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WO2022143993A1 - Vaccin contre neisseria meningitidis et son utilisation - Google Patents

Vaccin contre neisseria meningitidis et son utilisation Download PDF

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Publication number
WO2022143993A1
WO2022143993A1 PCT/CN2021/143737 CN2021143737W WO2022143993A1 WO 2022143993 A1 WO2022143993 A1 WO 2022143993A1 CN 2021143737 W CN2021143737 W CN 2021143737W WO 2022143993 A1 WO2022143993 A1 WO 2022143993A1
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Prior art keywords
fhbp
vaccine
subfamily
factor
seq
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PCT/CN2021/143737
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English (en)
Chinese (zh)
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郑宝
王洪山
祝歆
史伯谦
杨晓天
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基础治疗有限公司
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    • 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
    • A61K39/095—Neisseria
    • 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
    • A61K47/46—Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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

Definitions

  • the present invention relates to vaccines, compositions, uses and methods for preventing or treating Neisseria meningitidis infection or diseases caused by it.
  • Neisseria meningitidis can be divided into 13 serogroups according to their capsular polysaccharides, of which 6 serogroups (A, B, C, W, X , Y) can cause invasive meningococcal disease (IMD) (Harrison et al., n.d.).
  • IMD invasive meningococcal disease
  • the number of invasive disease cases worldwide is at least about 1.2 million annually, with 135,000 deaths associated with IMD (“Epidemics of meningococcal disease. African meningitis belt, 2001", 2001).
  • a tetravalent vaccine comprising a conjugate of a protein and a polysaccharide from serogroups A, C, W-135 and Y was introduced in the United States and is recommended for subjects 11 years of age and older routine use (Pace et al., 2009).
  • a more immunogenic tetravalent conjugate vaccine, and a conjugate vaccine against Haemophilus influenzae type b and meningococcal C and Y are in the pipeline for application In clinical stage development in infants (Nolan et al., 2007).
  • Serogroup B meningococcal disease accounts for more than 50% of all meningococcal disease in the United States and even higher in many European countries (Harrison et al., 2009; Trotter et al., 2007). However, there has been no widely effective vaccine for serogroup B meningococcal disease for a long time, and until recently the FDA approved GSK's BEXSERO and Pfizer's TRUMENBA.
  • meningococcal serogroup B is a homogeneous linear polymer of ⁇ -(2-8)N-acetylneuraminic acid (polysialic acid) and is an autoantigen (Finne et al., 1983); serogroup B
  • the polysaccharide is a weak immunogen even when conjugated to protein carriers (Jennings and Lugowski, 1981). To enhance immunogenicity, n-propionyl derivatization modifications of this polysaccharide have been attempted with limited success in mice but not in humans (Bruge et al., 2004; Jennings et al., 1987).
  • transferrin-binding protein West et al., 2001; Rokbi et al., 1997), opacity protein (Opc) (Perez et al., 2006; Jolley et al., 2001; Callaghan et al., 2008), GNA 2132 ( Giuliani et al., 2006; Plested and Granoff, 2008; Jacobsson et al., 2006; Welsch et al., 2003), fHBP (also known as GNA 1870 or LP2086) (Koeberling et al., 2008; Fletcher et al., 2004), FetA (iron-regulated outer membrane protein) (Thompson et al., 2003), Neisseria adhesin A (NadA, also known as GNA 1994) (Capecchi et al., 2005; Beernink et al., 2007; Comanducci et al., 2004) and other proteins
  • Protein- or polysaccharide-based subunit vaccines are known to have poor immunogenicity relative to vaccines based on inactivated or attenuated pathogens. Therefore, immune adjuvants are often used with antigens to enhance immune responses. Another approach is the conjugation of proteins to polysaccharide antigens as previously described.
  • BGs Bacterial ghosts
  • Bacterial ghosts are inanimate empty cell envelopes of bacteria, produced by the release of bacterial cytoplasm through channels in the bacterial cell envelope, which can be induced by phage phi X174 cleavage of protein E in the target protein. controlled expression in gram-negative bacteria, or in gram-positive bacteria by methods based on critical concentrations of compounds ("Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli
  • Bacterial ghosts still retain intact antigenic structures on the surface of native bacteria and can be used directly as vaccines (Langemann et al., 2010; Kudela et al., 2010; Riedmann et al., 2007). Bacterial ghosts are also good delivery vehicles for loading biomacromolecules such as antigens, drugs and DNA (Lubitz, 2001; Java et al., 2003; Mayr et al., 2005; Bengal et al., 2012). In addition, bacterial ghosts contain known innate immune stimulatory components and have the potential to act as effective adjuvants.
  • bacterial ghosts retain antigenic components on the surface of native bacteria, such as lipopolysaccharide (LPS), flagellin, peptidoglycan, and many other types of substances, which are various pattern recognition receptors , PRRs) ligands, collectively referred to as pathogen-associated molecular patterns (PAMPs) (Huter et al., 1999; Lubitz, 2001; Arabic et al., 2012).
  • LPS lipopolysaccharide
  • PRRs pattern recognition receptors
  • PAMPs pathogen-associated molecular patterns
  • TLR2 Toll-like receptor 2
  • TLR4 Toll-like receptor 4
  • the present invention provides a Neisseria meningitidis vaccine comprising a bacterial ghost and a factor H binding protein (fHBP), the fHBP comprising lipidated or non-lipidated fHBP.
  • the fHBP comprises at least one subfamily A fHBP and/or at least one subfamily B fHBP.
  • the fHBP of subfamily A is at least 83%, 84%, 85%, 86%, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.
  • the fHBP of subfamily B is at least 83%, 84%, 85%, 86%, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7% , 98.9% or 99.9% amino acid sequence identity of fHBP proteins; or 1, 2, 3 compared to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid insertions, substitutions and/or deletions of fHBP proteins.
  • the above substitutions are conservative substitutions.
  • the fHBP of subfamily A is from a serogroup B strain, eg, strains 961-5945, CDC1034, CDC2369, 870446.
  • the fHBP of subfamily B is from a serogroup A strain, eg, strain A4.
  • the fHBP of subfamily B is from a serogroup B strain, eg, strains CDC-1343, CDC-983, CDC-852.
  • the sequence of the factor H-binding protein of subfamily A is set forth in SEQ ID NO:5.
  • the sequence of the factor H-binding protein of subfamily B is set forth in SEQ ID NO:10.
  • fHBP is loaded within bacterial ghosts.
  • the vaccines of the present invention further comprise one or more adjuvants.
  • the adjuvant is a TLR9 agonist.
  • the adjuvant is loaded within bacterial ghosts.
  • the bacterial ghosts of the present invention are those of Lactobacillus acidophilus.
  • the vaccine is used to prevent or treat infection of Neisseria meningitidis serogroups A, B, C, W, X and/or Y, or a disease induced thereof.
  • the vaccine is used to prevent or treat infection of serogroups A and/or B, or disease induced thereof. In some embodiments, the vaccine is used to prevent or treat serogroup B infection or disease induced thereof. In some embodiments, the disease comprises invasive meningococcal disease (IMD). In some embodiments, the vaccine may be used in humans, poultry, livestock and/or mammals. In some embodiments, the vaccine is in a lyophilized dosage form.
  • IMD invasive meningococcal disease
  • the vaccine may be used in humans, poultry, livestock and/or mammals. In some embodiments, the vaccine is in a lyophilized dosage form.
  • the present invention provides the use of a composition comprising bacterial ghosts and factor H-binding protein (fHBP) in the preparation of a vaccine for the prevention or treatment of infection by Neisseria meningitidis or a disease caused by the same ).
  • the vaccine can be used to prevent or treat infection of serogroup B or a disease caused by it, infection of serogroup A or a disease caused by it, infection of serogroup C or a disease caused by it, infection of serogroup W or a disease caused by it Disease, infection of serogroup X or disease induced by it, and/or infection of serogroup Y or disease induced by it.
  • the disease comprises invasive meningococcal disease (IMD).
  • the fHBP is lipidated or non-lipidated fHBP.
  • the fHBP includes at least one subfamily A fHBP and at least one subfamily B fHBP.
  • the fHBP of subfamily A is at least 83%, 84%, 85%, 86% identical to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7% , 98.9% or 99.9% amino acid sequence identity of fHBP proteins; or 1, 2, 3 compared to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid insertions, substitutions and/or deletions of f
  • the factor H-binding protein (fHBP) of subfamily B is at least 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5% , 99.6%, 99.7%, 98.9% or 99.9% amino acid sequence identity of fHBP proteins; or compared to SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10, Insertion, substitution and/or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids the fHBP protein.
  • the above substitutions are conservative substitutions.
  • the fHBP of subfamily A is from a serogroup B strain, eg, strains 961-5945, CDCl034, CDC2369, 870446.
  • the fHBP of subfamily B is from a serogroup A strain, eg, strain A4.
  • the fHBP of subfamily B is from a serogroup B strain, eg, strains CDC-1343, CDC-983, CDC-852.
  • the composition further comprises one or more adjuvants.
  • the adjuvant is a TLR9 agonist.
  • the adjuvant is loaded within the bacterial ghost.
  • the bacterial ghost is a ghost of Lactobacillus acidophilus.
  • the vaccine may be used in humans, poultry, livestock and/or mammals.
  • the present invention provides a method of preventing or treating infection of Neisseria meningitidis serogroups A, B, C, W, X and/or Y, or a disease induced thereof, comprising administering to a subject an immunogenic An effective amount, a prophylactically effective amount or a therapeutically effective amount of the vaccine of the present invention; the mode of administration, the number of times and the dose are determined according to the condition of the subject.
  • the subject of the method is a human, poultry, livestock and/or mammal.
  • the present methods prevent or treat infection of Neisseria meningitidis serogroups A, B, C, W, X, and/or Y, or diseases caused by the same, including invasive meningococcus disease (IMD).
  • IMD invasive meningococcus disease
  • the subject of the method is a subject uninfected with Neisseria meningitidis serogroups A, B, C, W, X, and/or Y, but infected with Neisseria meningitidis serogroups Subjects from groups A, B, C, W, X and/or Y but not exhibiting symptoms of the disease they induce, or with Neisseria meningitidis serogroups A, B, C, W, X and/or or Y-induced disease subjects.
  • the present invention provides a method of loading fHBP and an adjuvant into bacterial ghosts, the method comprising resuspending the lyophilized bacterial ghosts with a protein solution, then freezing at about -80°C, and then lyophilizing , the protein solution contains one or more proteins to be loaded, optionally with an adjuvant.
  • the freezing time is 0.5 to 1.5 hours, preferably 0.6 to 1.4 hours, more preferably 0.7 to 1.3 hours, more preferably 0.8 to 1.2 hours, more preferably 0.9 to 1.1 hours hours, more preferably about 1 hour.
  • the freeze-drying time is 12-20 hours, preferably 13-19 hours, preferably 14-18 hours, preferably 15-17 hours.
  • the mixture of protein solution and ghosts can be incubated at 4°C prior to freezing.
  • the incubation time at 4°C is 1-3 hours, preferably 1.5-2.5 hours, preferably about 2 hours. In order to increase the loading of recombinant protein in the ghost, the above steps can be repeated.
  • the present invention significantly increases the antibody titer/titer against Neisseria meningitidis in serum by loading fHBP into bacterial ghosts.
  • the vaccine of the present invention can induce the production of antibodies against various strains expressing fHBP of subfamily A and antibodies against various strains expressing fHBP of subfamily B , provides broad-spectrum immune protection.
  • These strains include strains belonging to serogroups A, B, C, W, X or Y.
  • the examples of the present invention show that the combination of fHBP and bacterial ghosts significantly increases the serum antibody titers against fHBP of subfamily A and fHBP of subfamily B, as well as the bactericidal potency of serum against all tested strains.
  • the ghost of Lactobacillus acidophilus used in the present invention has an advantage in release kinetics, which is one of the reasons for its excellent effect of eliciting an immune response.
  • the ideal delivery vehicle cannot release the antigenic protein too quickly, and the examples of the present application show that the ghost of the present invention is engulfed by the antigen-presenting cell. Sufficient antigenic proteins can still be released during phagocytosis.
  • the vaccine of the present invention can be a freeze-dried product, so it can be transported or stored for a short period of time (within one month) at normal temperature, and does not require cold-chain transport or low-temperature storage.
  • Neisseria meningitidis (also known as “meningococcus”) is an encapsulated gram-negative bacterium that can be divided into 13 serogroups based on the polysaccharides on its capsule 6 serogroups (A, B, C, W, X, Y) cause invasive meningococcal disease (IMD), which commonly presents as meningococcal meningitis and/or meningococcemia/septicemia, less commonly, meningococcal pneumonia, septic arthritis, epiglottitis, or otitis media.
  • IMD invasive meningococcal disease
  • fHBP factor H binding protein
  • Lipidation refers to the covalent binding of lipids to proteins, which can be achieved by post-translational modification of proteins in vivo or by chemical synthesis in vitro.
  • Native fHBP usually contains an N-terminal cysteine to which a lipid group can be covalently attached, e.g.
  • the amino group of the cysteine residue at the N-terminus of fHBP is linked to a fatty acid (R1) by forming an amide bond
  • the cysteinyl sulfhydryl is connected to glycerol containing two ester-bonded fatty acids (R2 and R3)
  • R1, R2 and R3 are usually fatty acids containing 14-19 carbon atoms (see WO 2018/142280; Luo et al ., The Dual Role of Lipids of the Lipoproteins in Trumenba, a Self-Adjuvanting Vaccine against Meningococcal Meningitis B Disease, AAPS J., 2016 Nov;18(6):1562-1575 et al).
  • the cysteine residue at the N-terminus of fHBP is generally lipidated in naturally occurring fHBP, which may be lipidated in the fHBP of the subject of the present invention.
  • fHBP lipidated or non-lipidated.
  • Methods of producing lipidated fHBP using recombinant protein technology are well known to those skilled in the art and include the use of native fHBP nucleic acid sequences comprising lipidation signals, or fHBP nucleic acid sequences comprising modified or foreign lipidation signals , and purified from the membrane fraction of E. coli by the method of detergent extraction to obtain lipidated mature fHBP, the mature fHBP does not contain lipidation signal, and the lipid molecule is linked to the cysteine at the N-terminus of fHBP (see Andersson et al, J.
  • Methods of producing non-lipidated fHBP using recombinant protein technology are also well known to those skilled in the art and include the use of native fHBP nucleic acid sequences that do not contain a lipidation signal, or N-terminally altered ones that do not contain a lipidation signal.
  • fHBP nucleic acid sequence these changes include changes in the length of the "glycine/serine stem" downstream of the N-terminal cysteine residue, which can affect the stability or expression of non-lipidated fHBP (see CN103096920B , WO2012/032489, US20120093852, WO2013/132452, US20160030543, etc.).
  • Methods for in vitro conjugation of lipids to proteins are also well known to those skilled in the art.
  • Bacterial ghost (BG) (also known as “bacterial slough”) refers to the empty bacterial shell that does not contain bacterial cell contents such as nucleic acid and cytoplasm. Bacterial ghosts can originate from Gram-negative or Gram-positive bacteria. Methods of preparing bacterial ghosts are well known to those skilled in the art. For example, a method for expressing a lytic protein based on the lytic gene E of phage PhiX174 includes cloning the lytic gene E into an expression control system to achieve controllable expression of the lytic gene E, thereby lysing Gram-negative bacteria.
  • cleavage gene E has been successfully applied to various Escherichia coli strains, Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Helicobacter pylori, Radiobacterium pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolyticus, Pasteurella multocida, Edwardsiella blunt, Vibrio eel, Aeromonas hydrophila, etc.
  • a chemical preparation method that does not depend on cleavage gene E can also be used.
  • the chemical preparation method breaks through the limitation of relying on cleavage gene E to prepare ghosts, and can be effectively applied to Gram-positive bacteria, such as Staphylococcus aureus, Streptococcus Special bacteria, Streptococcus pneumoniae, Bacillus anthracis, Bacillus diphtheriae and Bacillus tetanus, etc.
  • Bacterial ghosts of the present invention include ghosts of various Gram-negative and Gram-positive bacteria, for example, various strains of Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Helicobacter pylori, R.
  • Bacterial ghosts maintain the basic structure of the bacterial cell envelope.
  • the bacterial cell envelope refers to the multi-layered structure including the cytoplasmic membrane that surrounds and protects the cytoplasm.
  • the cell envelope of Gram-negative bacteria includes three main structures from the inside to the outside: the cytoplasmic membrane, peptidoglycan, and the outer membrane; the cell envelope of Gram-positive bacteria includes two main layers from the inside to the outside. Structure: cytoplasmic membrane, peptidoglycan.
  • Adjuvant refers to an auxiliary substance that can enhance the immune response ability of the body to the antigen or change the type of immune response together with the antigen or pre-injected into the body.
  • TLR Toll-like receptor Toll-like receptor
  • Aluminum salt aluminum salt
  • calcium phosphate calcium phosphate
  • oil-in-water emulsion oil-in-water emulsion
  • Freund's adjuvant inactivated bacteria
  • cytokines IL-1, IL-2, IL-12 etc.
  • Antist refers to a substance that binds to a cellular receptor and induces a response. Such a response may be an increase in receptor-mediated activity. Agonists typically mimic the action of naturally occurring substances such as ligands.
  • TLR9 agonist refers to a Toll-like receptor 9 agonist.
  • TLR9 recognizes specific unmethylated CpG oligonucleotide (ODN) sequences to distinguish microbial DNA from mammalian DNA, so TLR9 agonists include various CpG ODNs.
  • ODN unmethylated CpG oligonucleotide
  • Exemplary TLR9 agonists are described in US Pat. Nos. 8,420,615, 7,566,702, 7,498,425, 7,498,426, 7,405,285, 7,427,405, including respective Tables 1 and 2A-2D, the entire contents of which are incorporated herein by reference in their entirety.
  • Vaccine refers to a biological or pharmaceutical preparation comprising an antigen.
  • the formulation can be a composition, and in addition to the antigen, other ingredients such as adjuvants, pharmaceutically acceptable carriers and the like can also be included.
  • the vaccines of the present invention include prophylactic vaccines and therapeutic vaccines.
  • Prophylactic vaccines can provide acquired immunity against specific pathogenic microorganisms, diseases, tumors or cancers before pathogenic microorganisms are infected, or before the appearance of diseases, tumors or cancers; therapeutic vaccines Vaccines can treat or prevent the progression of a disease, tumor or cancer after infection with a pathogenic microorganism, or after the development of the infection, disease, tumor or cancer. Treatment includes complete cure and remission of some or all symptoms.
  • the vaccine of the present invention is a lyophilized product, or a product to be injected obtained by resuspending the lyophilized product in a pharmaceutically acceptable solution or carrier.
  • the lyophilized product of the present invention may also contain the use of a pharmaceutically acceptable carrier or solution when prepared.
  • “Pharmaceutically acceptable” refers to those compounds, materials, compositions and/or dosage forms which, within the scope of sound medical judgment, are suitable for use in contact with human and animal tissue without undue toxicity, irritation, allergic reaction or other problems or complications with a reasonable benefit/risk ratio.
  • “Pharmaceutically acceptable carrier” refers to a pharmaceutically acceptable material, composition or vehicle such as liquid or solid fillers, diluents, excipients, formulation aids (eg lubricants, talc, stearic acid) magnesium, calcium stearate or zinc stearate or stearic acid). Each carrier must be “acceptable” in the meaning of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials that can be used as pharmaceutically acceptable carriers include: (1) carbohydrates such as lactose, glucose and sucrose; (2) starches such as corn starch and potato starch; (3) cellulose and derivatives thereof , such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers such as magnesium hydroxide and aluminum hydroxide (15) alginic acid; (16)
  • the vaccine of the present invention can exist in various dosage forms, including liquid dosage form, lyophilized dosage form, oral dosage form, etc., and the route of administration can be reasonably selected according to the corresponding dosage form, for example, oral administration, intradermal injection, subcutaneous injection, intramuscular injection , intravenous injection, nasal administration, etc.
  • the dosage, administration frequency, etc. of the vaccine of the present invention can be determined according to the specific conditions of the subject.
  • treating refers to alleviating some or all of the symptoms of a disease in a subject, or curing a disease; the alleviation refers to a condition in which the same therapeutic agent is not used.
  • a “therapeutically effective amount” as used herein refers to an amount that elicits an immune response in a subject and that achieves a therapeutic effect.
  • Prevention herein refers to preventing infection of a certain bacterial species or preventing the appearance of a certain disease in a subject.
  • prophylactically effective amount refers to an amount that elicits an immune response in a subject and is expected to achieve a prophylactic effect.
  • an “immunogenically effective amount” herein refers to an amount capable of eliciting an immune response in a subject.
  • an “immune response” herein includes a cellular (T cell) response or a humoral (B cell or antibody) response, or both.
  • a “subject” as used herein refers to a human or animal receiving a therapeutic or prophylactic treatment.
  • “Incubating” and “incubating” are sometimes used interchangeably herein, and both refer to subjecting a reaction to specific conditions for a period of time.
  • Freeze drying (“lyophilization”) refers to a drying process that freezes an aqueous material below the freezing point, converts the water to ice, and then removes the ice by converting it to vapor under a relatively high vacuum. Methods and equipment for freeze drying are well known to those skilled in the art.
  • amino acid sequence identity herein refers to the percentage of amino acids in a candidate sequence that are identical to amino acids in a reference sequence after alignment of the sequences and introduction of gaps where necessary (to achieve maximum percent sequence identity). Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways that are within the skill in the art, for example, using publicly available computer software such as Needle, BLAST, BLAST-2, ALIGN, ALIGN-2, CD-HIT or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment can be determined by known methods, including any algorithms needed to achieve maximal alignment over the full length of the sequences to be aligned.
  • amino acid sequence identity is determined using, for example, the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Heritage). Genet) 16:276-277) (preferably version 5.0.0 or newer) of the Needleman-Wunsch algorithm (Needleman and paper) implemented in the Needle program Wunsch, 1970, J. Mol. Biol. 48: 443-453).
  • the parameters used were a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the Needle output (obtained with the -nobrief option) marked as "longest identity” was used as percent identity and calculated as follows: (identical amino acid residues x 100)/(alignment length - total number of gaps in the alignment) .
  • Constant substitution refers to the replacement of amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (eg, lysine, arginine, histidine), acidic side chains (eg, aspartic acid, glutamic acid) ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine) acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g. threonine, valine, isoleucine) and aryl family side chains (eg tyrosine, phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, his
  • ABSOR as used herein means ⁇ 20%, ⁇ 18%, ⁇ 15%, ⁇ 12%, ⁇ 10%, ⁇ 9%, ⁇ 8%, ⁇ 7%, ⁇ 6%, ⁇ 10% of the stated value
  • a range of 5%, ⁇ 4%, ⁇ 3%, ⁇ 2%, ⁇ 1%, or ⁇ 0.5% includes the endpoints of the range and any value within the range.
  • compositions, system or method, etc. contains at least, but is not limited to, the structure, composition, element, component, feature or step, etc.; “Comprising”, “comprising” and “having” are intended to indicate the presence of the stated structure, ingredient, element, component, feature or step, etc., but do not exclude the presence of any other structure, ingredient, element, component, feature or step, etc. exist.
  • a compound contains, includes or has a certain structure or structures
  • compounds consisting of this or these structures when it is mentioned that a product or composition contains, When it includes or has one or some ingredients, it should be understood that it also covers a product or composition consisting of this or these ingredients; when it is mentioned that a method includes, includes or has one or some steps, it should be It is understood to encompass methods consisting of this or these steps as well.
  • Figure 1 A. Bacterial ghosts loaded with FITC-avidin under fluorescence microscope; B. Protein release kinetics of bacterial ghosts.
  • FIG. 1 Expression of recombinant proteins.
  • Part A is the expression of A19_001;
  • Part B is the expression of B22_001.
  • Leftmost lanes protein markers; lanes 1 and 4: no induction; lanes 2 and 5: induction for 3 hours; lanes 3 and 6: induction overnight.
  • Figure 3 Purification of recombinant protein.
  • Lane M molecular weight standards
  • Lane T crude cell lysate
  • Lane F flow-through fraction
  • Lane P purified recombinant protein.
  • Figure 4 Anti-fHBP-A19_001 antibody titers in mouse serum.
  • Three groups of mice were: immunized with BG+fHBP-A19_001+fHBP-B22_001, immunized with fHBP-A19_001+fHBP-B22_001+alum adjuvant, and not immunized.
  • the X-axis is the dilution factor of serum, and the Y-axis is the OD value at 450 nm.
  • Figure 5 Anti-fHBP-B22_001 antibody titers in mouse serum.
  • Three groups of mice were: immunized with BG+fHBP-A19_001+fHBP-B22_001, immunized with fHBP-A19_001+fHBP-B22_001+alum adjuvant, and not immunized.
  • the X-axis is the dilution factor of serum, and the Y-axis is the OD value at 450 nm.
  • Example 1 Preparation of bacterial ghosts and kinetics of protein release
  • Bacterial ghosts are from Lactobacillus acidophilus. The critical concentrations of several compounds (sodium hydroxide, calcium carbonate, sodium lauryl sulfate) were first determined, and then the ghosts were prepared by chemical methods. The basic experimental protocol is from Wu et al. (Production of Bacterial Ghosts from Gram-Positive Pathogen Listeria monocytogenes, FOODBORNE PATHOGENS AND DISEASE Volume 14, Number 1, 2017). The prepared ghosts were suspended in ddH2O, then frozen in an Eppendorf tube at -80°C for 1 hour, and then lyophilized overnight. The ghosts used in the examples were all prepared by this method.
  • Figure 1A is a bacterial ghost loaded with FITC-avidin under a fluorescence microscope.
  • step 2) The FITC-avidin-loaded bacterial ghosts obtained in step 1) were washed 3 times with ddH 2 O and resuspended in 200 ⁇ l ddH 2 O after the final wash. Prepare multiple 200 ⁇ l samples simultaneously as previously described. Every 30 minutes (ie at 0, 30, 60, 90, 120, 150 minutes), one of the samples was taken and the resuspended bacterial ghosts were centrifuged at 1000 xg for 5 minutes. After discarding the supernatant, the bacterial ghost pellet was dissolved with 100 ⁇ l of B-PER TM Bacterial Protein Extraction Reagent (Fisher Scientific) and incubated at room temperature for 15 min.
  • B-PER TM Bacterial Protein Extraction Reagent (Fisher Scientific)
  • Figure 1B The X-axis of Figure IB represents time points (minutes) and the Y-axis represents the amount of protein retained within bacterial ghosts.
  • Fig. 1B It can be clearly seen from Fig. 1B that although the release of the loaded protein is fast in the first 30 minutes, the release becomes very slow after that, which indicates that the bacterial strains loaded with biomolecules prepared by the method of the present invention
  • the film has the advantage of release kinetics, because it takes a certain period of time from the injection of the delivery carrier into the body to the phagocytosis by the antigen-presenting cells, the ideal delivery carrier cannot release the antigen protein too fast, and the antigen-loaded protein prepared in the present invention is loaded with the antigen.
  • the bacterial ghost of the protein still has enough antigenic protein when it is phagocytosed by the antigen-presenting cell.
  • fHBP fHBP-A19_001
  • strain 961-5945 serogroup B, subfamily A
  • fHBP fHBP-B22_001
  • strain A4 serogroup A, subfamily B
  • the underlined first part in SEQ ID NO: 1 is the lipidation signal, and the underlined second part is the sequence fragment encoding the 6 ⁇ His tag.
  • the main part of SEQ ID NO: 1 is derived from the DQ523568 sequence of GenBank, and the second part of the underlined mark is added, which is different from the sequence of the aforementioned Genbank.
  • Amino acid sequence of mature (without lipidation signal) fHBP-A19_001 (SEQ ID NO: 5):
  • Nucleic acid sequence encoding fHBP of Neisseria meningitidis strain A4 (fHBP-B22_001) + 6x His-tag nucleic acid sequence (SEQ ID NO: 6):
  • the underlined first part in SEQ ID NO: 6 is the lipidation signal, and the underlined second part is the sequence fragment encoding the 6 ⁇ His tag.
  • the main part of SEQ ID NO: 6 comes from the AY330381 sequence of GenBank, and the first part and the second part of the underlined mark are added different from the sequence of the aforementioned Genbank.
  • amino acid sequence of the protein obtained after removing the 6x His tag of SEQ ID NO:7 (SEQ ID NO:8):
  • SEQ ID NO: 1 and SEQ ID NO: 6 were synthesized by Genscript (Piscataway, NJ, USA), and inserted into the pUC57 plasmid through the EcoRV site, and then cut out by NdeI and XhoI enzymes, And inserted into pET26b bacterial expression vector cut with the same enzyme.
  • Example 3 The expression vector obtained in Example 3 was used to transform BL21 plysE(DE3) bacteria. A single colony was inoculated into 5 ml of LB medium containing ampicillin and chloramphenicol for overnight culture. 100 ⁇ l of the overnight culture was then added to 50 ml of LB medium containing ampicillin and chloramphenicol, and the culture was shaken until OD600 to about 0.6. At this point, IPTG was added at a final concentration of 1 mM and the cultures were shaken for an additional 3 hours or overnight.
  • Example 4 The culture obtained in Example 4 was centrifuged at 6000 to 9000 xg for 15 minutes at 4°C, the supernatant was discarded, and the cell pellet was obtained. To each gram of cell pellet was added 5 ml PBS containing 40 mM imidazole, 5 ⁇ l MgCl 2 (1 M), 50 ⁇ l non-toxic serine protease inhibitor Pefabloc (100 mM) (Sigma-Aldrich), 5 ⁇ l DNase (20 mg/ml) (NEB, Ipswich, MA, USA) and 80 ⁇ l of lysozyme (10 mg/ml) (Sigma-Aldrich), the cell pellet was resuspended and mixed until a homogeneous cell resuspension was obtained.
  • PBS containing 40 mM imidazole, 5 ⁇ l MgCl 2 (1 M), 50 ⁇ l non-toxic serine protease inhibitor Pefabloc (100 mM) (Sigma-Al
  • the crude cell lysate was loaded directly onto a 5 ml HisTrap TM Fast Flow Crude Cytiva column (Sigma-Aldrich) previously decontaminated with 10 column volumes of binding buffer (PBS, 40 mM imidazole, 0.1%) agent (eg DDM), pH 7.4). Elution purification was performed with an NGC Quest 10 chromatography system (Bio-rad, Hercules, CA, USA) with a flow rate set to 1 ml/min.
  • Example 6 Bacterial ghost loading of recombinant proteins
  • lyophilized bacterial ghosts were resuspended in 200 ⁇ l of recombinant protein solution containing 10 ⁇ g fHBP-A19_001, 10 ⁇ g fHBP-B22_001 and 6 ⁇ g TLR9 agonist ODN2395 (Invivogen, San Diego) in a microcentrifuge tube (Eppendorf tube). , CA, USA). The aforementioned microcentrifuge tubes were frozen at -80°C for 1 hour and then lyophilized overnight.
  • Example 7 Mouse immunization and serum antibody titer analysis
  • the recombinant protein-loaded bacterial ghosts prepared above were used to immunize a group of C57 mice by subcutaneous injection at a dose of 5 mg bacterial ghosts/mouse.
  • 10 ⁇ g fHBP-A19_001, 10 ⁇ g fHBP- B22_001 , 50 ⁇ L PBS and 50 ⁇ L Alum Adjuvant (Fisher science, Waltham, MA, USA) were mixed and injected intramuscularly in the hind leg for another Immunization of group C57 mice.
  • As a negative control another group of 6 C57 mice was bred under the same conditions as the treatment group without immunization, and sera were collected from the tails of the mice 28 days later for analysis of antibody titers.
  • Antibody titers against recombinant proteins fHBP-A19_001 and fHBP-B22_001 were determined using enzyme linked immunosorbent assay (ELISA). 100 ⁇ l of 1 ⁇ g/mL recombinant protein solution (fHBP-A19_001 or fHBP-B22_001) was used to coat each well of a 96-well Costar plate and the plate was left at 4°C overnight. After washing and incubation with blocking solution, 100ul of mouse serum at various dilutions was added to each well. Color was developed using a secondary antibody conjugated to alkaline phosphatase and TMB substrate.
  • ELISA enzyme linked immunosorbent assay
  • Figure 4 is the titer of anti-fHBP-A19_001 antibody in mouse serum
  • Figure 5 is the titer of anti-fHBP-B22_001 antibody in mouse serum. The results showed that the antibody titers in the serum of mice immunized with ghosts were significantly higher than those of mice immunized without ghosts.
  • Example 8 Mouse immunization and serum bactericidal titer analysis
  • the bacterial ghosts loaded with recombinant protein prepared according to Example 6 ((10 ⁇ g fHBP-A19_001+10 ⁇ g fHBP-B22_001+6 ⁇ g ODN2395)/5 mg bacterial ghosts) were prepared at a dose of 5 mg bacterial ghosts/mouse and with The way of subcutaneous injection was used to immunize a group of C57 mice (5 mice/group).
  • mice/group 10 ⁇ g fHBP-A19_001, 10 ⁇ g fHBP- B22_001 , 50 ⁇ L PBS and 50 ⁇ L Alum Adjuvant (Fisher science, Waltham, MA, USA) were mixed and injected intramuscularly in the hind leg for another Immunization of group C57 mice (5 mice/group). After 14 days, two groups of mice were given a second injection in the same manner as before. Fourteen days after the second injection, the mice were bled and serum samples were collected from each group of mice separately. As a negative control, another group of mice (5 mice/group) was bred under the same conditions as the treatment group without immunization, and 28 days later, the mice were bled and serum samples were collected.
  • SBA Serum Bactericidal Activity
  • Neisseria meningitidis serogroup B strains were streaked to obtain individual colonies and grown on Brain Heart Infusion (BHI) medium containing 10% Horse Blood Supplement. Incubate overnight at 37°C and 5% CO2 . A fraction of individual colonies were resuspended in 0.1% glucose in calcium and magnesium-containing PBS (PCM) buffer pH 7.4 at the appropriate density as required for the assay.
  • BHI Brain Heart Infusion
  • PCM calcium and magnesium-containing PBS
  • Serum from a human donor was used according to the method of Mountzouros et al. (Detection of complement mediated antibody-dependent bactericidal activity in a fluorescence-based serum bactericidal assay for group B Neisseria meningitidis. J. Clin. Microbiol. 38:2878-2884. Determination of complement-mediated bactericidal activity of serum was performed as a source of complement.
  • test solution consisting of 25 ⁇ l of PCM buffer, 5 ⁇ l of heat (56°C for 30 min) inactivated serially diluted (two-fold dilution) mouse serum to be tested, 10 ⁇ l of human complement and Mix 10 ⁇ l of PCM buffer containing approximately 1x10 3 to 3x10 3 live Neisseria meningitidis.
  • the test plates were incubated at 37°C for 30 minutes.
  • 200 ⁇ l of modified Frantz growth medium containing alamarBlue dye (Fisher Scientific) diluted 1:20 and 0.7% low melting point agarose was then added to each well containing the test solution, and the test Plates were incubated overnight at 37°C.
  • Fluorescent signals produced by the reaction of alamar blue dye and live bacteria were read with a Fluoroskan TM Microplate Fluorescence Analyzer (Fisher Scientific).
  • a Fluoroskan TM Microplate Fluorescence Analyzer (Fisher Scientific).
  • 30 ⁇ l of PCM was added to another well of the test plate, and 5 ⁇ l of the mouse serum to be tested was not added.
  • Other components and reaction conditions were the same as the above experimental groups.
  • different known amounts of Neisseria meningitidis and alamar blue dye were added to another group of wells of the test plate respectively, and no serum to be tested was added to generate a standard curve (Y-axis is the fluorescence signal value, X-axis is is the number of bacteria).
  • the number of bacteria after the reaction in the experimental group and the negative control was calculated.
  • the number of killed bacteria can be obtained by subtracting the number of bacteria after the reaction from the number of bacteria before the reaction. Using sera with known bactericidal titers as a positive control, the feasibility and accuracy of the above test were confirmed.
  • mice immunized with ghosts showed that for the mice immunized with ghosts, the bactericidal power of their sera against different strains was significantly higher than that of mice immunized without ghosts (see Table 1).
  • the bactericidal titer (bactericidal titer) in Table 1 refers to the reciprocal of the maximum serum dilution that can kill more than 50% of bacteria compared to the negative control. If a serum sample showed a bactericidal rate of ⁇ 50% at the lowest serum dilution (minimum serum dilution was 1:25), the bactericidal titer for that sample was reported as ⁇ 30.
  • Table 1 Bactericidal titers of mouse serum against different strains of Neisseria meningitidis serogroup B
  • Alexopoulou L., Holt, A.C., Medzhitov, R., Flavell, R.A., 2001. Recognition of double-stranded RNA and activation of NF- ⁇ B by Toil-like receptor 3. Nature 413, 732-738. https:// doi.org/10.1038/35099560
  • Neisseria meningitidis NadA is a new invasin which promotes bacterial adhesion to and penetration into human epithelial cells.Mol.Microbiol.55, 687-698.https://doi.org/10.1111/j.1365-2958.2004.04423.x
  • Grifantini R., Bartolini, E., Muzzi, A., Draghi, M., Frigimelica, E., Berger, J., Ratti, G., Petracca, R., Galli, G., Agnusdei, M., Giuliani, M.M., Santini, L., Brunelli, B., Tettelin, H., Rappuoli, R., Randazzo, F., Grandi, G., 2002.
  • Previously unrecognized vaccine candidates again group B meningococcus identified by DNA microarrays. Nat .Biotechnol.20, 914-921. https://doi.org/10.1038/nbt728
  • N-propionylated group B meningococcal polysaccharide mimics a unique epitope on group B Neisseria meningitidis.J.Exp.Med.165, 1207-1211. https://doi. org/10.1084/jem.165.4.1207
  • a novel combined Haemophilus influenzae type b- Neisseria meningitidis serogroups C and Y-tetanus-toxoid conjugate vaccine is immunogenic and induces immune memory when co-administered with DTPa-HBV-IPV and conjugate pneumococcal vaccines in infants.
  • Neisseria meningitidis transferrin binding protein A protects against experimental meningococcal infection.Infect.Immun.69, 1561-1567. https://doi.org/10.1128/IAI.69.3.1561-1567.2001

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Abstract

La présente invention concerne un vaccin pour la prévention ou le traitement d'une infection par Neisseria meningitidis ou de maladies induites par celle-ci, son utilisation et un procédé associé. La présente invention concerne un vaccin contre Neisseria meningitidis, qui comprend un fantôme bactérien et une protéine de liaison au facteur H (fHBP), et est utilisé pour prévenir ou traiter une infection par Neisseria meningitidis ou des maladies induites induites par celle-ci, telle que la méningococcie invasive (MI). La présente invention concerne également l'utilisation d'une composition comprenant un fantôme bactérien et une fHBP dans la préparation d'un vaccin pour prévenir ou traiter une infection par Neisseria meningitidis ou des maladies induites par celle-ci. En outre, la présente invention concerne un procédé pour prévenir ou traiter une infection par Neisseria meningitidis ou des maladies induites par celle-ci, tel que la MI, lequel procédé comprend l'administration d'une dose thérapeutiquement efficace ou d'une dose prophylactiquement efficace du vaccin contre Neisseria meningitidis à un sujet.
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CN1377415A (zh) * 1999-08-03 2002-10-30 史密丝克莱恩比彻姆生物有限公司 疫苗组合物
US20120040829A1 (en) * 1999-03-05 2012-02-16 Werner Lubitz Bacterial ghosts as carrier and targeting vehicles
CN105617372A (zh) * 2001-10-11 2016-06-01 惠氏控股有限公司 预防和治疗脑膜炎球菌性疾病的新型免疫原性组合物
CN110234658A (zh) * 2017-01-31 2019-09-13 辉瑞大药厂 脑膜炎奈瑟菌组合物及其使用方法
WO2020030782A1 (fr) * 2018-08-09 2020-02-13 Glaxosmithkline Biologicals Sa Polypeptides fhbp méningococciques modifiés

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WO2009038889A1 (fr) * 2007-08-02 2009-03-26 Children's Hospital And Research Center At Oakland Vaccins vésiculaires à base de fhbp et de lpxl1 pour une protection à large spectre contre les maladies à neisseria meningitidis
WO2010109323A1 (fr) * 2009-03-24 2010-09-30 Novartis Ag Protéine de liaison du facteur h méningococcique utilisée comme adjuvant
CN103275914B (zh) * 2013-06-03 2015-04-01 中国人民解放军军事医学科学院微生物流行病研究所 展示保护性抗原的细菌菌蜕及其应用
RU2662968C2 (ru) * 2013-09-08 2018-07-31 Пфайзер Инк. Иммуногенная композиция против neisseria meningitidis (варианты)
CN108939061A (zh) * 2018-08-03 2018-12-07 北京智飞绿竹生物制药有限公司 一种多组分b群脑膜炎球菌疫苗及其制备方法

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US20120040829A1 (en) * 1999-03-05 2012-02-16 Werner Lubitz Bacterial ghosts as carrier and targeting vehicles
CN1377415A (zh) * 1999-08-03 2002-10-30 史密丝克莱恩比彻姆生物有限公司 疫苗组合物
CN105617372A (zh) * 2001-10-11 2016-06-01 惠氏控股有限公司 预防和治疗脑膜炎球菌性疾病的新型免疫原性组合物
CN110234658A (zh) * 2017-01-31 2019-09-13 辉瑞大药厂 脑膜炎奈瑟菌组合物及其使用方法
WO2020030782A1 (fr) * 2018-08-09 2020-02-13 Glaxosmithkline Biologicals Sa Polypeptides fhbp méningococciques modifiés

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