) per 0.5 ml, methylparaben in an amount of 0.1 to 1.5 mg (w / v) per 0.5 ml, and propylparaben in an amount of 0.05 to 0.2 mg (w / v) per 0.5 ml.

[0489] A combination vaccine composition comprising reduced dose IPV, IRV, D, T, HepB, ViPs-TT, OSP, acellular pertussis and Hib antigens may comprise a detoxified (particularly genetically or chemically detoxified) form of Bordetella toxin, particularly an acellular pertussis antigen selected from pertussis toxoid, filamentous hemagglutinin, pertactin or fimbriae, in particular 1 to 50 micrograms (more particularly 8 μg) pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) pertactin and optionally 2 to 25 micrograms of fimbriae per 0.5 ml.

[0490] [Table 81]

[0491] [Table 82]

[0492] Example 5: Stability Data Stability of monovalent Salmonella typhi conjugates at 2-8°C for initial (0), 3 and 6 months, at 25°C for initial (0), 1 and 3 months, and at 40°C for initial (0), 14 and 28 days:

[0493] [Table 83]

[0494] [Table 84]

[0495] [Table 85]

[0496] observation: Free Ps (180-220 kDa Ps at 40°C / 2-8°C / 25°C) "Initial <4.5%, 7.5% or less after 6 months" vs. "Initial 5.4% for 388 / 80 / 45 kDa, 10.5% or more after 6 months").

[0497] For 180-220 kDa, the initial low free Ps, size of the conjugates was also maintained throughout the stability study, with little free Ps observed after 6 months compared to the other sizes. SE-HPLC stability data (VI-TT conjugate in Tris, Tris-NaCl and 0.17M NaCl)

[0498] [Table 86]

[0499] Conclusion: The Vi-TT conjugate is prepared in 10 mM Tris buffer containing 0.17 M NaCl. It was found to be stable in 10 mM Tris and in 0.17 M NaCl alone.

[0500] Data for different concentrations of Tris (5 mM, 10 mM, 20 mM, 25 mM, 30 mM, 50 mM) shows that 25 mM Tris is best. Vi-TT CJ concentrations were maintained in three different strengths of Tris, pH 7.2: 5 mM, 10 mM and 20 mM. This was maintained at various temperature conditions including -20°C, 2-8°C, room temperature and 37°C.

[0501] The study was conducted for two weeks, with pH monitored daily. · Samples were checked for particle size and zeta potential. pH result

[0502] [Table 87]

[0503] Conclusion: In Vi-TT CJ studies, the conjugate was diluted in 5, 10 and 20 mM Tris The pH was maintained at 7.2, with the pH ranging from 2 to 8° C. in 5, 10 and 20 mM concentrations. Viscosity and osmolality measurements of Vi-TT conjugates Part A: Viscosity and osmolality results for Tris buffer and sodium chloride without conjugate

[0504] [Table 88]

[0505] Part B: Viscosity and osmolality results of Tris buffer and sodium chloride containing conjugates

[0506] [Table 89]

[0507] Conclusion: Conjugates at various Tris concentrations were found to be isotonic, and the conjugate containing 150 mM NaCl was found to be isotonic.

[0508] The viscosities were found to be similar for the conjugates in different Tris buffers and NaCl concentrations, and are suitable for use as a parenteral formulation. Example 6: Immunogenicity Data A. Immunogenicity Studies of Monovalent Conjugate Vaccines: The immunogenic potential of the SIIP Vi TT vaccine was evaluated in a mouse model. Mouse Immunogenicity Study #1: Immunogenicity studies were performed in mice, where non-conjugated and TT-conjugated SIIPL vaccines (Vi TT) were administered intramuscularly to mice (8 animals per group). Animals were inoculated on days 1 and 14, and serum samples were collected on days 14 (data not shown) and 21. See Table 86 for details. Paired serum samples were available from all animals in each group. Serum samples were used for the determination of antibodies against the injected polysaccharide using a suitable serological and immunological method, such as an in-lab IgG ELISA. Serum from mice receiving non-conjugated SIIPL polysaccharide (Vi PS) was used as a comparative control for the induction of IgG responses in serum from mice receiving conjugated versions of Vi PS (of various PS sizes). Mouse Immunogenicity Study #2: Another similar study was performed in mice. Similar animal treatment protocols were used in both studies. The animal protocols are briefly described below. Sera from both studies were tested by the same immunological / serological analytical assay, i.e., IgG ELISA. Five- to six-week-old female mice (Balb C strain; housed in the laboratory) were used in the study using an IAEC-approved animal research protocol. All animals were specific pathogen-free and inoculated. During seeding and blood sample collection, mice were handled under sterile conditions in a biosafety cabinet. Mice weighed approximately 18-20 g at the start of the study. Each mouse received 2.5 μg of conjugated Vi TT vaccine via the intramuscular route. SIIPL Vi TT vaccine was diluted in phosphate-buffered saline (PBS) as a vehicle. No adjuvants were used in the study for any of the treatment groups.

[0509] Treatment Summary: The overall treatment plan for the study is summarized in the table below.

[0510] [Table 90]

[0511] Methodology and techniques used for serological analysis: Blood samples were collected from the retro-orbital vein of the experimental animals using sterile glass capillary tubes. Isoflurane was used as a safe anesthetic during the blood collection procedure. Serum was analyzed using an in-lab IgG ELISA with either unconjugated or conjugated Vi TT. The IgG antibodies produced in response to the injection of the vaccine were analyzed. The ELISA used NIBSC Vi PS reference standard (cat. no. 16 / 126; 1st International Standard for Vi PS of S.Typhi) as the coating antigen. The IgG levels were estimated by analyzing the optical density (OD) observed in serum from mice receiving conjugates of Vi PS (Vi TT with various PS sizes) compared to the OD observed in serum samples from mice receiving unconjugated SIIPL polysaccharide (Vi PS).

[0512] The table below shows the immunogenicity induction data (day 21) from the different Vi TT PS. Tables 87, 88, 89 and 90: Immunogenicity results

[0513] [Table 91]

[0514] [Table 92]

[0515] observation: Thus, the group of mice with Vi TT showing >4-fold higher induction of IgG (compared to mice receiving unconjugated Vi PS) strongly indicates the immunogenic potential of SIIPL VI TT across all PS sizes. Two separate studies were performed with Vi TT and both of these studies showed similar responses to Vi TT versus Vi PS.

[0516] The following parameters were compared across the various groups: a. >4-fold increase: The mean fold increase was >60-fold. b. GM of antibody titers: All groups showed GMs higher than those observed with unconjugated PS (>1.9).

[0517] c. % of mice with positive response: All mice in all Vi TT groups showed higher antibody responses (100% positive). Conclusions from the analysis of serological data: Immunogenicity studies in mice were performed with 8 (female) mice per group. In all groups, antibody induction was evaluated in response to Vi TT (various PS sizes) injected versus non-conjugated Vi PS. All Vi TT PS forms showed stronger induction of Vi TT-specific IgG antibodies compared to the Vi PS alone group (day 21; after the first booster). Induction of IgG in mouse serum is a strong determinant of serological response to Vi TT. Thus, monovalent Vi TT can induce strong immunogenic responses in mouse models, offering promising possibilities for future studies in higher animals.

[0518] [Table 93]

[0519] observation: 1. Immunogenicity data is evaluated with respect to IgG antibody levels observed in response to the SIIPL Vi TT conjugate in a mouse model.

[0520] 2. IgG induction is measured as the geometric mean of the colorimetric response (OD) observed in the IgG ELISA. 3. A GM observed in response to SIIPL Vi TT is considered immunogenic if the fold increase in OD upon VI TT treatment is 4-fold higher than the OD observed in response to unconjugated Vi PS.

[0521] Conclusion: 1. All monovalent Vi-TT conjugates were found to induce an immunogenic response that was observed via serology assays.

[0522] 2. All monovalent Vi-TT conjugates demonstrated higher IgG levels than were observed in the Typbar TCV vaccine group. 3. Induction of immunogenic responses (GM and >4-fold increase) was observed in the following order: G7 SIIPL Vi PS-TT (PS size 214) > G7 SIIPL Vi PS-TT (PS size 388) > Typbar TCV (commercial vaccine) B. Immunogenicity Studies of Bivalent Conjugate Vaccines The immunogenic potential of a bivalent (typhoid and paratyphoid) SIIPL vaccine was evaluated in a mouse model. Mouse Immunogenicity Study #1: Immunogenicity studies were performed in mice, and bivalent vaccines containing a combination of monovalent Vi TT vaccine and monovalent O-SP A (O-specific polysaccharide antigen of S. Paratyphi A) conjugated to carrier proteins such as TT, DT and CRM were used in the study. The monovalent and bivalent versions of these vaccines were administered intramuscularly to mice (8 animals per group). Animals were inoculated on days 1, 14 and 28, while serum samples were collected on days 14, 28 and 42. Paired serum samples were available from all animals in each group. For details regarding the treatment schedule, see Table 92 below. Serum samples were used for the determination of antibodies against the injected polysaccharides using a suitable serological and immunological method such as in-lab IgG ELISA. Each unconjugated SIIPL polysaccharide (Vi Sera from mice receiving O-PS) and O-SP alone were used as comparative controls for the induction of IgG responses.

[0523] The animal protocol is briefly described in the table below. Serum samples were tested by immunological / serological analytical assays, i.e., IgG ELISA. Five- to six-week-old female mice (Balb C strain; housed in a laboratory) were used in the study using an IAEC-certified animal research protocol. All animals were specific pathogen-free and handled under aseptic conditions in a biosafety cabinet during inoculation and blood sample collection. Mice ranging in weight from approximately 18 to 20 g were used in the study. Each mouse received 2.5 μg of monovalent conjugate Vi TT alone or O-SP A DT / TT / CRM alone and 2.5 μg of each bivalent vaccine (monovalent conjugate Vi TT mixed with O-SP A DT / TT / CRM) via the intramuscular route. The vaccine was diluted in phosphate-buffered saline (PBS) as a vehicle. No adjuvants were used in the study for any of the treatment groups.

[0524] Treatment Summary: The overall treatment plan for the study is summarized in the table below.

[0525] [Table 94]

[0526] Methodology and techniques used for serological analysis: Blood samples were collected from the retro-orbital vein of the experimental animals using sterile glass capillary tubes. Isoflurane was used as a safe anesthetic during the blood collection procedure. Serum was subjected to analysis of IgG antibodies produced in response to the injection of antigen using an in-laboratory IgG ELISA. IgG levels were estimated in serum samples from all study groups by colorimetric analysis. observation: The table below shows the immunogenicity induction data (day 21) from the different bivalent (typhoid and paratyphoid) SIIPL vaccines.

[0527] Tables 93, 94, 95, 96: Immunogenicity results

[0528] [Table 95]

[0529] [Table 96]

[0530] observation: a) SIIPL Vi PS-TT+SIIPL O-SP A DT, b) SIIPL Vi PS-TT+SIIPL O-SP A TT, and c) SIIPL Vi Groups of mice injected with a bivalent (typhoid and paratyphoid) SIIPL vaccine such as PS-TT+SIIPL O-SP A CRM Vi TT showed >4-fold higher induction of IgG (compared to mice receiving unconjugated Vi PS or unconjugated SIIPL O-SP A), thus demonstrating the immunogenic potential of a bivalent SIIPL vaccine containing the combination of Vi TT and SIIPL O-SP A (DT / TT / CRM).

[0531] IgG induction by bivalent (typhoid and paratyphoid) SIIPL vaccine This was higher than the IgG induction observed with a bivalent vaccine containing O-SP A CRM and the commercial Vi TT vaccine, Typbar TCV.

[0532] The following parameters were compared across the various groups: a. >4-fold increase: The mean fold increase was >10-fold. b. GM of antibody titers: All groups showed a higher GM than that observed with unconjugated PS.

[0533] c. % of mice with positive response: All mice in all Vi TT groups showed higher antibody responses (100% positive). Conclusions from the analysis of serological data: Immunogenicity studies were performed in mice with 8 (female) mice per group. In all groups, antibody induction in response to bivalent Vi (typhoid and paratyphoid) SIIPL vaccine injected against non-conjugated Vi PS and O-SP A PS was evaluated. All bivalent (typhoid and paratyphoid) SIIPL vaccines showed stronger induction of PS-specific IgG antibodies (day 21; after the first booster) compared to the PS alone (Vi and O-SP A) group. Induction of IgG in mouse serum is a strong determinant of serological response to bivalent (typhoid and paratyphoid) SIIPL vaccine. Thus, bivalent (typhoid and paratyphoid) SIIPL vaccine can induce strong immunogenic response in mouse model, offering promising possibilities for future studies in higher animals.

[0534] observation: 1. Immunogenicity data are evaluated with respect to IgG antibody levels observed in response to a bivalent (typhoid and paratyphoid) SIIPL vaccine in a mouse model.

[0535] 2. IgG induction is measured as the geometric mean of the colorimetric response (OD) observed in the IgG ELISA. 3. A GM observed in response to a bivalent (typhoid and paratyphoid) SIIPL vaccine is considered immunogenic if the fold increase in OD upon VI TT treatment is 4-fold higher than the OD observed in response to unconjugated Vi PS.

[0536] Conclusion: 1. All bivalent (typhoid and paratyphoid) SIIPL vaccines were found to induce immunogenic responses that were observed via serological assays.

[0537] 2. All bivalent (typhoid and paratyphoid) SIIPL vaccine conjugates demonstrated higher IgG levels than were observed in the non-conjugated controls. 3. IgG induction by the bivalent (typhoid and paratyphoid) SIIPL vaccine was higher than that observed with a bivalent vaccine containing SIIPL O-SP A CRM and the commercial Vi TT vaccine, Typvar TCV.

[0538] 4. Induction of immunogenic responses (GM and >4-fold increase) was observed in mice receiving the "Bivalent (Typhoid and Paratyphoid) SIIPL vaccine", therefore, it can be concluded that the Bivalent (Typhoid and Paratyphoid) SIIPL vaccine can induce strong immunogenic responses in a mouse model, suggesting its clinical immunogenic potential.

[0539] Example 7: Single-dose vaccine kit A) A single dose vaccine kit comprising: Lyophilized (freeze-dried) immunogenic composition: a) 5 μg Neisseria meningitidis A saccharide-TT conjugate antigen per 0.5 ml; b) 5 μg Neisseria meningitidis C saccharide-CRM197 conjugate antigen per 0.5 ml; c) 5 μg of Neisseria meningitidis Y glycoprotein per 0.5 ml CRM197 conjugated antigen, d) 5 μg Neisseria meningitidis W-135 saccharide-CRM197 conjugate antigen per 0.5 ml; e) 5 μg Neisseria meningitidis X-glyco-TT conjugate antigen per 0.5 ml; f) 1 to 12 mg of sucrose per 0.5 ml; g) 0.1-2 mg of sodium citrate (dihydrate) per 0.5 ml; h) 0.05 to 0.5 mg of Tris buffer per 0.5 ml a first container containing; and a) 1.25~50μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride; c) Water for injection (WFI) in appropriate volume A second container containing a liquid composition for the reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising:

[0540] interpretation: For protection against typhoid fever caused by Salmonella typhi and Neisseria meningitidis antigens, there is no antigenic interference of ViPs-TT with Neisseria meningitidis antigens, and the vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi in children under 2 years of age, teenagers, adults and the elderly with only one injection containing a complete vaccination schedule. B) A single dose vaccine kit comprising: Lyophilized (freeze-dried) immunogenic composition: a) 5 μg Neisseria meningitidis A saccharide-TT conjugate antigen per 0.5 ml; b) 5 μg Neisseria meningitidis C saccharide-CRM197 conjugate antigen per 0.5 ml; c) 5 μg Neisseria meningitidis Y glyco-CRM197 conjugate antigen per 0.5 ml; d) 5 μg Neisseria meningitidis W-135 saccharide-CRM197 conjugate antigen per 0.5 ml; e) 5 μg Neisseria meningitidis X-glyco-TT conjugate antigen per 0.5 ml; f) 1 to 12 mg of sucrose per 0.5 ml; g) 0.1-2 mg of sodium citrate (dihydrate) per 0.5 ml; h) 0.05 to 0.5 mg of Tris buffer per 0.5 ml a first container containing; and a) 1.25~50μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen, b) 1.25~50μg Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197), c) 1 to 10 mg of sodium chloride; d) Water for injection (WFI) in appropriate volume A second container containing a liquid composition for the reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising:

[0541] interpretation: For protection against typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi and Neisseria meningitidis antigens, there is no antigenic interference of ViPs-TT;OSP antigen with Neisseria meningitidis antigens, and the vaccine formulation is sufficient to induce the necessary T-dependent immune responses against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly, with only one injection comprising a complete vaccination schedule. C) A single dose vaccine kit comprising: Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; c) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; d) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen containing one or more modified adenylate cyclases, 1-50 μg pertussis toxin (PT), 1-50 μg filamentous hemagglutinin (FHA), 1-20 μg pertactin (P69 or PRN) or 2-25 μg fimbrial proteins (FIM1, 2 and 3) per 0.5 ml; e) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; f) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; And a) 1.25~50μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride, and / or c) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer, and / or d) Polysorbate 20, and / or e) 1 to 10 mg of 2-phenoxyethanol, and / or f) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising:

[0542] interpretation: For protection against Salmonella typhi and typhoid fever caused by hexavalent antigens, there is no antigenic interference of ViPs-TT with the hexavalent immune composition, and the vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi in children under 2 years of age, teenagers, adults and the elderly with only one injection comprising a complete vaccination schedule. D) A single dose vaccine kit comprising: Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; c) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; d) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen containing one or more modified adenylate cyclases, 1-50 μg pertussis toxin (PT), 1-50 μg filamentous hemagglutinin (FHA), 1-20 μg pertactin (P69 or PRN) or 2-25 μg fimbrial proteins (FIM1, 2 and 3) per 0.5 ml; e) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; f) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; And a) 1.25~50μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen, b) 1.25~50μg Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197), c) 1 to 10 mg of sodium chloride, and / or d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer, and / or g) a polysorbate selected from polysorbate 20, and / or h) 1 to 10 mg of 2-phenoxyethanol, and / or e) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising:

[0543] interpretation: For protection against typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi and the hexavalent antigen, there is no antigenic interference of the ViPs-TT;OSP antigen with the hexavalent antigen, and the vaccine formulation is sufficient to induce the necessary T-dependent immune responses against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly, with only one injection comprising the complete vaccination schedule. E) A single dose vaccine kit comprising: Complete liquid heptavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml; c) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; d) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; e) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen containing one or more modified adenylate cyclases, 1-50 μg pertussis toxin (PT), 1-50 μg filamentous hemagglutinin (FHA), 1-20 μg pertactin (P69 or PRN) or 2-25 μg fimbrial proteins (FIM1, 2 and 3) per 0.5 ml; f) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; g) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; and a) 1.25~50μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride; c) Water for injection (WFI) in appropriate volume a second container containing a completely liquid immunogenic composition comprising:

[0544] interpretation: For protection against Salmonella typhi and typhoid fever caused by heptavalent antigens, there is no antigenic interference of ViPs-TT with the heptavalent immune composition, and the vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi in children under 2 years of age, teenagers, adults and the elderly with only one injection containing a complete vaccination schedule. F) A single dose vaccine kit comprising: Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1 to 50 D antigenic units (DU) for IPV type 1, 1 to 50 D antigenic units (DU) for IPV type 2, or 1 to 50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml; c) diphtheria toxoid (D) antigen in an amount of 1 to 50 Lf per 0.5 ml; d) tetanus toxoid (T) antigen in an amount of 1 to 30 Lf per 0.5 ml; e) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen containing one or more modified adenylate cyclases, 1-50 μg pertussis toxin (PT), 1-50 μg filamentous hemagglutinin (FHA), 1-20 μg pertactin (P69 or PRN) or 2-25 μg fimbrial proteins (FIM1, 2 and 3) per 0.5 ml; f) Hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; g) Haemophilus influenzae type b antigen (Ηib) in an amount of 1 to 20 μg per 0.5 ml a first container containing; And a) 1.25~50μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen, b) 1.25~50μg Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197), c) 1 to 10 mg of sodium chloride, and / or d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer, and / or g) a polysorbate selected from polysorbate 20; h) 1 to 10 mg of 2-phenoxyethanol, and / or e) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising:

[0545] interpretation: There was no antigenic interference of ViPs-TT;OSP antigen with the heptavalent antigen for protection against typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi and the heptavalent antigen, and the vaccine formulation was found to be compatible with the complete vaccination schedule. A single injection containing the vaccinia virus is sufficient to induce the necessary T-dependent immune response against S. typhi and paratyphi in individuals, including children under 2 years of age, teenagers, adults, and the elderly.

Claims

1. i) a) Salmonella enterica serovar typhi saccharide-carrier protein conjugates; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate, c) Salmonella enterica serovar paratyphi B sugar-carrier protein conjugate, d) Salmonella enterica serovar paratyphi C sugar-carrier protein conjugate, e) Salmonella enterica serovar typhimurium sugar-carrier protein conjugates; f) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugates; g) Salmonella enterica serovar choleraesuis sugar-carrier protein conjugates; h) Salmonella enterica serovar dublin carbohydrate carrier protein conjugate; At least one antigen selected from the group consisting of ii) Optionally, Salmonella (non-typhoidal), diphtheria toxoid (D), tetanus toxoid (T), whole cell pertussis (wP), Hepatitis B surface antigen (HBsAg), Haemophilus influenzae b PRP-carrier protein conjugate (HB), Haemophilus influenzae (serotypes a, c, d, e, f, and non-capsular strains), poliovirus, N. meningitidis antigens (A, B, C, D, W135, X, Y, Z, and 29E), Streptococcus Pneumoniae antigen (1, 2, 3, 4, 5, 6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A, 9L, 9F, 9N, 9V, 10F, 10B, 10C, 10A, 11A, 11F, 11B, 11C, 11D, 11E , 12A, 12B, 12F, 13, 14, 15A, 15C, 15B, 15F, 16A, 16F, 17A, 17F, 18C, 18F, 18A, 18B, 19A, 19B, 19C, 19F, 20, 20A, 20B, 21, 22A, 22F, 23A, 23B , 23F, 24A, 24B, 24F, 25F, 25A, 27, 28F, 28A, 29, 31, 32F, 32A, 33A, 33C, 33D, 33E, 33F, 33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48), group A Streptococcus spp., group B Streptococcus spp. (groups Ia, Ib, II, III, IV, V, Vl, VII, VIII and IX), Neisseria meningitidis B vesicles or antigens (fHbp, PorA, chimeric fHbp-porA), Staphylococcus aureus antigens, splenic herpes, BCG, Hepatitis (strains A, C, D, E, F and G) antigens, human papillomavirus, HIV, acellular pertussis, modified adenylate cyclase, malaria antigens (RTS, S), measles, mumps, rubella, dengue, Zika, Ebola, chikungunya, Japanese encephalitis, rotavirus, diarrheal antigens (E. coli spp., Shigella spp., Campylobacter spp., Vibrio cholera), flavivirus, smallpox, yellow fever, varicella, varicella virus antigens.

20. An immunogenic composition comprising:

2. a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, b) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen The immunogenic composition of claim 1, comprising at least one bivalent combination of:

3. a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; c) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Salmonella enterica serovar typhimurium sugar-carrier protein conjugate antigen The immunogenic composition of claim 1, comprising at least one trivalent combination of:

4. a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen The immunogenic composition of claim 1, comprising a tetravalent combination of:

5. a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; f) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; g) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; g) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; h) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; g) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; h) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; i) Neisseria meningitidis X saccharide-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; g) Neisseria meningitidis X carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y carbohydrate-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C sugar-carrier protein conjugate antigen The immunogenic composition of claim 1, comprising at least one combination of:

6. a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; c) diphtheria toxoid (D) antigen; d) tetanus toxoid (T) antigen; e) whole cell pertussis (wP) antigen or acellular pertussis (aP); f) Hepatitis B virus surface antigen (HBsAg), and g) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; c) rotavirus antigens, d) diphtheria toxoid (D) antigen; e) tetanus toxoid (T) antigen; f) whole cell pertussis (wP) antigen or acellular pertussis (aP); g) Hepatitis B virus surface antigen (HBsAg), and h) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; d) diphtheria toxoid (D) antigen; e) tetanus toxoid (T) antigen; f) whole cell pertussis (wP) antigen or acellular pertussis (aP); g) Hepatitis B virus surface antigen (HBsAg), and h) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; e) diphtheria toxoid (D) antigen; f) tetanus toxoid (T) antigen; g) whole cell pertussis (wP) antigen or acellular pertussis (aP) antigen; h) Hepatitis B virus surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; e) diphtheria toxoid (D) antigen; f) tetanus toxoid (T) antigen; g) whole cell pertussis (wP) antigen or acellular pertussis (aP) antigen; h) Hepatitis B virus surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) rotavirus antigens, e) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; f) diphtheria toxoid (D) antigen; g) tetanus toxoid (T) antigen; h) whole cell pertussis (wP) antigen or acellular pertussis (aP) antigen; i) Hepatitis B virus surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; e) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; f) diphtheria toxoid (D) antigen; g) tetanus toxoid (T) antigen; h) whole cell pertussis (wP) antigen or acellular pertussis (aP) antigen; i) Hepatitis B virus surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritid is sugar-carrier protein conjugate antigen, e) rotavirus antigens, f) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; g) diphtheria toxoid (D) antigen; h) tetanus toxoid (T) antigen; i) whole cell pertussis (wP) antigen or acellular pertussis (aP), j) Hepatitis B virus surface antigen (HBsAg), and k) Haemophilus influenzae type b antigen (Ηib) The immunogenic composition of claim 1, comprising at least one combination of:

7. a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) rotavirus antigens, c) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; d) Shigella species antigen; e) Campylobacter jejuni antigen, f) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen; f) Campylobacter jejuni antigen, g) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen; f) Campylobacter jejun antigen, or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen; or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) rotavirus antigens, d) Shigella species antigen; or a) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; c) rotavirus antigens, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen; f) Campylobacter jejuni antigen, g) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) rotavirus antigens, e) diarrheagenic Escherichia coli spp. (enterotoxigenic and enterohaemorrhagic) antigens; f) Shigella species antigen; g) Campylobacter jejuni antigen; h) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; b) Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; e) rotavirus antigens, f) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; g) Shigella species antigen; h) Campylobacter jejuni antigen, i) Vibrio cholerae antigen The immunogenic composition of claim 1, comprising at least one combination of:

8. Carrier proteins (CPs) include tetanus toxin, tetanus toxoid (TT), fragments of tetanus toxoid, diphtheria toxoid (DT), CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, , Clostridium perfringens toxoid, E. coli LT, E. coli ST, Escherichia coli heat labile toxin-B subunit, Neisseria meningitidis outer membrane complex, rEPA, H. Protein D from H. influenzae, Flagellin FliC, horseshoe crab hemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porin, transferrin binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) from Bacillus anthracis and Bacillus purpura. The immunogenic composition according to claims 1 to 7, wherein the immunogenic protein is selected from the group comprising the detoxifying edema factor (EF) and lethal factor (LF) of C. anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins containing multiple human CD4+ T cell epitopes from various pathogens such as N19, iron uptake proteins, toxin A or B from C. difficile and S. agalactiae proteins with or without linkers, and fragments, derivatives, variants thereof.

9. Salmonella enterica serovar typhi sugar-carrier protein conjugate antigen; Salmonella enterica serovar 8. The immunogenic composition according to claims 1 to 7, wherein the Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; the Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen is in a dose range of 5 μg / dose to 30 μg / dose.

10. 8. The immunogenic composition of claims 1 to 7, comprising a pharma- ceutically acceptable buffer selected from the group of sodium chloride, acetate, carbonate, citrate, butyrate, gluconate, tartrate, phosphate buffered saline, borate, histidine buffer, succinate buffer, HEPES, Tris or citrate-phosphate.

11. 8. The immunogenic composition of claims 1 to 7, comprising a pharma- ceutically acceptable excipient selected from the group of sugars, surfactants, polymers, salts, amino acids or pH adjusters.

12. 12. The immunogenic composition of claim 11, comprising as excipient an amino acid or a protein selected from the group of L-histidine, lysine, isoleucine, methionine, glycine, aspartic acid, tricine, arginine, leucine, glutamine, alanine, peptides, hydrolyzed proteins or proteins such as serum albumin.

13. 12. The immunogenic composition of claim 11, comprising as an excipient a sugar selected from the group of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol or a combination thereof.

14. The excipient may be a non-ionic surfactant selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin, and poloxamer. The immunogenic composition of claim 11 .

15. The immunogenic composition of claim 11, comprising as an excipient a polymer selected from the group consisting of dextran, carboxymethylcellulose, hyaluronic acid, and cyclodextrin.

16. NaCl, KCl, KH as excipients ２ P.O. ４ , Na ２ H.P.O. ４ . 2H ２ O, CaCl ２ and MgCl ２ The immunogenic composition of claim 11, comprising a salt selected from:

17. The immunogenic composition of claims 1 to 7, wherein the single dose composition is preservative-free.

18. 8. The immunogenic composition of claims 1 to 7, wherein the multi-dose composition comprises one or more preservatives selected from the group of 2-phenoxyethanol, benzethonium chloride (femerol), phenol, thiomersal, formaldehyde, paraben esters (e.g. methyl-, ethyl-, propyl- or butyl-paraben), benzyl alcohol, m-cresol, benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chloro-m-cresol.

19. 8. The immunogenic composition of claims 1 to 7, further comprising an adjuvant selected from the group of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate and potassium aluminum sulfate or mixtures thereof.

20. 8. The immunogenic composition of claims 1 to 7, further comprising an immunostimulatory component selected from the group of: oil-in-water emulsion, MF-59, liposomes, lipopolysaccharide, saponin, lipid A, lipid A derivatives, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, oligonucleotides, oligonucleotides comprising at least one unmethylated CpG and / or liposomes, Freund's adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellin from gram-negative bacteria, dmLT, TLR-5 agonists, fragments of flagellin capable of binding to the TLR-5 receptor, QS-21, ISCOMS, chitosan, sterols and saponin combinations with lipids.

21. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1 to 10 mg of sodium chloride c) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

22. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1 to 10 mg of sodium chloride c) 1 to 10 mg of 2-phenoxyethanol d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

23. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, or histidine Succinate or succinate buffer c) 1 to 10 mg of sodium chloride d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

24. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer c) 1 to 10 mg of sodium chloride d) 1 to 10 mg of 2-phenoxyethanol e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

25. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

26. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 1 to 10 mg of 2-phenoxyethanol e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

27. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxyethyl ether, polysorbate 60, polysorbate 65 ... Cystearate, Polyoxyethylene-35 Ricinoleate, Soy Lecithin and Poloxamer f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

28. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

29. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

30. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is TT or D T or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

31. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

32. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 1 to 10 mg of 2-phenoxyethanol e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

33. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine in an amount of 25-500 μg; , triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

34. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

35. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

36. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

37. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

38. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 1 to 10 mg of 2-phenoxyethanol e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

39. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol in an amount of 25 to 500 μg; Toxoxyethanol, Octoxynol 40, Nonoxynol-9, Triethanolamine, Triethanolamine Polypeptide Oleate, Polyoxyethylene-660 Hydroxystearate, Polyoxyethylene-35 Ricinoleate, Soy Lecithin and Poloxamer f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

40. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

41. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

42. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

43. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

44. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 1 to 10 mg of 2-phenoxyethanol e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

45. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, or histidine Succinate or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

46. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

47. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

48. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

49. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

50. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 1 to 10 mg of 2-phenoxyethanol e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

51. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

52. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

53. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) Polysorbate 20, Polysorbate 40, Polysorbate 50 in an amount of 25-500 μg polysorbates selected from polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer. f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

54. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

55. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

56. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 1 to 10 mg of 2-phenoxyethanol e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

57. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

58. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

59. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

60. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1 to 10 mg of sodium chloride d) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

61. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

62. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 1 to 10 mg of 2-phenoxyethanol f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

63. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

64. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) a polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine in an amount of 25-500 μg; , triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer f) 2.5 to 25 mg of sucrose g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

65. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

66. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. f) 2.5 to 25 mg of sucrose g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

67. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

68. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 2.5 to 25 mg of sucrose h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

69. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is TT or D T or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

70. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 2.5 to 25 mg of sucrose h) 1 to 10 mg of 2-phenoxyethanol i) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

71. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is TT or DT or is either CRM197) d) 1 to 10 mg of sodium chloride e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

72. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 1 to 10 mg of 2-phenoxyethanol f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

73. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

74. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

75. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

76. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 2.5 to 25 mg of sucrose h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

77. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is TT or D T or CRM197) c) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

78. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 2.5 to 25 mg of sucrose h) 1 to 10 mg of 2-phenoxyethanol i) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

79. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

80. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 1 to 10 mg of 2-phenoxyethanol f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

81. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

82. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

83. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

84. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 2.5 to 25 mg of sucrose h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

85. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

86. 0.5 ml of the composition a) 1.25-50 μg of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1 to 10 mg of sodium chloride e) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer f) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in amounts of 25-500 μg. g) 2.5 to 25 mg of sucrose h) 1 to 10 mg of 2-phenoxyethanol i) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

87. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

88. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) 1 to 10 mg of 2-phenoxyethanol g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

89. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer g) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

90. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer g) 1 to 10 mg of 2-phenoxyethanol h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

91. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer g) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25-500 μg. h) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

92. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is TT or D T or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer g) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25-500 μg. h) 2.5 to 25 mg of sucrose i) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

93. 0.5 ml of the composition a) 1.25-50 μg Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer g) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25-500 μg. h) 1 to 10 mg of 2-phenoxyethanol i) Water for injection (WFI) in appropriate volume The immunogenic composition of claim 1 , comprising:

94. 0.5 ml of the composition a) 1.25-50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen; b) 1.25-50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen; (CP is either TT or DT or CRM197) c) 1.25-50 μg of Salmonella enterica serovar Typhimurium saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) d) 1.25-50 μg Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen; (CP is either TT or DT or CRM197) e) 1 to 10 mg of sodium chloride f) 0.1 mg to 1.6 mg of Tris buffer, citrate buffer, histidine buffer, or succinate buffer g) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25-500 μg. h) 2.5 to 25 mg of sucrose i) 1 to 10 mg of 2-phenoxyethanol j) Water for injection (WFI) in appropriate quantity The immunogenic composition of claim 1 , comprising:

95. Lyophilized (freeze-dried) immunogenic composition: a) 5 μg Neisseria meningitidis A saccharide-TT conjugate antigen per 0.5 ml; b) 5 μg Neisseria meningitidis C saccharide-CRM197 conjugate antigen per 0.5 ml; c) 5 μg Neisseria meningitidis Y saccharide-CRM197 conjugate antigen per 0.5 ml; d) 5 μg Neisseria meningitidis W-135 saccharide-CRM197 conjugate antigen per 0.5 ml; e) 5 μg Neisseria meningitidis X-saccharide-TT conjugate antigen per 0.5 ml; f) 1-12 mg of sucrose per 0.5 ml; g) 0.1-2 mg of sodium citrate (dihydrate) per 0.5 ml; h) Tris buffer, 0.05-0.5 mg per 0.5 ml a first container containing and a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride per 0.5 ml; c) Water for injection (WFI) in appropriate volume a second container containing a liquid composition for the reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising A single-dose vaccine kit comprising: a) a kit for preventing typhoid fever caused by Salmonella typhi and Neisseria meningitidis antigens, wherein there is no antigenic interference of ViPs-TT with Neisseria meningitidis antigens, and said vaccine preparation comprises a single dose of a vaccine comprising a complete vaccination schedule. A single-dose vaccine kit in which only one injection is sufficient to induce the necessary T-dependent immune response against S. typhi in individuals, including children under the age of 2, teenagers, adults, and the elderly.

96. Lyophilized (freeze-dried) immunogenic composition: a) 5 μg Neisseria meningitidis A saccharide-TT conjugate antigen per 0.5 ml; b) 5 μg Neisseria meningitidis C saccharide-CRM197 conjugate antigen per 0.5 ml; c) 5 μg Neisseria meningitidis Y saccharide-CRM197 conjugate antigen per 0.5 ml; d) 5 μg Neisseria meningitidis W-135 saccharide-CRM197 conjugate antigen per 0.5 ml; e) 5 μg Neisseria meningitidis X-saccharide-TT conjugate antigen per 0.5 ml; f) 1-12 mg of sucrose per 0.5 ml; g) 0.1-2 mg of sodium citrate (dihydrate) per 0.5 ml; h) Tris buffer, 0.05-0.5 mg per 0.5 ml a first container containing and a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197); c) 1 to 10 mg of sodium chloride per 0.5 ml; d) Water for injection (WFI) in appropriate volume a second container containing a liquid composition for the reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising 1. A single-dose vaccine kit comprising: a) a single-dose vaccine kit for the prevention of typhoid and paratyphoid caused by Salmonella typhi, S. paratyphi and Neisseria meningitidis antigens, wherein there is no antigenic interference of the ViPs-TT;OSP antigen with the Neisseria meningitidis antigen, and wherein the vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly, including those with a complete vaccination schedule, with only one injection.

97. Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1-50 D antigenic units (DU) for IPV type 1, 1-50 D antigenic units (DU) for IPV type 2, or 1-50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) diphtheria toxoid (D) antigen in an amount of 1-50 Lf per 0.5 ml; c) tetanus toxoid (T) antigen in an amount of 1-30 Lf per 0.5 ml; d) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen comprising one or more modified adenylate cyclases selected from pertussis toxin (PT) 1-50 μg, filamentous hemagglutinin (FHA) 1-50 μg, pertactin (P69 or PRN) 1-20 μg or fimbrial proteins (FIM1, 2 and 3) 2-25 μg per 0.5 ml; e) Hepatitis B virus surface antigen (HBsAg) in an amount of 1-20 μg per 0.5 ml; f) Haemophilus influenzae type b antigen (HB) in an amount of 1 to 20 μg per 0.5 ml a first container containing And a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride per 0.5 ml, and / or c) 0.1 mg to 1.6 mg per 0.5 ml of Tris buffer, citrate buffer, histidine buffer or succinate buffer, and / or d) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25-500 μg per 0.5 ml, and / or e) 1-10 mg of 2-phenoxyethanol per 0.5 ml, and / or f) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising wherein there is no antigenic interference of ViPs-TT with the hexavalent immune composition for protection against Salmonella typhi and typhoid fever caused by the hexavalent antigen, and said vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi in children under 2 years of age, teenagers, adults and the elderly with only one injection comprising a complete vaccination schedule.

98. Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1-50 D antigenic units (DU) for IPV type 1, 1-50 D antigenic units (DU) for IPV type 2, or 1-50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) diphtheria toxoid (D) antigen in an amount of 1-50 Lf per 0.5 ml; c) tetanus toxoid (T) antigen in an amount of 1-30 Lf per 0.5 ml; d) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml, or acellular pertussis (aP) antigen comprising one or more modified adenylate cyclases selected from pertussis toxin (PT) 1-50 μg, filamentous hemagglutinin (FHA) 1-50 μg, pertactin (P69 or PRN) 1-20 μg or fimbrial proteins (FIM1, 2 and 3) 2-25 μg per 0.5 ml; e) Hepatitis B virus surface antigen (HBsAg) in an amount of 1-20 μg per 0.5 ml; f) Haemophilus influenzae type b antigen (HB) in an amount of 1 to 20 μg per 0.5 ml a first container containing and a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197); c) 1-10 mg of sodium chloride per 0.5 ml, and / or d) 0.1 mg to 1.6 mg per 0.5 ml of Tris buffer, citrate buffer, histidine buffer or succinate buffer, and / or e) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25-500 μg per 0.5 ml, and / or f) 1-10 mg of 2-phenoxyethanol per 0.5 ml, and / or g) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising 1. A single-dose vaccine kit comprising: a) a single-dose vaccine kit for protection against typhoid and paratyphoid caused by Salmonella typhi, S. paratyphi and hexavalent antigen, wherein there is no antigenic interference of ViPs-TT;OSP antigen with the hexavalent antigen, and said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly, with only one injection comprising a complete vaccination schedule.

99. Complete liquid heptavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1-50 D antigenic units (DU) for IPV type 1, 1-50 D antigenic units (DU) for IPV type 2, or 1-50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml; c) diphtheria toxoid (D) antigen in an amount of 1-50 Lf per 0.5 ml; d) tetanus toxoid (T) antigen in an amount of 1-30 Lf per 0.5 ml; e) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml or acellular pertussis (aP) antigen comprising one or more modified adenylate cyclases selected from pertussis toxin (PT) 1-50 μg, filamentous hemagglutinin (FHA) 1-50 μg, pertactin (P69 or PRN) 1-20 μg or fimbrial proteins (FIM1, 2 and 3) 2-25 μg per 0.5 ml; f) Hepatitis B virus surface antigen (HBsAg) in an amount of 1-20 μg per 0.5 ml; g) Haemophilus influenzae type b antigen (HB) in an amount of 1 to 20 μg per 0.5 ml a first container containing and a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1 to 10 mg of sodium chloride per 0.5 ml; c) Water for injection (WFI) in appropriate volume a second container containing a completely liquid immunogenic composition comprising wherein there is no antigenic interference of ViPs-TT with the heptavalent immune composition for protection against Salmonella typhi and typhoid fever caused by the heptavalent antigen, and said vaccine formulation is sufficient to induce the necessary T-dependent immune response against S. typhi in children under 2 years of age, teenagers, adults and the elderly with only one injection comprising a complete vaccination schedule.

100. Complete liquid heptavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strains in a dose of 1-50 D antigenic units (DU) for IPV type 1, 1-50 D antigenic units (DU) for IPV type 2, or 1-50 D antigenic units (DU) for IPV type 3 per 0.5 ml; b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount ranging from 1 to 50 μg per 0.5 ml; c) diphtheria toxoid (D) antigen in an amount of 1-50 Lf per 0.5 ml; d) tetanus toxoid (T) antigen in an amount of 1-30 Lf per 0.5 ml; e) whole cell pertussis (wP) antigen in an amount of 1-50 IOU per 0.5 ml or acellular pertussis (aP) antigen comprising one or more modified adenylate cyclases selected from pertussis toxin (PT) 1-50 μg, filamentous hemagglutinin (FHA) 1-50 μg, pertactin (P69 or PRN) 1-20 μg or fimbrial proteins (FIM1, 2 and 3) 2-25 μg per 0.5 ml; f) Hepatitis B virus surface antigen (HBsAg) in an amount of 1-20 μg per 0.5 ml; g) Haemophilus influenzae type b antigen (HB) in an amount of 1 to 20 μg per 0.5 ml a first container containing And a) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar typhi ViPs-TT conjugate antigen, b) 1.25 to 50 μg of Salmonella enterica per 0.5 ml serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197); c) 1-10 mg of sodium chloride per 0.5 ml, and / or d) 0.1 mg to 1.6 mg per 0.5 ml of Tris buffer, citrate buffer, histidine buffer or succinate buffer, and / or g) polysorbates selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer in an amount of 25-500 μg per 0.5 ml, and / or h) 1 to 10 mg of 2-phenoxyethanol per 0.5 ml, and / or e) Water for injection (WFI) in appropriate quantity a second container containing a completely liquid immunogenic composition comprising 1. A single-dose vaccine kit comprising: a) a single-dose vaccine kit for protection against typhoid and paratyphoid caused by Salmonella typhi, S. paratyphi and heptavalent antigen, wherein there is no antigenic interference of ViPs-TT;OSP antigen with the heptavalent antigen, and said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi and paratyphi in children under 2 years of age, teenagers, adults and the elderly, with only one injection comprising a complete vaccination schedule.

101. 1. A method for producing a Salmonella enterica serovar typhi saccharide-carrier protein conjugate, comprising: a. A fed-batch mode cultivation step for obtaining high yield harvest of Salmonella typhi capsular Vi polysaccharide (ViPs), wherein during the cultivation, antifoam agent, 40-70 g / L The use of a combination of soy peptone in the range of 40-70 g / L and yeast extract in the range of 40-70 g / L resulted in improved yields (100-700 mg / L) and fermentation parameters consisted of pH maintained in the range of 6.7-7.1, temperature maintained in the range of 34.0-38.0° C., dissolved oxygen level maintained at 36-39%, agitation (rpm) maintained at 150-500, and osmolarity of 400-600 mOsmol / kg; b. Purifying the Salmonella typhi capsular ViPs by treatment with less than 2% anionic detergent, more preferably sodium dodecyl sulfate (SDS) and cationic detergent, preferably CTAB, ethylenediaminetetraacetic acid (EDTA) (4-10 mM), sodium acetate (5%-10%), alcohol precipitation (30%-60%), and concentration and 30 kDa cut-off membrane at various steps, the process resulting in the production of endotoxins (PS resulting in desirable O-acetyl levels (>2.0 mmol / g polysaccharide), purified Vi polysaccharide yields in the range of 100-4000 mg / L, molecular size distribution (>50% of PS elutes before a distribution coefficient (KD) of 0.25 is reached), together with significant reductions in impurities (<100 EU endotoxin / μg), protein (<1%) and nucleic acid (<2%) impurities, and higher capsular polysaccharide recoveries preferably in the range of 40%-65%, and the average molecular weight of the purified Vi polysaccharide may be in the range of 40-400 kDa, preferably 150-250 kDa; c. conjugating the purified ViPs to a carrier protein (CP) using carbodiimide, reductive amination or cyanylation conjugation reactions; d. Purifying the ViPs-carrier protein conjugate using gel filtration chromatography or ultrafiltration, which concentrates the conjugate bulk at least 3-fold and removes substantially all unreacted compounds, unconjugated polysaccharide, unconjugated protein, resulting in a purified Vi polysaccharide conjugate vaccine having a size of 1000-1500 kDa, with a conjugate yield of ≧50%. A method comprising:

102. Prior to conjugation, ViPs was reacted with FeCl3, H ２ O ２ 102. The method of claim 101, wherein the Salmonella enterica serovar is subjected to depolymerization / sizing by chemical means selected from the group consisting of sodium metaperiodate and sodium acetate, or mechanical means selected from the group consisting of high pressure cell disruption, homogenizer, and ultrasonic treatment. A method for producing a S. typhi saccharide-carrier protein conjugate.

103. 102. The method of claim 101, wherein the purified ViPs is covalently coupled to a carrier protein (CP) using a carbodiimide conjugation reaction in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC) mixed in a weight ratio of ViPs:EDAC of 1:0.5 to 1:2, the ratio of Vi polysaccharide and carrier protein being 0.5 to 1.5, the ViPs:CP:EDAC ratio being preferably 1:1:2 or 1:0.9:2 or 1:0.8:1.8 or 1:0.7:1.8, and the reaction is carried out at a pH of 5 to 7, at a temperature in the range of 2°C to 30°C.

104. 104. The method of claim 101 or 103, wherein prior to conjugation, the polysaccharide or carrier protein is derivatized with a hetero- or homobifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethylamine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propionamide, nitrophenylethylamine, haloalkyl halide, 6-aminocaproic acid, using carbodiimide, reductive amination or cyanylation reactions. A method for producing a S. typhi saccharide-carrier protein conjugate.

105. 105. A method for producing a Salmonella enterica serovar typhi saccharide-carrier protein conjugate according to claim 104, wherein prior to conjugation, the carrier protein (CP) is derivatized with an adipic acid dihydrazide (ADH) linker in the presence of a carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC), for a reaction duration of 1 hour carried out at a pH ranging from 5 to 7.

106. The carrier protein may be tetanus toxin, tetanus toxoid, a fragment of tetanus toxoid, diphtheria toxoid, CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, E. coli LT, E. coli ST, Escherichia coli heat-labile toxin-B subunit, Neisseria meningitidis outer membrane complex, rEPA, H. influenzae protein D, Flagellin FliC, horseshoe crab hemocyanin, Pseudomonas The antibodies have been identified from a variety of pathogens, including exotoxin A from P. aeruginosa, outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) from Bacillus anthracis and the detoxifying edema factor (EF) and lethal factor (LF) from Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, and multiple human CD4+ antigens from various pathogens, including N 19.

106. A method for producing a Salmonella enterica serovar typhi saccharide-carrier protein conjugate according to claim 101, 102, 103, 104 or 105, selected from the group consisting of artificial proteins comprising T cell epitopes, iron uptake proteins, toxins A or B from C. difficile and S. agalactiae proteins with or without linkers, and fragments, derivatives and variants thereof.

107. 107. The method of producing a Salmonella enterica serovar typhi saccharide-carrier protein conjugate according to claim 106, wherein the carrier protein is tetanus toxoid (TT).

108. 107. The method of producing a Salmonella enterica serovar typhi saccharide-carrier protein conjugate according to claim 106, wherein the carrier protein is diphtheria toxoid (DT).

109. 107. The method of producing a Salmonella enterica serovar typhi saccharide-carrier protein conjugate according to claim 106, wherein the carrier protein is CRM197.

110. 1. A method for producing a Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate, comprising: a. Extraction of lipopolysaccharide (LPS) from Salmonella P by acid hydrolysis of LPS, preferably with acetic acid (final concentration 0.5-5%) at pH about 2.0-3.0, neutralization to pH 7.0 with aqueous ammonia, sodium deoxycholate (final concentration 0.5-5%), and concentration and diafiltration using 10 kDa to 30 kDa cut-off membranes in various steps. purifying Aratyphi A O-specific polysaccharide (OSP), the process resulting in a significant reduction in endotoxin (<100 EU endotoxin / μg PS), protein (<1%) and nucleic acid (<2%) impurities, and a higher capsular polysaccharide recovery, preferably in the range of 40%-65%, with a desired O-acetyl level (>2.0 mmol / g polysaccharide), molecular size distribution (>50% of PS elutes before a distribution coefficient (KD) of 0.25 is reached), and the average molecular weight of the purified O-specific polysaccharide (OSP) may be 40-400 kDa; b. conjugating the purified OSP to a carrier protein (CP) using a carbodiimide, reductive amination or cyanylation conjugation reaction; c) purifying the OSP-carrier protein conjugate using gel filtration chromatography or ultrafiltration, which concentrates the conjugate bulk by at least 3-fold and removes substantially all unreacted compounds, unconjugated polysaccharides, and unconjugated proteins to obtain a purified OSP-carrier protein conjugate vaccine, with a conjugate yield of >50%. A method comprising:

111. Prior to conjugation, OPS was ３ , H ２ O ２ 111. The method of claim 110, wherein the Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate is subjected to depolymerization / sizing by chemical means selected from the group consisting of sodium metaperiodate and sodium acetate, or mechanical means selected from the group consisting of high pressure cell disruption, sonication and homogenizer.

112. 111. The method of producing a Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate of claim 110, wherein the purified OSP is covalently attached to the carrier protein (CP) using cyanylation conjugation chemistry, the cyanylation reagent being selected from the group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ("CDAP"), p-nitrophenyl cyanate and N-cyanotriethylammonium tetrafluoroborate ("CTEA") or 2-cyanopyridazin-3(2H)one (2-CPO).

113. 113. The method of producing a Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate according to claim 112, wherein the purified OSP is covalently attached to a carrier protein (CP) using cyanylation conjugation chemistry, the cyanylation reagent being 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) mixed in a weight ratio of OSP:CPPT of 1:0.5 to 1:2, the ratio of OSP and carrier protein may be 0.5 to 1.5, and the reaction is carried out at a pH ranging from 5 to 7, at a temperature ranging from 2°C to 30°C.

114. 114. A method for producing a Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate according to claim 112 or 113, wherein prior to conjugation the carrier protein is derivatized with a hetero- or homobifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethylamine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propionamide, nitrophenylethylamine, haloalkyl halide, 6-aminocaproic acid using carbodiimide, reductive amination or cyanylation reactions.

115. 115. The method of producing a Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate according to claim 114, wherein prior to conjugation, the carrier protein (CP) is derivatized with an adipic acid dihydrazide (ADH) linker in the presence of a carbodiimide, for example 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC) mixed in a weight ratio of OSP:ADH of 1:1 to 1:10, the ratio of OSP:EDAC may be 0.5 to 1.5, and the reaction is carried out at a pH ranging from 5 to 7, for a reaction duration of 1 to 2 hours, at a temperature ranging from 2° C. to 30° C.

116. 111. The method of producing a Salmonella enterica serovar Partyphi A saccharide-carrier protein conjugate of claim 110, wherein purified OSP is covalently attached to a carrier protein (CP) using a carbodiimide conjugation reaction in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC) mixed in a weight ratio of OSP:EDAC of 1:0.5 to 1:2, the ratio of OSP polysaccharide and carrier protein may be 0.5 to 1.5, and the reaction is carried out at a pH of 5 to 7 and a temperature ranging from 2° C. to 30° C.

117. 117. The method of producing a Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate according to claim 116, wherein prior to conjugation, the OSP is derivatized with a hetero- or homobifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethylamine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propionamide, nitrophenylethylamine, haloalkyl halide, 6-aminocaproic acid using carbodiimide, reductive amination or cyanylation reactions.

118. 118. The method of producing a Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate of claim 117, wherein prior to conjugation, the OSP is derivatized with an adipic acid dihydrazide (ADH) linker using a cyanylation conjugation chemistry, the cyanylation reagent being selected from the group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1-cyano-imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ("CDAP"), p-nitrophenyl cyanate, and N-cyanotriethylammonium tetrafluoroborate ("CTEA") or 2-cyanopyridazin-3(2H)one (2-CPO).

119. 119. The method of producing a Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate according to claim 118, wherein prior to conjugation, the OSP is derivatized with an adipic acid dihydrazide (ADH) linker using cyanylation conjugation chemistry, the cyanylation reagent being 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) mixed in a weight ratio of OSP:ADH of 1:1 to 1:10, the ratio of OSP:CPPT may be 0.5 to 1.5, and the reaction is carried out at a pH ranging from 7 to 10, for a reaction duration of 1 to 2 hours, at a temperature ranging from 2°C to 30°C.

120. The carrier protein may be tetanus toxin, tetanus toxoid, a fragment of tetanus toxoid, diphtheria toxoid, CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, E. coli LT, E. coli ST, Es cherichia coli heat-labile toxin-B subunit, Neisseria meningitidis outer membrane complex, rEPA, H. influenzae protein D, Flagellin FliC, horseshoe crab hemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porin, transferrin-binding protein, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, Bacillus anthracis protective antigen (PA) and Bacillus 120. A method for producing a Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate according to any one of claims 110 to 119, selected from the group consisting of the detoxifying edema factor (EF) and lethal factor (LF) of S. anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins containing multiple human CD4+ T cell epitopes from various pathogens such as N19, iron uptake proteins, toxins A or B from C. difficile and S. agalactiae proteins with or without linkers, and fragments, derivatives and variants thereof.

121. 121. The method of producing a Salmonella enterica serovar Partyphi A saccharide-carrier protein conjugate according to claim 120, wherein the carrier protein is tetanus toxoid (TT).

122. 121. The method of producing a Salmonella enterica serovar Partyphi A saccharide-carrier protein conjugate according to claim 120, wherein the carrier protein is diphtheria toxoid (DT).

123. 121. The method of producing a Salmonella enterica serovar Partyphi A sugar-carrier protein conjugate according to claim 120, wherein the carrier protein is CRM197.

124. 13. The immunogenic composition of any one of the preceding claims, wherein the immunogenic composition is formulated for use in a method for prophylaxis against typhoid, paratyphoid and non-typhoid infections, for reducing or preventing the onset of infections caused by Salmonella typhi, Salmonella paratyphi and Salmonella non-typhoid related genera spp, the method comprising the step of administering an effective amount of the immunogenic composition via parenteral, or subcutaneous, or intradermal, or intramuscular, or intraperitoneal, or intravenous, or injectable, or sustained release from an implant, or ophthalmic, or nasal, or buccal, or vaginal, oral, intragastric, or mucosal, or sublingual, alveolar, gingival, olfactory, or respiratory mucosal administration, or any other immunization route to a human subject under the age of 2, a teenager, an adult, or an elderly person.

125. 10. The immunogenic composition according to any one of the preceding claims, which is stable at 2-8°C, 25°C and 40°C for a period of 6 months.

126. 10. The immunogenic composition according to any one of the preceding claims, which is stable at 2-8°C, 25°C and 40°C and after 6 months has no more than 7.5% free polysaccharide for the 180-220 kDa polysaccharide and no more than 10.5% free polysaccharide for the 388 kDa, 80 kDa and 45 kDa polysaccharides.

127. 10. The immunogenic composition of any one of the preceding claims, wherein the immunogenic composition is formulated for administration to a human subject under 2 years of age, a teenager, an adult or an elderly person according to a dose regimen consisting of a single dose of the vaccine composition.

128. 10. The immunogenic composition of any one of the preceding claims, wherein the immunogenic composition is formulated for administration to a human subject aged 2 years or younger, a teenager, an adult or an elderly person according to a two-dose regimen consisting of a first dose and a second dose administered 3 months to 2 years after the first dose.

129. 13. The immunogenic composition of any one of the preceding claims, wherein the immunogenic composition is formulated for administration to a human subject aged 2 years or younger, a teenager, an adult or an elderly person according to a three-dose regimen consisting of a first dose, a second dose administered 3 months to 2 years after the first dose, and a third dose administered 3 months to 2 years after the second dose.

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