CARRIER PROTEIN WITH SITE-DIRECTED MUTATION AND USE THEREOF IN PREPARATION OF VACCINE

Abstract

The present invention relates to a carrier protein with site-directed mutation and use thereof in preparation of a vaccine, wherein the carrier protein is selected from fusion proteins formed by one, two or more of diphtheria toxoid, a non-toxic mutant of diphtheria toxin, a bacterial outer membrane protein and a bacterially expressed protein, wherein an amino acid at at least one site on the carrier protein is mutated into an unnatural amino acid, and the unnatural amino acid contains an azido or alkynyl terminal group. In a mutual reaction process of the carrier protein with site-directed mutation of the present invention and a polysaccharide antigen, a covalent bond is formed, and meanwhile a formed conjugate is in a bead-string state, so that the carrier protein and the polysaccharide antigen can be effectively prevented from being excessively crosslinked. Further, particle size distribution of the conjugate is significantly uniform and controllable, which provides an effective means for improving quality of a polysaccharide-protein conjugate vaccine.

Claims

1. A carrier protein with site-directed mutation, wherein the carrier protein is selected from fusion proteins formed by one, two or more of diphtheria toxoid, a non-toxic mutant of diphtheria toxin, a bacterial outer membrane protein and a bacterially expressed protein, wherein an amino acid at at least one site on the carrier protein is mutated into an unnatural amino acid, and the unnatural amino acid contains an azido or alkynyl terminal group.

2. The protein with site-directed mutation of claim 1, wherein the carrier protein is selected from fusion proteins formed by one, two or more of diphtheria toxoid, non-toxic mutant of diphtheria toxin CRM197, group B meningococcal outer membrane protein, recombinant Pseudomonas aeruginosa exotoxin A, Haemophilusinfluenzae lipoprotein HID and Haemophilusinfluenzae heat shock protein HIN47; preferably, the carrier protein is selected from non-toxic mutant CRM197 of diphtheria toxin, Haemophilusinfluenzae lipoprotein HID, fusion protein HIN47-HID and fusion protein HID-HIN47.

3. The protein with site-directed mutation of claim 1, wherein the unnatural amino acid is a phenylalanine derivative, a tyrosine derivative, a glutamine derivative, an alanine derivative, a cysteine derivative, a serine derivative or a lysine derivative.

4. The protein with site-directed mutation of claim 1, wherein the unnatural amino acid is ##STR00005##

5. The protein with site-directed mutation of claim 1, wherein a mutation site of HIN47-HID fusion protein may be one or more amino acids at any site in SEQ ID NO: 2; preferably, the mutation site is selected from position 174R, position 2171, position 223N, position 2261, position 313R, position 315K, position 522K, position 557D, position 653E and position 684Y on a sequence set forth in SEQ ID NO: 2.

6. The protein with site-directed mutation of claim 1, wherein a mutation site of HID-HIN47 fusion protein may be one or more amino acids at any site in SEQ ID NO: 3; preferably, the mutation site is selected from position 78K, position 113D, position 209E, position 240Y, position 527R, position 5701, position 576N, position 5791, position 666R and position 668K on a sequence set forth in SEQ ID NO: 3.

7. The protein with site-directed mutation of claim 1, wherein an amino acid at position N on an amino acid sequence of a protein before mutation is mutated into Lys-azido, and a linking mode of the mutated amino acid is shown as the following formula: ##STR00006## wherein, the N is a mutation site, and AA is an amino acid located before and after the mutation site, and R.sub.1 is ##STR00007##

8. A conjugate of a carrier protein with site-directed mutation, wherein the conjugate is prepared by the carrier protein with site-directed mutation of claim 1 and a saccharide, a polynucleotide, an amino acid, a polypeptide or a small molecule compound containing or modified to have an alkynyl terminal group.

9. The conjugate of the carrier protein with site-directed mutation of claim 8, wherein the amino acid at position N on the amino acid sequence of the protein before mutation is mutated into the following structure: ##STR00008## wherein, the N is a mutation site, AA is an amino acid located before and after the mutation site, and R.sub.2 is a saccharide, a polynucleotide, an amino acid, a polypeptide or a carboxyl terminal modifying group.

10. The conjugate of the carrier protein with site-directed mutation of claim 8, wherein the conjugate is a glycoconjugate formed by conjugating the carrier protein with site-directed mutation to a polysaccharide containing or modified to have an alkynyl terminal group.

11. The conjugate of the carrier protein with site-directed mutation of claim 10, wherein the ratio of the saccharide to the carrier protein (w/w) in the glycoconjugate is 0.3 to 3.

12. The conjugate of the carrier protein with site-directed mutation of claim 10, wherein at least one covalent bond is present between the polysaccharide and the carrier protein every 10 to 50 saccharide repeat units of the polysaccharide.

13. An immunogenic composition comprising the glycoconjugate of claim 11, and a pharmaceutically acceptable excipient, carrier or diluent.

14. The immunogenic composition of claim 13, wherein the glycoconjugate is a glycoconjugate of a capsular polysaccharide selected from one of or a combination of two or more of: pneumococcus serotype 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and 33F.

15. (canceled)

16. (canceled)

17. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] FIG. 1 shows SDS-PAGE electrophoretograms of the mutated protein Hin47-HID and the polysaccharide-protein conjugates obtained by Click reaction;

[0037] FIG. 2a shows a particle size distribution plot of the polysaccharide-protein conjugate obtained by Click reaction;

[0038] FIG. 2b shows a particle size distribution plot of the polysaccharide-protein conjugate obtained by conventional coupling method; and

[0039] FIG. 3 shows ELISA results of type 3 conjugates obtained by different coupling modes after triple immune of rabbits.

DETAILED DESCRIPTION

[0040] The present invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are merely intended to illustrate the present invention rather than limit the scope of the present invention.

Example 1

Construction of Expression Plasmid of Carrier Protein with Site-directed Mutation

1: Selection of Mutation Sites

[0041] The three-dimensional structures of HID and HIN47 were predicted separately using online software phyre2.

[0042] 10 key amino acids were selected on each of fusion proteins HIN47-HID and HID-HIN47. After the key amino acid sites were substituted, the fusion proteins can be bound to the polysaccharide at fixed points, and the residual protease activity of HIN47 is expected to be further reduced as well, which is beneficial to improve the stability of a final polysaccharide-binding product. The information of specific mutation sites is shown in Table 1 and Table 2, wherein an amino acid position refers to a position on sequences set forth in SEQ ID NOs: 2-3.

TABLE-US-00001 TABLE 1 HIN47-HID mutation sites Amino acid Amino Codon before Codon after position acid mutation mutation 522 K AAG TAG 557 D GAC 653 E GAA 684 Y TAT 174 R CGC 217 I ATC 223 N AAT 226 I ATT 313 R CGC 315 K AAA

TABLE-US-00002 TABLE 2 HID-HIN47 mutation sites Amino acid Amino Codon before Codon after position acid mutation mutation 78 K AAG TAG 113 D GAC 209 E GAA 240 Y TAT 527 R CGC 570 I ATC 576 N AAT 579 I ATT 666 R CGC 668 K AAA

2: Obtaining of Expression Plasmids

[0043] According to HID and HIN47 gene sequences published by NCBI GeneBank, full-length DNA fragments of the genes were each obtained through whole-gene synthesis. Then HIN47 and HID proteins were each fused and constructed, by using a GGSGGS six-amino-acid linker, in a pET9a vector in sequences of HIN47-linker-HID and HID-linker-HIN47, respectively, so that expression plasmids pET9a-HIN47-HID and pET9a-HID-HIN47 were obtained, respectively.

3: Site-Directed Mutation

[0044] The Fast Mutagenesis System site-directed mutation kit from TransGene Biotech was used, and according to the instruction of the kit, the expression plasmids pET9a-HIN47-HID and pET9a-HID-HIN47 were used as templates, and the mutation primers in Tables 3-4 were used for mutations. For the plasmids obtained after the mutation, sequencing verification was performed. Sequencing results showed that all genes at mutation sites were successfully mutated into TAG, and 10 plasmids with site-directed mutation were obtained.

[0045] The 10 mutant clones of the fusion protein pET9a-HIN47-HID were named: pET9a-HIN47-HID-R174, pET9a-HIN47-HID-I217, pET9a-HIN47-HID-N223, pET9a-HIN47-HID-I226, pET9a-HIN47-HID-R313, pET9a-HIN47-HID-K315, pET9a-HIN47-HID-K522, pET9a-HIN47-HID-D557, pET9a-HIN47-HID-E653, and pET9a-HIN47-HID-Y684.

[0046] The 10 mutant clones of the fusion protein pET9a-HID-HIN47 were named: pET9a-HID-HIN47-K78, pET9a-HID-HIN47-D113, pET9a-HID-HIN47-E209, pET9a-HID-HIN47-Y240, pET9a-HID-HIN47-R527, pET9a-HID-HIN47-1570, pET9a-HID-HIN47-N576, pET9a-HID-HIN47-I579, pET9a-HID-HIN47-R666, and pET9a-HID-HIN47-K668.

TABLE-US-00003 TABLE 3 List of mutation primers for HIN47-HID Mutation Primer Sequence site coding number Primer sequence 5′ to 3′ K522 522F SEQ ID CCGATGTAGCCtagAAATTTCCTCATAGGCATAGGAAAG NO: 4 522R SEQ ID TTTctaGGCTACATCGGTTAAGCCGTCCAGAA NO: 5 D557 557F SEQ ID GACCAAAtagGGGAAACAGGCTCAAGTGTATCC NO: 6 557R SEQ ID GTTTCCCctaTTTGGTCTCGAAATTTTCCGTC NO: 7 E653 653F SEQ ID AAAACCtagCTCCTTCCTCAGATGGGAATGGA NO: 8 653R SEQ ID GGAAGGAGctaGGTTTTTATGCGTTTCAGTTCATT NO: 9 Y684 684F SEQ ID CGAAAGGAtagTGGGTAAACTACAATTATGATTGGATG NO: 10 684R SEQ ID TACCCActaTCCTTTCGGATCTTTTTCCTGGG NO: 11 R174 174F SEQ ID tagAGTACCGGGTCAGACTCCGGCACCTACGA NO: 12 174R SEQ ID TCTGACCCGGTACTctaGCCCAGAGCGGAAACTATCC NO: 13 I217 217F SEQ ID TGCTATCtagTCACCGAGCGGCGGTAATGCGG NO: 14 217R SEQ ID TCGGTGActaGATAGCAGTATTGATACCGATCAATTC NO: 15 N223 223F SEQ ID TtagGCGGGAATTGCGTTCGCAATTCCCAGTA NO: 16 223R SEQ ID ACGCAATTCCCGCctaACCGCCGCTCGGTGAGAT NO: 17 I226 226F SEQ ID AATGCGGGAtagGCGTTCGCAATTCCCAGTAA NO: 18 226R SEQ ID AACGCctaTCCCGCATTACCGCCGCTCGGTGA NO: 19 R313 313F SEQ ID GCTGAAATTtagGCAAAAATTGCTACAACTGGTGC NO: 20 313R SEQ ID TTTGCctaAATTTCAGCAAACGAAGAGATTTTC NO: 21 K315 315F SEQ ID TCGCGCAtagATTGCTACAACTGGTGCAGGCA NO: 22 315R SEQ ID TAGCAATctaTGCGCGAATTTCAGCAAACGAA NO: 23

TABLE-US-00004 TABLE 4 List of mutation primers for HID-HIN47 Mutation Primer Sequence site coding number Primer sequence 5′ to 3′ K78 78F SEQ ID CCGATGTAGCCtagAAATTTCCTCATAGGCATAGGAAAG NO: 24 78R SEQ ID TTTctaGGCTACATCGGTTAAGCCGTCCAGAA NO: 25 D113 113F SEQ ID GACCAAAtagGGGAAACAGGCTCAAGTGTATCC NO: 26 113R SEQ ID GTTTCCCctaTTTGGTCTCGAAATTTTCCGTC NO: 27 E209 209F SEQ ID AAAACCtagCTCCTTCCTCAGATGGGAATGGA NO: 28 209R SEQ ID GGAAGGAGctaGGTTTTTATGCGTTTCAGTTCATT NO: 29 Y240 240F SEQ ID CGAAAGGAtagTGGGTAAACTACAATTATGATTGGATG NO: 30 240R SEQ ID TACCCActaTCCTTTCGGATCTTTTTCCTGGG NO: 31 R527 527F SEQ ID tagAGTACCGGGTCAGACTCCGGCACCTACGA NO: 32 527R SEQ ID TCTGACCCGGTACTctaGCCCAGAGCGGAAACTATCC NO: 33 I570 570F SEQ ID TGCTATCtagTCACCGAGCGGCGGTAATGCGG NO: 34 570R SEQ ID TCGGTGActaGATAGCAGTATTGATACCGATCAATTC NO: 35 N576 576F SEQ ID TtagGCGGGAATTGCGTTCGCAATTCCCAGTA NO: 36 576R SEQ ID ACGCAATTCCCGCctaACCGCCGCTCGGTGAGAT NO: 37 I579 579F SEQ ID AATGCGGGAtagGCGTTCGCAATTCCCAGTAA NO: 38 579R SEQ ID AACGCctaTCCCGCATTACCGCCGCTCGGTGA NO: 39 R666 666F SEQ ID GCTGAAATTtagGCAAAAATTGCTACAACTGGTGC NO: 40 666R SEQ ID TTTGCctaAATTTCAGCAAACGAAGAGATTTTC NO: 41 K668 668F SEQ ID TCGCGCAtagATTGCTACAACTGGTGCAGGCA NO: 42 668R SEQ ID TAGCAATctaTGCGCGAATTTCAGCAAACGAA NO: 43

Example 2

Expression and Purification of Mutant Carrier Protein

Lys-Azido Incorporation Expression and Purification of Mutant Proteins

[0047] The expression plasmid pET9a-HIN47-HID-K522 obtained in Example 1 was cultured in LB medium at 37° C. for 12 to 16 h, and then secondary amplification was carried out until the OD value of the bacterial solution was 0.6 to 1.0. Lys-azido was added until a final concentration of 1 mM was reached, and amplification was continued at 37° C. for 30 mM, and IPTG was added until a final concentration of 0.5 mM was reached. The final concentration of arabinose was 0.2%. Expression was induced at 24° C. for 12 h, and then bacteria were collected.

[0048] The collected bacteria were equilibrated and resuspended using Ni-NTA-Bind-Buffer, disrupted by ultrasonic, centrifuged to remove cell debris, purified by Ni-NTA metal chelating affinity chromatography, fully washed with Ni-NTA-Wash-Buffer and finally eluted with Ni-NTA-Elute-Buffer to obtain a preliminary purified protein sample HIN47-HID-K522 with a purity of about 90% (see FIG. 1, line 4).

Example 3

Site-Directed Coupling of Mutant Carrier Protein to Polysaccharide by Copper-Free Catalysis

[0049] The copper-free Click reaction needed to rely on the ring tension effect of cyclooctyne, and a modified substance was coupled to DIBO (polysaccharide with a cyclooctyne structure), so that the copper-free catalytic Click reaction with a group containing azido was carried out. The Click reaction system was as follows:

[0050] 1 μg/μL HIN47-HID-K522 protein and DIBO-pneumonia type 3 polysaccharide (a molecular weight of 200 KD and 400 KD, 2 mM each) were vertically suspended at 4° C. for 2 hours. The results showed that pneumonia type 3 polysaccharide with different molecular weights (200 KD and 400 KD) could be successfully conjugated to the protein by modification to obtain F3-HIN47-HID-K522-200 and F3-HIN47-HID-K522-400 (see FIG. 1, lines 1 and 2).

[0051] By the reaction conditions described above, about 90% of proteins could be site-directly coupled with polysaccharides within 1 h. The re-dissolved substance after the reaction was desalted and then subjected to ion exchange, so that a protein with site-directedly coupled polysaccharide that had a purity >95% could be obtained.

Example 4

Coupling to Polysaccharide by Copper-Catalyzed Click Reaction

[0052] The reaction system was as follows:

TABLE-US-00005 HIN47-HID-K522 protein 1 μg/μL Pneumonia type 1 polysaccharide-alkyne 20 μg/μL Cu.sup.2+ 1 mM BTTES 400 μmol PBS 0.01M (pH ≈ 7) Cu wire segment (sufficient amount) Note: (BTTES was short for (1,2,3-triazol-1-yl) ethanesulfonic acid)

[0053] The reaction conditions were as follows: the reaction system was vertically suspended for 30 min at 4 ° C., and after the reaction was finished, EDTA was added until a concentration of 1 mM was reached to stop the reaction. The resulting final product was a conjugate F3-HIN47-HID-K522-Cu that was formed by site-directly coupling the pneumonia type 3 polysaccharide to the HIN47-HID-K522 protein (see FIG. 1, line 3), wherein the coupling of polysaccharide to the target protein is carried out through a copper-catalyzed Click reaction, as can be seen in FIG. 1.

Example 5

Evaluation of Filterability of Protein with Site-Directedly Coupled Polysaccharide

[0054] According to analysis of the particle size distribution of the conjugates using the Malvern laser particle size analyzer Mastersizer 2000, the particle size distribution of the conjugate formulation obtained by site-directly coupling polysaccharide to protein was uniform and free of obvious aggregates, but the conjugate F3-HIN47-HID-K522-Cu obtained by the conventional polysaccharide-protein coupling method was not uniform and contained aggregates (see FIG. 2b). The conventional polysaccharide-protein coupling method was as follows: type 3 polysaccharide was hydrolyzed at high temperature of 60° C. and then subjected to activation reaction in the presence of 0.5 g/L sodium periodate to obtain an activated polysaccharide; the activated polysaccharide mixed with carrier protein HIN47-HID in 0.1 M phosphate buffer solution (pH 6.5), sodium cyanoborohydride was added until a final concentration of 2 g/L was reached, and the reaction mixture reacted for 56 h; then 2 g/L sodium borohydride, was added, and the reaction was continued for two hours; and then ultrafiltration with normal saline was performed to obtain a conjugate F3-HIN47-HID.

Example 6

Evaluation on Immunogenicity of Protein Conjugate with Site-Directedly Coupled Polysaccharide

[0055] In the rabbit models, vaccines obtained by different coupling modes were studied. The study related to the effects of the polysaccharide-protein conjugates of the type 3 capsular polysaccharide from Streptococcus pneumoniae and the HIN47-HID proteins in Examples 3, 4 and 5 and aluminium phosphate (A1PO.sub.4) adjuvant on the immune response of a monovalent vaccine in rabbits. These effects were characterized by serum IgG concentrations determined by antigen-specific ELISA and antibody functions determined by opsonophagocytic assay (OPA). FIG. 3 shows ELISA results of type 3 conjugates obtained by different coupling modes after triple immune of rabbits. The results show that polysaccharide-protein conjugates of the type 3 capsular polysaccharide from Streptococcus pneumoniae and H1N47-HID proteins in Examples 3, 4, and 5 showed significant antibody titers compared to the control.

[0056] At weeks 0, 2, and 4, New Zealand white rabbits were immunized intramuscularly with one-quarter dose (0.5 μg of polysaccharide) of the planned clinical dose (2 μg of polysaccharide) for human. Serums were collected at different time points. Serum-specific IgG was determined by ELISA and functional activity was assessed by bactericidal ability (OPA).

TABLE-US-00006 TABLE 5 Detection results of type 3 polysaccharide-protein conjugates Free polysac- Free Polysaccharide-protein Polysaccharide-protein charide protein conjugate conjugation ratio (%) (%) F3-HIN47-HID-K522-200 0.62 18 1.2 F3-HIN47-HID-K522-400 0.98 12 1 F3-HIN47-HID-K522-Cu 0.85 16 1.6 F3-HIN47-HID 0.89 23 2.8

TABLE-US-00007 TABLE 6 Bactericidal ability (OPA) results of type 3 polysaccharide-protein conjugates Polysaccharide-protein conjugate OPA50%) F3- HIN47-HID--K522-200 698 F3- HIN47-HID--K522-400 1098 F3- HIN47-HID--K522-Cu 773 F3-HIN47-HID 305 F3-prevenar 325

[0057] Table 5 shows the detection results of the type 3 polysaccharide-protein conjugates, and the results show that the type 3 polysaccharide-protein conjugates obtained by site-directed coupling have percentages of free polysaccharide and free protein that are significantly less than those of the unmutated protein carrier. Table 6 shows the bactericidal ability (OPA) results of the type 3 polysaccharide-protein conjugates, and the results show that the type 3 polysaccharide-protein conjugates obtained by site-directed coupling induce significantly higher antibody responses and specific bactericidal ability in rabbits.

Example 7

Study on Safety of Protein Conjugate with Site-Directedly Coupled Polysaccharide

[0058] 3 polysaccharide-protein conjugates prepared in Examples 3 and 4 were sampled. 5 mice weighed 18-22 g and 2 guinea pigs weighed 250-350 g were selected for each sample, wherein each mouse was subcutaneously injected with 0.5 mL of sample at ventral area and each guinea pig was subcutaneously injected with 5.0 mL of sample at ventral area. The animals were observed for 7 days, and the body weight changes thereof were recorded. The results show that all the mice and the guinea pigs were healthy and had no abnormal reaction in the observation period, and the body weight of each mouse and guinea pig was increased at the end of observation period compared to that before injection. This shows that the site-directed coupling conjugate vaccines prepared by the present invention haven't been contaminated by exogenous toxic substances and they are free of unexpected unsafe factors.

[0059] Examples 2-7 take HIN47-HID-K522 as an example to demonstrate effect experiments of preparation, characterization, detection methods, immunogenicity evaluation and the like of the mutant carrier protein. Other carrier proteins with site-directed mutation of HIN47-linker-HID and HID-linker-HIN47 are prepared by the methods above, and after immunogenicity and safety evaluation, they can reach a level equivalent to HIN47-HID-K522. The results show that the type 3 polysaccharide-protein conjugates obtained by site-directed coupling induce significantly higher antibody responses and specific bactericidal ability in rabbits.

[0060] Although the present invention has been described with the preferred embodiments above, they are not intended to limit the scope of the claims. Since many possible variations and modifications can be made by one skilled in the art without departing from the conception of the present invention, the scope of the present invention is to be determined by the claims.