Group B meningococcus vaccine composition as well as preparation method and application thereof

Abstract

The invention discloses a group B meningococcus vaccine composition as well as a preparation method and application thereof. The vaccine composition comprises a factor H binding protein (fHbp) fusion protein and an outer membrane vesicle (OMV) wherein the factor H binding protein fusion protein comprises a meningococcal heparin binding protein antigen (NHBA) or a variant thereof, and an operably linked fHbp or a variant thereof. The vaccine composition can induce an organism to generate a functional antibody with bactericidal activity, has bactericidal activity on three variant strains, can induce the organism to generate higher protective response, and improves the bactericidal coverage rate of the vaccine on domestic B group strains.

Description

Technical Field

[0001] This application relates to the field of biological products, and particularly to a group B meningococcal vaccine composition, a preparation method thereof, and an application thereof. Background Art

[0002] Epidemic cerebrospinal meningitis is a respiratory infectious disease mainly caused by meningococcal infection, featuring cerebrospinal meningitis and bacteremia. The vast majority of such diseases are caused by serogroups A / B / C / W / X / Y, and polysaccharide conjugate vaccines against serogroups A / C / W135 / Y have been widely used. However, due to the weak immunogenicity of the capsular polysaccharide of group B meningococcus and the cross-reaction between the epitopes of this polysaccharide and sialylproteins in human tissues, the components of group B meningococcal vaccines mainly include outer membrane vesicles (OMVs) and outer membrane proteins.

[0003] Since the 1970s, OMVs (outer membrane vesicles) based on the porin ProA of group B meningococcus have been widely studied and developed. However, this vaccine has an important limitation, that is, it is only specifically effective against meningitis diseases caused by the same genotype ProA and cannot provide broader protection against other numerous MenB strains with different genotypes of ProA. Therefore, such vaccines are more suitable for controlling the outbreak of endemic diseases.

[0004] Currently, two group B meningococcal vaccines (Bexsero™ and Trumenba®) have been marketed. Bexsero™ is a vaccine prepared by combining three recombinant antigens, namely factor H-binding protein (fHbp), Neisseria adhesin A (NadA), Neisseria heparin-binding protein (NHBA), and OMVs. Trumenba® is a bivalent recombinant lipidated fHbp vaccine. Both vaccines have shown good immune protection in the vaccinated populations of corresponding ages. However, the fHbp contained in the components of both vaccines is the V1 subvariant, which is different from the prevalent V1 strains and lacks broad cross-immunoreactivity against the mainly prevalent V2 strains. Therefore, it is particularly important to develop a group B meningococcal vaccine against prevalent strains. However, the vaccines developed by referring to the combination methods of these two marketed vaccines cannot provide good bactericidal activity and coverage against prevalent strains. Moreover, the inherent instability of V2 strains and their sensitivity to proteolysis further increase the difficulty of developing group B meningococcal vaccines. Summary of the Invention

[0005] Based on this, it is necessary to provide a group B meningococcal vaccine composition, a preparation method thereof, and an application thereof.

[0006] A first aspect of the present application provides a group B meningococcal vaccine composition, the vaccine composition comprising a factor H-binding protein (fHbp) fusion protein and outer membrane vesicles (OMV), wherein the factor H-binding protein fusion protein comprises a meningococcal heparin-binding protein antigen (NHBA) or a variant thereof, and an operably linked fHbp or a variant thereof.

[0007] In some embodiments, the factor H-binding protein fusion protein comprises the NHBA variant and the operably linked fHbp variant.

[0008] In some embodiments, the factor H-binding protein fusion protein sequentially comprises the NHBA variant, a linker, and the fHbp variant from the N-terminus to the C-terminus.

[0009] In some embodiments, the amino acid sequence of the factor H-binding protein fusion protein is as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0010] In some embodiments, the mass ratio of the factor H-binding protein fusion protein to the outer membrane vesicles is (2-15):1.

[0011] In some embodiments, the factor H-binding protein fusion protein comprises one or more of the fusion protein with the amino acid sequence as shown in SEQ ID NO:1 and the fusion protein with the amino acid sequence as shown in SEQ ID NO: 2.

[0012] In some embodiments, the outer membrane vesicles are from group B meningococcus.

[0013] In some embodiments, the vaccine composition further comprises an adjuvant; optionally, the adjuvant is selected from one or more of aluminum adjuvant, Freund's adjuvant, calcium phosphate, polynucleotide, muramyl peptide, saponin, RIBI adjuvant system, cholera toxin, polymers of acrylic acid or methacrylic acid, oil-in-water emulsion, and water-in-oil emulsion.

[0014] In some embodiments, the vaccine composition further comprises a buffer; optionally, the buffer is selected from one or more of histidine buffer, phosphate buffer, and the pH of the buffer is 6.5-7.5.

[0015] A second aspect of the present application provides a method for preparing the vaccine composition described in the first aspect of the present application, comprising: mixing the factor H-binding protein fusion protein and the outer membrane vesicles to prepare the vaccine composition.

[0016] In some embodiments, the step of obtaining the outer membrane vesicles comprises the following steps:

[0017] Culture Neisseria meningitidis group B, heat-inactivate the cultured Neisseria meningitidis group B bacterial solution, and centrifuge to collect the supernatant;

[0018] Concentrate and diafilter the supernatant, and replace it into a sucrose solution;

[0019] Wherein the temperature of the heat inactivation is 50°C to 55°C, and the time of the heat inactivation is 2h to 2.5h.

[0020] The third aspect of the present application provides the use of the vaccine composition described in the first aspect of the present application in the preparation of a medicament for preventing or treating cerebrospinal meningitis.

[0021] The Neisseria meningitidis group B vaccine composition provided in the embodiments of the present application can induce the body to produce functional antibodies with bactericidal activity, has bactericidal activity against 3 variant strains, can induce the body to produce a higher protective response, and improve the bactericidal coverage rate of the vaccine against domestic Neisseria meningitidis group B strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments and implementations of the present application and to more fully understand the present application and its beneficial effects, the following will briefly introduce the drawings required for the description of the embodiments or implementations. Obviously, the following drawings are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative efforts.

[0023] Figure 1 SDS-PAGE identification and HPLC identification of the purified fusion protein NF1 in an embodiment of the present application;

[0024] Figure 2 SDS-PAGE identification and HPLC identification of the purified fusion protein NF2 in an embodiment of the present application;

[0025] Figure 3 OMV size and morphology identification in an embodiment of the present application, where in the DLS graph, "size" represents the particle size and "intensity" represents the scattered light intensity;

[0026] Figure 4 OMV proteomics identification in an embodiment of the present application;

[0027] Figure 5 Specific antibody titer detection in an embodiment of the present application. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0028] To facilitate the understanding of the present application, the present application will be described more comprehensively below with reference to the relevant accompanying drawings. Preferred embodiments of the present application are shown in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the description of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0030] In this application, "optionally", "optional", and "option" mean that it is optional, that is, it refers to any one of the two parallel options of "having" or "not having". If the word "optional" appears multiple times in a technical solution, without special instructions and without contradictions or mutual restrictions, each "optional" is independent of each other.

[0031] In this application, "preferred", "better", "more preferable", and "preferably" are only used to describe embodiments or examples with better effects, and it should be understood that they do not constitute a limitation on the protection scope of this application.

[0032] The terms "have", "contain", "include", and "comprise" used in this application are synonyms, which are inclusive or open-ended and do not exclude additional, unrecited members or features. Members or features include, for example, materials or components, structures, elements, instruments, etc.; non-limiting examples of members or features also include actions, conditions for the occurrence of actions, timing, states, etc.

[0033] In this application, in a technical feature or technical solution described in an open language, a closed technical feature or technical solution composed of the listed content is included, and an open technical feature or technical solution including the listed content is also included.

[0034] In this application, for the unit of the data range, if only the unit is attached after the right endpoint, it means that the units of the left endpoint and the right endpoint are the same.

[0035] In this application, where a method flow involves multiple steps, unless there are explicit different descriptions herein, the execution of these steps has no strict order limitation, and they can be executed in an order other than the described one. Moreover, any step may include multiple sub-steps or multiple stages. These sub-steps or stages do not necessarily need to be completed at the same moment, but can be executed at different moments, and their execution order does not necessarily have to be sequential, but can be executed alternately or simultaneously with other steps or a part of the sub-steps or stages of other steps.

[0036] In this application, exemplary descriptions such as "in some embodiments" or "in one embodiment" may cover, but are not limited to, the following meaning: These solutions can be combined with other solutions in a suitable manner to form new technical solutions.

[0037] In this application, in "the first aspect", "the second aspect", "the third aspect", etc., the terms "first", "second", "third", etc. are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", etc. only serve the purpose of non-exhaustive enumerative description and should be understood not to constitute a closed limitation on quantity.

[0038] In this application, regarding numerical intervals (i.e., numerical ranges), unless otherwise specified, the distribution of the selectable numerical values within this numerical interval is considered continuous and includes the two numerical endpoints of this numerical interval (i.e., the minimum value and the maximum value), as well as each numerical value between these two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within this numerical interval, it includes the two endpoint integers of this numerical range, as well as each integer between the two endpoints, which is equivalent to directly listing each integer. When multiple numerical ranges are provided to describe features or characteristics, these numerical ranges can be combined. In other words, unless otherwise specified, the numerical ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein. The "numerical value" in this numerical interval can be any quantitative value, such as a number, a percentage, a ratio, etc. The "numerical interval" allows for a broad inclusion of numerical interval types such as percentage intervals, ratio intervals, ratio value intervals, etc.

[0039] Currently, the vaccines developed by combining the two traditional B group meningococcal vaccines cannot provide good bactericidal activity and coverage against the prevalent V1 and V2 strains. Moreover, the V2 strain has inherent instability and its sensitivity to proteolysis, which further increases the difficulty of developing B group meningococcal vaccines.

[0040] Based on this, at least one embodiment of the present application provides a group B meningococcal vaccine composition, its preparation method and application.

[0041] In some embodiments of the present application, the immunogenic composition has three components, including the fusion protein fHbp-V1, the fusion protein fHbp-V2, and meningococcal outer membrane vesicles (OMV). The fHbp fusion protein has an amino acid sequence of NH2-A-N-L-F-B-COOH, where N is the NHBA sequence of different genotypes, L is an optional linker amino acid sequence, F is the fHbp sequence of different variants, A is an optional N-terminal amino acid sequence, and B is an optional C-terminal amino acid sequence.

[0042] In some embodiments of the present application, the fHbp fusion protein contains NHBA-V1 (NF1) and NHBA-V2 (NF2), and the specific amino acid sequences are shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively.

[0043] In some embodiments of the present application, the OMV is from the sequence group CC4821 prevalent in domestic group B meningococci, and the PorA type of the OMV is P1.20, and its amino acid sequence is shown in SEQ ID NO: 3.

[0044] In some embodiments of the present application, the OMV is obtained by natural preparation or detergent extraction.

[0045] In some embodiments of the present application, the multi-component vaccine has good immunogenicity and can induce specific antibodies produced by all of the following 4 types of antigens: A) the polypeptide composed of SEQ ID NO: 4 (V1.13), B) the polypeptide composed of SEQ ID NO: 5 (V2.16), C) the polypeptide composed of SEQ ID NO: 6 (NHBA), D) OMV.

[0046] In the first aspect of the present application, a group B meningococcal vaccine composition is provided, which includes a factor H-binding protein (fHbp) fusion protein and outer membrane vesicles (OMV), wherein the factor H-binding protein fusion protein includes meningococcal heparin-binding protein antigen (NHBA) or its variant, and fHbp or its variant operably linked.

[0047] In the present application, unless otherwise specified, the term "variant" refers to a protein obtained by mutating the wild type through one or any combination of methods including but not limited to amino acid substitution, deletion (including truncation), insertion, translocation, permutation, and modification. Taking the variant of fHbp as an example, it contains some or all of the functions of the wild type fHbp, or it at least contains the functions of the wild type fHbp.

[0048] In the present application, unless otherwise specified, the term "operably linked" refers to the functional relationship between two regions of a fusion protein, namely, the Neisseria meningitidis heparin-binding protein antigen (NHBA) or a variant thereof, and fHbp or a variant thereof; wherein the two regions are linked to produce a fusion protein.

[0049] In some embodiments, the factor H-binding protein fusion protein comprises an NHBA variant and an operably linked fHbp variant.

[0050] In some embodiments, the factor H-binding protein fusion protein sequentially comprises an NHBA variant, a linker, and an fHbp variant from the N-terminus to the C-terminus.

[0051] In some embodiments, the amino acid sequence of the linker can be (GGGGS)n, (GGGS)n, (GGS)n, (GS)n, (AS)n, (G)n or (A)n, where n is selected from 1, 2, 3, 4, 5 or 6. Further, the amino acid sequence of the linker is GGGGS.

[0052] In some embodiments, the amino acid sequence of the factor H-binding protein fusion protein is as shown in SEQ ID NO: 1 or the amino acid sequence has at least 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO: 1.

[0053] In some embodiments, the amino acid sequence of the factor H-binding protein fusion protein is as shown in SEQ ID NO: 2 or the amino acid sequence has at least 80% (such as at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) identity to SEQ ID NO: 2.

[0054] In the present application, unless otherwise specified, the term "identity" refers to the percentage of amino acid residues in a first sequence that are identical to the amino acid residues in a second sequence when aligning the amino acid sequences (introducing gaps if necessary) to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. To determine the percentage of amino acid sequence identity, the alignment can be achieved in a variety of ways within the scope of those skilled in the art, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Those skilled in the art can determine the parameters suitable for measuring the alignment, including any algorithms required to achieve the maximum alignment over the entire length of the sequences being compared.

[0055] In some embodiments, in the vaccine composition, the factor H-binding protein fusion protein comprises one or more of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 1 and a fusion protein having an amino acid sequence as shown in SEQ ID NO: 2.

[0056] In some embodiments, the mass ratio of the factor H-binding protein fusion protein to the outer membrane vesicles is (2-15):1. Without limitation, the mass ratio of the factor H-binding protein fusion protein to the outer membrane vesicles can be, but is not limited to, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or a ratio or range between any two of the above ratios.

[0057] In some embodiments, the outer membrane vesicles are from Neisseria meningitidis serogroup B.

[0058] In some embodiments, the vaccine composition further comprises an adjuvant; the adjuvant is selected from one or more of aluminum adjuvants, Freund's adjuvants, calcium phosphate, polynucleotides, muramyl peptides, saponins, RIBI adjuvant systems, cholera toxin, polymers of acrylic acid or methacrylic acid, water-in-oil emulsions and oil-in-water emulsions. Further, the adjuvant is selected from aluminum adjuvants, and still further, the adjuvant is aluminum hydroxide adjuvant.

[0059] In the present application, unless otherwise specified, "adjuvant" refers to a substance that non-specifically enhances or potentiates the immune response to an immunogenic agent in an individual subject exposed to a mixture when added to the immunogenic agent such as an antigen.

[0060] In some embodiments, the vaccine composition further comprises a buffer; further, the buffer is selected from one or more of histidine buffer and phosphate buffer, and the pH of the buffer is 6.5-7.5.

[0061] In a second aspect of the present application, there is provided a method for preparing the vaccine composition of the first aspect of the present application, which comprises: mixing the factor H-binding protein fusion protein and the outer membrane vesicles to prepare the vaccine composition.

[0062] In some embodiments, the step of obtaining the outer membrane vesicles comprises the following steps:

[0063] S10: Culturing Neisseria meningitidis serogroup B, heat-inactivating the cultured Neisseria meningitidis serogroup B bacterial solution, and centrifuging to collect the supernatant;

[0064] S20: Concentrating and washing and filtering the supernatant, and replacing it into a sucrose solution;

[0065] Wherein the temperature of heat inactivation is 50°C-55°C, and the time of heat inactivation is 2h-2.5h.

[0066] In some embodiments, the temperature of heat inactivation can be, but is not limited to, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, or a value or range between any two of the above values.

[0067] In some embodiments, the time of heat inactivation can be 2 h, 2.1 h, 2.2 h, 2.3 h, 2.4 h, 2.5 h, or a value or range between any two of the above values.

[0068] In some embodiments, the concentration of the sucrose solution is 1 w / v% - 5 w / v%. Without limitation, the concentration of the sucrose solution can be, but is not limited to, 1 w / v%, 2 w / v%, 3 w / v%, 4 w / v%, 5 w / v%, or a value or range between any two of the above values.

[0069] In the third aspect of the present application, there is provided the use of the vaccine composition of the first aspect of the present application in the preparation of a drug for preventing or treating cerebrospinal meningitis.

[0070] In the fourth aspect of the present application, there is provided a method for prevention or treatment, which includes administering an effective dose of the vaccine composition to a subject.

[0071] The diseases to be prevented or treated are usually diseases caused by Neisseria meningitidis group B, such as epidemic cerebrospinal meningitis and meningococcemia, etc.

[0072] In the present application, unless otherwise specified, the term "prevention" refers to the prevention or protective treatment of a disease (such as epidemic cerebrospinal meningitis). Prevention can include reducing the risk of infection, transmission, and / or progression, or reducing the severity of the disease.

[0073] In the present application, unless otherwise specified, the term "treatment" refers to the cure of a disorder or disease (such as epidemic cerebrospinal meningitis), the alleviation of symptoms, or the reduction in the severity of the disease or disease symptoms.

[0074] When "administering", "giving", and "treating" are applied to an animal, a human, an experimental subject, a cell, a tissue, an organ, or a biological fluid, it refers to the contact of an exogenous drug, therapeutic agent, diagnostic agent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administering", "giving", and "treating" can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. The treatment of cells includes the contact of a reagent with the cells, as well as the contact of the reagent with a fluid, where the fluid contacts the cells. "Administering", "giving", and "treating" also mean the treatment of cells in vitro and ex vivo by a reagent, a diagnostic, a binding composition, or by another cell. When "treating" is applied to a human, veterinary medicine, or a research subject, it refers to therapeutic treatment, preventive or prophylactic measures, research, and diagnostic applications.

[0075] "Effective amount" or "effective dose" refers to the amount of a drug, compound, or pharmaceutical composition necessary to achieve any one or more beneficial or desired therapeutic outcomes. For prophylactic use, beneficial or desired outcomes include eliminating or reducing risk, alleviating severity, or delaying the onset of a disease or condition, including biochemical, histological, and / or behavioral symptoms of the disease or condition, its complications, and intermediate pathological phenotypes presented during the development of the disease or condition. For therapeutic applications, beneficial or desired outcomes include clinical outcomes such as reducing the incidence of various diseases associated with the target antigens of the present application or improving one or more symptoms of the disease or condition, reducing the dose of other agents required to treat the disease or condition, enhancing the efficacy of another agent, and / or delaying the progression of the disease or condition associated with the target antigens of the present application in a patient.

[0076] Some examples are provided below.

[0077] The embodiments of the present application will be described in detail below in conjunction with the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. For the experimental methods without specified conditions in the following examples, the guidelines given in the present application are preferably referred to, and it is also possible to follow the experimental manuals or conventional conditions in the art, or the conditions recommended by the manufacturers, or refer to the experimental methods known in the art.

[0078] In the following embodiments, for the measurement parameters of the raw material components, if not otherwise specified, there may be slight deviations within the weighing accuracy range. For the temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0079] The sequences involved in the following embodiments are shown as follows:

[0080] SEQ ID NO 1:

[0081] MNIFAPEGNYRYLTYGAEKLSGGSYALSVQGEPAKGEMLAGTAVYNGEVLHFHTENGRPYPSRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGGGGSVAADIGAGLADALTAPLDHKDKGLQSLTLDQSVSKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ*

[0082] SEQ ID NO 2:

[0083] MNIFAPEGNYRYLTYGAEKLSGGSYALSVQGEPAKGEMLAGTAVYNGEVLHFHTENGRPYPSRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQDGGGGSVAADIGAGLADALTAPLDHKDKSLQSLTLDQSVSKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ*

[0084] SEQ ID NO 3:

[0085] QPQTANTQQGGKVKVTKA

[0086] SEQ ID NO 4:

[0087] VAADIGAGLADALTAPLDHKDKGLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGKLITLESGEFQVYKQSHSALTALQTEQVQDSEDSGKMVAKRQFRIGDIAGEHTSFDKLPKGGSATYRGTAFGSDDAGGKLTYTIDFAAKQGHGKIEHLKSPELNVELATAYIKPDEKRHAVISGSVLYNQDEKGSYSLGIFGGQAQEVAGSAEVETANGIHHIGLAAKQ*

[0088] SEQ ID NO 5:

[0089] VAADIGAGLADALTAPLDHKDKSLQSLTLDQSVRKNEKLKLAAQGAEKTYGNGDSLNTGKLKNDKVSRFDFIRQIEVDGQLITLESGEFQIYKQDHSAVVALQIEKINNPDKIDSLINQRSFLVSGLGGEHTAFNQLPDGKAEYHGKAFSSDDAGGKLTYTIDFAAKQGHGKIEHLKTPEQNVELAAAELKADEKSHAVILGDTRYGSEEKGTYHLALFGDRAQEIAGSATVKIGEKVHEIGIAGKQ*

[0090] SEQ ID NO 6:

[0091] MFERSVIAMACIFALSACGGGGGGSPDVKSADTLSKPAAPVVAEKETEVKEDAPQAGSQGQGAPSTQGSQDMAAVSAENTGNGGAATTDKPKNEDEGPQNDMLQNSAESANQTGNNQPADSSDSAPASNPAPANGGSNFGRVDLANGVLIDGPSQNITLTHCKGDSCNGDNLLDEEAPSKSEFENLNESERIEKYKKDGKSDKFTNLVATAVQANGTNKYVIIYKDKSASSSFARFRRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKLSGGSYALSVQGEPAKGEMLAGTAVYNGEVLHFHTENGRPYPSRGRFAAKVDFGSKSVDGIIDSGDDLHMGTQKFKAAIDGNGFKGTWTENGSGDVSGKFYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKEQD*

[0092] Example 1

[0093] 1. Preparation of fHbp fusion protein

[0094] 1) The truncated NHBA was ligated to fHbp (two variants V1 and V2) by gene fusion, and a GGGGS linker was inserted between the two proteins. Then the fusion fragment was cloned into the expression vector pET24b with a 6×his tag at the N-terminus. The recombinant plasmid of the fusion fragment was obtained by commissioning General Biology Company for gene synthesis. The specific amino acid sequences of the fusion proteins are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0095] 2) The recombinant plasmid was transformed into the Escherichia coli expression strain BL21(DE3). Single colonies were selected and transferred into a shaking tube containing LB medium with the corresponding antibiotic, and shaken overnight at 37°C and 200 rpm. The next day, it was transferred to fresh LB medium with the corresponding antibiotic and shaken at 37°C and 200 rpm until the OD600 of the bacterial solution reached 0.6 - 0.8. 1 mM IPTG (isopropyl-β-D-thiogalactoside) was added and the culture was continued at 37°C and 180 rpm for 4 h. The cells were collected by centrifugation and identified by SDS-PAGE.

[0096] 3) After two-step purification by nickel column affinity chromatography and anion exchange chromatography, fHbp fusion protein with a purity of over 90% was obtained through SDS-PAGE and HPLC identification. The results are shown in Figure 1 andFigure 2 As shown, the concentrations of the fusion proteins NF1 and NF2 measured by the Lowary method were 0.95 mg / mL and 1.02 mg / mL, respectively.

[0097] 2. Preparation of native OMV

[0098] 1) Native OMV (nOMV) consists of native outer membrane vesicles and is spontaneously released into the culture supernatant during bacterial growth. A glycerol stock of a prevalent serogroup B meningococcus strain (CC4821, PorA genotype P1.20) was streaked onto a blood agar plate and cultured at 37 °C in 5% CO2 for 16 h.

[0099] 2) A single colony was inoculated into 7 mL of Mueller-Hinton medium and cultured at 37 °C until the mid-logarithmic phase (optical density [OD] of 0.6 - 0.7). It was then transferred to 100 mL of Mueller-Hinton medium and cultured at 37 °C for 6 h until the early stationary phase (OD value of 1.0 - 1.2).

[0100] 3) The culture temperature was raised to 50 °C and sterilized at 50 °C for 2 h, and then the bacterial suspension was left at 4 °C overnight.

[0101] 4) The supernatant was collected after centrifugation of the bacterial suspension, and then filtered through a 0.45 μm filter membrane to remove residual cells and concentrated using a 100 kDa membrane package.

[0102] 5) It was washed and filtered twice with deionized water using a 300 kDa membrane package and finally exchanged into a 3% sucrose solution.

[0103] 6) Qualitative and quantitative detection of outer membrane proteins of the prepared OMV: The particle size was detected by dynamic light scattering (DLS), the morphology and size were detected by transmission electron microscopy (TEM), and the proteomics was detected by Label Free. The detection results are as Figure 3 and Figure 4 shown, indicating the successful preparation of native OMV. The total protein concentration of OMV measured by the Lowary method was 1.36 mg / mL.

[0104] Example 2: Vaccine formulation

[0105] 1) Prepared according to the human dose, the fHbp fusion protein or OMV stored in physiological saline was adsorbed onto aluminum hydroxide adjuvant, where the fHbp fusion protein was 50 - 100 μg / mL, OMV was 10 - 25 μg / mL, aluminum hydroxide was 1 mg / mL, and the buffer system was histidine buffer (pH 6.0).

[0106] 2) For the two fHbp fusion protein combined with OMV vaccines, three groups (2:1, 4:1, 8:1) were set within the range of the mass ratio of the fusion protein to OMV from 2:1 to 10:1, and the other vaccine components were the same as above.

[0107] 3) The prepared preparations were subjected to particle size identification and endotoxin content detection.

[0108] Example 3: Animal Immunization

[0109] 1) Female mice of CD1 strain at 4 - 6 weeks old were selected, with 5 mice per group, and intraperitoneal injection was carried out at 1 / 5 of the human dose. The immunization groups are shown in Table 1.

[0110] Table 1 Immunization Grouping

[0111]

[0112] 2) Immunization was carried out on the 0th day and 21st day respectively, and blood was collected on the 35th day to collect serum.

[0113] Example 4: Detection of Specific and Bactericidal Antibody Titers

[0114] 1) The specific antibody titer of the serum was detected by conventional ELISA. That is, the purified fusion protein was first used to coat a 96-well plate. After blocking, the primary antibody serum with different dilution multiples was added successively for incubation, the enzyme-labeled secondary antibody was incubated, the enzyme detection substrate was added, and the results were judged and data analyzed.

[0115] 2) The bactericidal antibody titer of the serum was detected by SBA: First, the Neisseria meningitidis serogroup B strain was spread on a blood agar plate and cultured overnight at 37°C and 5% CO2. The single colony was inoculated into Mueller-Hinton medium to control the initial OD 620 of the bacterial solution at 0.05 - 0.08, and cultured in a shaker at 37°C until the OD620 reached 0.23 - 0.24, and the bacterial viability was measured. All the mouse sera to be tested were inactivated by heating at 56°C for 30 min. The total volume in each well was 50 μL, including 25 μL of serially two-fold diluted test serum, 12.5 μL of bacterial working solution, and 12.5 μL of human complement. The controls included: serum incubated with complement serum, immune serum incubated with bacteria, and inactivated complement. After adding complement, immediately take 10 μL of the control and spread it on a Mueller-Hinton agar plate, and incubate at 37°C and 5% CO2 for 1 h. Take 7 μL of each sample and spot it on a Mueller-Hinton agar plate, and incubate at 37°C and 5% CO2 for 18 h.

[0116] The results of the specific antibody titer detection are shown in Figure 5 , indicating that the specific antibody titer induced by the fHbp fusion protein in the body reached 1:106 As described above, it exhibits good immunogenicity. After the combination of the fHbp fusion protein and OMV, the specific antibody titer induced in the body is higher and the immunogenicity is better.

[0117] The results of the bactericidal antibody titer detection are shown in Table 2, indicating that the immunization with the fHbp fusion protein composition can induce the body to produce functional antibodies with bactericidal activity (titer above 1:4), which have bactericidal activity against all three variant strains. Moreover, the combined immunization of the two fHbp fusion proteins and OMV can induce a higher protective response, with a bactericidal coverage rate of about 80%.

[0118] Table 2: Detection of bactericidal antibody titer

[0119]

[0120] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0121] The above-described embodiments merely represent several implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the appended claims, and the description and drawings can be used to explain the scope of the claims.

Claims

1. A group B meningococcal vaccine composition, characterized in that: The vaccine composition comprises a factor H binding protein (fHbp) fusion protein and an outer membrane vesicle (OMV), wherein the factor H binding protein fusion protein comprises a meningococcal heparin binding protein antigen (NHBA) or a variant thereof, and an operably linked fHbp or a variant thereof.

2. The vaccine composition according to claim 1, characterized in that The factor H binding protein fusion protein comprises the NHBA variant and the operably linked fHbp variant; Optionally, the factor H binding protein fusion protein comprises the NHBA variant, a linker, and the fHbp variant in order from the N-terminus to the C-terminus; Further optionally, the amino acid sequence of the factor H binding protein fusion protein is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

3. The vaccine composition according to claim 2, characterized in that The factor H binding protein fusion protein includes one or more of a fusion protein having an amino acid sequence as shown in SEQ ID NO: 1 and a fusion protein having an amino acid sequence as shown in SEQ ID NO:

2.

4. The vaccine composition according to claim 1, characterized in that The mass ratio of the factor H binding protein fusion protein to the outer membrane vesicle is (2-15):

1.

5. The vaccine composition according to claim 4, characterized in that The outer membrane vesicles are from serogroup B meningococci.

6. The vaccine composition according to claim 1, characterized in that The vaccine composition also includes an adjuvant; Optionally, the adjuvant is selected from one or more of aluminum adjuvant, Freund's adjuvant, calcium phosphate, polynucleotide, cell wall peptide, saponin, RIBI adjuvant system, cholera toxin, polymer of acrylic acid or methacrylic acid, water-in-oil emulsion and oil-in-water emulsion.

7. The vaccine composition according to any one of claims 1 to 6, characterized in that The vaccine composition also includes a buffer; optionally, the buffer is selected from one or more of a histidine buffer and a phosphate buffer, and the pH of the buffer is 6.5-7.

5.

8. A method for preparing the vaccine composition according to any one of claims 1 to 7, characterized in that: include: The factor H binding protein fusion protein and the outer membrane vesicles are mixed to prepare the vaccine composition.

9. The method according to claim 8, characterized in that The step of obtaining the outer membrane vesicles comprises the following steps: Cultivating group B meningococci, heat-inactivating the cultured group B meningococcal bacterial solution, and collecting the supernatant by centrifugation; The supernatant is concentrated and filtered, and replaced with a sucrose solution; The temperature of the heat inactivation is 50°C to 55°C, and the time of the heat inactivation is 2h to 2.5h.

10. Use of the vaccine composition according to any one of claims 1 to 7 in the preparation of a medicament for preventing or treating cerebrospinal meningitis.

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