CN114681601A - Neisseria meningitidis vaccines and uses thereof - Google Patents

Abstract

The present invention relates to vaccines, uses and methods for preventing or treating infection by neisseria meningitidis or diseases induced thereby. In one aspect, the invention relates to a neisseria meningitidis vaccine comprising a bacterial ghost and a factor H binding protein (fHBP) for use in preventing or treating neisseria meningitidis infection or a disease induced thereby, such as Invasive Meningococcal Disease (IMD). In another aspect, the invention relates to the use of a composition comprising a bacterial ghost and fHBP in the manufacture of a vaccine for the prevention or treatment of neisseria meningitidis infection or disease induced thereby, such as IMD. In another aspect, the invention relates to a method of preventing or treating neisseria meningitidis infection or a disease, e.g., IMD, induced thereby, comprising administering to a subject a prophylactically or therapeutically effective amount of a neisseria meningitidis vaccine.

Description

Neisseria meningitidis vaccines and uses thereof

Technical Field

The present invention relates to vaccines, compositions, uses and methods for preventing or treating neisseria meningitidis infection or diseases caused thereby.

Background

As one of the major causes of global bacterial meningitis, Neisseria meningitidis (Neisseria meningitidis) is classified into 13 serogroups based on its capsular polysaccharide, 6 of which (a, B, C, W, X, Y) cause Invasive Meningococcal Disease (IMD) (Harrison et al, n.d.). The number of cases of invasive disease worldwide per year is at least about 1.2 million, with 135,000 deaths associated with IMDs ("Epidemics of medical diseases african medical diseases belt, 2001", 2001).

To combat IMD, many industrialized countries have developed different meningococcal vaccine formulations for routine immunization programs. For example, a tetravalent vaccine comprising a conjugate of a protein and a polysaccharide from A, C, W-135 and Y serogroups was introduced in the united states and recommended for routine use in subjects 11 years and older (Pace et al, 2009). In addition, a more immunogenic tetravalent conjugate vaccine, and a conjugate vaccine against Haemophilus influenzae type b and meningococcus C and Y, are under development in the clinical stage for adaptation to infants (Nolan et al, 2007). Serogroup B meningococcal disease accounts for over 50% of all meningococcal disease in the united states, with this proportion being even higher in many european countries (Harrison et al, 2009; Trotter et al, 2007). However, there has been no widely effective serogroup B meningococcal disease vaccine for a long time until recently the FDA approved BEXSERO by GSK and TRUMENBA by fevere.

Conjugation to carrier proteins is known to confer polysaccharide antigens immunogenic to infants and to elicit memory anti-capsular antibody responses (Granoff and polard, 2007), which is the rationale for all meningococcal serogroup vaccines except serogroup B. The polysaccharide of meningococcal serogroup B is a homogeneous linear polymer of alpha- (2-8) N-acetylneuraminic acid (polysialic acid), and is an autoantigen (Finne et al, 1983); the polysaccharides of serogroup B are still a weak immunogen even when conjugated to a protein carrier (Jennings and Lugowski, 1981). In order to enhance immunogenicity, n-propionyl-derivatized modifications of this polysaccharide have been attempted with only limited success in mice, not humans (Bruge et al, 2004; Jennings et al, 1987).

Current research on vaccines against meningococcal serogroup B has focused on non-capsular antigens, such as proteins or Lipopolysaccharides (LPS). A large number of novel vaccine candidates against serogroup B meningococcal disease have been identified by screening techniques using genomic, proteomic, and immunological methods, such as NspA (Martin et al, 1997; Moe et al, 1999; Halperin et al, 2007), transferrin binding protein (West et al, 2001; Rokbi et al, 1997), opaque protein (opacity protein, Opc) (Perez et al, 2006; Jolley et al, 2001; Callaghan et al, 2008), GNA 2132(Giuliani et al, 2006; pleded and Granoff, 2008; Jacobsson et al, 2006; Welsch et al, 2003), fHBP (also known as GNA1870 or 2086) (Koeberling et al, 2008; Fletcher et al, 2004), FetA (iron regulated outer membrane protein) (Thompson et al, 2003), Neisserial A (Nacca, adhesin A, also known as GNA et al (Cambodipy et al, 1994; Bezizan et al, 2000; and other proteins). Unfortunately, for a variety of reasons, the potential of almost all of the currently identified candidate proteins alone as antigens for vaccines against serogroup B meningococcus is very limited. These include antigen variability (FetA and Opc), lack of genes from certain strains of highly virulent lineages (NadA), phase variability (Opc), low constitutive expression levels of antigens by certain strains (fHbp, GNA 2132 and NspA), and difficulty in recombinant expression of certain important conformational epitopes (NspA) (Halperin et al, 2007; Hou et al, 2003). Thus, current methods focus on the use of multiple bacterial proteins to achieve broad protection against different strains of serogroup B. BEXSERO from GSK and TRUMENBA from pyroxene were formulated from a variety of proteins (fusion proteins).

Subunit vaccines based on proteins or polysaccharides are known to be less immunogenic than vaccines based on inactivated or deactivated pathogens. Therefore, immunological adjuvants are often used with antigens to enhance the immune response. Another approach is to conjugate the protein to a polysaccharide antigen as described previously.

Bacterial Ghosts (BG) are non-living empty cell envelopes of bacteria, resulting from the release of bacterial cytoplasm through channels in the bacterial cell envelope, which can be achieved by controlled expression of the bacteriophage phi X174 cleaved protein E in gram-negative bacteria, or by methods based on the critical concentration of compounds ("Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli | Springer Link", n.d.; Amara et al, 2013; Wu et al, 2017).

Bacterial ghosts still retain the complete antigenic structure of the native bacterial surface 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 that can be loaded with biological macromolecules such as antigens, drugs and DNA (Lubitz, 2001; Jalava et al, 2003; Mayr et al, 2005; Muhammad et al, 2012). In addition, bacterial ghosts contain known innate immune stimulatory components, and have the potential to act as potent adjuvants. In particular, bacterial ghosts retain antigenic components of the natural bacterial surface, such as Lipopolysaccharides (LPS), flagellins, peptidoglycans, and many other types of substances that are ligands of various Pattern Recognition Receptors (PRRs), collectively referred to as pathogen-associated molecular patterns (PAMP) (Huter et al, 1999; Lubitz, 2001; Muhammad et al, 2012). These structures are efficiently recognized and taken up by immune and non-immune cells (Ebensen et al, 2004; Abtin et al, 2010; Stein et al, 2013), which in turn will activate cells (Adam et al, 2010; Quevedo-Diaz et al, 2010) primarily through the Toll-like receptor 2 (TLR 2) and Toll-like receptor 4(TLR4) pathways, as well as achieve adjuvant activity through multiple Toll-like receptors (TLRs) present on many immune and non-immune cells.

Disclosure of Invention

In one aspect, the invention provides a neisseria meningitidis vaccine comprising a bacterial ghost and factor H binding protein (fHBP), including lipidated or non-lipidated fHBP. In some embodiments, the fHBP comprises at least one fHBP of subfamily a and/or at least one fHBP of subfamily B. In some embodiments, the fHBP of subfamily a is a polypeptide that is identical to SEQ ID NO: 2. SEQ ID NO: 3. the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 fHBP proteins having 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; or a sequence identical to SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: a fHBP protein having an 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 compared to 5. In some embodiments, the fHBP of subfamily B is a polypeptide that differs from SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 or SEQ ID NO: 10, a fHBP protein having 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; or a sequence identical to SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 or SEQ ID NO: a fHBP protein having an 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 compared to 10. In some embodiments, the substitution is a conservative substitution. In some embodiments, the fHBP of subfamily a is from a serogroup B strain, e.g., strains 961-5945, CDC1034, CDC2369, 870446. In some embodiments, the fHBP of subfamily B is from a serogroup a strain, e.g., strain a 4. In some embodiments, the fHBP of subfamily B is from a serogroup B strain, e.g., strains CDC-1343, CDC-983, CDC-852. In some embodiments, the subfamily a factor H binding protein has the sequence set forth in SEQ ID NO: 5, respectively. In some embodiments, the subfamily B factor H binding protein has the sequence set forth in SEQ ID NO: shown at 10. In some embodiments, fHBP is loaded within a bacterial ghost. In some embodiments, the vaccine of the present invention further comprises one or more adjuvants. In some embodiments, the adjuvant is a TLR9 agonist. In some embodiments, the adjuvant is loaded within a bacterial ghost. In some embodiments, the bacterial ghost of the present invention is a ghost of lactobacillus acidophilus. In some embodiments, the vaccine is for use in the prevention or treatment of infection by neisseria meningitidis serogroup A, B, C, W, X and/or Y or a disease induced thereby. In some embodiments, the vaccine is for use in the prevention or treatment of infection by serogroup a and/or B or a disease induced thereby. In some embodiments, the vaccine is for use in the prevention or treatment of infection by serogroup B or a disease induced thereby. In some embodiments, the disease comprises Invasive Meningococcal Disease (IMD). In some embodiments, the vaccine can be used in humans, poultry, livestock, and/or mammals. In some embodiments, the vaccine is in a lyophilized dosage form.

In another aspect, the invention provides the use of a composition comprising a bacterial ghost and a factor H binding protein (fHBP) in the manufacture of a vaccine for the prevention or treatment of infection by neisseria meningitidis or a disease induced thereby. The vaccine may be used for the prevention or treatment of infection by serogroup B or a disease induced thereby, infection by serogroup A or a disease induced thereby, infection by serogroup C or a disease induced thereby, infection by serogroup W or a disease induced thereby, infection by serogroup X or a disease induced thereby, and/or infection by serogroup Y or a disease induced thereby. In some embodiments, the disease comprises Invasive Meningococcal Disease (IMD). In some embodiments, the fHBP is lipidated or non-lipidated fHBP. In some embodiments, the fHBP comprises at least one fHBP of subfamily a and at least one fHBP of subfamily B. In some embodiments, the fHBP of subfamily a is a polypeptide that is identical to SEQ ID NO: 2. SEQ ID NO: 3. the amino acid sequence of SEQ ID NO: 4 or SEQ ID NO: 5 fHBP proteins having 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; or a sequence identical to SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4 or SEQ ID NO: a fHBP protein having an 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 compared to 5. In some embodiments, the factor H binding protein of subfamily B (fHBP) is a polypeptide that binds to SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 or SEQ ID NO: 10, a fHBP protein having 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; or a sequence identical to SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9 or SEQ ID NO: a fHBP protein having an 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 compared to 10. In some embodiments, the substitution is a conservative substitution. In some embodiments, the fHBP of subfamily a is from a serogroup B strain, e.g., strains 961-5945, CDC1034, CDC2369, 870446. In some embodiments, the fHBP of subfamily B is from a serogroup a strain, e.g., strain a 4. In some embodiments, the fHBP of subfamily B is from a serogroup B strain, e.g., strains CDC-1343, CDC-983, CDC-852. In some embodiments, wherein the sequence of the subfamily a factor H binding protein is as set forth in SEQ ID NO: 5, and/or wherein the sequence of the subfamily B factor H binding protein is set forth in SEQ ID NO: shown at 10. In some embodiments, fHBP is loaded within a bacterial ghost. In some embodiments, the composition further comprises one or more adjuvants. In some embodiments, the adjuvant is a TLR9 agonist. In some embodiments, the adjuvant is loaded within the bacterial ghost. In some embodiments, the bacterial ghost is a ghost of lactobacillus acidophilus. In some embodiments, the vaccine can be used in humans, poultry, livestock, and/or mammals.

In another aspect, the invention provides a method of preventing or treating infection by neisseria meningitidis serogroup A, B, C, W, X and/or Y or a disease induced thereby, comprising administering to a subject an immunogenically, prophylactically or therapeutically effective amount of a vaccine of the invention; the mode, number and dosage of administration will depend on the subject. In some embodiments, the subject of the present method is a human, poultry, livestock, and/or mammal. In some embodiments, the methods prevent or treat infection by neisseria meningitidis serogroup A, B, C, W, X and/or Y or diseases that result therefrom, including Invasive Meningococcal Disease (IMD). In some embodiments, the subject of the present methods is a subject not infected with neisseria meningitidis serogroup A, B, C, W, X and/or Y, a subject infected with neisseria meningitidis serogroup A, B, C, W, X and/or Y but not exhibiting symptoms of the disease it induces, or a subject having neisseria meningitidis serogroup A, B, C, W, X and/or Y induced disease.

In another aspect, the invention provides a method of loading fHBP and adjuvant into bacterial ghosts, the method comprising resuspending the lyophilized bacterial ghosts with a protein solution, then freezing at about-80 ℃, and then freeze-drying, the protein solution containing the one or more proteins to be loaded, optionally with adjuvant. In some embodiments, the freezing time is from 0.5 to 1.5 hours, preferably from 0.6 to 1.4 hours, more preferably from 0.7 to 1.3 hours, more preferably from 0.8 to 1.2 hours, more preferably from 0.9 to 1.1 hours, more preferably about 1 hour. In some embodiments, the freeze-drying time is 12 to 20 hours, preferably 13 to 19 hours, preferably 14 to 18 hours, preferably 15 to 17 hours. In some embodiments, the mixture of protein solution and bacterial ghost can be incubated at 4 ℃ prior to freezing. In some embodiments, the incubation at 4 ℃ is for a period of 1-3 hours, preferably 1.5-2.5 hours, preferably about 2 hours. The above steps can be repeated in order to increase the loading of recombinant protein in the bacterial ghost.

According to the invention, by loading fHBP into bacterial ghost, the antibody titer/titer against Neisseria meningitidis in serum is significantly improved. By combining fHBP of subfamily a with fHBP of subfamily B, the vaccine of the invention is able to induce the production of antibodies against various strains expressing fHBP of subfamily a and antibodies against various strains expressing fHBP of subfamily B, providing broad spectrum immunoprotection. These strains include strains belonging to serogroup A, B, C, W, X or Y. The examples of the invention show that the combination of fHBP and bacterial ghosts significantly increases the antibody titers in serum against fHBP of subfamily a and against fHBP of subfamily B, as well as significantly increases the bactericidal potency of the serum against all the test strains. In addition, the bacterial 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. In particular, because of the time required from injection of the delivery vehicle into the body to phagocytosis by antigen presenting cells, the release of antigenic protein by the ideal delivery vehicle cannot be too rapid, and the examples herein show that the bacterial ghosts of the present invention are phagocytosed by antigen presenting cells and still release sufficient antigenic protein. Further, the vaccine of the present invention may be a lyophilized product, and thus may be transported in a short time or stored in a short time (within one month) at normal temperature without cold chain transportation or cryopreservation.

Detailed Description

"Neisseria meningitidis" (also known as "meningococcus") is a gram-negative bacterium with a capsular membrane, which can be divided into 13 serogroups based on the polysaccharides in its capsule, of which 6 serogroups (a, B, C, W, X, Y) can lead to Invasive Meningococcal Disease (IMD), which is commonly manifested as meningococcal meningitis and/or meningococcal hematopathy (meningococcus em/septicemia), and less commonly manifested as meningococcal pneumonia (meningococcal pneumoniaa), suppurative arthritis (septoritis), epiglottitis (epiglottitis) or otitis media (meningitis media), and the like.

"factor H binding protein" (fHBP) (also known as "LP 2086", "GNA 1870", or "ORF 2086") is a lipoprotein expressed in almost all serogroup B strains, partial serogroup A, C, W, X, and Y strains that is anchored to the outer membrane of meningococcus (outer membrane) by a lipid molecule. fHBP can be divided into two distinct subfamilies (i.e., subfamily a and subfamily B) based on deduced amino acid sequence homology. There is at least 83% amino acid sequence similarity between variants belonging to the same subfamily. There is 60% to 75% amino acid sequence similarity between variants belonging to different subfamilies. Known Strains expressing the ffHBP variant of subfamily A or B are described in published papers (e.g., Flethcher et al, Vaccine Potential of the immunization prediction 2086Lipoprotein, infection Immun.2004Apr, 72 (4): 2088-2100; Murphy et al, Sequence Diversity of the Factor H Binding Protein Vaccine polypeptide in epidemic deletion variants of Serrogoup B immunization prediction, J infection prediction 2009Aug 1, 200 (3): 379-89), and those skilled in the art can also classify the newly found ffHBP variant into subfamily A or B by these published methods.

"lipidation" (also known as "lipidation") refers to the covalent attachment of lipids to proteins, either in vivo by post-translational modification of proteins or in vitro by chemical synthesis. Natural fHBP typically contains an N-terminal cysteine to which a lipid group can be covalently attached, e.g., The amino group of The N-terminal cysteine residue of fHBP is linked to one fatty acid (R1) by forming an amide bond and The cysteinylthiol group links glycerol containing two ester-bonded fatty acids (R2 and R3), R1, R2 and R3 are typically fatty acids containing 14-19 carbon atoms (see WO 2018/142280; Luo et al, The Dual Role of Lipids of The Lipids in Tromenba, a Self-Adjuvanting Vaccine ingredient Mennical B Disease, AAPS J., 2016 Nov; 18 (6): 1562 1575, etc.). The N-terminal cysteine residue of fHBP is typically lipidated in naturally occurring fHBP, which may be lipidated in fHBP of the subject invention. Thus, in the amino acid sequences described herein, reference to a cysteine at that particular position includes both an unmodified cysteine and a lipidated cysteine. Thus, fHBP of the subject of the invention may be lipidated or non-lipidated. Methods for producing lipidated fHBPs using recombinant protein technology are well known to those skilled in the art and include the use of native fHBP nucleic acid sequences containing lipidation signals, or fHBP nucleic acid sequences containing modified or foreign lipidation signals, and purification from the membrane fraction of E.coli by detergent extraction to obtain lipidated mature fHBP which does not contain lipidation signals, with lipid molecules attached to cysteines at the N-terminus of fHBP (see Andersson et al, J. immunological Methods, 2001, 255: 135-48; Fletcher et al, Infection and Immunity, 2004, 72: 2088-100; US20200138933A1, etc.). Methods for 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 comprise a lipidation signal, or fHBP nucleic acid sequences that do not comprise an N-terminal alteration of the lipidation signal, which alteration comprises altering the length of the "glycine/serine stem" downstream of the N-terminal cysteine residue, which length may affect the stability or amount of expression of the non-lipidated fHBP (see CN103096920B, WO2012/032489, US20120093852, WO2013/132452, US20160030543 etc.). Methods for binding lipids to proteins in vitro are also well known to those skilled in the art.

"bacterial ghost" (also known as bacterial ghost) refers to a bacterial ghost that does not contain bacterial cell contents such as nucleic acids and cytoplasm. The bacterial ghosts may be derived from gram-negative or gram-positive bacteria. Methods for preparing bacterial ghosts are well known to those skilled in the art. For example, a method for expressing lytic proteins based on lytic gene E of bacteriophage PhiX174, comprising cloning lytic gene E into an expression regulation system, allowing controlled expression of lytic gene E, thereby lysing gram-negative bacteria. The expression regulation system of the lytic gene E has been successfully applied to various Escherichia coli strains, Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, helicobacter pylori, actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, Edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophila, and the like. For example, a chemical preparation method independent of the lytic gene E can be adopted, which breaks through the limitation of preparation of bacterial ghost dependent on the lytic gene E and can be effectively applied to gram-positive bacteria, such as Staphylococcus aureus, St.sp.pneumoniae, Bacillus anthracis, Bacillus diphtheriae and Bacillus tetani. Bacterial ghosts of the present invention include bacterial ghosts of various gram-negative and gram-positive bacteria, for example, bacterial ghosts of various strains of Escherichia coli, Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, helicobacter pylori, Radiobacterium pleuropneumoniae, Haemophilus influenzae, Pasteurella haemolytica, Pasteurella multocida, Edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophila, Staphylococcus aureus, Statebacillus, Streptococcus pneumoniae, Bacillus anthracis, Diptheria, tetanus, and Lactobacillus acidophilus, and the like. The bacterial ghost retains the basic structure of bacterial cell envelope (bacterial cell envelope). The bacterial cell envelope refers to a multi-layered structure containing the cytoplasmic membrane that surrounds and protects the cytoplasm. The cell envelope of gram-negative bacteria comprises three main layers from inside to outside: cytoplasmic membrane, peptidoglycan, outer membrane (outer membrane); the cell envelope of gram-positive bacteria comprises two main structures from inside to outside: cytoplasmic membrane, peptidoglycan.

"adjuvant" refers to an auxiliary substance that enhances the body's ability to respond to an antigen or alters the type of immune response, either with or prior to injection into the body, and includes specific substances well known to those skilled in the art, such as: toll-like receptor (TLR) agonists, aluminium salts, calcium phosphates, oil-in-water emulsions, Freund's adjuvant, inactivated bacteria, cytokines IL-1, IL-2, IL-12 and the like.

"agonist" refers to a substance that binds to a cellular receptor and induces a response. Such a response may be an increase in activity mediated by the receptor. Agonists generally mimic the action of naturally occurring substances (e.g., ligands).

"TLR 9 agonist" refers to a Toll-like receptor 9 agonist. TLR9 recognizes specific unmethylated CpG Oligonucleotide (ODN) sequences to distinguish microbial DNA from mammalian DNA, and thus TLR9 agonists include a variety of CpG ODNs. Exemplary TLR9 agonists are described in U.S. patent 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 may be a composition, which may contain, in addition to the antigen, other ingredients such as adjuvants, pharmaceutically acceptable carriers, and the like. Vaccines of the invention include prophylactic and therapeutic vaccines that can provide acquired immunity against a particular pathogenic microorganism, disease, tumor or cancer prior to infection by the pathogenic microorganism, or prior to the occurrence of the disease, tumor or cancer; therapeutic vaccines can treat or prevent the progression of an infection, disease, tumor or cancer after it has been infected with a pathogenic microorganism, or after a disease, tumor or cancer has occurred, including complete cure and partial or complete remission. In some embodiments, 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 products of the present invention may also be prepared by using a pharmaceutically acceptable carrier or solution.

"pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

"pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, formulation aid (e.g., lubricant, talc, magnesium stearate, calcium or zinc stearate, or stearic acid). The carriers must each be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials that can be used as pharmaceutically acceptable carriers include: (1) sugars such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc powder; (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) buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) ringer's solution; (9) ethanol; (20) pH buffer solutions (e.g., PBS, etc.); (21) polyesters, polycarbonates and/or polyanhydrides; and (22) other non-toxic compatible materials for use in pharmaceutical formulations. The vaccine of the present invention may be presented in various dosage forms including liquid dosage forms, lyophilized dosage forms, oral dosage forms, etc., and the administration route may be appropriately selected according to the corresponding dosage forms, for example, oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, etc. The amount, number of administrations, etc. of the vaccine of the present invention may be decided according to the particular circumstances of the subject.

"treating" or "treatment" as used herein refers to alleviating some or all of the symptoms of a disease in a subject, or curing a disease; the relief is compared to the situation where the same therapeutic agent is not used.

As used herein, a "therapeutically effective amount" refers to an amount that elicits an immune response in a subject and achieves a therapeutic effect.

"prevention" as used herein refers to the prevention of infection by a bacterium or the prevention of the appearance of a disease in a subject.

As used herein, a "prophylactically effective amount" refers to an amount that elicits an immune response in a subject and is expected to provide a prophylactic effect.

An "immunogenically effective amount" herein refers to an amount capable of eliciting an immune response in a subject.

An "immune response" as used herein includes a cellular (T cell) response or a humoral (B cell or antibody) response, or both.

By "subject" herein is meant a human or animal subject to a therapeutic or prophylactic treatment. "incubation" and "culturing" are sometimes used interchangeably herein and refer to subjecting a reaction to specific conditions for a period of time.

"lyophilization" refers to a drying process in which an aqueous material is frozen below freezing to convert water to ice, and then the ice is removed by converting the ice to a vapor under a relatively high vacuum. Methods and apparatus for freeze-drying are well known to those skilled in the art.

"amino acid sequence identity" herein refers to: percentage of amino acids in a candidate sequence that are identical to amino acids in the reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for the purpose of determining percent sequence identity may be accomplished in a variety of ways within the skill in the art, for example, using publicly available computer software such as Needle, BLAST-2, ALIGN-2, CD-HIT, or megalign (dnastar) software. By known methods, suitable parameters for measuring alignment can be determined, including any algorithm required to achieve maximum alignment over the full length of the sequences to be aligned. In some embodiments, amino acid sequence identity is determined using the Needman-Wunsch algorithm (Needman-Wunsch) as implemented in the Nidel (Needle) program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite (European Molecular Biology Software Suite), Rice (Rice), et al, 2000, genetics Trends (Trends Genet) 16: 276-. The parameters used are the gap opening penalty of 10, the gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM 62) substitution matrix. The Needle output labeled "longest identity" (obtained using the-nobrief option) is used as the percent identity and is calculated as follows: (identical amino acid residues X100)/(alignment Length-total number of gaps in the alignment).

"conservative substitution" refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

"about" herein means a range of ± 20%, ± 18%, ± 15%, ± 12%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, ± 1% or ± 0.5% of the stated value, the range including the endpoints of the range and any value within the range.

As used herein, "comprising," "including," "having," or the like means that the material, composition, system, or method, etc. contains, but is not limited to, at least the recited structure, ingredient, element, component, feature or step; the terms "comprises," "comprising," "including," "has" and the like, are intended to mean that there is a stated structure, component, element, component, feature, or step, but not to preclude the presence or addition of any other structure, component, element, component, feature, or step. In this context, when a compound is referred to as comprising, including or having a certain structure or structures, it is to be understood that it also encompasses compounds consisting of this structure or structures; when a product or composition is referred to as comprising, including or having a certain ingredient or ingredients, it is to be understood that it also encompasses products or compositions consisting of that ingredient or ingredients; when a method is referred to as comprising, including or having a certain step or steps, it is to be understood that it also encompasses methods consisting of the step or steps.

As used herein, the term "and/or" is intended to include any possible combination of one or more of the listed items.

Drawings

The present invention will become more fully understood from the detailed description and the accompanying drawings, wherein:

FIG. 1: A. loading bacterial ghosts loaded with FITC-avidin under a fluorescent microscope; B. protein release kinetics of bacterial ghosts.

FIG. 2: and (4) expressing the recombinant protein. Part a is the expression of a19_ 001; part B is the expression of B22_ 001. Leftmost lane: molecular weight standards (protein marker); lanes 1 and 4: not inducing; lanes 2 and 5: inducing for 3 hours; lanes 3 and 6: induction was carried out overnight.

FIG. 3: and (4) purifying the recombinant protein. Lane M: a molecular weight standard; lane T: a crude cell lysate; lane F: flow-through fraction; lane P: purified recombinant protein.

FIG. 4: titers of anti-fHBP-a 19_001 antibodies in mouse sera. Three groups of mice were: immunization with BG + fHBP-A19_001+ fHBP-B22_001, with fHBP-A19_001+ fHBP-B22_001+ alum adjuvant, and no immunization. The X-axis is the dilution factor of the serum and the Y-axis is the OD value of 450 nm.

FIG. 5: titers of anti-fHBP-B22 _001 antibodies in mouse sera. Three groups of mice were: immunization with BG + fHBP-A19_001+ fHBP-B22_001, with fHBP-A19_001+ fHBP-B22_001+ alum adjuvant, and no immunization. The X-axis is the dilution factor of the serum and the Y-axis is the OD value of 450 nm.

Detailed Description

While the present invention has been particularly shown and described with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Example 1: preparation of bacterial ghost and kinetics of protein release

Preparing a bacterial ghost:

bacterial ghosts are derived from Lactobacillus acidophilus (Lactobacillus acidophilus). The bacterial ghost was prepared by first determining the critical concentration of several compounds (sodium hydroxide, calcium carbonate, sodium dodecyl sulfate) and then by chemical means. The basic protocol was derived from Wu et al (Production of Bacterial hosts from Gram-reactive Pathologens, FOODBORNE PATHOGENS AND DISEASE Volume 14, Number1, 2017). Suspending the prepared bacterial ghostIn ddH₂O, then frozen in a microcentrifuge tube (Eppendorftube) at-80 ℃ for 1 hour, followed by freeze-drying overnight. The ghost used in the examples was prepared by this method.

Kinetics of protein release:

1) mu.l FITC-avidin (sigma A2050) (200. mu.g/100. mu.l) was added to the lyophilized bacterial ghosts and incubated at 4 ℃ for 2 hours, then frozen at-80 ℃ for 30 minutes, and then freeze-dried overnight to load FITC-avidin into 5mg of the bacterial ghosts. FIG. 1A is a bacterial ghost loaded with FITC-avidin under a fluorescent microscope.

2) ddH for bacterial ghost loaded with FITC-avidin obtained in step 1)₂O washes 3 times and after the final wash resuspended in 200. mu.l ddH₂And (4) in O. A plurality of 200. mu.l samples were prepared simultaneously according to the above procedure. Every 30 minutes (i.e., at 0, 30, 60, 90, 120, 150 minutes), one of the samples was taken and the resuspended bacterial ghosts were centrifuged at 1000x g for 5 minutes. Discarding supernatant, and applying 100 μ l B-PER to the bacterial ghost precipitate^TMBacterial protein extraction reagents (Fisher Scientific) were solubilized and incubated at room temperature for 15 minutes, after centrifugation at 15,000 Xg for 5 minutes, the supernatant was collected and fluorescence was measured using a Wallac Victor 21420 multi-label counter, the results of which are shown in FIG. 1B. The X-axis of fig. 1B represents time points (minutes) and the Y-axis represents the amount of protein remaining within the bacterial ghost.

As is clear from fig. 1B, although the release of the loaded protein is faster in the first 30 minutes, the release becomes very slow thereafter, which indicates that the bacterial ghost loaded with the biomolecules prepared by the method of the present invention has an advantage in release kinetics because a certain time is required from the injection of the delivery vehicle into the body to the phagocytosis by the antigen presenting cells, the release of the antigen protein by the ideal delivery vehicle cannot be too fast, and the bacterial ghost loaded with the antigen protein prepared by the present invention still has sufficient antigen protein to be phagocytized by the antigen presenting cells.

Example 2: selection of fHBP variants

Previous studies have shown that there is a high degree of amino acid sequence similarity in each fHBP subfamily (subfamily a and subfamily B), the highest degree of genetic diversity exists between the two subfamilies, and antisera produced against a single fHBP variant have broad bactericidal activity against strains expressing different fHBP variants of the same subfamily, including strains expressing different serosubtype (serosubtype) antigens (Flethcher et al, Vaccine Potential of the neissemia meniginia meningitidis 2086 lipoproteins, infection immun.2004apr, 72 (4): 2088 + 2100). In this study, fHBP from strain 961-5945 (serogroup B, subfamily A) (fHBP-A19001) and fHBP from strain A4 (serogroup A, subfamily B) (fHBP-B22-001) were selected as vaccine antigens. The two aforementioned strains, which have been identified and exist in china for many years, also belong to two different fHBP subfamilies.

Example 3: sequence Synthesis

Nucleic acid sequence encoding fHBP (fHBP-A19001) of Neisseria meningitidis strain 961-5945 +6 XHis-tag (SEQ ID NO: 1):

the amino acid sequence of SEQ ID NO: the first part of the underlined tag in 1 is the lipidation signal and the second part of the underlined tag is the sequence fragment encoding the 6 × His tag. SEQ ID NO: the main part of 1 is derived from the sequence of DQ523568 from GenBank, and differs from the aforementioned sequence of GenBank by the second part underlined.

Converting SEQ ID NO: 1 amino acid sequence of the protein obtained after translation (SEQ ID NO: 2):

converting SEQ ID NO: 2 (SEQ ID NO: 3):

amino acid sequence +6 × His tag of mature (without lipidation signal) fHBP-A19_001 (SEQ ID NO: 4):

amino acid sequence of mature (without lipidation signal) fHBP-A19_001 (SEQ ID NO: 5):

nucleic acid sequence encoding the nucleic acid sequence of fHBP (fHBP-B22_001) of Neisseria meningitidis strain A4 +6 XHis tag (SEQ ID NO: 6):

SEQ ID NO: the first part of the underlined tag in 6 is the lipidation signal and the second part of the underlined tag is the sequence fragment encoding the 6 × His tag. SEQ ID NO: the main part of 6 is derived from the AY330381 sequence of GenBank, and the first and second parts with underlined marks are different from the aforementioned sequence of GenBank.

Converting SEQ ID NO: 6 amino acid sequence of the protein obtained after translation (SEQ ID NO: 7):

converting the amino acid sequence of SEQ ID NO: 7 amino acid sequence of the protein obtained after removal of the 6 × His tag (SEQ ID NO: 8):

amino acid sequence +6 × His tag of mature (without lipidation signal) fHBP-B22_001 (SEQ ID NO: 9):

amino acid sequence of mature (without lipidation signal) fHBP-B22_001 (SEQ ID NO: 10):

both of the above DNA sequences (SEQ ID NO: 1 and SEQ ID NO: 6) were synthesized by Genscript (Piscataway, NJ, USA), inserted into a pUC57 plasmid through the EcoRV site, then excised by NdeI and XhoI enzymes, and inserted into pET26b bacterial expression vector cut with the same enzyme.

Example 4: expression of recombinant proteins

BL21plysE (DE3) bacteria were transformed with the expression vector obtained in example 3. Individual colonies were inoculated into 5ml of LB medium containing ampicillin and chloramphenicol and cultured overnight. Then 100. mu.l of the overnight culture was added to 50m1 LB medium containing ampicillin and chloramphenicol, and the culture was shaken until the OD 600 was about 0.6. At this time, IPTG was added at a final concentration of 1mM and the culture was shaken for additional 3 hours or overnight.

Expression of the recombinant protein was detected by SDS-PAGE gel electrophoresis, Western blotting (Western Blot) and anti-6 XHis antibody (ab14923) (Abcam, Cambridge, MA, USA). The results showed that the lipidated recombinant protein was successfully expressed in BL21plysE (DE3) bacteria.

Example 5: purification of recombinant proteins

The culture obtained in example 4 was centrifuged at 6000 to 9000x g at 4 ℃ for 15 minutes, and the supernatant was discarded to obtain a cell pellet. 5ml PBS containing 40mM imidazole, 5. mu.l MgCl were added per gram of cell pellet₂(1M), 50. mu.l of the nontoxic serine protease inhibitor Pefabloc (100mM) (Sigma-Aldrich), 5. mu.l of DNase (20mg/ml) (NEB, Ipswich, MA, USA) and 80. mu.l of lysozymeEnzyme (10mg/ml) (Sigma-Aldrich), resuspend the cell pellet, mix until a uniform cell resuspension is obtained. The cell resuspension on ice was sonicated using a Q700 sonicator (Qsonica, Newtown, CT, USA) for 15 seconds with a 45 second off cycle for 12 minutes with an amplitude of 45%. Then, to this, the detergent DDM (n-dodecyl- β -D-maltoside) (Sigma-Aldrich) was added to a final concentration of 0.8%, and stirred on ice for 1.5 hours to obtain a crude cell lysate.

Crude cell lysates were directly loaded onto 5ml HisTrap^TMFast Flow blue Cytiva column (Sigma-Aldrich) which had been equilibrated beforehand with 10 column volumes of binding buffer (PBS, 40mM imidazole, 0.1% detergent (e.g. DDM), pH 7.4). The elution was purified using an NGC Quest 10 chromatography system (Bio-rad, Hercules, Calif., USA) with a flow rate set at 1 ml/min. Elution was first performed with a gradient of 0% to 12% elution buffer for 10 column volumes, and then with a gradient of 12% to 100% elution buffer for 5 to 10 column volumes (elution buffer: PBS, 1M imidazole, 0.1% detergent (e.g., DDM), pH 7.4).

Western blot analysis of crude cell lysates, flow-through fractions, and purified proteins were performed using anti-6 x His antibody (ab14923) (Abcam, Cambridge, MA, USA). The results showed that the lipidated recombinant protein was successfully purified.

Example 6: bacterial ghost loading of recombinant proteins

5mg of the lyophilized bacterial ghosts were resuspended in 200. mu.l of a recombinant protein solution containing 10. mu.g of fHBP-A19_001, 10. mu.g of fHBP-B22_001 and 6. mu.g of the TLR9 agonist ODN2395(Invivogen, San Diego, Calif., USA) in a microfuge tube (Eppendorf tube). The previously described microcentrifuge tubes were frozen at-80 ℃ for 1 hour and then freeze-dried overnight.

Example 7: mouse immunization and serum antibody titer analysis

The bacterial ghosts prepared above and loaded with recombinant protein were used for immunization of a group of C57 mice at a dose of 5mg bacterial ghosts/mouse and by subcutaneous injectionAnd (5) epidemic disease. As controls, 10. mu.g fHBP-A19_001, 10. mu.g fHBP-B22_001, 50. mu.L PBS and 50. mu.L alum adjuvant (Imject)^TMAlum Adjuvant) (Fisher science, Waltham, MA, USA) were mixed and used for immunization of another group of C57 mice by intramuscular injection of the hind legs. Each group had 6 mice. After 14 days, two groups of mice were each injected a second time in the same manner as before. Serum was collected from the tail of the mice for analysis of antibody titer 14 days after the second injection. As a negative control, another group of 6C 57 mice was incubated under the same conditions as the treated group without immunization, and serum was collected from the tail of the mice after 28 days to analyze the antibody titer.

Antibody titers against recombinant proteins fHBP-A19_001 and fHBP-B22_001 were determined using an enzyme-linked immunosorbent assay (ELISA). Each well of a 96-well Costar plate was coated with 100. mu.l of 1. mu.g/mL recombinant protein solution (fHBP-A19_001 or fHBP-B22_001) and the plate was left at 4 ℃ overnight. After washing and incubation with blocking solution, 100 μ l of mouse serum at different dilution folds was added to each well. Secondary antibodies coupled to alkaline phosphatase and TMB substrate were used for color development. OD 450nm was recorded by ELISA reader and the results are shown in FIGS. 4 and 5. FIG. 4 shows titers of anti-fHBP-A19 _001 antibodies in mouse sera; FIG. 5 shows the titers of anti-fHBP-B22 _001 antibodies in mouse sera. The results show that the antibody titers in the sera of mice immunized with ghosts were significantly higher than those of mice not immunized with ghosts.

Example 8: mouse immunization and serum bactericidal titer analysis

Bacterial ghosts prepared according to example 6 and loaded with recombinant protein ((10. mu.g fHBP-A19-001 + 10. mu.g fHBP-B22-001 + 6. mu.g ODN2395)/5mg bacterial ghosts) were used for immunization of a group of C57 mice (5 mice/group) at a dose of 5mg bacterial ghosts/mouse and by subcutaneous injection. As controls, 10. mu.g fHBP-A19_001, 10. mu.g fHBP-B22_001, 50. mu.L PBS and 50. mu.L alum adjuvant (Imject)^TMAlum Adjuvant) (Fisher science, Waltham, MA, USA) were mixed and used for immunization of another group of C57 mice (5 mice/group) by post-leg intramuscular injection. After 14 days, two groups were smallMice were given a second injection in the same manner as before. The mice were bled 14 days after the second injection and serum samples were collected from each group of mice. As a negative control, another group of mice (5 mice/group) was incubated under the same conditions as the treatment group without immunization, and the mice were bled 28 days later and serum samples were collected.

Serum Bactericidal activity test (SBA) Assay):

neisseria meningitidis serogroup B strains were streaked to obtain individual colonies and plated on Brain Heart Infusion (BHI) medium containing 10% Horse Blood Supplement (Horse Blood Supplement) at 37 ℃ and 5% CO₂The culture was performed overnight. A portion of the individual colonies were resuspended at the appropriate density as required for the assay in 0.1% glucose in calcium and magnesium containing pbs (pcm) buffer pH 7.4.

The measurement of the bactericidal activity of complement-mediated sera was carried out according to the method of Mountzorrouros et al (Detection of complete and differentiated antibody activity in a fluorescence-based serum activity for group B Neisseria meningitidis. J.Clin. Microbiol.38: 2878-2884.) using sera from human donors as source of complement. To the wells of the test plate were added test solutions consisting of 25. mu.l PCM buffer, 5. mu.l hot (56 ℃ for 30 minutes) inactivated serial diluted (two-fold dilution) test mouse serum, 10. mu.l human complement and 10. mu.l containing approximately 1X10³To 3x10³The PCM buffer solution of the live neisseria meningitidis is mixed. The test plates were incubated at 37 ℃ for 30 minutes. Subsequently 200. mu.l of modified Frantz growth medium containing Almarblue dye (Fisher Scientific) and 0.7% low melting agarose diluted 1: 20 were added to each well containing the test solution, and the test plate was incubated overnight at 37 ℃. With Fluoroskan^TMThe fluorescence signal (generated by the reaction of alamar blue dye and live bacteria) was read by a microplate fluorescence analyzer (Fisher Scientific). For negative control, 30. mu.l PCM was added to another well of the test plate, 5. mu.l of test mouse serum was not added, other components and reaction conditions were followed in the above experimentThe groups are identical. In addition, different known amounts of Neisseria meningitidis and Amar blue dye were added to the other set of wells of the test plate, respectively, without the addition of the test serum, to generate a standard curve (Y-axis is the value of the fluorescence signal and X-axis is the number of bacteria). According to the standard curve, the number of bacteria after reaction in the experimental group and the negative control is calculated. The number of killed bacteria was obtained by subtracting the number of bacteria after the reaction from the number of bacteria before the reaction. The feasibility and accuracy of the above assay was confirmed using sera of known bactericidal titres as positive controls.

The results of the SBA test showed that the sera of the mice immunized with the ghost produced significantly higher bactericidal power against the different strains than the mice immunized without the ghost (see table one).

The bactericidal titer (bactericidal titer) in table one refers to the reciprocal of the maximum serum dilution that kills greater than 50% of the bacteria compared to the negative control. If a serum sample shows a bactericidal rate of < 50% at the lowest serum dilution (lowest serum dilution of 1: 25), the bactericidal titer of that sample is reported to be < 30.

Table one: bactericidal titres of mouse serum against different strains of neisseria meningitidis serogroup B

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Claims (26)

1. a neisseria meningitidis vaccine comprising a bacterial ghost and factor H binding protein (fHBP).

2. The vaccine of claim 1, wherein the factor H binding protein (fHBP) is a lipidated or non-lipidated fHBP.

3. The vaccine of claim 1 or2, wherein the factor H binding protein (fHBP) comprises at least one fHBP of subfamily a and/or at least one fHBP of subfamily B.

4. The vaccine of claim 3, wherein the subfamily A factor H binding protein (fHBP) is a polypeptide that differs from the amino acid sequence of SEQ ID NO: 5 a fHBP protein having at least 85% amino acid sequence identity.

5. The vaccine of claim 3 or 4, wherein the subfamily B factor H binding protein (fHBP) is a polypeptide that differs from the amino acid sequence of SEQ ID NO: 10 a fHBP protein having at least 85% amino acid sequence identity.

6. The vaccine of any one of claims 3-5, wherein the sequence of the subfamily A factor H binding protein (fHBP) is as set forth in SEQ ID NO: 5, and/or wherein the sequence of the subfamily B factor H binding protein (fHBP) is as shown in SEQ ID NO: shown at 10.

7. The vaccine of any one of claims 1-6, further comprising one or more adjuvants.

8. The vaccine of claim 7, wherein the adjuvant is a TLR9 agonist.

9. The vaccine of any one of claims 1-8, wherein the factor H binding protein (fHBP) is loaded within the bacterial ghost.

10. The vaccine of any one of claims 7-9, wherein the adjuvant is loaded within the bacterial ghost.

11. The vaccine of any one of claims 1-10, which is useful for humans or mammals.

12. The vaccine of any one of claims 1-11, wherein the bacterial ghosts are of lactobacillus acidophilus.

13. Use of a composition comprising a bacterial ghost and a factor H binding protein (fHBP) in the manufacture of a vaccine for the prevention or treatment of infection by neisseria meningitidis or a disease induced thereby.

14. Use of a composition comprising a bacterial ghost and a factor H binding protein (fHBP) in the manufacture of a vaccine for the prevention or treatment of infection by neisseria meningitidis serogroup B or a disease induced thereby.

15. The use according to claim 13 or 14, wherein the factor H binding protein (fHBP) is a lipidated or non-lipidated fHBP.

16. The use of any one of claims 13-15, wherein the factor H binding protein (fHBP) comprises at least one fHBP of subfamily a and/or at least one fHBP of subfamily B.

17. The use according to claim 16, wherein the factor H binding protein of subfamily a (fHBP) is a polypeptide that differs from SEQ ID NO: 5 a fHBP protein having at least 85% amino acid sequence identity.

18. Use according to claim 16 or 17, wherein the subfamily B factor H binding protein (fHBP) is a polypeptide which differs from SEQ ID NO: 10 a fHBP protein having at least 85% amino acid sequence identity.

19. The vaccine of any one of claims 16-18, wherein the sequence of the subfamily a factor H binding protein (fHBP) is as set forth in SEQ ID NO: 5, and/or wherein the sequence of the subfamily B factor H binding protein (fHBP) is as shown in SEQ ID NO: shown at 10.

20. The use of any one of claims 13-19, the composition further comprising one or more adjuvants.

21. The use of claim 20, wherein the adjuvant is a TLR9 agonist.

22. The use of any one of claims 13-21, wherein the factor H binding protein (fHBP) is loaded within the bacterial ghost.

23. The use of any one of claims 20-22, wherein the adjuvant is loaded within the bacterial ghost.

24. The use of any one of claims 13-23, wherein the vaccine is for use in a human or a mammal.

25. The use of any one of claims 13 to 24, wherein the disease comprises Invasive Meningococcal Disease (IMD).

26. The use of any one of claims 13-25, wherein the bacterial ghost is a ghost of lactobacillus acidophilus.

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