Neisseria meningitidis vaccine and its application

Abstract

The present invention relates to vaccines, uses and methods for preventing or treating infection with Neisseria meningitidis or diseases induced therefrom. In one aspect, the present invention relates to a Neisseria meningitidis vaccine comprising bacterial ghosts and factor H binding protein (fHBP) for preventing or treating infection with Neisseria meningitidis or diseases induced therefrom, such as invasive meningococcal disease (IMD). In another aspect, the present invention relates to the use of a composition comprising bacterial ghosts and fHBP in the preparation of a vaccine for preventing or treating infection with Neisseria meningitidis or diseases induced therefrom, such as IMD. In another aspect, the present invention relates to a method for preventing or treating infection with Neisseria meningitidis or diseases induced therefrom, such as IMD, comprising administering to a subject a prophylactically effective amount or a therapeutically effective amount of a Neisseria meningitidis vaccine.

Description

Technical Field

[0001] The present invention relates to vaccines, compositions, uses and methods for preventing or treating Neisseria meningitidis infection or diseases caused by it. Background Art

[0002] As one of the main causes of global bacterial meningitis, Neisseria meningitidis (Neisseriameningitidis) can be divided into 13 serogroups according to its capsule polysaccharide, wherein 6 serogroups (A, B, C, W, X, Y) can cause invasive meningococcal disease (invasive meningococcal disease, IMD) (Harrison etc., nd).The invasive disease case number in the whole world every year is at least about 1.2 million, and wherein 135,000 deaths are relevant to IMD (" Epidemics of meningococcal disease.African meningitis belt, 2001 ", 2001).

[0003] To combat IMD, many industrialized countries have developed different meningococcal vaccine formulations for use in routine immunization programs. For example, a quadrivalent vaccine containing a conjugate of proteins and polysaccharides from serogroups A, C, W-135, and Y was introduced in the United States and is recommended for routine use in subjects aged 11 years and older (Pace et al., 2009). In addition, a more immunogenic quadrivalent conjugate vaccine and a conjugate vaccine targeting Haemophilus influenzae type b and meningococcal C and Y are in clinical development for use in infants (Nolan et al., 2007). In the United States, serogroup B meningococcal disease accounts for over 50% of all meningococcal disease, and this proportion is 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, until recently, when the FDA approved GlaxoSmithKline's BEXSERO and Pfizer's TRUMENBA.

[0004] Conjugation to a carrier protein is known to render polysaccharide antigens immunogenic in infants and to elicit anamnestic anti-capsular antibody responses (Granoff and Pollard, 2007), a principle underlying vaccines for all meningococcal serogroups except serogroup B. The polysaccharide of meningococcal serogroup B is a homogeneous linear polymer of α-(2-8)N-acetylneuraminic acid (polysialic acid) and is an autoantigen (Finne et al., 1983); even when conjugated to a protein carrier, serogroup B polysaccharide remains a weak immunogen (Jennings and Lugowski, 1981). Attempts to enhance immunogenicity have been made to derivatize the polysaccharide with n-propionyl groups, but this has met with limited success in mice, but not in humans (Bruge et al., 2004; Jennings et al., 1987).

[0005] Current research on vaccines against meningococcal serogroup B focuses on non-capsular antigens, such as proteins or lipopolysaccharide (LPS). By utilizing screening technologies such as genome mining, proteomics and immunological methods, a large number of novel vaccine candidates against serogroup B meningococcal disease have been identified, 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), 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), Neisserial adhesin A (NadA, also known as GNA 1994) (Capecchi et al., 2005; Beernink et al., 2007; Comanducci et al., 2004) and other proteins (Grifantini et al., 2002; Pizza et al., 2000). Unfortunately, for various reasons, the potential of almost all of the candidate proteins identified so far as antigens for vaccines against serogroup B meningococci alone is very limited. These reasons include antigenic variability (FetA and Opc), lack of genes from strains of certain highly toxic lineages (NadA), phase variability (Phase variability) (Opc), low levels of constitutive expression of antigens by certain strains (fHbp, GNA 2132 and NspA), and some important conformational epitopes that are difficult to express in recombinant form (NspA) (Halperin et al., 2007; Hou et al., 2003). Therefore, current approaches focus on using multiple bacterial proteins to achieve broad protection against different strains of serogroup B. BEXSERO from GSK and TRUMENBA from Pfizer are both formulated with multiple proteins (fusion proteins).

[0006] It is known that subunit vaccines based on proteins or polysaccharides have poor immunogenicity compared to vaccines based on inactivated or attenuated pathogens. Therefore, immune adjuvants are often used with antigens to enhance the immune response. Another approach is to conjugate proteins to polysaccharide antigens as described previously.

[0007] Bacterial ghosts (BGs) are the inanimate, empty cell envelopes of bacteria that result from the release of bacterial cytoplasm through channels in the bacterial cell envelope, which can be achieved by controlled expression of the bacteriophage PhiX174 lytic protein E in Gram-negative bacteria or by compound-based critical concentration methods in Gram-positive bacteria (“Dynamics of PhiX174 protein E-mediated lysis of Escherichia coli | SpringerLink,” n.d.; Amara et al., 2013; Wu et al., 2017).

[0008] Bacterial ghosts retain the intact antigenic structure of native bacterial surfaces and can be used directly as vaccines (Langemann et al., 2010; Kudela et al., 2010; Riedmann et al., 2007). Bacterial ghosts are also excellent delivery vehicles for biomacromolecules such as antigens, drugs, and DNA (Lubitz, 2001; Jalava et al., 2003; Mayr et al., 2005; Muhammad et al., 2012). Furthermore, bacterial ghosts contain known innate immune-stimulating components and have the potential to serve as effective adjuvants. Specifically, bacterial ghosts retain antigenic components of native bacterial surfaces, such as lipopolysaccharide (LPS), flagellin, peptidoglycan, and many other types of substances, which are ligands for various pattern recognition receptors (PRRs) and are collectively referred to as pathogen-associated molecular patterns (PAMPs) (Huter et al., 1999; Lubitz, 2001; Muhammad et al., 2012). These structures can be effectively recognized and taken up by immune and non-immune cells (Ebensen et al., 2004; Abtin et al., 2010; Stein et al., 2013), and then activate cells mainly through the Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) pathways (Adam et al., 2010; Quevedo-Diaz et al., 2010), as well as achieve adjuvant activity through multiple Toll-like receptors (TLRs) present on many immune and non-immune cells. Summary of the Invention

[0009] In one aspect, the present invention provides a Neisseria meningitidis vaccine comprising bacterial ghosts and factor H binding protein (fHBP), wherein the fHBP comprises 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 subfamily A fHBP is an 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 to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or an fHBP protein having 1, 2, 3, 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 compared to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, the fHBP of subfamily B is an 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 to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; or In some embodiments, the fHBP protein comprises an fHBP having 1, 2, 3, 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 compared to SEQ ID NO: 10. In some embodiments, the substitutions are conservative substitutions. In some embodiments, the fHBP of subfamily A is from a serogroup B strain, e.g., strain 961-5945, CDC1034, CDC2369, or 870446. In some embodiments, the fHBP of subfamily B is from a serogroup A strain, e.g., strain A4. In some embodiments, the fHBP of subfamily B is from a serogroup B strain, e.g., strain CDC-1343, CDC-983, or CDC-852. In some embodiments, the sequence of the factor H binding protein of subfamily A is as shown in SEQ ID NO: 5.In some embodiments, the sequence of the factor H binding protein of subfamily B is shown in SEQ ID NO: 10. In some embodiments, the fHBP is loaded into bacterial ghosts. 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 into bacterial ghosts. In some embodiments, the bacterial ghosts of the present invention are ghosts of Lactobacillus acidophilus. In some embodiments, the vaccine is used to prevent or treat infection with Neisseria meningitidis serogroups A, B, C, W, X, and / or Y, or diseases induced therefrom. In some embodiments, the vaccine is used to prevent or treat infection with serogroups A and / or B, or diseases induced therefrom. In some embodiments, the vaccine is used to prevent or treat infection with serogroups B, or diseases induced therefrom. In some embodiments, the disease includes invasive meningococcal disease (IMD). In some embodiments, the vaccine is suitable for use in humans, poultry, livestock, and / or mammals. In some embodiments, the vaccine is in a lyophilized dosage form.

[0010] On the other hand, the present invention provides a composition for the preparation of a vaccine for preventing or treating an infection with Neisseria meningitidis or a disease induced therefrom, the composition comprising bacterial ghosts and factor H binding protein (fHBP). The vaccine can be used to prevent or treat an infection with serogroup B or a disease induced therefrom, an infection with serogroup A or a disease induced therefrom, an infection with serogroup C or a disease induced therefrom, an infection with serogroup W or a disease induced therefrom, an infection with serogroup X or a disease induced therefrom, and / or an infection with serogroup Y or a disease induced therefrom. 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 subfamily A fHBP is an 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 to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5; or an fHBP protein having 1, 2, 3, 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 compared to SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5. In some embodiments, the subfamily B factor H binding protein (fHBP) is an 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 to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10; or an 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 to SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. fHBP proteins having 1, 2, 3, 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 compared to NO: 10. In some embodiments, the substitutions are conservative substitutions.In some embodiments, the fHBP of subfamily A is from a serogroup B strain, e.g., strains 961-5945, CDC1034, CDC2369, or 870446. In some embodiments, the fHBP of subfamily B is from a serogroup A strain, e.g., strain A4. In some embodiments, the fHBP of subfamily B is from a serogroup B strain, e.g., strains CDC-1343, CDC-983, or CDC-852. In some embodiments, the sequence of the factor H binding protein of subfamily A is shown in SEQ ID NO:5, and / or the sequence of the factor H binding protein of subfamily B is shown in SEQ ID NO:10. In some embodiments, the fHBP is encapsulated within bacterial ghosts. 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 encapsulated within the bacterial ghosts. In some embodiments, the bacterial ghosts are of Lactobacillus acidophilus. In some embodiments, the vaccine can be used for humans, poultry, livestock and / or mammals.

[0011] In another aspect, the present invention provides a method for preventing or treating infection by Neisseria meningitidis serogroups A, B, C, W, X, and / or Y, or a disease induced therefrom, comprising administering to a subject an immunogenically effective amount, a prophylactically effective amount, or a therapeutically effective amount of a vaccine of the present invention; the mode of administration, frequency of administration, and dosage are determined based on the subject's condition. In some embodiments, the subject of this method is a human, poultry, livestock, and / or mammal. In some embodiments, this method can prevent or treat infection by Neisseria meningitidis serogroups A, B, C, W, X, and / or Y, or a disease induced therefrom, including invasive meningococcal disease (IMD). In some embodiments, the subject of the present method is a subject not infected with Neisseria meningitidis serogroups A, B, C, W, X and / or Y, a subject infected with Neisseria meningitidis serogroups A, B, C, W, X and / or Y but not showing symptoms of disease caused by them, or a subject suffering from disease caused by Neisseria meningitidis serogroups A, B, C, W, X and / or Y.

[0012] In another aspect, the present invention provides a method for loading fHBP and an adjuvant into bacterial ghosts, the method comprising resuspending lyophilized bacterial ghosts with a protein solution containing one or more proteins to be loaded, optionally containing an adjuvant, and then freeze-drying. In some embodiments, 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, and more preferably about 1 hour. In some embodiments, the freeze-drying time is 12-20 hours, preferably 13-19 hours, preferably 14-18 hours, and preferably 15-17 hours. In some embodiments, the mixture of protein solution and bacterial ghosts can be incubated at 4°C prior to freezing. In some embodiments, the incubation time at 4°C is 1-3 hours, preferably 1.5-2.5 hours, and preferably about 2 hours. To increase the loading amount of recombinant protein in bacterial ghosts, the above steps can be repeated.

[0013] The present invention significantly increases the antibody titer / valence against Neisseria meningitidis in serum by loading fHBP into bacterial ghosts. By combining fHBP of subfamily A and fHBP of subfamily B, 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, providing 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 antibody titer against fHBP of subfamily A and fHBP of subfamily B in serum, and significantly increases the bactericidal power of serum against all tested strains. In addition, the ghosts of Lactobacillus acidophilus used in the present invention have advantages in release kinetics, which is one of the reasons why it has an excellent effect in eliciting an immune response. Specifically, since it takes a certain amount of time for the delivery vector to be injected into the body and engulfed by antigen-presenting cells, the ideal delivery vector cannot release the antigen protein too quickly. However, the examples of this application show that the bacterial ghosts of the present invention can still release sufficient antigen protein when engulfed by antigen-presenting cells. Furthermore, the vaccine of the present invention can be a freeze-dried product, so it can be transported over short distances or stored for a short period of time (within one month) at room temperature, without the need for cold chain transportation or low-temperature storage. Detailed Description of the Invention

[0015] Neisseria meningitidis (also known as "meningococcus") is a Gram-negative encapsulated bacterium that can be divided into 13 serogroups based on the polysaccharides on its capsule. Six serogroups (A, B, C, W, X, and Y) can cause invasive meningococcal disease (IMD). IMD commonly manifests as meningococcal meningitis and / or meningococcemia / septicemia, and less commonly as meningococcal pneumonia, septic arthritis, epiglottitis, or otitis media.

[0016] Factor H binding protein (fHBP) (also known as LP2086, GNA1870, or ORF2086) is a lipoprotein expressed in nearly all serogroup B strains and some serogroups A, C, W, X, and Y strains, anchored to the outer membrane of meningococci by lipid molecules. Based on deduced amino acid sequence homology, fHBP can be divided into two distinct subfamilies (i.e., subfamily A and subfamily B). Variants belonging to the same subfamily share at least 83% amino acid sequence similarity. Variants belonging to different subfamilies share 60%-75% amino acid sequence similarity. Known strains expressing fHBP variants of subfamily A or B are described in published papers (e.g., Flethcher et al., Vaccine Potential of the Neisseria meningitidis 2086 Lipoprotein, Infect Immun. 2004 Apr, 72(4): 2088-2100; Murphy et al., Sequence Diversity of the Factor H Binding Protein Vaccine Candidate in Epidemiologically Relevant Strains of Serogroup B Neisseria meningitidis, J Infect Dis. 2009 Aug 1, 200(3): 379-89). Those skilled in the art can also classify newly discovered fHBP variants into subfamily A or B using these published methods.

[0017] "Lipidation" (also known as "lipidation") refers to the covalent binding of lipids to proteins, which can be achieved in vivo through post-translational modification of proteins or in vitro through chemical synthesis. Natural fHBP usually contains an N-terminal cysteine to which a lipid group can be covalently attached. For example, the amino group of the cysteine residue at the N-terminus of fHBP is connected to a fatty acid (R1) by forming an amide bond, and the cysteine sulfhydryl group is connected to a 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, etc.). The cysteine residue at the N-terminus of fHBP is typically lipidated in naturally occurring fHBP, but may be lipidated in the fHBP of the present invention. Therefore, in the amino acid sequences described herein, references to cysteine at this specific position include both unmodified cysteine and lipidated cysteine. Therefore, the fHBP of the present invention may be lipidated or non-lipidated. The method of producing lipidated fHBP using recombinant protein technology is well known to those skilled in the art, which includes using a natural fHBP nucleic acid sequence containing a lipidation signal, or an fHBP nucleic acid sequence containing a modified or foreign lipidation signal, and purifying the lipidated mature fHBP from the membrane fraction of Escherichia coli by a detergent extraction method. The mature fHBP does not contain a lipidation signal, and the lipid molecule is linked to the cysteine at the N-terminus of the 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, including the use of a native fHBP nucleic acid sequence that does not contain a lipidation signal, or an fHBP nucleic acid sequence with an N-terminal alteration that does not contain a lipidation signal. These alterations include changing the length of the "glycine / serine stem" downstream of the N-terminal cysteine residue, which can affect the stability or expression level of non-lipidated fHBP (see CN103096920B, WO2012 / 032489, US20120093852, WO2013 / 132452, US20160030543, etc.). Methods for combining lipids with proteins in vitro are also well known to those skilled in the art.

[0018] "Bacterial ghost" (BG) (also known as "bacterial exuviae") refers to an empty bacterial shell that does not contain bacterial cell contents such as nucleic acids and cytoplasm. Bacterial ghosts can be derived from Gram-negative bacteria or Gram-positive bacteria. Methods for 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 bacteriophage PhiX174 comprises cloning the lytic gene E into an expression control system to achieve controllable expression of the lytic gene E, thereby lysing Gram-negative bacteria. The expression control 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 hemolytica, Pasteurella multocida, Edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophila, etc. For example, a chemical preparation method that does not rely on the lytic gene E can also be used. The chemical preparation method breaks through the limitations of relying on the lytic gene E to prepare bacterial ghosts and can be effectively applied to Gram-positive bacteria, such as Staphylococcus aureus, Sterility, Streptococcus pneumoniae, Bacillus anthracis, Corynebacterium diphtheriae, and Clostridium tetani. The bacterial ghosts of the present invention include ghosts of various Gram-negative and Gram-positive bacteria, such as ghosts of various Escherichia coli strains, Salmonella typhimurium, Salmonella enteritidis, Vibrio cholerae, Klebsiella pneumoniae, Helicobacter pylori, Actinobacillus pleuropneumoniae, Haemophilus influenzae, Pasteurella hemolytica, Pasteurella multocida, Edwardsiella tarda, Vibrio anguillarum, Aeromonas hydrophila, Staphylococcus aureus, Sterility, Streptococcus pneumoniae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium tetani, and Lactobacillus acidophilus. The bacterial ghosts retain the basic structure of the bacterial cell envelope. The bacterial cell envelope refers to the multilayered structure that surrounds and protects the cytoplasm, including the cytoplasmic membrane. The cell envelope of Gram-negative bacteria consists of three main layers from the inside out: the cytoplasmic membrane, peptidoglycan, and the outer membrane. The cell envelope of Gram-positive bacteria consists of two main layers from the inside out: the cytoplasmic membrane and peptidoglycan.

[0019] "Adjuvant" refers to an auxiliary substance that is injected into the body together with an antigen or in advance and can enhance the body's immune response to the antigen or change the type of immune response. The specific substances included are well known to those skilled in the art, for example: Toll-like receptor (TLR) agonists, aluminum salts, calcium phosphate, oil-in-water emulsions, Freund's adjuvant, inactivated bacteria, cytokines IL-1, IL-2, IL-12, etc.

[0020] An "agonist" is a substance that binds to a cell receptor and induces a response. Such a response may be an increase in activity mediated by the receptor. Agonists typically mimic the effects of a naturally occurring substance, such as a ligand.

[0021] "TLR9 agonist" refers to a Toll-like receptor 9 agonist. TLR9 recognizes specific unmethylated CpG oligonucleotide (ODN) sequences to distinguish between microbial DNA and mammalian DNA, and therefore TLR9 agonists include various 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, and 7,427,405, including respective Tables 1 and 2A-2D, the entire contents of which are incorporated herein by reference in their entirety.

[0022] "Vaccine" refers to a biological or pharmaceutical preparation containing an antigen. The preparation may be a composition that, in addition to the antigen, may also contain other ingredients such as an adjuvant and a pharmaceutically acceptable carrier. The vaccines of the present invention include preventive vaccines and therapeutic vaccines. Preventive vaccines can provide acquired immunity against specific pathogenic microorganisms, diseases, tumors or cancers before infection with pathogenic microorganisms, or before the occurrence of diseases, tumors or cancers; therapeutic vaccines can treat or prevent the worsening of the infection, disease, tumor or cancer after infection with pathogenic microorganisms, or after the occurrence of a certain disease, tumor or cancer. Treatment includes complete cure and relief of some or all symptoms. In some embodiments, the vaccine of the present invention is a freeze-dried product, or a product to be injected obtained by resuspending the freeze-dried product in a pharmaceutically acceptable solution or carrier. The freeze-dried product of the present invention may also include the use of a pharmaceutically acceptable carrier or solution during preparation.

[0023] "Pharmaceutically acceptable" refers to those compounds, materials, compositions and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0024] "Pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, formulation aid (such as a lubricant, talc, magnesium stearate, calcium stearate or zinc stearate or stearic acid). Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful 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 carboxymethylcellulose, ethylcellulose and cellulose acetate; (4) tragacanth; (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, For example, 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) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (9) ethanol; (20) pH buffer solutions (such as PBS, etc.); (21) polyesters, polycarbonates, and / or polyanhydrides; and (22) other non-toxic compatible substances used in pharmaceutical preparations. The vaccine of the present invention can be present in various dosage forms, including liquid dosage forms, lyophilized dosage forms, oral dosage forms, etc., and the route of administration can be reasonably selected according to the corresponding dosage form, such as oral administration, intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, nasal administration, etc. The dosage and number of administrations of the vaccine of the present invention can be determined according to the specific conditions of the subject.

[0025] As used herein, "treating" refers to alleviating some or all symptoms of a disease in a subject, or curing a disease; the relief is compared to the condition without the use of the same therapeutic agent.

[0026] The "therapeutically effective amount" herein refers to an amount that can induce an immune response in a subject and achieve a therapeutic effect.

[0027] The term "prevention" as used herein refers to preventing a bacterial infection or the occurrence of a disease in a subject.

[0028] The term "prophylactically effective amount" as used herein refers to an amount that can induce an immune response in a subject and is expected to achieve a preventive effect.

[0029] As used herein, an "immunogenically effective amount" refers to an amount that is capable of eliciting an immune response in a subject.

[0030] As used herein, an "immune response" includes a cellular (T cell) response or a humoral (B cell or antibody) response, or both.

[0031] As used herein, "subject" refers to a human or animal receiving therapeutic or prophylactic treatment. "Incubation" and "culturing" are sometimes used interchangeably herein to refer to placing reactants under specific conditions for a period of time.

[0032] "Freeze drying" ("lyophilization") refers to a drying process in which a water-containing material is frozen below freezing, the water is converted to ice, and then the ice is removed by converting it to vapor under a relatively high vacuum. Freeze drying methods and equipment are well known to those skilled in the art.

[0033] As used herein, "amino acid sequence identity" refers to the percentage of amino acids in a candidate sequence that are identical to the amino acids in the reference sequence, after aligning the sequences and, if necessary, introducing spaces (to achieve maximum sequence identity percentage). Alignment for purposes of determining percent sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as Needle, BLAST, BLAST-2, ALIGN, ALIGN-2, CD-HIT, or Megalign (DNASTAR) software. Suitable parameters for measuring the alignment can be determined by known methods, including any algorithm required for achieving maximum alignment over the full length of the sequence being compared. In some embodiments, amino acid sequence identity is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS program package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet 16: 276-277), preferably version 5.0.0 or later. The parameters used are 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 labeled "longest identity" (obtained using the -nobrief option) was used as the percent identity and was calculated as follows: (identical amino acid residues x 100) / (length of alignment - total number of gaps in the alignment).

[0034] "Conservative substitutions" refer to substitutions 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 and include 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), non-polar 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).

[0035] As used herein, “about” 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, including the endpoints of the range and any value within the range.

[0036] As used herein, “include,” “comprising,” and “having” mean that the substance, composition, system, or method at least contains, but is not limited to, the described structure, ingredient, element, component, feature, or step; “include,” “comprising,” and “having” are intended to indicate the presence of the described structure, ingredient, element, component, feature, or step, but do not exclude the presence of any other structure, ingredient, element, component, feature, or step. In this article, when a compound is mentioned as containing, including, or having one or more structures, it should be understood that it also covers compounds composed of this or these structures; when a product or composition is mentioned as containing, including, or having one or more ingredients, it should be understood that it also covers products or compositions composed of this or these ingredients; when a method is mentioned as containing, including, or having one or more steps, it should be understood that it also covers methods composed of this or these steps.

[0037] As used herein, the term "and / or" is intended to include any possible combination of one or more of the listed items. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The present invention will be more fully understood through the following detailed description taken in conjunction with the accompanying drawings, in which:

[0039] Figure 1 : A. Bacterial ghosts loaded with FITC-avidin under a fluorescence microscope; B. Protein release kinetics from bacterial ghosts.

[0040] Figure 2: Recombinant protein expression. Part A shows the expression of A19_001; Part B shows the expression of B22_001. Leftmost lane: Molecular weight marker; Lanes 1 and 4: Uninduced; Lanes 2 and 5: 3-hour induction; Lanes 3 and 6: Overnight induction.

[0041] Figure 3 : Purification of recombinant protein. Lane M: molecular weight standard; Lane T: crude cell lysate; Lane F: flow-through fraction; Lane P: purified recombinant protein.

[0042] 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 unimmunized. The X-axis represents the serum dilution factor, and the Y-axis represents the OD value at 450 nm.

[0043] 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 unimmunized. The X-axis represents the serum dilution factor, and the Y-axis represents the OD value at 450 nm. DETAILED DESCRIPTION

[0044] The technical solutions of the present invention are further described in detail below through examples and in conjunction with the accompanying drawings. However, the present invention is not limited to the following examples. Without departing from the spirit and scope of the present invention, ordinary technicians in this field can make various changes and improvements to the present invention in form and details, and these are all considered to fall within the scope of protection of the present invention.

[0045] Example 1: Preparation of bacterial ghosts and protein release kinetics

[0046] Preparation of bacterial shadows:

[0047] Bacterial ghosts are derived from Lactobacillus acidophilus. First, the critical concentrations of several compounds (sodium hydroxide, calcium carbonate, sodium lauryl sulfate) are determined, and then bacterial ghosts are prepared by chemical methods. The basic experimental protocol comes 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 bacterial ghosts are suspended in ddH2O, then frozen in an Eppendorf tube at -80°C for 1 hour, and then freeze-dried overnight. The bacterial ghosts used in the examples were all prepared by this method.

[0048] Protein release kinetics:

[0049] 1) Add 100 μl of FITC-avidin (Sigma A2050) (200-350 μg / 100 μl) to the lyophilized bacterial ghosts and incubate at 4°C for 2 hours, then freeze at -80°C for 30 minutes and then freeze-dry overnight to load FITC-avidin into 5 mg of bacterial ghosts. Figure 1 A is the bacterial shadow loaded with FITC-avidin under a fluorescence microscope.

[0050] 2) Wash the bacterial ghosts loaded with FITC-avidin obtained in step 1) three times with ddH2O and resuspend in 200μl ddH2O after the final wash. Prepare multiple 200μl samples at the same time according to the above steps. Take one of the samples every 30 minutes (i.e. at 0, 30, 60, 90, 120, and 150 minutes), and centrifuge the resuspended bacterial ghosts at 1000x g for 5 minutes. After discarding the supernatant, the bacterial ghost pellet was precipitated with 100μl B-PER TM The cells were dissolved with bacterial protein extraction reagent (Fisher Scientific) and incubated at room temperature for 15 min. After centrifugation at 15,000 × g for 5 min, the supernatant was collected and fluorescence was measured using a Wallac Victor21420 multilabel counter. The results were as follows: Figure 1 As shown in B. Figure 1 The X-axis of panel B represents the time point (minutes), and the Y-axis represents the amount of protein remaining in the bacterial ghost.

[0051] from Figure 1It can be clearly seen in Figure B that although the released protein is relatively fast in the first 30 minutes, the release becomes very slow thereafter. This indicates that the bacterial ghosts loaded with biomolecules prepared by the method of the present invention have advantages in release kinetics. Because it takes a certain amount of time for the delivery vector to be injected into the body and then be phagocytosed by antigen-presenting cells, an ideal delivery vector cannot release the antigen protein too quickly. However, the bacterial ghosts loaded with antigen proteins prepared by the present invention still have sufficient antigen protein when being phagocytosed by antigen-presenting cells.

[0052] Example 2: Selection of fHBP variants

[0053] Previous studies have shown that there is a high degree of amino acid sequence similarity within 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 a wide range of bactericidal activity against strains expressing different fHBP variants of the same subfamily, including strains expressing different serosubtype antigens (Flethcher et al., Vaccine Potential of the Neisseria meningitidis 2086 Lipoprotein, Infect Immun. 2004 Apr, 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 above two strains are strains that have existed and been identified in China for many years and they also belong to two different fHBP subfamilies.

[0054] Example 3: Sequence Synthesis

[0055] Nucleic acid sequence encoding fHBP (fHBP-A19001) of Neisseria meningitidis strain 961-5945 + nucleic acid sequence of 6x His tag (SEQ ID NO: 1):

[0056]

[0057] The underlined first portion of SEQ ID NO: 1 is the lipidation signal, and the underlined second portion is the sequence encoding the 6x His tag. The majority of SEQ ID NO: 1 is derived from GenBank sequence DQ523568; the difference from the GenBank sequence is the addition of the underlined second portion.

[0058] The amino acid sequence of the protein obtained by translating SEQ ID NO: 1 (SEQ ID NO: 2):

[0059]

[0060] The amino acid sequence of the protein obtained after removing the 6x His tag of SEQ ID NO: 2 (SEQ ID NO: 3):

[0061]

[0062] Amino acid sequence of mature (without lipidation signal) fHBP-A19_001 + 6x His tag (SEQ ID NO: 4):

[0063]

[0064] Amino acid sequence of mature (without lipidation signal) fHBP-A19_001 (SEQ ID NO: 5):

[0065]

[0066] Nucleic acid sequence encoding fHBP of Neisseria meningitidis strain A4 (fHBP-B22_001) + 6x His tag (SEQ ID NO: 6):

[0067]

[0068] The underlined first portion of SEQ ID NO:6 is the lipidation signal, and the underlined second portion is the sequence encoding the 6x His tag. The majority of SEQ ID NO:6 is derived from GenBank sequence AY330381; the difference from the GenBank sequence is the addition of the underlined first and second portions.

[0069] The amino acid sequence of the protein obtained by translating SEQ ID NO: 6 (SEQ ID NO: 7):

[0070]

[0071] The amino acid sequence of the protein obtained after removing the 6x His tag of SEQ ID NO: 7 (SEQ ID NO: 8):

[0072]

[0073] Amino acid sequence of mature (without lipidation signal) fHBP-B22_001 + 6x His tag (SEQ ID NO: 9):

[0074]

[0075] Amino acid sequence of mature (without lipidation signal) fHBP-B22_001 (SEQ ID NO: 10):

[0076]

[0077] The two DNA sequences (SEQ ID NO: 1 and SEQ ID NO: 6) were synthesized by Genscript (Piscataway, NJ, USA) and inserted into the pUC57 plasmid via the EcoRV site. They were then excised by NdeI and XhoI enzymes and inserted into the pET26b bacterial expression vector after being cut with the same enzymes.

[0078] Example 4: Expression of recombinant protein

[0079] BL21 plysE (DE3) bacteria were transformed using the expression vector obtained in Example 3. A single colony was inoculated into 5 ml of LB medium containing ampicillin and chloramphenicol and cultured overnight. 100 μl of the overnight culture was then added to 50 ml of LB medium containing ampicillin and chloramphenicol, and the culture was shaken until the OD 600 reached approximately 0.6. At this point, IPTG was added to a final concentration of 1 mM, and the culture was shaken for an additional 3 hours or overnight.

[0080] The expression of the recombinant protein was detected by SDS-PAGE gel electrophoresis, Western blotting and anti-6x His antibody (ab14923) (Abcam, Cambridge, MA, USA). The results showed that the lipidated recombinant protein was successfully expressed in BL21plysE(DE3) bacteria.

[0081] Example 5: Purification of recombinant protein

[0082] The culture obtained in Example 4 was centrifuged at 6000 to 9000 x g for 15 minutes at 4°C, and the supernatant was discarded to obtain a cell pellet. Per gram of cell pellet, 5 ml of PBS containing 40 mM imidazole, 5 μl of 1 M MgCl2, 50 μl of the non-toxic serine protease inhibitor Pefabloc (100 mM) (Sigma-Aldrich), 5 μl of 20 mg / ml DNase (NEB, Ipswich, MA, USA), and 80 μl of 10 mg / ml lysozyme (Sigma-Aldrich) were added. The cell pellet was resuspended and mixed until a homogenous cell suspension was obtained. The cell suspension was sonicated on ice using a Q700 ultrasonic disruptor (Qsonica, Newtown, CT, USA) with a 15-second on, 45-second off cycle for a total of 12 minutes at an amplitude of 45%. Subsequently, detergent DDM (n-dodecyl-β-D-maltoside) (Sigma-Aldrich) was added thereto to a final concentration of 0.8%, and the mixture was stirred on ice for 1.5 hours to obtain a crude cell lysate.

[0083] Load the crude cell lysate directly into a 5 ml HisTrap TM The protein was purified on a Fast Flow Crude Cytiva column (Sigma-Aldrich) pre-equilibrated with 10 column volumes of binding buffer (PBS, 40 mM imidazole, 0.1% detergent (e.g., DDM), pH 7.4). Elution was performed using an NGC Quest 10 chromatography system (Bio-rad, Hercules, CA, USA) at a flow rate of 1 ml / min. Elution was performed initially with a gradient from 0% to 12% elution buffer over 10 column volumes, followed by a gradient from 12% to 100% elution buffer over 5 to 10 column volumes (elution buffer: PBS, 1 M imidazole, 0.1% detergent (e.g., DDM), pH 7.4).

[0084] Western blot analysis of crude cell lysates, flow-through fractions, and purified proteins was performed using anti-6x His antibody (ab14923) (Abcam, Cambridge, MA, USA). The results showed that the lipidated recombinant protein was successfully purified.

[0085] Example 6: Bacterial ghost loading of recombinant proteins

[0086] 5 mg of lyophilized bacterial ghosts were resuspended in 200 μl of recombinant protein solution containing 10 μg fHBP-A19_001, 10 μg fHBP-B22_001, and 6 μg TLR9 agonist ODN2395 (Invivogen, San Diego, CA, USA) in an Eppendorf tube. The tube was frozen at -80°C for 1 hour and then freeze-dried overnight.

[0087] Example 7: Mouse immunization and serum antibody titer analysis

[0088] The bacterial ghosts loaded with recombinant proteins were injected subcutaneously at a dose of 5 mg bacterial ghosts / mouse to immunize a group of C57 mice. As a control, 10 μg fHBP-A19_001, 10 μg fHBP-B22_001, 50 μL PBS and 50 μL alum adjuvant were injected. TM Alum Adjuvant (Fisher Scientific, Waltham, MA, USA) was mixed and injected intramuscularly into the hind legs of another group of C57 mice. Each group consisted of 6 mice. 14 days later, both groups received a second injection in the same manner as before. 14 days after the second injection, serum was collected from the tails of the mice for antibody titer analysis. As a negative control, another group of 6 C57 mice was raised under the same conditions as the treated group without immunization. 28 days later, serum was collected from the tails of the mice for antibody titer analysis.

[0089] The antibody titer against the recombinant proteins fHBP-A19_001 and fHBP-B22_001 was determined using an enzyme-linked immunosorbent assay (ELISA). Each well in a 96-well Costar plate was coated with 100 μl of 1 μg / mL recombinant protein solution (fHBP-A19_001 or fHBP-B22_001), and the plate was placed at 4°C overnight. After washing and incubation with blocking solution, 100 μl of mouse serum at different dilutions was added to each well. Color development was performed using a secondary antibody coupled to alkaline phosphatase and TMB substrate. The OD value at 450 nm was recorded using an ELISA reader, and the results were as follows: Figure 4 and Figure 5 shown. Figure 4 is the titer of anti-fHBP-A19_001 antibodies in mouse serum; Figure 5The results showed that the antibody titer in the serum of mice immunized with bacterial ghosts was significantly higher than that of mice not immunized with bacterial ghosts.

[0090] Example 8: Mouse Immunization and Serum Bactericidal Titer Analysis

[0091] The bacterial ghosts loaded with recombinant proteins prepared according to Example 6 ((10 μg fHBP-A19_001 + 10 μg fHBP-B22_001 + 6 μg ODN2395) / 5 mg bacterial ghosts) were subcutaneously injected at a dose of 5 mg bacterial ghosts / mouse to immunize a group of C57 mice (5 mice / group). As a control, 10 μg fHBP-A19_001, 10 μg fHBP-B22_001, 50 μL PBS and 50 μL alum adjuvant (Immune TM Alum Adjuvant (Fisher Scientific, Waltham, MA, USA) was mixed and injected into the hind leg muscle to immunize another group of C57 mice (5 mice / group). 14 days later, both groups of mice received a second injection in the same manner as before. 14 days after the second injection, the mice were bled, and serum samples were collected from each group. As a negative control, another group of mice (5 mice / group) was raised under the same conditions as the treated group without immunization. 28 days later, the mice were bled, and serum samples were collected.

[0092] Serum Bactericidal Actitivity (SBA) Assay:

[0093] Neisseria meningitidis serogroup B strains were streaked to obtain single colonies and cultured overnight in Brain Heart Infusion (BHI) medium containing 10% Horse Blood Supplement at 37°C and 5% CO. A portion of the single colony was resuspended at an appropriate density as required for the assay in PBS (PCM) buffer containing 0.1% glucose at pH 7.4 and calcium and magnesium.

[0094] The bactericidal activity of complement-mediated serum was determined using serum from human donors as a complement source according to the method of Mountzoros 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.). A test solution consisting of 25 μl of PCM buffer, 5 μl of heat-inactivated (56°C for 30 minutes) serially diluted (two-fold dilutions) serum of the mouse to be tested, 10 μl of human complement, and 10 μl of a solution containing approximately 1×10 3 Up to 3x10 3 The test plate was incubated at 37°C for 30 minutes. 200 μl of modified Frantz growth medium containing alamar blue 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 plate was incubated at 37°C overnight. TM A microplate fluorescence analyzer (Fisher Scientific) reads the fluorescence signal (generated by the reaction of Alamy blue dye and live bacteria). For the negative control, 30 μl of PCM was added to another well of the test plate without adding 5 μl of the mouse serum to be tested. The other components and reaction conditions were consistent with the above experimental group. In addition, different known quantities of Neisseria meningitidis and Alamy blue dye were added to another set of wells on the test plate without adding the serum to be tested to generate a standard curve (the Y-axis is the fluorescence signal value and the X-axis is the number of bacteria). According to the standard curve, the number of bacteria after the reaction in the experimental group and the negative control was calculated. The number of bacteria killed can be obtained by subtracting the number of bacteria after the reaction from the number of bacteria before the reaction. Serum with a known bactericidal titer was used as a positive control to confirm the feasibility and accuracy of the above test.

[0095] The results of the SBA test showed that the bactericidal activity of the serum of mice immunized with bacterial ghosts against different strains was significantly higher than that of mice not immunized with bacterial ghosts (see Table 1).

[0096] The bactericidal titer (bactericidal titer) in Table 1 is the reciprocal of the highest serum dilution factor that kills more 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 factor (the lowest serum dilution factor is 1:25), the bactericidal titer of the sample is reported as <30.

[0097] Table 1: Bactericidal titers of mouse serum against different strains of Neisseria meningitidis serogroup B

[0098]

[0099] References:

[0100] Abtin, A., Kudela, P., Mayr, UB, Koller, VJ, Mildner, M., Tschachler, E., Lubitz, W., 2010. Escherichia coli ghosts promote innate immune responses in human keratinocytes.Biochem.Biophys.Res.Commun.400, 78-82. https: / / doi.org / 10.1016 / j.bbrc.2010.08.013

[0101] Adam, E., Delbrassine, L., Bouillot, C., Reynders, V., Mailleux, A.-C., Muraille, E., Jacquet, A., 2010. Probiotic Escherichia coli Nissle 1917activatesDC and prevents house dust mite allergy through a TLR4-dependentpathway.Eur.J.Immunol.40, 1995-2005.https: / / doi.org / 10.1002 / eji.200939913

[0102] Alexopoulou,L.,Holt,A.C.,Medzhitov,R.,Flavell,R.A.,2001.Recognitionof double-stranded RNA and activation of NF-κB by Toll-like receptor 3.Nature413,732-738.https: / / doi.org / 10.1038 / 35099560

[0103] Amara,A.,Salem-Bekh,M.M.,Alanazi,F.,2013.Preparation of BacterialGhosts for E.coli JM109Using“Sponge-like Reduced Protocol.”https: / / doi.org / 10.3923 / AJBS.2013.363.369

[0104] Beernink,P.T.,Welsch,J.A.,Harrison,L.H.,Leipus,A.,Kaplan,S.L.,Granoff,D.M.,2007.Prevalence of factor H-binding protein variants and NadAamong meningococcal group B isolates from the United States:implications forthe development of a multicomponent group B vaccine.J.Infect.Dis.195,1472-1479.https: / / doi.org / 10.1086 / 514821

[0105] Bruge,J.,Bouveret-Le Cam,N.,Danve,B.,Rougon,G.,Schulz,D.,2004.Clinical evaluation of a group B meningococcal N-propionylatedpolysaccharide conjugate vaccine in adult,male volunteers.Vaccine22,1087-1096.https: / / doi.org / 10.1016 / j.vaccine.2003.10.005

[0106] Callaghan,MJ,Buckee,CO,Jolley,KA,Kriz,P.,Maiden,MCJ,Gupta,S.,2008.The effect of immune selection on the structure of the meningococcalopa protein repertoire.PLoS Pathog.4,e1000020.https: / / doi.org / 10.1371 / journal.ppat.1000020

[0107] Capecchi, B., Adu-Bobie, J., Di Marcello, F., Ciucchi, L., Masignani, V., Taddei, A., Rappuoli, R., Pizza, M., Aricò, B., 2005. Neisseria meningitidis NadA is a new invader 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

[0108] Comanducci, M., Bambini, S., Caugant, DA, Mora, M., Brunelli, B., Capecchi, B., Ciucchi, L., Rappuoli, R., Pizza, M., 2004. NadA diversity and carriage in Neisseria meningitidis. Infect. Immun. 72, 4217-4223. https: / / doi.org / 10.1128 / IAI.72.7.4217-4223.2004

[0109] Dynamics of PhiX174protein E-mediated lysis of Escherichia coli|SpringerLink[WWW Document],n.d.URL https: / / link.springer.com / article / 10.1007 / BF00248685(accessed 10.19.20).

[0110] Ebensen,T.,Paukner,S.,Link,C.,Kudela,P.,de Domenico,C.,Lubitz,W.,Guznán,C.A.,2004.Bacterial ghosts are an efficient delivery system for DNA vaccines.J.Immunol.Baltim.Md1950 172,6858-6865.https: / / doi.org / 10.4049 / jimmunol.172.11.6858

[0111] Epidemics of meningococcal disease.African meningitis belt,2001,2001..Releve Epidemiol.Hebd.76,282-288.

[0112] Finne,J.,Leinonen,M., P.H.,1983.Antigenic similarities betweenbrain components and bacteria causing meningitis.Implications for vaccinedevelopment and pathogenesis.Lancet Lond.Engl.2,355-357.https: / / doi.org / 10.1016 / s0140-6736(83)90340-9

[0113] Fletcher,LD,Bernfield,L.,Barniak,V.,Farley,JE,Howell,A.,Knauf,M.,Ooi,P.,Smith ,RP,Weise,P.,Wetherell,M.,Xie,X.,Zagursky,R.,Zhang,Y.,Zlotnick,GW,2004.Vaccine potential of the Neisseria meningitidis2086lipoprotein.Infect.Immun.72,2088-2100.https: / / doi.org / 10.1128 / iai.72.4.2088-2100.2004

[0114] Giuliani,MM,Adu-Bobie,J.,Comanducci,M., B., Savino, S., Santini, L., Brunelli, B., Bambini, S., Biolchi, A., Capecchi, B., Cartocci, E., Ciucchi, L., DiMarcello, F., Ferlicca, F., Galli, B., Luzzi, E., Masignani, V., Serruto, D., Veggi, D., Contorni, M., Morandi, M., Bartalesi, A., Cinotti, V., Mannucci, D., Titta, F., Ovidi, E., Welsch, JA, Granoff, D., Rappuoli, R., Pizza, M., 2006. A universal vaccine forserogroup B meningococcus. Proc. Natl. Acad. Sci. USA 103, 10834-10839. https: / / doi.org / 10.1073 / pnas.0603940103

[0115] Granoff,DM,Pollard,AJ,2007.Reconsideration of the use of meningococcal polysaccharide vaccine.Pediatr.Infect.Dis.J.26,716-722.https: / / doi.org / 10.1097 / INF.0b013e3180cc2c25

[0116] 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.Previouslyunrecognized vaccine candidates against group B meningococcus identified byDNA microarrays.Nat.Biotechnol.20,914-921.https: / / doi.org / 10.1038 / nbt728

[0117] Halperin,S.A.,Langley,J.M.,Smith,B.,Wunderli,P.,Kaufman,L.,Kimura,A.,Martin,D.,2007.Phase 1 first-in-human studies of the reactogenicity andimmunogenicity of a recombinant meningococcal NspA vaccine in healthyadults.Vaccine 25,450-457.https: / / doi.org / 10.1016 / j.vaccine.2006.08.001

[0118] Harrison,L.H.,Trotter,C.L.,Ramsay,M.E.,2009.Global epidemiology ofmeningococcal disease.Vaccine 27 Suppl 2,B51-63.https: / / doi.org / 10.1016 / j.vaccine.2009.04.063

[0119] Harrison,O.B.,Claus,H.,Jiang,Y.,Bennett,J.S.,Bratcher,H.B.,Jolley,K.A.,Corton,C.,Care,R.,Poolman,J.T.,Zollinger,W.D.,Frasch,C.E.,Stephens,D.S.,Feavers,I.,Frosch,M.,Parkhill,J.,Vogel,U.,Quail,M.A.,Bentley,S.D.,Maiden,M.C.J.,n.d.Description and Nomenclature of Neisseria meningitidis CapsuleLocus-Volume 19,Number 4-April 2013-Emerging Infectious Diseases journal-CDC.https: / / doi.org / 10.3201 / eid1904.111799

[0120] Hou,V.C.,Moe,G.R.,Raad,Z.,Wuorimaa,T.,Granoff,D.M.,2003.Conformational epitopes recognized by protective anti-neisserial surfaceprotein A antibodies.Infect.Immun.71,6844-6849.https: / / doi.org / 10.1128 / iai.71.12.6844-6849.2003

[0121] Huter,V.,Szostak,M.P.,Gampfer,J.,Prethaler,S.,Wanner,G.,Gabor,F.,Lubitz,W.,1999.Bacterial ghosts as drug carrier and targetingvehicles.J.Control.Release Off.J.Control.Release Soc.61,51-63.https: / / doi.org / 10.1016 / s0168-3659(99)00099-1

[0122] Innate Immune Recognition|Annual Review of Immunology[WWW Document],n.d.URL https: / / www.annualreviews.org / doi / abs / 10.1146 / annurev.immunol.20.083001.084359(accessed 10.19.20).

[0123] Jacobsson,S.,Thulin,S., P.,Unemo,M.,Comanducci,M.,Rappuoli,R.,Olcén,P.,2006.Sequence constancies and variations in genes encoding three newmeningococcal vaccine candidate antigens.Vaccine 24,2161-2168.https: / / doi.org / 10.1016 / j.vaccine.2005.11.006

[0124] Jalava,K.,Eko,F.O.,Riedmann,E.,Lubitz,W.,2003.Bacterial ghosts ascarder and targeting systems for mucosal antigen delivery.Expert Rev.Vaccines2,45-51.https: / / doi.org / 10.1586 / 14760584.2.1.45

[0125] Jennings,H.J.,Gamian,A.,Ashton,F.E.,1987.N-propionylated group Bmeningococcal polysaccharide mimics a unique epitope on group B Neisseriameningitidis.J.Exp.Med.165,1207-1211.https: / / doi.org / 10.1084 / jem.165.4.1207

[0126] Jennings,H.J.,Lugowski,C.,1981.Immunochemistry of groups A,B,and Cmeningococcal polysaccharide-tetanus toxoid conjugates.J.Immunol.Baltim.Md1950 127,1011-1018.

[0127] Jolley,K.A.,Appleby,L.,Wright,J.C.,Christodoulides,M.,Heckels,J.E.,2001.Immunization with recombinant Opc outer membrane protein from Neisseriameningitidis:influence of sequence variation and levels of expression on thebactericidal immune response against meningococci.Infect.Immun.69,3809-3816.https: / / doi.org / 10.1128 / IAI.69.6.3809-3816.2001

[0128] Koeberling,O.,Seubert,A.,Granoff,D.M.,2008.Bactericidal antibodyresponses elicited by a meningococcal outer membrane vesicle vaccine withoverexpressed factor H-binding protein and genetically attenuatedendotoxin.J.Infect.Dis.198,262-270.https: / / doi.org / 10.1086 / 589308

[0129] Kudela,P.,Koller,V.J.,Lubitz,W.,2010.Bacterial ghosts(BGs)--advancedantigen and drug delivery system.Vaccine 28,5760-5767.https: / / doi.org / 10.1016 / j.vaccine.2010.06.087

[0130] Langemann,T.,Koller,V.J.,Muhammad,A.,Kudela,P.,Mayr,U.B.,Lubitz,W.,2010.The Bacterial Ghost platform system:production andapplications.Bioeng.Bugs 1,326-336.https: / / doi.org / 10.4161 / bbug.1.5.12540

[0131] Lubitz,W.,2001.Bacterial ghosts as carrier and targetingsystems.Expert Opin.Biol.Ther.1,765-771.https: / / doi.org / 10.1517 / 14712598.1.5.765

[0132] Martin,D.,Cadieux,N.,Hamel,J.,Brodeur,B.R.,1997.Highly conservedNeisseria meningifidis surface protein confers protection againstexperimental infection.J.Exp.Med.185,1173-1183.https: / / doi.org / 10.1084 / jem.185.7.1173

[0133] Mayr,U.B.,Walcher,P.,Azimpour,C.,Riedmann,E.,Haller,C.,Lubitz,W.,2005.Bacterial ghosts as antigen delivery vehicles.Adv.Drug Deliv.Rev.57,1381-1391.https: / / doi.org / 10.1016 / j.addr.2005.01.027

[0134] Moe,G.R.,Tan,S.,Granoff,D.M.,1999.Differences in surface expressionof NspA among Neisseria meningitidis group B strains.Infect.Immun.67,5664-5675.https: / / doi.org / 10.1128 / IAI.67.11.5664-5675.1999

[0135] Muhammad,A.,Champeimont,J.,Mayr,U.B.,Lubitz,W.,Kudela,P.,2012.Bacterial ghosts as carriers of protein subunit and DNA-encoded antigensfor vaccine applications.Expert Rev.Vaccines 11,97-116.https: / / doi.org / 10.1586 / erv.11.149

[0136] Nolan,T.,Lambert,S.,Roberton,D.,Marshall,H.,Richmond,P.,Streeton,C.,Poolman,J.,Boutriau,D.,2007.A novel combined Haemophilus influenzae type b-Neisseria meningitidis serogroups C and Y-tetanus-toxoid conjugate vaccine isimmunogenic and induces immune memory when co-administered with DTPa-HBV-IPVand conjugate pneumococcal vaccines in infants.Vaccine 25,8487-8499.https: / / doi.org / 10.1016 / j.vaccine.2007.10.013

[0137] Pace,D.,Pollard,A.J.,Messonier,N.E.,2009.Quadrivalent meningococcalconjugate vaccines.Vaccine 27 Suppl 2,B30-41.https: / / doi.org / 10.1016 / j.vaccine.2009.05.003

[0138] Pérez,R.E.,Lasa,A.M.,Rodriguez,R.S.,Menéndez,E.C.,Suárez,J.G.,Balaguer,H.D.,2006.Scale-up of recombinant Ope protein production inEscherichia coli for a meningococcal vaccine.J.Biotechnol.127,109-114.https: / / doi.org / 10.1016 / j.jbiotec.2006.06.005

[0139] Pizza,M.,Scarlato,V.,Masignani,V.,Giuliani,M.M., B., Comanducci, M., Jennings, GT, Baldi, L., Bartolini, E., Capecchi, B., Galeotti, CL, Luzzi, E., Manetti, R., Marchetti, E., Mora, M., Nuti, S., Ratti, G., Santini, L., Savino, S., Scarselli, M., Storni ,E.,Zuo,P.,Broeker,M.,Hundt,E.,Knapp,B.,Blair,E.,Mason,T.,Tettelin,H.,Hood,DW,Jeffries ,AC,Saunders,NJ,Granoff,DM,Venter,JC,Moxon,ER,Grandi,G.,Rappuoli,R.,2000.Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.Science 287,1816-1820.https: / / doi.org / 10.1126 / science.287.5459.1816

[0140] Plested,JS,Granoff,DM,2008.Vaccine-induced opsonophagocyticimmunity to Neisseria meningitidis group B.Clin.Vaccine Immunol.CVI 15,799-804.https: / / doi.org / 10.1128 / CVI.00036-08

[0141] Quevedo-Diaz,MA,Song,C.,Xiong,Y.,Chen,H.,Wahl,LM,Radulovic,S.,Medvedev,AE,2010.Involvement of TLR2 and TLR4 in cell responses to Rickettsia akari.J.Leukoc.Biol.88,675-685.https: / / doi.org / 10.1189 / jlb.1009674

[0142] Riedmann,EM,Kyd,JM,Cripps,AW,Lubitz,W.,2007.Bacterial ghosts asadjuvant particles.Expert Rev.Vaccines 6,241–253.https: / / doi.org / 10.1586 / 14760584.6.2.241

[0143] Rokbi,B.,Mignon,M.,Maitre-Wilmotte,G.,Lissolo,L.,Danve,B.,Caugant,DA,Quentin-Millet,MJ,1997.Evaluation of recombinant transferrin-binding protein B variants from Neisseria meningitidis for their ability to inducecross-reactive and bactericidal antibodies against a genetically diversecollection of serogroup B strains.Infect.Immun.65,55-63

[0144] Stein,E.,Inic-Canada,A.,Belij,S.,Montanaro,J.,Bintner,N.,Schlacher,S.,Ma yr,UB,Lubitz,W.,Stojanovic,M.,Najdenski,H.,Barisani-Asenbauer,T.,2013.In In vitro and in vivo uptake study of Escherichia coli Nissle 1917bacterial ghosts:a cell-based delivery system to target ocular surface diseases.Invest.Ophthalmol.Vis.Sci.54,6326-6333

[0145] Thompson,E.A.L.,Feavers,I.M.,Maiden,M.C.J.,2003.Antigenic diversityof meningococcal enterobactin receptor FetA,a vaccine component.Microbiol.Read.Engl.149,1849-1858.https: / / doi.org / 10.1099 / mic.0.26131-0

[0146] Trotter,C.L.,Chandra,M.,Cano,R.,Larrauri,A.,Ramsay,M.E.,Brehony,C.,Jolley,K.A.,Maiden,M.C.J.,Heuberger,S.,Frosch,M.,2007.A surveillance networkfor meningococcal disease in Europe.FEMS Microbiol.Rev.31,27-36.https: / / doi.org / 10.1111 / j.1574-6976.2006.00060.x

[0147] Welsch,J.A.,Moe,G.R.,Rossi,R.,Adu-Bobie,J.,Rappuoli,R.,Granoff,D.M.,2003.Antibody to genome-derived neisserial antigen 2132,a Neisseriameningitidis candidate vaccine,confers protection against bacteremia in theabsence of complement-mediated bactericidal activity.J.Infect.Dis.188,1730-1740.https: / / doi.org / 10.1086 / 379375

[0148] West,D.,Reddin,K.,Matheson,M.,Heath,R.,Funnell,S.,Hudson,M.,Robinson,A.,Gorringe,A.,2001.Recombinant Neisseria meningitidis transferrin bindingprotein A protects against experimental meningococcalinfection.Infect.Immun.69,1561-1567.https: / / doi.org / 10.1128 / IAI.69.3.1561-1567.2001

[0149] Wu,X.,Ju,X.,Du,L.,Yuan,J.,Wang,L.,He,R.,Chen,Z.,2017.Production ofBacterial Ghosts from Gram-Positive Pathogen Listeria monocytogenes.FoodbornePathog.Dis.14,1-7.https: / / doi.org / 10.1089 / fpd.2016.2184

Claims

1. A Neisseria meningitidis vaccine, comprising bacterial ghosts and a factor H binding protein (fHBP), wherein the factor H binding protein (fHBP) comprises fHBP of subfamily A and fHBP of subfamily B, wherein the sequence of the factor H binding protein (fHBP) of subfamily A is shown in SEQ ID NO: 5, and the sequence of the factor H binding protein (fHBP) of subfamily B is shown in SEQ ID NO: 10, and the bacterial ghosts are ghosts of Lactobacillus acidophilus.

2. The vaccine of claim 1, further comprising one or more adjuvants.

3. The vaccine of claim 2, wherein the adjuvant is a TLR9 agonist.

4. The vaccine of any one of claims 1-3, wherein the factor H binding protein (fHBP) is loaded into the bacterial ghost.

5. The vaccine of any one of claims 2-3, wherein the adjuvant is loaded within the bacterial ghost. The vaccine according to any one of claims 1 to 3, which can be used in humans or mammals.

7. Use of a composition in preparing a vaccine for preventing or treating infection by Neisseria meningitidis or a disease induced therefrom, the composition comprising bacterial ghosts and factor H binding protein (fHBP), the factor H binding protein (fHBP) comprising fHBP from subfamily A and fHBP from subfamily B, wherein the sequence of the factor H binding protein (fHBP) from subfamily A is shown in SEQ ID NO: 5, and the sequence of the factor H binding protein (fHBP) from subfamily B is shown in SEQ ID NO: 10, and the bacterial ghosts are ghosts of Lactobacillus acidophilus.

8. Use of a composition for preparing a vaccine for preventing or treating infection by Neisseria meningitidis serogroup B or a disease induced therefrom, the composition comprising bacterial ghosts and factor H binding protein (fHBP), the factor H binding protein (fHBP) comprising fHBP from subfamily A and fHBP from subfamily B, wherein the sequence of the factor H binding protein (fHBP) from subfamily A is shown in SEQ ID NO: 5, and the sequence of the factor H binding protein (fHBP) from subfamily B is shown in SEQ ID NO: 10, and the bacterial ghosts are ghosts of Lactobacillus acidophilus.

9. The use according to any one of claims 7 to 8, wherein the composition further comprises one or more adjuvants.

10. The use according to claim 9, wherein the adjuvant is a TLR9 agonist.

11. The use according to any one of claims 7 to 8, wherein the factor H binding protein (fHBP) is loaded into the bacterial ghost.

12. The use of claim 9, wherein the adjuvant is loaded into the bacterial ghost.

13. The use according to any one of claims 7 to 8, wherein the vaccine can be used for humans or mammals.

14. The use of any one of claims 7-8, wherein the disease comprises invasive meningococcal disease (IMD).

Images