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 严志红 晏巧玲 邵娟 史建明 隋秀文 李军强 朱涛 于 2020-07-07 设计创作，主要内容包括：本发明涉及定点突变、定点修饰的蛋白抗原。本发明还涉及定点突变和定点修饰蛋白抗原的方法,所述方法包括使用基因密码子扩展技术将非天然氨基酸定点引入到蛋白抗原特定位点,借助非天然氨基酸与修饰剂,所述修饰剂如三棕榈酰-S-甘油半胱氨酸、单磷酰脂质A等受体激动剂,与蛋白抗原定点修饰。本发明进一步涉及定点突变、定点修饰的蛋白抗原的应用,如作为疫苗等用途。(The invention relates to a site-directed mutant and site-directed modified protein antigen. The invention also relates to a method for site-directed mutagenesis and site-directed modification of a protein antigen, which comprises site-directed introduction of an unnatural amino acid into a specific site of the protein antigen using genetic codon expansion techniques, with the aid of the unnatural amino acid and a modifying agent, such as receptor agonists tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, etc., and site-directed modification of the protein antigen. The invention further relates to the application of the protein antigen of site-directed mutation and site-directed modification, such as the application as vaccine and the like.)

1. A site-directed mutant protein, wherein the protein is selected from one or more than two mutant proteins in group B meningococcal fHBP protein, the protein is selected from variant2, 3 in group B meningococcal fHBP protein Subfamily A and variant1 in Subfamily B, at least 1 amino acid position on the protein is mutated into an unnatural amino acid, and the mutation position is selected from: amino acids 2-10 of the sequence shown in SEQ ID NO. 1; the unnatural amino acid is

。

2. A site-directed mutant protein, wherein the protein is selected from one or more than two mutant proteins in group B meningococcal fHBP protein, the protein is selected from variant2, 3 in group B meningococcal fHBP protein Subfamily A and variant1 in Subfamily B, at least 1 amino acid position on the protein is mutated into an unnatural amino acid, and the mutation position is selected from: amino acids 2-10 of the sequence shown in SEQ ID NO 2; the unnatural amino acid is

。

3. A site-directed mutant protein, wherein the protein is selected from one or more than two mutant proteins in group B meningococcal fHBP protein, the protein is selected from variant2, 3 in group B meningococcal fHBP protein Subfamily A and variant1 in Subfamily B, at least 1 amino acid position on the protein is mutated into an unnatural amino acid, and the mutation position is selected from: amino acids 2-10 of the sequence shown in SEQ ID NO 3; the unnatural amino acid is

。

4. The site-directed mutant protein of any one of claims 1-3, wherein the N-th amino acid of the amino acid sequence of said protein is mutated to Lys-azido, and said 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.

5. A site-directed mutant group B meningococcal fHBP protein conjugate, wherein the conjugate is a site-directed mutant protein according to any one of claims 1 to 4 further conjugated to a modifying compound which is a TLR2 receptor agonist.

6. The conjugate of claim 5, wherein the agonist is selected from tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, dipalmitoyl-S-glycero-cysteine, or an analog thereof.

7. The conjugate of claim 5, wherein the amino acid at position N of the amino acid sequence of the protein is modified by mutation to the structure:

either the first or the second substrate is, alternatively,

wherein the content of the first and second substances,

n is a mutation site, AA is amino acid before and after the mutation site,

n=1~20，R₂are agonists of the TLR2 receptor.

8. The conjugate of claim 7, wherein R is₂Is tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, dipalmitoyl-S-glycero-cysteine or analogs thereof.

9. The conjugate of claim 8, wherein R is₂Is a tripalmitoyl-S-glycerocysteine analog selected from the group consisting of analogs of the following structural formula:

、orWherein n, m =1~ 6.

10. The conjugate of claim 8, wherein R is₂Are monophosphoryl lipid a receptor agonist derivatives.

11. The conjugate of claim 10, wherein the monophosphoryl lipid a receptor agonist has the formula:

n = 1-20, R end can be coupled with group B meningococcal fHBP protein of site-directed mutagenesis,

wherein R is₃Selected from phosphate or H;

R₄is selected fromN is 1,3, 5; or；

R₈Selected from H or OH;

R₅is selected from；

R₆Is selected from H or；

R₇Is selected fromOr。

12. The conjugate of any one of claims 5 to 11, wherein the group B meningococcal fHBP protein: the molar ratio of the modified compound is 1: 1-30.

13. The conjugate of any of claims 5-11, wherein the mutation site in the group B meningococcal fHBP protein is one or more of amino acids 2 to 30 of amino acid sequence No. 1.

14. The conjugate of any one of claims 5 to 11, wherein the mutation site of the group B meningococcal fHBP protein is selected from amino acids 2 to 10 of the sequence shown in SEQ ID No. 1.

15. The conjugate of any of claims 5-11, wherein the mutation site in the group B meningococcal fHBP protein is one or more amino acids from amino acid sequence 2 to amino acid sequence 30 of SEQ ID No. 2.

16. The conjugate of any of claims 5-11, wherein the mutation site of the group B meningococcal fHBP protein is selected from amino acids 2 to 10 of the sequence shown in SEQ ID No. 2.

17. The conjugate of any one of claims 5 to 11, wherein the mutation site in the group B meningococcal fHBP protein is one or more amino acids of amino acid sequence 2 to 30 of SEQ ID No. 3.

18. The conjugate of any one of claims 5 to 11, wherein the mutation site of the group B meningococcal fHBP protein is selected from amino acids 2 to 10 of the sequence shown in SEQ ID No. 3.

19. A vaccine or immunogenic composition comprising one or more of the site-directed mutant protein of any one of claims 1 to 4 or the conjugate of any one of claims 5 to 18.

20. The vaccine or immunogenic composition according to claim 19, wherein the vaccine or immunogenic composition comprises both three site-directed mutated proteins of claims 1 to 3 to form a multivalent vaccine or immunogenic composition, or both three conjugates of claims 14, 16, 18 to form a multivalent vaccine or immunogenic composition.

21. The vaccine or immunogenic composition of claim 19 or 20, wherein the group B meningococcal fHBP site-directed mutagenesis protein or conjugate is administered at a dose of 10 to 100 micrograms.

22. The vaccine or immunogenic composition of claim 19 or 20, further comprising a pharmaceutically acceptable excipient, carrier or diluent.

23. The vaccine or immunogenic composition of claim 19 or 20, further used in combination with an ACW135Y group epidemic encephalitis conjugate vaccine.

24. The vaccine or immunogenic composition of claim 19 or 20, wherein the vaccine or immunogenic composition comprises 5-10 micrograms of group a meningococcal polysaccharide antigen per dose, 5-10 micrograms of group C meningococcal polysaccharide antigen per dose, 5-10 micrograms of group W135 meningococcal polysaccharide antigen per dose, 5-10 micrograms of group Y meningococcal polysaccharide antigen per dose, and 10-100 micrograms of group B meningococcal fHBP site-directed mutant protein or conjugate per dose.

25. Use of a site-directed mutant protein according to any one of claims 1 to 4, a conjugate according to any one of claims 5 to 18, or an immunogenic composition according to any one of claims 19 to 24 for the preparation of a vaccine.

26. The use of claim 25, wherein the vaccine is a meningococcal vaccine.

27. A method for site-directed mutagenesis and site-directed modification of a protein antigen, the method comprising site-directed introduction of an unnatural amino acid into a specific site of a protein using gene codon expansion techniques to obtain a site-directed mutated protein, the site-directed mutated protein being further coupled to a modifying compound, the modifying compound being a lipoprotein receptor agonist.

28. The method of site-directed mutagenesis and site-directed modification of a protein antigen of claim 27, wherein the unnatural amino acid is。

29. The method for site-directed mutagenesis and site-directed modification of a protein antigen according to claim 27 or 28, wherein the lipoprotein receptor agonist is selected from the group consisting of tripalmitoyl-S-glycerocysteine, monophosphoryl lipid a, dipalmitoyl-S-glycero-cysteine, or an analog thereof.

30. The method for site-directed mutagenesis and site-directed modification of a protein antigen of claim 29, wherein the tripalmitoyl-S-glycerocysteine analog is selected from the group consisting of analogs of the following structural formula:

、orWherein n, m = 1-6.

31. A tripalmitoyl-S-glycerocysteine analog for use in site-directed mutagenesis and site-directed modification of a protein antigen, selected from the group consisting of analogs of the following structural formula:

、orWherein n, m =1, 3-6.

Technical Field

The invention relates to the field of biological pharmacy, in particular to a protein with site-directed mutation and site-directed modification and application thereof in preparing vaccines, especially meningococcal multivalent vaccines.

Background

Epidemic cerebrospinal meningitis (epidemic cerebrospinal meningitis) caused by Neisseria meningitidis (Nm) infection is a worldwide pathogenic acute respiratory infectious disease, and still seriously harms human health, especially children. Neisseria meningitidis can be divided into 13 serogroups based on differences in its capsular polysaccharide structure, and all serogroups can be pathogenic. A, B, C, Y, W135 Neisseria meningitidis group causes more than 95% of diseases related to Neisseria meningitidis.

Among them, A, C, Y, W135 group meningococcal capsular polysaccharide and polysaccharide-protein combined various monovalent (A or C), bivalent (A \ C) and tetravalent (ACYW 135) vaccines play a very key role and ideal effect in preventing invasive diseases and controlling disease prevalence and outbreak, and effectively control the occurrence of vaccine-type diseases.

Since the group B meningococcal capsular polysaccharide has weak immunogenicity due to its inclusion of epitopes potentially cross-immunized with human anti-stress agents, and can induce autoimmune diseases, vaccine research using group B Nm capsular polysaccharide as immunogen has been challenging.

Currently, international research on group B Nm vaccines mainly adopts two strategies, one is Outer membrane Vesicle (Outer membrane Vesicle OMV) vaccine based on Outer membrane protein, i.e. a clonal population for some specific circulating strains; the other is a recombinant protein vaccine based on reverse vaccinology technology.

In the last 70 s of the century, Zollinger, Frasch et al first reported the study of group B meningococcal Outer Membrane Vesicle (OMV) vaccines, followed by the emergence of three representative OMV vaccines, respectively.

In 1986, the epidemic of the B group epidemic in south America, Brazil, Guba, etc., began, and based on the B group meningococcal Cu385/83 strain (B4: P1.19, 15) which is an epidemic in Guba, the research institute of Guba Finlay developed the VA-ENGOC-BC vaccine containing the B group OMV, the C group CPS (capsular polysaccharide), and AL (OH) 3 adjuvant. Production permission was obtained in 1989, immunization program was incorporated in 1991, and 5500 ten thousand doses of vaccination had been accumulated.

In 1991, the popular B-group meningococcus 44/76-SL (B15: P1.7, 16) was used as a vaccine strain by Norwegian Institute of Public Health, NIPH, and a MenBvac group B OMV vaccine was developed, later assigned to Kailong (Chiron) and developed and scaled up.

In 1991, a group B epidemic in New Zealand appeared and a group B meningococcal vaccine, MeNZB, was developed based on the disease isolate NZ98/254 (CC 41/44, B4: P1.7-2.4) during the year 2004-.

In 2000, the reverse vaccinology technology was successfully applied to group B meningococcal vaccines for the first time. The technology is mainly characterized in that three proteins of fHbp, NadA and NHBA are respectively cloned and expressed through a gene recombination expression technology to serve as candidate components of a group B Nm protein vaccine. At present, two B-group epidemic encephalitis protein vaccines developed by reverse vaccinology techniques are available internationally, namely 4C MenB (Bexsero) from Nowa and bivalent fHBp (r-fHBp) from Perey. 4C MenB (Bexsero) contains FHBp1 type protein, NadA protein and NHBA protein, followed by addition of New Zealand group B OMV vaccine (MeNZB). Bivalent fHBp (r-fHBp) is a Bivalent protein vaccine containing fHBp1 type and 3 type.

In addition, the hui rLP2086 vaccine, two recombinant lipoproteins LP2086 containing a single lipoprotein (rLP 2086), namely two fHBp recombinant lipoproteins Subfamily a variant a05 and Subfamily B variant01, were also a component of 4C MenB, and the results of the experiment also showed that bactericidal antibodies were induced.

Other reports, such as Bruge et al, have produced polysaccharide-protein conjugate vaccines by substituting acetyl groups in purified group B Nm capsular polysaccharides with propionyl groups, and then conjugating the resulting polysaccharide-protein conjugate to a carrier protein, although the vaccine proved to be safe, the induced antibodies were not functionally active.

Although the development history of group B meningococcal vaccines is long, and a plurality of products are on the market or in research, problems still need to be solved.

1. There is currently no international universal group B meningococcal OMV vaccine

Because epidemic serogroups and subtypes of the B group epidemic in different countries and regions have obvious regional characteristics, the OMV vaccines developed so far have no universality. Therefore, for the research and development of group B epidemic encephalitis OMV vaccines, the selection of strains suitable for the pathogenesis characteristics of local diseases is particularly important. The European district group B Nm is dominated by the ST-41/44clonal complex serotype 15, and the American and oceania group B Nm is dominated by the ST-8CC, ST-11CC and ST-32CC serotype 4. According to the Chinese epidemic cerebrospinal meningitis monitoring and Nm molecular epidemiological research, the epidemic type B in China is different from the epidemic type B in China, mainly the serotype 3 ST-4821CC is taken as the main epidemic meningitis type, and the epidemic meningitis type B is planted in a large number in healthy people. According to B group epidemic: guba: VA-MENGOC-BC vaccine strain: cu385/83 (PorA: P1.19, 15, PorB: serotype 4); norway: MenBvac TM vaccine strain: 44/76-SL (PorA: P1.17, 16, PorB: serotype 15); new Zealand: MenZB vaccine strain: NZ98/254 (PorA: P1.7-2.4, PorB: serotype 4). GSK is subjected to the Guba VA-MENGOC-BC vaccine technology, the vaccine is popularized to other countries, and the clinical research protection rates in different regions are different. In 1987-1989, Sierra reported that the protection rate in cuba was 83% for 106000 students aged 10-14 years (6-8 weeks apart); in 1990 1989-1990 Moraes reported a protection rate of 47% in children at 24-47 months of age, performed in St.Paul; protection rate in children over 47 months of age was 74%; in 1990-1991, Mornha reported that multiple clinical studies in children over 47 months of age, performed in Riyowa, showed a protective rate of 58-74%. Epidemiological observation in brazil in 1992 showed a protection rate of 74% for people over 4 years of age and no protection for people under 2 years of age, thus the scholars suggested that the vaccine lacks protection for children under 4 years of age. Since the vaccine was approved for use in 1989, its use in the american countries cuba, brazil, colombia, yerba mate, etc. successfully controlled epidemic encephalitis, and it is particularly important that some of the epidemic encephalitis are caused by different group B strains. The conclusion from the experiments conducted by Cassio de Moraes is that "this finding suggests that the vaccine is able to provide protection against a proportion of group B meningococcal infections, not just vaccinal strains". From 1991, VA-MENGOC-BC was placed in a part of Guba infant vaccination program. The focus of controversy, the hypothesis of low protection for the population under 4 years old, includes argumentation by many scholars and experts of royal medical college of london, VA-MENGOC-BC can also cause appropriate immune response even if used for infant vaccination.

VA-MENGOC-BC is the only group B meningococcal OMV vaccine approved for many years and has been inoculated with a total of about 5500 doses in 15 countries, predominantly in Latin America and the Caribbean region, and has proven to be the first safe and effective commercial vaccine worldwide. Subsequently, important experiences were also obtained from the respective "tailored" OMV vaccines studied in various countries. To date, however, there is no international group B meningococcal OMV vaccine in common use.

2. Limitations of the protective coverage of OMV vaccines

Meningococcus group B is rather complex, based on its proteinic typing of PorA and PorB, and has low cross-protection between serotypes and subtypes, and therefore, the protective coverage of a limited number of "tailored" vaccines is limited.

PorA in group B meningococcal OMVs was identified as a major inducer of immune response and as a target for bactericidal serum antibodies, and many meningococci expressed this protein. However, different PorA proteins have different antigenic specificities, and thus, the immune response elicited by one PorA cannot defend against a strain of heterologous PorA antigen. Group B Nm strain epidemics surveys conducted in the united states found that combining 20 different PorA proteins would cover 80% of the epidemic strains. The MenB OMV vaccine has been successfully used for controlling the prevalence and outbreak of diseases, but the effectiveness is only limited to controlling the outbreak of vaccine type and subtype MenB diseases, and the effectiveness is limited for diseases caused by different types and subtypes MenB.

3. Process for producing MenB OMV

To date, MenB OMV vaccines have been produced in different processes in different countries, resulting in different degrees of differences and damage to the components, content, purity and natural conformational integrity of the antigen.

Since MenB is relatively complex in structure and composition, the OMV antigen component is obtained by elution and purification from the cell membrane. OMV is a multi-site antigen complex which is derived from bacterial cell membranes and has a vesicle-shaped structure consisting of multiple protein components with complex three-dimensional conformations, LPS and the like, and the synergistic effect of each antigen site can achieve better immune effect.

Early studies highlighted protein purity, but employed more complex physical, chemical combined repetitive processes that disrupted the natural conformation of OMVs and attenuated abundant antigenic sites, thereby affecting immunogenicity and antibody specificity of vaccines, resulting in poor protection rates and immune persistence.

4. Broad spectrum group B meningococcal vaccines (e.g. Nowa 4C MenB, Bexsero) have difficulty achieving broad protection

Since OMV vaccines have the geographical specificity of selected epidemic strains and the influence of the factors, and thus are difficult to have wide universality and effectiveness, norwa and fevered companies have developed 4C MenB (Bexsero), a B-group meningococcal vaccine containing a plurality of protein components, respectively, based on reverse vaccinology techniques in order to achieve a wider coverage rate against B-group strains.

4C MenB (Bexsero) contains 5 E.coli expressed protein antigens screened by reverse vaccinology techniques, which are: fHbp (factor H binding protein, originally designated LP 2086), NHBA (Neisseria heparin binding antigen), NadA (meningococcal adhesin A), and GNA2091 and GNA1030, which bind to fHbp and NHBA, respectively, to form fusion antigens GNA2091-fHbp and GNA1030-NHBA, which in turn increases the group B OMV (MeNZB) composition of a new strain of epidemic West orchid (NZ 98/254; P1.7-2.4, ST 41/44).

The broad protection of the 4C MenB vaccine against different group B Nm is constantly under debate as a broad spectrum group B meningococcal vaccine. The representative group B strains circulating in different countries are different, and the vaccine only contains OMV of PorA protein and only can generate protective force against the group B strains of P1.7-2.4. The B groups Nm PorA types popular in different countries are different, and the coverage rate of the PorA strains of the 4CMenB vaccine is influenced by the PorA types of the strains of the different popular B groups, so that the method has limitation.

The fHbp in the 4C MenB vaccine is of type Variant1, and immune sera are 95% effective against fHbp Variant1 strains, but only 56% protective against Variant2 or 3 strains. 35% of pathogenic strains of group B of the United states are Variant type 2 or 3. About 90% of the meningococcus in group B of China are Variant type 2.

In addition, NadA in the 4C MenB vaccine is the main antigen component, but most of the B-group meningococci epidemic in different regions do not express NadA, for example, 36% of the B-group strains in the united states do not contain NadA gene, and more than 90% of the B-group meningococcal strains in our country do not contain NadA gene. Therefore, the 4C MenB vaccine is not suitable for preventing diseases in China.

In general, reviewing the research and development of the group B Nm OMV protein vaccine in various countries over decades, the clinical application effect shows that the difference is large in different regions, and the following factors may exist for the analysis reason:

the group B has more Nm types and subtypes and weak cross protection;

the antigenicity of epidemic strains in each region has regional characteristic difference and is influenced by a plurality of factors which are not disclosed at present;

the complexity and excessive purification of foreign vaccine preparation process change the three-dimensional natural conformation (protein two, three and four spatial conformations) of the bacterial antigen, and the induced antibody lacks specificity for the invasion of the epidemic strains in the nature;

fourthly, the multi-step purification process also causes the loss, damage and singleness of rich antigenic sites and influences the antigenicity of the rich antigenic sites.

Modification in biomolecular systems by the Click reaction using azide and alkyne groups is a popular field of research in recent years. 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 (natie w. narinetal, 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 lipidization degree, complex separation and purification process and the like in the recombinant protein vaccine in the prior art, the recombinant protein vaccine with a specific modified functional group at a specific site is urgently needed in the field so as to ensure the consistency among product batches. In the field of vaccines, the technology is also applied, for example, CN106929482A describes site-directed mutated influenza virus, live vaccine thereof, and preparation method and application thereof, and non-natural amino acids are introduced into any gene of influenza virus at fixed points by using gene codon expansion technology and gene codon expansion technology, so as to improve the safety and immunogenicity thereof. CN110845587A describes site-directed mutant diphtheria toxoid, diphtheria toxin carrier protein and its use in preparing vaccine, the immunogenic composition introduces amber codon TAG at specific site of carrier protein gene, and utilizes orthogonal aminoacyl tRNA synthetase-tRNA to site-directed mutant unnatural amino acid with cross-linking property to specific site of carrier protein. 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. However, the above prior art is limited to site-directed mutagenesis of unnatural amino acids to specific sites in a target protein, and no further modification is made.

The pre-modified recombinant protein fHBP provided by the invention has been proved to be capable of preventing infection of group B meningococcus, and Bivalent fHBp (r-fHBp) of Hewlett-packard company is Bivalent protein vaccine containing fHBp1 type and 3 type, and has a bactericidal effect in teenagers, but in infant groups, the proportion of people with side effects such as fever and local red swelling is obviously increased, and the increase of the side effects is suspected due to non-uniform lipidization degree. Therefore, there is an urgent need for further modification of the recombinant protein fHBP of group B meningococci to improve its immunogenicity and reduce side effects.

Therefore, the invention provides a fHBP protein for introducing unnatural amino acids at fixed points and a method for modifying the same at fixed points, wherein the mutation method is that the unnatural amino acid Lys-azido introduced to a specific site in the protein has a specific azide group which can specifically react with a modifying agent to generate Click reaction, so that the unnatural amino acids are coupled to a target protein at fixed points.

Disclosure of Invention

The invention relates to an immunogenic composition with site-directed mutagenesis and site-directed modification, a preparation method and application thereof. The antigen protein and the liposome form covalent bonds through Click reaction, the obtained lipoprotein can effectively avoid the defect of inhomogeneous lipidization of the recombinant lipoprotein in the expression process, the obtained fixed-point modified lipoprotein has consistent liposome length and obviously controllable quality, and an effective means is provided for improving the quality of the lipoprotein vaccine.

Through the consideration and research of the prior art, the inventor utilizes a protein translation system of tRNA (tRNAPcyl) of the Archimedes and pyrrolysine-tRNA synthetase (tRNAcyl/PylRS) to ensure that unnatural amino acid is doped into protein in a fixed point manner, thereby obtaining target peptide or protein of fixed point mutation, such as variant2 and 3 in group B meningococcus fHBP protein Subfamily A and variant1 in Subfamily B, and then uses the antigen protein of fixed point mutation as a raw material which can be further modified in a fixed point manner, and conjugates the antigen protein of fixed point mutation with liposome to obtain the lipid protein vaccine of fixed point modification.

Accordingly, the invention provides a group B meningococcal fHBP protein antigen with site-directed mutagenesis, wherein the group B meningococcal fHBP protein antigen is selected from one or more than two protein antigens of variant2 and 3 in Subfamily A and variant1 in Subfamily B, wherein at least 1 amino acid on a carrier protein is mutated into an unnatural amino acid, and the unnatural amino acid contains an azido group or an alkynyl group.

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 invention, the mutation site of variant2 in subgroup B meningococcal fHBP protein antigen subunit A can be one or more amino acids at any site in SEQ ID NO. 1, and preferably, the mutation site is selected from positions 2-10 of the sequence shown in SEQ ID NO. 1 or other sites which do not affect the epitope.

In one embodiment of the present invention, the mutation site of variant3 in subgroup B meningococcal fHBP protein antigen subunit a may be one or more amino acids at any position in SEQ ID No. 2, and preferably, the mutation site is selected from: 2, or other sites that do not affect the epitope of the antigen.

In one embodiment of the invention, the mutation site of variant1 in the antigen subunit B of the group B meningococcal fHBP protein may be one or more amino acids at any position in SEQ ID No. 3, and preferably, the mutation site is selected from: 3 or other sites that do not affect the epitope of the antigen.

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 site-directed mutagenesis group B meningococcal fHBP protein antigen conjugate, which is prepared from the site-directed mutagenesis carrier protein and a compound containing or modifying an alkynyl end group, wherein the molecule is a modifier containing sugar, nucleic acid, amino acid, polypeptide or small molecule compound, or the sugar, nucleic acid, amino acid, polypeptide or small molecule compound obtained by modifying the alkynyl at the tail end.

The group B meningococcal fHBP protein of site-directed mutagenesis 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, in the conjugate of group B meningococcal fHBP protein with site-specific mutation according to the present invention, the N-th amino acid of the amino acid sequence of the protein before mutation is mutated into the following structure:

or is or

Wherein the content of the first and second substances,

n is a mutation site, AA is amino acid before and after the mutation site,

r2 is a sugar, nucleic acid, amino acid, polypeptide, or carboxy-terminal modifying group.

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 invention also provides the site-directed mutant group B meningococcal fHBP protein, a conjugate of the site-directed mutant group B meningococcal fHBP protein, or an application of the immunogenic composition in preparing a vaccine.

Preferably, the vaccine is a meningococcal vaccine.

Specifically, the invention relates to a site-directed mutant protein, wherein the protein is selected from one or more than two mutant proteins in group B meningococcal fHBP protein, wherein amino acids at least 1 site on the protein antigen are mutated into unnatural amino acids, and the unnatural amino acids contain azide groups, alkynyl end groups or other active groups.

The protein is selected from variant protein formed by one or more than two of group B meningococcal fHBP proteins, and preferably, the protein antigen is selected from variant2 and 3 in group B meningococcal fHBP protein Subfamily A and variant1 in Subfamily B.

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.

The unnatural amino acid is

。

The mutant site of the group B meningococcal fHBP protein is one or more amino acids in the 2 nd to 30 th amino acid sequences in SEQ ID NO. 1, and preferably, the mutant site is selected from: amino acids 2-10 of the sequence shown in SEQ ID NO. 1.

The mutant site of the group B meningococcal fHBP protein is one or more amino acids in the 2 nd to 30 th amino acid sequences in SEQ ID NO. 2, and preferably, the mutant site is selected from: amino acids 2-10 of the sequence shown in SEQ ID NO 2.

The mutation site of the group B meningococcal fHBP protein can be one or more amino acids in amino acid sequences 2-30 in SEQ ID NO. 3, and preferably, the mutation site is selected from the group consisting of: amino acids 2-10 of the sequence shown in SEQ ID NO 3.

The N-th amino acid of the amino acid sequence of the protein 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.

Specifically, the invention also relates to a conjugate of site-directed mutant group B meningococcal fHBP protein, wherein the conjugate is the site-directed mutant protein as described in any one of claims 1-8, and is further coupled with a modifying compound, and the modifying compound is a TLR2 receptor agonist.

The agonist is selected from tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, dipalmitoyl-S-glycero-cysteine or analogs thereof.

The N-th amino acid of the amino acid sequence of the protein is mutated and modified into the following structure:

either the first or the second substrate is, alternatively,

wherein the content of the first and second substances,

n is a mutation site, AA is amino acid before and after the mutation site,

n = 1-20, and R2 is a TLR2 receptor agonist.

R2 is tripalmitoyl-S-glycerocysteine, monophosphoryl lipid a, dipalmitoyl-S-glycero-cysteine or an analogue thereof, preferably a tripalmitoyl-S-glycero-cysteine analogue, selected from the analogues of the following structural formula:

、orWherein n, m =1~ 6.

R2 is a monophosphoryl lipid A receptor agonist derivative, preferably a monophosphoryl lipid A receptor agonist, having the structural formula:

n = 1-20, R end can be coupled with group B meningococcal fHBP protein of site-directed mutagenesis,

wherein R3 is selected from phosphate or H;

r4 is selected fromN is 1,3, 5; or；

R8 is selected from H or OH;

r5 is selected from；

R6 is selected from H or；

R7 is selected fromOr。

Wherein the group B meningococcal fHBP protein: the molar ratio of the modified compound is 1: 1-30.

The mutant site of the group B meningococcal fHBP protein is one or more amino acids in the 2 nd to 30 th amino acid sequences in SEQ ID NO. 1, and preferably, the mutant site is selected from: amino acids 2-10 of the sequence shown in SEQ ID NO. 1.

The mutant site of the group B meningococcal fHBP protein is one or more amino acids in the 2 nd to 30 th amino acid sequences in SEQ ID NO. 2, and preferably, the mutant site is selected from: amino acids 2-10 of the sequence shown in SEQ ID NO 2.

The mutation site of the group B meningococcal fHBP protein can be one or more amino acids in amino acid sequences 2-30 in SEQ ID NO. 3, and preferably, the mutation site is selected from the group consisting of: amino acids 2-10 of the sequence shown in SEQ ID NO 3.

In particular, the invention also relates to a vaccine or immunogenic composition comprising one or more of the site-directed mutant proteins of any one of claims 1 to 8 or the conjugates of any one of claims 9 to 17

Wherein the vaccine or immunogenic composition comprises simultaneously three site-directed mutated proteins of claims 5-7 to form a multivalent vaccine or immunogenic composition, or three conjugates of claims 15-17 to form a multivalent vaccine or immunogenic composition.

The dose of the group B meningococcus fHBP site-directed mutagenesis protein or conjugate is 10-100 micrograms. Further comprising a pharmaceutically acceptable excipient, carrier or diluent. The vaccine or immunogenic composition is further used in combination with an ACW135Y group epidemic encephalitis conjugate vaccine.

The vaccine or the immunogenic composition contains 5-10 micrograms of group A meningococcal polysaccharide antigen per dose, 5-10 micrograms of group C meningococcal polysaccharide antigen per dose, 5-10 micrograms of group W135 meningococcal polysaccharide antigen per dose, 5-10 micrograms of group Y meningococcal polysaccharide antigen per dose, and 10-100 micrograms of group B meningococcal fHBP site-directed mutant protein or conjugate per dose.

In particular, the invention also relates to the application of the site-directed mutant protein, the conjugate or the immunogenic composition in preparing vaccines. Wherein the vaccine is a meningococcal vaccine.

Specifically, the invention also relates to a method for site-directed mutagenesis and site-directed modification of a protein antigen, which comprises the step of introducing an unnatural amino acid into a specific site of a protein by using a gene codon expansion technology to obtain a site-directed mutated protein, wherein the site-directed mutated protein is further coupled with a modified compound, and the modified compound is a lipid protein receptor agonist.

The unnatural amino acid is。

The lipoprotein receptor agonist is selected from tripalmitoyl-S-glycerocysteine, monophosphoryl lipid A, dipalmitoyl-S-glycero-cysteine or analogs thereof.

The tripalmitoyl-S-glycerocysteine analog is selected from analogs of the following structural formula:

、orWherein n, m =1~ 6.

In particular, the invention also relates to a tripalmitoyl-S-glycerocysteine analogue used for site-directed mutagenesis and site-directed modification of a protein antigen, which is selected from the analogues of the following structural formula:

、orWherein n, m =1~ 6.

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 a protein, thereby creating a protein antigen 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. by optimizing modification conditions and utilizing the copper-free Click reaction mediated by cyclooctyne, the modification reaction which is efficient, harmless to protein and simple and feasible can be realized;

4. by introducing the modification group with confirmed structure, the obtained conjugate of group B meningococcal fHBP protein has uniform components and controllable quality, and the immunogenicity is ensured while the side reaction degree is remarkably reduced;

5. the conjugate prepared by the invention can be used together with ACW135Y epidemic encephalitis conjugate vaccine to improve the protective coverage of the vaccine.

Drawings

FIG. 1 MenB-V1.55 protein particle size plot;

FIG. 2 MenB-V2.16 protein particle size plot;

FIG. 3 is a graph showing the particle size of MenB-V1.55-G2-L1 protein;

FIG. 4 is a graph showing the particle size of MenB-V2.16-S3-L1 protein;

FIG. 5 is a graph showing the particle size of MenB-V2.16-S3-L1 protein.

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.