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

Abstract

Provided are a protein antigen subjected to site-directed mutation and site-directed modification, and a method for site-directed mutation and site-directed modification of the protein antigen. The method comprises: site-directedly introducing an unnatural amino acid into a specific site of the protein antigen by genetic codon expansion technique; and performing site-directed modification with the protein antigen by the unnatural amino acid and a modifier, wherein the modifier is a receptor agonist such as tripalmitoyl-S-glyceryl cysteine and monophosphoryl lipid A. Further provided is use of the protein antigen subjected to site-directed mutation and site-directed modification, such as use as a vaccine.

Claims

1. A site-directedly mutated protein, wherein the protein is selected from one or more mutant proteins in group B meningococcal fHBP proteins, and the amino acid of at least one site on the protein antigen is mutated into an unnatural amino acid comprising azido, alkynyl end group, or other active groups.

2. The site-directedly mutated protein according to claim 1, wherein the protein is selected from variant proteins formed by one or more in group B meningococcal fHBP proteins; and the protein antigen is selected from: variants 2 and 3 in subfamily A or variant 1 in subfamily B of B meningococcal fHBP proteins.

3. The site-directedly mutated protein according to claim 1, wherein the unnatural amino acid is at least one selected from the group consisting of: a phenylalanine derivative, a tyrosine derivative, a glutamine derivative, an alanine derivative, a cysteine derivative, a serine derivative, or a lysine derivative.

4. The site-directedly mutated protein according to claim 3, wherein the unnatural amino acid is a lysine derivative comprising an azido or the unnatural amino acid is ##STR00019##

5. The site-directedly mutated protein according to claim 2, wherein the protein antigen is variant 2 in subfamily A of group B meningococcal fHBP proteins, and the mutation site of the protein antigen is one or more amino acids in the amino acid sequence of positions 2-30 in SEQ ID NO: 1, or the mutation site is one or more amino acids in the amino acid sequence of positions 2-10 in SEQ ID NO: 1.

6. The site-directedly mutated protein according to claim 2, wherein the protein antigen is variant 3 in subfamily A of group B meningococcal fHBP protein antigen, and the mutation site of the protein antigen is one or more amino acids in the amino acid sequence of positions 2-30 in SEQ ID NO: 2, or the mutation site is one or more amino acids in the amino acid sequence of positions 2-10 in SEQ ID NO: 2.

7. The site-directedly mutated protein according to claim 2, wherein the protein antigen is variant 1 in subfamily B of group B meningococcal fHBP protein antigen, and the mutation site of the protein antigen is one or more amino acids in the amino acid sequence of positions 2-30 in SEQ ID NO: 3, or the mutation site is one or more amino acids in the amino acid sequence of positions 2-10 in SEQ ID NO: 3.

8. The site-directedly mutated protein according to claim 1, wherein the amino acid at position X in the amino acid sequence of the protein antigen is mutated into Lys-azido, and the connection mode of the mutated amino acid is as follows: ##STR00020## wherein X is the mutation site, and AA is the amino acid before or after the mutation site.

9. A conjugate of a site-directedly mutated group B meningococcal fHBP protein, wherein the conjugate is formed by coupling the site-directedly mutated protein according to claim 1 with a modification compound, and the modification compound is a compound with an end group comprising alkynyl group or a modified alkynyl group.

10. The conjugate according to claim 9, wherein the modification compound is selected from: a carbohydrate, a nucleic acid, an amino acid, a polypeptide or a small molecule compound which comprises a alkyne end group; or a modification product of a carbohydrate, a nucleic acid, an amino acid, a polypeptide or a small molecule compound which is obtained by modifying with a terminal alkynyl group.

11. The conjugate according to claim 9, wherein the modification compound is a lipoprotein receptor agonist, or the modification compound is a TLR2 receptor agonist, or the agonist is selected from: tripalmitoyl-S-glyceryl cysteine, monophosphoryl lipid A, dipalmitoyl-S-glyceryl-cysteine, or an analogue thereof.

12. The conjugate according to claim 9, wherein the amino acid at position X of the amino acid sequence of the group B meningococcal fHBP protein is mutated and modified into the following structure: ##STR00021## wherein X is a mutation site, AA is an amino acid before or after the mutation site, n=1-20, and R.sub.2 is a TLR2 receptor agonist.

13. The conjugate according to claim 12, wherein R.sub.2 is a tripalmitoyl-S-glyceryl cysteine analogue, and is selected from an analogue of the following structural formula: ##STR00022## wherein n, m=1-5.

14. The conjugate according to claim 12, wherein R2 is a monophosphoryl lipid A receptor agonist or a derivative thereof; and the monophosphoryl lipid A receptor agonist with a structural formula as follows: ##STR00023## n=1-20, the R terminal may be coupled with a site-directedly mutated group B meningococcal fHBP protein, wherein R.sub.3 is selected from phosphate or H; R.sub.4 is selected from ##STR00024## n is 1, 3, 5; or ##STR00025## R.sub.5 is selected from ##STR00026## R.sub.6 is selected from H or ##STR00027## R.sub.7 is selected from ##STR00028## R.sub.8 is selected from H or OH.

15. The conjugate according to claim 9, wherein the molar ratio of the group B meningococcal fHBP protein to the modification compound in the conjugate is 1:1-30.

16. A vaccine or immunogenic composition, wherein the vaccine or immunogenic composition comprises the site-directedly mutated proteins according to claim 1, or the conjugates, which is formed by coupling the site-directedly mutated protein with a modification compound.

17. The vaccine or immunogenic composition according to claim 16, wherein the vaccine or immunogenic composition simultaneously comprises three site-directedly mutated proteins to form a multivalent vaccine or immunogenic composition, or simultaneously comprise three conjugates to form a multivalent vaccine or immunogenic composition.

18. The vaccine or immunogenic composition according to claim 16, wherein the dose of site-directedly mutated group B meningococcal fHBP proteins or the conjugates is 10-100 μg.

19. The vaccine or immunogenic composition according to claim 16, wherein each dose of the vaccine or immunogenic composition comprises: 5-10 μg of group A meningococcal polysaccharide antigen, 5-10 μg of group C meningococcal polysaccharide antigen, 5-10 μg of group W135 meningococcal polysaccharide antigen, 5-10 μg of group Y meningococcal polysaccharide antigen, or 10-100 μg of site-directedly mutated group B meningococcal fHBP protein or a conjugate thereof.

20. A method for site-directed mutation and site-directed modification of the protein antigen of claim 1, wherein the method comprises: site-directedly introducing an unnatural amino acid into a specific site of the protein antigen by genetic codon expansion technique to obtain a site-directedly mutated protein which is further coupled with a modification compound, and wherein the modification compound is a compound with an end group comprising alkynyl group or a modified alkynyl group.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0071] FIG. 1 is a particle size diagram of MenB-V1.55 protein;

[0072] FIG. 2 is a particle size diagram of MenB-V2.16 protein;

[0073] FIG. 3 is a particle size diagram of MenB-V1.55-G2-L1 protein;

[0074] FIG. 4 is a particle size diagram of MenB-V2.16-S3-L1 protein;

[0075] FIG. 5 is a particle size diagram of MenB-V2.16-S3-L1 protein.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0076] The present disclosure will be further elaborated below in conjunction with particular examples. It should be understood that these examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure.

[0077] As used herein, the term “orthogonal” refers to a molecule (such as an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNA synthetase (O-RS)) that functions like an endogenous component of the cell, however, its activity is reduced as compared with its corresponding endogenous molecule in the cell or translation system, or it does not function as an endogenous component of the cell. When referring to tRNA and aminoacyl-tRNA synthetases, orthogonal means that the efficiency of cooperation of orthogonal tRNA and endogenous tRNA synthetase is reduced as compared with that of cooperation of endogenous tRNA and endogenous tRNA synthetase, for example decreasing to 20%, 10%, 5%, or 1%, or less. Orthogonal molecules lack the normal function of endogenous complementary molecules within the cell.

[0078] As used herein, the term “click reaction” performs the Huisgen [3+2] cyclization of azides and alkynes.

EXAMPLE 1

Construction of the Expression Plasmid of Site-directedly Mutated MenB Protein

[0079] 1. Selection of Mutation Site

[0080] Natural MenB undergoes lipidation modification at its N-terminus. This modification does not affect the three-dimensional structure of the MenB protein, but plays a role in anchoring the protein antigen to cell membrane. Structural studies have shown that the first 20 amino acids at the N-terminal of the MenB protein are not folded to form a secondary structure, but are stretched, and the function is to expose the antigen part through the bacterial outer membrane to the bacterial surface. Therefore, the 20 amino acids at the N-terminal are preferred for the mutation sites, particularly the positions 2-10 are preferred. The information of specific mutation sites is shown in Tables 1-3, wherein the amino acid positions refer to the positions on the sequences shown in SEQ ID NO: 1-3 respectively.

[0081] SEQ ID NO.1:

[0082] cgssggggsggggvtadigtgladaltapldhkdkglksltledsi sqngtltl saqgaektygngdslntgklkndkvsrfdfirqi evdgqlitlesgefqvykqshsaltalqteqeqdpehsekmvakrrfrigdiagehtsfdklpkdvmatyrgtafgsddaggkltytidfa akqghgkiehlkspelnvdlavayikpdekhhavisgsvlynqdekgsyslgifgekaqevagsaevetangihhiglaakq

[0083] SEQ ID NO.2:

[0084] Cgssggggvaadigagladaltapldhkdkslqsltldqsvrkneklklaaqgaektygngdslntgklkndkvsrfdfirqiev dgqlitlesgefqiykqdhsavvalqiekinnpdkidslinqrsflvsglggehtafnqlpdgkaeyhgkafssddaggkltytidfaakq ghgkiehlktpeqnvelaaaelkadekshavilgdtrygseekgtyhlalfgdraqeiagsatvkigekvheigiagkq

[0085] SEQ ID NO.3:

[0086] Cgssggggvaadigtgladaltapldhkdkglksltledsisqngtifisacigaektfkvgdkdnslntgklkndkisrfdfvqkie vdgqtitlasgefqiykqdhsavvalqiekinnpdkidslinqrsflvsglggehtafnqlpsgkaeyhgkafssddaggkltytidfaak qghgkiehlktpeqnvelasaelkadekshavilgdtrygseekgtyhlalfgdraqeiagsatvkirekvheigiagkq

TABLE-US-00001 TABLE 1 V1.55 mutation sites Amino acid Amino Codon before Codon after position acid mutation mutation 2 G GGT TAG 3 S AGC 4 S AGC 5 G GGT 6 G GGT 7 G GGT 8 G GGT 9 S AGT 10 G GGT

TABLE-US-00002 TABLE 2 V2.16 mutation sites Amino acid Amino Codon before Codon after position acid mutation mutation 2 G GGT TAG 3 S AGC 4 S AGC 5 G GGT 6 G GGT 7 G GGT 8 G GGC 9 V GTT 10 A GCA

TABLE-US-00003 TABLE 3 V3.45 mutation sites Amino acid Amino Codon before Codon after position acid mutation mutation 2 G GGT TAG 3 S AGC 4 S AGC 5 G GGT 6 G GGT 7 G GGT 8 G GGC 9 V GTT 10 A GCA

[0087] 2. Acquisition of Expression Plasmids

[0088] According to the MenB V.155, V2.16 and V3.45 gene sequences published by NCBI Gene Bank (genbank sequence numbers are AAR84481, AAR84445, AAR84435, respectively corresponding to SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3), the full-length DNA fragments of the genes were obtained by whole gene synthesis respectively, and then respectively fused and constructed between the NcoI and XhoI restriction sites of the pET28a vector; and the C-terminal His purification tag was retained respectively to obtain pET28a-MenB-V1.55, pET28a-MenB-V2.16, and pET28a-MenB-V3.45 expression plasmids.

[0089] 3. Site-Directed Mutation

[0090] The Fast Mutagenesis System site-directed mutation kit from TransGen Biotech Company was used according to its instructions to perform each mutation by using the above pET28a-MenB-V1.55, pET28a-MenB-V2.16 and pET28a-MenB-V3.45 expression plasmids as templates, and using the mutation primer pairs in Tables 4-6. For the plasmids obtained after mutation, sequencing was performed for verification. Sequencing results show that, each mutation site was successfully mutated into TAG, and 9 site-directedly mutated plasmids were obtained.

[0091] 9 mutant clones of MenB-V1.55 were named as follows: pET28a-MenB-V1.55-G2, pET28a-MenB-V1.55-53, pET28a-MenB-V1.55-54, pET28a-MenB-V1.55-G5, pET28a-MenB-V1.55-G6, pET28a-MenB-V1.55-G7, pET28a-MenB-V1.55-G8, pET28a-MenB-V1.55-59, pET28a-MenB-V1.55-G10.

[0092] 9 mutant clones of MenB-V2.16 were named as follows: pET28a-MenB-V2.16-G2, pET28a-MenB-V2.16-S3, pET28a-MenB-V2.16-S4, pET28a-MenB-V2.16-G5, pET28a-MenB-V2.16-G6, pET28a-MenB-V2.16-G7, pET28a-MenB-V2.16-G8, pET28a-MenB-V2.16-V9, pET28a-MenB-V2.16-A10.

[0093] 9 mutant clones of MenB-V3.45 were named as follows:

[0094] pET28a-MenB-V3.45-G2, pET28a-MenB-V3.45-S3, pET28a-MenB-V3.45-S4, pET28a-MenB-V3.45-G5, pET28a-MenB-V3.45-G6, pET28a-MenB-V3.45-G7, pET28a-MenB-V3.45-G8, pET28a-MenB-V3.45-V9, pET28a-MenB-V3.45-A10.

TABLE-US-00004 TABLE 4 Mutation primer pairs for MenB V1.55 Muta- tion Primer Primer sequence site No. SEQ ID NO: 5′ to 3′ G2  12F SEQ ID NO: 4 ggagatataccatgggtTGT tag AGCAGCGGTGGTGGTGGTAG  12R SEQ ID NO: 5 CTAACAACCCATGGTATATCTCC S3  13F SEQ ID NO: 6 gatataccatgggtTGTggt tag AGCGGTGGTGGTGGTAGTGG  13R SEQ ID NO: 7 CTAACCACAACCCATGGTATATC S4  14F SEQ ID NO: 8 ataccatgggtTGTggtAGC tag GGTGGTGGTGGTAGTGGTGG  14R SEQ ID NO: 9 CTAGCTACCACAACCCATGGTAT G5  15F SEQ ID NO: 10 ccatgggtTGTggtAGCAGC tag GGTGGTGGTAGTGGTGGCGG  15R SEQ ID NO: 11 CTAGCTGCTACCACAACCCATGG G6  16F SEQ ID NO: 12 tgggtTGTggtAGCAGCGGT tag GGTGGTAGTGGTGGCGGTGG  16R SEQ ID NO: 13 CTAACCGCTGCTACCACAACCCA G7  17F SEQ ID NO: 14 gtTGTggtAGCAGCGGTGGT tag GGTAGTGGTGGCGGTGGTGT  17R SEQ ID NO: 15 CTAACCACCGCTGCTACCACAAC G8  18F SEQ ID NO: 16 GTggtAGCAGCGGTGGTGGT tag AGTGGTGGCGGTGGTGTTAC  18R SEQ ID NO: 17 CTAACCACCACCGCTGCTACCAC S9  19F SEQ ID NO: 18 gtAGCAGCGGTGGTGGTGGT tag GGTGGCGGTGGTGTTACCGC  19R SEQ ID NO: 19 CTAACCACCACCACCGCTGCTAC G10 110F SEQ ID NO: 20 GCAGCGGTGGTGGTGGTAGTtagG GCGGTGGTGTTACCGCAGA 110R SEQ ID NO: 21 CTAACTACCACCACCACCGCTGC

TABLE-US-00005 TABLE 5 Mutation primer pairs for MenB V2.16 Muta- tion Primer Primer sequence site No. SEQ ID NO: 5′ to 3′ G2  22F SEQ ID NO: 22 ggagatataccatgggtTGT tag AGCAGCGGTGGTGGTGGCGT  22R SEQ ID NO: 23 CTAACAACCCATGGTATATCTCC S3  23F SEQ ID NO: 24 gatataccatgggtTGTGGT tag AGCGGTGGTGGTGGCGTTGC  23R SEQ ID NO: 25 CTAACCACAACCCATGGTATATC S4  24F SEQ ID NO: 26 ataccatgggtTGTGGTAGC tag GGTGGTGGTGGCGTTGCAGC  24R SEQ ID NO: 27 CTAGCTACCACAACCCATGGTAT G5  25F SEQ ID NO: 28 ccatgggtTGTGGTAGCAGC tag GGTGGTGGCGTTGCAGCAGA  25R SEQ ID NO: 29 CTAGCTGCTACCACAACCCATGG G6  26F SEQ ID NO: 30 tgggtTGTGGTAGCAGCGGT tag GGTGGCGTTGCAGCAGATAT  26R SEQ ID NO: 31 CTAACCGCTGCTACCACAACCCA G7  27F SEQ ID NO: 32 gtTGTGGTAGCAGCGGTGGT tag GGCGTTGCAGCAGATATTGG  27R SEQ ID NO: 33 CTAACCACCGCTGCTACCACAAC G8  28F SEQ ID NO: 34 GTGGTAGCAGCGGTGGTGGTtag GTTGCAGCAGATATTGGTGC  28R SEQ ID NO: 35 CTAACCACCACCGCTGCTACCAC V9  29F SEQ ID NO: 36 GTAGCAGCGGTGGTGGTGGCtag GCAGCAGATATTGGTGCAGG  29R SEQ ID NO: 37 CTAGCCACCACCACCGCTGCTAC A10 210F SEQ ID NO: 38 GCAGCGGTGGTGGTGGCGTTtag GCAGATATTGGTGCAGGTCT 210R SEQ ID NO: 39 CTAAACGCCACCACCACCGCTGC

TABLE-US-00006 TABLE 6 Mutation primer pairs for MenB V3.45 Muta- tion Primer Primer sequence site No. SEQ ID NO: 5′ to 3′ G2  32F SEQ ID NO: 40 ggagatataccatgggtTGT tag AGCAGCGGTGGTGGTGGCGT  32R SEQ ID NO: 41 CTAACAACCCATGGTATATCTCC S3  33F SEQ ID NO: 42 gatataccatgggtTGTGGT tag AGCGGTGGTGGTGGCGTTGC  33R SEQ ID NO: 43 CTAACCACAACCCATGGTATATC S4  34F SEQ ID NO: 44 ataccatgggtTGTGGTAGC tag GGTGGTGGTGGCGTTGCAGC  34R SEQ ID NO: 45 CTAGCTACCACAACCCATGGTAT G5  35F SEQ ID NO: 46 ccatgggtTGTGGTAGCAGC tag GGTGGTGGCGTTGCAGCAGA  35R SEQ ID NO: 47 CTAGCTGCTACCACAACCCATGG G6  36F SEQ ID NO: 48 tgggtTGTGGTAGCAGCGGT tag GGTGGCGTTGCAGCAGATAT  36R SEQ ID NO: 49 CTAACCGCTGCTACCACAACCCA G7  37F SEQ ID NO: 50 gtTGTGGTAGCAGCGGTGGT tag GGCGTTGCAGCAGATATTGG  37R SEQ ID NO: 51 CTAACCACCGCTGCTACCACAAC G8  38F SEQ ID NO: 52 GTGGTAGCAGCGGTGGTGGTtag GTTGCAGCAGATATTGGCAC  38R SEQ ID NO: 53 CTAACCACCACCGCTGCTACCAC V9  39F SEQ ID NO: 54 GTAGCAGCGGTGGTGGTGGCtag GCAGCAGATATTGGCACCGG  39R SEQ ID NO: 55 CTAGCCACCACCACCGCTGCTAC A10 310F SEQ ID NO: 56 GCAGCGGTGGTGGTGGCGTTtag GCAGATATTGGCACCGGTCT 310R SEQ ID NO: 57 CTAAACGCCACCACCACCGCTGC

EXAMPLE 2

Lys-Azido Incorporation Expression and Purification of the Mutation Protein

[0095] The expression plasmid vectors pET28a-MenB-V1.55-G2, pET28a-MenB-V2.16-S3 and pET28a-MenB-V3.45-S4 obtained in Example 1 were cultured in LB medium at 37° C. for 12-16 hours, performing secondary amplification to reach 0.6-1.0 of OD value of the bacterial solution, adding Lys-azido to a final concentration of 1 mM, and continuing the amplification at 37° C. for 30 minutes, then adding IPTG to a final concentration of 0.5 mM, and arabinose to a final concentration of 0.2%; cells were collected after induced expression at 24° C. for 12 hours.

[0096] The collected cells were balanced and resuspended with Ni-NTA-Bind-Buffer, then ultrasonically disrupted and centrifuged to remove cell debris, performing Ni-NTA metal chelate affinity chromatography, fully washing with Ni-NTA-Wash-Buffer, and finally eluting with Ni-NTA-Elute-Buffer to obtain primary purified protein samples pET28a-MenB-V1.55-G2, pET28a-MenB-V2.16-S3, and pET28a-MenB-V3.45-S4 having a purity of about 90%.

[0097] Other mutant proteins of V1.55, V2.16 and V3.45 were also prepared according to the above methods, but due to space limitations, not all of them are described in the description of this disclosure.

EXAMPLE 3

Synthesis of Tripalmitoyl-S-glyceryl Cysteine Analogue 8

[0098] The synthetic route of tripalmitoyl-S-glyceryl cysteine analogue 8 is as follows:

[0099] 1. Compound 1 (5 g) and 2,2-dimethoxypropane (5 g) were dissolved in dichloromethane (100 ml). After the dissolution is complete, PTSA (0.9 g) was slowly added in the solution in ice-water bath. After the addition is complete, the ice bath was removed to stir at room temperature for 2 hours. After the reaction, the solvent was distilled off under reduced pressure, and Compound 2 was obtained by purification with silica gel chromatography column.

[0100] 2. Compound 2 (5 g) was dissolved in DMF (100 ml), adding EDCI (5 g), HOBT (3.5 g), TEA (10 g) in sequence to stir for 3-5 minutes, then adding Compound a (6 g); after the addition, the solution was placed in an oil bath at 80° C. to react overnight. After the reaction, the solvent was distilled off under reduced pressure, and Compound 3 was obtained by purification with silica gel chromatography column.

[0101] 3. Compound 3 (5 g) was dissolved in dichloromethane (100 ml). After the dissolution was complete, 1N HCl methanol solution (20 ml) was added to the system to stir at room temperature for 3 hours. After the reaction, the solvent was distilled off under reduced pressure, and Compound 4 was obtained by purification with silica gel chromatography column.

[0102] 4. Compound 4 (5 g) was dissolved in DMF (100 ml). After the dissolution was complete, adding triphenylchlorosilane (8 g) and imidazole (1 g) to the system in sequence to stir overnight at 40° C. After the reaction, the solvent was distilled off under reduced pressure, and compound 5 was obtained by purification with silica gel chromatography column.

[0103] 5. Compound 5 (5 g) and Compound b (6 g) were dissolved in DMF (100 ml), adding molecular sieves (10 g) at the same time, then adding 3-5 drops of concentrated sulfuric acid to place in an oil bath at 80° C. to react overnight. After the reaction, the molecular sieves were removed by filtration, the solvent was distilled off under reduced pressure, and Compound 6 was obtained by purification with silica gel chromatography column.

[0104] 6. Compound 6 (5 g) was dissolved in acetic acid (100 ml). After the dissolution was complete, the solution was refluxed at 120° C. for 6 h. The reaction process was monitored by TLC. After the reaction of the raw materials was completed, the solvent was distilled off under reduced pressure, and Compound 7 was obtained by purification with silica gel chromatography column.

[0105] 7. Compound 7 (5 g) and Compound c (6 g) were dissolved in DMF (100 ml), adding molecular sieves (10 g) at the same time, then adding 3-5 drops of concentrated sulfuric acid to place in an oil bath at 80° C. to react overnight. After the reaction, the molecular sieves were removed by filtration, the solvent was distilled off under reduced pressure, and Compound 8 was obtained by purification with silica gel chromatography column.

[0106] The flow chart is as follows:

##STR00014## ##STR00015##

[0107] Using the same method, other analogues L1-L15 of tripalmitoyl-S-glyceryl cysteine may be obtained:

TABLE-US-00007 [00016] No. L1 L2 L3 L4 L5 m 1 2 3 4 5 n 1 2 3 4 5

TABLE-US-00008 [00017] No. L6 L7 L8 L9 L10 m 1 2 3 4 5 n 1 2 3 4 5

TABLE-US-00009 [00018] No. L11 L12 L13 L14 L15 m 1 2 3 4 5 n 1 2 3 4 5

EXAMPLE 4

Coupling of Mutant Protein pET28a-MenB-V1.55-G2 with Tripalmitoyl-S-Glyceryl Cysteine Analogue via Copper-Catalyzed Click Reaction

[0108] The reaction system is as follows:

TABLE-US-00010 pET28a-MenB-V1.55-G2 protein  1 μg/μl Tripalmitoyl-S-glyceryl cysteine Compound 8 20 μg/μl Cu.sup.2+ 1 mM BTTES 400 μmol PBS 0.01M (pH ≈ 7) Cu wire segment Sufficient Note: (1,2,3-triazol-1-yl)ethanesulfonic acid, referred to as BTTES)

[0109] Reaction conditions: 4° C., vertical suspension for 30 minutes, after the reaction was completed, EDTA was added to 1 mM to terminate the reaction to obtain a final product, i.e., a site-directedly coupling conjugate MenB-V1.55-G2-L1 of tripalmitoyl-S-glyceryl cysteine analogue and pET28a-MenB-V1.55-G2 protein.

EXAMPLE 5

Coupling of Mutant Protein pET28a-MenB-V2.16-S3 with Tripalmitoyl-S-Glyceryl Cysteine Analogue via Copper-Catalyzed Click Reaction

[0110] With the same operation steps as in Example 4, a site-directedly coupling conjugate MenB-V2.16-S3-L1 of tripalmitoyl-S-glyceryl cysteine analogue and pET28a-MenB-V2.16-S3 protein was obtained.

EXAMPLE 6

Coupling of Mutant Protein pET28a-MenB-V3.45-S4 with Tripalmitoyl-S-Glyceryl Cysteine Analogue via Copper-Catalyzed Click Reaction

[0111] With the same operation steps as in Example 4, a site-directedly coupling conjugate MenB-V3.45-S4-L1 of tripalmitoyl-S-glyceryl cysteine analogue and pET28a-MenB-V3.45 protein was obtained.

[0112] According to the method of Examples 4-6, site-directedly coupling conjugates of a protein with different liposomes were simultaneously prepared, as shown in the following table:

TABLE-US-00011   MenB-V1.55-G2-L2 MenB-V2.16-S3-L2 MenB-V3.45-S4-L2 MenB-V1.55-G2-L11 MenB-V2.16-S3-L11 MenB-V3.45-S4-L11 MenB-V1.55-G2-L15 MenB-V2.16-S3-L15 MenB-V3.45-S4-L15 MenB-V1.55-G2-L12 MenB-V2.16-S3-L12 MenB-V3.45-S4-L12 MenB-V1.55-G2-L14 MenB-V2.16-S3-L14 MenB-V3.45-S4-L14 MenB-V1.55-S3-L1 MenB-V2.16-S3-L1 MenB-V3.45-S4-L1 MenB-V1.55-S4-L2 MenB-V2.16-S4-L2 MenB-V3.45-S4-L2 MenB-V1.55-G5-L6 MenB-V2.16-G5-L6 MenB-V3.45-S4-L6 MenB-V1.55-G6-L11 MenB-V2.16-G6-L11 MenB-V3.45-S4-L11 MenB-V1.55-G7-L15 MenB-V2.16-G7-L15 MenB-V3.45-S4-L15 MenB-V1.55-G8-L1 MenB-V2.16-G8-L1 MenB-V3.45-S4-L1 MenB-V1.55-S9-L2 MenB-V2.16-G2-L2 MenB-V3.45-S4-L1 MenB-V1.55-G10-L1 MenB-V2.16-V9-L2 MenB-V3.45-S4-L3

EXAMPLE 7

Preparation of Trivalent fHBP Protein Vaccine

[0113] Three mutants were selected from the mutated proteins with modification prepared in this disclosure, and the three mutant recombinant group B fHBP lipoproteins MenB-V1.55-G2-L1, MenB-V2.16-S3-L1, MenB-V3.45-S4-L1 were adsorbed with aluminum hydroxide adjuvant respectively, stirring overnight at 4° C., and the adsorption rate was above 95%. The protein vaccine was prepared by diluting with 0.15 mol/1 sodium chloride, until the final concentration of the protein is 240 μg/ml. The final concentration of aluminum is 0.45-0.6 mg/ml, and the pH value is 5.8-7.2.

EXAMPLE 8

The Bactericidal Activity (SBA) Test of Popular Strains

[0114] The strain of group B meningococcus 440902 was used. The strain belongs to ST4821 sequence type and ST4821 sequence group, and is a recent epidemic strain of group B meningococcus in China, and the fHBP typing is a V2 variant.

[0115] Preparation of target bacteria: epidemic meningococcus 440902 strain was cultured on 8-12% blood-nourishing agar plate at 37° C., 6-10% CO.sub.2 for 16-18 hours, scraping bacterial lawn into normal saline, and counting the bacteria by turbidimetric method; the target bacteria were diluted to 1×10.sup.6 according to the count.

[0116] The mouse serum to be tested was inactivated at 56° C. for 1 hour to inactivate the intrinsic complement activity of the mouse serum. During the experiment, the Pel-Freez young rabbit complement was added to the serum of the mouse to be tested, and the inactivated complement and complement control were set at the same time, performing doubling dilution to a 96-well culture plate, and dropwise adding 10 μm freshly prepared target bacteria to shake and mix well, then incubating at 37° C. for 2-4 hours.

[0117] Sample application: after culturing, the mixed bacterial solution was taken to dropwise add to a solid nutrient agar comprehensive medium in an amount of 10 ml, incubating overnight at 37° C., 5% CO.sub.2.

[0118] Color development: the soft agar comprising 150-300 μg/m1 TTC was plated on the solid nutrient agar comprehensive medium cultured overnight, developing color at an appropriate temperature and appropriate time.

[0119] Counting: high-definition photos of colored colonies were taken, using image scanning technology, and analyzing with proprietary analysis software to count the number of bacterial colonies; bactericidal titer was calculated with the bactericidal activity calculation software, and the results are as follows:

[0120] Mouse Serum Bactericidal Titer

TABLE-US-00012 Strain 440902 MenB-V1.55-G2-L1 MenB-V2.16-S3-L1 MenB-V3.45-S4-L1 Trivalent fHBP Normal saline Bactericidal 26 ± 1.5 1373 ± 52.3 458 ± 15.1 1420 ± 67.1 2.2 ± 0.1 titer

EXAMPLE 9

Bactericidal Activity (SBA) Test Results of Other Conjugates

[0121]

TABLE-US-00013 Site-directedly coupling Bactericidal titer Bactericidal titer conjugate (Strain 440902, V2) (Strain H44/76, V1) MenB-V1.55-G2-L2 25 ± 1.3 800 ± 13   MenB-V2.16-S3-L2 1342 ± 55.1  20 ± 5.1 MenB-V3.45-S4-L2 451 ± 18.1 11 ± 1.1 MenB-V1.55-G2-L11 27 ± 1.3 1000 ± 13   MenB-V2.16-S3-L11 1360 ± 52.1  30 ± 5.1 MenB-V3.45-S4-L11 431 ± 28.1 116 ± 1.1  MenB-V1.55-G2-L15 23 ± 1.6 950 ± 13   MenB-V2.16-S3-L15 1337 ± 47.1  20 ± 5.1 MenB-V3.45-S4-L15 437 ± 28.4 12 ± 1.1 MenB-V1.55-G2-L12 27 ± 4.3 890 ± 13   MenB-V2.16-S3-L12 1357 ± 43.3  22 ± 5.1 MenB-V3.45-S4-L12 433 ± 22.4 17 ± 1.1 MenB-V1.55-G2-L14 30 ± 2.3 1220 ± 13   MenB-V2.16-S3-L14 1331 ± 33.7  20 ± 5.1 MenB-V3.45-S4-L14 418 ± 22.4 11 ± 1.1 MenB-V1.55-S3-L1 19 ± 6.3 980 ± 13   MenB-V2.16-S3-L1 1361 ± 23.7  20 ± 5.1 MenB-V3.45-S4-L1 397 ± 22.4 11 ± 1.1 MenB-V1.55-S4-L2 33 ± 6.1 1060 ± 13   MenB-V2.16-S4-L2 1412 ± 33.4  20 ± 5.1 MenB-V3.45-S4-L2 503 ± 28.3 11 ± 1.1 MenB-V1.55-G5-L6 23 ± 5.1 860 ± 13   MenB-V2.16-G5-L6 1452 ± 23.4  20 ± 5.1 MenB-V3.45-S4-L6 443 ± 26.3 11 ± 1.1 MenB-V1.55-G6-L11 31 ± 3.1 1220 ± 13   MenB-V2.16-G6-L11 1312 ± 33.1  20 ± 5.1 MenB-V3.45-S4-L11 403 ± 21.3 11 ± 1.1 MenB-V1.55-G7-L15 22 ± 3.1 1550 ± 13   MenB-V2.16-G7-L15 1512 ± 22.7  20 ± 5.1 MenB-V3.45-S4-L15 393 ± 24.5 11 ± 1.1 MenB-V1.55-G8-L1 40 ± 3.1 1020 ± 13   MenB-V2.16-G8-L1 1292 ± 22.7  20 ± 5.1 MenB-V3.45-S4-L1 473 ± 19.7 11 ± 1.1 MenB-V1.55-S9-L2 24 ± 3.1 1300 ± 13   MenB-V2.16-G2-L2 1492 ± 30.1  20 ± 5.1 MenB-V3.45-S4-L1 413 ± 22.6 11 ± 1.1 MenB-V1.55-G10-L1 29 ± 3.6 1700 ± 13   MenB-V2.16-V9-L2 1502 ± 20.8  20 ± 5.1 MenB-V3.45-S4-L3 393 ± 22.1 11 ± 1.1 MenB-V1.55-G10 29 ± 3.6 260 ± 13   (non-lipidation) MenB-V2.16-V9 302 ± 2.8  20 ± 5.1 (non-lipidation) MenB-V3.45-S4  20 ± 21.1 11 ± 1.1 (non-lipidation) Normal saline 2.2 ± 0.1  2.2 ± 0.1 

[0122] The above test results of bactericidal activity of different site-directedly coupling conjugates show that:

[0123] 1. The lipoproteins obtained by site-directedly coupling V1 and V2 variants of MenB protein with liposome has significant bactericidal activity as compared with normal saline in the negative control group, and has no significant difference in bactericidal activity as compared with the trivalent fHBP positive control group;

[0124] 2. The lipoproteins obtained by site-directedly coupling V1, V2 and V3 variants of MenB protein with liposome has significant increased bactericidal activity as compared with the non-lipidated protein without liposome modification;

[0125] 3. The V3 variant lipoprotein of MenB protein has a certain cross-protection effect on V2 strain.

[0126] This result shows that the liposome obtained by chemical synthesis in the present disclosure may be site-directedly coupled with MenB protein to obtain a lipoprotein with clear structure, uniform composition, controllable quality and high antigenic activity; and the product effectiveness and safety may be further improved by accurately controlling and adjusting the effective dosage.

EXAMPLE 10

Acute Toxicity and Abnormal Toxicity Test

[0127] This test utilizes the drug acute toxicity reaction of different doses, and certain dosage of testing solution (a trivalent group B meningococcal protein vaccine formed by MenB-V1.55-G2-L1, MenB-V2.16-S3-L1 and MenB-V3.45-S4-L1 prepared in Examples 4-6) was injected into the test animals (mice, guinea pigs), observing the symptoms of toxic reactions and death in the animals within a specified time, and judging whether the test product meets the specified quality requirements and the safety degree.

[0128] Experimental method: NIH mice, body weight: 18-22 g/mouse, 5 animals in each group; guinea pigs, body weight: 250-350 g/mouse, 2 animals in each group;

[0129] Injection Dose and Grouping

[0130] Abnormal toxicity test: the inoculation dose specified in the abnormal toxicity inspection method of item XIIF in the appendix of volume 3 of “Chinese Pharmacopoeia” (2015 edition) was adopted: mice, intraperitoneal injection of 0.5 ml (1 human dose); guinea pigs, intraperitoneal injection of 5 ml (10 human doses).

[0131] Repeated dosing test: after administration according to the above test, the tested animals were observed that no abnormal symptoms appeared within three days, continuing to raise until day 7, and the tested animals were healthy, normal with weight again; then the above doses were given repeatedly to continue to observe for 7 days to judge the result.

[0132] Acute toxicity test: 5 times of the inoculation dose specified in the abnormal toxicity inspection method of item XIIF in the appendix of volume 3 of “Chinese Pharmacopoeia” (2015 edition) was adopted, and a concentrated vaccine was prepared by various monovalent group B meningococcal protein stock solution; the dosage is as follows: mice, intraperitoneal injection of 0.5 ml (5 human doses), guinea pigs, intraperitoneal injection of 5 ml (50 human doses).

[0133] Judgment of Experimental Results

[0134] After the mice and guinea pigs were inoculated with the test product, they were observed continuously for 7 days. During the observation period, all the animals may be healthy and alive without any abnormal reaction. When the time expired, the weight of the animals were increased, and the test product was judged to be qualified.

[0135] Test Results

[0136] Abnormal Toxicity and Acute Toxicity Test of Trivalent Group B Meningococcal Protein Vaccine

TABLE-US-00014 Grouping Abnormal toxicity Repetitive dosing Acute toxicity (normal dose) (normal dose) (5 times of normal dose) Mouse Guinea pig Mouse Guinea pig Mouse Guinea pig Injection Before After Before After Before After Before After Before After Before After Body 1 19.2 23.1 300 322 19.3 23.3 303 325 19.4 23.2 310 352 weight 2 19 22.8 309 341 19.1 23 312 341 19.2 22.9 322 349 (g), and 3 20.2 23.8 / / 20.3 24 / / 20.4 23.9 / / No. 4 18.5 24 / / 18.6 24.2 / / 18.7 24.1 / / 5 20.1 22.9 / / 20.2 23.1 / / 20.3 23 / / Animal Eating, normal activity, No No Eating, normal activity, condition no abnormality abnormality abnormality no abnormality during after first after first observation injection, injection, normal normal activity after activity after injection injection again again

[0137] Result Analysis

[0138] Regardless of the abnormal toxicity of the normal dose, the repetitive dosing of the normal dose, and the acute toxicity test of 5 times the normal dose, after the inoculation, the animals in each group moved and ate normally without abnormal reactions, and all survived and gained weight. The tests confirm that the test product has reliable safety. It shows that the abnormal toxicity and acute toxicity tests of the trivalent group B meningococcal protein vaccine are qualified.

[0139] Conclusion: The abnormal toxicity and acute toxicity tests of the trivalent group B meningococcal protein vaccine provided by the present invention are qualified.

EXAMPLE 11

Comparison on the Particle Size Analysis of Site-Directedly Modified Lipoprotein and Wild-Type Lipoprotein

[0140] The size distribution of each lipoprotein MenB-V1.55-G2-L1, MenB-V2.16-S3-L1, and MenB-V3.45-S4-L1 was analyzed by using Dynamic Light Scattering (DLS) and Zetasizer Nano ZS. The lipoproteins obtained by site-directedly modifying and coupling the proteins has uniform particle size distribution, and good product uniformity; while the wild-type lipoproteins MenB-V1.55 and MenB-V2.16 obtained by traditional fermentation have uneven particle size distribution and aggregates. Referring to FIGS. 1-5, FIG. 1 is a diagram of the particle size of the MenB-V1.55 protein, including two main particle size ranges, e.g., about 20 nm and 90 nm respectively. FIG. 2 is a diagram of the particle size of MenB-V2.16 protein, including two main particle size ranges, e.g., about 30 nm and 400 nm respectively; FIG. 3 is a diagram of the particle size of MenB-V1.55-G2-L1 protein, the main particle size range of about 50 nm. FIG. 4 is a particle size diagram of MenB-V2.16-S3-L1 protein, and the main particle size range is about 60 nm. FIG. 5 is a diagram of the particle size of MenB-V2.16-S3-L1 protein, the main particle size range is about 50 nm.

[0141] In summary, the site-directedly modified lipoproteins obtained in this disclosure have consistent liposome length and significantly controllable quality, which can effectively avoid the disadvantage of heterogeneous lipidation in the expression process of recombinant lipoproteins, thereby ensuring immunogenicity and significantly reducing the degree of side effects.

[0142] Although the preferred examples are disclosed above in the present disclosure, they are not used to limit the claims. Any person skilled in the art may make some possible changes and modifications without departing from the concept of the present disclosure. Therefore, the protection scope of present disclosure should be based on the scope defined by the claims of the present disclosure.