WO2017054635A1 - 一种流感嗜血杆菌融合蛋白及其构建方法与应用 - Google Patents

一种流感嗜血杆菌融合蛋白及其构建方法与应用 Download PDF

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WO2017054635A1
WO2017054635A1 PCT/CN2016/098520 CN2016098520W WO2017054635A1 WO 2017054635 A1 WO2017054635 A1 WO 2017054635A1 CN 2016098520 W CN2016098520 W CN 2016098520W WO 2017054635 A1 WO2017054635 A1 WO 2017054635A1
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hin47
hid
protein
linker
haemophilus influenzae
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PCT/CN2016/098520
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French (fr)
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李军强
苗丹青
杨鸣鸣
邵忠琦
朱涛
宇学峰
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天津康希诺生物技术有限公司
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Priority to ES16850259T priority Critical patent/ES2784428T3/es
Priority to EP16850259.9A priority patent/EP3357933B1/en
Priority to CA3000549A priority patent/CA3000549C/en
Publication of WO2017054635A1 publication Critical patent/WO2017054635A1/zh
Priority to US15/940,871 priority patent/US10716847B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/102—Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the technical field of vaccine product development, in particular to a Haemophilus influenzae fusion protein and a construction method and application thereof.
  • Haemophilus influenzae (Hi) is a kind of non-motivated, spore-free, rod-shaped Gram-negative bacilli that reside in the respiratory tract of normal people and are conditional pathogens, mainly causing upper respiratory tract infections in children, otitis media and Pneumonia can also cause serious infections such as sepsis and meningitis.
  • Haemophilus influenzae is divided into capsular type (dividable type) and non-capsulated type (non-separable type).
  • the capsular type Haemophilus influenzae can be divided into a, b, c, d according to the composition of capsular polysaccharide. , e, f a total of 6 serotypes.
  • the non-capsular type is also called Nontypeable Haemophilus influenza (NTHI).
  • Haemophilus influenzae invasive diseases occur all over the world.
  • Haemophilus influenzae type b (Hib) is the most toxic, and is the main pathogen of bacterial meningitis in children under 5 years of age. With the worldwide spread of Hib conjugate vaccines, the incidence of Haemophilus influenzae type b has decreased dramatically.
  • the Hib conjugate vaccine was developed based on Haemophilus influenzae type b capsular polysaccharide.
  • Capsular polysaccharide is the main antigenic substance of Haemophilus influenzae capsular bacterium, which can induce protective immune response in human body, but capsular polysaccharide is a thymus-independent antigen, although it can produce weak IgM antibody but does not produce immune memory.
  • Vaccine developers bind capsular polysaccharides to protein carriers to form polysaccharide protein-binding vaccines, which convert polysaccharide antigens from thymus-independent antigens to thymus-dependent antigens, thereby activating T-helper lymphocytes and promoting B-cell-specific IgG production.
  • Antibodies which make Hib conjugate vaccines widely available.
  • the Hib conjugate vaccine is only effective against Haemophilus influenzae type B and cannot prevent other Haemophilus influenzae and non-typeable Haemophilus influenzae, which leads to infection with other Haemophilus influenzae and non-typeable Haemophilus influenzae.
  • non-typeable Haemophilus influenzae can cause otitis media (OM), and the non-type of Haemophilus influenzae Egyptian biota causes epidemic conjunctivitis and Brazilian purple fever.
  • OM otitis media
  • D protein is a lipoprotein (HiD) on the surface of Haemophilus influenzae with a relative molecular weight of 42 kDa.
  • the D protein is highly conserved among all Haemophilus influenzae strains, including both capsular and non-capsulated Haemophilus influenzae. It is a specific antigen of human immunoglobulin D. Although D protein can effectively prevent otitis media caused by NTHi, HiD has a relatively small molecular weight and weak immunogenicity.
  • Hin47 also known as HtrA is a heat shock protein expressed by Haemophilus influenzae under ambient pressure conditions with serine protease activity. Hin47 is an important immune antigen. It has been shown to stimulate the body to produce B cell and T cell responses, and its immunogenicity has been fully confirmed.
  • the technical problem to be solved by the present invention is to provide a fusion protein protein Hin47-HiD fusion protein.
  • Another technical problem to be solved by the present invention is to provide a method for constructing the above fusion protein.
  • Another technical problem to be solved by the present invention is to provide the use of the above fusion protein, and to develop a universal Haemophilus influenzae conjugate vaccine using Hin47-HiD as a protein carrier for Hia and Hib conjugate vaccines.
  • the technical solution of the present invention is:
  • a Haemophilus influenzae fusion protein which is a fusion of Haemophilus influenzae HiD protein and Haemophilus influenzae Hin47 protein, that is, Hin47-HiD
  • the amino acid sequence of Hin47 is the sequence shown in the sequence ⁇ 400>1
  • the amino acid of HiD The sequence is the sequence shown in Sequence Listing ⁇ 400>3.
  • the Haemophilus influenzae fusion protein is a fusion of a Haemophilus influenzae HiD protein mutant molecule and/or a Haemophilus influenzae Hin47 protein mutant molecule, and the mutant molecule has an identity of 80% or more with the wild type molecule.
  • the mutant molecule can maintain no less than 70% of the immunogenicity of the wild type protein.
  • the Haemophilus influenzae fusion protein described above the fusion gene expression system includes, without limitation, a prokaryotic expression system, a eukaryotic expression system, and a yeast expression system.
  • the Haemophilus influenzae fusion protein, the Haemophilus influenzae HiD protein gene and the Haemophilus influenzae Hin47 protein gene include, but are not limited to, wild type and mutant.
  • the Haemophilus influenzae fusion protein described above is formed by fusing a linker in a 1:1 manner, and the linker maintains the Hin47 protein and the HiD protein monomer in their own unique conformation.
  • the Haemophilus influenzae fusion protein described above including but not limited to G4S, DL or EL, maintains the Hin47 protein and the HiD protein monomer in their own unique conformation.
  • the Haemophilus influenzae fusion protein is the G4S, and the sequence is the sequence shown in the sequence ⁇ 400>2.
  • the Haemophilus influenzae fusion protein has a composition form of (n) Hin47-linker-(n)HiD, (n)HiD-linker-(n)Hin47, -linker-HiD-(Hin47 -linker-HiD)n-linker-Hin47-, -linker-Hin47-(HiD-linker-Hin47)n-linker-HiD-, where n is 1-3, representing the number of molecules of the monomer gene.
  • the Haemophilus influenzae fusion protein has a composition form of -linker-(Hin47-linker-HiD-linker)n-Hin47-, -linker-(HiD-linker-Hin47-linker)n- HiD-, where n is 1-3, represents the number of molecules of the monomeric gene.
  • the Haemophilus influenzae fusion protein, Hin47-HiD as a conjugate vaccine protein carrier, can effectively increase the immunogenicity of the polysaccharide antigen.
  • the Haemophilus influenzae fusion protein is used as a conjugate vaccine protein carrier in the development of a conjugate vaccine using a polysaccharide antigen as an active ingredient, and a polysaccharide protein conjugate vaccine developed as a protein carrier, It is not limited to Haemophilus influenzae, pneumonia, meningitis, dysentery, typhoid, and the like.
  • a broad-spectrum H. influenzae polysaccharide protein-binding vaccine preparation using one or more capsular polysaccharides of Haemophilus influenzae type a, b, c, d, e, f as an antigen, and Hin47- HiD is a protein carrier.
  • the above broad-spectrum Haemophilus influenzae polysaccharide protein-binding vaccine preparation the polysaccharide antigen component in the preparation is effective for preventing the corresponding serotype of Haemophilus influenzae, and the protein carrier component can prevent non-type of Haemophilus influenzae.
  • the above broad spectrum of Haemophilus influenzae polysaccharide protein conjugate vaccine formulation is a bivalent conjugate vaccine comprising a conjugate of Hia and Hin47-HiD protein carrier, a conjugate of Hib and Hin47-HiD protein carrier.
  • the above broad-spectrum Haemophilus influenzae polysaccharide protein-binding vaccine preparation is a liquid injection or a lyophilized powder injection, each dose containing a type-type polysaccharide having a content of 8-15 ⁇ g, a type B polysaccharide The content is 8-15 ⁇ g, and the Hin47-HiD content is 20-80 ⁇ g.
  • the broad-spectrum Haemophilus influenzae polysaccharide protein-binding vaccine preparation is a liquid injection, and the content of the a-type polysaccharide is 0.5-15 ml per dose, and the content of the b-type polysaccharide is It is 8-15 ⁇ g and the Hin47-HiD content is 20-80 ⁇ g.
  • the bivalent conjugate vaccine does not contain a preservative, may contain an adjuvant including not limited to aluminum phosphate, and the buffer in the preparation is PB and NaCl. The final concentration was 0.45% NaCl + 10 mM PB.
  • the method for constructing the above Haemophilus influenzae fusion protein is as follows:
  • the fusion gene includes a molecule of Hin47 gene and a molecule of HiD gene, and the anterior segment is Hin47, and the anterior segment is HiD; the designed cleavage site is preceded by NdeI, followed by BamHI;
  • the fusion gene was digested with NdeI and BamHI, and ligated with the T-vector ligated with the same endonuclease, and ligated with T4 DNA ligase; after successful ligation, DH5a was transformed, and the plasmid was extracted by PCR and double digestion.
  • the fusion gene was digested with NdeI and BamHI from the T clone, and ligated with pET9a digested with the same endonuclease. After successful ligation, the BL21 competent state was transformed, and then the expression vector was verified by double enzyme digestion and PCR.
  • the positive monoclonal colonies were picked and cultured in LB medium, and the precipitate was collected by centrifugation.
  • the precipitate was precipitated in PBS, and after ultrasonication, the supernatant was subjected to SDS-PAGE electrophoresis.
  • the method for constructing the Haemophilus influenzae fusion protein described above is as follows:
  • the Life technology synthesizes the Hin47-HiD fusion gene, and the selected linker is G4S (GGGGS).
  • the fusion gene includes one molecule of Hin47 gene and one molecule of HiD gene, and the anterior segment is Hin47 and the back end is HiD.
  • the cleavage site is preceded by NdeI, followed by BamHI, wherein the amino acid sequence of Hin47 is the sequence shown in the sequence ⁇ 400>1, the amino acid sequence of Linker is the sequence shown in the sequence ⁇ 400>2, and the amino acid of HiD
  • the sequence is the sequence shown in the sequence ⁇ 400>3, and it can be seen that Hin47-HiD contains 790 aa and the molecular weight is 87 KD;
  • the fusion gene was digested with NdeI and BamHI, and ligated with the T-vector ligated with the same endonuclease, and ligated with T4 DNA ligase; after successful ligation, DH5a was transformed, and the plasmid was extracted by PCR and double digestion.
  • the fusion gene was digested with NdeI and BamHI from the T clone, and ligated with pET9a digested with the same endonuclease. After successful ligation, the BL21 competent state was transformed, and then the expression vector was verified by double enzyme digestion and PCR.
  • Hin47-HiD fusion protein expression vector and the techniques used in the induction of expression are routine experimental manipulation methods known to those skilled in the art, and these methods can be found in published literature and books.
  • the above method for purifying the Hin47-HiD fusion protein has the following specific steps:
  • the bacterial fermentation is carried out by conventional test methods. These experimental methods can be found in the published literature, or the test methods are known to those skilled in the art; the frozen culture liquid is inoculated into the LB medium, and the bacteria are shaken at 37 ° C and 250 rpm. After overnight, the resuscitation was carried out; the resuscitation bacterial solution was transferred to a 2 L flask containing 1 L of medium for amplification culture, and when the OD 600 was 0.6-0.7, the final concentration was 1 mM IPTG, and after induction for 4 hours, the bacteria were collected by centrifugation at 8000 rpm for 30 minutes. Body precipitation
  • the collected bacteria were collected by centrifugation, reconstituted in PBS, sonicated, and the supernatant was collected by centrifugation.
  • the supernatant was sonicated and the Q column was collected, and the flow-through solution was collected (the target protein flowed through); the Q-column flow was passed through the CHT column, and then the target protein was eluted with 150 mM PB + 170 mM NaCl, and 175 mM PB + 1 M NaCl.
  • the target protein with a purity of more than 80% can be obtained for the development of the subsequent conjugate vaccine.
  • Hia, Hib bivalent conjugate vaccine preparation method the specific steps are as follows:
  • Polysaccharide antigens are prepared by common test methods, including Haemophilus influenzae fermentation culture, CTAB or ethanol fractionation and precipitation crude, CL-4B gel separation to further recover capsular polysaccharide antigen, dialysis lyophilization;
  • Hia conjugate vaccine After adding a type capsular polysaccharide to CDAP (which is cyano-4-dimethylamino-pyridine tetrafluoroboric acid) for 30 s, 0.2M TEA was added to adjust the pH to 9.5 for polysaccharide activation, and the activation lasted for 2.5 min; activation After the end, adjust the pH to 9.0, then add the carrier protein according to the glycoprotein ratio of 1:1, react at room temperature for 1h, overnight at 4°C, then add 2M glycine to terminate the reaction; after dialysis removes the added reagent, CL-4B condenses Separating and purifying the conjugate; or
  • Hib conjugate vaccine After adding b-type capsular polysaccharide to CDAP30s, 0.2M TEA was added to adjust the pH to 9.5 for polysaccharide activation, and the activation lasted for 2.5 min; after the end of activation, the pH was adjusted to 9.0, followed by a glycoprotein ratio of 1:1.
  • the carrier protein was added, reacted at room temperature for 1 h, overnight at 4 ° C, and then the reaction was terminated by adding 2 M glycine; after dialysis to remove the added reagent, the conjugate was separated and purified by CL-4B gel.
  • the recommended finished product ingredients for the test are Hia, Hib conjugate 10ug (polysaccharide content), 0.45% NaCl, 10 mM PB per dose; the finished preparation can ensure sufficient immunity for the immunized population.
  • the above Hia and Hib bivalent conjugate vaccine, 1/4 dose (125 ⁇ l) immunized animals can produce anti-Hia capsular polysaccharide and Hib Antibody response to capsular polysaccharides, and antibody responses to carrier proteins.
  • the anti-Hia capsular polysaccharide antibody can effectively prevent Haemophilus influenzae type A
  • the antibody against Hib capsular polysaccharide can effectively prevent Haemophilus influenzae type b
  • the anti-carrier protein (Hin47, HiD) antibody can effectively prevent non-invasive Haemophilus influenzae.
  • Hin47-HiD fusion protein can effectively enhance the immunogenicity of Hin47 and HiD single protein antigens; not only that, Hin47-HiD as a conjugate vaccine protein carrier can effectively enhance the immunogenicity of polysaccharide antigen; the fusion protein Hin47-HiD is used as a binding Vaccine protein carrier, developed Hia conjugate vaccine, Hib conjugate vaccine, and bivalent vaccine containing Hia conjugate vaccine and Hib conjugate vaccine.
  • Figure 1 is a fusion protein expression assay
  • Figure 2 is a purification diagram of the Hin47-HiD fusion protein
  • Figure 3 is a comparison of immunogenicity between Hin47 and HiD
  • Figure 4 is a comparison of the immunogenicity of Hin47 and Hin47-HiD against Hin47 antigen.
  • the immunization dose of Hin47 monomer in Hin47-HiD fusion protein is the same as that of the control group, and L, M and H are respectively expressed in Hin47-HiD fusion protein.
  • the Hin47 monomer antigen dose (including the control group Hin47) was 1 ⁇ g, 5 ⁇ g and 10 ⁇ g, respectively;
  • Figure 5 is a comparison of the immunogenicity of HiD and Hin47 against HiD, wherein the immune dose of HiD monomer in Hin47-HiD fusion protein is the same as that of the control HiD antigen, and L, M and H respectively represent HiD monomer in Hin47-HiD fusion protein.
  • the antigen dose (including the control group HiD) was 1 ⁇ g, 5 ⁇ g and 10 ⁇ g, respectively;
  • Figure 6 is a diagram showing the separation and purification of the Hia conjugate
  • Figure 7 is a diagram showing the separation and purification of Hib conjugates.
  • the Life technology synthesizes the Hin47-HiD fusion gene, and the selected linker is G4S (GGGGS).
  • the fusion gene includes one molecule of Hin47 and one molecule of HiD, and the anterior segment is Hin47 and the back end is HiD.
  • the position is NdeI in front and BamHI in the latter, wherein the amino acid sequence of Hin47 is the sequence shown in the sequence ⁇ 400>1, the amino acid sequence of Linker is the sequence shown in the sequence ⁇ 400>2, and the amino acid sequence of HiD is The sequence shown in the sequence table ⁇ 400>3 is shown in Table 1 below.
  • Hin47-HiD contains 790 aa and has a molecular weight of 87 KD.
  • the fusion gene was digested with NdeI and BamHI, and ligated with the T-vector ligated with the same endonuclease, and ligated with T4 DNA ligase; after successful ligation, DH5a was transformed, and the plasmid was extracted by PCR and double digestion. Identification.
  • the fusion gene was digested with NdeI and BamHI from the T clone, and ligated with pET9a digested with the same endonuclease. After successful ligation, the BL21 competent state was transformed, and then the expression vector was identified by double enzyme digestion and PCR.
  • Figure 1 illustrates that the fusion protein is expressed in the supernatant and the expression level is high.
  • Hin47-HiD fusion protein expression vector and the techniques used in inducing expression are routinely tested by those skilled in the art and can be found in published literature and books.
  • the PBS reconstituted precipitate was sonicated, and the supernatant was collected by centrifugation; the supernatant was passed through a Q column to collect a flow through (target protein flow through), and then the flow of the Q column was passed through a CHT column and washed with 150 mM PB + 170 mM NaCl.
  • the target protein with a purity of more than 90% can be collected.
  • Hin47-HiD fusion protein was used to immunize BALB/C mice, and the immunogenicity was studied. At the same time, Hin47 and HiD were used as controls, immunized three times, two weeks apart, and the whole blood was collected 14 days after the last time to detect the antibody level in serum.
  • the immunization doses of the Hin47 and HiD protein antigens in the control group were 1 ⁇ g, 5 ⁇ g, and 10 ⁇ g, while the effective doses of the protein monomers in the immunized group of the fusion protein were 1 ⁇ g, 5 ⁇ g, and 10 ⁇ g.
  • Hin47 and HiD antigens were immunized to mice at three doses of 1 ⁇ g, 5 ⁇ g and 10 ⁇ g, and the results of the antibody titer of the three immunos with the respective antigens are shown in Fig. 3.
  • the fusion protein Hin47-HiD was used for immunogenicity studies in three doses, and the actual immunization dose of the monomeric protein in the fusion protein was identical to that of the control antigen (Hin47 or HiD), in which the immunogenicity of Hin47-HiD and Hin47 was compared, L
  • the fusion protein Hin47-HiD was used for immunogenicity studies in three doses, and the actual immunization dose of the monomeric protein in the fusion protein was identical to that of the control antigen (Hin47 or HiD), in which the Hin47-HiD and HiD immunogenicity comparison test, L
  • the three groups of M and H indicate that the dose of HiD monomer antigen in the fusion protein was 1 ⁇ g, 5 ⁇ g and 10 ⁇ g.
  • the HIA polysaccharide and Hib polysaccharide protein conjugate were prepared by the same test method. Specifically, after the capsular polysaccharide was added to CDAP 30s, 0.2MTEA was added to adjust the pH to 9.5 for polysaccharide activation, and the activation was continued for 2.5 min. After the end of the activation, the pH was adjusted to 9.0, and then the Hin47-HiD fusion protein was added according to the glycoprotein ratio of 1:1, and reacted at room temperature for 1 hour, overnight at 4 ° C, and then the reaction was terminated by adding 2 M glycine. After dialysis to remove the added reagent, the conjugate was isolated and purified by CL-4B gel.
  • Example 4 Production of Hia, Hib bivalent conjugate vaccine, ie, influenza A haemogenide conjugate vaccine
  • the finished component contained Hia, Hib conjugate 10 ug (polysaccharide content), 0.45% NaCl, 10 mM PB per dose.
  • a, b-type Haemophilus influenzae conjugate vaccine both with Hin47-HiD as a carrier protein
  • the Bacillus conjugate vaccine immunized 10 12-14 g BALB/C mice at a dose of 1/4 person (each containing Hia polysaccharide and Hib polysaccharide 2.5 ⁇ g), and immunized 10 BALB/C mice with NS as a negative control, immunizing 2 times, 2 weeks apart, and 14 days after the last immunization, the eyes were harvested for serum collection.
  • the anti-a and b-type capsular polysaccharide antibody titers in the serum were detected by ELISA, and the serum seroconversion rate of the mice in the vaccine group was calculated by using the serum of the NS-immunized mice as the CUT off value.
  • the results are shown in Table 3.
  • the type A and type B Haemophilus influenzae conjugate vaccine is used to immunize mice at a dose of 1/4, and the seroconversion rate of the second sera is 100%, which meets the release standard of the Chinese Pharmacopoeia for the immunological efficacy of the Hib conjugate vaccine product.
  • Example 6 Protective study of Haemophilus influenzae conjugate vaccines of type a and b
  • the specific antibody content in serum can be effectively determined by ELISA, but not all antibodies with antigen binding ability have bactericidal protection function, and serum bactericidal antibody (SBA) can reflect the functional activity of the antibody, thereby directly reflecting the protective effect of the vaccine.
  • SBA serum bactericidal antibody
  • the combination of a and b type Haemophilus influenzae conjugate vaccine, the SBA of the blood of the immunized mouse is as high as 1300, which can effectively prevent Haemophilus influenzae type A and Haemophilus influenzae type b.
  • the Haemophilus influenzae type b conjugate vaccine after immunizing mice, simultaneously produced an effective antibody against the carrier protein, wherein the antibody titer against the HiD antigen was 5.65, while the antibody titer against Hin47 was as high as 6.34, these antibodies are effective in preventing non-invasive Haemophilus influenzae.
  • the protective results indicate that the antibodies against H. and Hab polysaccharides can effectively prevent Haemophilus influenzae type A and Haemophilus influenzae type b after immunizing mice with a combination of influenza A and b.
  • the antibody against the carrier protein Hin47-HiD is effective for preventing non-invasive Haemophilus influenzae.
  • the application principle of the present invention is as follows:
  • the polysaccharide protein-binding vaccine can promote the thymus-independent polysaccharide antigen to become thymus-dependent, thereby making the polysaccharide antigen suitable for immunoprevention of a group such as infants and young children.
  • the first-generation conjugate vaccine protein carrier is tetanus toxoid TT and diphtheria toxoid DT
  • the second-generation conjugate vaccine protein carrier is a non-toxic diphtheria toxin mutant CRM protein;
  • the invention is developed by a new generation of conjugate vaccine protein carrier,
  • the feature is that in addition to effectively improving the immunogenicity of the polysaccharide antigen, the antigen against the carrier protein itself can also resist certain diseases.
  • Hin47 and HiD have been shown to prevent non-invasive Haemophilus influenzae, and the Hin47-HiD fusion protein developed by the present invention can significantly increase the immunogenicity of the weak antigen HiD without affecting the immunogenicity of Hin47, and more importantly, The Hin47-HiD fusion protein is effective as a conjugate vaccine protein carrier. Therefore, the present invention A novel conjugated vaccine protein vector has been developed, and the conjugate vaccine developed as a vector can prevent non-invasive Haemophilus influenzae, in addition to preventing bacterial infection corresponding to the polysaccharide antigen.
  • Haemophilus influenzae fusion protein and its construction method and application in the above reference examples are illustrative and not limiting, and several examples can be enumerated according to the limited scope, and therefore Variations and modifications that are within the spirit of the invention are intended to be within the scope of the invention.

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Abstract

提供一种融合蛋白及其构建方法，所述融合蛋白由流感嗜血杆菌D蛋白和Hin47（Htra）蛋白组成，该融合蛋白作为流感嗜血杆菌多糖蛋白结合疫苗的蛋白载体，可增强多糖抗原的免疫原性。

Description

一种流感嗜血杆菌融合蛋白及其构建方法与应用 技术领域
本发明涉及疫苗产品开发技术领域，尤其是一种流感嗜血杆菌融合蛋白及其构建方法与应用。
发明背景
流感嗜血杆菌(Haemophilusinfluenza，Hi)是一类无动力、无芽孢，呈杆状的革兰氏阴性杆菌，寄居于正常人的呼吸道，属条件致病菌，主要引起儿童上呼吸道感染，中耳炎和肺炎，亦可引起败血症、脑膜炎等严重感染。
流感嗜血杆菌分为荚膜型(可分型)和无荚膜型(不可分型)，荚膜型的流感嗜血杆菌根据荚膜多糖的成分不同又可分为a、b、c、d、e、f共6种血清型。无荚膜型又称之为不可分型流感嗜血杆菌(Nontypeable Haemophilus influenza,NTHI)。
流感嗜血杆菌侵入性疾病在全世界均有发生。b型流感嗜血杆菌(Hib)的毒性最强，是5岁以下儿童细菌性脑膜炎的主要致病菌。随着Hib结合疫苗在全球范围的推广使用，b型流感嗜血杆菌的发病率大幅下降。Hib结合疫苗是基于b型流感嗜血杆菌荚膜多糖开发的。荚膜多糖是荚膜型流感嗜血杆菌的主要抗原物质，能在人体中诱发保护力免疫反应，但是荚膜多糖为胸腺非依赖性抗原，虽可产生较弱的IgM抗体但是不产生免疫记忆，不能对2岁以内婴幼儿产生有效保护。疫苗开发者将荚膜多糖结合到蛋白载体上形成多糖蛋白结合疫苗，蛋白载体使多糖抗原由胸腺非依赖性抗原变为胸腺依赖性抗原，从而激活T辅助淋巴细胞，促使B细胞产生特异性IgG抗体，这使得Hib结合疫苗得到广泛应用。然而Hib结合疫苗仅对B型流感嗜血杆菌有效，不能对其它型流感嗜血杆菌及不可分型流感嗜血杆菌产生预防作用，这导致其它型流感嗜血杆菌及不可分型流感嗜血杆菌的感染率大幅上升。例如，不可分型流感嗜血杆菌可导致中耳炎(OM)，不分型流感嗜血杆菌埃及生物群引起流行性结膜炎和巴西紫热。全球尚没有批准的用于预防b型以外的其它型流感嗜血杆菌或不可分型嗜血杆菌的疫苗。
D蛋白是流感嗜血杆菌表面的一种脂蛋白(HiD)，相对分子量为42KDa，D蛋白在所有流感嗜血杆菌菌株(包括有荚膜和无荚膜的流感嗜血杆菌)中高度保守，是人免疫球蛋白D的特异性抗原。尽管D蛋白可有效预防NTHi引起的中耳炎，但HiD相对分子量较小，其免疫原性较弱。
Hin47(又称HtrA)是流感嗜血杆菌在环境压力条件下表达的热休克蛋白，具丝氨酸蛋白酶活性。Hin47是重要的免疫抗原，实验证明其可刺激机体产生B细胞和T细胞反应，其免疫原性已得到充分证实。
发明内容
本发明所要解决的技术问题在于提供一种结合疫苗蛋白载体Hin47-HiD融合蛋白。
本发明所要解决的另一技术问题在于提供上述融合蛋白的构建方法。
本发明所要解决的另一技术问题在于提供上述融合蛋白的应用，以Hin47-HiD作为Hia和Hib结合疫苗的蛋白载体，开发通用性流感嗜血杆菌结合疫苗。
为解决上述技术问题，本发明的技术方案是：
一种流感嗜血杆菌融合蛋白，为流感嗜血杆菌HiD蛋白和流感嗜血杆菌Hin47蛋白融合而成，即Hin47-HiD，Hin47的氨基酸序列为序列表<400>1所示序列，HiD的氨基酸序列为序列表<400>3所示序列。
优选的，上述流感嗜血杆菌融合蛋白，为流感嗜血杆菌HiD蛋白突变分子和/或流感嗜血杆菌Hin47蛋白突变分子融合而成，突变分子与野生型分子具有80％及以上的相同性，突变分子可以维持不低于野生型蛋白70％的免疫原性。
优选的，上述流感嗜血杆菌融合蛋白，所述融合基因表达系统包括不限于原核表达系统、真核表达系统以及酵母表达系统。
优选的，上述流感嗜血杆菌融合蛋白，所述流感嗜血杆菌HiD蛋白基因和流感嗜血杆菌Hin47蛋白基因(组成融合蛋白的单体蛋白)包括但不限于野生型和突变体两种。
优选的，上述流感嗜血杆菌融合蛋白，所述Hin47-HiD由以1：1方式和linker融合而成，所述linker使Hin47蛋白和HiD蛋白单体维持自身特有的构象。
优选的，上述流感嗜血杆菌融合蛋白，所述linker包括但不限于G4S、DL或EL，所述linker使Hin47蛋白和HiD蛋白单体维持自身特有的构象。
优选的，上述流感嗜血杆菌融合蛋白，所述linker为G4S，序列为序列表<400>2所示序列。
优选的，上述流感嗜血杆菌融合蛋白，所述融合基因的组成形式为(n)Hin47-linker-(n)HiD，(n)HiD-linker-(n)Hin47，-linker-HiD-(Hin47-linker-HiD)n-linker-Hin47-，-linker-Hin47-(HiD-linker-Hin47)n-linker-HiD-，其中n为1-3、代表该单体基因的分子数。
优选的，上述流感嗜血杆菌融合蛋白，所述融合基因的组成形式为-linker-(Hin47-linker-HiD-linker)n-Hin47-，-linker-(HiD-linker-Hin47-linker)n-HiD-，其中n为1-3、代表该单体基因的分子数。
优选的，上述流感嗜血杆菌融合蛋白，Hin47-HiD，作为结合疫苗蛋白载体，可有效提高多糖抗原的免疫原性。
优选的，上述流感嗜血杆菌融合蛋白，Hin47-HiD，作为结合疫苗蛋白载体，在以多糖抗原作为有效成分的结合疫苗开发中进行应用，以其作为蛋白载体开发的多糖蛋白结合疫苗，包 括不限于流感嗜血杆菌、肺炎、脑膜炎、痢疾、伤寒等。
一种广谱的流感嗜血杆菌多糖蛋白结合疫苗制剂，该疫苗制剂以a、b、c、d、e、f型流感嗜血杆菌中一种或多种荚膜多糖为抗原，以Hin47-HiD为蛋白载体。
优选的，上述广谱的流感嗜血杆菌多糖蛋白结合疫苗制剂，制剂中的多糖抗原成分可有效预防相应血清型的流感嗜血杆菌，蛋白载体成分可以预防非分型流感嗜血杆菌。
优选的，上述广谱的流感嗜血杆菌多糖蛋白结合疫苗制剂，该制剂为包含Hia与Hin47-HiD蛋白载体的偶联物、Hib与Hin47-HiD蛋白载体的偶联物的二价结合疫苗。
优选的，上述广谱的流感嗜血杆菌多糖蛋白结合疫苗制剂，所述二价结合疫苗剂型为液体针剂或冻干粉针剂，每剂含有a型多糖的含量为8-15μg，b型多糖的含量为8-15μg，Hin47-HiD含量在20-80μg。
优选的，上述广谱的流感嗜血杆菌多糖蛋白结合疫苗制剂，所述二价结合疫苗剂型为液体针剂，每剂以0.5ml计含有a型多糖的含量为8-15μg，b型多糖的含量为8-15μg，Hin47-HiD含量在20-80μg。
优选的，上述广谱的流感嗜血杆菌多糖蛋白结合疫苗制剂，所述二价结合疫苗不含有防腐剂，可以含有包括不限于磷酸铝在内的佐剂，制剂中的缓冲液为PB和NaCl，终浓度为0.45％NaCl+10mM PB。
上述流感嗜血杆菌融合蛋白的构建方法，具体步骤如下：
(1)基因合成：
融合基因各包括一分子的Hin47基因和一分子的HiD基因，且其前段为Hin47，后端为HiD；所设计的酶切位点，前面为NdeI，后为BamHI；
(2)T克隆构建：
融合基因经NdeI和BamHI双酶切后，同以相同内切酶双酶切的T载体进行连接，连接采用T4DNA连接酶；成功连接后转化DH5a感受态，抽提质粒，以PCR和双酶切进行鉴定；
(3)表达载体构建：
从T克隆中以NdeI和BamHI双酶切得到融合基因，同以相同内切酶双酶切的pET9a进行连接，成功连接后转变BL21感受态，后以双酶切和PCR对表达载体进行检定；
(4)表达检定：
挑取阳性单克隆菌落LB培养基培养并离心收集沉淀，PBS复溶沉淀，超声破碎后，上清液进行SDS-PAGE电泳。
优选的，上述流感嗜血杆菌融合蛋白的构建方法，具体步骤如下：
(1)基因合成：
Life technology公司合成Hin47-HiD融合基因，其中选择的linker为G4S(GGGGS)，融合基因各包括一分子的Hin47基因和一分子的HiD基因，且其前段为Hin47，后端为HiD；所设计的酶切位点，前面为NdeI，后为BamHI，其中，所述Hin47的氨基酸序列为序列表<400>1所示序列，Linker的氨基酸序列为序列表<400>2所示序列，HiD的氨基酸序列为序列表<400>3所示序列，由此可以看出，Hin47-HiD含有790aa，分子量为87KD；
Hin47-HiD融合蛋白氨基酸序列
Figure PCTCN2016098520-appb-000001
(2)T克隆构建：
融合基因经NdeI和BamHI双酶切后，同以相同内切酶双酶切的T载体进行连接，连接采用T4DNA连接酶；成功连接后转化DH5a感受态，抽提质粒，以PCR和双酶切进行鉴定；
(3)表达载体构建：
从T克隆中以NdeI和BamHI双酶切得到融合基因，同以相同内切酶双酶切的pET9a进行连接，成功连接后转变BL21感受态，后以双酶切和PCR对表达载体进行检定；
(4)表达检定：
挑取阳性单克隆菌落，转接LB培养基，37℃、200rpm摇菌培养过夜，后转接50ml新鲜LB培养基中，待OD600＝0.6-0.7时，加入终浓度为1mM的IPTG，37℃诱导4h后离心收集沉淀；PBS复溶沉淀，超声破碎后，上清液进行SDS-PAGE电泳，结果表明，构建的表达载体经IPTG诱导，成功高表达目标蛋白。
需要说明的是，上述Hin47-HiD融合蛋白表达载体构建以及诱导表达过程中所使用的技术为本领域技术人员所掌握的常规试验操作方法，这些方法在公开的文献和书籍中均可查到。
上述Hin47-HiD融合蛋白纯化方法，具体步骤如下：
(1)菌体发酵
菌体发酵采用常规的试验方法，这些实验方法在公开的文献中均可以找到，或者为本领域内技术人员所掌握的试验方法；冻存菌液接种LB培养基中，37℃、250rpm摇菌过夜，进行复苏；复苏菌液转接至含有1L培养基的2L三角瓶中进行放大培养，待OD600＝0.6-0.7时，加入终浓度为1mM IPTG，诱导4h后，8000rpm离心30分钟收集菌体沉淀；
(2)蛋白纯化
发酵结束，离心收集的菌体沉淀，PBS复溶后，超声破碎，离心收集上清。超声破碎上清过Q柱，收集流传液(目的蛋白流穿)；将Q柱流穿液过CHT柱，此后用150mM PB+170mM NaCl洗杂蛋白，175mM PB+1M NaCl洗脱目标蛋白。
通过两步柱纯化，即可得到纯度在80％以上的目标蛋白，用于后续结合疫苗的开发。
Hia,Hib二价结合疫苗的制备方法，具体步骤如下：
(1)荚膜多糖的纯化和检定
多糖抗原采用常见的试验方法制备，具体包括流感嗜血杆菌发酵培养，CTAB或者乙醇分级沉淀粗纯，CL-4B凝胶分离进一步回收荚膜多糖抗原，透析冻干；
a型和b型流感嗜血杆菌荚膜多糖的纯化及检定方法，在公开的文献中均可以找到。
(2)Hia，Hib结合原液的制备
Hia结合疫苗的制备：a型荚膜多糖加入CDAP(是氰基-4-二甲氨基-吡啶四氟硼酸)30s后，加入0.2M TEA调节pH到9.5进行多糖活化，活化持续2.5min；活化结束后调pH到9.0，后按照糖蛋白比1:1的方式加入载体蛋白，室温反应1h，4℃过夜，后加入2M甘氨酸终止反应即可；透析去除加入的反应试剂后，CL-4B凝胶分离纯化结合物；或
Hib结合疫苗的制备：b型荚膜多糖加入CDAP30s后，加入0.2M TEA调节pH到9.5进行多糖活化，活化持续2.5min；活化结束后调pH到9.0，后按照糖蛋白比1:1的方式加入载体蛋白，室温反应1h，4℃过夜，后加入2M甘氨酸终止反应即可；透析去除加入的反应试剂后，CL-4B凝胶分离纯化结合物。
(3)Hia，Hib两价结合疫苗的制备
试验推荐的成品制剂成分为每剂含有Hia，Hib结合物10ug(多糖含量)、0.45％NaCl、10mM PB；该成品制剂可以保证免疫人群提供足够强的保护力。
根据成品中单个抗原的含量，以及结合物原液的浓度，计算结合物原液的需要量；按照规定取适当的结合物原液，加入PB和NaCl，然后补充WFI，使得终浓度中Hia，Hib结合物20ug/ml(多糖含量)、0.9％NaCl、20mM PB；分装即可。
(4)Hia，Hib两价结合疫苗的免疫原性研究
上述Hia和Hib二价结合疫苗，1/4剂量(125μl)免疫动物后可产生抗Hia荚膜多糖和Hib 荚膜多糖的抗体反应，以及针对载体蛋白的抗体反应。其中抗Hia荚膜多糖的抗体可有效预防a型流感嗜血杆菌，抗Hib荚膜多糖的抗体可有效预防b型流感嗜血杆菌，抗载体蛋白(Hin47，HiD)的抗体可有效预防非侵袭性流感嗜血杆菌。
本发明的有益效果：
Hin47-HiD融合蛋白可有效的增强Hin47和HiD单个蛋白抗原的免疫原性；不仅如此，Hin47-HiD作为结合疫苗蛋白载体，可有效增强多糖抗原的免疫原性；以融合蛋白Hin47-HiD作为结合疫苗蛋白载体，开发的Hia结合疫苗，Hib结合疫苗，以及包含Hia结合疫苗和Hib结合疫苗的二价疫苗。
附图简要说明
图1是融合蛋白表达检定；
图2是Hin47-HiD融合蛋白纯化图；
图3是Hin47和HiD免疫原性比较；
图4是Hin47和Hin47-HiD抗Hin47抗原免疫原性比较，其中，Hin47-HiD融合蛋白中Hin47单体的免疫剂量和对照组Hin47抗原相同，L、M、H分别表示Hin47-HiD融合蛋白中Hin47单体抗原剂量(包括对照组Hin47)分别为1μg、5μg和10μg；
图5是HiD和Hin47抗HiD免疫原性比较，其中，Hin47-HiD融合蛋白中HiD单体的免疫剂量和对照组HiD抗原相同，L、M、H分别表示Hin47-HiD融合蛋白中HiD单体抗原剂量(包括对照组HiD)分别为1μg、5μg和10μg；
图6是Hia结合物分离纯化图；
图7是Hib结合物分离纯化图。
实施本发明的方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。
实施例1：Hin47-HiD融合蛋白制备
(1)Hin47-HiD表达载体构建
Life technology公司合成Hin47-HiD融合基因，其中选择的linker为G4S(GGGGS)，融合基因各包括一分子的Hin47和一分子的HiD，且其前段为Hin47，后端为HiD；所设计的酶切位点，前面为NdeI，后为BamHI，其中，所述Hin47的氨基酸序列为序列表<400>1所示序列，Linker的氨基酸序列为序列表<400>2所示序列，HiD的氨基酸序列为序列表<400>3所示序列，具体见下表1。
表1 Hin47-HiD融合蛋白氨基酸序列
Figure PCTCN2016098520-appb-000002
Figure PCTCN2016098520-appb-000003
从表1中可以看出Hin47-HiD含有790aa，分子量为87KD。
融合基因经NdeI和BamHI双酶切后，同以相同内切酶双酶切的T载体进行连接，连接采用T4DNA连接酶；成功连接后转化DH5a感受态，抽提质粒，以PCR和双酶切进行鉴定。
从T克隆中以NdeI和BamHI双酶切得到融合基因，同以相同内切酶双酶切的pET9a进行连接，成功连接后转变BL21感受态，后以双酶切和PCR对表达载体进行检定。
挑取阳性单克隆菌落，转接LB培养基，37℃、200rpm摇菌培养过夜，后转接50ml新鲜LB培养基中，待OD600＝0.6-0.7时，加入终浓度为1mM的IPTG，37℃诱导4h后离心收集沉淀。PBS复溶沉淀，超声破碎后，上清液进行SDS-PAGE电泳，分析蛋白是否表达，结果见图1：
图1说明融合蛋白为上清表达，且表达量较高。
需要说明的是，Hin47-HiD融合蛋白表达载体构建以及诱导表达过程中所使用的技术为本领域技术人员所掌握的常规试验操作方法，这些方法在公开的文献和书籍中均可查到。
(2)Hin47-HiD融合蛋白纯化
Hin47-HiD冻存种子过夜复苏后，转接发酵培养基，待OD600＝0.8-1.0时加入IPTG，终浓度为1mM，37℃诱导4h后离心收集沉淀。
将PBS复溶的沉淀超声破碎，离心收集上清；上清过Q柱收集流穿液(目标蛋白流穿)，然后将Q柱的流穿液过CHT柱，用150mM PB+170mM NaCl洗杂蛋白，175mM PB+1M NaCl洗脱即可得到目标蛋白；本纯化工艺的优势为步骤简单，使用常规填料，仅通过两步就可以得到纯度在80％以上的目标蛋白，结果见图2。
从图2中可以看出，经过两步柱纯化，则可收集到纯度在90％以上的目标蛋白。
实施例2：Hin47-HiD融合蛋白免疫原性研究
Hin47-HiD融合蛋白免疫BALB/C小鼠，研究其免疫原性，同时以Hin47和HiD作为对照，免疫三次，间隔两周，最后一次免后14天采集全血，检测血清中的抗体水平。
对照组Hin47和HiD蛋白抗原的免疫剂量为1μg、5μg和10μg，而融合蛋白的免疫组，蛋白单体的有效剂量则为1μg、5μg和10μg。
Hin47和HiD抗原以1μg、5μg和10μg三个剂量免疫小鼠，三免血清分别以相应的抗原检测抗体效价结果见图3。
统计分析：
P(Hin47 1μg：HiD 1μg)＝0.000＜0.01
P(Hin47 5μg：HiD 5μg)＝0.000＜0.01
P(Hin47 10μg：HiD 10μg)＝0.000＜0.01
结果表明：同等剂量，Hin47免疫原性强于HiD。
融合蛋白Hin47-HiD以三个剂量进行免疫原性研究，且融合蛋白中单体蛋白的实际免疫剂量和对照抗原(Hin47或者HiD)相同，其中Hin47-HiD和Hin47免疫原性比较试验中，L、M和H三组表示融合蛋白中Hin47单体抗原的剂量为1μg、5μg和10μg。
Hin47与Hin47-HiD免疫原性比较见图4，
统计分析：
P(L组Hin47：Hin47-HiD)＝0.2820＞0.05
P(M组Hin47：Hin47-HiD)＝0.2820＞0.05
P(H组Hin47：Hin47-HiD)＝0.350＞0.05
结果表明：Hin47-HiD融合蛋白并没有影响Hin47单体的免疫原性。
融合蛋白Hin47-HiD以三个剂量进行免疫原性研究，且融合蛋白中单体蛋白的实际免疫剂量和对照抗原(Hin47或者HiD)相同，其中Hin47-HiD和HiD免疫原性比较试验中，L、M和H三组表示融合蛋白中HiD单体抗原的剂量为1μg、5μg和10μg。
HiD与Hin47-HiD免疫原性比较见图5，
经统计分析：
P(L组HiD：Hin47-HiD)＝0.000＜0.01
P(M组HiD：Hin47-HiD)＝0.000＜0.01
P(H组HiD：Hin47-HiD)＝0.000＜0.01
结果表明：Hin47-HiD融合蛋白可有效增强HiD免疫原性。
实施例3：Hia和Hib结合原液的制备
Hia多糖和Hib多糖蛋白结合物的制备采用相同的试验方法，具体为荚膜多糖加入CDAP30s后，加入0.2MTEA调节pH到9.5进行多糖活化，活化持续2.5min。活化结束后调pH到9.0，后按照糖蛋白比1:1的方式加入Hin47-HiD融合蛋白，室温反应1h，4℃过夜，然后加入2M甘氨酸终止反应即可。透析去除加入的反应试剂后，CL-4B凝胶分离纯化结合物。
Hia结合物CL-4B凝胶分离结果见图6。
Hib结合物CL-4B凝胶分离结果见图7。
表2 Hia，Hib两价结合疫苗的质检结果
Figure PCTCN2016098520-appb-000004
实施例4：Hia，Hib两价结合疫苗即a、b型流感嗜血杆菌结合疫苗成品的生产
以生产200只疫苗为例进行说明，200只疫苗理论上需要配置100ml半成品，按照结合物原液的浓度，需要的Hia结合原液的0.57ml，需要的Hib结合物原液的体积为0.72ml，需要1M的PB 1ml，需要NS 50ml，然后补充WFI 47.7ml，即可得到半成品，以0.5ml/dose的方式进行分装，即可得到成品。
成品组分为每剂含有Hia，Hib结合物10ug(多糖含量)、0.45％NaCl、10mM PB。
实施例5：a、b型流感嗜血杆菌结合疫苗的免疫原性研究
a、b型流感嗜血杆菌结合疫苗(均以Hin47-HiD作为载体蛋白)以中国药典b型流感嗜血杆菌结合疫苗效力试验的方法来评价其免疫原性，具体为a、b型流感嗜血杆菌结合疫苗以1/4人用剂量(各含Hia多糖和Hib多糖2.5μg)免疫10只12-14g BALB/C小鼠，同时以NS作为阴性对照免疫10只BALB/C小鼠，免疫2次，间隔2周，最后一次免疫后14天摘眼球采集血清。
ELISA检测血清中抗a、b型荚膜多糖抗体滴度，以NS免疫小鼠血清作为CUT off值，计算疫苗组小鼠血清阳转率，结果如表3。
表3 a、b流感嗜血杆菌结合疫苗阳转率测定结果
Figure PCTCN2016098520-appb-000005
结果表明：以Hin47-HiD融合蛋白作为蛋白载体可有效激发机体产生对多糖抗原的免疫反 应。本实施例中，a、b型流感嗜血杆菌结合疫苗以1/4剂量免疫小鼠，二免血清阳转率为100％，达到中国药典对Hib结合疫苗产品免疫效力的放行标准。
实施例6：a、b型流感嗜血杆菌结合疫苗保护性研究
通过ELISA可以有效测定血清中的特异性抗体含量，但是并非所有具有抗原结合能力的抗体均具有杀菌保护功能，血清杀菌抗体(SBA)能够反映抗体的功能活性，从而直接反映疫苗的保护作用。
a、b型流感嗜血杆菌结合疫苗，免疫小鼠血清中SBA测定结果如表4。
表4 a、b型流感嗜血杆菌结合疫苗SBA测定结果
Figure PCTCN2016098520-appb-000006
本实施例中a、b型流感嗜血杆菌结合疫苗，免疫鼠血中SBA高达1300，可有效预防a型流感嗜血杆菌和b型流感嗜血杆菌。
表5 a、b型流感嗜血杆菌结合疫苗抗载体蛋白抗体滴度
Figure PCTCN2016098520-appb-000007
从表5中可以看出，a、b型流感嗜血杆菌结合疫苗，免疫小鼠后，同时产生针对载体蛋白的有效抗体，其中针对HiD抗原的抗体滴度为5.65，而针对Hin47的则高达6.34，这些抗体可有效预防非侵袭性流感嗜血杆菌。
保护性结果表明：a、b型流感嗜血杆菌结合疫苗，免疫小鼠后，抗Hia和Hib多糖的抗体可有效预防a型流感嗜血杆菌和b型流感嗜血杆菌。而针对载体蛋白Hin47-HiD的抗体可有效预防非侵袭性流感嗜血杆菌。
本发明的应用原理如下：多糖蛋白结合疫苗可促使胸腺非依赖性的多糖抗原变为胸腺依赖性，从而使多糖抗原适用于婴幼儿等群体的免疫预防。第一代结合疫苗蛋白载体为破伤风类毒素TT和白喉类毒素DT，第二代结合疫苗蛋白载体为无毒的白喉毒素突变体CRM等蛋白；本发明研发的属于新一代结合疫苗蛋白载体，特点是除可有效提高多糖抗原的免疫原性外，针对载体蛋白本身的抗原也可以抵御某种疾病。Hin47和HiD已经被证明可预防非侵袭性流感嗜血杆菌，而本发明开发的Hin47-HiD融合蛋白可明显提高弱抗原HiD的免疫原性而不影响Hin47的免疫原性，更为关键的是Hin47-HiD融合蛋白可有效作为结合疫苗蛋白载体。因此，本发明 开发了一个新型的结合疫苗蛋白载体，以此作为载体开发的结合疫苗除可预防多糖抗原相对应的细菌感染外，针对载体本身的抗体可预防非侵袭性流感嗜血杆菌。
上述参照实施例对该一种流感嗜血杆菌融合蛋白及其构建方法与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
Figure PCTCN2016098520-appb-000008
Figure PCTCN2016098520-appb-000009
Figure PCTCN2016098520-appb-000010

Claims (11)

  1. 一种流感嗜血杆菌融合蛋白，其特征在于：为流感嗜血杆菌HiD蛋白和流感嗜血杆菌Hin47蛋白融合而成，即Hin47-HiD，Hin47的氨基酸序列为序列表<400>1所示序列，HiD的氨基酸序列为序列表<400>3所示序列。
  2. 如权利要求1所述的融合蛋白，其特征在于：所述的融合蛋白为流感嗜血杆菌HiD蛋白突变分子和/或流感嗜血杆菌Hin47蛋白突变分子融合而成，突变分子与野生型分子具有80％及以上的相同性。
  3. 如权利要求1所述的融合蛋白，其特征在于：所述的融合蛋白由Hin47和HiD以1：1方式和linker融合而成，所述linker使Hin47蛋白和HiD蛋白单体维持自身特有的构象。
  4. 如权利要求1或2所述的融合蛋白，其特征在于：所述融合基因的组成形式为(n)Hin47-linker-(n)HiD，(n)HiD-linker-(n)Hin47，-linker-HiD-(Hin47-linker-HiD)n-linker-Hin47-，-linker-Hin47-(HiD-linker-Hin47)n-linker-HiD-，其中n为1-3、代表该单体基因的分子数。
  5. 如权利要求1或2所述的融合蛋白，其特征在于：所述融合基因的组成形式为-linker-(Hin47-linker-HiD-linker)n-Hin47-，-linker-(HiD-linker-Hin47-linker)n-HiD-，其中n为1-3、代表该单体基因的分子数。
  6. 一种流感嗜血杆菌多糖蛋白结合疫苗制剂，其特征在于：所述疫苗制剂以a、b、c、d、e、f型流感嗜血杆菌中一种或多种荚膜多糖为抗原，以权利要求1所述的融合蛋白为蛋白载体。
  7. 如权利要求6所述的疫苗制剂，其特征在于：所述制剂中的多糖抗原成分可有效预防相应血清型的流感嗜血杆菌，蛋白载体成分可以预防非分型流感嗜血杆菌。
  8. 如权利要求6所述的疫苗制剂，其特征在于：所述疫苗制剂为包含Hia与Hin47-HiD蛋白载体的偶联物、Hib与Hin47-HiD蛋白载体的偶联物的二价结合疫苗。
  9. 如权利要求8所述的疫苗制剂，其特征在于：所述二价结合疫苗剂型为液体针剂或冻干粉针剂，每剂含有a型多糖的含量为8-15μg，b型多糖的含量为8-15μg，Hin47-HiD含量在20-80μg。
  10. 如权利要求8所述的疫苗制剂，其特征在于：所述二价结合疫苗剂型为液体针剂，每剂以0.5ml计含有a型多糖的含量为8-15μg，b型多糖的含量为8-15μg，Hin47-HiD含量为20-80μg。
  11. 如权利要求10所述的疫苗制剂，其特征在于：所述二价结合疫苗不含有防腐剂，制剂中的缓冲液为PB和NaCl，终浓度为0.45％NaCl+10mM PB。
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EP3357933A4 (en) 2018-08-29
CN105175549A (zh) 2015-12-23
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