Vaccine composition and application thereof

Abstract

The present disclosure provides a vaccine composition wherein the antigen components comprise a factor H binding protein (fHbp), a recombinant outer membrane vesicle (OMV) carrying porin A (PorA protein), and optionally a heparin binding protein (NHBA protein). The antigen components in the vaccine composition disclosed by the invention have a good synergistic effect, so that the vaccine composition has extremely high vaccine forming potential, is used for preventing epidemic of group B meningococcus, and plays a role in protecting infection of group B meningococcus.

Description

Technical Field

[0001] The present disclosure belongs to the field of biological products, and specifically relates to a multivalent group B meningococcal vaccine composition and application thereof. Background Art

[0002] Meningococcus, also known as Neisseria meningitidis (Nm), is a Gram-negative prokaryotic bacterium. Humans are the only host of the pathogen and about 8-25% of adults are carriers. Meningococcus causes meningococcal disease (MD), with common symptoms including meningitis, arthritis, pericarditis, etc. The mortality rate is about 15%, and 10-15% of the recovered patients have serious sequelae. According to the structure of the surface capsular polysaccharide of the pathogen, meningococci can be divided into different serotypes. The main pathogenic bacteria groups are A, B, C, Y and W135 groups, and the proportion of meningococcal disease caused by group B in all cases is increasing year by year.

[0003] Since the structure of the capsular polysaccharide of group B meningococcus is similar to that of human nerve cell proteins, its immunogenicity is not enough to be used as a vaccine preparation component, so the design of group B meningococcal vaccine must rely on its outer membrane protein. At present, there is no multivalent and multicomponent group B meningococcal vaccine that is more suitable for China's epidemiological characteristics. Summary of the invention

[0004] In response to the epidemic trend of group B meningococcus in recent years, the present disclosure provides a multivalent, multicomponent group B meningococcal vaccine composition. The antigenic components of the vaccine composition have good synergistic effects, can be effectively used to prevent the epidemic of group B meningococcus, play a protective role against group B meningococcal infection, and have extremely high vaccine potential.

[0005] In a first aspect, the present disclosure provides a vaccine composition comprising an antigenic component, wherein the antigenic component comprises a factor H binding protein (fHbp), a recombinant outer membrane vesicle (OMV) carrying porin A (PorA protein), and optionally a heparin binding protein (NHBA protein).

[0006] In some embodiments, the factor H binding protein includes factor H binding protein variant 1 (fHbp-v1 variant protein), factor H binding protein variant 2 (fHbp-v2 variant protein) and / or factor H binding protein variant 3 (fHbp-v3 variant protein).

[0007] In some embodiments, the fHbp-v1 variant protein has the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:1, or has one or more amino acid residues substituted, deleted or inserted with the amino acid sequence shown in SEQ ID NO:1.

[0008] In some embodiments, the fHbp-v2 variant protein has the amino acid sequence shown in SEQ ID NO:2, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:2, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence shown in SEQ ID NO:2.

[0009] In some embodiments, the fHbp-v3 variant protein has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:3, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence shown in SEQ ID NO:3.

[0010] In some embodiments, the NHBA protein has an amino acid sequence as shown in at least one of SEQ ID NOs:4-6, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as shown in at least one of SEQ ID NOs:4-6, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence as shown in at least one of SEQ ID NOs:4-6.

[0011] In some embodiments, the PorA protein has a variable region 1 (VR1) and a variable region 2 (VR2).

[0012] In some embodiments, the VR1 has an amino acid sequence as shown in at least one of SEQ ID NOs:7-9, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in at least one of SEQ ID NOs:7-9, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence shown in at least one of SEQ ID NOs:7-9.

[0013] In some embodiments, the VR2 has an amino acid sequence as shown in at least one of SEQ ID NOs: 10-12, or an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence as shown in at least one of SEQ ID NOs: 10-12, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence as shown in at least one of SEQ ID NOs: 10-12.

[0014] In some embodiments, the PorA protein includes a first PorA protein having VR1 shown in SEQ ID NO:7 and VR2 shown in SEQ ID NO:10, a second PorA protein having VR1 shown in SEQ ID NO:8 and VR2 shown in SEQ ID NO:11, and a third PorA protein having VR1 shown in SEQ ID NO:9 and VR2 shown in SEQ ID NO:12.

[0015] In some embodiments, the antigenic component does not contain Neisseria adhesin A (NadA).

[0016] In some embodiments, the antigenic component consists of fHbp protein, NHBA protein, and OMV carrying PorA protein.

[0017] In some embodiments, the proteins in the antigenic component are all from Neisseria meningitidis.

[0018] In some embodiments, the fHbp protein in the antigen component is a lipidated fHbp protein.

[0019] In some embodiments, the NHBA protein is a lipidated NHBA protein.

[0020] In some embodiments, the N-terminus of the lipidated fHbp protein and / or lipidated NHBA protein has a lipidation signal peptide, more preferably, the lipidation signal peptide has the amino acid sequence shown in SEQ ID NO: 13, or has one or more amino acid residues substituted, deleted or inserted with the amino acid sequence shown in SEQ ID NO: 13.

[0021] In some embodiments, in the vaccine composition, the weight ratio of the fHbp protein, NHBA protein and OMV is (1-5): (0-2): (0.25-1), preferably (1-5): (0.5-2): (0.25-1).

[0022] In some embodiments, in the fHbp protein, the weight ratio of fHbp-v1 variant protein, fHbp-v2 variant protein and fHbp-v3 variant protein is 1:(0-2):(0-2).

[0023] In some embodiments, in the vaccine composition, the ratio between fHbp-v1 variant protein, fHbp-v2 variant protein, fHbp-v3 variant protein, NHBA protein and OMV is 1:(0-2):(0-2):(0.5-2):(0.25-1), wherein the weight ratio of the first PorA protein, the second PorA protein and the third PorA protein carried on the OMV is 1:(0.9-1.1):(0.9-1.1).

[0024] In some embodiments, in the vaccine composition, the ratio between fHbp-v1 variant protein, fHbp-v2 variant protein, fHbp-v3 variant protein, NHBA protein and OMV components is 1:0.5:0.5:1:0.5, wherein the weight ratio of the first PorA protein, the second PorA protein and the third PorA protein carried on the OMV is 1:(0.9-1.1):(0.9-1.1).

[0025] In some embodiments, the antigen component includes 10-100 μg of lipidated fHbp-v1 variant protein, 0-100 μg of lipidated fHbp-v2 variant protein, 0-100 μg of lipidated fHbp-v3 variant protein, 0-100 μg of lipidated NHBA protein, and 10-100 μg of OMV vector carrying PorA protein.

[0026] In some embodiments, the vaccine composition is a liquid formulation.

[0027] In some embodiments, a 500 μL liquid formulation of a vaccine composition includes 10-100 μg of lipidated fHbp-v1 variant protein, 0-100 μg of lipidated fHbp-v2 variant protein, 0-100 μg of lipidated fHbp-v3 variant protein, 0-100 μg of lipidated NHBA protein, and 10-100 μg of an OMV vector carrying a PorA protein.

[0028] In some embodiments, the vaccine composition further comprises a vaccinologically acceptable carrier.

[0029] In some embodiments, the vaccinologically acceptable carrier includes one or more of aluminum adjuvant, MF59 adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG adjuvant, QS21 adjuvant or MPLA adjuvant.

[0030] In some embodiments, the vaccine composition further comprises a stabilizing formulation.

[0031] In some embodiments, the stabilizing formulation includes one or more of histidine, polysorbate 80, or sucrose.

[0032] In some embodiments, the vaccine composition further comprises other agents for stabilizing the antigen structure, such as sucrose.

[0033] In some embodiments, the vaccine composition further comprises other agents for stabilizing pH and osmotic pressure, such as histidine and sodium chloride.

[0034] The vaccine composition disclosed in the present invention has the following beneficial effects: the polyclonal antibodies induced by fHbp have a broad spectrum, and in particular, the N-terminal lipidation of the protein has a self-adjuvant effect, which will help to improve the immunogenicity of the antigen and the titer level of the antibody after immunization; the polyclonal antibodies induced by the lipidated NHBA antigen will supplement the broad spectrum of the fHbp-specific antibodies; the antibodies induced by the trivalent PorA will again supplement the broad spectrum of the vaccine, and the self-adjuvant effect of the OMV component itself can also improve the immunogenicity and specific antibody titer level of the fHbp antigen and the NHBA antigen. In particular, the antigen component sequence selection of the vaccine composition disclosed in the present invention is based on the epidemiological situation of group B meningococcus in China and the pathogenicity of the epidemic strain, which will be more helpful to the prevention of meningitis in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The SDS-PAGE electrophoresis patterns of the purified lipoproteins fHbp-v1, fHbp-v2, and fHbp-v3 are shown.

[0036] Figure 2 The SDS-PAGE electrophoresis patterns of the three purified lipoproteins NHBA-P503, NHBA-P669 and NHBA-P688 are shown.

[0037] Figure 3 The relative expression levels of different PorAs on the engineered OMV fractions are shown.

[0038] Figure 4 The results of measuring the bactericidal activity of serum after immunization of rabbits with monovalent vaccine compositions of fHbp-v1, fHbp-v2, and fHbp-v3 are shown.

[0039] Figure 5 The results of the determination of bactericidal activity of sera after immunization of rabbits with monovalent vaccine compositions of NHBA-P503, NHBA-P669, and NHBA-P688 are shown.

[0040] Figure 6 The results of the bactericidal activity test of serum from rabbits immunized with the monovalent OMV vaccine are shown.

[0041] Figure 7 Results are shown for serum antibody titers in rabbits of multivalent, multicomponent vaccine compositions of different combinations of fHbp, NHBA and OMV.

[0042] Figure 8 The results of the bactericidal activity test of serum of rabbits after immunization with a multivalent multicomponent vaccine composition composed of fHbp, NHBA and OMV are shown. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present disclosure clearer, the present disclosure is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present disclosure and are not intended to constitute any limitation to the present disclosure. In addition, in the following description, the description of the well-known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0044] definition

[0045] Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly used in the field to which this disclosure belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0046] Unless the context clearly dictates otherwise, the expressions "a", "an" and "an" as used herein include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0047] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0048] Reference herein to "homology" or "percent identity (% identity)" between two sequences can be determined using techniques known in the art. Some of the methods for determining identity are codified in publicly available computer programs. Sequence alignments and calculations of percent identity can be performed using, for example, the AlignX program of the VectorNTI software package (Invitrogen, Carlsbad, CA) or the MegAlignX program of the LASERGENE bioinformatics computing package (DNASTAR Inc., Madison, WI).TM Program is carried out. In addition, the mensuration of the percentage identity between two sequences can also be determined using mathematical algorithms known to those skilled in the art. Those skilled in the art can determine the appropriate parameters for the comparison sequence, including any algorithm required for maximum comparison on the compared sequence full length. In some embodiments, "at least 85% sequence identity" comprises about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 98.9% or 99.9% or the sequence identity of any value therebetween.

[0049] Factor H binding protein (fHbp), also known as GNA1870, LP2086, ORF2086, NMB1870. fHbp is a membrane surface lipoprotein, and many strains of meningococci carry its gene. According to the amino acid sequence, the protein can be divided into three variants: factor H binding protein variant 1 (fHbp-v1), factor H binding protein variant 2 (fHbp-v2), and factor H binding protein variant 3 (fHbp-v3). The fHbp-v1 variant, fHbp-v2 variant, and fHbp-v3 variant genes encode approximately 280 amino acid residues, respectively.

[0050] The fHbp-v1 variant protein that can be used in the present disclosure is not particularly limited, and can be any wild-type or mutant fHbp-v1 variant protein. Preferably, in the fHbp protein that can be used in the vaccine composition of the present disclosure, the sequence of the fHbp-v1 variant can be selected from SEQ ID NO: 1, and can also include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 1, and can also include an amino acid sequence in which one or more amino acids are added, deleted or substituted to SEQ ID NO: 1.

[0051] The fHbp-v2 variant protein that can be used in the present disclosure is not particularly limited, and can be any wild-type or mutant fHbp-v2 variant protein. Preferably, in the fHbp protein that can be used in the vaccine composition of the present disclosure, the sequence of the fHbp-v2 variant can be selected from SEQ ID NO: 2, and can also include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 2, and can also include an amino acid sequence in which one or more amino acids are added, deleted or substituted to SEQ ID NO: 2.

[0052] The fHbp-v3 variant protein that can be used in the present disclosure is not particularly limited, and can be any wild-type or mutant fHbp-v3 variant protein. Preferably, in the fHbp protein that can be used in the vaccine composition of the present disclosure, the sequence of the fHbp-v3 variant can be selected from SEQ ID NO: 3, and can also include an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 3, and can also include an amino acid sequence in which one or more amino acids are added, deleted or substituted to SEQ ID NO: 3.

[0053] Heparin binding protein (NHBA) is a lipoprotein unique to Neisseria and is expressed in almost all strains. NHBA consists of about 420-500 amino acid residues. In addition to single or multiple amino acid variations, there are also insertions or deletions of single amino acids or short peptides between different NHBA proteins.

[0054] The NHBA that can be used in the present disclosure is not particularly limited and can be any wild-type or mutant NHBA variant protein. Preferably, the sequence of the NHBA that can be used in the vaccine composition of the present disclosure can be selected from the sequence shown in at least one of SEQ ID NOs: 4-6, and can also include an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% sequence identity with the amino acid sequence shown in at least one of SEQ ID NOs: 4-6, and can also include an amino acid sequence in which one or more amino acids are added, deleted or substituted to the sequence shown in at least one of SEQ ID NOs: 4-6.

[0055] Outer membrane vesicles (OMVs) are lipid vesicles released from the outer membrane of Gram-negative bacteria that have a membrane structure and are able to carry and present outer membrane proteins.

[0056] Porin A (PorA) is a major inducer of immune response in meningococcal OMVs and is a target of serum bactericidal antibodies. Most of the variation between PorA proteins occurs in two of the eight putative surface-exposed loops, variable loops I and IV, named variable region 1 (VR1) and variable region 2 (VR2), respectively. Different PorA proteins have different antigenic specificities, and the immune response caused by one PorA usually cannot play a defensive role against strains of heterologous PorA antigens. Preferably, in the PorA protein that can be used in the vaccine composition of the present invention, the sequence of VR1 can be selected from the sequences shown in SEQ ID NOs: 7-9, and can also include the amino acid sequence in which one or more amino acids are added, deleted or substituted to the sequence shown in at least one of SEQ ID NOs: 7-9, and the sequence of VR2 can be selected from the sequences shown in SEQ ID NOs: 10-12, and can also include the amino acid sequence in which one or more amino acids are added, deleted or substituted to the sequence shown in at least one of SEQ ID NOs: 10-12. More preferably, the PorA protein includes a first PorA protein having VR1 shown in SEQ ID NO:7 and VR2 shown in SEQ ID NO:10, a second PorA protein having VR1 shown in SEQ ID NO:8 and VR2 shown in SEQ ID NO:11, and a third PorA protein having VR1 shown in SEQ ID NO:9 and VR2 shown in SEQ ID NO:12.

[0057] Neisserial Adhesin A (NadA), GNA1994, is highly conserved in group B meningococci, can produce protection in experimental animal immune evaluation, and is an effective antigen component of group B meningococcal vaccine.

[0058] As used herein, the term "antigen" refers to a substance that can stimulate the body to produce a (specific) immune response and can bind to the immune response product antibodies and sensitized lymphocytes in vitro to produce an immune effect (specific reaction). There are two basic characteristics of antigens: one is the ability to induce an immune response, that is, immunogenicity, and the other is to react with the product of the immune response, that is, antigenicity.

[0059] As used herein, the antigen component also includes active fragments, derivatives and analogs of the antigen component. As used herein, the terms "fragment", "derivative", "derivative protein" and "analog" refer to proteins that substantially maintain the activity of activating the body to produce an immune response to group B meningococci. The fragments, derivatives or analogs of the protein disclosed herein may be (i) a polypeptide in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, or (ii) a polypeptide having a substitution group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of the protein disclosed herein with another compound (such as a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (a derivative protein formed by fusion with a leader sequence, a secretory sequence or a tag sequence such as 6His). According to the teachings of this article, these fragments, derivatives and analogs may be wild-type or mutant, which are all within the scope known to those skilled in the art.

[0060] As used herein, the antigen component may also have a variant form of the sequence shown in SEQ ID NOs: 1-12 that activates the body to produce an immune response to group B meningococci. These variant forms include but are not limited to: deletion, insertion and / or substitution of 1-5 (usually 1-4, preferably 1-3, more preferably 1-2) amino acids, and addition or deletion of one or several (usually within 5, preferably within 3-4, more preferably within 1-2) amino acids at the C-terminus and / or N-terminus. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the structure and function of the protein.

[0061] As used herein, the antigenic components also include analogs of antigenic components. The difference between these analogs and the natural antigenic components of the present disclosure may be a difference in amino acid sequence, or a difference in modification form that does not affect the sequence, or both. Analogs also include analogs with residues different from natural L-amino acids (such as D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (such as β, γ-amino acids). It should be understood that the polypeptides of the present disclosure are not limited to the representative polypeptides exemplified above.

[0062] Valency refers to the serotype of the same bacteria. If a bacteria has multiple serotypes, there will be multiple valencies. The higher the "valency", the more serotypes it covers. Monovalent vaccine refers to a vaccine prepared by using the proliferation culture of a single serotype strain of the same microbial strain. Monovalent vaccines have good immune protection against diseases caused by the corresponding single serotype of microorganisms. Vaccines containing only a single antigen component are called "monovalent vaccines". Monovalent vaccines can only prevent one infectious disease or one type of pathogen infection. Polyvalent vaccine refers to a vaccine made by mixing two or more serotype antigen components in appropriate proportions. Polyvalent vaccine refers to a vaccine that can prevent multiple pathogenic serotypes of a bacterium. When multivalent vaccines are used for combined immunization, the immune effect is similar to or better than that of monovalent vaccines, and there is no increase in the side effects of the vaccine.

[0063] After in-depth and extensive research, the present inventors have discovered for the first time that a multivalent multicomponent antigen composition can effectively and broadly prevent and protect against the epidemic strains of group B meningitis in China. In the antigen composition, the multivalent lipidated fHbp antigen, NHBA antigen and the OMV carrier presenting the PorA protein have a good synergistic effect. Animal immunization experiments have verified that the vaccine composition can widely protect against a variety of epidemic strains of meningitis in China. The antigen composition has a very high potential for vaccine formation. In addition, in theory, the antigen composition disclosed in the present invention can also cover meningococci of other serogroups such as groups A, C, Y, W, and X to a certain extent, and has the value of a broad-spectrum anti-meningococcal vaccine.

[0064] The present disclosure designs a vaccine according to the epidemic trend of group B meningitis in China and the classification characteristics of its structure, so as to produce a multivalent and multi-component group B meningococcal vaccine that is more suitable for the epidemiological characteristics of China. Among them, fHbp adopts its natural lipoprotein design, and selects one sequence from its three variant sequence classifications (v1, v2 and v3) as a component of the vaccine, as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3; wherein, NHBA adopts its natural lipoprotein design, and selects three sequences as vaccine alternatives, as shown in SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6; wherein, NadA is not selected as a component of the vaccine preparation, because the pathogens in China contain almost no NadA targets; wherein, OMV will carry three PorAs, the VR1 sequences of PorA are shown in SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively, and the VR2 sequences are shown in SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12.

[0065] In some embodiments, the vaccine composition of the present disclosure comprises the following antigen components: lipidated fHbp-v1, with a protein content of 10-100 μg; lipidated fHbp-v2 and fHbp-v3, one or both, each with a protein content of 0-100 μg; lipidated NHBA, with a protein content of 0-100 μg; an OMV vector presenting trivalent PorA, wherein the total content of PorA is 10-100 μg.

[0066] In some embodiments, the present disclosure adopts a liquid preparation, dissolving the above-mentioned antigen components in a liquid volume of 500 μL. In addition to the antigen components, the vaccine composition also includes 0.25-1.5 mg of aluminum phosphate adjuvant and 0.01-0.1 g of polysorbate 80 emulsifier. In some embodiments, other reagents for stabilizing the antigen structure, such as sucrose, may be added to the vaccine composition. Other reagents for stabilizing pH and osmotic pressure, such as histidine and sodium chloride, may be added to the formulation prescription of the present disclosure.

[0067] In some embodiments, lipidated fHbp-v1, fHbp-v2 and fHbp-v3 may have amino acid sequences as shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and a lipidation signal peptide is added to its N-terminus, and the lipidation signal peptide has an amino acid sequence as shown in SEQ ID NO: 13. After codon optimization according to Escherichia coli preference, the gene sequence of lipidated fHbp protein is synthesized and connected to the pET28a (+) vector. The expression vector is transformed into Escherichia coli BL21 (DE3), and lipidated fHbp is generated after IPTG induction and post-translational modification.

[0068] In some embodiments, the lipidated NHBA may have an amino acid sequence as shown in SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6, and a lipidation signal peptide is added to its N-terminus, and the lipidation signal peptide has an amino acid sequence as shown in SEQ ID NO: 13. After codon optimization according to E. coli preference, the gene sequence of the lipidated NHBA protein is synthesized and connected to the pET28a (+) vector. The expression vector is transformed into E. coli BL21 (DE3), and the lipidated NHBA is produced after IPTG induction and post-translational modification.

[0069] In some embodiments, the OMV components can be obtained by knocking out the LpxL1, rmpM and SynX genes in sequence from wild-type group B meningococci through homologous recombination. The recombinant group B meningococci are screened for single clones and sequenced using a dual screening system containing 5-fluoroorotic acid (5-FOA) and uracil plates.

[0070] In some embodiments, three PorA genes are synthesized on a plasmid vector and arranged linearly, and the three PorA genes are inserted into the PorA expression region of the original bacteria. The three PorA genes are expressed and regulated by the same promoter to ensure the same expression level. The transformed strains are cultured in a liquid culture medium and harvested at the end of their exponential growth phase.

[0071] In the present disclosure, group B meningococci Nm-01, Nm-02 and Nm-03 containing three PorA protein coding sequences were donated from the National Center for Disease Control and Prevention, wherein group B meningococci Nm-01 has a coding sequence for expressing PorA proteins including VR1 shown in SEQ ID NO:7 and VR2 shown in SEQ ID NO:10; group B meningococci Nm-02 has a coding sequence for expressing PorA proteins including VR1 shown in SEQ ID NO:8 and VR2 shown in SEQ ID NO:11; group B meningococci Nm-03 has a coding sequence for expressing PorA proteins including VR1 shown in SEQ ID NO:9 and VR2 shown in SEQ ID NO:12.

[0072] In some embodiments, Nm-01 is used as a starting point for the transformation of pathogenic bacteria for OMV production. Generally, Nm-02 or Nm-03 can also be used as a starting point for the transformation of pathogenic bacteria for OMV production.

[0073] The modified strain disclosed in the present invention will be able to express three PorA proteins simultaneously and present them on OMVs extracted from it. The PorA antigens presented by its OMVs are all immunogenic.

[0074] Meningococci are in a naturally competent state and can be knocked out by homologous recombination. Generally, 100-500bp of homologous sequences are cloned upstream and downstream of the gene to be knocked out and cloned into the pUC vector. The target gene to be inserted or the resistance gene to be used for screening is cloned between the homologous sequences. In the experiment, group B meningococci were evenly spread on the screening plate before transformation, and the DNA for transformation was dropped on the bacteria and cultured at 37°C for 6-8 hours to grow the transformed bacteria.

[0075] In the present disclosure, genetic screening was performed using a dual screening system that utilized the differential growth of bacteria with or without the orotate phosphoribosyltransferase (pyrE) gene on plates containing uracil or 5-FOA.

[0076] In some embodiments, the present disclosure sequentially performs the following transformations: ① knocking out the original pyrE gene of meningococcal Nm-02; ② knocking out the lpxL1 gene; ③ inserting the PorA gene cloned in Nm-01 in the lpxL1 gene in situ; ④ knocking out the outer membrane protein rmpM expression gene; ⑤ inserting the PorA gene cloned in Nm-03 in the rmpM gene in situ; ⑥ knocking out the capsular polysaccharide synthesis gene synX; ⑦ backfilling the original pyrE gene. The resulting OMV production strain is named Nm-OMV, and after identification and preservation of the strain, it is used for fermentation, OMV extraction and purification research.

[0077] However, those skilled in the art should understand that the above transformation sequence disclosed in the present disclosure is not the only one, and any transformation sequence that can obtain the transformation of the strain for OMV production disclosed in the present disclosure belongs to the scope of the present disclosure. In the transformation sequence disclosed in the present disclosure, after performing the transformation step ②, the process of OMV purification can be explored; after performing the transformation step ③, the expression level of PorA antigen and its immunogenicity can be explored.

[0078] Examples and drawings are provided below to help understand the present disclosure. However, it should be understood that these examples and drawings are only used to illustrate the present disclosure and do not constitute any limitation. The actual scope of protection of the present disclosure is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present disclosure.

[0079] Example

[0080] Example 1: Construction and purification of lipidated fHbp vector

[0081] Three variants of fHbp, fHbp-v1 (SEQ ID NO: 1), fHbp-v2 (SEQ ID NO: 2) and fHbp-v3 (SEQ ID NO: 3), were selected respectively, and a lipidated signal peptide (SEQ ID NO: 13) was added to the N-terminus thereof. After codon optimization preferred by Escherichia coli according to the protein sequences of the full-length (lipidated) fHbp-v1 (SEQ ID NO: 14), the full-length (lipidated) fHbp-v2 (SEQ ID NO: 15) and the full-length (lipidated) fHbp-v3 (SEQ ID NO: 16), the gene sequences of lipidated fHbp-v1 protein, lipidated fHbp-v2 protein and lipidated fHbp-v3 protein (SEQ ID NO: 17-19) were synthesized respectively and directly synthesized on the pET28a (+) vector.

[0082] The synthesized vectors were transferred into Escherichia coli BL21 (DE3) by chemical transformation for expression. During the exponential growth phase of Escherichia coli BL21 (DE3), 0.5 mM IPTG was added, induced at 16°C for 16 h, and the cells were harvested by centrifugation at 6000 g. After freezing the cells at -70°C overnight, the cells were resuspended in buffer A (10 mM HEPES-NaOH, 1 mM Na2EDTA, 1 mM PMSF, pH = 7.4), crushed 3 times at 1200 Bar with a high-pressure homogenizer, and centrifuged at 150000 g for 30 min to obtain a precipitate. The precipitate was washed twice with buffer A, centrifuged at 150000 g for 30 min, and then frozen at -70°C overnight. The precipitate was resuspended in buffer B (10 mM HEPES-NaOH, 2 mM MgCl2, 1% Triton X-100, pH = 7.4), mixed at room temperature for 30 min to extract lipoprotein fHbp-v1, lipoprotein fHbp-v2 and lipoprotein fHbp-v3, respectively, and the supernatant was harvested after centrifugation at 150000g for 30 min. The extraction operation was repeated twice. The supernatant was loaded on an anion chromatography column (QHP FF chromatography filler, #17-1014-03, GE Healthcare LifeSciences), eluted with a NaCl salt ion gradient, and then passed through a cation chromatography column (Capto SPImpRes chromatography filler, #17546803, GE Healthcare Life Sciences), eluted with a pH 6-8 gradient, and finally further purified and impurities were removed with molecular sieves (S-200HR filler, #17-0584-10, Cytiva).

[0083] The purification results are as follows Figure 1 As shown, the purified protein was exchanged by ultrafiltration and stored in a stabilizing buffer (10 mM phosphate buffer, 30 mM NaCl, pH=6.0).

[0084] Example 2: Lipidated NHBA vector construction and purification

[0085] NHBA P503 (SEQ ID NO: 4), NHBA P669 (SEQ ID NO: 5) and NHBA P688 (SEQ ID NO: 6) were selected respectively, and a lipidated signal peptide (SEQ ID NO: 13) was added to the N-terminus thereof. After codon optimization preferred by Escherichia coli was performed according to the full-length (lipidated) NHBA P503 (SEQ ID NO: 20), full-length (lipidated) NHBA P669 (SEQ ID NO: 21) and full-length (lipidated) NHBA P688 (SEQ ID NO: 22) protein sequences, the gene sequences (SEQ ID NOs: 23-25) of lipidated NHBA P503, lipidated NHBA P669 and lipidated NHBA P688 proteins were synthesized respectively and directly synthesized on the pET28a (+) vector.

[0086] In the same manner as in Example 1, the synthesized vector was transferred into Escherichia coli BL21 (DE3) by chemical transformation for expression, and IPTG induction and post-translational modification were performed. The harvested bacteria were frozen at -70°C overnight, resuspended in buffer A (10mM HEPES-NaOH, 1mM Na2EDTA, 1mM PMSF, pH=7.4), crushed 3 times at 1200Bar with a high-pressure homogenizer, and centrifuged at 15000g for 30min to obtain a precipitate. The precipitate was washed twice with buffer A, centrifuged at 150000g for 30min, and then frozen at -70°C overnight. The precipitate was resuspended in buffer B (10mM HEPES-NaOH, 2mM MgCl2, 1% Triton X-100, pH=7.4), mixed at room temperature for 30min to extract lipoprotein NHBA, and the supernatant was harvested after centrifugation at 150000g for 30min. The extraction operation was repeated 2 more times. The supernatant was loaded onto an anion chromatography column (QHP FF chromatography filler, #17-1014-03, GE Healthcare Life Sciences), eluted with a NaCl salt ion gradient, then passed through a cation chromatography column (HR-S filler, #1560515, BioRad), eluted with a pH 6-8 gradient, and finally further purified and impurities were removed with molecular sieves (S-200HR filler, #17-0584-10, Cytiva). The purification results are shown in Figure 2 As shown, the purified protein was exchanged by ultrafiltration and stored in a stabilizing buffer (10 mM phosphate buffer, 30 mM NaCl, pH=6.0).

[0087] Example 3: Transformation of pathogenic bacteria for OMV production

[0088] The present invention discloses group B meningococci Nm-01, Nm-02 and Nm-03, which are respectively capable of expressing a first PorA protein, a second PorA protein and a third PorA protein, donated by the National Center for Disease Control and Prevention, wherein the amino acid sequences of the first PorA protein, the second PorA protein and the third PorA protein are shown in SEQ ID NO:26, SEQ ID NO:27 and SEQ ID NO:28, respectively.

[0089] The present invention uses Nm-02 as a starting point to transform pathogenic bacteria for OMV production, and uses a dual screening system with different growth conditions of bacteria with or without the pyrE gene on plates lacking uracil or containing 5-fluoroorotic acid (5-FOA) for genetic screening. The specific feature of the dual screening system is that when the pyrE gene is present, meningococci cannot grow on plates containing 5-fluoroorotic acid (5-FOA); and when the pyrE gene is missing, meningococci cannot grow on plates lacking uracil.

[0090] The transformation sequence disclosed in the present invention is as follows: ① knock out the original pyrE gene of meningococcus Nm-02 → ② knock out the lpxL1 gene → ③ insert the PorA gene cloned from Nm-01 in the lpxL1 gene in situ → ④ knock out the outer membrane protein rmpM expression gene → ⑤ insert the PorA gene cloned from Nm-03 in the rmpM gene in situ → ⑥ knock out the capsular polysaccharide synthesis gene synX → ⑦ restore the original pyrE gene. The final OMV production strain obtained by the transformation is named Nm-OMV, and after the strain is identified and preserved, it is used for fermentation, OMV extraction and purification research.

[0091] The group B meningococcus Nm-02 was gene-edited by homologous recombination, and the lpxL1, rmpM and synX genes were knocked out in sequence. The recombinant group B meningococcus was screened and sequenced for single clones by a dual screening system containing 5-FOA or plates lacking uracil. The results showed that the PorA genes from Nm-01 and Nm-03 were inserted into the sites of the original lpxL1 and rmpM genes, respectively, and the three PorA genes were expressed and regulated by the same promoter to ensure the same expression level.

[0092] The modified strain Nm-OMV obtained in this example can express three PorA proteins simultaneously and can present them on the OMVs extracted therefrom. The PorA antigens presented by its OMVs are all immunogenic.

[0093] Example 4: OMV fermentation and purification

[0094] The Nm-OMV strain obtained in Example 3 was fermented and cultured in GC medium, maintaining the temperature at 37°C and maintaining the carbon dioxide concentration at 8%. When the bacteria grew to the end of the exponential growth phase, the temperature was lowered and harvested. The bacteria were extracted from OMV in a buffer containing EDTA (0.1M Tris, 10mM EDTA, pH = 7.4). After centrifugation at a centrifugal force of 20,000g for 1h, the OMV component was separated from the supernatant and centrifuged at a centrifugal force of 125,000g for 2h to form a precipitate. The precipitate was resuspended and sterilized and filtered at 0.22μM for preservation to obtain a purified OMV component. Generally, further purification by molecular sieves can reduce the content of host protein impurities.

[0095] The relative expression of PorA in purified OMVs was examined using monoclonal antibodies specific for the three PorAs. The experimental results are shown in Figure 3 The results of multiple batches of experiments showed that the expression levels of the three PorAs were within 10%.

[0096] Example 5: Immunogenicity Assay

[0097] The purified lipidated fHbp protein, lipidated NHBA protein and OMV components were separately prepared as finished vaccines. The purified antigen was added to the base buffer (10mM histidine, 0.02% polysorbate 80, NaCl was added to make the osmotic pressure close to that of saline, pH = 6.0), and the addition ratio of the purified antigen to the base buffer was adjusted to adjust the protein content in the final immunogenic composition. 0.5-3mg of aluminum phosphate adjuvant was added to the final volume of 1mL of the immunogenic composition for adsorption. A small amount of sucrose was added as a stabilizer during the preparation of the immunogenic composition of the OMV component. The composition of each immunogenic composition (1mL) is shown in Table 1 below.

[0098] Table 1

[0099] protein Protein content Aluminum content (aluminum phosphate) Sucrose content Immunogenic composition 1 Lipidated fHbp-v1 100 μg 0.5mg 0 Immunogenic composition 2 Lipidated fHbp-v2 100 μg 0.5mg 0 Immunogenic composition 3 Lipidated fHbp-v3 100 μg 0.5mg 0 Immunogenic composition 4 Lipidated NHBA P503 100 μg 0.5mg 0 Immunogenic composition 5 Lipidated NHBA P688 100 μg 0.5mg 0 Immunogenic composition 6 Lipidated NHBA P669 100 μg 0.5mg 0 Immunogenic composition 7 OMV 50μg 0.5mg 3％

[0100] The prepared immunogenic composition was evaluated for immunogenicity by intramuscular injection in rabbits. Five 4-6 week old rabbits were selected for the titer verification of each group of experiments. Blood was collected before immunization to ensure that they did not contain antigen-specific antibodies to group B meningococci. The animals were immunized once every four weeks, for a total of four injections. Venous blood was collected and serological titer was evaluated three weeks after the fourth injection. The collected serum was separated and diluted to different multiples. The indicator bacteria carrying specific antigens were incubated with the diluted serum and then cultured. The number of colonies grown was used to calculate the titer of the serum bactericidal ability (SBA). The design of the experimental groups is shown in Table 2. The serum bactericidal results are shown in Figure 4-6As shown. The experimental results show that all fHbp and NHBA antigens are immunogenic, among which fHbp-v2 and fHbp-v3 have cross-reactions, and all NHBA antigens have cross-reactions. The three PorA proteins presented by OMV can induce antibodies with serum bactericidal activity. Therefore, it is possible to consider multi-component formulation of vaccines to make the vaccine broad-spectrum.

[0101] Table 2: Immunogenicity profile of monovalent vaccines in rabbits

[0102] antigen Dosage (μg) Experimental Group 1 Immunogenic composition 1 Lipidated fHbp-v1 50 Experimental Group 2 Immunogenic composition 2 Lipidated fHbp-v2 50 Experimental Group 3 Immunogenic composition 3 Lipidated fHbp-v3 50 Experimental Group 4 Immunogenic composition 4 Lipidated NHBA P503 50 Experimental Group 5 Immunogenic composition 5 Lipidated NHBA P688 50 Experimental Group 6 Immunogenic composition 6 Lipidated NHBA P669 50 Experimental Group 7 Immunogenic composition 7 OMV 25 Control group / Normal saline /

[0103] Example 6: Proportion of multi-component vaccines

[0104] The purified lipidated fHbp protein, lipidated NHBA protein and OMV components will be used to prepare the finished vaccine in a certain proportion to evaluate the immunogenicity of the multi-component vaccine. The purified antigen will be added to the base buffer (10mM histidine, 0.02% polysorbate 80, NaCl is added to make the osmotic pressure close to that of normal saline, pH = 6.0) in a certain proportion, and the antigen content in the final immunogenic composition is adjusted by adjusting the total protein amount of the purified antigen and the addition ratio of the base buffer. 0.5-3mg of aluminum phosphate adjuvant is added to each 1mL of the final volume for adsorption. A small amount of sucrose is added as a stabilizer during the preparation of the immunogenic composition of the OMV component. The composition of each immunogenic composition (1mL) is shown in Table 3 below.

[0105] Table 3

[0106]

[0107] The prepared immunogenic composition was evaluated for immunogenicity by intramuscular injection in rabbits. Five 4-6 week old rabbits were selected for the titer verification of each group of experiments. Blood was collected before immunization to ensure that they did not contain antigen-specific antibodies to group B meningococci. The animals were immunized once every four weeks, for a total of four injections. Venous blood was collected and serological titer was evaluated three weeks after the fourth injection. The collected serum was separated and diluted to different multiples. The titer of antibodies in the serum binding to specific antigens was measured by ELISA. The indicator bacteria carrying specific antigens were incubated with the diluted serum and then cultured, and the number of colonies grown was used to calculate the titer of serum bactericidal ability (SBA). The design of the experimental groups is shown in Table 4. The serum titer results are shown in Figure 7 As shown, the serum bactericidal effect of immunogenic composition 12 is shown in Figure 8 The results showed that all antigens in the multi-component group B meningococcal vaccine could induce binding antibodies, and animal serum had a good bactericidal effect on indicator bacteria.

[0108] Table 4: Immunogenicity validation scheme for multi-component vaccines in guinea pigs and rabbits.

[0109]

[0110]

[0111] The present disclosure formulates the described antigens so that the finished multi-component vaccine can induce high titer levels of polyclonal antibodies that have bactericidal effects on multiple genotypes of group B meningococci.

[0112] The technical solution of the present disclosure is not limited to the above-mentioned specific embodiments, and all technical variations made according to the technical solution of the present disclosure fall within the protection scope of the present disclosure.

Claims

1. A vaccine composition comprising an antigenic component, wherein the antigenic component comprises a factor H binding protein (fHbp), a recombinant outer membrane vesicle (OMV) carrying porin A (PorA protein), and an optional heparin binding protein (NHBA protein).

2. The vaccine composition according to claim 1, characterized in that The factor H binding protein includes factor H binding protein variant 1 (fHbp-v1 variant protein), factor H binding protein variant 2 (fHbp-v2 variant protein) and / or factor H binding protein variant 3 (fHbp-v3 variant protein); Preferably, the fHbp-v1 variant protein has the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence shown in SEQ ID NO: 1; Preferably, the fHbp-v2 variant protein has the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 2, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence shown in SEQ ID NO: 2; Preferably, the fHbp-v3 variant protein has the amino acid sequence shown in SEQ ID NO:3, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:3, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence shown in SEQ ID NO:

3.

3. The vaccine composition according to claim 1, characterized in that The NHBA protein has an amino acid sequence shown in at least one of SEQ ID NOs:4-6, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence shown in at least one of SEQ ID NOs:4-6, or has a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence shown in at least one of SEQ ID NOs:4-6.

4. The vaccine composition according to claim 1, characterized in that The PorA protein has a variable region 1 (VR1) and a variable region 2 (VR2), The VR1 has an amino acid sequence as shown in at least one of SEQ ID NOs: 7-9, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence as shown in at least one of SEQ ID NOs: 7-9, or having a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence as shown in at least one of SEQ ID NOs: 7-9; The VR2 has an amino acid sequence as shown in at least one of SEQ ID NOs: 10-12, or an amino acid sequence having at least 85% sequence identity with the amino acid sequence as shown in at least one of SEQ ID NOs: 10-12, or an amino acid sequence having a substitution, deletion or insertion of one or more amino acid residues with the amino acid sequence as shown in at least one of SEQ ID NOs: 10-12; Preferably, the PorA protein includes a first PorA protein having VR1 shown in SEQ ID NO:7 and VR2 shown in SEQ ID NO:10, a second PorA protein having VR1 shown in SEQ ID NO:8 and VR2 shown in SEQ ID NO:11, and a third PorA protein having VR1 shown in SEQ ID NO:9 and VR2 shown in SEQ ID NO:

12.

5. The vaccine composition according to claim 1, characterized in that The fHbp protein in the antigen component is a lipidated fHbp protein, and / or the NHBA protein is a lipidated NHBA protein, Preferably, the N-terminus of the lipidated fHbp protein and / or the lipidated NHBA protein has a lipidated signal peptide. More preferably, the lipidation signal peptide has the amino acid sequence shown in SEQ ID NO:13, or has one or more amino acid residues substituted, deleted or inserted with the amino acid sequence shown in SEQ ID NO:

13.

6. The vaccine composition according to claim 1, characterized in that In the vaccine composition, the weight ratio of the fHbp protein, NHBA protein and OMV is (1-5): (0-2): (0.25-1), preferably (1-5): (0.5-2): (0.25-1); Preferably, in the fHbp protein, the weight ratio of fHbp-v1 variant protein, fHbp-v2 variant protein and fHbp-v3 variant protein is 1:(0-2):(0-2); More preferably, in the vaccine composition, the ratio between fHbp-v1 variant protein, fHbp-v2 variant protein, fHbp-v3 variant protein, NHBA protein and OMV is 1:(0-2):(0-2):(0.5-2):(0.25-1), wherein the weight ratio of the first PorA protein, the second PorA protein and the third PorA protein carried on the OMV is 1:(0.9-1.1):(0.9-1.1); More preferably, in the vaccine composition, the ratio between fHbp-v1 variant protein, fHbp-v2 variant protein, fHbp-v3 variant protein, NHBA protein and OMV components is 1:0.5:0.5:1:0.5, wherein the weight ratio of the first PorA protein, the second PorA protein and the third PorA protein carried on the OMV is 1:(0.9-1.1):(0.9-1.1).

7. The vaccine composition according to claim 3, characterized in that The antigen component includes 10-100 μg of lipidated fHbp-v1 variant protein, 0-100 μg of lipidated fHbp-v2 variant protein, 0-100 μg of lipidated fHbp-v3 variant protein, 0-100 μg of lipidated NHBA protein and 10-100 μg of OMV vector carrying PorA protein; Preferably, the vaccine composition is a liquid preparation. More preferably, the vaccine composition of 500 μL of liquid preparation includes 10-100 μg of lipidated fHbp-v1 variant protein, 0-100 μg of lipidated fHbp-v2 variant protein, 0-100 μg of lipidated fHbp-v3 variant protein, 0-100 μg of lipidated NHBA protein and 10-100 μg of OMV vector carrying PorA protein.

8. The vaccine composition according to claim 1, characterized in that The vaccine composition further comprises a vaccinologically acceptable carrier and / or a stabilizing formulation, Preferably, the vaccinologically acceptable carrier comprises one or more of aluminum adjuvant, MF59 adjuvant, Freund's complete adjuvant, Freund's incomplete adjuvant, CpG adjuvant, QS21 adjuvant or MPLA adjuvant; Preferably, the stabilizing formulation comprises one or more of histidine, polysorbate 80 or sucrose.

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