Structural and immunological characterization of E. coli derived recombinant CRM197 protein used as carrier in conjugate vaccines

It is established that the immunogenicity of polysaccharides is enhanced by coupling them to carrier proteins. Cross reacting material (CRM197), a nontoxic variant of diphtheria toxin (DT) is widely used carrier protein for polysaccharide conjugate vaccines. Conventionally, CRM197 is isolated by fermentation of Corynebacterium diphtheriae C7 (β197) cultures, which often suffers from low yield. Recently, several recombinant approaches have been reported with robust processes and higher yields, which will improve the affordability of CRM197-based vaccines. Vaccine manufacturers require detailed analytical information to ensure that the CRM197 meets quality standards and regulatory requirements. In the present manuscript we have described detailed structural characteristics of Escherichia coli based recombinant CRM197 (rCRM197) carrier protein. The crystal structure of the E. coli based rCRM197 was found to be identical with the reported crystal structure of the C7 CRM197 produced in C. diphtheriae C7 strain (Protein Data Bank (PDB) ID: 4EA0). The crystal structure of rCRM197 was determined at 2.3 Å resolution and structure was submitted to the PDB with accession number ID 5I82. This is the first report of a crystal structure of E. coli derived recombinant CRM197 carrier protein. Furthermore, the rCRM197 was conjugated to Vi polysaccharide to generate Typhoid conjugate vaccine (Vi-rCRM197) and its immunogenicity was evaluated in Balb/C Mice. The Vi-rCRM197 conjugate vaccine was found to generate strong primary α-Vi antibody response and also showed a booster response after subsequent vaccination in mice. Overall data suggest that E. coli based recombinant CRM197 exhibits structural and immunological similarity with the C7 CRM197 and can be used as a carrier protein in conjugate vaccine development.


Introduction
Over the past three decades, many routine childhood and adult vaccines have been developed using conjugation technology. Conjugate vaccines are developed by covalent attachment of an antigenic polysaccharide to a nontoxic carrier protein. Conjugation to a nontoxic carrier protein enhances the immunogenicity of polysaccharide antigens, enabling host defence against diseases caused by encapsulated pathogens. Conjugation transforms the T cell-independent response of polysaccharide vaccines to a T cell-dependent one. Conjugate vaccines have been demonstrated to be immunogenic and capable of inducing immunological memory and high avidity antibodies. Unlike unconjugated polysaccharides, conjugate vaccines also elicit protective responses in the immature immune system of young infants and the senescent immune system of the elderly [1][2][3].

Cloning, expression and purification of CRM 197
The codon optimized sequence corresponding to CRM 197 gene was synthesized and cloned into pTWIN 1 vector (New England Biolabs, U.K.) using BamH1 and Sap1 restriction sites [18]. The pTWIN1 CRM 197 plasmid was transformed into chemically competent BL21-DE3 E. coli expression host. Transformants were screened on LB agar plates supplemented with ampicillin (100 μg/ml). Five to ten randomly selected colonies were purified by repeated subculturing on LB + ampicillin (100 μg/ml) plate. Finally, a few colonies were selected and a research cell bank was generated. For the expression analysis of rCRM 197 protein, clones were grown in 100-ml LB + Ampicillin (100 μg/ml) till mid log phase (OD 600 ∼1). At this stage, culture was induced with 1 mM IPTG and was further grown to reach late log phase (OD 600 ∼4-5). One millilitre culture from each transformant was centrifuged and pellet was resuspended into 100-μl SDS loading buffer and heated at 80 • C for 10 min. Twenty microlitres of lysate was loaded on 8-12% Tri-Glycine gel and SDS/PAGE was run using 80 V constant current for ∼2 h. Gel was stained with Coomassie Blue (make: Amresco, Cat No: 0472-25 G). The expression of rCRM 197 was confirmed by comparing a band appearing at ∼58 kDa on the gel. Further, Western blot was performed using anti-CRM 197 antibody (Make: Santa Cruz Biotechnology, Cat No: SC2054). One of the clones expressing rCRM 197 protein has been used for fermentation and large-scale production of protein.
Fermentation inoculum was generated by inoculating one vial of research cell bank into seed flask containing 200-ml LB + ampicillin (100 μg/ml). Seed flask (containing growth media and inoculum) was grown to achieve OD 600 ∼4 and inoculated into 20-l fermentation medium. Fermentation was performed in chemically defined medium. After fermentation was complete, cell mass was separated by centrifugation. The cell pellet was lysed by Micro fluidizer high pressure homogenization. Homogenized cell mass was centrifuged and pellet containing inclusion body protein was retained and used for rCRM 197 purification. The inclusion body was solubilized in buffer containing 6 M urea at 30 • C and rCRM 197 was purified by refolding followed by ion exchange chromatography [18]. The solubilized inclusion body protein was refolded and purified by ion exchange chromatography method described by Stefan et al. [19].
C7 CRM 197 used as a reference in the studies was procured as a lyophilized powder from Sigma-Aldrich (Cat# D2189).

Endonuclease assay for rCRM 197
The assay was performed as described by Stefan et al. (2011) [19] with minor modifications. In brief, 2 μg rCRM 197 was mixed with 1 μg pUC57 plasmid DNA using endonuclease reaction buffer (10 mM Tris/HCl pH 7.6, 2.5 mM CaCl 2 , 2.5 mM MgCl 2 ). Content was mixed by brief centrifugation (3000 g, 30 s) and incubated for different time points (0, 0.5, 1, 3, 5 and 18 h) at 25 • C. The 0-h time point sample was used as a control. At each time point, reaction was terminated by the addition of 5 mM EDTA (final concentration) to each tube and frozen at −20 • C. Thawed samples were mixed with DNA loading buffer and run on 1% agarose gel electrophoresis in 1× TAE buffer using 80 V constant voltage. DNA staining dye SYBR Safe gel stain (Invitrogen, Cat# S33102) was added to the agarose gel at the time of pouring. After the electrophoresis run was completed, the gel was analysed under UV transillumination (Bio-Rad ChemiDoc Gel imaging System).

SDS/PAGE and Western immunoblot analysis
Reducing and nonreducing SDS/PAGE and Western blot analyses were performed with purified rCRM 197 as per the standard protocol [20]. Four micrograms of purified rCRM 197 along with the reference CRM 197 was loaded on to 4-12% Tris-Glycine gels under reducing and nonreducing conditions. Gels were run at 80 V constant voltage until blue dye reached the bottom of the gel. SDS/PAGE molecular weight markers (New England Biolabs, Cat# P7712S) were used for molecular weight calibration. After Coomassie Brilliant Blue staining, the image was captured using transilluminator (Bio-Rad ChemiDoc system). For Western immunoblotting, 2 μg protein was separated by SDS/PAGE and transferred to nitrocellulose membrane (0.45 μm, Bio-Rad, Cat# 1620115). The blot was probed with α-CRM 197 rabbit polyclonal primary antibody (Abcam, Cat# ab151222) by incubating the membrane for 1 h. After the primary incubation, the membrane was further incubated in HRP-conjugated secondary antibody (rabbit) (make: Santa Cruz Biotechnology, Cat No: SC2054). Bands were visualized using 3,3 -diaminobenzidine (DAB) substrate and the image captured using Bio-Rad ChemiDoc gel imaging system.

Amino acid composition analysis
rCRM 197 was subjected to high sensitivity amino acid analysis (AAA). The analysis was performed on samples desalted by ultrafiltration (Vivaspin 6 spin columns with a 10-kDa polyethersulfone filter: Sartorius, Cat# VS0601). Samples were resuspended in 20% acetonitrile (ACN) containing 0.1% trifluoroacetic acid (TFA) and subjected to gas phase hydrolysis (6 N HCl at 110 • C). α-amino butyric acid (AABA) was run as an internal standard prior to sample analysis. Cysteine analysis was performed after performic acid oxidation followed by hydrolysis as above. Hydrolysates were analysed using a Waters Acquity UPLC system with AccQTag Ultra chemistry. Samples were analysed in duplicate and the mean reported [23,24].

Determination of crystal structure of rCRM 197 by X-ray crystallography
Center for the Structural Genomics of Infectious Diseases (CSGID) standard protocols were used to determine the structure of CRM 197 [25][26][27].
Sitting drop crystallization plates were set up at room temperature and crystals of rCRM 197 were obtained by mixing Harvested crystals were transferred to the reservoir solution before being flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K at the Life Sciences Collaborative Access Team at the Advance Photon Source, Argonne, Illinois (APS BEAMLINE 21-ID-F). Data were processed using HKL-3000 for indexing, integration and scaling. The crystal structure of recombinant CRM 197 (Protein Data Bank (PDB) ID: 5I82) was determined by molecular replacement using PDB entry 4AE0 as a search model. The structure was refined with Refmac 5 [28]. Models were displayed in Coot and manually corrected based on electron density maps [29]. All structure figures were prepared using Py-MOL Molecular Graphics System, version 1.3 (Schrödinger, LLC). The structure was submitted to the PDB (PDB ID: 5I82).

Generation of typhoid conjugate vaccine (Vi-rCRM 197 ) using rCRM 197 and immunogenicity assessment
rCRM 197 was conjugated with Vi polysaccharide antigen to generate Vi-rCRM 197 conjugate vaccine (typhoid conjugate vaccine). The Vi polysaccharide was produced by fermentation of Citrobacter freundii as per method described by Rondini et al. [30]. The C. freundii stain was provided by Dr Laura B. Martin, GSK Vaccine Institute of Global Health, Siena, Italy. The Vi-rCRM 197 conjugate was prepared according to the method reported by Kossaczka et al. [31] and as detailed by Micoli et al. [8]. Briefly, rCRM 197 was derivatized with adipic acid dihydrazine (ADH). Then, Vi (purified from C. freundii) was activated with N-hydroxy succinamide and 1-ethyl-3(3-dimethylaminopropyl) carbodiimide hydrochloride and linked to the derivatized rCRM 197 protein generating Vi-rCRM 197 conjugate vaccine. The ADH derivatized rCRM 197 (rCRM 197 -ADH) was mixed with activated Vi polysaccharides in 1:1 ratio and incubated at room temperature for 5-6 h. The resulting conjugate was purified by HIC (hydrophobic interaction chromatography) using phenyl sepharose (GE) resin.
The immunogenicity of Vi-rCRM 197 was performed as per procedures described by Rondini et al. [30]. Four to six weeks old female Balb/C mice were used in the experiment. A group of 30 mice were subcutaneously immunized with three doses of Vi-rCRM 197 , unconjugated Vi polysaccharide and PBS (placebo control). A 100μl dose of Vi-rCRM 197 conjugate vaccine containing 2.5 μg Vi, unconjugated Vi polysaccharide (2.5 μg) and PBS (control) were administrated in mice on day 1 followed by booster dose at days 14 and 21. Fourteen days after first immunization, ten randomly selected mice from each group were terminally bled to collect the post-dose 1 sera. Remaining animals (20 each) were given the second dose of vaccine. Seven days after second immunization (day 21), ten mice from each group were terminally bled and post-dose 2 sera was collected. Finally, remaining ten mice were given the third dose of vaccine. The final sera (post-dose 3) was collected at day 28 by bleeding of remaining mice from each group. Mice sera was subjected to ELISA analysis and anti-Vi IgG response was measured as per the procedure described by Micoli et al. [8].

Determination of molecular weight and identity of E. coli derived rCRM 197
rCRM 197 appears as a ∼58 kDa band in SDS/PAGE and Western blot analyses performed under both nonreducing and reducing conditions. This is consistent with the reported molecular weight of CRM 197 of 58.4 kDa [16]. Gels run under nonreducing conditions help to evaluate possible covalent and noncovalent aggregation in the samples and to detect impurities. rCRM 197 contains two disulfide bonds, one linking chains A and B and a second within the B chain. Gels run in reducing conditions allow detection of the proteolytically nicked form (if any) of the protein. β-mercaptoethanol (2-βME) was used to reduce disulfide bridges during sample preparation. Under these reducing conditions, rCRM 197 (test sample) showed no visible evidence of nicked and/or degraded forms in SDS/PAGE ( Figure  1A,B). Figure 1C,D summarizes the Western blot analysis of rCRM 197 and reference CRM 197 run in reducing and nonreducing conditions. The Western blot was probed with α-CRM 197 rabbit polyclonal antibody (make: Abcam; Cat No: ab151222).
Three independent manufacturing lots of rCRM 197 were evaluated in the present study and exhibited similar profiles under both nonreducing and reducing conditions, also supporting consistency of manufacturing for rCRM 197 .

Intact mass determination by MS
MS is a powerful analytical tool as it measures an intrinsic property of a molecule, its mass, with very high sensitivity and therefore it is used in a wide range of applications [22]. The intact mass of rCRM 197 was obtained by deconvolution of mass/charge (m/z) ratio. Data obtained by TOF-MS showed most abundant mass of 58413 Da compared with a theoretical calculated mass of 58421 Da based on amino acid sequence ( Figure 2). This result confirms the molecular weight of the purified rCRM 197 protein i.e. ∼58.4 kDa. Similar data have also been reported both for a recombinant CRM 197 produced in Pseudomonas fluorescens and C7 CRM 197 [21,32].

Endonuclease activity
Mild endonuclease activity is an inherent property of CRM 197 and its parent molecule DT [4] and is considered as an indicator of the correctly folded bioactive form of the protein. Incubation of CRM 197 with supercoiled plasmid DNA in the presence of divalent cations (e.g. Ca ++ and Mg ++ ) results in DNA cleavage [19,33]. Incubation of pUC57 plasmid DNA with rCRM 197 in a 2:1 ratio for different times and analysis of the DNA by agarose gel electrophoresis confirmed the positive nuclease activity in the protein. During incubation, rCRM 197 was able to linearize the supercoiled DNA. No endonuclease activity was seen in the 0-h time point sample but it increased with the incubation time ( Figure 3). This endonuclease assay can be used as an in-process control assay for CRM 197 protein produced from insoluble inclusion bodies. Similarly, earlier studies in which endonuclease activity was used to establish the purity and correctness of refolding of rCRM 197 produced in E. coli is also reported [19]. Previous reports also showed that both DT and CRM 197 should have fragment A-associated nuclease activity [33].

Determination of amino acid composition
Acid hydrolysis followed by high-sensitivity AAA has been performed with rCRM 197 and compared with reference CRM 197 . After acid hydrolysis, amino acid mixture was analysed by ultra performance liquid chromatography (UPLC) using the Waters AccQTag Ultra chemistry. All samples were analysed in duplicate and results expressed as an average. The amino acid composition of rCRM 197 closely matches (within 1-2% variability range) with reference and with the theoretical values of CRM 197 protein (Figure 4). AAA can be used to confirm the primary amino acid composition of protein. The overall data suggest that rCRM 197 produced in E. coli has similar amino acid composition as CRM 197 produced by C. diphtheriae C7 strain (reference CRM 197 ) [15].

Immunoblotting with α-CRM 197 mAbs
Three independent lots of rCRM 197 were run in SDS/PAGE and were probed with five different mAbs with specificity to different epitopes on the protein in Western immunoblotting experiments. All these mAbs recognized the protein at similar level and showed a major immune-reactive band at ∼58 kDa, the expected size of the protein ( Figure 5). The reference CRM 197 was recognized at similar level as rCRM 197 . However, a slight difference in the recognition pattern was observed with respect to different mAbs, which suggests differential affinity of different mAbs for recognition of CRM 197 . mAb mapping of CRM 197 has been found useful to assess potential nicking of A and B chains that could be seen in Western blot. A similar study was performed with different mAbs raised against CRM 197 for their ability to bind DT and six functional mutants CRM 197 , CRM 176 , CRM 228 , CRM 1001 , CRM 45 and CRM 30 by immunoblotting    data also confirm that rCRM 197 has attained the correct conformational structure after purification from insoluble inclusion bodies [35].

Determination of crystal structure of E. coli rCRM 197 by X-ray crystallography
The crystal structure of E. coli derived recombinant CRM 197 was determined at 2.3Å resolution and the structure was submitted to the PDB with the PDB ID: 5I82. Previous studies have elucidated the structural features of DT in different conditions, such as in the apo conformation, at acidic pH, in complex with NAD, with dinucleotide inhibitor adenylyl 3 -5 uridine 3 -monophosphate and with an extracellular fragment of its receptor [36][37][38][39]. More recently, the structures of C7 CRM 197 in the apo form and in complex with the NAD hydrolysis product nicotinamide have been determined [16]. This showed that the single amino acid substitution of G52E that is the difference between CRM 197 and DT, results in an intrinsic flexibility of the active-site loop [16]. All these studies were performed on DT and CRM 197 produced by C. diphtheriae. However, there is very limited information available on CRM 197 protein produced from the recombinant E. coli. To verify if the rCRM 197 produced from insoluble inclusion body protein have the overall similar 3D structure of the protein, we determined the X-ray crystal structure of rCRM 197 . Furthermore, the crystal structure of rCRM 197 confirms the presence of structural determinants that ablated the toxicity of DT and resulted in CRM 197 (G52E mutation).
The structure was determined at 2.3Å resolution by molecular replacement using a search model, the coordinates of the apo form of C7 CRM 197 (PDB entry 4EA0). Data collection, refinement and the final model validation statistics for the structure of rCRM197 (PDB ID: 5I82) have been given in Table 1   out of 504 Cα, RMSD of 0.10Å; AB and CD, 839 out of 1011 Cα, RMSD of 0.17Å) [40], demonstrating that these are very similar. The dimers in the crystal structure of rCRM 197 and C7-CRM 197 , are equivalent. By superimposing the A and B chains of rCRM 197 on to C7-CRM 197 and its symmetry-related molecule that forms the dimer, 924 out of 998 Cα superimposed with RMSD of 0.38Å (Figure 6). The overall fold of the monomer rCRM 197 is equivalent to C7-CRM 197 , with an overall RMSD of 0.34Å for 451 out of 498 superimposed Cα atoms ( Figure 6). Since overall structure is  preserved between C7 CRM 197 and rCRM 197 , it is unlikely that there will be any significant differences between these two molecules in terms of T epitopes that can potentially impact the efficacy of rCMR 197 .
Furthermore, previous studies showed that the substitution G52E induces the dislocation of the active-site loop CL2 in C7-CRM 197 with respect to DT [16]. The intrinsic flexibility of CL2 results in a localized low quality electron density map in the C7-CRM 197 structure [38,39]. In our crystal structure of rCRM 197, we have also observed a low electron density in the active loop CL2, consistent with the previous results (Figure 7). This confirms the structural basis of lack of toxicity in rCRM 197.
Another important biochemical and structural features of DT, as well as C7 CRM 197 , is the presence of the disulfide bond C186-C201 that maintains the A and B fragments bound together in the nicked form after the proteolytic cleavage of the chain. The structure revealed that the disulfide bond is conserved in rCRM 197 (Figure 8). To our knowledge, this is the first reported crystal structure of E. coli derived rCRM 197 and first described comparison with C7-CRM 197 . In conclusion, rCRM 197 conserves the overall folding, making it structurally equivalent to C7-CRM 197 , which is used in many licenced conjugate vaccine such as meningitis, pneumococcal conjugate vaccine etc. [10][11][12][13][14].

Immunogenicity of Vi-rCRM 197 conjugate vaccine in mice
Immunogenicity of typhoid conjugate vaccine (Vi-rCRM 197 ) generated using E. coli derived rCRM 197 as carrier protein was evaluated in Balb/c mice. Mice were immunized with three doses of conjugate vaccine Vi-rCRM 197 and anti-Vi IgG immune response was measured by ELISA. Sera from mice immunizied with PBS was used to generate the baseline. Vi-rCRM 197 immunized mice were able to elicit strong Anti-Vi IgG antibody response. Mice immunized with unconjugated Vi polysaccharide (Control) showed only basal level of Anti-Vi IgG. The Anti-Vi antibodies response also found to be bootable as seen in a steep increase in IgG titre after second dose of vaccine (Booster) ( Figure  9). The Anti-Vi IgG response was in mice immunized with Vi-rCRM 197 conjugate vaccine was found to be ∼16-fold higher than teh baseline response (PBS) after booster dose wheseas un-conjugated Vi showed only˜2 fold increase in the anti-Vi IgG over baseline. Noteworthy, the response of anti-Vi IgG in mice immunized with conjugate vaccine was 10-12 fold higher than the un-conjugated Vi.
As per the recommendation of World Health Organization-Technical Report Series (WHO-TRS) on typhoid conjugate vaccine, the conjugate vaccine should induce an immune response that is at least four-fold higher than the response induced by un-conjugated Vi, and a booster response should occur after the second dose in animals (WHO-TRS, 2013) [41]. The conjugate vaccine generated by using rCRM 197 as carrier protein comfortably meets the WHO immunogenicity criteria for typhoid conjugate vaccine, as it induces more than four-fold Anti-Vi IgG and also exhibits a booster response in mice. The data confirm the immunological functionality of rCRM 197 as carrier protein and suggests that it is able to convert T-independent response of polysaccharides in mice into T-dependent one and therefore booster antibody response is resulted. These data corroborate that rCRM 197 conjugation with Vi polysaccharide in mice can be resulted in immunogenic conjugates and therefore strengthening the suitability of rCRM 197 as a carrier protein for the development of conjugate vaccines.

Conclusion
The present study provides the detailed structural and immunological characteristics of CRM 197 carrier protein produced by heterologous expression in E. coli. In addition to reveal the scientific insights of rCRM 197 , the data presented in this work are also important in the context that regulatory guidelines on conjugate vaccines that emphasizes the need for in-depth characterization of carrier protein used in the conjugate vaccines development. Successful and broadly marketed carbohydrate conjugate vaccines are based on just a few FDA-approved carrier proteins. First-generation of carrier proteins such as DT and tetanus toxin require detoxification with formaldehyde, potentially eliminating part of the lysine residues needed for glycan attachment, thereby potentially compromising the conjugation efficacy. CRM 197 is one of the most widely used and highly effective carrier protein for conjuate vaccine. Licenced conjugate vaccines such as HibTITER (Haemophilus influenzae type b associated diseases), Prevnar (pneumococcal diseases), and Menveo (meningococcal diseases) are containing CRM 197 as a carrier protein. Majority of currently marketed glycoconjugate vaccines contain CRM 197 as a carrier protein and access to high quality material will support the development and production of new conjugate vaccines. The data presented herein demonstrate for the first time structural and immunological characterization of rCRM 197 produced in E. coli.
In a recent study, the recombinant CRM 197 has also been expressed in E. coli using pET28a expression vector [42]. Unlike our study, CRM 197 was expressed in Histidine-tagged form (His-CRM 197 ) which requires a histidine removal step by enterokinase treatment. This process often poses the limitation during the scale-up of production process and inconsistencies with respect to batch to batch purity/impurity profiles of protein. The protein was purified at small scale by immobilized metal affinity chromatography (IMAC) with reasonable purity [42]. However, the in-depth analytical characterization of protein was also lacking in this study. In the present study, rCRM 197 expression was done in pTWIN expression vector, which releases the expressed protein from fusion tag by self-splicing with high fidelity. CRM 197 protein was purified at high scale (∼20 l Fermentation Scale) using ion exchange chromatography and highly pure form of protein (>95%) was recovered as the final product.
Recently, we have studied the analytical comparability of five CRM 197 proteins produced in three different expression systems (E. coli, P. fluorescens and C. diphtheriae C7). A comprehensive physicochemical analysis of the CRM 197 molecules demonstrate that recombinant CRM 197 s expressed in E. coli are overall highly similar to those expressed in the traditional system (C. diphtheriae C7) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile and in vitro antigenicity [43]. However, the detailed structural analysis and its in vivo antigenicity/immunological characterization was lacking. As a part of further in-depth characterization, we have determined the 3D crystal structure of E. coli derived rCRM 197 . The rCRM 197 has shown the equivalence with C7 CRM 197 in terms of overall folding pattern, domain architecture and the presence of correct disulfide bonds. The absence of toxicity in CRM 197 relies on a point mutation at amino acid position 52 that replaces Glycine with Glutamate (G52E) and renders protein nontoxicity. In the present study, the genetic basis of nontoxicity of E. coli based rCRM 197 was also confirmed by X-ray crystallography. These data are important for scientific communities as well as industries working on conjugate vaccine development using CRM 197 as carrier protein. The in vivo functionality of E. coli expressed recombinant CRM 197 carrier protein has been demonstrated by conjugating it with Vi polysaccharide antigen of Salmonella Typhi thereby generating Vi-rCRM 197 conjugate vaccine. The data confirm that the use of rCRM 197 as a carrier protein in conjugate vaccine will be able to elicit the primary and a booster antibody response in animals. The Anti-Vi IgG elicited by Vi-rCRM 197 conjugate vaccine was more than four-fold higher than the response generated by unconjugated Vi which is one of the benchmark for a protective typhoid conjugate vaccine [41]. The comparative immunogenicity of Vi polysaccharide conjugated to other carrier protein like tetanus toxoid (TT), diphtheria toxoid (DT) and C7 CRM 197 is already published [44]. The immunogenicity of Vi-rCRM 197 showed the comparable result in terms of overall fold change in Anti-Vi IgG over Vi and booster impact. The overall data presented in our study will potentially catalyse the efforts for the production of CRM 197 protein in the recombinant system and its use as carrier protein in the conjugate vaccine development.