FHbp recombinant protein

Abstract

According to the invention, fHbp protein of V1 variant of group B meningococcus is artificially modified to obtain an fHbp (V1) monomer consensus sequence and recombinant protein and fusion protein designed based on the fHbp (V1) monomer consensus sequence, and the fHbp (V1) monomer consensus sequence and the recombinant protein and fusion protein are used as antigens for preparing group B meningococcus vaccines. The method comprises the following steps: firstly, constructing an fHbp (V1) monomer consensus sequence through sequence comparative analysis, and designing an fHbp (V1) monomer recombinant protein containing one fHbp (V1) monomer and an fHbp (V1-V1) fusion protein containing two fHbp (V1) monomers on the basis of the fHbp (V1) monomer consensus sequence; meanwhile, an NHBA recombinant protein is constructed, and the NHBA recombinant protein is covalently linked with an fHbp (V1) monomer to construct an NHBA-fHbp fusion protein. Experiments prove that the recombinant protein or the fusion protein can be efficiently expressed and modified through an escherichia coli expression system, has good immunogenicity and can be used for preparing a vaccine composition for preventing group B meningococcus.

Description

Technical Field

[0001] The present invention relates to the field of biological products. Specifically, the present invention relates to the field of meningococcal vaccines, particularly recombinant proteins, immunogenic compositions related to Neisseria meningitidis serogroup B, and their uses, preparation methods, etc. Background Art

[0002] Neisseria meningitidis, also known as Neisseria meningitidis, meningococcus, or simply meningococcus, is a Gram-negative coccus that is restricted to humans. It belongs to the family Neisseriaceae, is diplococcal in shape, and is a major pathogen that causes epidemic cerebrospinal meningitis and septicemia worldwide. Bacterial septicemia caused by meningococcus generally mainly presents as limited vascular endothelial colonization at lower bacterial levels, and some microcolonies may induce meningitis by disrupting the blood-brain barrier; while at higher bacterial levels, it leads to extensive colonization of endothelial cells, causing a significant increase in vascular permeability, and then resulting in rapid and severe vascular leakage, causing fulminant purpura and related septic shock. The high-risk population for invasive meningococcal disease caused by meningococcal infection is mainly infants and young children under 5 years old, but the incidence is also showing an increasing trend in the adolescent population. Due to the use of different serogroup vaccines in various countries and regions, different serogroup epidemic characteristics are shown.

[0003] According to different capsular polysaccharide structures, meningococci can be divided into a total of 13 serogroups, but only 6 of them (A, B, C, W-135, X, and Y) can cause most life-threatening diseases. Currently, polysaccharide or polysaccharide conjugate vaccines for preventing epidemic meningitis caused by serogroups A, C, W-135, and Y have been marketed in most countries and regions around the world. In July 2023, the pentavalent polysaccharide conjugate vaccine MenFive™ jointly developed by the Serum Institute of India and PATH Foundation for serogroups A, C, W, X, and Y also received WHO's priority recommendation for vaccination in the 1-85-year-old population. However, due to the great difficulty in developing a serogroup B meningococcal vaccine and its late market entry, it is currently only available in some countries and regions in Europe and the Americas, and there is no serogroup B meningococcal vaccine on the market in China. In addition, with the use of other serotype meningococcal vaccines in most parts of the world, serogroup B meningococcus has currently become the most prevalent meningococcal serogroup globally. The relative prevalence of serogroup B meningococcus in China is also gradually increasing, becoming the main pathogen causing meningitis. Therefore, there is an urgent need to develop a preventive vaccine against serogroup B meningococcus.

[0004] Compared with the marketed ACYW135 meningococcal vaccine, the R & D path of the group B meningococcal vaccine is quite different and the R & D difficulty is also greater. The latter is mainly developed based on the capsular polysaccharide on the surface of meningococcus, but this development strategy is not applicable to the group B meningococcal vaccine. Early studies showed that the vaccine developed based on the capsular polysaccharide of group B meningococcus not only had low immunogenicity and was almost difficult to induce an antibody response, but also had the risk of inducing autoimmune reactions. Currently, only two group B meningococcal vaccines have been approved for marketing in Europe and the United States, namely Bexsero from GSK and Trumenba from Pfizer. There is no domestic similar product approved for marketing. Both marketed vaccines are mainly based on the recombinant protein technology platform of the Escherichia coli prokaryotic expression system and use aluminum adjuvant to enhance the immune response. The Bexsero vaccine from GSK contains four antigen components, namely OMV (outer membrane vesicles of bacteria), NadA (Neisserial adhesin A) protein, NHBA (Neisseria Heparin Binding Antigen, also known as GNA2132) protein, and fHbp (factor H-binding protein, also known as GNA1870) protein. The fHbp protein comes from the MC58 strain (belonging to serogroup B) (refer to WO2004032958A1); the Trumenba vaccine from Pfizer only uses two different variants (serogroup A and serogroup B) of the fHbp protein as antigens. The fHbp protein of serogroup A comes from the M98 250771 strain, and the fHbp protein of serogroup B comes from the CDC1573 strain (refer to WO2015033251A2). Although these two vaccines have been approved for marketing, their antigen designs are mainly targeted at the group B meningococcal strains prevalent in Europe and the United States, and their potential reactivity to the group B meningococcal strains prevalent in China is relatively poor. For example, OMV, one of the main components of the Bexsero vaccine, the main antigen protein is derived from the PorA antigen protein on the bacterial surface, but the PorA sequence has poor conservation, resulting in poor broad-spectrum protection of the OMV vaccine. Generally, the single OMV component vaccine is mainly used in local areas. In addition, the vaccine based on OMV has weak immune persistence and poor protection effect in infants and young children in the lower age groups. Another component of the Bexsero vaccine, the NadA protein, mainly mediates the adhesion of group B meningococcus to nasal epithelial cells. However, through the analysis of the molecular epidemiological characteristics of group B meningococcus in China, it is found that more than 90% of the strains do not contain the NadA gene (Zhu Bingqing et al., Chinese Journal of Preventive Medicine, 2019). It can be seen that the marketed group B meningococcal vaccines abroad are not fully applicable to the Chinese population.

[0005] For the fHbp antigen protein, scientists at Wyeth (later acquired by Pfizer) initially found through genetic evolutionary tree analysis that the global fHbp protein sequences could be divided into two subfamilies (or subfamilies), namely A and B; subsequent further sequence analysis revealed that the global fHbp protein sequences could be subdivided into three variants: V1, V2, and V3. In addition, there are also certain degrees of sequence differences among the strains within each variant of fHbp. Therefore, the antigen designed based on fHbp from a single strain has the problem that its protection efficiency cannot cover the entire variant. However, whether it is the Bexsero vaccine of GSK or the Trumenba vaccine of Pfizer, the fHbp antigen peptides used only come from a single strain, so there is a potential risk of limited coverage, especially in countries or regions where the corresponding strain is not prevalent. Therefore, there is an urgent need for fHbp antigen peptides that can cover more different strains within the same variant.

[0006] In addition, the molecular weight of the fHbp monomer protein is relatively small, only about 27 kDa. Therefore, in the existing vaccine design, when using fHbp monomer as a separate antigen component, there is a problem that the molecular weight is small and thus the immunogenicity is low. Although there are prior arts that attempt to make tandem proteins from multiple fHbp monomers, their inventive purposes are not to solve the above problems. For example: (1) In the patent CN110804102B, the partial sequence at the N-terminus of the fHbp V1 subtype (including domains A, B, and part of domain C, denoted as "B01 ABpC ") was fused with the partial sequence of domain C of the V2 subtype (part of domain C, denoted as "A19 pC ") to obtain the chimeric protein B01 ABpC -A19 pC, however, the chimeric protein is only about 29 kDa and cannot overcome the problem of the small molecular weight of the fHbp monomer. (2) In the patent CN106661092B, the fHbp v2, v3, and v1 sequences were sequentially ligated to construct a fusion polypeptide. However, the invention point of this patent lies in the modification of the amino acid sequences of v2 and v3 type fHbp to improve its stability and reduce its affinity for fH. Only in the specific embodiment was it mentioned that the mutant v2, v3 could be fused with the mutant v1 (including R41S) to form a mutant fusion protein arranged in the order of v2-v3-v1 from the N-terminus to the C-terminus. This patent states that: "This fusion protein thus utilizes the observation that mutant #3 provides a greatly increased stability (Tm) and a greatly reduced fH affinity for both v2 and v3", and "For v1, the R41S mutation has a minor effect on thermal stability but strongly reduces fH binding". Thus, it can be seen that the purpose of sequence modification and the provision of the fusion protein in this patent is to improve the stability of fHbp and reduce its fH affinity, and its invention point does not lie in solving the problem that the fHbp monomer has a small molecular weight and thus low immunogenicity. (3) WO2011024072A2 constructed several different hybrid proteins based on the modified fHbp, but its main purpose is to improve the ability of the fHBP protein to induce antibodies that cross-react between families, rather than to improve its coverage of different strains within the family.

[0007] In addition, the fusion proteins constructed and verified in the aforementioned patents are all fHbp heterologous hybrid proteins (the sequences of more than two fused fHbp monomers are different), and the effects of fHbp homologous hybrid proteins (the sequences of more than two fused fHbp monomers are the same) are still unknown, and the effects of fusing the fHbp protein with other antigenic proteins of Neisseria meningitidis serogroup B are also unknown. For fusion proteins, since the spatial position relationship (distance, orientation, degree of freedom, etc.) and their interactions between each domain or functional fragment will have an important impact on the function and / or production process of the fusion protein, therefore, even though the fusion proteins disclosed in the above prior art have achieved beneficial technical effects to a certain extent, those skilled in the art still cannot predict whether other fusion proteins constructed based on fHbp monomers can also meet the requirements of the vaccine production process while having antigenicity. Therefore, to obtain fHbp fusion proteins with other structures and overcome the above problems at the same time, it is still expected to be achieved through a large number of creative experiments. Summary of the Invention

[0008] In view of the above problems, the object of the present invention is to provide a recombinant protein or fusion protein with a simple preparation method and capable of stimulating the body to produce a high-level antibody response against Neisseria meningitidis serogroup B strain V1 variant, and its uses, preparation methods, nucleic acid sequences encoding the recombinant protein or fusion protein, immunogenic compositions containing the recombinant protein or fusion protein, and uses of the immunogenic compositions, etc.

[0009] The present invention provides the Neisseria meningitidis serogroup B fHbp (V1) monomer consensus sequence or a combination thereof; the fHbp (V1) monomer consensus sequence is a synthetic polypeptide, and its amino acid sequence is selected from the following options (1) to (5):

[0010] (1) The sequence shown in SEQ ID NO: 1;

[0011] (2) A well-immunogenic fragment of the sequence shown in SEQ ID NO: 1, the well-immunogenic fragment is a truncated fragment of SEQ ID NO: 1, which is obtained by deleting the first X amino acids at the N-terminus of SEQ ID NO: 1, where X is a positive integer and 1≤X≤26; preferably, X is 19, 20, 21, 22, 23, 24, 25 or 26; more preferably, X is 19 or 20;

[0012] (3) A well-immunogenic fragment of the sequence shown in SEQ ID NO: 1, the well-immunogenic fragment is a truncated fragment of SEQ ID NO: 1, which at least contains amino acids 120-183 of SEQ ID NO: 1 starting from the N-terminus;

[0013] (4) A well-immunogenic variant of the sequence shown in SEQ ID NO: 1, the variant is obtained by substituting, deleting and / or adding Y amino acids outside the domain formed by amino acids 120-183 of SEQ ID NO: 1 starting from the N-terminus, where Y is a positive integer and 1≤Y≤5, preferably, 1≤Y≤2; the well-immunogenic variant has the same or similar immunogenicity as SEQ ID NO: 1;

[0014] (5) A well-immunogenic variant of the sequence shown in SEQ ID NO: 1, the variant is a sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% identity with SEQ ID NO: 1.

[0015] Regarding the fHbp (V1) monomer consensus sequence described above, it should be noted that:

[0016] i) The wild-type fHbp protein is 274 amino acids in length. The mature fHbp lipoprotein lacks the first 19 amino acids (signal peptide) from the N-terminus. The ΔG form of fHBP lacks the first 26 amino acids and still has good immunogenicity (WO2009104097A2). Therefore, based on the consensus sequence shown in SEQ ID NO: 1, deleting the sequence after the 1st to Xth amino acids from its N-terminus (X is a positive integer and 1 ≤ X ≤ 26, where X is preferably 19 or 20) can also achieve the same or very similar technical effects as the monomer consensus sequence shown in SEQ ID NO: 1.

[0017] ii) fHbp can be divided into three domains, A, B, and C, which correspond to the amino acid fragments at positions 1 - 119, 120 - 183, and 184 - 274 of the wild-type original sequence (WO2006024954A2), or the amino acids at positions 8 - 100, 101 - 164, and 165 - 255 of the mature sequence after removing the signal peptide (Francesca Cantini et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 11, pp. 7220 – 7227, March 17, 2006). Among them, the V1 variant expresses an antigenic epitope located in domain B, the antigenic epitopes of the V2 and V3 variants are mainly located in domain C, and domain A is highly conserved among the three variants of fHbp (CN110804102B); domain B is the most critical for the antigenicity of the fHbp protein V1 variant. Therefore, based on the fHbp(V1) monomer consensus sequence shown in SEQ ID NO: 1, a truncated fragment containing its domain B (amino acids 120 - 183 from the N-terminus) can also achieve the same or very similar technical effects as the monomer consensus sequence shown in SEQ ID NO: 1; similarly, based on the fHbp(V1) monomer consensus sequence shown in SEQ ID NO: 1, a variant sequence obtained by substituting, deleting, and / or adding Y amino acids (Y is a positive integer and 1 ≤ Y ≤ 5, where Y is preferably 1 or 2) outside its domain B, or a variant sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% identity thereto, can also achieve the same or very similar technical effects as the monomer consensus sequence shown in SEQ ID NO: 1.

[0018] The present invention also provides an fHbp(V1) recombinant protein constructed based on the above-mentioned fHbp(V1) monomer consensus sequence. The fHbp(V1) recombinant protein includes more than one (preferably one, two, three or four) fHbp(V1) monomer consensus sequences as described above. The present invention also provides two specific fHbp(V1) recombinant proteins, namely fHbp(V1) monomer recombinant protein and fHbp(V1-V1) fusion protein, which are as follows:

[0019] fHbp(V1) monomer recombinant protein: It contains one of the aforementioned fHbp(V1) monomer consensus sequences. Preferably, the sequence of the fHbp(V1) monomer consensus sequence is obtained by deleting the first X amino acids at the N-terminus of the sequence shown in SEQ ID NO: 1, where X is a positive integer and 1≤X≤26. Preferably, X is 19. Further preferably, the N-terminus of the fHbp(V1) monomer recombinant protein contains the P4 signal peptide sequence shown in SEQ ID NO: 3. Further preferably, the fHbp(V1) monomer recombinant protein contains the amino acid sequence shown in SEQ ID NO: 5.

[0020] fHbp(V1-V1) fusion protein: It contains two of the aforementioned fHbp(V1) monomer consensus sequences, which are tandemly formed into a homodimer recombinant protein. A linker peptide may or may not exist between the two monomer consensus sequences. Preferably, for the two fHbp(V1) monomer consensus sequences, they are obtained by deleting the first X amino acids at the N-terminus of the sequence shown in SEQ ID NO: 1, where X is a positive integer and 1≤X≤26. Further preferably, for the amino acid sequences of the first and second fHbp(V1) monomer consensus sequences from the N-terminus, the corresponding values of X are 19 and 20 respectively. Further preferably, the fHbp(V1-V1) fusion protein is added with the P4 signal peptide shown in SEQ ID NO: 3 at the N-terminus to further enhance its immunogenicity. Further preferably, the two fHbp(V1) monomers are covalently bound through a linker peptide, and the amino acid sequence of the linker peptide is preferably the sequence shown in SEQ ID NO: 4. Further preferably, the fHbp(V1-V1) fusion protein contains the amino acid sequence shown in SEQ ID NO: 6.

[0021] The present invention also provides an NHBA-fHbp fusion protein, which comprises an NHBA recombinant protein from Neisseria meningitidis serogroup B and an fHbp recombinant protein from Neisseria meningitidis serogroup B variant V1, V2 or V3; preferably, the NHBA recombinant protein is located at the N-terminus of the fusion protein, and the fHbp recombinant protein is located at the C-terminus of the fusion protein, and the two are tandemly formed into a heterodimeric recombinant protein, and a linker peptide may or may not exist between the two monomers. Preferably, the fHbp recombinant protein is from Neisseria meningitidis serogroup B variant V1, and further preferably, the sequence of the fHbp recombinant protein is the above-mentioned fHbp(V1) monomer consensus sequence, so the formed NHBA-fHbp fusion protein is also called "NHBA-fHbp(V1) fusion protein"; further preferably, the amino acid sequence of the fHbp(V1) monomer consensus sequence: is obtained by deleting the first - X amino acids at the N-terminus of the sequence shown in SEQ ID NO: 1, where X is a positive integer and 1 ≤ X ≤ 26, and further preferably, the value of X is 20. Preferably, in the NHBA-fHbp fusion protein: the amino acid sequence of the NHBA recombinant protein is as shown in SEQ ID NO: 7. Preferably, the NHBA and fHbp(V1) are covalently bound through a linker peptide, and the amino acid sequence of the linker peptide is preferably the sequence shown in SEQ ID NO: 4. Preferably, the NHBA-fHbp fusion protein comprises the amino acid sequence shown in SEQ ID NO: 8.

[0022] The present invention also provides a nucleic acid sequence or a combination thereof encoding the above-mentioned fHbp(V1) monomer consensus sequence, fHbp(V1) recombinant protein, and / or NHBA-fHbp fusion protein.

[0023] The present invention also provides the following specific nucleic acid sequences:

[0024] A nucleic acid sequence encoding the Neisseria meningitidis serogroup B fHbp(V1) monomer recombinant protein shown in SEQ ID NO: 5, characterized in that the nucleic acid sequence comprises SEQ ID NO: 9;

[0025] A nucleic acid sequence encoding the fHbp(V1-V1) fusion protein shown in SEQ ID NO: 6, characterized in that the nucleic acid sequence comprises SEQ ID NO: 10;

[0026] A nucleic acid sequence encoding the NHBA recombinant protein shown in SEQ ID NO: 7, characterized in that the nucleic acid sequence comprises SEQ ID NO: 11;

[0027] A nucleic acid sequence encoding the NHBA-fHbp fusion protein shown in SEQ ID NO: 8, characterized in that the nucleic acid sequence comprises SEQ ID NO: 12.

[0028] The present invention also provides an expression vector containing the nucleic acid sequence, a host cell containing the expression vector, etc. The expression vector can be a plasmid vector, such as pET-30a(+); the host cell can be Escherichia coli.

[0029] The present invention also provides a method for preparing the aforementioned fHbp(V1) recombinant protein and / or NHBA-fHbp fusion protein, comprising the following steps:

[0030] Obtaining a recombinant plasmid containing the coding sequence of the above recombinant protein or fusion protein through codon optimization and gene synthesis; transforming the recombinant plasmid into an Escherichia coli expression system respectively, and obtaining correct positive expression strains through the identification of the target antigen and the screening of strains; further expanding the fermentation culture of the positive expression strains, and separately isolating and purifying the target protein from the positive expression strains, so as to obtain the recombinant protein or fusion protein.

[0031] The present invention also provides the use of the aforementioned fHbp(V1) recombinant protein and / or NHBA-fHbp fusion protein in the preparation of a drug for inducing an immune response or alleviating or preventing an infection caused by Neisseria meningitidis in mammals; preferably, the Neisseria meningitidis is Neisseria meningitidis serogroup B.

[0032] The present invention also provides an immunogenic composition containing the aforementioned fHbp(V1) recombinant protein and / or NHBA-fHbp fusion protein; in an alternative embodiment, the immunogenic composition contains the aforementioned fHbp(V1-V1) fusion protein; in an alternative embodiment, the immunogenic composition contains the aforementioned NHBA-fHbp fusion protein; in another alternative embodiment, the immunogenic composition contains the aforementioned fHbp(V1) monomeric recombinant protein and the NHBA recombinant protein shown in SEQ ID NO: 7; an adjuvant, such as an aluminum adjuvant, preferably aluminum hydroxide adjuvant, can also be contained in the immunogenic composition.

[0033] The present invention also provides the use of the aforementioned immunogenic composition in the preparation of a drug for inducing an immune response or alleviating or preventing an infection caused by Neisseria meningitidis in mammals; preferably, the Neisseria meningitidis is Neisseria meningitidis serogroup B.

[0034] Definition:

[0035] Monomer (monomeric protein, monomeric recombinant protein): It only contains one protein or its immunogenic fragment; for example, the fHbp(V1) monomer consensus sequence, fHbp(V1) monomeric recombinant protein, and NHBA recombinant protein in the text belong to this category.

[0036] Homodimeric recombinant protein: It refers to a recombinant protein formed by tandemly linking two monomers derived from the same protein sequence or its immunogenic fragment. A linker, such as a connecting peptide, may or may not exist between the two monomers; "fHbp(V1-V1)" in the text belongs to this category.

[0037] Heterodimeric recombinant protein: It refers to a recombinant protein formed by tandemly linking two monomers derived from different protein sequences or their immunogenic fragments. A linker, such as a connecting peptide, may or may not exist between the two monomers; "NHBA-fHbp" in the text belongs to this category.

[0038] Fusion protein: It refers to an artificial multi-domain protein formed by fusing multiple functional fragments, which is obtained by using genetic engineering and other technologies to link two or more genes or gene fragments together, constructing a vector and expressing it in a host cell. Therefore, the homodimeric recombinant protein "fHbp(V1-V1)" and the heterodimeric recombinant protein "NHBA-fHbp" described in this text essentially belong to fusion proteins at the same time.

[0039] Advantages of the present invention:

[0040] (1) Regarding the problem of low coverage of fHbp protein from a single strain: Based on dozens of fHbp(V1) sequences from different strains, the fHbp(V1) monomer consensus sequence was constructed, and a recombinant protein antigen was constructed based on it, thus solving the problem of low strain coverage of fHbp antigen peptides from a single strain.

[0041] (2) Regarding the problem of the relatively small molecular weight and low immunogenicity of the fHbp antigen, the following aspects were optimized:

[0042] 1. The fHbp (V1) monomer is concatenated with another monomer to prepare a homologous or heterologous dimer recombinant protein (fusion protein) form, thereby increasing its molecular size. Immunogenicity results show that the homologous dimer recombinant protein fHbp (V1-V1) can improve the antigen-induced antibody response level compared with the fHbp (V1) monomer recombinant protein; although the heterologous dimer recombinant protein NHBA-fHbp (V1) does not have the best immunization effect, since it provides both the NHBA antigen of Neisseria meningitidis serogroup B and the fHbp (V1) antigen sequence at the same time, the antibody responses against both antigens can be induced by this one fusion protein. In the preparation process, only this one fusion protein needs to be expressed and purified instead of two independent recombinant proteins, so it helps to simplify the vaccine components and production process.

[0043] 2. The immunogenicity of the recombinant fHbp antigen sequence is enhanced by adding the P4 signal peptide to the N-terminus. The P4 signal peptide can further direct the recombinant protein to be located on the bacterial cell membrane after translation and expression in Escherichia coli, and then this signal peptide will be degraded and removed. At the same time, the mature fHbp antigen protein located on the membrane will undergo lipidation modification. Existing literature (Fletcher LD, et al., Infect Immun. 2004.) research shows that the lipidated fHbp antigen can have better immunization effects and is more suitable as a vaccine design.

[0044] (III) In addition, the nucleotide coding sequence of the recombinant protein of the present invention has been codon-optimized, which helps to efficiently express in the Escherichia coli expression system; the designed recombinant protein antigens can all obtain soluble proteins after being expressed in Escherichia coli, and moreover, experiments such as electrophoresis, Western blot, SEC-HPLC, mass spectrometry, ELISA, etc. have proved that the structure, purity, modification and function of the prepared recombinant or fusion proteins meet the requirements, which is beneficial to vaccine process scale-up and process optimization. Brief Description of the Drawings

[0045] Figure 1 : The distribution position of the amino acid consensus sequence of the fHbp (V1) protein monomer (shown as red circles in the figure) in the phylogenetic tree analysis results of all 63 fHbp (V1) type sequences.

[0046] Figure 2 : The sequence alignment result of the fHbp (V1) monomer consensus sequence shown in SEQ ID NO: 1 blasted by blastp on the NCBI website with the sequence ranked first (the closest sequence); Query: SEQ ID NO: 1; Sbjct: the closest sequence obtained by blastp; the different amino acids are shown as short horizontal lines in the figure.

[0047] Figure 3: Alignment result of the consensus sequence of the fHbp (V1) monomer shown in SEQ ID NO: 1 with SEQ ID NO: 8 (fHbp antigen used in GSK's Bexsero vaccine) in Patent WO2004032958A1; the short horizontal lines in the figure indicate the different amino acids.

[0048] Figure 4 : Alignment result of the consensus sequence of the fHbp (V1) monomer shown in SEQ ID NO: 1 with SEQ ID NOs: 1 and 2 (fHbp antigens used in Pfizer's Trumenba vaccine) in Patent WO2015033251A2; Left figure: Alignment result of the consensus sequence of the fHbp (V1) monomer in the present invention with SEQ ID NO: 1 in WO2015033251A2. Since there are many different amino acids, they are not all shown; Right figure: Alignment result of the consensus sequence of the fHbp (V1) monomer in the present invention with SEQ ID NO: 2 in WO2015033251A2. The short horizontal lines in the figure indicate the different amino acids.

[0049] Figure 5 : SDS-PAGE and Western Blot electrophoresis results of the fHbp (V1) monomer recombinant protein.

[0050] Figure 6 : SDS-PAGE and Western Blot electrophoresis results of the fHbp (V1-V1) fusion protein.

[0051] Figure 7 : SDS-PAGE and Western Blot electrophoresis results of the NHBA recombinant protein.

[0052] Figure 8 : SDS-PAGE and Western Blot electrophoresis results of the NHBA-fHbp (V1) fusion protein.

[0053] Figure 9 : Results of SEC-HPLC for detecting the purity of the purified fHbp (V1) monomer recombinant protein and fHbp (V1-V1) fusion protein.

[0054] Figure 10 : Results of mass spectrometry for detecting the N-terminal integrity and modification of the purified fHbp (V1) monomer recombinant protein and fHbp (V1-V1) fusion protein.

[0055] Figure 11 : Results of in vitro affinity ELISA of the fHbp (V1) monomer recombinant protein, fHbp (V1-V1) fusion protein, fHbp (V1-V1)(SL) fusion protein, and NHBA-fHbp (V1) fusion protein with complement factor H.

[0056] Figure 12 : In vitro affinity ELISA results of NHBA recombinant protein and NHBA-fHbp(V1) fusion protein with heparin. Detailed implementation manners

[0057] Example 1: Construction of the consensus sequence of Neisseria meningitidis serogroup B fHbp protein and selection of NHBA protein sequence.

[0058] Through sequence retrieval and analysis, the consensus sequences of fHbp(V1) monomer and NHBA protein were constructed respectively.

[0059] 1.1 Design scheme of the consensus sequence of fHbp(V1) monomer:

[0060] The amino acid sequences of fHbp proteins of 136 and 61 wild-type Neisseria meningitidis serogroup B strains prevalent in China were retrieved from the databases PubMLST (https: / / pubmlst.org / ) and GenBank (https: / / www.ncbi.nlm.nih.gov / genbank / ), respectively. After similarity analysis of the total 197 sequences, they were classified into three sequence clusters: fHbp(V1), fHbp(V2), and fHbp(V3). Among them, fHbp(V1) contained 63 sequences (PubMLST: 34; GenBank: 29). It can be calculated by the software MegAlign that the amino acid sequence similarity among these 63 fHbp(V1) is relatively high (≥81.3%). However, if a certain sequence is randomly selected as the antigen design sequence, it may not be able to induce a relatively balanced antibody response level against all strains in the body. The consensus sequence refers to the nucleotide or amino acid sequence that is theoretically the most representative for a cluster of sequences, and each nucleotide or amino acid is the nucleotide or amino acid that most frequently appears at this site in different sequences in nature. Usually, the consensus amino acid sequence is closer to the theoretically most conserved sequence of the protein and has a relatively moderate genetic evolutionary distance from each wild-type sequence. Based on the above 63 wild-type amino acid sequences of fHbp(V1), a consensus sequence was designed and used for vaccine development. Theoretically, it can induce a more balanced immune response level against each wild-type fHbp(V1) strain in the body, thus achieving a broader spectrum of vaccine protection.

[0061] By calculating the above 63 wild-type amino acid sequences of fHbp(V1) through the software MegAlign, the monomer consensus amino acid sequence of the fHbp(V1) sequence cluster can be constructed, as shown in SEQ ID NO: 1. Figure 1The results showed that the consensus sequence of the fHbp(V1) monomer was located at a relatively conserved position in the V1 serotype sequence cluster. The results of sequence similarity analysis showed that the amino acid similarity between the consensus sequence of the fHbp(V1) monomer and 63 wild-type sequences was between 87.3% and 97.6%.

[0062] For the consensus sequence of the fHbp(V1) monomer shown in SEQ ID NO: 1, ProteinBlast (blastp) verification was performed on the NCBI website. Select "Database" as "Non-redundant sequences (nr)" or "Patented protein sequences (pataa)", and other parameters as default. The SequenceIDs of the sequences ranked highest in the match were AHY94675.1 and AFQ69598.1 (sequence 55 in Novartis patent US8226960B2). Their identities with SEQ ID NO: 1 were 268 / 274 (98%) and 267 / 274 (97%) respectively, that is, there were 6 and 7 amino acid differences respectively (see Figure 2 )). Thus, it was confirmed that the consensus sequence of the fHbp(V1) monomer provided by the present invention was a newly constructed polypeptide sequence that was not publicly disclosed in the prior art.

[0063] Sequence alignment results between the consensus sequence of the fHbp(V1) monomer and the fHbp protein used in the Bexsero vaccine: The Bexsero vaccine of GSK used an fHbp recombinant protein (refer to WO2004032958A1), specifically the "936-741 hybrid" shown in SEQ ID NO: 8 in this patent (where the 741 protein is the fHbp protein, and the 936 protein is another protein antigen from Neisseria meningitidis). After aligning SEQ ID NO: 1 in the present invention with the sequence of the "936-741 hybrid" used in the Bexsero vaccine, it was found that their identity was only 58% (see Figure 3 ). Thus, it can be seen that the consensus sequence of the fHbp monomer provided in the present invention has a large difference from the fHbp antigen sequence used in the Bexsero vaccine.

[0064] Alignment results of the consensus sequence of fHbp (V1) monomers and the fHbp proteins used in the Trumenba vaccine: The Trumenba vaccine from Pfizer uses two antigenic proteins from fHbp protein subfamilies A and B (refer to WO2015033251A2), specifically the first lipidated polypeptide and the second lipidated polypeptide containing the amino acid sequences shown in SEQ ID NO:1 and SEQ ID NO:2 described in this patent. After aligning SEQ ID NO: 1 in the present invention with the two fHbp antigenic proteins used in the Trumenba vaccine respectively, it is found that the identities are only 60% and 84% respectively (see Figure 4 ). Thus, it can be seen that the consensus sequence of fHbp monomers provided in the present invention has a large difference from the fHbp antigen sequences used in the Trumenba vaccine.

[0065] 1.2 NHBA sequence design scheme:

[0066] Since the overall sequence of the NHBA sequence has a high degree of conservation and there is no subtype classification. The amino acid sequences of the NHBA proteins of 127 wild-type group B meningococcus strains prevalent in the Chinese region were retrieved from the PubMLST database. The NHBA sequences among the strains are highly similar, ranging from 79.1% to 100%. Therefore, the NHBA sequence of one wild strain "Nm510510" was selected as the antigen sequence, and its wild-type amino acid sequence is shown in SEQ ID NO: 2.

[0067] Example 2: Construction and screening of engineering strains

[0068] (I) Selection of antigen protein sequences: Based on the consensus sequence of fHbp (V1) monomers and the NHBA protein sequence obtained in Example 1, recombinant proteins and fusion proteins were further designed, specifically including:

[0069] 1) fHbp(V1) monomer recombinant protein: Considering that the original leader sequence of fHbp will be cleaved after maturation (the first 19 amino acids from the N-terminus, i.e., the sequence before the first cysteine - the lipidation site, refer to WO2009104097A2, WO2004032958A1), based on the consensus sequence of fHbp(V1) monomer shown in SEQ ID NO:1, the leader sequence at the N-terminus (amino acids 1 - 19) was replaced with the P4 signal peptide sequence from non-typeable Haemophilus influenzae to improve expression and enhance immunogenicity, thereby obtaining the fHbp(V1) monomer recombinant protein, whose amino acid sequence is shown in SEQ ID NO: 5 (where amino acids 1 - 20 come from the P4 signal peptide sequence shown in SEQ ID NO: 3, and amino acids 21 - 275 come from amino acids 20 - 274 in the fHbp(V1) monomer consensus sequence SEQ ID NO:1).

[0070] 2) NHBA recombinant protein: Based on the amino acid sequence of the NHBA protein of Nm510510 strain shown in SEQ ID NO:2, the first 1 - 24 amino acids at the N-terminus were removed and replaced with two amino acid residues "MA", thereby obtaining the NHBA recombinant protein, whose sequence is specifically shown in SEQ ID NO: 7.

[0071] 3) fHbp(V1-V1) fusion protein: Based on two fHbp(V1) monomer consensus sequences, covalently linked by a linker peptide and adding a P4 signal peptide sequence at the N-terminus, a homologous dimer recombinant protein, namely fHbp(V1-V1), was constructed; among them, the two fHbp(V1) monomer consensus sequences are respectively the sequence shown in SEQ ID NO:1 with the first 19 amino acids or the first 20 amino acids deleted. The amino acid sequence of the obtained fHbp(V1-V1) fusion protein is specifically shown in SEQ ID NO: 6 (where amino acids 1 - 20 come from the P4 signal peptide sequence shown in SEQ ID NO: 3, and the first and second fHbp(V1) monomer consensus sequences from the N-terminus come from amino acids 20 - 274 and 21 - 274 in SEQ ID NO: 1 respectively, and the two are sequentially linked by the linker peptide shown in SEQID NO: 4).

[0072] 4) NHBA-fHbp(V1) fusion protein: Based on the common sequence of an NHBA recombinant protein and an fHbp(V1) monomer, they are covalently concatenated through a linker peptide to construct a heterodimeric recombinant protein, namely NHBA-fHbp(V1); the common sequence of the fHbp(V1) monomer is the fHbp(V1) monomer common sequence shown in SEQ ID NO:1 with its first 20 amino acids deleted. The amino acid sequence of the obtained NHBA-fHbp(V1) fusion protein is specifically shown in SEQ ID NO: 8 (wherein, the amino acid sequence of the NHBA recombinant protein is from SEQ ID NO: 7, the fHbp(V1) monomer common sequence is from amino acids 21 to 274 of SEQ ID NO: 1, and the two are sequentially connected through the linker peptide shown in SEQ ID NO: 4).

[0073] (II) Construction of recombinant plasmids: After determining the amino acid sequence of the antigen protein, codon optimization is performed according to Escherichia coli as the expression host to obtain the DNA sequence encoding the target antigen protein. By means of genetic engineering, the DNA sequence encoding the target antigen protein is artificially synthesized. After confirming that the sequence is correct by checking the sequencing report (CoA), the synthesized DNA sequence is inserted between the NdeI and HindIII restriction enzyme sites in the expression plasmid pET-30a(+), respectively, to obtain recombinant expression plasmids that can express each antigen, and further transformed into cloning strains such as DH5α or TOP10 for amplification culture of the recombinant expression plasmids. After amplification culture, a certain amount of purified recombinant expression plasmids can be extracted using a plasmid extraction kit, and the concentration and purity of the recombinant plasmids are measured by NanoDrop, the recombinant plasmids are enzymatically verified by restriction enzyme mapping, or directly sequenced for target sequence identification. The DNA sequence information after the aforementioned codon optimization is as follows:

[0074] SEQ ID NO: 9: The coding DNA sequence of the aforementioned fHbp(V1) monomer recombinant protein;

[0075] SEQ ID NO:10: The coding DNA sequence of the aforementioned fHbp(V1-V1) fusion protein;

[0076] SEQ ID NO:11: The coding DNA sequence of the aforementioned NHBA recombinant protein;

[0077] SEQ ID NO:12: The coding DNA sequence of the aforementioned NHBA-fHbp(V1) fusion protein;

[0078] (III) Recombinant plasmid transformation: The recombinant plasmid with the identification result conforming to the expected sequence design was transformed into Escherichia coli BL21 competent cells to construct an engineering strain. Take about 100 ng of recombinant plasmid DNA and add it to 50 μl of competent cells placed on ice. Mix gently and incubate on ice for 30 min. Then, heat shock at 42 °C for 30 s in a water bath. Immediately transfer the sample tube to ice and incubate for 2 min. Add 250 μl of S.O.C. medium and culture at 37 °C, 220 rpm on a shaker for 45 min to 1 hour. After that, spread the bacterial solution on the LB solid medium with the corresponding resistance and culture overnight at 37 °C in a constant temperature incubator.

[0079] (IV) Strain PCR identification: Scrape 10 - 50 single colonies grown on the solid medium after overnight culture and directly dissolve them in about 10 μl of sterile water to mix into a bacterial solution. Take about 1 μl of this bacterial solution as a template for colony PCR to identify the target gene. The procedure of the colony PCR test was carried out according to the instructions of the kit (GoTaq® G2 Hot Start Master Mixes, Promega). The forward primer sequence: CGATGCGTCCGGCGTAGA; the reverse primer sequence: GCTAGTTATTGCTCAGCGG. Take about 5 - 10 μl of the colony PCR product and perform agarose gel electrophoresis with a concentration of 1%. After electrophoresis at 100 V for 50 min, compare the amplified product sequence with the gel to check if it conforms to the expected size. At the same time, the remaining PCR product can be sent for sequencing to further verify whether the target sequence conforms to the expectation.

[0080] (V) Small - scale expression of recombinant protein: Take an appropriate amount of the bacterial solution in the above sterile water and add it to about 2 ml of liquid LB medium. Culture overnight at 37 °C, 220 rpm. Inoculate the overnight - cultured bacterial solution into 10 ml of LB liquid medium at a certain ratio and culture at 37 °C, 220 rpm until the OD 600 value reaches 0.6 - 0.8; add IPTG inducer with a final concentration of 0.5 mM and induce for 4 h. Collect the bacterial solution after induction, centrifuge at 4 °C, 4000 g for 10 min, discard the supernatant, and store the bacterial cells at - 20 °C.

[0081] (6) SDS-PAGE identification: Resuspend the collected bacteria in the previous step with the buffer solution. Take the resuspended bacterial solution, add 5× Loading Buffer and mix well. After heating in a water bath at 100 °C, load the sample for routine SDS-PAGE detection. By comparing whether there is an obvious and thicker protein band near the size of the target antigen in the samples before and after induction. If so, it indicates that the strain screening is successful. Select the clone with the thickest band, which is the screened high-expression engineering strain. In addition, to increase the accuracy of identification, for some fHbp recombinant proteins, the conventional Western-Blot (immunoblotting) method is used to further identify the target recombinant protein. The corresponding results are shown in Figures 5 to 8 。

[0082] Example 3: Large-scale expression of engineering strains

[0083] Take a small amount of the preserved bacterial solution of the screened high-expression strain, streak it on an LB solid medium plate, and culture it overnight at 37 °C. After culturing, pick a single colony and inoculate it into an LB liquid medium, and culture it overnight at 37 °C and 220 rpm. Inoculate the overnight cultured bacterial solution into a larger volume of LB liquid medium at a certain ratio, and culture it at 37 °C and 220 rpm until the OD 600 value is 0.6 - 0.8, add IPTG with a final concentration of 0.5 - 1 mM, and induce at 37 °C and 220 rpm for 4 h. Centrifuge at 4000 g and 4 °C for about 10 min until the bacteria completely settle, discard the supernatant, and store the fermented bacteria at -20 °C.

[0084] Example 4: Extraction and purification of recombinant protein

[0085] Extraction of recombinant protein: Take out the collected bacteria and resuspend them in the lysis buffer (10 mM Tris-HCl, 100 mM NaCl, 1 mM EDTA). Break the bacterial suspension by an ultrasonic crusher or a homogenizer, and centrifuge to separate the bacterial debris precipitate and the supernatant of the lysed bacteria. Take a small amount of the bacterial debris precipitate and the supernatant of the lysed bacteria for SDS-PAGE identification of the target protein. If the target protein is mostly present in the supernatant of the lysed bacteria, then aliquot and store the supernatant of the lysed bacteria for downstream purification; if the target protein is mostly present in the bacterial debris precipitate, then add an appropriate amount of 10 mM Tris-HCl buffer containing a detergent to resuspend the precipitate, incubate at 4 °C for 1 - 2 h, overnight incubation, sonication or homogenization, and extract the target protein into the resuspended supernatant to ensure that all proteins exist in a soluble form for downstream protein purification.

[0086] Purification of recombinant protein:

[0087] After cleaning the nickel column, equilibrate the nickel column with 20 mM PB, 0.5 M NaCl, 20 mM imidazole (pH 7.4) buffer. Filter the supernatant containing the soluble target protein through a 0.22 μm membrane and load the sample. After washing away impurities with 5 column volumes of 20 mM PB, 0.5 M NaCl containing 50 mM imidazole, elute with 3 column volumes of 500 mM imidazole, 20 mM PB, 0.5 M NaCl solution, and collect the flow-through. Detect the purification effect of the target protein by SDS-PAGE electrophoresis and Western-Blot method (anti-His tag antibody). The results are as Figures 5 - 8 shown. After expression and purification of each recombinant protein through the Escherichia coli system, a highly pure expected protein product can be obtained. The eluted purified harvest solution is centrifugally exchanged to the required buffer system through a 50 KDa ultrafiltration membrane and concentrated to the required concentration and volume, and then filtered through a 0.22 μm membrane to sterilize to obtain the recombinant protein stock solution.

[0088] Example 5: Evaluation of the purified recombinant protein

[0089] Protein purity detection: The purity of the protein product can be further determined by size exclusion chromatography (SEC-HPLC) for the purified protein. Select a chromatographic column of Agilent AdvanceBio SEC 300A, and chromatographic purity analysis of the purified protein can be carried out in the mobile phase "0.1 mol / L phosphate buffer system containing 0.1 mol / L Na2SO4, pH 6.7 ± 0.3". The results are as Figure 9 shown that the purity of the purified fHbp(V1-V1) fusion protein can reach 92%.

[0090] Protein N-terminal modification detection: Since the fHbp-related recombinant proteins have post-expression N-terminal lipidation modification induced by the addition of the P4 signal peptide, it is necessary to detect and evaluate the N-terminal modification of the recombinant protein product. Use Intact-MS (Intact protein mass spec) technology to detect the exact molecular weight of the recombinant protein molecule, and the N-terminal modification can be directly evaluated. The results are as Figure 10 shown that there are a series of modified products with a molecular weight greater than the target molecular weight for the purified fHbp(V1-V1) fusion protein, indicating that the protein has a certain degree of lipidation modification.

[0091] Protein bioactivity detection: Both recombinant proteins fHbp and NHBA have their affinity host target proteins. fHbp can bind to complement factor H in vitro and in vivo; NHBA can bind to heparin-binding proteins in vitro and in vivo. Therefore, the bioactivity of the purified recombinant protein products can be detected by in vitro affinity Binding ELISA to preliminarily confirm the correct protein structure and biological function of the recombinant protein expression products. Add 100 μl of the recombinant protein diluted to a certain concentration into a 96-well enzyme-linked immunosorbent assay (ELISA) plate and coat it overnight at 2 - 8 °C. Discard the liquid in the wells, add approximately 200 μl of PBST buffer to wash the plate 5 times, and discard the liquid in the wells. Add 150 μl of milk blocking solution to each well and block it at room temperature for approximately 1 hour. After blocking, discard the blocking solution, add approximately 200 μl of PBST buffer to wash the plate 5 times, and discard the liquid in the wells. Add 100 μl of a series of concentration gradients of complement factor H or heparin-biotin solution to each well and incubate at room temperature for approximately 2 hours. Discard the liquid in the wells, add approximately 200 μl of PBST buffer to wash the plate 5 times, and discard the liquid in the wells. Add approximately 100 μl of anti-complement factor H antibody or avidin-HRP to each well and incubate at room temperature for approximately 1 hour. After washing as above, for the fHbp-related experiments, add approximately 100 μl of rabbit anti-IgG-HRP secondary antibody to each well and incubate at room temperature for 1 hour. After incubating with the antibody labeled with HRP, wash each well, then add approximately 100 μl of chromogenic substrate TMB to each well, develop color in the dark for approximately 15 minutes, add approximately 100 μl of stop solution to each well, and read the OD 450mm -OD 570mm absorbance value. The results are as shown in Figure 11 and Figure 12As shown, the in vitro affinity ELISA results of different recombinant proteins / fusion proteins with factor H of complement and heparin are shown respectively. The results show that: (1) The monomeric recombinant protein of fHbp(V1) and the fusion protein of fHbp(V1-V1) can both bind to factor H of complement, but the affinity of the latter is significantly higher than that of the former; (2) The NHBA-fHbp(V1) fusion protein has obvious affinity activities for both factor H of complement and heparin protein; (3) In addition, on the basis of the aforementioned fHbp(V1-V1) fusion protein, we also constructed a mutant and named it fHbp(V1-V1)(SL). Although the detection results of its physical and chemical analyses (such as purity, N-terminal lipid modification, etc.) are not much different from those before the mutation, it has no obvious binding activity with factor H of complement, which reflects from the side that the common sequence of the fHbp(V1) monomer constructed in the present invention and the structures and functions of the recombinant proteins constructed based on it are better. The above results indicate that after the monomeric recombinant protein of fHbp(V1), the fusion protein of fHbp(V1-V1), and the fusion protein of NHBA-fHbp(V1) constructed in the present invention are expressed solubly in Escherichia coli, they can all fold into correct protein structures and have obvious biological activities, and can be further used for other applications, such as developing vaccines as antigens: The fHbp(V1-V1) fusion protein can bind well to fH, indicating that the structure and function of its fH-binding site are intact, so it is beneficial to induce antibodies corresponding to the fH-binding site, thereby inhibiting the binding of fHbp and fH on the bacterial surface after infecting bacteria and reducing the immune escape of bacteria.

[0092] Example 6: Immunogenicity study

[0093] Animal immunization: The antigens prepared according to the aforementioned examples were inoculated into female BALB / c mice aged 6-8 weeks, with 8-10 mice in each group. The mice in each group were immunized intramuscularly three times at 1 / 10 human dose on days 0, 21, and 42 respectively. The blood of the mice in each group was collected 14 days after the last immunization, the serum was separated, and the antibody titer was detected. The experimental groups are as follows:

[0094] Group 1: Only the monomeric recombinant protein of fHbp(V1) was used as the antigen;

[0095] Group 2: Only the recombinant protein of NHBA was used as the antigen;

[0096] Group 3: The monomeric recombinant protein of fHbp(V1) and the recombinant protein of NHBA were used jointly as the antigen;

[0097] Group 4: Only the fusion protein of fHbp(V1-V1) was used as the antigen.

[0098] Group 5: Only the fusion protein of NHBA-fHbp(V1) was used as the antigen.

[0099] Serum separation: The collected peripheral blood of mice was allowed to stand at 37°C for 1 hour, then at 4°C for 1 hour, and centrifuged at 800 g for 20 min. After centrifugation, the supernatant was aspirated and transferred to a new 1.5 ml EP tube. It was stored in the dark at -20°C for later use.

[0100] Determination of specific antibody titer: The specific IgG titer in the immune serum was determined by whole-cell ELISA, that is: inactivated Neisseria meningitidis group B diluted by a certain multiple was coated on the enzyme-linked immunosorbent assay (ELISA) plate. After blocking with milk, the serially diluted serum separated from immunized mice was added. The goat anti-mouse IgG enzyme-labeled antibody labeled with horseradish peroxidase was used as the detection antibody. After the color reaction, the optical density (OD) was read on an enzyme-linked immunosorbent assay (ELISA) reader (Molecule Device). 450mm -OD 570mm Optical absorption value.

[0101] Taking the OD value of 0.105 as the cut-off value, that is, when the OD value of a certain dilution in the serum group is ≥0.105, it is positive. When the OD value <0.105, the antibody titer of the serum is the previous dilution.

[0102] Table 1 Results of serum anti-whole-cell IgG antibody titers induced by immunizing mice with different antigens

[0103]

[0104] The results showed that:

[0105] Immunization with the antigens shown in groups 1-5 alone could induce specific antibody immune responses against Neisseria meningitidis group B. Among them:

[0106] Comparison between group 4 and group 1 showed that, under the same dose condition, the antibody level induced by the fHbp(V1-V1) fusion protein was much higher than that of the fHbp(V1) monomeric recombinant protein, indicating that constructing a homologous dimeric recombinant protein on the basis of the fHbp(V1) monomer could significantly enhance the immunogenicity, thus overcoming the problem of low immunogenicity due to the small molecular weight of the fHbp(V1) monomer.

[0107] Comparison between group 3 and groups 1 and 2 showed that, when the total amount of antigen was the same, the combination of the fHbp(V1) monomeric recombinant protein and the NHBA recombinant protein had a better effect than using only one antigen, indicating that the combination of these two antigens could have a synergistic effect, so it could be used to prepare a composition for preventing Neisseria meningitidis group B infection.

[0108] Compared with Group 3, under the condition of the same dose, although the NHBA-fHbp(V1) fusion protein has not yet shown a technical effect equivalent to that of the combination of the two monomers, it still has an immunological effect; moreover, compared with the combination of the two monomers, the NHBA-fHbp(V1) fusion protein also has other potential advantages, such as: when preparing the vaccine, only this one recombinant protein antigen needs to be expressed and purified, without having to consider and formulate two antigen components at the same time, thus simplifying the vaccine preparation process. Therefore, the NHBA-fHbp(V1) fusion protein still has the potential to be used as an antigen for preparing meningococcal group B vaccine.

[0109] The above experimental data prove that the above-mentioned recombinant proteins, fusion proteins or their combinations can be used as antigens for preparing meningococcal group B vaccine.

Claims

1. The consensus sequence of the fHbp (V1) monomer or a combination thereof; the consensus sequence of the fHbp (V1) monomer is a synthetic polypeptide, and its amino acid sequence is selected from the following (1) - (5): (1) The sequence shown in SEQ ID NO: 1; (2) A highly immunogenic fragment of the sequence shown in SEQ ID NO: 1, the highly immunogenic fragment is a truncated fragment of SEQ ID NO: 1, which is obtained by deleting the first 1 - X amino acids at the N - terminus of SEQ ID NO: 1, where X is a positive integer and 1 ≤ X ≤ 26; preferably, X is 19, 20, 21, 22, 23, 24, 25 or 26; more preferably, X is 19 or 20; (3) A highly immunogenic fragment of the sequence shown in SEQ ID NO: 1, the highly immunogenic fragment is a truncated fragment of SEQ ID NO: 1, which at least contains the amino acids at positions 120 - 183 from the N - terminus of SEQ ID NO: 1; (4) A highly immunogenic variant of the sequence shown in SEQ ID NO: 1, the variant is obtained by substituting, deleting and / or adding Y amino acids outside the domain formed by the amino acids at positions 120 - 183 from the N - terminus of SEQ ID NO: 1 on the basis of SEQ ID NO: 1, where Y is a positive integer and 1 ≤ Y ≤ 5; preferably, 1 ≤ Y ≤ 2; (5) A highly immunogenic variant of the sequence shown in SEQ ID NO: 1, the variant is a sequence having more than 80%, more than 85%, more than 90%, more than 95%, or more than 99% identity with SEQ ID NO:

1.

2. The fHbp (V1) recombinant protein or a combination thereof; the fHbp (V1) recombinant protein contains more than one consensus sequence of the fHbp (V1) monomer as described in claim 1; preferably, the fHbp (V1) recombinant protein contains one, two, three or four consensus sequences of the fHbp (V1) monomer as described in claim 1.

3. The fHbp (V1) recombinant protein according to claim 2, wherein The fHbp (V1) recombinant protein is also called the fHbp (V1 - V1) fusion protein: it contains two consensus sequences of the fHbp (V1) monomer as described in claim 1 and they are in tandem.

4. The fHbp (V1) recombinant protein according to claim 3, wherein The amino acid sequences of both consensus sequences of the fHbp (V1) monomer are selected from option (2) in claim 1; preferably, the corresponding X values of the first and second fHbp (V1) monomer consensus sequences from the N - terminus of the fHbp (V1) recombinant protein are 19 and 20 respectively.

5. The fHbp (V1) recombinant protein according to any one of claims 3-4, characterized in that, The N - terminus of the fHbp (V1) recombinant protein contains the P4 signal peptide sequence shown in SEQ ID NO:

3.

6. The fHbp (V1) recombinant protein according to any one of claims 3-5, characterized in that, The two consensus sequences of the fHbp (V1) monomer are covalently linked by a linker peptide; preferably, the sequence of the linker peptide is as shown in SEQ ID NO:

4.

7. The fHbp (V1) recombinant protein according to any one of claims 3-6, characterized in that, The fHbp (V1) recombinant protein contains the amino acid sequence shown in SEQ ID NO:

6.

8. The fHbp (V1) recombinant protein according to claim 2, wherein The Hbp(V1) recombinant protein is also known as the fHbp(V1) monomer recombinant protein: it contains an fHbp(V1) monomer consensus sequence as described in claim 1.

9. The fHbp (V1) recombinant protein according to claim 8, characterized in that, The amino acid sequence of the fHbp(V1) monomer consensus sequence is selected from option (2) in claim 1; preferably, the corresponding X value of the fHbp(V1) monomer consensus sequence is 19.

10. The fHbp (V1) recombinant protein according to any one of claims 8-9, characterized in that, The N-terminus of the fHbp(V1) recombinant protein contains the P4 signal peptide sequence shown in SEQ ID NO:

3.

11. The fHbp (V1) recombinant protein according to any one of claims 8-10, characterized in that, The fHbp(V1) recombinant protein contains the amino acid sequence shown in SEQ ID NO:

5.

12. The NHBA-fHbp fusion protein, which contains an NHBA recombinant protein from Neisseria meningitidis serogroup B and an fHbp recombinant protein from Neisseria meningitidis serogroup B variant V1, V2 or V3; preferably, the NHBA recombinant protein is located at the N-terminus of the fusion protein, and the fHbp recombinant protein is located at the C-terminus of the fusion protein, and the two are tandemly linked.

13. The NHBA-fHbp fusion protein according to claim 12, wherein The fHbp recombinant protein is from Neisseria meningitidis serogroup B variant V1; preferably, the sequence of the fHbp recombinant protein is the fHbp(V1) monomer consensus sequence as described in claim 1.

14. The NHBA-fHbp fusion protein according to claim 13, wherein The amino acid sequence of the fHbp(V1) monomer consensus sequence is selected from option (2) in claim 1; preferably, the corresponding X value of the fHbp(V1) monomer consensus sequence is 20.

15. The NHBA-fHbp fusion protein according to any one of claims 12-14, characterized in that, The amino acid sequence of the NHBA recombinant protein is as shown in SEQ ID NO:

7.

16. The NHBA-fHbp fusion protein according to any one of claims 12-15, characterized in that, The NHBA recombinant protein and the fHbp recombinant protein are covalently linked through a linker peptide; preferably, the sequence of the linker peptide is as shown in SEQ ID NO:

4.

17. The NHBA-fHbp fusion protein according to any one of claims 12-16, characterized in that, The NHBA-fHbp fusion protein contains the amino acid sequence shown in SEQ ID NO:

8.

18. A nucleic acid sequence or a combination thereof, the nucleic acid sequence encoding the fHbp(V1) monomer consensus sequence as described in claim 1, the fHbp(V1) recombinant protein as described in any one of claims 2-11, and / or the NHBA-fHbp fusion protein as described in any one of claims 12-17.

19. A nucleic acid sequence encoding a recombinant protein of Neisseria meningitidis group B fHbp (V1) monomer, characterized in that, The nucleic acid sequence contains SEQ ID NO:

9.

20. A nucleic acid sequence encoding an fHbp (V1-V1) fusion protein, characterized in that, The nucleic acid sequence contains SEQID NO:

10.

21. A nucleic acid sequence encoding a recombinant Neisseria meningitidis serogroup B NHBA protein, characterized in that, The nucleic acid sequence contains SEQ ID NO:

11.

22. A nucleic acid sequence encoding an NHBA-fHbp fusion protein, characterized in that, The nucleic acid sequence contains SEQ IDNO:

12.

23. An expression vector, which contains the nucleic acid sequence as described in any one of claims 18-22.

24. A host cell, which contains the expression vector as described in claim 23.

25. A method for preparing the fHbp(V1) recombinant protein as described in any one of claims 2-11, and / or the NHBA-fHbp fusion protein as described in any one of claims 12-17, characterized in that: Obtain a recombinant plasmid containing the coding sequence of the above-mentioned recombinant protein or fusion protein through codon optimization and gene synthesis; transform the recombinant plasmid into an Escherichia coli expression system respectively, and obtain correct positive expression strains through the identification of the target antigen and the screening of strains; further expand the fermentation culture of the positive expression strains, and separately isolate and purify the target protein from the positive expression strains, so as to obtain the recombinant protein or fusion protein.

26. Use of the fHbp(V1) recombinant protein according to any one of claims 2-11, and / or the NHBA-fHbp fusion protein according to any one of claims 12-17 in the preparation of a drug for inducing an immune response or alleviating or preventing an infection caused by Neisseria meningitidis in a mammal; preferably, the Neisseria meningitidis is Neisseria meningitidis serogroup B.

27. An immunogenic composition, characterized in that, The immunogenic composition contains: the fHbp(V1) recombinant protein according to any one of claims 2-11, and / or the NHBA-fHbp fusion protein according to any one of claims 12-17.

28. The immunogenic composition according to claim 27, wherein The immunogenic composition contains the fHbp(V1) recombinant protein according to any one of claims 2-7, namely the fHbp(V1-V1) fusion protein.

29. The immunogenic composition according to claim 27, wherein, The immunogenic composition contains the NHBA-fHbp fusion protein according to any one of claims 12-17.

30. The immunogenic composition according to claim 27, wherein, The immunogenic composition contains the fHbp(V1) recombinant protein according to any one of claims 8-11, namely the fHbp(V1) monomeric recombinant protein, and the NHBA recombinant protein with the sequence shown in SEQ ID NO:

7.

31. The immunogenic composition according to any one of claims 27-30, characterized in that, The immunogenic composition further contains an adjuvant; preferably, the adjuvant is an aluminum adjuvant; more preferably, the adjuvant is an aluminum hydroxide adjuvant.

32. Use of the immunogenic composition according to any one of claims 27-31 in the preparation of a drug for inducing an immune response or alleviating or preventing an infection caused by Neisseria meningitidis in a mammal; preferably, the Neisseria meningitidis is Neisseria meningitidis serogroup B.

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