Field of the Invention
The present invention relates to an immunogenic composition comprising Vi capsular polysaccharide protein conjugate. More particularly, the present invention provides an immunogenic composition comprising fragmented Vi capsular polysaccharide having an average molecular weight between 100-200 KD conjugated to a carrier protein.
Background of the Invention
In developing countries, typhoid fever is common, serious, and increasingly difficult to treat because of resistance of the bacillus to antibiotics. Typhoid fever has been thought of as a disease of mostly older children and young adults. In children less than 5 years of age, typhoid fever was often unrecognized due to a typical clinical symptoms, difficulties in drawing blood and less-than-optimal culture media. Vaccination against typhoid fever caused due to Salmonella typhi is essential for protection against this disease due to increasing antibiotic resistance.
Purified capsular polysaccharide from Salmonella typhi (Vi) is used as a vaccine, providing about 70% protection against typhoid fever in individuals 5- to 45-years-old. However, the vaccine is unable to establish immunological memory and is ineffective in infants or toddlers.
The licensed vaccines such as Vivotif® (Live attenuated Ty21a), Typherix® (Salmonella typhi Vi polysaccharide), TyphimVi® (Vi Polysaccharide), showed moderate protections and have multiple deficiencies. Most important of all, none of the licensed typhoid vaccines demonstrated protection for children under 5 years old. These limitations impeded successful implementation of typhoid vaccination programs. Recently, two conjugate vaccines such as Peda typh™ (Vi polysaccharide conjugated with Tetanus toxoid), Typbar TCV ® (Vi Polysaccharide Tetanus Toxoid Conjugate vaccine), are licensed in India for all ages, starting as young as 3 month old.
Polysaccharide protein conjugate vaccines are primarily used for the prevention of bacterial infections. Some pathogenic bacteria are covered with a polysaccharide

capsule that primarily helps protect the bacteria from phagocytosis, or uptake of the bacteria by immune cells. The production of specific antibodies to the polysaccharide coat in an infected person can increase phagocytosis of bacteria, thus stimulating an immune response. Therefore, vaccination using polysaccharides from pathogenic bacteria is a potential strategy for boosting host immunity.
Although polysaccharides are immunogenic on their own, conjugation of polysaccharides to protein carriers has been used to improve immunogenicity. The carrier protein can be either a related protein antigen from the target pathogen, boosting the specific immune response to that pathogen, or generally an immunogenic protein that serves more as an adjuvant or general immune response stimulant.
Szu et. al, Infection and immunity; Dec. 1989, 57(12), 3823-27 disclosed the effect of molecular weight or size of the components on the immunogenicity of polysaccharide-protein conjugates prepared with the native Vi capsular polysaccharide (Vi) (~3 x 103 kilo Daltons) or lower molecular-weight Vi (Vis; ~46 kilo Daltons) abound to cholera toxin (CT) or to its B subunit (CTB) was studied.
Micoh et.al, PLoS One. 2012; 7(ll):e47039: November 7, 2012 disclosed new conjugation chemistry to link 0:2 and the carrier protein CRM197, using the terminus 3-deoxy-D-manno-octulosonic acid (KDO), thus leaving the 0:2 chain unmodified .
Szu et.al, (Expert Rev. Vaccines 12(11), 1273-1286 (2013)) disclosed to improve immunogenicity, Vi was conjugated to rEPA, a recombinant exoprotein A from Pseudomonas aeruginosa. Vi-rEPA showed higher and longer lasting anti-Vi IgG in adults and children than Vi alone in high endemic areas.
US 6,045,805 disclosed fragmentation of the Vi polysaccharide of Salmonella typhi in the presence of oxidation-reduction agent selected from ascorbic acid and hydrogen peroxide, and in the presence of an iron or copper salt, wherein the mean MW of the oligosaccharides formed after one and two hours corresponds to a KB of the order of 0.7

and 0.8 respectively; equivalent to 30,000 and 10,000 DEq. This patent further disclosed preparation of an oligosaccharide Vi of Salmonella typhi /B subunit of the cholera toxin conjugate.
WO 2009/150543 disclosed a method for producing a Vi-CRM197 conjugate, wherein Vi is combined with derivatised CRM197. The Vi moiety in the conjugate is preferably a low molecular weight Vi polysaccharide or an oligosaccharide, as defined above.
IN 1330/MUM/2010 disclosed a reproducible and cost-effective size reduction method of polysaccharides using a non-chemical method to yield fragments of desired size wherein desired structural features of the polysaccharides are retained.
WO 2015/029056 Al disclosed a stable conjugate vaccine formulation for protection against Salmonella typhi, and methods of conjugation between Vi-polysaccharide of Salmonella typhi to tetanus toxoid as the carrier protein. The molecular size of purified ViPs are of approximately 250 kDa.
US 2016/0263213 Al disclosed a conjugate comprising a fragmented Vi polysaccharide and a carrier protein selected from CRM197 or diphtheria toxoid, wherein the fragmented Vi polysaccharide has an average molecular weight of between 40 and 55 KDa. It discloses critical chain length (about 82 KDa) below which the Vi polysaccharide is no longer able to act as T-independent antigen.
None of the above references disclose fragmented Vi polysaccharide having an average molecular weight between 100-200 KD or purification of Vi polysaccharide and fragment thereof free from the use of organic solvents and detergents. The inventors of the present invention have found that the conjugate having Vi polysaccharide size between 100-200 KD, exhibits higher single dose and booster immune response in animals.

Objective of the Invention
It is the objective of the present invention to provide an immunogenic composition comprising fragmented Vi capsular polysaccharide-protein conjugate.
Yet another objective of the present invention is to provide protein-polysaccharide conjugate prepared from 100-200 KD of Vi polysaccharide with T-dependent (Memory) Booster response.
Summary of the Invention
The present invention provides an immunogenic composition comprising fragmented Vi capsular polysaccharide having an average molecular weight between 100-200 KD conjugated to a carrier protein.
The present invention also provides a process for manufacturing a conjugate comprising fragmented Vi capsular polysaccharide having an average molecular weight between 100-200 KD and a carrier protein.
Brief Description of the Drawings
Figure 1: Fragmentation process of Vi polysaccharide.
Figure 2 - HPLC profile of the fragmented Vi-polysaccharide used for preparing VI-polysaccharide-protein conjugate.
Figure 3 - HPLC profile of the of Vi-polysaccharide and CRM197 conjugate prepared from 100-200 KD Vi polysaccharide.
Figure 4 - Analysis of identity and O-Acetyl composition of fragmented Vi-polysaccharide used in the preparation of Vl-polysaccharide-protein conjugates by *H NMR.

Figure 5 -Immunization schedule of Vi-CRMi97 conjugate prepared from 150 KD polysaccharide.
Figure 6: Quantification of Anti-Vi IgG titre in mice immunized with Vi-CRMi97 conjugate vaccine prepared by using 150 KD polysaccharide.
Figure 7: Quantification of Anti-Vi IgG titre in rabbit immunized with Vi-CRMi97 conjugate vaccine prepared by using 150 KD polysaccharide.
Detailed Description of the Invention
The present invention provides an immunogenic composition comprising fragmented Vi capsular polysaccharide having an average molecular weight ranging between 100-200 KD.
The present invention also provides an immunogenic composition comprising fragmented Vi capsular polysaccharide having an average molecular weight ranging between 100-200 KD conjugated to a carrier protein.
The carrier protein according to the invention is selected from CRM197, tetanus toxoid or diphtheria toxoid.
The term "Vi" or "Vi polysaccharide" refers to the capsular polysaccharide of Salmonella enterica serovar Typhi.
The term "fragmented" as used herein refers to the Vi polysaccharide having undergone size reduction thus reducing the number of repeating units in the polysaccharide. Fragmented Vi therefore has a lower average molecular weight compared to native Vi.
The term "native polysaccharide" as used herein refers to a polysaccharide which has not been subjected to a process, the purpose of which is to reduce the size of said polysaccharide.

Vi polysaccharide in its native size, has an average molecular weight of about 350 KD when measured by HPLC size exclusion chromatography (HPLC-SEC). Fragmented Vi of the present invention has an average molecular weight (MW) of between 100 to 200 KD as measured by HPLC-SEC.
The average molecular weight is typically calculated by running the sample on a TSK gel 3000 PWXL column, (30 cmx7.8 mm; particle size 7 um; cod. 808021) with a TSK gel PWXL guard column (4.0 cmx6.0 mm; particle size 12 um; cod. 808033) (Tosoh Bioscience) using Pullulan as standards (5, 25, 50, 100, 150, 200, 400 & 800 kDa). The mobile phase is 0.1 M NaCl, 0.1 M NaH2P04, 5% CH3CN, pH 7.2, at the flow rate of 0.5 mL/min (isocratic method for 30 min). Void and bed volume calibration is performed with X-DNA (X-DNA Molecular Weight Marker III 0.12-21.2 kb; Roche) and sodium azide (NaN3; Merck), respectively.
The present invention provides an immunogenic composition comprising Vi-capsular polysaccharide - protein conjugate wherein the booster dose response of the fragmented Vi polysaccharide having an average molecular weight of between 100-200 KD is higher than the native polysaccharide conjugate.
Citrobacter freundii is commonly used as surrogate host for production of Vi polysaccharide. This is because of the fact that Vi polysacchirde produced by Citrobacter is structurally and immunologically identical to the Vi polysaccharide produced by wild type Salmonella Typhi strain.
The present invention also provides a process for manufacturing a conjugate comprising fragmented Vi polysaccharide and a carrier protein comprising the steps of:
I) culturing Citrobacter freundii or Salmonella typhi in suitable culture medium under suitable conditions,
II) isolating the concentrated Vi polysaccharide,
III) sizing Vi polysaccharide using hydrogen peroxide (H2O2),

IV) removal of protein impurities, nucleic acid impurities & endotoxins using anion exchange chromatography,
V) subjecting the purified polysaccharide obtained in step (IV) to ultrafiltration to obtain size reduced purified Vi polysaccharide having an average molecular weight of 100-200 KD,
VI) activating the fragmented Vi capsular polysaccharide obtained in step (v) using ED AC to form an activated polysaccharide and
VII) conjugating the activated capsular polysaccharide with the carrier protein.
In a preferred embodiment, the present invention provides an immunogenic composition comprising fragmented Vi capsular polysaccharide having an average molecular weight between 125-175 KD conjugated to a carrier protein.
In a more preferred embodiment, the present invention provides an immunogenic composition comprising fragmented Vi capsular polysaccharide having an average molecular weight between 130-160 KD conjugated to a carrier protein.
In yet another embodiment, the present invention provides a fragmented Vi capsular polysaccharide (fVi) - CRM197 conjugates synthesized using EDAC/NHS chemistry. CRMi97was derivatized with ADH in MES buffer pH 6.0. Then fVi carboxylates (-COOH) were activated with EDAC in presence of N-hydroxysuccinimide, by forming an active ester intermediate, which increases the efficiency of conjugation with CRM197 -ADH, avoiding fVi modification further.
The present invention provides an organic solvent and detergent free, industrially scalable process for the recovery of size reduced bacterial antigenic polysaccharides selected from Citrobacter freundii and Salmonella typhi.
In yet another preferred embodiment, the present invention provides a process for manufacturing a conjugate comprising fragmented Vi capsular polysaccharide having

an average molecular weight between 130-160 KD and a carrier protein comprising the
steps of:
I) culturing Citrobacter freundii or Salmonella typhi in suitable culture medium
under suitable conditions, isolation of concentrated crude antigenic
polysaccharides,
II) sizing polysaccharide using hydrogen peroxide (H2O2),
III) removal of protein impurities, nucleic acid impurities & endotoxins using anion exchange chromatography,
IV) the purified polysaccharide obtained in step (III) is subjected to ultrafiltration to obtain size reduced purified antigenic polysaccharide having a fragmented Vi capsular polysaccharide with an average molecular weight of 130-160 KD;
V) activating the fragmented Vi capsular polysaccharide to form an activated polysaccharide;
VI) conjugating the activated capsular polysaccharide with the carrier protein.
The present invention provides a process for preparing an immunogenic composition comprising fragmented Vi capsular polysaccharide having an average molecular weight between 100-200 KD conjugated to a CRM197 carrier protein.
The present invention also provides a process for preparing an immunogenic composition comprising 10-100 ug/ml Vi capsular polysaccharide CRM197 protein conjugate, wherein VI polysaccharide having an average molecular weight between 100-200 KD, preferably 25 to 50 ug/ml.
In an embodiment, the present invention provides a pharmaceutical composition comprising the conjugate of the invention in combination with one or more pharmaceutically acceptable carrier / excipients.
The present invention also provides a formulation comprising 10 to 100 ug/ml, preferably 25 to 50 ug/ml Vi capsular polysaccharide CRM197 protein conjugate

prepared by diluting the Vi capsular polysaccharide CRM197 protein conjugate in phosphate buffered saline at neutral pH.
The present invention also provides an immunogenic composition of a typhoid conjugate vaccine comprising Vi capsular polysaccharide-protein conjugate bulk wherein Vi capsular polysaccharide having an average molecular weight ranging from 100-200 KD, preferably, 125-175 KD and more preferably 130-160 KD.
In a most preferred embodiment, the present invention provides an immunogenic composition comprising Vi capsular polysaccharide having an average molecular weight of 150 KD conjugated to CRM197 as carrier protein.
Pharmaceutical compositions of the invention comprise an immunologically effective amount of Vi polysaccharide protein conjugate. The effective amount of Vi polysaccharide according to the present invention is about 1 ug to 40 ug and about between 1 ug to 20 ug of carrier protein.
The invention also provides a pharmaceutical composition comprising the conjugate of the present invention in combination with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes adjuvant, surfactants, antioxidants, preservatives, isotonic agents, salts, stabilizers, and the like and combinations thereof.
Immunogenic compositions of the present invention may be adjuvanted or unadjuvanted. The adjuvant may include aluminum hydroxide and aluminum phosphate.
The Vi capsular polysaccharide of the present invention is prepared by chemical hydrolysis with hydrogen peroxide, without altering the repeating units. When the fragmentation is carried out by chemical hydrolysis with hydrogen peroxide, addition of a catalytic amount of ferrous sulfate (FeS04) allows the reaction to work under milder conditions.

In yet another embodiment of the present invention, there is provided a method for fragmenting a polysaccharide comprising the step of reacting the native polysaccharide with hydrogen peroxide in the presence of catalytic amount of ferrous sulfate.
The various size of Vi-polysaccharides was conjugated to carrier protein CRM197 in order to prepare Typhoid conjugate vaccine Vi-CRMi97. The immunogenicity of conjugate vaccines prepared from different sizes of Vi polysaccharide has been evaluated in mice and rabbit animal model (Comparative Immunogenicity of Vi Polysaccharide-Protein Conjugates Composed of Cholera Toxin or Its B Subunit as a Carrier Bound to High or Lower Molecular Weight Vi. Infection and Immunity; page 3823-3827, Dec. 1989). In rabbit, protein-polysaccharide conjugate vaccine formulation containing a 25 ug of Vi antigen and in mice vaccine formulation containing 2.5ug Vi antigen (calculated based on single human dose of Typhoid conjugate vaccine) was administered in via either subcutaneous route (SC) or intramuscular route (EVI) using the schedule mentioned in Figure 5. Two doses of vaccine were given at day 0, Day 28 and sera was collected pre and post each vaccination. The antibody specific to Vi antigen (Anti-Vi IgG) immune response in sera was determined by ELISA (Enzyme Linked Immunosorbent Assay).
The Vi-CRMi97 conjugate vaccine prepared according to the present invention has been used for immunization in various animal models and it is found that this conjugate has been able to show strong anti-Vi antibody response in mice and rabbits. Noteworthy, the immune response obtained by conjugate of the present invention is significantly higher than the response obtained by conjugate prepared from Vi polysaccharide having lower molecular weight e.g. ~50 D and ~10 KD. Furthermore, the novel conjugate of the present invention is able to show a booster response in animal confirming the T-dependent nature of immune response prepared by 100-200 KD Vi conjugates.
Example below are intended to illustrate the invention and are not to be construed as being limitations thereon.

Example 1
Step (i): Preparation of Vi polysaccharide (Figure 1)
1. Fermentation process:
Vi polysaccharide was purified by pilot scale fermentation oi Citrobacter freundii sensu lato 3056 strain. For the production of Vi polysaccharide, glycerol stock oi Citrobacter freundii sensu lato 3056 strain was grown in Seed flask to OD600 > 1 and culture was transferred into fermentor.
Table 1. Pre-seed and Seed Media composition:
Fermentation was carried out at 37°C, maintaining the pH 6.0 ± 0.2 and dissolved oxygen (DO) >30%, respectively. Once culture attained the stationary stage, fermentation broth was harvested by centrifugation followed by filtration with a 0.2-um-pore-size filter. Vi polysaccharide was purified by a process that combined precipitation, resolubilization and filtration. The structural identity and O-acetylation level of Vi was confirmed by *H NMR (Figure 4). Molecular size of polysaccharide was determined by SEC-HPLC method. The nucleic acid and protein impurity in Vi was measured by A260 and micro BCA respectively.

2. Ultrafiltration Diafiltration-I:
Clarified fermenter harvest was concentrated and diafiltered using 100 / 300 kDa UF membrane.
3. Fragmentation:
100 kDa ultra filtered crude polysaccharide was used as starting material for fragmentation and purification of the Vi polysaccharide.
Fragmented Vi polysaccharide was obtained by means of fragmentation reaction wherein chemical hydrolysis were carried out with 0.5% w/v of hydrogen peroxide (H2O2) in the presence of Ferrous Sulphate as a catalyst. The temperature of the reaction was kept at 15±2°C and reaction was carried out for 2 hour. Further reaction was quenched by addition of EDTA (Ethylenediaminetetraacetic acid) at the concentration

of 10 mM to obtain targeted fragment size having an average molecular weight of 150 kDa.
4. Ultrafiltration Diafiltration (buffer exchange):
Quenched reaction mixture was concentrated 3-5X by maintaining transmembrane pressure (TMP) in range of 0.8-1.5 bar. After concentration, diafiltration (8-10DV) was performed with diafiltration Buffer to remove H2O2 and EDTA used during fragmentation reaction using 100 kDa membrane.
5. Stabilization:
Fragmented Vi was stabilized by heating at 80° C for 2 Hour.
6. Purification of fragmented Vi PS :
Stabilized fragmented Vi was further purified using ion exchange chromatography.
7. Desalting of Vi PS:
Purified fragmented Vi PS was concentrated and diafiltered using 30 kDa membrane with WFI.
Step (ii): Preparation of Vi - CRM197 conjugate bulk:
Vi- CRM197 conjugates were synthesized using EDAC/NHS chemistry. Briefly, CRM197 was derivatized with ADH in MES buffer pH 6.0. Then Vi carboxylates (-COOH) were activated with ED AC in presence of N-hydroxysuccinimide, by forming an active ester intermediate, increases the efficiency of conjugation with CRM197 -ADH, avoiding Vi modification further.

Characterization of Vi polysaccharide and Vi polysaccharide protein Conjugates Vi polysaccharide having an average molecular weight of 150 kDa were prepared and characterized using SEC-HPLC. The HPLC profile of the Vi-polysaccharide and Vi-polysaccharide and CRM197 protein conjugate is shown in Figures 2 & 3.
Step (iii): Immunization of Vi-CRMi97 protein-polysaccharide conjugate in Mice and Rabbit
Vi-CRMi97 protein polysaccharide conjugate having an average molecular weight of 150 kDa vaccine were injected in mice and rabbit by subcutaneous route. Two injection of vaccine were given to the animals two weeks apart. The immune sera was collected from the animal and anti-Vi IgG was quantified by ELIS A.
Step (iv): Evaluation of Immune response of Vi-CRMw conjugate vaccine by quantification of Anti-Vi IgG
For the quantification of Anti-Vi IgG in the immune sera generated against Vi-CRMi97 conjugate Vi-specific ELISA was used. Vi polysaccharide was immobilized in ELISA plate and incubated with the immune sera. The detection of Anti-Vi IgG was done following incubation of plate with secondary antibody conjugated with alkaline phosphatase.
Immunization schedule of Vi-CRMi97 conjugate, Quantification of Anti-Vi IgGtitre in mice and rabbit immunized with Vi-CRMi97 conjugate vaccine is shown in figures 5-7.
The data suggests that fragmented Vi polysaccharide having an average molecular weight of 100-200 KD conjugated with carrier protein gives better overall (Dose 1 and Dose 2) immune response than lower size of Vi polysaccharide. Further to this it has been found that booster dose response of the fragmented Vi polysaccharide is higher than the native conjugated polysaccharide.

The Vi polysaccharide protein conjugate of the present invention offers multiple benefits over existing conjugates:
1) The conjugate prepared from Vi ranging from 100-200 KD is more immunogenic as compared to conjugate made from lower size of fragmented Vi.
2) The above conjugates proffers, superior dose 1 and dose 2 response as compared to existing conjugates.
3) The conjugate of Vi polysaccharide having an average molecular weight ranging between 100-200 KD with CRM197 showed a booster response following second dose of vaccine.
4) The conjugate of Vi polysaccharide having an average molecular weight ranging between 100-200 KD with CRM197 showed T-dependent immune response in animals.
5) The conjugate of Vi polysaccharide having an average molecular weight ranging between 100-200 KD with CRM197 also offers manufacturing advantage as compared to the conjugate made from smaller size of polysaccharides or with the native polysaccharide.

We claim
1. An immunogenic composition comprising fragmented Vi capsular polysaccharide protein conjugate, wherein fragmented Vi capsular polysaccharide having an average molecular weight between 100-200 KD.
2. An immunogenic composition as claimed in claim 1, wherein the fragmented Vi capsular polysaccharide has an average molecular weight from 125-175 KD.
3. An immunogenic composition as claimed in claim 1, wherein the fragmented Vi capsular polysaccharide is conjugated to a carrier protein selected from CRM197, tetanus toxoid or diphtheria toxoid.
4. An immunogenic composition as claimed in claim 3, wherein the fragmented Vi capsular polysaccharide has an average molecular weight of 150 KD conjugated to CRM197.
5. An immunogenic composition as claimed in claim 3 and 4 wherein the amount of fragmented Vi capsular polysaccharide is about between 1 ug to 40 ug and the amount of carrier protein is about between 1 ug to 20 ug.
6. A process for manufacturing a conjugate comprising fragmented Vi capsular
polysaccharide and a carrier protein comprising the steps of:
I) culturing Citrobacter freundii or Salmonella typhi in suitable culture medium under suitable conditions,
II) isolating the concentrated Vi polysaccharide,

III) sizing Vi polysaccharide using hydrogen peroxide (H2O2),
IV) removal of protein impurities, nucleic acid impurities & endotoxins using anion exchange chromatography,
V) subjecting the purified polysaccharide obtained in step (IV) to ultrafiltration to obtain size reduced purified Vi polysaccharide having an average molecular weight of 100-200 KD,

VI) activating the fragmented Vi capsular polysaccharide obtained in step (v) using
ED AC to form an activated polysaccharide and VII) conjugating the activated capsular polysaccharide with the carrier protein.
7. The process as claimed in claim 6, wherein the fragmented Vi capsular
polysaccharide obtained has an average molecular weight of 130-160 KD.
8. The process as claimed in claims 6 and 7, wherein the carrier protein is selected from CRM197, tetanus toxoid or diphtheria toxoid.
9. A pharmaceutical formulation comprising 10 to 100 |ig/ml, preferably 25 to 50 ug/ml Vi capsular polysaccharide CRM197 protein conjugite prepared by diluting the Vi capsular polysaccharide CRM197 protein conjugate in phosphate buffered saline at neutral pH.