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

Abstract

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.

Keywords: CRM197; Carrier Protein; Conjugate Vaccine; E. coli; X-ray crystallography.

Figure 1. Determination of molecular weight, identity and purity of CRM₁₉₇ by SDS/AGE and Western immunoblot respectively

(A,B) Reducing SDS/PAGE and Western blot analysis of three different lots of rCRM₁₉₇, and reference CRM₁₉₇. (C,D) Nonreducing SDS/PAGE and Western blot analysis of three different lots of rCRM₁₉₇ and reference CRM₁₉₇ (Lane 1: protein molecular weight marker, Lane 2: reference CRM₁₉₇, Lane 3–5: rCRM₁₉₇ Lot 1–3). Purified recombinant CRM₁₉₇ protein appeared as ~58 kDa molecular weight protein on SDS/PAGE with ≥95% purity. This molecular weight matches the expected size of the protein and with reference standard used. Anti-CRM₁₉₇ antibodies recognized rCRM₁₉₇ in Western blot, thereby confirming the protein identity as CRM₁₉₇.

Figure 2. Determination of intact mass of rCRM₁₉₇ using MS (ESI-qTof)

The major abundant peak of 58.4 kDa has been observed which is similar to the expected/theoretical molecular weight of CRM₁₉₇ protein.

Figure 3. Demonstration of functional activity in rCRM₁₉₇ samples purified from inclusion body

pUC57 plasmid was incubated with rCRM₁₉₇ for different time points and the mix was run on 1% agarose gel. Native CRM₁₉₇ possesses a mild endonuclease activity and this assay is used to prove the restoration of biological activity and conformation integrity of the protein purified from insoluble inclusion body. The purified rCRM₁₉₇ exhibits the endonuclease activity as evident by nicking of supercoiled plasmid DNA incubated with the protein (Lanes 2–7).

Figure 4. High-sensitivity AAA profile of rCRM₁₉₇ and CRM₁₉₇

Both the proteins showed similar elution profile during AAA chromatogram of high-sensitivity AAA of CRM₁₉₇. (A) AAA chromatogram of BioE rCRM₁₉₇ and (B) AAA chromatogram of reference CRM₁₉₇. Both the proteins showed similar elution profile. The peak data were extrapolated and plotted in bar diagram (C). Blue bar: theoretical number of amino acid residues, Red bar: experimental amino acid residues of rCRM₁₉₇ and Green bar: experimental amino acid residues of reference CRM₁₉₇.

Figure 5. Mapping of rCRM₁₉₇ protein by structural mAbs against CRM₁₉₇ by Western immunoblot

All mAbs recognized rCRM₁₉₇ as single major band of ~58 kDa. This confirms the structural integrity and identity of rCRM₁₉₇ and its equivalence with reference. (A,B) Western blot using A chain specific α-CRM₁₉₇ mAb 7F2 and 8A4; (C) Western blot using B chain specific α-CRM₁₉₇ mAb 73B6; (D,E) Western blot using whole CRM₁₉₇ specific α-CRM₁₉₇ mAb 1H2 and Hyb. Lane 1: Reference CRM₁₉₇, 2–4: rCRM₁₉₇ Lot 1–3 and Lane 5: protein molecular weight marker.

Figure 6. Crystal structure of monomeric form of E. coli derived rCRM197 protein (PDB ID: 5I82)

(A) Superimposition of the monomer of rCRM₁₉₇ and C7 CRM₁₉₇. (B) Chain A (cartoon colored as ruby) and chain B (transparent, dark grey ribbon) of rCRM₁₉₇ are superimposed on the monomer of wild-type CRM₁₉₇ (PDB code: 4AE0, cartoon colored as yellow) and its symmetry-related molecule (transparent, yellow ribbon). (C) Catalytic domains consisting of residues 1–187, (T) Transmembrane domain consisting of residues 201–384, (R) Receptor domain consisting of residues 387–535. The dimers show the swapping of the T domains in the open conformation.

Figure 7. The glycine to glutamate mutation at amino acid position 52 (G52E) in rCRM₁₉₇.

Zoom in the active site loop CL2 where G52E mutation has taken place. The mutation creates a structural distortion in active site loop of DT thereby eliminating its toxicity (consequently CRM₁₉₇ is formed). The image depicts the structural superimposition of the DT (PDB code: 1SGK; thin blue ribbon), C7-CRM₁₉₇ (PDB code: 4AE0, yellow carton) and rCRM₁₉₇ (PDB code: 5I82: chain A as ruby cartoon, chain B as red cartoon, chain C as light red cartoon and chain D as firebrick color cartoon). The data demonstrate that rCRM₁₉₇ possesses G52E point mutation that converted DT into CRM₁₉₇. The same point mutation was also evident in C7-CRM₁₉₇.

Figure 8. The presence of correct dilsuphide bonds in E. coli derived rCRM₁₉₇.

Zoom in the disulfide bond C186-C201 (A) and C461-C471 (B) in rCRM₁₉₇ (PDB ID: 5I82). Two disulfide bonds has been identified in at the expected position of the resolved structure of rCRM₁₉₇.

Figure 9. Immunogenicity of typhoid conjugate vaccine (Vi-rCRM₁₉₇) generated by conjugating Vi polysaccharide with rCRM₁₉₇

Balb/C mice immunized with three dose of Vi-rCRM₁₉₇ conjugate vaccine and Anti Vi-IgG was measured by ELISA. The titre of Vi specific antibody was compared with Vi PS (unconjugated Vi polysaccharide) group. There was significant increase in the Vi antibody was observed in Vi-CRM₁₉₇ immunized mice and a booster response was also observed after second injection.