Immunogenic composition for preventing neisseria meningitidis

Abstract

The composition is prepared from group B meningococcus NHBA recombinant protein, fHbp (V1-V1) recombinant protein and fHbp (V2-V3) recombinant protein obtained through artificial modification, or three corresponding recombinant proteins obtained by further adding P4 signal peptide on the basis of the group B meningococcus NHBA recombinant protein, the fHbp (V1-V1) recombinant protein and the fHbp (V2-V3) recombinant protein, and animal experiments prove that the composition has good immunogenicity and can be used for preparing the immunopotentiator. The compound can be used for preparing vaccines for preventing group B meningococcus.

Description

Technical Field

[0001] The present invention relates to the field of biological products, in particular, the present invention relates to the field of meningococcal vaccines, especially recombinant proteins and immunogenic compositions related to group B meningococci. Background Art

[0002] Neisseria meningitidis, also known as Neisseria meningitidis, meningococcus, or simply meningococcus, is a Gram-negative coccus confined to humans. It belongs to the Neisseriaceae family and is a diplococcoid. It is a major pathogen causing epidemic meningitis and sepsis worldwide. Neisseria meningitidis is a Gram-negative coccus confined to humans. It is a diplococcoid, hence its name, meningococcus, or simply meningococcus. It is a major pathogen causing epidemic meningitis and bacteremia worldwide. Meningococci can be encapsulated or non-encapsulated, and their genome consists of approximately 2 million base pairs, containing approximately 2,000 coding genes. Meningococci can be divided into at least 13 serogroups based on the structure of their capsular polysaccharides, but only infection with six of these serogroups (A, B, C, W135, X, and Y) can cause more severe disease.

[0003] Currently, polysaccharide or polysaccharide conjugate vaccines for the prevention of epidemic meningitis caused by serogroups A, C, W-135, and Y are available in most countries and regions worldwide. In July 2023, MenFive™, a pentavalent polysaccharide conjugate vaccine targeting serogroups A, C, W, X, and Y, jointly developed by the Serum Institute of India and the PATH Foundation, received a WHO priority recommendation for vaccination for individuals aged 1-85 years. Due to the difficulty in developing serogroup B meningococcal vaccines and their delayed release, they are currently only available in select European and American countries and regions. There is no vaccine available in China for serogroup B meningococci. Furthermore, with the global adoption of other meningococcal vaccines, serogroup B meningococci have become the most prevalent meningococcal serogroup globally. The relative prevalence of serogroup B meningococci in China is also increasing, making it the leading cause of meningitis. Therefore, the development of a preventive vaccine against serogroup B meningococci is urgently needed.

[0004] Compared with the marketed ACYW135 group meningococcal vaccine, the research and development path of group B meningococcal vaccine is quite different and the research and development is also more difficult. The latter is mainly developed based on the surface capsular polysaccharide of meningococci, but this development strategy is not applicable to group B meningococcal vaccine. Early studies have shown that vaccines developed based on group B meningococcal capsular polysaccharides not only have low immunogenicity and are almost difficult to induce antibody responses, but also have the risk of inducing autoimmune reactions. Currently, only two group B meningococcal vaccines have been approved for marketing in Europe and the United States, namely Bexsero from GSK and Trumenba from Pfizer. No similar products have been approved for marketing in China. Both marketed vaccines are mainly based on the recombinant protein technology platform of the Escherichia coli prokaryotic expression system and use aluminum adjuvants to enhance immune response. GlaxoSmithKline's Bexsero vaccine contains four antigenic components: OMVs (bacterial outer membrane vesicles), NadA (Neisserial adhesin A) protein, NHBA (Neisseria Heparin Binding Antigen, also known as GNA2132) protein, and fHbp (factor H-binding protein, also known as GNA1870). The fHbp protein is derived from the MC58 strain (subfamily B) (reference WO2004032958A1). Pfizer's Trumenba vaccine uses only two different variants of the fHbp protein (subfamily A and subfamily B) as antigens: the subfamily A fHbp protein is derived from the M98 250771 strain, and the subfamily B fHbp protein is derived from the CDC1573 strain (reference WO2015033251A2). Although both vaccines have been approved for marketing, their antigens are designed primarily to target serotype B meningococcal strains prevalent in Europe and the United States, and have a lower potential reactivity to serotype B meningococcal strains prevalent in China. For example, OMV vaccines offer poor broad-spectrum protection, and single OMV component vaccines are generally used primarily in localized settings. Furthermore, OMV-based vaccines offer weak immunity, and their protective efficacy is poor in younger infants and young children. Another component of the Bexsero vaccine, the NadA protein, primarily mediates the adhesion of serogroup B meningococci to nasal epithelial cells. However, molecular epidemiological analysis of serogroup B meningococci in China revealed that over 90% of isolates lack the NadA gene (Zhu Bingqing et al., Chinese Journal of Preventive Medicine, 2019). Regarding the fHbp antigen protein, scientists at Wyeth (later acquired by Pfizer) initially discovered through genetic phylogenetic tree analysis that global fHbp protein sequences can be divided into two subfamilies (or subfamilies): A and B. Subsequent sequence analysis revealed that global fHbp protein sequences can be further subdivided into three variants: V1, V2, and V3. Subfamily A includes variants V2 and V3, while subfamily B includes variant V1.Both vaccines currently on the market contain fHbp protein components, but more specifically, Bexsero contains only the V1 variant, and Trumenba contains only the V1 and V3 variants. Neither vaccine contains the V2 variant component. However, epidemiological survey data on serogroup B meningococcal disease in China between 1968 and 2000 and 2005 and 2016 showed that the V2 variant accounted for more than half of the serogroup B strains, whether isolated from patients or carried by healthy individuals. This shows that the serogroup B meningococcal vaccines currently on the market abroad are not fully suitable for the Chinese population. Therefore, it is necessary to develop a vaccine that has relatively broad strain coverage and is more suitable for the prevention of serogroup B meningococcal disease in China. Summary of the Invention

[0005] In view of the above problems, the purpose of the present invention is to provide a new immunogenic composition that is suitable for the Chinese population, has simple components, and can stimulate the body to produce a high level of antibody response against group B meningococci, as well as the use of the immunogenic composition.

[0006] First, the applicant developed new recombinant antigen proteins for group B meningococci, including NHBA antigen protein and fHbp antigen protein, specifically NHBA recombinant protein, fHbp (V1-V1) recombinant protein and fHbp (V2-V3) recombinant protein, as well as three recombinant proteins to which the P4 lipoprotein signal peptide from nontypable Haemophilus influenzae (Green BA et al., INFECTION AND IMMUNITY, Sept. 1991, p. 3191-3198, referred to herein as P4 signal peptide, the sequence of which is shown in SEQ ID NO: 7) was further added, namely P4-NHBA recombinant protein, P4-fHbp (V1-V1) recombinant protein and P4-fHbp (V2-V3) recombinant protein. For the above-mentioned recombinant proteins, the applicant has demonstrated through ELISA tests that they can fold into the correct protein structure and have obvious biological activity, and has demonstrated through animal experiments that they have good immunogenicity and can induce the body to produce a good immune response (patent application numbers: 202410035415.6, 202411713143.7, 202411711203.1).

[0007] Based on the above work, in order to produce more comprehensive and effective immune protection against group B meningococci, the present invention further prepared an immunogenic composition based on the above recombinant protein to develop a group B meningococcal vaccine covering the three subtypes of fHbp (V1, V2, and V3), which includes the following three components:

[0008] NHBA antigen protein (Component 1): It is either NHBA recombinant protein or P4-NHBA recombinant protein. The difference between them lies mainly in whether the P4 signal peptide is added, and secondly in the adjustment of individual amino acid residues. Among them, the amino acid sequence of NHBA recombinant protein is shown in SEQ ID NO: 1; the amino acid sequence of P4-NHBA recombinant protein is shown in SEQ ID NO: 2.

[0009] fHbp V1 subtype homodimer tandem recombinant protein (Component 2): It is a fusion protein obtained by covalently concatenating two fHbp protein V1 variant consensus sequences via a connecting peptide (shown in SEQ ID NO: 8), expressed as fHbp (V1-V1) or P4-fHbp (V1-V1). The difference between the two lies mainly in whether the P4 signal peptide is added, and secondly in the adjustment of individual amino acid residues; the amino acid sequence of the fHbp (V1-V1) is shown in SEQ ID NO: 3; the amino acid sequence of the P4-fHbp (V1-V1) is shown in SEQ ID NO: 4.

[0010] Heterologous dimer tandem recombinant protein of fHbp V2 and V3 subtypes (component three): It is a fusion protein obtained by covalently concatenating the consensus sequence of the fHbp protein V2 variant and the consensus sequence of the V3 variant via a connecting peptide (shown in SEQ ID NO: 8), expressed as fHbp (V2-V3) or P4-fHbp (V2-V3). The difference between the two lies mainly in whether the P4 signal peptide is added, and secondly in the adjustment of individual amino acid residues; the amino acid sequence of the fHbp (V2-V3) is shown in SEQ ID NO: 5; the amino acid sequence of the P4-fHbp (V2-V3) is shown in SEQ ID NO: 6.

[0011] Preferably, in the immunogenic composition, the mass ratio of the three components is (0.5~2.0):(0.5~2.0):(0.5~2.0); further preferably, the mass ratio of the three components is 1:1:1.

[0012] Preferably, the immunogenic composition may further comprise an adjuvant, such as an aluminum adjuvant, preferably an aluminum phosphate adjuvant or an aluminum hydroxide adjuvant, more preferably an aluminum hydroxide adjuvant; optionally, the concentration of the aluminum hydroxide adjuvant is 50-1000 μg / ml, for example 240 μg / ml.

[0013] The present invention also provides a nucleic acid combination encoding the above three groups of antigenic proteins, which comprises the following (1) to (3):

[0014] (1) a nucleic acid encoding the NHBA recombinant protein shown in SEQ ID NO: 1 (preferably comprising the nucleic acid of SEQ ID NO: 9), or a nucleic acid encoding the P4-NHBA recombinant protein shown in SEQ ID NO: 2 (preferably comprising the nucleic acid of SEQ ID NO: 10);

[0015] (2) a nucleic acid encoding the fHbp (V1-V1) recombinant protein of SEQ ID NO: 3 (preferably comprising the nucleic acid of SEQ ID NO: 11), or a nucleic acid encoding the P4-fHbp (V1-V1) recombinant protein of SEQ ID NO: 4 (preferably comprising the nucleic acid of SEQ ID NO: 12);

[0016] (3) A nucleic acid encoding the fHbp (V2-V3) recombinant protein shown in SEQ ID NO: 5 (preferably comprising the nucleic acid of SEQ ID NO: 13), or a nucleic acid encoding the P4-fHbp (V2-V3) recombinant protein shown in SEQ ID NO: 6 (preferably comprising the nucleic acid of SEQ ID NO: 14).

[0017] The nucleic acid combination can be optionally selected from: a combination of nucleic acid sequences comprising SEQ ID NOs: 9, 11, and 13, or a combination of nucleic acid sequences comprising SEQ ID NOs: 9, 12, and 14, or a combination of nucleic acid sequences comprising SEQ ID NOs: 10, 12, and 14.

[0018] The present invention also provides the use of the immunogenic composition and the nucleic acid combination in the preparation of a drug for inducing an immune response in a mammal, preventing or alleviating Neisseria meningitidis infection and / or diseases caused by Neisseria meningitidis; preferably, the Neisseria meningitidis is group B Neisseria meningitidis.

[0019] Beneficial effects of the present invention:

[0020] (1) In view of the fact that there is currently no approved preventive vaccine against group B meningococcus in China, the present invention provides a novel immunogenic composition suitable for the Chinese population that can stimulate the body to produce high-level antibody responses against group B meningococcus, and is used to prevent or alleviate infectious diseases caused by group B meningococcus, such as cerebrospinal meningitis, pneumonia, suppurative arthritis, conjunctivitis, etc.

[0021] (2) In response to the problem of low strain coverage of single-component vaccines, the new immune composition described in the present invention not only contains two types of antigenic proteins, NHBA and fHbp, which are widely expressed by group B meningococci, but also covers the three variants of fHbp, V1, V2, and V3. It not only comprehensively covers the globally prevalent strains, but is also more suitable for the epidemic prevention of group B meningococci in China. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : SDS-PAGE electrophoresis results of recombinant proteins. Left: SDS-PAGE electrophoresis results of recombinant fHbp (V1-V1) and fHbp (V2-V3) dimer proteins; Center: SDS-PAGE electrophoresis results of recombinant P4-fHbp (V1-V1) and P4-fHbp (V2-V3) dimer proteins; Right: SDS-PAGE electrophoresis results of recombinant NHBA and P4-NHBA proteins.

[0023] Figure 2 : ELISA results of recombinant proteins. Left: In vitro affinity ELISA results of recombinant fHbp (V1-V1) and P4-fHbp (V1-V1) proteins with various concentrations of complement factor H; Center: In vitro affinity ELISA results of recombinant fHbp (V2-V3) and P4-fHbp (V2-V3) proteins with various concentrations of complement factor H; Right: In vitro affinity ELISA results of recombinant NHBA and P4-NHBA proteins with various concentrations of heparin. DETAILED DESCRIPTION

[0024] Example 1: Immunogenicity studies

[0025] First, through the construction and screening of engineering strains, the large-scale expression of engineering strains, the extraction and purification of recombinant proteins, the recombinant proteins NHBA, P4-NHBA, fHbp (V1-V1), P4-fHbp (V1-V1), fHbp (V2-V3) and P4-fHbp (V2-V3) were obtained ( Figure 1 ), and then respectively by fHbp-complement H factor in vitro affinity ELISA, NHBA-heparin in vitro affinity ELISA ( Figure 2 ), verified the in vitro activity evaluation of the purified recombinant protein, proving that the recombinant protein can fold into the correct protein structure after soluble expression in E. coli and has significant biological activity, and can be further used for other applications, such as as an antigen for vaccine development. The specific amino acid or nucleic acid sequences involved in this example are as follows:

[0026] SEQ ID NO: 1: NHBA recombinant protein amino acid sequence;

[0027] SEQ ID NO: 2: P4-NHBA recombinant protein amino acid sequence;

[0028] SEQ ID NO: 3: fHbp (V1-V1) recombinant protein amino acid sequence;

[0029] SEQ ID NO: 4: P4-fHbp (V1-V1) recombinant protein amino acid sequence;

[0030] SEQ ID NO: 5: fHbp (V2-V3) recombinant protein amino acid sequence;

[0031] SEQ ID NO: 6: P4-fHbp (V2-V3) recombinant protein amino acid sequence;

[0032] SEQ ID NO: 7: P4 signal peptide amino acid sequence;

[0033] SEQ ID NO: 8: connecting peptide amino acid sequence;

[0034] SEQ ID NO: 9: DNA sequence encoding NHBA recombinant protein;

[0035] SEQ ID NO: 10: DNA sequence encoding P4-NHBA recombinant protein;

[0036] SEQ ID NO: 11: DNA sequence encoding fHbp (V1-V1) recombinant protein;

[0037] SEQ ID NO: 12: DNA sequence encoding P4-fHbp (V1-V1) recombinant protein;

[0038] SEQ ID NO: 13: DNA sequence encoding fHbp (V2-V3) recombinant protein;

[0039] SEQ ID NO: 14: DNA sequence encoding P4-fHbp (V2-V3) recombinant protein.

[0040] Based on the above recombinant proteins, compositions were formulated for use in the development of a serogroup B meningococcal vaccine. Component formulation: The purified recombinant protein components were individually adsorbed with aluminum hydroxide adjuvant and then mixed to form a three-component solution. In this example, three combinations were formulated (see Table 1); the resulting three-component immune composition solutions contained 240 μg / ml of aluminum adjuvant.

[0041] Animal Immunization: 6-8 week old female BALB / c mice were inoculated with the antigen prepared according to the previous example, with 8-10 mice per group. Each group of mice was immunized three times intramuscularly on days 0, 21, and 42 at a dose of 100 μl per mouse. Blood was collected 14 days after the final immunization, and serum was isolated for antibody titer determination.

[0042] Serum separation: Incubate mouse peripheral blood at 37°C for 1 hour, then at 4°C for 1 hour. Centrifuge at 800g for 20 minutes. Transfer the supernatant to a new 1.5ml EP tube. Protect from light and store at -20°C until use.

[0043] Specific antibody titer determination: The specific IgG titer in the immune serum was determined by ELISA method, that is, the recombinant antigen protein to be detected was coated on a 96-well enzyme-labeled plate at 1 μg / ml, blocked with milk, and then a series of gradient dilutions of mouse immune serum were added. Horseradish peroxidase-labeled goat anti-mouse IgG enzyme-labeled antibody was used as the detection antibody. After color development, the OD was read on a microplate reader (Molecule Device). 450mm -OD 570mm Light absorption value.

[0044] The OD value of 0.105 was used as the cut-off value, that is, when the OD value of the serum group at a certain dilution was ≥ 0.105, it was positive; when the OD value was < 0.105, the previous dilution was the antibody titer of the serum.

[0045] Immunogenicity study results:

[0046] Table 1 Antibody titer test results after mice were immunized with three-component immune compositions of different combinations

[0047]

[0048] The results showed that the three-component recombinant protein immune composition induced antibody responses against NHBA and the three different fHbp variant detection antigens: fHbp (V1), fHbp (V2), and fHbp (V3). This suggests that the three-antigen combination immune composition can induce a broader spectrum of antibodies, targeting all variants and potentially providing wider strain coverage.

[0049] Comparison between combination 2 and combination 1 showed that the fHbp antigen protein showed an order of magnitude higher level of antibody response after modification with the P4 signal peptide. The slight decrease in the immunogenicity of NHBA may be due to a certain degree of immune interference between NHBA and the fHbp antigen protein. The latter's significantly enhanced immunogenicity after the addition of the P4 signal peptide suppressed the antigenicity of NHBA to a certain extent, and the antibody titer decreased slightly, but there was no order of magnitude difference.

[0050] The comparison between combination 3 and combination 2 showed that the antibody response to NHBA antigen protein after adding P4 signal peptide modification increased, although the fHbp antibody response decreased slightly at the same time. The possible reason is still that there is a certain degree of immune interference between NHBA and fHbp antigen protein. However, in general, combination 3 maintained a good immune protection effect against the three variants of fHbp while improving the antigenicity of NHBA (the corresponding antibody titers detected were all above 10 5 above).

[0051] Comparison between combination three and combination one showed that after the P4 signal peptide was added to all three antigen proteins, the immunogenicity of the fHbp antigen protein was significantly stronger (increased by about 1 order of magnitude), although the immunogenicity of the NHBA antigen protein decreased (decreased by less than 1 order of magnitude).

[0052] The above experimental data demonstrate that the aforementioned immune compositions can be used to develop a novel serogroup B meningococcal vaccine. Appropriate antigen combinations can be selected based on actual needs. For example, to achieve higher NHBA antigenicity, combination 1 can be selected, where none of the three antigen proteins are modified with the P4 signal peptide. To achieve higher fHbp antigenicity, combination 2 can be selected, where only the fHbp antigen protein is modified with the P4 signal peptide. To achieve both, combination 3 can be selected, where all three antigen proteins are modified with the P4 signal peptide.

Claims

1. An immunogenic composition comprising the following three components: (1) NHBA recombinant protein or P4-NHBA recombinant protein, comprising the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2, respectively; (2) fHbp (V1-V1) recombinant protein or P4-fHbp (V1-V1) recombinant protein, comprising the amino acid sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, respectively; (3) fHbp (V2-V3) recombinant protein or P4-fHbp (V2-V3) recombinant protein, comprising the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 6, respectively.

2. The immunogenic composition according to claim 1, characterized in that The immunogenic composition comprises one of the following combinations: Combination 1: the NHBA recombinant protein, fHbp (V1-V1) recombinant protein, and fHbp (V2-V3) recombinant protein; Combination 2: the NHBA recombinant protein, P4-fHbp (V1-V1) recombinant protein, and P4-fHbp (V2-V3) recombinant protein; Combination three: the P4-NHBA recombinant protein, P4-fHbp (V1-V1) recombinant protein and P4-fHbp (V2-V3) recombinant protein.

3. The immunogenic composition according to any one of claims 1 to 2, characterized in that The mass ratio of the three components is (0.5~2.0):(0.5~2.0):(0.5~2.0).

4. The immunogenic composition according to claim 3, characterized in that The mass ratio between the three components is 1:1:

1.

5. The immunogenic composition according to any one of claims 1 to 4, characterized in that The immunogenic composition also includes an adjuvant.

6. The immunogenic composition according to any one of claims 1 to 5, characterized in that The adjuvant is an aluminum adjuvant.

7. The immunogenic composition according to any one of claims 1 to 6, characterized in that The adjuvant is an aluminum hydroxide adjuvant; preferably, the concentration of the aluminum hydroxide adjuvant in the composition is 50-1000 μg / ml, more preferably 100-500 μg / ml, and further preferably 240 μg / ml.

8. A nucleic acid combination comprising the following three groups of nucleic acids: (1) A nucleic acid encoding the NHBA recombinant protein shown in SEQ ID NO: 1, or a nucleic acid encoding the P4-NHBA recombinant protein shown in SEQ ID NO: 2; (2) A nucleic acid encoding the fHbp (V1-V1) recombinant protein shown in SEQ ID NO: 3, or a nucleic acid encoding the P4-fHbp (V1-V1) recombinant protein shown in SEQ ID NO: 4; (3) A nucleic acid encoding the fHbp (V2-V3) recombinant protein shown in SEQ ID NO: 5, or a nucleic acid encoding the P4-fHbp (V2-V3) recombinant protein shown in SEQ ID NO:

6.

9. The nucleic acid combination according to claim 8, characterized in that The nucleic acid encoding the NHBA recombinant protein shown in SEQ ID NO: 1 comprises the sequence shown in SEQ ID NO:

9.

10. The nucleic acid combination according to claim 8, characterized in that The nucleic acid encoding the P4-NHBA recombinant protein shown in SEQ ID NO: 2 comprises the sequence shown in SEQ ID NO:

10.

11. The nucleic acid combination according to claim 8, characterized in that The nucleic acid encoding the fHbp (V1-V1) recombinant protein shown in SEQ ID NO: 3 comprises the sequence shown in SEQ ID NO:

11.

12. The nucleic acid combination according to claim 8, characterized in that The nucleic acid encoding the P4-fHbp (V1-V1) recombinant protein shown in SEQ ID NO: 4 comprises the sequence shown in SEQ ID NO:

12.

13. The nucleic acid combination according to claim 8, characterized in that The nucleic acid encoding the fHbp (V2-V3) recombinant protein shown in SEQ ID NO: 5 comprises the sequence shown in SEQ ID NO:

13.

14. The nucleic acid combination according to claim 8, characterized in that The nucleic acid encoding the P4-fHbp (V2-V3) recombinant protein shown in SEQ ID NO: 6 comprises the sequence shown in SEQ ID NO:

14.

15. The nucleic acid combination according to any one of claims 8 to 14, characterized in that The nucleic acid combination includes one of the following combinations: Combination 1: A nucleic acid comprising the sequence of SEQ ID NO: 9, a nucleic acid comprising the sequence of SEQ ID NO: 11, and a nucleic acid comprising the sequence of SEQ ID NO: 13; Combination 2: a nucleic acid comprising the sequence of SEQ ID NO: 9, a nucleic acid comprising the sequence of SEQ ID NO: 12, and a nucleic acid comprising the sequence of SEQ ID NO: 14; Combination three: comprising a nucleic acid having the sequence shown in SEQ ID NO: 10, a nucleic acid having the sequence shown in SEQ ID NO: 12, and a nucleic acid having the sequence shown in SEQ ID NO:

14.

16. Use of the immunogenic composition according to any one of claims 1 to 7, and / or the nucleic acid combination according to any one of claims 8 to 15, in the preparation of a medicament for preventing or alleviating Neisseria meningitidis infection and / or a disease caused by Neisseria meningitidis; preferably, the Neisseria meningitidis is serogroup B Neisseria meningitidis.

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