FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; Rule 13)
IMMUNOGENIC COMPOSITIONS AGAINST ENTERIC DISEASES AND METHODS FOR ITS PREPARATION THEREOF

FIELD
The present disclosure relates to the field of vaccine manufacturing, 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
The background information herein below relates to the present disclosure but is not necessarily a prior art.
Salmonella infection remains a serious health problem throughout the world, particularly in developing countries affecting millions of people each year. Salmonella infection can cause enteritis which may be complicated by bacteraemia (enteric fever) and gastroenteritis in both normal and immunocompromised individuals.
The genus Salmonella belongs to the family of Enterobacteriaceae and comprises Gram-negative, non-spore forming, facultative anaerobic bacilli. Salmonella enterica serovar typhi (S. typhi) and Salmonella enterica serovar paratyphi (S. paratyphi) A and B cause enteric fever, a systemic febrile illness, occurring only in humans that is distinguished from the more commonly self-limited acute gastroenteritis caused by other numerous Salmonella serotypes. Enteric fever caused by members of the genus Salmonella, including typhi and paratyphi, continues to constitute a significant disease and mortality burden among populations in developing countries (Lancet 2005; 366:749 762) and represents a notable risk for travellers (Lancet Infect Dis. 2005:5(10):623-628). Typhoid fever remains endemic in low- and middle-income countries (LMICs). Between 12.5–20.6 million cases of enteric fever occur each year in LMICs particularly in south Asia and sub-Saharan Africa (Lancet Glob Heal. 2014; 2: e570-e580 & J Glob Health. 2012; 2: 10401). Salmonella paratyphi is responsible for increasing proportion of enteric in parts of Asia, including in Nepal, Cambodia, and China. Salmonella paratyphi has highest burdens on the Indian subcontinent and South East Asia. In one of the studies in Nepal where typhoid fever is highly endemic, the municipal water was found to be contaminated with both S. typhi and Salmonella enterica serovar paratyphi A (PLoS Negl Trop Dis. 2016 Jan; 10(1):e0004346 & PLoS Negl Trop Dis. 2013; 7(8):e2391).
Non-typhoidal Salmonella enterica (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 percent of all human isolates of NTS

reported globally. Further, invasive non-typhoidal Salmonella (iNTS) infections caused by serovars enteritidis (SE) and typhimurium (STm) are major pediatric health problems in sub-Saharan Africa.NTS has been increasingly recognized recently as a major cause of invasive bacterial infections in young children and immunocompromised individuals, as well as elderly worldwide. These two serovars are also the major cause of gastroenteritis in healthy children and adults in industrialized countries. NTS can also cause severe extra-intestinal, invasive bacteremia, which is referred to as iNTS. It usually presents as a febrile illness. In fact, iNTS frequently occurs without gastrointestinal symptoms in both adults and children.
Of the more than 2,500 non-typhoidal serovars, Salmonella enterica subsp. enterica serovar typhimurium (S. typhimurium) and Salmonella enterica serovar enteritidis (S. enteritidis) account for nearly 80 percent of all human isolates of NTS reported globally. NTS has been increasingly recognized recently as a major cause of invasive bacterial infections in young children and HIV-infected individuals in sub-Saharan Africa, as well as elderly and immunocompromised individuals worldwide.
The global incidence of NTS gastroenteritis in 2010 was estimated to be 93 million cases, some 80.3 million of which were via food-borne transmission, 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 per year, with a loss of 17,000 quality-adjusted life years, the most of any food-borne pathogen. As mentioned previously, NTS can also cause severe extra-intestinal, invasive bacteremia, which is referred to as iNTS. Invasive infections of Salmonella 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. In fact, iNTS frequently occurs without gastrointestinal symptoms in both adults and children. Symptoms of iNTS are similar to malaria and include fever and sweats (more than 90 percent) as well as splenomegaly (40 percent). It is not clear why iNTS is such a problem in Africa, but this could be related to: increased invasiveness of the 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, e.g., through contaminated water supplies., and NTS bacteremia in HIV-infected African adults has an associated high mortality (up to 47 percent) and recurrence rate (43 percent) rate.

Antibiotics have been used to treat typhoidal and Non-typhoidal Salmonella related infections, and the choice of antimicrobials and length of treatment are determined by the cost and availability of antibiotics, local pattern of resistance, and a patient’s treatment response. It is becoming increasingly recognized both in the developed and developing world, that multiple antibiotic-resistant strains are emerging as important causes of invasive bacteraemia and gastroenteritis complications, resulting in hospitalizations and deaths.
As available tools for treatment become less effective, the problem of Typhoidal and Non-typhoidal Salmonella related 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 travelling into areas where Typhoidal and Non-typhoidal Salmonella related infections are endemic.
Currently three types of typhoid vaccines are licensed for use i)typhoid conjugate vaccine (TCV) ii) unconjugated Vi polysaccharide (ViPS) vaccine and iii) live attenuated Ty21a vaccine. World Health Organization (WHO) has recommended greater use of typhoid vaccines with preference given to Typhoid Conjugate Vaccines (TCV) (WHO position paper. Wkly Epidemiol Rec 2018; 93:153–72).
Vaccines for S. paratyphi are currently not available. Likewise, there is a need for an immunogenic composition/ vaccine which is able to simultaneously confer immunity against typhoidal and non-typhoidal Salmonella.
A drawback of the currently-available vaccines is that they are all directed against only S. typhi. S. paratyphi A causes enteric fever with the same geographic distribution as S. typhi, and the diseases are clinically indistinguishable. Hence, for South and South-East Asia, a vaccine that can protect against both serovars would be more valuable than a vaccine that is restricted to one. Also, in sub-Saharan Africa, the same is true for S. typhimurium and S. enteritidis, indicating the importance of a vaccine that can additionally protect against NTS serovars for this region.
It is unclear as to whether vaccine candidates in pipeline will be protective against both the gastroenteritis and invasive manifestations of iNTS. While understanding of the disease burden of typhoid in LMICs is growing, the global medical demand for a conjugate vaccine protecting against typhoidal and non-typhoidal Salmonella infections has attained significance. It is estimated that the peak demand for a typhoid conjugate vaccine is likely to
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occur between 2023 and 2026, approaching 300 million annual doses for 133 countries (Clin Infect Dis. 2019 Mar 15; 68(Suppl 2): S154–S160).
Further, Upstream, Downstream, conjugation and formulation development can often be the rate-limiting step in early introduction of biopharmaceuticals into the market and in meeting the demands of the population. Upstream includes the entire process from early cell isolation and cultivation, to cell banking, to the culture expansion of the bacterial fermentation process and the final harvest. The cell culture is scaled up from 100 to 500 millilitres to a bioreactor of 3 to 20,000 litres. Further steps include primary recovery of Salmonella polysaccharide, and elimination of cell and debris. Further, in order to facilitate cost-effective Salmonella polysaccharide based conjugate vaccine development, 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 Salmonella spp polysaccharides. Increasing capsular polysaccharide (CPS) "fermentation harvest stage yield" by employing novel feed strategies, and improved fermentation medium has been one of the preferable approaches to achieve said objective. Merritt et al. (2000) showed that fed batch culture at 500 litre manufacturing scale bioreactor increased the cell density and the yield of capsular polysaccharide approximately fourfold when compared to batch culture.
Studies of capsular polysaccharide production by other pathogenic bacteria such as Haemophilus influenzae Type B, and Neisseria meningitidis showed that production was dependent upon the fermentation conditions (temperature, pH, DO, osmolality) and the media components and those optimal conditions differed for each bacterium. Zhan et al. (2002) similarly showed that pH control and changing to fed batch fermentation increased the yield of cells and the production of capsular polysaccharide.
The expression of capsular polysaccharide is highly regulated in relation to certain fermentation conditions, such as osmolality wherein reduced synthesis of polysaccharide have been reported when osmolality was high.
The polysaccharides have been produced under different concentrations of glucose, casamino acids, and phosphate ions.
Baruque-Ramos et al., 2005 showed that higher yields of capsular polysaccharide were obtained when N. Meningitidis (serogroup C) was cultured in media when the glucose concentration was maintained below 1.0 g/l and that low oxygen tension favoured higher
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polysaccharide production. Further, it has been observed that concentrated casamino acids in the feed solution limited the final cell density. Further, growth and polysaccharide yield in a defined medium is also dependent on the ratio of carbohydrate to nitrogen source. In one of the Fed Batch Fermentation Process for S. typhi, ammonia was supplied as a nitrogen source along with the feed medium. However, it has been observed the Polysaccharide formation by Aureobasidium pullulans was affected by the ammonia nitrogen source in the medium, and its yield fell when excess ammonium ions were present, even under conditions which otherwise supported its synthesis (Appl Microbiol Biotechnol (1990)32:637–644).
Growth and polysaccharide synthesis in a defined medium were greatest when amino acids were substituted for ammonia as a nitrogen source.
Use of casamino acid as the nitrogen source in a defined medium has been reported. However, animal derived Casamino acid most probably from bovine casein has been reported as allergen and are restricted in use. Further, use of Casein Digest/tryptone medium as the nitrogen source in a defined medium has been reported however, it may not support the growth of fastidious organisms.
Addition of animal-component-free hydrolysates (Bacto TC Yeastolate, Phytone Peptone) to chemically defined media is one of the approaches to increase cell density, culture viability and productivity in a timely manner. Hydrolysates are protein digests composed of amino acids, small peptides, carbohydrates, vitamins and minerals that provide nutrient supplements to the media. Non- animal derived hydrolysates from soy, wheat and yeast are used commonly in cell culture media and feeds to improve polysaccharide yield (Refer US9284371). However, because of its composition complexity, lot-to-lot variations, undesirable attribute of making culture viscous, Yeast extract and hydrolysates can be a significant source of medium variability. Formation of foam during large scale fermentation could i) reduce capsular polysaccharide yield due to loss of cells and culture medium to the foam phase, ii) can be detrimental to cells since when bubbles burst they exert sheer forces iii) result in a loss of sterility if the foam escapes and iv) can lead to over-pressure if a foam-out blocks an exit filter.
The fermentation cell supernatant is subjected to different steps of purification to isolate purified polysaccharide and eliminate host cell impurities such as proteins, nucleic acid and lipopolysaccharide. Filtration techniques play an important role in downstream processing or purification of bacterial polysaccharides from host cell impurities. Downstream involves

inactivation of bacterial culture, separation of the cells from the media, isolation of the product, concentration, purification. Downstream processing is the most challenging part of the process because of its complexity.
Each bacterium has different capsular polysaccharides and different serotypes of the same bacteria further differ in chemical structure of bacterial capsular polysaccharides. A case in point is S typhi expresses a Vi polysaccharide capsule which is a linear homopolymer of α(1-4)-D-GalpA N-acetylated at C-2 and O-acetylated at C-3. The N and O acetyls dominate the surface and are essential for both antigenicity and immunogenicity of Vi, whereas in contrast, S paratyphi A and B and NTS (with rare exceptions) do not express capsular polysaccharides. Rather, 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-Gal p-(1→) (which serologically constitutes epitope 12). However, a dideoxy hexose saccharide linked α-(3→6) at the mannose of the repeating trisaccharide results in an immunodominant epitope that confers Salmonella group identity. In case of S typhimurium, the galactose of the trisaccharide backbone epitope 12 becomes α-(1→6) glucosylated. (Refer: Lindberg AA, Le Minor L. Serology of Salmonella In: Bergan T, ed. Methods in Microbiology: Academic Press, 1984:1-141).
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.
Previous method of purification that can be performed at large scale volumes includes lysing and precipitation of impurities such as nucleic acids and lipids using solvents, pH manipulation and using detergent such as sodium deoxycholate, Triton-X.
Sodium Deoxycholate (DOC) is a mild detergent and is one of the most commonly used detergent in polysaccharide purifications. Sodium Deoxycholate with a core steroidal structure is less denaturing and limited in its solubilising strength, it breaks the endotoxins without affecting the chemical structure; and hence upon removal of sodium deoxycholate, endotoxins regain their biological activity. Also, DOC based procedures do not work efficiently for removal of contaminants from polysaccharides, especially sialic acid containing polysaccharides. This could be due to weak detergent activity of DOC on Lipopolysaccharide- Protein association formed during the downstream processing, resulting in high level of Endotoxins and protein content in the final isolated polysaccharide. Further

Sodium deoxycholate being an animal-origin product, even its residual presence in final product may lead to non-acceptance of product by regulatory agencies and certain religious communities.
The disadvantage of using Triton-X is that the residual detergent persists in the extraction phase and elimination requires extensive washing to remove all the residues.
For some CPS types, precipitation with Zinc acetate/Ammonium sulphate/Sodium citrate for removal of protein contaminants is also included. However, in order to achieve the high level of purity one needs to perform repetitive ammonium sulfate precipitations making the process more tedious and labor intensive. Further, at times it also precipitates capsular polysaccharides, resulting in loss of total polysaccharide.
Some other methods make use of enzymes that help in degradation of proteins and nucleic acid contaminants; however the removal of enzymes and hydrolyzed material is a daunting task and may result in loss of the product of interest. Furthermore, regulatory agencies have restricted the use of animal enzymes in products for humans because of the risk of contamination with prions. The usage of enzymes, besides the fact of high cost, will introduce more regulatory issues in the cGMP framework e.g. the origins of enzymes (from animal or recombinant), enzyme activity variations between different vendors and lots, etc.
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 which can't be scaled up easily.
Some other methods made use of toxic organic solvents like Phenol, butanol, Toluene and chloroform for the separation of endotoxins of bacterial polysaccharides. This method is expensive and time consuming. Furthermore, it is unpleasant to work with toxic organic solvents that produce toxic waste.
The high purities required for polysaccharides specific to vaccine have led to the development of new purification methods based on fractional precipitation; ion exchange chromatography; gel filtration; and affinity chromatography. Chromatographic techniques like Size Exclusion chromatography, Ion exchange chromatography, and hydrophobic interaction chromatography have been successfully used for isolation of bacterial polysaccharides with effective removal of protein and nucleic acid contaminants. Despite the successful isolation of bacterial polysaccharide with WHO specifications, the use of

chromatographic techniques involves multistep labour and time consuming sample preparation, involves scalability issues, drastically compromises the recovery of the capsular polysaccharides and thus is not a feasible low cost option for industrial scale downstream processing. Further, the addition of new purification steps to eliminate these contaminants increases the complexity of the process, decreasing the final yields and increasing the economic costs.
The Salmonella typhi purified polysaccharides obtained by previously reported purification processes have endotoxin content between 25-50 EU/µg.
Hence, there exists a need of alternative purification methodologies, aimed to maximize recovery of Salmonella polysaccharides while removing impurities to acceptable levels.
Various methods such as acid hydrolysis, alkaline degradation, oxidation by periodate, ozonolysis (Wang et al. Carb. Res. 1999, 319, 1-4,141-147), enzymatic hydrolysis, sonication (Pawlowski et al. Vaccine, 2000, 18.18, 1873-1885), electron beam fragmentation (Pawlowski et al. Micro Lett, 1999, 174.2, 255-263) have been described for the depolymerisation (sizing) of bacterial and non-bacterial polysaccharides. However, acid hydrolysis, and alkaline degradation are time consuming and the resultant size reduced sample has high polydispersity. Also periodate oxidation has deleterious effects on labile antigenic epitopes of some polysaccharides. Further, ozonolysis can only be used with polysaccharides containing β-D-aldosidic linkages, and only few endoglycanases have been isolated till date.US 20090041802 discloses 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 wider deviations, less stable products and the need to run more passes or use higher pressures than should be required.
Treatments with ultrasounds have been used to depolymerise polysaccharides (see for example WO 2010/055250). However, ultrasonic depolymerization method is not suited for industrial depolymerization of a large bulk of polysaccharide, because of its low efficiency.
US20090234108 describe 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 destruction of immunogenic moieties which may affect immunogenicity of conjugates.

For all the reasons stated above, a simple and low-cost process for the depolymerisation of polysaccharides or polysaccharides derivatives is still desirable in the art.
Producing polysaccharide – protein conjugate vaccine is specific to the particular carrier protein and the native polysaccharide involved in the conjugation process.
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.
The activated polysaccharide is coupled directly or via a spacer (linker) group to an amino group on to the carrier protein. Linkers used for conjugation as disclosed in prior-art are N-succinimidyl-3-(2- pyridyldithio) propionate (SPDP) (SZU ET AL; 1987). Other linkers, B-propionamido (WO 00/10599), nitrophenyl - ethylamine (Gever et al (1979) Med Microbiol Immunol 165; 171-288), haloalkyl halide (U.S. Pat. No. 4,057,685) glycosidic bond (U.S. Pat. No. 4,673,574), hexane diamine and 6-aminocaproic acid (U.S. Pat. No. 4,459,286). Marburg et al., J. Am. Chem. Soc., 108, 5282 (1986), disclosed one means of conjugating polysaccharides and immunogenic proteins through bigeneric spacers. The Protein (PRO) was derivatized to exhibit pendant nucleophilic or electrophilic groups (PRO*), while a partner Polysaccharide (Ps) was functionalized so as to exhibit pendant groups of opposite reactivity (Ps*). Upon combining Ps* with PRO*, bigeneric spacers were formed, covalently joining Ps to PRO (Ps-PRO). Upon acid hydrolysis, the bigeneric spacer (linker) is released as an unusual amino acid, quantitated by amino acid analysis, and thereby providing a means of proving covalency.
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 which may adversely affect stability of product. Polysaccharide-carrier protein conjugates are known to release free polysaccharide after conjugation while further processing, lyophilization or storage in liquid as well as solid formulations. Only the Salmonella polysaccharide that is covalently bound to the carrier protein (i.e. Conjugated polysaccharide is immunologically important for clinical protection and excessive levels of unbound polysaccharide could potentially result in immunological hyporesponsiveness to polysaccharide (Refer WHO/TRS/924 Page No. 14, A.3.3.5). Particularly, the Salmonella

typhi conjugates reported in literature have a high free polysaccharide content upto 34% and high free protein content above 5% which indicates lower conjugation efficiency and lower stability of conjugates that is not desirable. Accordingly there is a need for vaccines demonstrating free polysaccharide content less than 10%.
Indeed, if it was possible to have a generic process that could be employed for manufacturing and formulating all vaccine candidates it would greatly reduce the time and resources needed for process development. This can have a significant impact on the number of clinical candidates that can be introduced into clinical trials. Also, processes developed for early stage clinical trials, including those developed using a platform, may be non-optimal with respect to process economics, yield, pool volumes, and throughput 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 given that process development needs to occur prior to introduction of a therapeutic candidate into clinical trials. Refer Abhinav A. Shukla et al Journal of Chromatography B, 848 (2007) 28–39.
Further, Salmonella disease burden is high in developing countries where availability of electrical power and refrigeration are often inadequate and therefore vaccine stability across temperature excursions assumes greater relevance for these regions.
Thus, there is a need for an efficient platform process for manufacturing an effective vaccine against Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis. that meets multiple criterion including good immunogenicity, safety and affordability, in particular a platform that provides i) improved polysaccharide yield across fermentation and purification processes; ii) improved purification processes showing optimal percentage recovery and minimum impurity levels; and iii) improved ratio of polysaccharide – protein conjugate in the vaccine iv) improved formulation showing low viscosity, devoid of aggregation; showing long-term stability across wide temperature ranges.
To overcome the aforementioned limitations of prior art and to resolve the long felt unmet global medical need, Applicant proposes improved, alternative fermentation, purification, conjugation processes, formulation(s) for preparing a monovalent Salmonella typhoid conjugate as well as multivalent vaccine(s) comprising of atleast one additional conjugates from Salmonella paratyphi (S. paratyphi A, B, C), and Non-typhoidal Salmonella enterica serovars typhimurium (S. typhimurium) and enteritidis (S. enteritidis)

OBJECTS
An object of the present disclosure is to ameliorate one or more problems of the prior art or to at least provide a useful alternative.
It is an object of the present disclosure to develop an effective vaccine formulation for prophylaxis and treatment of infections caused by Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in humans.
Another object of the present disclosure is to provide improved processes for the production of polysaccharide – protein conjugate vaccine comprising polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis that may be employed on an industrial scale.
Yet another object of the present disclosure is to provide a monovalent polysaccharide – protein conjugate vaccine comprising polysaccharide derived from either of Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis.
Yet another object of the present disclosure is to provide a bivalent polysaccharide – protein conjugate vaccine comprising polysaccharide derived from either of Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in any combination thereof.
Yet another object of the present disclosure is to provide a multivalent polysaccharide – protein conjugate vaccine comprising polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in any combination thereof.
Yet another objective of the present disclosure is to provide an improved fed-batch methods for production of polysaccharide of Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis.
Yet another objective of the present disclosure is to provide an improved purification method of polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis.
Yet another objective of the present disclosure is to provide an improved methods of conjugation of polysaccharide (with or without size reduction) derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis. to a carrier protein.

Yet another object of the present disclosure is to provide a method of conjugation of polysaccharide (with or without size reduction) derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis to a carrier protein with or without a linker (spacer) molecule.
Yet another object of the present disclosure is to provide immunogenic vaccine formulations comprising polysaccharide-protein conjugates in an appropriate single dose and multidose vials to be administered in infants and adults at appropriate concentrations effective to confer protection or treatment of infections against Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis or to prevent, ameliorate, or delay the onset or progression of the clinical manifestations thereof.
Other objects and advantages of the present disclosure will be more apparent from the following description, which is not intended to limit the scope of the present disclosure.
SUMMARY
The present disclosure provides:
a) an immunogenic composition comprising one or more of polysaccharide-protein conjugate wherein polysaccharide is derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis;
b) fed-batch methods for cultivation and processing of polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis;
c) downstream processing steps to obtain polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis;
d) Methods of Conjugation of polysaccharide (with or without size reduction) derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis to the carrier protein in the presence or absence of a linker molecule; and
e) methods for eliciting an immune response in a subject via administering a subject of a therapeutically effective amount of the immunogenic composition to confer protection or treatment of infections against Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis or to prevent, ameliorate, or delay the onset or progression of the clinical manifestations thereof.

DESCRIPTION
Although the present disclosure may be susceptible to different embodiments, certain embodiments are shown in the following detailed discussion, with the understanding that the present disclosure can be considered an exemplification of the principles of the disclosure and is not intended to limit the scope of disclosure to that which is illustrated and disclosed in this description.
Embodiments are provided so as to thoroughly and fully convey the scope of the present disclosure to the person skilled in the art. Numerous details are set forth, relating to specific components, and processes, to provide a complete understanding of embodiments of the present disclosure. It will be apparent to the person skilled in the art that the details provided in the embodiments should not be construed to limit the scope of the present disclosure. In some embodiments, well-known composition, well-known processes, and well-known techniques are not described in detail.
The terminology used, in the present disclosure, is only for the purpose of explaining a particular embodiment and such terminology shall not be considered to limit the scope of the present disclosure. As used in the present disclosure, the forms "a,” "an," and "the" may be intended to include the plural forms as well, unless the context clearly suggests otherwise.
The terms "comprises," "comprising," “including,” and “having,” are open ended transitional phrases and therefore specify the presence of stated features, integers, steps, operations, elements, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. The particular order of steps disclosed in the process of the present disclosure is not to be construed as necessarily requiring their performance as described or illustrated. It is also to be understood that additional or alternative steps may be employed.
The terms first, second, third, etc., should not be construed to limit the scope of the present disclosure as the aforementioned terms may be only used 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 disclosure. The present disclosure provides an immunogenic composition and a process for preparing the same.

The term "vaccine" is optionally substitutable with the term "immunogenic composition" and vice versa.
"D-antigen units" (also referred to as "international units" or IU): The D antigenic form of the poliovirus induces protective neutralising antibodies. D antigen units referred to herein (for instance in the vaccines of the invention) are the measured total D antigen units of each unadsorbed bulk IPV antigen type prior to formulation of the final vaccine which are added in each human dose of formulated vaccine (typically 0.5mL final volume). Reliable methods of measuring D-antigen units are well known in the art and are published, for instance, by the European Pharmacopoeia. For instance, D-antigen units may be measured using the ELISA test as described in Example 1 ("D-antigen quantification by ELISA") below. European Pharmacopoeia provides a test sample (European Pharmacopoeia Biological Reference Preparation - available from Ph. Eur. Secretariat, e.g. Code P 216 0000) for standardisation of such methods between manufacturers (Pharmeuropa Special Issue, Bio 96-2). Thus the D-antigen unit value is well understood in the art.
The term "dose" herein is typically one administration of the vaccine of the invention, which is typically one injection. A typical human dose is 0.5mL. Of course various doses may be administered in a vaccine administration schedule.
The term "IPV" or a immunogenic composition comprising these components herein is intended to mean inactivated polio virus type 1 (e.g. Mahoney, as preferably used), type 2 (e.g. MEF-1), or type 3 (e.g. Saukett), or a Sabin Serotype 1, 2, 3 combination of either two or all three of these types. An example of a full (or standard) dose (40-8-32 D antigen units of Salk based IPV types 1, 2 and 3 respectively) IPV immunogenic composition for the purposes of this invention could be Poliovac® (Serum Institute of India Pvt. Ltd.).
The term "saccharide" throughout this specification may indicate polysaccharide or oligosaccharide and includes both. The capsular saccharide antigen may be a full length polysaccharide or it may be extended to bacterial 'sized-saccharides' and 'oligosaccharides' (which naturally have a low number of repeat units, or which are polysaccharides reduced in size for manageability, but are still capable of inducing a protective immune response in a host.
According to a first embodiment of the present disclosure, the immunogenic composition may comprise one or more of the polysaccharide-protein conjugate, wherein the

polysaccharide is derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis.
According to a second embodiment of the present disclosure, the method of obtaining the polysaccharide derived from Salmonella serovar strains 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 a Fermentation medium compositions,
2. Inactivation,
3. Separation,
4. Clarification, and
5. Sterilized Filtration.
According to a first aspect of 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 can be selected from the group consisting of glucose, mannitol, sucrose, lactose, fructose, and trehalose, preferably Glucose. The magnesium salt may be selected from magnesium chloride, magnesium sulfate, preferably Magnesium sulfate heptahydrate. The potassium source may be selected from Di-sodium hydrogen phosphate heptahydrate, sodium di-hydrogen phosphate monohydrate, potassium phosphate, and dipotassium phosphate. Preferably the potassium source is a combination of Di-sodium hydrogen phosphate heptahydrate, sodium di-hydrogen phosphate. Preferably, the soy hydrolysate is hysoy.
Yet accordingly the fermentation medium may additionally comprise an anti-foam agent selected from the group of Antifoam 204, Antifoam C, SE‐15, Y‐30, Antifoam EX‐Cell, S184 (pure silicon oil), SLM54474 (polypropylene glycol: PPG), VP1133 (silicon oil/PPG mixture), BREOX (polyalkylene glycol), J673 STRUKTOL (Alkoxylated fatty acid esters on vegetable base) and SE9 (aqueous emulsion with 10% silicon oil component) of Wacker-Chemie Co. The anti-foam agent in combination with soy hydrolysate and yeast extract may aid in improved yield of the polysaccharides. Yet preferably the anti-foam agent may be Antifoam C or J673 STRUKTOL.

In accordance with the embodiments of the present disclosure, 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™, Difco™ 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 Difco™ Select Phytone™ UF. In another embodiment, the soy hydrolysate may be hysoy.
The combination of antifoam, soya peptone and yeast extract results in improved harvest yield as compared to other media.
According to a second aspect of second embodiment, the process may follow a two shot strategy by incorporating the feed contents at a fixed proportion at particular fixed time intervals during when the fermentation is already undergoing and/or allowing continuous feed throughout the fed-batch mode of fermentation comprising multiple stages with the proportionate increase in the batch size at every stage.
According to a third aspect of second embodiment, the fermentation parameters may comprise of:
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 – 600 nl/min
Osmolality: 400 – 600 mOsm/kg
According to a fourth aspect of second embodiment, the inactivation of the bacterial culture may be carried out using formalin.
Yet preferably the inactivation of the bacterial culture may be carried out by using formaldehyde in the range of 0.1 to 2 % v/v, preferably 0.5 % v/; incubated at 34 to 38 deg C , preferably 36 deg C; for 5-12 hrs, preferably 8 to 12 hrs.

According to a fifth aspect of second embodiment, the separation may be carried out by centrifugation. Yet preferably the separation may be carried out by centrifugation with parameters set at temperature 2-8 deg C; RPM – 7000-8000; Centrifuge time 40 -60 min.
According to a sixth aspect of second embodiment, the clarification may be carried out through depth filtration.
According to a seventh aspect of second embodiment, the clarified harvest may be sterilized through filtration using 0.2 µM sterile filters.
According to eighth aspect of second embodiment, the crude Salmonella enterica serovar typhi Vi-polysaccharide (ViPs) yield at the fermentation stage may be at least 40% and the average Vi-polysaccharide yield could be in the range of 100 mg/L to 5000 mg/L; more preferably 100-700 mg/L.
According to a third embodiment of the present disclosure, the fermentation harvest may be subjected to any subset or in any order or all of the following downstream purification steps to obtain 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 having 10 – 300 kDa or kD molecular weight cut off (MWCO);
c) treatment with anionic or cationic detergent, ethylene diamine tetra-acetic acid (EDTA) (4 to 10mM) and Sodium acetate (5% to 10%) for denaturation of proteins, nucleic acids and lipopolysaccharide;
d) alcohol precipitation (40% to 70%);
e) Centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 µM;
f) treatment with akali salt for removal of excess detergent followed by Centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 µM;
g) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 - 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
clarification filter having a pore size of about 0.45 micrometers to about 0.2 micrometers; j) removal of protein and nucleic acid impurities by washing pellet with alcohol (50% to
70%) in presence of sodium chloride (0.1M to 2M); k) selective precipitation of polysaccharide by utilizing alcohol (<75% OR>95% ); l) dissolving polysaccharide in WFI and subjecting to concentration by tangential flow
filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 -
300kDa or kD molecular weight cut off (MWCO); and m) Sterile Filtration through at least one sterile filter having a pore size of about 0.2
micrometers under sterile conditions.
According to a first aspect of third embodiment, the purification process may be devoid of any chromatography step.
According to a second aspect of third embodiment, the purification process may result in significant recovery of about 40% to 65% with the desired O-acetyl levels ( greater than 2.0 mmol/g polysaccharide), purified Vi polysaccharide yield could be in the range of 1000 to 4000 mg/L, average molecular weight could be in the range of 40 to 400 kDa, contains less than 1% proteins/peptides, less than 2% nucleic acids, less than 100 EU of endotoxins per µg of polysaccharide (PS), Molecular size distribution (greater than 50% of PS is eluted before a distribution coefficient (KD) of 0.25 is reached)
Yet preferably the average molecular weight of the purified Vi polysaccharide could be in the range of 40 to 400 kDa.
According to a third aspect of third embodiment, the anionic detergent may be selected from the group comprising of alkyl sulfates, sodium dodecyl sulfate (SDS), sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethylene ether sulfates, sodium oleyl sulfate, N-oleoyl poly (amino acid) sodium, sodium alkylbenzene sulfonates, sodium alpha olefin sulfonate , sodium alkyl sulfonates, alpha-sulfo monocarboxylic acid esters, fatty acid sulfoalkyl esters, succinate sulfonate, alkyl naphthalene sulfonates, sodium alkane sulfonates, sodium ligninsulfonate , and sodium alkyl glyceryl ether sulfonates.

Yet preferably, said anionic detergent could be alkyl sulphate, more preferably sodium dodecyl sulphate at a final concentration in the range of 0.1% to 20%, more preferably in the range of 1-20% may be added to the retentate and stirred at 25°C - 30°C for 2 hour.
According to a fourth aspect of third embodiment, the alcohol precipitation may be carried out using methanol, ethanol, n-propyl alcohol, isopropyl alcohol, acetone or t-butyl alcohol; or a combination thereof.
Yet preferably the said alcohol could be ethanol.
According to a fifth aspect of 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.
Yet preferably, the said potassium salt could be potassium chloride at a final concentration in the range of 0.1M to 2M mixed with the supernatant, and upon its dissolution the mixture was incubated at 2-8°C for >3 hours.
According to a sixth aspect of third embodiment, the cationic detergent may be selected from the group comprising of cetyltrimethylammonium salt, tetrabutylammonium salt, myristyltrimethylammonium salt and hexadimethrine bromide; or a combination thereof.
Yet preferably, said cationic detergent could be Cetyl trimethylammonium bromide (CTAB) at a final concentration in the range of 0.1% to 12%; preferably at 2% - 3% may be added to the retentate and stirred at 25°C - 30°C for 1 -2 hour.
According to a seventh aspect of third embodiment, the final purified polysaccharide bulk may be stored at less than or equal to -20°C.
According to a fourth embodiment of the present disclosure, the fermentation harvest may be subjected to any subset or in any order or all of the following downstream purification steps to obtain desired quality of O-specific polysaccharide from Salmonella Paratyphi A lipopolysaccharide (LPS):
a) Centrifugation and separation

octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) Sucrose
g) Water for Injection (WFI) q.s.
52. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) 2-Phenoxyethanol 1 – 10 mg
g) Water for Injection (WFI) q.s.
53. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer

f) Sucrose
g) 2-Phenoxyethanol 1 – 10 mg
h) Water for Injection (WFI) q.s.
54. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) Water for Injection (WFI) q.s.
55. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) 2-Phenoxyethanol 1 – 10 mg
e) Water for Injection (WFI) q.s.
56. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine

polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate,
soy lecithin and a poloxamer
f) Water for Injection (WFI) q.s.
57. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) Sucrose
g) Water for Injection (WFI) q.s.
58. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) 2-Phenoxyethanol 1 – 10 mg
g) Water for Injection (WFI) q.s.

58. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
c) Sodium chloride 1 – 10 mg
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) Sucrose
g) 2-Phenoxyethanol 1 – 10 mg
h) Water for Injection (WFI) q.s.
59. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Water for Injection (WFI) q.s.
60. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg

e) 2-Phenoxyethanol 1 – 10 mg
f) Water for Injection (WFI) q.s.
61. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) Water for Injection (WFI) q.s.
62. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) Sucrose
g) Water for Injection (WFI) q.s.

63. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) 2-Phenoxyethanol 1 – 10 mg h) Water for Injection (WFI) q.s.
64. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
f) Sucrose
g) 2-Phenoxyethanol 1 – 10 mg
h) Water for Injection (WFI) q.s.

65. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Water for Injection (WFI) q.s.
66. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Sucrose
h) Water for Injection (WFI) q.s.

67. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) 2-Phenoxyethanol 1 – 10 mg h) Water for Injection (WFI) q.s.
68. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Sucrose
h) 2-Phenoxyethanol 1 – 10 mg i) Water for Injection (WFI) q.s.

69. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Water for Injection (WFI) q.s.
70. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) 2-Phenoxyethanol 1 – 10 mg
f) Water for Injection (WFI) q.s.
71. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Water for Injection (WFI) q.s.
72. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;

b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) 2-Phenoxyethanol 1 – 10 mg
g) Water for Injection (WFI) q.s.
73. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Water for Injection (WFI) q.s.
74. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg

f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Sucrose
h) Water for Injection (WFI) q.s.
75. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) 2-Phenoxyethanol 1 – 10 mg h) Water for Injection (WFI) q.s.
75. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg

f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Sucrose
h) 2-Phenoxyethanol 1 – 10 mg i) Water for Injection (WFI) q.s.
76. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Water for Injection (WFI) q.s.
77. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) 2-Phenoxyethanol 1 – 10 mg
f) Water for Injection (WFI) q.s.
78. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:

a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Water for Injection (WFI) q.s.
79. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) 2-Phenoxyethanol 1 – 10 mg
g) Water for Injection (WFI) q.s.
80. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,

octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer g) Water for Injection (WFI) q.s.
81. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Sucrose
h) Water for Injection (WFI) q.s.
82. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,

octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer g) 2-Phenoxyethanol 1 – 10 mg h) Water for Injection (WFI) q.s.
83. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Sodium chloride 1 – 10 mg
e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer
g) Sucrose
h) 2-Phenoxyethanol 1 – 10 mg i) Water for Injection (WFI) q.s.
84. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg

f) Water for Injection (WFI) q.s.
85. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg
f) 2-Phenoxyethanol 1 – 10 mg
g) Water for Injection (WFI) q.s.
86. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg
f) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
g) Water for Injection (WFI) q.s.
87. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197

d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg
f) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
g) 2-Phenoxyethanol 1 – 10 mg
h) Water for Injection (WFI) q.s.
88. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg
f) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,
polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,
octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine
polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate,
soy lecithin and a poloxamer
h) Water for Injection (WFI) q.s.
89. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg

f) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,
polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,
octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine
polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate,
soy lecithin and a poloxamer
h) Sucrose
i) Water for Injection (WFI) q.s.
90. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg; wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg
f) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,
polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,
octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine
polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate,
soy lecithin and a poloxamer
h) 2-Phenoxyethanol 1 – 10 mg i) Water for Injection (WFI) q.s.
91. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition
comprises of:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
c) Salmonella enterica serovar typhimurium saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197

d) Salmonella enterica serovar enteritidis saccharide – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197
e) Sodium chloride 1 – 10 mg
f) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg
g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,
polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,
octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine
polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate,
soy lecithin and a poloxamer
h) Sucrose
i) 2-Phenoxyethanol 1 – 10 mg
j) Water for Injection (WFI) q.s.
92. A single dose vaccine kit comprising:
a first container containing a lyophilized (freeze-dried) immunogenic composition:
a) Neisseria meningitidis A saccharide – TT conjugate antigen 5 µg per 0.5 ml;
b) Neisseria meningitidis C saccharide – CRM197 conjugate antigen 5 µg per 0.5 ml;
c) Neisseria meningitidis Y saccharide – CRM197 conjugate antigen 5 µg per 0.5 ml;
d) Neisseria meningitidis W -135 saccharide – CRM197 conjugate antigen 5 µg per 0.5 ml;
e) Neisseria meningitidis X saccharide – TT conjugate antigen 5 µg per 0.5 ml;
f) Sucrose 1 – 12 mg per 0.5 ml;
g) Sodium citrate (Dihydrate) 0.1 – 2 mg per 0.5 ml;
h) Tris Buffer 0.05 – 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 – 50 µg;
b) Sodium chloride 1 – 10 mg;
c) Water for Injection (WFI) q.s.;
wherein there is no antigenic interference of ViPs-TT with Neisseria meningitidis antigens, for prophylaxis against typhoid caused by Salmonella typhi and Neisseria meningitidis antigens wherein the said vaccine formulation is sufficient to elicit the required T- dependent

immune response against S. typhi including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
93. A single dose vaccine kit comprising:
a first container containing a lyophilized (freeze-dried) immunogenic composition:
a) Neisseria meningitidis A saccharide – TT conjugate antigen 5 µg per 0.5 ml;
b) Neisseria meningitidis C saccharide – CRM197 conjugate antigen 5 µg per 0.5 ml;
c) Neisseria meningitidis Y saccharide – CRM197 conjugate antigen 5 µg per 0.5 ml;
d) Neisseria meningitidis W -135 saccharide – CRM197 conjugate antigen 5 µg per 0.5 ml;
e) Neisseria meningitidis X saccharide – TT conjugate antigen 5 µg per 0.5 ml;
f) Sucrose 1 – 12 mg per 0.5 ml;
g) Sodium citrate (Dihydrate) 0.1 – 2 mg per 0.5 ml;
h) Tris Buffer 0.05 – 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 – 50 µg;
b) Salmonella enterica serovar paratyphi A OSP – CP conjugate antigen 1.25 – 50 µg;
wherein the CP is either TT or DT or CRM197;
c) Sodium chloride 1 – 10 mg;
d) Water for Injection (WFI) q.s.;
wherein there is no antigenic interference of ViPs-TT; OSP antigen with Neisseria meningitidis antigens, for prophylaxis against typhoid and paratyphoid caused by Salmonella typhi, S. paratyphi and Neisseria meningitidis antigens wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against S. typhi and paratyphi including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
94. A single dose vaccine kit comprising:
a first container containing a fully liquid hexavalent immunogenic composition:
a) an inactivated polio virus (IPV) antigen selected from Sabin or Salk Strain; wherein IPV Type 1 at a dose 1-50 D-antigen units (DU), IPV Type 2 at a dose of 1-50 D-antigen unit (DU) or IPV Type 3 at a dose of 1-50 D-antigen unit (DU), per 0.5 ml;

b) a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml;
c) a tetanus toxoid, (T) antigen in an amount of 1 to 30 Lf per 0.5 ml;
d) a whole cell pertussis, (wP) antigen 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, Pertussis toxoid (PT) 1-50µg, Filamentous hemagglutinin (FHA) 1-50µg, Pertactin (P69 or PRN) 1-20µg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25µg; per 0.5ml;
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, (Ηib) in an amount of 1 to 20 µg per 0.5 ml;
and
a second container containing a fully liquid immunogenic composition comprising:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 µg;
b) Sodium chloride 1 - 10 mg; and/or
c) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg; and/or
d) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer; and/or
e) 2-Phenoxyethanol 1 - 10 mg; and/or
f) Water for Injection (WFI) q.s.
wherein there is no antigenic interference of ViPs-TT with Hexavalent immunogenic composition, for prophylaxis against typhoid caused by Salmonella typhi and Hexavalent antigens wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against S. typhi including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
95. A single dose vaccine kit comprising:
a first container containing a fully liquid hexavalent immunogenic composition:

a) an inactivated polio virus (IPV) antigen selected from Sabin or Salk Strain; wherein IPV Type 1 at a dose 1-50 D-antigen units (DU), IPV Type 2 at a dose of 1-50 D-antigen unit (DU) or IPV Type 3 at a dose of 1-50 D-antigen unit (DU), per 0.5 ml;
b) a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml;
c) a tetanus toxoid, (T) antigen in an amount of 1 to 30 Lf per 0.5 ml;
d) a whole cell pertussis, (wP) antigen 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, Pertussis toxoid (PT) 1-50µg, Filamentous hemagglutinin (FHA) 1-50µg, Pertactin (P69 or PRN) 1-20µg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25µg; per 0.5ml;
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, (Ηib) in an amount of 1 to 20 µg per 0.5 ml;
and
a second container containing a fully liquid immunogenic composition comprising:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 µg;
b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 µg; wherein the CP is either TT or DT or CRM197;
c) Sodium chloride 1 - 10 mg; and/or
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg; and/or
g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,
polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,
octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine
polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate,
soy lecithin and a poloxamer; and/or
h) 2-Phenoxyethanol 1 - 10 mg; and/or
e) Water for Injection (WFI) q.s.
wherein there is 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 the said vaccine formulation is sufficient to elicit the required T- dependent immune response against S. typhi and paratyphi including in

children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
96. A single dose vaccine kit comprising:
a first container containing a fully liquid heptavalent immunogenic composition:
a) an inactivated polio virus (IPV) antigen selected from Sabin or Salk Strain; wherein IPV Type 1 at a dose 1-50 D-antigen units (DU), IPV Type 2 at a dose of 1-50 D-antigen unit (DU) or IPV Type 3 at a dose of 1-50 D-antigen unit (DU), per 0.5 ml;
b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strains present in an amount in the range of 1 to 50 µg per 0.5 ml;
c) a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml;
d) a tetanus toxoid, (T) antigen in an amount of 1 to 30 Lf per 0.5 ml;
e) a whole cell pertussis, (wP) antigen 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, Pertussis toxoid (PT) 1-50µg, Filamentous hemagglutinin (FHA) 1-50µg, Pertactin (P69 or PRN) 1-20µg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25µg; per 0.5ml;
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, (Ηib) in an amount of 1 to 20 µg per 0.5 ml;
and
a second container containing a fully liquid immunogenic composition comprising:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 µg;
b) Sodium chloride 1 - 10 mg;
c) Water for Injection (WFI) q.s.;
wherein there is no antigenic interference of ViPs-TT with Heptavalent immunogenic composition, for prophylaxis against typhoid caused by Salmonella typhi and Heptavalent antigens wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against S. typhi including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
97. A single dose vaccine kit comprising:

a first container containing a fully liquid hexavalent immunogenic composition:
a) an inactivated polio virus (IPV) antigen selected from Sabin or Salk Strain; wherein IPV Type 1 at a dose 1-50 D-antigen units (DU), IPV Type 2 at a dose of 1-50 D-antigen unit (DU) or IPV Type 3 at a dose of 1-50 D-antigen unit (DU), per 0.5 ml;
b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strains present in an amount in the range of 1 to 50 µg per 0.5 ml;
c) a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml;
d) a tetanus toxoid, (T) antigen in an amount of 1 to 30 Lf per 0.5 ml;
e) a whole cell pertussis, (wP) antigen 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, Pertussis toxoid (PT) 1-50µg, Filamentous hemagglutinin (FHA) 1-50µg, Pertactin (P69 or PRN) 1-20µg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25µg; per 0.5ml;
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, (Ηib) in an amount of 1 to 20 µg per 0.5 ml;
and
a second container containing a fully liquid immunogenic composition comprising:
a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 µg;
b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 µg; wherein the CP is either TT or DT or CRM197;
c) Sodium chloride 1 - 10 mg; and/or
d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg; and/or
g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65,
polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,
octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer; and/or h) 2-Phenoxyethanol 1 - 10 mg; and/or
e) Water for Injection (WFI) q.s.

wherein there is 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 wherein the said vaccine formulation is sufficient to elicit the required T- dependent immune response against S. typhi and paratyphi including in children below 2 years of age, adolescent adult, and elders through only one injection to comprise a complete vaccination schedule.
98. A method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate comprises of:
a. Fed-batch mode of cultivation to obtain a high yield harvest of Salmonella typhi capsular
Vi-polysaccharide (ViPs), wherein the use of combination of an antifoam agent, soya
peptone at a range of 40 to 70 g/L and yeast extract at a range of 40 to 70 g/L during
cultivation results in improved harvest yield (100-700 mg/L), and the fermentation
parameters comprises of pH maintained in the range of 6.7 to 7.1, temperature maintained in
the range of 34.0- 38.0°C, dissolved oxygen level maintained between 36- 39%, Agitation
(rpm) maintained between 150 to 500 and osmolality 400 – 600 mOsmol/kg;
b. Purifying Salmonella typhi capsular ViPs by treatment with anionic detergent more
preferably sodium dodecyl sulfate (SDS) less than 2% and cationic detergent preferably
CTAB, ethylene diamine tetra-acetic acid (EDTA) (4 to 10mM), Sodium acetate (5% to
10%), alcohol precipitation (30% to 60%) and concentration and diafiltration using 30kDa
cut off membranes at different steps, wherein the process results in significant reduction of
endotoxin (< 100EU of endotoxin per µg of PS), protein (< 1%) and nucleic acid (< 2%)
impurities, higher recovery of capsular polysaccharide suitably in the range of 40% to 65%,
with the desired O-acetyl levels (> 2.0 mmol/g polysaccharide), purified Vi polysaccharide
yield in the range of 100 to 4000 mg/L, Molecular size distribution (>50% of PS is eluted
before a distribution coefficient (KD) of 0.25 is reached) and average molecular weight of the
purified Vi polysaccharide could be in the range of 40 to 400 kDa, preferably between 150 to
250kDa;
c. Conjugating Purified ViPs to carrier protein (CP) using a carbodiimide, reductive
amination, or cyanylation conjugation reaction
d. Purifying ViPs-carrier protein conjugate using Gel Filtration Chromatography or Ultra
filtration whereby the conjugate bulk is concentrated at least 3 fold and substantially all
unreacted compounds, unconjugated polysaccharides, unconjugated proteins are removed,

yielding a purified Vi polysaccharide conjugate vaccine having size between 1000 to 1500 kDa wherein the conjugate yield is ≥ 50%.
99. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier
protein conjugate as claimed in claim 98, wherein before conjugation the ViPs 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 , homogenizer, sonication.
100. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 98, wherein the Purified ViPs is covalently bound to carrier protein (CP) using carbodiimide conjugation reaction in presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC) mixed in a ratio of 1:0.5 to 1:2 by weight of ViPs: ED AC and the ratio of Vi polysaccharide and carrier protein is between 0.5 and 1.5, ViPs: CP: ED AC ratio is preferably 1:1:2 or 1:0.9:2 or 1:0.8:1.8 or 1:0.7:1.8, reaction carried out at a pH in range of 5-7, temperature in range of 2°C to 30°C.
101. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 100, wherein before conjugation the polysaccharide or carrier protein is derivatized with a hetero or homo-bifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethyl amine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propinamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid, and combinations thereof using a carbodiimide, reductive amination or cyanylation reaction.
102. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 101, wherein before conjugation the carrier protein (CP) is derivatized with an adipic acid dihydrazide (ADH) linker in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC), reaction duration for 1 hour, carried out at a pH in range of 5-7.
103. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 98, 99, 100, 101 and 102 wherein the carrier protein is selected from the group comprising of tetanus toxin, tetanus toxoid, fragment of tetanus toxoid, diphtheria toxoid, CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis

toxoid, Clostridium perfringens toxoid, E.coli LT, E. coli ST, Escherichia coli heat-labile toxin - B subunit, Neisseria meningitidis outer membrane complex, rEPA, protein D of H. influenzae, Flagellin FliC, Horseshoe crab Haemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of 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 from C. difficile and S.agalactiae proteins with or without linker and fragments, derivatives, modifications thereof.
104. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 103, wherein the carrier protein is tetanus toxoid (TT).
105. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 103, wherein the carrier protein is diphtheria toxoid (DT).
106. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 103, wherein the carrier protein is CRM197.
107. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate comprises of:
a. Purifying Salmonella Paratyphi A O-specific polysaccharide (OSP) of the lipopolysaccharide (LPS), by Acid hydrolysis of LPS preferably Acetic acid (final concentration 0.5 -5%) pH~2.0 - 3.0, neutralized to pH 7.0 with liquor ammonia, Sodium deoxycholate (final concentration 0.5 -5%), and concentration and diafiltration using 10kDa to 30kDa cut off membranes at different steps, wherein the process results in significant reduction of endotoxin (< 100EU of endotoxin per µg of PS), protein (< 1%) and nucleic acid (< 2%) impurities, higher recovery of capsular polysaccharide suitably in the range of 40% to 65%, with the 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 the purified O-specific polysaccharide (OSP) could be in the
range of 40 to 400 kDa;
b. Conjugating Purified OSP to carrier protein (CP) using a carbodiimide, reductive
amination, or cyanylation conjugation reaction
c) Purifying OSP-carrier protein conjugate using Gel Filtration Chromatography or Ultra
filtration whereby the conjugate bulk is concentrated at least 3 fold and substantially all
unreacted compounds, unconjugated polysaccharides, unconjugated proteins are removed,
yielding a purified OSP-carrier protein conjugate vaccine wherein the conjugate yield is ≥
50%.
108. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 107, wherein before conjugation the OSP is 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 ,sonication and homogenizer.
109. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 107, wherein the purified OSP is covalently bound to carrier protein (CP) using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1- cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).
110. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 109, wherein the purified OSP is covalently bound to 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 in between 0.5 and 1.5, reaction carried out at a pH in range of 5-7, temperature in range of 2°C to 30°C.
111. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 110, wherein before conjugation the carrier protein or OSP derivatized with a hetero or homo-bifunctional linker selected from the group

consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethyl amine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propinamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid, and combinations thereof using a carbodiimide, reductive amination or cyanylation reaction using a carbodiimide, reductive amination or cyanylation reaction.
112. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 111, wherein before conjugation the carrier protein (CP) is derivatized with an adipic acid dihydrazide (ADH) linker in the presence of carbodiimide, such as 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC), mixed in a ratio of 1:1 to 1:10 by weight of OSP: ADH and the ratio of OSP: EDAC may be in between 0.5 and 1.5, reaction carried out at a pH in range of 5-7, reaction duration for 1 - 2 hour, temperature in range of 2°C to 30°C.
113. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 107, wherein the purified OSP is covalently bound to carrier protein (CP) using carbodiimide conjugation reaction in presence of 1-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDAC) mixed in a ratio of 1:0.5 to 1:2 by weight of OSP: EDAC and the ratio of OSP polysaccharide and carrier protein may be in between 0.5 and 1.5, reaction carried out at a pH in range of 5-7, temperature in range of 2°C to 30°C.
114. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 113, wherein before conjugation the carrier protein or OSP is derivatized with a hetero or homo-bifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nitrophenylethyl amine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propinamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid, and combinations thereof using a carbodiimide, reductive amination or cyanylation reaction using a carbodiimide, reductive amination or cyanylation reaction.
115. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 114, wherein before conjugation the 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 (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).
116. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 115, wherein before conjugation the 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 in between 0.5 and 1.5, reaction carried out at a pH in range of 7-10, reaction duration for 1 – 2 hour, temperature in range of 2°C to 30°C.
117. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claims 107 to 116, wherein the carrier protein is selected from the group comprising of tetanus toxin, tetanus toxoid, fragment of tetanus toxoid, diphtheria toxoid, CRM197, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, E.coli LT, E. coli ST, Escherichia coli heat-labile toxin - B subunit, Neisseria meningitidis outer membrane complex, rEPA, protein D of H. influenzae, Flagellin FliC, Horseshoe crab Haemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumolysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesin A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-11, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and lethal factor (LF) of 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 from C. difficile and S.agalactiae proteins with or without linker and fragments, derivatives, modifications thereof.
118. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 117, wherein the carrier protein is tetanus toxoid (TT).

119. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 117, wherein the carrier protein is diphtheria toxoid (DT).
120. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugate as claimed in claim 117, wherein the carrier protein is CRM197.
121. The immunogenic composition according to any one of the preceding claim, wherein the immunogenic composition is formulated for use in a method for prophylaxis against typhoid, paratyphoid and Non-typhoid infections, reducing the onset of or preventing infections caused by Salmonella typhi, Salmonella paratyphi and Salmonella Non-typhi related spp. the method involving administration of an effective amount of the immunogenic composition to a human subject 2 years of age or below, adolescent, adults or elders via via parenteral or subcutaneous or intradermal or 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, peroral or intragastric or mucosal or perlinqual, alveolar or gingival or olfactory or respiratory mucosa administration or any other routes of immunization.
122. The immunogenic composition according to any one of the preceding claim, wherein the immunogenic composition is stable at 2-8°C, 25°C and 40°C for over a period of six months.
123. The immunogenic composition according to any one of the preceding claim, wherein the immunogenic composition is stable at 2-8°C, 25°C and 40°C and free polysaccharide after 6 months not more than 7.5% for 180-220 kDa polysaccharide and free polysaccharide not more than 10.5% for 388/80/45 polysaccharide.
124. The immunogenic composition according to any one of the preceding claim, wherein the immunogenic composition is formulated for administration to a human subject 2 years of age or below, adolescents, adults or elders according to a one dose regimen consisting of a single dose of the said vaccine composition.
125. The immunogenic composition according to any one of the preceding claim, wherein the immunogenic composition is formulated for administration to a human subject 2 years of age or below, adolescents, adults or elders according to a two dose regimens consisting of a first dose and a second dose to be administered between 3 months to 2 years after the first dose.

126. The immunogenic composition according to any one of the preceding claim, wherein the immunogenic composition is formulated for administration to a human subject 2 years of age or below, adolescents, adults or elders according to a three dose regimens consisting of a first dose, a second dose to be administered between 3 months to 2 years after the first dose and a third dose to be administered between 3 months to 2 years after the second dose.