OA20613A

OA20613A - Immunogenic compositions against enteric diseases and methods for its preparation thereof.

Info

Publication number: OA20613A
Application number: OA1202200079
Authority: OA
Inventors: Rajeev Mhalasakant DHERE; Sambhaji Shankar PISAL; Dattatreya Sarma Annamraju; Nikhil Dattatray Avalaskar; Yogesh Tukaram Hundekari; Anil Pirajirao TAKLIKAR; Sunil Kumar Goel; Chandrashekhar Dwarkanath KAMAT; Vishal Bharat Chavan
Original assignee: Serum Institute Of India Private Limited
Priority date: 2019-09-03
Filing date: 2020-09-02
Publication date: 2022-11-29

Abstract

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

Description

TITLE

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 préparation.

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 gastroenterîtis in both normal and immunocompromised individuals.

The genus Salmonella belongs to the famîly of Enterobacteriaceae and comprises Gramnegative, non-spore forming, facultative anaérobie bacilh. 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 gastroenterîtis 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 mortalîty 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 middleincome 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: e57O-e58O & J Glob Health. 2012; 2: 10401). Salmonella paratyphi is responsible for increasing proportion of enteric in parts of Asia, including in Népal, Cambodia, and China. Salmonella paratyphi has highest burdens on the Indîan subcontinent and South East Asia. In one of the studies in Népal where typhoid fever is highly endemic, the municipal water was found to be contaminated with both S. typhi and Salmonella enter ica serovar paratyphi A (PLoS Negl Trop Dis. 2016 Jan; 10(l):c0004346 & PLoS Negl Trop Dis. 2013; 7(8):e2391).

Non-typhoidal Salmonella enter ica (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 ail human isolâtes of NTS reported globally. Further, invasive non-typhoidal Salmonella (iNTS) infections caused by serovars enteritidis (SE) and typhimurium (STm) are major pédiatrie health problems in subSaharan 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 présents 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 ail human isolâtes 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 économie 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 extraintestinal, invasive bacteremia, which is referred to as iNTS. Invasive infections of Salmonella are more common throughout the developing world and hâve become the most common cause of bacteremia in tropical Africa, especially among young children and individuals with HIV. It usually présents 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 opportunités for human-to-human transmission,

e.g., through contammated water supplies., and NTS bacteremia m HIV-infected Afncan adults has an associated high mortality (up to 47 percent) and récurrence rate (43 percent) rate.

Antibiotics hâve been used to treat typhoidal and Non-typhoidal Salmonella related infections, and the choice of antimicrobiais and length of treatment are determined by the cost and availability of antibiotics, local pattern of résistance, 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 availabie tools for treatment become less effective, the problem of Typhoidal and Nontyphoidal 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 Ty2la vaccine. World Health Organization (WHO) has recommended greater use of typhoid vaccines with preference given to Typhoid Conjugale Vaccines (TCV) (WHO position paper. Wkly Epidemiol Rec 2018; 93:153-72).

Vaccines for S. paratyphi are currently not availabie. Lîkewise, 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 ail directed against only S. typhi. S. paratyphi A causes enteric lever with the same géographie 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 additionaily protect against NTS serovars for this région.

It is unclear as to whether vaccine candidates in pipeline will be protective against both the gastroenteritis and invasive manifestations of iNTS. While understandmg of the disease burden of typhoid in LMICs is growing, the global medical demand for a conjugale vaccine protecting against typhoidal and non-typhoidal Salmonella infections has attained significance. It is estimated that the peak demand for a typhoid conjugale vaccine is likely to 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 élimination of cell and débris. Further, in order to faciiitate cost-effective Salmonella polysaccharide based conjugale 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 employîng novel feed strategies, and improved fermentation medium has been one of the préférable approaches to achieve said objective. Merrill 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 înfluenzae Type B, and Neisseria meningitidis showed that production was dépendent upon the fermentation conditions (température, 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 hâve been reported when osmolality was high.

The polysaccharides hâve 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/1 and that low oxygen tension favoured higher polysaccharide production. Further, it has been observed that concentrâted casamino acids in the feed solution limited the final cell density. Further, growth and polysaccharide yield in a defined medium is also dépendent 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 pullulons was affected by the ammonia nitrogen source in the medium, and its yield fell when excess ammonium ions were présent, 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 vîability and productivity in a tîmely manner. Hydrolysates are protein digests composed of amino acids, small peptides, carbohydrates, vitamins and minerais that provide nutrient suppléments 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-Iot variations, undesirabie attribute of making culture viscous, Yeast ex tract and hydrolysates can be a significant source of medium variabiiity. 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 îîi) resuit in a loss of sterility if the foam escapes and iv) can lead to over-pressure if a foamout blocks an exit fïlter.

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 rôle in downstream processing or purification of bacterial polysaccharides from host cell impurities. Downstream involves inactivation of bacterial culture, séparation 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 a(l4)-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)-rx-D-Manρ-(1^4)-αL-Rhap-(1^3)-α-D-Galp-( 1—►) (which serologically construites epitope 12). However, a dideoxy hexose saccharide linked a-(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 a(1—*6) glucosylated. (Refer: Lindberg AA, Le Minor L. Seroiogy of Salmonella In: Bergan T, ed. Methods in Microbiology: Academie 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 încludes lysing and précipitation 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 animai-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 élimination requires extensive washing to remove ail the residues.

For some CPS types, précipitation 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 répétitive ammonium sulfate précipitations making the process more tedious and labor intensive. Further, at times it also précipitâtes capsular polysaccharides, resulting in loss of total polysaccharide.

Some other methods make use of enzymes that help in dégradation of proteins and nucleic acid contaminants; however the removal of enzymes and hydrolyzed material is a daunting task and may resuit in loss of the product of interest. Furthermore, regulatory agencies hâve 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, Protéinase K or Nargase for dégradation of residual proteins and/or nucleic acid materials followed by chromatographie purification resulting in high costs and process which can't be scaled up easily.

Some other methods made use of toxic organic solvents like Phénol, butanol, Toluene and chloroform for the séparation 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 purifies required for polysaccharides spécifie to vaccine hâve led to the development of new purification methods based on fractional précipitation; ion exchange chromatography; gel filtration; and affinity chromatography. Chromatographie techniques like Size Exclusion chromatography, Ion exchange chromatography, and hydrophobie interaction chromatography hâve 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 spécifications, the use of chromatographie techniques involves multistep labour and time consuming sample préparation, in volves 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 économie costs.

The Salmonella typhi purified polysaccharides obtained by previously reported purification processes hâve endotoxin content between 25-50 EU/pg.

Hence, there exists a need of alternative purification méthodologies, aimed to maximize recovery of Salmonella polysaccharides while removing impurities to acceptable levels.

Various methods such as acid hydrolysis, alkaline dégradation, 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), électron beam fragmentation (Pawlowski et al. Micro Lett, 1999, 174.2, 255-263) hâve been described for the depolyinerisation (sizing) of bacterial and non-bacterial polysaccharides. However, acid hydrolysis, and alkaline dégradation are time consuming and the résultant 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 hâve 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 déviations, less stable products and the need to run more passes or use higher pressures than should be required.

Treatments with ultrasounds hâve been used to depolymerise polysaccharides (see for example WO 2010/055250). However, ultrasomc 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 consumîng and prone to destruction of immunogenic moieties which may affect immunogenicity of conjugates.

For ail the reasons stated above, a simple and low-cost process for the depolymerisation of polysaccharides or polysaccharides dérivatives is still désirable in the art.

Producing polysaccharide - protein conjugate vaccine is spécifie 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, hydrazîdes, active esters, norborane, p- nitrobenzoic acid, N- hydroxysuccinimide, S-NHS, EDC, using TSTU. 1cyano-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 Nsuccinîmidyl-3-(2- pyridyldithio) propionate (SPDP) (SZU ET AL; 1987). Other linkers, Bpropionamido (WO 00/10599), nitrophenyl - ethylamine (Gever et al (1979) Med Microbiol Immunol 165; 171-288), haloalkyl halide (U.5. Pat. No. 4,057,685) glycosidic bond (U.5. 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 graduai depolymerization at a rate that dépends 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 conjugales 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 îmmunologically important for clinical protection and excessive levels of unbound polysaccharide could potentially resuit in immunological hyporesponsiveness to polysaccharide (Refer WHO/TRS/924 Page No. 14, A.3.3.5). Particularly, the Salmonella typhi conjugales reported in literature hâve a high free polysaccharide content upto 34% and high free protein content above 5% which indicates lower conjugation efficiency and lower stability of conjugales that is not désirable. Accordingly there is a need for vaccines demonstrating free polysaccharide content less than 10%.

Indeed, if it was possible to hâve a generic process that could be employed for manufacturing and formulating ali vaccine candidates it would greatly reduce the time and resources needed for process development. This can hâve 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 économies, yieid, pool volumes, and throughput and may not be suitable for producing the quantities required for late stage or commercial campaigns. Another important considération 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 counlries where availability of electrical power and réfrigération are often inadéquate and therefore vaccine stability across température excursions assumes greater relevance for these régions.

Thus, there is a need for an efficient platform process for manufacturing an effective vaccine against Salmonella serovar strains 5. typhi; S. paratyphi A; 5. typhimurium and S. enteritidis. that meets multiple criterion including good immunogenicity, safety and affordability, in particular a platform that provides i) improved polysaccharide yieid across fermentation and purification processes; ii) improved purification processes showing optimal percentage recovery and minimum impurity levels; and iii) improved ratio of polysaccharide - protein conjugale in the vaccine iv) improved formulation showing low viscosity, devoid of aggregation; showing long-term stability across wide température ranges,

To overcome the aforementioned limitations of prior art and to résolve 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 conjugales 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 présent 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 présent 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 în humans.

Another object of the présent 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 présent 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 5. enteritidis.

Yet another object of the présent disclosure is to provide a bivalent polysaccharide - protein conjugate vaccine comprising polysaccharide derived from either of Salmonella serovar strains 5. typhi; S. paratyphi A; S. typhimurium and S. enteritidis in any combination thereof.

Yet another object of the présent 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 5. enteritidis in any combination thereof.

Yet another objective of the présent 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 présent 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 présent disclosure is to provide an improved methods of conjugation of polysaccharide (with or without size réduction) derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis. to a carrier protein.

Yet another object of the présent disclosure is to provide a method of conjugation of polysaccharide (with or without size réduction) derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis to a carrier protein with or without a Iinker (spacer) molécule.

Yet another object of the présent disclosure is to provide immunogenic vaccine formulations comprising polysaccharide-protein conjugales 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 présent disclosure will be more apparent from the following description, which is not intended to limit the scope of the présent disclosure.

SUMMARY

The présent disclosure provides:

a) an immunogenic composition comprising one or more of polysaccharide-protein conjugale 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 réduction) 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 molécule; and

e) methods for eiiciting 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 spécifie components, and processes, to provide a complété 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, éléments, modules, units and/or components, but do not forbid the presence or addition of one or more other features, integers, steps, operations, éléments, components, and/or groups thereof. The particular order of steps disclosed in the process of the présent disclosure is not to be construed as necessarily requiring their performance as described or îllustrated. 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 présent disclosure as the aforementioned terms may be only used to distinguish one element, component, région, layer or section from another component, région, layer or section. Terms such as first, second, third etc., when used herein do not imply a spécifie sequence or order unless clearly suggested by the présent disclosure. The présent disclosure provides an immunogenic composition and a process for preparing the same.

The term vaccine is optionaliy 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). Reliabie 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 sampie (European Pharmacopoeia Biological Reference Préparation - available from Ph. Eut. Secrétariat, e.g. Code P 216 0000) for standardisation of such methods between manufacturers (Pharmeuropa Spécial Issue, Bio 96-2). Thus the Dantigen 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-I), or type 3 (e.g. Saukett), or a Sabin Serotype 1, 2, 3 combination of either two or ail three of these types. An example of a full (or standard) dose (40-8-32 D antigen units of

Salk based TPV types 1, 2 and 3 respectively) IPV immunogenic composition for the purposes of this invention could be Poliovac® (Sérum Institute of India Pvt. Ltd.).

The term saccharîde throughout this spécification may indicate polysaccharide or oligosaccharide and includes both. The capsular saccharîde antigen may be a full Iength polysaccharide or it may be extended to bacterial 'sized-saccharides' and 'oligosaccharides' (which naturally hâve 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 présent 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 présent disclosure, the method of obtaining the polysaccharide derived from Salmonella serovar strains 5. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis, by fed-batch process may comprise any subset or ail of the following steps:

1. Inoculation, cultivation and Harvesting of bacteria in a Fermentation medium compositions,

2. Inactivation,

3. Séparation,

4. Clarification, and

5. Sterilized Filtration.

According to a first aspect of second embodiment, the fermentation medium compositions may comprise a carbon source, a magnésium sait, 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 magnésium sait may be selected from magnésium chloride, magnésium sulfate, preferably Magnésium 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), VPH33 (silicon oil/PPG mixture), BREOX (polyalkylene glycol), J673 STRUKTOL (Alkoxylated fatty acid esters on vegetable base) and SE9 (aqueous émulsion with 10% Silicon oil component) of WackerChemie 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 continuons 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:

Température: 36.0 ± 2° C

Agitation: 150to600rpm 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 formaldéhyde 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 séparation may be carried out by centrifugation. Yet preferably the séparation may be carried out by centrifugation with parameters set at température 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 μΜ stérile 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 présent disclosure, the fermentation harvest may be subjected to any subset or in any order or ail 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 eut off (MWCO);

c) treatment with anionic or cationic detergent, ethylene diamine tetra-acetic acid (EDTA) (4 to lOmM) and Sodium acetate (5% to 10%) for dénaturation of proteins, nucleic acids and lipopolysaccharide;

d) alcohoi précipitation (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 μΜ;

f) treatment with akali sait 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 μΜ;

g) concentration by tangential flow filtration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 - 300 kDa or kD molecular weight eut 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 alcohoi (50% to 70%) in presence of sodium chloride (0.1M to 2M);

k) sélective précipitation of polysaccharide by utilizing alcohoi (<75% OR>95% );

1) 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 eut off (MWCO); and

m) Stérile Filtration through at least one stérile filter having a pore size of about 0.2 micrometers under stérile 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 res tilt 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 pg 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 précipitation may be carried out using methanol, éthanol, n-propyl alcohol, isopropyl alcohol, acetone or t-butyl alcohol; or a combination thereof.

Yet preferably the said alcohol could be éthanol.

According to a fifth aspect of third embodiment, the alkali sait may be selected from the group of sodium, potassium, calcium and magnésium sait. More preferably the alkali sait may be potassium sait 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 sait 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 cetyltri méthylammonium sait, tetrabutylammonium sait, myristyltrimethylarninonium sait and hexadimethrine bromide; or a combination thereof.

Yet preferably, said cationic detergent could be Cetyl trime thylammonium 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 présent disclosure, the fermentation harvest may be subjected to any subset or in any order or ail of the following downstream purification steps to obtain desired quality of O-specific polysaccharide from Salmonella Paratyphi A lipopolysaccharide (LPS):

a) Centrifugation and séparation

b) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiitration (DF) using a membrane having 10 - 300kDa molecular weight eut off (MWCO);

c) Acid hydrolysis of LPS

d) Centrifugation and séparation

e) Neutralization

f) Clarification of a LPS by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and 0.2 micrometers;

g) treatment with anionic or cationic detergent,

h) Centrifugation and séparation

i) Direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and 0.2 micrometers;

j) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiitration (DF) using a membrane having 10 - 300kDa molecular weight eut off (MWCO);

k) alcohol précipitation (40% to 70%);

1) Centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.2 μΜ;

m) treatment with akali sait 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 μΜ;

n) concentration by tangential flow filtration (TFF) and buffer exchange by diafiitration (DF) using a membrane having 10 - 300kDa molecular weight eut off (MWCO);

ο) removal of protein and nucleic acid impurities by washing pellet with alcohol (50% to

70%) m presence of sodium chloride (0.1M to 2M);

p) dissolving polysaccharide in WFI and subjecting to concentration by tangential flow filtration (TFF) and buffer exchange by diafiltralion (DF) using a membrane having 10 300kDa molecular weight eut off (MWCO); and

q) Stérile Filtration through at least one stérile filters having a pore size of about 0.2 micrometers under stérile conditions.

According to a first aspect of fourth embodiment, the Acid hydrolysis of LPS may be carried out preferably using acelic acid (final concentration 0.5 -5%) pH~2.0 - 3.0; température 30 to 90 deg Celsius and time for about 100 to 200 minutes.

According to a second aspect of fourth embodiment. Acid hydrolysis neutralization may be carried out preferably using liquor ammonia to achieve final pH of 7.0.

According to a third aspect of fourth embodiment, the anionic detergent may be selected from the group comprising of alkyl sulfates, sodium dodecyl sulfate, sodium deoxycholate, sodium dodecyl sulfonate, sodium s-alkyl sulfates, sodium fatty alcohol polyoxyethyiene 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 sodium deoxycholate at a final concentration in the range of 0.1% to 20%, more preferably in the range of 1-2% may be added to the retentate and stirred at 25°C - 30°C for 10- 120 minutes.

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

Yet preferably the said alcohol could be éthanol.

According to a fifth aspect of fourth embodiment, the alkali sait may be selected from the group of sodium, potassium, calcium and magnésium sait. More preferably the alkali sait may be potassium sait 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 preferabiy, the said potassium sait 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 fourth embodiment, the cationic detergent may be selected from the group comprising of cetyltrimethylammonium sait, tetrabutyl ammonium sait, myristyitrimethylammonium sait and hexadimethrine bromide; or a combination thereof.

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

According to an eighth aspect of fourth embodiment, the purification process may resulî in

According to a ninth aspect of fourth embodiment, the process results in significant réduction of endotoxin (< 100EU of endotoxin per μ g of PS), protein (< 1%) and nucleic acid (< 2%) împurities, 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 could be in the range of 40 to 200 kDa.

According to a tenth aspect of fourth embodiment, the final purified polysaccharide bulk may be stored at less than or equal to -20°C.

According to a fifth embodiment of the présent disclosure, the purified Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis, polysaccharide may be covalently bound to carrier protein (CP) using a carbodiimide, reductive amination, or cyanylation conjugation reaction.

According to a first aspect of fifth embodiment, the purified Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, polysaccharide may be covalently bound to carrier protein (CP) selected from the group comprising of tetanus toxin, tetanus toxoid (TT), diphtheria toxoid (DT), CRM 197, Pseudomonas aeruginosa toxoid, Bordeteila 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 FhC, Horseshoe crab Haemocyanin, exotoxin A from Pseudomonas aeruginosa, outer membrane complex c (OMPC), porins, transferrin binding proteins, pneumoiysin, pneumococcal surface protein A (PspA), pneumococcal surface adhesîn A (PsaA), pneumococcal PhtD, pneumococcal surface proteins BVH-3 and BVH-ll, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and léthal factor (LF) of Bacillus anthracis, ovalbumin, keyhole 1 impet hemocyanin (KLH), human sérum albumin, bovine sérum albumin (BSA), purified protein dérivative 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, ironuptake proteins, toxin A or B from C. difficile and S.agalactiae proteins with or without linker and fragments, dérivatives, modifications thereof.

Yet preferably the carrier protein used for conjugation with Salmonella typhi Vi polysaccharide may be tetanus toxoid. The polysaccharides derived from S. paratyphi A, S. typhimurium and Y enteritidis may be preferably individually conjugated to a carrier protein selected from tetanus toxoid, diphtheria toxoid or CRM 197. Immunogenic compositions comprising the following polysaccharide-carrier proteins conjugales are envisaged in accordance with the présent disclosure: 5. typhi conjugated to tetanus toxoid, S. paratyphi A conjugated to TT or DT or CRM 197; S. typhimurium conjugated to TT or DT or CRM 197 and S. enteritidis conjugated to TT or DT or CRM 197; S. typhi conjugated to tetanus toxoid, S. paratyphi A conjugated to tetanus toxoid; S. typhimurium conjugated to CRM 197 and 5. enteritidis conjugated to CRM197;and S. typhi conjugated to tetanus toxoid, S. paratyphi A conjugated to DT; S. typhimurium conjugated to CRM 197 and S. enteritidis conjugated to tetanus toxoid and Y typhi conjugated to tetanus toxoid, Y paratyphi A conjugated to CRM 197; S. typhimurium conjugated to CRM 197 and Y enteritidis conjugated to tetanus toxoid.

In accordance with the présent disclosure, CRM 197 is procured from Recombinant Strain CS463-003 (MB 101) of Pseudomonas fluorescens from Pfenex USA.

In accordance with the présent disclosure, TT is procured from Clostridium Tetani (Harvard No 49205) obtained from Central research Institute (CRI), National Control Authority, 23

Kasauli, Himachal Pradesh, India. Central research Institute (CRI) procured this strain from

NVI, Netherland.

In accordance with the présent disclosure, DT is produced from cultures of Cornynebacterium diphtheriae Park-Williams Number 8 strain, the strain is obtained from Central Research Institute (CRI), Kasauli, Himachal Pradesh, India.

According to a second aspect of fifth embodiment, before conjugation the purified Salmonella enterica serovar strains 5. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis, polysaccharide may be subjected to depolymerization/sizing by Chemical means selected from the group of FeCl3, H2O2, sodium metaperiodate and sodium acetate or mechanical means selected from the group of High pressure cell disruption and homogenizer.

Yet preferably, the purified Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, polysaccharide may be subjected to depolymerization/sizing by High pressure cell disruption.

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

Yet preferably, the purified Salmonella enterica serovar typhi polysaccharide (ViPs) may be subjected to depolymerization/sizing by sodium acetate (5% to 10%) wherein the average moiecular weight of the ViPs could be in the range of 100- 250 kDa.

Applicant has found that the conjugation efficiency/conjugate yield, immunogenicity of conjugales prepared from using partially size reduced polysaccharides was higher as compared to conjugales prepared from full length polysaccharides Also Size réduction of polysaccharides decreases the viscosity of the solution and increases the number of reactive end groups, both factors contribute to an increased frequency of covalent bond formation.

Yet alternatively the purified Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, polysaccharide may not be subjected to depolymerization/sizing.

According to a third aspect of fifth embodiment, before conjugation the carrier protein (CP) may be derivatized to comprise, amino and/or carboxyl groups via a hetero or homobifunctional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D glucuronolactone, cystamme 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.

Yet preferably the hetero or homo-bifunctional linker may be dihydrazide, more preferably adipic acid dihydrazide.

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

Yet preferably, reaction of carrier protein (CP) with adipic acid dihydrazide (ADH) in the presence of carbodiimide, such as l-ethyl~3(3-dimethylaminopropyi) carbodiimide (EDC) may be carried out at a pH of 5 to 7; more preferably 6.

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

Alternatively, after derivatization of carrier protein with ADH, the method may additionally comprise the step of buffer exchange by diafiltration (DF) using a membrane either of 10 kDa or 30 kDa or 50 kDa molecular weight eut off (MWCO) and stérile filtration using 0.2 u filter; whereby the ADH derivatized carrier protein is either buffer exchanged at least 10 volumes or passed through a suitable gel filtration column and substantiaily ail unreacted compounds, residual ADH and residual EDC are removed, yielding a purified ADH derivatized carrier protein.

Yet alternatively before conjugation the carrier protein may not be derivatized to comprise, amino and/or carboxyl groups via a hetero or homo-bifunctional linker.

According to a third aspect of fifth embodiment, before conjugation the purified

Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis, polysaccharide may be derivatized to comprise, amino and/or carboxyl groups via a hetero or homo-bi functional linker selected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, a mixture thereof, e-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and p-nîtrophenylethyl amine, hexanedi amine, 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.

Yet preferably the hetero or homo-bîfunctional linker may be dihydrazide more preferably adipic acid dihydrazide.

Hydrazide groups can be introduced into the Salmonella enterica serovar strains S. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis, polysaccharide by using a carbodiimide, reductive amination, cyanylation reaction for example reaction of polysaccharide with hydrazine, carbohydrazide, succinyl dihydrazide, adipic acid dihydrazide, hydrazine chloride (e.g., hydrazine dihydrochloride) or any other dîhydrazides in the presence of cyanogen bromide (CNBr) may form hydrazide dérivatives of Vi polysaccharide.

Yet preferably the Salmonella enterica serovar Paratyphi A OSP is derivatized with an adipic acid dihydrazide (ADH) linker using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1- cyano- imidazole ( 1-CI), 1-cyanobenzotriazole (1-CBT), l-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).

Yet preferably the Salmonella enterica serovar Paratyphi A OSP is derivatized with an adipic acid dihydrazide (ADH) linker using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) mixed in a ratio of 1:1 to 1:10 by weight of OSP: ADH and the ratio of OSP: CPPT may be in between 0.5 and 1.5, reaction carried out at a pH in range of 7-10, reaction duration for 1-2 hour, température in range of 2°C to 30°C.

Yet another aspect of fifth embodiment, before conjugation the polysaccharide may not be derivatized to comprise, amino and/or carboxyl groups via a hetero or homo-bifunctional linker.

A preferred aspect of fifth embodiment wherein, Salmonella enterica serovar typhi polysaccharide (ViPs) may be covalently bound to carrier protein using carbodiimide conjugation chemistry wherein any water-soluble carbodiimide more preferably l-ethyl-3(3dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.

More preferably the Vi polysaccharide may be covalently bound to ADH derivatized carrier protein using carbodiimide conjugation chemistry wherein any water-soluble carbodiimide more preferably l-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.

More preferably the Vi polysaccharide may be covalently bound to ADH derivatized tetanus toxoid using carbodiimide conjugation chemistry wherein any water-soluble carbodiimide more preferably l-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.

Yet alternatively the ADH derivatized Vi polysaccharide may be covalently bound to ADH derivatized tetanus toxoid using carbodiimide conjugation chemistry wherein any watersoluble carbodiimide more preferably l-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.

Yet alternatively the ADH derivatized Vi polysaccharide may be covalently bound to tetanus toxoid using carbodiimide conjugation chemistry wherein any water-soluble carbodiimide more preferably l-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) can be used as a catalyst.

Yet preferably the Purified ViPs is covalently bound to carrier protein (CP) using carbodiimide conjugation reaction in presence of l-ethyl-3(3-dimethylaniinopropyl) carbodiimide (EDAC) mixed in a ratio of 1:0.5 to 1:2 by weight of ViPs: EDAC and the ratio of Vi polysaccharide and carrier protein may be in between 0.5 and 1.5, réaction carried out at a pH in range of 5-7, température in range of 2°C to 30°C.

Yet another aspect of fifth embodiment, the Vi polysaccharide (ViPs) may be covalently bound to ADH derivatized tetanus toxoid (TT) in presence of l-ethyl-3(3 dimethylaminopropyl) carbodiimide (EDC) wherein the ratio by weight of ViPs:TT:EDC could be 1:1:2 and the concentration of Vi polysaccharide and tetanus toxoid may be between 0.1 mg/mL - 10.0 mg/mL and the ratio of Vi polysaccharide and tetanus toxoid may be in between 0.5 and 1.5.

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

Further the reaction of derivatized tetanus toxoid (TT) and Vi polysaccharide (ViPs) in the presence of carbodiimide, such as l-ethyl-3(3-dimethylaminopropyl) carbodiimide (EDC) may be stopped by raising the pH from about 6 to about 7 to 8.

According to one aspect of the fifth embodiment, S. paratyphi A polysaccharide (OSP) may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyrrolidinopyridinium tetrafiuoroborate (CPPT), 1- cyano- imidazole (1-CI), 1-cyanobenzotriazole (1-CBT), 1cyano-4-(dimethylamino)-pyridinium tetrafiuoroborate (‘CDAP’), p-nîtrophenylcyanate and N-cyanotriethylammonium tetrafiuoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2CPO).

More preferably S. paratyphi A OSP may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4-pyrrolidinopyridinium tetrafiuoroborate (CPPT) or (CPIP).

Yet preferably the purified S. paratyphi A OSP is covalently bound to carrier protein (CP) using cyanylation conjugation chemistry wherein the cyanylation reagent is 1-cyano- 4pyrrolidinopyridinium tetrafiuoroborate (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, température in range of 2°C to 30°C.

Yet preferably the carrier protein used for conjugation with S. paratyphi A polysaccharide OSP may be Tetanus toxoid.

Yet preferably the carrier protein used for conjugation with S. paratyphi A polysaccharide

OS P may be Diphtheria toxoid.

Yet preferably the carrier protein used for conjugation with 5. paratyphi A polysaccharide OSPmaybeCRM 197.

Yet another aspect of the fifth embodiment, S. typhimurium polysaccharide may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of I-cyano- 4-pyrroIidinopyridinium tetrafluoroborate (CPPT), 1- cyano- imidazole (I-CI), 1-cyanobenzotriazole (1-CBT), Icyano-4-(dimethylamino)-pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2CPO).

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

Yet preferably the carrier protein used for conjugation with S. typhimurium polysaccharide may be Tetanus toxoid.

Yet preferably the carrier protein used for conjugation with S. typhimurium polysaccharide may be Diphtheria toxoid.

Yet preferably the carrier protein used for conjugation with 5. typhimurium polysaccharide may be CRM 197.

Yet another aspect of the fifth embodiment, 5. enteritidis polysaccharide may be conjugated to a carrier protein using cyanylation conjugation chemistry wherein the cyanylation reagent is selected from a group of 1-cyano- 4-pyriOlidinopyridinium tetrafluoroborate (CPPT), 1cyano- 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).

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

Yet preferably the carrier protein used for conjugation with 5. enteritidis polysaccharide may be Tetanus toxoïd.

Yet preferably the carrier protein used for conjugation with S. enteritidis polysaccharide may be Diphtheria toxoid.

Yet preferably the carrier protein used for conjugation with 5. enteritidis polysaccharide may beCRM 197.

Applicant has found methods for stabilizing final polysaccharide - protein conjugate by utilizing the alternative methods of conjugation, ratio of polysaccharide to protein, ratio of polysaccharide to coupling agents, using appropriate linkers, appropriate size of the polysaccharide. The same can resuit in improvement in ratio of polysaccharide - protein conjugate in the vaccine which in turn can reduce the number of free saccharide and free protein in the conjugate, reduced carrier protein suppression, improved stérile filterability of the conjugate, better control of the conjugation, and greater intra-moiety cross-links indirectly provides a good immune response.

Applicant has found that varions factors influence the coupling of polysaccharides and proteins which dépend upon molecular weight of the ViPs, type of carrier protein selected, the ratio of the amount of polysaccharide: carrier protein used, activation of the functional groups, use of spacers and conjugation chemistry.

Yet another aspect of fifth embodiment, after conjugation réaction the method may comprise the step of concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane having either of 100 kDa or 300 kDa molecular weight eut off (MWCO); whereby the conjugate bulk is concentrated at least 3 fold and substantially ail unreacted compounds, unconjugated polysaccharide, unconjugated protein and residual EDC are removed, yielding a purified Vi polysaccharide conjugate vaccine.

Additionally the method may comprise of Gel filtration chromatography whereby the conjugate bulk is concentrated at least 3 fold and substantially ail unreacted compounds, unconjugated polysaccharides, unconjugated proteins and residual EDC are removed, yielding a purified S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, polysaccharide conjugate vaccine wherein the conjugate yield is > 50%.

Further method of purification may comprise of combination of ultrafiltration and gel filtration chromatography.

According to a sixth embodiment, the immunogenic composition may be a monovalent vaccine comprising either of 5. typhi Vi polysaccharide conjugated to a carrier proteîn or S. paratyphi A polysaccharide conjugated to a carrier proteîn or S. typhimurium conjugated to a carrier proteîn or 5. enteritidis conjugated to a carrier proteîn.

According to a seventh embodiment of the present disclosure, wherein the immunogenic composition may comprise of atleast one bivalent combination:

a) Salmonella enterica serovar typhi saccharide-carrier protein (CP) conjugale antigen;

b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein (CP) conjugale antigen;

or

b) Salmonella enterica serovar typhimurium saccharide-carrier protein (CP) conjugale antigen;

or

a) Salmonella enterica serovar typhi saccharide-carrier proteîn conjugale antigen;

b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugale antigen;

or

a) Salmonella enterica serovar Paratyphi A saccharide-carrier protein conjugale antigen;

b) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen; or

or

a) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen;

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi ViPs-TT conjugale antigen in a dose range of 1.25 - 50 gg;

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi A OSP-CP conjugale antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhimurium saccharide-CP conjugale antigen m a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis saccharide-CP conjugale antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM197

According to an eighth embodiment of the présent disclosure, wherein the immunogenic composition may comprise of atleast one trivalent combination:

b) Salmonella enterica serovar Paratyphi A saccharîde- carrier protein conjugale antigen;

c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugale antigen;

b) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen;

c) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugale antigen;

or

a) Salmonella enterica serovar Paratyphi A saccharîde- carrier protein conjugale antigen;

c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen;

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhimurium saccharide-CP conjugale antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRMI97

According to a ninth embodiment of the présent disclosure, wherein the tetravalent immunogenic composition may comprise of: i) 5. typhi Vi polysaccharide conjugated to a carrier protein (CP), ii) S. paratyphi A polysaccharide conjugated to a carrier protein, iii) S.

entérinais polysaccharide conjugated to a carrier protein, and iv) 5. typhimurium polysaccharide conjugated to a carrier protein.

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi A OSP-CP conjugale antigen in a dose range of l .25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhimurium saccharide-CP conjugale antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis saccharide-CP conjugale antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

According to a tenth embodiment of the présent disclosure, the immunogenic composition may further comprise one or more antigen selected from the group consisting of but not limited to Salmonella paratyphi B, Salmonella paratyphi C, Salmonella antigens such as Outer membrane vesicles, Outer membrane proteins (eg, OmpC, OmpD, OmpF), siderophores (enterobactin), type III sécrétion System proteins (eg, SipB, SipD, SseB, SseC, and PrgI), flagellin, Non-typhoidal Salmonella spp. Shigella, Shigella sonnet, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Escherichia coli, Enterohacter species, Yersinia species, Pseudomonas species, Pseudomonas aeruginosa, Haemophilus influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Hepatitis (A, C, D, E, F and G strains), , Influenza, Staphylococcus spp., Staphylococcus aureus, Staphylococcus aureus type 5, Staphylococcus aureus type 8, Streptococcus spp , Streptococcus pneumoniae (1, 2, 3, 4, 5,6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A,9L,9F,9N, 9V, 10F, 10B,10C, 10A, 11 A,UF,11B, 11C,11D,11E,12A,12B, 12F,13, 14, 15AJ5C ,15B,15F,16A, 16F, 17AJ7F, 18C,18F,18A,18B, 19A, 19B, 19C, 19F, 20, 20A,20B,21,22A, 22F, 23A,23B, 23F, 24A, 24B,24F , 25F, 25A,27,28F, 28A, 29, 31,32F, 32A,33A, 33C, 33D, 33E, 33F,33B, 34, 45,38,35A,35B,35C,35F,36,37,38, 39, 40,41F,41A,42,43,44,45,46,47F,47A,48), Group A Streptococcus, Group B Streptococcus( group la, Ib, II, III, IV, V, VI, VII Vil, VIII, and IX.), Neisseria meningitidis, Haemophilus pneumonia, Hélicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma pneumoniae,

Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus viridans,

Enterococcusfaecalis, Enterococcus faecium, Enterococcus faecalis Neisseria gonorrhoeae, Bacillus anthracis, Vibrio choieras. Pasteurella pestis, Campylobacter spp., Campylobacter jejuni, Clostridium spp., Clostridium tetani, Clostridium difficile, Mycobacterium spp., Mycobacterium tuberculosis, M. catarrhalis , Klebsiella pneumoniae .Treponema spp., Borrelia spp., Borrelia burgdorferi, Leptospira spp., Hemophilus ducreyi, Corynebacierium diphtheria, Bordetella pertussis, Bordetella parapertussis, Bordetella bronchiseptica, Shigella spp., Erlichia spp., Rickettsia spp and N. meningitidis polysaccharide (A, B, C, D, W135, X, Y, Z and 29E)acellular pertussis antigen, modified adenylate cyclase, Malaria Antigen (RTS, S), anthrax, dengue, malaria, measles, mumps, rubella, BCG, Human papilloma virus, Japanese encephalitis, Dengue, Zika, Ebola, Chikungunya, Poliovirus, Rotavirus, smallpox, yellow fever, Flavivirus, Shingles, and Varicella virus antigens.

According to a eleventh embodiment of the présent disclosure, wherein the composition comprises of atleast one combination:

b) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

c) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis Y saccharide - carrier protein conjugale antigen;

e) Neisseria meningitidis IV -135 saccharide - carrier protein conjugale antigen;

f) Neisseria meningitidis X saccharide - carrier protein conjugale antigen;

or

b) Salmonella enterica serovar Paralyphi A saccharide- carrier protein conjugale antigen;

c) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugale antigen;

e) Neisseria meningitidis Y saccharide - carrier protein conjugale antigen;

f) Neisseria meningitidis W-135 saccharide - carrier protein conjugale antigen;

g) Neisseria meningitidis X saccharide - carrier protein conjugale antigen;

or

b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugale antigen; c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen; d) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

e) Neisseria meningitidis C saccharide - carrier protein conjugale antigen;

f) Neisseria meningitidis V saccharide - carrier protein conjugale antigen;

g) Neisseria meningitidis IV -135 saccharide - carrier protein conjugale antigen;

h) Neisseria meningitidis X saccharide - carrier protein conjugale antigen; or

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen;

b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen;

c) Salmonella enterica serovar typhimurium saccharîde-carrier protein conjugate antigen;

d) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;

e) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

g) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

h) Neisseria meningitidis W -135 saccharide - carrier protein conjugate antigen;

i) Neisseria meningitidis X saccharide - carrier protein conjugate antigen; or

a) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;

b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;

c) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis IV -135 saccharide - carrier protein conjugate antigen;

g) Neisseria meningitidis X saccharide - carrier protein conjugate antigen

b) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

c) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis IV-135 saccharide - carrier protein conjugate antigen or

c) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis Y saccharide - carrier protein conjugale antigen;

f) Neisseria meningitidis W-135 saccharide - carrier protein conjugale antigen or

b) Salmonella enterica serovar enteritidis saccharîde-carrier protein conjugale antigen;

c) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugale antigen;

e) Neisseria meningitidis K saccharide - carrier protein conjugale antigen;

f) Neisseria meningitidis IV-135 saccharide - carrier protein conjugale antigen;

or

a) Salmonella enterica serovar typhi saccharîde-carrier protein conjugale antigen;

b) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

c) Neisseria meningitidis C saccharide - carrier protein conjugale antigen;

or

b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugale antigen;

c) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugale antigen; or

c) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugale antigen;

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi ViPs-TT conjugale antigen in a dose range of 1.25 - 50 pg;

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar Paratyphi A OSP-CP conjugale antigen in a dose range of 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

B Yet preferabiy 0.5 ml of the composition comprises of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen in a dose range of 1.25 - 50 pg; wherein the CP is either TT or DT or CRM197

Yet preferabiy 0.5 ml of the composition comprises of Salmonella enterica serovar 5 enteritidis saccharide-CP conjugate antigen in a dose range of 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

Yet preferabiy 0.5 ml of the composition comprises of Neisseria meningitidis A saccharide TT conjugate antigen in a dose of 5 pg

Yet preferabiy 0.5 ml of the composition comprises of Neisseria meningitidis C saccharide 10 CRM197 conjugate antigen in a dose of 5 pg

Yet preferabiy 0.5 ml of the composition comprises of Neisseria meningitidis Y saccharide CRM197 conjugate antigen in a dose of 5 pg

Yet preferabiy 0.5 mi of the composition comprises of Neisseria meningitidis IV saccharide CRM197 conjugate antigen in a dose of 5 pg

Yet preferabiy 0.5 ml of the composition comprises of Neisseria meningitidis X saccharide TT conjugate antigen in a dose of 5 pg

According to a twelfth embodiment of the présent disclosure, wherein the immunogenic composition comprises of atleast one combination:

b) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

c) a diphtheria toxoid, (D) antigen

d) a tetanus toxoid, (T) antigen

e) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

f) a hepatitis B virus surface antigen, (HBsAg) and

g) a Haemophilus influenzae type b antigen, (Hib); or

b) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

c) a rotavirus antigen;

d) a diphtheria toxoid, (D) antigen

e) a tetanus toxoid, (T) antigen

f) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

g) a hepatitis B virus surface antigen, (HBsAg) and

h) a Haemophilus influenzae type b antigen, (Hib);

or

c) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

d) a diphiheria toxoid, (D) antigen

e) a tetanus toxoid, (T) antigen

f) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

g) a hepatitis B virus surface antigen, (HBsAg) and

h) a Haemophilus influenzae type b antigen, (Hib); or

c) a rotavirus antigen;

d) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

e) a diphtheria toxoid, (D) antigen

f) a tetanus toxoid, (T) antigen

g) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

h) a hepatitis B virus surface antigen, (HBsAg) and

i) a Haemophilus influenzae type b antigen, (Hib); or

d) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

e) a diphtheria toxoid, (D) antigen

f) a tetanus toxoid, (T) antigen

g) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

h) a hepatitis B virus surface antigen, (HBsAg) and

i) a Haemophilus influenzae type b antigen, (Hib); or

c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen; d) a rotavirus antigen;

e) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

f) a diphtheria toxoid, (D) antigen

g) a tetanus toxoid, (T) antigen

h) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

i) a hepatitis B virus surface antigen, (HBsAg) and

j) a Haemophilus influenzae type b antigen, (Hib); or

d) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugale antigen;

e) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

f) a diphtheria toxoid, (D) antigen

g) a tetanus toxoid, (T) antigen

h) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

i) a hepatitis B virus surface antigen, (HBsAg) and

j) a Haemophilus influenzae type b antigen, (Hib); or

e) a rotavirus antigen;

f) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

g) a diphtheria toxoid, (D) antigen

h) a tetanus toxoid, (T) antigen

î) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

j) a hepatitis B virus surface antigen, (HBsAg) and

k) a Haemophilus influenzae type b antigen, (Hib);

Yet preferably 0.5 mi of the composition comprises of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in a dose range of l.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferably 0,5 ml of the composition comprises of Salmonella enterica serovar typhimunum sacchande-CP conjugate antigen in a dose range of 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis saccharîde-CP conjugate antigen in a dose range of 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

Yet preferably the composition comprises of dîphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml

Yet preferably the composition comprises of a tetanus toxoid, (T) in an amount of 1 to 30 Lf per 0.5 ml

Yet preferably the composition comprises of 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) I-50pg, Filamentous hemagglutinin (FHA) I-50pg, Pertactin (P69 or PRN) l-20pg or Fimbrial proteins (FIM 1 , 2 and 3) 225pg; per 0.5ml;

Yet preferably the composition comprises of a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 pg per 0.5 ml;

Yet preferably the composition comprises of a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 pg per 0.5 ml;

Yet preferably the composition comprises of an inactivated rota virus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strains présent in an amount in the range of 1 to 50 pg per 0.5 ml;

Yet preferably the composition comprises of 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;

According to a thirteenth embodiment of the présent disclosure, wherein the immunogenic composition comprises of atleast one combination:

b) a rotavirus antigen;

c) a diarrheo génie Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen; d) a Shigella spp. antigen;

e) a Campylobacter jejuni antigen;

f) a Vibrio cholerae antigen;

or

c) a rotavirus antigen;

d) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;

e) a Shigella spp. antigen;

f) a Campylobacter jejuni antigen;

g) a Vibrio cholerae antigen; or

c) a rotavirus antigen;

e) a Shigella spp. antigen;

f) a Campylobacter jejuni antigen or

c) a rotavirus antigen;

e) a Shigella spp. antigen; or

c) a rotavirus antigen;

d) a Shigella spp. antigen; or

c) a rotavirus antigen;

e) a Shigella spp. antigen;

f) a Campylobacter jejuni antigen;

g) a Vibrio cholerae antigen;

or

c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen;

d) a rotavirus antigen;

e) a diarrheogenic Escherichia coli spp. (enterotoxigénie and enterohemorragic) antigen;

f) a Shigella spp. antigen;

g) a Campylobacter jejuni antigen;

h) a Vibrio cholerae antigen; or

e) a rotavirus antigen;

f) a diarrheogenic Escherichia coli spp. (enterotoxigénie and enterohemorragic) antigen;

g) a Shigella spp. antigen;

h) a Campylobacter jejuni antigen;

i) a Vibrio cholerae antigen;

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar typhi ViPs-TT conjugate antigen in a dose range of 1.25 - 50 gg;

Yet preferably 0.5 mi of the composition comprises of Salmonella enterica serovar Paratyphi A OSP-CP conjugate antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferabiy 0.5 ml of the composition comprises of Salmonella enterica serovar typhimurium saccharide-CP conjugate antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferably 0.5 ml of the composition comprises of Salmonella enterica serovar enteritidis saccharide-CP conjugate antigen in a dose range of 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

Yet preferably the composition comprises of an inactivated rotavirus antigen selected from

CDC-9, CDC-66 or any other inactivated rotavirus strains présent in an amount in the range of 1 to 50 ug per 0.5 ml;

According to one aspect of the embodiment, the immunogenic composition may additionally comprise of a buffering agent selected from the group consisting of carbonate, phosphate, acetate, HEPES, Succinate, Histidine, TRIS, borate, citrate, lactate, gluconate and tartrate, as well as more complex organic buffering agents including a phosphate buffering agent that contains sodium phosphate and/or potassium phosphate in a ratio selected to achieve the desired pH. In another example, the buffering agent contains Tris (hydroxymethyl) aminomethane, or Tris, formulated to achieve the desired pH. Yet in another example, the buffering agent could be the minimum essential medium with Hanks salts. Other buffers, such as HEPES, piperazine-N, Ν'-bis (PIPES), and 2-ethanesulfonic acid (MES) are also envisaged by the présent disciosure. The buffer aids in stabilizing the immunogenic composition of the présent disciosure. The amount of the buffer may be in the range of 0.1 mM to 100 mM, preferably selected from 5mM, 6mM, 7mM, 22 mM, 23 mM, 24 mM, 25mM, 26 mM, 27 mM, 28 mM, 29 mM and 30 mM. The amount of the buffer may be in the range of 0.1 mg - 2.0 mg.

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

Preferably the immunogenic composition may additionally comprise of TRIS or Citrate buffer or Histidine buffer or Succinate Buffer in the range of 0.1 mg - 2.0 mg.

Preferably the immunogenic composition may additionally comprise of TRIS Buffer in the range of 0.61 mg - 1.52 mg.

Preferably the immunogenic composition may additionally comprise of Citrate buffer in the range of 10 mM to 25 mM.

Preferably the immunogenic composition may additionally comprise of Histidine buffer in the range of 0.78 to 1.94 mg.

Preferably the immunogenic composition may additionally comprise of Succinate Buffer in the range of 0.59 to 1.48 mg.

Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of pharmaceuticaliy acceptable excipients selected from the group consisting of sugars, surfactants, polymers, salts, aminoacids or pH modifiers.

Examples of Surfactants may include ionic and non-ionic surfactants such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethanol, t-Octylphenoxypolyethoxyethanol, oxtoxynol 40, nonoxynol-9, tri éthanol amine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer.

Preferably the immunogenic composition may comprise of polysorbate 20 as pharmaceuticaliy acceptable excipients.

Examples of the polymers may include dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin, etc.

Exampies of the salts may include NaCl, KC1, KH2PO4, NasHPO^EbO, CaCh, MgCh, etc. Preferably, the sait may be NaCl. Typically the amount of the sait may be in the range of 100 mM to 200 mM.

Preferably the immunogenic composition may comprise of Sodium chloride in the range of 1 - 10 mg.

Examples of the aminoacids as excipient selected from the group of L-Histidine, Lysine, Isoleucine, Méthionine, Glycine, Aspartic acid. Tricine, arginine, leucine, glutamine, alanine, peptide, hydrolysed protein or protein such as sérum albumin.

Preferably the immunogenic composition may comprise of Histidine.

Exampies of the sugars as excipient selected from the group of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionîc acid, glucose, maltulose, iso- maltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol, or a combination thereof.

Preferably the immunogenic composition may additionally comprise of Sucrose.

Examples of the polymers as excipient selected from the group of dextran, carboxymethylcellulose, hyaluronic acid, cyclodextrin,

Yet preferably the single dose composition is free of preservative.

Yet preferably the multî-dose immunogenic composition may additionally comprise of preservative selected from the group consisting of 2-phenoxy éthanol, Benzéthonium chloride (Phemerol), Phénol, m-cresol, Thiomersal, Formaldéhyde, paraben esters (e.g. methyl-, ethyl, propyl- or butyl- paraben), benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlorm-cresol, or benzyl alcohol or a combination thereof. A vaccine composition may include material for a single immunization, or may include material for multiple immunizations (i.e. a ‘multidose’ kit). The inclusion of a preservative is preferred in multidose arrangements. As an alternative (or in addition) to including a preservative in multidose compositions, the compositions may be contained in a container having an aseptie adaptor for removal of material. Preferably, the preservative may be 2-phenoxyethanol in the range of 0.1 mg to 50 mg; more preferably 1-10 mg.

Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of auxiliary substances such as wetting or emulsifying agents, diluent pH buffering agents, gelling or viscosity enhancing additives, preservatives, flavoring agents, colors, and the like, depending upon the route of administration and the préparation desired.

Yet préférable aspect of the embodiment, the immunogenic composition may additionally comprise of water for injection as diluent.

Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of an adjuvant selected from the group of aluminum sait, aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate.

Yet another aspect of the embodiment, the immunogenic composition may additionally comprise of an immunostimulatory component selected from the group consisting of an oil and water émulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A dérivatives, Monophosphoryl lipid A, 3-deacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a liposome, Freund’s adjuvant, Freund’s complété adjuvant, Freund’s incomplète adjuvant, polymers, co-polymers such as polyoxyethylene-polyoxypropylene copolymers, including block co-polymers, polymer p 1005, CRL-83OO adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flagellins derived from grain négative bacteria, TLR-5 agonists, fragments of flagellins capable of binding to TLR-5 receptors, Alpha-C-galactosylceramide, Chitosan, Interleukin-2, QS-21, ISCOMS, squalene mixtures (SAF-1), Quil A, choiera toxin B subunit, polyphosphazene and dérivatives, mycobacterium cell wall préparations, mycolic acid dérivatives, non-ionic block copolymer surfactants, OMV, fHbp, saponin combination with sterols and lipids.

Yet another aspect of the embodiment, the immunogenic composition may be fully liquid. Suitable forms of liquid préparation may include solutions, suspensions, émulsions, syrups, isotonie aqueous solutions, viscous compositions and élixirs that are buffered to a selected pH.

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

The immunogenic composition of the présent disclosure may be in the form of transdermal préparations including lotions, gels, sprays, ointments or other suitable techniques. If nasal or respiratory (mucosal) administration is desired (e.g., aérosol inhalation or insufflation), compositions can be in a form and dispensed by a squeeze spray dispenser, pump dispenser or aérosol dispenser. Aérosols are usually under pressure by means of a hydrocarbon. Pump dispensers can preferably dispense a metered dose or a dose having a particular particle size. When in the form of solutions, suspensions and gels, in some embodiments, the immunogenic compositions contain a major amount of water (preferably purified water) in addition to the active ingredient(s).Yet alternative aspect of the embodiment, the immunogenic composition could be lyophilized or freeze dried composition.

As used herein the terms Freeze-drying or “lyophilize” or lyophilization” involves lyophilization and refers to the process by which a suspension/solution is frozen, after which the water is removed by sublimation at low pressure. As used herein, the term sublimation refers to a change in the physical properties of a composition, wherein the composition changes directly from a solid State to a gaseous State without becoming a liquid.

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

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

According to a ninth embodiment of the présent disclosure, the immunogenic composition may be formulated for use in a method for reducing the onset of or preventing a health condition comprising Salmonella serovar strains 5. typhi; S. paratyphi A; S. typhimurium and S. enteritidis infection involving administration of an immunologically effective amount of the immunogenic composition to a human subject via patenterai or subcutaneous or intradermal, intramuscular or intraperitoneal or intravenous administration or injectable administration or sustained release from implants or administration by eye drops or nasal or rectal or buccal or vaginal, pérorai or intragastric or mucosal or perlinqual, alveolar or gingival or olfactory or respiratory mucosa administration or any other routes of immunization.

According to the preferred aspect of the embodiment, the immunogenic composition may be administered to a human subject via intramuscular route or subcutaneous.

According to the preferred aspect of the embodiment, an immunologically-effective amount of the immunogenic composition comprising the polysaccharide - protein conjugate for vaccination against Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis infection bacterial infection could be from about I pg/0.5ml of the Polysaccharide conjugate of Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium or S. enteritidis or less to about 100 pg/0.5ml of the Polysaccharide conjugate of Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium or S. enteritidis or more. In some other aspects, it could be from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 or 50 pg to about 55, 60, 65, 70, 75, 80, 85, 90, or 95 pg per 0.5ml single dose. In some embodîments, the immunologically-effective amount for vaccination against Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis infection bacterial infection is from 1 pg/0.5ml to 50 pg/0.5ml; more preferably Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen; Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen; is présent in a dose range of about 5 ug/0.5ml to about 30 ug/0.5ml; yet more preferably Salmonella enterica serovar typhi saccharide-carrier protein conjugale antigen; Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugale antigen; Salmonella enterica serovar typhimurium saccharide-carrier protein conjugale antigen; Salmonella enterica serovar enteritidis saccharide-carrier protein conjugale antigen; is présent in a dose of about 25 ug/0.5ml

According to tenth embodiment of the présent disclosure, the immunogenic composition may be administered intramuscularly or subcutaneous in a dose effective for the production of neutralizing antibody and protection. The vaccines are administered in a manner compatible wilh the dosage formulation, and in such amount as will be prophylactically and/or therapeutically effective against Typhoidal and NTS infection. The immunogenic composition of the présent disclosure can be administered as primary prophylactic agents in elders, adolescents, adults or children at the risk of infection, or can be used as secondary agents for treating infected patients. For example, the immunogenic composition as disclosed herein can be used in elders, adolescents, adults or children less than 2 years of âge or more than 2 years of âge at risk of Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and 5. enteritidis infection, or can be used as secondary agents for treating Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis infected patients.

Yet alternatively the vaccines for NTS may be administered in infants between the âges of two and four months old, before peak incidence occurs around the âge of 12 months. In addition, vaccine implémentation would likely also include populations infected with HIV, as they are at heightened risk of infection with NTS. In developed countries, NTS vaccines may target the elderly who expérience very high case-fatality rates (up to 50 percent). Il has been proposed that, in children, programmatic field implémentation would intégrale directly with existing Expanded Programme on Immunization schedules, perhaps at 6, 10, and 14 weeks.

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

According to eleventh embodiment of the présent disclosure, the immunogenic composition could be formulated as single dose vials or multidose vials (2 Dose or 5 Dose or 10 Dose vials) or multidose kit or as pre-filled syringes wherein the said immunogenic composition may be given in a single dose schedule, or preferably a multiple dose schedule in which a primary course of vaccination is followed by 1-3 separate doses given at subséquent time intervals after 1-3 years if needed. The dosage regimen will also, at least in part, be determined on the need of a booster dose required to confer protective immunity.

Yet preferably the immunogénie composition may be formulated for administration to a human subject elders, adolescents, adults or children less than 2 years of âge or more than 2 years of âge according to a one dose or two dose regimens or 3 dose regimens consisting of a first dose and/or a second dose to be administered between 3 months to 2 years after the first dose and/or a third dose to be administered between 3 months to 2 years after the second dose.

According to eleventh embodiment of the présent disclosure, the immunogenic composition may be administered concomitantly with other drugs or any other vaccine.

Yet according to one aspect of eleventh embodiment, wherein a single dose vaccine kit may comprise of:

a first container containing a lyophilized (freeze-dried) immunogenic composition:

a) Neisseria meningitidis A saccharide - TT conjugate antigen 5 pg per 0.5 ml;

b) Neisseria meningitidis C saccharide - CRM 197 conjugate antigen 5 μ g per 0.5 ml;

c) Neisseria meningitidis Y saccharide - CRM 197 conjugate antigen 5 μ g per 0.5 ml;

d) Neisseria meningitidis W -135 saccharide - CRM 197 conjugate antigen 5 pg per 0.5 ml;

e) Neisseria meningitidis X saccharide - TT conjugate antigen 5 pg 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 pg per 0.5 ml;

b) Sodium chloride 1-10 mg per 0.5 ml;

c) Water for Injection (WFI) q.s.;

wherein there is no antigenîc interférence 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- dépendent immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

Yet according to second aspect of eleventh embodiment, wherein a single dose vaccine kit may comprise of:

a) Neisseria meningitidis A saccharide - TT conjugate antigen 5 pg per 0.5 ml;

b) Neisseria meningitidis C saccharide - CRM 197 conjugate antigen 5 pg per 0.5 ml;

c) Neisseria meningitidis Y saccharide - CRM 197 conjugate antigen 5 pg per 0.5 ml;

d) Neisseria meningitidis W-135 saccharide - CRM 197 conjugate antigen 5 pg per 0.5 ml;

e) Neisseria meningitidis X saccharide - TT conjugate antigen 5 pg 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;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg per 0.5 ml; wherein the CP is either TT or DT or CRM197;

c) Sodium chloride 1-10 mg per 0.5 ml;

d) Water for Injection (WFI) q.s.;

wherein there is no antigénie interférence of ViPs-TT; OSP antigen with Neisseria meningitidis antigens, for prophylaxis against typhoid and paratyphoid caused by Salmonella typhi, S. paratyphi and Neisseria meningitidis an tige ns wherein the said vaccine formulation is sufficient to elicit the required T- dépendent immune response against S. typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

Yet according to third aspect of eleventh embodiment, wherein a single dose vaccine kit may comprise of:

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 l 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 selected from Pertussis toxoid (PT) l-50pg, Filamentous hemagglutinin (FHA) l-50pg, Pertactin (P69 or PRN) l-20pg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25pg; per 0.5ml;

e) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 pg per 0.5 ml;

f) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 pg per 0.5 ml;

and a second container containing a fully liquid immunogenic composition comprising:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 pg per 0.5 ml;

b) Sodium chloride I - 10 mg per 0.5 ml; and/or

c) Tris Buffer or Citrate buffer or Histidine briffer or Succinate Buffer 0.1 mg - 1.6 mg per 0.5 ml; and/or

d) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanol amine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg per 0.5 ml; and/or

e) 2-Phenoxyethanol 1 - 10 mg per 0.5 ml; and/or

f) Water for Injection (WFI) q.s.

wherein there is no antigenic interférence of ViPs-TT with Hexavalent immunogenic composition, for prophyIaxis against typhoid caused by Salmonella typhi and Hexavalent antigens wherein the said vaccine formulation is sufficient to elicit the required T- dépendent immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

Yet according to fourth aspect of eleventh embodiment, wherein a single dose vaccine kit may comprise of:

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 5 Type l 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 10 pertussis, (aP) antigen comprising one or more of modified adenylate cyclase selected from

Pertussis toxoid (PT) l-50gg, Filamentous hemagglutinin (FHA) l-50gg, Pertactin (P69 or PRN) l-20gg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25gg; per 0.5ml;

e) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 gg per 0.5 ml;

f) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 gg per 0.5 ml;

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 gg per 0.5ml;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 gg per 0.5 ml; wherein the CP is either TT or DT or CRM 197;

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 per 0.5ml; and/or

g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, 25 octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethyiene- 35 ricinoleate, soy lecithin and a poloxamer; in an amount of 25-500 gg per 0.5 ml; and/or

h) 2-Phenoxyethanol 1 - 10 mg per 0.5ml; and/or

e) Water for Injection (WFI) q.s.

wherein there is no antigenic interférence 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- dépendent immune response against S. typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

Yet according to fifth aspect of eleventh embodiment, wherein a single dose vaccine kit may comprise of:

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 I 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 unît (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 pg 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 selected from Pertussis toxoid (PT) l-50pg, Filamentous hemaggîutinin (FHA) l-50pg, Pertactin (P69 or PRN) l-20pg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25pg; per 0.5ml;

f) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 μ g per 0.5 ml;

g) a Haemophilus influenzae type b antigen, (Hib) in an amount of I to 20 pg per 0.5 ml;

b) Sodium chloride 1-10 mg per 0.5 ml;

c) Water for Injection (WFI) q.s.;

wherein there is no antigenic interférence of ViPs-TT with Heptavalent inimunogenic composition, for prophylaxis against typhoid caused by Salmonella typhi and Heptavalent antigens wherein the said vaccine formulation is sufficient to elicit the required T- dépendent immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through oniy one injection to comprise a complété vaccination schedule.

Yet according to sixth aspect of eleventh embodiment, wherein a single dose vaccine kit may comprise of:

a first container containing a fully liquid heptavalent inimunogenic composition:

a) an inactivated polio virus (IPV) antigen selected from Sabin or Salk S train; 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 présent in an amount in the range of 1 to 50 gg 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 selected from Pertussis toxoid (PT) l-50gg, Filamentous hemagglutinîn (FHA) l-50gg, Pertactin (P69 or PRN) l-20gg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25gg; per 0.5ml;

f) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 gg per 0.5 ml;

g) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 gg per 0.5 ml;

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 g g per 0.5 ml;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 — 50 gg per 0.5 ml; wherein the CP is either TT or DT or CRM 197;

c) Sodium chloride 1 - 10 mg per 0.5 ml; and/or

d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg per

0.5 ml; and/or

g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, trîethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg per 0.5 ml; and/or

h) 2-Phenoxyethanol 1 - 10 mg per 0.5 ml; and/or e) Water for Injection (WFI) q.s.

wherein there is no antigenic interférence 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- dépendent immune response against S. typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition of the présent disciosure may include: Salmonella enterica serovar typhi TY2 strain with tviB gene spécifie for Vi polysaccharide (Identified by Geneombio Technologies Private Limited, Pune and isolated from stool sample of typhoid confirnied patient at Villoo Poonawalla Memorial Hospital, Pune); S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150 procured from Chromachemie Laboratory Private Limited, Bangalore, CMCC5OO73, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; S. enteritidis RI 1; S. enteritidis D24359; S. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219A. typhimurium: S. typhimurium 2192; ATCC 14208; S. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2/SL134 (ST 19)); S.typhimurium I77(ST19) CDC 6516-60; ATCC 700720. Further any attenuated Salmonella serovar strain (S. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used for the préparation of the immunogenic composition of the présent disciosure.

Salmonella enterica serovar typhi deposited at NCMR-NCCS; Pune, an International Depositary Authority having assigned Accession No. MCC 0193; Strain désignation- PDL-1.

Other embodiments dîsclosed herein also encompasses vaccine kit comprising a first container containing a lyophilized (freeze-dried) immunogenic composition and a second container containing an aqueous solution optionally saline or WFI (water for injection) for the reconstitution oi the lyophilized (freeze-dried) immunogenic composition.

The foregoing description of the spécifie embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such spécifie embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of équivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein has been described in ter ms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

Throughout this spécification the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element, integer or step, or group of éléments, integers or steps, but not the exclusion of any other element, integer or step, or group of éléments, integers or steps.

The use of the expression “one or more” or “at least one” suggests the use of one or more éléments or ingrédients or quantities, as the use may be in the embodiment of the invention to achieve one or more of the desired objects or resu Its.

Any discussion of documents, acts, materials, devices, articles or the like that has been included in this spécification is solely for the purpose of providing a context for the disclosure. It is not to be taken as an admission that any or ail of these matters form a part of the prior art base or were common general knowledge in the field relevant to the disclosure as it existed anywhere before the priority date of this application.

The numerical values given for various physical parameters, dimensions and quantities are only approximate values and it is envisaged that the values higher than the numerical value assigned to the physical parameters, dimensions and quantities fall within the scope of the invention unless there is a statement in the spécification to the contrary.

While considérable emphasis has been placed herein on the spécifie features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustration of the disclosure and not as a limitation.

TECHNICAL ADVANTAGES:

1. The présent disclosure provides monovalent and multivalent 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 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.

2. Improved upstream, downstream and conjugation process

3. Upstream fermentation media is devoid of casein digest/Tryptone, casamino acids.

4. Use of i) combination of antifoam , soya peptone and yeast extract ii) particular DO 3639% and iii) osmolality 400 - 600 mOsmoI/kg thereby resulting in improvement in harvest stage polysaccharide yield of >50%

5. Improved purification method with low endotoxin <10 EU/pg, low protein <1% and low nucleic acid content <2% wherein purification process is i) devoid of Sodium deoxycholate (DOC) being an animal-origin product, even its residuai presence in final product may lead to non-acceptance of product by regulatory agencies and certain religions communities ii) devoid of Triton X iii) devoid of ammonium sulphate iv) devoid of any Adsorbents (hydroxylapatite, calcium phosphate or apatite)

6. Improved conjugation efficiency (> 60%), improved conjugate yield (>50%) and stability of conjugate wherein i) Ps:Pr; EDAC ratio is 1:1:2 ii) stability indicating free polysaccharide is < 5% (preferably < 3%) and free protein is < 5% (preferably < 4%)

7. Improved immunogenicity for conjugate attributed to i) Vi polysaccharide - protein conjugate having a peculiar size between 1200 kDa to 1600 kDa. and ii) Polysaccharide used for conjugation has average molecular weight between 150 and 300 kDa (preferably between 150 and 250 kDa)

Μ 8. The immunogenic composition is stable at 2-8°C, 25°C and 40°C for over a period of six months and free polysaccharide after 6 months not more than 7.5% for 180-220 kDa polysaccharide and free polysaccharide after 6 months not more than 10.5% for 388/80/45 kDa polysaccharide.

9. The mice groups injected with bivalent (typhoid and paratyphoid) SIIPL vaccine such as

a) SIIPL Vi PS-TT + SIIPL O-SP A DT, b) SIIPL Vi PS-TT + SIIPL O-SP A TT and c) SIIPL Vi PS-TT + SIIPL O-SP A CRM Vi TT exhibited greater than 4-fold higher induction of IgG (as compared to mice administered with unconjugated Vi PS or unconjugated SIIPL O-SP A) thus indicating the immunogenic potential of bivalent

SIIPL vaccine containing combination of Vi TT and SIIPL O-SP A (DT/TT/CRM).

10. Minimum components involved in the vaccine composition.

φ EXAMPLES

The following examples are included to demonstrate preferred embodiments of the invention.

It should be appreciated by those of skill in the art that the compositions and techniques disclosed in the examples which follow represent techniques discovered by the inventor to 5 function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the présent disclosure, appreciate that many changes can be made in the spécifie embodiments which are disclosed and still obtain a like or similar resuit without departing from the spirit and scope of the invention.

Example 1:

A) STRAINS:

Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis used for developing the immunogenic composition of the présent disclosure may include: Salmonella enterica serovar typhi TY2 strain with ”tviB gene spécifie for Vi polysaccharide (Identified by Geneombio Technologies Private Limited, Pune and isolated from stool sample of typhoid confïrmed patient at Villoo Poonawalla Memorial Hospital, Pune); Salmonella enterica serovar typhi deposited at NCMR-NCCS; Pune, an International Depositary Authority having assigned Accession No. MCC 0193; Strain désignation- PDL-1, S. typhi: ATCC 19430; C6524 (NICED, Kolkata, India); S. paratyphi A: ATCC 9150 procured from Chromachemie Laboratory Private Limited, Bangalore, CMCC5OO73, CMCC50973; S. enteritidis: ATCC 4931; ATCC 13076; 5. enteritidis RII; S. enteritidis D24359; 5. enteritidis 618; S. enteritidis 502; S. enteritidis IV3453219;S. typhimurium: S. typhimurium 2192; ATCC 14208; 5. typhimurium 2189; S. typhimurium D23580; ATCC 19585; ATCC 700408; (LT2/SL134 (ST 19)); S.typhimurium 177(ST19) CDC 6516-60; ATCC 700720. Further any attenuated Salmonella serovar strain (S. typhi, S. paratyphi A, S. enteritidis and S. typhimurium) may be used for the préparation of the immunogenic composition of the présent disclosure.

WO2018037365, WO2019016654 and WO2020075184 are being incorporated with reference to the préparation of diphtheria toxoid (DT), tetanus toxoid (TT), inactivated whole cell pertussis, (wP), acellular pertussis, (aP), hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae type b antigen (Hib) and inactivated polio virus (standard dose and dose reduced polîovirus).

WO2018037365 is being incorporated with reference to the préparation of inactivated polîovirus and inactivated rotavirus.

WO2013114268 is being incorporated with reference to the préparation of meningococcal polysaccharide antigens from serotypes A, C, W, X and Y.

B) 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 comprise of the following steps: (UPSTREAM FERMENTATION)

1) Inoculation and harvesting of SI stage:

0.5 mL culture from cell bank vial is dîluted to 5 mL with medium and loopful culture from this is streaked on SCDA plate and incubated at 36±0.5°C for 28 to 32 Hrs.

2) Inoculation and harvesting of S2 stage:

well isolated colonies from the SI stage plate are inoculate into the conical flask containing 40 mL medium. Incubate the flask at 36±0.5°C and 150±10 RPM until Optical Density (OD) of the culture reaches between 2.5 to 3.5 range.

3) Inoculation and harvesting of S3 stage:

When Optical Density (OD) of the S2 stage culture reaches between 2.5 to 3.5 range, Expand the 40ml culture to 800 ml and distribute 160 ml content in each five IL flasks. Incubate the flasks at 36±0.5°C and 150±I0 rpm until OD of the culture reaches between 2.5 to 3.5 range.

4) Inoculation and harvesting of S4 stage (20L Fermenter):

When OD of the S3 stage culture reaches between 2.5 to 3.5 range, pool the culture of ail five flasks in seed bottle assembly and inoculate in 20L fermenter. Continue the fermentation using following parameters,

Fermentation process parameters:

Table 1: Fermentation process parameters
Parameter	Set Point & Range
DO (%)	37 (30 to 90)
Agitation (RPM)	150 to 500
Air (nl/min)	2.0 to 10
Oxygen (nl/min)	0.0 to 15
pH	7.0 ±0.4
TEMP (°C)	36.0 ±2.0

Start the Feed media from 3rd hour on wards and increase in proportion with OD. Continue the fermentation for 13 to 15 hrs.

5) S4 Stage Formalin Inactivation:

After S4 stage is completed, add formaldéhyde solution to the fermenter making the final conc. of 0.5 to 1.0% and incubate the culture at 36°C ± 2°C for 8 to 10 hrs.

6) Cell Séparation by Centrifugation:

After Inactivation step is completed, clarify the broth by centrifugation and collect the supernatant.

Carryout centrifugation using following parameters,

Table 2: Centrifugation parameters
Parameter	Set value
Temp set point	2 - 8 °C
RPM	7000 - 8000
Centrifugation time	40 to 60 min

7) Clarification by Depth Filtration:

After centrifugation is over, supernatant is filtered using depth filter.

8) 0.2μ Filtration:

After the depth filtration, the filtrate is subjected to 0.2μ filtration.

Bl. Media optimization for growth of Salmonella typhi S. paratyphi A; S. typhimurium and S. enteritidis, Following experiments were carried out for Media optimization and batch parameters;

Experiment No.: 01 - Frantz media was selected for the experîment as it is the widely used for growth of Gram négative organisms and evaluated for the growth of Salmonella Typhi.

Frantz media was used for development of seed and fermentation. Glucose as carbon source was composed in Feed media.

Table 3: Components of Fermentation media for Experiment No.; 01
Material	Qty. (g/L)
D- Glucose monohydrate	4

Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5

Table 4:Components of Feed media for Experiment No.: 01
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625

Seed was developed in disposable flasks (125ml and 500ml) and inoculated in 2L fermenter, After inoculation, fermentation was carried out in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH, 25% dissolved oxygen (DO) and 150 to 500RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

Observations:

Table 5: OD590 values in Experiment No.: 01
Culture Age (Hrs)	ODS90
0	0.0
1	0.12
2	0.295
3	0.49
4	1.43
5	4.33
6	8.73
7	9.24
8	9.7
9	9.68
10	9.7
11	9.57
12	9.3

13	9.2
14	9.5

Conclusion: Frantz media was found to support the growth of Salmonella typhi organism to limited extent. Further optimization of media was required.

Experiment No.: 02 - Yeast ex tract was added to the fermentation and feed media to support the growth of cells.

Table 6:Components of Fermentation media for Experiment No.: 02
Material	Qty. (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast ex tract	14

Table 7:Components of Feed media for Experiment No.: 02
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast extract	50

Procedure:

Seed was developed in disposable flasks (125ml and 500ml) and inoculated in 2L fermenter.

After inoculation, fermentation was carried out in fed-batch mode. Feed were added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH, 25% dissolved oxygen (DO) and 150 to 500RPM agitation (in cascade 15 mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

Observations:

Table 8: OD590 values in Experiment No.: 02
Culture Age (Hrs)	OD590
0	0.0
l	0.12
2	0.295
3	0.49
4	1.43
5	4.33
6	8.73
7	9.24
8	9.7
9	9.68
IO	9.7
11	10.1
12	10.6
13	11.2
14	11.8

Conclusion: Addition of yeast extract was found to support the growth of Salmonella typhi organisai to limited extent. Further optimization of media was required.

Experiment No.: 03 - Soya peptone was added to the fermentation and feed media to support the growth of cells.

Table 9:Components of Fermentation media for Experiment No.: 03
Material	Qty. (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 10:Components of Feed media for Experîment No.: 03
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast extract	50
Soya peptone	50

Procedure:

Seed was developed în disposable flasks (125ml and 500ml) and inoculated in 2L fermenter.

After inoculation, fermentation was carried out in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH, 25% dissolved oxygen (DO) and 150 to 500RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittent!y to control foaming during the fermentation.

Observations:

Table 11: OD590 values in Experîment No.: 03

Culture Age (Hrs)	OD590
0	0.0
1	0.12
2	0.295
3	0.49
4	1.43
5	4.33
6	8.73
7	9.24
8	9.7
9	9.68
10	9.8
II	10.5

12	11.8
13	12.6
14	13.2

Conclusion: Addition of Soya peptone was found to support the growth of Salmonella typhi organism. Optîmizations of batch parameters for manufacturing were required.

Experiment No.: 04 - Antifoam Ctobe was replaced by antifoam Struktol for improved cell growth.

Table 12:Components of Fermentation media for Experiment No.: 04
Material	Qty. (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 13:Components of Feed media for Experiment No.: 04
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast extract	50
Soya peptone	50

Procedure:

Seed was developed in disposable flasks (125ml and 500ml) and inoculated in 2L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH, 25% dissolved oxygen (DO) and 150 to 500RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

Observations:

Table 14: ODsîw values in Experiment No.: 04
Culture Age (Hrs)	ODvw
0	0.0
1	0.12
2	0.295
3	0.49
4	1.43
5	4.33
6	8.73
7	9.24
8	10.6
9	11.4
10	12.9
11	13.2
12	13.8
13	14.4
14	15.2

Conclusion: Replacement of antifoam C with antifoam Struktol was found to improve the growth of Salmonella typhi organism.

Experiment No.: 05 - Growth pattern of Salmonella typhi at following fermentation parameters at 20 L scale batch was studied. For batches of Salmonella typhi, suitable parameters were defined. Dissolved oxygen and Osmolality were controlled around 15 experimental set points.

Table 15: Experimental set points for Experiment No.: 05

Parameters	Experimental Set Points
pH	7.0
Température (°C)	36
Dissolved Oxygen (%)	30
Agitation (rpm)	150 to 500
Osmolality (mOsmol/kg)	300

Table 16:Components of Fermentation media for Experiment No.: 05

Material	Qty· (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 17:Components of Feed media for Experiment No.: 05
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast ex tract	50
Soya peptone	50

Procedure: Seed was developed in disposable flasks (250ml and IL) and inoculated in 20L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH and 150 to 500RPM agitation (in cascade mode to maintain DO). Antifoam was added intérimttently to control foaming during the fermentation.

Observations:

Table 18: ODsw values in Experiment No.: 05
Culture Age (Hrs)	OD590
0	0.20
1	0.45
2	0.96
3	1.87
4	3.2
5	4.33
6	8.73
7	9.24
8	9.7
9	11.2
10	14.1
11	16.2
12	17.3
13	17.9
14	18.5

Conclusion: From the observations, it was concluded that the optimization of parameters mentioned in the table of experimental set points of this experiment were required for growth 5 of Salmonella typhi organ ism.

Experiment No.: 06 - Growth pattern of Salmonella typhi at following fermentation parameters at 20L scale batch was studîed. For batches of Salmonella typhi, suitable parameters were defined. Dissolved oxygen and Osmolality were controlled around 10 experimental set points.

Table 19: Experimental set points for Experiment No.: 06
Parameters	Experimental Set Points
pH	7.0
Température (°C)	36
Dissolved Oxygen (%)	40

Agitation (rpm)	150 to 500
Osmolality (mOsmol/kg)	400

Table 20:Components of Fermentation media for Experiment No.: 06
Material	Qty. (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 21:Components of Feed media for Experiment No.: 06
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast ex tract	50
Soya peptone	50

Procedure: Seed was developed in disposable flasks (250m] and IL) and inoculated in 20L 5 fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH and 150 to 500RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

Observations:

Table 22: OD590 values in Experiment No.: 06
Culture Age (Hrs)	OD590
0	0.20
1	0.45
2	0.96

3	1.87
4	3.2
5	4.33
6	8.73
7	11.8
8	15.2
9	17.8
10	19.5
11	20.5
12	21.1
13	21.9
14	22.8

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

Experiment No.: 07 - Growth pattern of Salmonella typhi at following fermentation parameters at 20 L scale batch was studied. For batches of Salmonella typhi, suitable parameters were defined. Dîssolved oxygen and Osmolality were controlled around experimental set points.

Table 23: Experimental set points for Experiment No.: 07
Parameters	Experimental Set Points
pH	7.0
Température (°C)	36
Dissolved Oxygen (%)	37
Agitation (rpm)	150 to 500
Osmolality (mOsmol/kg)	700

Table 24:Components of Fermentation media for Experiment No.: 07
Material	Qty. (g/L)
D- Glucose monohydrate	4

Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 25:Components of Feed media for Experiment No.: 07
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast extract	50
Soya peptone	50

Procedure: Seed was developed in disposable flasks (250ml and IL) and inoculated in 20L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was 5 added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH and 150 to 500RPM agitation (in cascade mode to maintain DO). Antifoam was added intermittently to control foaming during the fermentation.

Observations:

Table 26: ODs^u values in Experiment No.: 07
Culture Age (Hrs)	ODsso
0	0.21
1	0.49
2	0.29
3	1.43
4	2.81
5	4.73
6	7.1
7	9.2
8	12.4
9	16.1

10	19.2
11	23.4
12	26.3
13	28.2
14	28.4

Conclusion: From the observations, it was concluded that the optimization of parameters mentioned in the table of experimental set points of this ex périment were required for growth of Salmonella typhi organism.

Experiment No.: 08 - Growth pattern of Salmonella typhi at following fermentation parameters at 20 L scale batch was studied. For batches of Salmonella typhi, suitable parameters were defined. Dissolved oxygen and Osmolality were controlled around experimental set points.

Table 27: Experimental set points for Experiment No.: 08
Parameters	Experimental Set Points
pH	7.0
Température (°C)	36
Dissolved Oxygen (%)	40
Agitation (rpm)	150 to 500
Osmolality (mOsmol/kg)	700

Table 28:Components of Fermentation media for Experiment No.: 08
Material	Qty. (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 29:Components of Feed media for Experiment No.: 08
Material	Qty. (g/L)

Table 29:Components of Feed media for Experîment No.: 08
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium dî-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast extract	50
Soya peptone	50

Procedure: Seed was developed in disposable flasks (250ml and IL) and inoculated in 20L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated 5 at 36°C température, 7.00 pH and 150 to 500RPM agitation (in cascade mode to maintaîn

DO). An ti foam was added intermittendy to control foaming during the fermentation.

Observations:

Table 30: OD590 values in Experîment No.: 08
Culture Age (Hrs)	OD590
0	0.20
1	0.66
2	1.62
3	3.2
4	5.3
5	8.1
6	11.2
7	16.3
8	19.2
9	23.8
10	27.4
11	28.8
12	30.1
13	31.2
________7________	32.4

Experiment No.: 09 - Growth pattern of Salmonella typhi at following fermentation parameters at 20 L scale batch was studied. For batches of Salmonella typhi, suitable parameters were defined. Dissolved oxygen and Osmolality were controlled around experimental set points.

Table 31: Experimental set points for Experiment No.: 09
Parameters	Experimental Set Points
pH	7.0
Température (°C)	36
Dissolved Oxygen (%)	40
Agitation (rpm)	150 to 500
Osmolality (mOsmol/kg)	500

Table 32:Components of Fermentation media for Experiment No.: 09
Material	Qty. (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 33:Components of Feed media for Experiment No.: 09
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625

Table 33:Components of Feed media for Experiment No.: 09
Material	Qty. (g/L)
Yeast extract	50
Soya peptone	50

Procedure: Seed was developed in disposable flasks (250ml and IL) and inoculated in 20L fermenter. After inoculation, fermentation was carried out in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was opérâted 5 at 36°C température, 7.00 pHand 150 to 500RPM agitation (in cascade mode to maintain

DO). Antifoam was added intermittently to control foaming during the fermentation.

Observations:

Table 34: ODs9« values in Experiment No.: 09
Culture Age (Hrs)	ODs9Û
0	0.22
1	0.65
2	2.14
3	4.2
4	6.5
5	11.3
6	14.2
7	18.6
8	26.5
9	29.4
10	32.3
11	36.2
12	37.2
13	38.1
14	38.5

Conclusion: From the observations, it was concluded that the optimization of parameters 10 mentioned in the table of experimental set points of this experiment were required for growth of Salmonella typhi organism.

Experiment No.: 10 - Growth pattern of Salmonella typhi at following fermentation parameters at 20 L scale batch was studied. For batches of Salmonella typhi, suitabie parameters were defined. Dissolved oxygen and Osmolality were controlled around experimental set points.

Table 35: Experimental set points for Experiment No.: 10
Parameters	Experimental Set Points
pH	7.0
Température (°C)	36
Dissolved Oxygen (%)	37
Agitation (rpm)	150 to 500
Osmolality (mOsmol/kg)	500

Table 36:Components of Fermentation media for Experiment No.: 10
Material	Qty. (g/L)
D- Glucose monohydrate	4
Sodium di-hydrogen phosphate monohydrate	4.1
Di- sodium hydrogen phosphate heptahydrate	18.8
Magnésium sulfate heptahydrate	2.5
Yeast extract	14
Soya peptone	9

Table 37:Components of Feed media for Experiment No.: 10
Material	Qty. (g/L)
D- Glucose monohydrate	100
Sodium di-hydrogen phosphate monohydrate	1.025
Di- sodium hydrogen phosphate heptahydrate	0.94
Magnésium sulfate heptahydrate	0.625
Yeast extract	50
Soya peptone	50

Procedure: Seed was developed in disposable flasks (250ml and IL) and inoculated in 20L fermenter. After inoculation, fermentation was carried oui in fed-batch mode. Feed was added in fermenter to support the growth of Salmonella Typhi organism. Batch was operated at 36°C température, 7.00 pH and 150 to 500RPM agitation (in cascade mode to maintain 5 DO). Antifoam was added intermittently to control foaming during the fermentation.

Observations:

Table 38: OD590 values in Experiment No.: 10
Culture Age (Hrs)	OD590
0	0.21
1	0.87
2	2.75
3	5.3
4	8.30
5	14.12
6	18.5
7	24.6
8	30
9	33.3
10	38.6
11	42.4
12	43.1
13	43.7
14	44

Conclusion:

Table 39: USP - Harvest Yield (in percentage or other unit) obtained for each of the experiments 1 to 10
S.No.	Description	Harvest Yield (mg/L)
1	Experiment-1	142 mg/L
2	Experiment-2	180 mg/L
3	Experiment-3	210 mg/L
4	Experiment-4	452 mg/L

5	Experiment-5	463 mg/L
6	Experîment-6	480 mg/L
7	Experiment-7	492 mg/L
8	Experiment-8	510 mg/L
9	Experiment-9	537 mg/L
10	Experiment-10	600 mg/L

Fed-batch mode of cultivation to obtain a high yield harvest of polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis, by fed5 batch process involved the use of combination of an antifoam agent J673 STRUKTOL, soya peptone Difco™ Select Phytone™ UF at a range of 40 to 70 g/L and yeast extract Difco™ Yeast Extract, UF 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, température maintained in the range of 34.0- 38.0°C, dissolved oxygen 10 level maintained between 36- 39%, Agitation (rpm) maintained between 150 to 500 and osmolality 400 - 600 mOsmol/kg.

Example 2

C) The method of purifying the polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis (DOWNSTREAM PURIFICATION)

Cl) ViPs fermentation harvest subjected to following downstream purification steps to obtain desired quality of Vi-polysaccharide (ViPs):

n) 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;

o) concentration by tangential flow ultrafiltration (TFF) and buffer exchange (20mM Tris buffer pH-7.5 containing 5mM EDTA) by diafiitration (DF) using a membrane having 10 0 kDa molecular weight eut off (MWCO);

p) treatment with S DS (20% S DS stock input), ethylene diamine tetra-acetic acid (EDTA) (4 to lOmM) and Sodium acetate (5% to 10%) for dénaturation of proteins, nucleic acids and lipopolysaccharide ;

q) Ethanol précipitation (40% to 70%);

r) Centrifugation and filtration by direct flow filtration (DFF) through ai least one clarification filter having a pore size of about 0.2 μΜ;

s) treatment with 2M Potassium chloride (KC1) for removal of excess detergent followed by Centrifugation and filtration by direct flow filtration (DFF) through at least one 20 clarification filter having a pore size of about 0.2 μΜ;

t) concentration by tangential flow filtration (TFF) and buffer exchange by diafiitration (DF) using a membrane having 30kDa molecular weight eut off (MWCO);

u) Sélective précipitation of PS by CTAB (12% CTAB stock input);

v) centrifugation and filtration by direct flow filtration (DFF) through at least one clarification filter having a pore size of about 0.45 micrometers to about 0.2 micrometers;

w) Removal of protein and nucleic acid impurities by washing pellet with 60% éthanol (50% to 70%) in presence of IM NaCI (0.1M to 2M);

x) sélective précipitation of polysaccharide by utilizing 60% éthanol (<75% OR>95% );

y) dissolving polysaccharide in WFI or IM NaCI and subjecting to concentration by tangential flow filtration (TFF) and buffer exchange by diafiitration (DF) using a membrane having 30kDa molecular weight eut off (MWCO); and

z) Stérile Filtration through at least one stérile filter having a pore size of about 0.2 micrometers under stérile conditions and stored at < -20“C

C2) Salmonella paratyphi O-specific polysaccharide (OSP) of Lipopolysaccharide (LPS) fermentation harvest subjected to following downstream purification steps to obtain desired quality of O-specific polysaccharide from Salmonella Paratyphi A lipopolysaccharide (LPS):

r) Centrifugation at 7000 rpm for 30 min at 4°C, Collected cell pellet (~lKg) and supernatant and Cell pellet suspended in 15 L of IM NaCl and stirred for Ihr at Room temp.

s) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using 0.45gm Prostak cassette and membrane having 30kDa molecular weight eut off (MWCO);

t) Acid hydrolysis of LPS with 1% Acetic acid (pH~2.8 - 3.0) at 90°C température for 180 min

u) The mixture cooled to Room Température (RT) & Removal of impurity précipitation by Centrifugation at 7000 rpm for 45 min at 25°C

v) Neutralizatîon Collected the supernatant in glass bottle and neutralized to pH -7.0 with liquor ammonia

w) Clarification by direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and 0.2 micrometers;

x) Added Sodium deoxycholate stock solution to get final concentration of 1% and incubated for 30 min under stirring at 30°C température, Adjusted pH to 2.0 with Acetic acid and incubated for 15 min under stirring at 30°C

y) Centrifugation at 7000 rpm for 45 min at 25°C

z) Direct flow filtration (DFF) through at least one membrane having a pore size of about 0.45 and 0.2 micrometers;

aa) concentration by tangential flow ultrafiltration (TFF) and buffer exchange by diafiltration (DF) using a membrane having 10 kDa molecular weight eut off (MWCO);

bb) Stérile Filtration through at least one stérile filters having a pore size of about 0.2 micrometers under stérile conditions to obtain Purified O-Specific Polysaccharide (OSP).

Table 40: Analytical Methods used for testing at various stages
S.No *	Test	Test method	Units	References	Acceptance criteria
1	Vi Polysacchari de content	HPAEC-PAD	mg/mL	WHO TRS 987	Actual value
2	Identîty	NMR spectroscopy	NA	NA	Confirm the identîty/Compara ble to NIBSC Vi Polysaccharide Standard.
3	Protein impurity	Lowry method	mg/g	IP/BP/Ph Eur 0250/WHO/Inhouse	Shall contain less than 10.0 mg of Protein per gram of PS
4	Nucleic acid impurity	UV spectroscopy (Absorbance at 260nm)	mg/g	IP/BP/Ph Eur 0250/WHO/Inhouse	Shall contain less than 20.0 mg of Nucleic acid per gram of PS
5	0 acetyl content (Lyophilized)	Hestrin’s method	mmol/g	IP/BP/Ph Eur 0250/WHO/Inhouse	Shall be > 2.0 mmol/g polysaccharide
6	Moisture content	Thermogravimet ry	Percentag e (%)	IP/BP/EP/WHO/I n house	Actual value
7	Molecular size distribution	SEC-HPLC	Percentag e (%)	IP/BP/EP/WHO/I n-house	At least 50% of polysaccharide shall be eluted before a KD of 0.3
8	Endotoxin content	Kinetic turbidometric assay using Limulus amebolysate (LAL)	EU/gg	IP/BP/Ph Eur 0250/WHO/Inhouse	Less than 100 EU of Endotoxin per gg ofPS

9	pH	Potentiometric	Number	—	Actual value
10	Bioburden	—	CFU/mL	IP/BP/WHO/Inhou se	100 cfu/ 10 mL of PS
11	Total PS Content	HPAEC-PAD	mg/mL	WHO TRS 987	Actual value
12	Average Molecular Weight (AMW)	SE-HPLC	kDa	WHO TRS 987	Actual value
13	Identity	NMR	NA	In-house	Comparable to published data/NIBSC STD
14	Bacterial and mycotic Sterility	Suitable	NA	WHO TRS 987	Shall be comply
15	Vi strength	HPAEC-PAD	gg/mL	WHO TRS 987	25 gg /0.5 mL
16	Free Polysaccharid e	HPAEC-PADDOC	Percentag e(%)	WHO TRS 987/Inhouse *	<40%
17	O-Acetylation	Hestrin	mmoVg	WHO TRS 987/Inhouse	> 2.0 mmol/g of Vi
18	Molecular Size Distribution (MSD)	SE-HPLC	Percentag e(%)	WHO TRS 987/Inhouse	For information (but larger than Vi) Kd to be decided
19	Endotoxin or Pyrogen Content	Kinetic turbîdometric assay using Limulus amebolysate (LAL) and Animal Test	EU/gg	WHO TRS 987	<100 E.U./dose Agreed by NRA
20	Adjuvant Content and degree of adsorptîon	Suitable	mg/ml	WHO TRS 987	Actual value
21	Preservative Content	Instrument /Suitable Method	mg/ml	WHO TRS 987/Inhouse	Y

•	22	General Safety (Innocuity)	Animal Test	NA	WHO TRS 987/Inhouse	Agreed by NRA
23	PH	Potentiometric	Number	In-house	6.0 to 8.0
24	Osinolality	Osmometer	mOsmol/k g	WHO TRS 987/Inho u se	Agreed by NRA

Results and Interprétation:

Table 41: Purified Vi polysaccharide tested by IPQC
TEST	SPECIFICATION	Batch 1	Batch 2	Batch 3
Polysaccharide concentration	Actualvalue (mg/mL)	3.762	3.52	3.306
Identity test by NMR	Should compiles	Compiles	Compiles	Compiles
O-acetyl content	Not less than 2.0 mmol/g polysaccharide	2.395	2.943	3.336
Protein impurity	Not more than 1% by weight of PS	0.797	0,568	0.907
Nucleic acid impurity	Not more than 2% by weight of PS	0.984	1.136	1.180
Endotoxin content	Not more than 150EU of endotoxin per pg of PS	16.39	52.37	45.33
Molecular size distribution (MSD)	At least 50% of PS is eluted before a distribution coefficient (Kd) of 0.25 is reached.	93.38	94.35	94.47
Free formaldéhyde (Residual)	Not more than 0.02% w/v.	0.00	0.00	0.00
Residual Ethanol	Not more than 5,000 ppm.	Not detected	Not detected	Not detected
Bioburden	NMT 100 cfu/LOmL ofPS	0 cfu/lOmL of PS	0 cfu/lOmL of PS	0 cfu/lOmL of PS

Improved method of Vi PS purification has clear advantages în ternis of both, ease of operation as well as several other advantages such as, > Improved Polysaccharide purification (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, viscosity) when Sodium acetate (6%) used Vs Sodium acetate (2%)

When Sodium acetate (6%) used - Values for

DSP % Recovery - 50%

Ps Viscosity - Data not available

Ps Polydispersity - Data not available

O-acetyl - 2.9 mmol/gin of PS

When Sodium acetate (2%) used - Values for

DSP % Recovery - 40%

Ps Viscosity - Data not available

Ps Polydispersity - Data not available

O-acetyl - 2.6 mmol/gm of PS

Sodium acetate reacts with nucleic acids. It breaks up into Na+ and (CH3COO)-. The posîtively charged sodium ion neutralizes negatively charged PO3- of the nucleic acids; thus helps in précipitation of nucleic acids. So higher concentration i.e. 6% of sodium acetate effectively précipitâtes host cell impurities like nucleic acids.

> Improved Polysaccharide purification (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, viscosity) CT AB (3%) Vs CT AB (0.5, 1%). CT AB is an amine based cationic quaternary surfactant. It interact with anionic polysaccharide (ionic interaction) and then decreases their solubility hence it précipitâtes Polysaccharide from the solution. So higher concentration of CTAB (2%) always précipitâtes higher amounts polysaccharide which gives higher yields and removal of protein impurity is an added advantage.

> Improved Polysaccharide purification (in terms of recovery, O-acetyl, endotoxin, protein, nucleic acid content, polydispersity, viscosity) for current process without DOC Vs DOC based process.

Advantage of Improved method without DOC process

1. Regulatory issue: DOC is an animal origin component and is non HALAL compilant.

It is manutactured & supplied by a single vendor throughout the world, whereas SDS is a synthetic detergent, HALAL certified and with several suppliers avaîlable globally.

2. Functional issue: DOC breaks down the endotoxins without effecting the Chemical composition and once the detergent is removed it can regains its biological activity, whereas SDS owing to îts amphipathic nature and higher aggregation number dénaturés and solubilizes the proteins well and also disrupts endotoxins irreversibly to its monomeric units.

> The new improved method of Vi PS purification developed without using Sodium deoxycholate (DOC) which is an animal origin component or Phénol. Improved method based on the reverse purification of polysaccharides in which host cell impurities like proteins, nucleic acids and lipo-polysaccharides will be removed at initial steps by using Sodium acetate (6%), Sodium dodecyl sulfate (2%) and Ethanol (40%) and concentration and diafiltration using 30kDa eut off membranes at different steps, then polysaccharide precipitated by cationic detergent CTAB and performed polishing purification steps to achieve higher yields of Purified Vi Polysaccharide which is meeting WHO spécifications.

> The purification process results 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 in the range of 400 to 4000 mg/L, average molecular weight was found to 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 μ g of polysaccharide (PS), Molecular size distribution (greater than 50% of PS is eluted before a distribution coefficient (KD) of 0.25 is reached) > The O-specific polysaccharide (OSP) purification process results in significant réduction of endotoxin (< 100EU of endotoxin per pg 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) was found to be in the range of 40 to 200 kDa.

M Example 3:

D) The method of conjugating the polysaccharide derived from Salmonella serovar strains S. typhi; S. paratyphi A; S. typhimurium and S. enteritidis to carrier protein.

The carrier protein used for conjugation with Salmonella typhi Vi polysaccharide is tetanus toxoid. The polysaccharides derived from S. paratyphi A, S. typhimurium and S. enteritidis individually conjugated to a carrier protein selected from tetanus toxoid (TT), diphtheria toxoid (DT) or CRM 197.

In accordance with the present disclosure, CRM 197 is procured from Recombinant Strain CS463-003 (MB 101) of Pseudomonas fluorescens from Pfenex USA.

In accordance with the present disclosure, TT is procured from Clostridium Tetani (Harvard No 49205) obtained from Central research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. Central research Institute (CRI) procured this strain from NVI, Netherland.

In accordance with the present disclosure, DT is produced cured from cultures of 15 Corynebacterium diphtheriae Park-Williams Number 8 strain, the strain is obtained from Central research Institute (CRI), National Control Authority, Kasauli, Himachal Pradesh, India. Central research Institute (CRI) procured this strain from Wellcome Research Laboratories.

Dl) Préparation of Monovalent Salmonella typhi conjugate using Carbodiimide

Chemistry:

Step 1: Concentration and Derivatization of TT Procedure:

a) GFC purified TT (Monomeric Content More than 90%) was concentrated using lOkDa membrane to achieve NLT 12mg/ml Protein concentration (Lowry’s assay.)

b) Derivatization ratio was used as given below and calculated the required quantifies accordingly.

Pr:ADH:EDC as 1:6.0:1

Derivatization Scale: 13gm

c) The Concentrated TT (in 0.9% NaCl), IM MES pH 6.0, ADH solution (dissolved in 0.1M MES pH 6.0) and EDC solution (dissolved in 0.1M MES pH 6.0) were added sequentially and made final required volume by addition of 0.1M MES buffer pH 6.0. The final Pr conc. in reaction was ~4.5mg/ml.

d) The reaction mixture was stirred and allowed to continue about I hr at pH 5.90. Then derivatization reaction was quenched by adjusting pH above 7.5 using 0. IM Phosphate buffer containing EDTA pH 8.0.

e) Quenched reaction mixture was diafiltered using lOkDa eut Off membrane against lOmM Phosphate buffer using 22volumes followed by 0.1M MES buffer pH 6.0 at least 12 volumes to remove unreacted moieties and other residuals.

f) The derivatized sample was analyzed for total Pr conc. by Lowry’s assay and Extent of derivatization value by colorimétrie (TNBS) assay.

Results:

The Derivatized Pr Conc. by Lowry’s assay: 15mg/ml

The Extent of derivatization value (DOA): 19

Percentage recovery: ~75 %

Step 2: Conjugation of Vi Ps to Derivatization Of TT Procedure:

Vi PS-Derivatized TT conjugation performed using Carbodiimide chemistry.

Following parameters of PS and Prwere used for Conjugation.

a.

The ratio for conjugation reaction was used Ps:Pr:EDC::1:0.9:1.75 (+ 0.5 for Pr and

EDC) Conjugation scale lOgm.

b. The required batch volume of PS was added into pre-sterilize glass bottle.

c. IM MES buffer pH 6.0 (stock buffer) was added into the measured volume of PS to achieve 0.1M MES conc.

d. The mixture was stirred at -lOOrpm for homogeneous mixing.

e. Then measured volume of Derivatized TT was added into Ps containing bottle.

f. After addition of TT immediatelyfreshly prepared EDC (dîssolved in 0.1 M MES Buffer pH 6.0) was added into above reaction.

g. pH was observed and recorded (pH 6.0+0.5)

h. The sampie was analyzed at different time intervals using SE-HPLC. The conjugation conversion percentage and Protein consumption was monitored.

i. The reaction was quenched after 1.45 hrs by raising pH at 7.5 using 0.1.M Phosphate buffer containing EDTA, when <90% protein consumed.

j. The quenched reaction mixture was stored at 2-8°C till further use.

Step 3: Purification of Quenched Vi-TT Conjugate

Purification of quenched conjugate were performed by two methods carried out to remove unreacted Ps, Unreacted Pr, other residuals using following parameters;

1. Ultrafiltration method:

Following parameters used for diafiltration;

Diafiltration Scale: 8.5gm

Ps conc. during D/F: ~2mg/ml

Membrane Cut Off: 300kDa

Membrane Make: Pall

Membrane Area: 2.5m²

Buffer A Used: lOmM PBS least 25volumes

Buffer B Used: 0.9% NaCl 30 volumes.

Final volume: 6.5Lts

Purified Conjugate was filtered using 0.2μΜ filter.

P 2. Gel filtration Chromatography:

Quenched conjugate about 1.5gm was concentrated on O.lm2 300kDa eut off membrane - 2-4mg/ml. Conjugate purification performed to remove unbound PS, unbound TT and residuai EDC using Gel filtration chromatography.

Two separate GFC run were performed with same parameters for purification.

Following Chromatographie conditions used for Purification.

Table 42: Chromatographie conditions used for Purification of Conjugates
Sr. No	Parameters	Details
1	Chromatography used	Gel filtration
2	Coiumn used	B PG Sériés
3	Resin Used	Toyopearl ‘HW’65F
4	Elution buffer	0.17M NaCJ
5	Operating Linear flow rate	30cm/hr
6	Operating Volumétrie flow rate	40ml/min
7	Sample loading	3.5% of total bed volume
8	Fraction collection	1 min

Total 27 Fractions collected were(100ml each) analyzed on SE-HPLC and pooled together on the basis of chromatographie profile.

2 Fractions were analyzed for Ps content by HPAD method, Pr content by Lowry’s method, % free Ps by DOC-HPAD method.

Based on analysis Fractions were pooled (1 to 23) and filtered using 0.22μΜ filter.

Sample was analyzed for Ps content, Pr content and free Ps analysis and endotoxin, OAcetyl content, sterîlity, pH value and Endotoxin analysis.

RESULTS AND INTERPRETATION:

Table 43; IPQC évaluation of Vi-TT Conjugate Purified byUltra-filtration and GFC
Test	Vi-TT Conjugate Purified by Ultra-filtration	Vi-TT Conjugate Purified GFC
Ps Content	1.43mg/ml	1.36 mg/ml
Pr Content	1.04 mg/ml	1.20mg/ml
PS/Pr ratio	1.37	1.13
Recovery %	Γ 60	52

pH	6.67	5.63
Endotoxin	0.35Eu/pg 505.89Eu/ml	0.46 Eu/pg 619.61Eu/ml

Comparative Conjugation process data- Improved conjugation efficiency, improved conjugate yield and stability (Free Ps, Free Pr) of SIIPL typhoid conjugate wherein Ps:Pr: 5 EDAC ratio is 1:1:2 Vs Other Ps:Pr: EDAC ratios

Improved conjugation efficiency; Conjugation Conversion Percentage observed more than 90%.

Improved conjugate yield: The purification performed using different methods i.e Ultrafiltration method and Gel filtration method with variable conjugation purification scale 10 and recovery found 40 to 87%.

Ratio used 1:1:2 vs 1:1.5:1.2, 1:0.9:2, 1:0.9:1.75, 1:0.8:1.8:, 1:0.7:1.8, 1:0.9:0.6, 1:0.9:1, and

1:0.7:1.5 for Vi-TT Conjugation and it was found that lower protein with 1.5 EDC can give optimum conjugation conversion (%).

It was found that Vi Ps with variable sizes can be conjugated with ADH activated TT.

The used different ratio like Ps:Pr:EDC:: 1:0.7:1.8, 1:0.8:1.8,1:0.9:2 and 1:1:2 at lower pH at different purification techniques résulte in more than 0.5 PS/Pr ratio, lower free ps with good recovery of Conjugales.

D2) Préparation of Monovalent Salmonella paratyphi conjugales:

Two types of conjugation chemistries (cyanylation and carbodiimide chemistry) were applied for the conjugation of the Paratyphi OSP to Carrier protein Diphtheria Toxoid (DT), CRM197 and Tetanus Toxoid (TT):

1) Cyanylation chemistry based conjugation of the Paratyphi OSP to Carrier protein Diphtheria Toxoid (DT), CRM197 and Tetanus Toxoid (TT)

A) Derivatization of Diphtheria Toxoid (DT), (Addition of Linker ADH) using carbodiimide chemistry and conjugation with Concentrated OSP using cyanylation chemistry

B) Derivatization of CRM 197, Tetanus Toxoid (TT) (Addition of Linker ADH) using 10 carbodiimide chemistry and conjugation with Concentrated OSP using cyanylation chemistry

C) Derivatization of Tetanus Toxoid (TT) (Addition of Linker ADH) using carbodiimide chemistry and conjugation with Concentrated OSP using cyanylation chemistry

IA) Préparation of S. Paratyphi Conjugates using Diphtheria Toxoid as carrier protein. 1) Protein derivatization:

High Monomerîc Diphtheria toxoid (DT) was concentrated to (10-20mg/ml) on a lOkDa membrane and analysed for protein content

Table 44: The protein content measured by BCA (Bicinchoninic acid) assay.
Test Sample	Total Protein mg/ml
Concentrated DT	12.27

To the concentrated DT freshly prepared IM MES Buffer was added and Adipic acid 20 dihydrazide (ADH) (dissolved 75-100mg/ml in WOmM MES buffer pH:5.8) in the 1:10 by weight ratio and EDAC (Lethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer pH:5.8) in the 1:1 by weight ratio. The reaction was continued at 5.8 pH for about IHrs and then the reaction mixture was diafiltered on 10 kDa TFF in 50mM Borate buffer pH 9.0 to remove residuals and unreacted components. The final sample 25 was analysed for protein content and degree of derivatization

Table 45: Degree of Derivatization by TNBS assay
Test Sample	Total Protein mg/ml	DOD
ADH Derivatized DT	40	16.40

2) Conjugation of S. Paratyphi A polysaccharide (OSP) and ADH Derivatized DT:

Two Experiments were performed by change in 1-cyano- 4-pyrrolidinopyridinium 5 tetrafluoroborate (CPPT) (CPIP) ratio

Expt No:l - The OSP was concentrated on 10 kDa membrane to achieve a concentration of (10-15mg/ml).

Table 46: The Polysaccharide content was analysed by Anthrone assay
Test Sample	Total PS mg/ml
Concentrated OSP	13.48

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.3 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein was added in 1:0.8 by weight ratio PS: PR: CPIP as 1:0.8:1.3

Shodex columns SB-804 HQ and SB-805 HQ were used sequentiaily with PBS as mobile 15 phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 times to that of PS by weight.

Refer Figure 1: Comparison of Polysaccharide, Protein and Conjugate (PS: PR: CPIP 1:0.8:1.3)

3) Conjugate Purification by GFC: A Column XK16/70 was made ready using GFC resin (Tyopearl HW65F) with a bed height of 40cm. The column was packed at a flow rate of lOOcm/hr and allowed to settle and IM NaCl 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30cm/hr and the conjugate was loaded on the column and fractions collected at Imîn interval. Fractions 2-9 were pooled according to the profile on HPLC. The final pooled fractions were filtered and sent for analysis

•
	Table 47: Evaluation of Purified OSP-DT ADH Conjugale (PS:PR:CPIP as 1:0.8:1.3)
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio (Ps/pr)
OSP-DT ADH Conjugale 1	0.215	0.210	1.02

Refer Figure 2: Chromatogram of GFC purified OSP-DT ADH Conjugale (PS:PR:CPIP as 1:0.8:1.3) 5

Expt No: 2 To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.1 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein 10 was added in 1:0.8 by weight ratio PS:PR:CPIP as 1:0.8:1.1,

Shodex columns SB-804 HQ and SB-805 HQ were used sequentîally with PBS as mobile phase at 1 inl/min flow rate to monitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 times to that of PS by weight.

Refer Figure 3: Comparison of Polysaccharide, Protein and Conjugate (PS:PR:CPIP 1:0.8:1.1)

Conjugale Purification by GFC: A Column XK16/70 was made ready using GFC resin 20 (Tyoperal HW65F) with a bed heîght of 40cm. The column was packed at a flow rate of lOOcm/hr and allowed to settle and IM NaCL 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCL at 30cm/hr and the conjugale was loaded on the column and fractions were collected at 1min interval. Fractions 2-8 were pooled according to the profile on HPLC. The final pooled 25 fractions were filtered and sent for analysis

Table 48: Evaluation of Purified OSP-DT ADH Conjugate(PS:PR:CPIP 1:0.8:1.1)
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio (Ps/pr)
OSP-DT ADH Conjugale 2	0.113	0.100	1.13

Refer Figure 4: Chromatogram of GFC purified OSP-DT ADH

Conjugate (PS:PR:CPIP 1:0.8:1.1)

Conclusion: Conjugation of Paratyphi A PS using ADH derivatized DT was fonnd successful and the PS/PR ratio was found satisfactory.

IB. Préparation of S.Paratyphi Conjugates using CRM 197 as carrier protein

1) Protein derivatization: Cross reacting mutant (CRM 197) received from Production department (SIIPL) was taken for derivatization

Table 49: The protein content measured by BCA (Bicinchoninic acid) assay.
Test Sample	Total Protein mg/ml
Concentrated CRM 197	32

To the concentrated CRM 197 freshiy prepared IM MES Buffer was added and Adipic acid dihydrazide (ADH) (dissolved 75-100mg/ml in lOOmM MES buffer pH:6.5) in the 1:3.5 by weight ratio and ED AC ( l-ethyES-iS-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer pH:6.5) in the 1:0.25 by weight ratio. 5% Tween 80 was added. The final reaction volume was made up using 100mM MES Buffer to achieve a final concentration of 3-4mg/ml the reaction was continued at 6.5 pH for about 3Hrs and then the reaction mixture was diafiltered on 10 kDa TFF in 50mM Borate buffer and 0.005% Tween 80 pH 9.0 to remove residuals and unreacted components. The final sample was analysed for protein content and degree of derivatization

Brief description of assay or reference: The protein content was measured by BCA (Bicinchoninic acid) assay.

Table 50: Degree of Derivatization by TNBS assay
Test Sample	Total Protein mg/ml	DOD
ADH Derivatized CRM 197	32	6.74

2) Conjugation of S.Paratyphi A polysaccharide (OSP) and ADH Derivatized CRM197: The OSP received from DSP was concentrated on 10 kDa membrane to achieve a concentration of (10-15mg/ml).

Table 51: The Polysaccharide content analysed by Anthrone assay
Test Sample	Total PS mg/ml
Concentrated OSP	11.7

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (U4mg/ml in acetonitrile) was added into polysaccharide in the 1:1.3 by weight ratio. The pH was shifted to 9.5 immedîately with 2.5M NaOH and held for up to 3 min. Then protein 5 was added in 1:1 by weight ratio PS:PR:CPIP as 1:1:1.3

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate to monitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 times to that of PS by weight.

Refer Figure 5 and 6: Comparison of Polysaccharide, Protein and Conjugate (PS:PR:CPIP as 1:1:1.3)

3) Conjugate Purification by GFC: A Column XK16/70 was made ready using GFC resin (Tyoperal HW65F) with a bed height of 40cm. The column was packed at a flow rate of 15 lOOcm/hr and allowed to settle and IM NaCL 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCL at 30cm/hr and the conjugate was loaded on the column and fractions collected at 1min interval, Fractions 2-7 were pooled according to the profile on HPLC. The final pooled fractions were filtered and sent for analysis

Table 52: Evaluation of Purified OSP-DT ADH Conjugate(PS:PR:CPIP 1:1:1.3)
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio (Ps/pr)
OSP-CRM ADH Conjugate	0.150	0.220	0.681

Refer Figure 7: Chromatogram of GFC purified OSP-DT ADH Conjugate (PS:PR:CPIP as 1:1:1.3)

Conclusion: Conjugation of Paratyphi A PS using ADH derivatized CRM 197 was found successful and the PS/PR ratio was found satisfactory.

IC) Préparation of S.Paratyphi Conjugates using Tetanus Toxoid as carrier protein:

1) Protein derivatization: High Monomeric Tetanus toxoid (TT) received from Production department (SIIPL) was concentrated to (15-20mg/ml) on a 30 kDa membrane and analysed for protein content

Table 53: The protein content measured by Lowry assay
Test Sample	Total Protein mg/ml
Concentrated TT	16.29

The concentrated TT freshly prepared IM MES Buffer was added and Adipic acid dihydrazide (ADH) (dissolved 75-100mg/ml in lOOniM MES buffer pH:6.0)in the 1:10 by weight ratio and EDAC (l“ethyl-3-(3-dimelhyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES BufferpH;6.0) in the 1:1 by weight ratio.The reaction was continued at 6.0 pH for about IHrs and then the reaction mixture was diafiltered on 30 kDa TFF in lOmM Phosphate buffer pH 7.2 to remove residuals and unreacted components. The final sample was analysed for protein content and degree of derivatization

Brief description of assay or reference: The protein content was measured by Lowry assay.

Table 54: Degree of Derivatization by TNBS assay
Test Sample	Total Protein mg/ml	DOD
ADH Derivatized TT	16.29	12.9

2) Conjugation of SJParatyphi A polysaccharide(OSP) and ADH Derivatized TT:

The OSP received from DSP Team was concentrated on 10 kDa membrane to achieve a concentration of (I0-13mg/ml).

Table 55:The Polysaccharide content analysed by Anthrone assay
Test Sample	Total PS mg/ml
Concentrated OSP	13.20

Two Expérimenta were performed by tetrafluoroborate (CPPT) (CPÏP) ratio change in 1-cyano- 4-pyrrolidinopyridinium

4P ExptNo:l

To the Concentrated PS 0.9% NaCl was added and freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.25 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein was added in 1:0.8 by weight ratio PS:PR:CPIP asl:0.8:1.25

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PB S as mobile phase at 1 ml/mîn flow rate tomonitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 times to that of PS by weight.

Refer Figure 8: Comparison of Polysaccharide, Protein and Conjugate (PS:PR:CPIP 10 1:0.8:1.25)

3) Conjugate Purification by Ultrafiltration: Quenched conjugate was purified by diafîltration 300 kDa TFF membrane in lOmM PBS pH 7.2 followed by lOmM Tris Buffer pH7.2 The final concentrated sample was sent for analysis.

Table 56: Evaluation of Purified OSP-DT ADH Conjugate (PS:PR:CPIP as 1:0.8:1.25)
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio(PsZpr)
OSP-TT ADH Conjugate	0.098	0.120	0.816

Refer Figure 9: Chromatogram of GFC purified OSP-DT ADH Conjugate (PS:PR:CPIP as 1:0.8:1.25)

Expt No: 2

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.3 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein was added in 1:0.9 by weight ratio PS:PR:CPIP1:0.9:1.3

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 times to that of PS by weight.

Refer Figure 10: Comparison of Polysaccharide, Protein and Conjugate 30 (PS:PR:CPIP1:O.9:1.3)

3) Conjugale Purification by Ultrafiltration: Quenched conjugale was purified by diafillration 300 kDa TFF membrane in lOmM PBS pH7.2 followed by lOmM Tris Buffer pH7.2 The final concentrated sample was sent for analysis.

Table 57: Evaluation of Purified OSP-DT ADH Conjugate(PS:PR:CPIPl :0.9:1.3)
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio(Ps/pr)
OSP-TT ADH Conjugale	0.025	0.035	0.715

Refer Figure 11: Chromatogram of GFC purified OSP-DT ADH Conjugale (PS:PR:CPIP1:O.9:1.3)

Conclusion: Conjugation of Paratyphi A PS using ADH derivatized TT was found 10 successful, two different types of diafillration strategy were applied and both the process gave satisfactory PS/Pr ratio.

100

2) Carbodiimide chemistry based conjugation of the Paratyphi OSP to Carrier protein

Diphtheria Toxoid (DT), CRM197 and Tetanus Toxoid (TT)

A) Derivatization of Concentrated OSP (Addition of Linker ADH) using cyanylation chemistry and conjugation with Tetanus Toxoid (TT) using carbodiimide chemistry.

B) Derivatization of Concentrated OSP (Addition of Linker ADH) using cyanylation chemistry and conjugation with Diphtheria Toxoid (DT) using carbodiimide chemistry.

C) Derivatization of Concentrated OSP (Addition of Linker ADH) using cyanylation chemistry and conjugation with CRM197 using carbodiimide chemistry.

2A) Préparation of S.Paratyphi Conjugates using Tetanus Toxoid as carrier protein.

1) Polysaccharide derivatization: The OSP received from DSP Team was concentrated on kDa membrane to achieve a concentration of (10-13mg/ml).

Table 58:The Polysaccharide content analysed by Anthrone assay
Test Sample	Total PS mg/ml
Concentrated OSP	13.20

To the Concentrated PS 0.9% NaCl was added and freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:0.7 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then Adipic acid dihydrazide (ADH) (dissolved 75-100mg/ml in 0.5M Sodium bicarbonate buffer pH: 8.0) in the 1:10 by weight ratio PS: ADH: CPIP as 1:10:0.7 The reaction was continued at 9.5 pH for about 2Hrs, quenched using 2M Glycine 10 and then the réaction mixture was diafiltered on 10 kDa TFF in lOOmM MES buffer pH 6.0 to remove residuals and unreacted components. The final sample was analysed for polysaccharide content

Table 59:The Polysaccharide content analysed by Anthrone assay
Test Sample	PS Content (mg/ml)
PS Content	20.7

2) Protein Préparation: High Monomeric Tetanus toxoid (TT) received from Production department (SIIPL) was concentrated to (10-15mg/ml) on a 30 kDa membrane and analysed for protein content

101

B nef description of assay or reference: The protein content was measured by Lowry assay.

Table 60: The protein content measured by Lowry assay
Test Sample	Total Protein mg/ml
Concentrated TT	15.3

3) Conjugation of ADH Derivatized S.Paratyphi A polysaccharide (OSP) and Concentrated TT: Two experiments were performed at different températures to check the Pr conversion.

Expt No :1

To the ADH derivatized PS, protein was added in 1:0.9 by weight and freshly prepared EDAC (I-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer) in the 1:0.86 by weight ratio. The reaction was continued at 6.0 pH température 6°C ratio PS:PR:EDC as 1:0.9:0.86

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate tomonîtored for protein conversion. The reaction was quenched after 23 hours by adding lOOmM Phosphate buffer containing EDTA.

Refer Figure 12: Chromatogram depicting progression of conjugation reaction (Quenching of reaction at 23 hrs)

4) Conjugate Purification by Ultrafiltration: Quenched conjugate was purified by diafiltration 300 kDa TFF membrane in lOmM PBS pH7.2 foliowed by lOmM Tris Buffer pH7.2 The final concentrated sample was sent for analysis.

Table 61: Evaluation of Purified OSP ADH-TT Conjugate(PS:PR:EDC 1:0.9:0.86)
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio (Ps/pr)
ADH OSP-TT Conjugate	0.013	0.200	0.06

Expt No : 2

To the ADH derivatized PS, protein was added in 1:0.9 by weight and freshly prepared EDAC (l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer) in the 1:0.86 by weight ratio. The reaction was continued at 6.0 pH, température 10°C ratio PS:PR:EDC as 1:0.9:0.86

102

P Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 4 hours by adding lOOmM Phosphate buffer containing EDTA.

Refer Figure 13: Chromatogram depicting progression of conjugation reaction

4) Conjugate Purification by GFC: A Column XK 16/70 was made ready using GFC resin (Tyopérai HW65F) with a bed height of 40cm. The column was packed at a flow rate of lOOcm/hr and allowed to settle and IM NaCl 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30cm/hr and the conjugate was loaded on the column and fractions collected at 1min interval, Fractions 2-10 were pooled according to the profile on HPLC. The final pooled fractions were filtered and sent for analysis

Table 62: Evaluation of Purified ADH OSP-TT Conjugate
Test Sampie	TPS (mg/ml)	TPr (mg/ml)	Ratio (Ps/pr)
ADH OSP-TT Conjugate	0.017	0.270	0.06

Conclusion: Using the reverse conjugation chemistry (Activating PS), Conjugation of ADH 15 derivatized Paratyphi A PS and concentrated TT was found successful, by increase in température from 6 to 10°C. Conjugation rate of reaction was increased, PS/Pr ratio was very low in both the reactions.

2B) Préparation of S.Paratyphi Conjugales using Dîptheria Toxoid as carrier protein.

1) Polysaccharide derivatization; The OSP received from DSP Team was concentrated on kDa membrane to achieve a concentration of (10-13 mg/ml).

Table 63:The Polysaccharide content analysed by Anthrone assay
Test Sampie	Total PS mg/ml
Concentrated OSP	10.09

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution 25 (0.114mg/ml in acetonitrile) was added into polysaccharide in the 1:0.7 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then Adipic acid dihydrazide (ADH) (dissolved 75-100mg/ml in 0.5M Sodium bicarbonate buffer pH:8.0)

103 in the 1:10 by weight ratio PS:ADH:CPIP as 1:10:0.7 The reaction was continued at 9.5 pH for about 2Hrs,quenched using 2M Glycine 10 and then the reaction mixture was diafiltered on 10 kDa TFF in lOOmM MES buffer pH 6.0 to remove residuais and unreacted components. The final sample was analysed for polysaccharide content

Table 64: The Polysaccharide content analysed by Anthrone assay
Test Sample	PS Content (mg/ml)
PS Content	18.20

2) Protein Préparation: High Monomeric Diptheria toxoid (DT) received from Production department (SIIPL) was concentrated to (10-20mg/ml) on a 10 kDa membrane and analysed for protein content

Table 65: The protein content measured by Lowry assay
Test Sample	Total Protein mg/ml
Concentrated DT	16.09

3) Conjugation of ADH Derivatized S.Paratyphi A polysaccharide(OSP) and Concentrated DT: Same conditions of TT conjugate were applied to DT to check the Pr Conversion.

Expt No :1

To the ADH derivatized PS, protein was added in 1:0.8 by weight and freshly prepared EDAC (l-ethyl-3-(3-diinethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer) in the 1:0.86 by weight ratio. The reaction was continued at 6.0 pH température 6°C ratio PS:PR:EDC as 1:0.8:0.86

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 20 hours by adding lOOmM Phosphate buffer containing EDTA.

Refer Figure 14: Chromatogram depicting progression of conjugation reaction (Quenching of reaction at 20 hrs)

4) Conjugate Purification by GFC: A Column XK16/70 was made ready using GFC resin (Tyoperal HW65F) with a bed height of 40cm. The column was packed at a flow rate of

104 lOOcm/hr and allowed to seule and IM NaCl 1% of the column volume was passed to assess the mtegnty of the packed column. The column was equihbrated using 0.9% NaCl at 30cm/hr and the conjugate was loaded on the column and fractions collected at 1min interval, Fractions 2-12 were pooled according to the profile on HPLC. The final pooled fractions were filtered and sent for analysis

Table 66: Evaluation of Purified OSP ADH-DT Conjugate(PS:PR:EDC 1:0.9:0.86)
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio (Ps/pr)
ADH OSP-DT Conjugate	0.020	0.300	0.060

Refer Figure 15: Chromatogram depicting purified conjugate (pooled fractions) Expt No :2

To the ADH derivatized PS, protein was added in 1:1.0 by weight and freshly prepared EDAC (i-ethyl-3-(3-dimethyI-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer) in the 1:0.9 by weight ratio. The reaction was continued at 6.0 pH, température 10“C ratio PS:PR:EDC as 1:1.0:0.9

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PB S as mobile phase at I ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 4 hours by adding lOOmM Phosphate buffer containing EDTA.

Refer Figure 16: Chromatogram depicting progression of conjugation reaction (Quenching of reaction at 4 hrs)

4) Conjugate Purification by Ultrafiltration: Quenched conjugate was purified by diafiltration 300 kDa TFF membrane in lOmM PB S pH 7.2 folio wed by lOmM Tris Buffer pH 7.2 The final concentrated sample was sent for analysis.

Refer Figure 17: Chromatogram depicting purified conjugate

Table 67: Evaluation of Purified ADH OSP-DT Conjugate
Test Sample	TPS (mg/ml)	TPr (mg/ml)	Ratio (Ps/pr)
ADH OSP-DT Conjugate	0.019	0.230	0.08

105

Conclusion: The Conjugation of ADH derivatized Paratyphi A PS using Carbodiimide chemistry was found successful using concentrated DT. Rate of conjugation reaction was increased by increasing the température but PS/Pr ratio was low in both experiments

106

D3) The method of conjugating the polysaccharide derived from Salmonella serovar strains S. typhimurium and S. enteritidis to carrier protein selected from tetanus toxoid (TT), diphtheria toxoid (DT) or CRM 197.

1) Cyanylation chemistry based conjugation of the S. typhimurium and S. enteritidis polysaccharide to Carrier protein Diphtheria Toxoid (DT), CRM197 and Tetanus Toxoid (TT)

A) Derivatization of Diphtheria Toxoid (DT), (Addition of Linker ADH) using carbodiimide chemistry and conjugation with Concentrated S. typhimurium and S. enteritidis polysaccharide using cyanylation chemistry

B) Derivatization of CRM 197, (Addition of Linker ADH) using carbodiimide chemistry and conjugation with Concentrated S. typhimurium and S. enteritidis using cyanylation chemistry

C) Derivatization of Tetanus Toxoid (TT) (Addition of Linker ADH) using carbodiimide chemistry and conjugation with Concentrated S. typhimurium and S. enteritidis using cyanylation chemistry

D) Cyanylation chemistry (CPPT) based conjugation of the Paratyphi OSP to Carrier protein Diphtheria Toxoid (DT), CRM197 and Tetanus Toxoid (TT) without derivatization of Polysaccharide or Carrier protein.

Procedure followed:

A) Préparation of S. typhimurium and S. enteritidis polysaccharide conjugales using Diphtheria Toxoid (DT) as carrier protein.

1) Protein derivatization:

High Monomeric Diphtheria toxoid (DT) was concentrated to (10-20mg/ml) on a 10 kDa membrane and analysed for protein content

To the concentrated DT freshly prepared IM MES Buffer was added and Adipic acid dihydrazide (ADH) (dissolved 75-100mg/ml in lOOmM MES buffer pH: 5.8) in the 1:10 by weight ratio and ED AC (l-ethyl-3-(3-dimethyTaminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer pH: 5.8) in the 1:1 by weight ratio. The reaction was continued at 5.8 pH for about IHrs and then the reaction mixture was diafiltered on 10 kDa TFF in 50mM Borate buffer pH 9.0 to remove residuals and unreacted components. The final sample was analysed for protein content and degree of derivatization

107

2) Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) and ADH Derivatized DT :

Two Experiments were performed by change in 1-cyano- 4-pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP) ratio

Expt No:l - The S. typhimurium and S. enteritidis polysaccharide was concentrated on 10 kDa membrane to achieve a concentration of (10-15mg/ml).

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution! 114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.3 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then 10 protein was added in 1:0.8 by weight ratio PS:PR:CPIP as 1:0.8:1.3

3) Conjugate Purification by GFC: A Column XK16/70 was made ready using GFC resin (Tyopearl HW65F) with a bed height of 40cm. The column was packed at a flow rate of lOOcm/hr and allowed to settle and IM NaCl 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30cm/hr and the conjugate was loaded on the column and fractions collected at 1min interval. The final pooled fractions were filtered and sent for analysis

Expt No: 2 To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.1 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein was added in 1:0.8 by weight ratio PS:PR:CPIP asl:0.8:l.l.

Conjugate Purification by GFC: A Column XK 16/70 was made ready using GFC resin (Tyoperal HW65F) with a bed height of 40cm. The column was packed at a flow rate of lOOcm/hr and allowed to settle and IM NaCL 1% of the column volume was passed to assess

108 the integrity of the packed column. The column was equilibrated using 0.9% NaCL at

30cm/hr and the conjugate was loaded on the column and fractions were collected at 1min interval. The final pooled fractions were filtered and sent for analysis

Conclusion: Conjugation of S. typhimurium and S. enteritidis polysaccharide using ADH derivatized DT was found successful and the PS/PR ratio was found satisfactory.

B. Préparation of S. typhimurium and S. enteritidis polysaccharide Conjugales using CRM197 as carrier protein

1) Protein derivatization; Cross reactîng mutant (CRM 197) received from Production department (SIIPL) was taken for derivatization

To the concentrated CRM 197 freshly prepared IM MES Buffer was added and Adipic acid dihydrazide (ADH) (dissolved 75-100mg/ml in lOOmM MES buffer pH:6.5) in the 1:3.5 by weight ratio and EDAC (l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in 100mM MES Buffer pH:6.5) in the 1:0.25 by weight ratio. 5% Tween 80 was added. The final reaction volume was made up using 100mM MES Buffer to achieve a final concentration of 3-4mg/ml the reaction was continued at 6.5 pH for about 3Hrs and then the reaction mixture was diafiltered on 10 kDa TFF in 50mM Borate buffer and 0.005% Tween 80 pH 9.0 to remove residuals and unreacted components. The final sample was analysed for protein content and degree of derivatization

2) Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) and ADH Derivatized CRM197:

The PS received from DSP was concentrated on 10 kDa membrane to achieve a concentration of (10-I5mg/ml).

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (114mg/ml in acetonitriie) was added into polysaccharide in the 1:1.3 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein was added in 1:1 by weight ratio PS:PR:CPIP as 1:1:1.3

Shodex column s SB-804 HQ and SB-805 HQ were used sequentially with PB S as mobile phase at 1 ml/min flow rate to monitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 times to that of PS by weight.

109

3) Conjugate Purification by GFC: A Column XK 16/70 was made ready using GFC resin (Tyoperal HW65F) with a bed height of 40cm. The column was packed at a flow rate of lOOcm/hr and allowed to settle and IM NaCL 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCL at 5 30cm/hr and the conjugate was loaded on the column and fractions collected at Imin interval, The final pooled fractions were filtered and sent for analysis.

Conclusion: Conjugation of S. typhimurium and S. enteritidis PS using ADH derivatized CRM 197 was found successful and the PS/PR ratio was found satisfactory.

C) Préparation of S.Paratyphi Conjugates using Tetanus Toxoid as carrier protein:

1) Protein derivatization: High Monomeric Tetanus toxoid (TT) received from Production department (SIIPL) was concentrated to (15-20mg/ml) on a 30 kDa membrane and analysed for protein content. The concentrated TT freshly prepared IM MES Buffer was added and 15 Adîpic acid dihydrazide (ADH) (dissolved 75-100mg/ml in lOOmM MES buffer pH:6.0)in the 1:10 by weight ratio and EDAC (l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in 100mM MES BufferpH:6.0) in the 1:1 by weight ratio.The reaction was continued at 6.0 pH for about IHrs and then the reaction mixture was diafiltered on 30 kDa TFF in lOmM Phosphate buffer pH 7.2 to remove residuals and unreacted components. The final sample was analysed for protein content and degree of derivatization

2) Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) and ADH Derivatized TT :

The OSP received from DSP Team was concentrated on 10 kDa membrane to achieve a 25 concentration of (10~13mg/ml).

Two Experiments were performed by change in 1-cyano- 4-pyrrolidinopyridinmm tetrafiuoroborate (CPPT) (CPIP) ratio Expt No:l

To the Concentrated PS 0.9% NaCl was added and freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.25 by weight ratio. The pH was shifted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein was added in 1:0.8 by weight ratio PS:PR:CPIP as 1:0.8:1.25

110

B Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 limes to that of PS by weight.

3) Conjugate Purification by Ultrafiltration: Quenched conjugate was purified by diafiltratîon 300 kDa TFF membrane in lOmM PBS pH7.2 followed by lûmM Tris Buffer pH7.2 The final concentrated sample was sent for analysis.

Expt No: 2

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:1.3 by weight ratio. The pH was shîfted to 9.5 immediately with 2.5M NaOH and held for up to 3 min. Then protein was added in 1:0,9 by weight ratio PS:PR:CPIP 1:0.9:1.3

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile 15 phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 3-4hours by adding 2M Glycine 10 limes to that of PS by weight.

3) Conjugate Purification by Ultrafiltration: Quenched conjugate was purified by diafiltratîon 300 kDa TFF membrane in lOmM PBS pH7.2 followed by 10mM Tris Buffer pH7.2 The final concentrated sample was sent for analysis.

Conclusion: Conjugation of S. typhimurium and S. enteritidis polysaccharide (PS) using ADH derivatized TT was found successful, two different types of diafiltratîon strategy were applied and both the process gave satisfactory PS/Pr ratio.

111

Μ 2) Carbodiimide chemistry based conjugation of the 5. typhimurium and S. enteritidis polysaccharide to Carrier protein Diphtheria Toxoid (DT), CRM 197 and Tetanus Toxoid (TT)

A) Derivatization of Concentrated S. typhimurium and S. enteritidis polysaccharide (Addition 5 of Linker ADH) using cyanylation chemistry and conjugation with Tetanus Toxoid (TT) using carbodiimide chemistry.

B) Derivatization of Concentrated S. typhimurium and S. enteritidis polysaccharide (Addition of Linker ADH) using cyanylation chemistry and conjugation with Diphtheria Toxoid (DT) using carbodiimide chemistry.

C) Derivatization of Concentrated S. typhimurium and S. enteritidis polysaccharide (Addition of Linker ADH) using cyanylation chemistry and conjugation with CRM 197 using carbodiimide chemistry.

D) Carbodiimide chemistry (EDAC) based conjugation of the Paratyphi OSP to Carrier protein Diphtheria Toxoid (DT), CRM 197 and Tetanus Toxoid (TT) without derivatization of 15 Polysaccharide or Carrier protein.

Procedure foliowed:

1) Polysaccharide (PS) derivatization: The S. typhimurium and S. enteritidis polysaccharide received was concentrated on 10 kDa membrane to achieve a concentration of (10-13mg/ml).

To the Concentrated PS 0.9% NaCl was added and Freshly prepared CPIP solution (114mg/ml in acetonitrile) was added into polysaccharide in the 1:0.5 to 1: 2 (1:0.7) by weight ratio. The pH was shifted to 9.5 îmmediately with 2.5M NaOH and held for up to 3 min. Then Adipic acid dihydrazide (ADH) (dissolved 75-100mg/ml in 0.5M Sodium bicarbonate buffer pH:8.0) in the 1:10 by weight ratio PS:ADH:CPIP as 1:2:0.7 to 1:10:0.7

The reaction was continued at 9.5 pH for about 2Hrs,quenched using 2M Glycine 10 and then the reaction mixture was diafiltered on 10 kDa TFF in lOOmM MES buffer pH 6.0 to remove residuals and unreacted components. The final sample was analysed for polysaccharide content

112

2) Protein Préparation:

2A) Protein Préparation: High Monomeric Tetanus toxoid (TT) received front Production department (SIIPL) was concentrated to (10-15 mg/ml) on a 30 kDa membrane and analysed for protein content

2B) Protein Préparation: High Monomeric Diptheria toxoid (DT) received from Production department (SIIPL) was concentrated to (10-20mg/ml) on a 10 kDa membrane and analysed for protein content

2C) Protein Préparation: High Monomeric CRM197 received from Production department (SIIPL) was concentrated to (10-20mg/ml) on a 10 kDa membrane and analysed for protein 10 content

3) Conjugation

3A ) Conjugation of ADH Derivatized S. typhimurium and S. enteritidis polysaccharide and Concentrated TT:

To the ADH derivatized PS, protein was added in 1:0.9 by weight and freshly prepared 15 EDAC (l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer) in the 1:0.86 by weight ratio. The reaction was continued at 6.0 pH température 6“C ratio PS:PR:EDC as 1:0.9:0.86

Shodex columns SB-804 HQ and S B-805 HQ were used sequentially with PB S as mobile phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched 20 after 23 hours by adding 100mM Phospahte buffer containing EDTA.

3B) Conjugation of ADH Derivatized S. typhimurium and S. enteritidis polysaccharide and Concentrated DT: Same conditions of TT conjugate were applied to DT to check the Pr Conversion.

To the ADH derivatized PS, protein was added in 1:0,8 by weight and freshly prepared EDAC (l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer) in the 1:0.86 by weight ratio. The reaction was continued at 6.0 pH température 6°C ratio PS:PR:EDC as 1:0.8:0.86

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile 30 phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched after 20 hours by adding lOOmM Phosphate buffer containing EDTA.

113

P 3C) Conjugation of ADH Derivatized S. typhimurium and S. enteritidis polysaccharide and Concentrated CRM 197: Same conditions of TT and DT conjugate were applied to CRM197 to check the Pr Conversion.

To the ADH derivatized PS, protein was added in 1:0.8 by weight and freshly prepared 5 EDAC ( l-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide) (dissolved 30-40 mg/ml in lOOmM MES Buffer) in the 1:0.86 by weight ratio. The reaction was continued at 6.0 pH température 6°C ratio PS:PR:EDC as 1:0.8:0.86

Shodex columns SB-804 HQ and SB-805 HQ were used sequentially with PBS as mobile phase at 1 ml/min flow rate tomonitored for protein conversion. The reaction was quenched 10 after 20 hours by adding 100mM Phosphate buffer containing EDTA.

4) Conjugate Purification

4A ) Conjugate Purification by Ultrafiltration: Quenched conjugale was purified by diafiltration 300 kDa TFF membrane in lOmM PBS pH7.2 followed by 10mM Tris Buffer 15 pH7.2 The final concentrated sample was sent for analysis.

4B) Conjugate Purification by GFC: A Column XK16/70 was made ready using GFC resin (Tyoperal HW65F) with a bed height of 40cm. The column was packed at a flow rate of lOOcm/hr and allowed to settle and IM NaCl 1% of the column volume was passed to assess the integrity of the packed column. The column was equilibrated using 0.9% NaCl at 30cm/hr 20 and the conjugate was loaded on the column and fractions collected at 1min interval, Fractions 2-12 were pooled according to the profile on HPLC. The final pooled fractions were filtered and sent for analysis

Conclusion: Using the reverse conjugation chemistry (Activating PS), Conjugation of ADH 25 derivatized S. typhimurium and S. enteritidis polysaccharide and concentrated TT, DT and

CRM 197 was found successful, by increase in température from 6 to 10°C. Conjugation rate of reaction was increased; PS/Pr ratio was very low in ali the reactions.

The Conjugation of ADH derivatized S. typhimurium and S. enteritidis polysaccharide and concentrated TT, DT and CRM 197 using Carbodiimide chemistry was found successful. Rate 30 of conjugation reaction was increased by increasing the température but PS/Pr ratio was low in both experiments.

114

Example 4: Immunogenic Compositions

Table 68A: Monovalent Immunogenic Compositions comprising Salmonella enterica serovar typhi ViPs conjugate antigen
S. No.	Formulation Components	Immunogenic composition in accordance with the present disclosure [per 0.5ml DoseJ
1	Salmonella enterica serovar typhi ViPs— TT conjugate antigen'.	25 pg (1.25-50 pg)
2	Salmonella enterica serovar typhi ViPs— DT conjugate antigen;	25 pg (1.25-50 pg)
3	Salmonella enterica serovar typhi ViPs— CRM 197 conjugate antigen;	25 pg (1.25-50 pg)
4	Salmonella enterica serovar paratyphi A OSP - TT conjugate antigen;	25 pg (1.25-50 pg)
5	Salmonella enterica serovar paratyphi A OSP -DT conjugate antigen;	25 pg (1.25-50 pg)
6	Salmonella enterica serovar paratyphi A OSP - CRM 197 conjugate antigen ;	25 pg (1.25- 50 pg)
7	Salmonella enterica serovar typhimurium saccharide - 7T conjugate antigen;	25 pg (1.25-50 pg)
8	Salmonella enterica serovar typhimurium saccharide — DT conjugate antigen;	25 pg (1.25-50 pg)
9	Salmonella enterica serovar typhimurium saccharide - CRM 197 conjugate antigen;	25 pg (1.25-50 pg)
10	Salmonella enterica serovar enteritidis saccharide — TT conjugale antigen;	25 pg (1.25-50 pg)
11	Salmonella enterica serovar enteritidis saccharide — DTconjugate antigen;	25 pg (1.25-50 pg)
12	Salmonella enterica serovar enteritidis saccharide — CRM 197 conjugate antigen;	25 pg (1.25-50 pg)

Single Dose doesn’l comprise of préservât! ve 2-Phenoxyethanol

Muki-dose composition may additionally comprise of 2-Phenoxyéthanol - 5 mg (I - 10 mg)

115

Table 68B: Bivalent Immunogenic Compositions
S. No.	Formulation Components	Immunogenic composition in accordance with the présent disclos are [per 0.5ml Dose]
1	2	3	4	5	6
1	Salmonella enterica serovar typhi ViPs— CP conjugale antigen; the CP is either TT or DT or CRMI97	25 pg (1.25 50 pg)	25 Pg (1.25 -50 pg)	25 pg (1.25-50 pg)
2	Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen-, the CP is either TT or DT or CRM 197	25 pg (1.2550 pg)			25 pg (1.25 -50 pg)	25 pg (1.25 -50 Pg)
3	Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen; the CP is either TT or DT or CRM 197		25 pg (1.25 -50 pg)		25 Pg (1.25 -50 pg)		25 pg (1.25-50 Pg)
4	Salmonella enterica serovar enteritidis saccharide - CP conjugale antigen; the CP is either TT or DT or CRM197			25 pg (1.25-50 Pg)		25 pg (1.25 -50 Pg)	25 pg (1.25-50 Pg)
Table 68C: Trivalent Immunogenic Compositions
S. No.	Formulation Components	Immunogenic composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3	4
1	Salmonella enterica serovar typhi ViPs— CP conjugale antigen; the CP is either TT or DT or CRM 197	25 pg (1.25 -50 Kg)		25 pg (1.25 -50 pg)	25 pg (1.25- 50 pg)
2	Salmonella enterica serovar paratyphi A OSP CP conjugale antigen; the CP is either TT or DT or CRM 197	25 Pg (1.25 -50 pg)	25 pg (1.25 - 50 pg)	25 pg (1.25 -50 Pg)
3	Salmonella enterica serovar typhimurium	25 pg (1.25	25 pg (1.25-50 pg)		25 pg (1.25-50 pg)

116

	saccharide - CP conjugate antigen-, the CP is either TT or DT or CRM 197	-50 pg)
4	Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen', the CP is either TT or DT or CRM 197		25 pg (1.25-50 pg)	25 Pg (1.25 -50 Pg)	25 pg (1.25-50 pg)

Table 68D: Tetra valent Immunogenic Compositions

S. No.	Formulation Components	Immunogenic composition in accordance with the présent disclosure [per 0.5ml Dose]
1	Salmonella enterica serovar typhi ViPs— CP conjugate antigen; the CP is either TT or DT or CRM 197	25 pg (1.25-50 pg)
2	Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen; the CP is either TT or DT or CRM 197	25 pg (1.25-50 pg)
3	Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen; the CP is either TT or DT or CRM 197	25 pg (1.25-50 pg)
4	Salmonella enterica serovar enteritidis saccharide CP conjugate antigen; the CP is either TT or DT or CRM197	25 pg (1.25-50 pg)

117

Table 68E: Combination of Excipient Tested in accordance with the Antïgenic Component disclosed above

S. No.

Excipient

1

2

3

4

5

6

7

8

9

10

11

12

13

2

Sodium chloride (140-160 mM)

4.5 mg (1 - 10 mg)

4,5 mg (1 - 10 mg)

4.5 mg (1 - 10 mg)

4,5 mg (1 - 10 mg)

4.5 mg (1 - 10 mg)

3

Tris Buffer (10mM to 25mM)

-

0.61 to 1.52 mg

4

Citrate buffer (lOmM to 25mM) Prepared by dissolving citric acid monohydrate (CAM) and trisodïum citrate dehydrate (TCD)

-

CAM- 1.05 to 2.63 mg TCD1.473.68 mg

CAM- 1.05 to 2.63 mg TCD- 1.47-3.68 mg

CAM- 1.05 to 2.63 mg 1X2D- 1.47- 3.68 mg

5

Histidine buffer (lOmM to 25mM)

-

0.78 to 1.94 mg

6

Suce in aie Buffer (10mM to 25niM)

-

0.59 to 1.48 mg

7

Polysorbate-20 (0.005 to 0.1 %w/v)

|

25-500 Bg

25-500 BS

25-500 Bg

118

8

Sucrose (0.5% to 5.0% w/v)

2.5 to 25 mg

9

Water for Injection (WFI)

q.S.

q.s.

V = included in the composition

Table 69: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Salk Strain type 1 (Mahoney) or type 2 (MEF) or type 3 (Saukett)), D, T, HepB, wP, ViPs- TT and Hib antigen IPV

S. No.	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3	4	5	6	7
1	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15IOU	I5IOU	15IOU	15IOU	15IOU	15IOU	15IOU	15IOU	15IOU
4	HBs antigen	12.5 gg	12.5 gg	12.5 gg	12.5 gg	12.5 gg	12.5 gg	12.5 gg	12.5 gg	12.5 gg
5	Hib PRP-TT conjugale antigen	10 gg of PRP	10 gg of PRP	10 gg of PRP	10 gg of PRP	10 gg of PRP	10 gg of PRP	10 gg of PRP	10 gg of PRP	10 gg of PRP
6	Inactivated Polio Virus (IPV)
Type 1(D antigen units)	7.5	8	5	10	10	10	10	4	40
Type 2 (D antigen units)	16	2	2	2	2	2	2	0.5	8
Type 3(D antigen units)	10	5	5	10	5	12	16	3.2	32

119

7	Salmonella enterica serovar typhi ViPs— TT conjugate antigen;	25 pg (1.25-50 Fg)	25 pg (1.25-50 Pg)	25 pg (1.25-50 με)	25 μg (1.25-50 Pg)	25 pg (1.25-50 με)	25 pg (1.25-50 με)	25 μg (1.25-50 με)	25 pg (1.25-50 με)	25 pg (1.25-50 με)
8	Total Aluminium Content (AP⁺)	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg

Table 70: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Salk Strain type 1 (Mahoney) or type 2 (MEF) or type 3 (Saukett)), D, T, HepB, wP, ViPs—TT, OSP conjugate and Hib antigen IPV
S. No.	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3	4	5	6	7
1	Dîphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU
4	HBs antigen	12-5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg
5	Hib PRP-TT conjugate antigen	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP
6	Inactivated Polio Virus (IPV)

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	Type 1(D antigen units)	7.5	8	5	10	10	10	10	4	40
Type 2 (D antigen units)	16	2	2	2	2	2	2	0.5	8
Type 3(D antigen units)	10	5	5	10	5	12	16	3.2	32
7	Salmonella enterica serovar typhi ViPs—TT conjugate antigen;	25 pg (1.25-50 pg)	25 pg (1.25-50 Pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 Pg)	25 pg (1.25-50 pg)	25 pg (1,25-50 Pg)	25 pg (1.25-50 Pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)
8	OSP-CP Conjugate; the CP is either TT or DT or CRM 197	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 Pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)	25 pg ( 1.25 - 50 pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 Pg)
9	Total Aluminium Content (AP*)	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0,5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg

Additionally adjListing the pH of the composition as disclosed above to about 6.0 to 7.0 with Sodium Hydroxide / Sodium Carbonate and make up the volume by adding normal saline (0.9%).

May additionally comprise of one of the preservative combination

i. 2-Phenoxyethanol in an amount of l to 10 mg per 0.5 ml (v/v) ii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or iîî. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or iv. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 - 0,2 mg per 0.5 ml (w/v).

121

2061

The Combination Vaccine Compositions comprising Dose reduced IPV, D, T, HepB, ViPs—TT, OSP conjugale, acellular pertussis, and Hib antigen may comprise of acellular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular either genetically or chemically detoxified), in particular Pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly Pertussis toxoid: l to 50 micrograms (More particularly 8pg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particularly 8 μ g); - Pertactin: l to 20 micrograms (More particularly 2.5pg); - Optionaîly, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

Table 71: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Sabin Strain type 1 or type 2 or type 3), D, T, HepB, wP, ViPs— TT and Hib antigen IPV
S. No.	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3
1	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15 IOU	15 IOU	15 IOU
4	HBs antigen	12.5 pg	12.5 pg	12.5 pg
5	Hib PRP-TT conjugale antigen	10 μ g ofPRP	10 pg of PRP	10 pg ofPRP
6	Inactivated Polio Virus (IPV)
Type 1(D antigen units)	5	2.5	7.5
Type 2 (D antigen units)	16	8	16
Type 3(D antigen units)	10	5	10
7	Salmonella enterica serovar typhi ViPs— TT conjugale antigen;	25 pg (1.25 -50 pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)
8	Total Aluminium Content (Al³⁺)	Not more than 0.9 mg	Not more than 0.9 mg	Not more than 0.9 mg

122

2061

Table 72: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Sabin Strain type 1 or type 2 or type 3), D, T, HepB, wP, ViPs—TT, OSP conjugate and Hib antigen IPV
S. No.	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3
1	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15 IOU	15 IOU	15 IOU
4	HBs antigen	12.5 pg	12.5 pg	12.5 pg
5	Hib PRP-TT conjugate antigen	10 pg of PRP	10 pg ofPRP	10 pg ofPRP
6	Inactivated Polio Virus (IPV)
Type 1(D antigen units)	5	2.5	7.5
Type 2 (D antigen units)	16	8	16
Type 3(D antigen units)	10	5	10
7	Salmonella enterica serovar typhi ViPs— TT conjugate antigen;	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)
8	OSP-CP Conjugate; the CP is eiLher TT or DT or CRM 197	25 pg (1.25-50 pg)	25 μg (1.25-50 pg)	25 μg (1.25 -50 μ_δ)
⁹	Total Aluminium Content (AI³⁺)	Not more than 0.9 mg	Not more than 0.9 mg	Not more than 0.9 mg

AdditionalIy adjusting the pH of the composition as disclosed above to about 6.0 to 7.0 with Sodium Hydroxide ! Sodium Carbonate and make up the volume by adding normal saline (0.9%).

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2061

May additionally comprise of one of the preservative combination

i. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0,5 ml (v/v) ii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or iiL 2-Phenoxyethanol in an amount of I to 10 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or îv. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v).

The Combination Vaccine Compositions comprising Dose reduced IPV, D, T, HepB, ViPs—TT, OSP Conjugate, acellular pertussis, and Hib antigen may comprise of acellular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular either genetîcally or Chemically detoxified), in particular Pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly Pertussis toxoid: 1 to 50 micrograms (More particularly 8pg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particularly 8pg); - Pertactin: 1 to 20 micrograms (More particularly 2.5pg); - Optionaliy, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

124

2061

Table 73: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Salk Strain type 1 (Mahoney) or type 2 (MEF) or type 3 (Saukett)), Inactivated Rotavirus (IRV), D, T, HepB, wP, ViPs- TT and Hib antigen IPV
S. No.	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3	4	5	6	7
1	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU
4	HBs antigen	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg
5	Hib PRP-TT conjugale antigen	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP
6	Inactivated Polio Virus (IPV)
Type 1(D antigen units)	7.5	8	5	10	10	10	10	4	40
Type 2 (D antigen units)	16	2	2	2	2	2	2	0.5	8
Type 3(D antigen units)	10	5	5	10	5	12	16	3.2	32
7	IRV	10 pg 1 to 50 Pg	10 pg 1 to 50 Pg	10 pg 1 to 50 Pg	10 pg 1 to 50 pg	10 pg 1 to 50 Pg	10 pg 1 to 50 pg	10 pg 1 to 50 pg	10 pg 1 to 50 Pg	10 pg 1 to 50 Pg
8	Salmonella enterica serovar typhi ViPs— TT conjugale	25 pg (1.25-50	25 pg (1.25-50	25 pg (1.25-50	25 pg (1.25-50	25 pg (1.25-50	25 pg (1.25-50	25 pg (1.25-50	25 pg (1.25-50	25 pg (1.25-50

125

2061

	antigen;	pg)	μg)	μ?)	με)	με)	με)	με)	με)	με)
9	Total Aluminium Content (Al³⁺)	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg

Table 74: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Salk Strain type 1 (Mahoney) or type 2 (MEF) or type 3 (Saukett)), Inactivated Rotavirus (IRV), D, T, HepB, wP, ViPs- TT and Hib antigen IPV

S. No.	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3	4	5	6	7
1	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU	15 IOU
4	HBs antigen	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12.5 pg	12,5 pg
5	Hib PRP-TT conjugate antigen	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP	10 pg of PRP
6	Inactivated Polio Virus (IPV)
Type 1 (D antigen units)	7.5	8	5	10	10	10	10	4	40
Type 2 (D antigen units)	16	2	2	2	2	2	2	0.5	8
Type 3(D antigen units)	10	5	5	10	5	12	16	3.2	32

126

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7	1RV	10 pg 1 to 50 pg	10 pg 1 to 50 pg	10 pg 1 to 50 pg	10 pg 1 to 50 Pg	10 pg 1 to50 Pg	10 pg I to 50 pg	io pg 1 to50 pg	10 pg 1 to 50 pg	io pg 1 to 50 pg
8	Salmonella enterica serovar typhi ViPs— TT conjugate antigen;	25 gg (1.25-50 Pg)	25 gg (1.25-50 pg)	25 gg (1.25-50 pg)	25 gg (1.25-50 pg)	25 pg (1.25-50 pg)	25 gg (1.25-50 Pg)	25 gg (1.25-50 Pg)	²⁵ Pg (1.25-50 pg)	25 gg (1.25-50 gg)
9	OSP-CP Conjugate; the CP is either TT or DT or CRM 197	25 μg (1.25-50 Pg)	25 gg (1.25-50 pg)	25 gg (1.25-50 pg)	25 gg (1.25-50 pg)	25 gg (1.25-50 pg)	25 gg (1.25-50 pg)	25 gg (1.25-50 pg)	25 pg (1.25-50 pg)	25 gg (1.25-50 pg)
10	Total Aluminium Content (AP⁺)	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg	Not more than 0.5 mg

Additionally adjusting the pH of the composition as disclosed above to about 6.0 to 7.0 with Sodium Hydroxide / Sodium Carbonate and make up the volume by adding normal saline (0.9%).

May additionally comprise of one of the preservative combination

v. 2-PhenoxyethanoI in an amount of l to 10 mg per 0.5 ml (v/v) vi. 2-Phenoxyethanol in an amount of l to 10 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or vii. 2-Phenoxyethanol in an amount of l to 10 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or viii. 2-Phenoxyethanol in an amount of l to 10 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v).

127

The Combination Vaccine Compositions comprising Dose reduced IPV, IRV, D, T, HepB, ViPs—TT, acellular pertussis, and Hib antigen may comprise of acellular pertussis antigen selected from - Borde tell a toxin in detoxified form (in particular either genetically or chemically detoxified), in particular Pertussis toxoid; Filamentous Haemagglutinin; Pertactin; or Fimbriae. Particuiarly Pertussis toxoid: l to 50 micrograms (More particuiarly 8pg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particuiarly 8gg); - Pertactin: 1 to 20 micrograms (More particuiarly 2.5gg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0,5 ml.

Table 75: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Sabin Strain type 1 or type 2 or type 3), D, T, HepB, wP, ViPs— TT and Hib antigen IPV
S. No.	Formulation Components	Combination composition in accordance with the présent disclosure [per 0.5ml Dose]
1	2	3
!	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15 IOU	15 IOU	15 IOU
4	HBs antigen	12.5 gg	12.5 gg	12.5 gg
5	Hib PRP-TT conjugate antigen	WugofPRP	10 μ g of PRP	10 gg ofPRP
6	Inactivated Polio Virus (IPV)
Type 1(D antigen units)	5	2.5	7.5
Type 2 (D antigen units)	16	8	16
Type 3(D antigen units)	10	5	10
7	IRV	10 Pg 1 to 50 pg	io pg 1 to 50 gg	10 pg 1 to 50 gg
8	Salmonella enterica serovar typhi ViPsTT conjugate antigen;	25 gg (1.25-50 gg)	25 gg (1.25-50 gg)	25 gg (1.25-50 gg)

128

9

Total Aluminium Content (AP⁺)

Not more than 0.9 mg

Table 76: Combination Vaccine Compositions comprising Standard Dose and Dose reduced IPV (Sabin Strain type 1 or type 2 or type 3), D, T, HepB, wP, ViPs— TT and Hib antigen IPV
S. No.	Formulation Components	Combination composition in accordance with the present disclosure [per 0.5ml Dose]
1	2	3
1	Diphtheria Toxoid (D)	22.5 Lf	22.5 Lf	22.5 Lf
2	Tetanus toxoid (T)	7.5 Lf	7.5 Lf	7.5 Lf
3	Inactivated B. pertussis antigen (wP)	15 IO U	15IOU	I5IOU
4	HBs antigen	12.5 pg	12.5 pg	12.5 pg
5	Hib PRP-TT conjugate antigen	10 pg of PRP	10 pg ofPRP	10 pg of PRP
6	Inactivated Polio Virus (IPV)
Type 1 (D antigen units)	5	2.5	7.5
Type 2 (D antigen units)	16	8	16
Type 3(D antigen units)	10	5	10
7	IRV	10 pg 1 to 50 pg	10 Fg 1 to 50 pg	10 pg 1 to 50 pg
8	Salmonella enterica serovar typhi ViPsTT conjugate antigen;	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)	25 pg (1.25 -50 pg)
9	OSP-CP Conjugate; the CP is either TT or DT or CRM 197	25 pg (1.25-50 pg)	25 pg (1.25 - 50 pg)	25 pg (1.25-50 pg)

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10

Total Aluminium Content (AP⁺)

Not more than 0.9 mg

Addîtionally adjusting the pH of the composition as disclosed above to about 6.0 to 7.0 with Sodium Hydroxide / Sodium Carbonate and make up the volume by adding normal saline (0.9%).

May addîtionally comprise of one of the preservative combination

v. 2-Phenoxyethanol in an amount of l to 10 mg per 0.5 ml (v/v) vi. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml (v/v) and methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v); or vii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml(v/v)and propylparaben in an amount of 0.05 - 0.2 mg per 0.5 ml (w/v); or viii. 2-Phenoxyethanol in an amount of 1 to 10 mg per 0.5 ml(v/v), methylparaben in an amount of 0.1 - 1.5 mg per 0.5 ml (w/v) and propylparaben in an amount of 0.05 - 0.2 mg per 0,5 ml (w/v).

The Combination Vaccine Compositions comprising Dose reduced IPV, IRV D, T, HepB, ViPs—TT, aceliular pertussis, and Hib antigen may comprise of aceliular pertussis antigen selected from - Bordetella toxin in detoxified form (in particular either genetically or chemically detoxified), in particular Pertussis toxoid; FÎIamentous Haemagglutinin; Pertactin; or Fimbriae. Particularly Pertussis toxoid: 1 to 50 micrograms (More particularly 8pg); - Filamentous Haemagglutinin: 1 to 50 micrograms (More particularly 8 μ g); - Pertactin: 1 to 20 micrograms (More particularly 2.5μg); - Optionally, Fimbriae: 2 to 25 micrograms; per 0.5 ml.

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Table 77: Fully Liquid Immunogenic Composition
	Single Dose(0.5mL)	Single Dose(0.5mL)	Single Dose(0.5mL)	Single Dose(0.5mL)	Single Dose(O.SmL)
Men A-TT Conjugale	5 gg(l -30 gg)	5 gg (1 -30 gg)	5gg(l-30 gg)	5gg(l-30 gg)	5 gg (1 -30 gg)
Men C-CRM197 Conjugale	5 gg (1 -30 gg)	5 gg (1 -30 gg)	5 gg (1 -30 gg)	5 gg (1 -30 gg)	5 gg (1 -30 gg)
Men Y-CRM197 Conjugale	5 gg (1 - 30 gg)	5 gg (1 - 30 gg)	5gg(l-30gg)	5 gg (1 - 30 gg)	5gg(l-30 gg)
Men W-CRM197 Conjugale	5gg(l-30gg)	5 gg (1 - 30 gg)	5 gg (1-30 gg)	5 gg (1-30 gg)	5 gg (1 -30 gg)
Men X-TT Conjugale	5gg(l-30 gg)	5gg(l-3O gg)	5 gg(l -30 gg)	5gg(l-30 gg)	5 gg (1-30 gg)
VÎ-TT Conjugale	25 gg (1.25-50 gg)	25 gg (1.25-50 gg)	25 gg (1.25 - 50 gg)	25 gg (1.25-50 gg)	-
OSP-CP Conjugale; the CP is either TT or DT or CRM 197	-	25 gg (1.25-50 gg)	25 gg (1.25 - 50 gg)	25 gg (1.25-50 gg)	25 gg (1.25-50 pg)
Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen; the CP is either TT or DT or CRM 197	-	-	25 gg (1.25 -50gg)	25 gg (1.25-50 gg)	25 gg (1.25-50 μg)
Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen', the CP is either TT or DT or CRM 197	-	-	-	25 gg (1.25-50 gg)	25 gg (1.25-50 μg)
Sodium Chloride	4.5 mg (1 - 10 mg)	4.5 mg (1 - 10 mg)	4.5 mg (1 - 10 mg)	4.5 mg (1 - 10 mg)	4.5 mg (1-10 mg)
2-Phenoxyethanol	5.0 mg (l - 10 mg)	5.0 mg (l - 10 mg)	5.0 mg (1 - 10 mg)	5.0 mg (I - io mg)	5.0 mg (1 - 10 mg)

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Water for Injection

Qs. 0.5mL

Table 78: Lyophilized (Freeze-Dried) Immunogenic Composition
	Single Dose(0.5mL)	Single Dose(0.5mL)	Single Dose(O.SmL)	Single Dose(0.5mL)	Single Dose(0.5mL)
Men A-TT Conjugate	5 pg (1 - 30 pg)	5 pg (1 -30 pg)	5 pg (1 - 30 pg)	5 pg (1 - 30 pg)	5pg(l-3O pg)
Men C-CRM197 Conjugate	5 pg (1 ~ 30 pg)	5pg(l-3O pg)	5pg(l-30pg)	5pg(l-30 pg)	5 pg (1 -30pg)
Men Y-CRM 197 Conjugate	5pg(l-3O pg)	5pg(l-3O pg)	5pg(l-30 pg)	5pg(l -30 pg)	5pg(l-3O pg)
Men W-CRM197 Conjugate	5pg(l-30 pg)	5 pg (1 -30 pg)	5 pg (1 - 30 pg)	5pg(l-30 pg)	5 pg (1 -30pg)
Men X-TT Conjugate	5pg(l-30 pg)	5 pg (1 - 30 pg)	5 pg (1 — 30 pg)	5 pg (1 - 30 pg)	5 pg (1 - 30 pg)
Vi~TT Conjugate	25 pg (1.25 - 50 pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 pg)	25 pg(L25-50pg)	-
OSP-CP Conjugate; the CP is either TT or DT or CRM 197	-	25 pg (1.25-50 pg)	25 pg (1.25 - 50 pg)	25 pg (1.25 - 50 pg)	25 pg (1.25-50 Fg)
Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen; the CP is either TT or DT or CRM 197	-	-	25 pg (1.25 - 50 pg)	25 pg (1.25-50 pg)	25 pg (1.25-50 Hg)
Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen; the CP is either TT or DT or CRM 197	-	-	-	25 pg (1.25-50 pg)	25 pg (1.25-50 Kg)

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Sucrose	11.90 mg (1- 50mg)	11.90 mg(l-50mg)	11.90 mg(l-50mg)	11.90 mg (1- 50mg)	11.90 mg (150mg)
Sodium citrate (Dihydrate)	1.98 mg (1- 50mg)	1.98 mg (1- 50mg)	1.98 mg (1- 50mg)	1.98 mg (1- 50mg)	1.98 mg (1- 50mg)
Tris Buffer	0.48 mg (0.1-5 mg)	0.48 mg (0.1- 5 mg)	0.48 mg (0.1- 5 mg)	0.48 mg (0.1-5 mg)	0.48 mg (0.1- 5 mg)

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Example 5: Stability Data

Stability of Monovalent Salmonella typhi conjugate at 2-8 deg C for initial (0), 3, 6 months; at 25degC for initial (0), 1,3 months and at

40degC for initial (0), 14, 28days:

Table 79: Stability of Monovalent Salmonella typhi conjugate at 2-8degC for initial (0), 3, 6 months
Storage Température (Cekius): 2-8degC	0 month	3 months	6 months
Sr. No.	Sample	Target PS Size (kDa)	SIIPL PS Size (kDa)	Bat ch No.	pH	Appearance	Free PS (%)	SIIPL PS Conjugate Size (kDa)	pH	Appearance	Free PS (%)	SIIPL PS Conjugate Size (kDa)	pH	Appearance	Free PS (%)	SIIPL PS Conjugate Size (kDa)
D	SIIPL Conjugale (main)	180- 220	214 (Shoedex)	G7 SIIPL Vi PS-TT (Conj ugated) 2B#10	6,9	cl car yellowish liquid	4.4	1389	6.8	clear yellowish liquid	6.6	1398	7.2	clear yellowish liquid	5.8	1380
2)	SIIPL Conjugale	300	388 (Shoedex)	G6 SIIPL Vi PS-TT (Conj ugated) 1 B#9	7.3	clear yellowish liquid	7.5	1359	6.9	clear yellowish liquid	9.7	1350	7.3	clear yellowish liquid	8.8	1345
3)	Γ SIIPL Conjugate	120	80 (Shoedex)	G9 SIIPL Vi PS-TT (Conj ugated) 4B#12	6.9	clear yellowish liquid	8.0	1352	7.2	clear yellowish liquid	8.5	1300	6.9	clear yellowish liquid	6.7	1310
4)	SIIPL Conjugate	45	42 (TSK 3000 PWXL)	68 SIIPL Vi PS-TT (Conj ugated) 3B#12	6.8	clear yellowish liquid	5.4	1293	7.1	clear yellowish liquid	6.5	1200	6.6	clear yellowish liquid	7.7	1187

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Table 80: Stability of Monovalent Salmonella typhi conjugate at 25degC for initial (0), 1,3 months
Storage Température (Celeius): 25degC	0 month	1 months	3 months
Sr. No.	Sampie	Target PS Size (kDa)	SIIPL PS Size (kDa)	Batch No.	PH	Appearance	Free PS (%)	SIIPL PS Conjugate Size (kDa)	pH	Appea rance	Free PS (%)	SIIPL PS Conjugate Size (kDa)	pH	Appearance	Free PS (%)	SIIPL PS Conjugate Size (kDa)
1)	SIIPL Conjugate (main)	180- 220	214 (Shoedex)	G7 SIIPL Vi PS-TT (Conjugated) 2 B#10	6.9	clear yellowish liquid	4.4	1392	6.8	clear yellowish liquid	5.4	1380	6.7	clear yellowish liquid	6.1	1387
2)	SIIPL Conjugate	300	388 (Shoedex)	G6 SIIPL Vi PS-TT (Conjugated) 1 B#9	7.3	clear yellowish liquid	7.5	1360	7.2	clear yellowish liquid	8.2	1370	7.1	clear yellowish liquid	7.3	1350
3)	^H SIIPL Conjugate	120	80 (Shoedex)	G9 SIIPL Vi PS-TT (Conjugated) 4 B# 12	6.9	clear yellowish liquid	8.0	1350	7.1	clear yellowish liquid	8.1	1280	7.2	clear yellowish liquid	8.5	1268
4)	SIIPL Conjugale	45	42 (TSK 3000 PWXL)	68 SIIPL Vi PS-TT (Conjugated) 3 B#12	6.8	clear yellowish liquid	5.4	1285	6.9	clear yellowish liquid	5.9	1180	6.6	clear yellowish liquid	5.2	1162

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Table 81: Stability of Monovalent Salmonella typhi conjugale at 40degC for initial (0), 14,28 days
Storage Température (Celcius): 40°C	0 month	14 days (2 weeks)	28 days (4 weeks)
Sr. No.	Sample	Target PS Size (kDa)	SIIPL PS Size (kDa)	Batch No.	pH	Appearance	Free PS (%)	SIIPL PS Conjugale Size (kDa)	PH	Appearance	Free PS (%)	SIIPL PS Conjugale Size (kDa)	pH	Appearance	Free PS (%)	SIIPL PS Conjugale Size (kDa)
D	SIIPL Conjugale (main)	180- 220	214 (Shoedex)	G7 SIIPL Vi PS-TT (Conjugated) 2B#10	6.9	clear yellowish liquid	4.4	1388	6.7	clear yellowish liquid	5.8	1348	6.4	clear yellowish liquid	7.5	1355
2)	SIIPL Conjugale	300	388 (Shoedex)	G6 SIIPL Vi PS-TT (Conjugated) 1 B#9	7.3	clear yellowish liquid	7.5	1356	7.1	clear yellowish liquid	9.7	1310	6.8	clear yellowish liquid	10.5	1320
3)	SIIPL Conjugale	120	80 (Shoedex)	G9 SIIPL Vi PS-TT (Conjugated) 4B#12	6.9	clear yellowish liquid	8.0	1355	.6.8	clear yellowish liquid	12.3	1266	6.6	clear yellowish liquid	14.7	1270
4)	SIIPL Conjugale	45	42 (TSK 3000 PWXL)	68 SIIPL Vi PS-TT (Conjugated) 3 B# 12	6.8	clear yellowish liquid	5.4	1290	6.7	clear yellowish liquid	8.8	1150	6.5	clear yellowish liquid	11.5	1168

Observations:

Free Ps(“initial< 4.5%, after 6 months NMT 7.5%” for 180-220 kDa Ps Vs “initial 5.4 % after 6 months NLT 10.5% for 388/80/45 kDa” at 40 deg C/2 to 8 deg C/25 deg C).

For 180-220 kDa, initial low free Ps, size of conjugale was also maintained over stability study, less free Ps after 6 months were observed as compared to other sizes.

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SE-HPLC Stability data (VI-TT Conjugate in Tris, Tris-NaCl and 0.17M NaCl)

Table 82: SE-HPLC Stability data of Vi-TT Conjugate in Tris, Tris-NaCI and 0.17M NaCl
Sr. No.	Sample Name	HPLC-SEC Size (in kDa)
0 Day	Week 2
1	B#9 Vi-TT CJ (in lOmM Tris) 1:1 Dil in IX PBS -20°C	1049	1099
2	B#9 Vi-TT CJ (in lOmM Tris + 0.17 M NaCl) 1:1 Dil in IXPBS-20°C	1108	1035
3	B# 13 Vi-TT CJ (in 0.17 M NaCl) 1:1 Dil in IX PBS -20°C	996	1009
4	B#13 Vi-TT CJ (0.17 M NaCl + lOmMTris) 1:1 Dil in IX PBS -20°C	1004	1006
5	B#9 Vi-TT CJ (in lOmM Tris) Native 2-8°C 1:1 Dil in IX PBS	1049	996
6	B#13 Vi-TT CJ (in 0.17 M NaCl) Native 2-8°C 1:1 Dil in IX PBS	996	965

Conclusion:

The Vi-TT Conjugate found stable in lOmM tris buffer, lOmM tris containing 0.17M NaCl and in 0.17M NaCl alone.

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Data for different concentrations of TRIS ( 5mM, 10 mM, 20 mM, 25 mM, 30 mM,50 mM) showîng 25 mM TRIS as best case.

• Vi-TT CJ concentration was maintained in three different strengths of Tris, pH 7.2 such as 5mM, 10mM, and 20mM. This was kept in various température conditions such as -20°C, 2-8°C, Room Température and 37°C.

• Study was carried out for 2weeks and it was monitored daily for pH.

• Samples were checked for particle size and zêta potentiai.

pH Results

Table 83: pH of Vi-TT conjugate in 5mM, lOmM, 20mM Tris at -20°C, 2-8°C, Room Température and 37°C
Day	Vi TT CJ in 5mM Tris pH 7.0	Vi TT CJ in 10mM Tris pH 7.14	Vi TT CJ in 20mM Tris pH 7.21
-20°C	2-8°C	RT	37°C	-20°C	2-8°C	RT	37°C	-20C	2-8°C	RT	37°C
0 Day	7.0	7.0	7.0	7.0	7.14	7.14	7.14	7.14	7.21	7.21	7,21	7.21
After 2 weeks	6.58	6.95	6.76	6.94	6.56	7.08	6.98	7.06	7.06	7.10	7.19	7.00

Conclusion:

• In Vi-TT CJ study, Conjugate was maintained in 5, 10 and 20 mM Tris pH 7.2, pH was within range at 2-8°C in 5, 10, and 20 mM concentration.

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Viscosity and Osmolality Measurement of Vi-TT Conjugate

Part A: Viscosity and Osmolality Results of Tris Buffer and Sodium Chloride without Conjugate

Table 84: Part A: Viscosity and Osmolality of Tris Buffer and Sodium Chloride without Conjugate
Buffer Strength (in mM)	Osmolality (mOsm/Kg)	Density (g/cm³)	Viscosity
Kinematic (mm²/s)	Dynamic (mPa.s)
1) Tris Buffer pH 7.2
5	13	0.9986	1.030	1.028
30	61	0.9999	1.020	1.020
50	81	1.0005	1.031	1.031
75	113	1.0014	1.028	1.030
2) Sodium Chloride
150	270	1.0043	1.013	1.017
308	562	1.0117	1.020	1.031
350	607	1.0110	1.023	1.035

Part B: Viscosity and Osmolality Results of Tris Buffer and Sodium Chloride with Conjugate

Table 85: Part B: Viscosity and Osmolality of Tris Buffer and Sodium Chloride with
Buffer Strength (in mM)	Osmolality (mOsm/Kg)	Density (g/cm³)	Viscosity
Kinematic	Dynamic (mPa.s)
I) Tris Buffer + Conjugate
5	18	0.9985	1.058	1.057
30	53	0.9997	1.061	1.061
50	84	1.0005	1.066	1.067
75	124	1.0017	1.079	1.081
II) Sodium Chloride + Conjugate
150	281	1.0046	1.048	1.052
308	550	1.0107	1.053	1.064
350	1253	1.0122	1.053	1.066

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Conclusion:

The Conjugate with variable tris concentrations found isotonie and conjugate with 150mM NaCl found isotonie.

Conjugate with different tris buffer and NaCl concentrations viscosity found similar and the 5 viscosity is suitable for use as parentéral formulations.

140

Example 6: Immunogenicity Data

A. Immunogenicity study of Monovalent conjugale vaccine:

Immunogenic potential of SIIP Vi TT vaccine was assessed in mouse model.

Mice Immunogenicity Study #1:

An immunogenicity study was conducted in mice wherein unconjugated and TT-conjugated SIIPL vaccine (Vi TT) was administered intramuscularly to mice (8 animais per group). The animais were inoculated on day 1 and day 14 and the sera sample was collected on day 14 (data not shown) and day 21. Please refer to Table 86 for details. Paired sera sample was available from ail the animais in each group. The sera sample was used for détermination of antibodies against the injected polysaccharide using a suitable serological immunochemical method such as an in-house IgG ELISA. The sera from mice which received unconjugated SIIPL polysaccharide (Vi PS) was used a comparative control for induction of IgG response in sera from mice which received the conjugated version of Vi PS (of varying PS size).

Mice Immunogenicity Study #2:

Another similar study was performed in mice. Both the studies employed a similar animal treatment protocol. The animal protocol is briefly described below. The sera from both the studies were tested by the identical immunological / serological analytical assay i.e. IgG ELISA. The 5-6 weeks old, female mice (Balb C strain; bred in-house) were used in the study using an IAEC approved animal study protocol. Ail the animais were Spécial Pathogen Free and were handled under aseptie conditions under a bio-safety cabinet during inoculation and blood sample collection. The mice weighed a-18-20 g. at the start of the study. Each mouse received 2.5pg of conjugated Vi TT vaccine via intramuscular route. The SIIPL Vi TT vaccine was diluted in phosphate buffered saline (PBS) as a vehicie. No adjuvant was used in the study for any of the treatment groups.

Treatment Schematic: The following table summarizes the overall treatment plan for the study.

Table 86: Mice treatment Plan
No	Grou p ID	Treatment Arms (Actual Plan)	Animals/Grou P	Vi PS dose per anima 1	Injectio n Volume	Day of Injectio n (IM)	Blood (serum) collectio n day
Balb/C (Female) Mice
1	G1	Vehicie alone (PBS)	8	PBS	IOOuL	1, 14	21

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2	G2	Typbar (Unconjugated)	8	2.5ug	lOÛuL	L 14	21
3	G3	Typbar TCV(Conjugate d)	8	2.5ug	lOOuL	L 14	21
4	G4	NIBSC Vi PS Standard	8	2.5ug	lOOuL	L 14	21
5	G5	SIIPL Vi PS (Unconjugated)	8	2.5ug	lOOuL	1, 14	21
6	G6	G6 SIIPL Vi PS- TT	8	2.5ug	lOOuL	1, 14	21
7	G7	G7 SIIPL Vi PS-	8	2.5ug	lOOuL	1, 14	21
8	G8	G8 SIIPL Vi PS- TT	8	2.5ug	lOOuL	L 14	21
9	G9	G9 SIIPL Vi PS-	8	2.5ug	lOOuL	L M	21

Methodology and techniques used for serological analysis:

The blood sample was collected from the experimental animais from the retro-orbital vein using stérile, glass capillary tubes. The isoflurane was used as a safe anesthetic during the 5 blood collection procedure. The sera were subjected to analysis of IgG antibodies produced in response to injected unconjugated or conjugated Vi TT vaccine using an in-house IgG ELIS A. The ELIS A used NIBSC Vi PS Reference Standard the (Catalog No. I6/126; first international standard for Vi PS of S. Typhi) as a coating antigen. The IgG levels were estimated by analyzing the optical density (OD) observed in sera from mice which received 10 the conjugated of Vi PS (Vi TT of varying PS size) as compared to the OD observed in sera samples from mice which received unconjugated SIIPL polysaccharide (Vi PS).

The following tables indicate the immunogenicity induction data (on day 21) from different Vi TT PS.

Table 87, 88, 89 and 90: Immunogenicity Results

Table 87: Immunogenicity Results Table 88: Immunogenicity Results
Immunogenicity Study 1		Immunogenicity Study 1

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I

G7 SIIPL Vi PS-TT		G6 SIIPL Vi PS-TT
No. of Mice	Gende r	Fold Increase		No. of Mice	Gende r	Fold Increase
8	F	Conjugated over Unconjugated		8	F	Conjugated over Unconjugated
>4 Fold ri se	63.2		>4 Fold rise	67.23
n/N %	100% (8 of8)		n/N %	100% (8 of8)
Géométrie Mean	1.8		Géométrie Mean	1.8
Minimum	1.45		Minimum	1.57
Maximum	1.95		Maximum	1.98

Table 89: Immunogenicity Results Table 90: Immunogenicity Results
Immunogenicity Study 1		Immunogenicity Study 1
G9 SIIPL Vi PS-TT		G8 SIIPL Vi PS-TT
No. of Mice	Gende r	Fold Increase		No. of Mice	Gende r	Fold Increase
8	F	Conjugated over Unconjugated		8	F	Conjugated over Unconjugated
>4 Fold rise	65.4		>4 Fold rise	62.5
n/N %	100% (8 of8)		n/N%	100% (8 of 8)
Géométrie Mean	1.7		Géométrie Mean	1.7
Minimum	1.50		Minimum	1.05
Maximum	2.287		Maximum	1.95

Observations:

The mice groupe with Vi TT exhibited >4-fold higher induction of IgG (as compared mice 5 administered with unconjugated Vi PS) thus strongly indicating the immunogenic potential of

SIIPL VI TT across ail the PS sizes. Two separate studies were performed with Vi TT and both these studies indicated similar response to Vi TT over Vi PS.

143

The following parameters were compared across the various groups:

a. >4-fold rise: average fold rise was >60 fold

b. GM in antibody titer: ail groups exhibited higher GM than observed in unconjugated PS (>l.9)

c. % of mice with positive response: ail the mice in ail the Vi TT groups exhibited higher antibody response (100% positives).

Conclusions from analysis of serological data:

The immunogenicity study in mice was performed with 8 (female) mice in each group. The antibody induction was assessed, in ail the groups, in response to the injected Vj TT (varying PS size) versus the unconjugated Vi PS. Ail the Vi TT PS forms strongly indicated induction of Vi TT spécifie IgG antibodîes (day 21; after first booster) as compared to Vi PS alone group. The induction of IgG in mice sera is a strong déterminant of serological response to the Vi TT. Therefore, monovalent Vi TT is able to induce a strong immunogenicity response in mouse model and provides a promising potential for future studies in higher animais.

Table 91: Summary of Immunogenicity Potential of Monovalent SIIPL Vï TT Vaccine in Mouse Model
Sr. No.	Sample	Target PS Size (kDa)	SIIPL PS Size (kDa)	Conjugate Changes	Batch No.	Immunogenicity (GM Data)	Fold Rise in GM	% O-Acetylation (by NMR)
υ	SIIPL Conjugate (main)	180-220	214	Purification: GFC	G7 SIIPL Vi PSTT	1.94	69	105%
2)	SIIPL Conjugate	300	388	Purification: GFC	G6 SIIPL Vi PS-TT	1,79	64	107%
3)	SIIPL Conjugate	120	80	Purification: GFC	G9 SIIPL Vi PS-TT	1.79	64	102%

144

4)	SIIPL Conjugate	45	42	Purification: Diafiltration	G8 SIIPL Vi PS-TT	1.75	63	102%
5)	Typbar TCV	250-300	NA	NA	NA	1.69	60	-

Observations:

1. The immunogenicity data is assessed in terms of IgG antibody levels observed in response to the SIIPL Vi TT conjugales in mouse model.

2. The IgG induction is measured as Géométrie Mean (géométrie mean) of colorimétrie response (OD) observed in IgG ELISA.

3. The GM observed in response to SIIPL Vi TT is considered as immunogenic if the fold rîse in OD in VI TT treatment is higher than 4 fold over OD observed în response to unconjugated Vi PS.

Conclusions:

1. Ail of the monovalent Vi-TT conjugales were found to induce immunogenic response observed via a serological assay.

2. Ail of monovalent Vi-TT Conjugales exhibited higher IgG levels than observed in Typbar TCV vaccine group.

3. The induction of immunogenicity response (GM and >4 fold rise) was observed in following order: G7 SIIPL Vi PS-TT (PS Size 214) > G7 SIIPL Vi PS-TT (PS Size 388) >Typbar TCV (Commercial vaccine)

145

B. Immunogenicity study of Bivalent conjugate vaccine

Immunogenic potential of bivalent (typhoid and paratyphoîd) SIIPL vaccine was assessed in mouse model.

Mice Immunogenicity Study #1:

An immunogenicity study was conducted in mice wherein a bivalent vaccine containing a combination of monovalent Vi TT vaccine and monovalent O-SP A (O Spécifie Polysaccharide antigen of 5, Paratyphi A) conjugated to carrier proteins such as TT, DT and CRM was employed in the study. The monovalent and bivalent versions of these vaccines were administered intramuscularly to mice (8 animais per group). The animais were inoculated on days 1, 14 and 28 whiie the sera samples were collected on days 14, 28 and 42. Paired sera sample were availabié from ail the animais in each group. Please refer to below Table 92 for details regarding the treatment plan. The sera samples were used for détermination of antibodies against the înjected polysaccharide using a suitable serological immunochemical method such as an in-house IgG ELISA. The sera from mice which received respective unconjugated SIIPL polysaccharide (Vi PS) and O-SP alone were used a comparative control for induction of IgG response.

The animal protocol is briefly described in Table below.

The sera samples were tested by immunological / serological analytical assay i.e. IgG ELISA. The 5-6 weeks old, female mice (Balb C strain; bred in-house) were used in the study using an IAEC approved animal study protocol. Ail the animais were Spécial Pathogen Free and were handled under aseptie conditions under a biosafety cabinet during inoculation and blood sample collection. The mice weighing in the range of -18-20 g. were used in the study. Each mouse received 2.5pg of monovalent conjugated Vi TT alone or O-SP A DT/TT/CRM alone and 2.5pg of each in bivalent vaccine (monovalent conjugated Vi TT mixed with O-SP A DT/TT/CRM) via intramuscular route. The vaccines were diluted in phosphate buffered saline (PBS) as a vehicle. No adjuvant was used in the study for any of the treatment groups.

146

Table 92: Mice treatment Plan
Sr.No.	Group ID	Treatment Arms (Actual Plan)	Animais ! Group Balb/C (Female) Mice	Vi PS / 0SP dose per animal	Injection Volume	Day of Injection (IM)	Btood (sérum) collection day
1	G1	Vehicle alone (PBS)	8	PBS	50pL	l, 14, 28	14, 28,42
2	G2	Typbar TCV (Conjugated)	8	2-5gg	50pL	1, 14, 28	14, 28,42
3	G3	SIIPL Vi PS (Unconjugated)	8	2.5pg	50pL	1, 14, 28	14, 28,42
4	G4	SIIPL Vi PS-TT	8	2.5pg	50pL	I, 14,28	14, 28, 42
5	G5	SIIPL O-SP A (Unconjugated)	8	2..W	50pL	L 14,28	14, 28, 42
6	G6	SIIPL O-SP A DT/ TT/ CRM	8	2.5pg	50pL	1, 14, 28	14, 28,42
7	G7	SIIPL Vi PS-TT + SIIPL O-SP A DT	8	2.5pg of each component	50pL	1, 14,28	14, 28, 42
8	G8	SIIPL Vi PS-TT + SIIPL O-SP A TT	8	2.5μg of each comoonent	50pL	1, 14, 28	14, 28, 42
9	G9	SIIPL Vi PS-TT + SIIPL O-SP A CRM	8	2.5pg of each comnonent	50pL	1, 14, 28	14, 28, 42
10	G10	SIIPL O-SP A CRM + Typbar TCV (Conjugated)	8	2.5pg of each component	50pL	1, 14, 28	14, 28, 42

147

Methodology and techniques used for serological analysis:

The blood samples were collected from the experimental animais from the retro-orbital vein using stérile, glass capillary tubes. The isoflurane was used as a safe anesthetic during the blood collection procedure. The sera was subjected to analysis of IgG antibodies produced in 5 response to injected antigen using an in-house IgG ELISA. The IgG levels were estimated in sera samples from ail the study groups by colorimétrie analysis.

Observations:

The following tables indicate the immunogenicity induction data (on day 21) from different bivalent (typhoid and paratyphoid) SIIPL vaccines.

Table 93, 94, 95,96: Immunogenicity Results

Table 93: Immunogenicity Results Table 94: Immunogenicity Results
Immunogenicity Study 1		Immunogenicity Study 1
SIIPL Vi PS-TT + SIIPL O-SP A DT		SIIPL Vi PS-TT + SIIPL O-SP A TT
No. of Mice	Gender	Fold Increase		No. of Mice	Gender	Fold Increase
8	F	Conjugated over Unconjugated		8	F	Conjugated over Unconjugated
>4 Fold rise	Yes		>4 Fold rise	Yes
n/N %	100% (8 of 8)		n/N %	100% (8 of8)

Table 95: Immunogenicity Results Table 96: Immunogenicity Results
Immunogenicity Study 1		Immunogenicity Study 1
SIIPL Vi PS-TT + SIIPL O-SP A CRM		SIIPL O-SP A CRM + Typbar TCV
No. of Mice	Gender	Fold Increase		No. of Mice	Gender	Fold Increase
8	F	Conjugated over Unconjugated		8	F	Conjugated over Unconjugated
>4 Fold rise	Yes		>4 Fold rise	Yes
n/N %	100% (8 of 8)		n/N %	100% (8 of 8)

148

Observations:

The mice groups injected with bivalent (typhoid and paratyphoid) SIIPL vaccine such as a) SIIPL Vi PS-TT + SIIPL O-SP A DT, b) SIIPL Vi PS-TT + SIIPL O-SP A TT and c) SIIPL Vi PS-TT + SILPL O-SP A CRM Vi TT exhibited >4-fold higher induction of IgG (as 5 compared to mice administered with unconjugated Vi PS or unconjugated SIIPL O-SP A) thus indicating the immunogenic potential of bivalent SIIPL vaccine containing combination of Vi TT and SIIPL O-SP A (DT/TT/CRM).

The IgG induction by bivalent (typhoid and paratyphoid) SIIPL vaccine was higher than the IgG induction observed in bivalent vaccine containing SIIPL O-SP A CRM and commercial 10 Vi TT vaccine Typbar TCV.

The following parameters were compared across the various groups;

a. >4-fold rise: average fold rise was >10 fold

b. GM in antibody titer; ail groups exhibited higher GM than observed in unconjugated PS c, % of mice with positive response: ail the mice in ail the Vi TT groups exhibited higher antibody response (100% positives).

Conclusions from analysis of serological data:

The immunogenicity study in mice was performed with 8 (female) mice in each group. The antibody induction was assessed, in ail the groups, in response to the injected bivalent 20 (typhoid and paratyphoid) SIIPL vaccine versus the unconjugated Vi PS and O-SP A PS. Ail the bivalent (typhoid and paratyphoid) SIIPL vaccines strongly indicated induction of PS spécifie IgG antibodies (day 21; after first booster) as compared to PS alone (Vi and O-SP A) group. The induction of IgG in mice sera is a strong déterminant of serological response to the bivalent (typhoid and paratyphoid) SIIPL vaccine. Therefore, bivalent (typhoid and 25 paratyphoid) SIIPL vaccine is able to induce a strong immunogenicity response in mouse model and provides a promising potential for future studies in higher animais.

Observations:

1. The immunogenicity data is assessed in terms of IgG antibody levels observed in response to the bivalent (typhoid and paratyphoid) SIIPL vaccine in mouse model.

3. The GM observed in response to bivalent (typhoid and paratyphoid) SIIPL vaccine is considered as immunogenic if the fold rise in OD in VI TT treatment is higher than 4 fold over OD observed in response to unconjugated Vi PS.

149

Conclusions:

1. Ali of the bivalent (typhoid and paratyphoid) SIIPL vaccine conjugales were found to induce immunogenic response observed via a serological assay.

2. Ail of bivalent (typhoid and paratyphoid) SIIPL vaccine conjugales exhibited higher

IgG levels than observed in unconjugated counterparts.

3. The IgG induction by bivalent (typhoid and paratyphoid) SIIPL vaccine was higher than the IgG induction observed in bivalent vaccine containing SIIPL O-SP A CRM and commercial Vi TT vaccine Typvar TCV.

4. The induction of immunogenicity response (GM and >4 fold rise) was observed in mice receiving “bivalent (typhoid and paratyphoid) SIIPL vaccine” and hence it can be concluded that bivalent (typhoid and paratyphoid) SIIPL vaccine is able to induce a strong immunogenicity response in mouse model suggestive of its clinical immunogenic potential.

150

Example 7: Single dose vaccine kit

A) Single dose vaccine kit comprising of:

a) Neisseria meningitidis A saccharîde - TT conjugate antigen 5 pg per 0.5 ml;

b) Neisseria meningitidis C saccharîde - CRM 197 conjugate antigen 5 pg per 0.5 ml;

c) Neisseria meningitidis Y saccharîde - CRM 197 conjugate antigen 5 pg per 0.5 ml;

d) Neisseria meningitidis VF -135 saccharîde - CRM 197 conjugate antigen 5 pg per 0.5 ml;

e) Neisseria meningitidis X saccharîde - TT conjugate antigen 5 pg per 0.5 ml;

f) Sucrose 1-12 mg per 0.5 ml;

g) Sodium citrate (Dihydrate) 0.1 - 2 mg per 0.5 mi;

h) Tris Buffer 0.05 - 0.5 mg per 0.5 ml;

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Sodium chloride 1 - 10 mg;

c) Water for Injection (WFI) q.s.;

Interprétation:

No antigenic interférence 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- dépendent immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

151

B) Single dose vaccine kit comprising of:

a) Neisseria meningitidis A saccharide - TT conjugate antigen 5 pg per 0.5 ml;

d) Neisseria meningitidis W-135 saccharide — CRM 197 conjugate antigen 5 pg per 0.5 ml;

e) Neisseria meningitidis X saccharide - TT conjugate antigen 5 pg 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;

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Salmonella enterica serovar paratyphi A OS P - CP conjugate antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197;

c) Sodium chloride 1-10 mg;

d) Water for Injection (WFI) q.s.;

Interprétation:

No antigenic interférence of ViPs-TT; OSP antigen with Neisseria meningitidis antigens, for prophylaxie 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- dépendent immune response against S. typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

152

C) Single dose vaccine kit comprising of:

a first container contaîning a fully liquid hexavalent immunogenic composition:

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 I 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) l-50pg, Filamentous hemagglutinin (FHA) l-50pg, Pertactin (P69 or PRN) 120pg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25pg; per 0.5ml;

and a second container contaîning a fully liquid immunogenic composition comprising:

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

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 20; and/or

e) 2-Phenoxyethanol 1-10 mg; and/or

f) Water for Injection (WFI) q.s.

Interprétation:

No antîgenic interférence 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- dépendent immune response against S. typhi încluding in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

153

D) Single dose vaccine kit comprising of:

a first container containing a fully liquid hexavalent immuno génie 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) l-50qg, Filamentous hemagglutinin (FHA) L50μg, Pertactin (P69 or PRN) 120μg 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 ug per 0.5 ml;

f) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 μ g per 0.5 ml; and a second container containing a fully liquid immunogenic composition comprising:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 gg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 qg; wherein the CP is either TT or DT or CRM 197;

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; and/or

h) 2-Phenoxyethanol 1 — 10 mg; and/or

e) Water for Injection (WFI) q.s.

Interprétation:

No antigenic interférence of ViPs-TT; OSP antigen with Hexavalent antigens, for prophyIaxis 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- dépendent immune response against S, typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

154

E) Single dose vaccine kit comprising of:

a first container containing a fully liquid heptavaient immunogenic composition:

a) an inactivated polio virus (IPV) antigen selected from Sabin or Saik 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 rota virus strains present in an amount in the range of 1 to 50 μ g 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) l-50pg, Filamentous hemagglutinin (FHA) l-50gg, Pertactin (P69 or PRN) 120gg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25 μ g; per 0.5ml;

f) a hepatitis B virus surface antigen, (HBsAg) in an amount of I to 20 μ§ per 0.5 ml;

g) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 μ g per 0.5 ml;

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.;

Interprétation:

wherein there is no antigenic interférence of ViPs-TT with Heptavaient immunogenic composition, for prophylaxis against typhoid caused by Salmonella typhi and Heptavaient antigens wherein the said vaccine formulation is sufficient to elicit the required T- dépendent immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

155

B F) Single dose vaccine kit comprising of:

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 l at a dose 1-50 D-antigen units (DU), IPV Type 2 at a dose of 1-50 D-antigen unit 5 (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 présent in an amount in the range of 1 to 50 pg 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) l-50pg, Filamentous hemagglutinin (FHA) l-50pg, Pertactin (P69 or PRN) 120pg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25pg; per 0.5ml;

f) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 pg per 0.5 ml;

g) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 pg per 0.5 ml;

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg; 20 wherein the CP is either TT or DT or CRM 197;

c) Sodium chloride 1-10 mg; and/or

g) Polysorbate selected from polysorbate 20;

h) 2-Phenoxyethanol 1-10 mg; and/or

e) Water for Injection (WFI) q.s.

156

Interprétation:

No antigenic interférence of ViPs-TT; OSP antigen with Heptaveilent 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 5 T- dépendent immune response against S. typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

Claims

1. An immunogenic composition comprising of:

i) at least one antigen selected from:

a) Salmonella enterica serovar typhi saccharide-carrier protein conjugale;

b) Salmonella enterica serovar paratyphi A saccharide- carrier protein conjugale;

c) Salmonella enterica serovar paratyphi B saccharide- carrier protein conjugale;

d) Salmonella enterica serovar paratyphi C saccharide- carrier protein conjugale;

e) Salmonella enterica serovar typhimurium saccharide- carrier protein conjugate;

f) Salmonella enterica serovar enteritidis saccharide- carrier protein conjugate;

g) Salmonella enterica serovar choleraesuis saccharide- carrier protein conjugate;

h) Salmonella enterica serovar dublin saccharide- carrier protein conjugate; and ii) optionally an antigen selected from group comprising of Salmonella (non-typhoidal), Diphtheria toxoid (D), Tetanus toxoid (T), Whole cell pertussis (wP), hepatitis B virus surface antigen (HBsAg), Haemophilus influenzae b PRP-Carrier protein conjugate (Hib), Haemophilus influenzae (a, c, d, e, f serotypes and the unencapsulated strains), Polio virus, Conjugate comprising of N. meningitidis antigens (A, B, C, D, WI35, X, Y, Z and 29E), Streptococcus Pneumoniae antigen(s) (1, 2, 3, 4, 5,6A, 6B, 6C, 6D, 6E, 6G, 6H, 7A, 7B, 7C, 7F, 8, 9A,9L,9F,9N, 9V, 10F, 10B,10C, 10A, H A,11F,11B, 11C,11D,11E,12A, 12B, 12F,13, 14, 15A.15C ,15B,15F,16A, 16F, 17A,17F, 18C,18F,18A,18B, 19A, 19B, 19C, 19F, 20, 20 A, 20B,21,22 A, 22F, 23A,23B, 23F, 24A, 24B,24F , 25F, 25A,27,28F, 28A, 29, 31,32F, 32A,33A, 33C, 33D, 33E, 33F,33B, 34, 45,38,35A,35B,35C,35F,36,37,38, 39, 40,41F,41A,42,43,44,45,46,47F,47A,48),, Group A Streptococcus spp , Group B Streptococcus (group la, Ib, II, III, IV, V, VI, VII, VIII, and IX.) Neisseria meningitidis B bleb or antigen(s)(fHbp , PorA, chimeric fHbp-porA), Staphylococcus aureus antigen(s), Anthrax, BCG, Hepatitis (A, C, D, E, F and G strains) antigen(s), Human papilloma virus, HIV, aceilular pertussis, modified adenylate cyclase, Malaria Antigen (RTS,S), Measles, Mumps, Rubella, Dengue, Zika, Ebola, Chikungunya, Japanese encephalitis, Rotavirus, Diarrheal antigens (E. coli spp,, Shigella spp., Campylobacter spp. Vibrio choiera), Fiavivirus, smallpox, yellow fever, Shingles, Varicella virus antigens.

2. The immunogenic composition as claimed in claim 1, wherein the composition comprises of atleast one bivalent combination:

158

or

b) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugale antigen; or

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, température in range of 2°C to 30°C.

3. The immunogenic composition as claimed in claim ], wherein the composition comprises of atleast one trivalent combination:

or

a) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugale antigen;

4. The immunogenic composition as claimed in claim l, wherein the composition comprises of tetravalent combination:

159

5 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 ali unreacted compounds, unconjugated polysaccharides, unconjugated proteins are removed, yielding a purified OSP-carrier protein conjugate vaccine wherein the conjugate yield is > 50%.

10

5 b) an inactivated rotavirus antigen selected from CDC-9, CDC-66 or any other inactivated rotavirus strains présent in an amount in the range of 1 to 50 pg 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 I to 50 IOU per 0.5 ml or acellular 10 pertussis, (aP) antigen comprising one or more of modified adenylate cyclase selected from

Pertussis toxoid (PT) l-50pg, Filamentous hemagglutinin (FHA) l-50pg, Pertactin (P69 or PRN) l-20pg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25pg; per 0.5ml;

f) a hepatitis B virus surface antigen, (HBsAg) in an amount of I to 20 pg per 0.5 ml;

5 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 présent in an amount in the range of 1 to 50 qg per 0.5 ml;

5 c) a tetanus toxoid, (T) antigen in an amount of 1 to 30 Lf per 0.5 ml;

5 Pertussis toxoid (PT) L50pg, Fîlamentous hemagglutinin (FHA) l-50pg, Pertactin (P69 or PRN) l-20pg or Fimbrial proteins (FIM 1 , 2 and 3) 2-25pg; per 0.5ml;

and

5 h) Water for Injection (WFI) q.s.

5 c) Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197

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.

10

5 a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197

c) Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

5 octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 μg

f) Sucrose 2.5 - 25 mg

g) 2~Phenoxyethanol 1 - 10 mg

5 wherein the CP is either TT or DT or CRM 197

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, 10 octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25 - 500 gg

f) Sucrose 2.5 - 25 mg

g) Water for Injection (WFI) q.s.

15

5 d) 2-Phenoxyethanol 1-10 mg

e) Water for Injection (WFI) q.s.

5. The immunogenic composition as ciaimed in claim 1, wherein the composition comprises of atleast one combination:

b) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

c) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis W -135 saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis X saccharide - carrier protein conjugate antigen;

or

c) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis W-l 35 saccharide - carrier protein conjugate antigen;

g) Neisseria meningitidis X saccharide - carrier protein conjugate antigen; or

d) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis T saccharide - carrier protein conjugate antigen;

g) Neisseria meningitidis W -135 saccharide - carrier protein conjugate antigen;

h) Neisseria meningitidis X saccharide - carrier protein conjugate antigen; or

160

e) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

g) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

h) Neisseria meningitidis W-135 saccharide — carrier protein conjugate antigen;

i) Neisseria meningitidis X saccharide - carrier protein conjugate antigen;

or

b) Salmonella enterica serovar enteritidis saccharide-carrier protein conj ugate antigen;

c) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis W -135 saccharide - carrier protein conjugate antigen;

g) Neisseria meningitidis X saccharide - carrier protein conjugate antigen or

b) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

c) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis Y saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis W-135 saccharide - carrier protein conjugate antigen or

c) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugate antigen;

e) Neisseria meningitidis K saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis W -135 saccharide - carrier protein conjugate antigen or

c) Neisseria meningitidis A saccharide - carrier protein conjugate antigen;

d) Neisseria meningitidis C saccharide — carrier protein conjugate antigen;

e) Neisseria meningitidis K saccharide - carrier protein conjugate antigen;

f) Neisseria meningitidis W-135 saccharide - carrier protein conjugate antigen;

or

161

b) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

c) Neisseria meningitidis C saccharide - carrier protein conjugale antigen; or

c) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugale antigen;

or

c) Neisseria meningitidis A saccharide - carrier protein conjugale antigen;

d) Neisseria meningitidis C saccharide - carrier protein conjugale antigen;

6. The immunogenic composition as claimed in claim l, wherein the composition comprises of atleast one combination:

b) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

c) a diphtheria toxoid, (D) antigen

d) a tetanus toxoid, (T) antigen

e) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

f) a hepatitis B virus surface antigen, (HBsAg) and

g) a Haemophilus influenzae type b antigen, (Hib); or

b) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

c) a rotavirus antigen;

d) a diphtheria toxoid, (D) antigen

e) a tetanus toxoid, (T) antigen

f) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

g) a hepatitis B virus surface antigen, (HBsAg) and

h) a Haemophilus influenzae type b antigen, (Hib); or

162

c) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

d) a diphtheria toxoid, (D) antigen

e) a tetanus toxoid, (T) antigen

f) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

g) a hepatitis B virus surface antigen, (HBsAg) and

h) a Haemophilus influenzae type b antigen, (Hib); or

c) a rotavirus antigen;

d) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

e) a diphtheria toxoid, (D) antigen

f) a tetanus toxoid, (T) antigen

g) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

h) a hepatitis B virus surface antigen, (HBsAg) and

i) a Haemophilus influenzae type b antigen, (Hib); or

d) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

e) a diphtheria toxoid, (D) antigen

f) a tetanus toxoid, (T) antigen

g) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

h) a hepatitis B virus surface antigen, (HBsAg) and

i) a Haemophilus influenzae type b antigen, (Hib); or

c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; d) a rotavirus antigen;

e) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

f) a diphtheria toxoid, (D) antigen

163

;) a tetanus toxoid, (T) antigen

h) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

i) a hepatitis B virus surface antigen, (HBsAg) and

j) a Haemophilus influenzae type b antigen, (Hib); or

e) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

f) a diphtheria toxoid, (D) antigen

g) a tetanus toxoid, (T) antigen

h) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

i) a hepatitis B virus surface antigen, (HBsAg) and

j) a Haemophilus influenzae type b antigen, (Hib); or

e) a rota virus antigen;

f) an inactivated polio virus (IPV) antigen, selected from Salk or Sabin strain

g) a diphtheria toxoid, (D) antigen

h) a tetanus toxoid, (T) antigen

i) a whole cell pertussis, (wP) antigen or acellular pertussis, (aP);

j) a hepatitis B virus surface antigen, (HBsAg) and

k) a Haemophilus influenzae type b antigen, (Hib);

7. The immunogenic composition as claimed in claim 1, wherein the composition comprises of atleast one combination:

b) a rotavirus antigen;

c) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohémorragie) antigen;

d) a Shigella spp. antigen;

164

e) a Campylobacter jejuni antigen;

f) a Vibrio cholerae antigen;

or

b) Salmonella enterica serovar Paratyphi A saccharîde- carrier protein conjugate antigen;

c) a rotavirus antigen;

d) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohémorragie) antigen;

e) a Shigella spp. antigen;

f) a Campylobacter jejuni antigen;

g) a Vibrio cholerae antigen;

or

c) a rotavirus antigen;

e) a Shigella spp. antigen;

f) a Campylobacter jejuni antigen or

c) a rotavirus antigen;

e) a Shigella spp. antigen;

or

c) a rotavirus antigen;

d) a Shigella spp. antigen;

or

c) a rotavirus antigen;

e) a Shigella spp. antigen;

165

f) a Campylobacter jejuni antigen;

g) a Vibrio cholerae antigen;

or

e) a diarrheo génie Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;

f) a Shigella spp. antigen;

g) a Campylobacter jejuni antigen;

h) a Vibrio cholerae antigen;

or

b) Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen; c) Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; d) Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen;

e) a rotavirus antigen;

f) a diarrheogenic Escherichia coli spp. (enterotoxigenic and enterohemorragic) antigen;

g) a Shigella spp. antigen;

h) a Campylobacter jejuni antigen;

i) a Vibrio cholerae antigen;

8. The immunogenic composition as claimed in claims l to 7, wherein the carrier protein (CP) is selected from the group comprising of tetanus toxin, tetanus toxoid (TT), fragment of tetanus toxoid, diphtheria toxoid (DT), CRM197, Pseudomonas aeruginosa toxoid, Bordeteila pertussis toxoid, Clostridium perfringens toxoid, E.coli LT, E. coli ST, Escherichia coli heat-labile toxin - B subunit, Neisseria meningitidis outer membrane compiex, rEPA, protein D of H. influenzae, Flagellîn FliC, Horseshoe crab Haemocyanîn, exotoxin A from Pseudomonas aeruginosa, outer membrane compiex 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-l l, protective antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and léthal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human sérum albumin, bovine sérum albumin (BSA), purified protein

166 dérivative of tuberculin (PPD), synthetic peptides, beat 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, ironuptake proteins, toxin A or B from C. difficile and S.agalactiae proteins with or without linker and fragments, dérivatives, modifications thereof.

9. The immunogenic composition as claimed in daims 1 to 7, wherein Salmonella enterica serovar typhi saccharide-carrier protein conjugate antigen; Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate antigen; Salmonella enterica serovar typhimurium saccharide-carrier protein conjugate antigen; Salmonella enterica serovar enteritidis saccharide-carrier protein conjugate antigen; is présent in a dose range of 5 pg/dose to 30 pg/dose.

10 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 acelluiar pertussis, (aP) antigen comprising one or more of modified adenylate cyclase selected from Pertussis toxoid (PT) l-50qg, Filamentous hemagglutinin (FHA) l-50qg, Pertactin (P69 or 15 PRN) l-20qg or Fimbrial proteins (FIM 1,2 and 3) 2-25qg; per 0.5ml;

f) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 qg per 0.5 ml;

g) a Haemophilus influenzae type b antigen, (Hib) in an amount of 1 to 20 qg per 0.5 ml;

10 e) a hepatitis B virus surface antigen, (HBsAg) in an amount of 1 to 20 pg per 0.5 ml;

10 a second container containing a fully liquid immunogenic composition comprising:

b) Sodium chloride 1 - 10 mg per 0.5 ml; and/or

c) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg per 0.5 ml; and/or

10 b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM197

c) Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1 - 10 mg

10 wherein the CP is either TT or DT or CRM 197

c) Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen 1.25 - 50 pg; 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

10 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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

10 d) Sodium chloride I - 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, 15 soy lecithin and a poloxamer in an amount of 25-500 pg

f) Water for Injection (WFI) q.s.

10 h) Water for Injection (WFI) q.s.

10 d) Water for Injection (WFI) q.s.

10 c) Sodium chloride 1 — 10 mg

d) 2-Phenoxyethanol 1-10 mg

e) Water for Injection (WFI) q.s.

10 b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg; 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, 15 polysorbate S0, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxy stéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25 - 500 pg

f) Water for Injection (WFI) q.s.

20

10. The immunogenic composition as claimed in daims 1 to 7, wherein the composition comprises of a pharmaceutically acceptable buffer selected from the group of sodium chloride, acetate, carbonate, citrate, lactate, gluconate, tartrate, phosphate buffer saline, borate, Histidine buffer, Succinate buffer, HEPES, TRIS or Citrate-phosphate.

11. The immunogenic composition as claimed in daims 1 to 7, wherein the composition comprises of a pharmaceutically acceptable excipient selected from the group of sugars, surfactants, polymers, salts, aminoacids or pH modifiers.

12. The immunogenic composition as claimed in daim 11, wherein the composition comprises of aminoacids or proteins as excipient selected from the group of L-Histidine, Lysine, Isoleucine, Méthionine, Glycine, Aspartic acid. Tricine, arginine, leucine, glutamine, alanine, peptide, hydrolysed protein or protein such as sérum albumin.

13. The immunogenic composition as claimed in daim 11, wherein the composition comprises of sugars as excipient selected from the group of sucrose, mannitol, trehalose, mannose, raffinose, lactitol, lactobionic acid, glucose, maltulose, iso- maltulose, maltose, lactose sorbitol, dextrose, fructose, glycerol, or a combination thereof.

14. The immunogenic composition as claimed in daim 11, wherein the composition comprises of non-ionic surfactants as excipient selected from the group of polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonyiphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9,

167 triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer.

15 toxîn - 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

15 and a second container containing a fully liquid immunogenic composition comprising:

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 — 50 pg per 0.5 ml;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 — 50 pg per 0.5 ml; wherein the CP is either TT or DT or CRM 197;

15 b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg per 0.5 ml; wherein the CP is either TT or DT or CRM 197;

c) Sodium chloride 1 - 10 mg per 0.5 ml; and/or

d) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg per 0.5 ml; and/or

15 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 in an amount of 25-500 pg per 0.5 ml; and/or

15 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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, 20 soy lecithin and a poloxamer in an amount of 25-500 pg

g) Sucrose 2.5 - 25 mg

h) 2-Phenoxyethanol 1-10 mg

i) Water for Injection (WFI) q.s.

15 f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanoiamine, triethanoiamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

15 wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 mg

e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1,6 mg

15 g) Water for Injection (WFI) q.s.

15 b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 μ§; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

d) Water for Injection (WFI) q.s.

15 wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

d) 2-Phenoxyethanol 1 - 10 mg

e) Water for Injection (WFI) q.s.

15 a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 pg;

b) Salmonella enterica serovar typhimurium saccharide— CP conjugale antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

15 d) Water for Injection (WFI) q.s.

15. The immunogenic composition as claimed in claim 11, wherein the composition comprises of polymers as excipient seiected from the group of dextran, 5 carboxymethylceHulose, hyaiuronic acid, cyclodextrin.

16. The immunogenic composition as claimed in claim 11, wherein the composition comprises of salts as excipient seiected from NaCl, KC1, KH2PO4, Na2HPO4.2H2O, CaCh and MgC12.

17. The immunogenic composition as claimed in claims 1 to 7, wherein the single dose 10 composition is free of preservative.

18. The immunogenic composition as claimed in claims 1 to 7, wherein the multi-dose composition comprises of one or more preservative seiected from the group of 2phenoxyethanol. Benzéthonium chloride (Phemerol), Phénol, Thîomersal, Formaldéhyde, paraben esters (e.g. methyl-, ethyl-, propyl- or butyl- paraben), benzyl alcohol, m-cresol, 15 benzalkonium chloride, benzyl alcohol, chlorobutanol, p-chlor-m-cresol.

19. The immunogenic composition as claimed in claims 1 to 7, wherein the composition additionally comprises an adjuvant seiected from the group of aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate, and potassium aluminum sulfate or a mixture thereof.

20

20 antigen (PA) of Bacillus anthracis and detoxified edema factor (EF) and léthal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human sérum albumin, bovine sérum albumin (BSA), purified protein dérivative of tubercuiin (PPD), synthetic peptides, heat shock proteins, pertussis proteins, cytokines, lymphokines, hormones, growth factors, artificial proteins comprising multiple human CD4+ T cell epitopes from various

20 c) Sodium chloride 1 - 10 mg per 0.5 ml; and/or

d) Tris Buffer or Citrate buffer or Histidine buffer or Succînate Buffer 0.1 mg - 1.6 mg per 0.5 ml; and/or

g) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol, 25 octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg per 0.5 ml; and/or

h) 2-Phenoxyethanol 1 - 10 mg per 0.5 ml; and/or

e) Water for Injection (WFI) q.s.

203 wherein there is no antigenic interférence 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- dépendent immune response against S. typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

20 a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 qg per 0.5 ml;

b) Sodium chloride 1 - 10 mg per 0.5 ml;

c) Water for Injection (WFI) q.s.;

wherein there is no antigenic interférence of ViPs-TT with Heptavalent immunogenic composition, for prophylaxis against typhoid caused by Salmonella typhi and Heptavalent 25 antigens wherein the said vaccine formulation is sufficient to elicit the required T- dépendent immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

20 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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg per 0.5 ml; and/or

20 e) 2-Phenoxyethanol 1 - 10 mg per 0.5 ml; and/or

f) Water for Injection (WFI) q.s.

wherein there is no antigenic interférence 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- dépendent 25 immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

20 g) Sucrose 2.5 - 25 mg

h) Water for Injection (WFI) q.s.

20 f) 2-Phenoxyethanol 1 - 10 mg

g) Water for Injection (WFI) q.s.

20 b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 — 50 pg;

wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 mg

20 e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyelhoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynoi- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyelhylene- 660 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithîn and a poloxamer in an amount of 25-500 pg

20 c) 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 25 polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

f) Sucrose 2.5 - 25 mg

g) 2-Phenoxyethanol 1 - 10 mg

h) Water for Injection (WFI) q.s.

30

20 c) Sodium chloride 1-10 mg

d) 2-Phenoxyethanol 1-10 mg

e) Water for Injection (WFI) q.s.

20. The immunogenic composition as claimed in claims 1 to 7, wherein the composition additionally comprises an immunostimulatory component seiected from the group of an oil and water émulsion, MF-59, a liposome, a lipopolysaccharide, a saponin, lipid A, lipid A dérivatives, Monophosphoryl lipid A, 3Meacylated monophosphoryl lipid A, AS01, AS03, an oligonucleotide, an oligonucleotide comprising at least one unmethylated CpG and/or a

21. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition 30 comprises of:

168

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen l .25 - 50 pg;

b) Sodium chloride l - 10 mg

c) Water for Injection (WFI) q.s.

22. The immunogenic composition as claimed in daim l, wherein 0.5 ml of the composition 5 comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Sodium chloride 1 - 10 mg

c) 2-Phenoxyethanol 1-10 mg

d) Water for Injection (WFI) q.s.

10

23. The immunogenic composition as claimed in daim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0.1 mg - 1.6 mg

c) Sodium chloride 1 - 10 mg

24. 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 pg;

25 pathogen-derived antigens such as N 19, iron-uptake proteins, toxin A or B from C. difficile and S.agalactiae protein s with or without linker and fragments, dérivatives, modifications thereof.

25 f) 2-Phenoxyethanol 1-10 mg per 0.5 ml; and/or

g) Water for Injection (WFI) q.s.

wherein there is no antigenic interférence 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 30 the requîred T- dépendent immune response against S. typhi and paratyphi including in

201 children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

25 wherein the CP is either TT or DT or CRM 197

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

25 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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

25 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 in an amount of 25-500 pg

25 c) Sodium chloride 1 - 10 mg

d) 2-Phenoxyethanol 1 - 10 mg

e) Water for Injection (WFI) q.s.

25 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, 30 soy lecithin and a poloxamer in an amount of 25-500 gg

f) Water for Injection (WFI) q.s.

174

25 f) Water for Injection (WFI) q.s.

25 c) 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 30 polypeptide oleate, polyoxyethylene- 660 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

f) 2-Phenoxyethanol 1 - 10 mg

g) Water for Injection (WFI) q.s.

170

25 a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Salmonella enterica serovar paratyphi A OS P - CP conjugate antigen 1.25 - 50 pg;

wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1-10 mg

d) Water for Injection (WFI) q.s.

30

25. The immunogenic composition as claimed in daim 1, wherein 0.5 ml of the composition comprises of:

25 liposome, Freund’s adjuvant, Freund’s complété adjuvant, Freund’s incomplète adjuvant, CRL-8300 adjuvant, muramyl dipeptide, TLR-4 agonists, flagellin, flageliins derived from gram négative bacteria,dmLT, TLR-5 agonists, fragments of flageliins capable of binding to TLR-5 receptors, QS-21, ISCOMS, Chitosan, saponin combination with sterols and lipids.

26. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

169

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride I - 10 mg

27. 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 pg;

28. 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 pg;

29. 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 pg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 gg;

30 d) Sodium chloride 1-10 mg

f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,

193

R octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

g) 2-Phenoxyethanol 1 - 10 mg

30 d) Sodium chloride 1-10 mg

f) Polysorbate seiected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,

192 octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, trîethanolamine, triethanoiamine polypeptide oleate, polyoxyethylene- 660 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 μ g g) Water for Injection (WFI) q.s.

5

30 g) Sucrose 2.5 - 25 mg

h) Water for Injection (WFI) q.s.

186

30 f) Sucrose 2.5 - 25 mg

g) Water for Injection (WFI) q.s.

184

30 a) Salmonella enterica serovar enteritidis saccharîde - CP conjugate antigen 1.25 - 50 μg; wherein the CP is either TT or DT or CRM i 97

176

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen l .25 - 50 pg;

wherein the CP is either TT or DT or CRM 197

c) 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 in an amount of 25-500 pg

f) Water for Injection (WFI) q.s.

30 wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

172

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 in an amount of 25-500 qg

f) Sucrose 2.5 - 25 mg

g) Water for Injection (WFI) q.s.

30. 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 gg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

31. 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;

171

b) Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

d) Water for Injection (WFI) q.s.

5

32. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 pg;

33. The immunogenic composition as claimed in claim i, wherein 0.5 ml of the composition comprises of:

34. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 pg;

b) Salmonella enterica serovar typhimurium saccharide- CP conjugale antigen 1.25 - 50 pg;

35. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 qg;

b) Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen 1.25 - 50 qg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

f) 2-Phenoxyethanol 1-10 mg

g) Water for Injection (WFI) q.s.

36. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 qg;

c) Sodium chloride 1 - 10 mg

173

B f) Sucrose 2.5 - 25 mg

g) 2-Phenoxyethanol 1 - 10 mg

h) Water for Injection (WFI) q.s.

37. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition 5 comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 gg;

b) Salmonella enterica serovar enteritidis saccharide - CP conjugale antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

38. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 gg;

b) Salmonella enterica serovar enteritidis saccharide - CP conjugale antigen 1.25 - 50 gg;

39. The immunogenic composition as claimed in daim I, wherein 0.5 ml of the composition 20 comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 gg;

b) Salmonella enterica serovar enteritidis saccharide- CP conjugale antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

40. 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 gg;

b) Salmonella enterica serovar enteritidis saccharide- CP conjugate antigen 1.25 - 50 g g; wherein the CP is either TT or DT or CRM 197

c) 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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 gg

f) Sucrose 2.5 - 25 mg

g) Water for Injection (WFI) q.s.

41. 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 gg;

b) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

c) 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 in an amount of 25-500 gg

f) 2-Phenoxyethanol 1 - 10 mg

g) Water for Injection (WFI) q.s.

42. 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 gg;

175

B c) Sodium chloride I - 10 mg

d) Tris Buffer or Citrate buffer or Histidine buffer or Succînate Buffer 0.1 mg -1.6 mg

e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethoxethanol,

43. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar enteritidis saccharîde — CP conjugate antigen 1.25 — 50 μg; wherein the CP is either TT or DT or CRM 197

44. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition 20 comprises of:

a) Salmonella enterica serovar enteritidis saccharîde - CP conjugate antigen 1.25 - 50 μg; wherein the CP is either TT or DT or CRM 197

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 μ§; wherein the CP is either TT or DT or CRM 197

45. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

46. The immunogenic composition as claimed in claim I, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

f) Sucrose 2.5 - 25 mg

g) Water for Injection (WFI) q.s.

47. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

c) Sodium chloride 1-10 mg

177

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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

f) 2-Phenoxyethanol 1 - 10 mg

g) Water for Injection (WFI) q.s.

48. The immunogenic composition as claîmed in claim 1, wherein 0.5 ml of the composition comprises of:

b) Salmonella enterica serovar paratyphi A OSP — CP conjugate antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

d) Tris Buffer or Citrate buffer or Histidîne buffer or Succinate Buffer 0.1 mg - 1.6 mg

f) Sucrose 2.5 - 25 mg

g) 2-Phenoxyethanol 1 - 10 mg

h) Water for Injection (WFI) q.s.

49. The immunogenic composition as claîmed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhimurium. saccharide - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1-10 mg

d) Water for Injection (WFI) q.s.

178

50. 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 pg; 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.

51. 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 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethox éthanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

f) Water for Injection (WFI) q.s.

52. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

c) Sodium chloride 1 - 10 mg

e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonylphenoxypolyethox éthanol,

179 octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 pg

f) Sucrose 2.5 - 25 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:

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM197

c) Sodium chloride 1 - 10 mg

f) 2-Phenoxyethanol 1-10 mg

g) Water for Injection (WFI) q.s.

54. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

c) Sodium chloride 1 - 10 mg

180

f) Sucrose 2.5 - 25 mg

g) 2-Phenoxyethanol 1 - 10 mg

h) 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 conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM197

b) Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM197

c) Sodium chloride 1-10 mg

d) 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 conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1-10 mg

d) 2-Phenoxyethanol I - 10 mg

e) Water for Injection (WFI) q.s.

57. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

c) Sodium chloride 1-10 mg

e) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, nonyiphenoxypolyethoxethanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine

181 polypeptide oleate, polyoxyethylene- 660 hydroxystearate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 gg

f) 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 gg; wherein the CP is either TT or DT or CRM 197

b) Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

c) Sodium chloride 1 - 10 mg

f) Sucrose 2.5 - 25 mg

g) Water for Injection (WFI) q.s.

59. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

c) Sodium chloride 1-10 mg

f) 2-Phenoxyethanol I - 10 mg

g) Water for Injection (WFI) q.s.

182

60. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

b) Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM197

c) Sodium chloride 1 - 10 mg

f) Sucrose 2.5 - 25 mg

g) 2-Phenoxyethanol 1 - 10 mg

h) Water for Injection (WFI) q.s.

61. The immunogenic composition as claimed in claim I, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 gg;

c) Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1 - 10 mg

e) 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 gg;

d) Sodium chloride 1 - 10 mg

183

e) 2-Phenoxyethanol 1-10 mg

f) Water for Injection (WFI) q.s.

63, The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

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 pg;

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 saccharîde - CP conjugate antigen 1.25 - 50 μg; wherein the CP is either TT or DT or CRM197

d) Sodium chloride 1-10 mg

f) 2-Phenoxyethanol 1-10 mg

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 pg;

c) Salmonella enterica serovar typhimurium saccharîde - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 mg

f) Sucrose 2.5 - 25 mg

g) 2-Phenoxyethanol 1 - 10 mg

h) Water for Injection (WFI) q.s.

185

B

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 pg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg; 5 wherein the CP is either TT or DT or CRM197

d) Sodium chloride 1 - 10 mg

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 pg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 ~ 50 pg; 20 wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1 - 10 mg

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 conjugale antigen 1.25 - 50 gg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM197

c) Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 mg

f) Polysorbate selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 85, non ylphenoxypolyethox éthanol, octylphenoxypolyethoxethanol, oxtoxynol 40, nonoxynol- 9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene- 660 hydroxy stéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 gg

g) 2-Phenoxyethanol I - 10 mg

h) 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 conjugale antigen 1.25 - 50 gg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 gg

g) Sucrose 2.5 - 25 mg

h) 2-Phenoxyethanol 1-10 mg

i) Water for Injection (WFI) q.s.

187

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;

c) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 μg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 mg

e) 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 CRM 197

d) Sodium chloride 1 - 10 mg

e) 2-Phenoxyethanol 1 - 10 mg

f) 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 CRM 197

d) Sodium chloride 1-10 mg

e) Tris Buffer or Citrate buffer or Hîstidine buffer or Succinate Buffer 0.1 mg - 1.6 mg

f) 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;

188

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 qg;

wherein the CP is either TT or DT or CRM 197

c) Salmonella enterica serovar enteritidis saccharide - CP conjugale antigen 1.25 - 50 qg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 mg

f) 2-Phenoxyethanol 1 - 10 mg

g) 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 conjugale antigen 1.25 - 50 qg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 qg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1 - 10 mg

e) Tris Buffer or Citrate buffer or Histidine buffer or Succinate Buffer 0. i 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 in an amount of 25-500 qg

g) 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 typhi ViPs-TT conjugale antigen 1.25 - 50 qg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 qg; wherein the CP is either Tl' or DT or CRM 197

d) Sodium chloride 1-10 mg

189

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 in an amount of 25-500 pg g) Sucrose 2.5 - 25 mg h) 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 typhi VÎPs-ΤΤ conjugate antigen 1.25 - 50 pg;

c) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197

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 in an amount of 25-500 pg g) 2-Phenoxyethanol 1-10 mg h) 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 typhi ViPs-TT conjugate antigen 1.25 - 50 pg;

c) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1-10 mg

190

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 in an amount of 25-500 pg

g) Sucrose 2.5 - 25 mg

h) 2-Phenoxyethanol l - 10 mg

i) Water for Injection (WFI) q.s.

79. The immunogenic composition as claîmed in claim I, wherein 0.5 ml of the composition comprises of:

c) Salmonella enterica serovar typhimurium saccharide — CP conjugate antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197

d) Sodium chloride 1 - 10 mg

e) Water for Injection (WFI) q.s.

80. The immunogenic composition as claîmed in claim 1, wherein 0.5 ml of the composition comprises of:

d) Sodium chloride 1 - 10 mg

e) 2-Phenoxyethanol 1-10 mg

f) Water for Injection (WFI) q.s.

81. The immunogenic composition as claîmed in claim 1, wherein 0.5 ml of the composition comprises of:

191

82. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 pg;

83. The immunogenic composition as claimed in claim 1, wherein 0.5 mi of the composition comprises of:

a) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 — 50 pg;

84. 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 pg; wherein the CP is either TT or DT or CRM197

85. 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 pg; 25 wherein the CP is either TT or DT or CRM197

b) Salmonella enterica serovar paratyphi A OSP - CP conjugale antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM197

86. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition comprises of:

a) Salmonella enterica serovar enteritidis saccharide — CP conjugale antigen 1.25 — 50 pg; wherein the CP is either TT or DT or CRM 197

87. The immunogenic composition as claimed in claim 1, wherein 0.5 ml of the composition 25 comprises of:

a) Salmonella enterica serovar typhi ViPs-TT conjugale antigen 1.25 - 50 pg;

c) Salmonella enterica serovar typhimurium saccharide - CP conjugale antigen 1.25 - 50 pg; 30 wherein the CP is either TT or DT or CRM 197

d) Salmonella enterica serovar enteritidis saccharide - CP conjugale antigen 1.25 - 50 pg;

wherein the CP is either TT or DT or CRM 197

e) Sodium chloride 1-10 mg

194

f) 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 gg;

c) Salmonella enterica serovar typhimurium saccharide - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRMI97

d) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRM 197

e) Sodium chloride 1 - 10 mg

f) 2-Phenoxyethanol 1 - 10 mg

g) 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 gg;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 gg; wherein the CP is either TT or DT or CRMI97

c) Salmonella enterica serovar typhimurium saccharide — CP conjugate antigen 1.25 — 50 gg; wherein the CP is either TT or DT or CRM 197

d) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 gg; 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.

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 gg;

195

d) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen l .25 - 50 pg;

wherein the CP is either TT or DT or CRM 197

e) Sodium chloride I - 10 mg

g) 2-Phenoxyethanol 1 - 10 mg

h) 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 pg;

d) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

e) Sodium chloride 1-10 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 in an amount of 25-500 pg

h) Water for Injection (WFI) q.s.

92. 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 pg;

e) Sodium chloride 1-10 mg

196

h) Sucrose 2.5 - 25 mg

i) Water for Injection (WFI) q.s.

93. 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 pg;

c) Salmonella enterica serovar typhimurium saccharîde — CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

d) Salmonella enterica serovar enteritidis saccharîde - CP conjugate antigen 1.25 - 50 pg; wherein the CP is either TT or DT or CRM 197

e) Sodium chloride 1 - 10 mg

h) 2-Phenoxyethanol 1 - 10 mg

i) Water for Injection (WFI) q.s.

94. 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 pg;

197

d) Salmonella enterica serovar enteritidis saccharide - CP conjugate antigen 1.25 - 50 gg;

wherein the CP is either TT or DT or CRM 197

e) Sodium chloride 1-10 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 hydroxystéarate, polyoxyethylene- 35 ricinoleate, soy lecithin and a poloxamer in an amount of 25-500 gg

h) Sucrose 2.5-25 mg

i) 2-Phenoxyethanol 1-10 mg

j) Water for Injection (WFI) q.s.

95. A single dose vaccine kit comprising:

a) Neisseria meningitidis A saccharide - TT conjugate antigen 5 gg per 0.5 ml;

b) Neisseria meningitidis C saccharide - CRM 197 conjugate antigen 5 gg per 0.5 ml;

c) Neisseria meningitidis Y saccharide - CRM 197 conjugate antigen 5 gg per 0.5 ml;

d) Neisseria meningitidis IV -135 saccharide - CRM 197 conjugate antigen 5 gg per 0.5 ml;

e) Neisseria meningitidis X saccharide - TT conjugate antigen 5 gg 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;

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 gg per 0.5 ml;

b) Sodium chloride 1-10 mg per 0.5 ml;

c) Water for Injection (WFI) q.s.;

wherein there is no antigenic interférence of ViPs-TT with Neisseria meningitidis antigens, for prophy Iaxis against typhoid caused by Salmonella typhi and Neisseria meningitidis antigens wherein the said vaccine formulation is sufficient to elicit the required T- dépendent

198 immune response against S. typhi including in children below 2 years of âge, adolescent adult, and elders through oniy one injection to comprise a complété vaccination schedule.

96. A single dose vaccine kit comprising:

a) Neisseria meningitidis A saccharide — TT conjugate antigen 5 μg per 0.5 ml;

b) Neisseria meningitidis C saccharide - CRM 197 conjugate antigen 5 μg per 0.5 ml;

c) Neisseria meningitidis Y saccharide - CRM 197 conjugate antigen 5 μg per 0.5 ml;

d) Neisseria meningitidis IV -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;

a) Salmonella enterica serovar typhi ViPs-TT conjugate antigen 1.25 - 50 μg per 0.5 ml;

b) Salmonella enterica serovar paratyphi A OSP - CP conjugate antigen 1.25 - 50 gg per 0.5 ml; wherein the CP is either TT or DT or CRM 197;

c) Sodium chloride 1-10 mg per 0.5 ml;

d) Water for Injection (WFI) q.s.;

wherein there is no antigenic interférence 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- dépendent immune response against S. typhi and paratyphi including in children below 2 years of âge, adolescent adult, and elders through only one injection to comprise a complété vaccination schedule.

97. A single dose vaccine kit comprising:

a first container containing a fully liquid hexavalent immunogenic composition:

199

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 selected from

98. A single dose vaccine kit comprising:

a first container containing a fully liquid hexavalent immunogenic composition:

200

b) a diphtheria toxoid, (D) antigen in an amount of 1 to 50 Lf per 0.5 ml;

99. A single dose vaccine kit comprising:

100. A single dose vaccine kit comprising:

202 a first container containing a fully liquid heptavaient immunogenic composition:

101. A method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugale 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, température maintaîned 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 CT AB, ethylene di amine tetra-acetic acid (EDTA) (4 to lOmM), Sodium acetate (5% to 10%), alcohol précipitation (30% to 60%) and concentration and diafiltration using 30kDa cul off membranes at different steps, wherein the process results in significant réduction of endotoxin (< 100EU of endotoxin per gg of PS), protein (< 1%) and nucleic acid (< 2%) impurities, higher recovery of capsular polysaccharide suitabiy 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 conjugale using Gel Filtration Chromatography or Ultra filtration whereby the conjugale bulk is concentrated at least 3 fold and substantiaily ail unreacted compounds, unconjugated polysaccharides, unconjugated proteins are removed,

204 yielding a purified Vi polysaccharide conjugate vaccine having size between 1000 to 1500 kDa wherein the conjugate yield is > 50%.

102. The method of manufacturîng the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claîmed in claim 101, wherein before conjugation the ViPs subjected to depolymerization/sizing by Chemical means selected from the group of FeC13, H2O2, sodium metaperiodate and sodium acetate or mechanical means selected from the group of High pressure cell disruption , homogenizer, sonication.

103. The method of manufacturîng the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claîmed in claim 101, wherein the Purified ViPs is covalently bound to carrier protein (CP) using carbodiimide conjugation reaction in presence of l-ethyl-3(3dimethylaminopropyl) carbodiimide (EDAC) mixed in a ratio of 1:0.5 to 1:2 by weight of ViPs: EDAC and the ratio of Vi polysaccharide and carrier protein is between 0.5 and 1.5, ViPs: CP: EDAC 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, température in range of 2°C to 30°C.

104. The method of manufacturîng the Salmonella enterica serovar typhi saccharide-carrier protein conjugale as claîmed in claim 101 and 103, 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, 1,6diaminooxyhexane or β-propinamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid, using a carbodiimide, reductîve amination or cyanylation reaction.

105. The method of manufacturîng the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claîmed in claim 104, wherein before conjugation the carrier protein (CP) is derivatized with an adipic acid dihydrazide (ADH) linker in the presence of carbodiimide, such as l-ethyl-3(3-dimethyIaminopropyl) carbodiimide (EDAC), reaction duration for 1 hour, carried out at a pH in range of 5-7.

106. The method of manufacturîng the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claîmed in claim 101, 102, 103, 104 and 105 wherein the carrier protein is selected from the group comprising of tetanus toxin, tetanus toxoid, fragment of tetanus toxoid, dîphtheria toxoid, CRM 197, Pseudomonas aeruginosa toxoid, Bordetella pertussis

205 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 léthal factor (LF) of Bacillus anthracis, ovalbumin, keyhole limpet hemocyanin (KLH), human sérum albumîn, bovine sérum albumin (BSA), purified protein dérivative 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, dérivatives, modifications thereof.

107. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 106, wherein the carrier protein is tetanus toxoid (TT).

108. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 106, wherein the carrier protein is diphtheria toxoid (DT).

109. The method of manufacturing the Salmonella enterica serovar typhi saccharide-carrier protein conjugate as claimed in claim 106, wherein the carrier protein is CRMI97.

110. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate comprises of:

a. Purifying Salmonella Paratyphi A O-specific polysaccharide (OSP) of the lipopolysaccharide (LPS), by Acid hydroiysis 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 lOkDa to 30kDa eut off membranes at different steps, wherein the process results in significant réduction of endotoxin (< 100EU of endotoxin per pg 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)

206 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

111. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate as claimed in claim 110, wherein before conjugation the OSP is subjected to depolymerization/sizing by Chemical means selected from the group of FeCh, H2O2, sodium metaperiodate and sodium acetate or mechanical means selected from the group of High pressure cell disruption ,sonication and homogenizer.

15

112. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate as claimed in claim 110, 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), I- cyano- îmidazole ( 1-CI), 1-cyanobenzotriazole (1-CBT), 20 l-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).

113. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide carrier protein conjugate as claimed in claim 112, wherein the purified OSP is covalently 25 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, température in range of 2°C to 30°C.

30

114. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate as claimed in claims 112 and 113, wherein before conjugation the carrier protein is derivatized with a hetero or homo-bifunctional linker selected from the

207 group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, εamînohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine and pnitrophenylethyl amine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or βpropinamido, nitrophenyl ethylamine, haloalkyl halide, 6-amino caproic acid, using a 5 carbodiimide, reductive amination or cyanylation reaction

115. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate as claimed in claim 114, wherein before conjugation the carrier protein (CP) is derivatized with an adipic acid dihydrazide (ADH) linker in the presence of carbodiimide, such as l-ethyl-3(3-dimethylammopropyl) carbodiimide (EDAC), mixed in a 10 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, température in range of 2°C to 30°C.

116. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate as claimed in claim 110, wherein the purified OSP is covalently 15 bound to carrier protein (CP) using carbodiimide conjugation reaction in presence of 1-ethyl-

117. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide20 carrier protein conjugate as claimed in claim 116, wherein before conjugation the OSP is derivatized with a hetero or homo-bifunctional linker seiected from the group consisting of hydrazine, carbohydrazide, hydrazine chloride, a dihydrazide, ε-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronoiactone, cystamine and p-nitrophenylethyl amine, hexanediamine, ethylenediamine, 1,6-diaminooxyhexane or β-propinamido, nitrophenyl 25 ethylamine, haloalkyl halide, 6-amino caproic acid, using a carbodiimide, reductive amination or cyanylation reaction.

118. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide carrier protein conjugate as claimed in claim 117, wherein before conjugation the OSP is derivatized with an adipic acid dihydrazide (ADH) linker using cyanylation conjugation 30 chemistry wherein the cyanylation reagent is seiected from a group of 1-cyano- 4pyrrolidinopyridinium tetrafluoroborate (CPPT) (CPIP), 1- cyano- imidazole (I-CI), 1cyanobenzotriazole (1-CBT), l-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate

208 (‘CDAP’), p-nitrophenylcyanate and N-cyanotriethylammonium tetrafluoroborate (‘CTEA’) or 2-cyanopyridazine -3(2H) one (2-CPO).

119. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate as claimed in claim 118, wherein before conjugation the OSP is 5 derivatized with an adipic acid dihydrazide (ADH) linker using cyanyiation conjugation chemistry wherein the cyanyiation reagent is 1-cyano- 4-pyrroIidinopyridinium 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, température in range of 2°C to 30°C.

10

120. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharide- carrier protein conjugate as claimed in claims 110 to 119, wherein the carrier protein is selected from the group comprising of tetanus toxîn, 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-iabile

121. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate as claimed in claim 120, wherein the carrier protein is tetanus toxoid 30 (TT).

209

122. The method of manufacturing the Salmonella enterica serovar Paratyphi A saccharidecarrier protein conjugate as claimed in claim 120, wherein the carrier protein is diphtheria toxoid (DT).

123. The method of manufacturing the Salmonella enterica sera var Paratyphi A saccharide 5 carrier protein conjugate as claimed in claim 120, wherein the carrier protein is CRM 197.

124. The immunogenic composition according to any one of the preceding daim, 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 10 method involving administration of an effective amount of the immunogenic composition to a human subject 2 years of âge or below, adolescent, adults or elders via via parentéral 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, pérorai or intragastric or mucosal or perlînqual, 15 alveolar or gingival or olfactory or respiratory mucosa administration or any other routes of immunization.

125. 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.

20

126. 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 kDa, 80 kDa and 45 kDa polysaccharide.

127. The immunogenic composition according to any one of the preceding claim, wherein the 25 immunogenic composition is formulated for administration to a human subject 2 years of âge or below, adolescents, adults or elders according to a one dose regimen consisting of a single dose of the said vaccine composition.

128. 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 âge 30 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.

210

129. 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 âge or below, adolescents, adulis 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 5 third dose to be administered between 3 months to 2 years after the second dose.