Site-directed mutagenesis carrier protein and application thereof in preparation of vaccine
阅读说明：本技术 一种定点突变的载体蛋白及其在制备疫苗中的用途 (Site-directed mutagenesis carrier protein and application thereof in preparation of vaccine ) 是由 王浩猛 晏巧玲 巢守柏 毛慧华 朱涛 于 2019-10-15 设计创作，主要内容包括：本发明涉及一种定点突变的载体蛋白及其在制备疫苗中的应用,其中所述的载体蛋白选自白喉类毒素、白喉毒素的无毒突变体、细菌的外膜蛋白质和细菌表达蛋白中的一种或两种以上形成的融合蛋白,其中所述载体蛋白上至少1个位点上的氨基酸被突变为非天然氨基酸,所述非天然氨基酸含有叠氮基或炔基端基。本发明所述的定点突变的载体蛋白与多糖抗原相互反应过程中,形成共价键,同时结合物呈现连珠式状态,可有效避免载体蛋白与多糖抗原发生过度交联,结合物粒径分布显著均匀可控,为提高多糖蛋白结合疫苗质量提供了有效的手段。(The invention relates to a site-directed mutant carrier protein and application thereof in preparing vaccines, wherein the carrier protein is selected from one or more fusion proteins formed by diphtheria toxoid, a non-toxic mutant of diphtheria toxin, bacterial outer membrane protein and bacterial expression protein, wherein amino acid on at least 1 site on the carrier protein is mutated into unnatural amino acid, and the unnatural amino acid contains an azido group or alkynyl end group. In the mutual reaction process of the carrier protein with site-directed mutagenesis and the polysaccharide antigen, a covalent bond is formed, meanwhile, the conjugate is in a bead-connected state, the carrier protein and the polysaccharide antigen can be effectively prevented from being excessively crosslinked, the particle size distribution of the conjugate is obviously uniform and controllable, and an effective means is provided for improving the quality of the polysaccharide protein conjugate vaccine.)
1. A site-directed mutagenesis carrier protein, wherein the carrier protein is selected from the group consisting of diphtheria toxoid, a non-toxic mutant of diphtheria toxin, a bacterial outer membrane protein, and a fusion protein formed from one or more than two bacterial expression proteins, wherein at least 1 amino acid position on the carrier protein is mutated to an unnatural amino acid, and the unnatural amino acid has an azido or alkynyl end group.
2. The site-directed mutant protein of claim 1, wherein the carrier protein is selected from the group consisting of diphtheria toxoid, non-toxic mutant CRM197 of diphtheria toxin, group B meningococcal outer membrane protein, recombinant pseudomonas aeruginosa exotoxin a, haemophilus influenzae lipoprotein HID, and haemophilus influenzae heat shock protein HIN47, and preferably the carrier protein is selected from the group consisting of non-toxic mutant CRM197 of diphtheria toxin, haemophilus influenzae lipoprotein HID, fusion protein HIN47-HID, and fusion protein HID-HIN 47.
3. The site-directed mutant protein of claim 1, wherein said 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 site-directed mutant protein of claim 1, wherein the unnatural amino acid is
5. The site-directed mutant protein according to claim 1, wherein the mutation site of the HIN47-HID fusion protein is one or more amino acids at any position in SEQ ID No. 2, preferably the mutation site is selected from the group consisting of: position 174R, 217I, 223N, 226I, 313R, 315K, 522K, 557D, 653E or 684Y of the sequence shown in SEQ ID NO. 2.
6. The site-directed mutant protein according to claim 1, wherein the mutation site of the HID-HIN47 protein is one or more amino acids at any position in SEQ ID No. 3, preferably the mutation site is selected from the group consisting of: shown in SEQ ID NO:3, bit 78K, bit 113D, bit 209E, bit 240Y, bit 527R, bit 570I, bit 576N, bit 579I, bit 666R, or bit 668K.
7. The site-directed mutant protein of any one of claims 1-7, wherein the amino acid at position N of the amino acid sequence of the protein prior to mutation is mutated to Lys-azido, and wherein the mutated amino acid is linked as shown in the following formula:
wherein, N is a mutation site, and AA is amino acid before and after the mutation site.
8. A conjugate of a site-directed mutagenesis carrier protein, which is prepared from the site-directed mutagenesis carrier protein according to any one of claims 1 to 8 and a sugar, nucleic acid, amino acid, polypeptide or small molecule compound containing or modifying an alkynyl terminal group.
9. The conjugate of a site-directed mutagenesis carrier protein of claim 8, wherein the amino acid at position N of the amino acid sequence of the pre-mutagenesis protein is mutated to the following structure:
alternatively, the first and second electrodes may be,
wherein the content of the first and second substances,
n is a mutation site, AA is amino acid before and after the mutation site,
R2is sugar, nucleic acid, amino acid, polypeptide or carboxyl terminal modified group.
10. The conjugate of site-directed mutagenesis carrier protein according to claim 8 or 9, wherein the conjugate is a glycoconjugate, and the glycoconjugate is the site-directed mutagenesis carrier protein according to any one of claims 1 to 8 conjugated with a polysaccharide containing or modified with an alkynyl end group.
11. The conjugate of site-directed mutagenesis carrier protein according to claim 10, wherein the ratio of saccharide to carrier protein (w/w) in the saccharide conjugate is 0.3 to 3.
12. The conjugate of a site-directed mutagenesis carrier protein of claim 10 or 11 wherein the polysaccharide has between 10 and 50 saccharide repeat units per polysaccharide.
13. An immunogenic composition comprising the glycoconjugate of any one of claims 11 or 12 and a pharmaceutically acceptable excipient, carrier or diluent.
14. The immunogenic composition of claim 14, wherein the glycoconjugate is a glycoconjugate of a capsular polysaccharide selected from pneumococcus, one or a combination of two or more of serotypes 1,2,3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, or 33F.
15. A multivalent conjugate pneumonia vaccine, characterized by: the multivalent conjugate pneumonia vaccine is formed by multivalent pneumococcal polysaccharide and two or more site-directed mutant carrier proteins according to any one of claims 1-8.
16. Use of a site-directed mutagenesis carrier protein according to any one of claims 1-8, a conjugate of a site-directed mutagenesis carrier protein according to any one of claims 9-13, or an immunogenic composition according to any one of claims 14-15 for the preparation of a vaccine.
17. The use of claim 16, wherein the vaccine is an influenza vaccine or a pneumonia vaccine.
Technical Field
The invention relates to the field of biological pharmacy, in particular to a site-directed mutagenesis carrier protein and application thereof in preparing vaccines, especially pneumonia multivalent conjugate vaccines.
Background
Infection by streptococcus pneumoniae is one of the leading causes of morbidity and mortality worldwide. Pneumonia, febrile bacteraemia and meningitis are the most common manifestations of invasive streptococcus pneumoniae disease, while bacterial dissemination in the respiratory tract can lead to middle ear infections, sinusitis or recurrent bronchitis.
Polysaccharide protein conjugate vaccines are prepared using polysaccharides linked to protein carriers, the chemical bonds of which to the protein carriers can induce an immune response against bacteria that display on their surface the polysaccharides contained by the vaccine, thereby preventing disease, and therefore vaccination with polysaccharides from pathogenic bacteria is a potential strategy to enhance host immunity.
Although polysaccharides are themselves immunogenic, conjugation of polysaccharides to carrier proteins has been used to improve immunogenicity, which may be associated protein antigens from a target pathogen that enhance a specific immune response to the pathogen, or general immunogenic proteins that act primarily as adjuvants or general immune response stimulators.
The traditional polysaccharide-protein binding technology, Chinese patent ZL02159032.X, discloses a preparation method of polysaccharide-protein binding vaccine, which is also one of the most commonly used technologies for preparing polysaccharide binding vaccines at present. In the technique, adipic Acid Dihydrazide (ADH) is used as a linker to bind the polysaccharide to the protein. The combination mode firstly needs to activate the polysaccharide by cyanogen bromide, namely cyanogen bromide is used for acting on hydroxyl groups on polysaccharide molecules under alkaline conditions to form cyanate ester, and then the cyanate ester reacts with ADH; one carbon-oxygen bond in the cyanate is broken, and the cyanate and the amino at one end of the ADH generate addition reaction, so that ester hydrazide (AH) groups are introduced into polysaccharide molecules to form polysaccharide-AH derivatives; polysaccharide-AH derivatives form stable conjugates with carrier proteins mediated by carbodiimide (EDAC). The combination mode can reduce the steric hindrance of the combination of the polysaccharide and the carrier protein, retain the epitope of the polysaccharide, avoid the solubility of the polysaccharide, and reduce the side effect of the reaction of the polysaccharide and antiserum, but has some disadvantages: (1) the polysaccharide-AH derivative can continuously react with cyanogen bromide activated polysaccharide to form self-polymer of the polysaccharide, so that the combination efficiency of polysaccharide protein is reduced; (2) while mediating the binding of the ADH-derived polysaccharide to the carrier protein, EDAC easily causes self-crosslinking of the carrier protein, thereby reducing the binding efficiency of the polysaccharide protein, and thus, the immunogenicity and antibody duration of the polysaccharide protein conjugate vaccine still need to be further improved, for example, the polysaccharide conjugate vaccine needs to be immunized three times to generate an immune effect. These deficiencies limit the further development of polysaccharide conjugate vaccines.
Natalie W.Nairn et al introduce an unnatural amino acid containing an azide group into interferon β using a methionine deficient bacterium, and then perform single-site PEG modification using a copper-catalyzed Click reaction (Natalie W.Narin et al, Bioconjugate chem.2012).
The genetic code expansion technology is developed rapidly in recent years, an amber stop codon is used as a sense codon, and a designed unnatural amino acid can be finally introduced into protein by introducing corresponding orthogonal tRNA and aminoacyltRNA synthetase, so that the protein can be endowed with special functions according to the properties of the unnatural amino acid. To date, this technology has successfully expressed several dozen unnatural amino acids in a site-specific manner among proteins in living cells, and the involved unnatural amino acids include alkynyl and azide, and specific site-specific modifications of proteins can be made by using specific groups that are not present in the body weight of these organisms. In order to solve the problems of non-uniform combined vaccine combining process, complex separation and purification process and the like in the prior art, a method capable of modifying functional groups at any site specificity is urgently needed in the field.
CN106929482A describes site-directed mutant influenza virus, its live vaccine and its preparation method and application, while the modified carrier protein Hin47-HID of the present invention has proved to be capable of preventing non-invasive haemophilus influenzae, in a specific embodiment of the present invention, the present invention provides a site-directed mutant carrier protein Hin47-HID for introducing non-natural amino acid, and its site-directed modification method, the mutation method is to introduce non-natural amino acid Lys-azido for specific site in the protein, its specific azide group can specifically react with modifier to generate Click reaction, thereby site-directed coupling the non-natural amino acid on the target protein.
Disclosure of Invention
The invention relates to an immunogenic composition with site-directed mutagenesis and site-directed modification, a preparation method and application thereof. In the mutual reaction process of the carrier protein and the polysaccharide antigen, a covalent bond is formed, and meanwhile, the conjugate is in a bead-connected state, so that the carrier protein and the polysaccharide antigen can be effectively prevented from being excessively crosslinked, the particle size distribution of the conjugate is obviously uniform and controllable, and an effective means is provided for improving the quality of the polysaccharide protein conjugate vaccine.
The inventors of the present invention have made extensive studies and studies on the utilization of tRNA (tRNA) from Methanococcus archaeaPyl) And pyrrolysine-tRNA synthetase (tRNA)PylPylRS) protein translation SystemThen amino acid is site-directed incorporated into protein so as to obtain target peptide or protein of site-directed mutation, such as HID-Hin47, Hin47-HID or CRM197, then said carrier protein of site-directed mutation is used as raw material which can be further site-directed modified, and said carrier protein of site-directed mutation is conjugated with polysaccharide so as to obtain the polysaccharide vaccine of carrier protein of single site-directed modification.
Accordingly, the present invention provides a site-directed mutagenesis carrier protein, wherein the carrier protein is selected from a fusion protein formed by one or more than two of diphtheria toxoid, a non-toxic mutant of diphtheria toxin, a bacterial outer membrane protein and a bacterial expression protein, wherein at least 1 amino acid position on the carrier protein is mutated into an unnatural amino acid, and the unnatural amino acid contains an azido group or an alkynyl end group.
The carrier protein is selected from one or more than two fusion proteins of diphtheria toxoid, non-toxic mutant CRM197(SEQ ID NO:1) of diphtheria toxin, group B meningococcal outer membrane protein, recombinant pseudomonas aeruginosa exotoxin A, haemophilus influenzae lipoprotein HID and haemophilus influenzae heat shock protein HIN 47.
Preferably, the carrier protein is selected from haemophilus influenzae lipoprotein HID, fusion protein HIN47-HID or fusion protein HID-HIN 47.
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. Preferably, the unnatural amino acid is an azido-containing lysine derivative. More preferably, the unnatural amino acid is:
in one embodiment of the present invention, the carrier protein is HIN47-HID fusion protein, and the mutation site may be one or more amino acids at any position in SEQ ID No. 2, preferably, the mutation site is selected from: position 174R, position 217I, position 223N, position 226I, position 313R, position 315K, position 522K, position 557D, position 653E, position 684Y or other positions of the sequence shown in SEQ ID NO. 2 which have a minor effect on the activity.
In one embodiment of the present invention, the carrier protein is HID-HIN47 protein, and the mutation site can be one or more amino acids at any position in SEQ ID No. 3, preferably, the mutation site is selected from: shown in SEQ ID NO:3, position 78K, position 113D, position 209E, position 240Y, position 527R, position 570I, position 576N, position 579I, position 666R, position 668K or other sites that have a minor effect on activity.
After mutation, the amino acid sequence of the site-directed mutant protein differs from that of the target protein before mutation in that: the N-th amino acid of the amino acid sequence of the protein before mutation is mutated into Lys-azido, and the connection mode of the mutated amino acid is shown as the following formula:
wherein, N is a mutation site, and AA is amino acid before and after the mutation site
The invention also provides a conjugate of the site-directed mutagenesis carrier protein, wherein the conjugate is prepared from the site-directed mutagenesis carrier protein and a molecule containing or modified alkynyl end groups, and the molecule is a modifier containing sugar, nucleic acid, amino acid, polypeptide or small molecular compounds or obtained by modifying the sugar, nucleic acid, amino acid, polypeptide or small molecular compounds through the alkynyl end groups.
The site-directed mutagenesis carrier protein is prepared by a Click reaction with molecules containing or modified alkynyl end groups. The Click reaction can be monovalent copper mediated Click reaction, and can also be cyclooctyne or derivatives thereof mediated copper-free Click reaction.
The sugar, nucleic acid, amino acid, polypeptide or small molecular compound can be a modifier of a terminal alkynyl group of the sugar, nucleic acid, amino acid, polypeptide or small molecular compound, and the conjugate is prepared by site-specific coupling under the catalysis of monovalent copper, or the sugar, nucleic acid, amino acid, polypeptide or small molecular compound is a modifier taking cyclooctyne or a derivative thereof as a modifier, and site-specific coupling is directly realized.
Preferably, the conjugate of site-directed mutagenesis carrier protein of the present invention has the following structure that the N-th amino acid of the amino acid sequence of the protein before mutagenesis is mutated:
Wherein the content of the first and second substances,
n is a mutation site, AA is amino acid before and after the mutation site,
R2is sugar, nucleic acid, amino acid, polypeptide or carboxyl terminal modified group.
In one embodiment of the present invention, the conjugate is a glycoconjugate, wherein the glycoconjugate is obtained by conjugating the site-directed mutagenesis carrier protein of the present invention with a polysaccharide containing or modified with an alkynyl end group.
Preferably, the site-directed mutant protein of the present invention is conjugated to a polysaccharide, wherein the ratio (w/w) of polysaccharide to carrier protein is 0.3 to 3.
Preferably, there is at least one covalent bond between the carrier protein and the polysaccharide directly per 10 to 50 saccharide repeat units of the polysaccharide.
The polysaccharide can be a modifier of a terminal alkynyl group, and can be used for realizing site-directed coupling to prepare a glycoconjugate under the catalytic action of monovalent copper, or the polysaccharide is a modifier taking cyclooctyne or derivatives thereof as a modifying group, and can be used for directly realizing site-directed coupling to target polysaccharide protein.
The present application also provides an immunogenic composition wherein the glycoconjugate of the invention is in association with a pharmaceutically acceptable excipient, carrier or diluent.
The polysaccharide is selected from capsular polysaccharides, for example capsular polysaccharides are from
The invention also provides a pneumonia multivalent conjugate vaccine which is formed by multivalent pneumococcal polysaccharide and two or more than two site-directed mutant carrier proteins.
The invention also provides the site-directed mutagenesis carrier protein, the conjugate of the site-directed mutagenesis carrier protein or the application of the immunogenic composition in the preparation of vaccines.
Preferably, the vaccine is an influenza vaccine or a pneumonia vaccine.
Advantages of the invention over other approaches may be realized in one or more of the following:
1. unnatural amino acids can be introduced at any site of the protein, thereby creating a carrier protein that can be specifically modified at only that site;
2. the purposes of high efficiency and specificity modification can be realized by utilizing the specific active groups on the unnatural amino acids;
3. modification of specific sites can weaken protease activity with specific functions, reduce side effects and realize the effect of carrier protein
4. By optimizing modification conditions and utilizing the copper-free Click reaction mediated by cyclooctyne, the modification reaction which is efficient, harmless to protein, simple and feasible can be realized.
Drawings
FIG. 1 shows the SDS-PAGE gel of the mutated proteins Hin47-HID, polysaccharide protein conjugates obtained by click chemistry;
FIG. 2a shows a particle size distribution plot of the resulting polysaccharide protein conjugate as a click chemistry;
FIG. 2b shows a particle size distribution of a polysaccharide protein conjugate obtained by a conventional coupling method;
FIG. 3 shows Elisa results of rabbit triple immune in different coupling modes to obtain
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
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