Immunogenic compositions for intestinal diseases and methods for their preparation - Patents.com

Abstract

The present disclosure relates to novel immunogenic monovalent and multivalent polysaccharide-protein conjugate vaccine compositions comprising polysaccharides selected from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium, and S. enteritidis, as well as alternative and improved methods of polysaccharide fermentation, polysaccharide purification, polysaccharide-protein conjugation, and stable formulation. The disclosure also relates to methods for eliciting an immune response against Salmonella typhi and non-typhoid-related disease in a subject and / or for reducing or preventing Salmonella typhi and non-typhoid-related disease in a subject using the compositions disclosed herein. The vaccines induce bactericidal antibodies and are useful for preventing gastroenteritis, enteric fever, and typhoid fever.

Description

[Technical Field]

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

[0002] The following background information herein is relevant to the present disclosure, but is not necessarily prior art. Salmonella infection remains a significant health problem affecting millions of people annually throughout the world, especially in developing countries. Salmonella infection 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 Enterobacteriaceae family and includes gram-negative, nonspore-forming, facultative anaerobic bacilli. Salmonella enterica serovar Typhi (S. typhi) and Salmonella enterica serovar Paratyphi (S. paratyphi) A and B cause enteric fever, a systemic, febrile illness occurring exclusively in humans, distinct from the more common, self-limited acute gastroenteritis caused by numerous other Salmonella serovars. Enteric fever caused by members of the Salmonella genus, including typhoid and paratyphoid, continues to constitute a significant burden of illness and mortality in developing country populations (Lancet 2005;366:749-762) and represents a significant risk for travelers (Lancet Infect Dis. 2005:5(10):623-628). Typhoid fever remains endemic in low- and middle-income countries (LMICs). Between 12.5 and 20.6 million cases of enteric fever occur annually in LMICs, particularly 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 supplies were 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 recently been increasingly recognized 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 cause of gastroenteritis in healthy children and adults in industrialized countries. NTS can also cause severe extraintestinal invasive bacteremia, referred to as iNTS. iNTS typically 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 serovars, 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 leading 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 non-steroidal anti-inflammatory (NTS) gastroenteritis was estimated at 93 million cases in 2010, of which approximately 80.3 million were foodborne, resulting in 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 and accounts for 17,000 quality-adjusted life-years lost, most of which are due to some foodborne pathogen. As previously mentioned, NTS can also cause severe extraintestinal invasive bacteremia, referred to as iNTS. Invasive Salmonella infections are more common throughout developing countries and are the most common cause of bacteremia in tropical Africa, particularly among young children and individuals with HIV. This usually manifests as a fever. In fact, iNTS often occurs without gastrointestinal symptoms in both adults and children. Symptoms of iNTS are similar to those of malaria and include fever and sweats (over 90 percent) and splenomegaly (40 percent). The reasons why iNTS is such a problem in Africa are unclear, 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 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 death.

[0008] Due to the decreasing effectiveness of available treatment tools, the problem of typhoidal and nontyphoidal Salmonella-associated infections is likely to continue to increase, making vaccine development an important priority for disease control efforts. Vaccines are also an important protective tool for persons 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) recommends the increased use of typhoid vaccines, with the typhoid conjugate vaccine (TCV) being preferred (WHO position paper. Wkly Epidemiol Rec 2018;93:153-72).

[0010] No vaccine for S. paratyphi is currently available. 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 all target only 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 can protect against both serotypes would be more valuable than a vaccine limited to one. The same is also true for S. typhimurium and S. enteritidis in sub-Saharan Africa, highlighting the importance of vaccines that can additionally protect against NTS serotypes for this region.

[0012] It is unclear whether vaccine candidates in the pipeline will protect against both gastroenteritis and the invasive manifestations of iNTS. While our understanding of the disease burden of typhoid in LMICs increases, the global medical need for conjugate vaccines that protect against typhoidal and nontyphoidal Salmonella infections is becoming increasingly significant. Peak demand for typhoid conjugate vaccines is likely to occur between 2023 and 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 meeting population demand. The upstream process includes the entire process from initial cell isolation and cultivation, through cell banking, culture growth in the bacterial fermentation process, and final harvest. Cell cultures are scaled up from 100–500 milliliters to 3–20,000 liter bioreactors. Further steps include primary recovery of Salmonella polysaccharides and removal of cells and debris. Furthermore, to facilitate the development of cost-effective Salmonella polysaccharide-based conjugate vaccines, structurally intact polysaccharides must be obtained in high yield and purity. Yields of less than 40% have previously been reported for Salmonella polysaccharides. Increasing capsular polysaccharide (CPS) "fermentation harvest stage yield" by using novel feeding strategies and improved fermentation media is one preferred approach to achieve this goal. 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 pathogenic bacteria, such as 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 differ for each bacterium. Zhan et al. (2002) similarly showed that pH control and modification 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 cultured in a medium containing glucose at a concentration below 1.0 g / L, and that low oxygen tension favored higher polysaccharide production. Furthermore, they observed that the final cell density was limited by the concentration of casamino acids in the feed solution. Furthermore, growth and polysaccharide yield in a specific medium also depend on the ratio of carbohydrate to nitrogen source. In one fed-batch fermentation method for S. typhi, ammonia was supplied as a nitrogen source along with the feed medium. However, they observed that polysaccharide formation by Aureobasidium pullulans was affected by the ammonia nitrogen source in the medium, and the presence of excess ammonium ions reduced the yield even under conditions that otherwise supported polysaccharide synthesis (Appl Microbiol Biotechnol (1990) 32:637-644).

[0017] Growth and polysaccharide synthesis in defined media were 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 approach to timely increase cell density, culture viability, and productivity. 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 soybeans, 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 yield 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 bubbles disappear; and iv) lead to overpressure if the outlet filter becomes clogged with foam.

[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, product isolation, concentration, and purification. Downstream processing is the most challenging part of the process due to its complexity.

[0020] Each bacterium has a different capsular polysaccharide and a different serotype of the same bacterium, further differing in the chemical structure of the bacterial capsular polysaccharide. A relevant example is S. typhi, which expresses the Vi polysaccharide capsule, a linear homopolymer of α(1-4)-D-GalpA that is N-acetylated at C-2 and O-acetylated at C-3. The N- and O-acetyls occupy the surface and are essential for both the antigenicity and immunogenicity of Vi. Conversely, 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 lipopolysaccharides. They share the common trisaccharide backbone →2)-α-D-Manp-(1→4)-α-L-Rhap-(1→3)-α-D-Galp-(1→), which constitutes serological epitope 12. However, the α-(3→6)-linked dideoxyhexose sugar at the repeating trisaccharide mannose results 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: 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, and vaccines containing polysaccharides must meet certain quality standards. Previous purification methods that can be performed in large volumes include dissolving and precipitating impurities such as nucleic acids and lipids using solvents, manipulating pH, and using 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 its core steroid structure, is resistant to denaturation and has limited solubilizing strength, which destroys endotoxins without affecting their chemical structure. Therefore, upon removal of sodium deoxycholate, endotoxins regain their biological activity. Furthermore, DOC-based procedures are ineffective at 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 polysaccharide. Furthermore, sodium deoxycholate is an animal-derived product, and even its residual presence in the final product can render it unacceptable 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 residue. For some CPS types, precipitation with zinc acetate / ammonium sulfate / sodium citrate is also included to remove protein contaminants. However, to reach a high level of purity, repeated ammonium sulfate precipitations are required, making the process more cumbersome and labor-intensive. Furthermore, this sometimes also precipitates capsular polysaccharides, resulting in 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 overwhelming and lead to the loss of the desired product. Furthermore, regulatory agencies restrict the use of animal enzymes in human products due to the risk of prion contamination. In addition to being costly, the use of enzymes introduces many regulatory issues within 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 to separate endotoxins from bacterial polysaccharides. This method is expensive and time-consuming. Furthermore, working with toxic organic solvents produces toxic waste, which is undesirable.

[0027] The high purity required for vaccine-specific polysaccharides has led to the development of new purification methods based on fractional 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 to isolate bacterial polysaccharides by effectively removing protein and nucleic acid contaminants. Despite the success of bacterial polysaccharide isolation according to WHO standards, the use of chromatographic techniques involves laborious and time-consuming sample preparation steps, posing challenges for scale-up, and dramatically impairing capsular polysaccharide recovery, making them unsuitable as low-cost options for downstream processing on an industrial scale. Furthermore, the addition of new purification steps to remove these contaminants increases process complexity, reduces final yield, and increases economic costs.

[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 polysaccharides while removing impurities to acceptable levels.

[0029] Various methods have been described for the depolymerization (sizing) of bacterial and nonbacterial polysaccharides, including acid hydrolysis, alkaline hydrolysis, periodate oxidation, 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). However, acid hydrolysis and alkaline hydrolysis are time-consuming, and the resulting size-reduced samples have high polydispersity. Furthermore, periodate oxidation has a detrimental effect on labile antigenic epitopes of some polysaccharides. Furthermore, ozonolysis can only be used with polysaccharides containing β-D-aldoside linkages, and only a few endoglycanases have been isolated to date. US20090041802 discloses fragmentation of meningococcal polysaccharides 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 products, requiring more runs or higher pressures than would otherwise be required.

[0030] Ultrasonic treatment has been used to depolymerize polysaccharides (see, for example, WO2010 / 055250). However, the ultrasonic depolymerization method is not suitable for industrial depolymerization of large amounts of polysaccharides due to its 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 destroying immunogenic moieties, which may affect the immunogenicity of the conjugate.

[0032] For all the reasons stated above, there remains a need in the art for a simple, low-cost method for the depolymerization of polysaccharides or polysaccharide derivatives. Producing polysaccharide-protein conjugate vaccines 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] Activated polysaccharides are coupled to the amino groups of carrier proteins 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). Other linkers include B-propionamide (WO 00 / 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 for conjugating polysaccharides and immunogenic proteins via dimeric spacers. A protein (PRO) is 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 Ps, a digene spacer was formed that covalently linked Ps to PRO (Ps-PRO). Upon acid hydrolysis, the digene spacer (linker) was released as an unusual amino acid, which was 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 dependent on the type of conjugate, formulation components, and storage conditions. This leads to an increase in free polysaccharide, which can adversely affect product stability. Polysaccharide-carrier protein conjugates are known to release free polysaccharide 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; excessive levels of unbound polysaccharides can potentially result in a decreased immune response to the polysaccharide (see WHO / TRS / 924, p. 14, A.3.3.5). Notably, Salmonella typhi conjugates reported in the literature have high free polysaccharide contents (up to 34%) and high free protein contents (>5%), indicating lower conjugation efficiency and 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 can be introduced into clinical trials. Furthermore, 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 producing the quantities required for later-phase or commercial campaigns. Another important consideration is the speed of process development, given that process development must occur before therapeutic candidates are introduced 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 the availability of electricity and refrigeration is often inadequate and therefore vaccine stability over temperature ranges is presumed to be more relevant in these regions.

[0038] Therefore, there is a need for an efficient platform process for producing an effective vaccine against Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis that meets multiple criteria including good immunogenicity, safety and affordability, and in particular a platform process that provides i) improved polysaccharide yield throughout the fermentation and purification process, ii) an improved purification process that exhibits optimal percentage recovery and minimal impurity levels, and iii) an improved ratio of polysaccharide-protein conjugate in the vaccine, and iv) an improved formulation that exhibits low viscosity, avoidance of aggregation and long-term stability over a wide temperature range.

[0039] In order to overcome the aforementioned limitations of the prior art and to solve a long-felt unmet global medical need, the Applicant proposes improved and alternative fermentation, purification, conjugation processes, formulations for the preparation of monovalent Salmonella typhi conjugates, and 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 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 method for the production of polysaccharide-protein conjugate vaccines comprising polysaccharides from Salmonella enterica 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 improved methods for conjugation of polysaccharides (with or without size reduction) from Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis to carrier proteins.

[0048] Yet another object of the present disclosure is to provide a method for conjugation of polysaccharides (with or without size reduction) from Salmonella enterica 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 at 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 provides: a) an immunogenic composition comprising one or more polysaccharide-protein conjugates, wherein the polysaccharides are derived from Salmonella enterica 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, in the presence or absence of 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 explanation of the drawings]

[0052] [Figure 1] See Figure 1: Comparison of polysaccharides, proteins and conjugates (PS:PR:CPIP 1:0.8:1.3). [Figure 2] Figure 2: Chromatogram of GFC-purified OSP-DT ADH conjugate (PS:PR:CPIP = 1:0.8:1.3). [Figure 3] See Figure 3: Comparison of polysaccharide, protein and conjugate (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). [Figure 5] See Figure 5: Comparison of polysaccharide, protein and conjugate (PS:PR:CPIP at 1:1:1.3). [Figure 6] See Figure 6: Comparison of polysaccharide, protein and conjugate (PS:PR:CPIP at 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 polysaccharide, protein and conjugate (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 polysaccharide, protein and conjugate (PS:PR:CPIP 1:0.9:1.3). [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 purified conjugate. DETAILED DESCRIPTION OF THE INVENTION

[0053] While the present disclosure may be capable of various embodiments, certain embodiments are set forth in the following detailed discussion, which can be considered as an exemplification of the principles of the disclosure, and it is understood 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 set forth 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 construed 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 terminology is 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 to be construed as necessarily required to perform them as described or illustrated. It is also understood that additional or alternative steps may be employed.

[0057] Terms such as first, second, third, etc. should not be construed as limiting the scope of the present disclosure, and these terms may only be used to distinguish one element, component, region, layer, or section from another component, region, layer, or section. When used herein, terms such as first, second, third, etc. do not imply a specific sequence or order unless clearly indicated by the present disclosure. The present disclosure provides immunogenic compositions and methods 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 present invention) is the total D antigen unit measurement of each unadsorbed bulk IPV antigen type prior to formulation into the final vaccine, which is added to each human dose of the formulated vaccine (typically a final volume of 0.5 mL). Reliable methods for measuring D antigen units are well known in the art and are 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 across 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, various 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., the preferably used Mahoney), 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 would be Poliovac® (Serum Institute of India Pvt. Ltd.).

[0061] The term "saccharide" throughout this specification may refer to polysaccharides or oligosaccharides, 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 eliciting 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 enterica serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis.

[0063] According to a second embodiment of the present disclosure, a method for obtaining polysaccharides from Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis by a fed-batch process 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 include a carbon source, a magnesium salt, a phosphate source, yeast extract, and 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 and sodium dihydrogen phosphate. Preferably, the soy hydrolysate is hysoybean.

[0065] Further in accordance with this, the fermentation medium may further contain an antifoaming agent selected from the group consisting of 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 (vegetable-based alkoxylated fatty acid ester), and SE9 (aqueous emulsion containing 10% silicone oil) from Wacker-Chemie Co. Combining the antifoaming agent with soy hydrolysate and yeast extract may help improve the yield of polysaccharides. More preferably, the antifoaming agent may be Antifoam C or J673 STRUKTOL.

[0066] According to embodiments 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, soybean 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 hysoybean.

[0067] The combination of antifoam, soy peptone and yeast extract improves harvest yield 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 fixed rates at specific fixed time intervals when the fermentation is already underway and / or by allowing continuous feeding in fed-batch mode of fermentation, including multiple stages with proportionally increasing batch sizes between stages.

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

[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, by 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, preferably with parameters set to a temperature of 2 to 8°C, an RPM of 7000 to 8000, and a centrifugation time of 40 to 60 minutes.

[0071] According to a sixth aspect of the second embodiment, the clarification may be carried out 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 sterilizing filter.

[0072] According to an eighth aspect of the second embodiment, the crude Salmonella enterica serovar Typhi Vi polysaccharide (ViPs) yield in 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 kD; 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 alkaline salts 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; 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 kD; 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 a second aspect of the third embodiment, the purification method can result in a large recovery of approximately 40% to 65% with desired O-acetyl levels (greater than 2.0 mmol / g polysaccharide), purified Vi polysaccharide yields can range from 1000 to 4000 mg / L, average molecular weights can range from 40 to 400 kDa, contain less than 1% protein / peptides, less than 2% nucleic acids, less than 100 EU endotoxin per μg polysaccharide (PS), and a molecular size distribution (greater than 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 alkanesulfonate, sodium lignosulfonate and sodium alkyl glyceryl ether sulfonate.

[0076] Preferably, the anionic detergent may be an alkyl sulfate, more preferably sodium dodecyl sulfate, added to the retentate at a final concentration in the range of 0.1% to 20%, more preferably 1 to 20%, 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, 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 all of the following downstream purification steps to obtain O-specific polysaccharide of the desired quality from 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 a molecular weight cut-off (MWCO) of 10–300 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 a molecular weight cut-off (MWCO) of 10–300 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 alkaline salts to remove excess detergent, followed by centrifugation and filtration by direct flow filtration (DFF) through at least one clarifying filter with a pore size of about 0.2 μM; n) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using membranes with a molecular weight cut-off (MWCO) of 10 to 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 cut-off (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 using preferably acetic acid (final concentration 0.5-5%), pH about 2.0-3.0, at a temperature of 30-90°C, 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 may be selected from the group comprising alkyl sulfates, sodium dodecyl sulfate, 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 alkanesulfonate, sodium lignosulfonate and sodium alkyl glyceryl ether sulfonate.

[0085] 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, 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 a desired O-acetyl level (>2.0 mmol / g polysaccharide) along with a significant reduction in endotoxin (<100 EU endotoxin / µg PS), protein (<1%) and nucleic acid (<2%) impurities, preferably a higher capsular polysaccharide recovery in the range of 40%-65%, with a 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 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, the purified Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis polysaccharides are selected from the group consisting of 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, H. influenzae protein D, Flagellin FliC, horseshoe crab hemocyanin, Pseudomonas Multiple human CD4+ antibodies were detected from various pathogen-derived antigens, including exotoxin A from Streptococcus 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, Bacillus anthracis protective antigen (PA), and detoxified Bacillus anthracis edema factor (EF) and lethal factor (LF), 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 N19. It may be covalently linked to a carrier protein (CP) selected from the group including artificial proteins containing T cell epitopes, 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] More preferably, the carrier protein used for conjugation with the 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 contemplated in accordance with 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 A conjugated to tetanus toxoid; 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 and 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 the 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) obtained this strain from NVI, Netherlands.

[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, HO, 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, 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 to 400 kDa.

[0101] More preferably, 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. Additionally, size reduction of polysaccharides reduces solution viscosity and increases the number of reactive end groups, both of which contribute to an increased frequency of covalent bond formation.

[0103] Still alternatively, 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 acid 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 acid 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 a membrane of either 10 kDa, 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, and obtaining 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 using carbodiimide, reductive amination or cyanylation reactions to contain amino and / or carboxyl groups 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 acid dihydrazide. Hydrazide groups can be introduced into the polysaccharides of Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium, and S. enteritidis by using carbodiimide, reductive amination, and 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 acid 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, wherein the cyanylation reagent is 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] More preferably, Salmonella enterica serovar Paratyphi A 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, and 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, where 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), can 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), can 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), can be used as a catalyst.

[0120] More preferably, the purified ViPs are covalently conjugated to a carrier protein (CP) using a carbodiimide conjugation reaction in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDAC) mixed at a weight ratio of ViPs:EDAC of 1:0.5 to 1:2, the ratio of Vi polysaccharide to carrier protein may be 0.5 to 1.5, and the reaction is carried out at a pH ranging from 5 to 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 conjugated to the ADH-derivatized tetanus toxoid (TT) in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC), wherein 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, wherein 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 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, wherein 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, S. paratyphi A OSP may be conjugated to a carrier protein using cyanylation conjugation chemistry, where the cyanylation reagent is 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) or (CPIP).

[0126] More preferably, purified S. paratyphi A OSP is covalently conjugated 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 to carrier protein may be 0.5 to 1.5, and the reaction is carried out at a pH ranging from 5 to 7 and a temperature ranging from 2°C to 30°C.

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

[0128] 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, wherein 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).

[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] 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, wherein 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).

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

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

[0134] More preferably still, the carrier protein used for conjugation with the S. enteritidis polysaccharide may be CRM197. Applicant has discovered methods for stabilizing the final polysaccharide-protein conjugate by using appropriate linkers, appropriately sized polysaccharides, and utilizing alternative methods of conjugation, polysaccharide-to-protein ratios, and polysaccharide-to-coupling agent ratios, which 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 the steps of concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using membranes having either a 100 kDa or a 300 kDa molecular weight cut-off (MWCO), which concentrates the conjugate bulk by at least three times and removes substantially all unreacted compounds, unconjugated polysaccharide, unconjugated protein 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 provide 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 involve 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 carbohydrate-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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar Paratyphi A carbohydrate-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] Even more preferably, 0.5 ml of the composition contains a Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in the dose range of 1.25 to 50 μg, 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 to 50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, wherein the CP is either TT or DT or CRM197.

[0143] Even more preferably, 0.5 ml of the composition contains a Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen in the dose range of 1.25 to 50 μg, wherein 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 trivalent combination of: a) Salmonella enterica serovar typhi glyco-carrier protein (CP) conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen; c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi carbohydrate-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 carbohydrate-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] 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.

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

[0148] Even more preferably, 0.5 ml of the composition contains a Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen in the dose range of 1.25 to 50 μg, wherein 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] Even more preferably, 0.5 ml of the composition contains a Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in the dose range of 1.25 to 50 μg, 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 to 50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, wherein the CP is either TT or DT or CRM197.

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

[0154] According to a tenth embodiment of the present disclosure, the immunogenic composition may comprise any of a variety of antibodies, including, but 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 spp., Shigella flexneri, Shigella boydii, Escherichia coli, Enterobacter spp., Yersinia spp., Pseudomonas spp., Pseudomonas aeruginosa, Haemophilus influenzae (serotypes a, c, d, e, f and non-capsulated strains), hepatitis (strains A, C, D, E, F and G), influenza, Staphylococcus species, Staphylococcus 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, 2 1, 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, 35 A, 35B, 35C, 35F, 36, 37, 38, 39, 40, 41F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48), group A streptococcus, group B streptococcus (group Ia, Ib, II, III, IV, V, Vl, VII VII, VIII and IX), Neisseria meningitidis, Haemophiluspneumonia, 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 ulcers, 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 carbohydrate-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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A 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; or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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 carbohydrate-carrier protein conjugate antigen; 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 carbohydrate-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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A 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; 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; or a) Salmonella enterica serovar typhi carbohydrate-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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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] Even more preferably, 0.5 ml of the composition contains a Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in the dose range of 1.25 to 50 μg, 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 to 50 μg of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen, wherein the CP is either TT or DT or CRM197.

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

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

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

[0162] Even more preferably, 0.5 ml of the composition contains a dose of 5 μg 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 carbohydrate-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 carbohydrate-carrier protein conjugate antigen, b) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; c) rotavirus antigen, 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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen; c) rotavirus 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), h) Hepatitis B virus surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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), h) Hepatitis B virus surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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 strain; f) diphtheria toxoid (D) antigen, g) tetanus toxoid (T) antigen, h) whole cell pertussis (wP) antigen or acellular pertussis (aP), i) Hepatitis B virus surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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), i) Hepatitis B virus surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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 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).

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

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

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

[0168] Even more 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 to 20 μg per 0.5 ml. Even more preferably, the composition contains Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 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] Preferably, the composition comprises an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strain, with IPV type 1 being administered at a dose of 1 to 50 D antigenic units (DU), IPV type 2 being administered at a dose of 1 to 50 D antigenic units (DU), or IPV type 3 being administered at a dose of 1 to 50 D antigenic units (DU) 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 carbohydrate-carrier protein conjugate antigen, b) rotavirus antigen, 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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen; c) rotavirus antigen, 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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen; c) rotavirus antigen, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen, f) Campylobacter jejuni antigen, or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen; c) rotavirus antigen, d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen, or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen; c) rotavirus antigen, 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 antigen, 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 carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-carrier protein conjugate antigen; c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; d) rotavirus antigen, e) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; f) Shigella species antigen; g) Campylobacter jejuni antigen, h) Vibrio cholerae antigen, or a) Salmonella enterica serovar typhi carbohydrate-carrier protein conjugate antigen, b) Salmonella enterica serovar Paratyphi A carbohydrate-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] 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.

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

[0176] Even more preferably, 0.5 ml of the composition contains a Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen in the dose range of 1.25 to 50 μg, wherein 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 and / or potassium phosphate in ratios selected to achieve the desired pH. In another example, the buffer contains tris(hydroxymethyl)aminomethane, or "Tris," formulated to achieve the desired pH. In yet another example, the buffer may be minimal essential medium containing Hank's salts. 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 the range 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 to 2.63 mg and trisodium citrate anhydrous (TCD) in the range of 1.47 to 3.68 mg.

[0180] Preferably, the immunogenic composition may further comprise a Tris or citrate buffer or histidine buffer or 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 pharmaceutically 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, soybean lecithin, and poloxamer.

[0184] Preferably, the immunogenic composition may include polysorbate 20 as a pharmaceutically 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 be in the range of 100 mM to 200 mM.

[0186] Preferably, the immunogenic composition may contain sodium chloride in the range of 1 to 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, and cyclodextrin.

[0189] Even more preferably, the single dose composition is preservative-free. 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 a combination thereof. The vaccine composition may contain material for a single immunization or may contain material for multiple immunizations (i.e., a "multi-dose" kit). The inclusion of a preservative is preferred in multi-dose configurations. Alternatively (or in addition) to including a preservative in the multi-dose composition, the composition may be contained in a container with a sterile adapter 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 a still 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 aluminum potassium sulfate.

[0192] According to yet another aspect of the embodiment, the immunogenic composition comprises an oil-in-water emulsion, MF-59, liposomes, lipopolysaccharides, 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, 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, fragment 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 for a period of 6 months, with free polysaccharides 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 compositions of the present disclosure may be in the form of transdermal preparations, including lotions, gels, sprays, ointments, or other suitable techniques. When nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), the compositions are in a form that can be administered by a squeeze spray dispenser, pump dispenser, or aerosol dispenser. Aerosols are typically under pressure with hydrocarbons. Pump dispensers preferably administer metered doses or doses with specific particle sizes. In the form of solutions, suspensions, and gels, in some embodiments, the immunogenic compositions contain a large amount of water (preferably purified water) in addition to the active ingredient. According to yet another aspect of the embodiment, the immunogenic compositions may be lyophilized or freeze-dried compositions.

[0196] As used herein, the terms "freeze-drying" or "lyophilizing" or "lyophilization" include lyophilization and refer 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 a change in the physical properties of a composition, where the composition changes directly from a solid state to a gaseous state without becoming a liquid.

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

[0198] According to one aspect of the embodiment, the method for reconstituting the lyophilized immunogenic composition may comprise 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 infection with Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium, and S. enteritidis, 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 ocular 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 Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis may be about 1 μg / 0.5 ml or less of polysaccharide conjugate of 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 Salmonella serovars S. typhi, S. paratyphi A, S. typhimurium or S. enteritidis. In some other embodiments, 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 0.5 ml single dose.In some embodiments, an immunologically effective amount for vaccination against Salmonella serovar strains S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis bacterial infection is between 1 μg / 0.5 ml and 50 μg / 0.5 ml, more preferably the Salmonella enterica serovar Typhi sugar-carrier protein conjugate antigen, Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, Salmonella enterica serovar Typhimurium sugar-carrier protein conjugate antigen, Salmonella enterica serovar Enteritidis sugar-carrier protein conjugate antigen is present in a dose range of about 5 μg / 0.5 ml to about 30 μg / 0.5 ml, even more preferably the Salmonella enterica serovar Typhi sugar-carrier protein conjugate antigen, Salmonella enterica serovar Paratyphi A sugar-carrier protein conjugate antigen, Salmonella enterica serovar Enteritidis sugar-carrier protein conjugate antigen Typhimurium sugar-carrier protein conjugate antigen, Salmonella enterica serovar enteritidis sugar-carrier protein conjugate antigen are present at a dose of approximately 25ug / 0.5ml.

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

[0202] Alternatively, an NTS vaccine could be administered to infants aged 2 to 4 months, prior to the peak incidence occurring at approximately 12 months of age. Furthermore, vaccine implementation could potentially include HIV-infected populations, as they are at increased risk of NTS infection. In developed countries, an NTS vaccine could target the elderly, who experience significantly higher mortality rates (up to 50 percent). It has been proposed that planned field implementation in children could be directly integrated into existing immunization expansion program schedules, possibly at 6, 10, and 14 weeks of age.

[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 in a single-dose 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, consisting of a primary course of vaccination followed, if necessary, by 1 to 3 divided doses administered at subsequent time intervals of 1 to 3 years later. The dosage regimen will also be determined, at least in part, by 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, such as an elderly person, a teenager, an adult, or a child under or over two years of age, according to a one- or two-dose regimen consisting of a first dose and / or a second dose administered three months to two years after the first dose and / or a third dose administered three months to two 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 saccharide-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) 1.25-50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen per 0.5 ml; 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 saccharide-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) 1.25-50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen per 0.5 ml; b) 1.25 to 50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197) per 0.5 ml; 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 For protection against typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi, and Neisseria meningitidis antigens, 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 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.

[0207] Yet 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 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; 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 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 to 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) 1.25-50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen per 0.5 ml; 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 in an amount of 25 to 500 μg per 0.5 ml, nonylphenoxypolyethoxyethanol, octylphenoxypolyethoxyethanol, octoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and poloxamer, and / or e) 1 to 10 mg of 2-phenoxyethanol per 0.5 ml, and / or f) Water for injection (WFI) in appropriate volume a second container containing an entirely liquid immunogenic composition comprising 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 only one injection comprising a complete vaccination schedule, including in children under 2 years of age, teenagers, adults and the elderly.

[0208] Still according to a fourth aspect of the eleventh embodiment, the single dose vaccine kit comprises: Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strain in a dose of 1 to 50 D antigen units (DU) for IPV type 1, 1 to 50 D antigen units (DU) for IPV type 2, or 1 to 50 D antigen 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 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 to 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) 1.25-50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen per 0.5 ml; b) 1.25 to 50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197) per 0.5 ml; c) 1 to 10 mg of sodium chloride, 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 in an amount of 25 to 500 μg per 0.5 ml, 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 volume a second container containing an entirely liquid immunogenic composition comprising and 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 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] Yet 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 strain in a dose of 1 to 50 D antigen units (DU) for IPV type 1, 1 to 50 D antigen units (DU) for IPV type 2, or 1 to 50 D antigen 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 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 to 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) 1.25-50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen per 0.5 ml; 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 in only one injection comprising a complete vaccination schedule, including in children under 2 years of age, teenagers, adults and the elderly.

[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 strain in a dose of 1 to 50 D antigen units (DU) for IPV type 1, 1 to 50 D antigen units (DU) for IPV type 2, or 1 to 50 D antigen 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 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 to 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) 1.25-50 μg of Salmonella enterica serovar typhi ViPs-TT conjugate antigen per 0.5 ml; b) 1.25 to 50 μg of Salmonella enterica serovar paratyphi A OSP-CP conjugate antigen (CP is either TT or DT or CRM197) per 0.5 ml; c) 1 to 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 in an amount of 25 to 500 μg per 0.5 ml, 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 volume a second container containing an entirely liquid immunogenic composition comprising and for protection against typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi and the heptavalent antigen, there is no antigenic interference of the ViPs-TT;OSP antigen with the heptavalent antigen, and the 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] 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 TY2 strain (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 "tviB" gene specific for the Vi polysaccharide; 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; ATCC 19585; 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 preparing the immunogenic compositions of the present disclosure.

[0212] Salmonella enterica serovar typhi, assigned accession number MCC 0193, deposited with the international depository authority NCMR-NCCS, Pune; 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, can easily modify and / or adapt such specific embodiments to various uses without departing from the general concept; therefore, such adaptations and modifications should be understood and are intended to be within the meaning and range of equivalents of the embodiments of the present disclosure. It is understood that the phraseology or terminology used herein is for the purpose of description and not limitation. Thus, while 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" implies the use of one or more elements or ingredients or amounts by the use of which one or more desired objects or results can be achieved in 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 matter forms part of the prior art body or is general 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 physical parameters, dimensions and amounts are assumed to be within the scope of the present invention.

[0219] While particular 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 merely as illustrative of the present disclosure, and not as limiting. Technical advantages: 1. The present disclosure provides monovalent and multivalent polysaccharide-protein conjugate vaccines comprising polysaccharides from Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium and S. enteritidis, in any combination thereof, effective to confer protection against or treatment of infection with, or to 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.

[0220] 2. Improved upstream, downstream and conjugation processes. 3. The upstream fermentation medium does not contain casein digest / tryptone or casamino acids. 4. The 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, resulted 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 i) does not contain sodium deoxycholate (DOC), which is an animal-derived product and whose even residual presence in the final product can result in a product that is unacceptable by regulatory authorities and certain religious communities; ii) does not contain Triton X; iii) does not contain ammonium sulfate; iv) does not contain any adsorbents (hydroxyapatite, calcium phosphate or apatite).

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

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

[0224] 8. The immunogenic composition is stable at 2-8°C, 25°C and 40°C for a period of 6 months, with free polysaccharides after 6 months being no more than 7.5% for the 180-220 kDa polysaccharide and no more than 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 four-fold higher IgG induction (compared to mice receiving unconjugated Vi PS or unconjugated SIIPL O-SP A), demonstrating 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. [Example]

[0227] The following examples are included to demonstrate preferred embodiments of the invention. Those of skill in the art will recognize that the compositions and techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and can therefore be considered to constitute preferred modes of practice thereof. However, those of skill in the art should, in light of the present disclosure, recognize that many changes can be made in the specific embodiments which are disclosed and still obtain like or similar results without departing from the spirit and scope of the 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 accession number MCC 0193; strain designation PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC obtained from Chromachemie Laboratory Private Limited, Bangalore. 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; ATCC 19585; 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 preparing 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 virus 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. Streak a loopful of this culture onto an SCDA plate and incubate at 36 ± 0.5 °C for 28-32 hours.

[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 of medium. Incubate the flask at 36 ± 0.5 °C and 150 ± 10 RPM until the optical density (OD) of the culture reaches a range of 2.5-3.5.

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

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

[0234] Fermentation process parameters:

[0235] [Table 1]

[0236] After the third hour, start feeding the medium and increase the OD proportionally. 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) Depth filtration clarification: 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 carried out for medium optimization and batch parameters. Experiment No.: 01 - Frantz medium was selected for the experiment to evaluate the growth of Salmonella Typhi due to its widespread use for the growth of Gram-negative microorganisms. Frantz medium was used for the development and fermentation of the original strain. Glucose was included in the feed medium as the carbon source.

[0241] [Table 3]

[0242] [Table 4]

[0243] The starter stock was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 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 organisms. 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 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 stock was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 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 organisms. 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 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 stock was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 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 organisms. 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 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 stock was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 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 organisms. 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 fermentation.

[0262] observation:

[0263] [Table 14]

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

[0265] [Table 15]

[0266] [Table 16]

[0267] [Table 17]

[0268] Procedure: Starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermentor. After inoculation, fermentation was performed in fed-batch mode. Feed was added to the fermentor to support growth of the Salmonella Typhi organism. 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 organisms, optimization of the parameters mentioned in the experimental set points table of this experiment was required.

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

[0273] [Table 19]

[0274] [Table 20]

[0275] [Table 21]

[0276] Procedure: Starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermentor. After inoculation, fermentation was performed in fed-batch mode. Feed was added to the fermentor to support growth of the Salmonella Typhi organism. 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.

[0277] observation:

[0278] [Table 22]

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

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

[0281] [Table 23]

[0282] [Table 24]

[0283] [Table 25]

[0284] Procedure: Starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermentor. After inoculation, fermentation was performed in fed-batch mode. Feed was added to the fermentor to support growth of the Salmonella Typhi organism. 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.

[0285] observation:

[0286] [Table 26]

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

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

[0289] [Table 27]

[0290] [Table 28]

[0291] [Table 29]

[0292] Procedure: Starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermentor. After inoculation, fermentation was performed in fed-batch mode. Feed was added to the fermentor to support growth of the Salmonella Typhi organism. 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 organisms, optimization of the parameters mentioned in the experimental set points table of this experiment was required.

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

[0297] [Table 31]

[0298] [Table 32]

[0299] [Table 33]

[0300] Procedure: Starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermentor. After inoculation, fermentation was performed in fed-batch mode. Feed was added to the fermentor to support growth of the Salmonella Typhi organism. 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.

[0301] observation:

[0302] [Table 34]

[0303] Conclusion: From the observations, it was concluded that for the growth of Salmonella typhi organisms, 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 in 10-20 L scale batches with the following fermentation parameters: 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: Starter seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermentor. After inoculation, fermentation was performed in fed-batch mode. Feed was added to the fermentor to support growth of the Salmonella Typhi organism. 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.

[0309] observation:

[0310] [Table 38]

[0311] Conclusion:

[0312] [Table 39]

[0313] The fed-batch mode of cultivation to obtain high yields of polysaccharides from Salmonella enterica serovars S. typhi, S. paratyphi A, S. typhimurium, and S. enteritidis by a fed-batch process involving the 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 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 osmolality of 400-600 mOsmol / kg.

[0314] Example 2: C) Methods for Purifying Polysaccharides from Salmonella Enterica 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 polysaccharides (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 with 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 30 kDa molecular weight cut-off (MWCO); 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 30 kDa molecular weight cut-off (MWCO); z) Sterile filtration under sterile conditions through at least one sterile filter having a pore size of about 0.2 micrometers 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 O-specific polysaccharide of desired quality from Salmonella Paratyphi A lipopolysaccharide (LPS): r) centrifuging at 7000 rpm at 4°C for 30 minutes to collect the cell pellet (approximately 1 kg), suspending the supernatant and cell pellet in 15 L of 1 M 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 approximately 2.8-3.0) at a temperature of 90°C for 180 minutes; u) cooling the mixture to room temperature (RT) and removing impure precipitate by centrifugation at 7000 rpm at 25°C for 45 minutes; v) neutralizing the supernatant in a glass bottle and collecting it, 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 polysaccharides (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] An improved method for Vi PS purification would have clear advantages both in terms of ease of operation and several other advantages, including: 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 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 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 ion neutralizes 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, and viscosity) with CTAB (3%) versus CTAB (0.5%, 1%). CTAB is an amine-based cationic quaternary surfactant. CTAB interacts with anionic polysaccharides (ionic interaction), which then reduces their solubility, causing them to precipitate from solution. Therefore, a higher concentration of CTAB (2%) consistently precipitates a larger amount of polysaccharide, which results in a higher yield, and the removal of protein impurities is an additional benefit.

[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, while 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, while SDS, due to its amphiphilic nature and higher aggregation number, denatures and solubilizes proteins well and irreversibly destroys endotoxins into their monomeric units.

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

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

[0326] The O-specific polysaccharide (OSP) purification method resulted in significant reductions in endotoxin (<100 EU endotoxin / µg PS), protein (<1%), and nucleic acid (<2%) impurities, and higher capsular polysaccharide recoveries, preferably in the range of 40%-65%, along with desirable O-acetyl levels (>2.0 mmol / g polysaccharide), molecular size distributions (>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) 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. 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 the 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, Netherlands.

[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, which 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) Derivatization 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), 1 M MES pH 6.0, ADH solution (dissolved in 0.1 M MES pH 6.0), and EDC solution (dissolved in 0.1 M MES pH 6.0) were added sequentially, and brought to the final required volume by adding 0.1 M MES buffer pH 6.0. The final Pr concentration in the reaction was approximately 4.5 mg / ml.

[0333] d) The reaction mixture was stirred and continued at pH 5.90 for approximately 1 hour, and 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. Conjugation reaction ratio 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 approximately 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. Once <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 The quenched conjugate was purified 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 size: 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 g of the quenched conjugate was concentrated to approximately 2-4 mg / ml using a 0.1 m 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. Improvement of conjugate yield: Purification was performed using different methods, namely 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 a 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 1:0.7:1.8, 1:0.8:1.8, 1:0.9:2 and 1:1:2 Ps:Pr:EDC used at lower pH with different purification techniques resulted in better 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 (addition of the linker ADH) and conjugation with concentrated OSP using cyanylation chemistry. B) Derivatization of CRM197, tetanus toxoid (TT) (addition of the linker ADH) using carbodiimide chemistry and conjugation with concentrated OSP using cyanylation chemistry. 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, freshly prepared 1 M MES buffer, adipic acid dihydrazide (ADH) (75-100 mg / ml dissolved in 100 mM MES buffer, pH 5.8) at a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (30-40 mg / ml dissolved in 100 mM MES buffer, pH 5.8) at a weight ratio of 1:1 were added. The reaction was continued at pH 5.8 for approximately 1 hour, 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 a maximum of 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP = 1:0.8:1.3).

[0361] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight 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 / h, and 1% column volume of 1M NaCl was passed through to assess the integrity of the packed column. The column was equilibrated with 0.9% NaCl at 30 cm / h, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals. Fractions 2–9 were pooled according to the 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 = 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 a maximum of 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP = 1:0.8:1.1).

[0365] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS.

[0366] See Figure 3: Comparison of polysaccharide, protein and conjugate (PS:PR:CPIP 1:0.8:1.1). 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 with 0.9% NaCL at 30 cm / hr, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals. Fractions 2–8 were pooled according to their HPLC profile. The final pooled fractions were 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] 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-dimethylaminopropyl)carbodiimide) (dissolved in 100 mM MES buffer, pH 6.5, at 30-40 mg / ml) at a weight ratio of 1:0.25 were added to the concentrated CRM197. 5% Tween 80 was added. The final reaction volume was adjusted to reach a final concentration of 3-4 mg / ml using 100 mM MES buffer. The reaction was continued at pH 6.5 for approximately 3 hours. 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 a maximum of 3 minutes. Protein was then added in a weight ratio of 1:1 (PS:PR:CPIP = 1:1:1.3).

[0376] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS.

[0377] See Figures 5 and 6: Comparison of polysaccharide, protein and conjugate (PS:PR:CPIP = 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 with 0.9% NaCl at 30 cm / hr, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals, and fractions 2–7 were pooled according to the HPLC profile. The final pooled fractions were 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 division (SIIPL) was concentrated to (15-20 mg / ml) on a 30 kDa membrane and analyzed for protein content.

[0380] [Table 53]

[0381] Freshly prepared 1 M MES buffer, adipic acid dihydrazide (ADH) (75-100 mg / ml dissolved in 100 mM MES buffer, pH 6.0) at a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (30-40 mg / ml dissolved in 100 mM MES buffer, pH 6.0) at a weight ratio of 1:1 were added to the concentrated TT. The reaction was continued at pH 6.0 for approximately 1 hour, 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 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] 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 a maximum of 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP = 1:0.8:1.25).

[0387] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS.

[0388] See Figure 8: Comparison of polysaccharide, protein and conjugate (PS:PR:CPIP 1:0.8:1.25). 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration using 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 = 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 a maximum of 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 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS.

[0392] See Figure 10: Comparison of polysaccharide, protein and conjugate (PS:PR:CPIP 1:0.9:1.3). 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration using 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.

[0393] [Table 57]

[0394] See Figure 11: Chromatogram of GFC purified OSP-DT ADH conjugate (PS:PR:CPIP 1:0.9:1.3). Conclusion: Conjugation of Paratyphi A PS with ADH-derivatized TT was successful, applying two different types of diafiltration strategies, and both processes resulted in 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 linker ADH) using cyanylation chemistry and conjugation with tetanus toxoid (TT) using carbodiimide chemistry.

[0395] B) Derivatization of concentrated OSP (addition of linker ADH) using cyanylation chemistry and conjugation with diphtheria toxoid (DT) using carbodiimide chemistry. C) Derivatization of concentrated OSP using cyanylation chemistry (addition of linker ADH) and conjugation with CRM197 using carbodiimide chemistry. 2A) Preparation of S. Paratyphi conjugates using tetanus toxoid as a 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 at 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 minutes. Adipic acid dihydrazide (ADH) (dissolved at 75-100 mg / ml in 0.5 M sodium bicarbonate buffer, pH 8.0) was then added at a weight ratio of 1:10 (PS:ADH:CPIP 1:10:0.7). The reaction was continued at pH 9.5 for approximately 2 hours and quenched using 2 M glycine. 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 department (SIIPL) was concentrated to (10-15 mg / ml) with a 30 kDa membrane and analyzed for protein content.

[0400] Brief description or reference of the assay: Protein content was measured by 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 in a weight ratio of 1:0.9, and freshly prepared EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (dissolved at 3040 mg / ml in 100 mM MES buffer) in a weight ratio of 1:0.86. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio of 1:0.9:0.86).

[0403] Protein conversion was monitored using Shodex columns SB-804 HQ and SB-805 HQ sequentially 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 using 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.

[0405] [Table 61]

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

[0407] Protein conversion was monitored using Shodex columns SB-804 HQ and SB-805 HQ sequentially 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 with 0.9% NaCl at 30 cm / hr, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals, and fractions 2–10 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis.

[0409] [Table 62]

[0410] Conclusion: Using reverse conjugation chemistry (to activate PS) and increasing the temperature from 6°C to 10°C, the conjugation of ADH-derivatized Paratyphi A PS and concentrated TT was successful. 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: The 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 at 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 minutes. Adipic acid dihydrazide (ADH) (dissolved at 75-100 mg / ml in 0.5 M sodium bicarbonate buffer, pH 8.0) was then added at a weight ratio of 1:10 (PS:ADH:CPIP = 1:10:0.7). The reaction was continued at pH 9.5 for approximately 2 hours and quenched using 2 M glycine. The reaction mixture was then diafiltered through 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.

[0413] [Table 64]

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

[0415] Brief description or reference of the assay: Protein content was measured by 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 in a weight ratio of 1:0.8, and freshly prepared EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) was added in a weight ratio of 1:0.86. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio 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 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 with 0.9% NaCl at 30 cm / hr, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals, and fractions 2–12 were pooled according to the HPLC profile. The final pooled fractions were 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 in a weight ratio of 1:1.0, and freshly prepared EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) in a weight ratio of 1:0.9. The reaction was continued at pH 6.0 and 10°C (PS:PR:EDC ratio 1:1.0:0.9).

[0423] Protein conversion was monitored using Shodex columns SB-804 HQ and SB-805 HQ sequentially 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 using 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.

[0425] See Figure 17: chromatogram showing 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 for conjugating polysaccharides from Salmonella serovars S. typhimurium and S. enteritidis to a carrier protein selected from tetanus toxoid (TT), diphtheria toxoid (DT) or CRM197. 1) Conjugation of S. typhimurium and S. enteritidis polysaccharides to the carrier proteins diphtheria toxoid (DT), CRM197, and tetanus toxoid (TT) based on cyanylation chemistry A) Derivatization of diphtheria toxoid (DT) (attachment of the linker ADH) using carbodiimide chemistry and conjugation with 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 with concentrated S. typhimurium and S. enteritidis using cyanylation chemistry.

[0430] D) Conjugation of Paratyphoid OSP to the carrier proteins diphtheria toxoid (DT), CRM197, and tetanus toxoid (TT) based on cyanylation chemistry without derivatization of the 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, freshly prepared 1 M MES buffer, adipic acid dihydrazide (ADH) (75-100 mg / ml dissolved in 100 mM MES buffer, pH 5.8) at a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (30-40 mg / ml dissolved in 100 mM MES buffer, pH 5.8) at a weight ratio of 1:1 were added. The reaction was continued at pH 5.8 for approximately 1 hour, 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 polysaccharides (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 a maximum of 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP = 1:0.8:1.3).

[0434] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS. 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 with 0.9% NaCl at 30 cm / hr, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals. The final pooled fractions were 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 a maximum of 3 min. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP = 1:0.8:1.1).

[0436] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS.

[0437] 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 with 0.9% NaCL at 30 cm / hr, and the conjugate was loaded onto the column, with fractions collected at 1-minute intervals. The final pooled fractions were 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] Freshly prepared 1 M 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-dimethylaminopropyl)carbodiimide) (dissolved in 100 mM MES buffer pH 6.5 at 30-40 mg / ml) at a weight ratio of 1:0.25 were added to the concentrated CRM197. 5% Tween 80 was added. The final reaction volume was adjusted to reach a final concentration of 3-4 mg / ml using 100 mM MES buffer. The reaction was continued at pH 6.5 for approximately 3 hours. 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 polysaccharides (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 a maximum of 3 minutes. Protein was then added in a weight ratio of 1:1 (PS:PR:CPIP = 1:1:1.3).

[0441] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight 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 with 0.9% NaCl at 30 cm / hr, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals, and the final pooled fractions were 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-monomer tetanus toxoid (TT) received from the manufacturing department (SIIPL) was concentrated to 15-20 mg / ml using a 30 kDa membrane and analyzed for protein content. Freshly prepared 1 M MES buffer, adipic acid dihydrazide (ADH) (dissolved in 100 mM MES buffer, pH 6.0, at 75-100 mg / ml) at a weight ratio of 1:10, and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (dissolved in 100 mM MES buffer, pH 6.0, at 30-40 mg / ml) at a weight ratio of 1:1 were added to the concentrated TT. The reaction was continued at pH 6.0 for approximately 1 hour, and then the reaction mixture was diafiltered using 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. 2) Conjugation of S. typhimurium and S. enteritidis polysaccharides (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 a maximum of 3 minutes. Protein was then added in a weight ratio of 1:0.8 (PS:PR:CPIP = 1:0.8:1.25).

[0444] 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS. 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration using 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.

[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 a maximum of 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 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. After 3–4 h, the reaction was quenched by adding 2 M glycine in an amount 10 times the weight of PS. 3) Conjugate purification by ultrafiltration: The quenched conjugate was purified by diafiltration using 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.

[0447] Conclusion: Conjugation of S. typhimurium and S. enteritidis polysaccharides (PS) using ADH-derivatized TT was successful, applying two different types of diafiltration strategies, and both processes resulted in 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 (addition 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 using a 10 kDa membrane to 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 at a weight ratio of 1:0.5–1:2 (1:0.7). The pH was immediately shifted to 9.5 with 2.5 M NaOH and held for a maximum of 3 min. Next, adipic acid dihydrazide (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 = 1:2:0.7–1:10:0.7). The reaction was continued for approximately 2 hours at pH 9.5 and quenched using 2 M glycine 10. 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 department (SIIPL) was concentrated (10-15 mg / ml) on a 30 kDa membrane and analyzed for protein content.

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

[0453] 2C) Protein Preparation: High monomer CRM197 received from the manufacturing department (SIIPL) was concentrated (10-20 mg / ml) on a 30 kDa 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 in a weight ratio of 1:0.9, and freshly prepared EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) in a weight ratio of 1:0.86. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio of 1:0.9:0.86).

[0454] Protein conversion was monitored using Shodex columns SB-804 HQ and SB-805 HQ sequentially 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 in a weight ratio of 1:0.8, and freshly prepared EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) was added in a weight ratio of 1:0.86. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio of 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 in a weight ratio of 1:0.8, and freshly prepared EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (dissolved at 30-40 mg / ml in 100 mM MES buffer) was added in a weight ratio of 1:0.86. The reaction was continued at pH 6.0 and 6°C (PS:PR:EDC ratio of 1:0.8:0.86).

[0460] 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. 4) Conjugate purification 4A) 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.

[0461] 4B) 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 with 0.9% NaCl at 30 cm / hr, and the conjugate was loaded onto the column. Fractions were collected at 1-minute intervals, and fractions 2–12 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis.

[0462] Conclusion: Using reverse conjugation chemistry (to activate PS) and increasing the temperature from 6 to 10°C, ADH-derivatized S. typhimurium and S. enteritidis polysaccharides and concentrated TT, DT, and CRM197 were successfully conjugated. 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] 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:

[0474] i. 2-phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v / v); ii. 2-phenoxyethanol in an amount of 1 to 10 mg (v / v) per 0.5 ml and methylparaben in an amount of 0.1 to 1.5 mg (w / v) per 0.5 ml, or iii. 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 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) of pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) of filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) of 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 to 10 mg per 0.5 ml (v / v); ii. 2-phenoxyethanol in an amount of 1 to 10 mg (v / v) per 0.5 ml and methylparaben in an amount of 0.1 to 1.5 mg (w / v) per 0.5 ml, or iii. 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 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) of pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) of filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) of pertactin and optionally 2 to 25 micrograms of fimbriae per 0.5 ml.

[0481] [Table 77]

[0482] [Table 78]

[0483] 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:

[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 to 10 mg (v / v) per 0.5 ml 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 to 10 mg (v / v) per 0.5 ml and propylparaben in an amount of 0.05 to 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 doses of 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) of pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) of filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) of 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 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 preservative combinations: 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 to 10 mg (v / v) per 0.5 ml 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 to 10 mg (v / v) per 0.5 ml and propylparaben in an amount of 0.05 to 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) of pertussis toxoid, 1 to 50 micrograms (more particularly 8 μg) of filamentous hemagglutinin, 1 to 20 micrograms (more particularly 2.5 μg) of 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 conjugate 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 (at 40°C / 2-8°C / 25°C, for 180-220 kDa Ps, "initial <4.5% and after 6 months ≤7.5%" vs. "initial 5.4% and after 6 months ≥10.5% for 388 / 80 / 45 kDa Ps").

[0497] For 180–220 kDa, the initial low free Ps, size of the conjugate, 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 was found to be stable in 10 mM Tris buffer, in 10 mM Tris containing 0.17 M NaCl, 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 the best. Vi-TT CJ concentrations were maintained in three different strengths of Tris, pH 7.2: 5 mM, 10 mM, and 20 mM, at various temperatures, 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 conjugates were maintained in 5, 10 and 20 mM Tris pH 7.2, with pH ranging from 2 to 8°C at 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 conjugates in Tris buffer and sodium chloride

[0506] [Table 89]

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

[0508] Viscosity was found to be similar for conjugates in different Tris buffers and NaCl concentrations, and is 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 conducted in mice, where unconjugated 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 determination of antibodies against the injected polysaccharide using a suitable serological and immunological method, such as an in-house IgG ELISA. Serum from mice receiving unconjugated 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 (with various PS sizes). Mouse Immunogenicity Study #2: Another similar study was conducted in mice. Both studies used similar animal treatment protocols. The animal protocol is briefly described below. Sera from both studies were tested by the same immunological / serological 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-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 weighed approximately 18 to 20 g at the start of the study. Each mouse received 2.5 μg of conjugated Vi TT vaccine via the intramuscular route. The SIIPL Vi TT vaccine was diluted in phosphate-buffered saline (PBS) as a vehicle. No adjuvants were used in the study for any treatment group.

[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 experimental animals using sterile glass capillary tubes. Isoflurane was used as a safe anesthetic during the blood collection procedure. Serum was analyzed for IgG antibodies produced in response to injection of unconjugated or conjugated Vi TT vaccine using an in-house IgG ELISA. The ELISA used the NIBSC Vi PS Reference Standard (catalog no. 16 / 126; First International Standard for Vi PS in S. Typhi) as the coating antigen. 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 with 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 receiving Vi TT exhibiting a >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 demonstrated similar responses to Vi TT versus Vi PS.

[0516] The following parameters were compared across the different 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 eight female mice per group. In all groups, antibody induction in response to Vi TT (various PS sizes) injected versus unconjugated Vi PS was evaluated. All Vi TT PS forms showed stronger induction of Vi TT-specific IgG antibodies compared with the Vi PS alone group (day 21; after the first booster). The induction of IgG in mouse serum is a strong determinant of the serological response to Vi TT. Therefore, monovalent Vi TT can induce a strong immunogenic response in a mouse model, offering promising possibilities for future studies in higher animals.

[0518] [Table 93]

[0519] observation: 1. Immunogenicity data will be evaluated regarding 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 immunogenic responses that were observed via serological assays.

[0522] 2. All monovalent Vi-TT conjugates demonstrated higher IgG levels than those 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 conducted 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 studies. 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 to determine antibodies against the injected polysaccharides using a suitable serological and immunological method, such as an in-house IgG ELISA. Sera from mice receiving each unconjugated SIIPL polysaccharide (Vi 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 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 weighing approximately 18 to 20 g were used in the study. Each mouse received 2.5 μg of the 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 treatment group.

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

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

[0528] [Table 95]

[0529] [Table 96]

[0530] observation: 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 >4-fold higher IgG induction (compared to mice receiving unconjugated Vi PS or unconjugated SIIPL O-SP A), demonstrating the immunogenic potential of the bivalent SIIPL vaccine containing the combination of Vi TT and SIIPL O-SP A (DT / TT / CRM).

[0531] IgG induction with 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, Typbar TCV.

[0532] The following parameters were compared across the different 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 conducted in mice, with eight female mice per group. In all groups, antibody induction in response to the bivalent Vi (typhoid and paratyphoid) SIIPL vaccine injected against unconjugated Vi PS and O-SP A PS was evaluated. All bivalent (typhoid and paratyphoid) SIIPL vaccines showed a stronger induction of PS-specific IgG antibodies (day 21 after the first booster) compared with the PS-only (Vi and O-SP A) group. The induction of IgG in mouse serum is a strong determinant of the serologic response to the bivalent (typhoid and paratyphoid) SIIPL vaccine. Therefore, the bivalent (typhoid and paratyphoid) SIIPL vaccine can induce a strong immunogenic response in a mouse model, offering promising possibilities for future studies in higher animals.

[0534] observation: 1. Immunogenicity data will be evaluated in terms of 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 those 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 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 saccharide-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 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 comprising the 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 saccharide-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 serovar typhi ViPs-TT conjugate antigen; b) 1.25 to 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 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 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. C) A single-dose vaccine kit comprising: Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strain in a dose of 1 to 50 D antigen units (DU) for IPV type 1, 1 to 50 D antigen units (DU) for IPV type 2, or 1 to 50 D antigen 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 (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 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 volume a second container containing an entirely 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 only one injection containing a complete vaccination schedule, including children under 2 years of age, teenagers, adults, and the elderly. D) A single dose vaccine kit comprising: Complete liquid hexavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from the Sabin or Salk strain in a dose of 1 to 50 D antigen units (DU) for IPV type 1, 1 to 50 D antigen units (DU) for IPV type 2, or 1 to 50 D antigen 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 (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 serovar typhi ViPs-TT conjugate antigen; b) 1.25 to 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, 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 volume a second container containing an entirely liquid immunogenic composition comprising:

[0543] interpretation: For protection against typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi, and hexavalent antigens, 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 antigen units (DU) for IPV type 1, 1 to 50 D antigen units (DU) for IPV type 2, or 1 to 50 D antigen 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 to 50 IOU per 0.5 ml, or acellular pertussis (aP) antigen containing one or more modified adenylate cyclases, 1 to 50 μg pertussis toxin (PT), 1 to 50 μg filamentous hemagglutinin (FHA), 1 to 20 μg pertactin (P69 or PRN), or 2 to 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 (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 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 an entirely 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 comprising the 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 antigen units (DU) for IPV type 1, 1 to 50 D antigen units (DU) for IPV type 2, or 1 to 50 D antigen 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 to 50 IOU per 0.5 ml, or acellular pertussis (aP) antigen containing one or more modified adenylate cyclases, 1 to 50 μg pertussis toxin (PT), 1 to 50 μg filamentous hemagglutinin (FHA), 1 to 20 μg pertactin (P69 or PRN), or 2 to 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 (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 serovar typhi ViPs-TT conjugate antigen; b) 1.25 to 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, 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 volume a second container containing an entirely liquid immunogenic composition comprising:

[0545] interpretation: For protection against typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi, and the heptavalent antigen, there is no antigenic interference of the ViPs-TT;OSP antigen with the heptavalent 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.

Claims

1. 1. An immunogenic composition comprising at least two antigens, a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen, and b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; An immunogenic composition comprising:

2. 2. The immunogenic composition of claim 1, a) 1.25 μg to 50 μg of Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen, and b) 1.25 μg to 50 μg of Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; An immunogenic composition comprising at least a bivalent combination of:

3. The immunogenic composition of claim 2, wherein diphtheria toxoid (DT) is derivatized with an adipic acid dihydrazide (ADH) linker prior to conjugation with Salmonella enterica serovar Paratyphi A OSP polysaccharide.

4. a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen, and c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen; The immunogenic composition of claim 1, comprising at least one trivalent combination of:

5. a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; c) Salmonella enterica serovar typhimurium carbohydrate-carrier protein conjugate antigen, and d) Salmonella enterica serovar enteritidis carbohydrate-carrier protein conjugate antigen The immunogenic composition of claim 1, comprising a tetravalent combination of:

6. a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y sugar-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 Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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 Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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 sugar-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 typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; e) Neisseria meningitidis Y sugar-carrier protein conjugate antigen; f) Neisseria meningitidis W-135 saccharide-carrier protein conjugate antigen; or a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; c) Neisseria meningitidis A carbohydrate-carrier protein conjugate antigen; d) Neisseria meningitidis C carbohydrate-carrier protein conjugate antigen; The immunogenic composition of claim 1, comprising at least one combination of:

7. a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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 Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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); h) hepatitis B virus surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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); h) hepatitis B virus surface antigen (HBsAg), and i) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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); i) hepatitis B virus surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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); i) hepatitis B virus surface antigen (HBsAg), and j) Haemophilus influenzae type b antigen (Ηib), or a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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 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:

8. a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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 Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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 Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; c) rotavirus antigens; d) diarrheagenic Escherichia coli species (enterotoxigenic and enterohemorrhagic) antigens; e) Shigella species antigen; or a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; c) rotavirus antigens; d) Shigella species antigen; or a) Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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 Vi polysaccharide-tetanus toxoid (TT) conjugate antigen; b) Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) 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:

9. Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate 9. The immunogenic composition of any one of claims 5 to 8, wherein the antigens; Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen; Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen are in a dose range of 5 μg / dose to 30 μg / dose.

10. The immunogenic composition of any one of claims 1 to 8, comprising a pharmaceutically acceptable buffer selected from acetate, carbonate, citrate, butyrate, gluconate, tartrate, phosphate buffered saline, borate, histidine buffer, succinate buffer, HEPES, Tris, or citrate-phosphate.

11. a pharmaceutically acceptable excipient selected from a sugar, a surfactant, a polymer, a salt, an amino acid, or a pH adjuster; the amino acid or protein is selected from L-histidine, lysine, isoleucine, methionine, glycine, aspartic acid, tricine, arginine, leucine, glutamine, alanine, peptides, hydrolyzed proteins or proteins including serum albumin; the sugar is selected from sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol or a combination thereof; the surfactant is a nonionic 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, soybean lecithin and poloxamer; The polymer is selected from the group of dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, and Salts include NaCl, KCl, and KH ２ P.O. ４ , Na ２ HPO ４ . 2H ２ O, CaCl ２ and MgCl ２ Selected from: The immunogenic composition according to any one of claims 1 to 8.

12. A single dose composition that does not contain a preservative, or 9. The immunogenic composition of any one of claims 1 to 8, which is a multi-dose composition comprising one or more preservatives selected from the group consisting of 2-phenoxyethanol, benzethonium chloride (femerol), phenol, thiomersal, formaldehyde, paraben esters including methyl-, ethyl-, propyl-, or butyl-paraben, benzyl alcohol, m-cresol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and p-chloro-m-cresol.

13. 9. The immunogenic composition of claim 1, further comprising an adjuvant selected from the group consisting of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, aluminum potassium sulfate, and mixtures thereof.

14. 9. The immunogenic composition of any one of claims 1 to 8, further comprising an immunostimulatory component selected from the group consisting of an oil-in-water emulsion, MF-59, liposomes, lipopolysaccharides, 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, a TLR-4 agonist, flagellin, flagellin from Gram-negative bacteria, dmLT, a TLR-5 agonist, a fragment of flagellin capable of binding to the TLR-5 receptor, QS-21, ISCOMS, chitosan, a sterol, and a saponin combination with a lipid.

15. 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 saccharide-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) 0.05-0.5 mg Tris buffer per 0.5 ml a first container containing and a) 1.25-50 μg of Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen per 0.5 ml; b) 1.25-50 μg of Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen per 0.5 ml; 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 reconstitution of a lyophilized (freeze-dried) immunogenic composition comprising 1. A single-dose vaccine kit comprising: a ViPs-TT;OSP antigen for the prevention of typhoid and paratyphoid fever 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 the 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.

16. 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 1-30 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 pilus 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 (Hib) in an amount of 1-20 μg per 0.5 ml a first container containing and a) 1.25-50 μg of Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen per 0.5 ml; b) 1.25-50 μg of Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen per 0.5 ml; c) 1-10 mg 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 to 10 mg of 2-phenoxyethanol per 0.5 ml, and / or g) Water for injection (WFI) in appropriate volume a second container containing an entirely liquid immunogenic composition comprising 1. A single-dose vaccine kit comprising: a ViPs-TT;OSP antigen for the prevention of typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi, and a hexavalent antigen; wherein 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 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.

17. 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 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 pilus 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 (Hib) in an amount of 1-20 μg per 0.5 ml a first container containing and a) 1.25-50 μg of Salmonella enterica serovar typhi Vi polysaccharide-tetanus toxoid (TT) conjugate antigen per 0.5 ml; b) 1.25-50 μg of Salmonella enterica serovar Paratyphi A OSP polysaccharide-diphtheria toxoid (DT) conjugate antigen per 0.5 ml; c) 1-10 mg 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 in an amount of 25 to 500 μg per 0.5 ml, 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 volume a second container containing an entirely liquid immunogenic composition comprising 1. A single-dose vaccine kit comprising: a single-dose vaccine kit for the prevention of typhoid and paratyphoid fever caused by Salmonella typhi, S. paratyphi, and the heptavalent antigen, wherein there is no antigenic interference of the ViPs-TT;OSP antigen with the heptavalent antigen, and the 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.

18. 15. The immunogenic composition of any one of claims 1 to 14, wherein the immunogenic composition is 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 non-typhoid related Salmonella spp., wherein the method comprises the step of administering an effective amount of the immunogenic composition to a human subject under 2 years of age, a teenager, an adult or an elderly human subject via parenteral, or subcutaneous, or intradermal, or intramuscular, or intraperitoneal, or intravenous, or injectable, or sustained release from an implant, or ocular, or nasal, or buccal, or intravaginal, oral, intragastric, or mucosal, or sublingual, alveolar, or gingival, or olfactory, or respiratory mucosal administration, or any other immunization route.

19. Stable at 2-8°C, 25°C and 40°C for a period of 6 months; Stable at 2-8°C, 25°C and 40°C, with free polysaccharides of 7.5% or less for the 180-220 kDa polysaccharide and 10.5% or less for the 388 kDa, 80 kDa and 45 kDa polysaccharides after 6 months. The immunogenic composition according to any one of claims 1 to 14.

20. 15. The immunogenic composition of any one of claims 1 to 14, wherein the immunogenic composition is for administration to a human subject aged 2 years or younger, a teenager, an adult or an elderly person according to a dose regimen consisting of a single dose of said immunogenic composition.

21. 15. The immunogenic composition of any one of claims 1 to 14, 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.

22. 15. The immunogenic composition of any one of claims 1 to 14, 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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