Fusion protein containing NHBA and fHbp, immune preparation containing fusion protein, and preparation method and application of fusion protein

Abstract

The invention discloses a fusion protein containing NHBA and fHbp, an immune preparation containing the fusion protein, and a preparation method and application of the fusion protein. The fusion protein comprises NHBA or a variant thereof, and operably linked fHbp or a variant thereof. According to the fusion protein, the stability of fHbp and NHBA can be remarkably improved, and meanwhile, the fusion body is endowed with excellent bifunctional activity, so that the negative influence on the stability of fHbp and the immunogenicity of the fHbp is little or no.

Description

Technical Field

[0001] The present application relates to the field of protein engineering, and specifically to a fusion protein comprising NHBA and fHbp, an immune preparation comprising the fusion protein, and a preparation method and use thereof, and further to a fusion protein comprising NHBA and fHbp, a nucleic acid encoding the fusion protein, a recombinant expression vector comprising the nucleic acid, a host cell expressing the fusion protein, an immune preparation comprising the fusion protein, a kit comprising at least one of the above, a method for preparing the fusion protein, and the like. Background Art

[0002] Neisseria meningitidis is a human pathogen and the leading cause of sepsis and meningitis worldwide. Neisseria meningitidis is a commensal bacterium of the human nasopharynx that can occasionally cross the epithelial and blood-brain barriers to cause invasive meningococcal disease (IMD). Based on the biochemical properties of the capsular polysaccharide, Neisseria meningitidis can be divided into 12 serotypes, of which groups A, B, C, W, X, and Y are the main serotypes causing invasive meningitis diseases worldwide. Studies have shown that immunotherapy is one of the most effective methods for preventing and controlling meningitis diseases. Capsular polysaccharides, polysaccharide vaccines, and polysaccharide-protein conjugate vaccines based on meningococcal polysaccharides are effective in preventing meningococcal infections caused by groups A, C, W, and Y. The capsular polysaccharide of group B meningococci is similar to the polysialylated capsular polysaccharide present on human nerve cells, resulting in its poor immunogenicity. Therefore, the focus of its vaccine design is on protein antigens other than capsular polysaccharides, including the outer membrane protein (OMP) expressed on the surface.

[0003] Factor H binding protein (fHbp) is one of the OMPs expressed on the surface of group B meningococci. A large number of studies have shown that fHbp is a key virulence factor of Neisseria meningitidis. It specifically binds to human complement factor H, allowing meningococci to survive and grow in human blood, and can induce serum bactericidal activity in the body after immunization. Currently, fHbp is the active component of two group B meningococcal vaccines on the market (Bexsero™ and Trumenba®).

[0004] fHbp is a 27kDa lipoprotein composed of two β-barrels, which is located on the surface of Neisseria meningitidis. fHbp can be divided into three variants (V1 / V2 / V3), or two subfamilies (A / B) based on the diversity of the amino acid sequence. The amino acid sequence homology of subvariant fHbp within the same variant is more than 85%, while the amino acid sequence similarity of fHbp between different variants is only 60-70%. In addition, immunization with fHbp of the same variant family can induce the body to produce cross-immune responses against some fHbp within the same variant, but there is no immune protection response to other variant families (except for some cross-immune responses between V2 and V3).

[0005] V2 variant is one of the main fHbp variants in China, but compared with V1 variant, V2 variant has inherent instability and sensitivity to protein degradation. It has been reported that this instability is caused by the N-terminal β-barrel domain, and any substitution or truncation of this region may promote its instability (Yee WX, Barnes G, Lavender H, Tang CM. Meningococcal factor H-binding protein: implications for diseasesusceptibility, virulence, and vaccines. Trends Microbiol. 2023 Aug;31(8):805-815. doi: 10.1016 / j.tim.2023.02.011. Epub 2023 Mar 20. PMID: 36941192;PMCID: PMC10914675.). In the Bexsero™ vaccine component developed by GSK, fHbp-V1 improves its stability by fusing GNA2091 (another antigen in group B meningococci) to its N-terminus, but fusing it to the N-terminus of V2 does not effectively improve its stability, and GNA2091 has weak immunogenicity and cannot induce the body to produce bactericidal antibodies. In 2015, GSK improved protein stability by performing site-directed mutations on V2 or V3, and ensured its immunogenicity by reducing its binding to fH factor, but the results of serum bactericidal tests showed that the bactericidal activity produced by V2 or V3 mutant immunization was lower than that of wild-type immunization, indicating that this technology of improving the stability of fHbp by site-directed mutation of fHbp amino acids has certain risks. In addition, there are many studies to improve the immunogenicity of fHbp by fusing different fHbp variants or constructing chimeras, but they are still basically in preclinical research. Summary of the invention

[0006] Based on this, it is necessary to provide at least a fusion protein comprising NHBA and fHbp, an immune preparation comprising the fusion protein, and a preparation method and use thereof.

[0007] In a first aspect of the present application, a fusion protein is provided, which comprises a meningococcal heparin binding protein antigen (NHBA) or a variant thereof, and an operably linked factor H binding protein (fHbp) or a variant thereof.

[0008] In some embodiments, the fHbp or variant thereof is linked to the N-terminus, C-terminus or loop of the NHBA or variant thereof.

[0009] In some embodiments, the amino acid sequence of the NHBA is as shown in SEQ ID NO: 1.

[0010] In some embodiments, the variant of the NHBA comprises a truncation of the NHBA.

[0011] In some embodiments, the N-terminus of the NHBA is truncated, and at least the conserved functional domain at the C-terminus is retained.

[0012] In some embodiments, the highly variable N-terminal domain is truncated.

[0013] In some embodiments, the NHBA is truncated to a length of 1 to 200 amino acid residues, for example, the truncated length is the first 179 amino acid residues at the N-terminus.

[0014] In some embodiments, the variant of the fHbp comprises a truncation of the fHbp.

[0015] In some embodiments, the fHbp is truncated at the N-terminus.

[0016] In some embodiments, the length of the truncated fHbp is 1 to 40 amino acids.

[0017] In some embodiments, what is truncated is the leader peptide at the N-terminus of fHbp, and the length of the leader peptide is optionally 26 amino acid residues.

[0018] In some embodiments, the fHbp are of the same subvariant.

[0019] In some embodiments, the amino acid sequence of the fHbp variant is shown in any one of SEQ ID NOs: 2 to 4.

[0020] In some embodiments, the fusion protein comprises a variant of the NHBA and a variant of the fHbp.

[0021] In some embodiments, the fusion protein comprises a variant of NHBA, a linker, and a variant of fHbp in sequence from N-terminus to C-terminus.

[0022] In some embodiments, the linker is optionally (G4S)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)n or (A)n, wherein n is an integer greater than or equal to 1.

[0023] In some embodiments, the amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs: 5 to 7.

[0024] In the second aspect of the present application, a nucleic acid molecule is provided, which encodes the fusion protein as described in the first aspect.

[0025] In the third aspect of the present application, a recombinant expression vector is provided, which comprises the nucleic acid molecule as described in the second aspect.

[0026] In the fourth aspect of the present application, a host cell is provided, which expresses the fusion protein as described in the first aspect.

[0027] In some embodiments, it comprises one or more of the nucleic acid molecule described in the second aspect and the recombinant expression vector described in the third aspect.

[0028] In some embodiments, the host cell is selected from the group consisting of bacteria and fungi.

[0029] In some embodiments, the bacteria is Escherichia coli.

[0030] In the fifth aspect of the present application, an immune preparation is provided, which comprises the fusion protein as described in the first aspect.

[0031] In some embodiments, the immunizing formulation further comprises an adjuvant.

[0032] In some embodiments, the adjuvant includes one or more of aluminum hydroxide, aluminum phosphate, MPLA, AS03, AS04, CpG-ODN, and FM59.

[0033] In some embodiments, in the immune preparation, the concentration of the fusion protein is 10 μg / mL to 100 μg / mL, and the concentration of the adjuvant is 0.5 mg / mL to 2 mg / mL.

[0034] In some embodiments, the immunizing formulation comprises a vaccine formulation.

[0035] In the sixth aspect of the present application, a kit is provided, comprising one or more of the fusion protein as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant expression vector as described in the third aspect, the host cell as described in the fourth aspect, and the immune preparation as described in the fifth aspect, and a container.

[0036] In a seventh aspect of the present application, a method for preparing a fusion protein is provided, comprising:

[0037] Cultivating the host cell as described in the fourth aspect to obtain a culture fluid; and,

[0038] The fusion protein is isolated from the culture fluid.

[0039] In the eighth aspect of the present application, there is provided use of the fusion protein as described in the first aspect, the nucleic acid molecule as described in the second aspect, the recombinant expression vector as described in the third aspect, or the host cell as described in the fourth aspect in the preparation of an immune preparation.

[0040] The present application uses heparin binding protein (NHBA) or its variant on the surface of meningococcal strains and fHbp or its variant to form a fusion protein. The NHBA is present on the surface of almost all meningococcal strains. In some embodiments, the use of truncates of NHBA and truncates of fHbp can significantly improve the stability of fHbp and NHBA, while giving the fusion excellent bifunctional activity (including two functions of fHbp and NHBA), so that the stability of fHbp and its immunogenicity are less or not negatively affected. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions in the implementation methods and examples of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for use in the description of the implementation methods or examples. Obviously, the drawings described below are only some embodiments of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without creative work. It should also be noted that the drawings are all drawn in a simplified form and are only used to conveniently and clearly assist in explaining the present application.

[0042] Figure 1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify the results of NF1, NF2 and NF3 induced expression in one embodiment of the present application; lanes 1-3 are NF1 fusion proteins, lanes 4-6 are NF2 fusion proteins, and lanes 7-9 are NF3 fusion proteins.

[0043] Figure 2 In order to identify the purified NF1 in one embodiment of the present application, SDS-PAGE and high performance liquid chromatography (HPLC) were used.

[0044] Figure 3 In order to identify the purified NF2 in one embodiment of the present application, SDS-PAGE and HPLC were used.

[0045] Figure 4 In order to identify the purified NF3 in one embodiment of the present application, SDS-PAGE and HPLC were used.

[0046] Figure 5 For stability comparison and identification in one embodiment of the present application, SDS-PAGE was used; lanes 1, 2, and 3 respectively represent fHbp and fHbp fusion protein incubated at 4° C., 25° C., and 37° C. for 7 days (d).

[0047] FIG. 6A to FIG. 6C This is a specific antibody titer detection in one embodiment of the present application. DETAILED DESCRIPTION

[0048] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0050] In this application, unless otherwise specified, "one or more" means any one of the listed items or any combination of the listed items. Similarly, "one or more" and the like, when used in other ways to mean "one or more", are also understood in the same way unless otherwise specified.

[0051] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0052] In this application, the word "suitable" in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0053] In this application, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent different technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this application. In this application, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0054] In this application, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent. Unless otherwise specified, the descriptions of "optionally include", "optionally contain", etc. in this application, taking "optionally include" as an example, mean "may include or not include".

[0055] The terms "contain", "include" and "include" used in this application are synonymous, which are inclusive or open-ended and do not exclude additional, uncited members or features. Members or features include materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features include actions, conditions for the occurrence of actions, timing, states, etc.

[0056] In this application, the technical features or technical solutions described in open language include closed technical features or technical solutions composed of the listed contents, and also include open technical features or technical solutions containing the listed contents.

[0057] In the present application, exemplary descriptions such as "in some implementation modes (or examples)" and "in one implementation mode (or example)" may include but are not limited to the following meanings: these solutions may be combined with other solutions in a suitable manner to form new technical solutions.

[0058] In the present application, in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc., the terms "first", "second", "third", "fourth", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0059] In the present application, when it comes to a numerical interval (i.e., a numerical range), unless otherwise specified, the distribution of the optional numerical values ​​in the numerical interval is considered to be continuous, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical interval, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to an integer in the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be merged. In other words, unless otherwise specified, the numerical range disclosed herein should be understood to include any and all sub-ranges included therein. The "numerical value" in the numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. "Numerical interval" allows broadly including numerical interval types such as percentage intervals, ratio intervals, and ratio intervals.

[0060] In this application, if there are multiple steps involved in the method flow, unless there is a clear different description in this document, there is no strict order restriction for the execution of these steps, and they can be executed in other orders than described. Moreover, any step can include multiple sub-steps or multiple stages, and these sub-steps or stages are not necessarily executed at the same time, but can be executed at different times, and their execution order is not necessarily sequential, but can be executed in turn, alternating or simultaneously with other steps or parts of sub-steps or stages of other steps.

[0061] The present application provides a fusion protein comprising a meningococcal heparin binding protein antigen (NHBA) or a variant thereof, and an operably linked factor H binding protein (fHbp) or a variant thereof.

[0062] In some embodiments, the fHbp or variant thereof is linked to the N-terminus, C-terminus or loop of the NHBA or variant thereof.

[0063] As mentioned above, the prior art clearly states that the V2 variant of fHbp has inherent instability and sensitivity to proteolysis, and there are literature reports that this instability is caused by the N-terminal β-barrel domain, and any substitution or truncation of this region may promote its instability. However, the inventors unexpectedly discovered that after truncating its N-terminus and preparing it into a fusion protein, its stability was improved, and there was almost no negative impact on its immunogenicity.

[0064] Unless otherwise specified, the term "variant" in this application refers to a protein obtained by mutating the wild type by one or any combination of methods including but not limited to amino acid substitution, deletion (including truncation), insertion, displacement, replacement and modification. Taking the variant of NHBA as an example, it contains part or all of the functions of the wild type NHBA, or it at least contains the functions of the wild type NHBA. Such functions include, for example, good stability and significant immunogenicity.

[0065] Unless otherwise specified, the term "operably linked" in the present application refers to the functional relationship between two regions of a fusion protein, namely NHBA or a variant thereof, and fHbp or a variant thereof; wherein the two regions are linked to produce a fusion protein.

[0066] In some embodiments, the amino acid sequence of the NHBA is as shown in SEQ ID NO: 1.

[0067] In some embodiments, the variant of the NHBA comprises truncating the NHBA. The truncated portion may be, for example, the N-terminus of the NHBA. In some embodiments, the truncated variant retains at least the conserved functional domain at the C-terminus.

[0068] In some embodiments, the truncated portion includes a highly variable N-terminal domain. Exemplarily, the length of the truncation is the first 1 to 200 amino acid residues of the N-terminus. In some embodiments, the truncation is the first 179 amino acid residues of the N-terminus.

[0069] In some embodiments, the variant of the fHbp comprises a truncation of the fHbp.

[0070] Exemplarily, the N-terminus of the fHbp is truncated. The length of the truncation may be, for example, the first 1 to 40 amino acids at the N-terminus.

[0071] In some embodiments, the truncated guide peptide is the N-terminal guide peptide. Exemplarily, the guide peptide is 26 amino acid residues in length;

[0072] In the present application, the fHbp may belong to the same subvariant. In some embodiments, the amino acid sequence of the fHbp variant is shown in any one of SEQ ID NOs: 2 to 4.

[0073] In some embodiments, the NHBA or a variant thereof and the fHbp or a variant thereof are connected via a connecting peptide (or referred to as a linker).

[0074] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, a variant of NHBA, a linker, and a variant of fHbp.

[0075] In some embodiments, the linker comprises one or more flexible amino acids.

[0076] In some embodiments, the linker is a flexible amino acid linker comprising glycine (G) and serine (S).

[0077] In some embodiments, the linker is a flexible amino acid linker comprising alanine (A) and lysine (K), and optionally glutamic acid (E).

[0078] In some embodiments, the linker is selected from the group consisting of (G4S)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)n and (A)n, wherein any n is independently an integer greater than or equal to 1, for example, n may be 1, 2, 3, 4, 5, 6, 7, 8 or 9. In some embodiments, n is an integer greater than or equal to 1 and less than or equal to 6.

[0079] In some embodiments, the linker comprises 1 to 20 amino acids (eg, 1 to 15 amino acids, 1 to 8 amino acids, 2 to 6 amino acids, or about 4).

[0080] In addition, the present application also provides functional variants of the present application fusion protein described in the present application within the scope of the present application. The term "functional variant" used in the present application refers to a recombinant protein, polypeptide or protein having a large amount of or significant sequence identity or similarity with the parent fusion protein, and the functional variant retains the biological activity of the fusion protein. Functional variants encompass, and the amino acid sequence of the functional variant may have, for example, at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99% or higher identity with the amino acid sequence of the parent fusion protein.

[0081] The functional variant may comprise, for example, an amino acid sequence of a parent fusion protein having at least one conservative amino acid substitution. Alternatively or additionally, the functional variant may comprise an amino acid sequence of a parent fusion protein having at least one non-conservative amino acid substitution. In this case, non-conservative amino acid substitutions that do not interfere with or inhibit the biological activity of the functional variant are preferred. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased compared to the parent fusion protein.

[0082] The amino acid substitutions of the fusion protein of the present application are preferably conservative amino acid substitutions. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with certain physical and / or chemical properties is exchanged for another amino acid with the same or similar chemical or physical properties. For example, conservative amino acid substitutions can be substitutions of an acidic / negatively charged polar amino acid with another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain with another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Tip, Cys, Val, etc.), a basic / positively charged polar amino acid with another basic / positively charged polar amino acid (e.g., Lys, His, Arg, etc.), an uncharged amino acid with a polar side chain with another uncharged amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr, etc.), an amino acid with a β-branched side chain with another amino acid with a β-branched side chain (e.g., Ile, Thr and Val), an amino acid with an aromatic side chain with another amino acid with an aromatic side chain (e.g., His, Phe, Trp and Tyr), etc.

[0083] The fusion proteins of the embodiments of the present application (including the functional parts and functional variants of the present application) may contain synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2- carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-tert-butylglycine.

[0084] Mutants having a certain amino acid homology with the amino acid sequence of the fusion protein as described above, for example, a homology between 70% and 99%, a further homology between 80% and 99%, a further homology between 90% and 99%, and a homology of 99%, should also fall within the scope of protection of the present application.

[0085] "Homology" (percentage (%) of sequence identity) of an amino acid sequence (or nucleic acid sequence) is defined as the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to the amino acid (or nucleic acid) residues in a reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum number of identical amino acids (or nucleic acids). In other words, the percentage (%) of sequence identity of an amino acid sequence (or nucleic acid sequence) can be calculated by dividing the number of identical amino acid residues (or bases) relative to the reference sequence to which it is compared by the total number of amino acid residues (or bases) in the candidate sequence or the reference sequence, whichever is shorter. Conservative substitutions of amino acid residues may or may not be considered identical residues. For example, publicly available tools such as BLASTN, BLASTp (available on the website of the US National Center for Biotechnology Information (NCBI), see also Altschul SF et al., Journal of Mol. Biol., 215:403-410 (1990); Stephen F et al., Nucleic Acids Res., 25:3389-3402 (1997)), Clustal W2 (available on the website of the European Bioinformatics Institute, see also Higgins DG et al., Methods in Enzymology, 266:383-402 (1996); Larkin et al., Methods in Enzymology, 267:383-402 (1997); MA et al. Bioinformatics (Bioinformatics) (Cambridge, England), 23 (21): 2947-8 (2007)) and ALIGN or Megalign (DNASTAR) software are used to determine the comparison of amino acid (or nucleic acid) sequence identity percentage. Those skilled in the art can use the default parameters provided by the tool or can appropriately customize the parameters according to the needs of the comparison, for example, by selecting a suitable algorithm.

[0086] As used herein, the term "amino acid" refers to an organic compound that includes amino (-NH2) and carboxyl (-COOH) functional groups and side chains unique to each amino acid. Amino acid names are also represented in this disclosure as standard single-letter or three-letter codes, which are summarized below.

[0087]

[0088] This application targets the inherent instability of fHbp (e.g., V2), one of the candidate antigens of group B meningococcus, and uses the functional domain of another candidate antigen, NHBA, to perform gene fusion with fHbp to form a stable fusion protein that ensures the dual functions of fHbp and NHBA, as one of the potential group B meningococcal vaccine components. The fusion protein finally prepared has good stability and immunogenicity, and can better induce the body to produce bactericidal activity. As a candidate antigen of group B meningococcus, it provides an important reference for the development of group B meningococcal vaccines. The following is a partial technical solution of this application.

[0089] The present application also provides a nucleic acid molecule, which encodes the fusion protein as described above.

[0090] In the present application, unless otherwise specified, "nucleic acid" has the commonly known meaning in the technical field, also known as "polynucleotide", which is a molecule formed by multiple nucleotide monomers.

[0091] The present application also provides a recombinant expression vector, which comprises the nucleic acid molecule as described above.

[0092] If not otherwise specified, the term "vector" in this application refers to a vehicle into which a genetic element (e.g., the aforementioned nucleic acid molecule) can be operatively inserted and expressed. A vector can be, for example, a plasmid, a cosmid, a virus (e.g., a slow virus, a retrovirus, an adenovirus, and an adeno-associated virus), an RNA vector, or a linear or circular DNA or RNA molecule, which can include a chromosome, a non-chromosomal, semisynthetic, or synthetic nucleic acid molecule. The term includes vectors as self-replicating nucleic acid structures and vectors incorporated into the host cell genome into which they have been introduced. Certain vectors can direct the expression of nucleic acids to which they are operatively connected.

[0093] The nucleic acid molecules of the present application mainly refer to isolated nucleic acid molecules. "Isolated" refers to molecules that are substantially free of other biomolecules, such as nucleic acids, proteins, lipids, carbohydrates or other materials, such as cell debris and growth medium. Generally, the term "isolated" is not intended to refer to the complete absence of these materials or the absence of water, buffer or salts, unless they are present in an amount that significantly interferes with the experimental or therapeutic use of the compounds as described herein.

[0094] The term "vector", which may also be referred to as "nucleic acid construct", refers to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop in which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors can replicate autonomously in the host cells into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, and thus replicated together with the host genome. In addition, certain vectors can direct the expression of genes to which they are effectively connected. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, expression vectors useful in recombinant DNA technology are usually present in the form of plasmids. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which play equivalent functions.

[0095] The present application also provides a host cell, which expresses the fusion protein as described above.

[0096] In some embodiments, the host cell comprises one or more of the nucleic acid molecules and recombinant expression vectors described above.

[0097] The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, fungal, plant or animal cells. Easily transformed bacteria include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable fungi include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells, COS cells, NSO cells, HeLa cells, BHK cells or HEK293 cells.

[0098] In one example, the method of introduction is transfection.

[0099] The term "transfection" refers to the process of introducing nucleic acids into eukaryotic cells, particularly mammalian cells. Protocols and techniques for transfection include, but are not limited to, lipid transfection and chemical and physical methods such as electroporation. Many transfection techniques are well known in the art and disclosed herein. See, for example, Graham et al., 1973, Virology 52:456; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier; Chu et al, 1981, Gene 13:197.

[0100] The present application also provides a method for preparing a fusion protein, which comprises culturing the host cell as described above to obtain a culture fluid, and isolating the fusion protein therefrom.

[0101] In some examples, the fusion protein of the present application is obtained by biosynthesis, such as culturing the host cell. It is understood by those skilled in the art that, if the amino acid sequence of the fusion protein is known, it can also be obtained by other methods such as solid phase synthesis.

[0102] The present application also provides an immune preparation, which comprises the fusion protein as described above.

[0103] In some embodiments, the immune preparation further comprises an adjuvant; the adjuvant comprises one or more of aluminum hydroxide, aluminum phosphate, MPLA, AS03, AS04, CpG-ODN, and FM59.

[0104] In the immune preparation, the concentration of the fusion protein may be 10 μg / mL to 100 μg / mL, and the concentration of the adjuvant may be 0.5 mg / mL to 2 mg / mL. Exemplarily, the concentration of the fusion protein may be 10 μg / mL, 11 μg / mL, 12 μg / mL, 13 μg / mL, 14 μg / mL, 15 μg / mL, 17 μg / mL, 18 μg / mL, 20 μg / mL, 25 μg / mL, 30 μg / mL, 35 μg / mL, 40 μg / mL, 45 μg / mL, 50 μg / mL, 55 μg / mL, 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL, 100 μg / mL, or a range or value between any two values. Illustratively, the concentration of the adjuvant can be 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL, or a range or value between any two values.

[0105] In the present application, the types of immune preparations may include vaccine preparations, for example.

[0106] The present application also provides a kit, which comprises one or more of the fusion protein, nucleic acid molecule, recombinant expression vector, host cell and immune preparation as described above, and a container.

[0107] On the other hand, the present application also provides a method for inducing an immune response in a subject and / or for preventing and / or treating meningitis in a subject, comprising: administering an immunologically effective amount of the fusion protein or immune preparation described in the present application to a subject in need thereof.

[0108] As used herein, the term "immunologically effective amount" is an amount sufficient to provide a protective immune response against a bacterial infection (eg, an infection caused by Neisseria meningitidis) or to induce a protective immune response against an immunogen.

[0109] "Administering," "giving," and "treating" when applied to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, immunogenic agent, or composition with an animal, human, subject, cell, tissue, organ, or biological fluid. "Administering," "giving," and "treating" may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental procedures. Treatment of cells includes contact of an agent with a cell, and contact of an agent with a fluid, wherein the fluid is in contact with the cell. "Administering," "giving," and "treating" also mean in vitro and ex vivo treatment of, for example, a cell, by an agent, a diagnosis, a combination composition, or by another cell. "Treatment," when applied to humans, veterinary medicine, or research subjects, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.

[0110] In some embodiments, the immune preparation further comprises a pharmaceutically acceptable carrier and / or excipient. As used in the present application, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, ionic strength enhancers, agents that maintain osmotic pressure, agents that delay absorption, diluents, preservatives, stabilizers, etc. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cations, anions or nonionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Agents that maintain osmotic pressure include but are not limited to sugars, NaCl and the like. Agents that delay absorption include but are not limited to monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0111] Some examples are provided below.

[0112] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which the conditions are not specified in the following examples are preferably referred to the guidance given in the present application, and can also be based on the experimental manual or normal conditions in the art, or can also be based on the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0113] Example 1: Design and preparation of fHbp fusion protein, and stability evaluation

[0114] According to the fHbp amino acid sequence and stability characteristics, three variant fHbp amino acid sequences, V1.13, V2.16 and V3.45, were selected from the PubMLST database, and the leading peptide sequence AA1-AA26 at the fHbp-N terminus was deleted. The specific amino acid sequences are shown in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

[0115] Considering that NHBA is also one of the candidate antigens of group B meningococcus, its functional region is retained, that is, its N-terminal AA1-AA179 is deleted. The specific sequence is shown in SEQ ID NO: 1.

[0116] The truncated NHBA (SEQ ID NO: 1) was connected to the truncated fHbp (SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4) as above by gene fusion, and a G4S linker was inserted between the two proteins, and then the fusion fragment was cloned into the expression vector pET24b, and the N-terminus was labeled with a histidine tag 6×his. The recombinant plasmid of the fusion fragment was obtained by gene synthesis commissioned by General Biotechnology Company, and the specific amino acid sequences are shown in SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7.

[0117] The recombinant plasmid was transformed into the E. coli expression strain BL21 (DE3), and a single clone was selected and placed in a shaking tube containing LB medium containing the corresponding antibiotics, and shaken at 37°C, 200 rpm overnight. The next day, it was transferred to fresh LB medium containing the corresponding antibiotics and shaken at 37°C, 200 rpm until the bacterial solution OD 600 The pH value was 0.6-0.8, 1 mM isopropylthio-β-galactoside (IPTG) was added, and the culture was continued at 37°C and 180 rpm for 4 h. The cells were collected by centrifugation and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the modified fHbp fusion protein was almost not degraded during the purification process.

[0118] After two-step purification by nickel column affinity chromatography and anion exchange chromatography, fHbp fusion proteins with a purity of more than 90% were obtained by SDS-PAGE and HPLC identification. The concentrations of the fusion proteins measured by the Lowary method were 0.95 mg / mL for NF1, 1.02 mg / mL for NF2, and 0.78 mg / mL for NF3. Figure 1~Figure 4 .

[0119] Take equal amounts of fHbp and fHbp fusion protein and place them at 4°C, 25°C, and 37°C, respectively. Take samples on the 7th day (d) and perform SDS-PAGE identification. Figure 5 The results showed that no obvious degradation was observed after stability monitoring (identified by SDS-PAGE after being placed at 4°C, 25°C and 37°C for 7 days).

[0120] Example 2: Vaccine preparation, animal immunization test and antibody level detection

[0121] The fHbp fusion protein, NHBA, V1.13, V2.16 and V3.45 prepared in Example 1 and stored in physiological saline were respectively adsorbed on aluminum hydroxide adjuvant, wherein the recombinant protein was 50 μg / mL each, aluminum hydroxide was 1 mg / mL, and the buffer system was histidine buffer (pH 6.0).

[0122] The prepared preparations were tested for particle size identification and endotoxin content.

[0123] Female mice of the CD1 strain aged 4-6 weeks were selected, 5 mice per group, and injected intraperitoneally at 1 / 5 of the human dose.

[0124] Immunization was performed on day 0 and day 21, and blood was collected on day 35 to collect serum.

[0125] Conventional ELISA tests the specific antibody titer of serum, that is, first coat the 96-well plate with purified fusion protein, and after blocking, add primary antibody serum of different dilution multiples for incubation, enzyme-labeled secondary antibody for incubation, add enzyme detection substrate, and interpret the results and analyze the data.

[0126] SBA was used to detect the bactericidal antibody titer of serum: first, group B meningococcal strains were spread on blood plates and cultured overnight at 37°C and 5% CO2. Single colonies were inoculated into Mueller-Hinton medium and the initial OD of the bacterial solution was 620 Controlled at 0.05-0.08, cultured in a shaking incubator at 37°C until OD 620Reach 0.23-0.24, and measure bacterial activity. All mouse sera required for testing were first heated and inactivated at 56°C for 30 min. The total volume in each well was 50 μL, including 25 μL of test serum diluted two-fold, 12.5 μL of bacterial working solution and 12.5 μL of human complement. Controls included: serum incubated with complement serum, immune serum incubated with bacteria, and inactivated complement. After adding complement, immediately take 10 μL of the control and spread it on a Mueller-Hinton agar plate and incubate at 37°C and 5% CO2 for 1 hour. Take 7 μL of each sample and spot it on a Mueller-Hinton agar plate and incubate it at 37°C and 5% CO2 for 18 hours.

[0127] The results can be found in Table 1. FIG. 6A to FIG. 6C NHBA, V1.13, V2.16 and V3.45 were used as the immune control group. The specific antibody titers induced by immunization with the three fHbp fusion proteins NF1, NF2 and NF3 alone reached 1:10. 6 It can be seen that the prepared fusion protein has bifunctional activity and has little or no negative impact on immunogenicity.

[0128] Table 1 Bactericidal antibody titer detection

[0129]

[0130] The results showed that fHbp fusion protein immunization could induce the body to produce functional antibodies with bactericidal activity (titer above 1:4), and NF2 immune serum had cross-bactericidal activity against V3 strain.

[0131] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0132] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description and drawings may be used to interpret the contents of the claims.

Claims

1. A fusion protein, characterized in that It comprises a meningococcal heparin binding protein antigen (NHBA) or a variant thereof, and an operably linked factor H binding protein (fHbp) or a variant thereof; Optionally, the fHbp or its variant is connected to the N-terminus, C-terminus or loop of the NHBA or its variant.

2. The fusion protein according to claim 1, characterized in that The amino acid sequence of the NHBA is shown in SEQ ID NO:

1.

3. The fusion protein according to claim 1 or 2, characterized in that Variants of the NHBA include truncations of the NHBA; Optionally, the N-terminus of the NHBA is truncated, and at least the conserved functional domain at the C-terminus is retained; Further optionally, the highly variable N-terminal domain is truncated; Optionally, the truncation length is 1 to 200 amino acid residues; In a further alternative, the truncated portion is the first 179 amino acid residues at the N-terminus.

4. The fusion protein according to any one of claims 1 to 3, characterized in that The variants of the fHbp include truncating the fHbp; Optionally, the N-terminus of the fHbp is truncated; the length of the truncation is optionally 1 to 40 amino acids; Further optionally, the truncated one is the N-terminal leader peptide, and the length of the leader peptide is optionally 26 amino acid residues; Optionally, the fHbp belongs to the same subvariant; Further optionally, the amino acid sequence of the fHbp variant is shown in any one of SEQ ID NOs: 2 to 4.

5. The fusion protein according to any one of claims 1 to 4, characterized in that It comprises a variant of said NHBA and a variant of said fHbp; Optionally, the fusion protein comprises a variant of NHBA, a linker, and a variant of fHbp from the N-terminus to the C-terminus; the linker is optionally (G4S)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)n or (A)n, wherein any n is independently an integer greater than or equal to 1 and less than or equal to 6; Further optionally, the amino acid sequence of the fusion protein is shown in any one of SEQ ID NOs: 5 to 7.

6. A nucleic acid molecule, characterized in that It encodes the fusion protein according to any one of claims 1 to 5.

7. A recombinant expression vector, characterized in that: It comprises the nucleic acid molecule according to claim 6.

8. A host cell, characterized in that It expresses the fusion protein according to any one of claims 1 to 5; Optionally, it comprises one or more of the nucleic acid molecule according to claim 6 and the recombinant expression vector according to claim 7; Optionally, the host cell is selected from the group consisting of bacteria and fungi, and the bacteria is optionally Escherichia coli.

9. An immune preparation, characterized in that It comprises the fusion protein according to any one of claims 1 to 5; Optionally, the immune preparation further comprises an adjuvant; the adjuvant comprises one or more of aluminum hydroxide, aluminum phosphate, MPLA, AS03, AS04, CpG-ODN, and FM59; Further optionally, in the immune preparation, the concentration of the fusion protein is 10 μg / mL to 100 μg / mL, and the concentration of the adjuvant is 0.5 mg / mL to 2 mg / mL.

10. The immune preparation according to claim 9, characterized in that The immune preparations include vaccine preparations.

11. A kit, characterized in that: It comprises one or more of the fusion protein according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant expression vector according to claim 7, the host cell according to claim 8, and the immune preparation according to claim 9 or 10, and a container.

12. A method for preparing a fusion protein, characterized in that: The method comprises: Cultivating the host cell according to claim 8 to obtain a culture fluid; and, The fusion protein is isolated from the culture fluid.

13. Use of the fusion protein according to any one of claims 1 to 5, the nucleic acid molecule according to claim 6, the recombinant expression vector according to claim 7 or the host cell according to claim 8 in preparing an immune preparation; Optionally, the immune preparation comprises a vaccine preparation.

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