Mutant factor h binding protein (fHbp) of neisseria meningitidis, compositions thereof and uses thereof

Abstract

The invention relates to biological pharmacy. In particular, the present invention relates to a mutant factor H binding protein (fHbp) of Neisseria meningitidis serogroup B. In addition, the present invention relates to a composition for the prevention of the Factor H binding protein (fHbp) of Neisseria meningitidis. In particular, the present invention relates to mutant factor H binding proteins (fHbp) of Neisseria meningitidis, compositions thereof, and uses thereof. The invention also relates to the use of said mutant factor H binding protein (fHbp) in the treatment or prevention or diagnosis of neisseria meningitidis disease caused by at least one of serogroup A, C, B, Y and W diseases. In particular, the invention relates to a pharmaceutical composition comprising said mutant Factor H binding protein (fHbp) for use in the treatment or prevention or diagnosis of Neisseria meningitidis serogroup B disease.

Description

Technical Field

[0001] The present invention relates to biopharmaceuticals. Specifically, the present invention relates to a mutant factor H-binding protein (fHbp) of Neisseria meningitidis. In addition, the present invention relates to a composition for preventing the mutant factor H-binding protein (fHbp) of Neisseria meningitidis (N. meningitidis). The present invention particularly relates to the mutant factor H-binding protein (fHbp) of Neisseria meningitidis, a composition thereof, and uses thereof. The present invention also relates to the use of the mutant factor H-binding protein (fHbp) in treating, preventing or diagnosing Neisseria meningitidis diseases caused by at least one of diseases of serogroups A, C, B, Y, X and W. The present invention specifically relates to a pharmaceutical composition comprising the mutant factor H-binding protein (fHbp) for treating, preventing or diagnosing Neisseria meningitidis serogroup B disease. The present invention specifically relates to a pharmaceutical composition comprising the mutant factor H-binding protein (fHbp) for use as a prophylactic or therapeutic vaccine. Background Art

[0002] The background description contains information that may be helpful for understanding the present invention. However, it is not admitted that any information provided herein is prior art or relevant to the invention claimed herein, nor is it admitted that any publication explicitly or implicitly cited is prior art.

[0003] Neisseria meningitidis (Men) is a Gram-negative capsulated bacterium that can cause diseases such as sepsis, meningitis, and death. The bacterium has 13 serogroups, among which the most common (serogroups) are A, B, C, W, Y, and X (MenA, MenB, MenC, MenW, MenY, and MenX) [McNeil et al., Vaccine; 27, 2009, 3417 - 3421; Tapia et al., N Engl J Med; 2021 Jun 3; 384(22):2115 - 2123]. MenB is globally prevalent [Garcia et al., npj Vaccines 6, 130 (2021)].

[0004] The polysaccharide (PS) capsule present in bacteria is considered an important virulence factor and has been successfully used to develop PS-based conjugate vaccines against MenA, MenC, MenW, MenY, and MenX which is under development. However, the MenB PS capsule has poor immunogenicity in humans and also resembles human neuronal cells [Goldblatt, Clin Exp Immunol. 2000;119:1-3; Luo et al., AAPS J. 2016 Nov;18(6):1562-1575; Tapia et al., N Engl J Med; 2021 Jun 3;384(22):2115-2123; McNeil et al., Vaccine; 27, 2009, 3417-3421]. However, there are also multiple other (surface) virulence factors of Neisseria meningitidis [Caesar et al., Microbial Pathogenesis, 57, 2013, 33-40].

[0005] Factor H-binding protein (fHbp), an outer membrane native lipoprotein of approximately 30-32 kDa in size, has been shown to be a virulence factor of Neisseria meningitidis and is also a target of functional bactericidal antibodies. Also known as lipoprotein 2086 (LP2086) and Neisserial antigen from genome (GNA1870), fHbp binds to human factor H (fH), and this binding enables the bacteria to resist complement-mediated killing and evade immune attack [Yee WX, Barnes G et al., Trends Microbiol. 2023 Aug;31(8):805-815. Abad et al., J Infect. 2021 Apr;82(4):37-44; Fletcher et al., Infect Immun. 2004 Apr;72(4):2088-100; Gandhi et al., Postgrad Med. 2016 Aug;128(6):548-56; McNeil et al., Microbiol Mol Biol Rev. 2013 Jun;77(2):234-52].

[0006] Sequencing of the fHbp gene in different MenB strains has revealed the presence of three major variants (var1, var2, var3) [Masignani et al., J Exp Med. 2003 Mar 17; 197(6):789-99; Brehony et al., N. Microbiology (Reading). 2009 Dec; 155(Pt12):4155-4169;], or two subfamilies (A and B: corresponding to variants 2 / 3 and 1 respectively) [Gandhi et al., Postgrad Med. 2016 Aug; 128(6):548-56].

[0007]

[0008] Pfizer's and GSK's are two approved fHbp-containing vaccines. Although both contain fHbp, their compositions are different. is a recombinantly produced bivalent fHbp lipoprotein-based vaccine that has two fHbps, from subfamily A (A05) and subfamily B (B01) respectively [Gandhi et al., Postgrad Med. 2016 Aug; 128(6):548-56; Feavers and Maiden, Clin Vaccine Immunol. 2017 May 5; 24(5):e00566-16].

[0009] Also known as 4CMenB, it has four antigens, namely Neisseria adhesin A (NadA), Neisseria heparin-binding antigen (NHBA) fused to protein GNA1030, fHbp (non-lipidated, subfamily B and variant B24) fused to protein GNA2091, and outer membrane vesicles (OMV) [Gandhi et al., Postgrad Med. 2016 Aug; 128(6):548-56; Feavers and Maiden, Clin Vaccine Immunol. 2017 May 5; 24(5):e00566-16].

[0010] The potency of meningococcal vaccines can be measured by the Serum Bactericidal Assay (SBA) (correlate of protection) and is considered the gold standard for immunity [Borrow et al., J Infect. 2020 Dec;81(6):862 - 872; Dretler et al., Hum Vaccin Immunother. 2018 May 4;14(5):1146 - 1160]. Preclinical studies have demonstrated that fHBP - based vaccines can elicit serum bactericidal antibodies and are capable of killing MenB strains in the SBA. The results of preclinical studies have prompted fHBP - based vaccines to enter the clinical trial phase and have shown that they can also generate bactericidal antibodies in human subjects [Caesar et al., Microbial Pathogenesis, 57, 2013, 33 - 40; McNeil et al., Microbiol Mol Biol Rev. 2013 Jun;77(2):234 - 52]. There is still an unmet need for a broader range of fHbp polypeptides that can elicit an effective antigenic response.

[0011] In the present invention, A02, A42, A46, B44, and B107 in the fHbp family were selected to mutate their amino acid sequences and produce them as the recombinant proteins disclosed herein.

[0012] The advantage of including fHbp antigens from different sub - families is a broader coverage. It has been observed that in order to achieve a broader coverage, fHbp from two sub - families (A and B) is required because no SBA killing of sub - family B isolates was observed when using sub - family A sera [McNeil et al., Microbiol Mol Biol Rev. 2013 Jun;77(2):234 - 52]. Therefore, in order to achieve a broader coverage, it is desirable and prudent to use fHbp from two sub - families.

[0013] The development of serogroup B meningococcal vaccines will potentially fill the unmet need for meningococcal B disease through coverage in the global population [https: / / www.who.int / es / publications / i / item / 9789240026407 accessed on November 1, 2023; Safadi et al., Expert Rev Vaccines. 2021 Apr;20(4):401 - 414].

[0014] Although different companies are researching Neisseria meningitidis, new methods and new compositions for Neisseria meningitidis serogroup B vaccines are still needed that can be more economical, easier to produce, more scalable, and can overcome the deficiencies associated with the prior art.

[0015] The present invention provides new, specific and industrially advantageous mutant factor H-binding proteins (fHbp) of Neisseria meningitidis. The object of the present invention is to modify / mutate the amino acid (aa) sequences of the fHbp proteins of both subfamilies A and B. These modifications should be antigenic and thus capable of eliciting the desired immune response to prevent Neisseria meningitidis serogroup B disease. It is preferred to include fHbp proteins from both subfamily A and subfamily B to provide a broader coverage. The vaccine will meet the unmet medical needs for the prevention of Neisseria meningitidis.

[0016] Object of the invention

[0017] One object of the present invention is to provide a mutant factor H-binding protein (fHbp) of Neisseria meningitidis.

[0018] One object of the present invention is to provide mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44 and B107.

[0019] One object of the present invention is to provide mutant factor H-binding proteins (fHbp) of Neisseria meningitidis families A02, A42, A46, B44 and B107, which are respectively selected from SEQ ID No. 1, 2, 3, 4 and 5.

[0020] One object of the present invention is to provide a pharmaceutical composition comprising at least two of the following:

[0021] a) A mutant protein of Neisseria meningitidis fHbp family A02 (the protein of SEQ ID.1);

[0022] b) A mutant protein of Neisseria meningitidis fHbp family A42 (the protein of SEQ ID.2);

[0023] c) A mutant protein of Neisseria meningitidis fHbp family A46 (the protein of SEQ ID.3);

[0024] d) A mutant protein of Neisseria meningitidis fHbp family B44 (the protein of SEQ ID.4); and

[0025] e) A mutant protein of Neisseria meningitidis fHbp family B107 (the protein of SEQ ID.5).

[0026] One object of the present invention is to provide a pharmaceutical composition comprising at least two of the following:

[0027] a) A mutant protein of the Neisseria meningitidis fHbp family A02 (having at least 90% sequence identity with SEQ ID No. 1);

[0028] b) A mutant protein of the Neisseria meningitidis fHbp family A42 (having at least 90% sequence identity with SEQ ID No. 2);

[0029] c) A mutant protein of the Neisseria meningitidis fHbp family A46 (having at least 90% sequence identity with SEQ ID No. 3);

[0030] d) A mutant protein of the Neisseria meningitidis fHbp family B44 (having at least 90% sequence identity with SEQ ID No. 4); and

[0031] e) A mutant protein of the Neisseria meningitidis fHbp family B107 (having at least 90% sequence identity with SEQ ID No. 5).

[0032] Another object of the present invention is to provide a pharmaceutical composition as described above herein, which is used for preventing meningococcal diseases caused by pathogenic Neisseria meningitidis.

[0033] Another object of the present invention is to provide a pharmaceutical composition as described above herein, which is used for preventing meningococcal diseases caused by all six prevalent serogroups of Neisseria meningitidis (MenA, MenB, MenC, MenW, MenY and MenX), and these serogroups can cause invasive meningitis and sepsis.

[0034] Another object of the present invention is to provide a pharmaceutical composition as described above herein, which is used for preventing meningococcal diseases caused by the Neisseria meningitidis serogroup B antigen.

[0035] Another object of the present invention is to provide a pharmaceutical composition as described above herein, which is used as a prophylactic or therapeutic vaccine.

[0036] Another object of the present invention is to provide a pharmaceutical composition as described above herein, which is used as a prophylactic or therapeutic vaccine against meningococcal diseases caused by the Neisseria meningitidis serogroup B antigen.

[0037] Another object of the present invention is to provide a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44 and B107.

[0038] Another object of the present invention is to provide a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44 and B107, said method being selected from recombinant methods, synthetic methods or a combination thereof.

[0039] Another object of the present invention is to provide a method for preparing a pharmaceutical composition comprising at least two of the following:

[0040] a) a mutant protein of Neisseria meningitidis fHbp family A02 (having at least 90% sequence identity with SEQ ID No. 1);

[0041] b) a mutant protein of Neisseria meningitidis fHbp family A42 (having at least 90% sequence identity with SEQ ID No. 2);

[0042] c) a mutant protein of Neisseria meningitidis fHbp family A46 (having at least 90% sequence identity with SEQ ID No. 3);

[0043] d) a mutant protein of Neisseria meningitidis fHbp family B44 (having at least 90% sequence identity with SEQ ID No. 4); and

[0044] e) a mutant protein of Neisseria meningitidis fHbp family B107 (having at least 90% sequence identity with SEQ ID No. 5). Summary of the Invention

[0046] This summary is provided to introduce some concepts in a simplified form that will be further described in the "Detailed Description of the Invention" section below. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to assist in determining the scope of the claimed subject matter.

[0047] The present invention relates to mutant factor H-binding proteins (fHbp) of Neisseria meningitidis.

[0048] In one aspect, the present invention relates to mutant factor H-binding proteins (fHbp) of A02, A42, A46, B44 and B107 of Neisseria meningitidis serogroup B.

[0049] In another aspect, the present invention relates to mutant factor H-binding proteins (fHbp) of A02, A42, A46, B44 and B107 of Neisseria meningitidis serogroup B, which are SEQ ID No. 1, 2, 3, 4 and 5 respectively.

[0050] In one aspect, the present invention relates to a pharmaceutical composition comprising at least two of the following:

[0051] a) A mutant protein of Neisseria meningitidis fHbp family A02 (protein of SEQ ID.1);

[0052] b) A mutant protein of Neisseria meningitidis fHbp family A42 (protein of SEQ ID.2);

[0053] c) A mutant protein of Neisseria meningitidis fHbp family A46 (protein of SEQ ID.3);

[0054] d) A mutant protein of Neisseria meningitidis fHbp family B44 (protein of SEQ ID.4); and

[0055] e) A mutant protein of Neisseria meningitidis fHbp family B107 (protein of SEQ ID.5).

[0056] In another aspect, the present invention relates to a pharmaceutical composition as described above herein, which is used for preventing meningococcal disease caused by pathogenic Neisseria meningitidis.

[0057] In another aspect, the present invention relates to a pharmaceutical composition as described above herein, which is used as a prophylactic or therapeutic vaccine.

[0058] In another aspect, the present invention relates to a pharmaceutical composition as described above herein, which is used as a prophylactic or therapeutic vaccine against meningococcal disease caused by Neisseria meningitidis serogroup B antigen.

[0059] In one aspect, the present invention relates to a pharmaceutical composition as described above herein, which is used for preventing meningococcal disease caused by Neisseria meningitidis serogroups MenA, MenB, MenC, MenW, MenY and MenX.

[0060] In one aspect, the present invention relates to a pharmaceutical composition as described above herein, which is used for preventing meningococcal disease caused by Neisseria meningitidis serogroup B antigen, and these serogroups can cause invasive meningitis and sepsis.

[0061] In another aspect, the present invention relates to a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44 and B107.

[0062] In another aspect, the present invention relates to a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44 and B107, and the method is selected from recombinant methods, synthetic methods or a combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] The following figures form a part of this specification and are included to further illustrate aspects of the present disclosure. The present disclosure can be better understood by referring to the figures and in conjunction with the detailed description of the specific embodiments presented herein.

[0064] Figure 1 For SDS PAGE, showing the induction of mutant subfamily B fHbp sequence ID 5.

[0065] Figure 2 For SDS PAGE, showing the induction of mutant subfamily B fHbp sequence ID 4.

[0066] Figure 3 For SDS PAGE, showing the induction of mutant subfamily A fHbp sequence ID 2.

[0067] Figure 4 For SDS PAGE, showing the induction of mutant subfamily A fHbp sequence ID 1 and sequence ID 3.

[0068] Figure 5 For SDS PAGE, showing the images of purified sequence ID 3 and sequence ID 5.

[0069] Figure 6 Showing the Western blot images of purified sequence ID 3 and sequence ID 5.

[0070] Figure 7 Showing a graph of the IgG response against mutant subfamily A fHbp.

[0071] Figure 8 Showing a graph of the IgG response against mutant subfamily B fHbp. DETAILED DESCRIPTION OF THE INVENTION

[0073] The following is a detailed description of the embodiments of the present disclosure. These embodiments are very detailed to clearly convey the present disclosure. However, the amount of details provided is not intended to limit the expected variations of the embodiments; on the contrary, it is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

[0074] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. If the definition or use of a term in the incorporated reference is inconsistent with or contrary to the definition of that term provided herein, then the definition of that term provided herein shall apply, and not the definition of that term in the reference.

[0075] Throughout this specification, the reference to "one embodiment" or "an embodiment" means that the particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" that appear throughout this specification do not necessarily all refer to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0076] In some embodiments, the numbers that are used to describe and claim certain embodiments of the present invention, representing the amounts of components, properties (such as concentrations), reaction conditions, etc., should be understood to be modified by the term "about" in some cases. Thus, in some embodiments, the numerical parameters set forth in the written description and the appended claims are approximations, which may vary depending on the properties desired to be obtained in a particular embodiment. In some embodiments, the numerical parameters should be construed in accordance with the number of significant figures reported and by applying ordinary rounding techniques. Although the numerical ranges and parameters set forth in the broad scope of some embodiments of the present invention are approximations, the numerical values set forth in the specific examples should be reported as precisely as possible. The numerical values presented in some embodiments of the present invention may contain certain errors, which are necessarily caused by the standard deviation in their corresponding test measurements.

[0077] As used throughout this specification and in the claims that follow, a noun that is not qualified by a quantity word includes a singular and / or plural reference, unless the context clearly dictates otherwise.

[0078] Furthermore, as used in this specification, the meaning of "in" includes "in" and "on", unless the context clearly dictates otherwise.

[0079] Unless the context requires otherwise, throughout the following specification, the term "comprising" and its variations such as "comprises" and "comprising" shall be construed in an open, inclusive sense, i.e., "including but not limited to".

[0080] The recitation of numerical ranges herein is merely intended to be a shorthand method of referring individually to each separate numerical value that falls within the range. Unless otherwise indicated herein, each separate numerical value is incorporated into the specification as if it were recited individually herein. All of the methods described herein may be performed in any suitable order, unless otherwise indicated herein or the context clearly dictates otherwise. Any and all examples or exemplary language (e.g., "such as") provided herein for certain embodiments are only intended to better illustrate the invention and do not limit the scope of the invention otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0081] The following description and the embodiments described herein are provided in the form of one or more examples of specific embodiments that illustrate the principles and aspects of the present disclosure. These examples are provided to explain these principles and the present disclosure, and not for limiting purposes.

[0082] The title and abstract of the invention provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0083] The various terms used herein are as follows. If a term used in a claim is not defined below, the broadest definition of that term given in published publications and issued patents by those skilled in the relevant art at the time of the application shall be adopted.

[0084] According to the present invention, the term "protein" means, but is not limited to, large biomolecules and macromolecules comprising one or more long chains of amino acid residues. The chain of amino acid residues can have any length and can be straight or branched. The term encompasses all naturally occurring or modified / recombinant proteins.

[0085] According to the present invention, the term "immunological composition" means, but is not limited to, a composition that elicits an immune response. The term "immunological composition" may be used interchangeably with other terms having the same meaning as the definition of the immunological composition, such as "composition", "pharmaceutical composition", "vaccine composition" or "vaccine preparation".

[0086] According to the present invention, the term "Neisseria meningitidis" ("Neisseria Meningitidis" or "N. meningitidis") means, but is not limited to, a diplococcal, Gram-negative human upper respiratory tract commensal bacterium. This pathogen can invade the mucosa and enter the bloodstream, causing meningitis, severe sepsis or local infections of the joints and heart.

[0087] According to the present invention, the term "mutant factor H-binding protein (fHbp)" means, but is not limited to, an altered or modified version of the factor H-binding protein. These mutant fHbp proteins have been engineered to have alterations or mutations in their amino acid sequences.

[0088] According to the present invention, the term "adjuvant / adjuvant component" means, but is not limited to, an adjuvant or adjuvant component in the broadest sense, and generally refers to a (e.g., pharmacological or immunological) agent or composition that can modify (e.g., enhance) the efficacy of other agents (e.g., drugs or vaccines). Generally, in the context of the present invention, the term refers to a compound or composition that serves as a carrier or auxiliary substance for an immunogen and / or other pharmaceutically active compound. The term should be interpreted broadly and refers to a broad spectrum of substances capable of enhancing the immunogenicity of an antigen introduced into or co-administered with the adjuvant in question. In the context of the present invention, the adjuvant preferably enhances the specific immunogenic effect of the active agent of the present invention. Generally, "adjuvant" or "adjuvant component" have the same meaning and can be used interchangeably. Adjuvants can be classified, for example, as immunopotentiators, antigen delivery systems or even combinations thereof. The term "adjuvant" is generally understood not to include substances that confer immunity by themselves. Adjuvants non-specifically assist the immune system to enhance the antigen-specific immune response, for example, by facilitating the presentation of antigens to the immune system or inducing a non-specific innate immune response. In addition, the adjuvant can preferably, for example, modulate the antigen-specific immune response, for example, by shifting a dominant Th2-based antigen-specific response to a more Th1-based antigen-specific response and vice versa, and / or by inducing a mucosal immune response and / or increasing the IgA titre. Thus, the adjuvant can advantageously modulate the expression / secretion of cytokines, antigen presentation, the type of immune response, etc.

[0089] Trehalose or α-D-glucopyranosyl α-D-glucopyranoside is a disaccharide known for its protective effect on proteins at high temperatures (especially during drying or lyophilization procedures). According to the teachings of U.S. Patent No. 4,891,319, its protective effect can be explained by the replacement of water molecules by trehalose molecules, both compounds containing OH functional groups. Trehalose is also known in the prior art as a cytoprotectant.

[0090] Advantages of adjuvants include enhancing the immunogenicity of antigens, altering the nature of the immune response, reducing the amount of antigen required for successful immunization, reducing the frequency of booster immunizations required, and improving the immune response in the elderly and immunocompromised populations. These can be co-administered by any route (e.g., intramuscular, subcutaneous, IV or intradermal injection).

[0091] The term "therapeutically effective amount" according to the present invention means, but is not limited to, the amount of an active ingredient (i.e., a therapeutic protein or antibody) sufficient to produce a desired therapeutic effect in a human or animal, e.g., the amount required to treat, cure, prevent or inhibit the development and progression of a disease or its symptoms, and / or the amount required to improve the symptoms or cause the disease to regress. Such a therapeutically effective amount may vary depending on the structure and potency of the active ingredient and the intended mode of administration.

[0092] According to the present invention, the term "treatment" means, but is not limited to, therapeutic treatment and prophylactic or preventive measures. Individuals in need of treatment include individuals already suffering from a disease or disorder to be treated, such as humans and animals, as well as individuals predisposed to a disease or in need of preventing a disease. "Treatment" as used herein also includes reducing the likelihood of contracting a disease, alleviating the severity of a disease in an individual already suffering therefrom, and inducing regression of a disease or its symptoms.

[0093] According to the present invention, the term "pharmaceutically acceptable carrier" means, but is not limited to, liquid fillers, diluents or encapsulating substances that can be safely used for systemic administration. Depending on the particular route of administration, various pharmaceutically acceptable carriers known in the art can be used. These carriers can be selected from the group including the following: sugars, starches, cellulose and its derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions (including phosphate buffered saline), emulsifiers, isotonic saline and pyrogen-free water. In particular, a pharmaceutically acceptable carrier can contain different components, such as buffers, sterile water for injection, physiological saline or phosphate buffered saline, sucrose, histidine, salts and polysorbates. Terms such as "physiologically acceptable", "diluent" or "excipient" can be used interchangeably.

[0094] Mutant fHbp:

[0095] The present invention provides a mutant factor H-binding protein (fHbp) of Neisseria meningitidis.

[0096] The present invention provides a mutant factor H-binding protein (fHbp) of Neisseria meningitidis serogroup B.

[0097] In one embodiment, the present invention provides a mutant factor H-binding protein (fHbp) of Neisseria meningitidis families A02, A42, A46, B44 and B107.

[0098] In one embodiment, the present invention provides a mutant factor H-binding protein (fHbp) of Neisseria meningitidis fHbp families A02, A42, A46, B44 and B107, which have at least 90% sequence identity with SEQ ID Nos. 1, 2, 3, 4 and 5, respectively.

[0099] In a further embodiment, the present invention provides a mutant factor H-binding protein (fHbp) of Neisseria meningitidis families A02, A42, A46, B44 and B107, which is used as a drug or an active pharmaceutical ingredient or a drug substance. In addition, the present invention also provides a pharmaceutical composition of the present invention for preventing Neisseria meningitidis infection.

[0100] Method for preparing mutant fHbp:

[0101] In one embodiment, the present invention provides a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107.

[0102] In one embodiment, the present invention provides a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107, wherein the mutant factor H-binding proteins (fHbp) have at least 90% sequence identity with SEQ ID No. 1, 2, 3, 4, and 5, respectively.

[0103] In another embodiment, the present invention provides a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107, the method being selected from recombinant methods, synthetic methods, or a combination of both.

[0104] Another embodiment of the present invention provides a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis serogroup B fHbp A02, A42, A46, B44, and B107, comprising the following steps:

[0105] a) cloning a single fHbp gene with a signal sequence into an expression vector;

[0106] b) plasmid extraction;

[0107] c) transformation;

[0108] d) inducing the corresponding fHbp;

[0109] e) purifying the corresponding fHbp.

[0110] Another embodiment of the present invention provides a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis serogroup B fHbp A02, A42, A46, B44, and B107, wherein the cloning is carried out in a pET303 vector.

[0111] Another embodiment of the present invention provides a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis serogroup B fHbp A02, A42, A46, B44, and B107, wherein the cloning step (a) further involves codon optimization, gene synthesis, subcloning, and transformation.

[0112] Another embodiment of the present invention provides a method for preparing mutant factor H-binding proteins (fHbp) of Neisseria meningitidis serogroup B fHbp A02, A42, A46, B44 and B107, wherein the purification step (e) is carried out by a method selected from but not limited to: affinity chromatography, ion exchange chromatography, size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), immunoaffinity chromatography, His-tag affinity chromatography, Protein A / G purification, precipitation method, two-phase partitioning, ultrafiltration, differential solubility.

[0113] Composition:

[0114] In one embodiment, the present invention provides a pharmaceutical composition comprising at least two of the following:

[0115] a) A mutant protein of Neisseria meningitidis fHbp family A02 (the protein of SEQ ID. No. 1);

[0116] b) A mutant protein of Neisseria meningitidis fHbp family A42 (the protein of SEQ ID. No. 2);

[0117] c) A mutant protein of Neisseria meningitidis fHbp family A46 (the protein of SEQ ID. No. 3);

[0118] d) A mutant protein of Neisseria meningitidis fHbp family B44 (the protein of SEQ ID. No. 4); and

[0119] e) A mutant protein of Neisseria meningitidis fHbp family B107 (the protein of SEQ ID. No. 5).

[0120] In another embodiment, the present invention provides a pharmaceutical composition comprising at least two of the following:

[0121] a) A mutant protein of Neisseria meningitidis fHbp family A02 (having at least 90% sequence identity with SEQ ID No. 1);

[0122] b) A mutant protein of Neisseria meningitidis fHbp family A42 (having at least 90% sequence identity with SEQ ID No. 2);

[0123] c) A mutant protein of Neisseria meningitidis fHbp family A46 (having at least 90% sequence identity with SEQ ID No. 3);

[0124] d) A mutant protein of Neisseria meningitidis fHbp family B44 (having at least 90% sequence identity with SEQ ID No. 4); and

[0125] e) A mutant protein of the fHbp family B107 of Neisseria meningitidis (having at least 90% sequence identity with SEQ ID No. 5).

[0126] In another embodiment, the present invention provides a method for producing mutant factor H-binding proteins (fHbp) of A02, A42, A46, B44 and B107 of Neisseria meningitidis serogroup B, said mutant factor H-binding proteins (fHbp) having at least 90% sequence identity with SEQ ID No. 1, 2, 3, 4 and 5 respectively, said method comprising using host cells selected from but not limited to cells of mammalian, plant, insect, fungal or bacterial origin. The host cells of the present invention include but are not limited to eukaryotic cells, such as mammalian cells, such as hamster, rabbit, rat, pig, mouse, etc.; avian cells, such as duck, chicken, quail, etc.; insect cells or other animal cells; plant cells and fungal cells, such as corn, tobacco, Saccharomyces cerevisiae, Pichia pastoris; prokaryotic cells, such as Escherichia coli; and other cells used in the art for producing monoclonal antibodies and other binding proteins.

[0127] Composition / Preparation:

[0128] In one embodiment, the present invention provides a pharmaceutical composition comprising at least two of the following:

[0129] a) A mutant protein of the fHbp family A02 of Neisseria meningitidis (having at least 90% sequence identity with SEQ ID No. 1);

[0130] b) A mutant protein of the fHbp family A42 of Neisseria meningitidis (having at least 90% sequence identity with SEQ ID No. 2);

[0131] c) A mutant protein of the fHbp family A46 of Neisseria meningitidis (having at least 90% sequence identity with SEQ ID No. 3);

[0132] d) A mutant protein of the fHbp family B44 of Neisseria meningitidis (having at least 90% sequence identity with SEQ ID No. 4); and

[0133] e) A mutant protein of the fHbp family B107 of Neisseria meningitidis (having at least 90% sequence identity with SEQ ID No. 5).

[0134] In one embodiment, the present invention provides a pharmaceutical composition comprising at least one of the mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107, which can be used for treating, preventing, or diagnosing Neisseria meningitidis diseases caused by Neisseria meningitidis serogroups MenA, MenB, MenC, MenW, MenY, and MenX.

[0135] In another embodiment, the present invention provides a pharmaceutical composition comprising at least one of the mutant factor H-binding proteins (fHbp) of A02, A42, A46, B44, and B107 of Neisseria meningitidis serogroup B, which can be used for treating, preventing, or diagnosing Neisseria meningitidis diseases.

[0136] In yet another embodiment, the present invention provides a pharmaceutical composition comprising at least one of the mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107 of the present invention, and further comprising a pharmaceutically acceptable carrier, diluent, or adjuvant.

[0137] In yet another embodiment, the present invention provides a pharmaceutical composition comprising at least one of the mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107 of the present invention, and further comprising a pharmaceutically acceptable carrier selected from, but not limited to, buffers, antioxidants, preservatives, isotonic agents, and chelating agents.

[0138] In yet another embodiment, the present invention provides a pharmaceutical composition comprising a fully liquid and / or lyophilized modified fHbp of subfamily A of Neisseria meningitidis serogroup B and / or a modified fHbp of subfamily B, and / or mixed with a fully liquid and / or lyophilized conjugated capsular polysaccharide from Neisseria meningitidis serogroups A, C, W135, X, and / or Y.

[0139] Method for preparing composition / preparation:

[0140] In one embodiment, the present invention provides a method for preparing a pharmaceutical composition, the pharmaceutical composition comprising at least two of the following:

[0141] a) A mutant protein of Neisseria meningitidis fHbp family A02 (having at least 90% sequence identity with Sequence ID No. 1);

[0142] b) A mutant protein of Neisseria meningitidis fHbp family A42 (having at least 90% sequence identity with Sequence ID No. 2);

[0143] c) A mutant protein of Neisseria meningitidis fHbp family A46 (having at least 90% sequence identity with SEQ ID No. 3);

[0144] d) A mutant protein of Neisseria meningitidis fHbp family B44 (having at least 90% sequence identity with SEQ ID No. 4); and

[0145] e) A mutant protein of Neisseria meningitidis fHbp family B107 (having at least 90% sequence identity with SEQ ID No. 5).

[0146] Diagnosis

[0147] In one embodiment, the present invention provides a diagnostic kit comprising at least one of at least two of the following:

[0148] a) A mutant protein of Neisseria meningitidis fHbp family A02 (having at least 90% sequence identity with SEQ ID No. 1);

[0149] b) A mutant protein of Neisseria meningitidis fHbp family A42 (having at least 90% sequence identity with SEQ ID No. 2);

[0150] c) A mutant protein of Neisseria meningitidis fHbp family A46 (having at least 90% sequence identity with SEQ ID No. 3);

[0151] d) A mutant protein of Neisseria meningitidis fHbp family B44 (having at least 90% sequence identity with SEQ ID No. 4); and

[0152] e) A mutant protein of Neisseria meningitidis fHbp family B107 (having at least 90% sequence identity with SEQ ID No. 5).

[0153] In one embodiment, the present invention provides a pharmaceutical composition of mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107, which has immunogenicity.

[0154] In one embodiment, the present invention provides a pharmaceutical composition of mutant factor H-binding proteins (fHbp) of Neisseria meningitidis A02, A42, A46, B44, and B107, which is a vaccine.

[0155] In one embodiment, the present invention further provides a method for eliciting an antibody response in a mammal, which comprises administering the pharmaceutical composition of the present invention to the mammal. The antibody response is preferably a protective and / or bactericidal antibody response.

[0156] In another embodiment, the present invention also provides a method for protecting a mammal from Neisseria meningitidis infection, which comprises administering to the mammal the pharmaceutical composition of the present invention.

[0157] In another embodiment, the mammal is preferably a human. The human can be an adult or preferably a child.

[0158] In one embodiment, the present invention provides a pharmaceutical composition of mutant factor H-binding protein (fHbp), which is generally directly administered to a patient. The direct delivery can be by parenteral injection (such as subcutaneous injection, intraperitoneal injection, intradermal injection, intravenous injection, intramuscular injection or injection into the tissue interstitium), or by rectal, oral, vaginal, topical, transdermal, intranasal, ocular, aural, pulmonary or other mucosal routes. Intramuscular administration to the thigh or upper arm is preferred. The injection can be carried out by a needle (such as a subcutaneous injection needle), but alternatively needleless injection can also be used. A typical intramuscular dose is 0.5 ml. The present invention can be used to elicit systemic and / or mucosal immunity.

[0159] The dosing regimen can be a single-dose regimen or a multi-dose regimen. Multiple doses can be used in the primary immunization regimen and / or the booster immunization regimen. A booster dose regimen can be carried out after the primary dose regimen. The appropriate time between priming doses (such as between 4 - 16 weeks) and between the priming dose and the booster dose can be determined routinely.

[0160] In one embodiment, the present invention provides a pharmaceutical composition of mutant factor H-binding protein (fHbp), which comprises an immunologically effective amount of the fHbp protein, and any other specified components as required. An immunologically effective amount means an amount that is effective for treatment or prevention when administered to an individual as a single dose or as part of a series of doses. This amount varies according to the health and physical condition of the individual to be treated, age, taxon of the individual to be treated (such as non-human primate, primate, etc.), the ability of the individual's immune system to synthesize antibodies, the desired degree of protection, the formulation of the vaccine, the assessment of the condition by the attending physician, and other relevant factors. It is expected that this amount will be within a relatively wide range, which can be determined by routine tests. The dosing regimen can be a single-dose regimen or a multi-dose regimen (such as including booster doses). The composition can be administered in combination with other immunomodulators. Examples

[0161] The present invention will be further explained in the form of the following examples. However, it should be understood that the following examples are for illustration only and should not be regarded as limiting the scope of the present invention.

[0162] Method for preparing mutant factor H-binding protein (fHbp) of Neisseria meningitidis serogroup B of A02 (SEQ ID NO: 1), A42 (SEQ ID NO: 2), A46 (SEQ ID NO: 3), B44 (SEQ ID NO: 4) and B107 (SEQ ID NO: 5)

[0163] Clone the respective fHbp genes (with P4 signal sequence at the N-terminal start) into the expression vector pET303 (Thermo, USA). Further processes include codon optimization, gene synthesis, subcloning, transformation of Escherichia coli DH5α (NEB, USA) strain, and extraction of positive clone strains to obtain plasmids. All five corresponding His-Tag [6His-Tag] (at the C-terminal of fHbp) fHbps are expressed in Escherichia coli BL21DE3 [NEB, USA] cells in LB medium (Sigma, USA) containing 100 μg / ml ampicillin. Induce all five fHbps (of subfamilies A and B) (OD 600nm between 0.6 - 0.8), using 1 mM IPTG (Sigma, USA) in a culture shaker at 37 °C and 250 rpm for 4 hours. Before induction, take uninduced samples and store them under the same conditions as described for induction. After induction, centrifuge the total cell pellet at 6000 rpm for 20 minutes, store the pellet and discard the supernatant. Analyze the expression of the corresponding fHbp protein in the uninduced (not shown) and induced fractions using SDS PAGE ( Figures 1-4 ). Store the induced fraction or pellet at -80 °C for further processing and protein purification.

[0164] Example 2: Purification of mutant fhbp

[0165] Purification of mutant proteins by Ni-NTA chromatography. The bacterial pellet from 1 L of culture was resuspended in 50 ml of lysis buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 10 mM imidazole, 10% glycerol) containing lysozyme, protease inhibitors and Benzonase. The suspended pellet was incubated on ice for 30 minutes, sonicated for 3 minutes (30-second pulse on, 30-second pulse off, amplitude 30%), and then centrifuged at 14000 rpm for 20 minutes at 10 °C. The pellet was dissolved in 2.5% sarkosyl buffer (prepared in lysis buffer). The resuspended and dissolved pellet was left at room temperature for 15 minutes and then centrifuged at 13000 rpm for 10 minutes at 10 °C. The supernatant was collected and diluted (about 9-10 times) in lysis buffer containing 1% NP 40 and then interacted with Ni-NTA resin. The column was washed with 5 resin bed volumes of Wash buffer 1 (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 25 mM imidazole, 10% glycerol, 1% NP 40) and Wash buffer 2 (20 mM Tris-Cl pH 8.0, 500 mM NaCl, 25 mM imidazole, 10% glycerol, 1% NP 40). The bound His-tagged protein was eluted with 2 resin bed volumes of elution buffer (20 mM Tris-Cl pH 8.0, 300 mM NaCl, 250 mM imidazole, 10% glycerol, 1% NP 40). The eluted fractions were analyzed by SDS-PAGE and the relevant fractions were pooled. The purified fHbp proteins from two subfamilies (A and B) were desalted and placed in phosphate buffered saline using a PD 10 column, and then the purified fHbp was analyzed by SDS PAGE and Western blotting ( Figure 5 and Figure 6 ).

[0166] Example 2: Toxicity study

[0167] A repeated dose subcutaneous toxicity / immunogenicity study was conducted in Swiss Albino Mice using the fHbp of the present invention. The aim of the study was to evaluate the toxicity / immunogenicity when administered to mice via the subcutaneous route at dosing intervals of day 0 and day 28.

[0168] In this study, the reference product was used at a low dose level. This would help to compare the toxic effects between the test product and the reference product.

[0169] This study provides information on the safety profile and potential toxic effects of the fHbp of the invention under repeated exposure (Day 0 and Day 28) over an extended period of time.

[0170] (a) Blood samples were drawn from the retroorbital plexus using a glass capillary tube.

[0171] (b) The blood sample was centrifuged and the serum was separated.

[0172] (c) Blood samples were collected from all animals on day 0 (before treatment), day 14, day 28 and day 42 (before termination) for immunogenicity analysis. Blood samples were collected in Eppendorf tubes and centrifuged. After centrifugation, serum was separated from the blood for immunogenicity analysis. Serum samples were stored at -20°C until shipped. Serum samples have been shipped to the sponsor for immunogenicity analysis and were analyzed and reported in Example 3 below.

[0173] All animals survived until the end of the scheduled treatment period (day 42), at which time they were weighed, sacrificed, and subjected to necropsy.

[0174] Conclusion: The fHbp of the present invention was administered to Swiss albino mice on day 0 and day 28. No adverse reactions related to the treatment were observed in terms of clinical signs, body weight, body weight change, feed consumption and macroscopic observation.

[0175] Therefore, under experimental conditions and based on the results of the study entitled "Repeated-dose subcutaneous toxicity / immunogenicity study in Swiss albino mice using fHbp of the present invention", it was concluded that the fHbp of the present invention was safe and non-toxic to mice at the test doses shown in Table 1.

[0176] Example 3: Immunogenicity studies

[0177] The composition containing fHbp of Neisseria meningitidis serogroup B composition was administered to the animal model on day 0 and day 28. Eight groups were set up and then administered to the animal model to examine the effects of the A / B subfamily or both subfamilies together in the composition.

[0178]

[0179] Procedure for indirect ELISA:

[0180] The first and second doses were given on days 0 and 28, respectively, and sera were collected on days 0, 28, and 42.

[0181] Indirect enzyme-linked immunosorbent assay (ELISA) was performed on the pooled sera of each group at day 0, day 28, and day 42 according to the method of Gheesling et al. with slight modification [Gheesling et al., J Clin Microbiol. 1994 Jun;32(6):1475-82.]. MaxiSorp 96-well plates were coated with fHbp of subfamily A or fHbp of subfamily B in 1X PBS buffer (pH 7.3 ± 0.1), with 100 μl per well containing 100 ng of antigen. After coating, the plates were incubated overnight at 2 - 8°C. The next day, the plates were washed with wash buffer (1X PBS buffer [pH 7.3 ± 0.1] + 0.1% Tween20 [v / v]). After washing, blocking was performed with blocking buffer (1X PBS buffer [pH 7.3 ± 0.1] + 0.1% Tween 20 v / v + 5% fetal bovine serum [FBS]) at room temperature (RT) [25 ± 2°C] for 1 hour. Blocking was performed with 200 μl of blocking buffer per well. Meanwhile, pre-dilutions of serum samples (1:800) and pre-dilutions of quality control (QC) sera (1:1600) were prepared using the assay. The ELISA performed against fHbp of subfamily A or subfamily B had the same pre-dilution for serum samples and QC sera [QC sera were prepared in-house using groups 7 and 8 at day 42. Equal amounts of sera from each mouse in the two groups (groups 7 and 8) at day 42 were pooled to prepare QC sera]. After blocking, the plates were washed, and assay buffer (1X PBS buffer [pH 7.3 ± 0.1] + 0.1% Tween 20 v / v + 5% FBS) at 100 μl / well was added to the coated plates in rows B to H, while 200 μl / well of QC sera (in duplicate) and serum samples (in duplicate) were added to row A, respectively. Subsequently, six two-fold serial dilutions were made in the corresponding wells until row G. All wells in row H had only assay buffer and were designated as blanks as they contained no serum. The plates were incubated at room temperature for 2 hours. After incubation, the plates were washed with wash buffer and incubated with the secondary antibody (anti-mouse antibody conjugated to horseradish peroxidase (HRP)) at room temperature for 1 hour. The secondary antibody was 100 μl / well, with a dilution of 1:16000 for plates coated with subfamily A fHbp and a dilution of 1:8000 for plates coated with subfamily B fHbp. The secondary antibody diluent was prepared in assay buffer. After incubation was complete, the plates were washed, and TMB substrate (3,3′,5,5′-tetramethylbenzidine) was added at 100 μl / well at room temperature for 10 minutes (min). After 10 minutes, 50 μl / well of 2M H2SO4 was added to terminate the reaction, and the absorbance of the corresponding plates was read at 450 nm / 620 nm.The prepared QC serum was assigned an arbitrary value of 5000 ELISA units / ml or EU / ml and used as a standard in each plate. A standard ELISA curve was generated using the standards in each plate to estimate the IgG concentration of the corresponding samples / groups using the combistats software ( Figure 7 and Figure 8 ).

[0182] Conclusion: As Figure 7 and Figure 8 shown, the studies conducted indicate that both fHbp A and fHbp B of the present invention have immunogenic potential after two doses (day 42). The IgG concentration (EU / ml) of group 7 (higher dose combination of 60 μg each of fHbp A and fHbp B) was higher than that of group 5 (dose of 12 μg each of fHbp A and fHbp B). In addition, it was also observed that the IgG concentration of the combined groups with both fHbps (subfamilies A and B) was also higher than that of the groups with fHbps from one subfamily (i.e., subfamily A or subfamily B).

[0183] Sequence Listing

[0184]

[0185]

[0186] Although the preferred embodiments of the present invention and their respective variations have been described, various modifications to these embodiments can be envisioned by those of ordinary skill in the art.

[0187] Therefore, the present invention should not be limited to the exact forms and manners disclosed and described above, but should be by way of example only. Accordingly, modifications and variations can be made to the present invention without departing from its true scope and spirit as defined by the appended claims.

[0188] Advantages of the Present Invention

[0189] The present invention provides mutant factor H-binding proteins (fHbps) of serogroup B Neisseria meningitidis of A02, A42, A46, B44, and B107.

[0190] The present invention provides an immunocomposition as described above herein, which comprises a combination of mutant subfamily A and subfamily B proteins, and thus can provide a broader coverage and can be used for the complete prevention of meningococcal diseases caused by pathogenic Neisseria meningitidis.

[0191] The present invention provides an immunocomposition as described above herein, which is used for the complete prevention of meningococcal diseases caused by pathogenic Neisseria meningitidis.

[0192] The present invention provides novel, specific and industrially advantageous mutant factor H binding proteins (fHbp) of serogroup B Neisseria meningitidis of A02, A42, A46, B44 and B107, which are capable of completely preventing meningococcal diseases. These compositions should be able to meet the unmet medical needs in meningitis prevention and treatment.

Claims

1. A mutant factor H-binding protein (fHbp) of Neisseria meningitidis families A02, A42, A46, B44, and B107.

2. A mutant factor H-binding protein (fHbp) of Neisseria meningitidis fHbp families A02, A42, A46, B44, and B107, wherein the mutant factor H-binding protein (fHbp) has at least 90% sequence identity with SEQ ID No. 1, 2, 3, 4, and 5, respectively.

3. A method for preparing the mutant factor H-binding protein (fHbp) as claimed in claim 1 or 2.

4. A method for preparing the mutant factor H-binding protein (fHbp) as claimed in claim 1 or 2, wherein the method is selected from recombinant methods, synthetic methods, or a combination of both.

5. A method for preparing mutant factor H-binding proteins (fHbp) of A02, A42, A46, B44, and B107 of Neisseria meningitidis serogroup B fHbp, comprising the following steps: a) Cloning a single fHbp gene with a signal sequence into an expression vector; b) Plasmid extraction; c) Transformation; d) Inducing the corresponding fHbp; e) Purifying the corresponding fHbp.

6. A pharmaceutical composition comprising at least two of the following: a) A mutant protein of Neisseria meningitidis fHbp family A02 (the protein of SEQ ID.1); b) A mutant protein of Neisseria meningitidis fHbp family A42 (the protein of SEQ ID.2); c) A mutant protein of Neisseria meningitidis fHbp family A46 (the protein of SEQ ID.3); d) A mutant protein of Neisseria meningitidis fHbp family B44 (the protein of SEQ ID.4); and e) A mutant protein of Neisseria meningitidis fHbp family B107 (the protein of SEQ ID.5).

7. The composition as claimed in claim 5, which can be used for treating, preventing, or diagnosing Neisseria meningitidis diseases caused by Neisseria meningitidis serogroups MenA, MenB, MenC, MenW, MenY, and MenX.

8. The composition as claimed in claim 5, which can be used for treating, preventing, or diagnosing Neisseria meningitidis diseases caused by MenB.

9. The composition as claimed in claim 5, further comprising a pharmaceutically acceptable carrier, diluent, or adjuvant.

10. The composition as claimed in claim 8, wherein the pharmaceutically acceptable carrier is selected from, but not limited to, buffers, antioxidants, preservatives, isotonic agents, and chelating agents.

11. The composition as claimed in claim 5, wherein the composition is a fully liquid and / or lyophilized modified fHbp of Neisseria meningitidis serogroup B subfamily A and / or subfamily B, and / or mixed with a fully liquid and / or lyophilized conjugated capsular polysaccharide of Neisseria meningitidis serogroups A, C, W135, X, and / or Y.

12. The composition as claimed in claim 5, wherein the composition is a vaccine.

13. The composition according to claim 5, wherein the composition is administered subcutaneously, intraperitoneally, intradermally, intravenously, intramuscularly, or into the tissue interstitium, or is administered rectally, orally, vaginally, topically, transdermally, intranasally, ophthalmically, aurally, via the lungs or other mucosal routes.

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