BR122024027321A2 - VACCINE IMMUNOGENIC COMPOSITIONS - Google Patents

Abstract

The present invention relates to monovalent and multivalent immunogenic polysaccharide-protein conjugate vaccine compositions comprising a polysaccharide selected from Salmonella serovar strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis and improved alternative methods of polysaccharide fermentation, polysaccharide purification, polysaccharide-protein conjugation and stable formulation. The present invention further relates to methods for inducing an immune response in individuals against Salmonella Typhi and non-Typhi related diseases and/or for reducing or preventing Salmonella Typhi and non-Typhi related diseases in individuals using the compositions described herein. The vaccine induces bactericidal antibodies and is useful for the prevention of gastroenteritis, enteric fever and typhoid fever.

Description

FIELD

[001] The present invention relates to the field of manufacturing vaccines, more particularly, it relates to an immunogenic composition for prophylaxis against infections caused by Salmonella and non-typhoidal Salmonella infections and processes for its preparation.

BACKGROUND

[002] The background information below relates to the present invention, but is not necessarily prior art.

[003] Salmonella infection remains a serious health problem worldwide, particularly in developing countries, affecting millions of people each year. Salmonella infection can cause enteritis that may be complicated by bacteremia (enteric fever) and gastroenteritis in both normal and immunocompromised individuals.

[004] The genus Salmonella belongs to the family Enterobacteriaceae and comprises Gram-negative, non-spore-forming, facultatively 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 only in humans that is distinct from the self-limiting acute gastroenteritis more commonly caused by several other Salmonella serotypes. Enteric fever caused by members of the genus Salmonella, including typhi and Paratyphi, continues to constitute a significant burden of disease and mortality among populations in developing countries (Lancet 2005; 366:749-762) and poses a notable risk to travelers (Lancet InfectDis. 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 each year in LMICs, particularly in South Asia and sub-Saharan Africa (Lancet GlobHeal. 2014;2:e570-e580 & J Glob Health. 2012;2:10401). Salmonella Paratyphi is responsible for the increasing burden of enteric infections in parts of Asia, including Nepal, Cambodia, and China. Salmonella Paratyphi has the highest burdens in the Indian subcontinent and Southeast Asia. In one study in Nepal, where typhoid fever is highly endemic, municipal water was contaminated with S. typhi and Salmonella enterica serovar ParatyphiA (PLoSNegl- TropDis. 2016 Jan;10(1):e0004346 &PLoSNeglTropDis. 2013;7(8):e2391).

[005] Enteric nontyphoidal Salmonella (NTS) serovars are important causes of invasive Salmonella disease worldwide. Of the more than 2,500 NTS serovars, NTS serovars typhimurium and enteritidis account for nearly 80% of all human NTS isolates reported globally. Furthermore, invasive nontyphoidal Salmonella (iNTS) infections caused by serovars enteritidis (SE) and typhimurium (STm) are major pediatric health problems in sub-Saharan Africa. NTS has increasingly recently been recognized as the leading cause of invasive bacterial infections in children and immunocompromised individuals, as well as the elderly, worldwide. These two serovars are also the leading cause of gastroenteritis in healthy children and adults in industrialized countries. NTS can also cause severe extraintestinal invasive bacteremia, termed iNTS. It usually presents as a febrile illness. In fact, NTS often occurs without gastrointestinal symptoms in adults and children.

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

[007] The global incidence of NTS gastroenteritis in 2010 was estimated at 93 million cases, of which approximately 80.3 million were foodborne, with 155,000 deaths. The economic burden of NTS is significant in the developed world. In the United States alone, NTS costs US$3.3 billion annually, with a loss of 17,000 quality-adjusted life years, the highest of any foodborne pathogen. As mentioned previously, NTS can also cause severe invasive extraintestinal bacteremia, termed iNTS. Invasive Salmonella infections are more common throughout the developing world and have become the most common cause of bacteremia in tropical Africa, especially among young children and individuals with HIV. It usually presents as a febrile illness. Indeed, iNTS frequently occurs without gastrointestinal symptoms in both adults and children. Symptoms of iNTS are similar to those of malaria and include fever and sweats (more than 90 percent), as well as splenomegaly (40 percent). It is unclear why iNTS is such a problem in Africa, but this may be related to: increased invasiveness of distinct clades of iNTS bacteria (such as S. typhimurium ST313) that are found in Africa and not elsewhere; decreased host immunity related to HIV infection, malaria, and malnutrition; and increased opportunities for human-to-human transmission, for example through contaminated water supplies; and NTS bacteremia in HIV-infected African adults has a high associated mortality (up to 47 percent) and recurrence rate (43 percent).

[008] Antibiotics have been used to treat typhoidal and non-typhoidal Salmonella-related infections, and the choice of antimicrobials and duration of treatment are determined by the cost and availability of antibiotics, local resistance patterns, and patient response to treatment. It is becoming increasingly recognized, in both the developed and developing world, that multiple antibiotic-resistant strains are emerging as important causes of invasive bacteremia and complications of gastroenteritis, resulting in hospitalizations and deaths.

[009] As available treatment tools become less effective, the problem of Salmonella-related and non-typhoidal infections is likely to continue to increase, making vaccine development an important priority for disease control efforts. It is also an important protective tool for people traveling to areas where Salmonella-related and non-typhoidal infections are endemic.

[0010] Currently, three types of typhoid vaccines are licensed for use i) typhoid conjugate vaccine (TCV) ii) unconjugated Vi polysaccharide vaccine (ViPs) and iii) live attenuated Ty21a vaccine. The World Health Organization (WHO) has recommended increased use of typhoid vaccines, giving preference to typhoid conjugate vaccines (TCV) (WHO position paper. WklyEpidemiolRec 2018; 93:153 - 72).

[0011] Vaccines for S. Paratyphi are not currently available. Similarly, there is a need for an immunogenic composition/vaccine that is capable of simultaneously conferring immunity against both typhoidal and non-typhoidal Salmonella.

[0012] A disadvantage of currently available vaccines is that they are all directed only against S. typhi. S. Paratyphi A causes enteric fever with the same geographic distribution as S. typhi, and the diseases are clinically indistinguishable. Thus, for South and Southeast Asia, a vaccine that can protect against both serovars would be more valuable than a vaccine restricted to one. Furthermore, in sub-Saharan Africa, both S. typhimurium and S. enteritidis are protected, indicating the importance of a vaccine that can additionally protect against NTS serovars for this region.

[0013] It is unclear whether the vaccine candidates in the pipeline will protect against both gastroenteritis and invasive manifestations of NTS. Although understanding of the disease burden of typhoid fever in LMICs is growing, the global medical demand for a conjugate vaccine that protects against both typhoidal and non-typhoidal Salmonella infections has reached significance. Peak demand for a typhoid conjugate vaccine is estimated to occur between 2023 and 2026, approaching 300 million doses annually for 133 countries (ClinInfectDis. 2019 Mar 15;68(Suppl 2):S154–S160).

[0014] Furthermore, upstream conjugation and formulation development can often be the rate-limiting step in the early introduction of biopharmaceuticals to the market and meeting population demands. Upstream includes the entire process from initial cell isolation and cultivation, to cell banking, culture expansion through bacterial fermentation process, and final harvest. The cell culture is scaled up from 100 to 500 milliliters to a 3 to 20,000 liter bioreactor. Other steps include primary recovery of Salmonella polysaccharide and elimination of cells and debris. Furthermore, in order to facilitate the development of low-cost Salmonella polysaccharide-based conjugate vaccines, it is necessary to obtain structurally intact polysaccharides with higher yields as well as high purity. Less than 40% yield has been previously reported for polysaccharides from Salmonella spp. Increasing the “fermentation harvest stage yield” of capsular polysaccharide (CPS) by employing novel feeding strategies and improved fermentation media has been one of the preferable approaches to achieve the said goal. Merritte et al. (2000) showed that fed-batch culture in 500-liter manufacturing-scale bioreactor increased the cell density and capsular polysaccharide yield approximately fourfold when compared to batch culture.

[0015] Studies of capsular polysaccharide production by other pathogenic bacteria, such as Haemophilus influenzae Type B and Neisseria meningitidis, showed that production depended on fermentation conditions (temperature, pH, DO, osmolarity) and medium components, and that these optimal conditions differed for each bacterium. Zhan et al. (2002) also showed that pH control and switching to fed-batch fermentation increased cell yield and capsular polysaccharide production.

[0016] Capsular polysaccharide expression is highly regulated in relation to certain fermentation conditions, such as osmolarity, where reduced polysaccharide synthesis has been reported when osmolarity was high.

[0017] Polysaccharides were produced under different concentrations of glucose, casamino acids and phosphate ions.

[0018] Baruque-Ramos et al., 2005 showed that higher yields of capsular polysaccharide were obtained when N. meningitidis (serogroup C) was grown in medium where the glucose concentration was kept below 1.0 g/l and that low oxygen tension favored higher polysaccharide production. Furthermore, it was observed that the concentrated casamino acids in the feeding solution limited the final cell density. Furthermore, growth and polysaccharide yield in a defined medium also depend on the ratio of carbohydrate to nitrogen source. In one of the fed-batch fermentation processes for S. typhi, ammonia was supplied as a nitrogen source along with the feeding medium. However, it was observed that polysaccharide formation by Au- reobasidiumpullulans was affected by the ammonium nitrogen source in the medium, and its yield dropped when excess ammonium ions were present, even under conditions that otherwise supported its synthesis (ApplMicrobiolBiotechnol (1990)32 :637-644).

[0019] Growth and polysaccharide synthesis in a defined medium were enhanced when amino acids were replaced by ammonia as the nitrogen source.

[0020] The use of casamino acid as a nitrogen source in a defined medium has been reported. However, animal-derived casamino acid, likely from bovine casein, has been reported to be an allergen and its use is restricted. Furthermore, the use of CaseinDigest/tryptone medium as the nitrogen source in a defined medium has been reported, however, it may not support the growth of fastidious organisms.

[0021] Addition of animal-free hydrolysates (Bacto TC Yeastolate, PhytonePeptone) to chemically defined media is one of the approaches to increase cell density, culture viability and productivity in a timely manner. Hydrolysates are digested proteins composed of amino acids, small peptides, carbohydrates, vitamins and minerals that provide nutritional supplements to the medium. Non-animal-derived hydrolysates of soybeans, wheat and yeast are commonly used in cell culture media and feeds to improve polysaccharide yields (see US9284371). However, due to their compositional complexity, batch-to-batch variations, undesirable attribute of making the culture viscous, yeast extract and hydrolysates can be a significant source of medium variability. Foam formation during large-scale fermentation can i) reduce the yield of capsular polysaccharide due to loss of cells and culture medium to the foam phase, ii) be detrimental to the cells as when bubbles burst they exert shear forces, iii) result in loss of sterility if the foam escapes, and iv) can lead to overpressure if a foam outlet blocks an outlet filter.

[0022] The fermentation cell supernatant is subjected to different purification steps to isolate the purified polysaccharide and eliminate host cell impurities such as proteins, nucleic acid and lipopolysaccharide. Filtration techniques play an important role in the downstream processing or purification of bacterial polysaccharides from host cell impurities. Downstream involves inactivation of bacterial culture, separation of cells from the medium, isolation of product, concentration, purification. Downstream processing is the most challenging part of the process due to its complexity.

[0023] Each bacterium has different capsular polysaccharides, and different serotypes of the same bacterium also differ in the chemical structure of the bacterial capsular polysaccharides. A case in point is that S. typhi expresses a capsule polysaccharide Vi that is a linear homopolymer of α(1-4)-D-GalpA N-acetylated at C-2 and O-acetylated at C-3. The N- and O-acetyls dominate the surface and are essential for both the antigenicity and immunogenicity of Vi, whereas S. Paratyphi A and B and NTS (with rare exceptions) do not express capsular polysaccharides. Instead, their surface polysaccharides are the O-polysaccharide (OPS) of lipopolysaccharide. They share a common trisaccharide backbone →2)-α-D-Manp-(1→4)-α-L-Rhap-(1→3)-α-D-Galp-(1→) (which serologically constitutes epitope 12). However, an α-(3→6)-linked dideoxyhexose saccharide on the mannose of the repeating trisaccharide results in an immunodominant epitope that confers identity to the Salmonella group. In the case of S. typhimurium, the galactose of epitope 12 of the trisaccharide backbone becomes α-(1→6) glycosylated. (See: Lindberg AA, Le Minor L. Serology of Salmonella In: Bergan T, ed. Methods in Microbiology: Academic Press, 1984:1-141.)

[0024] This diversity of bacterial polysaccharide structure makes the purification of these polysaccharides more challenging and difficult. The vaccine comprising a polysaccharide needs to meet a certain quality standard.

[0025] The prior method of purification that can be performed in large scale volumes includes lysis and precipitation of impurities such as nucleic acids and lipids using solvents, pH manipulation and use of detergent such as sodium deoxycholate, Triton-X.

[0026] Sodium deoxycholate (DOC) is a mild detergent and is one of the most commonly used detergents in polysaccharide purifications. Sodium deoxycholate with a central steroidal structure is less denaturing and limited in its solubilizing power, it breaks down endotoxins without affecting the chemical structure; and therefore, after removal of sodium deoxycholate, endotoxins regain their biological activity. Furthermore, DOC-based procedures do not work efficiently for the removal of polysaccharide contaminants, especially polysaccharides containing sialic acid. This may be due to the weak detergent activity of DOC on the lipopolysaccharide-protein association formed during downstream processing, resulting in high endotoxin level and protein content in the final isolated polysaccharide. Furthermore, sodium deoxycholate being a product of animal origin, even its residual presence in the final product may lead to non-acceptance of the product by regulatory agencies and certain religious communities.

[0027] The disadvantage of using Triton-X is that residual detergent persists in the extraction phase and disposal requires extensive washing to remove all residue.

[0028] For some types of CPS, zinc acetate/ammonium sulfate/sodium citrate precipitation for removal of protein contaminants is also included. However, in order to achieve the high level of purity, it is necessary to perform repetitive ammonium sulfate precipitations, making the process more tedious and laborious. In addition, it sometimes also precipitates capsular polysaccharides, resulting in the loss of total polysaccharide.

[0029] Some other methods use enzymes that aid in the degradation of protein and nucleic acid contaminants; however, removing enzymes and hydrolyzed material is a difficult task and may result in loss of the product of interest. Furthermore, regulatory agencies have restricted the use of animal enzymes in human products due to the risk of prion contamination. The use of enzymes, in addition to the high cost, will introduce more regulatory issues into the cGMP framework, for example, the origins of the enzymes (from animals or recombinants), variations in enzyme activity between different suppliers and batches, etc.

[0030] Some other methods made use of Benzonase, Proteinase K or Nargase for degradation of residual proteins and/or nucleic acid materials followed by chromatographic purification resulting in high costs and process that cannot be scaled up easily.

[0031] Some other methods have made use of toxic organic solvents such as phenol, butanol, toluene and chloroform for the separation of endotoxins from bacterial polysaccharides. This method is expensive and time consuming. In addition, it is unpleasant to work with toxic organic solvents that produce toxic residues.

[0032] The high purities required for vaccine-specific polysaccharides have 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 for the isolation of bacterial polysaccharides with effective removal of protein and nucleic acid contaminants. Despite the successful isolation of bacterial polysaccharides to WHO specifications, the use of chromatographic techniques involves multi-step work and time-consuming sample preparation, involves scalability issues, drastically compromises the recovery of capsular polysaccharides, and is therefore not a viable low-cost option for downstream processing on an industrial scale. Furthermore, the addition of additional purification steps to eliminate these contaminants increases the complexity of the process, decreasing final yields and increasing economic costs.

[0033] Purified Salmonella typhi polysaccharides obtained by previously reported purification processes have endotoxin content between 25 to 50 EU/μg.

[0034] Therefore, there is a need for alternative purification methodologies aimed at maximizing the recovery of Salmonella polysaccharides while removing impurities to acceptable levels.

[0035] Various methods such as acid hydrolysis, alkaline degradation, 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), electron beam fragmentation (Pawlowski et al. Micro Lett, 1999, 174.2, 255-263) have been described for the depolymerization (sizing) of bacterial and nonbacterial polysaccharides. However, acid hydrolysis and alkaline degradation are time-consuming and the resulting size-reduced sample has high polydispersity. Periodate oxidation also has deleterious effects on the labile antigenic epitopes of some polysaccharides. Furthermore, ozonolysis can only be used with polysaccharides containing β-D-aldoside linkages, and only a few endoglucanases have been isolated to date. US 20090041802 discloses the fragmentation of meningococcal polysaccharides by Emulsiflex C-50 (conventional homogenizer) (Avestin). However, conventional homogenizers operate at peak pressures for mere moments (approximately 7%) of each cycle, leading to larger biases, less stable products, and the need to perform more passes or use higher pressures than necessary.

[0036] Ultrasonic treatments have been used to depolymerize polysaccharides (see, for example, WO 2010/055250). However, the ultrasonic depolymerization method is not suitable for the industrial depolymerization of a large volume of polysaccharide due to its low efficiency.

[0037] US20090234108 describes a method of partial deacetylation of pneumococcal serotype 1 polysaccharide by chemical treatment with sodium carbonate buffer (pH 9.0). This process is time-consuming and prone to the destruction of immunogenic moieties that may affect the immunogenicity of the conjugates.

[0038] For all the reasons stated above, a simple and low-cost process for the depolymerization of polysaccharides or polysaccharide derivatives is still desirable in the art.

[0039] Polysaccharide-protein conjugate vaccine production is specific to the specific carrier protein and native polysaccharide involved in the conjugation process.

[0040] Various conjugation techniques are known in the art. Conjugates can be prepared by direct reductive amination methods, carbodiimide conjugation chemistry, hydrazides, active esters, norborane, p-nitrobenzoic acid, N-hydroxysuccinimide, S-NHS, EDC, using TSTU. 1-Cyano-4-dimethylamino pyridinium tetrafluoroborate (CDAP) conjugation chemistry.

[0041] The activated polysaccharide is coupled directly or via a spacer group (linker) to an amino group on the carrier protein. The linkers used for conjugation as disclosed in the prior art are N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (SZU ET AL; 1987). Other linkers, B-propionamido (WO 00/10599), nitrophenyl-ethylamine (Geveret al (1979) Med MicrobiolImmunol 165; 171288), haloalkyl halide (U.S. Pat. No. 4,057,685) glycosidic linkage (U.S. Pat. No. 4,057,685) 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 a means of conjugating polysaccharides and immunogenic proteins via bigeneric spacers. Protein (PRO) was derivatized to exhibit pendant nucleophilic or electrophilic groups (PRO*), while a partner polysaccharide (Ps) was functionalized to exhibit pendant groups of opposite reactivity (Ps*). By combining Ps* with PRO*, bigeneric spacers were formed, covalently linking Ps to PRO (Ps-PRO). Upon acid hydrolysis, the bigeneric spacer (linker) is released as an unusual amino acid, quantified by amino acid analysis and thus providing a means of proving covalency.

[0042] The polysaccharide component of polysaccharide-protein conjugate vaccines undergoes gradual depolymerization at a rate that depends on the type of conjugate, formulation components, and storage conditions. This results in an increase in free polysaccharide that may adversely affect product stability. Polysaccharide-carrier protein conjugates are known to release free polysaccharide after conjugation during processing, lyophilization, or storage in liquid or solid formulations. Only the Salmonella polysaccharide that is covalently linked to the carrier protein (i.e. the conjugated polysaccharide) is immunologically important for clinical protection and excessive levels of unbound polysaccharide may result in immunological hyporesponsiveness to the polysaccharide (See WHO/TRS/924 Page No. 14, A. 3.3.5). In particular, the Salmonella typhi conjugates reported in the literature have a high free polysaccharide content of up to 34% and high free protein content of over 5%, which indicates lower conjugation efficiency and lower conjugate stability which is not desirable. Therefore, there is a need for vaccines that demonstrate free polysaccharide content of less than 10%.

[0043] Indeed, if it were possible to have a generic process that could be employed to manufacture and formulate all vaccine candidates, this would greatly reduce the time and resources required for process development. This could have a significant impact on the number of clinical candidates that can be introduced into clinical trials. Furthermore, processes developed for early-stage clinical trials, including those developed using a platform, may not be optimal with respect to process economics, yield, pooling and production volumes and may not be suitable for producing the quantities required for late-stage or commercial campaigns. Another important consideration is the speed of process development, since process development needs to occur before a therapeutic candidate is introduced into clinical trials. See Abhinav A. Shuklaet al., Journal of Chromatography B, 848 (2007) 28 - 39.

[0044] Furthermore, the disease burden of Salmonella is high in developing countries, where availability of electricity and refrigeration are often inadequate and therefore vaccine stability in temperature variations assumes greater relevance for these regions.

[0045] Thus, there is a need for an efficient platform process for the manufacture of an effective vaccine against the Salmonella strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis that meets several criteria including good immunogenicity, safety and affordability, in particular a platform that provides i) improved polysaccharide yield in the fermentation and purification processes; ii) improved purification processes showing optimal percentage recovery and minimal impurity levels; and iii) improved polysaccharide-protein conjugate ratio in the vaccine iv) improved formulation showing low viscosity, devoid of aggregation; showing long-term stability over wide temperature ranges.

[0046] To overcome the above-mentioned limitations of the prior art and to address the long-felt unmet global medical need, Applicant proposes improved alternative fermentation, purification, conjugation processes, formulations for preparing a monovalent conjugate of Salmonella typhoidal as well as multivalent vaccines comprising at least one additional conjugate of Salmonella Paratyphi (S. Paratyphi A, B, C), and of non-typhoidal enteric Salmonella serovars typhimurium (S. typhimurium) and enteritidis (S. enteritidis).

OBJECTIVES

[0047] An object of the present invention is to improve one or more problems of the prior art or at least provide a useful alternative.

[0048] It is an objective of the present invention to develop an effective vaccine formulation for prophylaxis and treatment of infections caused by Salmonella spp. strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis in humans.

[0049] Another object of the present invention is to provide improved processes for the production of polysaccharide-protein conjugate vaccine comprising polysaccharide derived from Salmonella strains of the following species: S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis which can be employed on an industrial scale.

[0050] Yet another object of the present invention is to provide a monovalent polysaccharide-protein conjugate vaccine comprising polysaccharide derived from any of the Salmonella sorovar strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis.

[0051] Yet another object of the present invention is to provide a bivalent polysaccharide-protein conjugate vaccine comprising polysaccharide derived from any of the Salmonella strains of the var. S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis in any combination thereof.

[0052] Yet another object of the present invention is to provide a multivalent polysaccharide-protein conjugate vaccine comprising polysaccharide derived from Salmonella strains of the following species: S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis in any combination thereof.

[0053] Yet another object of the present invention is to provide improved fed-batch methods for the production of polysaccharide from Salmonella spp. strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis.

[0054] Yet another object of the present invention is to provide an improved purification method of polysaccharide derived from Salmonella strains of the following species: S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis.

[0055] Yet another object of the present invention is to provide improved methods of conjugating polysaccharide (with or without size reduction) derived from Salmonella sorovar strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis to a carrier protein.

[0056] Yet another object of the present invention is to provide a method of conjugating polysaccharide (with or without size reduction) derived from Salmonella strains of the S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis vars to a carrier protein with or without a linker (spacer) molecule.

[0057] Yet another object of the present invention is to provide immunogenic vaccine formulations comprising polysaccharide-protein conjugates in single-dose and multi-dose vials suitable for administration to infants and adults at appropriate concentrations effective to confer protection against or treatment of infections against strains of Salmonella spp. S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis or to prevent, ameliorate or delay the onset or progression of their clinical manifestations.

[0058] Other objects and advantages of the present invention will become more apparent from the following description, which is not intended to limit the scope of the present invention.

SUMMARY

[0059] The present invention provides: a) an immunogenic composition comprising one or more polysaccharide-protein conjugates wherein the polysaccharide is derived from strains of Salmonella sorovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis ; b) fed-batch methods for cultivating and processing polysaccharides derived from strains of Salmonella sorovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis ; c) downstream processing steps for obtaining polysaccharides derived from strains of Salmonella sorovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis ; d) methods for conjugating polysaccharides (with or without size reduction) derived from strains of Salmonella sorovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis to the carrier protein in the presence or absence of a binding molecule; and (e) methods for inducing an immune response in an individual by administering to an individual a therapeutically effective amount of the immunogenic composition to confer protection or treatment against infections with strains of Salmonella sorovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis or to prevent, ameliorate or delay the onset or progression of their clinical manifestations.

DESCRIPTION

[0060] Although the present invention may be susceptible to different embodiments, certain embodiments are shown in the detailed discussion below, with the understanding that the present invention may be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of the invention to what is illustrated and disclosed in this description.

[0061] Embodiments are provided in order to fully and completely convey the scope of the present invention to one skilled in the art. Numerous details are presented, relating to specific components and processes, to provide a complete understanding of the embodiments of the present invention. It will be apparent to one skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present invention. In some embodiments, well-known composition, well-known processes, and well-known techniques are not described in detail.

[0062] The terminology used herein is for the sole purpose of explaining a specific embodiment and such terminology should not be construed as limiting the scope of the present invention. As used herein, the forms "a", "an" and "the" may also include the plural forms unless the context clearly suggests otherwise.

[0063] The terms "comprises", "comprising", "including" and "having" are open transitional phrases and therefore 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 the steps described in the process of the present invention should not be construed as necessarily requiring their performance as described or illustrated. It should also be understood that additional or alternative steps may be employed.

[0064] The terms first, second, third, etc., should not be construed to limit the scope of the present invention, as the aforementioned terms may be used only to distinguish one element, component, region, layer or section from another component, region, layer or section. Terms such as first, second, third, etc., when used herein, do not imply a specific sequence or order unless clearly suggested by the present invention. The present invention provides an immunogenic composition and a process for preparing the same.

[0065] The term "vaccine" is optionally substitutable for the term "immunogenic composition" and vice versa.

[0066] "D antigen units" (also called "international units" or IU): The D antigenic form of poliovirus induces protective neutralizing antibodies. The D antigen units referred to herein (e.g., in the vaccines of the invention) are the total D antigen units measured from each type of unadsorbed bulk IPV antigen prior to final vaccine formulation that are added to each human dose of formulated vaccine (typically 0.5 mL final volume). Reliable methods of 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 the ELISA test as described in Example 1 ("D antigen quantification by ELISA") below. The European Pharmacopoeia provides a test sample (European Pharmacopoeia Biological Reference Preparation - available from the Ph. Eur. Secretariat, e.g. Code P 216 0000) for standardization of such methods among manufacturers (PharmeuropaSpecialIssue, Bio 96-2). Thus, the unit value of the D antigen is well understood in the art.

[0067] The term "dose" herein is typically an administration of the vaccine of the invention, which is typically an injection. A typical human dose is 0.5 mL. Of course, multiple doses may be administered in a vaccine administration schedule.

[0068] The term "IPV" or an immunogenic composition comprising these components herein is intended to mean inactivated poliovirus type 1 (e.g., Mahoney, as preferably used), type 2 (e.g., MEF-1), or type 3 (e.g., Saukett), or a combination of Sabin serotype 1, 2, 3 of two or all three of these types. An example of a full-dose (or standard) IPV immunogenic composition (Salk-based IPV types 1, 2, and 3 antigen 40-8-32 D units respectively) for the purposes of this invention could be Poliovac® (SerumInstituteofIndiaPvt. Ltd.).

[0069] The term "saccharide" throughout this specification may indicate either polysaccharide or oligosaccharide and includes both. The capsular saccharide antigen may be a full-length polysaccharide or may be extended to bacterial 'size saccharides' and 'oligosaccharides' (which naturally have a low number of repeating units, or which are size-reduced polysaccharides for manageability, but are still capable of inducing a protective immune response in a host.

[0070] According to a first embodiment of the present invention, the immunogenic composition may comprise one or more polysaccharide-protein conjugates, wherein the polysaccharide is derived from Salmonella sorovar strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis.

[0071] According to a second embodiment of the present invention, the method of obtaining the polysaccharide derived from strains of Salmonella sorovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, by fed-batch process, may comprise any subset or all of the following steps: 1. Inoculation, cultivation and harvesting of bacteria in fermentation medium compositions, 2. Inactivation, 3. Separation, 4. Clarification, and 5. Sterile Filtration.

[0072] According to a first aspect of the second embodiment, the fermentation medium compositions may comprise 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 sulphate, preferably magnesium sulphate heptahydrate. The potassium source may be selected from sodium dihydrogen phosphate heptahydrate, sodium dihydrogen phosphate monohydrate, potassium phosphate and dipotassium phosphate. Preferably, the potassium source is a combination of sodium dihydrogen phosphate heptahydrate, sodium dihydrogen phosphate. Preferably, the soy hydrolysate is hisso.

[0073] Further, the fermentation medium may additionally comprise an antifoam agent selected from the group of Antifoam 204, Antifoam C, SE-15, Y-30, AntifoamEX-Cell, S184 (pure silicon oil), SLM54474 (polypropylene glycol:PPG), VP1133 (silicon oil/PPG blend), BREOX (polyalkylene glycol), J673 STRUKTOL (vegetable-based alkoxylated fatty acid esters) and SE9 (aqueous emulsion with 10% silicon oil component) from Wacker-Chemie Co. The antifoam agent in combination with soy hydrolysate and yeast extract may aid in improved yield of the polysaccharides. Further preferably, the antifoam agent may be Antifoam C or J673 STRUKTOL.

[0074] According to embodiments of the present invention, the yeast extract may be a yeast autolysate, an ultrafiltered yeast extract, or a synthetic yeast extract. The yeast extract may be selected from BD BBL™, BD BACTO™, Disco™, and the like. In a preferred embodiment, the yeast extract may be an ultrafiltered yeast extract, such as Difco™ Yeast Extract, UF. The soy hydrolysate may be selected from, but not limited to, soybean meal, soy peptone, and soy flour. In one embodiment, the soy hydrolysate may be Disco™ SelectPhytone™ UF. In another embodiment, the soy hydrolysate may be hisso.

[0075] The combination of antifoam, soy peptone and yeast extract results in improved crop yield compared to other media.

[0076] According to a second aspect of the second embodiment, the process may follow a two-shot strategy incorporating the feed content in a fixed ratio at specific fixed time intervals during when fermentation is already in progress and/or allowing continuous feeding throughout a fed-batch fermentation mode comprising several stages with proportional increase in batch size at each stage.

[0077] According to a third aspect of the second embodiment, the fermentation parameters may comprise: Temperature: 36.0 ± 2°C Agitation: 150 to 600 rpm pH: 7.0 ± 0.5 Dissolved oxygen: 30% to 90% Air (nl/min): 2 to 10 Gas flow: 60 to 600 nl/min Osmolarity: 400 to 600 mOsm/kg

[0078] According to a fourth aspect of the second embodiment, the inactivation of the bacterial culture can be performed using formalin.

[0079] Still preferably, the inactivation of the bacterial culture may be carried out using formaldehyde in the range of 0.1 to 2% v/v, preferably 0.5% v/; incubated at 34 to 38°C, preferably 36°C; for 5 to 12 hours, preferably 8 to 12 hours.

[0080] According to a fifth aspect of the second embodiment, the separation may be performed by centrifugation. Still preferably, the separation may be performed by centrifugation with parameters set at temperature of 2 to 8°C; RPM - 7000 to 8000; Centrifugation time 40 to 60 min.

[0081] According to a sixth aspect of the second embodiment, the clarification may be accomplished by depth filtration.

[0082] According to a seventh aspect of the second embodiment, the clarified crop can be sterilized by filtration using 0.2 μM sterile filters.

[0083] According to the eighth aspect of the second embodiment, the crude yield of Vi polysaccharide (ViPs) from Salmonella enterica serovar typhi in the fermentation phase may be at least 40% and the average yield of Vi polysaccharide may be in the range of 100 mg/L to 5000 mg/L; more preferably 100 to 700 mg/L.

[0084] According to a third embodiment of the present invention, the fermentation harvest may be subjected to any subset or in any order or all of the following downstream purification steps to obtain the desired quality of Vi polysaccharide (ViPs): a) clarification of a 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 a membrane with 10 to 300 kDa or kD molecular weight cut-off (MWCO); c) treatment with anionic or cationic detergent, ethylene diamine tetraacetic acid (EDTA) (4 to 10 mM) and sodium acetate (5% to 10%) for denaturation of proteins, nucleic acids and lipoproteins; polysaccharides; d) precipitation with alcohol (40% to 70%); e) centrifugation and filtration by direct flow filtration (DFF) through at least one clarifying filter having a pore size of about 0.2 μM; f) treatment with alkaline salt 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 a membrane with 10 to 300 kDa or kD molecular weight cut-off (MWCO); h) treatment with anionic or cationic detergent; i) centrifugation and filtration by direct flow filtration (DFF) through at least one clarifying 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% to 70%) in the presence of sodium chloride (0.1M to 2M);k) selective precipitation of polysaccharide using alcohol (<75% or >95%);l) dissolving polysaccharide in WFI and subjecting it to concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 to 300 kDa or kD molecular weight cut-off (MWCO); and (m) sterile filtration through at least one sterile filter with a pore size of about 0.2 micrometers under sterile conditions.

[0085] According to a first aspect of the third embodiment, the purification process may be devoid of any chromatography step.

[0086] According to a second aspect of the third embodiment, the purification process may result in significant recovery of about 40% to 65% with desired O-acetyl levels (greater than 2.0 mmol/g polysaccharide), yield of purified Vi polysaccharide may be in the range of 1,000 to 4,000 mg/L, average molecular weight may be in the range of 40 to 400 kDa, contains less than 1% proteins/peptides, less than 2% nucleic acids, less than 100 EU endotoxins per μg polysaccharide (PS), molecular size distribution (greater than 50% PS is eluted before a distribution coefficient (KD) of 0.25 is reached)

[0087] However, preferably, the average molecular weight of the purified Vi polysaccharide may be in the range of 40 to 400 kDa.

[0088] According to a third aspect of the third embodiment, the anionic detergent can 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 sulfates, sodium oleyl sulfate, sodium N-oleoyl poly(amino acid), sodium alkylbenzene sulfonates, sodium alpha olefin sulfonate, sodium alkyl sulfonates, alpha-sulfomonocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkanesulfonates, sodium ligninsulfonate, and sodium alkylglyceryl ether sulfonates.

[0089] Still preferably, said anionic detergent may be alkyl sulfate, more preferably sodium dodecyl sulfate at a final concentration in the range of 0.1% to 20%, more preferably in the range of 1 to 20% may be added to the retentate and stirred at 25°C to 30°C for 2 hours.

[0090] According to a fourth aspect of the third embodiment, the precipitation of alcohol can be carried out using methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone, or t-butyl alcohol; or a combination thereof.

[0091] However, preferably said alcohol may be ethanol.

[0092] According to a fifth aspect of the third embodiment, the alkali salt may be selected from the group of sodium, potassium, calcium and magnesium salt. More preferably, the alkali salt may be 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.

[0093] Still preferably, said potassium salt may be potassium chloride at a final concentration in the range of 0.1 M to 2 M mixed with the supernatant and, after its dissolution, the mixture was incubated at 2 to 8°C for >3 hours.

[0094] According to a sixth aspect of the third embodiment, the cationic detergent may be selected from the group comprising cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt and hexadimethine bromide; or a combination thereof.

[0095] Still preferably, said cationic detergent may be cetyltrimethylammonium bromide (CTAB) at a final concentration in the range of 0.1% to 12%; preferably 2% to 3% may be added to the retentate and stirred at 25°C to 30°C for 1 to 2 hours.

[0096] According to a seventh aspect of the third embodiment, the final mass of purified polysaccharide can be stored at less than or equal to -20°C.

[0097] According to a fourth embodiment of the present invention, the fermentation harvest may be subjected to any subset or in any order or all of the following downstream purification steps to obtain the desired quality of Salmonella Paratyphi A lipopolysaccharide O-specific polysaccharide (LPS): a) centrifugation and separation b) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a molecular weight cutoff of 10 to 300 kDa (MWCO); c) acid hydrolysis of LPS d) centrifugation and separation e) neutralization f) clarification of an 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 detergent, h) centrifugation and separation i) direct flow filtration (DFF) through at least one a membrane having a pore size of about 0.45 and 0.2 micrometers; j) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane having a molecular weight cutoff of 10 to 300 kDa (MWCO); k) precipitation with alcohol (40% to 70%); l) centrifugation and filtration by direct flow filtration (DFF) through at least one clarifying filter having a pore size of about 0.2 μM; m) treatment with alkaline salt 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; n) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane having a molecular weight cutoff of 10 to 300 kDa (MWCO);o) removal of protein and nucleic acid impurities by washing the pellet with alcohol (50% to 70%) in the presence of sodium chloride (0.1M to 2M);p) dissolving 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 of 10 to 300 kDa (MWCO); andq) sterile filtration through at least one sterile filter with a pore size of about 0.2 micrometers under sterile conditions.

[0098] According to a first aspect of the fourth embodiment (the acid hydrolysis of LPS may be carried out preferably using acetic acid concentration 0.5 to 5%) pH~2.0 to 3.0; temperature 30 to 90 degrees Celsius and time for about 100 to 200 minutes.

[0099] According to a second aspect of the fourth embodiment, the neutralization of the acid hydrolysis may preferably be carried out using ammonia liquor to achieve the final pH of 7.0.

[00100] According to a third aspect of the fourth embodiment, the anionic detergent can 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 sulfates, sodium oleyl sulfate, sodium N-oleoyl poly(amino acid), sodium alkylbenzene sulfonates, sodium alpha olefin sulfonate, sodium alkyl sulfonates, alpha-sulfomonocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate, and sodium alkylglyceryl ether sulfonates.

[00101] Still preferably, said 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% may be added to the retentate and stirred at 25°C to 30°C for 10 to 120 minutes.

[00102] According to a fourth aspect of the fourth embodiment, the precipitation of alcohol may be carried out using methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone, or t-butyl alcohol; or a combination thereof.

[00103] However, preferably said alcohol may be ethanol.

[00104] According to a fifth aspect of the fourth embodiment, the alkaline salt may be selected from the group of sodium, potassium, calcium and magnesium salt. More preferably, the alkaline salt may be 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.

[00105] However, preferably, said potassium salt may be potassium chloride at a final concentration in the range of 0.1 M to 2 M mixed with the supernatant and, after its dissolution, the mixture was incubated at 2 to 8°C for >3 hours.

[00106] According to a sixth aspect of the fourth embodiment, the cationic detergent may be selected from the group comprising cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt, and hexadimethrine bromide; or a combination thereof.

[00107] According to a seventh aspect of the fourth embodiment, the purification process may be devoid of any chromatography step.

[00108] According to an eighth aspect of the fourth embodiment, the purification process may result in

[00109] According to a ninth aspect of the fourth embodiment, the process results in significant reduction of endotoxin (<100EU endotoxin per μg PS), protein (<1%) and nucleic acid (<2%) impurities, increased recovery of capsular polysaccharide in the range of 40% to 65%, desired O-acetyl levels (>2.0 mmol/L), molecular size distribution (>50% of PS and polysaccharide before a distribution coefficient (KD) of 0.25 is achieved) and the average molecular weight of the purified O-specific polysaccharide can be in the range of 40 to 200 kDa.

[00110] According to a tenth aspect of the fourth embodiment, the final mass of the purified polysaccharide can be stored at less than or equal to -20°C.

[00111] According to a fifth embodiment of the present invention, the polysaccharide from purified strains of Salmonella enterica serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, can be covalently linked to the carrier protein (CP) using a carbodiimide conjugation, reductive amination or cyanylation reaction.

[00112] According to a first aspect of the fifth embodiment, the polysaccharide of purified strains of salmonella enterica serovar S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, can be covalently bound to carrier protein (CP) selected from the group comprising 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-subunit B, Neisseria meningitidis outer membrane complex, rEPA, H. influenzae protein D, FlagellinFliC, horseshoe crab hemocyanin, Pseudomonas aeruginosa exotoxin A, outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) from Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) from Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derived from tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N 19, iron uptake proteins, toxin A or B proteins from C. difficile and S. agalactiae with or without linker, and fragments, derivatives, modifications thereof.

[00113] Still preferably, the carrier protein used for conjugation with the Salmonella typhi Vi polysaccharide may be tetanus toxoid. The polysaccharides derived from S. Paratyphi A, S. typhimurium and S. enteritidis may preferably be conjugated individually with a carrier protein selected from tetanus toxoid, diphtheria toxoid or CRM197. Immunogenic compositions comprising the following polysaccharide-carrier protein conjugates are envisaged in accordance with the present disclosure: S. typhi conjugated with tetanus toxoid, S. Paratyphi A conjugated with TT or DT or CRM197; S. typhimurium conjugated with TT or DT or CRM197 and S. enteritidis conjugated with TT or DT or CRM197; S. typhiconjugated with tetanus toxoid, S. Paratyphi A conjugated with tetanus toxoid; S. typhimurium conjugated with CRM197 and S. enteritidis conjugated with CRM197; and S. typhiconjugated with tetanus toxoid, S. Paratyphi A conjugated with DT; S. typhimurium conjugated with CRM197 and S. enteritidis conjugated with tetanus toxoid and S. typhi conjugated with tetanus toxoid, S. Paratyphi A conjugated with CRM197; S. typhimurium conjugated with CRM197 and S. enteritidis conjugated with tetanus toxoid.

[00114] In accordance with the present disclosure, CRM197 is acquired from Pseudomonas fluorescens Recombinant Strain CS463-003 (MB101) from Pfenex USA.

[00115] According to the present disclosure, TT is acquired from Clostridium Tetani (Harvard No 49205) obtained from Central Research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. Central Research Institute (CRI) acquired this strain from NVI, Netherlands.

[00116] According to the present disclosure, DT is produced from cultures of the strain Cornynebacteriumdiphtheriae Park-Williams Number 8, the strain is obtained from the Central Research Institute (CRI), Kasauli, Himachal Pradesh, India.

[00117] According to a second aspect of the fifth embodiment prior to conjugation, polysaccharides from purified Salmonella enterica strains serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, may be subjected to depolymerization/sizing by chemical means selected from the group of FeCl3, H2O2, sodium metaperiodate and sodium acetate or mechanical means selected from the group of high pressure cell disruption and homogenizer.

[00118] Still preferably, polysaccharides from purified strains of Salmonella enterica serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, may be subjected to depolymerization/sizing by high-pressure cell disruption.

[00119] Still preferably, the purified polysaccharide of Salmonella enterica serovar typhi (ViPs) may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the ViPs may be in the range of 40 to 400 kDa.

[00120] Still preferably, the purified polysaccharide of Salmonella enterica serovar typhi (ViPs) may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average molecular weight of the ViPs may be in the range of 100 to 250 kDa.

[00121] It was found that the conjugation efficiency/conjugate yield, immunogenicity of conjugates prepared from the use of partially reduced polysaccharides was higher compared to conjugates prepared from full-length polysaccharides. of reactive end groups, both factors contribute to an increased frequency of covalent bond formation.

[00122] Alternatively, polysaccharides from purified strains of Salmonella enterica serovar S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, cannot be subjected to depolymerization/sizing.

[00123] According to a third aspect of the fifth embodiment, prior to conjugation, the carrier protein (CP) may be derivatized to comprise amino and/or carboxyl groups via a hetero- or homobifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, ε-aminohexanoic acid, dimethylacetalchlorohexanol, D-glucuronolactone, cystamine and p-nitrophenylethylamine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propynamido, nitrophenylethylamine, haloalkyl halide, 6-aminocaproic acid, and combinations thereof using a carbodiimide reaction, reductive amination, or cyanylation.

[00124] Still preferably, the hetero- or homo-bifunctional linker may be dihydrazide, most preferably adipic acid dihydrazide.

[00125] Hydrazide groups can be introduced into proteins via the carboxyl groups of aspartic acid and glutamic acid residues in the protein using a carbodiimide, reductive amination, cyanylation, reaction, for example, by reaction with hydrazine, carbohydrazide, succinyldihydrazide, adipic acid dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride), or any other dihydrazides in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC). EDC is employed as a catalyst to activate and modify the protein reactant with hydrazine or dihydrazide.

[00126] Still preferably, the reaction of the carrier protein (CP) with adipic acid dihydrazide (ADH) in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be carried out at a pH of 5 to 7; more preferably 6.

[00127] Accordingly, 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), can be stopped by raising the pH from about 6 to about 7 to 8.

[00128] Alternatively, following derivatization of the carrier protein with ADH, the method may further comprise the step of buffer exchange by diafiltration (DF) using a 10 kDa or 30 kDa or 50 kDa molecular weight cut-off (MWCO) membrane and sterile filtration with a 0.2 µm filter; wherein the ADH-derived carrier protein is buffer exchanged by at least 10 volumes or passed through a suitable gel filtration column and substantially all unreacted compounds, residual ADH and residual EDC are removed, producing a purified ADH-derived carrier protein.

[00129] Alternatively, prior to conjugation, the carrier protein cannot be derivatized to comprise amino and/or carboxyl groups via a hetero- or homobifunctional linker.

[00130] According to a third aspect of the fifth embodiment, prior to conjugation the polysaccharide from purified strains of salmonella enterica serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, may be derivatized to comprise, amino and/or carboxyl groups via a hetero- or homo-bifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, a mixture thereof, ε-aminohexanoic acid, dimethylacetalchlorohexanol, D-glucuronolactone, cystamine and p-nitrophenylethylamine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propynamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid and combinations thereof using a carbodiimide, reductive amination or cyanylation reaction.

[00131] Still preferably, the hetero- or homo-bifunctional linker may be dihydrazide, most preferably adipic acid dihydrazide.

[00132] Hydrazide groups may be introduced into the polysaccharide of Salmonella enterica strains serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, using a carbodiimide, reductive amination, cyanylation reaction, e.g. reaction of polysaccharide with hydrazine, carbohydrazide, succinyldihydrazide, adipic acid dihydrazide, hydrazine chloride (e.g. hydrazine dihydrochloride) or any other dihydrazides in the presence of cyanogen bromide (CNBr) may form hydrazide derivatives of the polysaccharide Vi.

[00133] 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 a group of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1-cyano-imidazole (1CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate ('CDAP'), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate ('CTEA') or 2-cyanopyridazine-3(2H)one (2-CPO).

[00134] 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 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) mixed in a ratio of 1:1 to 1:10 by weight of OSP:ADH and the ratio of OSP:CPPT may be between 0.5 and 1.5, reaction carried out at a pH in the range of 7 to 10, reaction duration of 1 to 2 hours, temperature in the range of 2°C to 30°C.

[00135] Still another aspect of the fifth embodiment, prior to conjugation, the polysaccharide cannot be derivatized to comprise amino and/or carboxyl groups via a hetero- or homo-bifunctional linker.

[00136] A preferred aspect of the fifth embodiment wherein, Salmonella enterica serovar typhi polysaccharide (ViPs) can be covalently linked to the carrier protein using carbodiimide conjugation chemistry wherein any water soluble carbodiimide most preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) can be used as a catalyst.

[00137] More preferably, the Vi polysaccharide may be covalently linked to the ADH-derived carrier protein using carbodiimide conjugation chemistry in which any water-soluble carbodiimide most preferably 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) may be used as a catalyst.

[00138] More preferably, the Vi polysaccharide can be covalently linked to the ADH-derived tetanus toxoid using carbodiimide conjugation chemistry in which any water-soluble carbodiimide most preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) can be used as a catalyst.

[00139] Still alternatively, the ADH-derived Vi polysaccharide can be covalently linked to the ADH-derived tetanus toxoid using carbodiimide conjugation chemistry in which any water-soluble carbodiimide most preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) can be used as a catalyst.

[00140] Still alternatively, the ADH-derived Vi polysaccharide can be covalently linked to tetanus toxoid using carbodiimide conjugation chemistry in which any water-soluble carbodiimide most preferably 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) can be used as the catalyst.

[00141] Still preferably, the purified ViPs are covalently linked to the carrier protein (CP) using the carbodiimide conjugation reaction in the presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC) mixed in a ratio of 1:0.5 to 1:2 by weight of ViPs:EDAC and the ratio of Vi polysaccharide and carrier protein may be between 0.5 and 1.5, reaction carried out at a pH in the range of 5 to 7, temperature in the range of 2°C to 30°C.

[00142] Still another aspect of the fifth embodiment, the Vi polysaccharide (ViPs) can be covalently linked to tetanus toxoid (TT) derived from ADH in the presence of 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) wherein the weight ratio of ViPs:TT:EDC can be 1:1:2 and the concentration of Vi polysaccharide and tetanus toxoid can be between 0.1 mg/mL to 10.0 mg/mL and the ratio of Vi polysaccharide and tetanus toxoid can be between 0.5 and 1.5.

[00143] Yet another aspect of the fifth embodiment, the reaction of the Vi polysaccharide (ViPs) with the derived tetanus toxoid (TT) in the presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be carried out at a pH in the range of 5 to 7; more preferably 6; temperature in the range of 2°C to 30°C; more preferably 10 to 25°C and the conjugation conversion efficiency is >70% more preferably >90% and the molecular size of the conjugate is preferably between 1000 to 1600 kDa

[00144] Furthermore, the reaction of derived tetanus toxoid (TT) and Vi polysaccharide (ViPs) in the presence of carbodiimide such as 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) can be stopped by raising the pH from about 6 to about 7 to 8.

[00145] According to an aspect of the fifth embodiment, S. Paratyphi A polysaccharide (OSP) can be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a 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-nitrophenylcyanate and N-cyanotriethylammoniumtetrafluoroborate ('CTEA') or 2-cyanopyridazine-3(2H)one (2-CPO).

[00146] More preferably, S. Paratyphi OSP may be conjugated to a protein carrier using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) or (CPIP).

[00147] Still preferably, the purified S. Paratyphi OSP is covalently linked to the carrier protein (CP) using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) mixed in a ratio of 1:0.5 to 1:2 by weight of OSP:CPPT and the ratio of OSP and carrier protein may be between 0.5 and 1.5, reaction carried out at a pH in the range of 5 to 7, temperature in the range of 2°C to 30°C.

[00148] Still preferably, the carrier protein used for conjugation with S. Paratyphi A OSP polysaccharide may be tetanus toxoid.

[00149] Still preferably, the carrier protein used for conjugation with S. Paratyphi A OSP polysaccharide may be diphtheria toxoid.

[00150] Still preferably, the carrier protein used for conjugation with S. ParatyphiA OSP polysaccharide may be CRM 197.

[00151] Still another aspect of the fifth embodiment, S. typhimurium polysaccharide can be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a 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-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate ('CTEA') or 2-cyanopyridazine-3(2H)one (2-CPO).

[00152] More preferably, the S. typhimurium polysaccharide may be conjugated to a protein carrier using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT).

[00153] Still preferably, the carrier protein used for conjugation with the S. typhimurium polysaccharide may be tetanus toxoid.

[00154] Still preferably, the carrier protein used for conjugation with the S. typhimurium polysaccharide may be diphtheria toxoid.

[00155] Still preferably, the carrier protein used for conjugation with the S. typhimurium polysaccharide may be CRM 197.

[00156] Still another aspect of the fifth embodiment, S. enteritidis polysaccharide can be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a 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-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate ('CTEA') or 2-cyanopyridazine-3(2H)one (2-CPO).

[00157] More preferably, the S. enteritidis polysaccharide may be conjugated to a protein carrier using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT).

[00158] Still preferably, the carrier protein used for conjugation with the S. enteritidis polysaccharide may be tetanus toxoid.

[00159] Still preferably, the carrier protein used for conjugation with the S. enteritidis polysaccharide may be diphtheria toxoid.

[00160] Still preferably, the carrier protein used for conjugation with the S. enteritidis polysaccharide may be CRM 197.

[00161] Methods have been found to stabilize the final polysaccharide-protein conjugate using the alternative methods of conjugation, polysaccharide to protein ratio, polysaccharide to coupling agents ratio, using appropriate linkers, appropriate size of polysaccharide. The same may result in improvement in the polysaccharide-protein conjugate ratio in the vaccine which in turn may reduce the number of free saccharide and free protein in the conjugate, reduced carrier protein suppression, improved sterile filterability of the conjugate, better control of conjugation, and increased intra-portion cross-links indirectly provide good immune response.

[00162] Several factors have been found to influence the coupling of polysaccharides and proteins that depend on the molecular weight of the ViPs, type of carrier protein selected, the ratio of the amount of polysaccharide:carrier protein used, activation of functional groups, use of spacers, and conjugation chemistry.

[00163] Still another aspect of the fifth embodiment, following the conjugation reaction, the method may comprise the step of concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane with 100 kDa or 300 kDa molecular weight cut-off (MWCO); wherein the bulk of the conjugate is concentrated at least 3-fold and substantially all unreacted compounds, unconjugated polysaccharide, unconjugated protein, and residual EDC are removed, producing a purified Vi polysaccharide conjugate vaccine.

[00164] Further, the method may comprise gel filtration chromatography wherein the volume of the conjugate is concentrated at least 3-fold and substantially all unreacted compounds, unconjugated polysaccharides, unconjugated proteins and residual EDC are removed, producing a purified polysaccharide conjugate vaccine from S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, wherein the yield of the conjugate is >50%.

[00165] Another purification method may comprise a combination of ultrafiltration and gel filtration chromatography.

[00166] According to a sixth embodiment, the immunogenic composition may be a monovalent vaccine comprising either S. Typhi 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.

[00167] According to a seventh embodiment of the present invention, wherein the immunogenic composition may comprise at least one bivalent combination: a) the saccharide conjugate antigen of salmonella enterica serovar typhi - carrier protein (CP); b) the saccharide conjugate antigen of salmonella enterica serovar Paratyphi A - carrier protein (CP); or a) the saccharide conjugate antigen of salmonella enterica serovar typhi - carrier protein (CP); b) the saccharide conjugate antigen of salmonella enterica serovar typhimurium - carrier protein (CP); or a) the saccharide conjugate antigen of salmonella enterica serovar typhi - carrier protein; b) the saccharide conjugate antigen of salmonella enterica serovar enteritidis - carrier protein; or a) the saccharide conjugate antigen of salmonella enterica serovar Paratyphi A - carrier protein; saccharide of salmonella enterica serovar typhimurium-carrier protein; or a) saccharide conjugate antigen of salmonella enterica serovar Paratyphi A-carrier protein; or b) saccharide conjugate antigen of salmonella enterica serovar enteritidis-carrier protein; or a) saccharide conjugate antigen of salmonella enterica serovar typhimurium-carrier protein; or b) saccharide conjugate antigen of salmonella enterica serovar enteritidis-carrier protein;

[00168] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhi ViPs-TT conjugate antigen in a dose range of 1.25 to 50 μg;

[00169] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar Paratyphi A - CP conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00170] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhimurium saccharide conjugate antigen - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00171] Further preferably 0.5 ml of the composition comprises saccharide conjugate antigen of salmonella enterica serovar enteritidis - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00172] According to an eighth embodiment of the present invention, wherein the immunogenic composition may comprise at least one trivalent combination: a) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein (CP); b) Salmonella enterica serovar Paratyphi A saccharide conjugate antigen - carrier protein; c) Salmonella enterica serovar typhimurium saccharide conjugate antigen - carrier protein; or a) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein; b) Salmonella enterica serovar typhimurium saccharide conjugate antigen - carrier protein; c) saccharide conjugate antigen of Salmonella enterica serovar enteritidis - carrier protein; ora) saccharide conjugate antigen of Salmonella enterica serovar Paratyphi A - carrier protein; b) saccharide conjugate antigen of Salmonella enterica serovar Enteritidis - carrier protein;

[00173] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhi ViPs - TT conjugate antigen in a dose range of 1.25 to 50 μg;

[00174] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00175] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhimurium-CP saccharide conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00176] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar enteritidis-CP saccharide conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00177] According to a ninth embodiment of the present invention, wherein 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.

[00178] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhi ViPs - TT conjugate antigen in a dose range of 1.25 to 50 μg;

[00179] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00180] Further preferably 0.5 ml of the composition comprises saccharide conjugate antigen of salmonella enterica serovar typhimurium - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00181] Further preferably 0.5 ml of the composition comprises saccharide conjugate antigen of salmonella enterica serovar enteritidis - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00182] According to a tenth embodiment of the present invention, the immunogenic composition may also comprise one or more antigens selected from the group consisting of, but not limited to, SalmonellaParatyphi B, SalmonellaParatyphi 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, Shigella sonnei, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Escherichia coli, Enterobacter species, Yersinia species, Pseudomonas species, Pseudomonas aeruginosa, Haemophilus influenzae (serotypes a, c, d, e, f and non-encapsulated strains), Hepatitis (A, C, D, E, F and G), Influenza, Staphylococcus spp., Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8, Streptococcusspp, Streptococcus pneumoniae (1, 2, 3, 4, 5,6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A,9L,9F,9N, 9V, 10F, 10B,10C, 10A, 11A,11F,11B, 11C, 11D,11E,12A,12B, 12F,13, 14, 15A,15C ,15B,15F,16A, 16F, 17A,17F, 18C,18F,18A,18B, 19A, 19B, 19C, 19F, 20, 20A, 20B,21,22A, 22F, 23A,23B, 23F, 24A, 24B,24F, 25F, 25A,27,28F, 28A, 29, 31,32F, 32A,33A, 33C, 33D, 33E, 33F,33B, 34, 45, 38, 35A, 35B, 35C, 35F, 36, 37,38, 39, 40, 1F, 41A, 42, 43, 44, 45, 46, 47F, 47A, 48), Streptococcus Group A, Streptococcus Group B(group Ia, Ib, II, III, IV, V, Vl, VII VII, VIII, and IX.), Neisseria meningitidis, Haemophilus pneumonia, Helicobacterpylori, Chlamydiapneumoniae, Chlamydiatrachomatis, Ureaplasmaurealyticum, Mycoplasmapneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans, Enterococcusfaecalis, Enterococcusfaecium, EnterococcusfaecalisNeisseriae, Pasteurellapestis, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium tetani, Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, M. catarrhalis, Klebsiellapneumoniae, Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilusducreyi, Corynebacteriumdiphtheria, Bordetellapertussis, Bordetellaparapertussis, Bordetellabronchiseptica, Shigella spp., Erlichia spp., Rickettsia spp and N. me-ningitidis polysaccharide (A, B, C, D, W135, X, Y, Z and 29E) acellular pertussis antigen, modified adenylate cyclase, malaria antigen (RTS, S), anthrax, dengue, malaria, measles, mumps, rubella, BCG, human papillomavirus, Japanese encephalitis, dengue, zika, ebola, chikungunya, poliovirus, rotavirus, smallpox, yellow fever, flavivirus, Shingles and varicella virus antigens.

[00183] According to an eleventh embodiment of the present invention, wherein the composition comprises at least one combination: a) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein; b) Neisseria meningi-tidis saccharide conjugate antigen A - carrier protein; c) Neisseria meningi-tidis saccharide conjugate antigen C - carrier protein; d) Neisseria meningi-tidis saccharide conjugate antigen Y - carrier protein; e) Neisseria meningi-tidis saccharide conjugate antigen W-135 - carrier protein; f) Neisseria meningitidis saccharide conjugate antigen X - carrier protein; or a) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein; b) Salmonella enterica serovar Paratyphi saccharide conjugate antigen A - carrier protein; c) Neisseria meningi-tidis saccharide conjugate antigen Neisseria meningi-tidis saccharide A - carrier protein;d) Neisseria meningi-tidis saccharide conjugate antigen C - carrier protein;e) Neisseria meningi-tidis saccharide conjugate antigen Y - carrier protein;f) Neisseria meningitidis saccharide conjugate antigen W-135 - carrier protein;g) Neisseria meningi-tidis saccharide conjugate antigen X - carrier protein;ora) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein;b) Salmonella enterica serovar Paratyphi saccharide conjugate antigen A - carrier protein;c) Salmonella enterica serovar typhimurium saccharide conjugate antigen - carrier protein;d) Neisseria meningi-tidis saccharide conjugate antigen A - carrier protein;e) Neisseria meningi-tidis saccharide conjugate antigen C - carrier protein;f) Neisseria meningitidis saccharide conjugate antigen Y - carrier protein;g) Neisseria meningitidis saccharide conjugate antigen W-135 - carrier protein;h) Neisseria meningitidis saccharide conjugate antigen X - carrier protein;ora) Salmonella enterica serovar Typhi saccharide conjugate antigen - carrier protein;b) Salmonella enterica serovar Paratyphi A saccharide conjugate antigen - carrier protein;c) Salmonella enterica serovar Typhimurium saccharide conjugate antigen - carrier protein;d) Salmonella enterica serovar Enteritidis saccharide conjugate antigen - carrier protein;e) Neisseria meningitidis saccharide conjugate antigen A - carrier protein;f) Neisseria meningitidis saccharide conjugate antigen C - carrier protein;g) Neisseria meningitidis saccharide conjugate antigen meningi-tidis Y - carrier protein;h) Neisseria meningi-tidis W-135 saccharide conjugate antigen - carrier protein; (i) Neisseria meningitidis saccharide conjugate antigen X - carrier protein; or (a) Salmonella enterica serovar typhimurium saccharide conjugate antigen - carrier protein; (b) Salmonella enterica serovar enteritidis saccharide conjugate antigen - carrier protein; (c) Neisseria meningi-tidis saccharide conjugate antigen A - carrier protein; (d) Neisseria meningi-tidis saccharide conjugate antigen C - carrier protein; (e) Neisseria meningi-tidis saccharide conjugate antigen Y - carrier protein; (f) Neisseria meningitidis saccharide conjugate antigen W-135 - carrier protein; (g) Neisseria meningi-tidis saccharide conjugate antigen X - carrier protein (a) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein; (b) Neisseria meningitidis saccharide conjugate antigen meningi-tidis A - carrier protein;c) Neisseria meningi-tidis saccharide conjugate antigen C - carrier protein;d) Neisseria meningi-tidis saccharide conjugate antigen Y - carrier protein;e) Neisseria meningi-tidis saccharide conjugate antigen W-135 - carrier proteinora) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein; b) salmonella enterica serovar Paratyphi A saccharide conjugate antigen - carrier protein;c) neisseria meningi-tidis saccharide conjugate antigen A - carrier protein;d) neisseria meningi-tidis saccharide conjugate antigen C - carrier protein;e) neisseria meningi-tidis saccharide conjugate antigen Y - carrier protein;f) neisseria meningitidis W-135 saccharide conjugate antigen - carrier protein ora) salmonella enterica serovar typhimurium saccharide conjugate antigen - carrier protein;b) salmonella enterica serovar enteritidis saccharide conjugate antigen - carrier protein;c) neisseria meningi-tidis saccharide conjugate antigen A - carrier protein;d) neisseria meningi-tidis saccharide conjugate antigen C - carrier protein;e) neisseria meningi-tidis saccharide conjugate antigen Neisseria meningi-tidis Y - carrier protein;f) Neisseria meningitidis W-135 saccharide conjugate antigen - carrier protein;ora) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein;b) Neisseria meningi-tidis A saccharide conjugate antigen - carrier protein;c) Neisseria meningi-tidis C saccharide conjugate antigen - carrier protein; (a) salmonella enterica serovar typhi saccharide conjugate antigen—carrier protein; (b) salmonella enterica serovar Paratyphi A saccharide conjugate antigen—carrier protein; (c) Neisseria meningitidis saccharide conjugate antigen A—carrier protein; (d) Neisseria meningi-tidis saccharide conjugate antigen C—carrier protein; (oua) salmonella enterica serovar typhimurium saccharide conjugate antigen—carrier protein; (b) salmonella enterica serovar enteritidis saccharide conjugate antigen—carrier protein; (c) Neisseria meningitidis saccharide conjugate antigen A—carrier protein; (d) Neisseria meningi-tidis saccharide conjugate antigen C—carrier protein;

[00184] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhi ViPs - TT conjugate antigen in a dose range of 1.25 to 50 μg;

[00185] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00186] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhimurium saccharide conjugate antigen - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00187] Further preferably 0.5 ml of the composition comprises saccharide conjugate antigen of salmonella enterica serovar enteritidis - CP in a dose range of 1.25 to 50 µg; wherein the CP is either TT or DT or CRM197

[00188] Further preferably 0.5 ml of the composition comprises Neisseria meningitidis A - TT saccharide conjugate antigen in a dose of 5 µg

[00189] Further preferably 0.5 ml of the composition comprises Neisseria meningitidis C saccharide conjugate antigen - CRM197 in a dose of 5 µg

[00190] Further preferably 0.5 ml of the composition comprises Neisseria meningitidis Y saccharide conjugate antigen - CRM197 in a dose of 5 µg

[00191] Further preferably 0.5 ml of the composition comprises Neisseria meningitidis W - CRM197 saccharide conjugate antigen in a dose of 5 µg

[00192] Further preferably 0.5 ml of the composition comprises Neisseria meingitidis saccharide conjugate antigen X - TT in a dose of 5 µg

[00193] According to a twelfth embodiment of the present invention, wherein the immunogenic composition comprises at least one combination: a) Salmonella enterica serovar typhi saccharide conjugate antigen-carrier protein; b) an inactivated polio virus (IPV) antigen selected from the Salk or Sabin strain; c) a diphtheria toxoid antigen (D); d) a tetanus toxoid antigen (T); e) a whole cell pertussis antigen (wP) or acellular pertussis (aP); f) a hepatitis B virus surface antigen (HBsAg) and g) a Haemophilus influenzae type b antigen (Hib); or a) Salmonella enterica serovar typhi saccharide conjugate antigen-carrier protein; b) an inactivated polio virus (IPV) antigen selected from the Salk or Sabin strain; rotavirus;d) a diphtheria toxoid, antigen (D)e) a tetanus toxoid, antigen (T)f) a whole-cell pertussis, antigen (wP) or acellular pertussis, (aP);g) a hepatitis B virus surface antigen, (HBsAg) andh) a Haemophilus influenzae type b antigen, (Hib);ora) salmonella enterica serovar typhi saccharide conjugate antigen-carrier protein;b) salmonella enterica serovar paratyphi A saccharide conjugate antigen-carrier protein;c) an inactivated poliovirus (IPV) antigen, selected from the Salk or Sabin straind) a diphtheria toxoid, antigen (D)e) a tetanus toxoid, antigen (T)f) a whole-cell pertussis, antigen (wP) or acellular pertussis, (aP);g) a hepatitis B virus surface antigen, hepatitis B virus (HBsAg) and (h) a Haemophilus influenzae type b antigen (Hib); or (a) salmonella enterica serovar typhi saccharide conjugate antigen-carrier protein; b) salmonella enterica serovar paratyphi A saccharide conjugate antigen-carrier protein; c) a rotavirus antigen; d) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabine strain; a diphtheria toxoid antigen (D) (f) a tetanus toxoid antigen (T) (g) a whole-cell pertussis (wP) or acellular pertussis (aP) antigen; h) hepatitis B virus surface antigen (HBsAg) and (i) a Haemophilus influenzae type b antigen (Hib); or (a) salmonella enterica serovar typhi saccharide conjugate antigen-carrier protein; b) salmonella enterica serovar Paratyphi A saccharide-carrier protein;c) salmonella enterica serovar Typhimurium saccharide-carrier protein conjugate antigen;d) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabine strain;f) a diphtheria toxoid, antigen (D);f) a tetanus toxoid, antigen (T);g) a whole-cell pertussis, antigen (wP) or acellular pertussis, antigen (aP);h) a hepatitis B virus surface antigen, (HBsAg) and i) a Haemophilus influenzae type b antigen, (Hib); (a) Salmonella enterica serovar Typhi saccharide conjugate antigen-carrier protein; (b) Salmonella enterica serovar Paratyphi A saccharide conjugate antigen-carrier protein; (c) Salmonella enterica serovar Typhimurium saccharide conjugate antigen-carrier protein; (d) a rotavirus antigen; (e) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain; (f) a diphtheria toxoid antigen (D); (g) a tetanus toxoid antigen (T); (h) a whole-cell pertussis antigen (wP) or acellular pertussis antigen (aP); (i) a hepatitis B virus surface antigen (HBsAg); and (j) a Haemophilus influenzae type b antigen (Hib); (oua) Salmonella enterica serovar Typhi saccharide conjugate antigen-carrier protein; (b) conjugated antigen of saccharide from salmonella enterica serovar Paratyphi A - carrier protein;c) saccharide conjugate antigen from salmonella enterica serovar Typhimurium - carrier protein;d) saccharide conjugate antigen from salmonella enterica serovar Enteritidis - carrier protein;e) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabin strain;f) a diphtheria toxoid antigen (D) (g) a tetanus toxoid antigen (T) (h) a whole-cell pertussis antigen (wP) or acellular pertussis antigen (aP);i) a hepatitis B virus surface antigen (HBsAg); andj) a Haemophilus influenzae type b antigen (Hib);ora) saccharide conjugate antigen from salmonella enterica serovar Typhi - carrier protein;b) saccharide conjugate antigen from salmonella enterica serovar Paratyphi A - carrier protein carrier;c) salmonella enterica serovar typhimurium saccharide conjugate antigen-carrier protein;d) salmonella enterica serovar enteritidis saccharide conjugate antigen-carrier protein;e) a rotavirus antigen;f) an inactivated poliovirus (IPV) antigen selected from the Salk or Sabing strain; a diphtheria toxoid antigen (D);h) a tetanus toxoid antigen (T);i) a whole-cell pertussis antigen (wP) or acellular pertussis antigen (aP);j) a hepatitis B virus surface antigen (HBsAg); andk) a Haemophilus influenzae type b antigen (Hib);

[00194] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhi ViPs - TT conjugate antigen in a dose range of 1.25 to 50 μg;

[00195] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00196] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhimurium saccharide conjugate antigen - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00197] Further preferably 0.5 ml of the composition comprises saccharide conjugate antigen of salmonella enterica serovar enteritidis - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00198] Further preferably the composition comprises diphtheria toxoid antigen, (D) in an amount of 1 to 50 Lf per 0.5 ml

[00199] Further preferably the composition comprises a tetanus toxoid, (T) in an amount of 1 to 30 Lf per 0.5 ml.

[00200] Still preferably the composition comprises a whole cell pertussis antigen, (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis antigen, (aP) comprising one or more of modified adenylate cyclase, pertussis toxoid (PT) (PT) 1 to 50 μg, Filamentous Hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial Proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml;

[00201] Still preferably the composition comprises a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;

[00202] Still preferably the composition comprises a Haemophilus influenzae type b (Hib) antigen in an amount of 1 to 20 μg per 0.5 ml;

[00203] Still preferably the composition comprises an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount in the range of 1 to 50 μg per 0.5 ml;

[00204] Still preferably the composition comprises an inactivated poliovirus antigen (IPV) selected from the Sabin or Salk strain; wherein the IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU) or IPV type 3 in a dose of 1 to 50 D antigen units (DU), per 0.5 ml;

[00205] According to a thirteenth embodiment of the present invention, wherein the immunogenic composition comprises at least one combination: a) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein; b) a rotavirus antigen; c) an Escherichia coli spp. antigen (enterotoxigenic and enterohemorrhagic); d) a Shigella spp. antigen; e) a Campylobacter jejuni antigen; f) a Vibrio cholerae antigen; or a) Salmonella enterica serovar typhi saccharide conjugate antigen - carrier protein; b) Salmonella enterica serovar Paratyphi A saccharide conjugate antigen - carrier protein; c) a rotavirus antigen; d) an Escherichia coli spp. antigen (enterotoxigenic and enterohemorrhagic);e) an antigen from Shigella spp;f) an antigen from Campylobacter jejuni;g) an antigen from Vibrio cholerae;oua) saccharide conjugate antigen from Salmonella enterica serovar typhi - carrier protein;b) saccharide conjugate antigen from Salmonella enterica serovar Paratyphi A - carrier protein;c) a rotavirus antigen;d) an antigen from Escherichia coli spp. (enterotoxigenic and enterohemorrhagic);e) an antigen from Shigella spp;f) an antigen from Campylobacter jejuni;oua) saccharide conjugate antigen from Salmonella enterica serovar typhi - carrier protein;b) saccharide conjugate antigen from Salmonella enterica serovar Paratyphi A - carrier protein;c) a rotavirus antigen;d) an antigen from Escherichia coli spp. (enterotoxigenic and enterohemorrhagic);e) an antigen from Shigella spp;ora) saccharide conjugate antigen from Salmonella enterica serovar Typhi - carrier protein;b) saccharide conjugate antigen from Salmonella enterica serovar Paratyphi A - carrier protein;c) a rotavirus antigen;d) an antigen from Shigella spp;ora) saccharide conjugate antigen from Salmonella enterica serovar Typhimurium - carrier protein;b) saccharide conjugate antigen from Salmonella enterica serovar enteritidis - carrier protein;c) a rotavirus antigen;d) an antigen from Escherichia coli spp. (enterotoxigenic and enterohemorrhagic);e) an antigen from Shigella spp;f) an antigen from Campylobacter jejuni;g) an antigen from Vibrio cholerae;ora) saccharide conjugate antigen from Salmonella enterica serovar Typhi - carrier protein;b) saccharide conjugate antigen from Salmonella enterica serovar Paratyphi A - carrier protein;c) saccharide conjugate antigen from Salmonella enterica serovar Typhimurium - carrier protein;d) a rotavirus antigen;e) an antigen from Escherichia coli spp. (enterotoxigenic and enterohemorrhagic);f) an antigen from Shigella spp;g) an antigen from Campylobacter jejuni;h) an antigen from Vibrio cholerae;ora) saccharide conjugate antigen from Salmonella enterica serovar Typhi-carrier protein;b) saccharide conjugate antigen from Salmonella enterica serovar Paratyphi A-carrier protein;c) saccharide conjugate antigen from Salmonella enterica serovar Typhimurium-carrier protein;d) saccharide conjugate antigen from Salmonella enterica serovar enteritidis-carrier protein;e) a rotavirus antigen;f) an antigen from Escherichia coli spp. (enterotoxigenic and enterohemorrhagic);g) an antigen from Shigella spp;h) an antigen from Campylobacter jejuni;i) an antigen from Vibrio cholerae;

[00206] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar typhi ViPs - TT conjugate antigen in a dose range of 1.25 to 50 μg;

[00207] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00208] Further preferably 0.5 ml of the composition comprises salmonella enterica serovar typhimurium saccharide conjugate antigen - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00209] Further preferably 0.5 ml of the composition comprises Salmonella enterica serovar enteritidis saccharide conjugate antigen - CP in a dose range of 1.25 to 50 μg; wherein the CP is either TT or DT or CRM197

[00210] Still preferably the composition comprises an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strain present in an amount in the range of 1 to 50 μg per 0.5 ml;

[00211] According to one aspect of the embodiment, the immunogenic composition may further comprise a buffering agent selected from the group consisting of carbonate, phosphate, acetate, HEPES, succinate, histidine, TRIS, borate, citrate, lactate, gluconate, and tartrate, as well as more complex organic buffering agents, including a phosphate buffering agent containing sodium phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH. In another example, the buffering agent contains Tris(hydroxymethyl)aminomethane, or "Tris", formulated to achieve the desired pH. In yet another example, the buffering agent may be minimum essential medium with Hanks' salts. Other buffers, such as HEPES, piperazine-N,N'-bis (PIPES), and 2-ethanesulfonic acid (MES) are also contemplated by the present invention. The buffer aids in stabilizing an immunogenic composition of the present invention. 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.

[00212] Citrate buffer can be prepared by dissolving citric acid monohydrate (CAM) in the range of 1.05 to 2.63 mg and trisodium citrate dehydrate (TCD) in the range of 1.47 to 3.68 mg

[00213] Preferably an immunogenic composition may further comprise TRIS or citrate buffer or histidine buffer or succinate buffer in the range of 0.1 mg to 2.0 mg.

[00214] Preferably an immunogenic composition may additionally contain TRIS buffer in the range of 0.61 mg to 1.52 mg.

[00215] Preferably the immunogenic composition may additionally comprise citrate buffer in the range 10 mM to 25 mM.

[00216] Preferably the immunogenic composition may additionally comprise histidine buffer in the range of 0.78 to 1.94 mg.

[00217] Preferably the immunogenic composition may additionally comprise succinate buffer in the range of 0.59 to 1.48 mg.

[00218] Still another aspect of the embodiment, the immunogenic composition may additionally comprise pharmaceutically acceptable excipients selected from the group consisting of sugars, surfactants, polymers, salts, amino acids, or pH modifiers.

[00219] Examples of surfactants may include ionic and nonionic surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethanol, t-octylphenoxypolyethoxyethanol, oxoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin, and a poloxamer.

[00220] Preferably the immunogenic composition may comprise polysorbate 20 as pharmaceutically acceptable excipients.

[00221] Examples of the polymers may include dextran, carboxymethyl cellulose, hyaluronic acid, cyclodextrin, etc.

[00222] Examples of the salts may include NaCl, KCl, KH2PO4, Na2HPO4.2H2O, CaC12, MgC12, etc. Preferably, the salt may be NaCl. Typically, the amount of salt may be in the range of 100 mM to 200 mM.

[00223] Preferably the immunogenic composition may comprise sodium chloride in the range of 1 to 10 mg.

[00224] Examples of amino acids as excipient selected from the group of L-Histidine, Lysine, Isoleucine, Methionine, Glycine, Aspartic Acid, Tricine, arginine, leucine, glutamine, alanine, peptide, hydrolyzed protein or protein such as serum albumin.

[00225] Preferably the immunogenic composition may comprise Histidine.

[00226] Examples of sugars as excipients selected from the group of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol or a combination thereof.

[00227] Preferably the immunogenic composition may additionally comprise sucrose.

[00228] Examples of polymers as excipient selected from the group of dextran, carboxymethyl cellulose, hyaluronic acid, cyclodextrin.

[00229] Further preferably the single dose composition is free of preservatives.

[00230] Still preferably the multidose immunogenic composition may additionally comprise preservatives 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-chlor-m-cresol, or benzyl alcohol or a combination thereof. A vaccine composition may include material for a single immunization or may include material for multiple immunizations (i.e., a 'multidose' kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to the inclusion of a preservative in multidose compositions, the compositions may be contained in a container with an aseptic 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.

[00231] Still another aspect of the embodiment, the immunogenic composition may additionally comprise auxiliary substances such as wetting or emulsifying agents, pH buffering diluent agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, coloring agents, and the like, depending on the route of administration and the desired preparation.

[00232] Still a preferable aspect of the embodiment, the immunogenic composition may additionally comprise Water for Injection as a diluent.

[00233] In yet another aspect of the embodiment, the immunogenic composition may further comprise an adjuvant selected from the group of aluminum salt, aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate.

[00234] Still another aspect of the embodiment, the immunogenic composition may further comprise an immunostimulatory component selected from the group consisting of an oil and water emulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A derivatives, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund's adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers including block copolymers, p 1005 polymer, CRL-8300 adjuvant, muramyl dipeptide, β-agonists, and/or a peptide-1 receptor antagonist. TLR-4, flagellin, flagellins derived from Gram-negative bacteria, TLR-5 agonists, flagellin fragments capable of binding to TLR-5 receptors, alpha-C-galactosylceramide, chitosan, interleukin-2, QS-21, ISCOMS, squalene mixtures (SAF-1), Quil A, cholera toxin subunit B, polyphosphazene and derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, OMV, fHbp, combination of saponin with sterols and lipids.

[00235] In yet another aspect of the embodiment, the immunogenic composition may be entirely liquid. Suitable forms of liquid preparation may include solutions, suspensions, emulsions, syrups, isotonic aqueous solutions, viscous compositions, and elixirs that are buffered to a selected pH.

[00236] Still preferably the immunogenic composition may be entirely liquid, may be stable at 2 to 8°C, 25°C and 40°C for a period of six months and free polysaccharide after 6 months not more than 7.5% for polysaccharide of 180 to 220 kDa and free polysaccharide after 6 months not more than 10.5% for polysaccharide of 388/80/45 kDa.

[00237] The immunogenic composition of the present invention may be in the form of transdermal preparations, including lotions, gels, sprays, ointments, or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aerosol inhalation or insufflation), the compositions may be in a form and dispensed by a compression spray dispenser, pump dispenser, or aerosol dispenser. Aerosols are generally under pressure by means of a hydrocarbon. Pump dispensers may preferably dispense a metered dose or a dose with a given particle size. When 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 ingredients. The immunogenic composition may be a lyophilized or freeze-dried composition.

[00238] As used herein, the terms "freeze drying" or "lyophilize" or "lyophilization" involve lyophilization and refer to the process by which a suspension/solution is frozen, after which the water is removed by sublimation at low pressure. As used herein, the term "sublimation" refers to a change in the physical properties of a composition, whereby the composition changes directly from a solid to a gaseous state without becoming a liquid.

[00239] Accordingly, the lyophilized immunogenic composition may be stable at 2 to 8°C for 12 to 36 months; at 25°C for 2 to 6 months; at 37°C for 1 week to 4 weeks, at 42°C for 2 to 7 days, and at 55°C for 2 to 7 days.

[00240] According to one aspect of the embodiment, the method for reconstituting a lyophilized immunogenic composition may comprise the step of reconstituting the lyophilized immunogenic composition with an aqueous solution optionally saline or Water for Injection (WFI) wherein, the final pH of the immunogenic composition after reconstitution is in the range of pH 6.0 to pH 8.0; more preferably in the range of pH 7.0 to pH 8.0; even more preferably in the range of pH 7.2 to pH 7.9; and most preferably in the range of pH 7.5 to pH 7.9.

[00241] According to a ninth embodiment of the present invention, the immunogenic composition may be formulated for use in a method for reducing the onset or preventing a health condition comprising infection by strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis involving the administration of an immunologically effective amount of an immunogenic composition to a human subject by means of parenteral or subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or injectable administration or sustained release from implants or administration by eye drops or nasal or rectal or buccal or vaginal, administration by oral or intragastric or mucosal or perlinqual, alveolar or gingival or olfactory or respiratory mucosa or any other route of immunization.

[00242] According to the preferred aspect of the embodiment, the immunogenic composition may be administered to a human subject intramuscularly or subcutaneously.

[00243] According to the preferred aspect of the embodiment, an immunologically effective amount of an immunogenic composition comprising the polysaccharide-protein conjugate for vaccination against bacterial infection by strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis can be from about 1 μg/0.5 ml of the polysaccharide conjugate of strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium or S. enteritidis or less to about 100 μg/0.5 ml of the polysaccharide conjugate of strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium or S. enteritidis or more. In some other aspects, it can 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, the immunologically effective amount for vaccinating against bacterial infections by strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium, and S. enteritidis is from 1 μg/0.5 ml to 50 μg/0.5 ml; more preferably Salmonella enterica serovar typhi saccharide antigen-carrier protein conjugate; salmonella enterica serovar Paratyphi A saccharide conjugate antigen - carrier protein; salmonella enterica serovar Typhimurium saccharide conjugate antigen - carrier protein; salmonella enterica serovar Enteritidis saccharide conjugate antigen - carrier protein; is present in a dose range of about 5 ug/0.5 ml to about 30 ug/0.5 ml; even more preferably salmonella enterica serovar Typhi saccharide conjugate antigen - carrier protein; salmonella enterica serovar Paratyphi A saccharide conjugate antigen - carrier protein; salmonella enterica serovar Typhimurium saccharide conjugate antigen - carrier protein; salmonella enterica serovar Enteritidis saccharide conjugate antigen - carrier protein; is present in a dose range of about 25 ug/0.5 ml

[00244] According to the tenth embodiment of the present invention, the immunogenic composition can be administered intramuscularly or subcutaneously in a dose effective for the production of neutralizing antibodies and protection. The vaccines are administered in a manner compatible with the dosage formulation, and in such an amount that it will be prophylactically and/or therapeutically effective against typhoid and NTS infection. The immunogenic composition of the present invention can be administered as primary prophylactic agents in elderly, adolescents, adults or children at risk of infection, or can be used as secondary agents in the treatment of infected patients. For example, the immunogenic composition as disclosed herein can be used in elderly, adolescents, adults or children under 2 years of age or over 2 years of age at risk of infection by strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, or may be used as secondary agents for the treatment of patients infected with strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis.

[00245] Alternatively, NTS vaccines could be administered to infants between the ages of two and four months, before peak incidence occurs around 12 months of age. In addition, vaccine rollout will likely also include HIV-infected populations, as they are at higher risk of NTS infection. In developed countries, NTS vaccines could target the elderly, who have very high case fatality rates (up to 50%). It has been proposed that in children, programmatic field rollout could integrate directly with existing Expanded Program on Immunization schedules, perhaps at 6, 10, and 14 weeks.

[00246] More preferably the immunogenic composition may be administered intramuscularly or subcutaneously in a dosage volume of about 0.5 ml or 1 ml.

[00247] According to the eleventh embodiment of the present invention, the immunogenic composition may be formulated as single dose vials or multidose vials (2-Dose or 5-Dose or 10-Dose vials) or multidose kit or as pre-filled syringes wherein said immunogenic composition may be administered in a single dose schedule, or preferably a multiple dose schedule wherein a primary vaccination schedule is followed by 1 to 3 separate doses administered at subsequent time intervals after 1 to 3 years, if necessary. The dosage regimen will also be, at least in part, determined by the need for a booster dose necessary to confer protective immunity.

[00248] Still preferably the immunogenic composition may be formulated for administration to an elderly human subject, adolescent, adult or child under 2 years of age or over 2 years of age according to a one dose or two dose regimens or 3 dose regimens consisting of a first dose and/or a second dose to be administered between 3 months to 2 years after the first dose and/or a third dose to be administered between 3 months to 2 years after the second dose.

[00249] According to the eleventh embodiment of the present invention, the immunogenic composition can be administered concomitantly with other drugs or any other vaccine.

[00250] Still according to an aspect of the eleventh embodiment, wherein a single dose vaccine kit can comprise: a first container containing a lyophilized (freeze-dried) immunogenic composition: a) Neisseria meningitidis saccharide conjugate antigen A - TT 5 μg per 0.5 ml; b) Neisseria meningitidis saccharide conjugate antigen C - CRM197 5 μg per 0.5 ml; c) Neisseria meningitidis saccharide conjugate antigen Y - CRM197 5 μg per 0.5 ml; d) Neisseria meningitidis saccharide conjugate antigen W-135 - CRM197 5 μg per 0.5 ml; e) Neisseria meningitidis saccharide conjugate antigen X - TT 5 μg per 0.5 ml; ml;f) Sucrose 1 to 12 mg per 0.5 ml;g) Sodium citrate (dihydrate) 0.1 to 2 mg per 0.5 ml;h) Tris buffer 0.05 to 0.5 mg per 0.5 ml; and a second container containing a liquid composition for the reconstitution of the lyophilized (freeze-dried) immunogenic composition comprising: a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 to 50 μg per 0.5 ml; b) Sodium chloride 1 to 10 mg per 0.5 ml; c) Water for Injection (WFI) q.s.; in which there is no antigenic interference of ViPs-TT with Neisseria meningitidis antigens, for prophylaxis against typhoid caused by Salmonella Typhi and Neisseria meningitidis antigens in which said vaccine formulation is sufficient to elicit the required T-dependent immune response against S. typhi, including in children under 2 years of age, adults, adolescents, and the elderly by means of just one injection to comprise a complete vaccination schedule.

[00251] Still according to the second aspect of the eleventh embodiment, wherein a single dose vaccine kit can comprise: a first container containing a lyophilized (freeze-dried) immunogenic composition: a) Neisseria meningitidis saccharide conjugate antigen A - TT 5 μg per 0.5 ml; b) Neisseria meningitidis saccharide conjugate antigen C - CRM197 5 μg per 0.5 ml; c) Neisseria meningitidis saccharide conjugate antigen Y - CRM197 5 μg per 0.5 ml; d) Neisseria meningitidis saccharide conjugate antigen W-135 - CRM197 5 μg per 0.5 ml; e) Neisseria meingitidis saccharide conjugate antigen X-TT 5 μg per 0.5 ml; f) Sucrose 1 to 12 mg per 0.5 ml; g) Sodium citrate (dihydrate) 0.1 to 2 mg per 0.5 ml; h) Tris buffer 0.05 to 0.5 mg per 0.5 ml; and a second container containing a liquid composition for the reconstitution of the lyophilized (freeze-dried) immunogenic composition comprising: a) Salmonella enterica serovar typhi ViPs conjugate antigen - TT1, 25 to 50 μg per 0.5 ml; b) Salmonella enterica serovar Paratyphi A OSP conjugate antigen - CP 1, 25 to 50 μg per 0.5 ml; wherein the CP is either TT or DT or CRM197;c) Sodium chloride 1 to 10 mg per 0.5 ml;d) Water for Injection (WFI) q.s.;in which there is no antigenic interference from ViPs-TT; OSP antigen with Neisseria meningitidis antigens, for prophylaxis against typhoid and paratyphoid caused by antigens of Salmonella Typhi, S. Paratyphi and Neisseria meningitidis in which said vaccine formulation is sufficient to elicit the required T-dependent immune response against S. Typhi and Paratyphi, including in children under 2 years of age, adults, adolescents, and the elderly through just one injection to comprise a complete vaccination schedule.

[00252] Still according to the third aspect of the eleventh embodiment, wherein a single dose vaccine kit may comprise: a first container containing an all-liquid hexavalent immunogenic composition: a) an inactivated poliovirus antigen (IPV) selected from the Sabin or Salk strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU) or IPV type 3 in a dose of 1 to 50 D antigen units (DU), per 0.5 ml; (b) a diphtheria toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (c) a tetanus toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (d) a whole cell pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis (aP) antigen comprising one or more of a modified adenylate cyclase selected from pertussis toxoid (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) English: 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml;e) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;f) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 μg per 0.5 ml;anda second container containing an entirely liquid immunogenic composition comprising:a) Salmonella enterica serovar typhi ViPs - TT1 conjugate antigen, 25 to 50 μg per 0.5 ml;b) Sodium chloride 1 to 10 mg per 0.5 ml; and/or c) Tris buffer or Citrate buffer or Histidine buffer or Succinate buffer 0.1 mg to 1.6 mg per 0.5 ml; and/or d) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin, and a poloxamer in an amount of 25 to 500 μg per 0.5 ml; and/or e) 2-Phenoxyethanol 1 to 10 mg per 0.5 ml; and/or f) Water for Injection (WFI) q.s. in which there is no antigenic interference from ViPs-TT with hexavalent immunogenic composition, for prophylaxis against typhoid caused by Salmonella Typhi antigens Hexavalent in which said vaccine formulation is sufficient to elicit the required T-dependent immune response against S. typhi including in children under 2 years of age, adults, adolescents, and the elderly through just one injection to comprise a complete vaccination schedule.

[00253] Still according to the fourth aspect of the eleventh embodiment, wherein a single dose vaccine kit can comprise: a first container containing an all-liquid hexavalent immunogenic composition: a) an inactivated poliovirus antigen (IPV) selected from the Sabin or Salk strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU) or IPV type 3 in a dose of 1 to 50 D antigen units (DU), per 0.5 ml; (b) a diphtheria toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (c) a tetanus toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (d) a whole cell pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis (aP) antigen comprising one or more of a modified adenylate cyclase selected from pertussis toxoid (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1,2 and 3) 2 to 25 μg; per 0.5 ml;e) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;f) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml;anda second container containing an entirely liquid immunogenic composition comprising:a) salmonella enterica serovar typhi ViPs conjugate antigen - TT1, 25 to 50 μg per 0.5 ml;b) salmonella enterica serovar Paratyphi A OSP conjugate antigen - CP 1, 25 to 50 μg per 0.5 ml; wherein CP is either TT or DT or CRM197;c) sodium chloride 1 to 10 mg; and/or d) Tris buffer or Citrate buffer or Histidine buffer or Succinate buffer 0.1 mg to 1.6 mg per 0.5 ml; and/or g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and a poloxamer; in an amount of 25 to 500 μg per 0.5 ml; and/or h) 2-Phenoxyethanol 1 to 10 mg per 0.5 ml; and/or e) Water for Injection (WFI) q.s. in which there is no antigenic interference from ViPs-TT; OSP antigen with hexavalent antigens, for prophylaxis against typhoid and paratyphoid fever caused by Salmonella Typhi, S. Paratyphi and hexavalent antigens in which said vaccine formulation is sufficient to elicit the required T-dependent immune response against S. Typhi and Paratyphi including in children under 2 years of age, adults, adolescents, and the elderly through just one injection to comprise a complete vaccination schedule.

[00254] Still according to the fifth aspect of the eleventh embodiment, wherein a single dose vaccine kit may comprise: a first container containing an entirely liquid heptavalent immunogenic composition: a) an inactivated poliovirus antigen (IPV) selected from the Sabin or Salk strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU) or IPV type 3 in a dose of 1 to 50 D antigen units (DU) per 0.5 ml; (b) an inactivated rotavirus antigen selected from CDC9, CDC-66 or any other inactivated rotavirus strain present in an amount in the range of 1 to 50 μg per 0.5 ml; (c) a diphtheria toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (d) a tetanus toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (e) a whole cell pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis (aP) antigen comprising one or more of modified adenylate cyclase selected from pertussis toxoid (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml;f) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;g) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml;and a second container containing a liquid composition for the reconstitution of the lyophilized (freeze-dried) immunogenic composition comprising:a) salmonella enterica serovar typhi ViPs - TT conjugate antigen 1.25 to 50 μg per 0.5 ml;b) sodium chloride 1 to 10 mg per 0.5 ml;c) water for injection (WFI) q.s.;in which there is no antigenic interference of ViPs-TT with a heptavalent immunogenic composition, for prophylaxis against typhoid caused by Salmonella typhi and heptavalent antigens in that said vaccine formulation is sufficient to elicit the required T-dependent immune response against S. typhi including in children under 2 years of age, adolescent adults, and the elderly through just one injection to comprise a complete vaccination schedule.

[00255] Still according to the sixth aspect of the eleventh embodiment, wherein a single dose vaccine kit may comprise: a first container containing an entirely liquid heptavalent immunogenic composition: a) an inactivated poliovirus antigen (IPV) selected from the Sabin or Salk strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU) or IPV type 3 in a dose of 1 to 50 D antigen units (DU), per 0.5 ml; (b) an inactivated rotavirus antigen selected from CDC9, CDC-66 or any other inactivated rotavirus strain present in an amount in the range of 1 to 50 μg per 0.5 ml; (c) a diphtheria toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (d) a tetanus toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (e) a whole cell pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis, (aP) antigen comprising one or more of modified adenylate cyclase selected from pertussis toxoid (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml; f) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml; g) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml; and a second container containing an entirely liquid immunogenic composition comprising: a) Salmonella enterica serovar typhi ViPs conjugate antigen - TT 1, 25 to 50 μg per 0.5 ml; b) Salmonella enterica serovar Paratyphi A OSP conjugate antigen - CP1, 25 to 50 μg per 0.5 ml; wherein CP is either TT or DT or CRM197; c) Sodium chloride 1 to 10 mg per 0.5 ml; and/or d) Tris buffer or Citrate buffer or Histidine buffer or Succinate buffer 0.1 mg to 1.6 mg per 0.5 ml; and/or g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxoxynol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and a poloxamer in an amount of 25 to 500 μg per 0.5 ml; and/or h) 2-Phenoxyethanol 1 to 10 mg per 0.5 ml; and/or e) Water for Injection (WFI) q.s. in which there is no antigenic interference from ViPs-TT; OSP antigen with heptavalent antigens, for prophylaxis against typhoid and paratyphoid fever caused by Salmonella Typhi, S. Paratyphi and heptavalent antigens in which said vaccine formulation is sufficient to elicit the required T-dependent immune response against S. Typhi and Paratyphi including in children under 2 years of age, adults, adolescents, and the elderly through just one injection to comprise a complete vaccination schedule.

[00256] Strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis used for developing an immunogenic composition of the present invention may include: Salmonella enterica serovar typhi TY2 strain with “tviB” gene specific for Vi polysaccharide (Identified by Geneombio Technologies Private Limited, Pune and isolated from stool sample of confirmed typhoid patient at Villoo Poonawalla Memorial Hospital, Pune); S. typhi : ATCC 19430; C6524 (NICED, Kolkata, India); S. Paratyphi A : ATCC 9150 purchased from Chromachemie Laboratory Private Limited, Bangalore, 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. typhimurium177(ST19) CDC 6516-60; ATCC 700720. In addition, any attenuated strain of Salmonella sorovar (S. typhi, S. Paratyphi A, S. enteritidis and S. typhimurium) can be used for the preparation of the immunogenic composition of the present invention.

[00257] Salmonella enterica serovar typhi deposited with NCMR- NCCS; Pune, an International Depository Authority assigned Accession No. MCC 0193; Strain designation - PDL-1.

[00258] Other embodiments described herein also encompass vaccine kits comprising a first container containing a lyophilized (freeze-dried) immunogenic composition and a second container containing an optionally saline aqueous solution or WFI (water for injection) for reconstituting the lyophilized (freeze-dried) immunogenic composition.

[00259] The foregoing description of the specific embodiments so fully discloses the general nature of the embodiments contained herein that others may, applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and therefore such adaptations and modifications should and must be understood within the meaning and scope of equivalents of the embodiments described. It should be understood that the phraseology or terminology employed herein is for purposes of description and not limitation. However, although the embodiments described herein have been described in terms of preferred embodiments with modification, those skilled in the art will recognize that the embodiments described herein may be practiced within the spirit and scope of the embodiments described herein.

[00260] Throughout this specification, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[00261] The use of the expression "one or more" or "at least one" suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or results.

[00262] Any discussion of documents, acts, materials, devices, articles or the like that have been included in this specification is for the purpose only of providing a context for the disclosure. It should not be construed as an admission that any or all of such matters form part of the prior art basis or were common general knowledge in the field relevant to the disclosure as they existed anywhere prior to the priority date of this application.

[00263] The numerical values given for various physical parameters, dimensions and quantities are approximate values only and values greater than the numerical value assigned to the physical parameters, dimensions and quantities are intended to fall within the scope of the invention unless otherwise stated in the specification.

[00264] While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features may be added and that many changes may be made in the preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiment of the invention will be apparent to those skilled in the art from the description herein, and it is therefore clearly understood that the foregoing descriptive matter is to be construed merely as illustrating the invention and not as limiting it. TECHNICAL ADVANTAGES: 1. The present invention provides monovalent and multivalent polysaccharide-protein conjugate vaccines comprising polysaccharide derived from strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis in any combination thereof effective for conferring protection against or treatment of infections against strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis or to prevent, ameliorate or delay the onset or progression of their clinical manifestations. 2. Improved upstream, downstream conjugation process. 3. Upstream fermentation medium is devoid of casein/tryptone digestion, Cas amino acids. 4. Use of i) combination of antifoam, Soy Peptone and Yeast Extract ii) particular OD 36 to 39% and iii) osmolarity 400 to 600 mOsmol/kg resulting in improvement in polysaccharide yield at harvest stage of >50%5. Improved purification method with low endotoxin <10 EU/μg, low protein <1% and low nucleic acid content <2% in which the purification process is i) free of sodium deoxycholate (DOC) being a product of animal origin, even its residual presence in the final product may lead to non-acceptance of the product by regulatory agencies and certain religious communities ii) free of Triton X, iii) free of ammonium sulfate iv) without any adsorbents (hydroxylapatite, calcium phosphate or apatite)6. Improved conjugation efficiency (>60%), improved conjugate yield (>50%) and conjugate stability in which i) Ps:Pr:EDAC ratio is 1:1:2, ii) stability indicating free polysaccharide is <5% (preferably <3%) and free protein is <5% (preferably <4%)7. Best immunogenicity for conjugate attributed to i) Vi polysaccharide - protein conjugate having a peculiar size between 1200 kDa to 1600 kDa. and ii) polysaccharide used for conjugation has average molecular weight between 150 and 300 kDa (preferably between 150 and 250 kDa)8. The immunogenic composition is stable at 2 to 8°C, 25°C and 40°C for a period of six months and free polysaccharide after 6 months not exceeding 7.5% for polysaccharide 180 to 220 kDa and free polysaccharide after 6 months not exceeding 10.5% for polysaccharide of 388/80/45 kDa.9. The groups of mice injected with bivalent (typhoid and paratyphoid) SIIPL vaccine 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 exhibited greater than 4-fold higher IgG induction (compared to mice administered with unconjugated Vi PS or unconjugated SIIPL O-SP A), thus indicating the immunogenic potential of the bivalent SIIPL vaccine containing the combination of Vi TT and SIIPL O-SP A (DT/TT/CRM).10. Minimum components involved in the vaccine composition.

EXAMPLES

[00265] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those skilled in the art that the compositions and techniques described in the following examples represent techniques discovered by the inventor to function well in the practice of the invention and therefore may be considered as constituting preferred modes for its practice. However, those skilled in the art should, in light of the present invention, realize that many changes can be made in the specific embodiments that are described and still obtain a similar or similar result without departing from the spirit and scope of the invention.

Example 1: A) STRAINS:

[00266] Strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition of the present invention may include: Salmonella enterica serovar typhi TY2 strain having “tviB” gene specific for Vi polysaccharide (Identified by Geneombio Technologies Private Limited, Pune and isolated from stool sample of typhoid patient at Villoo Memorial Hospital, Poonawalla, Pune); Salmonella enterica serovar typhi deposited with NCMR-NCCS; Pune, an International Depository Authority which has assigned Accession No. MCC 0193; Strain designation-PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. Paratyphi A: ATCC 9150 purchased from Chromachemie Laboratory Private Limited, Bangalore, CMCC50073, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis R11; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV 3453219; 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. In addition, any attenuated strain of Salmonella serovars (S. typhi, S. Paratyphi A, S. enteritidis and S. typhimurium) can be used for the preparation of the immunogenic composition of the present invention.

[00267] WO 2018037365, WO 2019016654 and WO 2020075184 are being incorporated by reference to the preparation of diphtheria toxoid (DT), tetanus toxoid (TT), inactivated whole cell (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).

[00268] WO 2018037365 is being incorporated by reference to the preparation of inactivated poliovirus and inactivated rotavirus.

[00269] WO2013114268 is being incorporated by reference to the preparation of meningococcal polysaccharide antigens of serotypes A, C, W, X and Y.B) The method for obtaining the polysaccharide derived from Salmonella serovar strains S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, by fed-batch process, comprises the following steps: (UPWARD FERMENTATION)1) Inoculation and harvesting of stage S1:

[00270] The 0.5 mL culture from the cell bank vial is diluted to 5 mL with medium and the loop culture from this is streaked onto the SCDA plate and incubated at 36 ± 0.5°C for 28 to 32 hours.2) Inoculation and harvesting of S2 stage:

[00271] Colonies from 10 wells isolated from the S1 stage plate are inoculated into the 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 between 2.5 to 3.5.3) Inoculation and harvesting of the S3 stage:

[00272] When the optical density (OD) of the S2 stage culture reaches between 2.5 to 3.5, expand the culture from 40 ml to 800 ml and distribute the contents of 160 ml into five 1 liter flasks each. Incubate the flasks at 36 ± 0.5 °C and 150 ± 10 rpm until the OD of the culture reaches a range of 2.5 to 3.5.4) Inoculation and harvest of S4 stage (20 L fermenter):

[00273] When the OD of the S3 stage culture reaches between 2.5 to 3.5, pool the culture from all five vials into the seed bottle assembly and inoculate into a 20 L fermenter. Continue fermentation using the following parameters:Fermentation Process Parameters:

[00274] Start the feeding medium from the 3rd hour onwards and increase proportionally with the DO. Continue the fermentation for 13 to 15 hours.5) Formalin inactivation of stage S4:

[00275] After the S4 stage is completed, add the formaldehyde solution to the fermenter making the final concentration 0.5 to 1.0% and incubate the culture at 36°C ± 2°C for 8 to 10 hours.6) Cell separation by centrifugation:

[00276] After the inactivation step is complete, clarify the broth by centrifugation and collect the supernatant. Perform centrifugation using the following parameters,

[00277] After centrifugation, the supernatant is filtered using a depth filter.8) 0.2μ Filtration:

[00278] After depth filtration, the filtrate is subjected to a 0.2 μ filtration. 81. Optimization of media for growth of Salmonella typhi S. Paratyphi A; S. typhimurium and S. enteritidis.

[00279] The following experiments were performed to optimize the medium and batch parameters; Experiment No.: 01 - Frantz medium was selected for the experiment because it is the most commonly used medium for the growth of Gram negative organisms and was evaluated for the growth of Salmonella Typhi. Frantz medium was used for seed development and fermentation. Glucose as a carbon source was composed in the feeding medium.

[00280] The seed was grown in disposable flasks (125 ml and 500 ml) and inoculated into a 2 L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Food was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and agitation at 150 to 500 RPM (in cascade mode to maintain DO). Antifoam was added intermittently to control foaming during fermentation. Observations: Conclusion: Frantz medium was found to support the growth of Salmonella typhi organism to a limited extent. Further optimization of the medium was required. Experiment No.: 02 - Yeast extract was added to the fermentation and feeding medium to support cell growth. Procedure:

[00281] The seed was grown in disposable flasks (125ml and 500ml) and inoculated in 2L of fermenter. After inoculation, fermentation was carried out in fed-batch mode. Food was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and agitation at 150 to 500 RPM (in cascade mode to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: The addition of yeast extract was found to support the growth of Salmonella typhi to a limited extent. Further optimization of the medium was required. Experiment No.: 03 - Soy peptone was added to the fermentation and feeding medium to support cell growth. Procedure:

[00282] The seed was grown in disposable flasks (125ml and 500ml) and inoculated in a 2L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Food was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and agitation at 150 to 500RPM (in cascade mode to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: It was found that the addition of soy peptone supports the growth of the organism Salmonella Typhi. Optimizations of batch parameters were necessary for manufacturing. Experiment No.: 04 - Ctobe antifoam was replaced by Struktol antifoam for better cell growth. Procedure:

[00283] The seed was grown in disposable flasks (125ml and 500ml) and inoculated into a 2 L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Food was added to the fermenter to support the growth of the Salmonella Typhi organism. The batch was operated at a temperature of 36°C, pH 7.00, 25% dissolved oxygen (DO) and agitation at 150 to 500RPM (in cascade mode to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: Replacement of antifoam C with antifoam Struktol was found to improve the growth of Salmonella Typhi organism. Experiment No.: 05 - Standard growth of Salmonella typhi at the following fermentation parameters at 20 L scale batch was studied. For the Salmonella Typhi batches, suitable parameters were set. Dissolved oxygen and osmolarity were controlled around experimental set points. Procedure: The seed was grown in disposable flasks (250ml and 1L) and inoculated into a 20L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi. The batch was operated at 36°C temperature, 7.0 pH and 150 to 500 RPM agitation (cascade to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: From the observations, it was concluded that optimization of parameters mentioned in the experimental set point table of this experiment were required for growth of Salmonella Typhi organism. Experiment No.: 06 - Standard growth of Salmonella typhi at the following fermentation parameters at 20L scale batch was studied. For the batches of Salmonella Typhi, suitable parameters were set. Dissolved oxygen and osmolarity were controlled around experimental set points. Procedure: The seed was grown in disposable flasks (250ml and 1L) and inoculated into a 20L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi. The batch was operated at 36°C temperature, 7.0 pH and 150 to 500 RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: From the observations, it was concluded that optimization of parameters mentioned in the experimental set point table of this experiment were required for growth of Salmonella Typhi organism. Experiment No.: 07 - Standard growth of Salmonella typhi at the following fermentation parameters at 20 L scale batch was studied. For the batches of Salmonella Typhi, suitable parameters were set. Dissolved oxygen and osmolarity were controlled around experimental set points. Procedure: Seed was grown in disposable flasks (250 ml and 1 L) and inoculated into 20 L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi organism. The batch was operated at 36°C temperature, 7.0 pH and 150 to 500 RPM agitation (cascade to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: From the observations, it was concluded that optimization of parameters mentioned in the experimental set point table of this experiment were required for growth of Salmonella Typhi organism. Experiment No.: 08 - Standard growth of Salmonella typhi at the following fermentation parameters at 20 L batch scale was studied. For the batches of Salmonella Typhi, suitable parameters were set. Dissolved oxygen and osmolarity were controlled around experimental set points. Procedure: The seed was grown in disposable flasks (250ml and 1L) and inoculated into a 20L fermenter. After inoculation, fermentation was performed in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi. The batch was operated at 36°C temperature, 7.0 pH and 150 to 500 RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: From the observations, it was concluded that optimization of parameters mentioned in the experimental set point table of this experiment were required for growth of Salmonella Typhi organism. Experiment No. 09 - Standard growth of Salmonella Typhi at the following fermentation parameters at 20 L scale batch was studied. For the batches of Salmonella Typhi, suitable parameters were set. Dissolved oxygen and osmolarity were controlled around experimental set points. Procedure: The seed was grown in disposable flasks (250 ml and 1 L) and inoculated into a 20 L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi. The batch was operated at a temperature of 36 °C, pH 7.00, and agitation at 150 to 500 RPM (in cascade mode to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion: From the observations, it was concluded that optimization of parameters mentioned in the experimental set point table of this experiment were required for growth of Salmonella Typhi organism. Experiment No. 10 - Standard growth of Salmonella Typhi at the following fermentation parameters at 20 L scale batch was studied. For the batches of Salmonella Typhi, suitable parameters were set. Dissolved oxygen and osmolarity were controlled around experimental set points. Procedure: Seed was grown in disposable flasks (250 ml and 1 L) and inoculated into 20 L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added to the fermenter to support the growth of Salmonella Typhi organism. The batch was operated at 36°C temperature, 7.0 pH and 150 to 500 RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittently to control foam formation during fermentation. Observations: Conclusion:

[00284] A fed-batch cultivation method for obtaining a high-yield crop of polysaccharide derived from strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis, by fed-batch process involved the use of the combination of an antifoaming agent J673 STRUKTOL, Difco™ SelectPhytone™ Soy Peptone UF in a range of 40 to 70 g/L and Difco™ Yeast Extract Yeast Extract, UF in a range of 40 to 70 g/L during cultivation results in better harvest yield (100 to 700 mg/L), and the fermentation parameters comprise pH maintained in the range of 6.7 to 7.1, temperature maintained in the range of 34.0 to 38.0°C, Dissolved Oxygen Level maintained between 36 to 39%, Agitation (rpm) maintained between 150 to 500 and osmolarity 400 to 600 mOsmol/kg. Example 2C) The method of purification of polysaccharide derived from strains of Salmonella serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis (DOWNSTREAM PURIFICATION)C1) ViPs fermentation crop subjected to the following downstream purification steps to obtain the desired quality of Vi polysaccharide (ViPs):n) Clarification of a bacterial capsular polysaccharide crop by direct flow filtration (DFF) through at least one membrane with a pore size of about 0.2 micrometers;o) concentration by tangential flow ultrafiltration (TFF) and buffer exchange (20 mM Tris buffer pH-7.5 containing 5 mM EDTA) by diafiltration (DF) using a membrane with a 100 kDa molecular weight cutoff (MWCO);p) treatment with SDS (20% SDS feedstock input), ethylene diamine tetraacetic acid (EDTA) (4 to 10 mM), and sodium acetate (5% to 10%) to denaturation of proteins, nucleic acids and lipopolysaccharides;q) Ethanol precipitation (40% to 70%);r) Centrifugation and filtration by direct flow filtration (DFF) through at least one clarifying filter with a pore size of about 0.2 μM;s) treatment with 2M 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 30 kDa molecular weight cut-off (MWCO) membrane;u) u) Selective precipitation of PS by CTAB (12% CTAB feedstock input);v) centrifugation and filtration by direct flow filtration (DFF) through at least one clarifying filter with a pore size of about 0.2 μM; direct flow filtration (DFF) through at least one clarifying filter with 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% to 70%) in the presence of 1M NaCl (0.1M to 2M);x) Selective precipitation of polysaccharide using 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 30 kDa molecular weight cut-off (MWCO) membrane; ez) Sterile filtration through at least one sterile filter with a pore size of about 0.2 micrometers under sterile conditions and stored at <- 20 °C2) Salmonella Paratyphi O-specific polysaccharide (OSP) from Lipopolysaccharide (LPS) fermentation harvest subjected to the following downstream purification steps to obtain the desired quality of Salmonella Paratyphi Lipopolysaccharide A (LPS) O-specific polysaccharide:2) Centrifugation at 7000 rpm for 30 min at 4 °C, collected cell pellet (~1Kg) and supernatant and cell pellet suspended in 15 L of 1M NaCl and shaken for 1 hour at room temperature.3) Concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using 0.45 µm Prostak cassette μm and 30kDa molecular weight cut-off (MWCO) membrane;4) Acid hydrolysis of LPS with 1% acetic acid (pH ~ 2.8 to 3.0) at 90°C temperature for 180 minutes5) The mixture cooled to room temperature (RT) and removal of impurity precipitation by centrifugation at 7000 rpm for 45 minutes at 25°C6) Neutralization collected the supernatant in glass bottle and neutralized to pH - 7.0 with ammonia liquor7) Clarification by direct flow filtration (DFF) through at least one membrane with a pore size of about 0.45 and 0.2 micrometers;8) Added sodium deoxycholate raw material solution to obtain 1% final concentration and incubated for 30 minutes under shaking at 30°C temperature, pH adjusted to 2.0 with acetic acid and incubated for 15 minutes under shaking at 30°C9) Centrifugation at 7000 rpm for 45 minutes at 25°C10) Direct flow filtration (DFF) through at least one membrane with a pore size of about 0.45 and 0.2 micrometers;11) Concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane with a 10 kDa molecular weight cut-off (MWCO)12) Sterile filtration through at least one sterile filter with a pore size of about 0.2 micrometers under sterile conditions to obtain purified O-specific polysaccharide (OSP). Results and Interpretation:

[00285] The improved ViPS purification method has clear advantages in terms of ease of operation as well as several other advantages such as, > Improved purification of polysaccharides (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, viscosity) when sodium acetate (6%) used vs sodium acetate (2%) When sodium acetate (6%) used - Values for % recovery of DSP - 50% Viscosity of Ps - Data not available Polydispersity of Ps - Data not available O-acetyl - 2.9 mmol/gm of PS When sodium acetate (2%) used - Values for % recovery of DSP - 40% Viscosity of Ps - Data not available Polydispersity of Ps - Data not available O-acetyl - 2.6 mmol/gm of PS

[00286] Sodium acetate reacts with nucleic acids. It decomposes into Na+ and (CH3COO)- . The positively charged sodium ion neutralizes the negatively charged PO3- of nucleic acids; thus helps in precipitation of nucleic acids. Therefore, a higher concentration i.e. 6% sodium acetate effectively precipitates host cell impurities like nucleic acids.> Improved purification of polysaccharides (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, viscosity) CTAB (3%) vs CTAB (0.5, 1%). CTAB is a cationic quaternary amine based surfactant. It interacts with anionic polysaccharide (ionic interaction) and then decreases its solubility, hence precipitates the polysaccharide from the solution. Hence, higher concentration of CTAB (2%) always precipitates higher amount of polysaccharide which gives higher yields and removal of protein impurities is an added advantage.> Improved purification of polysaccharides (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, poly-dispersity, viscosity) for current DOC Vs DOC based process-free process.Advantage of Improved DOC process-free method1. Regulatory Issue: DOC is an animal-derived component and is not HALAL compliant. It is manufactured and supplied by a single supplier worldwide whereas SDS is a synthetic detergent, HALAL certified and with multiple suppliers available globally.2. Functional issue: DOC decomposes endotoxins without affecting the chemical composition and once the detergent is removed it can recover its biological activity, while SDS, due to its amphipathic nature and higher aggregation number, denatures and solubilizes proteins well and also breaks endotoxins irreversibly into their monomeric units.> The new improved Vi PS purification method developed without the use of sodium deoxycholate (DOC), which is a component of animal origin or phenol. Improved method based on reverse purification of polysaccharides in which host cell impurities such as proteins, nucleic acids and lipopolysaccharides will be removed in initial steps using sodium acetate (6%), sodium dodecyl sulfate (2%) and ethanol (40%) and concentration and diafiltration using 30kDa cutoff membranes in different steps, then polysaccharide precipitated by CTAB cationic detergent and carried out polishing purification steps to obtain higher yields of purified Vi polysaccharide meeting WHO specifications.> The purification process results in significant recovery of about 40% to 65% with desired O-acetyl levels (greater than 2.0 mmol/g polysaccharide), purified Vi polysaccharide with yield in the range of 400 to 4000 mg/L, average molecular weight found to be in the range of 40 to 400 kDa, contains less than 1% protein nas/peptides, less than 2% nucleic acids, less than 100 EU endotoxins per μg polysaccharide (PS), molecular size distribution (greater than 50% of PS is eluted before a distribution coefficient (KD) of 0.25 is reached)> The O-specific polysaccharide (OSP) purification process results in significant reduction of endotoxin (<100EU endotoxin per μg PS), protein (<1%) and nucleic acid (<2%) impurities, increased recovery of capsular polysaccharide suitably in the range of 40% to 65%, with desired O-acetyl levels (>2.0 mmol/g polysaccharide), molecular size distribution (>50% of PS is eluted before a distribution coefficient (KD) of 0.25 is reached) and average molecular weight of O-specific polysaccharide (OSP) purified was found in the range of 40 to 200 kDa.Example 3:D) The method of conjugating the polysaccharide derived from Salmonella strains serovars S. typhi; S. Paratyphi A; S. typhimurium and S. enteritidis to a carrier protein.

[00287] The carrier protein used for conjugation with the Salmonella Typhi Vi polysaccharide is tetanus toxoid. Polysaccharides derived 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.

[00288] In accordance with the present invention, CRM197 is purchased from Pseudomonas fluorescens Recombinant Strain CS463-003 (MB101) from Pfenex USA.

[00289] According to the present invention, TT is purchased from Clostridium Tetani (Harvard No 49205) obtained from Central Research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. Central Research Institute (CRI) purchased this strain from NVI, Netherlands.

[00290] According to the present invention, DT is produced cured from cultures of Corynebacteriumdiphtheriae strain ParkWilliams Number 8, the strain is obtained from Central Research Institute (CRI), NationalControlAuthority, Kasauli, Himachal Pradesh, India. Central Research Institute (CRI) purchased this strain from Wellcome Research Laboratories.D1) Preparation of monovalent conjugate of Salmonella typhi using Carbodiimide Chemistry:Step 1: Concentration and Derivatization of TTProcedure:a) GFC purified TT (monomeric content more than 90%) was concentrated using 10 kDa membrane to reach protein concentration of 12 mg/ml NLT (Lowry assay).b) Derivatization ratio was used as given below and calculated required quantities accordingly.Pr:ADH:EDC as 1:6.0:1Derivatization Scale: 13 gmc) Concentrated TT (in 0.9% NaCl), 1M MES pH 6.0, ADH solution (dissolved in 0.1M MES pH 6.0) and EDC solution (dissolved in 0.1M MES pH 6.0) were added sequentially and made the required final volume by addition of 0.1 M MES buffer pH 6.0. The final concentration of Pr in reaction was ~4.5mg/ml.d) The reaction mixture was stirred and allowed to continue for about 1 h at pH 5.90. Then, the derivatization reaction was quenched by adjusting the pH above 7.5 using 0.1 M phosphate buffer containing EDTA pH 8.0. e) The quenched reaction mixture was diafiltered using 10 kDa cutoff membrane against 10 mM phosphate buffer using 22 volumes followed by 0.1 M MES buffer pH 6.0 at least 12 volumes to remove unreacted portions and other residues. f) The derivatized sample was analyzed for total Pr concentration by Lowry assay and Extent of derivatization value by colorimetric assay (TNBS). Results: Derivatized Pr concentration by Lowry assay: 15 mg/ml Extent of derivatization value (DOA): 19 Percent recovery: ~75% Step 2: Conjugation of ViPs for TT Derivatization Procedure: Conjugation of ViPS derived TT performed using chemistry Carbodiimide.The following parameters of PS and Pr were used for Conjugation.a. The ratio for conjugation reaction was used on the conjugation scale of 10gmPs:Pr:EDC::1:0.9:1.75 (+0.5 for Pr and EDC).b. The required batch volume of PS was added into a pre-sterilized glass bottle.c. 1M MES buffer pH 6.0 (raw material buffer) was added to the measured volume of PS to reach 0.1M MES concentration.d. The mixture was shaken at ~100 rpm for homogeneous mixing.e. Then the measured volume of Derivatized TT was added to the bottle containing Ps.f. f. After addition of TT, immediately freshly prepared EDC (dissolved in 0.1 M MES buffer pH 6.0) was added to the above reaction.g. The pH was observed and recorded (pH 6.0+0.5) h. The sample was analyzed at different time intervals using SE-HPLC. The percentage of conjugation conversion and protein consumption were monitored.i. The reaction was quenched after 1.45 h by increasing the pH to 7.5 using 0.1 M phosphate buffer containing EDTA, when <90% protein consumed.j. The quenched reaction mixture was stored at 2 to 8°C until further use.Step 3: Purification of Quenched Vi-TT Conjugate

[00291] Purification of the quenched conjugate was performed by two methods performed to remove unreacted Ps, unreacted Pr, other residues using the following parameters; 1 . Ultrafiltration method: Following parameters used for diafiltration; Diafiltration Scale: 8.5 g Ps concentration during D/F: ~2mg/ml Membrane Cutoff: 300 kDa Membrane Throughput: Pall Membrane Area: 2.5 m2 Buffer A used: 10 mM PBS minus 25 volumes Buffer B used: 0.9% NaCl 30 volumes Final volume: 6.5 liters The purified conjugate was filtered using 0.2 μM filter. 2 . Gel filtration chromatography:

[00292] The quenched conjugate of about 1.5 g was concentrated onto 0.1 m2 of 300 kDa cutoff membrane ~2 to 4 mg/ml. Conjugate purification performed to remove unbound PS, unbound TT and residual EDC using gel filtration chromatography.

[00293] Two separate GFC runs were performed with the same parameters for purification.

[00294] Following the chromatographic conditions used for purification. 1 Total 27 collected fractions were (100 ml each) analyzed on SE-HPLC and pooled on the basis of chromatographic profile.3 The fractions were analyzed for Ps content by HPAD method, Pr content by Lowry method, % free Ps by DOC-HPAD method.4 Based on the analysis, the fractions were pooled (1 to 23) and filtered using 0.22μM filter.5 The sample was analyzed for Ps content, Pr content and free Ps and endotoxin analysis, O-acetyl content, sterility, pH value and endotoxin analysis.RESULTS AND INTERPRETATION: Comparative data of conjugation process- Improved conjugation efficiency, improved conjugate yield and stability (Free Ps, Free Pr) of SIIPL typhoid conjugate where Ps:Pr:EDAC ratio is 1:1:2 Vs other Ps:Pr:EDAC ratios Improved conjugation efficiency: Observed conjugation conversion percentage more than 90%. Improved conjugate yield: Purification carried out using different methods i.e. ultrafiltration method and gel filtration method with varying conjugation purification range and recovery found from 40 to 87%. Ratio used 1:1:2 vs 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 for Vi-TT conjugation and it was found that the lower protein with 1.5 EDC can provide an optimal conjugation conversion (%).

[00295] It was found that Vi Ps with variable sizes can be conjugated to ADH-activated TT.

[00296] Different ratio used as Ps:Pr:EDC::1:0.7:1.8, 1:0.8:1.8,1:0.9:2 and 1:1:2 at lower pH in different purification techniques results in more than 0.5 PS/Pr ratio, lower free ps with good recovery of conjugates.D2) Preparation of monovalent conjugates from Salmonella Paratyphi:

[00297] Two types of conjugation chemistry (cyanylation and carbodiimide chemistry) were applied for the conjugation of Paratyphi OSP with Diphtheria Toxoid Carrier Protein (DT), CRM197, and Tetanus Toxoid (TT): 1) Cyanylation chemistry-based conjugation of Paratyphi OSP with Diphtheria Toxoid Carrier Protein (DT), CRM197, and Tetanus Toxoid (TT) A) Derivatization of Diphtheria Toxoid (DT), (Addition of ADH Ligand) using carbodiimide chemistry and conjugation with OSP Concentrate using cyanylation chemistry B) Derivatization of CRM197, Tetanus Toxoid (TT) (Addition of ADH Ligand) using carbodiimide chemistry and conjugation with OSP Concentrate using cyanylation chemistry C) Derivatization of Tetanus Toxoid (TT) (Addition of ADH Ligand) using carbodiimide chemistry and conjugation with OSP Concentrate using cyanylation chemistry1A) Preparation of S. Paratyphi Conjugates using Diphtheria Toxoid as carrier protein.1) Protein Derivatization:

[00298] High monomeric diphtheria toxoid (DT) was concentrated to (10 to 20 mg/ml) on a 10kDa membrane and analyzed for protein content

[00299] To the freshly prepared concentrated DT 1 M MES Buffer was added adipic acid dihydrazide (ADH) (dissolved 75 to 100 mg/ml in 100 mM MES buffer pH:5.8) in the ratio of 1:10 by weight and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved 30 to 40 mg/ml in 100 mM MES buffer pH:5.8) in the ratio of 1:1 by weight. The reaction was continued at pH 5.8 for about 1 hour and then the reaction mixture was diafiltered in 10 kDa TFF in 50 mM borate buffer pH 9.0 to remove residues and unreacted components. The final sample was analyzed for protein content and degree of derivatization 2) Conjugation of S. Paratyphi A polysaccharide (OSP) and ADH-derivatized DT: Two experiments were performed by altering the ratio of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) Experiment No: 1 - OSP was concentrated on 10 kDa membrane to reach a concentration of (10 to 15 mg/ml).

[00300] To the PS Concentrate was added 0.9% NaCl 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 changed to 9.5 immediately with 2.5 M NaOH and held for up to 3 minutes. Then, the protein was added in a weight ratio of 1:0.8 PS:PR:CPIP as 1:0.8:1.3

[00301] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min for monitoring protein conversion. The reaction was quenched after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. See Figure 1: Comparison of Polysaccharide, Protein, and Conjugate (PS:PR:CPIP 1:0.8:1.3)3) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyopearl HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/h and allowed to settle and 1M NaCl at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/h and the conjugate was loaded onto the column and fractions collected at 1 min interval. Fractions 2 to 9 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis. See Figure 2: Chromatogram of GFC-purified OSP-DT ADH Conjugate (PS:PR:CPIP as 1:0.8:1.3)Experiment No: 2 -

[00302] To the Concentrated PS was added 0.9% NaCl and freshly prepared CPIP solution was added (114 mg/ml in acetonitrile) to the polysaccharide at a weight ratio of 1:1.1. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 min. Then, protein was added at a ratio of 1:0.8 by weight PS:PR:CPIP as 1:0.8:1.1.

[00303] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min to monitor protein conversion. The reaction was quenched after 3 to 4 hours by the addition of 2M Glycine at 10 times that of PS by weight. See Figure 3: Comparison of Polysaccharide, Protein, and Conjugate (PS:PR:CPIP 1:0.8:1.1) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyoperal HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/h and allowed to settle and 1M NaCl at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/h and the conjugate was loaded onto the column and fractions were collected at 1 min interval. Fractions 2 to 8 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis. Refer to Figure 4: Chromatogram of GFC-purified OSP-DT ADH Conjugate (PS:PR:CPIP 1:0.8:1.1)Conclusion: Conjugation of Paratyphi A PS using DT derived from ADH was successful and PS/PR ratio was found to be satisfactory.1B. Preparation of S. Paratyphi Conjugate using CRM197 as carrier protein1) Protein derivatization: Cross-reactive mutant (CRM197) received from production department (SIIPL) was taken for derivatization

[00304] To the freshly prepared concentrated CRM197, 1M MES buffer was added and adipic acid dihydrazide (ADH) (dissolved 75 to 100 mg/ml in 100 mM MES buffer pH:6.5) in the ratio of 1:3.5 by weight and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved 30 to 40 mg/ml in 100 mM MES buffer pH:6.5) in the ratio of 1:0.25 by weight. 5% Tween 80 was added. The final reaction volume was completed using 100 mM MES buffer to reach a final concentration of 3 to 4 mg/ml, the reaction continued at pH 6.5 for about 3 hours, and then the reaction mixture was diafiltered into 10 kDa TFF in 50 mM borate buffer and 0.005% Tween 80 pH 9.0 to remove residues and unreacted components. The final sample was analyzed for protein content and degree of derivatization. Brief description of assay or reference: Protein content was measured by BCA (bicinchoninic acid) assay. 2) Conjugation of S. Paratyphi A polysaccharide (OSP) and ADH-derivatized CRM197:

[00305] The OSP received from the DSP was concentrated on the 10 kDa membrane to reach a concentration of (10 to 15 mg/ml).

[00306] To the PS Concentrate was added 0.9% NaCl and freshly prepared CPIP solution was added (114 mg/ml in acetonitrile) to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, the protein was added in a 1:1 by weight ratio PS:PR:CPIP as 1:1:1.3

[00307] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min to monitor protein conversion. The reaction was quenched after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. See Figure 5 and 6: Comparison of Polysaccharide, Protein, and Conjugate (PS:PR:CPIP as 1:1:1.3)3) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyoperal HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/h and allowed to settle and 1M NaCL at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/h and the conjugate was loaded onto the column and fractions collected at 1 min interval. Fractions 2 to 7 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis. Refer to Figure 7: Chromatogram of GFC purified OSP-DT ADH Conjugate (PS:PR:CPIP as 1:1:1.3)Conclusion: Conjugation of Paratyphi A to PS using CRM197 derivative ADH was found to be successful and PS/PR ratio was found to be satisfactory.1C) Preparation of S. Paratyphi Conjugates using Tetanus Toxoid as carrier protein:1) Protein derivatization: High monomeric tetanus toxoid (TT) received from production department (SIIPL) was concentrated to (15 to 20mg/ml) on 30 kDa membrane and analyzed for protein content

[00308] Freshly prepared concentrated 1 M TT MES buffer was added to adipic acid dihydrazide (ADH) (dissolved 75 to 100 mg/ml in 100 mM MES buffer pH:6.0) at a ratio of 1:10 by weight and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30 to 40 mg/ml in 100 mM MES buffer pH:6.0) at a ratio of 1:1 by weight. The reaction was continued at pH 6.0 for about 1 hour and then the reaction mixture was diafiltered into 30 kDa TFF in 10 mM phosphate buffer pH 7.2 to remove residues and unreacted components. The final sample was analyzed for protein content and degree of derivatizationBrief description of the assay or reference: Protein content was measured by the Lowry assay. 2) Conjugation of S. Paratyphi A polysaccharide (OSP) and ADH-derivatized TT:

[00309] The OSP received from the DSP Team was concentrated on the 10 kDa membrane to achieve a concentration of (10 to 13mg/ml). Two experiments were performed by changing the ratio of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) Experiment No. 1

[00310] To the concentrated PS was added 0.9% NaCl and freshly prepared CPIP solution was added (114 mg/ml in acetonitrile) to the polysaccharide in a weight ratio of 1:1.25. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, protein was added in a ratio of 1:0.8 by weight PS:PR:CPIP as 1:0.8:1.25

[00311] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min for monitoring protein conversion. The reaction was quenched after 3 to 4 hours by addition of 2M Glycine 10 times that of PS by weight. 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 300 kDa TFF membrane diafiltration in 10 mM PBS pH 7.2 followed by 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis. See Figure 9: Chromatogram of GFC-purified OSP-DT ADH Conjugate (PS:PR:CPIP as 1:0.8:1.25)Experiment No: 2

[00312] To the PS Concentrate was added 0.9% NaCl and freshly prepared CPIP solution was added (114 mg/ml in acetonitrile) to the polysaccharide in a weight ratio of 1:1.3. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 min. Then, the protein was added in a weight ratio of 1:0.9 PS:PR:CPIP1:0.9:1.3

[00313] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min for monitoring protein conversion. The reaction was quenched after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. See Figure 10: Comparison of Polysaccharide, Protein, and Conjugate (PS:PR:CPIP1:0.9:1.3)3) Conjugate Purification by Ultrafiltration: The quenched conjugate was purified by 300 kDa TFF membrane diafiltration in 10 mM PBS pH 7.2 followed by 10 mM Tris buffer pH 7.2. The final concentrated sample was sent for analysis. See Figure 11: Chromatogram of GFC-purified OSP-DT ADH Conjugate (PS:PR:CPIP1:0.9:1.3)Conclusion: Conjugation of Paratyphi A to PS using ADH-derivatized TT was successful, two different types of diafiltration strategy were applied and both processes gave satisfactory PS/Pr ratio.2) Carbodiimide chemistry based conjugation of Paratyphi OSP to carrier protein Diphtheria Toxoid (DT), CRM197 and Tetanus Toxoid (TT)A) Derivatization of OSP Concentrate (Addition of ADH Ligand) using cyanylation chemistry and conjugation with Tetanus Toxoid (TT) using carbodiimide chemistry. B) Derivatization of Concentrated OSP (Addition of ADH Ligand) using cyanylation chemistry and conjugation with Diphtheria Toxoid (DT) using carbodiimide chemistry. C) Derivatization of Concentrated OSP (Addition of ADH Ligand) using cyanylation chemistry and conjugation with CRM197 using carbodiimide chemistry. 2A) Preparation of S. Paratyphi Conjugates using Tetanus Toxoid as carrier protein. 1) Derivatization of Polysaccharides: The OSP received from DSP Team was concentrated on 10 kDa membrane to reach a concentration of (10 to 13 mg/ml).

[00314] To the Concentrated PS was added 0.9% NaCl and freshly prepared CPIP solution (114 mg/ml in acetonitrile) was added to the polysaccharide in a ratio of 1:0.7 by weight. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, adipic acid dihydrazide (ADH) (dissolved 75 to 100 mg/ml in 0.5 M sodium bicarbonate buffer pH: 8.0) at the ratio of 1:10 by weight PS:ADH:CPIP as 1:10:0.7 The reaction was continued at pH 9.5 for about 2 hours, quenched using 2M Glycine 10, and then the reaction mixture was diafiltered through 10 kDa TFF in 100 mM MES buffer pH 6.0 to remove residues and unreacted components. The final sample was analyzed for polysaccharide content. 2) Protein preparation: High monomeric tetanus toxoid (TT) received from the production department (SIIPL) was concentrated to (10 to 15 mg/ml) on a 30 kDa membrane and analyzed for protein contentBrief description of the assay or reference: Protein content was measured by Lowry assay. 3) Conjugation of ADH-derivatized S. Paratyphi A polysaccharide (OSP) and concentrated TT: Two experiments were performed at different temperatures to verify the conversion of Pr. Experiment No. 1

[00315] To the ADH derivatized PS, protein was added at 1:0.9 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved 30 to 40 mg/ml in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0 Temperature 6°C ratio of PS:PR:EDC as 1:0.9:0.86

[00316] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min for monitoring protein conversion. The reaction was quenched after 23 hours by the addition of 100 mM phosphate buffer containing EDTA. See Figure 12: Chromatogram depicting the progression of the conjugation reaction (Abrupt cessation of the reaction at 23 hours) 4) Conjugate Purification by Ultrafiltration: The quenched conjugate was purified by diafiltration 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. Experiment No. 2

[00317] To the ADH-derivatized PS, protein was added in the ratio of 1:0.9 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved 30 to 40 mg/ml in 100 mM MES buffer) in the ratio of 1:0.86 by weight. The reaction was continued at pH 6.0, temperature 10°C, ratio of PS:PR:EDC as 1:0.9:0.86

[00318] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min to monitor protein conversion. The reaction was quenched after 4 h by the addition of 100 mM phosphate buffer containing EDTA. See Figure 13: Chromatogram depicting the progression of the conjugation reaction. 4) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyoperal HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/h and allowed to settle and 1 M NaCl 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/h and the conjugate was loaded onto the column and fractions collected at 1 min interval. Fractions 2 to 10 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis. Conclusion: Using reverse conjugation chemistry (PS activation), conjugation of Paratyphi A to PS derived from ADH and concentrated TT was successful by increasing the temperature from 6 to 10°C. The conjugation rate of the reaction was increased, the PS/Pr ratio was very low in both reactions. 2B) Preparation of S. Paratyphi Conjugates using Diphytheria Toxoid as carrier protein. 1) Polysaccharide Derivatization: The OSP received from the DSP Team was concentrated on 10 kDa membrane to achieve a concentration of (10 to 13mg/ml).

[00319] To the Concentrated PS was added 0.9% NaCl and freshly prepared CPIP solution (0.114 mg/ml in acetonitrile) was added to the polysaccharide in a weight ratio of 1:0.7. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, adipic acid dihydrazide (ADH) (dissolved 75 to 100 mg/ml in 0.5 M sodium bicarbonate buffer pH:8.0) in a 1:10 weight ratio of PS:ADH:CPIP as 1:10:0.7. The reaction was continued at pH 9.5 for about 2 hours, stopped abruptly using 2 M Glycine 10, and then the reaction mixture was diafiltered through 10 kDa TFF in 100 mM MES buffer pH 6.0 to remove residues and unreacted components. The final sample was analyzed for polysaccharide content. 2) Protein preparation: High monomeric diphtheria toxoid (DT) received from the production department (SIIPL) was concentrated to (10 to 20mg/ml) on a 10 kDa membrane and analyzed for protein content. Brief description of assay or reference: Protein content was measured by Lowry assay. 3) Conjugation of ADH-derived S. Paratyphi A polysaccharide (OSP) and concentrated DT: The same conditions as for TT conjugate were applied to DT to verify the conversion of Pr.Experiment No. 1

[00320] To the ADH-derived PS, protein was added at 1:0.8 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved 30 to 40 mg/ml in 100 mM MES buffer) at 1:0.86 by weight ratio. The reaction was continued at pH 6.0, temperature 6°C, ratio of PS:PR:EDC as 1:0.8:0.86

[00321] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min for monitoring protein conversion. The reaction was quenched after 20 hours by the addition of 100 mM phosphate buffer containing EDTA. See Figure 14: Chromatogram depicting the progression of the conjugation reaction (Abrupt cessation of reaction at 20 hours) 4) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyoperal HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/h and allowed to settle and 1M NaCl at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/h and the conjugate was loaded onto the column and fractions collected at 1 min interval. Fractions 2 to 12 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis. See Figure 15: Chromatogram representing the purified conjugate (pooled fractions)Experiment No. 2

[00322] To the ADH-derived PS, protein was added at 1:1.0 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (dissolved at 30 to 40 mg/ml in 100 mM MES buffer) at a ratio of 1:0.9 by weight. The reaction was continued at pH 6.0, temperature 10°C, ratio of PS:PR:EDC as 1:1.0:0.9

[00323] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min for monitoring protein conversion. The reaction was quenched after 4 hours by the addition of 100 mM phosphate buffer containing EDTA. See Figure 16: Chromatogram depicting the progression of the conjugation reaction (Abrupt cessation of the reaction at 4 hours) 4) Conjugate Purification by Ultrafiltration: The quenched conjugate was purified by diafiltration of 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. See Figure 17: Chromatogram depicting the purified conjugate Conclusion: Conjugation of Paratyphi A PS derived from ADH using Carbodiimide chemistry was found successful using concentrated DT. The rate of conjugation reaction was increased by increasing the temperature, but the PS/Pr ratio was low in both experimentsD3) The method of conjugation of polysaccharide derived from Salmonella serovar S. typhimurium and S. enteritidis strains to a selected carrier protein of tetanus toxoid (TT), diphtheria toxoid (DT), or CRM197.1) Cyanylation chemistry-based conjugation of S. typhimurium and S. enteritidis polysaccharide to the carrier protein of Diphtheria toxoid (DT), CRM197, and Tetanus toxoid (TT)A) Derivatization of Diphtheria Toxoid (DT), (Addition of ADH ligand) using carbodiimide chemistry and conjugation with concentrated S. typhimurium and S. enteritidis polysaccharide using cyanylation chemistryB) Derivatization of CRM197, (Addition of ADH ligand) using carbodiimide chemistry and conjugation with S. typhimurium and S. enteritidis concentrate using cyanylation chemistryC) Derivatization of Tetanus Toxoid (TT) (Addition of ADH ligand) using carbodiimide chemistry and conjugation with S. typhimurium and S. enteritidis concentrate using cyanylation chemistryD) Cyanylation chemistry based conjugation (CPPT) of Paratyphi OSP with Diphtheria Toxoid (DT) carrier protein, CRM197 and Tetanus Toxoid (TT) without derivatization of polysaccharide or carrier protein.Procedure followed:A) Preparation of Polysaccharide Conjugates of S. typhimurium and S. enteritidis using Diphtheria Toxoid (DT) as carrier protein.1) Protein Derivatization:

[00324] High monomeric Diphtheria Toxoid (DT) was concentrated to (10 to 20 mg/ml) on a 10 kDa membrane and analyzed for protein content. To the freshly prepared concentrated DT 1 M MES buffer was added adipic acid dihydrazide (ADH) (75 to 100 mg/ml dissolved in 100 mM MES buffer pH: 5.8) in the ratio of 1:10 by weight and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (30 to 40 mg/ml dissolved in 100 mM MES buffer pH: 5.8) in the ratio of 1:1 by weight. The reaction was continued at pH 5.8 for about 1 hour and then the reaction mixture was diafiltered through 10 kDa TFF in 50 mM borate buffer pH 9.0 to remove residues and unreacted components. The final sample was analyzed for protein content and degree of derivatization. 2) Conjugation of S. typhi murium and S. enteritidis polysaccharide (PS) and DT-derivatized ADH: Two experiments were performed by changing the ratio of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) Exp. No.: 1 - S. typhimurium and S. enteritidis polysaccharide was concentrated on 10 kDa membrane to reach a concentration of (10 to 15mg/ml).

[00325] To the concentrated PS was added 0.9% NaCl and freshly prepared CPIP solution (114 mg/ml in acetonitrile) in polysaccharide was added in a ratio of 1:1.3 by weight. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, protein was added in a ratio of 1:0.8 by weight PS:PR:CPIP as 1:0.8:1.3

[00326] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/minute to monitor protein conversion. The reaction was stopped abruptly after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. 3) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyopearl HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/hour and allowed to settle and 1M NaCl at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/hour and the conjugate was loaded onto the column and fractions collected at 1 minute interval. The final pooled fractions were filtered and sent for analysis Exp. No.: 2 -

[00327] To the PS Concentrate was added 0.9% NaCl and a freshly prepared CPIP solution (114 mg/ml in acetonitrile) was added to the polysaccharide at a ratio of 1:1.1 by weight. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, protein was added at a ratio of 1:0.8 by weight PS:PR:CPIP as 1:0.8:1.1.

[00328] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/minute to monitor protein conversion. The reaction was stopped abruptly after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyoperal HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/hour and allowed to settle and 1M NaCl at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/hour and the conjugate was loaded onto the column and fractions were collected at 1 minute interval. The final pooled fractions were filtered and sent for analysis Conclusion: Conjugation of S. typhimurium and S. enteritidis polysaccharide using ADH-derived DT was found successful and PS/PR ratio was found satisfactory.B. Preparation of S. typhimurium and S. enteritidis polysaccharide conjugates using CRM197 as carrier protein 1) Derivatization of protein: Cross-reactive mutant (CRM197) received from production department (SIIPL) was taken for derivatization

[00329] To the freshly prepared concentrated CRM197 1M MES buffer was added adipic acid dihydrazide (ADH) (75 to 100mg/ml dissolved in 100mM MES buffer pH:6.5) in a ratio of 1:3.5 by weight and EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) (30 to 40 mg/ml dissolved in 100mM MES buffer pH:6.5) in a ratio of 1:0.25 by weight. 5% Tween 80 was added. The final reaction volume was completed using 100 mM MES buffer to reach a final concentration of 3 to 4 mg/ml, the reaction was continued at pH 6.5 for approximately 3 hours, and then the reaction mixture was diafiltered through 10 kDa TFF in 50 mM borate buffer and 0.005% Tween 80 pH 9.0 to remove residues and unreacted components. The final sample was analyzed for protein content and degree of derivatization. 2) Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) and CRM197-derivatized ADH:

[00330] The PS received from DSP was concentrated on 10 kDa membrane to reach a concentration of (10 to 15mg/ml).

[00331] To the PS Concentrate was added 0.9% NaCl and freshly prepared CPIP solution (114 mg/ml in acetonitrile) in polysaccharide was added in a weight ratio of 1:1.3. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, protein was added in a ratio of 1:1 by weight PS:PR:CPIP as 1:1:1.3

[00332] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/minute to monitor protein conversion. The reaction was stopped abruptly after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. 3) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyoperal HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/hour and allowed to settle and 1M NaCl at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/hour and the conjugate was loaded onto the column and fractions collected at 1 minute interval. The final pooled fractions were filtered and sent for analysis.Conclusion: Conjugation of S. typhimurium and S. enteritidis PS using ADH derived CRM197 was found successful and PS/PR ratio was found satisfactory.C) Preparation of S.Paratyphi Conjugates using Tetanus Toxoid as carrier protein:1) Protein derivatization: High Monomeric Tetanus Toxoid (TT) received from production department (SIIPL) was concentrated to (15 to 20 mg/ml) on 30 kDa membrane and analyzed for protein content. Freshly prepared concentrated 1 M MES TT buffer was added to adipic acid dihydrazide (ADH) (75 to 100 mg/ml dissolved in 100 mM MES buffer pH:6.0) in a ratio of 1:10 by weight and EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (30 to 40 mg/ml dissolved in 100 mM MES buffer pH:6.0) in a ratio of 1:1 by weight. The reaction was continued at pH 6.0 for about 1 hour and then the reaction mixture was diafiltered into 30 kDa TFF in 10 mM phosphate buffer pH 7.2 to remove residues and unreacted components. The final sample was analyzed for protein content and degree of derivatization2) Conjugation of S. typhi murium and S. enteritidis polysaccharide (PS) and ADH-derived TT:

[00333] The OSP received from the DSP team was concentrated on the 10 kDa membrane to reach a concentration of (10 to 13mg/ml). Two experiments were performed by changing the ratio of 1-cyano-4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) Exp. No.:1

[00334] To the concentrated PS was added 0.9% NaCl and freshly prepared CPIP solution (114 mg/ml in acetonitrile) was added to the polysaccharide at a ratio of 1:1.25 by weight. The pH was immediately changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, protein was added at a ratio of 1:0.8 by weight PS:PR:CPIP as 1:0.8:1.25

[00335] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/minute to monitor protein conversion. The reaction was stopped abruptly after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. 3) Conjugate Purification by Ultrafiltration: The quenched conjugate was purified by diafiltration 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. Exp. No.: 2

[00336] 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 changed to 9.5 with 2.5 M NaOH and held for up to 3 minutes. Then, the protein was added in a ratio of 1:0.9 by weight PS:PR:CPIP 1:0.9:1.3

[00337] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/minute to monitor protein conversion. The reaction was stopped abruptly after 3 to 4 hours by the addition of 2M Glycine 10 times that of PS by weight. 4) Conjugate Purification by Ultrafiltration: The quenched conjugate was purified by diafiltration 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. Conclusion: Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) using ADH-derived TT was successfully achieved, two different types of diafiltration strategy were applied and both processes gave satisfactory PS/Pr ratio. 2) Carbodiimide chemistry based conjugation of S. typhimurium and S. enteritidis polysaccharide to Diphtheria Toxoid (DT) carrier protein, CRM197 and Tetanus Toxoid (TT) A) Derivatization of S. typhimurium and S. enteritidis polysaccharide concentrate (Addition of ADH ligand) using cyanylation chemistry and conjugation with Tetanus Toxoid (TT) using carbodiimide chemistry. B) Derivatization of S. typhimurium and S. enteritidis polysaccharide concentrate (Addition of ADH ligand) using cyanylation chemistry and conjugation with Diphtheria Toxoid (DT) using carbodiimide chemistry.C) Derivatization of S. typhimurium and S. enteritidis polysaccharide concentrate (Addition of ADH Ligand) using cyanylation chemistry and conjugation with CRM197 using carbodiimide chemistry.D) Carbodiimide chemistry (EDAC) based conjugation of Paratyphi OSP with Diphtheria Toxoid (DT) carrier protein, CRM197 and Tetanus Toxoid (TT) without derivatization of polysaccharide or carrier protein.Procedure followed:1) Polysaccharide (PS) Derivatization: The received S. typhimurium and S. enteritidis polysaccharide was concentrated on 10 kDa membrane to reach a concentration of (10 to 13mg/ml). To the concentrated PS was added 0.9% NaCl and freshly prepared CPIP solution (114 mg/ml in acetonitrile) was added to the polysaccharide in a ratio of 1:0.5 to 1:2 (1:0.7) by weight. The pH was immediately changed to 9.5 with 2.5 M NaOH and maintained for up to 3 min. Then, adipic acid dihydrazide (ADH) (75 to 100 mg/ml dissolved in 0.5 M sodium bicarbonate buffer pH: 8.0) in a ratio of 1:10 by weight PS:ADH:CPIP as 1:2:0.7 to 1:10:0.7. The reaction was continued at pH 9.5 for about 2 hours, stopped abruptly using 2M Glycine 10, and then the reaction mixture was diafiltered into 10 kDa TFF in 100 mM MES buffer pH 6.0 to remove residues and unreacted components. The final sample was analyzed for polysaccharide content. 2) Protein Preparation: 2A) Protein Preparation: High Monomeric Tetanus Toxoid (TT) received from production department (SIIPL) was concentrated (10 to 15 mg/ml) on a 30 kDa membrane and analyzed for protein content. 2B) Protein Preparation: High Monomeric Diphtheria Toxoid (DT) received from production department (SIIPL) was concentrated (10 to 20 mg/ml) on a 10 kDa membrane and analyzed for protein content. 2C) Protein Preparation: High Monomeric CRM197 received from production department (SIIPL) was concentrated (10 to 20 mg/ml) on a 10 kDa membrane and analyzed for protein content. 3) Conjugation3A) Conjugation of S. typhimurium polysaccharide and S. enteritidis derivative of concentrated ADH and TT:

[00338] To the ADH-derived PS, protein was added at 1:0.9 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (30 to 40 mg/ml dissolved in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0, temperature 6°C and PS:PR:EDC ratio as 1:0.9:0.86

[00339] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min to monitor protein conversion. The reaction was stopped abruptly after 23 h by the addition of 100 mM Phosphate buffer containing EDTA.3B) Conjugation of S. typhimurium and S. enteritidis polysaccharide derived from ADH and concentrated DT: The same conditions as for TT conjugate were applied to DT to check Pr conversion.

[00340] To the ADH-derived PS, protein was added at 1:0.8 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimide) (30 to 40 mg/ml dissolved in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0, temperature 6°C and PS:PR:EDC ratio as 1:0.8:0.86

[00341] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/min to monitor protein conversion. The reaction was stopped abruptly after 20 h by the addition of 100 mM phosphate buffer containing EDTA.3C) Conjugation of ADH-derived S. typhimurium and S. enteritidis polysaccharide and concentrated CRM197: The same conditions as for TT and DT conjugate were applied to CRM197 to check Pr conversion.

[00342] To the ADH-derived PS, protein was added at 1:0.8 by weight and freshly prepared EDAC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (30 to 40 mg/ml dissolved in 100 mM MES buffer) at a ratio of 1:0.86 by weight. The reaction was continued at pH 6.0, temperature 6°C and PS:PR:EDC ratio as 1:0.8:0.86

[00343] Shodex SB-804 HQ and SB-805 HQ columns were used sequentially with PBS as the mobile phase at a flow rate of 1 ml/minute to monitor protein conversion. The reaction was stopped abruptly after 20 hours by the addition of 100 mM phosphate buffer containing EDTA. 4) Conjugate Purification 4A) Conjugate Purification by Ultrafiltration: The quenched conjugate was purified by diafiltration 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. 4B) Conjugate Purification by GFC: An XK16/70 Column was prepared using GFC resin (Tyoperal HW65F) with a bed height of 40 cm. The column was packed at a flow rate of 100 cm/hr and allowed to settle and 1M NaCl at 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30 cm/hr and the conjugate was loaded onto the column and fractions collected at 1 min interval. Fractions 2 to 12 were pooled according to the HPLC profile. The final pooled fractions were filtered and sent for analysis Conclusion: Using reverse conjugation chemistry (Activating PS), conjugation of S. typhimurium polysaccharide and S. enteritidis derived from ADH and concentrated TT, DT and CRM197 was successfully achieved by increasing the temperature from 6 to 10 °C. The conjugation rate of the reaction was increased; the PS/Pr ratio was very low in all reactions.

[00344] Conjugation of S. typhimurium and S. enteritidis polysaccharide derived from ADH and concentrated TT, DT and CRM197 using Carbodiimide chemistry was found to be successful. The conjugation reaction rate was increased by raising the temperature, but the PS/Pr ratio was low in both experiments. Example 4: Immunogenic Compositions

[00345] Single Dose does not include preservative 2-Phenoxyethanol

[00346] The multidose composition may additionally comprise 2-Phenoxyethanol - 5 mg (1 to 10 mg) √ = included in the composition

[00347] Further adjust the pH of the composition as described above to about 6.0 to 7.0 with sodium hydroxide/sodium carbonate and make up to volume by adding normal saline (0.9%).

[00348] It may additionally include one of the combinations of preservativesi. 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 per 0.5 ml (v/v) and methylparaben in an amount of 0.1 to 1 to 5 mg per 0.5 ml (w/v); oriii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v) and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v); oriv. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v), methylparaben in an amount of 0.1 to 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v).

[00349] Combination Vaccine Compositions comprising Reduced Dose IPV, D, T, HepB, ViPs—TT, OSP conjugate, acellular pertussis and Hib antigen may comprise the acellular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular genetically or chemically detoxified), in particular pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly pertussis toxoid: 1 to 50 micrograms (more particularly 8 μg); - Filamentous Haemagglutinin: 1 to 50 micrograms (more particularly 8 μg); - Pertactin: 1 to 20 micrograms (more particularly 2.5 μg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

[00350] Further adjust the pH of the composition as described above to about 6.0 to 7.0 with sodium hydroxide/sodium carbonate and make up to volume by adding normal saline (0.9%).

[00351] It may additionally include one of the combinations of preservativesi. 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 per 0.5 ml (v/v) and methylparaben in an amount of 0.1 to 1.5 mg per 0.5 ml (w/v); oriii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v) and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v); oriv. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v), methylparaben in an amount of 0.1 to 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v).

[00352] Combination Vaccine Compositions comprising Reduced Dose IPV, D, T, HepB, ViPs—TT, OSP Conjugate, acellular pertussis and Hib antigen may comprise the acellular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular genetically or chemically detoxified), in particular pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly pertussis toxoid: 1 to 50 micrograms (more particularly 8 μg); - Filamentous Haemagglutinin: 1 to 50 micrograms (more particularly 8 μg); - Pertactin: 1 to 20 micrograms (more particularly 2.5 μg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

[00353] Further adjust the pH of the composition as described above to about 6.0 to 7.0 with sodium hydroxide/sodium carbonate and make up to volume by adding normal saline (0.9%). It may additionally include one of the preservative combinationsv. 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 per 0.5 ml (v/v) and methylparaben in an amount of 0.1 to 1.5 mg per 0.5 ml (w/v); heari. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v) and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v); hearii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v), methylparaben in an amount of 0.1 to 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v).

[00354] Combination Vaccine Compositions comprising Reduced Dose IPV, IRV, D, T, HepB, ViPs—TT, acellular pertussis and Hib antigen may comprise the acellular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular genetically or chemically detoxified), in particular pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly pertussis toxoid: 1 to 50 micrograms (more particularly 8 μg); - Filamentous Haemagglutinin: 1 to 50 micrograms (more particularly 8 μg); - Pertactin: 1 to 20 micrograms (more particularly 2.5 μg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

[00355] Further adjust the pH of the composition as described above to about 6.0 to 7.0 with sodium hydroxide/sodium carbonate and make up to volume by adding normal saline (0.9%).

[00356] It may additionally include one of the combinations of preservativesv. 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 per 0.5 ml (v/v)and methylparaben in an amount of 0.1 to 1.5 mg per 0.5 ml (w/v); heari. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v)and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v); hearii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v), methylparaben in an amount of 0.1 to 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 to 0.2 mg per 0.5 ml (w/v).

[00357] The Combination Vaccine Compositions comprising Reduced Dose IPV, IRV D, T, HepB, ViPs-TT, acellular pertussis and Hib antigen may comprise the acellular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular genetically or chemically detoxified), in particular pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly pertussis toxoid: 1 to 50 micrograms (more particularly 8 μg); - Filamentous Haemagglutinin: 1 to 50 micrograms (more particularly 8 μg); - Pertactin: 1 to 20 micrograms (more particularly 2.5 μg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml. Example 5: Stability dataStability of monovalent Salmonella Typhi conjugate at 2 to 8ºC for initial (0), 3, 6 months; at 25 ºC for initial (0), 1, 3 months and at 40 ºC for initial (0), 14, 28 days: Remarks: Free Ps ("initial < 4.5 %, after 6 months NMT 7.5 %" for 180-220 kDa Ps Vs "initial 5.4 % after 6 months NLT 10.5 % for 388/80/45 kDa" at 40 °C/2 to 8 °C/25 °C).

[00358] For 180-220 kDa, low initial free Ps, the conjugate size was also maintained during the stability study, less free Ps was observed after 6 months compared to other sizes. SE-HPLC stability data (Vi-TT conjugate in Tris, Tris-NaCl and 0.17M NaCl) Conclusion:

[00359] The Vi-TT Conjugate was found to be stable in 10 mM Tris Buffer, 10 mM Tris containing 0.17 M NaCl and in 0.17 M NaCl alone.

[00360] Data for different concentrations of TRIS (5 mM, 10 mM, 20 mM, 25 mM, 30 mM, 50 mM) showing 25 mM TRIS as best case. • The concentration of Vi-TT CJ was maintained at three different dosages of Tris, pH 7.2, such as 5 mM, 10 mM and 20 mM. This was maintained at various temperature conditions such as -20 °C, 2 to 8 °C, room temperature and 37 °C. • The study was carried out for 2 weeks and was monitored daily for pH. • Samples were checked for particle size and zetal potential. pH results Conclusion: In the Vi-TT CJ study, the Conjugate was maintained in Tris at 5, 10 and 20 mM pH 7.2, the pH was within the range of 2 to 8°C at concentrations of 5, 10 and 20 mM. Measurement of Viscosity and Osmolality of Vi-TT Conjugate Part A: Viscosity and Osmolality Results of Tris Buffer and Sodium Chloride without Conjugate Part B: Viscosity and Osmolality Results of Tris and Sodium Chloride Buffer with Conjugate Conclusion:

[00361] The conjugate with varying Tris concentrations was found to be isotonic and the conjugate with 150 mM NaCl was found to be isotonic.

[00362] Conjugated with different concentrations of tris buffer and NaCl found similar viscosity and the viscosity is suitable for use as parenteral formulations.Example 6: Immunogenicity DataA. Immunogenicity study of monovalent conjugate vaccine:

[00363] The immunogenic potential of the SIIP Vi TT vaccine was evaluated in a mouse model. Mouse Immunogenicity Study #1:

[00364] An immunogenicity study was performed in mice in which unconjugated and TT-conjugated SIIPL vaccine (Vi TT) was administered intramuscularly to mice (8 animals per group). Animals were inoculated on day 1 and day 14 and serum sample was collected on day 14 (data not shown) and day 21. See Table 86 for details. Paired serum sample was available from all animals in each group. The serum sample was used for determination of antibodies against the injected polysaccharide using a suitable serological immunochemical method, such as an in-house IgG ELISA. Sera from mice receiving unconjugated SIIPL polysaccharide (Vi PS) were used as a comparative control for induction of IgG response in sera from mice receiving the conjugated version of Vi PS (variable PS size). Mouse Immunogenicity Study #2:

[00365] Another similar study was performed in mice. Both studies employed a similar animal treatment protocol. The animal protocol is briefly described below. Sera from both studies were tested by the identical immunological/serological analytical assay, i.e., IgG ELISA. 5- to 6-week-old female mice (Balb C strain; bred in-house) were used in the study using an IAEC-approved animal study protocol. All animals were free of special pathogens and were handled under aseptic conditions in a biosafety cabinet during inoculation and blood sample collection. Mice weighed ~18 to 20 g at the start of the study. Each mouse received 2.5 μg of Vi TT conjugate vaccine intramuscularly. The SIIPL Vi TT vaccine was diluted in phosphate-buffered saline (PBS) as the vehicle. No adjuvants were used in the study for either treatment group.Treatment Schedule: The following table summarizes the general treatment plan for the study. Methodology and techniques used for serological analysis:

[00366] Blood sample was collected from the experimental animals from the retro-orbital vein using sterile glass capillary tubes. Isoflurane was used as a safe anesthetic during the blood collection procedure. Sera were subjected to analysis for IgG antibodies produced in response to the injected 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 from S. typhi) as the coating antigen. IgG levels were estimated by analyzing the optical density (OD) observed in sera from mice administered the Vi PS conjugate (Vi TT variable-size PS) compared to the OD observed in serum samples from mice administered unconjugated SIIPL polysaccharide (Vi PS).

[00367] The following tables indicate the immunogenicity induction data (at day 21) of different Vi TT PS. Table 87, 88, 89 and 90: Immunogenicity Results Notes:

[00368] Mice groups with Vi TT exhibited >4-fold greater IgG induction (compared to mice administered unconjugated Vi PS), strongly indicating the immunogenic potential of SIIPL VI TT across all PS sizes. Two separate studies were performed with Vi TT and both studies indicated similar response to Vi TT over Vi PS.B. Immunogenicity Study of Bivalent Conjugate Vaccine

[00369] The immunogenic potential of the bivalent (typhoid and paratyphoid) SIIPL vaccine was evaluated in a mouse model. Mouse Immunogenicity Study #1:

[00370] An Immunogenicity Study was performed in mice where a bivalent vaccine containing a combination of monovalent Vi TT and monovalent O-SP A (S. Paratyphi A specific O polysaccharide antigen) vaccine conjugated with carrier proteins such as TT, DT and CRM was employed in the study. The monovalent and bivalent versions of these vaccines were administered intramuscularly to mice (8 animals per group). Animals were inoculated on days 1, 14 and 28 while serum samples were collected on days 14, 28 and 42. Paired serum samples were available from all animals in each group. See Table 92 below for details of the treatment schedule. Serum samples were used for determination of antibodies against the injected polysaccharide using a suitable serological immunochemical method such as an in-house IgG ELISA. Sera from mice receiving the respective unconjugated SIIPL polysaccharide (Vi PS) and O-SP alone were used as comparative controls for induction of IgG response.

[00371] The animal protocol is briefly described in the Table below.

[00372] Serum samples were tested by immunological/serological analytical assay, i.e., IgG ELISA. 5- to 6-week-old female mice (Balb C strain; bred in-house) were used in the study using an IAEC-approved animal study protocol. All animals were free of special pathogens and were handled under aseptic conditions in a biosafety cabinet during inoculation and blood sample collection. Mice weighing in the range of ~18 to 20 g were used in the study. Each mouse received 2.5μg of monovalent Vi TT conjugate alone or O-SP A DT/TT/CRM alone and 2.5μg of each bivalent vaccine (monovalent Vi TT conjugate mixed with O-SP A DT/TT/CRM) via intramuscular route. Vaccines were diluted in phosphate buffered saline (PBS) as vehicle. No adjuvants were used in the study for either treatment group.Treatment Schedule: The following table summarizes the general treatment plan for the study. Methodology and techniques used for serological analysis:

[00373] Blood samples were collected from the experimental animals from the retro-orbital vein using sterile glass capillary tubes. Isoflurane was used as a safe anesthetic during the blood collection procedure. Sera were subjected to analysis for IgG antibodies produced in response to the injected antigen using an in-house IgG ELISA. IgG levels were estimated in serum samples from all study groups by colorimetric analysis.Notes:

[00374] The following tables indicate the immunogenicity induction data (at day 21) of different bivalent (typhoid and paratyphoid) SIIPL vaccines. Table 93, 94, 95, 96: Immunogenicity Results Notes:

[00375] The groups of mice injected with bivalent (typhoid and paratyphoid) SIIPL vaccine 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 exhibited >4-fold higher IgG induction (compared to mice administered with unconjugated Vi PS or unconjugated SIIPL O-SP A), thus indicating the immunogenic potential of bivalent SIIPL vaccine containing combination of Vi TT and SIIPL O-SP A (DT/TT/CRM).

[00376] IgG induction by the bivalent (typhoid and paratyphoid) SIIPL vaccine was greater than the IgG induction observed in the bivalent vaccine containing SIIPL O-SP A CRM and the commercial vaccine Vi TT Typbar TCV.

[00377] The following parameters were compared between the various groups:a. >4-fold increase: mean fold increase was >10-foldb. GM antibody titer: all groups exhibited higher GM than that observed with unconjugated PSc. % mice with positive response: all mice in all Vi TT groups exhibited higher antibody response (100% positive).Conclusions from analysis of serological data:

[00378] The Immunogenicity Study in mice was performed with 8 mice (female) in each group. Antibody induction was evaluated, in all groups, in response to the injected bivalent (typhoid and paratyphoid) SIIPL vaccine versus the unconjugated Vi PS and O-SP A PS. All bivalent (typhoid and paratyphoid) SIIPL vaccines strongly indicated the induction of PS-specific IgG antibodies (day 21; after the first booster) compared to the PS alone group (Vi and O-SP A). IgG induction in mouse sera is a strong determinant of the serological response to the bivalent (typhoid and paratyphoid) SIIPL vaccine. Therefore, the bivalent (typhoid and paratyphoid) SIIPL vaccine is capable of inducing a strong immunogenicity response in a mouse model and provides a promising potential for future studies in higher animals. Observations: 1. Immunogenicity data are evaluated in terms of IgG antibody levels observed in response to bivalent SIIPL (typhoid and paratyphoid) vaccine in mouse model. 2. IgG induction is measured as Geometric Mean (Mean) of colorimetric response (OD) observed in IgG ELISA. 3. GM observed in response to bivalent SIIPL (typhoid and paratyphoid) vaccine is considered immunogenic if the increase in OD in VI TT treatment is greater than 4-fold over the OD observed in response to unconjugated Vi PS. Conclusions: 1. All bivalent SIIPL (typhoid and paratyphoid) vaccine conjugates induce immunogenic response as observed through serological assay. 2. All bivalent SIIPL (typhoid and paratyphoid) vaccine conjugates exhibited higher IgG levels than those observed in unconjugated counterparts. 3. IgG induction by the bivalent SIIPL vaccine (typhoid and paratyphoid) was greater than the IgG induction observed by the bivalent vaccine containing SIIPL O-SP A CRM and commercial vaccine Vi TT Typvar TCV.4. The induction of immunogenicity response (GM and > 4-fold increase) was observed in mice receiving "Bivalent SIIPL (Typhoid and Paratyphoid) vaccine" and therefore it can be concluded that Bivalent SIIPL (Typhoid and Paratyphoid) vaccine is capable of inducing a strong immunogenicity response in mouse model suggestive of its clinical immunogenic potential. Example 7: Single dose vaccine kit A) Single dose vaccine kit comprising: a first container containing a lyophilized (freeze-dried) immunogenic composition: a) Neisseria meningitidis saccharide conjugate antigen A - - TT 5 μg per 0.5 ml; b) Neisseria meningitidis saccharide conjugate antigen C -CRM197 5 μg per 0.5 ml; c) Neisseria meningitidis saccharide conjugate antigen Y -CRM197 5 μg per 0.5 ml;d) Neisseria meningitidis W-135 saccharide conjugate antigen - CRM197 5 μg per 0.5 ml;e) Neisseria meningitidis X saccharide conjugate antigen - 5 μg per 0.5 ml;f) Sucrose 1 to 12 mg per 0.5 ml;g) Sodium citrate (dihydrate) 0.1 to 2 mg per 0.5 ml;h) Tris buffer 0.05 to 0.5 mg per 0.5 ml;and a second container containing a liquid composition for the reconstitution of the lyophilized (freeze-dried) immunogenic composition comprising:i) Salmonella enterica serovar typhi ViPs conjugate antigen - TT 1.25 to 50 μg;j) Sodium chloride 1 to 10 mg;k) Water for Injection (WFI) q.s.;Interpretation:

[00379] There is no antigenic interference of ViPs-TT with Neisseria meningitidis antigens, for prophylaxis against typhoid and activation by Salmonella Typhiiti with Neisseria meningiti antigens in which said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi including in children under 2 years of age, adult adolescents, and elderly people through just one injection to comprise a complete vaccination schedule. B) Single-dose vaccine kit comprising: a first container containing a lyophilized (freeze-dried) immunogenic composition: a) Neisseria meningitidis saccharide conjugate antigen A - TT 5 μg per 0.5 ml; b) Neisseria meningitidis saccharide conjugate antigen C - CRM197 5 μg per 0.5 ml; c) Neisseria meningitidis saccharide conjugate antigen Y - CRM197 5 μg per 0.5 ml; d) Neisseria meningitidis saccharide conjugate antigen W-135 - CRM197 5 μg per 0.5 ml; e) Neisseria meningitidis saccharide conjugate antigen X - 5 μg per 0.5 ml; f) Sucrose 1 to 12 mg per 0.5 ml;g) Sodium citrate (dihydrate) 0.1 to 2 mg per 0.5 ml;h) Tris buffer 0.05 to 0.5 mg per 0.5 ml;and a second container containing a liquid composition for the reconstitution of the lyophilized (freeze-dried) immunogenic composition comprising:i) Salmonella enterica serovar typhi ViPs conjugate antigen - TT 1.25 to 50 μg;j) Salmonella enterica serovar Paratyphi A OSP conjugate antigen - CP 1.25 to 50 μg; wherein CP is either TT or DT or CRM197;k) Sodium chloride 1 to 10 mg;l) Water for Injection (WFI) q.s.;Interpretation:

[00380] No antigenic interference of ViPs-TT; OSP antigen with Neisseria meningitidis antigens, for prophylaxis against typhoid and paratyphoid caused by antigens of Salmonella Typhi, S. Paratyphi and Neisseria meningitidis wherein said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi and paratyphi including in children under 2 years of age, adult adolescent, and elderly by means of only one injection to comprise a complete vaccination schedule. C) Single dose vaccine kit comprising: a first container containing a fully liquid hexa-valent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from Sabin or Salk Strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU) or IPV type 3 in a dose of 1 to 50 D antigen units (DU), per 0.5 ml; (b) a Diphtheria Toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (c) a Tetanus Toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (d) a Whole cell Pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular Pertussis antigen (aP) comprising one or more of modified adenylate cyclase, Pertussis toxoid (PT) (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml;e) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;f) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml;anda second container containing an entirely liquid immunogenic composition comprising:g) Salmonella enterica serovar typhi ViPs - TT conjugate antigen 1.25 to 50 μg;h) Sodium chloride 1 to 10 mg; and/ori) Tris buffer or Citrate buffer or Histidine buffer or Succinate buffer 0.1 mg to 1.6 mg; and/orj) Polysorbate 20; and/ork) 2-Phenoxyethanol 1 to 10 mg; and/orl) Water for Injection (WFI) q.s.Interpretation:

[00381] No antigenic interference of Vis-TT with hexavalent immunogenic composition, for prophylaxis against typhoid and paratyphoid caused by Salmonella Typhi and hexavalent antigens in which said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi including in children under 2 years of age, adult adolescent, and elderly by means of only one injection to comprise a complete vaccination schedule. D) Single dose vaccine kit comprising: a first container containing a fully liquid hexavalent immunogenic composition: a) an inactivated poliovirus antigen (IPV) selected from Sabin or Salk Strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU) or IPV type 3 in a dose of 1 to 50 D antigen units (DU), per 0.5 ml; (b) a Diphtheria Toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (c) a Tetanus Toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (d) a Whole cell Pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular Pertussis antigen (aP) comprising one or more of modified adenylate cyclase, Pertussis toxoid (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml;e) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;f) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml;anda second container containing an entirely liquid immunogenic composition comprising:a) Salmonella enterica serovar typhi ViPs conjugate antigen - TT 1.25 to 50 μg;b) Salmonella enterica serovar Paratyphi A OSP conjugate antigen - CP 1.25 to 50 μg; wherein CP is either TT or DT or CRM197;c) Sodium chloride 1 to 10 mg; and/ord) Tris buffer or Citrate buffer or Histidine buffer or Succinate buffer 0.1 mg to 1.6 mg; and/org) Polysorbate selected from polysorbate 20; and/orh) 2-Phenoxyethanol 1 to 10 mg; and/ore) Water for Injection (WFI) q.s.Interpretation:

[00382] No antigenic interference of ViPs-TT; OSP antigen with hexavalent antigens, for prophylaxis against typhoid and paratyphoid caused by Salmonella Typhi, S. Paratyphi and hexavalent antigens wherein said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi and paratyphi including in children under 2 years of age, adult adolescents, and the elderly by means of only one injection to comprise a complete vaccination schedule. E) Single dose vaccine kit comprising: a first container containing a fully liquid heptavalent immunogenic composition: a) an inactivated poliovirus (IPV) antigen selected from Sabin or Salk strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU), or IPV type 3 in a dose of 1 to 50 D antigen units (DU), 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 in the range of 1 to 50 μg per 0.5 ml; (c) a diphtheria toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (d) a tetanus toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (e) a whole-cell pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or pertussis acellular, antigen (aP) comprising one or more of modified adenylate cyclase, pertussis toxoid (PT) (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml;f) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;g) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml;anda second container containing an entirely liquid immunogenic composition comprising:h) Salmonella enterica serovar typhi ViPs - TT conjugate antigen 1.25 to 50 μg; i) Sodium chloride 1 to 10 mg;j) Water for Injection (WFI) q.s.;Interpretation: in which there is no antigenic interference of ViPs-TT with heptavalent immunogenic composition, for prophylaxis against typhoid caused by Salmonella Typhi antigens and Heptavalent antigens in which said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi including in children under 2 years of age, adult adolescents, and the elderly by means of just one injection to comprise a complete vaccination schedule.F) Single-dose vaccine kit comprising: a first container containing a fully liquid hexavalent immunogenic composition:a) an inactivated poliovirus antigen (IPV) selected from Sabin or Salk strain; wherein IPV type 1 in a dose of 1 to 50 D antigen units (DU), IPV type 2 in a dose of 1 to 50 D antigen units (DU), or IPV type 3 in a dose of 1 to 50 D antigen units (DU), 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 in the range of 1 to 50 μg per 0.5 ml; (c) a diphtheria toxoid antigen (D) in an amount of 1 to 50 Lf per 0.5 ml; (d) a tetanus toxoid antigen (T) in an amount of 1 to 30 Lf per 0.5 ml; (e) a whole-cell pertussis antigen (wP) in an amount of 1 to 50 IOU per 0.5 ml or acellular pertussis antigen (aP) comprising one or more of modified adenylate cyclase, pertussis toxoid (PT) (PT) 1 to 50 μg, Filamentous hemagglutinin (FHA) 1 to 50 μg, Pertactin (P69 or PRN) 1 to 20 μg or Fimbrial proteins (FIM 1, 2 and 3) 2 to 25 μg; per 0.5 ml;f) a hepatitis B virus surface antigen (HBsAg) in an amount of 1 to 20 μg per 0.5 ml;g) a Haemophilus influenzae type b antigen (Hib) in an amount of 1 to 20 μg per 0.5 ml;anda second container containing an entirely liquid immunogenic composition comprising:a) Salmonella enterica serovar typhi ViPs conjugate antigen - TT 1.25 to 50 μg;b) Salmonella enterica serovar Paratyphi A OSP conjugate antigen - CP 1.25 to 50 μg; wherein CP is either TT or DT or CRM197;c) Sodium chloride 1 to 10 mg; and/ord) Tris buffer or Citrate buffer or Histidine buffer or Succinate buffer 0.1 mg to 1.6 mg; and/org) Polysorbate selected from polysorbate 20;h) 2-Phenoxyethanol 1 to 10 mg; and/ore) Water for Injection (WFI) q.s.Interpretation:

[00383] No antigenic interference of ViPs-TT; OSP antigen with heptavalent antigens, for prophylaxis against typhoid and paratyphoid caused by Salmonella Typhi, S. paratyphi and heptavalent antigens in which said vaccine formulation is sufficient to induce the required T-dependent immune response against S. typhi and paratyphi including in children under 2 years of age, adolescent adults and the elderly through just one injection to comprise a complete vaccination schedule.

Claims (13)

1. Immunogenic vaccine composition, characterized by the fact that it comprises: a) a bivalent polysaccharide-protein conjugate vaccine comprising or consisting of Salmonella enterica serovar typhi antigen-carrier protein (CP) conjugate and Salmonella enterica serovar Paratyphi A antigen-carrier protein (CP) conjugate; b) a sugar selected from the group of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, isomaltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol or a combination thereof; c) a buffering agent selected from the group of carbonate, phosphate, acetate, HEPES, succinate, histidine, TRIS, borate, citrate, lactate, gluconate, tartrate, HEPES, piperazine-N, PIPES and 2-ethanesulfonic acid (MES); and d) an aqueous solution. 2. Immunogenic vaccine composition according to claim 1, characterized in that the composition comprises a free polysaccharide content of less than 10%. 3. Immunogenic vaccine composition according to claim 1, characterized in that the composition comprises a free polysaccharide content of less than 5%. 4. Immunogenic vaccine composition according to claim 1, characterized in that the composition is free from aggregation. 5. Immunogenic vaccine composition according to claim 1, characterized in that the carrier protein (CP) is selected from the group consisting of tetanus toxin, tetanus toxoid (TT), tetanus toxoid fragment, diphtheria toxoid (DT), CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, E. coli LT, E. coli ST, Escherichia coli heat-labile toxin - subunit B, Neisseria meningitidis outer membrane complex, rEPA, H. influenzae protein D, Flagellin FliC, Horseshoe crab hemocyanin, Pseudomonas aeruginosa exotoxin A, outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolysin, surface protein pneumococcal surface adhesin A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) from Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) from Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human serum albumin, bovine serum albumin (BSA), purified protein derivative of tuberculin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various pathogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B proteins from C. difficile and S. agalactiae with or without ligand and fragments, derivatives, modifications thereof. 6. Immunogenic vaccine composition according to claim 5, characterized in that the CP is selected from the group consisting of tetanus toxin, tetanus toxoid (TT), tetanus toxoid fragment, diphtheria toxoid (DT) and CRM197. 7. Immunogenic vaccine composition according to claim 1, characterized in that the composition comprises ViPs-TT conjugate antigen of salmonella enterica serovar typhi in the range of 1.25 to 50 μg and OSP-CP conjugate antigen of salmonella enterica serovar Paratyphi A in the range of 1.25 to 50 μg per dose of 0.5 ml. 8. Immunogenic vaccine composition according to claim 6, characterized in that the ViPs-TT conjugate antigen of salmonella enterica serovar typhi and the OSP-CP conjugate antigen of salmonella enterica serovar Paratyphi A are present in a concentration of 25 μg per 0.5 ml dose. 9. Immunogenic vaccine composition according to claim 7, characterized in that the TRIS buffer is present in a concentration of 0.1 to 2.0 mg, preferably between 0.61 and 1.52 mg. 10. Immunogenic vaccine composition according to claim 1, characterized in that the aqueous solution is selected from a group consisting of saline and water for injection (WFI). 11. Immunogenic vaccine composition according to claim 1, characterized in that the composition is free of preservatives. 12. Immunogenic vaccine composition according to claim 1, characterized in that the composition further comprises:- a preservative selected from the group consisting of 2-phenoxyethanol, benzethonium chloride (Femerol), phenol, m-cresol, thiomer- salt, formaldehyde, paraben esters (e.g. methyl-, ethyl-, propyl- or butyl-paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol, benzyl alcohol, or a combination thereof, and/or- an adjuvant selected from the group consisting of aluminum salt, aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate and potassium aluminum sulfate, and/or- an immunostimulatory component selected from the group consisting of an oil and water emulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, derivatives of lipid A, monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund's adjuvant, complete Freund's adjuvant, incomplete Freund's adjuvant, polymers, copolymers such as polyoxyethylene-polyoxypropylene copolymers including block copolymers, polymer p 1005, adjuvant CRL-8300, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from gram negative bacteria, TLR-5 agonists, flagellin fragments capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, ISCOMS, mixtures of squalene (SAF-1), Quil A, cholera toxin subunit B, polyphosphazene and derivatives, mycobacterial cell wall preparations, mycolic acid derivatives, nonionic block copolymer surfactants, OMV, fHbp, combination of saponin with sterols and lipids, and/or surfactants selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethanol, t-octylphenoxypolyethoxyethanol, oxtoxinol 40, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene-35 ricinoleate, soy lecithin and a poloxamer, and/or- amino acids selected from the group consisting of L-Histidine, Lysine, Isoleucine, Methionine, Glycine, Aspartic Acid, Tricine, arginine, leucine, glutamine, alanine, peptide, hydrolyzed protein or protein such as serum albumin. 13. Immunogenic vaccine composition according to claim 1, characterized in that the final pH of the composition is in the range of pH 6.0 to 8.0.